Dr. Sheibani is a Professor in the Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health. He is interested in mechanisms that keep angiogenesis in check and alterations under pathological conditions, such as ischemia or diabetes, that result in ocular neovascularization. He has developed in-vivo mouse models (developing retinal vasculature, oxygen-induced ischemic retinopathy, diabetes, and laser-induced Choroidal neovascularization) and in-vitro cell culture models (retinal endothelial cells, pericytes, astrocytes, choroidal endothelial cells, retinal pigment epithelial cells, corneal endothelial cells, corneal epithelial cells, and trabecular meshwork cells) to study normal and pathological angiogenesis, the molecular and cellular mechanisms that contribute to normal ocular development and function, as well as pathologies associated with eye diseases at the cellular level.
Steered molecular dynamic simulations of conformational lock of Cu, Zn-superoxide dismutase.
Sci Rep. 2019 Mar 13;9(1):4353
Authors: Xiao BL, Ning YN, Niu NN, Li D, Moosavi-Movahedi AA, Sheibani N, Hong J
The conformational lock was a bio-thermodynamic theory to explain the characteristics of interfaces in oligomeric enzymes and their effects on catalytic activity. The previous studies on superoxide dismutases (Cu, Zn-SODs) showed that the dimeric structure contributed to the high catalytic efficiency and the stability. In this study, steered molecular dynamics simulations were used firstly to study the main interactions between two subunits of Cu, Zn-SODs. The decomposition process study showed that there were not only four pairs of hydrogen bonds but also twenty-five residue pairs participating hydrophobic interactions between A and B chains of SOD, and van der Waals interactions occupied a dominant position among these residue pairs. Moreover, the residue pairs of hydrogen bonds played a major role in maintaining the protein conformation. The analysis of the energy and conformational changes in the SMD simulation showed that there were two groups (two conformational locks) between A and B chains of SOD. The first group consisted of one hydrogen-bond residues pair and seven hydrophobic interactions residues pairs with a total average energy of -30.10 KJ/mol, and the second group of three hydrogen-bond residues pair and eighteen hydrophobic interactions residues pairs formed with a total average energy of -115.23 KJ/mol.
PMID: 30867507 [PubMed - in process]
Cyp1b1-deficient retinal astrocytes are more proliferative and migratory, and are protected from oxidative stress and inflammation.
Am J Physiol Cell Physiol. 2019 Mar 20;:
Authors: Falero-Perez J, Sorenson CM, Sheibani N
Astrocytes (AC) are the most abundant cells in the central nervous system. Retinal AC play an important role in maintaining the integrity of retinal neurovascular function, and their dysfunction contributes to the pathogenesis of various eye diseases including diabetic retinopathy. Cytochrome p450 1B1 (CYP1B1) expression in the neurovascular structures of the central nervous system including AC has been reported. We previously showed that CYP1B1 expression is a key regulator of redox homeostasis in retinal vascular cells. Its deficiency in mice resulted in increased oxidative stress, and attenuation of angiogenesis in vivo and proangiogenic activity of retinal vascular cells in vitro. Here using retinal AC prepared from wild type ( Cyp1b1+/+) and Cyp1b1-deficient ( Cyp1b1-/-) mice, we determined the impact of Cyp1b1 expression on retinal AC function. We showed that Cyp1b1-/- retinal AC were more proliferative and migratory. These cells also produced increased amounts of fibronectin and its receptors αvβ3 and α5β1 integrins. These results were consistent with the increased adhesive properties of Cyp1b1-/- AC, and their lack of ability to form a network in Matrigel. This was reversed by re-expression of Cyp1b1 in Cyp1b1-/- AC. Although no significant changes were observed in AKT/SRC/MAPK signaling pathways, the production of inflammatory mediators BMP7 and MCP1 were decreased in Cyp1b1-/- AC. Cyp1b1-/- AC also showed increased levels of connexin 43 phosphorylation, and CD38 expression when challenged with H2O2. These results are consistent with increased proliferation and diminished oxidative stress in Cyp1b1-/- cells. Thus, Cyp1b1 expression in AC plays an important role in retinal neurovascular homeostasis.
PMID: 30892936 [PubMed - as supplied by publisher]
The impact of water molecules on binding affinity of the anti-diabetic thiazolidinediones for catalase: Kinetic and mechanistic approaches.
Arch Biochem Biophys. 2019 Feb 06;:
Authors: Yekta R, Dehghan G, Rashtbari S, Sadeghi L, Baradaran B, Sheibani N, Moosavi-Movahedi AA
Water molecules play a vital role in efficient drug binding to its target. Thiazolidinediones (TZDs), a class of anti-diabetic drugs, are widely used for treatment of type 2 diabetes mellitus. In the present study, the possible contribution of water molecules to the binding of TZDs to catalase, a potential target in the liver, was investigated by different experimental and theoretical methods. These studies indicated that TZDs could significantly improve the catalase catalytic function with a significant contribution from water molecules. As a probe for the differential number of released water molecules during the catalase transition from E to E* states, the activity of TZDs-catalase complexes was demonstrated to be mainly dependent on water activity. However, free catalase decomposed the substrate more independently. In addition, the spectrofluorimetry studies showed that the binding of TZDs to catalase needed the release of water molecules from the enzyme's binding pocket. The thermodynamic studies indicated that the binding enthalpy and entropy of TZDs for catalase were decreased with lower water activity. The favorable process contributes to release of water molecules from the binding pocket through the formation of hydrophobic interactions between catalase and TZDs in an enthalpic manner. Molecular docking simulations confirmed that the depletion of water molecules from the binding cavity is essential for effective interactions between TZDs and catalase.
PMID: 30738039 [PubMed - as supplied by publisher]
Novel Paracrine Functions of Smooth Muscle Cells in Supporting Endothelial Regeneration Following Arterial Injury.
Circ Res. 2019 Feb 11;:
Authors: Ren J, Zhou T, Pilli VS, Phan NM, Wang Q, Gupta K, Liu Z, Sheibani N, Liu B
RATIONALE: Regeneration of denuded or injured endothelium is an important component of vascular injury response. Cell-cell communication between endothelial cells (ECs) and smooth muscle cells (SMCs) plays a critical role not only in vascular homeostasis but also in disease. We have previously demonstrated that protein kinase C-delta (PKCδ) regulates multiple components of vascular injury response including apoptosis of SMCs and production of chemokines, thus is an attractive candidate for a role in SMC-EC communication.
OBJECTIVE: To test whether PKCδ-mediated paracrine functions of SMCs influence reendothelialization in rodent models of arterial injury.
METHODS AND RESULTS: Femoral artery wire injury was performed in SMC-conditional Prkcd knockout mice, and carotid angioplasty was conducted in rats receiving transient Prkcd knockdown or overexpression. SMC-specific knockout of Prkcd impaired reendothelialization, reflected by a smaller Evans blue-excluding area in the knockout compared to the wildtype controls. A similar impediment to reendothelialization was observed in rats with SMC-specific knockdown of Prkcd. In contrast, SMC-specific gene transfer of Prkcd accelerated reendothelialization. In vitro, medium conditioned by AdPKCδ-infected SMCs increased endothelial wound closure without affecting their proliferation. A PCR-based array analysis identified Cxcl1 and Cxcl7 among others as PKCδ-mediated chemokines produced by SMCs. Mechanistically, we postulated that PKCδ regulates Cxcl7 expression through signal transducer and activator of transcription 3 (STAT3) as knockdown of STAT3 abolished Cxcl7 expression. The role of CXCL7 in SMC-EC communication was demonstrated by blocking CXCL7 or its receptor CXCR2, both significantly inhibited endothelial wound closure. Furthermore, insertion of a Cxcl7 cDNA in the lentiviral vector that carries a Prkcd shRNA overcame the adverse effects of Prkcd knockdown on reendothelialization.
CONCLUSIONS: SMCs promote reendothelialization in a PKCδ-dependent paracrine mechanism, likely through CXCL7-mediated recruitment of ECs from uninjured endothelium.
PMID: 30739581 [PubMed - as supplied by publisher]
Extended Intravitreal Rabbit Eye Residence of Nanoparticles Conjugated With Cationic Arginine Peptides for Intraocular Drug Delivery: In Vivo Imaging.
Invest Ophthalmol Vis Sci. 2018 Aug 01;59(10):4071-4081
Authors: Melgar-Asensio I, Kandela I, Aird F, Darjatmoko SR, de Los Rios C, Sorenson CM, Albert DM, Sheibani N, Henkin J
Purpose: Drug delivery by intravitreal injection remains problematic, small agents and macromolecules both clearing rapidly. Typical carriers use microparticles (>2 μm), with size-related liabilities, to slow diffusion. We recently described cationic nanoparticles (NP) where conjugated Arg peptides prolonged residence in rat eyes, through ionic interaction with vitreal poly-anions. Here we extended this strategy to in vivo tracking of NP-conjugate (NPC) clearance from rabbit eyes. Relating t1/2 to zeta potential, and varied dose, we estimated the limits of this charge-based delivery system.
Methods: NPC carried covalently attached PEG8-2Arg or PEG8-3Arg pentapeptides, having known sequences from human eye proteins. Peptides were conjugated (61-64 per NPC); each NP/NPC also carried a cyanine7 tag (<0.5 dye/particle). In vivo imaging system (IVIS), after intravitreal injection, estimated NPC loss by 800-nm photon emission (745-nm excitation) at 1 to 3-week intervals following initial scan at day 10.
Results: NPC of 2Arg-peptides or 3Arg-peptides showed clearance t1/2 of 7 days and 17 days respectively, unconjugated NP t1/2 was <<5 days. Doses of 90, 180, and 360 μg of PEG8-2Arg NPC were compared. The lower doses showed dose-proportional day-10 concentration, and similar clearance. Higher early loss was seen with a 360-μg dose, exceeding rabbit vitreal binding capacity. No inflammation was observed.
Conclusions: This type of cationic NPC can safely increase residence t1/2 in a 1 to 3-week range, with dose <100 μg per mL vitreous. Human drug load may then range from 10 to 100 μg/eye, usefulness depending on individual drug potency and release rate, superimposed on extended intravitreal residence.
PMID: 30098194 [PubMed - in process]
Mechanistic investigation of sulfonamide ligands as human carbonic anhydrase II inhibitors.
Int J Biol Macromol. 2018 Aug 31;:
Authors: Alaei L, Khodarahmi R, Sheikh-Hasani V, Sheibani N, Moosavi-Movahedi AA
The effect of some sulfonamide ligands on the structure and function of human carbonic anhydrase II (HCA II) was investigated using different spectroscopic techniques including UV-Vis, fluorescence, circular dichroism and molecular dynamics simulation tools. Kinetic measurements were performed in 50 mM Tris-HCl, pH 7.4 at 27 °C. Kinetic data revealed that sulfonamide ligands inhibit the HCA II esterase activity in a linear competitive manner with Ki in the nanomolar range. Fluorescence measurements illustrated that ligands act as the enzyme quenchers. Stern-Volmer analysis of the quenching data at different temperatures demonstrated that the quenching of the HCA II intrinsic fluorescence occurred through static and dynamic quenching mechanisms. Analysis of the binding thermodynamic parameters showed that hydrogen bonding and hydrophobic interactions play an important role in the stabilization of enzyme-drug complex. Job plot confirmed the 1:1 stoichiometry of ligand-protein complex, and therefore, the existence of one binding site for the ligand. Molecular simulations confirmed that acetazolamide induced impressive conformational changes in two domains adjacent to the active site, including amino acids 19-25 and 61-67. RMSF studies showed sharp changes in three distinct regions near the active site including amino acids 15-25, 160-180 and 190-210 upon drug binding.
PMID: 30176324 [PubMed - as supplied by publisher]
Attenuation of Retinal Vascular Development in Neonatal Mice Subjected to Hypoxic-Ischemic Encephalopathy.
Sci Rep. 2018 Jun 15;8(1):9166
Authors: Zaitoun IS, Cikla U, Zafer D, Udho E, Almomani R, Suscha A, Cengiz P, Sorenson CM, Sheibani N
A significant proportion of children that survive hypoxic-ischemic encephalopathy (HIE) develop visual impairment. These visual deficits are generally attributed to injuries that occur in the primary visual cortex and other visual processing systems. Recent studies suggested that neuronal damage might also occur in the retina. An important structure affecting the viability of retinal neurons is the vasculature. However, the effects of HIE on the retinal neurovasculature have not been systemically evaluated. Here we investigated whether exposure of postnatal day 9 (P9) neonatal mice to HIE is sufficient to induce neurovascular damage in the retina. We demonstrate that the blood vessels on the surface of the retina, from mice subjected to HIE, were abnormally enlarged with signs of degeneration. The intermediate and deep vascular layers in these retinas failed to form normally, particularly in the periphery. All the vascular damages observed here were irreversible in nature up to 100 days post HIE. We also observed loss of retinal neurons, together with changes in both astrocytes and Müller cells mainly in the inner retina at the periphery. Collectively, our findings suggest that HIE results in profound alterations in the retinal vasculature, indicating the importance of developing therapeutic strategies to protect neurovascular dysfunction not only in the brain but also in the retina for infants exposed to HIE.
PMID: 29907863 [PubMed - in process]
Time-lapse microscopy of oxidative stress demonstrates metabolic sensitivity of retinal pericytes under high glucose condition.
J Biophotonics. 2018 Mar 25;:e201700289
Authors: Ghanian Z, Mehrvar S, Jamali N, Sheibani N, Ranji M
Hyperglycemia affects retinal vascular cell function, promotes the development and progression of diabetic retinopathy, and ultimately causes vision loss. Oxidative stress, reactive oxygen species (ROS) in excess, is a key biomarker for diabetic retinopathy. Using time-lapse fluorescence microscopy, ROS dynamics was monitored and the metabolic resistivity of retinal endothelial cells (REC) and pericytes (RPC) was compared under metabolic stress conditions including high glucose (HG). In the presence of a mitochondrial stressor REC exhibited a significant increase in the rate of ROS production compared with RPC. Thus, under normal glucose (NG), REC may utilize oxidative metabolism as the bioenergetic source, while RPC metabolic activity is independent of mitochondrial respiration. In HG condition, the rate of ROS production in RPC was significantly higher, whereas this rate remained unchanged in REC. Thus, under HG condition RPC may preferentially utilize oxidative metabolism, which results in increased rate of ROS production. In contrast, REC use glycolysis as their major bioenergetic source for ATP production, and consequently HG minimally affect their ROS levels. These observations are consistent with our previous studies where we showed HG condition has minimal effect on apoptosis of REC, but results in increased rate of apoptosis in RPC. Collectively, our results suggest that REC and RPC exhibit different metabolic activity preferences under different glucose conditions. Thus, protection of RPC from oxidative stress may provide an early point of intervention in development and progression of diabetic retinopathy.
PMID: 29577636 [PubMed - as supplied by publisher]
Mice dental pulp and periodontal ligament endothelial cells exhibit different proangiogenic properties.
Tissue Cell. 2018 Feb;50:31-36
Authors: Saghiri MA, Asatourian A, Sorenson CM, Sheibani N
Dental pulp is a highly vascularized tissue with a high regenerative capacity. This is attributed to its unique blood supply and the presence of progenitor or postnatal dental pulp stem cells. Here we aimed to isolate and compare the angiogenic properties of endothelial cells (EC) prepared from mouse dental pulp and periodontal ligament (PDL). EC were isolated from 4-week-old wild type immorto mice. Mice were sacrificed and after mandible isolation, the molar and incisor teeth and the PDL from molar teeth were dissected. EC were prepared by collagenase digestion of tissues and affinity purification using magnetic beads coated with platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31) antibody. EC prepared from incisor and molar pulps and PDL were examined for expression of appropriate markers by fluorescence-activated cell sorting (FACS) analysis. The proliferation, migration, and capillary morphogenesis of EC were evaluated. Ex vivo sprouting angiogenesis from various tissues was also compared. Data were analyzed at the level of significance of P<0.05. Pulp EC prepared from incisors proliferated and migrated significantly faster than molar and PDL EC (P<0.05). In addition, molar and PDL EC formed a more extensive capillary network when plated on Matrigel. This is consistent with the lower proliferative and migratory characteristics of these cells compared with incisor EC (P<0.05). However, PDL tissue showed significantly more sprouting area than molar and incisor pulp tissues (P<0.05). Thus, pulp EC from molar and incisor and PDL EC present different proangiogenic properties. Collectively our results suggest that EC from different tooth tissue have unique characteristics related to their target tissue and function.
PMID: 29429515 [PubMed - in process]
Paclitaxel inhibited lysozyme fibrillation by increasing colloidal stability through formation of "off-pathway" oligomers.
Int J Biol Macromol. 2018 Jan 15;:
Authors: Kachooei E, Mozaffarian F, Khodagholi F, Sedeghi P, Karami L, Ghasemi A, Vahdat E, Saboury AA, Sheibani N, Moosavi-Movahedi AA
Protein fibrillation is a challenging issue in medicine, causing many diseases, and an impediment to pharmaceutics and protein industry. Many chemicals, especially polyphenol compounds and aromatic small molecules, have been widely used as an effective strategy to combat protein fibril formation. Hence, understanding mechanisms of fibrillation inhibition and contributing forces in this process are significant. In this study, the inhibitory effect of paclitaxel on lysozyme fibrillation was investigated with respect to thermal and colloidal stability. Fibrillation was monitored with ThT fluorescence, circular dichroism, and AFM; paclitaxel-lysozyme interaction with isothermal titration calorimetry and docking; thermal and colloidal stability with differential scanning calorimetry and zeta-pulse, respectively. Paclitaxel inhibited lysozyme fibrillation, and interacted with lysozyme through hydrogen bonds and van der Waals' interactions. The viability of PC12 cells retrieved as a result of fibrillation inhibition by paclitaxel. Hydrophobic forces dominantly shielded the aggregation-prone region of lysozyme and suppressed the effective interactions between lysozyme monomers. Although paclitaxel did not affect lysozyme's thermal stability, it increased lysozyme's colloidal stability by either increasing the surface charge density or charge distribution on lysozyme. In conclusion, our results suggest a model for paclitaxel's inhibitory role through two complementary steps driving to "off-pathway" oligomer formation and attenuation of fibril formation.
PMID: 29352977 [PubMed - as supplied by publisher]
PEDF Expression Affects the Oxidative and Inflammatory State of Choroidal Endothelial Cells.
Am J Physiol Cell Physiol. 2018 Jan 10;:
Authors: Farnoodian M, Sorenson CM, Sheibani N
Age related macular degeneration (AMD) is the leading cause of vision loss among the elderly population, and is associated with severe macular degeneration and choroidal neovascularization (CNV). Although the pathogenesis of AMD is associated with choroidal dysfunction and CNV, the detailed underlying mechanisms remain unresolved. Altered production of pigment epithelium derived factor (PEDF), a neuroprotective and anti-angiogenic factor, contributes to CNV. Furthermore, exogenous PEDF mitigates angiogenesis in preclinical CNV models. How PEDF expression affects choroidal endothelial cell (ChEC) function is unknown. Here we isolated ChEC from PEDF+/+ and PEDF-deficient (PEDF-/-) mice and determined the impact of PEDF expression on the proangiogenic and proinflammatory properties of ChEC. We showed that PEDF expression significantly affects the proliferation, migration, adhesion, and oxidative and inflammatory state of ChEC. The PEDF-/- ChEC were, however, more sensitive to H2O2 challenge and exhibited increased rate of apoptosis and oxidative stress. We also observed a significant increase in production of cytokines with a primary role in inflammation and angiogenesis including VEGF and osteopontin, and a reprograming of chemokines and cytokines expression profiles in PEDF-/- ChEC. Collectively, our results indicate that PEDF expression has a significant impact on oxidative and inflammatory properties of ChEC, whose alteration could contribute to pathogenesis of chronic inflammatory diseases including exudative AMD.
PMID: 29351407 [PubMed - as supplied by publisher]
Temporal diabetes-induced biochemical changes in distinctive layers of mouse retina.
Sci Rep. 2018 Jan 18;8(1):1096
Authors: Aboualizadeh E, Sorenson CM, Schofield AJ, Unger M, Sheibani N, Hirschmugl CJ
To discover the mechanisms underlying the progression of diabetic retinopathy (DR), a more comprehensive understanding of the biomolecular processes in individual retinal cells subjected to hyperglycemia is required. Despite extensive studies, the changes in the biochemistry of retinal layers during the development of DR are not well known. In this study, we aimed to determine a more detailed understanding of the natural history of DR in Akita/+ (type 1 diabetes model) male mice with different duration of diabetes. Employing label-free spatially resolved Fourier transform infrared (FT-IR) chemical imaging engaged with multivariate analysis enabled us to identify temporal-dependent reproducible biomarkers of the individual retinal layers from mice with 6 weeks,12 weeks, 6 months, and 10 months of age. We report, for the first time, the nature of the biochemical alterations over time in the biochemistry of distinctive retinal layers namely photoreceptor retinal layer (PRL), inner nuclear layer (INL), and plexiform layers (OPL, IPL). Moreover, we present the molecular factors associated with the changes in the protein structure and cellular lipids of retinal layers induced by different duration of diabetes. Our paradigm provides a new conceptual framework for a better understanding of the temporal cellular changes underlying the progression of DR.
PMID: 29348593 [PubMed - in process]
O-Linked β-N-acetylglucosamine (O-GlcNAc) modification: a new pathway to decode pathogenesis of diabetic retinopathy.
Clin Sci (Lond). 2018 Jan 31;132(2):185-198
Authors: Gurel Z, Sheibani N
The incidence of diabetes continues to rise among all ages and ethnic groups worldwide. Diabetic retinopathy (DR) is a complication of diabetes that affects the retinal neurovasculature causing serious vision problems, including blindness. Its pathogenesis and severity is directly linked to the chronic exposure to high glucose conditions. No treatments are currently available to stop the development and progression of DR. To develop new and effective therapeutic approaches, it is critical to better understand how hyperglycemia contributes to the pathogenesis of DR at the cellular and molecular levels. We propose alterations in O-GlcNAc modification of target proteins during diabetes contribute to the development and progression of DR. The O-GlcNAc modification is regulated through hexosamine biosynthetic pathway. We showed this pathway is differentially activated in various retinal vascular cells under high glucose conditions perhaps due to their selective metabolic activity. O-GlcNAc modification can alter protein stability, activity, interactions, and localization. By targeting the same amino acid residues (serine and threonine) as phosphorylation, O-GlcNAc modification can either compete or cooperate with phosphorylation. Here we will summarize the effects of hyperglycemia-induced O-GlcNAc modification on the retinal neurovasculature in a cell-specific manner, providing new insight into the role of O-GlcNAc modification in early loss of retinal pericytes and the pathogenesis of DR.
PMID: 29352075 [PubMed - in process]
The effect of Tmem135 overexpression on the mouse heart.
PLoS One. 2018;13(8):e0201986
Authors: Lewis SA, Takimoto T, Mehrvar S, Higuchi H, Doebley AL, Stokes G, Sheibani N, Ikeda S, Ranji M, Ikeda A
Tissues with high-energy demand including the heart are rich in the energy-producing organelles, mitochondria, and sensitive to mitochondrial dysfunction. While alterations in mitochondrial function are increasingly recognized in cardiovascular diseases, the molecular mechanisms through which changes in mitochondria lead to heart abnormalities have not been fully elucidated. Here, we report that transgenic mice overexpressing a novel regulator of mitochondrial dynamics, transmembrane protein 135 (Tmem135), exhibit increased fragmentation of mitochondria and disease phenotypes in the heart including collagen accumulation and hypertrophy. The gene expression analysis showed that genes associated with ER stress and unfolded protein response, and especially the pathway involving activating transcription factor 4, are upregulated in the heart of Tmem135 transgenic mice. It also showed that gene expression changes in the heart of Tmem135 transgenic mice significantly overlap with those of aged mice in addition to the similarity in cardiac phenotypes, suggesting that changes in mitochondrial dynamics may be involved in the development of heart abnormalities associated with aging. Our study revealed the pathological consequence of overexpression of Tmem135, and suggested downstream molecular changes that may underlie those disease pathologies.
PMID: 30102730 [PubMed - in process]
Negative Regulators of Angiogenesis, Ocular Vascular Homeostasis, and Pathogenesis and Treatment of Exudative AMD.
J Ophthalmic Vis Res. 2018 Oct-Dec;13(4):470-486
Authors: Farnoodian M, Sorenson CM, Sheibani N
Angiogenesis, the formation of new blood vessels from pre-existing capillaries, is very tightly regulated and normally does not occur except during developmental and reparative processes. This tight regulation is maintained by a balanced production of positive and negative regulators, and alterations under pathological conditions such as retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration can lead to growth of new and abnormal blood vessels. Although the role of proangiogenic factors such as vascular endothelial growth factor has been extensively studied, little is known about the roles of negative regulators of angiogenesis in the pathogenesis of these diseases. Here, we will discuss the role of thrombospondin-1 (TSP1), one of the first known endogenous inhibitors of angiogenesis, in ocular vascular homeostasis, and how its alterations may contribute to the pathogenesis of age-related macular degeneration and choroidal neovascularization. We will also discuss its potential utility as a therapeutic target for treatment of ocular diseases with a neovascular component.
PMID: 30479719 [PubMed]
Vitamin D and regulation of vascular cell function.
Am J Physiol Heart Circ Physiol. 2017 Dec 22;:
Authors: Jamali N, Sorenson CM, Sheibani N
Vitamin D deficiency is linked to pathogenesis of many diseases including cardiovascular, cancer, and various eye diseases. In recent years, important roles for vitamin D in regulation of immune function, inflammation, angiogenesis, and aging have been demonstrated. Thus, vitamin D and its analogs have been evaluated for treatment of various types of cancer and chronic diseases. We previously showed that the active form of vitamin D (1, 25(OH)2D3) is a potent inhibitor of angiogenesis. This activity is consistent with the important role proposed for vitamin D and its analogs in the mitigation of tumor growth through inhibition of angiogenesis. Here we review the important nutritional value of vitamin D and the abnormalities linked to its deficiency. We will explore its potential role as a regulator of angiogenesis and vascular cell function, and the role vitamin D receptor (VDR) expression plays in these activities during vascular development and neovascularization. Our studies have established an important role for 1, 25(OH)2D3 and VDR in regulation of perivascular supporting cell function. In addition, interaction of 1, 25(OH)2D3 and VDR is essential for these activities and inhibition of neovascularization. Delineating the signaling pathways involved and identification of genes that are the target of 1, 25(OH)2D3 regulation in vascular cells will allow us to identify novel pathways that are targets for regulation of vascular function and angiogenesis.
PMID: 29351464 [PubMed - as supplied by publisher]
Sustaining Intravitreal Residence With L-Arginine Peptide-Conjugated Nanocarriers.
Invest Ophthalmol Vis Sci. 2017 Oct 01;58(12):5142-5150
Authors: Li H, Liu W, Sorenson CM, Sheibani N, Albert DM, Senanayake T, Vinogradov S, Henkin J, Zhang HF
Purpose: Intravitreal injection of antiangiogenic agents is becoming a standard treatment for neovascular retinal diseases. Sustained release of therapeutics by injecting colloidal carriers is a promising approach to reduce the injection frequency, which reduces treatment burdens and the risk of complications on patients. Such sustained release often requires carriers to have micrometer-scale dimension that, however, can potentially promote glaucoma and inflammation. Small, polycationic particles can be immobilized in vitreous through multiple cooperative ionic interactions with hyaluronic acid of the vitreous interior, but such particles are generally toxic. Here, we synthesized and examined a biocompatible dextran-based nanocarrier (<50 nm in diameter) conjugated with cationic peptides containing L-arginine with minimal toxicity, aiming to provide sustained release of therapeutic drugs in vitreous.
Methods: We synthesized the nanocarriers with condensed cholesteryl dextran (CDEX) as core material. Cationic peptides containing 1 to 4 arginine groups, along with fluorescence tags, were conjugated to the CDEX surface. We monitored the carrier diffusion rate ex vivo and half-lives in vivo in rodent vitreous using fluorescence imaging. We evaluated the toxicity by histological examinations at the second, third, eighth, and thirty-sixth week.
Results: The diffusion rate of nanocarriers was inversely related to zeta potential values in freshly isolated vitreous humor. We observed increased half-lives in vivo with increasing zeta potential (up to 240 days). Histological examinations confirmed no adverse effects on ocular morphology and organization.
Conclusions: We demonstrated the potential of L-arginine peptide-conjugated nanocarriers toward safe and sustained therapeutic release system for posterior eye diseases.
PMID: 28986592 [PubMed - in process]
Augmentation of the cytotoxic effects of zinc oxide nanoparticles by MTCP conjugation: Non-canonical apoptosis and autophagy induction in human adenocarcinoma breast cancer cell lines.
Mater Sci Eng C Mater Biol Appl. 2017 Sep 01;78:949-959
Authors: Mozdoori N, Safarian S, Sheibani N
Zinc oxide nanoparticles are very toxic, but their agglomeration reduces their lethal cytotoxic effects. Here we tested the hypothesis that conjugation of ZnO nanoparticles via Meso-Tetra (4-Carboxyphenyl) Porphyrin (MTCP) could provide electrostatic or steric stabilization of ZnO nanoparticles and increase their cytotoxic effects. The cytotoxicity and cell death induction were assessed using two human breast adenocarcinoma cell lines (MCF-7 and MDA-MB-468). The MTT results indicated that the toxicity of ZnO nanoparticles was significantly increased upon MTCP conjugation. Annexin/PI and real time RT-PCR results demonstrated that the ZnO-MTCP nanoparticles induced cell death via different non-canonical pathways that are under ca(2+) control. Calcium signaling could regulate lysosomal dependent apoptosis and death autophagy, and killing of the two selected types of breast cancer cells.
PMID: 28576071 [PubMed - in process]
Storage Medium Affects the Surface Porosity of Dental Cements.
J Clin Diagn Res. 2017 Aug;11(8):ZC116-ZC119
Authors: Saghiri MA, Shabani A, Asatourian A, Sheibani N
INTRODUCTION: Calcium silicate-based cements physical properties is influenced by environmental changes.
AIM: Here, we intended to evaluate the effect of storage medium on surface porosity of root Mineral Trioxide Aggregate (MTA) and Biodentine cement.
MATERIALS AND METHODS: A total of 40 polyethylene tubes were selected and divided into two groups: Group A (MTA) and Group B (Biodentine). Each group was subdivided into two subgroups (n=10). In subgroups A1 and B1, tubes were transferred to Distilled Water (DW), while samples of subgroup A2 and B2 were transferred to Synthetic Tissue Fluid (STF) as storage medium and samples were stored for three days. All specimens were then placed in a desiccator for 24 hours and then subject to surface porosity evaluation by Scanning Electron Microscopy (SEM) at ×500, ×1000, ×2000 and ×5000 magnifications. The number and the surface porosities were determined by Image J analysis. Data were analyzed by ANOVA at level of significance of p<0.05.
RESULTS: The lowest surface porosity was observed in MTA samples stored in STF and the highest was in Biodentine samples stored in DW. Significant differences were noted between groups and subgroups of each group (p< 0.05). MTA samples stored in DW and STF showed significantly lower surface porosities compared to Biodentine samples (p < 0.05).
CONCLUSION: Storage medium can drastically affect the surface porosity of tested calcium silicate-based cements. However, MTA showed lower surface porosity compared to Biodentine cement, which can result in lower microleakage in applied area.
PMID: 28969288 [PubMed]
PEDF Expression Affects Retinal Endothelial Cell Pro-angiogenic Properties through Alterations in Cell Adhesive Mechanisms.
Am J Physiol Cell Physiol. 2017 Jul 26;:ajpcell.00004.2017
Authors: Falero-Perez J, Park S, Sorenson CM, Sheibani N
Pigment epithelium derived factor (PEDF) is an endogenous inhibitor of angiogenesis. Although various ocular cell types including retinal endothelial cells (EC) produce PEDF, we know very little about cell autonomous effects of PEDF in these cell types. Here we determined how PEDF expression affects retinal EC proangiogenic properties. Retinal EC were prepared from wild type (PEDF+/+) and PEDF-deficient (PEDF-/-) mice. The identity of EC was confirmed by staining for specific markers including VE-cadherin, CD31, and B4-lectin. Retinal EC also expressed VEGF-R1 and endoglin, as well as ICAM-1, ICAM-2, and VCAM-1. PEDF-/- retinal EC were more proliferative, less apoptotic when challenged with H2O2, less migratory, and less adherent compared with PEDF+/+ EC. These changes could be associated, at least in part, with increased levels of tenascin C, fibronectin, thrombospondin-1 and collagen IV, and lower amounts of osteopontin. PEDF-/- EC also exhibited alterations in expression of a number of integrins including α2, αv, β1, β8, and αvβ3, and cell-cell adhesion molecules including CD31, ZO-1, and occludin. These observations correlated with attenuation of capillary morphogenesis and increased levels of oxidative stress in PEDF-/- EC. PEDF -/- EC also produced lower levels of VEGF compared with PEDF+/+ cells. Thus, PEDF deficiency has a significant impact on retinal EC adhesion and migration, perhaps through altered production of ECM and junctional proteins in response to increased oxidative stress affecting their proangiogenic activity.
PMID: 28747334 [PubMed - as supplied by publisher]
Detection of Guanine and Adenine Using an Aminated Reduced Graphene Oxide Functional Membrane-Modified Glassy Carbon Electrode.
Sensors (Basel). 2017 Jul 18;17(7):
Authors: Li D, Yang XL, Xiao BL, Geng FY, Hong J, Sheibani N, Moosavi-Movahedi AA
A new electrochemical sensor based on a Nafion, aminated reduced graphene oxide and chitosan functional membrane-modified glassy carbon electrode was proposed for the simultaneous detection of adenine and guanine. Fourier transform-infrared spectrometry (FTIR), transmission electron microscopy (TEM), and electrochemical methods were utilized for the additional characterization of the membrane materials. The prepared electrode was utilized for the detection of guanine (G) and adenine (A). The anodic peak currents to G and A were linear in the concentrations ranging from 0.1 to 120 μM and 0.2 to 110 μM, respectively. The detection limits were found to be 0.1 μM and 0.2 μM, respectively. Moreover, the modified electrode could also be used to determine G and A in calf thymus DNA.
PMID: 28718793 [PubMed - in process]
Targeting of 12/15-Lipoxygenase in retinal endothelial cells, but not in monocytes/macrophages, attenuates high glucose-induced retinal leukostasis.
Biochim Biophys Acta. 2017 Jun;1862(6):636-645
Authors: Ibrahim AS, Saleh H, El-Shafey M, Hussein KA, El-Masry K, Baban B, Sheibani N, Wang MH, Tawfik A, Al-Shabrawey M
AIMS: Our previous studies have established a role for 12/15-lipoxygenase (LO) in mediating the inflammatory response in diabetic retinopathy (DR). However, the extent at which the local or systemic induction of 12/15-LO activity involved is unclear. Thus, the current study aimed to characterize the relative contribution of retinal endothelial versus monocytic/macrophagic 12/15-LO to inflammatory responses in DR.
MATERIALS & METHODS: We first generated a clustered heat map for circulating bioactive lipid metabolites in the plasma of streptozotocin (STZ)-induced diabetic mice using liquid chromatography coupled with mass-spectrometry (LC-MS) to evaluate changes in circulating 12/15-LO activity. This was followed by comparing the in vitro mouse endothelium-leukocytes interaction between leukocytes isolated from 12/15-LO knockout (KO) versus those isolated from wild type (WT) mice using the myeloperoxidase (MPO) assay. Finally, we examined the effects of knocking down or inhibiting endothelial 12/15-LO on diabetes-induced endothelial cell activation and ICAM-1 expression.
RESULTS: Analysis of plasma bioactive lipids' heat map revealed that the activity of circulating 12/15-LO was not altered by diabetes as evident by no significant changes in the plasma levels of major metabolites derived from 12/15-lipoxygenation of different PUFAs, including linoleic acid (13-HODE), arachidonic acid (12- and 15- HETEs), eicosapentaenoic acid (12- and 15- HEPEs), or docosahexaenoic acid (17-HDoHE). Moreover, leukocytes from 12/15-LO KO mice displayed a similar increase in adhesion to high glucose (HG)-activated endothelial cells as do leukocytes from WT mice. Furthermore, abundant proteins of 12-LO and 15-LO were detected in human retinal endothelial cells (HRECs), while it was undetected (15-LO) or hardly detectable (12-LO) in human monocyte-like U937 cells. Inhibition or knock down of endothelial 12/15-LO in HRECs blocked HG-induced expression of ICAM-1, a well-known identified important molecule for leukocyte adhesion in DR.
CONCLUSION: Our data support that endothelial, rather than monocytic/macrophagic, 12/15-LO has a critical role in hyperglycemia-induced ICAM-1 expression, leukocyte adhesion, and subsequent local retinal barrier dysfunction. This may facilitate the development of more precisely targeted treatment strategies for DR.
PMID: 28351645 [PubMed - in process]
Bcl-2 Expression is Essential for Development and Normal Physiological Properties of Tooth Hard Tissue and Saliva Production.
Exp Cell Res. 2017 Jun 10;:
Authors: Saghiri MA, Asatourian A, Gurel Z, Sorenson CM, Sheibani N
BACKGROUND: Apoptosis plays a fundamental role in appropriate tissue development and function. Although expression of Bcl-2 has been reported during tooth and submandibular gland (SMG) development, the physiological role Bcl-2 plays during these processes has not been addressed. This study was performed to evaluate the impact of Bcl-2 expression on the formation and properties of tooth hard tissue, and saliva production.
METHODS: Twenty-four mice (12 males and 12 females) were divided into three groups of eight (n=8): group A (Bcl-2 +/+), group B (Bcl-2 +/-), and group C (Bcl-2 -/-) and subjected to micro-CT analyses. The mineral content of first molars was analyzed by X-Ray diffraction (XRD) and scanning electron microscopy (SEM) color dot map. The surface microhardness was determined by Vickers test on labial surfaces of incisors. Saliva was collected from different groups of mice after subcutaneous injection of pilocarpine.
RESULTS: Samples from Bcl-2 -/- mice showed significantly smaller micro-CT values, lower and poor crystallinity of hydroxyapatite (HA), and lowest surface micro hardness. SMG from Bcl-2 -/- mice showed remarkable reduction in size, consistent with reduced saliva accumulation.
CONCLUSIONS: The absence of Bcl-2 expression in SMG did not affect the expression of other Bcl-2 family members. Thus, Bcl-2 expression influence on the formation and properties of tooth hard tissue, and saliva accumulation.
SIGNIFICANCE: Bcl-2 expression has a significant impact on the mineralogical content of enamel crystals of tooth structure. Lack of Bcl-2 expression led to impaired production of enamel ACP crystals.
PMID: 28610838 [PubMed - as supplied by publisher]
Activation of catalase by pioglitazone: Multiple spectroscopic methods combined with molecular docking studies.
J Mol Recognit. 2017 Jun 19;:
Authors: Yekta R, Dehghan G, Rashtbari S, Sheibani N, Moosavi-Movahedi AA
Pioglitazone is an important prescription antidiabetic drug with positive roles in controlling high blood sugar in patients with type 2 diabetes. In the present study, we investigated the effects of pioglitazone on the structure and function of bovine liver catalase (BLC) using different spectroscopic and theoretical methods. UV-Vis absorption, fluorescence spectroscopy, synchronous fluorescence, and circular dichroism studies revealed conformational changes in the BLC structure and heme group in the presence of different concentrations of pioglitazone. Kinetic studies indicated that pioglitazone can increase BLC activity by approximately threefold compared with free enzyme. The fluorescence quenching data showed one binding site for pioglitazone, and the binding constants at 298, 304, and 310 K were calculated as 5.01 × 10(7) M(-1) , 5.8 × 10(7) M(-1) , and 6.6 × 10(7) M(-1) , respectively. The static type of quenching mechanism was mainly involved in the quenching of intrinsic emission of the enzyme. Thermodynamic data suggested that hydrophobic interactions played a major role in the binding reaction of pioglitazone with BLC. The molecular docking studies indicated that pioglitazone interacts with the cavity in the middle of the β-barrel and wrapping domain of BLC. Thus, pioglitazone can increase catalase activity by changing the BLC structure.
PMID: 28626866 [PubMed - as supplied by publisher]
Microglia activation is essential for BMP7-mediated retinal reactive gliosis.
J Neuroinflammation. 2017 Apr 05;14(1):76
Authors: Dharmarajan S, Fisk DL, Sorenson CM, Sheibani N, Belecky-Adams TL
BACKGROUND: Our previous studies have shown that BMP7 is able to trigger activation of retinal macroglia. However, these studies showed the responsiveness of Müller glial cells and retinal astrocytes in vitro was attenuated in comparison to those in vivo, indicating other retinal cell types may be mediating the response of the macroglial cells to BMP7. In this study, we test the hypothesis that BMP7-mediated gliosis is the result of inflammatory signaling from retinal microglia.
METHODS: Adult mice were injected intravitreally with BMP7 and eyes harvested 1, 3, or 7 days postinjection. Some mice were treated with PLX5622 (PLX) to ablate microglia and were subsequently injected with control or BMP7. Processed tissue was analyzed via immunofluorescence, RT-qPCR, or ELISA. In addition, cultures of retinal microglia were treated with vehicle, lipopolysaccharide, or BMP7 to determine the effects of BMP7-isolated cells.
RESULTS: Mice injected with BMP7 showed regulation of various inflammatory markers at the RNA level, as well as changes in microglial morphology. Isolated retinal microglia also showed an upregulation of BMP-signaling components following treatment. In vitro treatment of retinal astrocytes with conditioned media from activated microglia upregulated RNA levels of gliosis markers. In the absence of microglia, the mouse retina showed a subdued gliosis and inflammatory response when exposed to BMP7.
CONCLUSIONS: Gliosis resulting from BMP7 is mediated through an inflammatory response from retinal microglia.
PMID: 28381236 [PubMed - in process]
Retinal oxidative stress at the onset of diabetes determined by synchrotron FTIR widefield imaging: towards diabetes pathogenesis.
Analyst. 2017 Mar 27;142(7):1061-1072
Authors: Aboualizadeh E, Ranji M, Sorenson CM, Sepehr R, Sheibani N, Hirschmugl CJ
Diabetic retinopathy is a microvascular complication of diabetes that can lead to blindness. In the present study, we aimed to determine the nature of diabetes-induced, highly localized biochemical changes in the neuroretina at the onset of diabetes. High-resolution synchrotron Fourier transform infrared (s-FTIR) wide field microscopy coupled with multivariate analysis (PCA-LDA) was employed to identify biomarkers of diabetic retinopathy with spatial resolution at the cellular level. We compared the retinal tissue prepared from 6-week-old Ins2(Akita/+) heterozygous (Akita/+, N = 6; a model of diabetes) male mice with the wild-type (control, N = 6) mice. Male Akita/+ mice become diabetic at 4-weeks of age. Significant differences (P < 0.001) in the presence of biomarkers associated with diabetes and segregation of spectra were achieved. Differentiating IR bands attributed to nucleic acids (964, 1051, 1087, 1226 and 1710 cm(-1)), proteins (1662 and 1608 cm(-1)) and fatty acids (2854, 2923, 2956 and 3012 cm(-1)) were observed between the Akita/+ and the WT samples. A comparison between distinctive layers of the retina, namely the photoreceptor retinal layer (PRL), outer plexiform layer (OPL), inner nucleus layer (INL) and inner plexiform layer (IPL) suggested that the photoreceptor layer is the most susceptible layer to oxidative stress in short-term diabetes. Spatially-resolved chemical images indicated heterogeneities and oxidative-stress induced alterations in the diabetic retina tissue morphology compared with the WT retina. In this study, the spectral biomarkers and the spatial biochemical alterations in the diabetic retina and in specific layers were identified for the first time. We believe that the conclusions drawn from these studies will help to bridge the gap in our understanding of the molecular and cellular mechanisms that contribute to the pathobiology of diabetic retinopathy.
PMID: 28210739 [PubMed - in process]
Increased Retinal Oxygen Metabolism Precedes Microvascular Alterations in Type 1 Diabetic Mice.
Invest Ophthalmol Vis Sci. 2017 Feb 01;58(2):981-989
Authors: Liu W, Wang S, Soetikno B, Yi J, Zhang K, Chen S, Linsenmeier RA, Sorenson CM, Sheibani N, Zhang HF
Purpose: To investigate inner retinal oxygen metabolic rate (IRMRO2) during early stages of type 1 diabetes in a transgenic mouse model.
Methods: In current study, we involved seven diabetic mice (Akita/+, TSP1-/-) and seven control mice (TSP1-/-), and applied visible-light optical coherence tomography (vis-OCT) to image functional parameters including retinal blood flow rate, oxygen saturation (sO2) and the IRMRO2 value longitudinally from 5 weeks of age to 13 weeks of age. After imaging at 13 weeks of age, we analyzed the imaging results, and examined histology of mouse retina.
Results: Between diabetic mice and the control group, we observed significant differences in venous sO2 from 9 weeks of age (P = 0.006), and significant increment in IRMRO2 from 11 weeks of age (P = 0.001) in diabetic mice compared with control group. We did not find significant differences in retinal blood flow rate as well as arterial sO2 during imaging between diabetic and control mice. Histologic examination of diabetic and control mice at 13 weeks of age also revealed no anatomical retinal alternations.
Conclusions: In diabetic retinopathy, complications in retinal oxygen metabolism may occur before changes of retinal anatomical structure.
PMID: 28535269 [PubMed - in process]
Regulation of high glucose-induced apoptosis of brain pericytes by mitochondrial CA VA: A specific target for prevention of diabetic cerebrovascular pathology.
Biochim Biophys Acta. 2017 Jan 26;1863(4):929-935
Authors: Price TO, Sheibani N, Shah GN
Events responsible for cerebrovascular disease in diabetes are not fully understood. Pericyte loss is an early event that leads to endothelial cell death, microaneurysms, and cognitive impairment. A biochemical mechanism underlying pericyte loss is rapid respiration (oxidative metabolism of glucose). This escalation in respiration results from free influx of glucose into insulin-insensitive tissues in the face of high glucose levels in the blood. Rapid respiration generates superoxide, the precursor to all reactive oxygen species (ROS), and results in pericyte death. Respiration is regulated by carbonic anhydrases (CAs) VA and VB, the two isozymes expressed in mitochondria, and their pharmacologic inhibition with topiramate reduces respiration, ROS, and pericyte death. Topiramate inhibits both isozymes; therefore, in the earlier studies, their individual roles were not discerned. In a recent genetic study, we showed that mitochondrial CA VA plays a significant role in regulation of reactive oxygen species and pericyte death. The role of CA VB was not addressed. In this report, genetic knockdown and overexpression studies confirm that mitochondrial CA VA regulates respiration in pericytes, whereas mitochondrial CA VB does not contribute significantly. Identification of mitochondrial CA VA as a sole regulator of respiration provides a specific target to develop new drugs with fewer side effects that may be better tolerated and can protect the brain from diabetic injury. Since similar events occur in the capillary beds of other insulin-insensitive tissues such as the eye and kidney, these drugs may also slow the onset and progression of diabetic disease in these tissues.
PMID: 28131914 [PubMed - as supplied by publisher]
Versatile synthetic alternatives to Matrigel for vascular toxicity screening and stem cell expansion.
Nat Biomed Eng. 2017;1:
Authors: Nguyen EH, Daly WT, Le NNT, Farnoodian M, Belair DG, Schwartz MP, Lebakken CS, Ananiev GE, Saghiri MA, Knudsen TB, Sheibani N, Murphy WL
The physiological relevance of Matrigel as a cell-culture substrate and in angiogenesis assays is often called into question. Here, we describe an array-based method for the identification of synthetic hydrogels that promote the formation of robust in vitro vascular networks for the detection of putative vascular disruptors, and that support human embryonic stem cell expansion and pluripotency. We identified hydrogel substrates that promoted endothelial-network formation by primary human umbilical vein endothelial cells and by endothelial cells derived from human induced pluripotent stem cells, and used the hydrogels with endothelial networks to identify angiogenesis inhibitors. The synthetic hydrogels show superior sensitivity and reproducibility over Matrigel when evaluating known inhibitors, as well as in a blinded screen of a subset of 38 chemicals, selected according to predicted vascular disruption potential, from the Toxicity ForeCaster library of the US Environmental Protection Agency. The identified synthetic hydrogels should be suitable alternatives to Matrigel for common cell-culture applications.
PMID: 29104816 [PubMed]
Bim expression in endothelial cells and pericytes is essential for regression of the fetal ocular vasculature.
PLoS One. 2017;12(5):e0178198
Authors: Wang S, Zaitoun IS, Johnson RP, Jamali N, Gurel Z, Wintheiser CM, Strasser A, Lindner V, Sheibani N, Sorenson CM
Apoptosis plays a central role in developmental and pathological angiogenesis and vessel regression. Bim is a pro-apoptotic Bcl-2 family member that plays a prominent role in both developmental and pathological ocular vessel regression, and neovascularization. Endothelial cells (EC) and pericytes (PC) each play unique roles during vascular development, maintenance and regression. We recently showed that germline deletion of Bim results in persistent hyaloid vasculature, increased retinal vascular density and prevents retinal vessel regression in response to hyperoxia. To determine whether retinal vascular regression is attributable to Bim expression in EC or PC we generated mice carrying a conditional Bim allele (BimFlox/Flox) and VE-cadherin-cre (BimEC mice) or Pdgfrb-cre (BimPC mice). BimEC and BimPC mice demonstrated attenuated hyaloid vessel regression and postnatal retinal vascular remodeling. We also observed decreased retinal vascular apoptosis and proliferation. Unlike global Bim -/- mice, mice conditionally lacking Bim in EC or PC underwent hyperoxia-mediated vessel obliteration and subsequent retinal neovascularization during oxygen-induced ischemic retinopathy similar to control littermates. Thus, understanding the cell autonomous role Bim plays in the retinal vascular homeostasis will give us new insight into how to modulate pathological retinal neovascularization and vessel regression to preserve vision.
PMID: 28552963 [PubMed - in process]
Antichaperone activity and heme degradation effect of methyl tert-butyl ether (MTBE) on normal and diabetic hemoglobins.
J Mol Recognit. 2016 Dec 05;:
Authors: Najdegerami IH, Maghami P, Sheikh-Hasani V, Hosseinzadeh G, Sheibani N, Moosavi-Movahedi AA
Because of the extensive use of methyl tert-butyl ether (MTBE) as an additive to increase the octane quality of gasoline, the environmental pollution by this compound has increased in recent decades. Environmental release of MTBE may lead to its entry to the blood stream through inhalation or drinking of contaminated water, and its interactions with biological molecules such as proteins. The present study was proposed to comparatively investigate the interactions of MTBE with hemoglobin (Hb) from diabetic and nondiabetic individuals using various spectroscopic methods including UV-visible, fluorescence, chemiluminescence, and circular dichroism. These results demonstrated the effects of MTBE on heme degradation of Hb and the reaction of these degradation products with water generating reactive oxygen species. Interaction of Hb with MTBE enhanced its aggregation rate and decreased lag time, indicating the antichaperone activity of MTBE upon interaction with Hb. Furthermore, the diabetic Hb showed more severe effects of MTBE, including heme degradation, reactive oxygen species production, unfolding, and antichaperone behavior than the nondiabetic Hb. The results from molecular docking suggested that the special interaction site of MTBE in the vicinity of Hb heme group is responsible for heme degradation.
PMID: 27917590 [PubMed - as supplied by publisher]
Antioxidant and Anticancer Activities of Walnut (Juglans regia L.) Protein Hydrolysates Using Different Proteases.
Plant Foods Hum Nutr. 2016 Dec;71(4):402-409
Authors: Jahanbani R, Ghaffari SM, Salami M, Vahdati K, Sepehri H, Sarvestani NN, Sheibani N, Moosavi-Movahedi AA
Walnut (Juglans regia L.) contains approximately 20-25 % protein with abundant essential amino acids. The enzymatic hydrolysate of Persian walnut (Chandler) seed proteins was prepared by incubation with three different proteases, including pancreatic chymotrypsin and trypsin, and a microbial enzyme proteinase K. The hydrolysates were found to possess excellent antioxidant capacities. The peptide fractions scavenged the 2, 2'-anizo-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) free radicals and inhibited the activity of reactive oxygen species. Walnut protein hydrolysates were also tested, for the first time, against the viability of human breast (MDA-MB231) and colon (HT-29) cancer cell lines. MTT, [3-(4, 5dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide], assay was used to assess in vitro cancer cell viability upon treatment with the peptide fractions. The peptide fractions showed cell growth inhibition of 63 ± 1.73 % for breast cancer and 51 ± 1.45 % for colon cancer cells. Thus, a direct correlation between antioxidant and anticancer activities of walnut peptide fractions exists and supports their potential therapeutic benefit.
PMID: 27679440 [PubMed - in process]
Studies to reveal the nature of interactions between catalase and curcumin using computational methods and optical techniques.
Int J Biol Macromol. 2016 Nov 16;95:550-556
Authors: Mofidi Najjar F, Ghadari R, Yousefi R, Safari N, Sheikhhasani V, Sheibani N, Moosavi-Movahedi AA
Curcumin is an important antioxidant compound, and is widely reported as an effective component for reducing complications of many diseases. However, the detailed mechanisms of its activity remain poorly understood. We found that curcumin can significantly increase catalase activity of BLC (bovine liver catalase). The mechanism of curcumin action was investigated using a computational method. We suggested that curcumin may activate BLC by modifying the bottleneck of its narrow channel. The molecular dynamic simulation data showed that placing curcumin on the structure of enzyme can increase the size of the bottleneck in the narrow channel of BLC, and readily allow the access of substrate to the active site. Because of the increase of the distance between amino acids of the bottleneck in the presence of curcumin, the entrance space of substrate increased from 250Å(3) to 440Å(3). In addition, the increase in emission of intrinsic fluorescence of BLC in presence of curcumin demonstrated changes in tertiary structure of catalase, and possibility of less quenching. We also used circular dichroism (CD) spectropolarimetry to determine how curcumin may alter the enzyme secondary structure. Catalase spectra in the presence of various concentrations of curcumin showed an increase in the amount of α-helix content.
PMID: 27865955 [PubMed - as supplied by publisher]
Calcium silicate-based cements and functional impacts of various constituents.
Dent Mater J. 2016 Oct 22;
Authors: Saghiri MA, Orangi J, Asatourian A, Gutmann JL, Garcia-Godoy F, Lotfi M, Sheibani N
Calcium silicate-based cements have superior sealing ability, bioactivity, and marginal adaptation, which make them suitable for different dental treatment applications. However, they exhibit some drawbacks such as long setting time and poor handling characteristics. To overcome these limitations calcium silicates are engineered with various constituents to improve specific characteristics of the base material, and are the focus of this review. An electronic search of the PubMed, MEDLINE, and EMBASE via OVID databases using appropriate terms and keywords related to the use, application, and properties of calcium silicate-based cements was conducted. Two independent reviewers obtained and analyzed the full texts of the selected articles. Although the effects of various constituents and additives to the base Portland cement-like materials have been investigated, there is no one particular ingredient that stands out as being most important. Applying nanotechnology and new synthesis methods for powders most positively affected the cement properties.
PMID: 27773894 [PubMed - as supplied by publisher]
The role of acetoacetate in Amadori product formation of human serum albumin.
J Photochem Photobiol B. 2016 Oct;163:345-51
Authors: Bohlooli M, Ghaffari-Moghaddam M, Khajeh M, Shahraki-Fallah G, Haghighi-Kekhaiye B, Sheibani N
Amadori product is an important and stable intermediate, which is produced during glycation process. It is a marker of hyperglycemia in diabetes mellitus, and its accumulation in the body contributes to microvascular complication of diabetes including diabetic nephropathy and retinopathy. In this study, the effect of acetoacetate on the formation of Amadori products and biophysical properties of human serum albumin (HSA), after incubation with glucose, was investigated using various methods. These included circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, and UV-visible and fluorescence spectroscopy. Our results indicated that the production of Amadori products in HSA incubated with glucose (GHSA) was increased in the presence of acetoacetate. We also detected alterations in the secondary and tertiary structure of GHSA, which was increased in the presence of acetoacetate. These changes were attributed to the formation of covalent bonds between the carbonyl group of acetoacetate and the nucleophilic groups (lysine residues) of HSA. Thus, acetoacetate can enhance the production of Amadori products through formation of covalent bonds with biomaterials.
PMID: 27614245 [PubMed - in process]
Effect of biomaterials on angiogenesis during vital pulp therapy.
Dent Mater J. 2016 Oct 01;35(5):701-709
Authors: Saghiri MA, Asatourian A, Garcia-Godoy F, Sheibani N
This review intended to provide an overview of the effects of dental materials, used in dentin-pulp complex and dental pulp regeneration, on angiogenesis processes during regenerative endodontic procedures. An electronic search was performed in PubMed and MEDLINE databases via OVID using the keywords mentioned in the PubMed and MeSH headings for English language published articles from January 2005-April 2014 that evaluated the angiogenic properties of different dental materials used in regenerative endodontic procedures. Of the articles identified in an initial search, only 40 articles met the inclusion criteria set for this review. Vital pulp therapy materials might have positive effects on angiogenesis events, while most of the canal irrigating solutions and antibiotic pastes have anti-angiogenic activity except for EDTA. Future clinical studies will be helpful in defining the mechanisms of action for dental materials that promote or inhibit angiogenesis events at applied areas.
PMID: 27546854 [PubMed - in process]
High glucose promotes the migration of retinal pigment epithelial cells through increased oxidative stress and PEDF expression.
Am J Physiol Cell Physiol. 2016 Sep 01;311(3):C418-36
Authors: Farnoodian M, Halbach C, Slinger C, Pattnaik BR, Sorenson CM, Sheibani N
Defects in the outer blood-retinal barrier have significant impact on the pathogenesis of diabetic retinopathy and macular edema. However, the detailed mechanisms involved remain largely unknown. This is, in part, attributed to the lack of suitable animal and cell culture models, including those of mouse origin. We recently reported a method for the culture of retinal pigment epithelial (RPE) cells from wild-type and transgenic mice. The RPE cells are responsible for maintaining the integrity of the outer blood-retinal barrier whose dysfunction during diabetes has a significant impact on vision. Here we determined the impact of high glucose on the function of RPE cells. We showed that high glucose conditions resulted in enhanced migration and increased the level of oxidative stress in RPE cells, but minimally impacted their rate of proliferation and apoptosis. High glucose also minimally affected the cell-matrix and cell-cell interactions of RPE cells. However, the expression of integrins and extracellular matrix proteins including pigment epithelium-derived factor (PEDF) were altered under high glucose conditions. Incubation of RPE cells with the antioxidant N-acetylcysteine under high glucose conditions restored normal migration and PEDF expression. These cells also exhibited increased nuclear localization of the antioxidant transcription factor Nrf2 and ZO-1, reduced levels of β-catenin and phagocytic activity, and minimal effect on production of vascular endothelial growth factor, inflammatory cytokines, and Akt, MAPK, and Src signaling pathways. Thus high glucose conditions promote RPE cell migration through increased oxidative stress and expression of PEDF without a significant effect on the rate of proliferation and apoptosis.
PMID: 27440660 [PubMed - in process]
Mechanistic role of cytochrome P450 (CYP)1B1 in oxygen-mediated toxicity in pulmonary cells: A novel target for prevention of hyperoxic lung injury.
Biochem Biophys Res Commun. 2016 Aug 05;476(4):346-51
Authors: Dinu D, Chu C, Veith A, Lingappan K, Couroucli X, Jefcoate CR, Sheibani N, Moorthy B
Supplemental oxygen, which is routinely administered to preterm infants with pulmonary insufficiency, contributes to bronchopulmonary dysplasia (BPD) in these infants. Hyperoxia also contributes to the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) in adults. The mechanisms of oxygen-mediated pulmonary toxicity are not completely understood. Recent studies have suggested an important role for cytochrome P450 (CYP)1A1/1A2 in the protection against hyperoxic lung injury. The role of CYP1B1 in oxygen-mediated pulmonary toxicity has not been studied. In this investigation, we tested the hypothesis that CYP1B1 plays a mechanistic role in oxygen toxicity in pulmonary cells in vitro. In human bronchial epithelial cell line BEAS-2B, hyperoxic treatment for 1-3 days led to decreased cell viability by about 50-80%. Hyperoxic cytotoxicity was accompanied by an increase in levels of reactive oxygen species (ROS) by up to 110%, and an increase of TUNEL-positive cells by up to 4.8-fold. Western blot analysis showed hyperoxia to significantly down-regulate CYP1B1 protein level. Also, there was a decrease of CYP1B1 mRNA by up to 38% and Cyp1b1 promoter activity by up to 65%. On the other hand, CYP1B1 siRNA appeared to rescue the cell viability under hyperoxia stress, and overexpression of CYP1B1 significantly attenuated hyperoxic cytotoxicity after 48 h of incubation. In immortalized lung endothelial cells derived from Cyp1b1-null and wild-type mice, hyperoxia increased caspase 3/7 activities in a time-dependent manner, but endothelial cells lacking the Cyp1b1 gene showed significantly decreased caspase 3/7 activities after 48 and 72 h of incubation, implying that CYP1B1 might promote apoptosis in wild type lung endothelial cells under hyperoxic stress. In conclusion, our results support the hypothesis that CYP1B1 plays a mechanistic role in pulmonary oxygen toxicity, and CYP1B1-mediated apoptosis could be one of the mechanisms of oxygen toxicity. Thus, CYP1B1 could be a novel target for preventative and/or therapeutic interventions against BPD in infants and ALI/ARDS in adults.
PMID: 27235555 [PubMed - in process]
CD40 in Retinal Müller Cells Induces P2X7-Dependent Cytokine Expression in Macrophages/Microglia in Diabetic Mice and Development of Early Experimental Diabetic Retinopathy.
Diabetes. 2016 Jul 29;:
Authors: Portillo JC, Corcino YL, Miao Y, Tang J, Sheibani N, Kern TS, Dubyak GR, Subauste CS
Müller cells and macrophages/microglia are likely important for development of diabetic retinopathy. However, the interplay between these cells in this disease is not well understood. An inflammatory process is linked to the onset of experimental diabetic retinopathy. CD40 deficiency impairs this process and prevents diabetic retinopathy. Using mice with CD40 expression restricted to Müller cells, we identified a mechanism by which Müller cells trigger pro-inflammatory cytokine expression in myeloid cells. During diabetes, mice with CD40 expressed in Müller cells upregulated Müller TNF-α, IL-1β, ICAM-1 and NOS2, developed leukostasis and capillary degeneration. However, CD40 did not cause TNF-α or IL-1β secretion in Müller cells. TNF-α was not detected in Müller cells from diabetic mice with CD40(+) Müller cells. Rather, TNF-α was upregulated in macrophages/microglia. CD40 ligation in Müller cells triggered phospholipase C-dependent ATP release that caused P2X7-dependent production of TNF-α and IL-1β by macrophages. P2X7(-/-) mice and mice treated with a P2X7 inhibitor were protected from diabetes-induced TFN-α, IL-1β, ICAM-1 and NOS2 upregulation. Our studies indicate that CD40 in Müller cells is sufficient to upregulate retinal inflammatory markers and appears to promote experimental diabetic retinopathy, and they indicate that Müller cells orchestrate inflammatory responses in myeloid cells through a CD40-ATP-P2X7 pathway.
PMID: 27474370 [PubMed - as supplied by publisher]
Differential regulation of angiogenesis using degradable VEGF-binding microspheres.
Biomaterials. 2016 Mar 16;93:27-37
Authors: Belair DG, Miller MJ, Wang S, Darjatmoko SR, Binder BY, Sheibani N, Murphy WL
Vascular endothelial growth factor (VEGF) spatial and temporal activity must be tightly controlled during angiogenesis to form perfusable vasculature in a healing wound. The native extracellular matrix (ECM) regulates growth factor activity locally via sequestering, and researchers have used ECM-mimicking approaches to regulate the activity of VEGF in cell culture and in vivo. However, the impact of dynamic, affinity-mediated growth factor sequestering has not been explored in detail with biomaterials. Here, we sought to modulate VEGF activity dynamically over time using poly(ethylene glycol) microspheres containing VEGF-binding peptides (VBPs) and exhibiting varying degradation rates. The degradation rate of VBP microspheres conferred a differential ability to up- or down-regulate VEGF activity in culture with primary human endothelial cells. VBP microspheres with fast-degrading crosslinks reduced VEGF activity and signaling, while VBP microspheres with no inherent degradability sequestered and promoted VEGF activity in culture with endothelial cells. VBP microspheres with degradable crosslinks significantly reduced neovascularization in vivo, but neither non-degradable VBP microspheres nor bolus delivery of soluble VBP reduced neovascularization. The covalent incorporation of VBP to degradable microspheres was required to reduce neovascularization in a mouse model of choroidal neovascularization in vivo, which demonstrates a potential clinical application of degradable VBP microspheres to reduce pathological angiogenesis. The results herein highlight the ability to modulate the activity of a sequestered growth factor by changing the crosslinker identity within PEG hydrogel microspheres. The insights gained here may instruct the design and translation of affinity-based growth factor sequestering biomaterials for regenerative medicine applications.
PMID: 27061268 [PubMed - as supplied by publisher]
Andrographolide Ameliorates Abdominal Aortic Aneurysm Progression by Inhibiting Inflammatory Cell Infiltration through Downregulation of Cytokine and Integrin Expression.
J Pharmacol Exp Ther. 2016 Jan;356(1):137-47
Authors: Ren J, Liu Z, Wang Q, Giles J, Greenberg J, Sheibani N, Kent KC, Liu B
Abdominal aortic aneurysm (AAA), characterized by exuberant inflammation and tissue deterioration, is a common aortic disease associated with a high mortality rate. There is currently no established pharmacological therapy to treat this progressive disease. Andrographolide (Andro), a major bioactive component of the herbaceous plant Andrographis paniculata, has been found to exhibit potent anti-inflammatory properties by inhibiting nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activity in several disease models. In this study, we investigated the ability of Andro to suppress inflammation associated with aneurysms, and whether it may be used to block the progression of AAA. Whereas diseased aortae continued to expand in the solvent-treated group, daily administration of Andro to mice with small aneurysms significantly attenuated aneurysm growth, as measured by the diminished expansion of aortic diameter (165.68 ± 15.85% vs. 90.62 ± 22.91%, P < 0.05). Immunohistochemistry analyses revealed that Andro decreased infiltration of monocytes/macrophages and T cells. Mechanistically, Andro inhibited arterial NF-κB activation and reduced the production of proinflammatory cytokines [CCL2, CXCL10, tumor necrosis factor α, and interferon-γ] in the treated aortae. Furthermore, Andro suppressed α4 integrin expression and attenuated the ability of monocytes/macrophages to adhere to activated endothelial cells. These results indicate that Andro suppresses progression of AAA, likely through inhibition of inflammatory cell infiltration via downregulation of NF-κB-mediated cytokine production and α4 integrin expression. Thus, Andro may offer a pharmacological therapy to slow disease progression in patients with small aneurysms.
PMID: 26483397 [PubMed - in process]
Quantitative Assessment of Retinopathy Using Multi-parameter Image Analysis.
J Med Signals Sens. 2016 Apr-Jun;6(2):71-80
Authors: Ghanian Z, Staniszewski K, Jamali N, Sepehr R, Wang S, Sorenson CM, Sheibani N, Ranji M
A multi-parameter quantification method was implemented to quantify retinal vascular injuries in microscopic images of clinically relevant eye diseases. This method was applied to wholemount retinal trypsin digest images of diabetic Akita/+, and bcl-2 knocked out mice models. Five unique features of retinal vasculature were extracted to monitor early structural changes and retinopathy, as well as quantifying the disease progression. Our approach was validated through simulations of retinal images. Results showed fewer number of cells (P = 5.1205e-05), greater population ratios of endothelial cells to pericytes (PCs) (P = 5.1772e-04; an indicator of PC loss), higher fractal dimension (P = 8.2202e-05), smaller vessel coverage (P = 1.4214e-05), and greater number of acellular capillaries (P = 7.0414e-04) for diabetic retina as compared to normal retina. Quantification using the present method would be helpful in evaluating physiological and pathological retinopathy in a high-throughput and reproducible manner.
PMID: 27186534 [PubMed]
Ocular Safety of Intravitreal Propranolol and Its Efficacy in Attenuation of Choroidal Neovascularization.
Invest Ophthalmol Vis Sci. 2015 Dec 1;56(13):8228-35
Authors: Nourinia R, Rezaei Kanavi M, Kaharkaboudi A, Taghavi SI, Aldavood SJ, Darjatmoko SR, Wang S, Gurel Z, Lavine JA, Safi S, Ahmadieh H, Daftarian N, Sheibani N
PURPOSE: Determine the safe dose of intravitreal propranolol (IVP), and evaluate its inhibitory effect on laser-induced choroidal neovascularization (CNV).
METHODS: To determine the IVP safe dose, 32 rabbits were divided into 4 groups. Three of these groups received IVP (15 μL) corresponding to 15 μg (group B), 30 μg (group C), and 60 μg (group D). The control group (A) received 15 μL saline. Safety was assessed by ocular examination, electroretinography (ERG), routine histopathologic evaluation, immunohistochemistry for glial fibrillary acidic protein (GFAP), and real-time qPCR for GFAP, VEGF, thrombospondin 1 (TSP1), and pigment epithelium-derived factor (PEDF). A similar experiment was performed in 24 mice by using a 100-fold lower amount of propranolol (0.15, 0.3, and 0.6 μg in 2 μL) based on vitreous volume. For assessment of the angioinhibitory effects of IVP, CNV was induced in 42 mice via laser burns. Mice were divided into two groups: group 1 received the safe dose of IVP (0.3 μg in 2 μL) and group 2 received saline. Neovascularization area was quantified by intercellular adhesion molecule (ICAM)-2 immunostaining of choroidal-scleral flat mounts by using ImageJ software.
RESULTS: According to clinical, ERG, and histopathologic findings, 30 μg IVP was chosen as the safe dose in rabbit eyes, comparable to 0.3 μg IVP in mouse eyes. As compared to the control eyes, the development of CNV was attenuated (4.8-fold) in mice receiving 0.3 μg IVP.
CONCLUSIONS: Intravitreal propranolol injection up to the final dose of 30 μg in rabbits and 0.3 μg in mice was safe, and was effective in attenuation of CNV in mice.
PMID: 26720475 [PubMed - in process]
Sensitization of breast cancer cells to doxorubicin via stable cell line generation and overexpression of DFF40.
Biochem Cell Biol. 2015 Dec;93(6):604-10
Authors: Bagheri F, Safarian S, Baghaban Eslaminejad M, Sheibani N
There are a number of reports demonstrating a relationship between the alterations in DFF40 expression and development of some cancers. Here, increased DFF40 expression in T-47D cells in the presence of doxorubicin was envisaged for therapeutic usage. The T-47D cells were transfected with an eukaryotic expression vector encoding the DFF40 cDNA. Following incubation with doxorubicin, propidium iodide (PI) staining was used for cell cycle distribution analysis. The rates of apoptosis were determined by annexin V/PI staining. Apoptosis was also evaluated using the DNA laddering analysis. The viability of DFF40-transfected cells incubated with doxorubicin was significantly decreased compared with control cells. However, there were no substantial changes in the cell cycle distribution of pIRES2-DFF40 cells incubated with doxorubicin compared to control cells. The expression of DFF40, without doxorubicin incubation, had also no significant effect on the cell cycle distribution. There was no DNA laddering in cells transfected with the empty pIRES2 vector when incubated with doxorubicin. In contrast, DNA laddering was observed in DFF40 transfected cells in the presence of doxorubicin after 48 h. Also, the expression of DFF40 and DFF45 was increased in DFF40 transfected cells in the presence of doxorubicin enhancing cell death. Collectively our results indicated that co-treatment of DFF40-transfected cells with doxorubicin can enhance the killing of these tumor cells via apoptosis. Thus, modulation of DFF40 level may be a beneficial strategy for treatment of chemo-resistant cancers.
PMID: 26529233 [PubMed - in process]
Mechanism and behavior of silver nanoparticles in aqueous medium as adsorbent.
Talanta. 2015 Nov 1;144:1377-86
Authors: Dastafkan K, Khajeh M, Bohlooli M, Ghaffari-Moghaddam M, Sheibani N
In recent years there has been a rising interest in the application of nanomaterial such as silver nanoparticles (AgNps) in many areas of scientific research. The most active areas of investigation are related to the study and application of optical, biomedical, and newly applied adsorption properties of AgNps. The recent evaluation of adsorptive properties of AgNPs has added new areas to their wide range of applications in analytical separations and pre-concentrating steps. They have also been used for removal of several environmental organic pollutants and uptake of heavy metals. The main objective of this review is to describe the characteristics, mechanisms, and behavior of AgNPs as adsorbent in aquatic systems and sample solutions.
PMID: 26452972 [PubMed - in process]
Visible light optical coherence tomography measures retinal oxygen metabolic response to systemic oxygenation.
Light Sci Appl. 2015 Sep;4(9)
Authors: Yi J, Liu W, Chen S, Backman V, Sheibani N, Sorenson CM, Fawzi AA, Linsenmeier RA, Zhang HF
The lack of capability to quantify oxygen metabolism noninvasively impedes both fundamental investigation and clinical diagnosis of a wide spectrum of diseases including all the major blinding diseases such as age-related macular degeneration, diabetic retinopathy, and glaucoma. Using visible light optical coherence tomography (vis-OCT), we demonstrated accurate and robust measurement of retinal oxygen metabolic rate (rMRO2) noninvasively in rat eyes. We continuously monitored the regulatory response of oxygen consumption to a progressive hypoxic challenge. We found that both oxygen delivery, and rMRO2 increased from the highly regulated retinal circulation (RC) under hypoxia, by 0.28 ± 0.08 μL min(-1) (p < 0.001), and 0.20 ± 0.04 μL min(-1) (p < 0.001) per 100 mmHg systemic pO2 reduction, respectively. The increased oxygen extraction compensated for the deficient oxygen supply from the poorly regulated choroidal circulation. Results from an oxygen diffusion model based on previous oxygen electrode measurements corroborated our in vivo observations. We believe that vis-OCT has the potential to reveal the fundamental role of oxygen metabolism in various retinal diseases.
PMID: 26658555 [PubMed - as supplied by publisher]
Interaction of insulin with methyl tert-butyl ether promotes molten globule-like state and production of reactive oxygen species.
Int J Biol Macromol. 2015 Sep;80:610-4
Authors: Valipour M, Maghami P, Habibi-Rezaei M, Sadeghpour M, Khademian MA, Mosavi K, Sheibani N, Moosavi-Movahedi AA
Interaction of methyl tert-butyl ether (MTBE) with proteins is a new look at its potential adverse biological effects. When MTBE is released to the environment it enters the blood stream through inhalation, and could affect the properties of various proteins. Here we investigated the interaction of MTBE with insulin and its effect on insulin structural changes. Our results showed that insulin formed a molten globule (MG)-like structure in the presence of 8μM MTBE under physiological pH. The insulin structural changes were studied using spectroscopy methods, viscosity calculation, dynamic light scattering and differential scanning calorimetry. To delineate the mechanisms involved in MTBE-protein interactions, the formation of reactive oxygen specious (ROS) and formation of protein aggregates were measured. The chemiluminscence experiments revealed an increase in ROS production in the presence of MTBE especially in the MG-like state. These results were further confirmed by the aggregation tests, which indicated more aggregation of insulin at 40μM MTBE compared with 8μM. Thus, the formation of initial aggregates and exposure of the hydrophobic patches upon formation of the MG-like state in the presence of MTBE drives protein oxidation and ROS generation.
PMID: 26193678 [PubMed - in process]
Repair of bone defect by nano-modified white mineral trioxide aggregates in rabbit: A histopathological study.
Med Oral Patol Oral Cir Bucal. 2015 Sep 01;20(5):e525-31
Authors: Saghiri MA, Orangi J, Tanideh N, Asatourian A, Janghorban K, Garcia-Godoy F, Sheibani N
BACKGROUND: Many researchers have tried to enhance materials functions in different aspects of science using nano-modification method, and in many cases the results have been encouraging. To evaluate the histopathological responses of the micro-/nano-size cement-type biomaterials derived from calcium silicate-based composition with addition of nano tricalcium aluminate (3CaO.Al2O3) on bone healing response.
MATERIAL AND METHODS: Ninety mature male rabbits were anesthetized and a bone defect was created in the right mandible. The rabbits were divided into three groups, which were in turn subdivided into five subgroups with six animals each based on the defect filled by: white mineral trioxide aggregate (WMTA), Nano-WMTA, WMTA without 3CaO.Al2O3, Nano-WMTA with 2% Nano-3CaO.Al2O3, and empty as control. Twenty, forty and sixty days postoperatively the animals were sacrificed and the right mandibles were removed for histopathological evaluations. Kruskal-Wallis test with post-hoc comparisons based on the LSMeans procedure was used for data analysis.
RESULTS: All the experimental materials provoked a moderate to severe inflammatory reaction, which significantly differed from the control group (p< 0.05). Statistical analysis of bone formation and bone regeneration data showed significant differences between groups at 40- and 60- day intervals in all groups. Absence of 3CaO.Al2O3 leads to more inflammation and foreign body reaction than other groups in all time intervals.
CONCLUSIONS: Both powder nano-modification and addition of 2% Nano-3CaO.Al2O3 to calcium silicate-based cement enhanced the favorable tissue response and osteogenesis properties of WMTA based materials.
PMID: 26034924 [PubMed - indexed for MEDLINE]
Role of Angiogenesis in Endodontics: Contributions of Stem Cells and Proangiogenic and Antiangiogenic Factors to Dental Pulp Regeneration.
J Endod. 2015 Jun;41(6):797-803
Authors: Saghiri MA, Asatourian A, Sorenson CM, Sheibani N
INTRODUCTION: Dental pulp regeneration is a part of regenerative endodontics, which includes isolation, propagation, and re-transplantation of stem cells inside the prepared root canal space. The formation of new blood vessels through angiogenesis is mandatory to increase the survival rate of re-transplanted tissues. Angiogenesis is defined as the formation of new blood vessels from preexisting capillaries, which has great importance in pulp regeneration and homeostasis. Here the contribution of human dental pulp stem cells and proangiogenic and antiangiogenic factors to angiogenesis process and regeneration of dental pulp is reviewed.
METHODS: A search was performed on the role of angiogenesis in dental pulp regeneration from January 2005 through April 2014. The recent aspects of the relationship between angiogenesis, human dental pulp stem cells, and proangiogenic and antiangiogenic factors in regeneration of dental pulp were assessed.
RESULTS: Many studies have indicated an intimate relationship between angiogenesis and dental pulp regeneration. The contribution of stem cells and mechanical and chemical factors to dental pulp regeneration has been previously discussed.
CONCLUSIONS: Angiogenesis is an indispensable process during dental pulp regeneration. The survival of inflamed vital pulp and engineered transplanted pulp tissue are closely linked to the process of angiogenesis at sites of application. However, the detailed regulatory mechanisms involved in initiation and progression of angiogenesis in pulp tissue require investigation.
PMID: 25649306 [PubMed - as supplied by publisher]
Effect of particle size on calcium release and elevation of pH of endodontic cements.
Dent Traumatol. 2015 Jun;31(3):196-201
Authors: Saghiri MA, Asatourian A, Orangi J, Lotfi M, Soukup JW, Garcia-Godoy F, Sheibani N
BACKGROUND/AIM: Elevation of pH and calcium ion release are of great importance in antibacterial activity and the promotion of dental soft and hard tissue healing process. In this study, we evaluated the effect of particle size on the elevation of pH and the calcium ion release from calcium silicate-based dental cements.
MATERIAL AND METHODS: Twelve plastic tubes were divided into three groups, filled with white mineral trioxide aggregate (WMTA), WMTA plus 1% methylcellulose, and nano-modified WMTA (nano-WMTA), and placed inside flasks containing 10 ml of distilled water. The pH values were measured using a pH sensor 3, 24, 72, and 168 h after setting of the cements. The calcium ion release was measured using an atomic absorption spectrophotometer with same sample preparation method. Data were subjected to two-way analysis of variance (anova) followed by post hoc Tukey tests with significance level of P < 0.05.
RESULTS: Nano-WMTA showed significant pH elevation only after 24 h (P < 0.05) compared with WMTA, and after 3, 24, and 72 h compared with WMTA plus 1% methylcellulose (P < 0.05). Nano-WMTA showed significantly higher calcium ion release values compared to the other two groups (P < 0.05).
CONCLUSIONS: Nano-modification of WMTA remarkably increased the calcium ion release at all time intervals postsetting, which can significantly influence the osteogenic properties of human dental pulp cells and as a consequence enhance mineralized matrix nodule formation to achieve desirable clinical outcomes. However, the increase in pH values mainly occurred during the short time postsetting. Addition of 1% methylcellulose imposed a delay in elevation of pH and calcium ion release by WMTA.
PMID: 25571910 [PubMed - in process]
Borane-protected phosphines are redox-active radioprotective agents for endothelial cells.
Redox Biol. 2015 Jun 26;6:73-79
Authors: Crowe ME, Lieven CJ, Thompson AF, Sheibani N, Levin LA
Exposure to radiation can damage endothelial cells in the irradiated area via the production of reactive oxygen species. We synthesized phosphine-borane complexes that reduce disulfide bonds and had previously been shown to interfere with redox-mediated signaling of cell death. We hypothesized that this class of drugs could interfere with the downstream effects of oxidative stress after irradiation and rescue endothelial cells from radiation damage. Cultured bovine aortic endothelial cells were plated for clonogenic assay prior to exposure to varying doses of irradiation from a (137)Cs irradiator and treated with various concentrations of bis(3-propionic acid methyl ester)phenylphosphine borane complex (PB1) at different time points. The clone-forming ability of the irradiated cells was assessed seven days after irradiation. We compared the radioprotective effects of PB1 with the aminothiol radioprotectant WR1065 and known superoxide scavengers. PB1 significantly protected bovine aortic endothelial cells from radiation damage, particularly when treated both before and after radiation. The radioprotection with 1µM PB1 corresponded to a dose-reduction factor of 1.24. Radioprotection by PB1 was comparable to the aminothiol WR1065, but was significantly less toxic and required much lower concentrations of drug (1µM vs. 4mM, respectively). Superoxide scavengers were not radioprotective in this paradigm, indicating the mechanisms for both loss of clonogenicity and PB1 radioprotection are independent of superoxide signaling. These data demonstrate that PB1 is an effective redox-active radioprotectant for endothelial cells in vitro, and is radioprotective at a concentration approximately 4 orders of magnitude lower than the aminothiol WR1065 with less toxicity.
PMID: 26188467 [PubMed - as supplied by publisher]
Effect of mobile phone use on metal ion release from fixed orthodontic appliances.
Am J Orthod Dentofacial Orthop. 2015 Jun;147(6):719-24
Authors: Saghiri MA, Orangi J, Asatourian A, Mehriar P, Sheibani N
INTRODUCTION: The aim of this study was to evaluate the effect of exposure to radiofrequency electromagnetic fields emitted by mobile phones on the level of nickel in saliva.
METHODS: Fifty healthy patients with fixed orthodontic appliances were asked not to use their cell phones for a week, and their saliva samples were taken at the end of the week (control group). The patients recorded their time of mobile phone usage during the next week and returned for a second saliva collection (experimental group). Samples at both times were taken between 8:00 and 10:00 pm, and the nickel levels were measured. Two-tailed paired-samples t test, linear regression, independent t test, and 1-way analysis of variance were used for data analysis.
RESULTS: The 2-tailed paired-samples t test showed significant differences between the levels of nickel in the control and experimental groups (t  = 9.967; P <0.001). The linear regression test showed a significant relationship between mobile phone usage time and the nickel release (F [1, 48] = 60.263; P <0.001; R(2) = 0.577).
CONCLUSIONS: Mobile phone usage has a time-dependent influence on the concentration of nickel in the saliva of patients with orthodontic appliances.
PMID: 26038076 [PubMed - in process]
Enhancement of thermal reversibility and stability of human carbonic anhydrase II by mesoporous nanoparticles.
Int J Biol Macromol. 2015 Apr;75:67-72
Authors: Khatibi A, Ma'mani L, Khodarahmi R, Shafiee A, Maghami P, Ahmad F, Sheibani N, Moosavi-Movahedi AA
Aminopropyl functionalized PEGylated mesoporous silica nanoparticles [H2N-Pr@PEGylated SBA-15] were synthesized and evaluated as a promising biocompatible additive to study the activity and thermal reversibility and stability of human carbonic anhydrase II (HCA II). For this purpose, the additive was prepared by covalent amino propyl functionalization of mesoporous silica nanoparticles (MSNs) bearing PEG moiety as linker. The MSNs was fully characterized using different techniques including transmission electron microscopy, N2 adsorption-desorption measurements, thermal gravimetric analysis, Fourier transform infrared spectroscopy and dynamic light scattering. The average particle size of [H2N-Pr@PEGylated SBA-15] was about 80 nm and showed high loading capacity for HCA II at pH 7.75 as a target protein. The efficiency of [H2N-Pr@PEGylated SBA-15] in improving reversibility of HCA II was investigated by various techniques including UV-vis, 1,8-Anilinonaphtalene Sulfonate (ANS) fluorescence, circular dichroism (CD), and differential scanning calorimetry. Our results showed that [H2N-Pr@PEGylated SBA-15] can increase the protein thermal reversibility and stability. Herein, kinetic studies were applied to confirm the ability of [H2N-Pr@PEGylated SBA-15] in increasing the activity of HCA II at high temperatures. Together our results present the [H2N-Pr@PEGylated SBA-15] as a water-dispersible and efficient additive for improving the activity, and thermal reversibility and stability of enzyme.
PMID: 25600988 [PubMed - in process]
Cytochrome P450 1B1: An unexpected modulator of liver fatty acid homeostasis.
Arch Biochem Biophys. 2015 Apr 1;571:21-39
Authors: Larsen MC, Bushkofsky JR, Gorman T, Adhami V, Mukhtar H, Wang S, Reeder SB, Sheibani N, Jefcoate CR
Cytochrome P450 1b1 (Cyp1b1) expression is absent in mouse hepatocytes, but present in liver endothelia and activated stellate cells. Increased expression during adipogenesis suggests a role of Cyp1b1 metabolism in fatty acid homeostasis. Wild-type C57BL/6j (WT) and Cyp1b1-null (Cyp1b1-ko) mice were provided low or high fat diets (LFD and HFD, respectively). Cyp1b1-deletion suppressed HFD-induced obesity, improved glucose tolerance and prevented liver steatosis. Suppression of lipid droplets in sinusoidal hepatocytes, concomitant with enhanced glycogen granules, was a consistent feature of Cyp1b1-ko mice. Cyp1b1 deletion altered the in vivo expression of 560 liver genes, including suppression of PPARγ, stearoyl CoA desaturase 1 (Scd1) and many genes stimulated by PPARα, each consistent with this switch in energy storage mechanism. Ligand activation of PPARα in Cyp1b1-ko mice by WY-14643 was, nevertheless, effective. Seventeen gene changes in Cyp1b1-ko mice correspond to mouse transgenic expression that attenuated diet-induced diabetes. The absence of Cyp1b1 in mouse hepatocytes indicates participation in energy homeostasis through extra-hepatocyte signaling. Extensive sexual dimorphism in hepatic gene expression suggests a developmental impact of estrogen metabolism by Cyp1b1. Suppression of Scd1 and increased leptin turnover support enhanced leptin participation from the hypothalamus. Cyp1b1-mediated effects on vascular cells may underlie these changes.
PMID: 25703193 [PubMed - indexed for MEDLINE]
Rheumatoid arthritis and the prevalence of diabetic retinopathy.
Rheumatology (Oxford). 2015 Mar 2;
Authors: Bartels CM, Wong JC, Johnson SL, Thorpe CT, Barney NP, Sheibani N, Smith MA
OBJECTIVE: RA increases vascular disease and angiogenesis, yet a 1964 Lancet report paradoxically linked RA to lower diabetic retinopathy. Our objective was to examine RA as a risk factor for diabetic retinopathy compared with other vascular risk factors.
METHODS: This cohort study compared the prevalence of diabetic retinopathy in diabetes patients with and without RA in a 5% Medicare sample. We analysed the impact of RA on the prevalence of diabetic retinopathy using multivariate logistic regression calculating adjusted rate ratios (ARRs) controlling for sociodemographics, co-morbidity and health utilization. Sensitivity analysis examined eye exam rates.
RESULTS: Among 256 331 Medicare diabetes patients, 5572 (2%) had RA. Diabetic retinopathy was less prevalent in patients with RA compared with those without RA (13.7% vs 16.1%, P ≤ 0.01). Compared with patients without RA, the adjusted model demonstrated that patients with diabetes and RA were 28% less likely to have diabetic retinopathy and 4% more likely to receive an eye exam [ARR 0.72 (95% CI 0.67, 0.77), ARR 1.04 (95% CI 1.02, 1.06)].
CONCLUSION: Findings support the 1964 paradox observing decreased diabetic retinopathy in patients with RA. These findings pose new questions regarding whether RA physiology or treatments protect against diabetic retinopathy and how intraocular factors vary in contrast to adverse vascular changes elsewhere.
PMID: 25731768 [PubMed - as supplied by publisher]
Correlation of hypocalcemia with serum parathyroid hormone and calcitonin levels in pediatric intensive care unit.
Indian J Pediatr. 2015 Mar;82(3):217-20
Authors: Haghbin S, Serati Z, Sheibani N, Haghbin H, Karamifar H
OBJECTIVES: To investigate factors involved in causing hypocalcemia in critically ill patients.
METHODS: The patients aged 1 mo to 18 y, admitted to PICU at Nemazee Hospital, from May through November 2012, were reviewed. Those with impaired calcium hemostasis or on vitamin-D supplement were excluded. Calcitonin and parathyroid hormone levels were checked if ionized calcium level was less than 3.2 mg/d. Patient's demographic data, length of stay, Pediatric Risk of Mortality-III (PRISM-III) score, the need for mechanical ventilation, inotropic drug administration and outcome were recorded.
RESULTS: Among the 294 patients enrolled in the study, the incidence of ionized hypocalcemia was 20.4 %. The mortality rate was 45 % in hypocalcemic groups and 24.8 % in normocalcemic patients. Highly significant negative correlations were found between serum ionized calcium, PRISM-III score (r = -0.371, P = 0.004), and calcitonin level (r = -0.256, P = 0.049), but no significant correlation between hypocalcemia and parathyroid hormone level (P = 0.206) was found. A significant difference was observed between survivor and non-survivor groups regarding PRISM-III score (P = 0.00), ionized calcium (P = 0.00), and calcitonin (P = 0.022) but not parathyroid hormone level (P = 0.206).
CONCLUSIONS: Hypocalcemia was associated with increased mortality rate in PICU patients. A negative correlation was found between ionized calcium level and calcitonin. There was also a link between PTH level and severity of illness. It can therefore be concluded that evaluating serum ionized calcium, calcitonin, and PTH levels can be used as prognostic factors in critically ill patients.
PMID: 25183240 [PubMed - in process]
Optical cryoimaging of mitochondrial redox state in bronchopulmonary-dysplasia injury models in mice lungs.
Quant Imaging Med Surg. 2015 Feb;5(1):159-62
Authors: MasoudiMotlagh M, Sepehr R, Sheibani N, Sorenson CM, Ranji M
BACKGROUND: Bronchopulmonary dysplasia (BPD) is a major cause of morbidity and mortality in premature infants exposed to high levels of oxygen. This is mainly attributed to increased oxidative stress and angiogenesis defects impacting lung alveolarization.
METHODS: Here we use optical imaging to investigate the role of Bcl-2 in modulation of oxidative stress and angiogenesis and pathogenesis of BPD. Cryoimaging of the mitochondrial redox state of mouse lungs was applied to determine the metabolic state of the lungs from Bcl-2 +/+ (control), Bcl-2-deleted in the endothelium (Bcl-2 VE-cad) and Bcl-2-deficient (Bcl-2 -/-; global null) using mitochondrial metabolic coenzymes NADH (Nicotinamide Adenine Dinucleotide), and FADH2 (Flavin Adenine Dinucleotide) as the primary electron carriers in oxidative phosphorylation.
RESULTS: We observed a 47% and 26% decrease in the NADH redox in Bcl-2 deficient lungs, Bcl-2 -/- and Bcl-2 VE-cad, respectively.
CONCLUSIONS: Thus, Bcl-2 deficiency is associated with a significant increase in oxidative stress contributing to reduced angiogenesis and enhanced pathogenesis of BPD.
PMID: 25694965 [PubMed]
Caseoperoxidase, mixed β-casein-SDS-hemin-imidazole complex: a nano artificial enzyme.
J Biomol Struct Dyn. 2015 Feb 11;:1-14
Authors: Moosavi-Movahedi Z, Gharibi H, Hadi-Alijanvand H, Akbarzadeh M, Esmaili M, Atri MS, Sefidbakht Y, Bohlooli M, Nazari K, Javadian S, Hong J, Saboury AA, Sheibani N, Moosavi-Movahedi AA
A novel peroxidase-like artificial enzyme, named "caseoperoxidase", was biomimetically designed using a nano artificial amino acid apo-protein hydrophobic pocket. This four-component nano artificial enzyme containing heme-imidazole-β-casein-SDS exhibited high activity growth and kcat performance toward the native horseradish peroxidase demonstrated by the steady state kinetics using UV-vis spectrophotometry. The hydrophobicity and secondary structure of the caseoperoxidase were studied by ANS fluorescence and circular dichroism spectroscopy. Camel β-casein (Cβ-casein) was selected as an appropriate apo-protein for the heme active site because of its innate flexibility and exalted hydrophobicity. This selection was confirmed by homology modeling method. Heme docking into the newly obtained Cβ-casein structure indicated one heme was mainly incorporated with Cβ-casein. The presence of a main electrostatic site for the active site in the Cβ-casein was also confirmed by experimental methods through Wyman binding potential and isothermal titration calorimetry. The existence of Cβ-casein protein in this biocatalyst lowered the suicide inactivation and provided a suitable protective role for the heme active-site. Additional experiments confirmed the retention of caseoperoxidase structure and function as an artificial enzyme.
PMID: 25562503 [PubMed - as supplied by publisher]
Radiopacifier particle size impacts the physical properties of tricalcium silicate-based cements.
J Endod. 2015 Feb;41(2):225-30
Authors: Saghiri MA, Gutmann JL, Orangi J, Asatourian A, Sheibani N
INTRODUCTION: The aim of this study was to evaluate the impact of radiopaque additive, bismuth oxide, particle size on the physical properties, and radiopacity of tricalcium silicate-based cements.
METHODS: Six types of tricalcium silicate cement (CSC) including CSC without bismuth oxide, CSC + 10% (wt%) regular bismuth oxide (particle size 10 μm), CSC + 20% regular bismuth oxide (simulating white mineral trioxide aggregate [WMTA]) as a control, CSC + 10% nano bismuth oxide (particle size 50-80 nm), CSC + 20% nano-size bismuth oxide, and nano WMTA (a nano modification of WMTA comprising nanoparticles in the range of 40-100 nm) were prepared. Twenty-four samples from each group were divided into 4 groups and subjected to push-out, surface microhardness, radiopacity, and compressive strength tests. Data were analyzed by 1-way analysis of variance with the post hoc Tukey test.
RESULTS: The push-out and compressive strength of CSC without bismuth oxide and CSC with 10% and 20% nano bismuth oxide were significantly higher than CSC with 10% or 20% regular bismuth oxide (P < .05). The surface microhardness of CSC without bismuth oxide and CSC with 10% regular bismuth oxide had the lowest values (P < .05). The lowest radiopacity values were seen in CSC without bismuth oxide and CSC with 10% nano bismuth oxide (P < .05). Nano WMTA samples showed the highest values for all tested properties (P < .05) except for radiopacity.
CONCLUSIONS: The addition of 20% nano bismuth oxide enhanced the physical properties of CSC without any significant changes in radiopacity. Regular particle-size bismuth oxide reduced the physical properties of CSC material for tested parameters.
PMID: 25492489 [PubMed - in process]
Expression of pigment epithelium-derived factor and thrombospondin-1 regulate proliferation and migration of retinal pigment epithelial cells.
Physiol Rep. 2015 Jan 1;3(1)
Authors: Farnoodian M, Kinter JB, Yadranji Aghdam S, Zaitoun I, Sorenson CM, Sheibani N
Age-related macular degeneration (AMD) is the leading cause of vision loss among elderly. Although the pathogenesis of AMD is associated with retinal pigmented epithelium (RPE) dysfunction and abnormal neovascularization the detailed mechanisms remain unresolved. RPE is a specialized monolayer of epithelial cells with important functions in ocular homeostasis. Pathological RPE damage contributes to major ocular conditions including retinal degeneration and irreversible loss of vision in AMD. RPE cells also assist in the maintenance of the ocular angiogenic balance by production of positive and negative regulatory factors including vascular endothelial growth factor (VEGF), thrombospondin-1 (TSP1), and pigment epithelium-derived factor (PEDF). The altered production of PEDF and TSP1, as endogenous inhibitors of angiogenesis and inflammation, by RPE cells have been linked to pathogenesis of AMD and choroidal and retinal neovascularization. However, lack of simple methods for isolation and culture of mouse RPE cells has resulted in limited knowledge regarding the cell autonomous role of TSP1 and PEDF in RPE cell function. Here, we describe a method for routine isolation and propagation of RPE cells from wild-type, TSP1, and PEDF-deficient mice, and have investigated their impact on RPE cell function. We showed that expression of TSP1 and PEDF significantly impacted RPE cell proliferation, migration, adhesion, oxidative state, and phagocytic activity with minimal effect on their basal rate of apoptosis. Together, our results indicated that the expression of PEDF and TSP1 by RPE cells play crucial roles not only in regulation of ocular vascular homeostasis but also have significant impact on their cellular function.
PMID: 25602019 [PubMed]
Antiproliferative effects of ZnO, ZnO-MTCP, and ZnO-CuMTCP nanoparticles with safe intensity UV and X-ray irradiation.
Biotechnol Appl Biochem. 2015 Jan 9;
Authors: Sadjadpour S, Safarian S, Zargar SJ, Sheibani N
In photodynamic therapy (PDT) of cancer both the light and the photosensitizing agent are normally harmless, but in combination they could result in selective tumor killing. Zinc oxide nanoparticles were synthesized and coated with the amino acid cysteine to provide an adequate arm for conjugation with porphyrin photosensitizers (meso-tetra (4-carboxyphenyl) porphyrin [MTCP] and CuMTCP). Porphyrin-conjugated nanoparticles were characterized by TEM, FTIR, and UV-vis, and fluorescence spectrophotometry. The 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay was used to measure cell viability in the presence or absence of porphyrin conjugates following UV and X-ray irradiation. The uptake of the porphyrin-conjugated ZnO nanoparticles by cells was detected using fluorescence microscopy. Our results indicated that the survival of T-47D cells was significantly compromised in the presence of ZnO-MTCP-conjugated nanostructures with UV light exposure. Exhibition of cytotoxic activity of ZnO-MTCP for human prostate cancer (Du145) cells occurred at a higher concentration, indicating the more resistant nature of these tumor cells. ZnO-CuMTCP showed milder cytotoxic effects in human breast cancer (T-47D) and no cytotoxic effects in Du145 with UV light exposure, consistent with its lower cytotoxic potency as well as cellular uptake. Surprisingly, none of the ZnO-porphyrin conjugates exhibited cytotoxic effects with X-ray irradiation, whereas ZnO alone exerted cytotoxicity. Thus, ZnO and ZnO-porphyrin nanoparticles with UV or X-ray irradiation may provide a suitable treatment option for various cancers.
PMID: 25581219 [PubMed - as supplied by publisher]
Implication of disulfide bridge induced thermal reversibility, structural and functional stability for luciferase.
Protein Pept Lett. 2015;22(1):23-30
Authors: Naderi M, Moosavi-Movahedi AA, Hosseinkhani S, Nazari M, Bohlooli M, Hong J, Hadi-Alijanvand H, Sheibani N
Firefly luciferase is a relatively unstable protein and commonly loses its activity at room temperature because of structural changes. The structural and functional stability of this protein is critical for its enzymatic applications. Different approaches are applied to increase the stability of this enzyme such as designing of covalent cross-links (disulfide bonds). In this study, luciferase mutants containing one or two disulfide bonds were compared to the native protein for their for their structural, thermodynamic, and functional properties. Mutant forms of P. Pyralis luciferase A²⁹⁶C-A³²⁶C and A²⁹⁶C-A³²⁶C/P⁴⁵¹C-V⁴⁶⁹C were used. Thermodynamic and biophysical studies were carried out using UV-Vis, fluorescence, circular dichroism, luminescence spectroscopy and differential scanning calorimetry (DSC). We observed that both mutant forms of the protein were more stable than the wild-type enzyme. However, the single disulfide bond containing mutant was structurally and functionally more stable than the mutant protein containing two disulfide bonds. Furthermore, the enzymatic activity of the single disulfide bond containing mutant protein was 7-folds greater than the wild type and the double disulfide bond proteins. The A²⁹⁶C-A³²⁶C mutation also increased the reversibility and disaggregation of the protein. The enhanced activity of the single disulfide bond mutant protein was contributed to the expansion of its active site cleft, which was confirmed by bioinformatics tools.
PMID: 25159509 [PubMed - in process]
Curcumin mitigates the fibrillation of human serum albumin and diminishes the formation of reactive oxygen species.
Protein Pept Lett. 2015;22(4):348-53
Authors: Mazaheri M, Moosavi-Movahedi AA, Saboury AA, Rezaei MH, Shourian M, Farhadi M, Sheibani N
The formation of amyloid fibrils are thought to contribute to pathogenesis of many amyloids associated human diseases. Here the impact of curcumin on amyloid formation of human serum albumin (HSA) was studied. Incubation of HSA at 68°C under physiologic pH led to amyloid fibril formation. Thioflavin T (ThT) fluorescence was used for determination of amyloid fibril formation. Atomic force microscopy experiments indicated different fibril structure of HSA incubated with or without curcumin. The monitoring of the changes in reactive oxygen species (ROS) levels upon incubation of curcumin with HSA showed a significant decrease in ROS levels. Similar experiments were also carried out in the presence of aflatoxin M1 (AFM1) and lead (Pb) ions. Our results indicated that AFM1 and Pb ions promote the fibrillation of HSA and accelerate ROS production, which were inhibited in the presence of curcumin. Thus, curcumin mitigates protein fibrillation activity and diminishes ROS generation.
PMID: 25666039 [PubMed - in process]
Retinal pericytes and cytomegalovirus infectivity: implications for HCMV-induced retinopathy and congenital ocular disease.
J Neuroinflammation. 2015;12(1):2
Authors: Wilkerson I, Laban J, Mitchell JM, Sheibani N, Alcendor DJ
BACKGROUND: Human cytomegalovirus (HCMV) is the leading infectious cause of vision loss among congenitally infected children. Retinal pericytes play an essential role in maintaining retinal vascular and endothelial cell proliferation. However, the role of retinal pericytes in ocular HCMV pathogenesis is unknown.
METHODS: Retinal pericytes were exposed to clinical (SBCMV) and lab strains of HCMV; infectivity was analyzed by microscopy, immunofluorescence and qRT-PCR (reverse transcription polymerase chain reaction). Cytokine expression was examined by Luminex assay. Recombinant HCMV-GPF was used to examine viral replication kinetics. A Tricell culture model of the inner blood-retinal barrier (IBRB) was examined for cell type infectivity using immunohistochemistry.
RESULTS: Retinal pericytes expressed the biomarker neuron-glial antigen 2. Antigenic expression profiles for several cytoskeletal, cell adhesion and inflammatory proteins were shared by both retinal and brain pericytes. Infected pericytes showed cytomegalic cytopathology and expressed mRNAs for the major immediate protein (MIE) and HCMV phosphorylated envelop protein 65. qRT-PCR analysis showed full lytic replication of HCMV in retinal pericytes. Pericytes exposed to SBCMV for 9 days expressed higher levels of vascular endothelial cell growth factor mRNA compared to controls. Luminex analysis of supernatants from SBCMV-infected retinal pericytes had increased levels of macrophage inflammatory protein-1α, beta-2 microglobulin (B2-m), matrix metalloproteinase-3 and -9 (MMP3/9), and lower levels of IL-6 and IL-8 compared to controls. At 24 hours post infection, pericytes expressed higher levels of IL-8, TIMP-1 (tissue inhibitor of metalloproteinase-1), and RANTES (regulated upon activation normal T cell-expressed and presumably secreted) but lower levels of MMP9. Time course analysis showed that both brain and retinal pericytes were more permissive for HCMV infection than other cellular components of the BBB (blood-brain barrier) and IBRB. Using a Tricell culture model of the IBRB (retinal endothelial, pericytes, Müller cells), retinal pericytes were most permissive for SBCMV infection. SBCMV infection of this IBRB Tricell mixture for 96 hours resulted in increased levels of IL-6, MMP9, and stem cell factor with a concomitant decrease in granulocyte-macrophage colony-stimulating factor and TNF-alpha.
CONCLUSION: In retinal pericytes, HCMV induces proinflammatory and angiogenic cytokines. In the IBRB, pericytes likely serve as an amplification reservoir which contributes to retinal inflammation and angiogenesis.
PMID: 25573478 [PubMed - in process]
Simultaneous application of bevacizumab and anti-CTGF antibody effectively suppresses proangiogenic and profibrotic factors in human RPE cells.
Mol Vis. 2015;21:378-90
Authors: Bagheri A, Soheili ZS, Ahmadieh H, Samiei S, Sheibani N, Astaneh SD, Kanavi MR, Mohammadian A
PURPOSE: Retinal pigment epithelial (RPE) cells play key roles in the development of choroidal neovascularization and subsequent fibrosis. We investigated the impact of bevacizumab, antihuman vascular endothelial growth factor (VEGF) antibody, and anticonnective tissue growth factor (anti-CTGF) neutralizing antibody, individually or in combination, on proangiogenic and profibrotic properties of RPE cells.
METHODS: Primary cultures of human RPE cells were incubated with different concentrations of bevacizumab (0.25, 0.5, and 0.8 mg/ml) and/or anti-CTGF (10 μg/ml), and cell proliferation and apoptosis were determined. Expression and activity of proangiogenic and profibrotic genes including matrix metalloproteinases (MMP)-2 and 9, VEGFA, CTGF, vascular endothelial growth factor receptor-1 (VEGFR-1), cathepsin D, tissue inhibitor of metalloproteinases (TIMP) -1 and -2, and alpha smooth muscle actin (α-SMA) were assessed with slot blot, real-time RT-PCR, and zymography.
RESULTS: Bevacizumab alone inhibited proliferation of RPE cells while anti-CTGF or bevacizumab and anti-CTGF combined had no inhibitory effect in this regard. Bevacizumab increased MMP-2, MMP-9, and cathepsin D but decreased VEGFA and VEGFR-1 expression. The CTGF level was increased by using 0.25 mg/ml bevacizumab but decreased at the 0.8 mg/ml concentration of bevacizumab. Treatment with anti-CTGF antibody decreased MMP-2 expression whereas combined treatment with bevacizumab and anti-CTGF resulted in decreased expression of MMP-2, TIMP-1, cathepsin D, VEGFA, CTGF, and α-SMA in the treated cultures.
CONCLUSIONS: Treatment of RPE cells with the combination of bevacizumab and anti-CTGF could effectively suppress the proangiogenic and profibrotic activity of RPE cells.
PMID: 25883524 [PubMed - in process]
Curcumin Protects β-Lactoglobulin Fibril Formation and Fibril-Induced Neurotoxicity in PC12Cells.
PLoS One. 2015;10(7):e0133206
Authors: Mazaheri M, Moosavi-Movahedi AA, Saboury AA, Khodagholi F, Shaerzadeh F, Sheibani N
In this study the β-lactoglobulin fibrillation, in the presence or absence of lead ions, aflatoxin M1 and curcumin, was evaluated using ThT fluorescence, Circular dichroism spectroscopy and atomic force microscopy. To investigate the toxicity of the different form of β-Lg fibrils, in the presence or absence of above toxins and curcumin, we monitored changes in the level of reactive oxygen species and morphology of the differentiated neuron-like PC12 cells. The cell viability, cell body area, average neurite length, neurite width, number of primary neurites, percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different form of β-Lg fibrils. Incubation of β-Lg with curcumin resulted in a significant decrease in ROS levels even in the presence of lead ions and aflatoxin M1. The β-Lg fibrils formed in the presence of lead ions and aflatoxin M1 attenuated the growth and complexity of PC12 cells compared with other form of β-Lg fibrils. However, the adverse effects of these toxins and protein fibrils were negated in the presence of curcumin. Furthermore, the antioxidant and inhibitory effects of curcumin protected PC12 cells against fibril neurotoxicity and enhanced their survival. Thus, curcumin may provide a protective effect toward β-Lg, and perhaps other protein, fibrils mediated neurotoxicity.
PMID: 26186474 [PubMed - in process]
ATF4 is a novel regulator of MCP-1 in microvascular endothelial cells.
J Inflamm (Lond). 2015;12:31
Authors: Huang H, Jing G, Wang JJ, Sheibani N, Zhang SX
BACKGROUND: Monocyte chemoattractant protein-1 (MCP-1) is a major chemokine that recruits monocyte/macrophage to the site of tissue injury and plays a critical role in microvascular complications of diabetes. However, the mechanisms underlying the regulation of MCP-1 are not fully understood. The present study aims to explore the role of activating transcription factor 4 (ATF4), an ER stress-inducible transcription factor, in regulation of MCP-1 expression and production in brain and retinal microvascular endothelial cells.
METHODS: For in vitro study, primary brain microvascular endothelial cells isolated from ATF4 knockout mice or mouse retinal endothelial cells were treated with lipopolysaccharide (LPS) to induce MCP-1 expression. ATF4 expression/function was manipulated by adenoviruses expressing wild type ATF4 (Ad-ATF4) or a dominant negative mutant of the protein (Ad-ATF4DN). For in vivo study, MCP-1 expression was induced by intravitreal injection of LPS or Ad-ATF4 in heterozygous ATF4 knockout or wild type mice.
RESULTS: LPS treatment induced a dose- and time-dependent increase in ATF4 expression, ER stress and MCP-1 production in brain and retinal microvascular endothelial cells. Overexpression of ATF4 in endothelial cells significantly increased the secretion of MCP-1 and promoted THP-1 monocyte-endothelial cell adhesion. Conditioned medium from ATF4-overexpressiing endothelial cells significantly enhanced THP-1 cell migration. Consistently, intravitreal injection of Ad-ATF4 remarkably enhanced retinal levels of MCP-1 and promoted inflammatory cell infiltration into the vitreous and retina. In contrast, LPS-induced MCP-1 upregulation was markedly attenuated in ATF4-deficient endothelial cells and in retinas of ATF4 knockout mice, suggesting that ATF4 is essential for LPS-induced MCP-1 production in endothelial cells and in the retina. Mechanistically, overexpression of ATF4 enhanced, while inhibition of ATF4, attenuated the basal and LPS-stimulated phosphorylation of NF-κB, P38, and JNK. Furthermore, pharmacological inhibition of NF-κB, P38, or JNK significantly reduced ATF4-stimulated MCP-1 secretion from endothelial cells.
CONCLUSIONS: Taken together, our results suggest a critical role of ATF4 in the regulation of MCP-1 production in retinal and brain microvascular endothelial cells, which may contribute to inflammation-related endothelial injury in diseases such as diabetic retinopathy.
PMID: 25914608 [PubMed]
Topiramate Protects Pericytes from Glucotoxicity: Role for Mitochondrial CA VA in Cerebromicrovascular Disease in Diabetes.
J Endocrinol Diabetes. 2015;2(2)
Authors: Patrick P, Price TO, Diogo AL, Sheibani N, Banks WA, Shah GN
Hyperglycemia in diabetes mellitus causes oxidative stress and pericyte depletion from the microvasculature of the brain thus leading to the Blood-Brain Barrier (BBB) disruption. The compromised BBB exposes the brain to circulating substances, resulting in neurotoxicity and neuronal cell death. The decline in pericyte numbers in diabetic mouse brain and pericyte apoptosis in high glucose cultures are caused by excess superoxide produced during enhanced respiration (mitochondrial oxidative metabolism of glucose). Superoxide is precursor to all Reactive Oxygen Species (ROS) which, in turn, cause oxidative stress. The rate of respiration and thus the ROS production is regulated by mitochondrial carbonic anhydrases (mCA) VA and VB, the two isoforms expressed in the mitochondria. Inhibition of both mCA: decreases the oxidative stress and restores the pericyte numbers in diabetic brain; and reduces high glucose-induced respiration, ROS, oxidative stress, and apoptosis in cultured brain pericytes. However, the individual role of the two isoforms has not been established. To investigate the contribution of mCA VA in ROS production and apoptosis, a mCA VA overexpressing brain pericyte cell line was engineered. These cells were exposed to high glucose and analyzed for the changes in ROS and apoptosis. Overexpression of mCA VA significantly increased pericyte ROS and apoptosis. Inhibition of mCA VA with topiramate prevented increases both in glucose-induced ROS and pericyte death. These results demonstrate, for the first time, that mCA VA regulates the rate of pericyte respiration. These findings identify mCA VA as a novel and specific therapeutic target to protect the cerebromicrovascular bed in diabetes.
PMID: 26167540 [PubMed - as supplied by publisher]
Stable overexpression of DNA fragmentation factor in T-47D cells: sensitization of breast cancer cells to apoptosis in response to acetazolamide and sulfabenzamide.
Mol Biol Rep. 2014 Nov;41(11):7387-94
Authors: Bagheri F, Safarian S, Eslaminejad MB, Sheibani N
Alterations in expression of the DFF40 gene have been reported in some cancers. This study is an in vitro study of the therapeutic effects of gene transfer that lead to elevation in DFF40 expression within T-47D cells in the presence of sulfonamide drugs. In this study, we have constructed a eukaryotic expression vector for DFF40 and transfected it into T-47D cancer cells. We used real time RT-PCR to detect the expression of DFF40 and the MTT assay to determine effects of the sulfonamide drugs acetazolamide, sulfabenzamide, sulfathiazole and sulfacetamide on cell viability in the presence of increased and normal DFF40 levels. Cell cycle distribution was assessed by propidium iodide (PI) staining and the rates of apoptosis by annexin V/PI staining. The DNA laddering analysis was employed to evaluate apoptosis. We observed that overexpression of DFF40 was only effective in decreasing viability in cells incubated with acetazolamide and sulfabenzamide. There was enhanced apoptosis in these groups, particularly with acetazolamide. The cell cycle distribution analysis showed that in the presence of sulfonamide drugs there were no substantial changes in empty-vector or DFF40-transfected cells, except for those cells treated with sulfabenzamide or sulfathiazole. There was no DNA laddering in cells that expressed the empty vector when incubated with sulfonamide drugs. In contrast, we observed DNA laddering in cells that expressed DFF40 in the presence of acetazolamide. Our results have demonstrated that combinatorial use of some sulfonamides such as acetazolamide along with increased expression of DFF40 can potently kill tumor cells via apoptosis and may be beneficial for treatment of some chemoresistant cancers.
PMID: 25086620 [PubMed - in process]
CD40 promotes the development of early diabetic retinopathy in mice.
Diabetologia. 2014 Oct;57(10):2222-31
Authors: Portillo JA, Greene JA, Okenka G, Miao Y, Sheibani N, Kern TS, Subauste CS
AIMS/HYPOTHESIS: Microangiopathy is a leading complication of diabetes that commonly affects the retina. Degenerate capillaries are a central feature of diabetic retinopathy. An inflammatory process has been linked to the development of diabetic retinopathy but its regulation is incompletely understood. Cluster of differentiation (CD) 40 is a member of the TNF receptor superfamily that promotes the development of certain inflammatory disorders. The role of CD40 in diabetic microangiopathy is unknown.
METHODS: B6 and Cd40−/− mice were administered streptozotocin to induce diabetes. Leucostasis was assessed using fluorescein isothiocyanate-conjugated concanavalin A. Retinal Icam1 and Cd40 mRNA levels were examined using real-time PCR. Protein nitration was assessed by immunohistochemistry. Histopathology was examined in the retinal vasculature. CD40 expression was assessed by flow cytometry and immunohistochemistry. Intercellular adhesion molecule 1 (ICAM-1) and nitric oxide synthase 2 (NOS2) were examined by immunoblot and/or flow cytometry. Nitric oxide production was examined by immunoblot and Griess reaction.
RESULTS: In mouse models of diabetes, Cd40−/− mice exhibited reduced retinal leucostasis and did not develop capillary degeneration in comparison with B6 mice. Diabetic Cd40−/− mice had diminished ICAM-1 upregulation and decreased protein nitration. Cd40 mRNA levels were increased in the retinas of diabetic B6 mice compared with non-diabetic controls. CD40 expression increased in retinal Müller cells, endothelial cells and microglia of diabetic animals. CD40 stimulation upregulated ICAM-1 in retinal endothelial cells and Müller cells. CD40 ligation upregulated NOS2 and nitric oxide production by Müller cells.
CONCLUSIONS/INTERPRETATION: CD40-deficient mice were protected fromthe development of diabetic retinopathy. These mice exhibited diminished inflammatory responses linked to diabetic retinopathy. CD40 stimulation of retinal cells triggered these pro-inflammatory responses.
PMID: 25015056 [PubMed - in process]
Direct electrochemistry of glucose oxidase and glucose biosensing on a hydroxyl fullerenes modified glassy carbon electrode.
Biosens Bioelectron. 2014 Oct 15;60:30-4
Authors: Gao YF, Yang T, Yang XL, Zhang YS, Xiao BL, Hong J, Sheibani N, Ghourchian H, Hong T, Moosavi-Movahedi AA
Direct electrochemistry of glucose oxidase (GOD) was achieved when GOD-hydroxyl fullerenes (HFs) nano-complex was immobilized on a glassy carbon (GC) electrode and protected with a chitosan (Chit) membrane. The ultraviolet-visible absorption spectrometry (UV-vis), transmission electron microscopy (TEM), and circular dichroism spectropolarimeter (CD) methods were utilized for additional characterization of the GOD, GOD-HFs and Chit/GOD-HFs. Chit/HFs may preserve the secondary structure and catalytic properties of GOD. The cyclic voltammograms (CVs) of the modified GC electrode showed a pair of well-defined quasi-reversible redox peaks with the formal potential (E°') of 353 ± 2 mV versus Ag/AgCl at a scan rate of 0.05 V/s. The heterogeneous electron transfer constant (ks) was calculated to be 2.7 ± 0.2s(-1). The modified electrode response to glucose was linear in the concentrations ranging from 0.05 to 1.0mM, with a detection limit of 5 ± 1 μM. The apparent Michaelis-Menten constant (Km(app)) was 694 ± 8 μM. Thus, the modified electrode could be applied as a third generation biosensor for glucose with high sensitivity, selectivity and low detection limit.
PMID: 24768859 [PubMed - indexed for MEDLINE]
Sodium dodecyl sulphate modulates the fibrillation of human serum albumin in a dose-dependent manner and impacts the PC12 cells retraction.
Colloids Surf B Biointerfaces. 2014 Oct 1;122:341-9
Authors: Movaghati S, Moosavi-Movahedi AA, Khodagholi F, Digaleh H, Kachooei E, Sheibani N
Protein aggregation is impacted by many factors including temperature, pH, and the presence of surfactants, electrolytes, and metal ions. The addition of sodium dodecyl sulphate (SDS) at different concentrations may play a significant role in the human serum albumin (HSA) fibrillation pathway. Here the heat induction of HSA fibrillation incubated with different concentrations of SDS was evaluated using a variety of techniques. These included ThT fluorescence, Congo red absorbance, circular dichroism, dynamic light scattering, and atomic force microscopy (AFM). To explore HSA surface properties, the surface tension of solutions was measured using Du Noüy Ring method tensiometry. In addition, the criteria of neurite outgrowth and complexity were monitored by exposing PC12 cells to different forms of HSA amyloid intermediates. ThT fluorescence kinetic studies indicated that SDS at low concentrations induced more fibrillation of HSA, while SDS at high concentrations inhibited the fibrillation of HSA. At higher SDS concentrations hydrophobic forces had a significant role whereas at lower SDS concentrations electrostatic forces were dominant. The cell culture studies demonstrated the significant impact of SDS concentration on HSA fibrillation and subsequent neuronal cell morphology. The HSA incubated with low concentrations of SDS inhibited neurite outgrowth and complexity of the PC12 cells, whereas high concentrations of SDS had lesser effect. Thus, SDS acts as a salt at lower concentrations, while at higher concentrations acts as a chaperon, with significant impact on fibrillation of HSA.
PMID: 25073074 [PubMed - indexed for MEDLINE]
Organ specific optical imaging of mitochondrial redox state in a rodent model of hereditary hemorrhagic telangiectasia-1.
J Biophotonics. 2014 Oct;7(10):799-809
Authors: Ghanian Z, Maleki S, Park S, Sorenson CM, Sheibani N, Ranji M
Hereditary Hemorrhagic Telangiectasia-1 (HHT-1) is a vascular disease caused by mutations in the endoglin (Eng)/CD105 gene. The objective of this study was to quantify the oxidative state of a rodent model of HHT-1 using an optical imaging technique. We used a cryofluorescence imaging instrument to quantitatively assess tissue metabolism in this model. Mitochondrial redox ratio (FAD/NADH), FAD RR, was used as a quantitative marker of the metabolic status and was examined in the kidneys, and eyes of wild-type and Eng +/- mice. Kidneys and eyes from wild-type P21, 6W, and 10M old mice showed, respectively, a 9% (±2), 24% (±0.4), 15% (±1), and 23% (±4), 33% (±0.6), and 30% (±2) change in the mean FAD RR compared to Eng +/- mice at the same age. Thus, endoglin haploinsufficiency is associated with less oxidative stress in various organs and mitigation of angiogenesis.
PMID: 23740865 [PubMed - in process]
Matricellular protein thrombospondins: influence on ocular angiogenesis, wound healing and immuneregulation.
Curr Eye Res. 2014 Aug;39(8):759-74
Authors: Masli S, Sheibani N, Cursiefen C, Zieske J
Thrombospondins are a family of large multi-domain glycoproteins described as matricelluar proteins based on their ability to interact with a broad range of receptors, matrix molecules, growth factors or proteases, and to modulate array of cellular functions including intracellular signaling, proliferation and migration. Two members of the thrombospondin family, thrombospondin 1 (TSP-1) and thrombospondin 2 (TSP-2) are studied extensively to determine their structure and function. While expressed at low levels in normal adult tissues, their increased expression is seen predominantly in response to cellular perturbations. Despite structural similarities, a notable functional difference between TSP-1 and TSP-2 includes the ability of former to activate of latent TGF-β and its competitive inhibition by the latter. Both these thrombospondins are reported to play important roles in TGF-β rich ocular environment with most reports related to TSP-1. They are expressed by many ocular cell types and detectable in the aqueous and vitreous humor. TSP-1 and TSP-2 influence many cellular interactions in the eye such as angiogenesis, cell migration, wound healing, TGF-β activation and regulation of inflammatory immune responses. Together, these processes are known to contribute to the immune privilege status of the eye. Emerging roles of TSP-1 and TSP-2 in ocular functions and pathology are reviewed here.
PMID: 24559320 [PubMed - indexed for MEDLINE]
The influence of endodontic broken stainless steel instruments on the urinary levels of iron.
Biol Trace Elem Res. 2014 Jun;158(3):330-3
Authors: Saghiri MA, Asatourian A, Haraji A, Ramezani GH, Garcia-Godoy F, Scarbecz M, Sheibani N
Previous studies on the endodontic broken stainless steel instruments have mainly focused on retrieval of specimens. However, the systemic consequences of the retained separated instruments need an investigation. The current study aimed to evaluate the correlation between broken stainless steel instruments inside the root canal space and the urinary level of iron (Fe) in patients. Sixty near-aged and same-gender patients were selected. Thirty patients in the control group had no endodontic treatment in their history, while the other 30 patients in the endodontic group had broken stainless steel instrument. The urine samples were collected in iron-free containers. All specimens were refrigerated for 1 day and then subjected to Fe level measurement by electrothermal atomic absorption spectrometry. Data were analyzed by Kolmogorov-Smirnov and t tests at P < 0.05. The correlation coefficients of age and sex were also evaluated in relation with Fe levels in the urine. The level of Fe did not show any significant increase in the experimental group (P > 0.05). There was a positive correlation between age and Fe levels of endodontic and control groups. However, the joint effects of age and sex on Fe levels were different for the two groups. The presence of broken stainless steel instruments inside the root canal space did not elevate the level of Fe in the urine of patients. However, this value was positively correlated with the patient age.
PMID: 24699830 [PubMed - indexed for MEDLINE]
Effect of endodontic cement on bone mineral density using serial dual-energy x-ray absorptiometry.
J Endod. 2014 May;40(5):648-51
Authors: Saghiri MA, Orangi J, Tanideh N, Janghorban K, Sheibani N
INTRODUCTION: Materials with new compositions were tested in order to develop dental materials with better properties. Calcium silicate-based cements, including white mineral trioxide aggregate (WMTA), may improve osteopromotion because of their composition. Nano-modified cements may help researchers produce ideal root-end filling materials. Serial dual-energy x-ray absorptiometry measurement was used to evaluate the effects of particle size and the addition of tricalcium aluminate (C3A) to a type of mineral trioxide aggregate-based cement on bone mineral density and the surrounding tissues in the mandible of rabbits.
METHODS: Forty mature male rabbits (N = 40) were anesthetized, and a bone defect measuring 7 × 1 × 1 mm was created on the semimandible. The rabbits were divided into 2 groups, which were subdivided into 5 subgroups with 4 animals each based on the defect filled by the following: Nano-WMTA (patent application #13/211.880), WMTA (as standard), WMTA without C3A, Nano-WMTA + 2% Nano-C3A (Fujindonjnan Industrial Co, Ltd, Fujindonjnan Xiamen, China), and a control group. Twenty and forty days postoperatively, the animals were sacrificed, and the semimandibles were removed for DXA measurement.
RESULTS: The Kruskal-Wallis test followed by the Mann-Whitney U test showed significant differences between the groups at a significance level of P < .05. P values calculated by the Kruskal-Wallis test were .002 for bone mineral density at both intervals and P20 day = .004 and P40 day = .005 for bone mineral content.
CONCLUSIONS: This study showed that bone regeneration was enhanced by reducing the particle size (nano-modified) and C3A mixture. This may relate to the existence of an external supply of minerals and a larger surface area of nano-modified material, which may lead to faster release rate of Ca(2+), inducing bone formation. Adding Nano-C3A to Nano-WMTA may improve bone regeneration properties.
PMID: 24767558 [PubMed - in process]
Correlation between long-term in vivo amalgam restorations and the presence of heavy elements in the dental pulp.
J Trace Elem Med Biol. 2014 Apr;28(2):200-4
Authors: Saghiri MA, Banava S, Sabzian MA, Gutmann JL, Asatourian A, Ramezani GH, Garcia-Godoy F, Sheibani N
PROJECT: To measure the levels of heavy metals (Hg, Sn) in the dental pulp and blood samples of patients with long-term amalgam restorations.
PROCEDURE: 12 amalgam restored and 12 non-restored, sound teeth were chosen and access cavity preparation to the pulp chamber was made. The contents were transferred and dissolved in 5mL of concentrated nitric acid followed by placement in an oven at 180°C for tissue digestion. After cooling the tubes each digested sample was transferred to an atomic absorption system to measure the levels of heavy metals. The blood samples of five patients in each group were randomly analyzed to determine the levels of these heavy metals in the blood and if there were a correlation between these levels in blood and pulp. Data were analyzed by t-test at a P<0.05 level of significance.
RESULTS: No significant difference was seen between the levels of Hg and Sn in pulp tissues (P>0.05); however, the blood analysis showed higher level of Hg amalgam group (P=0.009). The analysis between the pulp and blood samples showed positive correlations for both Hg and Sn elements in dental pulp and the blood (P=1.000) (P=0.900).
CONCLUSIONS: The long-term presence of dental amalgam (at least 5 years) did not result in any remarkable changes in the levels of mercury and tin in the pulp tissue; however, there were increases in the level of mercury in the blood circulation even five years following the placement of the restoration.
PMID: 24731778 [PubMed - indexed for MEDLINE]
PEDF expression regulates the proangiogenic and proinflammatory phenotype of the lung endothelium.
Am J Physiol Lung Cell Mol Physiol. 2014 Apr 1;306(7):L620-34
Authors: Shin ES, Sorenson CM, Sheibani N
Pigment epithelium-derived factor (PEDF) is a multifunctional protein with important roles in regulation of inflammation and angiogenesis. It is produced by various cell types, including endothelial cells (EC). However, the cell autonomous impact of PEDF on EC function needs further investigation. Lung EC prepared from PEDF-deficient (PEDF-/-) mice were more migratory and failed to undergo capillary morphogenesis in Matrigel compared with wild type (PEDF+/+) EC. Although no significant differences were observed in the rates of apoptosis in PEDF-/- EC compared with PEDF+/+ cells under basal or stress conditions, PEDF-/- EC proliferated at a slower rate. PEDF-/- EC also expressed increased levels of proinflammatory markers, including vascular endothelial growth factor, inducible nitric oxide synthase, vascular cell adhesion molecule-1, as well as altered cellular junctional organization, and nuclear localization of β-catenin. The PEDF-/- EC were also more adhesive, expressed decreased levels of thrombospondin-2, tenascin-C, and osteopontin, and increased fibronectin. Furthermore, we showed lungs from PEDF-/- mice exhibited increased expression of macrophage marker F4/80, along with increased thickness of the vascular walls, consistent with a proinflammatory phenotype. Together, our data suggest that the PEDF expression makes significant contribution to modulation of the inflammatory and angiogenic phenotype of the lung endothelium.
PMID: 24318110 [PubMed - indexed for MEDLINE]
The effect of pH on solubility of nano-modified endodontic cements.
J Conserv Dent. 2014 Jan;17(1):13-7
Authors: Saghiri MA, Godoy FG, Gutmann JL, Lotfi M, Asatourian A, Sheibani N, Elyasi M
AIMS: To evaluate the effect of storage pH on solubility of white mineral trioxide aggregate (WMTA), bioaggregate (BA), and nano WMTA cements.
MATERIALS AND METHODS: Forty-eight moulds randomly allocated into three groups of pH 4.4 (group A), 7.4 (group B), and 10.4 (group C); and one empty as control in each group. Each group was further divided into three subgroups according to the material studied; WMTA, BA, and nano WMTA. The specimens in subgroup A were soaked in butyric acid buffered with synthetic tissue fluid (STF) (pH 4.4), while the samples in subgroups B (pH 7.4) and C (pH 10.4) buffered in potassium hydroxide for 24 h and then the loss of cement was determined. A two-way analysis of variance (ANOVA) and Tukey post-hoc statistical tests were used to detect any statistically significant differences among the groups/subgroups.
RESULTS: Statistical analysis has showed the highest solubility in acidic pH for all tested materials. Nano WMTA samples in pH = 10.4 had the lowest and BA samples in pH = 4.4 showed the highest cement loss.
CONCLUSION: The solubility of all tested cements can be jeopardized in acidic environment which might affect on their sealing characteristic in clinical scenario. However, nano WMTA cement due to its small size particles and different additives was capable of producing lower porosity in set material, which resulted in showing more resistance in acidic environment.
PMID: 24554853 [PubMed]
Molecular hydrogen in drinking water protects against neurodegenerative changes induced by traumatic brain injury.
PLoS One. 2014;9(9):e108034
Authors: Dohi K, Kraemer BC, Erickson MA, McMillan PJ, Kovac A, Flachbartova Z, Hansen KM, Shah GN, Sheibani N, Salameh T, Banks WA
Traumatic brain injury (TBI) in its various forms has emerged as a major problem for modern society. Acute TBI can transform into a chronic condition and be a risk factor for neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, probably through induction of oxidative stress and neuroinflammation. Here, we examined the ability of the antioxidant molecular hydrogen given in drinking water (molecular hydrogen water; mHW) to alter the acute changes induced by controlled cortical impact (CCI), a commonly used experimental model of TBI. We found that mHW reversed CCI-induced edema by about half, completely blocked pathological tau expression, accentuated an early increase seen in several cytokines but attenuated that increase by day 7, reversed changes seen in the protein levels of aquaporin-4, HIF-1, MMP-2, and MMP-9, but not for amyloid beta peptide 1-40 or 1-42. Treatment with mHW also reversed the increase seen 4 h after CCI in gene expression related to oxidation/carbohydrate metabolism, cytokine release, leukocyte or cell migration, cytokine transport, ATP and nucleotide binding. Finally, we found that mHW preserved or increased ATP levels and propose a new mechanism for mHW, that of ATP production through the Jagendorf reaction. These results show that molecular hydrogen given in drinking water reverses many of the sequelae of CCI and suggests that it could be an easily administered, highly effective treatment for TBI.
PMID: 25251220 [PubMed - in process]
Diabetes and retinal vascular dysfunction.
J Ophthalmic Vis Res. 2014 Jul-Sep;9(3):362-73
Authors: Shin ES, Sorenson CM, Sheibani N
Diabetes predominantly affects the microvascular circulation of the retina resulting in a range of structural changes unique to this tissue. These changes ultimately lead to altered permeability, hyperproliferation of endothelial cells and edema, and abnormal vascularization of the retina with resulting loss of vision. Enhanced production of inflammatory mediators and oxidative stress are primary insults with significant contribution to the pathogenesis of diabetic retinopathy (DR). We have determined the identity of the retinal vascular cells affected by hyperglycemia, and have delineated the cell autonomous impact of high glucose on function of these cells. We discuss some of the high glucose specific changes in retinal vascular cells and their contribution to retinal vascular dysfunction. This knowledge provides novel insight into the molecular and cellular defects contributing to the development and progression of diabetic retinopathy, and will aid in the development of innovative, as well as target specific therapeutic approaches for prevention and treatment of DR.
PMID: 25667739 [PubMed]
Caspase-14 expression impairs retinal pigment epithelium barrier function: potential role in diabetic macular edema.
Biomed Res Int. 2014;2014:417986
Authors: Beasley S, El-Sherbiny M, Megyerdi S, El-Shafey S, Choksi K, Kaddour-Djebbar I, Sheibani N, Hsu S, Al-Shabrawey M
We recently showed that caspase-14 is a novel molecule in retina with potential role in accelerated vascular cell death during diabetic retinopathy (DR). Here, we evaluated whether caspase-14 is implicated in retinal pigment epithelial cells (RPE) dysfunction under hyperglycemia. The impact of high glucose (HG, 30 mM D-glucose) on caspase-14 expression in human RPE (ARPE-19) cells was tested, which showed significant increase in caspase-14 expression compared with normal glucose (5 mM D-glucose + 25 mM L-glucose). We also evaluated the impact of modulating caspase-14 expression on RPE cells barrier function, phagocytosis, and activation of other caspases using ARPE-19 cells transfected with caspase-14 plasmid or caspase-14 siRNA. We used FITC-dextran flux assay and electric cell substrate impedance sensing (ECIS) to test the changes in RPE cell barrier function. Similar to HG, caspase-14 expression in ARPE-19 cells increased FITC-dextran leakage through the confluent monolayer and decreased the transcellular electrical resistance (TER). These effects of HG were prevented by caspase-14 knockdown. Furthermore, caspase-14 knockdown prevented the HG-induced activation of caspase-1 and caspase-9, the only activated caspases by HG. Phagocytic activity was unaffected by caspase-14 expression. Our results suggest that caspase-14 contributes to RPE cell barrier disruption under hyperglycemic conditions and thus plays a role in the development of diabetic macular edema.
PMID: 25121097 [PubMed - in process]
High glucose alters retinal astrocytes phenotype through increased production of inflammatory cytokines and oxidative stress.
PLoS One. 2014;9(7):e103148
Authors: Shin ES, Huang Q, Gurel Z, Sorenson CM, Sheibani N
Astrocytes are macroglial cells that have a crucial role in development of the retinal vasculature and maintenance of the blood-retina-barrier (BRB). Diabetes affects the physiology and function of retinal vascular cells including astrocytes (AC) leading to breakdown of BRB. However, the detailed cellular mechanisms leading to retinal AC dysfunction under high glucose conditions remain unclear. Here we show that high glucose conditions did not induce the apoptosis of retinal AC, but instead increased their rate of DNA synthesis and adhesion to extracellular matrix proteins. These alterations were associated with changes in intracellular signaling pathways involved in cell survival, migration and proliferation. High glucose conditions also affected the expression of inflammatory cytokines in retinal AC, activated NF-κB, and prevented their network formation on Matrigel. In addition, we showed that the attenuation of retinal AC migration under high glucose conditions, and capillary morphogenesis of retinal endothelial cells on Matrigel, was mediated through increased oxidative stress. Antioxidant proteins including heme oxygenase-1 and peroxiredoxin-2 levels were also increased in retinal AC under high glucose conditions through nuclear localization of transcription factor nuclear factor-erythroid 2-related factor-2. Together our results demonstrated that high glucose conditions alter the function of retinal AC by increased production of inflammatory cytokines and oxidative stress with significant impact on their proliferation, adhesion, and migration.
PMID: 25068294 [PubMed - in process]
The species and heme pocket properties of sturgeon hemoglobins upon interaction with N-dodecyl trimethylammonium bromide.
Protein Pept Lett. 2014;21(2):171-8
Authors: Ariaeenejad S, Moosavi-Movahedi AA, Kavousi K, Dayer MR, Hong J, Yousefi R, Sheibani N, Habibi-Rezaei M
The variations in fish hemoglobin (Hb) structures play a vital role in their respiratory performance under various environmental conditions and are impacted by their physiological properties. The major hemoglobins from two species of sturgeon were studied upon interaction with n-dodecyl trimethyl ammonium bromide (DTAB) using the UV-vis absorption, circular dichroism (CD), fluorescence spectroscopy, and oxygen affinity measurement methods as well as chemometric analysis. The UV-Vis absorption spectra between 500 and 650 nm was used to identify each species of hemoglobin, and to show that the concentration of oxyHb and metHb decreases, while that of deoxyHb increases upon interaction with DTAB. Both reduced oxyHb and oxidized hemichrome of the two Hbs were studied to obtain information about the DTAB efects on their structural features. The circular dichroism (CD) was utilized to obtain secondary structure and compactness for Hb upon interaction with DTAB. Binding of DTAB molecules induced the unfolding of Hb, and was accompanied with exposure of the heme pocket facilitating its oxidation. The differences between unfolding processes for the two Acipenser species were indicated by fluorescence spectroscopy. The chemometric analysis of Hbs was investigated upon interaction with DTAB under titration, using fluorescence spectra allowing determination of the number of components and mole fractions of the oxidized Hb. Our data showed that Acipenser persicus Hb had a more hyperchromic character, more surface area, more loosely folded structure, and therefore, exposed region of heme group compared with Acipenser stellatus oxyHb. In addition, with increasing DTAB the transition of Acipenser stellatus oxyHb to the state of hemichrome occurred at a slower speed than Acipenser persicus oxyHb, and finally more oxygen affinity and compactness. Our results suggest that these differences aroused from the inherent differences between the heme groups which fulfil a potentially important physiological role in these fish.
PMID: 24000823 [PubMed - indexed for MEDLINE]
Identification of O-GlcNAc modification targets in mouse retinal pericytes: implication of p53 in pathogenesis of diabetic retinopathy.
PLoS One. 2014;9(5):e95561
Authors: Gurel Z, Zaro BW, Pratt MR, Sheibani N
Hyperglycemia is the primary cause of the majority of diabetes complications, including diabetic retinopathy (DR). Hyperglycemic conditions have a detrimental effect on many tissues and cell types, especially the retinal vascular cells including early loss of pericytes (PC). However, the mechanisms behind this selective sensitivity of retinal PC to hyperglycemia are undefined. The O-linked β-N-acetylglucosamine (O-GlcNAc) modification is elevated under hyperglycemic condition, and thus, may present an important molecular modification impacting the hyperglycemia-driven complications of diabetes. We have recently demonstrated that the level of O-GlcNAc modification in response to high glucose is variable in various retinal vascular cells. Retinal PC responded with the highest increase in O-GlcNAc modification compared to retinal endothelial cells and astrocytes. Here we show that these differences translated into functional changes, with an increase in apoptosis of retinal PC, not just under high glucose but also under treatment with O-GlcNAc modification inducers, PUGNAc and Thiamet-G. To gain insight into the molecular mechanisms involved, we have used click-It chemistry and LC-MS analysis and identified 431 target proteins of O-GlcNAc modification in retinal PC using an alkynyl-modified GlcNAc analog (GlcNAlk). Among the O-GlcNAc target proteins identified here 115 of them were not previously reported to be target of O-GlcNAc modification. We have identified at least 34 of these proteins with important roles in various aspects of cell death processes. Our results indicated that increased O-GlcNAc modification of p53 was associated with an increase in its protein levels in retinal PC. Together our results suggest that post-translational O-GlcNAc modification of p53 and its increased levels may contribute to selective early loss of PC during diabetes. Thus, modulation of O-GlcNAc modification may provide a novel treatment strategy to prevent the initiation and progression of DR.
PMID: 24788674 [PubMed - indexed for MEDLINE]
Expression of thrombospondin-1 modulates the angioinflammatory phenotype of choroidal endothelial cells.
PLoS One. 2014;9(12):e116423
Authors: Fei P, Zaitoun I, Farnoodian M, Fisk DL, Wang S, Sorenson CM, Sheibani N
The choroidal circulation plays a central role in maintaining the health of outer retina and photoreceptor function. Alterations in this circulation contribute to pathogenesis of many eye diseases including exudative age-related macular degeneration. Unfortunately, very little is known about the choroidal circulation and its molecular and cellular regulation. This has been further hampered by the lack of methods for routine culturing of choroidal endothelial cells (ChEC), especially from wild type and transgenic mice. Here we describe a method for isolation and culturing of mouse ChEC. We show that expression of thrombospondin-1 (TSP1), an endogenous inhibitor of angiogenesis and inflammation, has a significant impact on phenotype of ChEC. ChEC from TSP1-deficient (TSP1-/-) mice were less proliferative and more apoptotic, less migratory and less adherent, and failed to undergo capillary morphogenesis in Matrigel. However, re-expression of TSP1 was sufficient to restore TSP1-/- ChEC migration and capillary morphogenesis. TSP1-/- ChEC expressed increased levels of TSP2, phosphorylated endothelial nitric oxide synthase (NOS) and inducible NOS (iNOS), a marker of inflammation, which was associated with significantly higher level of NO and oxidative stress in these cells. Wild type and TSP1-/- ChEC produced similar levels of VEGF, although TSP1-/- ChEC exhibited increased levels of VEGF-R1 and pSTAT3. Other signaling pathways including Src, Akt, and MAPKs were not dramatically affected by the lack of TSP1. Together our results demonstrate an important autocrine role for TSP1 in regulation of ChEC phenotype.
PMID: 25548916 [PubMed - in process]
A novel method to evaluate the neurocompatibility of dental implants.
Int J Oral Maxillofac Implants. 2014 Jan-Feb;29(1):41-50
Authors: Saghiri MA, Ghasemi M, Moayer AR, Sheibani N, Garcia-Godoy F, Asatourian A, Aslroosta H
PURPOSE: To evaluate the neurocompatibility of different types of dental implant surface treatments using the P19 neural cell line.
MATERIALS AND METHODS: P19 cells were plated and supplemented with retinoic acid to grow as aggregates for 4 days. Twenty dental implants were selected from four different implant systems with five different surface treatments. The implants were divided into four groups (n=5), placed inside medical rings, and fixed by injection of warm gutta-percha using a thermoplastic injection technique. Implant molds were placed inside graded culture dishes, and culture medium containing P19 neural cells were plated on the dishes for 4 days. After 24 hours, the surfaces of the implant molds were covered with self-curing resin to make a replica of each mold surface. Replicas were assessed under a scanning electron microscope, and the number of cells and the total cells covering the areas were evaluated. Data were analyzed by a post hoc Tukey test.
RESULTS: There were significant differences in P19 cell counts between all modified and electropolished surfaces. The highest P19 cell counts were shown on OsseoSpeed and Laser-Lok surfaces. The lowest counts were shown on the Nanotite surface at the collar. OsseoSpeed and Laser-Lok surfaces showed higher counts at the collar than on the body; the opposite was seen for SLActive and Nanotite surfaces. Cell-covered areas on Laser-Lok surfaces showed significantly higher values than the resorbable blasted media (RBM)-treated surfaces, while it was not significantly different from the OsseoSpeed surface of collar regions. SLActive collar regions showed larger cell-covered areas than the SLActive body surfaces, but this was not significant.
CONCLUSIONS: All test surface treatments in this study showed better neurocompatibility than control group surfaces. The Laser-Lok, RBM, and OsseoSpeed surfaces were superior to the Nanotite and SLActive surfaces in terms of neurocompatibility.
PMID: 24451852 [PubMed - indexed for MEDLINE]
The sustained delivery of resveratrol or a defined grape powder inhibits new blood vessel formation in a mouse model of choroidal neovascularization.
Authors: Kanavi MR, Darjatmoko S, Wang S, Azari AA, Farnoodian M, Kenealey JD, van Ginkel PR, Albert DM, Sheibani N, Polans AS
The objective of this study was to determine whether resveratrol or a defined, reconstituted grape powder can attenuate the formation of new blood vessels in a mouse model of choroidal neovascularization (CNV). To accomplish this objective, C57BL/6J mice were randomized into control or treatment groups which received either resveratrol or grape powder by daily oral gavage, resveratrol or grape powder delivered ad libitum through the drinking water, or resveratrol by slow release via implanted osmotic pumps. A laser was used to rupture Bruch's membrane to induce CNV which was then detected in sclerochoroidal eyecups stained with antibodies against intercellular adhesion molecule-2. CNV area was measured using fluorescence microscopy and Image J software. Ad libitum delivery of both resveratrol and grape powder was shown to significantly reduce the extent of CNV by 68% and 57%, respectively. Parallel experiments conducted in vitro demonstrated that resveratrol activates p53 and inactivates Akt/protein kinase B in choroidal endothelial cells, contributing to its anti-proliferative and anti-migratory properties. In addition resveratrol was shown to inhibit the formation of endothelial cell networks, augmenting its overall anti-angiogenic effects. The non-toxic nature of resveratrol makes it an especially attractive candidate for the prevention and/or treatment of CNV.
PMID: 25361423 [PubMed - in process]
A combined method to quantify the retinal metabolic rate of oxygen using photoacoustic ophthalmoscopy and optical coherence tomography.
Sci Rep. 2014;4:6525
Authors: Song W, Wei Q, Liu W, Liu T, Yi J, Sheibani N, Fawzi AA, Linsenmeier RA, Jiao S, Zhang HF
Quantitatively determining physiological parameters at a microscopic level in the retina furthers the understanding of the molecular pathways of blinding diseases, such as diabetic retinopathy and glaucoma. An essential parameter, which has yet to be quantified noninvasively, is the retinal oxygen metabolic rate (rMRO2). Quantifying rMRO2 is challenging because two parameters, the blood flow rate and hemoglobin oxygen saturation (sO2), must be measured together. We combined photoacoustic ophthalmoscopy (PAOM) with spectral domain-optical coherence tomography (SD-OCT) to tackle this challenge, in which PAOM measured the sO2 and SD-OCT mapped the blood flow rate. We tested the integrated system on normal wild-type rats, in which the measured rMRO2 was 297.86 ± 70.23 nl/minute. This quantitative method may shed new light on both fundamental research and clinical care in ophthalmology in the future.
PMID: 25283870 [PubMed - in process]
Bone morphogenetic protein 7 regulates reactive gliosis in retinal astrocytes and Müller glia.
Mol Vis. 2014;20:1085-108
Authors: Dharmarajan S, Gurel Z, Wang S, Sorenson CM, Sheibani N, Belecky-Adams TL
PURPOSE: The focus of this study was to determine whether bone morphogenetic proteins (BMPs) trigger reactive gliosis in retinal astrocytes and/or Müller glial cells.
METHODS: Retinal astrocytes and the Müller glial cell line MIO-M1 were treated with vehicle, BMP7, or BMP4. Samples from the treated cells were analyzed for changes in gliosis markers using reverse transcriptase - quantitative PCR (RT-qPCR) and western blotting. To determine potential similarities and differences in gliosis states, control and BMP-treated cells were compared to cells treated with sodium peroxynitrite (a strong oxidizing agent that will bring about some aspects of gliosis). Last, mature mice were microinjected intravitreally with BMP7 and analyzed for changes in gliosis markers using RT-qPCR, western blotting, and immunohistochemistry.
RESULTS: Treatment of retinal astrocyte cells and Müller glial cells with BMP7 regulated various reactive gliosis markers. When compared to the response of cells treated with sodium peroxynitrite, the profiles of gliosis markers regulated due to exposure to BMP7 were similar. However, as expected, the profiles including the oxidative agent and growth factor were not identical. Treatment of cells with BMP4, however, showed an attenuated response in comparison to peroxynitrite and BMP7 treatment. Injection of BMP7 into the mouse retina also triggered a reactive gliosis response 7 days after injection.
CONCLUSIONS: BMP7 induced changes in levels of mRNA and protein markers typically associated with reactive gliosis in retinal astrocytes and Müller glial cells, including glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), a subset of chondroitin sulfate proteoglycans (CSPGs), matrix metalloproteinases (MMPs), and other molecules.
PMID: 25253985 [PubMed - in process]
The effect of electrical treatment on cyclic fatigue of NiTi instruments.
Scanning. 2014 Sep-Oct;36(5):507-11
Authors: Saghiri MA, Asatourian A, Garcia-Godoy F, Gutmann JL, Lotfi M, Sheibani N
Dentists desire to use NiTi rotary instruments, which do not break inside the root canals of teeth, since the pieces from broken files are difficult to remove. The NiTi rotary instrument breakage is because of cyclic and torsional fatigue. Here the low-voltage (12 V) and high voltage (24 V) electrical treatments were used to enhance the cyclic fatigue of NiTi rotary instruments and increase their durability. In excremental groups, following electrical treatment samples of the NiTi instruments were rotated inside artificial root canals until they broke. Our results showed that electrical treatment with 12-V DC was effective in restoring NiTi instrument's resistance to cyclic fatigue. The scanning electron microscopy images and fractograph of samples exposed to 12-V electrical treatment showed a more regular texture over the surface with less dimpling on fractured site. These patterns can improve the super elasticity of tested devices during rotational movement, and delay the NiTi instruments separation in root canal preparations.
PMID: 24798116 [PubMed - indexed for MEDLINE]
siRNA-mediated knock-down of DFF45 amplifies doxorubicin therapeutic effects in breast cancer cells.
Cell Oncol (Dordr). 2013 Dec;36(6):515-26
Authors: Bagheri F, Safarian S, Eslaminejad MB, Sheibani N
PURPOSE: RNA interference (RNAi) has become a promising tool for cancer therapy. Small interfering RNAs (siRNAs) can synergistically enhance the cell killing effects of drugs used in cancer treatment. Here we examined the effects of siRNA-mediated DNA fragmentation factor 45 (DFF45) gene silencing on breast cancer cell viability, cell cycle arrest, and apoptosis in the presence and absence of doxorubicin.
METHODS: We designed three siRNAs, which target different regions of the DFF45 mRNA. Gene silencing was confirmed by real time RT-PCR and Western blot analyses. The impact of DFF45 siRNA, doxorubicin, and their combination on the viability, cell cycle and apoptosis of T-47D and MDA-MB-231 breast cancer cells were determined by MTT, PI staining, annexin V binding, caspase-3 activity, DNA laddering, and chromatin condensation assays.
RESULTS: Based on flow cytometric analyses, we found that silencing of DFF45 alone had little effect on apoptosis, especially in T-47D cells. However, when used in combination with doxorubicin (0.33 μM) a significant increase (P < 0.05) in apoptosis was observed in T-47D and MDA-MB-231 cells, i.e., ~2.5- and 3-fold, respectively. Caspase-3 activity, chromatin condensation, as well as DNA laddering supported increased apoptosis in the combinatorial treatment. Cell cycle arrest in both cell lines occurred at lower levels after siRNA + doxorubicin treatment compared to doxorubicin only.
CONCLUSIONS: Our data indicate that DFF45 gene silencing, when applied in combination with doxorubicin, may offer a novel therapeutic strategy for the treatment of breast cancer.
PMID: 24277473 [PubMed - indexed for MEDLINE]
Cyp1B1 expression promotes angiogenesis by suppressing NF-κB activity.
Am J Physiol Cell Physiol. 2013 Dec 1;305(11):C1170-84
Authors: Palenski TL, Gurel Z, Sorenson CM, Hankenson KD, Sheibani N
Nuclear factor-κB (NF-κB) is a master regulator of genes that control a large number of cellular processes, including angiogenesis and inflammation. We recently demonstrated that cytochrome P-450 1B1 (Cyp1B1) deficiency in endothelial cells (EC) and pericytes (PC) results in increased oxidative stress, alterations in migration, attenuation of capillary morphogenesis, sustained activation of NF-κB, and increased expression of thrombospondin-2 (TSP2), an endogenous inhibitor of angiogenesis. On the basis of a growing body of evidence that phenethyl isothiocyanate (PEITC) and pyrrolidine dithiocarbamate (PDTC) function as antioxidants and suppressors of NF-κB activation, we investigated their potential ability to restore a normal phenotype in Cyp1B1-deficient (cyp1b1(-/-)) vascular cells. PEITC and PDTC inhibited NF-κB activity and expression in cyp1b1(-/-) EC and PC. We also observed restoration of migration and capillary morphogenesis of cyp1b1(-/-) EC and decreased cellular oxidative stress in cyp1b1(-/-) EC and PC without restoration to normal TSP2 levels. In addition, expression of a dominant-negative inhibitor κBα, a suppressor of NF-κB activation, decreased NF-κB activity without affecting TSP2 expression in these cells. In contrast, knockdown of TSP2 expression resulted in attenuation of NF-κB activity in cyp1b1(-/-) vascular cells. Furthermore, expression of TSP2 in wild-type (cyp1b1(+/+)) cells resulted in increased NF-κB activity. Together, our results demonstrate an important role for TSP2 in modulation of NF-κB activity and attenuation of angiogenesis. Thus Cyp1B1 expression in vascular cells plays an important role in the regulation of vascular homeostasis through modulation of the cellular reductive state, TSP2 expression, and NF-κB activation.
PMID: 24088896 [PubMed - indexed for MEDLINE]
Cyp1b1 mediates periostin regulation of trabecular meshwork development by suppression of oxidative stress.
Mol Cell Biol. 2013 Nov;33(21):4225-40
Authors: Zhao Y, Wang S, Sorenson CM, Teixeira L, Dubielzig RR, Peters DM, Conway SJ, Jefcoate CR, Sheibani N
Mutation in CYP1B1 has been reported for patients with congenital glaucoma. However, the underlying mechanisms remain unknown. Here we show increased diurnal intraocular pressure (IOP) in Cyp1b1-deficient (Cyp1b1(-/-)) mice. Cyp1b1(-/-) mice presented ultrastructural irregular collagen distribution in their trabecular meshwork (TM) tissue along with increased oxidative stress and decreased levels of periostin (Postn). Increased levels of oxidative stress and decreased levels of Postn were also detected in human glaucomatous TM tissues. Furthermore, Postn-deficient mice exhibited TM tissue ultrastructural abnormalities similar to those of Cyp1b1(-/-) mice. Administration of the antioxidant N-acetylcysteine (NAC) restored structural abnormality of TM tissue in Cyp1b1(-/-) mice. In addition, TM cells prepared from Cyp1b1(-/-) mice exhibited increased oxidative stress, altered adhesion, and decreased levels of Postn. These aberrant cellular responses were reversed in the presence of NAC or by restoration of Cyp1b1 expression. Cyp1b1 knockdown or inhibition of CYP1B1 activity in Cyp1b1(+/+) TM cells resulted in a Cyp1b1(-/-) phenotype. Thus, metabolic activity of CYP1B1 contributes to oxidative homeostasis and ultrastructural organization and function of TM tissue through modulation of Postn expression.
PMID: 23979599 [PubMed - indexed for MEDLINE]
Correlation between endodontic broken instrument and nickel level in urine.
Biol Trace Elem Res. 2013 Oct;155(1):114-8
Authors: Saghiri MA, Sheibani N, Garcia-Godoy F, Asatourian A, Mehriar P, Scarbecz M
This study aims to evaluate the correlation between the presences of separated endodontic instrument inside the dental canal and the nickel (Ni) level in the urine samples of subjected patients. Same-gendered and near-aged participants were selected and were instructed to collect their urine in sterile nickel-free plastic containers. The procedures were carried out in the office, and samples were stored in low-temperature cooler for 1 day and then they were transferred to the laboratory for electrothermal atomic absorption spectrometry. The level of Ni was measured and the correlation coefficient was calculated. Data were analyzed using t tests, Pearson's correlation coefficients, and linear regression analysis, at a level of significance P < 0.05. The statistical analysis has showed significant difference in Ni level between endodontic and control groups (P < 0.05). There was no correlation between Ni level in urine and the age or time period of broken instrument inside the canal; however, Ni level of urine and the age of participants in experimental group has demonstrated a positive correlation. The amount of Ni element can be increased in the urine of patients who have experienced broken endodontic instrument inside the dental canal. However, there is no positive correlation between the remaining pieces of instruments inside the canal and the elevation of nickel amount in urine during the tested time period. This issue suggested that the aging of remaining broken instrument inside the canal does not show any remarkable concern regarding the Ni elevation in the urine excreted by an individual.
PMID: 23861099 [PubMed - indexed for MEDLINE]
Effects of 940 Hz EMF on luciferase solution: structure, function, and dielectric studies.
Bioelectromagnetics. 2013 Sep;34(6):489-98
Authors: Sefidbakht Y, Hosseinkhani S, Mortazavi M, Tavakkolnia I, Khellat MR, Shakiba-Herfeh M, Saviz M, Faraji-Dana R, Saboury AA, Sheibani N, Moosavi-Movahedi AA
We designed a rectangular waveguide exposure system to study the effects of mobile phone frequency (940 MHz) electromagnetic fields (EMF) on luciferase structure and activity. The luciferase activity of exposed samples was significantly higher than that of unexposed samples. Dynamic light scattering of the exposed samples showed smaller hydrodynamic radii compared to unexposed samples (20 nm vs. 47 nm ± 5%). The exposed samples also showed less tendency to form aggregates, monitored by turbidity measurements at l = 360 nm. A microwave dielectric measurement was performed to study the hydration properties of luciferase solutions with a precision network analyzer over frequency ranges from 0.2 to 20 GHz before and after exposure. The change in the dielectric properties of the exposed luciferase solution was related to the disaggregation potency of the applied field. Together, our results suggested that direct interactions with luciferase molecules and its dipole moment were responsible for the reduced aggregation and enhanced luciferase activity upon exposure to the EMF.
PMID: 23633149 [PubMed - indexed for MEDLINE]
Inflammatory cytokine-specific alterations in retinal endothelial cell function.
Microvasc Res. 2013 Sep;89:57-69
Authors: Palenski TL, Sorenson CM, Sheibani N
Diabetic retinopathy (DR) is recognized as a chronic low-grade inflammatory disease. Retinal microvascular cell dysfunction and loss play an important role in the pathogenesis of DR. However, the basic mechanisms underlying the development and progression of DR are poorly understood. Many recent studies indicate that increased production of inflammatory factors either systemically and/or locally, is strongly associated with vascular dysfunction during diabetes. Here we sought to determine the specific impact of different inflammatory mediators on retinal endothelial cell (EC) function. Inflammatory mediators TNF-α and IL-1β attenuated the migration and capillary morphogenesis of retinal EC. These dysfunctions were associated with an increased production of reactive oxygen species, expression of inducible nitric oxide synthase, and production of total nitrate/nitrite. Incubation of retinal EC with TNF-α and IL-1β altered VE-cadherin localization, as well as the expression of other junctional proteins. In addition, TNF-α and IL-1β also altered the production of various ECM proteins including osteopontin, collagen IV, and tenascin-C. Mechanistically, these changes were concomitant with the activation of the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. In contrast, incubation of retinal EC with MCP-1 minimally affected their migratory, junctional, and ECM properties. Together our results indicate that the presence of inflammatory mediators in diabetes may have specific and significant impact on vascular cell function, and contribute to the pathogenesis of DR.
PMID: 23806781 [PubMed - indexed for MEDLINE]
Screening assay for blood vessel maturation inhibitors.
Biochem Biophys Res Commun. 2013 Aug 23;438(2):364-9
Authors: Fu C, van der Zwan A, Gerber S, Van Den Berg S, No E, Wang WC, Sheibani N, Carducci MA, Kachhap S, Hammers HJ
In cancer patients, the development of resistance to anti-angiogenic agents targeting the VEGF pathway is common. Increased pericyte coverage of the tumor vasculature undergoing VEGF targeted therapy has been suggested to play an important role in resistance. Therefore, reducing the pericytes coverage of the tumor vasculature has been suggested to be a therapeutic approach in breaking the resistance to and increasing the efficacy of anti-angiogenic therapies. To screen compound libraries, a simple in vitro assay of blood vessel maturation demonstrating endothelial cells and pericytes association while forming lumenized vascular structures is needed. Unfortunately, previously described 3-dimensional, matrix based assays are laborious and challenging from an image and data acquisition perspective. For these reasons they generally lack the scalability needed to perform in a high-throughput environment. With this work, we have developed a novel in vitro blood vessel maturation assay, in which lumenized, vascular structures form in one optical plane and mesenchymal progenitor cells (10T1/2) differentiate into pericyte-like cells, which associate with the endothelial vessels (HUVECs). The differentiation of the 10T1/2 cells into pericyte-like cells is visualized using a GFP reporter controlled by the alpha smooth muscle actin promoter (SMP-8). The organization of these vascular structures and their recruited mural cells in one optical plane allows for automated data capture and subsequent image analysis. The ability of this assay to screen for inhibitors of pericytes recruitment was validated. In summary, this novel assay of in vitro blood vessel maturation provides a valuable tool to screen for new agents with therapeutic potential.
PMID: 23892038 [PubMed - indexed for MEDLINE]
The ubiquitin-proteasome system and microvascular complications of diabetes.
J Ophthalmic Vis Res. 2013 Jul;8(3):244-56
Authors: Aghdam SY, Sheibani N
The ubiquitin-proteasome system (UPS) is the mainstay of protein quality control which regulates cell cycle, differentiation and various signal transduction pathways in eukaryotic cells. The timely and selective degradation of surplus and/or aberrant proteins by the UPS is essential for normal cellular physiology. Any disturbance, delay or exaggeration in the process of selection, sequestration, labeling for degradation and degradation of target proteins by the UPS will compromise cellular and tissue homeostasis. High blood glucose or hyperglycemia caused by diabetes disrupts normal vascular function in several target organs including the retina and kidney resulting in the development of diabetic retinopathy (DR) and diabetic nephropathy (DN). We and others have shown that hyperglycemia and oxidative stress modulate UPS activity in the retina and kidney. The majority of studies have focused on the kidney and provided insights into the contribution of dysregulated UPS to microvascular damage in DN. The eye is a unique organ in which a semi-fluid medium, the vitreous humor, separates the neural retina and its anastomosed blood vessels from the semi-solid lens tissue. The complexity of the cellular and molecular components of the eye may require a normal functioning and well tuned UPS for healthy vision. Altered UPS activity may contribute to the development of retinal microvascular complications of diabetes. A better understanding of the molecular nature of the ocular UPS function under normal and diabetic conditions is essential for development of novel strategies targeting its activity. This review will discuss the association of retinal vascular cell UPS activity with microvascular damage in DR with emphasis on alterations of the PA28 subunits of the UPS.
PMID: 24349668 [PubMed]
Lack of Cyp1b1 promotes the proliferative and migratory phenotype of perivascular supporting cells.
Lab Invest. 2013 Jun;93(6):646-62
Authors: Palenski TL, Sorenson CM, Jefcoate CR, Sheibani N
Perivascular supporting cells, including pericytes and smooth muscle cells (PC/SMC), have an integral role during angiogenesis and control vascular remodeling, maturation, and stabilization of neoteric vessels. We recently showed that a Cyp1B1 deficiency in mice results in the attenuation of angiogenesis in vivo and the pro-angiogenic activity of endothelial cells in vitro. However, the contribution of PC/SMC, and more specifically the cell autonomous effects of Cyp1B1 in these processes, needs further investigation. Here we demonstrate that PC constitutively expressed Cyp1B1, and that a deficiency in Cyp1B1 was associated with enhanced proliferation, and decreased apoptosis. Mechanistically, the lack of Cyp1B1 was associated with increased oxidative stress and sustained NF-κB activation, which was reversed by the antioxidant N-acetylcysteine. These changes were also concomitant with alterations in PC migration, adhesion, and expression of various extracellular matrix proteins, including thrombospondin-2. Cyp1B1-deficient PC also expressed decreased levels of vascular endothelial growth factor. Together, our results suggest an important role for Cyp1B1 expression in the regulation of PC proliferation, migration, and survival through modulation of the intracellular oxidative state and NF-κB expression and/or activity. Thus, a lack of Cyp1B1 in PC may have a significant role in vascular dysfunction and integrity, contributing to the attenuation of angiogenesis.
PMID: 23568032 [PubMed - indexed for MEDLINE]
Thrombospondin-1 Deficiency Exacerbates the Pathogenesis of Diabetic Retinopathy.
J Diabetes Metab. 2013 May 25;Suppl 12
Authors: Sorenson CM, Wang S, Gendron R, Paradis H, Sheibani N
Diabetic retinopathy is a leading cause of blindness in the United States. Access to new animal models that exhibit retinal vasculopathies with short duration of diabetes, are vital for understanding the underlying mechanisms and examining the efficacy of new treatment modalities. Our previous studies demonstrated decreased expression of Thrombospondin-1 (TSP1), an endogenous inhibitor of angiogenesis, in eyes from both patients and rodents with diabetes. Here we examined whether TSP1 deficiency could exacerbate diabetic retinal vasculopathies. Akita/+ male mice reproducibly develop diabetes by 4 weeks of age. These mice demonstrated the early non-proliferative stages of diabetic retinopathy, including decreased numbers of pericytes and increased glial cell activation. However, Akita/+ male mice deficient in TSP1 (Akita/+; TSP1-/-) demonstrated more advanced stages of diabetic retinopathy with a 4-fold increase in acellular capillaries and increased fibronectin and GFAP expression. These vascular changes were not attributable to aberrant retinal vascular development in the absence of TSP1, since down-regulation of TSP1 postnatally in the endothelium also resulted in more severe retinopathy. In addition, lack of another endogenous inhibitor of angiogenesis, pigment epithelium derived factor (PEDF), also enhanced diabetic retinopathy in Akita/+ mice. Akita/+; PEDF-/- male mice demonstrated increased numbers of acellular capillaries compared to controls but at a level lower than that observed in Akita/+; TSP1-/- mice. Thus, the exacerbation of diabetic retinopathy in Akita/+; TSP1-/- mice will allow the study of retinal vasculopathies with a shorter duration of diabetes and facilitate future testing of treatment modalities that protect the retinal vasculature and preserve sight.
PMID: 24224119 [PubMed - as supplied by publisher]
Death inducing and cytoprotective autophagy in T-47D cells by two common antibacterial drugs: sulphathiazole and sulphacetamide.
Cell Biol Int. 2013 Apr;37(4):348-58
Authors: Mohammadpour R, Safarian S, Sheibani N, Norouzi S, Razazan A
The broad spectrum of the pharmacological effects of sulphonamide family of drugs motivated us to investigate the cellular mechanisms for anti-cancer effects of sulphathiazole and sulphacetamide on T-47D breast cancer cells. Fluorescent microscopy, flow cytometric analysis, caspase-3 activity and DNA fragmentation assays were used to detect apoptosis. The distribution of the cells among different phases of the cell cycle was measured by flow cytometry. The expression of several genes with important roles in some critical cellular pathways including apoptosis, mTOR/AKT pathway and autophagy were determined by real-time RT-PCR analysis. Sulphathiazole and sulphacetamide induced anti-proliferative effects on T-47D cells were independent of apoptosis and cell cycle arrest. The overexpression of critical genes involved in autophagy including ATG5, p53 and DRAM indicated that the main effect of the drug-induced anti-proliferative effects was through induction of autophagy. This process was induced in two different forms, including death inducing and cytoprotective autophagy. Sulphathiazole treatment was followed by higher expression of p53/DRAM and downregulation of Akt/mTOR pathway resulting in death autophagy. In contrast, sulphacetamide treatment lowered expression of p53/DRAM pathway in parallel with upregulation of Akt/mTOR pathway promoting cytoprotective autophagy. The results indicated that autophagy is the main mechanism mediating the anti-cancer effects of sulphathiazole and sulphacetamide on T-47D cells. Alignment of the p53 and DRAM expression along with activation level of Akt survival pathway therefore determines the type of autophagy that occurs.
PMID: 23450781 [PubMed - in process]
Pharmacological inhibition of mitochondrial carbonic anhydrases protects mouse cerebral pericytes from high glucose-induced oxidative stress and apoptosis.
J Pharmacol Exp Ther. 2013 Mar;344(3):637-45
Authors: Shah GN, Price TO, Banks WA, Morofuji Y, Kovac A, Ercal N, Sorenson CM, Shin ES, Sheibani N
Diabetes-associated complications in the microvasculature of the brain are caused by oxidative stress, generated by overproduction of reactive oxygen species from hyperglycemia-induced accelerated oxidative metabolism of glucose. Pericytes, essential for the viability of the microvasculature, are especially susceptible to oxidative stress. Mitochondrial carbonic anhydrases, regulators of the oxidative metabolism of glucose, determine the rate of reactive oxygen species production and inhibition of mitochondrial carbonic anhydrases rescues glucose-induced pericyte loss in the diabetic mouse brain. We hypothesized that high glucose induces intracellular oxidative stress and pericyte apoptosis and that inhibition of mitochondrial carbonic anhydrases protects pericytes from oxidative stress-induced apoptosis. To validate our hypothesis, conditionally immortalized cerebral pericyte (IPC) cultures were established from Immortomice to investigate the effect of high glucose on oxidative stress and pericyte apoptosis. The IPCs expressed pericyte markers and induced high transendothelial electrical resistance and low permeability in brain endothelial cell monolayers comparable with pericytes in primary cultures. The IPCs also secreted cytokines constitutively and in response to lipopolysaccharide similar to pericytes. High glucose caused oxidative stress and apoptosis of these cells, with both oxidative stress and apoptosis significantly reduced after mitochondrial carbonic anhydrase inhibition. These results provide the first evidence that pharmacological inhibition of mitochondrial carbonic anhydrases attenuates pericyte apoptosis caused by high glucose-induced oxidative stress. Carbonic anhydrase inhibitors have a long history of safe clinical use and can be immediately evaluated for this new indication in translational research. Thus, mitochondrial carbonic anhydrases may provide a new therapeutic target for oxidative stress-related illnesses of the brain.
PMID: 23249625 [PubMed - indexed for MEDLINE]
Endoglin regulates the activation and quiescence of endothelium by participating in canonical and non-canonical TGF-β signaling pathways.
J Cell Sci. 2013 Mar 15;126(Pt 6):1392-405
Authors: Park S, Dimaio TA, Liu W, Wang S, Sorenson CM, Sheibani N
Endoglin (Eng) is an auxiliary receptor for transforming growth factor-β (TGFβ), with important roles in vascular function. TGFβ regulates angiogenesis through balancing the pro-proliferative and pro-differentiation signaling pathways of endothelial cells (EC). However, the contribution of endoglin to these TGFβ activities, and more specifically modulation of EC phenotype, remains elusive. Mutations in endoglin cause hereditary hemorrhagic telangiectasia-1 in humans. The Eng+/- mice are viable and exhibit some of the vascular defects seen in humans with endoglin haploinsufficiency. In the present study we show that haploinsufficiency of endoglin results in attenuation of retinal neovascularization during oxygen-induced ischemic retinopathy. Although the importance of endoglin expression in angiogenesis and vascular development has been demonstrated, the underlying mechanisms remain obscure. To gain detailed insight into the cell autonomous regulatory mechanisms that affect angiogenic properties of EC, we prepared retinal EC from Eng+/+ and Eng+/- Immorto mice. The Eng+/- EC were more adherent, less migratory, and failed to undergo capillary morphogenesis. Aortic sprouting angiogenesis was similarly attenuated in aortas from Eng+/- mice. In addition, Eng+/- EC expressed increased levels of VEGF but reduced expression of endothelial NO synthase and NO production. Mechanistically, these changes were consistent with sustained activation of mitogen-activated protein kinase (MAPK) pathways, and aberrant Smad-dependent signaling pathways in Eng+/- EC. Taken together, our results underscore the importance of endoglin in both canonical and non-canonical TGFβ signaling pathways modulating both the activation and quiescence of the endothelium during angiogenesis.
PMID: 23418351 [PubMed - indexed for MEDLINE]
High glucose and diabetes modulate cellular proteasome function: Implications in the pathogenesis of diabetes complications.
Biochem Biophys Res Commun. 2013 Mar 8;432(2):339-44
Authors: Aghdam SY, Gurel Z, Ghaffarieh A, Sorenson CM, Sheibani N
The precise link between hyperglycemia and its deleterious effects on retinal and kidney microvasculature, and more specifically loss of retinal perivascular supporting cells including smooth muscle cell/pericytes (SMC/PC), in diabetes are not completely understood. We hypothesized that differential cellular proteasome activity contributes to sensitivity of PC to high glucose-mediated oxidative stress and vascular rarefaction. Here we show that retinal endothelial cells (EC) have significantly higher proteasome peptidase activity compared to PC. High glucose treatment (HGT) increased the level of total ubiquitin-conjugated proteins in cultured retinal PC and EC, but not photoreceptor cells. In addition, in vitro proteasome activity assays showed significant impairment of proteasome chymotrypsin-like peptidase activity in PC, but not EC. The PA28-α/-β and PA28-β/-γ protein levels were also higher in the retina and kidney glomeruli of diabetic mice, respectively. Our results demonstrate, for the first time, that high glucose has direct biological effects on cellular proteasome function, and this modulation might be protective against cellular stress or damage induced by high glucose.
PMID: 23391566 [PubMed - indexed for MEDLINE]
Purification and autolysis of the ficin isoforms from fig (Ficus carica cv. Sabz) latex.
Phytochemistry. 2013 Mar;87:16-22
Authors: Zare H, Moosavi-Movahedi AA, Salami M, Mirzaei M, Saboury AA, Sheibani N
Ficin (EC 18.104.22.168), a cysteine endoproteolytic protease in fig trees' latex, has multiple isoforms. Until now, no data on autolysis of individual ficins (ficin isoforms) are available. Following purification, ficins' autolysis was determined by HPLC chromatogram changes and ultrafiltrations at different temperatures and storage times. These results showed that the number of HPLC peaks in latex proteins purification of Ficus carica cv. Sabz varied from previous fig varieties or cultivars. Proteolytic activity of ficins was inhibited by specific cysteine protease inhibitors, confirming the participation of the cysteine residue in the active site. The zeta potential of the first two eluted peaks (I and II) was negative, while that of other peaks were positive. All ficins were susceptible to autolysis when stored at high temperatures. In contrast, only the last two ficins (B, C) were prone to autolysis at cold temperature after long storage period. The rate of degradation of the ficins was significantly increased with the increased storage time. The ficin (A) related to peak (III) had the highest and the lowest surface hydrophobic patches and ratio of autolytic to proteolytic activity, respectively.
PMID: 23312458 [PubMed - indexed for MEDLINE]
Attenuation of choroidal neovascularization by β(2)-adrenoreceptor antagonism.
JAMA Ophthalmol. 2013 Mar;131(3):376-82
Authors: Lavine JA, Sang Y, Wang S, Ip MS, Sheibani N
OBJECTIVES: To determine whether β-adrenergic blockade inhibits choroidal neovascularization (CNV) in a mouse model of laser-induced CNV and to investigate the mechanism by which β-adrenoreceptor antagonism blunts CNV.
DESIGN: Mice were subjected to laser burns, inducing CNV, and were treated with daily intraperitoneal injections of propranolol hydrochloride. Neovascularization was measured on choroidal-scleral flat mounts using intercellular adhesion molecule 2 immunofluorescence staining. The effect of β-adrenoreceptor signaling on expression of vascular endothelial growth factor (VEGF) was investigated using primary mouse choroidal endothelial cells (ChECs) and retinal pigment epithelial (RPE) cells. These cells were incubated with β-adrenoreceptor agonists and/or antagonists and assayed for Vegf messenger RNA and protein levels.
SETTING: University of Wisconsin School of Medicine and Public Health.
PARTICIPANTS: Wild-type 6-week-old female C57BL/6j mice.
MAIN OUTCOME MEASURES: Inhibition of CNV after propranolol treatment and Vegf messenger RNA and protein expression after treatment with β-adrenoreceptor agonists and antagonists.
RESULTS: Propranolol-treated mice demonstrated a 50% reduction in laser-induced CNV. Treatment with norepinephrine bitartrate stimulated Vegf messenger RNA expression and protein secretion in ChECs and RPE cells. This effect was blocked by β2-adrenoreceptor antagonism and mimicked by β2-adrenoreceptor agonists.
CONCLUSIONS: Attenuation of CNV is achieved by β-adrenergic blockade. The β2-adrenoreceptors regulate VEGF expression in ChECs and RPE cells.
CLINICAL RELEVANCE: Antagonists of β-adrenoreceptors are safe and well tolerated in patients with glaucoma and cardiovascular disease. Thus, blockade of β-adrenoreceptors may provide a new avenue to inhibit VEGF expression in CNV.
PMID: 23303344 [PubMed - indexed for MEDLINE]
Denaturation and intermediates study of two sturgeon hemoglobins by n-dodecyl trimethylammonium bromide.
Int J Biol Macromol. 2013 Feb;53:107-13
Authors: Ariaeenejad S, Habibi-Rezaei M, Kavousi K, Jamili S, Fatemi MR, Hong J, Poursasan N, Sheibani N, Moosavi-Movahedi AA
Varieties of hemoglobin (Hb) forms exist in fish, which are usually well adapted to the different ecological conditions or various habitats. In the current study, Hbs from two Sturgeon species of the Southern Caspian Sea Basin were purified and studied upon interaction with n-dodecyl trimethylammonium bromide (DTAB; as a cationic surfactant) by various methods including UV-visible absorption, dynamic light scattering (DLS), and ANS fluorescence spectrophotometry. The chemometric analysis of Hbs was investigated upon interaction with DTAB under titration, using UV-visible absorption spectra. The chemometric resolution techniques were used to determine the number of the components and mole fraction of the oxidized Hbs. These results provided the evidence for the existence of three different molecular components including native (N), intermediate (I) and denatured (D) in sturgeon Hbs. According to the distribution of intermediates, which were broadened in a range of DTAB concentration, the aggregation states, DLS experiments, and thermal stability (T(m) obtained by differential scanning calorimetry (DSC)), the Acipenser stellatus Hb was more stable compared to Acipenser persicus Hb. These results demonstrate a significant relationship between the stability of fish Hbs and the habitat depth requirements.
PMID: 23142155 [PubMed - indexed for MEDLINE]
Aberrant production of extracellular matrix proteins and dysfunction in kidney endothelial cells with a short duration of diabetes.
Am J Physiol Renal Physiol. 2013 Jan 1;304(1):F19-30
Authors: Grutzmacher C, Park S, Zhao Y, Morrison ME, Sheibani N, Sorenson CM
Diabetic nephropathy is the most common cause of end-stage renal disease and is a major risk factor for cardiovascular disease. In the United States, microvascular complications during diabetic nephropathy contribute to high morbidity and mortality rates. However, the cell-autonomous impact of diabetes on kidney endothelial cell function requires further investigation. Male Akita/+ [autosomal dominant mutation in the insulin II gene (Ins2)] mice reproducibly develop diabetes by 4 wk of age. Here, we examined the impact a short duration of diabetes had on kidney endothelial cell function. Kidney endothelial cells were prepared from nondiabetic and diabetic mice (4 wk of diabetes) to delineate the early changes in endothelial cell function. Kidney endothelial cells from Akita/+ mice following 4 wk of diabetes demonstrated aberrant expression of extracellular matrix proteins including decreased osteopontin and increased fibronectin expression which correlated with increased α5-integrin expression. These changes were associated with the attenuation of migration and capillary morphogenesis. Kidney endothelial cells from Akita/+ mice had decreased VEGF levels but increased levels of endothelial nitric oxide synthase(eNOS) and NO, suggesting uncoupling of VEGF-mediated NO production. Knocking down eNOS expression in Akita/+ kidney endothelial cells increased VEGF expression, endothelial cell migration, and capillary morphogenesis. Furthermore, attenuation of sprouting angiogenesis of aortas from Akita/+ mice with 8 wk of diabetes was restored in the presence of the antioxidant N-acetylcysteine. These studies demonstrate that aberrant endothelial cell function with a short duration of diabetes may set the stage for vascular dysfunction and rarefaction at later stages of diabetes.
PMID: 23077100 [PubMed - indexed for MEDLINE]
Modulation of vascular cell function by bim expression.
Int J Cell Biol. 2013;2013:297537
Authors: Morrison ME, Palenski TL, Jamali N, Sheibani N, Sorenson CM
Apoptosis of vascular cells, including pericytes and endothelial cells, contributes to disease pathogenesis in which vascular rarefaction plays a central role. Bim is a proapoptotic protein that modulates not only apoptosis but also cellular functions such as migration and extracellular matrix (ECM) protein expression. Endothelial cells and pericytes each make a unique contribution to vascular formation and function although the details require further delineation. Here we set out to determine the cell autonomous impact of Bim expression on retinal endothelial cell and pericyte function using cells prepared from Bim deficient (Bim(-/-)) mice. Bim(-/-) endothelial cells displayed an increased production of ECM proteins, proliferation, migration, adhesion, and VEGF expression but, a decreased eNOS expression and nitric oxide production. In contrast, pericyte proliferation decreased in the absence of Bim while migration, adhesion, and VEGF expression were increased. In addition, we demonstrated that the coculturing of either wild-type or Bim(-/-) endothelial cells with Bim(-/-) pericytes diminished their capillary morphogenesis. Thus, our data further emphasizes the importance of vascular cell autonomous regulatory mechanisms in modulation of vascular function.
PMID: 24288535 [PubMed]
Modified β-casein restores thermal reversibility of human carbonic anhydrase II: the salt bridge mechanism.
Biotechnol Appl Biochem. 2013 May-Jun;60(3):298-304
Authors: Fallah-Bagheri A, Moosavi-Movahedi AA, Taghizadeh M, Khodarahmi R, Ma'mani L, Bijari N, Bohlooli M, Shafiee A, Sheibani N, Saboury AA
Modified β-casein (mβ-CN) was investigated as an efficient additive for thermal reversibility of human carbonic anhydrase II (HCA II) at pH 7.75. The mβ-CN was obtained via modification of β-casein (β-CN) acidic residues using Woodward's reagent K. The effects of mβ-CN on the reversibility and stability of HCA II were determined by differential scanning calorimetry, UV-vis, and 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopic methods. The mβ-CN, as an additive, enhanced thermal reversibility of HCA II by 33%. Together, our results indicated that mβ-CN is very efficient in decreasing thermal aggregation and enhancing the stability of HCA II. Using theoretical studies, we propose that the mechanism for thermal reversibility is mediated through formation of a salt bridge between the Woodward part of mβ-CN and the Zn ion of HCA II.
PMID: 23621563 [PubMed - indexed for MEDLINE]
Deletion of aldose reductase from mice inhibits diabetes-induced retinal capillary degeneration and superoxide generation.
PLoS One. 2013;8(4):e62081
Authors: Tang J, Du Y, Petrash JM, Sheibani N, Kern TS
PURPOSE: Pharmacologic inhibition of aldose reductase (AR) previously has been studied with respect to diabetic retinopathy with mixed results. Since drugs can have off-target effects, we studied the effects of AR deletion on the development and molecular abnormalities that contribute to diabetic retinopathy. Since recent data suggests an important role for leukocytes in the development of the retinopathy, we determined also if AR in leukocytes contributes to leukocyte-mediated death of retinal endothelial cells in diabetes.
METHODS: Wild-type (WT; C57BL/6J) and AR deficient (AR(-/-)) mice were made diabetic with streptozotocin. Mice were sacrificed at 2 and 10 months of diabetes to evaluate retinal vascular histopathology, to quantify retinal superoxide production and biochemical and physiological abnormalities in the retina, and to assess the number of retinal endothelial cells killed by blood leukocytes in a co-culture system.
RESULTS: Diabetes in WT mice developed the expected degeneration of retinal capillaries, and increased generation of superoxide by the retina. Leukocytes from diabetic WT mice also killed more retinal endothelial cells than did leukocytes from nondiabetic animals (p<0.0001). Deletion of AR largely (P<0.05) inhibited the diabetes-induced degeneration of retinal capillaries, as well as the increase in superoxide production by retina. AR-deficiency significantly inhibited the diabetes-induced increase in expression of inducible nitric oxide synthase (iNOS) in retina, but had no significant effect on expression of intercellular adhesion molecule-1 (ICAM-1), phosphorylated p38 MAPK, or killing of retinal endothelial cells by leukocytes.
CONCLUSIONS: AR contributes to the degeneration of retinal capillaries in diabetic mice. Deletion of the enzyme inhibits the diabetes-induced increase in expression of iNOS and of superoxide production, but does not correct a variety of other pro-inflammatory abnormalities associated with the development of diabetic retinopathy.
PMID: 23614016 [PubMed - indexed for MEDLINE]
Removal of white mineral trioxide aggregate cement: a promising approach.
Biomed Res Int. 2013;2013:469164
Authors: Saghiri MA, Garcia-Godoy F, Gutmann JL, Sheibani N, Asatourian A, Lotfi M, Elyasi M
Removal of MTA from dentin by applying 37% hydrochloric acid (HCl) to reduce microhardness and push-out bond strength. Forty dentin slices were filled with WMTA and divided into two groups (n = 20). Ten slices remained untreated while others were exposed to either HCl or phosphate buffer saline (PBS) and all samples were subjected to pushout test. The mode of bond failures was determined by SEM analysis. Later, twenty glass tubes were filled with WMTA and divided into two groups (n = 10). One side of tube was exposed to HCl or PBS while the other side remained untreated and the microhardness was analyzed by testing machine. HCl showed significantly lower pushout strength and microhardness values (P = 0.0001), (P = 0.0001). HCl treated samples showed mixed bond failures dominantly, while PBS samples mostly showed adhesive failures. The results of this study can suggest the 37% HCl as an effective solution to aid the removal of MTA from the dentin surfaces.
PMID: 24089680 [PubMed - indexed for MEDLINE]
The impact of thermocycling process on the dislodgement force of different endodontic cements.
Biomed Res Int. 2013;2013:317185
Authors: Saghiri MA, Asatourian A, Garcia-Godoy F, Gutmann JL, Sheibani N
To evaluate the effects of thermocycling (500 cycles, 5°C/55°C) on the push-out bond strength of calcium silicate based cements including WMTA, Nano-WMTA, and Bioaggregate to root dentin. Forty-eight dentin slices were prepared and divided into 3 groups (n = 16) and filled with Angelus WMTA, Nano-WMTA, or Bioaggregate. After incubation, half of the samples were thermocycled while the other half remained untreated. Push-out bond strength was calculated, and the modes of the bond failures were determined by SEM. The highest bond strength was seen in nonthermocycled Nano-WMTA samples and the lowest in thermocycled Bioaggregate samples. The significant differences between nonthermocycled and thermocycled samples were only noticed in WMTA and Nano-WMTA groups (P < 0.001). The mode of failure for thermocycled samples of all three cements was mostly cohesive. Thermocycling process can drastically affect the push-out bond strength of calcium silicate based cements. The intrastructural damages occurred due to the thermal stresses, causing cohesive failures in set materials. Sealing property of endodontic cements which have experienced the thermal stresses can be jeopardized due to occlusal forces happening in furcation cites.
PMID: 24063004 [PubMed - indexed for MEDLINE]
Direct electrochemistry of hemoglobin immobilized on a functionalized multi-walled carbon nanotubes and gold nanoparticles nanocomplex-modified glassy carbon electrode.
Sensors (Basel). 2013;13(7):8595-611
Authors: Hong J, Zhao YX, Xiao BL, Moosavi-Movahedi AA, Ghourchian H, Sheibani N
Direct electron transfer of hemoglobin (Hb) was realized by immobilizing Hb on a carboxyl functionalized multi-walled carbon nanotubes (FMWCNTs) and gold nanoparticles (AuNPs) nanocomplex-modified glassy carbon electrode. The ultraviolet-visible absorption spectrometry (UV-Vis), transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) methods were utilized for additional characterization of the AuNPs and FMWCNTs. The cyclic voltammogram of the modified electrode has a pair of well-defined quasi-reversible redox peaks with a formal potential of -0.270 ± 0.002 V (vs. Ag/AgCl) at a scan rate of 0.05 V/s. The heterogeneous electron transfer constant (ks) was evaluated to be 4.0 ± 0.2 s(-1). The average surface concentration of electro-active Hb on the surface of the modified glassy carbon electrode was calculated to be 6.8 ± 0.3 × 10(-10) mol cm(-2). The cathodic peak current of the modified electrode increased linearly with increasing concentration of hydrogen peroxide (from 0.05 nM to 1 nM) with a detection limit of 0.05 ± 0.01 nM. The apparent Michaelis-Menten constant (K(m)(app)) was calculated to be 0.85 ± 0.1 nM. Thus, the modified electrode could be applied as a third generation biosensor with high sensitivity, long-term stability and low detection limit.
PMID: 23881129 [PubMed - indexed for MEDLINE]
MyD88-dependent pathways in leukocytes affect the retina in diabetes.
PLoS One. 2013;8(7):e68871
Authors: Tang J, Allen Lee C, Du Y, Sun Y, Pearlman E, Sheibani N, Kern TS
BACKGROUND: Previous studies by us and other have provided evidence that leukocytes play a critical role in the development of diabetic retinopathy, suggesting a possible role of the innate immune system in development of the retinopathy. Since MyD88 is a convergence point for signaling pathways of the innate immune system (including Toll-Like Receptors (TLRs) and interleukin-1ß (IL-1ß)), the purpose of this study was to assess the role of MyD88 and its dependent pathways on abnormalities that develop in retina and white blood cells related to diabetic retinopathy.
METHODS: C57BL/6J mice were made diabetic with streptozotocin. Chimeric mice were generated in which MyD88-dependent pathways were deleted from bone marrow-derived only. Mice were sacrificed at 2 mos of diabetes for assessment of, leukostasis, albumin accumulation in neural retina, leukocyte-mediated killing of retinal endothelial cells, and cytokine/chemokine generation by retinas of diabetic mice in response to TLR agonists.
RESULTS: IL-6 and CXCL1 were generated in retinas from diabetic (but not nondiabetic mice) following incubation with Pam3CysK/TLR2, but incubation with other TLR ligands or IL-1ß did not induce cytokine production in retinas from nondiabetic or diabetic mice. Diabetes-induced abnormalities (leukostasis, ICAM-1 expression on the luminal surface of the vascular endothelium, retinal superoxide generation) were significantly inhibited by removing either MyD88 or the signaling pathways regulated by it (TLRs 2 and 4, and IL-1ß) from bone marrow-derived cells only. Leukocyte-mediated killing of endothelial cells tended to be decreased in the marrow-derived cells lacking TLR2/4, but the killing was significantly exacerbated if the marrow cells lacked MyD88 or the receptor for IL-1ß (IL-1ßr).
CONCLUSIONS: MyD88-dependent pathways play an important role in the development of diabetes-induced inflammation in the retina, and inhibition of MyD88 might be a novel target to inhibit early abnormalities of diabetic retinopathy and other complications of diabetes.
PMID: 23874797 [PubMed - indexed for MEDLINE]
Retinal O-linked N-acetylglucosamine protein modifications: implications for postnatal retinal vascularization and the pathogenesis of diabetic retinopathy.
Mol Vis. 2013;19:1047-59
Authors: Gurel Z, Sieg KM, Shallow KD, Sorenson CM, Sheibani N
PURPOSE: Hyperglycemia activates several metabolic pathways, including the hexosamine biosynthetic pathway. Uridine diphosphate N-acetylglucosamine (GlcNAc) is the product of the hexosamine biosynthetic pathway and the substrate for O-linked GlcNAc (O-GlcNAc) modification. This modification affects a wide range of proteins by altering their activity, cellular localization, and/or protein interactions. However, the role O-GlcNAcylation may play in normal postnatal retinal vascular development and in the ocular complications of diabetes, including diabetic retinopathy, requires further investigation.
METHODS: The total levels of O-GlcNAc-modified proteins were evaluated by western blot analysis of lysates prepared from retinas obtained at different days during postnatal retinal vascularization and oxygen-induced ischemic retinopathy. Similar experiments were performed with retinal lysate prepared from diabetic Ins2(Akita/+) mice with different durations of diabetes and retinal vascular cells cultured under various glucose conditions. The localization of O-GlcNAc-modified proteins in the retinal vasculature was confirmed by immunofluorescence staining. The impact of altered O-GlcNAcylation on the migration of retinal vascular cells was determined using scratch wound and transwell migration assays.
RESULTS: We detected an increase in protein O-GlcNAcylation during mouse postnatal retinal vascularization and aging, in part through the regulation of the enzymes that control this modification. The study of the diabetic Ins2(Akita/+) mouse retina showed an increase in the O-GlcNAc modification of retinal proteins. We also observed an increase in retinal O-GlcNAcylated protein levels during the neovascularization phase of oxygen-induced ischemic retinopathy. Our fluorescence microscopy data confirmed that the alterations in retinal O-GlcNAcylation are similarly represented in the retinal vasculature and in retinal pericytes and endothelial cells. Particularly, the migration of retinal pericytes, but not retinal endothelial cells, was attenuated by increased O-GlcNAc modification.
CONCLUSIONS: The O-GlcNAc modification pattern changes during postnatal retinal vascular development and neovascularization, and its dysregulation under hyperglycemia and/or ischemia may contribute to the pathogenesis of the diabetic retinopathy and retinal neovascularization.
PMID: 23734074 [PubMed - indexed for MEDLINE]
Effects of silica nanoparticle supported ionic liquid as additive on thermal reversibility of human carbonic anhydrase II.
Int J Biol Macromol. 2012 Dec;51(5):933-8
Authors: Fallah-Bagheri A, Saboury AA, Ma'mani L, Taghizadeh M, Khodarahmi R, Ranjbar S, Bohlooli M, Shafiee A, Foroumadi A, Sheibani N, Moosavi-Movahedi AA
Silica nanoparticle supported imidazolium ionic liquid [SNImIL] was synthesized and utilized as a biocompatible additive for studying the thermal reversibility of human carbonic anhydrase II (HCA II). For this purpose, we prepared additive by modification of nanoparticles through the grafting of ionic liquids on the surface of nanoparticles (SNImIL). The SNImIL were fully characterized by Fourier transform infrared spectroscopy, scanning electron microscopy and thermo gravimetric analysis. The characterization of HCA II was investigated by various techniques including UV-vis and ANS fluorescence spectrophotometry, differential scanning calorimetry, and docking study. SNImIL induced disaggregation, enhanced protein stability and increased thermal reversibility of HCA II by up to 42% at pH 7.75.
PMID: 22829053 [PubMed - indexed for MEDLINE]
Marrow-derived cells regulate the development of early diabetic retinopathy and tactile allodynia in mice.
Diabetes. 2012 Dec;61(12):3294-303
Authors: Li G, Veenstra AA, Talahalli RR, Wang X, Gubitosi-Klug RA, Sheibani N, Kern TS
The hypothesis that marrow-derived cells, and specifically proinflammatory proteins in those cells, play a critical role in the development of diabetes-induced retinopathy and tactile allodynia was investigated. Abnormalities characteristic of the early stages of retinopathy and allodynia were measured in chimeric mice lacking inducible nitric oxide synthase (iNOS) or poly(ADP-ribosyl) polymerase (PARP1) in only their marrow-derived cells. Diabetes-induced capillary degeneration, proinflammatory changes, and superoxide production in the retina and allodynia were inhibited in diabetic animals in which iNOS or PARP1 was deleted from bone marrow cells only. Of the various marrow cells, neutrophils (and monocytes) play a major role in retinopathy development, because retinal capillary degeneration likewise was significantly inhibited in diabetic mice lacking the receptor for granulocyte colony-stimulating factor in their marrow-derived cells. Immunodepletion of neutrophils or monocytes inhibited the endothelial death otherwise observed when coculturing leukocytes from wild-type diabetic animals with retinal endothelium. iNOS and PARP1 are known to play a role in inflammatory processes, and we conclude that proinflammatory processes within marrow-derived cells play a central role in the development of diabetes complications in the retina and nerve.
PMID: 22923475 [PubMed - indexed for MEDLINE]
Lack of thrombospondin 1 and exacerbation of choroidal neovascularization.
Arch Ophthalmol. 2012 May;130(5):615-20
Authors: Wang S, Sorenson CM, Sheibani N
OBJECTIVES: To assess the impact of thrombospondin 1(TSP1) deficiency on choroidal neovascularization (CNV)and to determine whether administration of a TSP1 antiangiogenic mimetic peptide attenuates CNV.
METHODS: The impact of TSP1 deficiency on laser induced CNV was assessed using wild-type (TSP1 +/+) and TSP1-deficient (TSP1 −/−) mice. Three laser burns were placed in each eye of TSP1 +/+ and TSP1 −/− mice to induce CNV. Intravitreal injection of the TSP1 mimetic peptide was performed on days 1 and 7 postlaser in the mice.For quantitative measurements of neovascularization, intercellular adhesion molecule 2 staining was performed at 14 days postlaser of the choroidal-sclera flat mounts. The recruitment of macrophages to the sites of damage was investigated by immunohistochemistry. The CNV area was measured by intercellular adhesion molecule 2 staining and use of ImageJ software.
RESULTS: The TSP1 −/− mice exhibited significantly larger areas of neovascularization on choroidal flat mounts compared with TSP1 +/ mice. This was consistent with enhanced recruitment of macrophages in TSP1 −/− mice compared with TSP1 +/+ mice 3 days postlaser. The development of CNV was significantly attenuated in mice receiving the TSP1 antiangiogenic mimetic peptide compared with those receiving vehicle alone.
CONCLUSIONS: Deficiency of TSP1 contributes to enhanced choroidal neovascularization. This is consistent with the anti-inflammatory and antiangiogenic activity of TSP1. The TSP1 antiangiogenic peptide was effective in attenuation of CNV.
CLINICAL RELEVANCE: Intravitreal injection of TSP1 antiangiogenic mimetic peptides may provide alternative treatment for CNV.
PMID: 22232368 [PubMed - indexed for MEDLINE]
BIM deficiency differentially impacts the function of kidney endothelial and epithelial cells through modulation of their local microenvironment.
Am J Physiol Renal Physiol. 2012 Apr 1;302(7):F809-19
Authors: Sheibani N, Morrison ME, Gurel Z, Park S, Sorenson CM
The extracellular matrix (ECM) acts as a scaffold for kidney cellular organization. Local secretion of the ECM allows kidney cells to readily adapt to changes occurring within the kidney. In addition to providing structural support for cells, the ECM also modulates cell survival, migration, proliferation, and differentiation. Although aberrant regulation of ECM proteins can play a causative role in many diseases, it is not known whether ECM production, cell adhesion, and migration are regulated in a similar manner in kidney epithelial and endothelial cells. Here, we demonstrate that lack of BIM expression differentially impacts kidney endothelial and epithelial cell ECM production, migration, and adhesion, further emphasizing the specialized role of these cell types in kidney function. Bim -/- kidney epithelial cells demonstrated decreased migration, increased adhesion, and sustained expression of osteopontin and thrombospondin-1 (TSP1). In contrast, bim -/- kidney endothelial cells demonstrated increased cell migration, and decreased expression of osteopontin and TSP1. We also observed a fivefold increase in VEGF expression in bim -/- kidney endothelial cells consistent with their increased migration and capillary morphogenesis. These cells also had decreased endothelial nitric oxide synthase activity and nitric oxide bioavailability. Thus kidney endothelial and epithelial cells make unique contributions to the regulation of their ECM composition, with specific impact on adhesive and migratory properties that are essential for their proper function.
PMID: 22169007 [PubMed - indexed for MEDLINE]
Suppression of thrombospondin-1 expression during uveal melanoma progression and its potential therapeutic utility.
Arch Ophthalmol. 2012 Mar;130(3):336-41
Authors: Wang S, Neekhra A, Albert DM, Sorenson CM, Sheibani N
OBJECTIVES: To determine whether expression of thrombospondin-1 (TSP1), an endogenous inhibitor of angiogenesis, is downregulated during progression of uveal melanoma and whether administration of TSP1 and/or its antiangiogenic peptides attenuate tumor growth.
METHODS: Tyrosinase-SV40 T-antigens (Tyr Tag) transgenic mice were used for evaluation of TSP1 expression during tumor progression using immunohistological methods. The therapeutic potential of TSP1 on tumor progression was evaluated either by crossing Tyr Tag mice with a line of transgenic mice overexpressing TSP1 in the eye or by administration of TSP1-mimetic peptide with known antiangiogenic, antitumor activity. Tumor areas were measured in histological sections using Optima software (Media Cybernetics, Inc).
RESULTS: The Tyr Tag tumors from 3-week-old mice showed significant TSP1 expression, which was dramatically downregulated in tumors from 12-week-old mice. Furthermore, the development and progression of tumor was significantly delayed in Tyr Tag TSP1 transgenic mice or Tyr Tag mice receiving TSP1-mimetic peptide (100 mg/kg/d).
CONCLUSIONS: Expression of TSP1 was decreased with the angiogenic switch during progression of uveal melanoma, and TSP1 and/or its antiangiogenic peptides were effective in attenuation of tumor growth.
CLINICAL RELEVANCE: Modulation of TSP1 expression and/or activity may be beneficial in treating uveal melanoma.
PMID: 22411663 [PubMed - indexed for MEDLINE]
Mitochondrial redox studies of oxidative stress in kidneys from diabetic mice.
Biomed Opt Express. 2012 Feb 1;3(2):273-81
Authors: Maleki S, Sepehr R, Staniszewski K, Sheibani N, Sorenson CM, Ranji M
Chronic hyperglycemia during diabetes leads to increased production of reactive oxygen species (ROS) and increased oxidative stress (OS). Here we investigated whether changes in the metabolic state can be used as a marker of OS progression in kidneys. We examined redox states of kidneys from diabetic mice, Akita(/+) and Akita(/+);TSP1(-/-) mice (Akita mice lacking thrombospondin-1, TSP1) with increasing duration of diabetes. OS as measured by mitochondrial redox ratio (NADH/FAD) was detectable shortly after the onset of diabetes and further increased with the duration of diabetes. Thus, cryo fluorescence redox imaging was used as a quantitative marker of OS progression in kidneys from diabetic mice and demonstrated that alterations in the oxidative state of kidneys occur during the early stages of diabetes.
PMID: 22312580 [PubMed]
Biochemical characterization of hemoglobins from Caspian Sea sturgeons (Acipenser persicus and Acipenser stellatus).
Cell Biochem Biophys. 2012 Jan;62(1):73-81
Authors: Ariaeenejad S, Habibi-Rezaei M, Jamili S, Fatemi MR, Poursasan N, Ahmad F, Sheibani N, Kavousi K, Moosavi-Movahedi AA
Hemoglobin (Hb) variability is a commonly used index of phylogenetic differentiation and molecular adaptation in fish enabling them to adapt to different ecological conditions. In this study, the characteristics of Hbs from two Sturgeon species of the Southern Caspian Sea Basin were investigated. After extraction and separation of hemoglobin from whole blood, the polyacrylamide gel electrophoresis (SDS-PAGE), cellulose acetate electrophoresis, and isoelectric focusing (IEF) were used to confirm Hb variabilities in these fishes. We showed that although both species have variable Hbs with different isoelectric points, their dominant Hbs can be identified. Ion exchange on CM-cellulose chromatography was used for purification of the dominant Hbs from these fishes. The accuracy of the methods was confirmed by IEF and SDS-PAGE. Spectral studies using fluorescence spectrophotometery indicated that although the Hbs from these fishes had similar properties they exhibited clear differences with human Hb. A comparative study of Hbs alpha-helix secondary substructures was performed by circular dichroism spectropolarimetry (CD) analysis. UV-vis spectrophotometery was also utilized to measure oxygen affinity of Hbs by sodium dithionite. Oxygen affinities of these Hbs were compared using Hb-oxygen dissociation curves. Together, these results demonstrate a significant relationship between oxygen affinity of fish hemoglobins and environmental partial pressure of oxygen.
PMID: 21833671 [PubMed - indexed for MEDLINE]
Antibody-mediated retinal pericyte injury: implications for diabetic retinopathy.
Invest Ophthalmol Vis Sci. 2012;53(9):5520-6
Authors: Li Y, Smith D, Li Q, Sheibani N, Huang S, Kern T, Nagaraj RH, Lin F
PURPOSE: To test the hypothesis that autoantibodies against retinal pericytes could develop in diabetic retinopathy, and that these autoantibodies could induce retinal pericyte dysfunction/death via complement.
METHODS: Human primary retinal pericytes cultured in media containing normal (5 mM) or high (30 mM) glucose concentrations were incubated with normal human sera in the presence of a retinal pericyte-reactive antibody, then their viability was assessed by a BCECF-based cytotoxicity assay, and their function was assessed by a T-cell proliferation assay. The pericytes were also analyzed by RT-PCR and flow cytometry to detect CD38, an established diabetes-associated cell surface autoantigen. The potential of the anti-CD38 antibodies in inducing pericyte cellular injury was evaluated using the same cytotoxicity assays. In addition, autoantibody-mediated cytotoxicity in mouse retinal pericytes sensitized by sera from mice with developing diabetic retinopathy or control normal mice were also studied.
RESULTS: Retinal pericyte-reactive antibodies induced cellular damage by activating complement in the serum. The antibody-injured pericytes had reduced efficacy in inhibiting T cells. Hyperglycemic culture conditions rendered pericytes more susceptible to antibody-mediated attack. CD38 was expressed in retinal pericytes, and upregulated by TNF-α and IFN-γ, and anti-CD38 antibodies induced pericyte cytotoxicity. Retinal pericytes sensitized with sera from chronic diabetic mice suffered significantly augmented cytotoxicity compared with those sensitized with sera from the control mice.
CONCLUSIONS: The autoantibody-initiated complement activation could be a mechanism underlying the loss of function, and eventually, death of retinal pericytes in diabetic patients, suggesting that inhibiting complement activation could be a novel therapeutic approach.
PMID: 22786897 [PubMed - indexed for MEDLINE]
Comparative study of immunological and structural properties of two recombinant vaccine candidates against botulinum neurotoxin type E.
Iran Biomed J. 2012;16(4):185-92
Authors: Rostamian M, Mousavy SJ, Ebrahimi F, Ghadami SA, Sheibani N, Minaei ME, Arefpour Torabi MA
BACKGROUND: Recently, botulinum neurotoxin (BoNT)-derived recombinant proteins have been suggested as potential botulism vaccines. Here, with concentrating on BoNT type E (BoNT/E), we studied two of these binding domain-based recombinant proteins: a multivalent chimer protein, which is composed of BoNT serotypes A, B and E binding subdomains, and a monovalent recombinant protein, which contains 93 amino acid residues from recombinant C-terminal heavy chain of BoNT/E (rBoNT/E-HCC). Both proteins have an identical region (48 aa) that contains one of the most important BoNT/E epitopes (YLTHMRD sequence).
METHODS: The recombinant protein efficiency in antibody production, their structural differences, and their BoNT/E-epitope location were compared by using ELISA, circular dichroism, computational modeling, and hydrophobicity predictions.
RESULTS: Immunological studies indicated that the antibody yield against rBoNT/E-HCC was higher than chimer protein. Cross ELISA confirmed that the antibodies against the chimer protein recognized rBoNT/E-HCC more efficiently. However, both antibody groups (anti-chimer and anti-rBoNT/E-HCC antibodies) were able to recognize other proteins. Structural studies with circular dichroism showed that chimer proteins have slightly more secondary structures than rBoNT/E-HCC.
CONCLUSION: The immunological results suggested that the above-mentioned identical region in rBoNT/E-HCC is more exposed. Circular dichroism, computational protein modeling and hydrophobicity predictions indicated a more exposed location for the identical region in rBoNT/E-HCC than the chimer protein, which is strongly in agreement with immunological results.
PMID: 23183617 [PubMed - indexed for MEDLINE]
Pericytes regulate vascular basement membrane remodeling and govern neutrophil extravasation during inflammation.
PLoS One. 2012;7(9):e45499
Authors: Wang S, Cao C, Chen Z, Bankaitis V, Tzima E, Sheibani N, Burridge K
During inflammation polymorphonuclear neutrophils (PMNs) traverse venular walls, composed of the endothelium, pericyte sheath and vascular basement membrane. Compared to PMN transendothelial migration, little is known about how PMNs penetrate the latter barriers. Using mouse models and intravital microscopy, we show that migrating PMNs expand and use the low expression regions (LERs) of matrix proteins in the vascular basement membrane (BM) for their transmigration. Importantly, we demonstrate that this remodeling of LERs is accompanied by the opening of gaps between pericytes, a response that depends on PMN engagement with pericytes. Exploring how PMNs modulate pericyte behavior, we discovered that direct PMN-pericyte contacts induce relaxation rather than contraction of pericyte cytoskeletons, an unexpected response that is mediated by inhibition of the RhoA/ROCK signaling pathway in pericytes. Taking our in vitro results back into mouse models, we present evidence that pericyte relaxation contributes to the opening of the gaps between pericytes and to the enlargement of the LERs in the vascular BM, facilitating PMN extravasation. Our study demonstrates that pericytes can regulate PMN extravasation by controlling the size of pericyte gaps and thickness of LERs in venular walls. This raises the possibility that pericytes may be targeted in therapies aimed at regulating inflammation.
PMID: 23029055 [PubMed - indexed for MEDLINE]
Caspase-14: a novel caspase in the retina with a potential role in diabetic retinopathy.
Mol Vis. 2012;18:1895-906
Authors: Al-Shabrawey M, Ahmad S, Megyerdi S, Othman A, Baban B, Palenski TL, Shin ES, Gurel Z, Hsu S, Sheibani N
PURPOSE: The purpose of this study was to evaluate caspase-14 expression in the retina under normal and diabetic conditions, and to determine whether caspase-14 contributes to retinal microvascular cell death under high glucose conditions.
METHODS: Quantitative real-time polymerase chain reaction and western blot analysis were used to evaluate caspase-14 expression in retinal cells, including pericytes (PCs), endothelial cells (ECs), astrocytes (ACs), choroidal ECs, and retinal pigment epithelium (RPE) cells. We also determined caspase-14 expression in the retinas of human subjects with or without diabetic retinopathy (DR) and in experimental diabetic mice. Retinal ECs and PCs were infected with adenoviruses expressing human caspase-14 or green fluorescent protein. Caspase-14 expression was also assessed in retinal vascular cells cultured under high glucose conditions. The number of apoptotic cells was determined with terminal deoxynucleotidyl transferase dUTP nick end labeling staining and confirmed by determining the levels of cleaved poly (ADP-ribose) polymerase-1 and caspase-3.
RESULTS: Our experiments demonstrated that retinal ECs, PCs, ACs, choroidal ECs, and RPE cells expressed caspase-14, and DR was associated with upregulation and/or activation of caspase-14 particularly in retinal vasculature. High glucose induced marked elevation of the caspase-14 level in retinal vascular cells. There was a significant increase in the apoptosis rate and the levels of cleaved poly (ADP-ribose) polymerase-1 and caspase-3 in retinal ECs and PCs overexpressing caspase-14.
CONCLUSIONS: Our findings indicate that caspase-14 might play a significant role in the pathogenesis of DR by accelerating retinal PC and EC death. Further investigations are required to elaborate the underlying mechanisms.
PMID: 22876114 [PubMed - indexed for MEDLINE]
Oligomeric forms of insulin amyloid aggregation disrupt outgrowth and complexity of neuron-like PC12 cells.
PLoS One. 2012;7(7):e41344
Authors: Kachooei E, Moosavi-Movahedi AA, Khodagholi F, Ramshini H, Shaerzadeh F, Sheibani N
Formation of protein amyloid fibrils consists of a series of intermediates including oligomeric aggregates, proto-fibrillar structures, and finally mature fibrils. Recent studies show higher toxicity for oligomeric and proto-fibrillar intermediates of protein relative to their mature fibrils. Here the kinetic of the insulin amyloid fibrillation was evaluated using a variety of techniques including ThT fluorescence, Congo red absorbance, circular dichroism, and atomic force microscopy (AFM). The solution surface tension changes were attributed to hydrophobic changes in insulin structure and were detected by Du Noüy Ring method. Determination of the surface tension of insulin oligomeric, proto-fibrillar and fibrillar forms indicated that the hydrophobicity of solution is enhanced by the formation of the oligomeric forms of insulin compared to other forms. In order to investigate the toxicity of the different forms of insulin we monitored morphological alterations of the differentiated neuron-like PC12 cells following incubation with native, oligomeric aggregates, proto-fibrillar, and fibrillar forms of insulin. The cell body area, average neurite length, neurite width, number of primary neurites, and percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different forms of insulin. We observed that the oligomeric form of insulin impaired the growth and complexity of PC12 cells compared to other forms. Together our data suggest that the lower surface tension of oligomers and their perturbation affects the morphology of PC12 cells, mainly due to their enhanced hydrophobicity and detergent-like structures.
PMID: 22848469 [PubMed - indexed for MEDLINE]
Retinal pericytes inhibit activated T cell proliferation.
Invest Ophthalmol Vis Sci. 2011 Nov;52(12):9005-10
Authors: Tu Z, Li Y, Smith DS, Sheibani N, Huang S, Kern T, Lin F
PURPOSE: To test the hypothesis that retinal pericytes (RPCs) are immunosuppressive; therefore, their loss of function under hyperglycemic conditions favors retinal inflammation and contributes to the pathogenesis of diabetic retinopathy (DR).
METHODS: Isolated mouse and human RPCs were tested in T cell function assays to evaluate their capability of inhibiting T cell responses. To elucidate the underlying mechanisms, transwell systems, blocking mAbs against PD-L1 and IL-10 were used. The efficacy of RPCs in protecting retinal endothelial cells (RECs) from inflammation-induced apoptosis was assessed by apoptosis detection staining. Finally, to test whether hyperglycemic conditions impair the immunomodulatory activity of RPCs, RPCs pre-incubated in high glucose or methylglyoxal (MGO) were evaluated using the T cell proliferation assays.
RESULTS: RPCs profoundly inhibited activated T cell proliferation and inflammatory cytokine production. The T cell inhibitory activity of RPCs was decreased, but was not abolished, in transwell experiments. RPCs express PD-L1, and blocking PD-L1 reduced RPCs' efficacy of T cell inhibition. RPCs also produce IL-10, and neutralization of IL-10 reduced their immunosuppressive activity. There were significantly reduced numbers of inflammation-induced apoptosis-detected RECs in the presence of RPCs. Incubation of RPCs with either high glucose or MGO reduced the activity of RPCs to inhibit activated T cell proliferation.
CONCLUSIONS: RPCs are highly immunosuppressive and they protected RECs from inflammation-mediated apoptosis. Hyperglycemic conditions impaired the T cell inhibitory activity of RPCs. These results reveal a new function of RPCs, and its regulation under hyperglycemic conditions. This may represent a novel mechanism by which RPCs contribute to preservation of retinal integrity in diseases, including DR.
PMID: 22003106 [PubMed - indexed for MEDLINE]
Integrin-linked kinase regulates integrin signaling in human trabecular meshwork cells.
Invest Ophthalmol Vis Sci. 2011 Mar;52(3):1684-92
Authors: Faralli JA, Newman JR, Sheibani N, Dedhar S, Peters DM
PURPOSE: To determine whether integrin-linked kinase (ILK) controls the organization of the actin cytoskeleton in the trabecular meshwork (TM) by regulating integrin co-signaling.
METHODS: The cell binding domain and the Heparin II (Hep II) domain of fibronectin were used to activate α5β1 and α4β1 integrin signaling, respectively, in differentiated human TM (HTM) cells. The role of ILK was determined using either ILK small interfering RNA (siRNA) to knockout ILK expression or the ILK inhibitors, KP392 and QLT0267. The knockdown of ILK expression was verified by Western blot analysis. The presence of actin stress fibers and focal adhesions was determined by labeling cultures with phalloidin and anti-talin or ILK antibodies, respectively.
RESULTS: Cell spreading in differentiated HTM cells required ILK, since ILK siRNA and the ILK inhibitors significantly reduced cell spreading, actin polymerization, and the localization of talin and ILK in focal adhesions (FAs). Both cell spreading and the localization of talin and ILK to FAs in differentiated HTM cells could be rescued by inducing α4β1 integrin signaling with a recombinant Hep II domain of fibronectin, even though α4β1 integrins were not found in FAs. In the absence of ILK inhibition, the Hep II domain had minimal effect on α5β1 integrin-mediated spreading.
CONCLUSIONS: This study suggests that cooperative α5β1/α4β1 integrin signaling may be regulated by ILK trans-dominantly and that alterations in ILK activity may affect actin cytoskeleton organization and contractility in the TM.
PMID: 21071740 [PubMed - indexed for MEDLINE]
Mesangial cell integrin αvβ8 provides glomerular endothelial cell cytoprotection by sequestering TGF-β and regulating PECAM-1.
Am J Pathol. 2011 Feb;178(2):609-20
Authors: Khan S, Lakhe-Reddy S, McCarty JH, Sorenson CM, Sheibani N, Reichardt LF, Kim JH, Wang B, Sedor JR, Schelling JR
Integrins are heterodimeric receptors that regulate cell adhesion, migration, and apoptosis. Integrin αvβ8 is most abundantly expressed in kidney and brain, and its major ligand is latent transforming growth factor-β (TGF-β). Kidney αvβ8 localizes to mesangial cells, which appose glomerular endothelial cells and maintain glomerular capillary structure by mechanical and poorly understood paracrine mechanisms. To establish kidney αvβ8 function, mice with homozygous Itgb8 deletion (Itgb8(-/-)) were generated on outbred and C57BL/6 congenic backgrounds. Most Itgb8(-/-) mice died in utero, and surviving Itgb8(-/-) mice failed to gain weight, and rarely survived beyond 6 weeks. A renal glomerular phenotype included azotemia and albuminuria, as well as increased platelet endothelial cell adhesion molecule-1 (PECAM-1) expression, which was surprisingly not associated with conventional functions, such as endothelial cell hyperplasia, hypertrophy, or perivascular inflammation. Itgb8(-/-) mesangial cells demonstrated reduced latent TGF-β binding, resulting in bioactive TGF-β release, which stimulated glomerular endothelial cell apoptosis. Using PECAM-1 gain and loss of function strategies, we show that PECAM-1 provides endothelial cytoprotection against mesangial cell TGF-β. These results clarify a singular mechanism of mesangial-to-endothelial cell cross-talk, whereby mesangial cell αvβ8 homeostatically arbitrates glomerular microvascular integrity by sequestering TGF-β in its latent conformation. Under pathological conditions associated with decreased mesangial cell αvβ8 expression and TGF-β secretion, compensatory PECAM-1 modulation facilitates glomerular endothelial cell survival.
PMID: 21281793 [PubMed - indexed for MEDLINE]
Increased expression and activity of 12-lipoxygenase in oxygen-induced ischemic retinopathy and proliferative diabetic retinopathy: implications in retinal neovascularization.
Diabetes. 2011 Feb;60(2):614-24
Authors: Al-Shabrawey M, Mussell R, Kahook K, Tawfik A, Eladl M, Sarthy V, Nussbaum J, El-Marakby A, Park SY, Gurel Z, Sheibani N, Maddipati KR
OBJECTIVE: Arachidonic acid is metabolized by 12-lipoxygenase (12-LOX) to 12-hydroxyeicosatetraenoic acid (12-HETE) and has an important role in the regulation of angiogenesis and endothelial cell proliferation and migration. The goal of this study was to investigate whether 12-LOX plays a role in retinal neovascularization (NV).
RESEARCH DESIGN AND METHODS: Experiments were performed using retinas from a murine model of oxygen-induced ischemic retinopathy (OIR) that was treated with and without the LOX pathway inhibitor, baicalein, or lacking 12-LOX. We also analyzed vitreous samples from patients with and without proliferative diabetic retinopathy (PDR). Western blotting and RT-PCR were used to assess the expression of 12-LOX, vascular endothelial growth factor (VEGF), and pigment epithelium-derived factor (PEDF). Liquid chromatography-mass spectrometry was used to assess the amounts of HETEs in the murine retina and human vitreous samples. The effects of 12-HETE on VEGF and PEDF expression were evaluated in Müller cells (rMCs), primary mouse retinal pigment epithelial cells, and astrocytes.
RESULTS: Retinal NV during OIR was associated with increased 12-LOX expression and 12-, 15-, and 5-HETE production. The amounts of HETEs also were significantly higher in the vitreous of diabetic patients with PDR. Retinal NV was markedly abrogated in mice treated with baicalein or mice lacking 12-LOX. This was associated with decreased VEGF expression and restoration of PEDF levels. PEDF expression was reduced in 12-HETE-treated rMCs, astrocytes, and the retinal pigment epithelium. Only rMCs and astrocytes showed increased VEGF expression by 12-HETE.
CONCLUSIONS: 12-LOX and its product HETE are important regulators of retinal NV through modulation of VEGF and PEDF expression and could provide a new therapeutic target to prevent and treat ischemic retinopathy.
PMID: 21228311 [PubMed - indexed for MEDLINE]
Regulation of angiogenesis by histone chaperone HIRA-mediated incorporation of lysine 56-acetylated histone H3.3 at chromatin domains of endothelial genes.
J Biol Chem. 2010 Dec 31;285(53):41567-77
Authors: Dutta D, Ray S, Home P, Saha B, Wang S, Sheibani N, Tawfik O, Cheng N, Paul S
Angiogenesis is critically dependent on endothelial cell-specific transcriptional mechanisms. However, the molecular processes that regulate chromatin domains and thereby dictate transcription of key endothelial genes are poorly understood. Here, we report that, in endothelial cells, angiogenic signal-mediated transcriptional induction of Vegfr1 (vascular endothelial growth factor receptor 1) is dependent on the histone chaperone, HIRA (histone cell cycle regulation-defective homolog A). Our molecular analyses revealed that, in response to angiogenic signals, HIRA is induced in endothelial cells and mediates incorporation of lysine 56 acetylated histone H3.3 (H3acK56) at the chromatin domain of Vegfr1. HIRA-mediated incorporation of H3acK56 is a general mechanism associated with transcriptional induction of several angiogenic genes in endothelial cells. Depletion of HIRA inhibits H3acK56 incorporation and transcriptional induction of Vegfr1 and other angiogenic genes. Our functional analyses revealed that depletion of HIRA abrogates endothelial network formation on Matrigel and inhibits angiogenesis in an in vivo Matrigel plug assay. Furthermore, analysis in a laser-induced choroidal neovascularization model showed that depletion of HIRA significantly inhibits neovascularization. Our results for the first time decipher a histone chaperone (HIRA)-dependent molecular mechanism in endothelial gene regulation and indicate that histone chaperones could be new targets for angiogenesis therapy.
PMID: 21041298 [PubMed - indexed for MEDLINE]
PECAM-1 regulates proangiogenic properties of endothelial cells through modulation of cell-cell and cell-matrix interactions.
Am J Physiol Cell Physiol. 2010 Dec;299(6):C1468-84
Authors: Park S, DiMaio TA, Scheef EA, Sorenson CM, Sheibani N
Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a member of the immunoglobulin superfamily of cell adhesion molecules with important roles in angiogenesis and inflammation. However, the molecular and cellular mechanisms, and the role that specific PECAM-1 isoforms play in these processes, remain elusive. We recently showed attenuation of retinal vascular development and neovascularization in PECAM-1-deficient (PECAM-1-/-) mice. To gain further insight into the role of PECAM-1 in these processes, we isolated primary retinal endothelial cells (EC) from wild-type (PECAM-1+/+) and PECAM-1-/- mice. Lack of PECAM-1 had a significant impact on endothelial cell-cell and cell-matrix interactions, resulting in attenuation of cell migration and capillary morphogenesis. Mechanistically these changes were associated with a significant decrease in expression of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) bioavailability in PECAM-1-/- retinal EC. PECAM-1-/- retinal EC also exhibited a lower rate of apoptosis under basal and challenged conditions, consistent with their increased growth rate. Furthermore, reexpression of PECAM-1 was sufficient to restore migration and capillary morphogenesis of null cells in an isoform-specific manner. Thus PECAM-1 expression modulates proangiogenic properties of EC, and these activities are significantly influenced by alternative splicing of its cytoplasmic domain.
PMID: 20810911 [PubMed - indexed for MEDLINE]
A new role for the muscle repair protein dysferlin in endothelial cell adhesion and angiogenesis.
Arterioscler Thromb Vasc Biol. 2010 Nov;30(11):2196-204
Authors: Sharma A, Yu C, Leung C, Trane A, Lau M, Utokaparch S, Shaheen F, Sheibani N, Bernatchez P
OBJECTIVE: Ferlins are known to regulate plasma membrane repair in muscle cells and are linked to muscular dystrophy and cardiomyopathy. Recently, using proteomic analysis of caveolae/lipid rafts, we reported that endothelial cells (EC) express myoferlin and that it regulates membrane expression of vascular endothelial growth factor receptor 2 (VEGFR-2). The goal of this study was to document the presence of other ferlins in EC.
METHODS AND RESULTS: EC expressed another ferlin, dysferlin, and that in contrast to myoferlin, it did not regulate VEGFR-2 expression levels or downstream signaling (nitric oxide and Erk1/2 phosphorylation). Instead, loss of dysferlin in subconfluent EC resulted in deficient adhesion followed by growth arrest, an effect not observed in confluent EC. In vivo, dysferlin was also detected in intact and diseased blood vessels of rodent and human origin, and angiogenic challenge of dysferlin-null mice resulted in impaired angiogenic response compared with control mice. Mechanistically, loss of dysferlin in cultured EC caused polyubiquitination and proteasomal degradation of platelet endothelial cellular adhesion molecule-1 (PECAM-1/CD31), an adhesion molecule essential for angiogenesis. In addition, adenovirus-mediated gene transfer of PECAM-1 rescued the abnormal adhesion of EC caused by dysferlin gene silencing.
CONCLUSIONS: Our data describe a novel pathway for PECAM-1 regulation and broaden the functional scope of ferlins in angiogenesis and specialized ferlin-selective protein cargo trafficking in vascular settings.
PMID: 20724702 [PubMed - indexed for MEDLINE]
Opposing effects of bim and bcl-2 on lung endothelial cell migration.
Am J Physiol Lung Cell Mol Physiol. 2010 Nov;299(5):L607-20
Authors: Grutzmacher C, Park S, Elmergreen TL, Tang Y, Scheef EA, Sheibani N, Sorenson CM
Integration of cell adhesive, survival, and proliferative processes is essential for capillary morphogenesis of endothelial cells (EC) in vitro and vascular development and function in vivo. Unfortunately, the molecular and cellular mechanisms that impact these processes are poorly defined. Here we examined how lack of bim and/or bcl-2 expression impact lung EC function. The absence of bcl-2 or bim had a significant impact on EC adhesion and migration. Lack of bcl-2 expression decreased lung EC migration, whereas lack of bim expression increased migration compared with their wild-type counterparts. Decreased adhesion to fibronectin and vitronectin was observed in both bcl-2-/- and bim-/- lung EC, with bcl-2-/- EC having very little adhesion to either matrix protein. Capillary morphogenesis was greatly diminished in bcl-2-/- EC, which correlated with decreased lung alveolarization in vivo, an angiogenesis-dependent process. We also observed aberrant production of extracellular matrix proteins, eNOS expression, and nitric oxide production in bcl-2-/- lung EC, which could contribute to inability to undergo capillary morphogenesis. The changes in cell adhesion and migration noted in the absence of bim or bcl-2 were independent of their impact on apoptosis. We observed no significant affect on the steady-state rate of apoptosis of lung EC in the absence of bim or bcl-2. Thus, bcl-2 family members, bim and bcl-2, play a central role in modulation of EC proangiogenic properties, which goes beyond their role as simple mediators of mitochondrial homeostasis and apoptosis.
PMID: 20656893 [PubMed - indexed for MEDLINE]
Heparin II domain of fibronectin mediates contractility through an alpha4beta1 co-signaling pathway.
Exp Cell Res. 2010 May 15;316(9):1500-12
Authors: Schwinn MK, Gonzalez JM, Gabelt BT, Sheibani N, Kaufman PL, Peters DM
In the trabecular meshwork (TM) of the eye, regulation of tissue contractility by the PPRARI sequence within the Heparin II (HepII) domain of fibronectin is believed to control the movement of aqueous humor and dictate the level of intraocular pressure. This study shows that the HepII domain utilizes activated alpha4beta1 integrin and collagen to mediate a co-signaling pathway that down-regulates contractility in TM cells. siRNA silencing of alpha4beta1 integrin blocked the actin disrupting effects of both PPRARI and the HepII domain. The down-regulation of the actin cytoskeleton and contractility did not involve syndecan-4 or other heparan sulfate proteoglycans (HSPGs) since siRNA silencing of syndecan-4 expression or heparitinase removal of cell surface HSPGs did not prevent the HepII-mediated disruption of the actin cytoskeleton. HepII-mediated disruption of the cytoskeleton depended upon the presence of collagen in the extracellular matrix, and cell binding studies indicated that HepII signaling involved cross-talk between alpha4beta1 and alpha1/alpha2beta1 integrins. This is the first time that the PPRARI sequence in the HepII domain has been shown to serve as a physiological alpha4beta1 ligand, suggesting that alpha4beta1 integrin may be a key regulator of tissue contractility.
PMID: 20302860 [PubMed - indexed for MEDLINE]
Increased synthesis of leukotrienes in the mouse model of diabetic retinopathy.
Invest Ophthalmol Vis Sci. 2010 Mar;51(3):1699-708
Authors: Talahalli R, Zarini S, Sheibani N, Murphy RC, Gubitosi-Klug RA
PURPOSE: Evidence suggests that capillary degeneration in early diabetic retinopathy results from chronic inflammation, and leukotrienes have been implicated in this process. The authors investigated the cellular sources of leukotriene biosynthesis in diabetic retinas and the effects of hyperglycemia on leukotriene production.
METHODS: Retinas and bone marrow cells were collected from diabetic and nondiabetic mice. Mouse retinal glial cells and retinal endothelial cells (mRECs) were cultured under nondiabetic and diabetic conditions. Production of leukotriene metabolites was assessed by mass spectrometry, and Western blot analysis was used to quantitate the expression of enzymes and receptors involved in leukotriene synthesis and signaling.
RESULTS: Bone marrow cells from nondiabetic mice expressed 5-lipoxygenase, the enzyme required for the initiation of leukotriene synthesis, and produced leukotriene B(4) (LTB(4)) when stimulated with the calcium ionophore A23187. Notably, LTB(4) synthesis was increased threefold over normal (P < 0.03) in bone marrow cells from diabetic mice. In contrast, retinas from nondiabetic or diabetic mice produced neither leukotrienes nor 5-lipoxygenase mRNA. Despite an inability to initiate leukotriene biosynthesis, the addition of exogenous leukotriene A(4) (LTA(4); the precursor of LTB(4)) to retinas resulted in robust production of LTB(4). Similarly, retinal glial cells synthesized LTB(4) from LTA(4), whereas mRECs produced both LTB(4) and the cysteinyl leukotrienes. Culturing the retinal cells in high-glucose concentrations enhanced leukotriene synthesis and selectively increased expression of the LTB(4) receptor BLT1. Antagonism of the BLT1 receptor inhibited LTB(4)-induced mREC cell death.
CONCLUSIONS: Transcellular delivery of LTA(4) from marrow-derived cells to retinal cells results in the generation of LTB(4) and the death of endothelial cells and, thus, might contribute to chronic inflammation and retinopathy in diabetes.
PMID: 19834040 [PubMed - indexed for MEDLINE]
CYP1B1 and endothelial nitric oxide synthase combine to sustain proangiogenic functions of endothelial cells under hyperoxic stress.
Am J Physiol Cell Physiol. 2010 Mar;298(3):C665-78
Authors: Tang Y, Scheef EA, Gurel Z, Sorenson CM, Jefcoate CR, Sheibani N
We have recently shown that deletion of constitutively expressed CYP1B1 is associated with attenuation of retinal endothelial cell (EC) capillary morphogenesis (CM) in vitro and angiogenesis in vivo. This was largely caused by increased intracellular oxidative stress and increased production of thrombospondin-2, an endogenous inhibitor of angiogenesis. Here, we demonstrate that endothelium nitric oxide synthase (eNOS) expression is dramatically decreased in the ECs prepared from retina, lung, heart, and aorta of CYP1B1-deficient (CYP1B1(-/-)) mice compared with wild-type (CYP1B1(+/+)) mice. The eNOS expression was also decreased in retinal vasculature of CYP1B1(-/-) mice. Inhibition of eNOS activity in cultured CYP1B1(+/+) retinal ECs blocked CM and was concomitant with increased oxidative stress, like in CYP1B1(-/-) retinal ECs. In addition, expression of eNOS in CYP1B1(-/-) retinal ECs or their incubation with a nitric oxide (NO) donor enhanced NO levels, lowered oxidative stress, and improved cell migration and CM. Inhibition of CYP1B1 activity in the CYP1B1(+/+) retinal ECs resulted in reduced NO levels and attenuation of CM. In contrast, expression of CYP1B1 increased NO levels and enhanced CM of CYP1B1(-/-) retinal ECs. Furthermore, attenuation of CYP1B1 expression with small interfering RNA proportionally lowered eNOS expression and NO levels in wild-type cells. Together, our results link CYP1B1 metabolism in retinal ECs with sustained eNOS activity and NO synthesis and/or bioavailability and low oxidative stress and thrombospondin-2 expression. Thus CYP1B1 and eNOS cooperate in different ways to lower oxidative stress and thereby to promote CM in vitro and angiogenesis in vivo.
PMID: 20032512 [PubMed - indexed for MEDLINE]
New model for polymerization of oligomeric alcohol dehydrogenases into nanoaggregates.
Appl Biochem Biotechnol. 2010 Feb;160(4):1188-205
Authors: Barzegar A, Moosavi-Movahedi AA, Kyani A, Goliaei B, Ahmadian S, Sheibani N
Polymerization and self-assembly of proteins into nanoaggregates of different sizes and morphologies (nanoensembles or nanofilaments) is a phenomenon that involved problems in various neurodegenerative diseases (medicine) and enzyme instability/inactivity (biotechnology). Thermal polymerization of horse liver alcohol dehydrogenase (dimeric) and yeast alcohol dehydrogenase (tetrameric), as biotechnological ADH representative enzymes, was evaluated for the development of a rational strategy to control aggregation. Constructed ADH nuclei, which grew to larger amorphous nanoaggregates, were prevented via high repulsion strain of the net charge values. Good correlation between the variation in scattering and lambda(-2) was related to the amorphousness of the nanoaggregated ADHs, shown by electron microscopic images. Scattering corrections revealed that ADH polymerization was related to the quaternary structural changes, including delocalization of subunits without unfolding, i.e. lacking the 3D conformational and/or secondary-ordered structural changes. The results demonstrated that electrostatic repulsion was not only responsible for disaggregation but also caused a delay in the onset of aggregation temperature, decreasing maximum values of aggregation and amounts of precipitation. Together, our results demonstrate and propose a new model of self-assembly for ADH enzymes based on the construction of nuclei, which grow to formless nanoaggregates with minimal changes in the tertiary and secondary conformations.
PMID: 19444390 [PubMed - indexed for MEDLINE]
Development of choroidal neovascularization in rats with advanced intense cyclic light-induced retinal degeneration.
Arch Ophthalmol. 2010 Feb;128(2):212-22
Authors: Albert DM, Neekhra A, Wang S, Darjatmoko SR, Sorenson CM, Dubielzig RR, Sheibani N
OBJECTIVES: To study the progressive changes of intense cyclic light-induced retinal degeneration and to determine whether it results in choroidal neovascularization (CNV).
METHODS: Albino rats were exposed to 12 hours of 3000-lux cyclic light for 1, 3, or 6 months. Fundus examination, fundus photography, fluorescein and indocyanine green angiography, and optical coherence tomography were performed prior to euthanization. Light-exposed animals were euthanized after 1, 3, or 6 months for histopathological evaluation. Retinas were examined for the presence of 4-hydroxy-2-nonenal- and nitrotyrosine-modified proteins by immunofluorescence staining.
RESULTS: Long-term intense cyclic light exposure resulted in retinal degeneration with loss of the outer segments of photoreceptors and approximately two-thirds of the outer nuclear layer as well as development of subretinal pigment epithelium neovascularization after 1 month. Almost the entire outer nuclear layer was absent with the presence of CNV, which penetrated the Bruch membrane and extended into the outer retina after 3 months. Absence of the outer nuclear layer, multiple foci of CNV, retinal pigment epithelial fibrous metaplasia, and connective tissue bands containing blood vessels extending into the retina were observed after 6 months. All intense light-exposed animals showed an increased presence of 4-hydroxy-2-nonenal and nitrotyrosine staining. Optical coherence tomographic and angiographic studies confirmed retinal thinning and leakiness of the newly formed blood vessels.
CONCLUSIONS: Our results suggest that albino rats develop progressive stages of retinal degeneration and CNV after long-term intense cyclic light exposure, allowing the detailed study of the pathogenesis and treatment of age-related macular degeneration.
CLINICAL RELEVANCE: The ability to study the progressive pathogenesis of age-related macular degeneration and CNV will provide detailed knowledge about the disease and aid in the development of target-specific therapy.
PMID: 20142545 [PubMed - indexed for MEDLINE]
Inhibitory effects of arresten on bFGF-induced proliferation, migration, and matrix metalloproteinase-2 activation in mouse retinal endothelial cells.
Curr Eye Res. 2010 Jan;35(1):45-55
Authors: Boosani CS, Nalabothula N, Sheibani N, Sudhakar A
PURPOSE: The potential role of arresten (alpha1(IV)NC1) as an endogenous angiogenesis inhibitor in the prevention of bFGF mediated retinal angiogenesis and regulation of matrix metalloproteinase-2 activation has not been explored.
METHODS: Mouse retinal endothelial cells (MREC) were cultured on type IV collagen and treated with basic fibroblast growth factor (bFGF) alone or in the presence of arresten at concentrations ranging from 1 to 10 microg/ml. The proliferation of MRECs were evaluated using 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay, and bFGF stimulated endothelial cell migration was assessed using Boyden chamber. Expression of matrix metalloproteinase-2 (MMP-2) was assessed by reverse transcription polymerase chain reaction (RT-PCR) analysis using RNA isolated from MRECs. Secretion and activation of MMP-2 in arresten-treated conditioned MREC growth medium was determined by gelatin zymography and Western blotting.
RESULTS: Different doses of bFGF induced MREC proliferation was significantly inhibited upon arresten treatment (P < 0.005). The bFGF-induced migration was significantly inhibited by arresten at 1 and 10 microg/ml concentrations (P < 0.01). The bFGF stimulated expression of MMP-2 mRNA and secretion of MMP-2 in MREC was not affected and interestingly activation of MMP-2 was suppressed by arresten in a dose and time dependent manner.
CONCLUSIONS: Inhibitory effects of arresten on proliferation, migration and MMP-2 activation but not on expression and secretion of MMP-2 in MREC; this early work with arresten supports potential therapeutic action in retinal neovascularization dependent disorders.
PMID: 20021254 [PubMed - indexed for MEDLINE]