Nader Sheibani

Exp Eye Res. 2024 Sep 20;248:110107. doi: 10.1016/j.exer.2024.110107. Online ahead of print.

ABSTRACT

Apoptosis plays prominent roles during organ development, maturation and homeostasis. In the retina, Bcl-2 family members function through the intrinsic cell death pathway with vital roles during vascular development and hyperoxia-mediated vessel obliteration during oxygen induced ischemic retinopathy (OIR). Bim, a BH3 only protein Bcl-2 family member, binds and activates Bax and/or Bak to facilitate apoptosis. In some systems deletion of both Bax and Bak are required to prevent cell loss, such as regression of ocular hyaloid vasculature. We previously showed Bim expression significantly impacts normal retinal vascular development and sensitivity to hyperoxia. Mice deficient in Bim (Bim^-/-) show increased retinal vascular density and are protected from hyperoxia mediated vessel obliteration. Since Bim activates Bax, here we determined the impact lack of Bax expression has on these processes. Compared to Bax^+/+ mice, retinas from Bax^-/- mice had significantly increased numbers of retinal endothelial cells and pericytes. We also demonstrated that hyperoxia-mediated vessel obliteration during OIR was significantly decreased in the absence of Bax. Although the increased endothelial cell numbers were comparable to that of Bim^-/- mice, the increased numbers of pericytes were not to the extent noted in Bim^-/- mice. These changes were supported by partial protection of retinal vessels from hyperoxia in Bax^-/- mice compared to that noted in Bim^-/- mice. Thus, Bim-Bax driven pathway is sufficient to remove excess endothelial cells but not pericytes during postnatal retinal vascularization and hyperoxia-mediated vessel obliteration. Thus, additional Bim-mediated pathway(s) are required for removal of pericytes and hyperoxia-mediated vessel obliteration.

PMID:39307450 | DOI:10.1016/j.exer.2024.110107

Front Ophthalmol (Lausanne). 2024 Jan 25;4:1362601. doi: 10.3389/fopht.2024.1362601. eCollection 2024.

NO ABSTRACT

PMID:38984111 | PMC:PMC11182198 | DOI:10.3389/fopht.2024.1362601

Heliyon. 2024 Mar 23;10(7):e28695. doi: 10.1016/j.heliyon.2024.e28695. eCollection 2024 Apr 15.

ABSTRACT

In this study, a very sensitive fluorescence nano-biosensor was developed using CeO₂ nanoparticles for the rapid detection of DNA methylation. The characteristics of CeO₂ nanoparticles were determined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD) spectroscopy, UV-visible spectroscopy, and fluorescence spectroscopy. The CeO₂ nanoparticles were reacted with a single-stranded DNA (ssDNA) probe, and then methylated and unmethylated target DNAs hybridized with an ssDNA probe, and the fluorescence emission was measured. Upon adding the target unmethylated and methylated ssDNA, the fluorescence intensity increased in the linear range of concentration from 2 × 10^-13 - 10^-18 M. The limit of detection (LOD) was 1.597 × 10^-6 M for methylated DNA and 1.043 × 10^-6 M for unmethylated DNA. The fluorescence emission intensity of methylated sequences was higher than of that unmethylated sequences. The fabricated DNA nanobiosensor showed a fluorescence emission at 420 nm with an excitation wavelength of 280 nm. The impact of CeO₂ binding on methylated and unmethylated DNA was further demonstrated by agarose gel electrophoresis. Finally, the actual sample analysis suggested that the nanobiosensor could have practical applications for detecting methylation in the human plasma samples.

PMID:38586346 | PMC:PMC10998132 | DOI:10.1016/j.heliyon.2024.e28695

Biomed Opt Express. 2024 Feb 12;15(3):1571-1584. doi: 10.1364/BOE.510351. eCollection 2024 Mar 1.

ABSTRACT

Mitochondrial morphology provides unique insights into their integrity and function. Among fluorescence microscopy techniques, 3D super-resolution microscopy uniquely enables the analysis of mitochondrial morphological features individually. However, there is a lack of tools to extract morphological parameters from super-resolution images of mitochondria. We report a quantitative method to extract mitochondrial morphological metrics, including volume, aspect ratio, and local protein density, from 3D single-molecule localization microscopy images, with single-mitochondrion sensitivity. We validated our approach using simulated ground-truth SMLM images of mitochondria. We further tested our morphological analysis on mitochondria that have been altered functionally and morphologically in controlled manners. This work sets the stage to quantitatively analyze mitochondrial morphological alterations associated with disease progression on an individual basis.

PMID:38495683 | PMC:PMC10942681 | DOI:10.1364/BOE.510351

PLoS One. 2024 Feb 26;19(2):e0297135. doi: 10.1371/journal.pone.0297135. eCollection 2024.

ABSTRACT

Age-related macular degeneration (AMD) is a vision threatening disease in older adults. Anti-VEGF treatment is effective for the majority of neovascular AMD (nAMD) patients, although approximately 30% of nAMD patients have an incomplete response for unknown reasons. Here we assessed the contribution of single nucleotide polymorphisms (SNPs) in key angioinflammatory regulatory genes in nAMD patients with an incomplete response compared to those responsive to anti-VEGF treatment. A total of 25 responsive and 30 nAMD patients with an incomplete response to anti-vascular endothelial growth factor (anti-VEGF) treatment were examined for known SNPs that impact the structure and function of thromobospondin-1 (TSP1), Bcl-2-interacting mediator of cell death (BIM) and complement factor H (CFH). Plasma levels of C-C motif chemokine ligand 2 (CCL2/MCP1), TSP1 and VEGF were assessed by ELISA. Patients responsive to anti-VEGF treatment showed a significant increase in the TSP1 rs2228262 AA allele and a trend for the BIM (rs724710) CT allele. Consistent with previous reports, 42% of the patients responsive to anti-VEGF expressed the CC allele for CFH rs1061170. Although the CFH TT allele had similarly low prevalence in both groups, the TC allele tended to be more prevalent in patients with an incomplete response. Patients with an incomplete response also had increased plasma CCL2/MCP1 levels, consistent with the role increased inflammation has in the pathogenesis of nAMD. Our studies point to new tools to assess the potential responsiveness of nAMD patients to anti-VEGF treatment and suggest the potential use of anti-CCL2 for treatment of nAMD patients with an incomplete response to anti-VEGF.

PMID:38408093 | PMC:PMC10896504 | DOI:10.1371/journal.pone.0297135

Cells. 2023 Dec 26;13(1):50. doi: 10.3390/cells13010050.

ABSTRACT

Age-related macular degeneration (AMD) remains a leading cause of vision loss in elderly patients. Its etiology and progression are, however, deeply intertwined with various cellular and molecular interactions within the retina and choroid. Among the key cellular players least studied are choroidal mast cells, with important roles in immune and allergic responses. Here, we will review what is known regarding the pathophysiology of AMD and expand on the recently proposed intricate roles of choroidal mast cells and their activation in outer retinal degeneration and AMD pathogenesis. We will focus on choroidal mast cell activation, the release of their bioactive mediators, and potential impact on ocular oxidative stress, inflammation, and overall retinal and choroidal health. We propose an important role for thrombospondin-1 (TSP1), a major ocular angioinflammatory factor, in regulation of choroidal mast cell homeostasis and activation in AMD pathogenesis. Drawing from limited studies, this review underscores the need for further comprehensive studies aimed at understanding the precise roles changes in TSP1 levels and choroidal mast cell activity play in pathophysiology of AMD. We will also propose potential therapeutic strategies targeting these regulatory pathways, and highlighting the promise they hold for curbing AMD progression through modulation of mast cell activity. In conclusion, the evolving understanding of the role of choroidal mast cells in AMD pathogenesis will not only offer deeper insights into the underlying mechanisms but will also offer opportunities for development of novel preventive strategies.

PMID:38201254 | PMC:PMC10778483 | DOI:10.3390/cells13010050

Int J Mol Sci. 2023 Nov 28;24(23):16884. doi: 10.3390/ijms242316884.

ABSTRACT

Polycyclic aromatic hydrocarbon (PAH) pollutants and microbiome products converge on the aryl hydrocarbon receptor (AhR) to redirect selective rapid adherence of isolated bone marrow (BM) cells. In young adult mice, Cyp1b1-deficiency and AhR activation by PAH, particularly when prolonged by Cyp1a1 deletion, produce matching gene stimulations in these BM cells. Vascular expression of Cyp1b1 lowers reactive oxygen species (ROS), suppressing NF-κB/RelA signaling. PAH and allelic selectivity support a non-canonical AhR participation, possibly through RelA. Genes stimulated by Cyp1b1 deficiency were further resolved according to the effects of Cyp1b1 and Cyp1a1 dual deletions (DKO). The adherent BM cells show a cluster of novel stimulations, including select developmental markers; multiple re-purposed olfactory receptors (OLFR); and α-Defensin, a microbial disruptor. Each one connects to an enhanced specific expression of the catalytic RNA Pol2 A subunit, among 12 different subunits. Mesenchymal progenitor BMS2 cells retain these features. Cyp1b1-deficiency removes lymphocytes from adherent assemblies as BM-derived mesenchymal stromal cells (BM-MSC) expand. Cyp1b1 effects were cell-type specific. In vivo, BM-MSC Cyp1b1 expression mediated PAH suppression of lymphocyte progenitors. In vitro, OP9-MSC sustained these progenitors, while Csf1 induced monocyte progenitor expansion to macrophages. Targeted Cyp1b1 deletion (Cdh5-Cre; Cyp1b1^fl/fl) established endothelium control of ROS that directs AhR-mediated suppression of B cell progenitors. Monocyte Cyp1b1 deletion (Lyz2-Cre; Cyp1b1^fl/fl) selectively attenuated M1 polarization of expanded macrophages, but did not enhance effects on basal M2 polarization. Thus, specific sources of Cyp1b1 link to AhR and to an OLFR network to provide BM inflammatory modulation via diverse microbiome products.

PMID:38069208 | PMC:PMC10706615 | DOI:10.3390/ijms242316884

Pharmaceuticals (Basel). 2023 Nov 2;16(11):1555. doi: 10.3390/ph16111555.

ABSTRACT

Neovascular age-related macular degeneration (nAMD) is a leading cause of irreversible visual impairment in the elderly. The current management of nAMD is limited and involves regular intravitreal administration of anti-vascular endothelial growth factor (anti-VEGF). However, the effectiveness of these treatments is limited by overlapping and compensatory pathways leading to unresponsiveness to anti-VEGF treatments in a significant portion of nAMD patients. Therefore, a system view of pathways involved in pathophysiology of nAMD will have significant clinical value. The aim of this study was to identify proteins, miRNAs, long non-coding RNAs (lncRNAs), various metabolites, and single-nucleotide polymorphisms (SNPs) with a significant role in the pathogenesis of nAMD. To accomplish this goal, we conducted a multi-layer network analysis, which identified 30 key genes, six miRNAs, and four lncRNAs. We also found three key metabolites that are common with AMD, Alzheimer's disease (AD) and schizophrenia. Moreover, we identified nine key SNPs and their related genes that are common among AMD, AD, schizophrenia, multiple sclerosis (MS), and Parkinson's disease (PD). Thus, our findings suggest that there exists a connection between nAMD and the aforementioned neurodegenerative disorders. In addition, our study also demonstrates the effectiveness of using artificial intelligence, specifically the LSTM network, a fuzzy logic model, and genetic algorithms, to identify important metabolites in complex metabolic pathways to open new avenues for the design and/or repurposing of drugs for nAMD treatment.

PMID:38004422 | PMC:PMC10674956 | DOI:10.3390/ph16111555

Exp Eye Res. 2023 Nov;236:109666. doi: 10.1016/j.exer.2023.109666. Epub 2023 Oct 4.

ABSTRACT

Angiogenesis, although required during eye development, has a causative effect in many ocular diseases. Aberrant neovascularization contributes to the progression of neovascular age-related macular degeneration (nAMD), a vision-threaten disease in aging Americans. Since increased amounts of vascular endothelial growth factor (VEGF) drives neovascularization during the pathogenesis of nAMD the standard of care are anti-VEGF therapies attempt to disrupt this vicious cycle. These current anti-VEGF therapies try to maintain vascular homeostasis while abating aberrant neovascularization but regrettably don't prevent fibrosis or scar formation. In addition, some patients demonstrate an incomplete response to anti-VEGF therapy as demonstrated by progressive vision loss. Here, we show choroidal endothelial cells (ChEC) incubated with artesunate demonstrated decreased migration and inflammatory and fibrotic factor expression, which corresponded with decreased sprouting in a choroid/retinal pigment epithelium (RPE) explant sprouting angiogenesis assay. To assess the efficacy of artesunate to curtail neovascularization in vivo, we utilized laser photocoagulation-induced rupture of the Bruch's membrane to induce choroidal neovascularization (CNV). Artesunate significantly inhibited CNV and the accompanying fibrotic scar, perhaps due in part to its ability to inhibit mononuclear phagocyte (MP) recruitment. Thus, artesunate shows promise in inhibiting both CNV and fibrosis.

PMID:37783334 | DOI:10.1016/j.exer.2023.109666

Diabetologia. 2023 Nov;66(11):2170-2185. doi: 10.1007/s00125-023-05995-4. Epub 2023 Sep 5.

ABSTRACT

AIMS/HYPOTHESIS: The loss of pericytes surrounding the retinal vasculature in early diabetic retinopathy underlies changes to the neurovascular unit that lead to more destructive forms of the disease. However, it is unclear which changes lead to loss of retinal pericytes. This study investigated the hypothesis that chronic increases in one or more inflammatory factors mitigate the signalling pathways needed for pericyte survival.

METHODS: Loss of pericytes and levels of inflammatory markers at the mRNA and protein levels were investigated in two genetic models of diabetes, Ins2^Akita/+ (a model of type 1 diabetes) and Lepr^db/db (a model of type 2 diabetes), at early stages of diabetic retinopathy. In addition, changes that accompany gliosis and the retinal vasculature were determined. Finally, changes in retinal pericytes chronically incubated with vehicle or increasing amounts of IFNγ were investigated to determine the effects on pericyte survival. The numbers of pericytes, microglia, astrocytes and endothelial cells in retinal flatmounts were determined by immunofluorescence. Protein and mRNA levels of inflammatory factors were determined using multiplex ELISAs and quantitative reverse transcription PCR (qRT-PCR). The effects of IFNγ on the murine retinal pericyte survival-related platelet-derived growth factor receptor β (PDGFRβ) signalling pathway were investigated by western blot analysis. Finally, the levels of cell death-associated protein kinase C isoform delta (PKCδ) and cleaved caspase 3 (CC3) in pericytes were determined by western blot analysis and immunocytochemistry.

RESULTS: The essential findings of this study were that both type 1 and 2 diabetes were accompanied by a similar progression of retinal pericyte loss, as well as gliosis. However, inflammatory factor expression was dissimilar in the two models of diabetes, with peak expression occurring at different ages for each model. Retinal vascular changes were more severe in the type 2 diabetes model. Chronic incubation of murine retinal pericytes with IFNγ decreased PDGFRβ signalling and increased the levels of active PKCδ and CC3.

CONCLUSIONS/INTERPRETATION: We conclude that retinal inflammation is involved in and sustains pericyte loss as diabetic retinopathy progresses. Moreover, IFNγ plays a critical role in reducing pericyte survival in the retina by reducing activation of the PDGFRβ signalling pathway and increasing PKCδ levels and pericyte apoptosis.

PMID:37670018 | PMC:PMC10541343 | DOI:10.1007/s00125-023-05995-4

Behav Brain Res. 2023 Oct 2;454:114657. doi: 10.1016/j.bbr.2023.114657. Epub 2023 Sep 7.

ABSTRACT

The hippocampus is a part of the brain's medial temporal lobe that is located under the cortex. It belongs to the limbic system and helps to collect and transfer information from short-term to long-term memory, as well as spatial orientation in each mammalian brain hemisphere. After more than two centuries of research in brain asymmetry, the hippocampus has attracted much attention in the study of brain lateralization. The hippocampus is very important in cognitive disorders, related to seizures and dementia, such as epilepsy and Alzheimer's disease. In addition, the motivation to study the hippocampus has increased significantly due to the asymmetry in the activity of the left and right hippocampi in healthy people, and its disruption during some neurological diseases. After a general review of the hippocampal structure and its importance in related diseases, the asymmetry in the brain with a focus on the hippocampus during the growth and maturation of healthy people, as well as the differences created in patients at the molecular, functional, and physiological levels are discussed. Most previous work indicates that the hippocampus is lateralized in healthy people. Also, lateralization at different levels remarkably changes in patients, and it appears that the most complex cognitive disorder is caused by a new dominant asymmetric system.

PMID:37683813 | DOI:10.1016/j.bbr.2023.114657

Nat Commun. 2023 Sep 25;14(1):5534. doi: 10.1038/s41467-023-41095-y.

ABSTRACT

Mesenchymal activation, characterized by dense stromal infiltration of immune and mesenchymal cells, fuels the aggressiveness of colorectal cancers (CRC), driving progression and metastasis. Targetable molecules in the tumor microenvironment (TME) need to be identified to improve the outcome in CRC patients with this aggressive phenotype. This study reports a positive link between high thrombospondin-1 (THBS1) expression and mesenchymal characteristics, immunosuppression, and unfavorable CRC prognosis. Bone marrow-derived monocyte-like cells recruited by CXCL12 are the primary source of THBS1, which contributes to the development of metastasis by inducing cytotoxic T-cell exhaustion and impairing vascularization. Furthermore, in orthotopically generated CRC models in male mice, THBS1 loss in the TME renders tumors partially sensitive to immune checkpoint inhibitors and anti-cancer drugs. Our study establishes THBS1 as a potential biomarker for identifying mesenchymal CRC and as a critical suppressor of antitumor immunity that contributes to the progression of this malignancy with a poor prognosis.

PMID:37749092 | PMC:PMC10520015 | DOI:10.1038/s41467-023-41095-y

Semin Cell Dev Biol. 2024 Mar 1;155(Pt B):32-44. doi: 10.1016/j.semcdb.2023.07.011. Epub 2023 Jul 27.

ABSTRACT

Angiogenesis is vital to developmental, regenerative and repair processes. It is normally regulated by a balanced production of pro- and anti-angiogenic factors. Alterations in this balance under pathological conditions are generally mediated through up-regulation of pro-angiogenic and/or downregulation of anti-angiogenic factors, leading to growth of new and abnormal blood vessels. The pathological manifestation of many diseases including cancer, ocular and vascular diseases are dependent on the growth of these new and abnormal blood vessels. Thrompospondin-1 (TSP1) was the first endogenous angiogenesis inhibitor identified and its anti-angiogenic and anti-inflammatory activities have been the subject of many studies. Studies examining the role TSP1 plays in pathogenesis of various ocular diseases and vascular dysfunctions are limited. Here we will discuss the recent studies focused on delineating the role TSP1 plays in ocular vascular development and homeostasis, and pathophysiology of various ocular and vascular diseases with a significant clinical relevance to human health.

PMID:37507331 | PMC:PMC10811293 | DOI:10.1016/j.semcdb.2023.07.011

J Ophthalmic Vis Res. 2023 Feb 21;18(1):51-59. doi: 10.18502/jovr.v18i1.12725. eCollection 2023 Jan-Mar.

ABSTRACT

PURPOSE: Adenosine signaling modulates ocular inflammatory processes, and its antagonism mitigates neovascularization in both newborns and preclinical models of ocular neovascularization including age-related macular degeneration (AMD). The adenosine receptor expression patterns have not been well characterized in the human retina and choroid.

METHODS: Here we examined the expression of adenosine receptor subtypes within the retina and choroid of human donor eyes with and without AMD. Antibodies specifically targeting adenosine receptor subtypes A1, A2A, A2B, and A3 were used to assess their expression patterns. Quantitative real-time PCR analysis was used to confirm gene expression of these receptors within the normal human retina and choroid.

RESULTS: We found that all four receptor subtypes were expressed in several layers of the retina, and within the retinal pigment epithelium and choroid. The expression of A1 receptors was more prominent in the inner and outer plexiform layers, where microglia normally reside, and supported by RNA expression in the retina. A2A and A2B showed similar expression patterns with prominent expression in the vasculature and retinal pigment epithelium. No dramatic differences in expression of these receptors were observed in eyes from patients with dry or wet AMD compared to control, with the exception A3 receptors. Eyes with dry AMD lost expression of A3 in the photoreceptor outer segments compared with eyes from control or wet AMD.

CONCLUSION: The ocular presence of adenosine receptors is consistent with their proposed role in modulation of inflammation in both the retina and choroid, and their potential targeting for AMD treatment.

PMID:36937188 | PMC:PMC10020792 | DOI:10.18502/jovr.v18i1.12725

Int J Mol Sci. 2023 Jan 26;24(3):2420. doi: 10.3390/ijms24032420.

ABSTRACT

Cytochrome P450 (CYP) 1B1 is a heme-containing monooxygenase found mainly in extrahepatic tissues, including the retina. CYP1B1 substrates include exogenous aromatic hydrocarbons, such as dioxins, and endogenous bioactive compounds, including 17β-estradiol (E2) and arachidonic acid. The endogenous compounds and their metabolites are mediators of various cellular and physiological processes, suggesting that CYP1B1 activity is likely important in maintaining proper cellular and tissue functions. We previously demonstrated that lack of CYP1B1 expression and activity are associated with increased levels of reactive oxygen species and oxidative stress in the retinal vasculature and vascular cells, including retinal endothelial cells (ECs). However, the detailed mechanism(s) of how CYP1B1 activity modulates redox homeostasis remained unknown. We hypothesized that CYP1B1 metabolism of E2 affects bone morphogenic protein 6 (BMP6)-hepcidin-mediated iron homeostasis and lipid peroxidation impacting cellular redox state. Here, we demonstrate retinal EC prepared from Cyp1b1-deficient (Cyp1b1^-/-) mice exhibits increased estrogen receptor-α (ERα) activity and expresses higher levels of BMP6. BMP6 is an inducer of the iron-regulatory hormone hepcidin in the endothelium. Increased hepcidin expression in Cyp1b1^-/- retinal EC resulted in decreased levels of the iron exporter protein ferroportin and, as a result, increased intracellular iron accumulation. Removal of excess iron or antagonism of ERα in Cyp1b1^-/- retinal EC was sufficient to mitigate increased lipid peroxidation and reduce oxidative stress. Suppression of lipid peroxidation and antagonism of ERα also restored ischemia-mediated retinal neovascularization in Cyp1b1^-/- mice. Thus, CYP1B1 expression in retinal EC is important in the regulation of intracellular iron levels, with a significant impact on ocular redox homeostasis and oxidative stress through modulation of the ERα/BMP6/hepcidin axis.

PMID:36768740 | PMC:PMC9916835 | DOI:10.3390/ijms24032420

Cells. 2023 Jan 16;12(2):335. doi: 10.3390/cells12020335.

ABSTRACT

The integrity of retinal endothelial cell (EC) is essential for establishing and maintaining the retinal blood barrier to ensure proper vision. Vitamin D is a hormone with known protective roles in EC function. The majority of vitamin D action is mediated through the vitamin D receptor (VDR). VDR is a nuclear receptor whose engagement by vitamin D impacts the expression of many genes with important roles in regulation of angiogenesis and inflammation. Although many studies have investigated vitamin D-VDR action in cardiovascular protection and tumor angiogenesis, its impact on retinal EC function and regulation of ocular angiogenesis and inflammation is exceedingly limited. We previously showed calcitriol, the active form of vitamin D, is a potent inhibitor of retinal neovascularization in vivo and retinal EC capillary morphogenesis in vitro. Here, using retinal EC prepared from wild-type (Vdr^+/+) and VDR-deficient (Vdr^-/-) mice, we show that retinal EC express VDR and its expression is induced by calcitriol. The lack of VDR expression had a significant impact on endothelial cell-cell and cell-matrix interactions. Vdr^-/- retinal EC proliferated at a slower rate and were more adherent and less migratory. They also exhibited increased expression levels of inflammatory markers driven in part by sustained activation of STAT1 and NF-κB pathways and were more sensitive to oxidative challenge. These changes were attributed, in part, to down-regulation of endothelial nitric oxide synthetase, enhanced hepcidin expression, and increased intracellular iron levels. Taken together, our results indicate that VDR expression plays a fundamental role in maintaining the proper angiogenic and inflammatory state of retinal EC.

PMID:36672270 | PMC:PMC9856450 | DOI:10.3390/cells12020335

J Ophthalmic Vis Res. 2022 Nov 29;17(4):449-452. doi: 10.18502/jovr.v17i4.12294. eCollection 2022 Oct-Dec.

NO ABSTRACT

PMID:36620701 | PMC:PMC9806321 | DOI:10.18502/jovr.v17i4.12294

Cells. 2022 Oct 21;11(20):3329. doi: 10.3390/cells11203329.

ABSTRACT

The visualization of choroidal vasculature and innate immune cells in the eyes of pigmented mice has been challenging due to the presence of a retinal pigment epithelium (RPE) layer separating the choroid and retina. Here, we established methods for visualizing the choroidal macrophages, mast cells, and vasculature in eyes of albino and pigmented mice using cell type-specific staining. We were able to visualize the choroidal arterial and venous systems. An arterial circle around the optic nerve was found in mice similar to the Zinn-Haller arterial circle that exists in humans and primates. Three different structural patterns of choriocapillaris were observed throughout the whole choroid: honeycomb-like, maze-like, and finger-like patterns. Choroidal mast cells were relatively few but dense around the optic nerve. Mast cell distribution in the middle and periphery was different among strains. Macrophages were found in all layers of the choroid. Thus, utilizing the simple and reliable methods described herein will allow the evaluation of transgenic and preclinical mouse models of ocular diseases that affect the choroid, including age-related macular degeneration (AMD), diabetic choroidopathy, and retinopathy of prematurity. These studies will advance our understanding of the pathophysiology, and molecular and cellular mechanisms that can be targeted therapeutically, in these diseases.

PMID:36291198 | PMC:PMC9600292 | DOI:10.3390/cells11203329

Cells. 2022 Sep 20;11(19):2930. doi: 10.3390/cells11192930.

ABSTRACT

Cytochrome P450 (CYP) 1B1 belongs to the superfamily of heme-containing monooxygenases. Unlike other CYP enzymes, which are highly expressed in the liver, CYP1B1 is predominantly found in extrahepatic tissues, such as the brain, and ocular tissues including retina and trabecular meshwork. CYP1B1 metabolizes exogenous chemicals such as polycyclic aromatic hydrocarbons. CYP1B1 also metabolizes endogenous bioactive compounds including estradiol and arachidonic acid. These metabolites impact various cellular and physiological processes during development and pathological processes. We previously showed that CYP1B1 deficiency mitigates ischemia-mediated retinal neovascularization and drives the trabecular meshwork dysgenesis through increased levels of oxidative stress. However, the underlying mechanisms responsible for CYP1B1-deficiency-mediated increased oxidative stress remain largely unresolved. Iron is an essential element and utilized as a cofactor in a variety of enzymes. However, excess iron promotes the production of hydroxyl radicals, lipid peroxidation, increased oxidative stress, and cell damage. The retinal endothelium is recognized as a major component of the blood-retinal barrier, which controls ocular iron levels through the modulation of proteins involved in iron regulation present in retinal endothelial cells, as well as other ocular cell types including trabecular meshwork cells. We previously showed increased levels of reactive oxygen species and lipid peroxidation in the absence of CYP1B1, and in the retinal vasculature and trabecular meshwork, which was reversed by administration of antioxidant N-acetylcysteine. Here, we review the important role CYP1B1 expression and activity play in maintaining retinal redox homeostasis through the modulation of iron levels by retinal endothelial cells. The relationship between CYP1B1 expression and activity and iron levels has not been previously delineated. We review the potential significance of CYP1B1 expression, estrogen metabolism, and hepcidin-ferroportin regulatory axis in the local regulation of ocular iron levels.

PMID:36230892 | PMC:PMC9563809 | DOI:10.3390/cells11192930

Biomolecules. 2022 Sep 14;12(9):1295. doi: 10.3390/biom12091295.

ABSTRACT

Branching morphogenesis is a key developmental process during organogenesis, such that its disruption frequently leads to long-term consequences. The kidney and eye share many etiologies, perhaps, due to similar use of developmental branching morphogenesis and signaling pathways including cell death. Tipping the apoptotic balance towards apoptosis imparts a ureteric bud and retinal vascular branching phenotype similar to one that occurs in papillorenal syndrome. Here, to compare ureteric bud and retinal vascular branching in the context of decreased apoptosis, we investigated the impact of Bim, Bcl-2's rival force. In the metanephros, lack of Bim expression enhanced ureteric bud branching with increases in ureteric bud length, branch points, and branch end points. Unfortunately, enhanced ureteric bud branching also came with increased branching defects and other undesirable consequences. Although we did see increased nephron number and renal mass, we observed glomeruli collapse. Retinal vascular branching in the absence of Bim expression had similarities with the ureteric bud including increased vascular length, branching length, segment length, and branching interval. Thus, our studies emphasize the impact appropriate Bim expression has on the overall length and branching in both the ureteric bud and retinal vasculature.

PMID:36139134 | PMC:PMC9496469 | DOI:10.3390/biom12091295

Int J Biol Macromol. 2022 Jul 31;213:166-194. doi: 10.1016/j.ijbiomac.2022.05.156. Epub 2022 May 26.

ABSTRACT

The advances in producing multifunctional lipid-polymer hybrid nanoparticles (LPHNs) by combining the biomimetic behavior of liposomes and architectural advantages of polymers have provided great opportunities for selective and efficient therapeutics delivery. The constructed LPHNs exhibit different therapeutic efficacies for special uses based on characteristics of different excipients. However, the high mechanical/structural stability of hybrid nano-systems could be viewed as both a negative property and a positive feature, where the concomitant release of drug molecules in a controllable manner is required. In addition, difficulties in scaling up the LPHNs production, due to involvement of several criteria, limit their application for biomedical fields, especially in monitoring, bioimaging, and drug delivery. To address these challenges bio-modifications have exhibited enormous potential to prepare reproducible LPHNs for site-specific therapeutics delivery, diagnostic and preventative applications. The ever-growing surface bio-functionality has provided continuous vitality to this biotechnology and has also posed desirable biosafety to nanoparticles (NPs). As a proof-of-concept, this manuscript provides a crucial review of coated lipid and polymer NPs displaying excellent surface functionality and architectural advantages. We also provide a description of structural classifications and production methodologies, as well as the biomedical possibilities and translational obstacles in the development of surface modified nanocarrier technology.

PMID:35644315 | DOI:10.1016/j.ijbiomac.2022.05.156

Arch Oral Biol. 2022 Jul;139:105434. doi: 10.1016/j.archoralbio.2022.105434. Epub 2022 Apr 18.

ABSTRACT

OBJECTIVE: This study was performed to evaluate the effect of type 1 diabetes mellitus (T1DM) on the microhardness of tooth enamel and dentine in mice.

DESIGN: Seventy male C57BL/6 J mice were used in this study. Thirty-five mice were rendered diabetic by administration of streptozotocin (STZ), and the remaining animals received citrate buffer (normal/non-diabetic). In each group, specimens were divided into 7 subgroups of 5 mice based on the time points 0, 1, 4, 8, 12, 20, and 28 weeks. The microhardness value (MHV) of the second molars' enamel and root dentine were tested with a Vickers microhardness tester. Five specimens from each subgroup were evaluated for dentinal tubular density by scanning electron microscope (SEM) and color dot map analysis to determine the color intensity of strontium (Sr) and magnesium (Mg) by using ImageJ software.

RESULTS: The MHV of enamel was significantly reduced in STZ specimens in time points of 12 weeks (STZ: 274.39 ± 15.42, normal: 291.22 ± 15.28), 20 weeks (STZ: 247.28 ± 19.65, normal: 290.68 ± 11.52), and 28 weeks (STZ: 232.87 ± 15.07, normal: 282.76 ± 10.36) (P < 0.05). When comparing the MHV of dentine in subgroups of the normal group, after 20 weeks (169.1 ± 7.5) and 28 weeks (168.6 ± 7.81), the MHV increased significantly (P < 0.05). However, in the STZ group, a significant reduction of MHV was noticed between 28 weeks (131.69 ± 6.2) specimens with other subgroups (P < 0.05).

CONCLUSIONS: T1DM negatively affected enamel and dentine microhardness, and enamel was influenced much more negatively and rapidly compared with dentine in diabetic groups.

PMID:35525015 | DOI:10.1016/j.archoralbio.2022.105434

Life (Basel). 2022 Jan 29;12(2):208. doi: 10.3390/life12020208.

ABSTRACT

Inflammation is increasingly recognized as an important modulator in the pathogenesis of neovascular age-related macular degeneration (nAMD). Although significant progress has been made in delineating the pathways that contribute to the recruitment of inflammatory cells and their contribution to nAMD, we know little about what drives the resolution of these inflammatory responses. Gaining a better understanding of how immune cells are cleared in the choroid will give a novel insight into how sustained inflammation could influence the pathogenesis of nAMD. The pro-apoptotic Bcl-2 family member Bim is a master regulator of immune cell homeostasis. In its absence, immune cell lifespan and numbers increase. Most therapeutic regimes that squelch inflammation do so by enhancing immune cell apoptosis through enhanced Bim expression and activity. To test the hypothesis that Bim expression tempers inflammation during the pathogenesis of nAMD, we used the mouse laser-induced choroidal neovascularization (CNV) model in which inflammation acts as a facilitator of CNV. Here, we showed minimal to no change in the recruitment of F4/80-, CD80-, CD11b-, and Iba1-positive myeloid-derived mononuclear phagocytes to the site of laser photocoagulation in the absence of Bim expression. However, the resolution of these cells from the choroid of Bim-deficient (Bim -/-) mice was significantly diminished following laser photocoagulation. With time, we noted increased scar formation, demonstrated by collagen I staining, in Bim -/- mice with no change in the resolution of neovascularization compared to wild-type littermates. We also noted that mice lacking Bim expression in mononuclear phagocytes (Bim^Flox/Flox; Lyz2-Cre (Bim^MP) mice) had delayed resolution of F4/80-, CD80-, CD11b-, and Iba1-positive cells, while those lacking Bim expression in endothelial cells (Bim^Flox/Flox; Cad5-Cre (Bim^EC) mice) had delayed resolution of only CD11b- and Iba1-positive cells. Both Bim^MP and Bim^EC mice demonstrated increased scar formation, albeit to differing degrees. Thus, our studies show that resolving inflammation plays an important role in moderating scar formation in nAMD, and it is impacted by Bim expression in both the endothelium and mononuclear phagocyte lineages.

PMID:35207495 | PMC:PMC8878746 | DOI:10.3390/life12020208

PLoS Negl Trop Dis. 2022 Jan 5;16(1):e0010074. doi: 10.1371/journal.pntd.0010074. eCollection 2022 Jan.

ABSTRACT

The protozoan parasite, Trypanosoma cruzi, causes severe morbidity and mortality in afflicted individuals. Approximately 30% of T. cruzi infected individuals present with cardiac pathology. The invasive forms of the parasite are carried in the vascular system to infect other cells of the body. During transportation, the molecular mechanisms by which the parasite signals and interact with host endothelial cells (EC) especially heart endothelium is currently unknown. The parasite increases host thrombospondin-1 (TSP1) expression and activates the Wnt/β-catenin and hippo signaling pathways during the early phase of infection. The links between TSP1 and activation of the signaling pathways and their impact on parasite infectivity during the early phase of infection remain unknown. To elucidate the significance of TSP1 function in YAP/β-catenin colocalization and how they impact parasite infectivity during the early phase of infection, we challenged mouse heart endothelial cells (MHEC) from wild type (WT) and TSP1 knockout mice with T. cruzi and evaluated Wnt signaling, YAP/β-catenin crosstalk, and how they affect parasite infection. We found that in the absence of TSP1, the parasite induced the expression of Wnt-5a to a maximum at 2 h (1.73±0.13), P< 0.001 and enhanced the level of phosphorylated glycogen synthase kinase 3β at the same time point (2.99±0.24), P<0.001. In WT MHEC, the levels of Wnt-5a were toned down and the level of p-GSK-3β was lowest at 2 h (0.47±0.06), P< 0.01 compared to uninfected control. This was accompanied by a continuous significant increase in the nuclear colocalization of β-catenin/YAP in TSP1 KO MHEC with a maximum Pearson correlation coefficient of (0.67±0.02), P< 0.05 at 6 h. In WT MHEC, the nuclear colocalization of β-catenin/YAP remained steady and showed a reduction at 6 h (0.29±0.007), P< 0.05. These results indicate that TSP1 plays an important role in regulating β-catenin/YAP colocalization during the early phase of T. cruzi infection. Importantly, dysregulation of this crosstalk by pre-incubation of WT MHEC with a β-catenin inhibitor, endo-IWR 1, dramatically reduced the level of infection of WT MHEC. Parasite infectivity of inhibitor treated WT MHEC was similar to the level of infection of TSP1 KO MHEC. These results indicate that the β-catenin pathway induced by the parasite and regulated by TSP1 during the early phase of T. cruzi infection is an important potential therapeutic target, which can be explored for the prophylactic prevention of T. cruzi infection.

PMID:34986160 | PMC:PMC8730400 | DOI:10.1371/journal.pntd.0010074

PLoS One. 2021 Dec 2;16(12):e0260793. doi: 10.1371/journal.pone.0260793. eCollection 2021.

ABSTRACT

Retinopathy of prematurity (ROP) is one of the main causes of blindness in children worldwide. Brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), play critical protective roles in the development and function of neurons and vasculature. Lack of BDNF expression results in increased endothelial cell apoptosis and reduced endothelial cell-cell contact. Premature babies who develop ROP tend to have lower serum BDNF levels. BDNF expression is also significantly lower in mouse retinas following exposure to hyperoxia compared to those reared in room air. Specifically, BDNF promotes angiogenic tube formation of endothelial cells (EC), and it is considered an EC survival factor required for stabilization of intramyocardial vessels. We hypothesized that the activation of TrkB receptor protects retinal vasculature in the mice during oxygen-induced ischemic retinopathy (OIR), a model of ROP. To test this hypothesis, we treated neonatal mice with 7,8-dihydroxyflavone (DHF) (5 mg/kg body weight), a TrkB receptor agonist. We examined its potential protective effects on retinal vessel obliteration and neovascularization, two hallmarks of ROP and OIR. We found that retinas from DHF treated postnatal day 8 (P8) and P12 mice have similar levels of vessel obliteration as retinas from age-matched control mice subjected to OIR. Similarly, DHF showed no significant effect on mitigation of retinal neovascularization during OIR in P17 mice. Collectively, our studies demonstrate that the TrkB receptor agonist DHF provides no significant protective effects during OIR.

PMID:34855884 | PMC:PMC8638941 | DOI:10.1371/journal.pone.0260793

Front Cell Dev Biol. 2021 Oct 14;9:737426. doi: 10.3389/fcell.2021.737426. eCollection 2021.

ABSTRACT

Adenosine receptors (AR) are widely expressed in a variety of tissues including the retina and brain. They are involved in adenosine-mediated immune responses underlying the onset and progression of neurodegenerative diseases. The expression of AR has been previously demonstrated in some retinal cells including endothelial cells and retinal pigment epithelial cells, but their expression in the choroid and choroidal cells remains unknown. Caffeine is a widely consumed AR antagonist that can influence inflammation and vascular cell function. It has established roles in the treatment of neonatal sleep apnea, acute migraine, and post lumbar puncture headache as well as the neurodegenerative diseases such as Parkinson and Alzheimer. More recently, AR antagonism with caffeine has been shown to protect preterm infants from ischemic retinopathy and retinal neovascularization. However, whether caffeine impacts the development and progression of ocular age-related diseases including neovascular age-related macular degermation remains unknown. Here, we examined the expression of AR in retinal and choroidal tissues and cells. We showed that antagonism of AR with caffeine or istradefylline decreased sprouting of thoracic aorta and choroid/retinal pigment epithelium explants in ex vivo cultures, consistent with caffeine's ability to inhibit endothelial cell migration in culture. In vivo studies also demonstrated the efficacy of caffeine in inhibition of choroidal neovascularization and mononuclear phagocyte recruitment to the laser lesion sites. Istradefylline, a specific AR 2A antagonist, also decreased choroidal neovascularization. Collectively, our studies demonstrate an important role for expression of AR in the choroid whose antagonism mitigate choroidal inflammatory and angiogenesis activities.

PMID:34722519 | PMC:PMC8551619 | DOI:10.3389/fcell.2021.737426

Int J Mol Sci. 2021 Aug 28;22(17):9356. doi: 10.3390/ijms22179356.

ABSTRACT

Age-related macular degeneration (AMD) is a leading cause of vision loss. Elevated homocysteine (Hcy) (Hyperhomocysteinemia) (HHcy) has been reported in AMD. We previously reported that HHcy induces AMD-like features. This study suggests that N-Methyl-d-aspartate receptor (NMDAR) activation in the retinal pigment epithelium (RPE) is a mechanism for HHcy-induced AMD. Serum Hcy and cystathionine-β-synthase (CBS) were assessed by ELISA. The involvement of NMDAR in Hcy-induced AMD features was evaluated (1) in vitro using ARPE-19 cells, primary RPE isolated from HHcy mice (CBS), and mouse choroidal endothelial cells (MCEC); (2) in vivo using wild-type mice and mice deficient in RPE NMDAR (NMDAR^R-/-) with/without Hcy injection. Isolectin-B4, Ki67, HIF-1α, VEGF, NMDAR1, and albumin were assessed by immunofluorescence (IF), Western blot (WB), Optical coherence tomography (OCT), and fluorescein angiography (FA) to evaluate retinal structure, fluorescein leakage, and choroidal neovascularization (CNV). A neovascular AMD patient's serum showed a significant increase in Hcy and a decrease in CBS. Hcy significantly increased HIF-1α, VEGF, and NMDAR in RPE cells, and Ki67 in MCEC. Hcy-injected WT mice showed disrupted retina and CNV. Knocking down RPE NMDAR improved retinal structure and CNV. Our findings underscore the role of RPE NMDAR in Hcy-induced AMD features; thus, NMDAR inhibition could serve as a promising therapeutic target for AMD.

PMID:34502266 | PMC:PMC8431693 | DOI:10.3390/ijms22179356

Anticancer Agents Med Chem. 2022;22(10):1897-1912. doi: 10.2174/1871520621666210906101421.

ABSTRACT

Lipid-based nanoparticles, as drug delivery carriers, are commonly used for the delivery of anti-cancer therapeutic agents. Due to their smaller particle size and similarity to cell membranes, Lipid-based nanoparticles are readily internalized into cancer cells. Cancer cells also overexpress receptors for specific ligands, including folic acid, hyaluronic acid, and transferrin, on their surface, thus, allowing the use of their ligands for surface modification of the lipid-based nanoparticles for their specific recognition by receptors on cancer cells. This would also allow the gradual intracellular accumulation of the targeted functionalized nanoplatforms. These ligand-receptor interactions eventually enhance the internalization of desired drugs by increasing the nanoplatforms cellular uptake. The cellular internalization of the nanoplatforms varies and depends on their physicochemical properties, including particle size, zeta potential, and shape. The cellular uptake is also influenced by the types of ligand internalization pathways utilized by cells, such as phagocytosis, macropinocytosis, and multiple endocytosis pathways. This review classifies and discusses lipidbased nanoparticles engineered to carry specific ligands, their recognition by receptors on cancer cells, and their cellular internalization pathways. Moreover, the intracellular fate of nanoparticles decorated with specific ligands and their best internalization pathway (caveolae-mediated endocytosis) for safe cargo delivery are also discussed.

PMID:34488605 | DOI:10.2174/1871520621666210906101421

J Ophthalmic Vis Res. 2021 Jul 29;16(3):317-319. doi: 10.18502/jovr.v16i3.9427. eCollection 2021 Jul-Sep.

NO ABSTRACT

PMID:34394859 | PMC:PMC8358751 | DOI:10.18502/jovr.v16i3.9427

Genes (Basel). 2021 Jul 20;12(7):1102. doi: 10.3390/genes12071102.

ABSTRACT

The transcription factor high mobility group protein A2 (HMGA2) plays an important role in the pathogenesis of some cancers including breast cancer. Polyamidoamine dendrimer generation 4 is a kind of highly branched polymeric nanoparticle with surface charge and highest density peripheral groups that allow ligands or therapeutic agents to attach it, thereby facilitating target delivery. Here, methotrexate (MTX)- modified polyamidoamine dendrimer generation 4 (G4) (G4/MTX) was generated to deliver specific small interface RNA (siRNA) for suppressing HMGA2 expression and the consequent effects on folate receptor (FR) expressing human breast cancer cell lines (MCF-7, MDA-MB-231). We observed that HMGA2 siRNA was electrostatically adsorbed on the surface of the G4/MTX nanocarrier for constructing a G4/MTX-siRNA nano-complex which was verified by changing the final particle size and zeta potential. The release of MTX and siRNA from synthesized nanocomplexes was found in a time- and pH-dependent manner. We know that MTX targets FR. Interestingly, G4/MTX-siRNA demonstrates significant cellular internalization and gene silencing efficacy when compared to the control. Besides, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay demonstrated selective cell cytotoxicity depending on the folate receptor expressing in a dose-dependent manner. The gene silencing and protein downregulation of HMGA2 by G4/MTX-siRNA was observed and could significantly induce cell apoptosis in MCF-7 and MDA-MB-231 cancer cells compared to the control group. Based on the findings, we suggest that the newly developed G4/MTX-siRNA nano-complex may be a promising strategy to increase apoptosis induction through HMGA2 suppression as a therapeutic target in human breast cancer.

PMID:34356120 | PMC:PMC8303903 | DOI:10.3390/genes12071102

Epilepsy Res. 2021 Oct;176:106727. doi: 10.1016/j.eplepsyres.2021.106727. Epub 2021 Jul 16.

ABSTRACT

Epilepsy is one of the foremost medical disorders. Oxidative stress is a well-known mechanism in epileptogenesis, and many studies suggest that oxidative stress affects the onset and evolution of epilepsy. Here we evaluated the walnut peptide extracts' anti-seizure property in three different mouse seizure models including pentylenetetrazole-induced clonic seizure, chemical kindling, and maximal electroshock. Walnut peptides (20 mg/Kg) were administered by intraperitoneal (IP) injection of mice 60 min before seizure induction in the three models. To delineate the mechanisms of walnut peptides anti-seizure activity, we evaluated the impact of diazepam, flumazenil, and a NOS inhibitor on this activity. Intraperitoneal administration of walnut peptides significantly increased the seizure threshold. Our results also demonstrated that walnut peptides exert their anti-seizure properties through the modulation of benzodiazepine receptors. Thus, walnut peptides may be considered as a new anti-convulsion agent, which can reduce seizure occurrence and slow down seizure progression.

PMID:34333374 | DOI:10.1016/j.eplepsyres.2021.106727

Pediatr Res. 2022 Jun;91(7):1677-1685. doi: 10.1038/s41390-021-01646-9. Epub 2021 Jul 20.

ABSTRACT

BACKGROUND: Pathologic ocular neovascularization in retinopathy of prematurity (ROP) and other proliferative retinopathies are characterized by dysregulation of vascular endothelial growth factor-A (VEGF-A). A study of Vegfa isoform expression during oxygen-induced ischemic retinopathy (OIR) may enhance our understanding of Vegf dysregulation.

METHODS: Following induction of OIR, immunohistochemistry and polymerase chain reaction (PCR) was performed on room air (RA) and OIR mice.

RESULTS: Total Vegfa messenger RNA (mRNA) expression was stable in RA mice, but increased in OIR mice with a peak at postnatal day 17 (P17), before returning to RA levels. Vegfa_164a expression was similar in both OIR and RA mice at P10 (Phase 1 OIR), but 2.4-fold higher in OIR mice compared to RA mice at P16 (Phase 2 OIR). At P10, Vegfa_164b mRNA was similar in OIR vs RA mice, but was expressed 2.5-fold higher in OIR mice compared to RA mice at P16. At P10 and P16, Vegfr2/Vegfr1 expression was increased in OIR mice compared to RA mice. Increased activation of microglia was seen in OIR mice.

CONCLUSIONS: Vegfa_164a, Vegfa_164b, and Vegfr1 were overexpressed in OIR mice, leading to abnormal signaling and angiogenesis. Further studies of mechanisms of Vegf dysregulation may lead to novel therapies for ROP and other proliferative retinopathies.

IMPACT: Vegfa₁₆₄ has two major isoforms, a proangiogenic, Vegfa_164a, and an antiangiogenic, Vegfa_164b, with opposing receptors, inhibitory Vegfr1, and stimulatory Vegfr2, but their role in OIR is unclear. In Phase 1 OIR, both isoforms and receptors are expressed similarly. In Phase 2 OIR, both isoforms are overexpressed, with an increased ratio of inhibitory Vegfr1. Modulation of angiogenesis by Vegf regulation enables pruning of excess angiogenesis during physiology, but results in ineffective angiogenesis during OIR. Knowledge of VEGF dysregulation may have novel therapeutic implications in the management of ROP and retinal proliferative diseases.

PMID:34285351 | PMC:PMC8770670 | DOI:10.1038/s41390-021-01646-9

Sci Rep. 2021 Jun 16;11(1):12670. doi: 10.1038/s41598-021-90447-5.

ABSTRACT

Ischemic stroke is a major cause of long-term disabilities, including vision loss. Neuronal and blood vessel maturation can affect the susceptibility of and outcome after ischemic stroke. Although we recently reported that exposure of neonatal mice to hypoxia-ischemia (HI) severely compromises the integrity of the retinal neurovasculature, it is not known whether juvenile mice are similarly impacted. Here we examined the effect of HI injury in juvenile mice on retinal structure and function, in particular the susceptibility of retinal neurons and blood vessels to HI damage. Our studies demonstrated that the retina suffered from functional and structural injuries, including reduced b-wave, thinning of the inner retinal layers, macroglial remodeling, and deterioration of the vasculature. The degeneration of the retinal vasculature associated with HI resulted in a significant decrease in the numbers of pericytes and endothelial cells as well as an increase in capillary loss. Taken together, these findings suggest a need for juveniles suffering from ischemic stroke to be monitored for changes in retinal functional and structural integrity. Thus, there is an emergent need for developing therapeutic approaches to prevent and reverse retinal neurovascular dysfunction with exposure to ischemic stroke.

PMID:34135369 | PMC:PMC8209038 | DOI:10.1038/s41598-021-90447-5

Front Cell Dev Biol. 2021 Apr 21;9:671989. doi: 10.3389/fcell.2021.671989. eCollection 2021.

ABSTRACT

Tight regulation of positive and negative regulators of angiogenesis is essential, particularly in the eye where their dysregulation can lead to vision loss. Thrombospondin-1 (TSP1) is a matricellular protein that negatively regulates angiogenesis and inflammation in the eye. It aids ocular vascular homeostasis such that its loss contributes to increased retinal vascular density and pathologic ocular neovascularization. Our previous studies demonstrated that mice globally lacking TSP1 expression had increased retinal vascular density, decreased hyperoxia-induced retinal vessel loss, and increased choroidal neovascularization. Here we determined the impact to the ocular vasculature of endothelial cell, pericyte, or astrocyte loss of TSP1 expression. Only lack of TSP1 expression in endothelial cells was sufficient to increase choroidal neovascularization with mice lacking expression in pericytes or astrocytes not demonstrating a significant impact. Although the global TSP1 knockout mice demonstrated increased retinal vascular density, individual cell type loss of TSP1 resulted in decreased retinal endothelial cell numbers before and/or after vascular maturation in a cell type specific fashion. Retinas from mice lacking TSP1 expression in endothelial cells, pericytes or astrocytes were not protected from retinal vessel regression in response to hyperoxia as we previously observed in the global knockout. Thus, modulation of TSP1 expression in individual cell types demonstrates a response that is unique to the role TSP1 plays in that cell type of interest, and their coordinated activity is critical for vision.

PMID:33968943 | PMC:PMC8097095 | DOI:10.3389/fcell.2021.671989

Int J Pharm. 2021 Apr 15;599:120421. doi: 10.1016/j.ijpharm.2021.120421. Epub 2021 Mar 4.

ABSTRACT

Aiming to simultaneous target of methotrexate (MTX), as folate antagonist, and conferone (CON) in various cancer cells, the newly lipid/polymer hybrid nanoparticle containing an albumin targeted succinylchitosan shell and lipoid bilayer core composed of hydrogenated soy phosphatidylcholine and cholesterol was synthesized. The covalently conjugating albumin to the external surface of chitosan was accomplished using N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and N- hydroxyl succinimide as an activating carboxylic group, and nanoliposomes were fabricated via thin film hydration-sonication method. The molecular structure of MTX@CON-targeted lipid/polymer hybrid nanoparticle (MTX@CON-TLPN) were characterized using FTIR spectroscopy, ¹H NMR, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and dynamic light scattering (DLS). The newly nanoparticle with high encapsulation efficiency (85.12%, and 78.4%), acceptable loading capacity (9.8% and 4.6% for MTX and CON) and the stimuli responsiveness drug release behavior in simulated physiologic tumor tissue condition (pH 5.4, 40 °C) was successfully synthetized in the spherical shape with mean average size of approximately 290 nm and ζ-potential of +21 mv. The enhanced efficiency of the targeted nanoparticle was further confirmed using MTT endpoints, cell cycle modulation, apoptosis assessment, and cellular internalization assessments. Collectively, these findings establish the utility of our newly prepared nanoparticle for simultaneous delivery of multiple anti-cancer drugs.

PMID:33676992 | DOI:10.1016/j.ijpharm.2021.120421

J Endod. 2021 Apr;47(4):612-620. doi: 10.1016/j.joen.2020.12.007. Epub 2020 Dec 23.

ABSTRACT

INTRODUCTION: Endodontic sealers play a vital role in the obturation of root canal space. The aim of this study was to evaluate the utility of a recently developed polyurethane expandable sealer (PES), along with its cytotoxicity and dimensional changes.

METHODS: L929 fibroblasts and an cell viability assay (MTS assay) were used to determine the cytotoxicity of dental sealers (AH Plus [Dentsply Maillefer, Ballaigues, Switzerland], Sure-Seal Root [Sure Dent Corporation, Gyeonggi-do, South Korea], and the PES) at 24, 48, 72, and 96 hours. An advanced choroidal neovascularization model was used to assess the effect of these sealers on angiogenesis. Thirty-six extracted single-rooted human teeth were prepared and randomly divided into 3 groups (n = 12). Obturation was performed with gutta-percha and a sealer using lateral compaction as follows: group 1, AH Plus; group 2, Sure-Seal; and group 3, PES. The average depth of sealer penetration into dentinal tubules was measured with a scanning electron microscope. Data were analyzed using 1-way analysis of variance and post hoc Tukey tests (level of significance, P < .05).

RESULTS: The values of MTS, choroidal neovascularization, and the penetration depth of PES were significantly higher than in other experimental groups (P < .05). The lowest values were noted in specimens of AH Plus, whereas the highest were detected in the PES group.

CONCLUSIONS: PES showed promising results in terms of biocompatibility and dentinal tubule adaptation and penetration.

PMID:33359533 | PMC:PMC8764616 | DOI:10.1016/j.joen.2020.12.007

Luminescence. 2021 May;36(3):658-667. doi: 10.1002/bio.3982. Epub 2020 Dec 2.

ABSTRACT

Metformin (MET), as an oral antidiabetic and antihyperglycemic agent, is widely used to treat type II diabetes mellitus. Because of its increasing consumption, developing a fast, simple, and selective method to determine its concentration in biological samples (serum and urine) and pharmaceutical formulations (tablets) is of great interest. In this study, we used a FRET-based fluorescent nanosensor (Tb-phen-AgNPs system) for sensitive detection of MET in tablet and serum samples. This method is based on the enhancing effect of MET on the emission intensity of the Tb-phen complex, which is quenched by AgNPs via energy transfer process (turn off-on mode). A good linear relationship between the MET concentration and enhanced emission intensity of the Tb-phen-AgNPs system was observed in the range of (0.75-3.7) × 10^-6 M under optimum conditions. Limit of detection and limit of quantitation were calculated to be 0.43 × 10^-6 M and 1.31 × 10^-6 M, respectively. This method was successfully used to determine MET concentrations in pharmaceutical dosage form and in spiked serum sample. The obtained recoveries from pharmaceutical formulation and serum sample were in the range 86.75-98.97% and 85.10-100.96%, respectively. Collectively, our results indicated that the method described here is simple, sensitive, cost effective, and free from interference. Therefore, it can be used as an effective and routine method for the direct and rapid determination of MET levels in biological samples such as serum.

PMID:33185014 | DOI:10.1002/bio.3982

Sci Rep. 2020 Oct 15;10(1):17370. doi: 10.1038/s41598-020-73486-2.

ABSTRACT

Diabetes associated complications, including diabetic retinopathy and loss of vision, are major health concerns. Detecting early retinal vascular changes during diabetes is not well documented, and only few studies have addressed this domain. The purpose of this study was to noninvasively evaluate temporal changes in retinal vasculature at very early stages of diabetes using fundus images from preclinical models of diabetes. Non-diabetic and Akita/+ male mice with different duration of diabetes were subjected to fundus imaging using a Micron III imaging system. The images were obtained from 4 weeks- (onset of diabetes), 8 weeks-, 16 weeks-, and 24 weeks-old male Akita/+ and non-diabetic mice. In total 104 fundus images were subjected to analysis for various feature extractions. A combination of Canny Edge Detector and Angiogenesis Analyzer plug-ins in ImageJ were utilized to quantify various retinal vascular changes in fundus images. Statistical analyses were conducted to determine significant differences in the various extracted features from fundus images of diabetic and non-diabetic animals. Our novel image analysis method led to extraction of over 20 features. These results indicated that some of these features were significantly changed with a short duration of diabetes, and others remained the same but changed after longer duration of diabetes. These patterns likely distinguish acute (protective) and chronic (damaging) associated changes with diabetes. We show that with a combination of various plugging one can extract over 20 features from retinal vasculature fundus images. These features change during diabetes, thus allowing the quantification of quality of retinal vascular architecture as biomarkers for disease progression. In addition, our method was able to identify unique differences among diabetic mice with different duration of diabetes. The ability to noninvasively detect temporal retinal vascular changes during diabetes could lead to identification of specific markers important in the development and progression of diabetes mediated-microvascular changes, evaluation of therapeutic interventions, and eventual reversal of these changes in order to stop or delay disease progression.

PMID:33060607 | PMC:PMC7567079 | DOI:10.1038/s41598-020-73486-2

Arterioscler Thromb Vasc Biol. 2020 Dec;40(12):e350-e366. doi: 10.1161/ATVBAHA.120.314913. Epub 2020 Oct 8.

ABSTRACT

OBJECTIVE: Abdominal aortic aneurysm is characterized by the progressive loss of aortic integrity and accumulation of inflammatory cells primarily macrophages. We previously reported that global deletion of matricellular protein TSP1 (thrombospondin-1) protects mice from aneurysm formation. The objective of the current study is to investigate the cellular and molecular mechanisms underlying TSP1's action in aneurysm. Approach and Results: Using RNA fluorescent in situ hybridization, we identified macrophages being the major source of TSP1 in human and mouse aneurysmal tissues, accounting for over 70% of cells that actively expressed Thbs1 mRNA. Lack of TSP1 in macrophages decreased solution-based gelatinase activities by elevating TIMP1 (tissue inhibitor of metalloproteinases-1) without affecting the major MMPs (matrix metalloproteinases). Knocking down Timp1 restored the ability of Thbs1^-/- macrophages to invade matrix. Finally, we generated Thbs1^flox/flox mice and crossed them with Lyz2-cre mice. In the CaCl₂-induced model of abdominal aortic aneurysm, lacking TSP1 in myeloid cells was sufficient to protect mice from aneurysm by reducing macrophage accumulation and preserving aortic integrity.

CONCLUSIONS: TSP1 contributes to aneurysm pathogenesis, at least in part, by suppressing TIMP1 expression, which subsequently enables inflammatory macrophages to infiltrate vascular tissues.

PMID:33028100 | PMC:PMC7686278 | DOI:10.1161/ATVBAHA.120.314913

Int J Mol Sci. 2020 Jul 12;21(14):4912. doi: 10.3390/ijms21144912.

ABSTRACT

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease. This neglected tropical disease causes severe morbidity and mortality in endemic regions. About 30% of T. cruzi infected individuals will present with cardiac complications. Invasive trypomastigotes released from infected cells can be carried in the vascular endothelial system to infect neighboring and distant cells. During the process of cellular infection, the parasite induces host cells, to increase the levels of host thrombospondin-1 (TSP-1), to facilitate the process of infection. TSP-1 plays important roles in the functioning of vascular cells, including vascular endothelial cells with important implications in cardiovascular health. Many signal transduction pathways, including the yes-associated protein 1 (YAP)/transcriptional coactivator, with PDZ-binding motif (TAZ) signaling, which are upstream of TSP-1, have been linked to the pathophysiology of heart damage. The molecular mechanisms by which T. cruzi signals, and eventually infects, heart endothelial cells remain unknown. To evaluate the importance of TSP-1 expression in heart endothelial cells during the process of T. cruzi infection, we exposed heart endothelial cells prepared from Wild Type and TSP-1 Knockout mouse to invasive T. cruzi trypomastigotes at multiple time points, and evaluated changes in the hippo signaling cascade using immunoblotting and immunofluorescence assays. We found that the parasite turned off the hippo signaling pathway in TSP-1KO heart endothelial cells. The levels of SAV1 and MOB1A increased to a maximum of 2.70 ± 0.23 and 5.74 ± 1.45-fold at 3 and 6 h, respectively, in TSP-1KO mouse heart endothelial cells (MHEC), compared to WT MHEC, following a parasite challenge. This was accompanied by a significant continuous increase in the nuclear translocation of downstream effector molecule YAP, to a maximum mean nuclear fluorescence intensity of 10.14 ± 0.40 at 6 h, compared to wild type cells. Furthermore, we found that increased nuclear translocated YAP significantly colocalized with the transcription co-activator molecule pan-TEAD, with a maximum Pearson's correlation coefficient of 0.51 ± 0.06 at 6 h, compared to YAP-Pan-TEAD colocalization in the WT MHEC, which decreased significantly, with a minimum Pearson's correlation coefficient of 0.30 ± 0.01 at 6 h. Our data indicate that, during the early phase of infection, upregulated TSP-1 is essential for the regulation of the hippo signaling pathway. These studies advance our understanding of the molecular interactions occurring between heart endothelial cells and T. cruzi, in the presence and absence of TSP-1, providing insights into processes linked to parasite dissemination and pathogenesis.

PMID:32664627 | PMC:PMC7403984 | DOI:10.3390/ijms21144912

J Endod. 2020 Aug;46(8):1113-1119. doi: 10.1016/j.joen.2020.04.005. Epub 2020 Jun 24.

ABSTRACT

INTRODUCTION: This study evaluated the effect of different pH values of 4.4, 5.4, 6.4, 7.4, 8.4, and 9.4 on angiogenesis.

METHODS: Endothelial cells were isolated from the mice molar teeth and placed in 42 Matrigel (Corning, NY)-coated wells, which were prepared and divided into 6 groups (n = 7). Synthetic tissue fluid was prepared and divided into 6 parts, and their pH values were adjusted to 4.4, 5.4, 6.4, 7.4, 8.4, and 9.4. A 2-mL volume from each group was diluted in the growth medium at a ratio of 1:3 and used for tubulogenesis assay. Forty-two 6-week-old mice in 6 groups (n = 7) were used for choroidal neovascularization (CNV). A 2-μL volume from each group or saline (control) was delivered by intravitreal injection on the day of laser application and 1 week later. Data on the number of nodes, the total length of the branches, and CNV areas (μm²) were determined using ImageJ software (National Institutes of Health, Bethesda, MD) and analyzed with 1-way analysis of variance and post hoc Tukey tests. The correlation was assessed between the tested variables.

RESULTS: The number of nodes decreased with changes in pH values as follows: 6.4 > 5.4 > 7.4 > 8.4 > 9.4 > 4.4. The total branch length decreased with pH value changes as follows: 6.4 > 4.4 > 6.4 > 7.4 > 8.4 > 9.4, and the CNV areas decreased with pH value changes as follows: 6.4 > 5.4 > 4.4 > 7.4 > 8.4 > 9.4.

CONCLUSIONS: Moderately acidic pH values (5.4 and 6.4) enhanced angiogenesis, whereas moderately alkaline pH values (8.4 and 9.4) suppressed angiogenesis.

PMID:32593435 | PMC:PMC8774255 | DOI:10.1016/j.joen.2020.04.005

Int J Mol Sci. 2020 Jun 1;21(11):3983. doi: 10.3390/ijms21113983.

ABSTRACT

We have shown that a high fat diet (HFD) induces the activation of retinal NOD-like receptor protein (NLRP3)-inflammasome that is associated with enhanced expression and interaction with thioredoxin-interacting protein (TXNIP). Here, the specific contribution of TXNIP and the impact of HFD on retinal leukostasis, barrier dysfunction and microvascular degeneration were investigated. Wild-type (WT) and TXNIP knockout (TKO) mice were fed with normal diet or 60% HFD for 8-18 weeks. TXNIP was overexpressed or silenced in human retinal endothelial cells (REC). At 8 weeks, HFD significantly induced retinal leukostasis and breakdown of the blood-retina barrier in WT mice, but not in TKO mice. In parallel, HFD also induced retinal expression of adhesion molecules and cleaved IL-1β in WT mice, which were also abrogated in TKO mice. In culture, TXNIP overexpression induced NLRP3, IL-1b, and adhesion molecules expression, while TXNIP silencing inhibited them. Blocking the IL-1β receptor significantly suppressed TXNIP-induced expression of NLRP3-inflammasome and adhesion molecules in HREC. Ex-vivo assay showed that leukocytes isolated from WT-HFD, but not from TKO-HFD, induced leukostasis and cell death. At 18 weeks, HFD triggered development of degenerated (acellular) capillaries and decreased branching density in WT but not in TKO mice. Together, HFD-induced obesity triggered early retinal leukostasis and microvascular dysfunction at least in part via TXNIP-NLRP3-inflammasome activation.

PMID:32492941 | PMC:PMC7312035 | DOI:10.3390/ijms21113983

PLoS One. 2020 May 4;15(5):e0232779. doi: 10.1371/journal.pone.0232779. eCollection 2020.

ABSTRACT

Apoptosis of neurovascular cells, including astroglial cells, contributes to the pathogenesis of diseases in which neurovascular disruption plays a central role. Bim is a pro-apoptotic protein that modulates not only apoptosis but also various cellular functions such as migration and extracellular matrix protein expression. Astroglial cells act as an intermediary between neural and vascular cells facilitating retinal vascular development and remodeling while maintaining normal vascular function and neuronal integrity. We previously showed that Bim deficient (Bim -/-) mice were protected from hyperoxia mediated vessel obliteration and ischemia-mediated retinal neovascularization. However, the underlying mechanisms and more specifically the role Bim expression in astroglial cells play remains elusive. Here, using retinal astroglial cells prepared from wild-type and Bim -/- mice, we determined the impact of Bim expression in retinal astroglial cell function. We showed that astroglial cells lacking Bim expression demonstrate increased VEGF expression and altered matricellular protein production including increased expression of thrombospondin-2 (TSP2), osteopontin and SPARC. Bim deficient astroglial cells also exhibited altered proliferation, migration, adhesion to various extracellular matrix proteins and increased expression of inflammatory mediators. Thus, our data emphasizes the importance of Bim expression in retinal astroglia cell autonomous regulatory mechanisms, which could influence neurovascular function.

PMID:32365083 | PMC:PMC7197808 | DOI:10.1371/journal.pone.0232779

PLoS One. 2020 Apr 24;15(4):e0231752. doi: 10.1371/journal.pone.0231752. eCollection 2020.

ABSTRACT

Astrocytes (AC) are the most abundant cells in the central nervous system. In the retina, astrocytes play important roles in the development and integrity of the retinal neurovasculature. Astrocytes dysfunction contributes to pathogenesis of a variety of neurovascular diseases including diabetic retinopathy. Recent studies have demonstrated the expression of Cyp1b1 in the neurovascular cells of the central nervous system including AC. We recently showed retinal AC constitutively express Cyp1b1, and global Cyp1b1-deficiency (Cyp1b1-/-) attenuates retinal ischemia-mediated neovascularization in vivo and the pro-angiogenic activity of retinal vascular cells in vitro. We also demonstrated that Cyp1b1 expression is a key regulator of retinal AC function. However, the underlying mechanisms involved need further investigation. Here we determined changes in the transcriptome profiles of Cyp1b1+/+ and Cyp1b1-/- retinal AC by RNA sequencing. We identified 585 differentially expressed genes, whose pathway enrichment analysis revealed the most significant pathways impacted in Cyp1b1-/- AC. These genes included those of axon guidance, extracellular matrix proteins and their receptors, cancer, cell adhesion molecules, TGF-β signaling, and the focal adhesion modulation. The expression of a selected set of differentially expressed genes was confirmed by RT-qPCR analysis. To our knowledge, this is the first report of RNAseq investigation of the retinal AC transcriptome and the molecular pathways impacted by Cyp1b1 expression. These results demonstrated an important role for Cyp1b1 expression in the regulation of various retinal AC functions, which are important in neurovascular development and integrity.

PMID:32330152 | PMC:PMC7182235 | DOI:10.1371/journal.pone.0231752

Appl In Vitro Toxicol. 2019 Jun 1;5(2):92-110. doi: 10.1089/aivt.2018.0025. Epub 2019 Jun 17.

ABSTRACT

Introduction: Human-induced pluripotent stem cells (iPSCs) represent a promising cell source for the construction of organotypic culture models for chemical toxicity screening and characterization. Materials and Methods: To characterize the effects of chemical exposure on the human neurovasculature, we constructed neurovascular unit (NVU) models consisting of endothelial cells (ECs) and astrocytes (ACs) derived from human-iPSCs, as well as human brain-derived pericytes (PCs). The cells were cocultured on synthetic poly(ethylene glycol) (PEG) hydrogels that guided the self-assembly of capillary-like vascular networks. High-content epifluorescence microscopy evaluated dose-dependent changes to multiple aspects of NVU morphology. Results: Cultured vascular networks underwent quantifiable morphological changes when incubated with vascular disrupting chemicals. The activity of predicted vascular disrupting chemicals from a panel of 38 compounds (U.S. Environmental Protection Agency) was ranked based on morphological features detected in the NVU model. In addition, unique morphological neurovascular disruption signatures were detected per chemical. A comparison of PEG-based NVU and Matrigel^™-based NVU models found greater sensitivity and consistency in chemical detection by the PEG-based NVU models. Discussion: We suspect that specific morphological changes may be used for discerning adverse outcome pathways initiated by chemical exposure and rapid mechanistic characterization of chemical exposure to neurovascular function. Conclusion: The use of human stem cell-derived vascular tissue and PEG hydrogels in the construction of NVU models leads to rapid detection of adverse chemical effects on neurovascular stability. The use of multiple cell types in coculture elucidates potential mechanisms of action by chemicals applied to the model.

PMID:32292797 | PMC:PMC6594538 | DOI:10.1089/aivt.2018.0025

Mol Vis. 2020 Apr 1;26:257-276. eCollection 2020.

ABSTRACT

PURPOSE: Retinopathy of prematurity (ROP) is a condition of aberrant retinal vascularization in premature infants in response to high levels of oxygen used for critical care that can potentially cause blindness. Although therapies to mitigate vascular abnormalities are being evaluated, functional deficits often remain in patients with treated or regressed ROP. This study investigated long-term outcomes of hyperoxia on retinal morphology and function using a mouse model of oxygen-induced ischemic retinopathy (OIR).

METHODS: Twenty-two mice were exposed to 77% oxygen to induce OIR, while 23 age-matched control mice were raised in room air (RA). In vivo fluorescein angiography (FA), spectral-domain optical coherence tomography (SD-OCT), and focal electroretinography (fERG) were performed at P19, P24, P32, and P47, followed by histological assessments of retinal morphology, gliosis, microglia activation, and apoptosis.

RESULTS: FA in OIR mice showed capillary attrition despite peripheral revascularization. Inner retina thinning was detected with SD-OCT; outer and inner retinal dysfunction were demonstrated with fERG. Histology of the OIR mice exhibited a thin, disorganized structure. Immunohistochemistry showed increased gliosis, microglial activation, and apoptosis with increasing age from P19 to P47. The synapses between rod photoreceptor cells and rod bipolar cells were ectopically localized in the OIR mice.

CONCLUSIONS: We demonstrated histological evidence of persistent ectopic synapses, prolonged cellular apoptosis, and gliosis in the OIR retina that corresponded with long-term in vivo evidence of capillary attrition, inner retinal thinning, and dysfunction despite full peripheral revascularization. Further studies on the mechanisms underlying these persistent phenotypes could enhance our understanding of ROP pathogenesis and lead to new therapeutic targets to preserve visual function in premature infants.

PMID:32256029 | PMC:PMC7127927

Invest Ophthalmol Vis Sci. 2020 Feb 7;61(2):23. doi: 10.1167/iovs.61.2.23.

ABSTRACT

PURPOSE: To validate the ability of visible-light optical coherence tomography (vis-OCT) in imaging the full Schlemm's canal (SC) and its surrounding limbal vascular network in mice in vivo through a compound circumlimbal scan.

METHODS: We developed an anterior segment vis-OCT system and a compound circumlimbal scanning method, which montages eight rotated raster scans. We calibrated the circumlimbal scan geometry using a three-dimensional printed phantom eyeball before imaging wild-type C57BL/6J mice. We measured SC size by segmenting SC cross sections from vis-OCT B-scan images and imaged the limbal microvascular network using vis-OCT angiography (vis-OCTA). To introduce changes in SC size, we used a manometer to adjust the intraocular pressure (IOP) to different levels. To create additional optical scattering contrast to enhance SC imaging, we surgically increased the episcleral venous pressure (EVP) and caused blood reflux into SC.

RESULTS: Using the compound circumlimbal scan, our anterior segment vis-OCT noninvasively imaged the full SC and limbal microvascular network in mouse for the first time. We observed an average 123% increase in SC volume when we decreased the IOP by 10 mm Hg from the baseline IOP of 7 to 10 mm Hg and an average 72% decrease in SC volume when the IOP level was elevated by 10 mm Hg from the baseline IOP. We also observed location-dependent SC size responses to IOP changes. Blood reflux caused by increased EVP enabled vis-OCTA to directly visualize SC, which matched well with the segmented SC.

CONCLUSIONS: Vis-OCT and vis-OCTA can accurately image the entire SC and limbal microvascular network in vivo using the compound circumlimbal scan. Vis-OCT is also able to quantitatively measure SC responses to changing IOP levels.

PMID:32068793 | PMC:PMC7326574 | DOI:10.1167/iovs.61.2.23

Spectrochim Acta A Mol Biomol Spectrosc. 2020 Apr 5;230:118063. doi: 10.1016/j.saa.2020.118063. Epub 2020 Jan 11.

ABSTRACT

In the present study the binding of diversin (DIV), a prenylated coumarin isolated from Ferula diversivittata, to bovine serum albumin (BSA) was investigated using surface plasmon resonance (SPR), spectrofluorimetry, and molecular docking approaches. Following the activation of carboxylic groups, via NHS/EDC, BSA was immobilized on the carboxymethyl dextran (CMD) hydrogel coated Au sensor, and was used for real-time monitoring of the interactions between DIV and BSA. K_D value of DIV binding to BSA increased with increasing temperature, confirmed that the affinity between BSA and DIV decreases with rising temperature. In addition, the fluorescence and synchronous fluorescence spectroscopic data revealed that the intrinsic emission intensity of BSA was quenched via a dynamic mechanism. In addition, the micro-region around BSA tyrosine residue was changed upon interaction with DIV. The thermodynamic parameter findings suggested that the hydrophobic interactions were dominant in the binding and formation of the BSA and DIV complex. The molecular docking outputs indicated that there is only one binding site on BSA for DIV, in agreement with experimental data, and DIV bind BSA in subdomain IB.

PMID:32000060 | DOI:10.1016/j.saa.2020.118063

Bioorg Chem. 2019 Dec;93:103329. doi: 10.1016/j.bioorg.2019.103329. Epub 2019 Sep 30.

ABSTRACT

3-Amino-1-aryl-1H-benzo[f]chromene-2-carbonitrile derivatives were synthesized from three-component reaction of arylaldehyde, malononitrile and 2-naphthol in the presence of 1, 4-bis(4-ferrocenylbutyl)piperazine as a new catalyst. Cytotoxic potencies of the compounds were tested on HT-29 cells. 3-Amino-1-(4-fluorophenyl)-1H-benzo[f]chromene-2-carbonitrile (4c) was more active among these compounds and was selected for further studies. Apoptosis was investigated by acridine orange/ethidium bromide (AO/EtBr) double staining and flow cytometry. The qRT-PCR was used to analyze the expression of pro- and anti-apoptotic genes. The binding attributes of 4c with calf thymus DNA (ctDNA) was examined using multi-spectroscopic measurements. We found that 4c had potent cytotoxic activity against HT-29 cells with an IC₅₀ value of 60 µM through induction of cell cycle arrest in the sub-G1 phase and apoptosis. RT-PCR analysis demonstrated down-regulation of Bcl-2 expression, while the expression of Bax, caspase-3, -8 and -9 genes was up-regulated in HT-29 cells incubated with 4c compared with control cells. These studies revealed that 4c interacts with DNA through groove binding mode with the intrinsic binding constant (K_b) of 3 × 10² M^-1. Thus, 4c is a valuable candidate for further evaluation as a new series of potent chemotherapeutic family in colon cancer treatment.

PMID:31590040 | DOI:10.1016/j.bioorg.2019.103329

Exp Eye Res. 2019 Oct;187:107756. doi: 10.1016/j.exer.2019.107756. Epub 2019 Aug 14.

ABSTRACT

Endoplasmic reticulum (ER) stress is recognized as a contributing factor to various ocular neurovascular pathologies including retinitis pigmentosa, glaucoma, and diabetic retinopathy (DR). ER stress in particular is implicated in the development of DR, which is significantly influenced by inflammation driven retinal vascular degeneration and dysfunction. Ultimately, loss of vision occurs if left untreated. However, the identity of the target cells and their temporal involvement in diabetes-mediated dysfunction need further investigation. Early diabetes-induced stress in photoreceptor cells is proposed as the driver of inflammatory mediated neurovascular changes during diabetes. Although tunicamycin induced ER stress results in photoreceptor loss, its consequences for retinal vascular degeneration and retinal ganglion (RGC) and pigment epithelium (RPE) cell loss remains unclear. Here we show intravitreal delivery of tunicamycin primarily induced ER stress in photoreceptor cells resulting in their loss by apoptosis. This was concomitant with induced expression of the unfolded protein response marker CHOP in these cells. We also demonstrated significant degeneration of retinal capillaries following the loss of photoreceptor cells with minimal impact on loss of RGC and RPE cells. However, activation of retinal microglial and Muller cells were noticeable. Thus, our data support the notion that ER stress mediated dysfunction and/or loss of photoreceptor cells in response to inflammation and oxidative stress could precede retinal vascular and neuronal dysfunction and degeneration.

PMID:31421136 | PMC:PMC7412575 | DOI:10.1016/j.exer.2019.107756

FASEB Bioadv. 2019 Jul;1(7):415-434. doi: 10.1096/fba.2018-00067. Epub 2019 May 3.

ABSTRACT

We have previously demonstrated that the active form of vitamin D (calcitriol; 1,25(OH)₂D₃) is a potent inhibitor of retinal neovascularization. However, the underlying molecular and cellular mechanisms involved remained poorly understood. Perivascular supporting cells including pericytes (PC) play important roles during angiogenesis, vascular maturation, and stabilization of blood vessels. How 1,25(OH)₂D₃ affects retinal PC proliferation and migration, and whether these effects are mediated through vitamin D receptor (VDR), are unknown. Here, we determined the impact of 1,25(OH)₂D₃ on retinal PC prepared from wild-type (Vdr+/+) and VDR-deficient (Vdr-/-) mice. Retinal PC expressed significantly higher VDR levels compared to retinal endothelial cells (EC). Unlike retinal EC, 1,25(OH)₂D₃ significantly decreased PC proliferation and migration and resulted in a G₀/G₁ cell cycle arrest. Although 1,25(OH)₂D₃ did not inhibit the proliferation of Vdr-/- PC, it did inhibit their migration. PC adhesion to various extracellular matrix (ECM) proteins and ECM production were also affected by incubation of PC with 1,25(OH)₂D₃. Vdr-/- PC were more adherent compared with Vdr+/+ cells. Mechanistically, incubation of Vdr+/+ PC with 1,25(OH)₂D₃ resulted in an increased expression of vascular endothelial growth factor (VEGF) and attenuation of signaling through VEGF-R2 and platelet-derived growth factor receptor-beta. Incubation with soluble VEGF-R1 (sFlt-1) partially reversed the effect of VEGF on Vdr+/+ PC. In addition, incubation of Vdr+/+ PC with VEGF or inhibition of VEGF-R2 increased VDR expression. Together, these results suggest an important role for retinal PC as a target for vitamin D and VDR action for attenuation of angiogenesis.

PMID:31396585 | PMC:PMC6687334 | DOI:10.1096/fba.2018-00067

Spectrochim Acta A Mol Biomol Spectrosc. 2019 Dec 5;223:117286. doi: 10.1016/j.saa.2019.117286. Epub 2019 Jul 3.

ABSTRACT

The binding of sitagliptin (SIT), an anti-diabetic drug, to human and bovine serum albumin (HSA and BSA; main serum transport proteins) was investigated using various spectroscopic and molecular docking techniques. The fluorescence data demonstrated that SIT quenched inherent fluorescence of these proteins through the formation of SIT-HSA/BSA complexes. The number of binding sites was obtained (~1) and binding constant (K_b) and effective quenching constant (K_a) were calculated as 10⁴ for both systems. Based on thermodynamic parameters, the van der Waals forces and hydrogen bonding were the most important forces in the interactions between HSA/BSA and SIT, and the complex formation processes were spontaneous. The results of UV-vis absorption and FT-IR spectroscopic revealed that SIT induces small conformational changes in the structure of the proteins (HSA/BSA). The synchronous fluorescence (SF) spectroscopy demonstrated that the binding of SIT with HSA/BSA had no effect on the polarity around Trp and Tyr residues. The CD spectra showed changes in the secondary and tertiary structures of both proteins with a decrease in α-helices contents and an increase in β-turn structures. The molecular docking and spectroscopic data verified the binding mechanisms between SIT and HSA/BSA, and revealed that SIT completely fits into the hydrophobic cavity between domain II and domain III of these proteins.

PMID:31302563 | DOI:10.1016/j.saa.2019.117286

Chem Biol Interact. 2019 Sep 25;311:108746. doi: 10.1016/j.cbi.2019.108746. Epub 2019 Jul 11.

ABSTRACT

Utilizing food additives at their optimized concentration is believed to be relatively safe, but their combinatorial effects remain largely unexplored. The influence of mixed food additives on the macromolecules may be altered by synergistic or antagonistic effects. It is previously shown that curcumin enhances the catalase activity by affecting its structural pocket in the active site. The aim of this study was to investigate the combination effects of food colorants sunset yellow FCF (SNY) and curcumin on the activation and/or inactivation of catalase activity using multispectral (fluorescence, FTIR, and UV-vis) analysis and simultaneous docking simulations. Kinetic studies demonstrated that SNY could significantly decrease catalase activity through a non-competitive inhibition mechanism. Fluorescence data indicated that SNY reduces intrinsic emission of catalase via a static quenching mechanism. Thermodynamic and molecular docking investigations suggested that catalase has one binding site for SNY, and hydrogen binding plays a main role in the binding reaction of catalase -SNY complex. Molecular dynamic simulation data indicated that the curcumin binding to the cavity, in the middle of the catalase helical domain, facilitates SNY binding to the enzyme pocket. For this purpose, the equilibrium dialysis system was used to study the stability and reversibility of SNY-catalase in the absence or presence of curcumin. The obtained data indicated that the binding of SNY-catalase is reversible and the stability of the complex is time-dependent. However, curcumin could make the complex more stable enhancing the SNY inhibition of catalase activity.

PMID:31301288 | DOI:10.1016/j.cbi.2019.108746

Sci Rep. 2019 Jul 4;9(1):9700. doi: 10.1038/s41598-019-45915-4.

ABSTRACT

B-cell lymphoma 2 (Bcl-2) protein is the founding member of a group of proteins known to modulate apoptosis. Its discovery set the stage for identification of family members with either pro- or anti-apoptotic properties. Expression of Bcl-2 plays an important role during angiogenesis by influencing not only vascular cell survival, but also migration and adhesion. Although apoptosis and migration are postulated to have roles during vascular remodeling and regression, the contribution of Bcl-2 continues to emerge. We previously noted that the impaired retinal vascularization and an inability to undergo pathologic neovascularization observed in mice globally lacking Bcl-2 did not occur when mice lacked the expression of Bcl-2 only in endothelial cells. To further examine the effect of Bcl-2 expression during vascularization of the retina, we assessed its contribution in pericytes or astrocytes by generating mice with a conditional Bcl-2 allele (Bcl-2^Flox/Flox) and Pdgfrb-cre (Bcl-2^PC mice) or Gfap-cre (Bcl-2^AC mice). Bcl-2^PC and Bcl-2^AC mice demonstrated increased retinal vascular cell apoptosis, reduced numbers of pericytes and endothelial cells and fewer arteries and veins in the retina. Bcl-2^PC mice also demonstrated delayed advancement of the superficial retinal vascular layer and aberrant vascularization of the deep vascular plexus and central retina. Although pathologic neovascularization in oxygen-induced ischemic retinopathy (OIR) was not affected by lack of expression of Bcl-2 in either pericytes or astrocytes, laser-induced choroidal neovascularization (CNV) was significantly reduced in Bcl-2^PC mice compared to littermate controls. Together these studies begin to reveal how cell autonomous modulation of apoptosis in vascular cells impacts development and homeostasis.

PMID:31273232 | PMC:PMC6609701 | DOI:10.1038/s41598-019-45915-4

Biochem Biophys Res Commun. 2019 Jun 25;514(2):475-481. doi: 10.1016/j.bbrc.2019.04.172. Epub 2019 May 2.

ABSTRACT

Liver sinusoidal endothelial cells are the border patrol in the liver. Their open transcellular fenestrations allow the transfer of small and dissolved substances from the blood into the liver parenchymal cells. Fenestrations are dynamic structures, and many drugs and diseases alter their size and number, thus making them an important target for modulation. There is an urgent need to understand how various diseases, toxic substances, and physiological conditions influence liver endothelial cell fenestrations, and how these changes affects liver function. This work represents a straightforward quantitative proteomics study of the in vivo arsenic-stressed liver sinusoidal endothelial cells using a reverse super-SILAC based method. The aim of this study was to identify proteins, which are up- or down-regulated in response to arsenic. This knowledge will aid in identification of potential targets and mechanisms of arsenic toxicity and novel ways to reverse these changes.

PMID:31056257 | PMC:PMC6924266 | DOI:10.1016/j.bbrc.2019.04.172

Am J Physiol Cell Physiol. 2019 Jun 1;316(6):C767-C781. doi: 10.1152/ajpcell.00021.2019. Epub 2019 Mar 20.

ABSTRACT

Astrocytes (ACs) are the most abundant cells in the central nervous system. Retinal ACs 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 ACs 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 ACs 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 ACs were more proliferative and migratory. These cells also produced increased amounts of fibronectin and its receptors, α_vβ₃- and α₅β₁-integrin. These results were consistent with the increased adhesive properties of Cyp1b1^-/- ACs and their lack of ability to form a network in Matrigel. This was reversed by reexpression of Cyp1b1 in Cyp1b1^-/- ACs. Although no significant changes were observed in Akt/SRC/MAPK signaling pathways, production of inflammatory mediators bone morphogenetic protein-7 (BMP-7) and monocyte chemoattractant protein-1 (MCP-1) was decreased in Cyp1b1^-/- ACs. Cyp1b1^-/- ACs also showed increased levels of connexin 43 phosphorylation and cluster of differentiation 38 expression when challenged with H₂O₂. These results are consistent with increased proliferation and diminished oxidative stress in Cyp1b1^-/- cells. Thus, Cyp1b1 expression in ACs plays an important role in retinal neurovascular homeostasis.

PMID:30892936 | PMC:PMC6620579 | DOI:10.1152/ajpcell.00021.2019

Sci Rep. 2019 Mar 13;9(1):4353. doi: 10.1038/s41598-019-40892-0.

ABSTRACT

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 | PMC:PMC6416402 | DOI:10.1038/s41598-019-40892-0

Circ Res. 2019 Apr 12;124(8):1253-1265. doi: 10.1161/CIRCRESAHA.118.314567.

ABSTRACT

RATIONALE: Regeneration of denuded or injured endothelium is an important component of vascular injury response. Cell-cell communication between endothelial cells and smooth muscle cells (SMCs) plays a critical role not only in vascular homeostasis but also in disease. We have previously demonstrated that PKCδ (protein kinase C-delta) 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-endothelial cells 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 with the wild-type 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 polymerase chain reaction-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 STAT3 (signal transducer and activator of transcription 3) as knockdown of STAT3 abolished Cxcl7 expression. The role of CXCL7 in SMC-endothelial cells 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 endothelial cells from uninjured endothelium.

PMID:30739581 | PMC:PMC6459708 | DOI:10.1161/CIRCRESAHA.118.314567

Arch Biochem Biophys. 2019 Mar 30;664:110-116. doi: 10.1016/j.abb.2019.02.002. Epub 2019 Feb 7.

ABSTRACT

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 | DOI:10.1016/j.abb.2019.02.002