Dr. Raghunathan is a Bioengineer in the Department of Surgical and Radiological Sciences at the University of California, Davis. He is cross trained in biomaterials, biomechanics, and cell and molecular biology with research interests and expertise in drug delivery, force microscopy, toxicology, tissue engineering and medical device development for orthopedic, wound healing, and ocular applications.
Mice Deficient in TAZ (Wwtr1) Demonstrate Clinical Features of Late-Onset Fuchs' Endothelial Corneal Dystrophy
Invest Ophthalmol Vis Sci. 2023 Apr 3;64(4):22. doi: 10.1167/iovs.64.4.22.
PURPOSE: We sought to define the role of Wwtr1 in murine ocular structure and function and determine the role of mechanotransduction in Fuchs' endothelial corneal dystrophy (FECD), with emphasis on interactions between corneal endothelial cells (CEnCs) and Descemet's membrane (DM).
METHODS: A Wwtr1 deficient mouse colony was established, and advanced ocular imaging, atomic force microscope (AFM), and histology/immunofluorescence were performed. Corneal endothelial wound healing was assessed using cryoinjury and phototherapeutic keratectomy in Wwtr1 deficient mice. Expression of WWTR1/TAZ was determined in the corneal endothelium from normal and FECD-affected patients; WWTR1 was screened for coding sequence variants in this FECD cohort.
RESULTS: Mice deficient in Wwtr1 had reduced CEnC density, abnormal CEnC morphology, softer DM, and thinner corneas versus wildtype controls by 2 months of age. Additionally, CEnCs had altered expression and localization of Na/K-ATPase and ZO-1. Further, Wwtr1 deficient mice had impaired CEnC wound healing. The WWTR1 transcript was highly expressed in healthy human CEnCs comparable to other genes implicated in FECD pathogenesis. Although WWTR1 mRNA expression was comparable between healthy and FECD-affected patients, WWTR1/TAZ protein concentrations were higher and localized to the nucleus surrounding guttae. No genetic associations were found in WWTR1 and FECD in a patient cohort compared to controls.
CONCLUSIONS: There are common phenotypic abnormalities seen between Wwtr1 deficient and FECD-affected patients, suggesting that Wwtr1 deficient mice could function as a murine model of late-onset FECD. Despite the lack of a genetic association between FECD and WWTR1, aberrant WWTR1/TAZ protein subcellular localization and degradation may play critical roles in the pathogenesis of FECD.
PMID:37074694 | PMC:PMC10132321 | DOI:10.1167/iovs.64.4.22
The TGM2 inhibitor cysteamine hydrochloride does not impact corneal epithelial and stromal wound healing in vitro and in vivo
Exp Eye Res. 2023 Jan;226:109338. doi: 10.1016/j.exer.2022.109338. Epub 2022 Dec 5.
Corneal wound healing is integral for resolution of corneal disease or for post-operative healing. However, corneal scarring that may occur secondary to this process can significantly impair vision. Tissue transglutaminase 2 (TGM2) inhibition has shown promising antifibrotic effects and thus holds promise to prevent or treat corneal scarring. The commercially available ocular solution for treatment of ocular manifestations of Cystinosis, Cystaran®, contains the TGM2 inhibitor cysteamine hydrochloride (CH). The purpose of this study is to assess the safety of CH on corneal epithelial and stromal wounds, its effects on corneal wound healing, and its efficacy against corneal scarring following wounding. Quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC) were first used to quantify and localize TGM2 expression in the cornea. Subsequently, (i) the in vitro effects of CH at 0.163, 1.63, and 16.3 mM on corneal epithelial cell migration was assessed with an epithelial cell migration assay, and (ii) the in vivo effects of application of 1.63 mM CH on epithelial and stromal wounds was assessed in a rabbit model with ophthalmic examinations, inflammation scoring, color and fluorescein imaging, optical coherence tomography (OCT), and confocal biomicroscopy. Post-mortem assessment of corneal tissue post-stromal wounding included biomechanical characterization (atomic force microscopy (AFM)), histology (H&E staining), and determining incidence of myofibroblasts (immunostaining against α-SMA) in wounded corneal tissue. TGM2 expression was highest in corneal epithelial cells. Application of the TGM2 inhibitor CH did not affect in vitro epithelial cell migration at the two lower concentrations tested. At 16.3 mM, decreased cell migration was observed. In vivo application of CH at 57 mM was well tolerated and did not adversely affect wound healing. No difference in corneal scarring was found between CH treated and vehicle control eyes. This study shows that the TGM2 inhibitor CH, at the FDA-approved dose, is well tolerated in a rabbit model of corneal wound healing and does not adversely affect epithelial or stromal wound healing. This supports the safe use of this medication in Cystinosis patients with open corneal wounds. CH did not have an effect on corneal scarring in this study, suggesting that Cystaran® administration to patients with corneal wounds is unlikely to decrease corneal fibrosis.
PMID:36470430 | PMC:PMC10120528 | DOI:10.1016/j.exer.2022.109338
Metallic Engineered Nanomaterials and Ocular Toxicity: A Current Perspective
Pharmaceutics. 2022 May 3;14(5):981. doi: 10.3390/pharmaceutics14050981.
The ocular surface, comprised of the transparent cornea, conjunctiva, and protective tear film, forms a protective barrier defending deeper structures of the eye from particulate matter and mechanical trauma. This barrier is routinely exposed to a multitude of naturally occurring and engineered nanomaterials (ENM). Metallic ENMs are particularly ubiquitous in commercial products with a high risk of ocular exposure, such as cosmetics and sunscreens. Additionally, there are several therapeutic uses for metallic ENMs owing to their attractive magnetic, antimicrobial, and functionalization properties. The increasing commercial and therapeutic applications of metallic ENMs come with a high risk of ocular exposure with poorly understood consequences to the health of the eye. While the toxicity of metallic ENMs exposure has been rigorously studied in other tissues and organs, further studies are necessary to understand the potential for adverse effects and inform product usage for individuals whose ocular health may be compromised by injury, disease, or surgical intervention. This review provides an update of current literature on the ocular toxicity of metallic ENMs in vitro and in vivo, as well as the risks and benefits of therapeutic applications of metallic ENMs in ophthalmology.
PMID:35631569 | PMC:PMC9145553 | DOI:10.3390/pharmaceutics14050981
Cross-linked actin networks (CLANs) affect stiffness and/or actin dynamics in transgenic transformed and primary human trabecular meshwork cells
Exp Eye Res. 2022 Jul;220:109097. doi: 10.1016/j.exer.2022.109097. Epub 2022 May 13.
Cross-linked actin networks (CLANs) in trabecular meshwork (TM) cells may contribute to increased IOP by altering TM cell function and stiffness. However, there is a lack of direct evidence. Here, we developed transformed TM cells that form spontaneous fluorescently labelled CLANs. The stable cells were constructed by transducing transformed glaucomatous TM (GTM3) cells with the pLenti-LifeAct-EGFP-BlastR lentiviral vector and selection with blasticidin. The stiffness of the GTM3-LifeAct-GFP cells were studied using atomic force microscopy. Elastic moduli of CLANs in primary human TM cells treated with/without dexamethasone/TGFβ2 were also measured to validate findings in GTM3-LifeAct-GFP cells. Live-cell imaging was performed on GTM3-LifeAct-GFP cells treated with 1 μM latrunculin B or pHrodo bioparticles to determine actin stability and phagocytosis, respectively. The GTM3-LifeAct-GFP cells formed spontaneous CLANs without the induction of TGFβ2 or dexamethasone. The CLAN containing cells showed elevated cell stiffness, resistance to latrunculin B-induced actin depolymerization, as well as compromised phagocytosis, compared to the cells without CLANs. Primary human TM cells with dexamethasone or TGFβ2-induced CLANs were also stiffer and less phagocytic. The GTM3-LifeAct-GFP cells are a novel tool for studying the mechanobiology and pathology of CLANs in the TM. Initial characterization of these cells showed that CLANs contribute to at least some glaucomatous phenotypes of TM cells.
PMID:35569518 | DOI:10.1016/j.exer.2022.109097
Endogenous expression of Notch pathway molecules in human trabecular meshwork cells
Exp Eye Res. 2022 Mar;216:108935. doi: 10.1016/j.exer.2022.108935. Epub 2022 Jan 14.
PURPOSE: Cells in the trabecular meshwork sense and respond to a myriad of physical forces through a process known as mechanotransduction. Whilst the effect of substratum stiffness or stretch on TM cells have been investigated in the context of transforming growth factor (TGF-β), Wnt and YAP/TAZ pathways, the role of Notch signaling, an evolutionarily conserved pathway, recently implicated in mechanotransduction, has not been investigated in trabecular meshwork (TM) cells. Here, we compare the endogenous expression of Notch pathway molecules in TM cells from glaucomatous and non-glaucomatous donors, segmental flow regions, and when subjected to cyclical strain, or grown on hydrogels of varying rigidity.
METHODS: Primary TM from glaucomatous (GTM), non-glaucomatous (NTM) donors, and from segmental flow regions [high flow (HF), low flow (LF)], were utilized between passages 2-6. Cells were (i) plated on tissue culture plastic, (ii) subjected to cyclical strain (6 h and 24 h), or (iii) cultured on 3 kPa and 80 kPa hydrogels. mRNA levels of Notch receptors/ligands/effectors in the TM cells was determined by qRT-PCR. Phagocytosis was determined as a function of substratum stiffness in NTM-HF/LF cells in the presence or absence of 100 nM Dexamethasone treatment.
RESULTS: Innate expression of Notch pathway genes were significantly overexpressed in GTM cells with no discernible differences observed between HF/LF cells in either NTM or GTM cells cultured on plastic substrates. With 6 h of cyclical strain, a subset of Notch pathway genes presented with altered expression. Expression of Notch receptors/ligands/receptors/inhibitors progressively declined with increasing stiffness and this correlated with phagocytic ability of NTM cells. Dexamethasone treatment decreased phagocytosis regardless of stiffness or cells isolated from segmental outflow regions.
CONCLUSIONS: We demonstrate here that the Notch expression in cultured TM cells differ intrinsically between GTM vs NTM, and by substratum cues (cyclical strain and stiffness). Of import, the most apparent differences in gene expression were observed as a function of substratum stiffness which closely followed phagocytic ability of cells. Interestingly, on soft substrates (mimicking normal TM stiffness) Notch expression and phagocytosis was highest, while both expression and phagocytosis was significantly lower on stiffer substrates (mimicking glaucomatous stiffness) regardless of DEX treatment. Such context dependent changes suggest Notch pathway may play differing roles in disease vs homeostasis. Studies focused on understanding the mechanistic role of Notch (if any) in outflow homeostasis are thus warranted.
PMID:35033558 | PMC:PMC8885976 | DOI:10.1016/j.exer.2022.108935
Organic Semiconductor Nanotubes for Electrochemical Devices
Adv Funct Mater. 2021 Dec 2;31(49):2105358. doi: 10.1002/adfm.202105358. Epub 2021 Jul 30.
Electrochemical devices that transform electrical energy to mechanical energy through an electrochemical process have numerous applications ranging from soft robotics and micropumps to autofocus microlenses and bioelectronics. To date, achievement of large deformation strains and fast response times remains a challenge for electrochemical actuator devices operating in liquid wherein drag forces restrict the actuator motion and electrode materials/structures limit the ion transportation and accumulation. We report results for electrochemical actuators, electrochemical mass transfers, and electrochemical dynamics made from organic semiconductors (OSNTs). Our OSNTs electrochemical device exhibits high actuation performance with fast ion transport and accumulation and tunable dynamics in liquid and gel-polymer electrolytes. This device demonstrates an excellent performance, including low power consumption/strain, a large deformation, fast response, and excellent actuation stability. This outstanding performance stems from enormous effective surface area of nanotubular structure that facilitates ion transport and accumulation resulting in high electroactivity and durability. We utilize experimental studies of motion and mass transport along with the theoretical analysis for a variable-mass system to establish the dynamics of the electrochemical device and to introduce a modified form of Euler-Bernoulli's deflection equation for the OSNTs. Ultimately, we demonstrate a state-of-the-art miniaturized device composed of multiple microactuators for potential biomedical application. This work provides new opportunities for next generation electrochemical devices that can be utilized in artificial muscles and biomedical devices.
PMID:34924917 | PMC:PMC8673914 | DOI:10.1002/adfm.202105358
An ex vivo model of human corneal rim perfusion organ culture
Exp Eye Res. 2022 Jan;214:108891. doi: 10.1016/j.exer.2021.108891. Epub 2021 Dec 9.
The human anterior segment perfusion culture model is a valuable tool for studying the trabecular meshwork (TM) and aqueous humor outflow in glaucoma. The traditional model relies on whole eye globes resulting in high cost and limited availability. Here, we developed a glue-based method which enabled us to use human corneal rims for perfusion culture experiments. Human corneal rim perfusion culture plates were 3D printed. Human corneal rims containing intact TM were attached and sealed to the plate using low viscosity and high viscosity glues, respectively. The human corneal rims were perfused using the constant flow mode, and the pressure changes were recorded using a computerized system. Outflow facility, TM stiffness, and TM morphology were evaluated. When perfused at rates from 1.2 to 3.6 μl/min, the outflow facility was 0.359 ± 0.216 μl/min/mmHg among 10 human corneal rims. The stiffness of the TM in naïve human corneal rim was similar to that of perfusion cultured human corneal rim. Also, the stiffness of TM of corneal rims perfused with dexamethasone was significantly higher than the control. Human corneal rims with glue contamination in the TM could be differentiated by high baseline intraocular pressure as well as high TM stiffness. Histology studies showed that the TM tissues perfused with plain medium appeared normal. We believed that our glued-based method is a useful tool and low-cost alternative to the traditional anterior segment perfusion culture model.
PMID:34896309 | PMC:PMC8792355 | DOI:10.1016/j.exer.2021.108891
Animal models of corneal endothelial dysfunction to facilitate development of novel therapies
Ann Transl Med. 2021 Aug;9(15):1271. doi: 10.21037/atm-20-4389.
Progressive corneal endothelial disease eventually leads to corneal edema and vision loss due to the limited regenerative capacity of the corneal endothelium in vivo and is a major indication for corneal transplantation. Despite the relatively high success rate of corneal transplantation, there remains a pressing global clinical need to identify improved therapeutic strategies to address this debilitating condition. To evaluate the safety and efficacy of novel therapeutics, there is a growing demand for pre-clinical animal models of corneal endothelial dysfunction. In this review, experimentally induced, spontaneously occurring and genetically modified animal models of corneal endothelial dysfunction are described to assist researchers in making informed decisions regarding the selection of the most appropriate animal models to meet their research goals.
PMID:34532408 | PMC:PMC8421955 | DOI:10.21037/atm-20-4389
The Canonical Wnt Signaling Pathway Inhibits the Glucocorticoid Receptor Signaling Pathway in the Trabecular Meshwork
Am J Pathol. 2021 Jun;191(6):1020-1035. doi: 10.1016/j.ajpath.2021.02.018. Epub 2021 Mar 8.
Glucocorticoid-induced glaucoma is a secondary open-angle glaucoma. About 40% of the general population may develop elevated intraocular pressure on prolonged glucocorticoid treatment secondary to damages in the trabecular meshwork (TM), a tissue that regulates intraocular pressure. Therefore, identifying the key molecules responsible for glucocorticoid-induced ocular hypertension is crucial. In this study, Dickkopf-related protein 1 (Dkk1), a canonical Wnt signaling inhibitor, was found to be elevated in the aqueous humor and TM of glaucoma patients. At the signaling level, Dkk1 enhanced glucocorticoid receptor (GR) signaling, whereas Dkk1 knockdown or Wnt signaling activators decreased GR signaling in human TM cells as indicated by luciferase assays. Similarly, activation of the GR signaling inhibited Wnt signaling. At the protein level, glucocorticoid-induced extracellular matrix was inhibited by Wnt activation using Wnt activators or Dkk1 knockdown in primary human TM cells. In contrast, inhibition of canonical Wnt signaling by β-catenin knockdown increased glucocorticoid-induced extracellular matrix proteins. At the physiological level, adenovirus-mediated Wnt3a expression decreased glucocorticoid-induced ocular hypertension in mouse eyes. In summary, Wnt and GR signaling inhibit each other in the TM, and canonical Wnt signaling activators may prevent the adverse effect of glucocorticoids in the eye.
PMID:33705750 | PMC:PMC8176142 | DOI:10.1016/j.ajpath.2021.02.018
Glucocorticoid-induced cell-derived matrix modulates transforming growth factor β2 signaling in human trabecular meshwork cells
Sci Rep. 2020 Sep 24;10(1):15641. doi: 10.1038/s41598-020-72779-w.
Aberrant remodeling of trabecular meshwork (TM) extracellular matrix (ECM) may induce ocular hypertensive phenotypes in human TM (hTM) cells to cause ocular hypertension, via a yet unknown mechanism. Here, we show that, in the absence of exogenous transforming growth factor-beta2 (TGFβ2), compared with control matrices (VehMs), glucocorticoid-induced cell-derived matrices (GIMs) trigger non-Smad TGFβ2 signaling in hTM cells, correlated with overexpression/activity of structural ECM genes (fibronectin, collagen IV, collagen VI, myocilin), matricellular genes (connective tissue growth factor [CTGF], secreted protein, acidic and rich in cysteine), crosslinking genes/enzymes (lysyl oxidase, lysyl oxidase-like 2-4, tissue transglutaminase-2), and ECM turnover genes/enzymes (matrix metalloproteinases-MMP2,14 and their inhibitors-TIMP2). However, in the presence of exogenous TGFβ2, VehMs and GIMs activate Smad and non-Smad TGFβ2 signaling in hTM cells, associated with overexpression of α-smooth muscle actin (α-SMA), and differential upregulation of aforementioned ECM genes/proteins with new ones emerging (collagen-I, thrombospondin-I, plasminogen activator inhibitor, MMP1, 9, ADAMTS4, TIMP1); with GIM-TGFβ2-induced changes being mostly more pronounced. This suggests dual glaucomatous insults potentiate profibrotic signaling/phenotypes. Lastly, we demonstrate type I TGFβ receptor kinase inhibition abrogates VehM-/GIM- and/or TGFβ2-induced upregulation of α-SMA and CTGF. Collectively, pathological TM microenvironments are sufficient to elicit adverse cellular responses that may be ameliorated by targeting TGFβ2 pathway.
PMID:32973273 | PMC:PMC7518434 | DOI:10.1038/s41598-020-72779-w
Intrastromal Injection of Hyaluronidase Alters the Structural and Biomechanical Properties of the Corneal Stroma
Transl Vis Sci Technol. 2020 May 21;9(6):21. doi: 10.1167/tvst.9.6.21. eCollection 2020 May.
PURPOSE: Glycosaminoglycans (GAGs) are important components of the corneal stroma, and their spatiotemporal arrangement regulates the organization of collagen fibrils and maintains corneal transparency. This study was undertaken to determine the consequences of hyaluronidase (HAse) injected into the corneal stroma on stromal stiffness and ultrastructure.
METHODS: Equal volumes of HAse or balanced salt solution (vehicle) were injected intrastromally into the corneas of New Zealand white rabbits. Ophthalmic examination and multimodal imaging techniques, including Fourier-domain optical coherence tomography and in vivo confocal microscopy (IVCM), were performed at multiple time points to evaluate the impact of HAse treatment in vivo. Atomic force microscopy and transmission electron microscopy (TEM) were used to measure corneal stiffness and collagen's interfibrillar spacing, respectively.
RESULTS: Central corneal thickness progressively decreased after HAse injection, reaching its lowest value at day 7, and then returned to normal by day 42. The HAse did not impact the corneal endothelium but transiently altered keratocyte morphology at days 1 and 7, as measured by IVCM. HAse-injected corneas became stiffer by day 1 postinjection, were stiffest at day 7, and returned to preinjection values by day 90. Changes in stromal stiffness correlated with decreased interfibrillar spacing as measured by TEM.
CONCLUSIONS: Degradation of GAGs by HAse decreases the corneal thickness and increases stromal stiffness through increased packing of the collagen fibrils in a time-dependent manner.
TRANSLATIONAL RELEVANCE: Intrastromal HAse injection appears relatively safe in the normal cornea, but its impact on corneal biomechanics and structure under pathologic conditions requires further study.
PMID:32821518 | PMC:PMC7409307 | DOI:10.1167/tvst.9.6.21
Crosslinked Extracellular Matrix Stiffens Human Trabecular Meshwork Cells Via Dysregulating β-catenin and YAP/TAZ Signaling Pathways
Invest Ophthalmol Vis Sci. 2020 Aug 3;61(10):41. doi: 10.1167/iovs.61.10.41.
PURPOSE: The purpose of this study was to determine whether genipin-induced crosslinked cell-derived matrix (XCDM) precipitates fibrotic phenotypes in human trabecular meshwork (hTM) cells by dysregulating β-catenin and Yes-associated protein (YAP)/ transcriptional coactivator with PDZ-binding motif (TAZ) signaling pathways.
METHODS: Cell-derived matrices were treated with control or genipin for 5 hours to obtain respective uncrosslinked (CDM) and XCDMs and characterized. hTM cells were seeded on these matrices with/without Wnt pathway modulators in serum-free media for 24 hours. Elastic modulus, gene, and protein (whole cell and subcellular fractions) expressions of signaling mediators and targets of Wnt/β-catenin and YAP/TAZ pathways were determined.
RESULTS: At the highest genipin concentration (10% XCDM), XCDM had increased immunostaining of N-ε(γ-glutamyl)-lysine crosslinks, appeared morphologically fused, and was stiffer (5.3-fold, P < 0.001). On 10% XCDM, hTM cells were 7.8-fold (P < 0.001) stiffer, total β-catenin was unchanged, pβ-catenin was elevated, and pGSK3β was suppressed. Although 10% XCDM had no effect on cytoplasmic β-catenin levels, it reduced nuclear β-catenin, cadherin 11, and key Wnt target genes/proteins. The 10% XCDM increased total TAZ, decreased pTAZ, and increased cytoplasmic TAZ levels in hTM cells. The 10% XCDM increased total YAP, reduced nuclear YAP levels, and critical YAP/TAZ target genes/proteins. Wnt activation rescued hTM cells from 10% XCDM-induced stiffening associated with increased nuclear β-catenin.
CONCLUSIONS: Increased cytoplasmic TAZ may inhibit β-catenin from its nuclear shuttling or regulating cadherin 11 important for aqueous homeostasis. Elevated cytoplasmic TAZ may inhibit YAP's probable homeostatic function in the nucleus. Together, TAZ's cytoplasmic localization may be an important downstream event of how increased TM extracellular matrix (ECM) crosslinking may cause increased stiffness and ocular hypertension in vivo. However, Wnt pathway activation may ameliorate ocular hypertensive phenotypes induced by crosslinked ECM.
PMID:32832971 | PMC:PMC7452853 | DOI:10.1167/iovs.61.10.41
Stromal Collagen Arrangement Correlates with Stiffness of the Canine Cornea
Bioengineering (Basel). 2019 Dec 25;7(1):4. doi: 10.3390/bioengineering7010004.
The cornea is the most external layer of the eye and serves two important roles in (1) the refraction of light and (2) protection from the outside environment, both of which are highly dependent on the collagen assembly of the corneal stroma. This study sought to determine the collagen fiber arrangement of the canine corneal stroma and correlate the stromal organization with tissue stiffness in the anterior and posterior cornea. Collagen organization of the canine cornea was visualized through second-harmonic generation (SHG) imaging, and tissue stiffness of the anterior and posterior corneal stroma was determined by atomic force microscopy. Analysis of the canine anterior corneal stroma using SHG imaging documented intertwining of the collagen fibers with a high degree of fiber branching, with a more lamellar and non-branching posterior stroma. The anterior stroma had significantly higher tissue stiffness in both dogs and humans, when compared with the posterior corneal stroma (canine median: 1.3 kPa vs. 0.3 kPa; human median: 14.6 kPa vs. 2.1 kPa, respectively). There was a direct correlation between corneal collagen stromal organization and tissue stiffness in the dog, which was consistent with other mammalian species previously examined and likely reflects the need for maintenance of rigidity and corneal curvature.
PMID:31881719 | PMC:PMC7175282 | DOI:10.3390/bioengineering7010004
Ocular phenotypic consequences of a single copy deletion of the <em>Yap1</em> gene (<em>Yap1</em> <sup>+/-</sup>) in mice
Mol Vis. 2019 Feb 17;25:129-142. eCollection 2019.
PURPOSE: To identify the effects of a single copy deletion of Yap1 (Yap1 +/-) in the mouse eye, the ocular phenotypic consequences of Yap1 +/- were determined in detail.
METHODS: Complete ophthalmic examinations, as well as corneal esthesiometry, the phenol red thread test, intraocular pressure, and Fourier-domain optical coherence tomography were performed on Yap1 +/- and age-matched wild-type (WT) mice between eyelid opening (2 weeks after birth) and adulthood (2 months and 1 year after birth). Following euthanasia, enucleated eyes were characterized histologically.
RESULTS: Microphthalmia with small palpebral fissures, corneal fibrosis, and reduced corneal sensation were common findings in the Yap1 +/- mice. Generalized corneal fibrosis precluded clinical examination of the posterior structures. Histologically, thinning and keratinization of the corneal epithelium were observed in the Yap1 +/- mice in comparison with the WT mice. Distorted collagen fiber arrangement and hypercellularity of keratocytes were observed in the stroma. Descemet's membrane was extremely thin and lacked an endothelial layer in the Yap1 +/- mice. The iris was adherent to the posterior cornea along most of its surface creating a distorted contour. Most of the Yap1 +/- eyes were microphakic with swollen fibers and bladder cells. The retinas of the Yap1 +/- mice were normal at 2 weeks and 2 months of age, but the presence of retinal abnormalities, including retinoschisis and detachment, was markedly increased in the Yap1 +/- mice at 1 year of age.
CONCLUSIONS: The results show that the heterozygous deletion of the Yap1 gene in mice leads to complex ocular abnormalities, including microphthalmia, corneal fibrosis, anterior segment dysgenesis, and cataract.
PMID:30820148 | PMC:PMC6382475
Transforming Growth Factor Beta 3 Modifies Mechanics and Composition of Extracellular Matrix Deposited by Human Trabecular Meshwork Cells
ACS Biomater Sci Eng. 2015 Feb 9;1(2):110-118. doi: 10.1021/ab500060r. Epub 2015 Jan 8.
Pseudoexfoliation syndrome is a systemic disorder of the extracellular matrix (ECM) with ocular manifestations in the form of chronic open angle glaucoma. Elevated levels of TGFβ3 in the aqueous humor of individuals with pseudoexfoliation glaucoma (PEX) have been reported. The influence of TGFβ3 on the biochemical composition and biomechanics of ECM of human trabecular meshwork (HTM) cells was investigated. HTM cells from eye bank donor eyes were isolated, plated on aminosilane functionalized glass substrates and cultured in the presence or absence of 1 ng/mL TGFβ3 for 4 weeks. After incubation, samples were decellularized and decellularization was verified by immunostaining. The mechanics of the remaining ECM that was deposited by the treated or the control cells were measured by atomic force microscopy (AFM). Imaged by AFM, the surface features of the ECM from both sets of samples had a similar roughness/topography (as determined by RMS values) suggesting surface features of the ECM were similar in both cases; however, the ECM from the HTM cells treated with TGFβ3 was between 3- and 5-fold stiffer than that produced by the control HTM cells. Proteins present in the ECM were solubilized and analyzed using liquid chromatography tandem mass spectroscopy (LC-MS/MS). Data indicate that multiple proteins previously reported to be altered in glaucoma were changed in the ECM as a result of the presence of TGFβ3, including inhibitors of the BMP and Wnt signaling pathways. Gremlin1and 4, SERPINE1 and 2, periostin, secreted frizzled related protein (SFRP) 1 and 4, and ANGPTL4 were among those proteins that were overexpressed in the ECM after TGFβ3 treatment.
PMID:30882039 | PMC:PMC6419775 | DOI:10.1021/ab500060r
YAP and TAZ are distinct effectors of corneal myofibroblast transformation
Exp Eye Res. 2019 Mar;180:102-109. doi: 10.1016/j.exer.2018.12.009. Epub 2018 Dec 19.
PURPOSE: Transforming growth factor β1 (TGFβ1) is elevated in wounds after injury and promotes the transdifferentiation of quiescent cells in the stroma (keratocytes, to activated fibroblasts and subsequently myofibroblasts-KFM transformation). Coactivators of transcription, YAP (Yes-associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif), are mechanotransducers that intersect with the TGFβ pathway via interactions with Smad proteins. Here, we examined the distinct role of YAP and TAZ on TGFβ1 induced myofibroblast transformation of primary human corneal fibroblasts (HCFs).
METHODS: A knockdown approach was used to silence YAP and TAZ individually in HCFs. Forty-eight hours post siRNA transfection, cells were cultured in the presence or absence of 2 ng/ml TGFβ1 for 24h. The cells were subjected to nuclear and cytoplasmic fractionation. The expression of α-smooth muscle actin (αSMA), Smad 2, 3 and 4, CTGF and phospho-Smad2, 3, and 4 were assessed by qPCR and Western blotting.
RESULTS: TGFβ1 stimulation resulted in the decreased phosphorylation of YAP in the cytosol, and increased levels of phosphorylated TAZ and Smad2/3/4 in the nucleus. Knockdown of TAZ resulted in elevated YAP expression but not vice versa. Additionally, knockdown of TAZ but not YAP resulted in upregulation of αSMA expression in the presence and absence of TGFβ1. In the presence of TGFβ1 YAP knockdown increased Smad2/3/4 expression and Smad4 phosphorylation, while TAZ knockdown had no effect on Smad2/3/4 expression and phosphorylation. YAP knockdown inhibited CTGF expression while TAZ knockdown resulted in its increased expression. Finally, simultaneous knockdown of YAP and TAZ resulted in cell death.
CONCLUSION: Our findings suggest that YAP and TAZ function as distinct modulators of TGFβ1 induced myofibroblast transformation and have different roles in signalling. Specifically, TAZ limits YAP's ability to mediate KFM transformation via Smad proteins. The data also suggest that while having distinct effects, YAP and TAZ have redundant or combinatorial functions critical to cell survival. These results suggest that a loss of TAZ may help drive corneal haze and fibrosis and that the balance between YAP/TAZ is essential in controlling myofibroblast differentiation.
PMID:30578787 | PMC:PMC6455812 | DOI:10.1016/j.exer.2018.12.009
Biomechanical changes to Descemet's membrane precede endothelial cell loss in an early-onset murine model of Fuchs endothelial corneal dystrophy
Exp Eye Res. 2019 Mar;180:18-22. doi: 10.1016/j.exer.2018.11.021. Epub 2018 Nov 22.
Early-onset Fuchs endothelial corneal dystrophy (FECD) has been associated with nonsynonymous mutations in collagen VIII α2 (COL8A2), a key extracellular matrix (ECM) protein in Descemet's membrane (DM). Two knock-in strains of mice have been generated to each express a mutant COL8A2 protein (Col8a2L450W/L450W and Col8a2Q455K/Q455K) that recapitulate the clinical phenotype of early-onset FECD including endothelial cell loss, cellular polymegathism and pleomorphism, and guttae. Due to abnormalities in ECM protein composition and structure in FECD, the stiffness of DM in Col8a2 knock-in mice and wildtype (WT) controls was measured using atomic force microscopy at 5 and 10 months of age, coinciding with the onset of FECD phenotypic abnormalities. At 5 months, only sporadic guttae were identified via in vivo confocal microscopy (IVCM) in Col8a2Q455K/Q455K mice, otherwise both strains of Col8a2 transgenic mice were indistinguishable from WT controls in terms of endothelial cell density and size. By 10 months of age, Col8a2L450W/L450W and Col8a2Q455K/Q455K mice developed reduced corneal endothelial density, increased endothelial cell area and guttae, with the Col8a2Q455K/Q455K strain exhibiting a more severe phenotype. However, at 5 months of age, prior to the development endothelial cell abnormalities, Col8a2L450W/L450W and Col8a2Q455K/Q455K mice knock-in mice had reduced tissue stiffness of DM that was statistically significant in the Col8a2Q455K/Q455K mice when compared with wildtype controls. These data indicate that alterations in the tissue compliance of DM precede phenotypic changes in endothelial cell count and morphology, and may play a role in onset and progression of FECD.
PMID:30471280 | PMC:PMC6389414 | DOI:10.1016/j.exer.2018.11.021
Consensus recommendations for trabecular meshwork cell isolation, characterization and culture
Exp Eye Res. 2018 Jun;171:164-173. doi: 10.1016/j.exer.2018.03.001. Epub 2018 Mar 9.
Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.
PMID:29526795 | PMC:PMC6042513 | DOI:10.1016/j.exer.2018.03.001
Glaucomatous cell derived matrices differentially modulate non-glaucomatous trabecular meshwork cellular behavior
Acta Biomater. 2018 Apr 15;71:444-459. doi: 10.1016/j.actbio.2018.02.037. Epub 2018 Mar 7.
Ocular hypertension is a causal risk-factor to developing glaucoma. This is associated with stiffening of the trabecular meshwork (TM), the primary site of resistance to aqueous-humor-outflow. The mechanisms underlying this stiffening or how pathologic extracellular matrix (ECM) affects cell function are poorly understood. It is recognized that mechanotransduction systems allow cells to sense and translate the intrinsic biophysical properties of ECM into intracellular signals to control gene transcription, protein expression, and cell behavior. Using an anterior segment perfusion model, we document that there are significantly more low flow regions that are much stiffer, and fewer high flow regions that are less stiff in glaucomatous TM (GTM) when compared to non-glaucomatous TMs (NTM). GTM tissue also has fewer cells overall when compared with NTM tissue. In order to study the role of pathologic ECM in glaucoma disease progression, we conducted studies using cell derived matrices (CDM). First, we characterized the mechanics, composition and organization of fibronectin in ECM deposited by GTM and NTM cells treated with glucocorticosteroids. Then, we determined that these GTM-derived ECM are able to induce stiffening of normal NTM cells, and alter their gene/protein expression to resemble that of a glaucomatous phenotype. Further, we demonstrate that GTM-derived ECM causes endoplasmic reticular stress in NTM. They also became resistant to being reorganized by these NTM cells. These phenomena were exacerbated by ECMs obtained from steroid treated glaucoma model groups. Collectively, our data demonstrates that CDMs represent a novel tool for the study of bidirectional interactions between TM cells and their immediate microenvironment.
STATEMENT OF SIGNIFICANCE: Extracellular matrix (ECM) changes are prevalent in a number of diseases. The precise mechanisms by which changes in the ECM contribute to disease progression is unclear, primarily due to absence of appropriate models. Here, using glaucoma as a disease model, we document changes in cell derived matrix (CDM) and tissue mechanics that contribute to the pathology. Subsequently, we determine the effect that ECMs from diseased and healthy individuals have on healthy cell behaviors. Data emanating from this study demonstrate that CDMs are a potent tool for the study of cell-ECM interactions.
PMID:29524673 | PMC:PMC5899949 | DOI:10.1016/j.actbio.2018.02.037
Latrunculin B and substratum stiffness regulate corneal fibroblast to myofibroblast transformation
Exp Eye Res. 2018 May;170:101-107. doi: 10.1016/j.exer.2018.02.003. Epub 2018 Feb 6.
The transformation of keratocytes and fibroblasts to myofibroblasts is important to corneal wound healing as well as formation of stromal haze. The purpose of this study was to determine the effect of latrunculin B, an actin cytoskeleton disruptor in conjunction with a fundamental biophysical cue, substrate stiffness, on myofibroblast transformation in vitro and in vivo. Rabbit corneal fibroblasts were cultured on substrates of differing compliance (1.5, 22, and 71 kPa) and tissue culture plastic (TCP; > 1 GPa) in media containing 0 or 10 ng/ml TGFβ1 for 72 h. Cells were treated with 0.4 μM Lat-B or DMSO for 30 min every 24 h for 72 h. RNA was collected from cells and expression of alpha-smooth muscle actin (α-SMA), keratocan, and ALDH1A1 determined using qPCR; immunocytochemistry was used to assess α-SMA protein expression. A rabbit phototherapeutic keratectomy (PTK) model was used to assess the impact of 0.1% Lat-B (n = 3) or 25% DMSO (vehicle control, n = 3) on corneal wound healing by assessment of epithelial wound size with fluorescein stain and semi-quantitative stromal haze scoring by an observer masked to treatment group as well as Fourier-domain optical coherence tomography (FD-OCT) at set time points. Statistical analysis was completed using one-way or two-way analysis of variance. Treatment with Lat-B versus DMSO resulted in significantly less αSMA mRNA (P ≤ 0.007) for RCF cells grown on 22 and 71 kPa substrates as well as TCP without or with TGFβ1, and significantly decreased α-SMA protein expression in RCFs cultured on the intermediate (22 kPa) stiffness in the absence (P = 0.028) or presence (P = 0.018) of TGFβ1. Treatment with Lat-B versus DMSO but did not significantly alter expression of keratocan or ALDH1A1 mRNA in RCFs (P > 0.05) in the absence or presence of TGFβ1, but RCFs grown on stiff hydrogels (71 kPa) had significantly more keratocan mRNA expression versus the 22 kPa hydrogel or TCP (P < 0.001) without TGFβ1. Administration of topical Lat-B BID was well tolerated by rabbits post-PTK but did not significantly alter epithelial wound closure, stromal haze score, stromal haze thickness as measured by FD-OCT in comparison to DMSO-treated rabbits. When corneal stromal cells are cultured on substrates possessing biologically relevant substratum stiffnesses, Lat-B modulates mRNA and protein expression of α-SMA and thus modulates myofibroblast transformation. At a dose and dose-frequency that reduced IOP in human glaucoma patients, Lat-B treatment did not substantially impact corneal epithelial or stromal wound healing in a rabbit PTK model. While a significant impact on wound healing was observed at the concentration and dose frequency reported here was not found, encouraging in vitro data support further investigations of topically applied Lat-B to determine if this compound can reduce stromal fibrosis.
PMID:29421383 | PMC:PMC5924616 | DOI:10.1016/j.exer.2018.02.003
Biomechanical Rigidity and Quantitative Proteomics Analysis of Segmental Regions of the Trabecular Meshwork at Physiologic and Elevated Pressures
Invest Ophthalmol Vis Sci. 2018 Jan 1;59(1):246-259. doi: 10.1167/iovs.17-22759.
PURPOSE: The extracellular matrix (ECM) of the trabecular meshwork (TM) modulates resistance to aqueous humor outflow, thereby regulating IOP. Glaucoma, a leading cause of irreversible blindness worldwide, is associated with changes in the ECM of the TM. The elastic modulus of glaucomatous TM is larger than age-matched normal TM; however, the biomechanical properties of segmental low (LF) and high flow (HF) TM regions and their response to elevated pressure, are unknown.
METHODS: We perfused human anterior segments at two pressures using an ex vivo organ culture system. After extraction, we measured the elastic modulus of HF and LF TM regions by atomic force microscopy and quantitated protein differences by proteomics analyses.
RESULTS: The elastic modulus of LF regions was 2.3-fold larger than HF regions at physiological (1×) pressure, and 7.4-fold or 3.5-fold larger than HF regions at elevated (2×) pressure after 24 or 72 hours, respectively. Using quantitative proteomics, comparisons were made between HF and LF regions at 1× or 2× pressure. Significant ECM protein differences were observed between LF and HF regions perfused at 2×, and between HF regions at 1× compared to 2× pressures. Decorin, TGF-β-induced protein, keratocan, lumican, dermatopontin, and thrombospondin 4 were common differential candidates in both comparisons.
CONCLUSIONS: These data show changes in biomechanical properties of segmental regions within the TM in response to elevated pressure, and levels of specific ECM proteins. Further studies are needed to determine whether these ECM proteins are specifically involved in outflow resistance and IOP homeostasis.
PMID:29340639 | PMC:PMC5770183 | DOI:10.1167/iovs.17-22759
Tissue and cellular biomechanics during corneal wound injury and repair
Acta Biomater. 2017 Aug;58:291-301. doi: 10.1016/j.actbio.2017.05.051. Epub 2017 May 27.
Corneal wound healing is an enormously complex process that requires the simultaneous cellular integration of multiple soluble biochemical cues, as well as cellular responses to the intrinsic chemistry and biophysical attributes associated with the matrix of the wound space. Here, we document how the biomechanics of the corneal stroma are altered through the course of wound repair following keratoablative procedures in rabbits. Further we documented the influence that substrate stiffness has on stromal cell mechanics. Following corneal epithelial debridement, New Zealand white rabbits underwent phototherapeutic keratectomy (PTK) on the right eye (OD). Wound healing was monitored using advanced imaging modalities. Rabbits were euthanized and corneas were harvested at various time points following PTK. Tissues were characterized for biomechanics with atomic force microscopy and with histology to assess inflammation and fibrosis. Factor analysis was performed to determine any discernable patterns in wound healing parameters. The matrix associated with the wound space was stiffest at 7days post PTK. The greatest number of inflammatory cells were observed 3days after wounding. The highest number of myofibroblasts and the greatest degree of fibrosis occurred 21days after wounding. While all clinical parameters returned to normal values 400days after wounding, the elastic modulus remained greater than pre-surgical values. Factor analysis demonstrated dynamic remodeling of stroma occurs between days 10 and 42 during corneal stromal wound repair. Elastic modulus of the anterior corneal stroma is dramatically altered following PTK and its changes coincide initially with the development of edema and inflammation, and later with formation of stromal haze and population of the wound space with myofibroblasts. Factor analysis demonstrates strongest correlation between elastic modulus, myofibroblasts, fibrosis and stromal haze thickness, and between edema and central corneal thickness.
STATEMENT OF SIGNIFICANCE: Tissue biomechanics during the course of corneal wound healing is documented for the first time through atomic force microscopy, and is correlated with advanced clinical imaging and immunohistochemistry. Parameters obtained from the study are applied in a multivariate statistical model to cluster the data for better classification and monitor the wound repair process. Elastic modulus of the anterior corneal stroma is dramatically altered following wounding and correlates initially with the development of edema and inflammation, and later with formation of stromal haze and population of the wound space with myofibroblasts. Importantly, the occurrence of myofibroblasts is preceded by changes in tissue mechanics, which is important to consider in light of crosslinking procedures applied to treat corneal diseases.
PMID:28559158 | PMC:PMC5560898 | DOI:10.1016/j.actbio.2017.05.051
Species variation and spatial differences in mucin expression from corneal epithelial cells
Exp Eye Res. 2016 Nov;152:43-48. doi: 10.1016/j.exer.2016.09.001. Epub 2016 Sep 8.
Mucins are large glycoproteins expressed by epithelial cells of both the conjunctiva and cornea, and principle components of the glycocalyx. They are thought to play an important role in determining the interactions between the cornea/conjunctiva and the overlying tear film. The purpose of this study was to characterize the membrane-associated corneal mucin expression pattern from multiple species commonly used in ophthalmic research and drug development to better define the biochemical attributes of the ocular surface. Humans, rhesus macaques and dogs were found to have a very similar pattern of mucin expression, with mucin 16 (MUC16) being the most prevalent mucin transcript. In contrast, the rabbit had a unique mucin expression pattern with all mucin transcripts expressed at relatively similar levels. To determine if there were spatial differences in expression, peripheral and central corneal epithelium were individually isolated and evaluated for mucin expression. In all species examined, MUC1, MUC4 and MUC16 had higher peripheral corneal expression when compared with central, which reached statistical significance in MUC1 (rhesus and dog). The data demonstrated variation in corneal epithelial membrane-associated mucin expression between species, with the rabbit having a distinct expression pattern. These differences may be reflective of the environment, pathogen exposure or tear film dynamics of the respective species. The species differences, as well as regional mucin expression patterns, characterized in this study further define the biochemical composition of the ocular surface and may play an important role in tear film stability.
PMID:27614208 | PMC:PMC6697374 | DOI:10.1016/j.exer.2016.09.001
Sensing Conductive Hydrogels for Rapid Detection of Cytokines in Blood
Adv Healthc Mater. 2016 Mar;5(6):659-64, 627. doi: 10.1002/adhm.201500571. Epub 2016 Jan 22.
Conducting polymer hydrogel is fabricated atop gold or ITO electrodes and is functionalized with monoclonal antibodies. Binding of interferon-γ molecules causes redox properties of conductive hydrogel to change in a concentration-dependent fashion without the need for washing or sample handling steps. This conductive hydrogel remains functional in a fouling media such as whole blood.
PMID:26799538 | DOI:10.1002/adhm.201500571
Effect of Stratification on Surface Properties of Corneal Epithelial Cells
Invest Ophthalmol Vis Sci. 2015 Dec;56(13):8340-8. doi: 10.1167/iovs.15-17468.
PURPOSE: The purpose of this study was to determine the influence of mucin expression in an immortalized human corneal epithelial cell line (hTCEpi) on the surface properties of cells, such as wettability, contact angle, and surface heterogeneity.
METHODS: hTCEpi cells were cultured to confluence in serum-free medium. The medium was then replaced by stratification medium to induce mucin biosynthesis. The mucin expression profile was analyzed using quantitative PCR and Western blotting. Contact angles were measured using a two-immiscible liquid method, and contact angle hysteresis was evaluated by tilting the apparatus and recording advancing and receding contact angles. The spatial distribution of mucins was evaluated with fluorescently labeled lectin.
RESULTS: hTCEpi cells expressed the three main ocular mucins (MUC1, MUC4, and MUC16) with a maximum between days 1 and 3 of the stratification process. Upon stratification, cells caused a very significant increase in contact angle hysteresis, suggesting the development of spatially discrete and heterogeneously distributed surface features, defined by topography and/or chemical functionality. Although atomic force microscopy measurements showed no formation of appreciable topographic features on the surface of the cells, we observed a significant increase in surface chemical heterogeneity.
CONCLUSIONS: The surface chemical heterogeneity of the corneal epithelium may influence the dynamic behavior of tear film by "pinning" the contact line between the cellular surface and aqueous tear film. Engineering the surface properties of corneal epithelium could potentially lead to novel treatments in dry eye disease.
PMID:26747762 | PMC:PMC4699408 | DOI:10.1167/iovs.15-17468
Dexamethasone Stiffens Trabecular Meshwork, Trabecular Meshwork Cells, and Matrix
Invest Ophthalmol Vis Sci. 2015 Jul;56(8):4447-59. doi: 10.1167/iovs.15-16739.
PURPOSE: Treatment with corticosteroids can result in ocular hypertension and may lead to the development of steroid-induced glaucoma. The extent to which biomechanical changes in trabecular meshwork (TM) cells and extracellular matrix (ECM) contribute toward this dysfunction is poorly understood.
METHODS: Primary human TM (HTM) cells were cultured for either 3 days or 4 weeks in the presence or absence of dexamethasone (DEX), and cell mechanics, matrix mechanics and proteomics were determined, respectively. Adult rabbits were treated topically with either 0.1% DEX or vehicle over 3 weeks, and mechanics of the TM were determined.
RESULTS: Treatment with DEX for 3 days resulted in a 2-fold increase in HTM cell stiffness, and this correlated with activation of extracellular signal-related kinase 1/2 (ERK1/2) and overexpression of α-smooth muscle actin (αSMA). Further, the matrix deposited by HTM cells chronically treated with DEX is approximately 4-fold stiffer, more organized, and has elevated expression of matrix proteins commonly implicated in glaucoma (decorin, myocilin, fibrillin, secreted frizzle-related protein [SFRP1], matrix-gla). Also, DEX treatment resulted in a 3.5-fold increase in stiffness of the rabbit TM.
DISCUSSION: This integrated approach clearly demonstrates that DEX treatment increases TM cell stiffness concurrent with elevated αSMA expression and activation of the mitogen-activated protein kinase (MAPK) pathway, stiffens the ECM in vitro along with upregulation of Wnt antagonists and fibrotic markers embedded in a more organized matrix, and increases the stiffness of TM tissues in vivo. These results demonstrate glucocorticoid treatment can initiate the biophysical alteration associated with increased resistance to aqueous humor outflow and the resultant increase in IOP.
PMID:26193921 | PMC:PMC4509060 | DOI:10.1167/iovs.15-16739
A Comparative Study of Vertebrate Corneal Structure: The Evolution of a Refractive Lens
Invest Ophthalmol Vis Sci. 2015 Apr;56(4):2764-72. doi: 10.1167/iovs.15-16584.
PURPOSE: Although corneal curvature plays an important role in determining the refractive power of the vertebrate eye, the mechanisms controlling corneal shape remain largely unknown. To address this question, we performed a comparative study of vertebrate corneal structure to identify potential evolutionarily based changes that correlate with the development of a corneal refractive lens.
METHODS: Nonlinear optical (NLO) imaging of second-harmonic-generated (SHG) signals was used to image collagen and three-dimensionally reconstruct the lamellar organization in corneas from different vertebrate clades.
RESULTS: Second-harmonic-generated images taken normal to the corneal surface showed that corneal collagen in all nonmammalian vertebrates was organized into sheets (fish and amphibians) or ribbons (reptiles and birds) extending from limbus to limbus that were oriented nearly orthogonal (ranging from 77.7°-88.2°) to their neighbors. The slight angular offset (2°-13°) created a rotational pattern that continued throughout the full thickness in fish and amphibians and to the very posterior layers in reptiles and birds. Interactions between lamellae were limited to "sutural" fibers in cartilaginous fish, and occasional lamellar branching in fish and amphibians. There was a marked increase in lamellar branching in higher vertebrates, such that birds ≫ reptiles > amphibians > fish. By contrast, mammalian corneas showed a nearly random collagen fiber organization with no orthogonal, chiral pattern.
CONCLUSIONS: Our data indicate that nonmammalian vertebrate corneas share a common orthogonal collagen structural organization that shows increased lamellar branching in higher vertebrate species. Importantly, mammalian corneas showed a different structural organization, suggesting a divergent evolutionary background.
PMID:26066606 | PMC:PMC4416662 | DOI:10.1167/iovs.15-16584
The intrinsic stiffness of human trabecular meshwork cells increases with senescence
Oncotarget. 2015 Jun 20;6(17):15362-74. doi: 10.18632/oncotarget.3798.
Dysfunction of the human trabecular meshwork (HTM) plays a central role in the age-associated disease glaucoma, a leading cause of irreversible blindness. The etiology remains poorly understood but cellular senescence, increased stiffness of the tissue, and the expression of Wnt antagonists such as secreted frizzled related protein-1 (SFRP1) have been implicated. However, it is not known if senescence is causally linked to either stiffness or SFRP1 expression. In this study, we utilized in vitro HTM senescence to determine the effect on cellular stiffening and SFRP1 expression. Stiffness of cultured cells was measured using atomic force microscopy and the morphology of the cytoskeleton was determined using immunofluorescent analysis. SFRP1 expression was measured using qPCR and immunofluorescent analysis. Senescent cell stiffness increased 1.88±0.14 or 2.57±0.14 fold in the presence or absence of serum, respectively. This was accompanied by increased vimentin expression, stress fiber formation, and SFRP1 expression. In aggregate, these data demonstrate that senescence may be a causal factor in HTM stiffening and elevated SFRP1 expression, and contribute towards disease progression. These findings provide insight into the etiology of glaucoma and, more broadly, suggest a causal link between senescence and altered tissue biomechanics in aging-associated diseases.
PMID:25915531 | PMC:PMC4558157 | DOI:10.18632/oncotarget.3798
Wnt inhibition induces persistent increases in intrinsic stiffness of human trabecular meshwork cells
Exp Eye Res. 2015 Mar;132:174-8. doi: 10.1016/j.exer.2015.01.025. Epub 2015 Jan 30.
Wnt antagonism has been linked to glaucoma and intraocular pressure regulation, as has increased stiffness of human trabecular meshwork (HTM) tissue. We have shown culturing HTM cells on substrates that mimic the elevated stiffness of glaucomatous tissue leads to elevated expression of the Wnt antagonist secreted frizzled related protein 1 (SFRP1), suggesting a linkage between SFRP1 and HTM mechanobiology. In this study, we document biomechanical consequences of Wnt antagonism on HTM cells. Cells were treated with the Wnt antagonists (SFRP1, KY02111, and LGK-974) for 8 days and allowed to recover for 4 days. After recovery, intrinsic cell stiffness and activation of the Wnt pathway via β-catenin staining and blotting were assayed. Basal cell stiffness values were 3.71 ± 0.37, 4.33 ± 3.07, and 3.07 ± kPa (median ± S.D.) for cells derived from 3 donors. Cell stiffness increased after 0.25 μg/mL (4.32 ± 5.12, 8.86 ± 8.51, 4.84 ± 3.15 kPa) and 0.5 μg/mL (16.75 ± 5.59, 13.18 ± 7.99, and 8.54 ± 5.77 kPa) SFRP1 treatment. Stiffening was observed after 10 μM KY02111 (10.72 ± 5.63 and 6.57 ± 5.53 kPa) as well as LGK-974 (9.60 ± 7.41 and 11.40 ± 9.24 kPa) treatment compared with controls (3.79 ± 1.01 and 5.16 ± 2.14 kPa). Additionally, Wnt inhibition resulted in decreased β-catenin staining and increased phosphorylation at threonine 41 after recovery. In conclusion, this work demonstrates a causal relationship between Wnt inhibition and cell stiffening. Additionally, these findings suggest transient Wnt inhibition resulted in durable modulation of the mechanical phenotype of HTM cells. When placed in context with previous results, these findings provide a causal link between Wnt antagonism and cell stiffness and suggest a feedback loop contributing to glaucoma progression.
PMID:25639201 | PMC:PMC4352377 | DOI:10.1016/j.exer.2015.01.025
Assessment of platelet-derived growth factor using A splinted full thickness dermal wound model in bearded dragons (Pogona vitticeps)
J Zoo Wildl Med. 2014 Dec;45(4):866-74. doi: 10.1638/2014-0037.1.
Wounds in reptiles are a common reason for presentation to a veterinarian. At this time there is limited information on effective topical medications to aid in wound closure. The objectives of this study were to translate the splinted, full-thickness dermal wound model, validated in mice, to the bearded dragon (Pogona vitticeps) and to determine the effect of topical becaplermin (BP), a platelet-derived growth factor (0.01%), on the rate of wound closure. Ten bearded dragons were anesthetized and two full-thickness cutaneous wounds were made on the dorsum of each lizard. Encircling splints were applied surrounding each wound and subsequently covered by a semi-occlusive dressing. Five lizards had one wound treated with BP and the adjacent wound treated with a vehicle control. Five additional lizards had one wound treated with saline and the second wound treated with a vehicle control. Wounds were imaged daily, and the wound area was measured using digital image analysis. The change in percentage wound closure over 17 days and the time to 50% wound closure was compared among the four treatment groups. There was no significant difference in wound closure rates between BP-treated and saline-treated wounds or in the time to 50% wound closure between any treatments. Vehicle-treated wounds adjacent to saline-treated wounds closed significantly slower than did BP (P < 0.010), saline (P < 0.001), and vehicle-treated wounds adjacent to BP-treated wounds (P < 0.013). Our preliminary study indicates that the splinted wound model, with modifications, may be used to determine wound closure rates in bearded dragons. When compared with saline, BP did not have a significant effect on wound closure rates, while the vehicle alone delayed wound closure. Histologic analysis of experimentally created wounds throughout the wound healing process is needed to further evaluate the effects of these treatments on reptile dermal wound healing.
PMID:25632675 | DOI:10.1638/2014-0037.1
Biomimetic stochastic topography and electric fields synergistically enhance directional migration of corneal epithelial cells in a MMP-3-dependent manner
Acta Biomater. 2015 Jan;12:102-112. doi: 10.1016/j.actbio.2014.10.007. Epub 2014 Oct 13.
Directed migration of corneal epithelial cells (CECs) is critical for maintenance of corneal homeostasis as well as wound healing. Soluble cytoactive factors and the intrinsic chemical attributes of the underlying extracellular matrix (ECM) participate in stimulating and directing migration. The central importance of the intrinsic biophysical attributes of the microenvironment of the cell in modulating an array of fundamental epithelial behaviors including migration has been widely documented. Among the best measures of these attributes are the intrinsic topography and stiffness of the ECM and electric fields (EFs). How cells integrate these multiple simultaneous inputs is not well understood. Here, we present a method that combines the use of (i) topographically patterned substrates (mean pore diameter 800nm) possessing features that approximate those found in the native corneal basement membrane; and (ii) EFs (0-150mVmm(-1)) mimicking those at corneal epithelial wounds that the cells experience in vivo. We found that topographic cues and EFs synergistically regulated directional migration of human CECs and that this was associated with upregulation of matrix metalloproteinase-3 (MMP3). MMP3 expression and activity were significantly elevated with 150mVmm(-1) applied-EF while MMP2/9 remained unaltered. MMP3 expression was elevated in cells cultured on patterned surfaces against planar surfaces. The highest single-cell migration rate was observed with 150mVmm(-1) applied EF on patterned and planar surfaces. When cultured as a confluent sheet, EFs induced collective cell migration on stochastically patterned surfaces compared with dissociated single-cell migration on planar surfaces. These results suggest significant interaction of biophysical cues in regulating cell behaviors and will help define design parameters for corneal prosthetics and help to better understand corneal wound healing.
PMID:25311684 | PMC:PMC4798428 | DOI:10.1016/j.actbio.2014.10.007
Involvement of YAP, TAZ and HSP90 in contact guidance and intercellular junction formation in corneal epithelial cells
PLoS One. 2014 Oct 7;9(10):e109811. doi: 10.1371/journal.pone.0109811. eCollection 2014.
The extracellular environment possesses a rich milieu of biophysical and biochemical signaling cues that are simultaneously integrated by cells and influence cellular phenotype. Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (WWTR1; TAZ), two important signaling molecules of the Hippo pathway, have been recently implicated as nuclear relays of cytoskeletal changes mediated by substratum rigidity and topography. These proteins intersect with other important intracellular signaling pathways (e.g. Wnt and TGFβ). In the cornea, epithelial cells adhere to the stroma through a 3-dimensional topography-rich basement membrane, with features in the nano-submicron size-scale that are capable of profoundly modulating a wide range of fundamental cell behaviors. The influences of substratum-topography, YAP/TAZ knockdown, and HSP90 inhibition on cell morphology, YAP/TAZ localization, and the expression of TGFβ2 and CTGF, were investigated. The results demonstrate (a) that knockdown of TAZ enhances contact guidance in a YAP dependent manner, (b) that CTGF is predominantly regulated by YAP and not TAZ, and (c) that TGFβ2 is regulated by both YAP and TAZ in these cells. Additionally, inhibition of HSP90 resulted in nuclear localization and subsequent transcriptional-activation of YAP, formation of cell-cell junctions and co-localization of E-cadherin and β-catenin at adherens junctions. Results presented in this study reflect the complexities underlying the molecular relationships between the cytoskeleton, growth factors, heat shock proteins, and co-activators of transcription that impact mechanotransduction. The data reveal the importance of YAP/TAZ on the cell behaviors, and gene and protein expression.
PMID:25290150 | PMC:PMC4188597 | DOI:10.1371/journal.pone.0109811
Impact of Nanotopography, Heparin Hydrogel Microstructures, and Encapsulated Fibroblasts on Phenotype of Primary Hepatocytes
ACS Appl Mater Interfaces. 2015 Jun 17;7(23):12299-308. doi: 10.1021/am504614e. Epub 2014 Sep 23.
Hepatocytes, the main epithelial cell type in the liver, perform most of the biochemical functions of the liver. Thus, maintenance of a primary hepatocyte phenotype is crucial for investigations of in vitro drug metabolism, toxicity, and development of bioartificial liver constructs. Here, we report the impact of topographic cues alone and in combination with soluble signals provided by encapsulated feeder cells on maintenance of the primary hepatocyte phenotype. Topographic features were 300 nm deep with pitches of either 400, 1400, or 4000 nm. Hepatocyte cell attachment, morphology and function were markedly better on 400 nm pitch patterns compared with larger scale topographies or planar substrates. Interestingly, topographic features having biomimetic size scale dramatically increased cell adhesion whether or not substrates had been precoated with collagen I. Albumin production in primary hepatocytes cultured on 400 nm pitch substrates without collagen I was maintained over 10 days and was considerably higher compared to albumin synthesis on collagen-coated flat substrates. In order to investigate the potential interaction of soluble cytoactive factors supplied by feeder cells with topographic cues in determining cell phenotype, bioactive heparin-containing hydrogel microstructures were molded (100 μm spacing, 100 μm width) over the surface of the topographically patterned substrates. These hydrogel microstructures either carried encapsulated fibroblasts or were free of cells. Hepatocytes cultured on nanopatterned substrates next to fibroblast carrying hydrogel microstructures were significantly more functional than hepatocytes cultured on nanopatterned surfaces without hydrogels or stromal cells significantly elevated albumin expression and cell junction formation compared to cells provided with topographic cues only. The simultaneous presentation of topographic biomechanical cues along with soluble signaling molecules provided by encapsulated fibroblasts cells resulted in optimal functionality of cultured hepatocytes. The provision of both topographic and soluble signaling cues could enhance our ability to create liver surrogates and inform the development of engineered liver constructs.
PMID:25247391 | PMC:PMC4372509 | DOI:10.1021/am504614e
PDGF-BB does not accelerate healing in diabetic mice with splinted skin wounds
PLoS One. 2014 Aug 14;9(8):e104447. doi: 10.1371/journal.pone.0104447. eCollection 2014.
Topical application of platelet-derived growth factor-BB (PDGF-BB) is considered to accelerate tissue repair of impaired chronic wounds. However, the vast literature is plagued with conflicting reports of its efficacy in animal models and this is often influenced by a wide array of experimental variables making it difficult to compare the results across the studies. To mitigate the confounding variables that influence the efficacy of topically applied PDGF-BB, we used a controlled full thickness splinted excisional wound model in db/db mice (type 2 diabetic mouse model) for our investigations. A carefully-defined silicone-splinted wound model, with reduced wound contraction, controlled splint and bandage maintenance, allowing for healing primarily by reepithelialization was employed. Two splinted 8 mm dorsal full thickness wounds were made in db/db mice. Wounds were topically treated once daily with either 3 µg PDGF-BB in 30 µl of 5% PEG-PBS vehicle or an equal volume of vehicle for 10 days. Body weights, wound contraction, wound closure, reepithelialization, collagen content, and wound bed inflammation were evaluated clinically and histopathologically. The bioactivity of PDGF-BB was confirmed by in vitro proliferation assay. PDGF-BB, although bioactive in vitro, failed to accelerate wound healing in vivo in the db/db mice using the splinted wound model. Considering that the predominant mechanism of wound healing in humans is by re-epithelialization, the most appropriate model for evaluating therapeutics is one that uses splints to prevent excessive wound contraction. Here, we report that PDGF-BB does not promote wound closure by re-epithelialization in a murine splinted wound model. Our results highlight that the effects of cytoactive factors reported in vivo ought to be carefully interpreted with critical consideration of the wound model used.
PMID:25121729 | PMC:PMC4133340 | DOI:10.1371/journal.pone.0104447
Automated AFM force curve analysis for determining elastic modulus of biomaterials and biological samples
J Mech Behav Biomed Mater. 2014 Sep;37:209-18. doi: 10.1016/j.jmbbm.2014.05.027. Epub 2014 Jun 5.
The analysis of atomic force microscopy (AFM) force data requires the selection of a contact point (CP) and is often time consuming and subjective due to influence from intermolecular forces and low signal-to-noise ratios (SNR). In this report, we present an automated algorithm for the selection of CPs in AFM force data and the evaluation of elastic moduli. We propose that CP may be algorithmically easier to detect by identifying a linear elastic indentation region of data (high SNR) rather than the contact point itself (low SNR). Utilizing Hertzian mechanics, the data are fitted for the CP. We first detail the algorithm and then evaluate it on sample polymeric and biological materials. As a demonstration of automation, 64 × 64 force maps were analyzed to yield spatially varying topographical and mechanical information of cells. Finally, we compared manually selected CPs to automatically identified CPs and demonstrated that our automated approach is both accurate (< 10nm difference between manual and automatic) and precise for non-interacting polymeric materials. Our data show that the algorithm is useful for analysis of both biomaterials and biological samples.
PMID:24951927 | PMC:PMC4465402 | DOI:10.1016/j.jmbbm.2014.05.027
Full-thickness splinted skin wound healing models in db/db and heterozygous mice: implications for wound healing impairment
Wound Repair Regen. 2014 May-Jun;22(3):368-80. doi: 10.1111/wrr.12172.
The excisional dorsal full-thickness skin wound model with or without splinting is widely utilized in wound healing studies using diabetic or normal mice. However, the effects of splinting on dermal wound healing have not been fully characterized, and there are limited data on the direct comparison of wound parameters in the splinted model between diabetic and normal mice. We compared full-thickness excisional dermal wound healing in db/db and heterozygous mice by investigating the effects of splinting, semi-occlusive dressing, and poly(ethylene glycol) treatment. Two 8-mm full-thickness wounds were made with or without splinting in db/db and heterozygous mice. Body weights, splint maintenance, wound contraction, wound closure, and histopathological parameters including reepithelialization, wound bed collagen deposition, and inflammation were compared between groups. Our results show that silicone splint application effectively reduced wound contraction in heterozygous and db/db mice. Splinted wounds, as opposed to nonsplinted wounds, exhibited no significant differences in wound closure between heterozygous and db/db mice. Finally, polyethylene glycol and the noncontact dressing had no significant effect on wound healing in heterozygous or db/db mice. We believe these findings will help investigators in selection of the appropriate wound model and data interpretation with fully defined parameters.
PMID:24844336 | DOI:10.1111/wrr.12172
A cell culture substrate with biologically relevant size-scale topography and compliance of the basement membrane
Langmuir. 2014 Mar 4;30(8):2101-8. doi: 10.1021/la403590v. Epub 2014 Feb 21.
A growing body of literature broadly documents that a wide array of fundamental cell behaviors are modulated by the physical attributes of the cellular microenvironment, yet in vitro assays are typically carried out using tissue culture plastic or glass substrates that lack the 3-dimensional topography present in vivo and have stiffness values that far exceed that of cellular and stromal microenvironments. This work presents a method for the fabrication of thin hydrogel films that can replicate arbitrary topographies with a resolution of 400 nm that possess an elastic modulus of approximately 250 kPa. Material characterization including swelling behavior and mechanics were performed and reported. Cells cultured on these surfaces patterned with anisotropic ridges and grooves react to the biophysical cues present and show an alignment response.
PMID:24524303 | PMC:PMC3983385 | DOI:10.1021/la403590v
Human trabecular meshwork cells exhibit several characteristics of, but are distinct from, adipose-derived mesenchymal stem cells
J Ocul Pharmacol Ther. 2014 Mar-Apr;30(2-3):254-66. doi: 10.1089/jop.2013.0175. Epub 2014 Jan 23.
PURPOSE: To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs).
METHODS: HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue.
RESULTS: Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue.
CONCLUSIONS: HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities.
PMID:24456002 | PMC:PMC3991981 | DOI:10.1089/jop.2013.0175
Robust and artifact-free mounting of tissue samples for atomic force microscopy
Biotechniques. 2014 Jan;56(1):40-2. doi: 10.2144/000114126.
Immobilization of tissue-samples for atomic for microscopy (AFM) is typically done using either semi-dry tissue or by gluing the tissue sample down, both of which can introduce artifacts. Here, we describe the design of a Soft- Clamping Immobilizing Retainer of Tissue (SCIRT) for consistent and nondestructive immobilization of tissues for AFM analysis. We compare the performance of our SCIRT method with glue-immobilization for two difficult to handle tissue types: human trabecular meshwork (HTM) and rabbit cornea (RC). Our results demonstrate that the SCIRT method has several advantages, including: (i) allowing for small sample sizes, (ii) enabling continuous hydration, (iii) eliminating contact with glue or associated solvents, (iv) permitting sample recovery following measurement, and (v) ease of use. In conclusion, the SCIRT method is a simple and effective means of immobilizing soft, hydrated tissue samples consistently and without artifacts.
PMID:24447138 | PMC:PMC4322673 | DOI:10.2144/000114126
Photopatternable and photoactive hydrogel for on-demand generation of hydrogen peroxide in cell culture
Biomaterials. 2014 Feb;35(5):1762-70. doi: 10.1016/j.biomaterials.2013.11.030. Epub 2013 Nov 28.
Oxidative stress, largely mediated by reactive oxygen species (ROS), is a nearly ubiquitous component in complex biological processes such as aging and disease. Optimal in vitro methods used in elucidating disease mechanisms would deliver of low levels of hydrogen peroxide, emulating the in vivo pathological state, but current methods are limited by kinetic stability or accurate measurement of the dose administered. Here we present an in vitro platform that exploits anthraquinone catalysts for the photocatalytic production of hydrogen peroxide. This system can be dynamically tuned to provide constant generation of hydrogen peroxide at a desired physiologic rate over at least 14 days and is described using a kinetic model. Material characterization and stability is discussed along with a proof-of-concept in vitro study that assessed the viability of cells as they were oxidatively challenged over 24 h at different ROS generation rates.
PMID:24290809 | PMC:PMC3992930 | DOI:10.1016/j.biomaterials.2013.11.030
Elastic modulus and collagen organization of the rabbit cornea: epithelium to endothelium
Acta Biomater. 2014 Feb;10(2):785-91. doi: 10.1016/j.actbio.2013.09.025. Epub 2013 Sep 29.
The rabbit is commonly used to evaluate new corneal prosthetics and study corneal wound healing. Knowledge of the stiffness of the rabbit cornea would better inform the design and fabrication of keratoprosthetics and substrates with relevant mechanical properties for in vitro investigations of corneal cellular behavior. This study determined the elastic modulus of the rabbit corneal epithelium, anterior basement membrane (ABM), anterior and posterior stroma, Descemet's membrane (DM) and endothelium using atomic force microscopy (AFM). In addition, three-dimensional collagen fiber organization of the rabbit cornea was determined using nonlinear optical high-resolution macroscopy. The elastic modulus as determined by AFM for each corneal layer was: epithelium, 0.57 ± 0.29 kPa (mean ± SD); ABM, 4.5 ± 1.2 kPa, anterior stroma, 1.1 ± 0.6 kPa; posterior stroma, 0.38 ± 0.22 kPa; DM, 11.7 ± 7.4 kPa; and endothelium, 4.1 ± 1.7 kPa. The biophysical properties, including the elastic modulus, are unique for each layer of the rabbit cornea and are dramatically softer in comparison to the corresponding regions of the human cornea. Collagen fiber organization is also dramatically different between the two species, with markedly less intertwining observed in the rabbit vs. human cornea. Given that the substratum stiffness considerably alters the corneal cell behavior, keratoprosthetics that incorporate mechanical properties simulating the native human cornea may not elicit optimal cellular performance in rabbit corneas that have dramatically different elastic moduli. These data should allow for the design of substrates that better mimic the biomechanical properties of the corneal cellular environment.
PMID:24084333 | PMC:PMC4280096 | DOI:10.1016/j.actbio.2013.09.025
Substratum compliance modulates corneal fibroblast to myofibroblast transformation
Invest Ophthalmol Vis Sci. 2013 Aug 28;54(8):5901-7. doi: 10.1167/iovs.12-11575.
PURPOSE: The transformation of fibroblasts to myofibroblasts is critical to corneal wound healing, stromal haze formation, and scarring. It has recently been demonstrated that the provision of biomimetic substratum topographic cues inhibits the progression toward the myofibroblast phenotype under the influence of transforming growth factor β1 (TGF-β1). The objective of this study was to determine the effect of another fundamental biophysical cue, substrate compliance, on TGF-β1-induced myofibroblast transformation of primary corneal cells isolated from human and rabbit corneas.
METHODS: Human and rabbit corneal fibroblasts were cultured on surfaces of varying substrate compliance (4-71 kPa) and tissue culture plastic (TCP) (> 1 gigapascal [GPa]). Cells were cultured in media containing TGF-β1 at concentrations of 0, 1, or 10 ng/mL for 72 hours. RNA and protein were collected from cells cultured on polyacrylamide gels and TCP and were analyzed for the expression of α-smooth muscle actin (α-SMA), a key marker of myofibroblast transformation, using quantitative PCR, immunocytochemistry, and Western blot.
RESULTS: Cells grown on more compliant substrates demonstrated significantly reduced amounts of α-SMA mRNA compared with TCP. Immunocytochemistry and Western blot analysis determining the presence of α-SMA corroborated this finding, thus confirming a reduced transformation to the myofibroblast phenotype on more compliant substrates compared with cells on TCP in the presence of TGF-β1.
CONCLUSIONS: These data indicate that substrate compliance modulates TGF-β1-induced expression of α-SMA and thus influences myofibroblast transformation in the corneal stroma. This provides further evidence that biomimetic biophysical cues inhibit myofibroblast transformation and participate in stabilizing the native cellular phenotype.
PMID:23860754 | PMC:PMC3757908 | DOI:10.1167/iovs.12-11575
Characterizing the effects of heparin gel stiffness on function of primary hepatocytes
Tissue Eng Part A. 2013 Dec;19(23-24):2655-63. doi: 10.1089/ten.TEA.2012.0681. Epub 2013 Aug 16.
In the liver, hepatocytes are exposed to a large array of stimuli that shape hepatic phenotype. This in vivo microenvironment is lost when hepatocytes are cultured in standard cell cultureware, making it challenging to maintain hepatocyte function in vitro. Our article focused on one of the least studied inducers of the hepatic phenotype-the mechanical properties of the underlying substrate. Gel layers comprised of thiolated heparin (Hep-SH) and diacrylated poly(ethylene glycol) (PEG-DA) were formed on glass substrates via a radical mediated thiol-ene coupling reaction. The substrate stiffness varied from 10 to 110 kPa by changing the concentration of the precursor solution. ELISA analysis revealed that after 5 days, hepatocytes cultured on a softer heparin gel were synthesizing five times higher levels of albumin compared to those on a stiffer heparin gel. Immunofluorescent staining for hepatic markers, albumin and E-cadherin, confirmed that softer gels promoted better maintenance of the hepatic phenotype. Our findings point to the importance of substrate mechanical properties on hepatocyte function.
PMID:23815179 | PMC:PMC3856597 | DOI:10.1089/ten.TEA.2012.0681
Influence of particle size and reactive oxygen species on cobalt chrome nanoparticle-mediated genotoxicity
Biomaterials. 2013 May;34(14):3559-70. doi: 10.1016/j.biomaterials.2013.01.085. Epub 2013 Feb 20.
Patients with cobalt chrome (CoCr) metal-on-metal (MOM) implants may be exposed to a wide size range of metallic nanoparticles as a result of wear. In this study we have characterised the biological responses of human fibroblasts to two types of synthetically derived CoCr particles [(a) from a tribometer (30 nm) and (b) thermal plasma technology (20, 35, and 80 nm)] in vitro, testing their dependence on nanoparticle size or the generation of oxygen free radicals, or both. Metal ions were released from the surface of nanoparticles, particularly from larger (80 nm) particles generated by thermal plasma technology. Exposure of fibroblasts to these nanoparticles triggered rapid (2 h) generation of reactive oxygen species (ROS) that could be eliminated by inhibition of NADPH oxidase, suggesting that it was mediated by phagocytosis of the particles. The exposure also caused a more prolonged, MitoQ sensitive production of ROS (24 h), suggesting involvement of mitochondria. Consequently, we recorded elevated levels of aneuploidy, chromosome clumping, fragmentation of mitochondria and damage to the cytoskeleton particularly to the microtubule network. Exposure to the nanoparticles resulted in misshapen nuclei, disruption of mature lamin B1 and increased nucleoplasmic bridges, which could be prevented by MitoQ. In addition, increased numbers of micronuclei were observed and these were only partly prevented by MitoQ, and the incidence of micronuclei and ion release from the nanoparticles were positively correlated with nanoparticle size, although the cytogenetic changes, modifications in nuclear shape and the amount of ROS were not. These results suggest that cells exhibit diverse mitochondrial ROS-dependent and independent responses to CoCr particles, and that nanoparticle size and the amount of metal ion released are influential.
PMID:23433773 | DOI:10.1016/j.biomaterials.2013.01.085
Role of substratum stiffness in modulating genes associated with extracellular matrix and mechanotransducers YAP and TAZ
Invest Ophthalmol Vis Sci. 2013 Jan 14;54(1):378-86. doi: 10.1167/iovs.12-11007.
PURPOSE: Primary open-angle glaucoma is characterized by increased resistance to aqueous humor outflow and a stiffer human trabecular meshwork (HTM). Two Yorkie homologues, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif, encoded by WWTR1 (TAZ), are mechanotransducers of the extracellular-microenvironment and coactivators of transcription. Here, we explore how substratum stiffness modulates the YAP/TAZ pathway and extracellular matrix genes in HTM cells and how this may be play a role in the onset and progression of glaucoma.
METHODS: HTM cells from normal donors were cultured on hydrogels mimicking the stiffness of normal (5 kPa) and glaucomatous (75 kPa) HTM. Changes in expression of YAP/TAZ related genes and steroid responsiveness were determined. Additionally, transglutaminase-2 expression was determined after YAP silencing.
RESULTS: YAP and TAZ are both expressed in human trabecular meshwork cells. In vitro, YAP and TAZ were inversely regulated by substratum stiffness. YAP and 14-3-3σ were downregulated to different extents on stiffer substrates; TAZ, tissue transglutaminase (TGM2), and soluble frizzled-related protein-1 (sFRP-1) were significantly upregulated. CTGF expression appeared to be altered differentially by both YAP and TAZ. Myocilin and angiopoietin-like 7 expression in response to dexamethasone was more pronounced on stiffer substrates. We demonstrated a direct effect by YAP on TGM2 when YAP was silenced by small interfering RNA.
CONCLUSIONS: The expression of YAP/TAZ and ECM-related-genes is impacted on physiologically relevant substrates. YAP was upregulated in cells on softer substrates. Stiffer substrates resulted in upregulation of canonical Wnt modulators, TAZ and sFRP-1, and thus may influence the progression of glaucoma. These results demonstrate the importance of YAP/TAZ in the HTM and suggest their role in glaucoma.
PMID:23258147 | PMC:PMC3594895 | DOI:10.1167/iovs.12-11007
Nuclear and cellular alignment of primary corneal epithelial cells on topography
J Biomed Mater Res A. 2013 Apr;101(4):1069-79. doi: 10.1002/jbm.a.34417. Epub 2012 Sep 11.
The basement membrane of the corneal epithelium presents biophysical cues in the form of topography and compliance that can modulate cytoskeletal dynamics, which, in turn, can result in altering cellular and nuclear morphology and alignment. In this study, the effect of topographic patterns of alternating ridges and grooves on nuclear and cellular shape and alignment was determined. Primary corneal epithelial cells were cultured on either planar or topographically patterned (400-4000 nm pitch) substrates. Alignment of individual cell body was correlated with respective nucleus for the analysis of orientation and elongation. A biphasic response in alignment was observed. Cell bodies preferentially aligned perpendicular to the 800 nm pitch; and with increasing pitch, cells increasingly aligned parallel to the substratum. Nuclear orientation largely followed this trend with the exception of those on 400 nm. On this biomimetic size scale, some nuclei oriented perpendicular to the topography while their cytoskeleton elements aligned parallel. Both nuclei and cell bodies were elongated on topography compared to those on flat surfaces. Our data demonstrate that nuclear orientation and shape are differentially altered by topographic features that are not mandated by alignment of the cell body. This novel finding suggests that nuanced differences in alignment of the nucleus versus the cell body exist and that these differences could have consequences on gene and protein regulation that ultimately regulate cell behaviors. A full understanding of these mechanisms could disclose novel pathways that would better inform evolving strategies in cell, stem cell, and tissue engineering as well as the design and fabrication of improved prosthetic devices.
PMID:22965583 | PMC:PMC3581752 | DOI:10.1002/jbm.a.34417
Focal adhesion kinase knockdown modulates the response of human corneal epithelial cells to topographic cues
Acta Biomater. 2012 Dec;8(12):4285-94. doi: 10.1016/j.actbio.2012.07.004. Epub 2012 Jul 17.
A rapidly expanding literature broadly documents the impact of biophysical cues on cellular behaviors. In spite of increasing research efforts in this field, the underlying signaling processes are poorly understood. One of the candidate molecules for being involved in mechanotransduction is focal adhesion kinase (FAK). To examine the role of FAK in the response of immortalized human corneal epithelial (hTCEpi) cells to topographic cues, FAK was depleted by siRNA transfection. Contrary to expectations, FAK knockdown resulted in an enhanced response with a greater number of hTCEpi cells aligned to the long axis of anisotropically ordered surface ridges and grooves. Both underlying topographic features and FAK depletion modulated the migration of corneal epithelial cells. The impact of FAK knockdown on both migration and alignment varied depending on the topographic cues to which the cells were exposed, with the most significant change observed on the biologically relevant size scale (400nm). Additionally, a change in expression of genes encoding perinuclear Nesprins 1 and 2 (SYNE1, 2) was observed in response to topographic cues. SYNE1/2 expression was also altered by FAK depletion, suggesting that these proteins might represent a link between cytosolic and nuclear signaling processes. The data presented here have relevance to our understanding of the fundamental processes involved in corneal cell behavior to topographic cues. These results highlight the importance of incorporating biophysical cues in the conduction of in vitro studies and into the design and fabrication of implantable prosthetics.
PMID:22813850 | PMC:PMC3634558 | DOI:10.1016/j.actbio.2012.07.004
Topographic modulation of the orientation and shape of cell nuclei and their influence on the measured elastic modulus of epithelial cells
Biophys J. 2011 Nov 2;101(9):2139-46. doi: 10.1016/j.bpj.2011.09.042. Epub 2011 Nov 1.
The influence of nucleus shape and orientation on the elastic modulus of epithelial cells was investigated with atomic force microscopy. The shape and orientation were controlled by presenting the epithelial cells with anisotropic parallel ridges and grooves of varying pitch at the cell substratum. As the cells oriented to the underlying topography, the volume of the nucleus increased as the pitch of the topography increased from 400 nm to 2000 nm. The increase in nucleus volume was reflected by an increase in the measured elastic modulus of the topographically aligned cells. Significant alterations in the shape of the nucleus, by intimate contact with the topographic ridge and grooves of the underlying cell, were also observed via confocal microscopy, indicating that the nucleus may also act as a direct mechanosensor of substratum topography.
PMID:22067151 | PMC:PMC3207178 | DOI:10.1016/j.bpj.2011.09.042
Changes in protein expression associated with chronic in vitro exposure of hexavalent chromium to osteoblasts and monocytes: a proteomic approach
J Biomed Mater Res A. 2010 Feb;92(2):615-25. doi: 10.1002/jbm.a.32396.
Cr (VI) is a well-recognized environmental toxin and carcinogen. It is known to be released from orthopedic metal implants in-situ by biocorrosion and is speculated to play a role in periprosthetic osteolysis. It is hence essential to understand its long-term biological effects. We have assessed the in vitro responses of osteoblasts and monocytes to chronic exposure (3 weeks) to Cr (VI), at concentrations that have been measured in patients with metal implants, using two-dimensional gel electrophoresis. Cr (VI) exposure resulted in a differential time-dependent regulation of glycolytic, stress, and cytoskeletal proteins. The proteins that have been found to be altered in expression play an essential role in normal cellular functioning such as energy metabolism, cell signaling, and proliferation. The results highlight the complex molecular changes that occur in both cell types with long-term exposure to Cr and may be useful in establishing a series of clinically useful biomarkers to monitor long-term use of metallic implants.
PMID:19235221 | DOI:10.1002/jbm.a.32396