Method to Assess the Intracellular Fate and Bioavailability of Clusterin Using Live Cell Confocal Microscopy Imaging.

Clusterin, also known as apolipoprotein J, is an ATP-independent holdase chaperone protein. Clusterin is involved in various functions including protein quality control and lipid transport. Though clusterin is secreted upon stress, the intracellular fate of clusterin after a stress response is not well understood. The protocol described here utilizes clusterin tagged to fluorescent proteins like green fluorescent protein and red fluorescent protein to understand the intracellular fate of clusterin.

Proteomic analysis uncovers clusterin-mediated disruption of actin-based contractile machinery in the trabecular meshwork to lower intraocular pressure.

Glaucoma, a major cause of blindness, is characterized by elevated intraocular pressure (IOP) due to improper drainage of aqueous humor via the trabecular meshwork (TM) outflow pathway. Our recent work identified that loss of clusterin resulted in elevated IOP. This study delves deeper to elucidate the role of clusterin in IOP regulation. Employing an ex vivo human anterior segment perfusion model, we established that constitutive expression and secretion as well as exogenous addition of clusterin can significantly lower IOP. Interestingly, clusterin significantly lowered transforming growth factor ß2 (TGFß2)-induced IOP elevation. This effect was linked to the suppression of extracellular matrix (ECM) deposition and, highlighting the crucial role of clusterin in maintaining ECM equilibrium. A comprehensive global proteomic approach revealed the broad impact of clusterin on TM cell structure and function by identifying alterations in protein expression related to cytoskeletal organization, protein processing, and cellular mechanics, following clusterin induction. These findings underscore the beneficial modulation of TM cell structure and functionality by clusterin. Specifically, clusterin influences the actin-cytoskeleton and focal adhesion dynamics, which are instrumental in cell contractility and adhesion processes. Additionally, it suppresses the activity of proteins critical in TGFß2, G-protein, and JAK-STAT signaling pathways, which are vital for the regulation of ocular pressure. By delineating these targeted effects of clusterin within the TM outflow pathway, our findings pave the way for novel treatment strategies aimed at mitigating the progression of ocular hypertension and glaucoma through targeted molecular interventions.

Regulatory role of cholesterol in modulating actin dynamics and cell adhesive interactions in the trabecular meshwork.

The trabecular meshwork (TM) tissue plays a crucial role in maintaining intraocular pressure (IOP) homeostasis. Increased TM contractility and stiffness are directly correlated with elevated IOP. Although cholesterol is known to be a determinant of glaucoma occurrence and elevated IOP, the underlying mechanisms remain elusive. In this study, we used human TM (HTM) cells to unravel the effects of cholesterol on TM stiffness. We achieved this by performing acute cholesterol depletion with Methyl-ß-cyclodextrin (MßCD) and cholesterol enrichment/replenishment with MßCD cholesterol complex (CHOL). Interestingly, cholesterol depletion triggered notable actin depolymerization and decreased focal adhesion formation, while enrichment/replenishment promoted actin polymerization, requiring the presence of actin monomers. Using a specific reporter of phosphatidylinositol 4,5-bisphosphate (PIP2), we demonstrated that cholesterol depletion decreases PIP2 levels on the cell membrane, whereas enrichment increases them. Given the critical role of PIP2 in actin remodeling and focal adhesion formation, we postulate that cholesterol regulates actin dynamics by modulating PIP2 levels on the membrane. Furthermore, we showed that cholesterol levels regulate integrin α5ß1 and αVß3 distribution and activation, subsequently altering cell-extracellular matrix (ECM) interactions. Notably, the depletion of cholesterol, as a major lipid constituent of the cell membrane, led to a decrease in HTM cell membrane tension, which was reversed upon cholesterol replenishment. Overall, our systematic exploration of cholesterol modulation on TM stiffness highlights the critical importance of maintaining appropriate membrane and cellular cholesterol levels for achieving IOP homeostasis.

Identification of the novel role of sterol regulatory element binding proteins (SREBPs) in mechanotransduction and intraocular pressure regulation.

Trabecular meshwork (TM) cells are contractile and mechanosensitive, and they aid in maintaining intraocular pressure (IOP) homeostasis. Lipids are attributed to modulating TM contractility, with poor mechanistic understanding. In this study using human TM cells, we identify the mechanosensing role of the transcription factors sterol regulatory element binding proteins (SREBPs) involved in lipogenesis. By constitutively activating SREBPs and pharmacologically inactivating SREBPs, we have mechanistically deciphered the attributes of SREBPs in regulating the contractile properties of TM. The pharmacological inhibition of SREBPs by fatostatin and molecular inactivation of SREBPs ex vivo and in vivo, respectively, results in significant IOP lowering. As a proof of concept, fatostatin significantly decreased the SREBPs responsive genes and enzymes involved in lipogenic pathways as well as the levels of the phospholipid, cholesterol, and triglyceride. Further, we show that fatostatin mitigated actin polymerization machinery and stabilization, and decreased ECM synthesis and secretion. We thus postulate that lowering lipogenesis in the TM outflow pathway can hold the key to lowering IOP by modifying the TM biomechanics.

Identification of the novel role of sterol regulatory element binding proteins (SREBPs) in mechanotransduction and intraocular pressure regulation.

Trabecular meshwork (TM) cells are highly contractile and mechanosensitive to aid in maintaining intraocular pressure (IOP) homeostasis. Lipids are attributed to modulating TM contractility with poor mechanistic understanding. In this study using human TM cells, we identify the mechanosensing role of the transcription factors sterol regulatory element binding proteins (SREBPs) involved in lipogenesis. By constitutively activating SREBPs and pharmacologically inactivating SREBPs, we have mechanistically deciphered the attributes of SREBPs in regulating the contractile properties of TM. The pharmacological inhibition of SREBPs by fatostatin and molecular inactivation of SREBPs ex vivo and in vivo respectively results in significant IOP lowering. As a proof of concept, fatostatin significantly decreased the SREBPs responsive genes and enzymes involved in lipogenic pathways as well as the levels of the phospholipid, cholesterol, and triglyceride. Further, we show that fatostatin mitigated actin polymerization machinery and stabilization, and decreased ECM synthesis and secretion. We thus postulate that lowering lipogenesis in the TM outflow pathway can hold the key to lowering IOP by modifying the TM biomechanics. Synopsis: In this study, we show the role of lipogenic transcription factors sterol regulatory element binding proteins (SREBPs) in the regulation of intraocular pressure (IOP). ( Synopsis Figure - Created using Biorender.com ) SREBPs are involved in the sensing of changes in mechanical stress on the trabecular meshwork (TM). SREBPs aid in transducing the mechanical signals to induce actin polymerization and filopodia/lamellipodia formation.SREBPs inactivation lowered genes and enzymes involved in lipogenesis and modified lipid levels in TM.SREBPs activity is a critical regulator of ECM engagement to the matrix sites.Inactivation of SCAP-SREBP pathway lowered IOP via actin relaxation and decreasing ECM production and deposition in TM outflow pathway signifying a novel relationship between SREBP activation status and achieving IOP homeostasis.

Profiling the miRNA from Exosomes of Non-Pigmented Ciliary Epithelium-Derived Identifies Key Gene Targets Relevant to Primary Open-Angle Glaucoma.

Oxidative stress (OS) on tissues is a major pathological insult leading to elevated intraocular pressure (IOP) and primary open-angle glaucoma (POAG). Aqueous humor (AH) produced by the non-pigmentary ciliary epithelium (NPCE) drains out via the trabecular meshwork (TM) outflow pathway in the anterior chamber. The exosomes are major constituents of AH, and exosomes can modulate the signaling events, as well as the responses of their target TM tissue. Despite the presence of molecular mechanisms to negate OS, oxidative damage directly, as well as indirectly, influences TM health, AH drainage, and IOP. We proposed that the expression of microRNA (miRNAs) carried by exosomes in the AH can be affected by OS, and this can modulate the pathways in target cells. To assess this, we subjected NPCE to acute and chronic OS (A-OS and C-OS), enriched miRNAs, performed miRNA microarray chip analyses, and miRNA-based gene targeting pathway prediction analysis. We found that various miRNA families, including miR27, miR199, miR23, miR130b, and miR200, changed significantly. Based on pathway prediction analysis, we found that these miRNAs can regulate the genes including Nrf2, Keap1, GSK3B, and serine/threonine-protein phosphatase2A (PP2A). We propose that OS on the NPCE exosomal miRNA cargo can modulate the functionality of the TM tissue.

Analysis of Lipid Contents in Human Trabecular Meshwork Cells by Multiple Reaction Monitoring (MRM) Profiling Lipidomics.

Lipids are among the major constituents of cells and play many important cellular functions. Lipid levels in the trabecular meshwork (TM) aqueous humor outflow pathway play an important role in the maintenance of aqueous humor drainage and intraocular pressure (IOP) homeostasis. Therefore, it is important to characterize the changes in the lipid contents in the aqueous humor outflow pathway tissues to better understand their functional significance in the maintenance of IOP. The multiple reaction monitoring (MRM)-based profiling aids in the analysis of the metabolome as a collection of functional groups and is utilized as an exploratory metabolomics and lipidomics approach. The MRM-based profiling utilizes tandem mass spectrometry experiments carried out on a commercial triple quadrupole mass spectrometer with three aligned quadrupole mass filters (Q1, Q2, and Q3). This screening methodology can be utilized for targeted lipidomics screening. This chapter focuses on the methodology for isolation and culturing of the TM cells, lipid extraction, and the MRM-based lipidomics approach with data analysis.

Multiomics analysis reveals the mechanical stress-dependent changes in trabecular meshwork cytoskeletal-extracellular matrix interactions.

Trabecular meshwork (TM) tissue is subjected to constant mechanical stress due to the ocular pulse created by the cardiac cycle. This brings about alterations in the membrane lipids and associated cell-cell adhesion and cell-extracellular matrix (ECM) interactions, triggering intracellular signaling responses to counter mechanical insults. A loss of such response can lead to elevated intraocular pressure (IOP), a major risk factor for primary open-angle glaucoma. This study is aimed to understand the changes in signaling responses by TM subjected to mechanical stretch. We utilized multiomics to perform an unbiased mRNA sequencing to identify changes in transcripts, mass spectrometry- (MS-) based quantitative proteomics for protein changes, and multiple reaction monitoring (MRM) profiling-based MS and high-performance liquid chromatography (HPLC-) based MS to characterize the lipid changes. We performed pathway analysis to obtain an integrated map of TM response to mechanical stretch. The human TM cells subjected to mechanical stretch demonstrated an upregulation of protein quality control, oxidative damage response, pro-autophagic signal, induction of anti-apoptotic, and survival signaling. We propose that mechanical stretch-induced lipid signaling via increased ceramide and sphingomyelin potentially contributes to increased TM stiffness through actin-cytoskeleton reorganization and profibrotic response. Interestingly, increased phospholipids and diacylglycerol due to mechanical stretch potentially enable cell membrane remodeling and changes in signaling pathways to alter cellular contractility. Overall, we propose the mechanistic interplay of macromolecules to bring about a concerted cellular response in TM cells to achieve mechanotransduction and IOP regulation when TM cells undergo mechanical stretch.

Novel insight into the role of clusterin on intraocular pressure regulation by modifying actin polymerization and extracellular matrix remodeling in the trabecular meshwork.

This study provides comprehensive mechanistic evidence for the role of clusterin, a stress-response secretory chaperone protein, in the modulation of intraocular pressure (IOP) by regulating the trabecular meshwork (TM) actin cytoskeleton and the extracellular matrix (ECM). The pathological stressors on TM known to elevate IOP significantly lowered clusterin protein levels indicating stress-related clusterin function loss. Small interfering RNA-mediated clusterin loss in human TM cells in vitro induced actin polymerization and stabilization via protein kinase D1, serine/threonine-protein kinase N2 (PRK2), and LIM kinase 1 (LIMK1), and the recruitment and activation of adhesome proteins including paxillin, vinculin, and integrin αV and ß5. A complete loss of clusterin as seen in clusterin knockout mice (Clu-/- ) led to significant IOP elevation at postnatal Day 70. Contrarily, constitutive clusterin expression using adenovirus (AdCLU) in HTM cells resulted in the loss of actin polymerization via decreased PRK2, and LIMK1 and negative regulation of integrin αV and ß5. Furthermore, we found that AdCLU treatment in HTM cells significantly decreased the ECM protein expression and distribution by significantly increasing matrix metalloprotease 2 (MMP2) activity and lowering the levels of pro-fibrotic proteins such as transforming growth factor-ß2 (TGFß2), thrombospondin-1 (TSP-1), and plasminogen activator inhibitor-1 (PAI-1). Finally, we found that HTM cells supplemented with recombinant human clusterin attenuated the pro-fibrotic effects of TGFß2. For the first time this study demonstrates the importance of clusterin in the regulation of TM actin cytoskeleton - ECM interactions and the maintenance of IOP, thus making clusterin an interesting target to reverse elevated IOP.

Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

Cathepsin K Regulates Intraocular Pressure by Modulating Extracellular Matrix Remodeling and Actin-Bundling in the Trabecular Meshwork Outflow Pathway.

The homeostasis of extracellular matrix (ECM) and actin dynamics in the trabecular meshwork (TM) outflow pathway plays a critical role in intraocular pressure (IOP) regulation. We studied the role of cathepsin K (CTSK), a lysosomal cysteine protease and a potent collagenase, on ECM modulation and actin cytoskeleton rearrangements in the TM outflow pathway and the regulation of IOP. Initially, we found that CTSK was negatively regulated by pathological stressors known to elevate IOP. Further, inactivating CTSK using balicatib, a pharmacological cell-permeable inhibitor of CTSK, resulted in IOP elevation due to increased levels and excessive deposition of ECM-like collagen-1A in the TM outflow pathway. The loss of CTSK activity resulted in actin-bundling via fascin and vinculin reorganization and by inhibiting actin depolymerization via phospho-cofilin. Contrarily, constitutive expression of CTSK decreased ECM and increased actin depolymerization by decreasing phospho-cofilin, negatively regulated the availability of active TGFß2, and reduced the levels of alpha-smooth muscle actin (αSMA), indicating an antifibrotic action of CTSK. In conclusion, these observations, for the first time, demonstrate the significance of CTSK in IOP regulation by maintaining the ECM homeostasis and actin cytoskeleton-mediated contractile properties of the TM outflow pathway.

Outflow Facility Effects of 3 Schlemm's Canal Microinvasive Glaucoma Surgery Devices.

PURPOSE: To study the effect of 3 Schlemm's canal (SC) microinvasive glaucoma surgery (MIGS) devices on outflow facility. DESIGN: Paired comparisons, randomized design, baseline-controlled study. PARTICIPANTS: Thirty-six pairs of dissected anterior segments from donated human eye bank eyes without glaucoma were studied. A baseline measurement was collected from each eye to serve as its control. METHODS: Using a constant pressure perfusion method, outflow facility was measured in paired eyes from human donors. Measurements were made at perfusion pressures of 10 mmHg, 20 mmHg, 30 mmHg, and 40 mmHg. Outflow facility was measured before (baseline control) and after the implantation of an SC glaucoma drainage device or sham procedure. Three sets of experiments were carried out comparing 1 and 2 iStent Trabecular Micro-Bypass Stents and 2 iStent Inject implants with the Hydrus Microstent. MAIN OUTCOME MEASURES: Change in outflow facility from baseline or contralateral eye. RESULTS: After Hydrus placement, the outflow facility increased from 0.23±0.03 µl/minute per millimeter of mercury at baseline to 0.38±0.03 µl/minute per millimeter of mercury (P < 0.001). The percent increase in outflow facility was 79±21% for the Hydrus and 11±16% for the 2 iStent Inject devices, a difference that was significant (P = 0.018). Outflow facility with 1 iStent (0.38±0.07 µl/minute per millimeter of mercury) was greater than baseline (0.28±0.03 µl/minute per millimeter of mercury; P = 0.031). The 1 iStent showed a greater increase in outflow facility from baseline (0.10±0.04 µl/minute per millimeter of mercury) compared with the sham procedure (-0.08±0.05 µl/minute per millimeter of mercury; P = 0.042). No other significant differences were found. CONCLUSIONS: The longer the MIGS device, and thus the more SC that it dilates, the greater the outflow facility.

Consensus recommendations for trabecular meshwork cell isolation, characterization and culture.

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.

Quantitative Proteomic Analysis of Human Aqueous Humor Using iTRAQ 4plex Labeling.

iTRAQ 4plex method enables multiplexing of up to four samples for simultaneous quantitation to improve sensitivity and scope of proteomic analysis. Here, we describe iTRAQ 4plex labeling of human aqueous humor specimens followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and data analysis for peptide identification and quantitation using Proteome Discoverer software. The protocol can be applied for other animals as well; however, pooling of specimens may be required to obtain sufficient amount of protein for labeling.

Discovery and Preclinical Development of Netarsudil, a Novel Ocular Hypotensive Agent for the Treatment of Glaucoma.

PURPOSE: Rho-associated protein kinase (ROCK) inhibitors lower intraocular pressure (IOP) by increasing aqueous outflow through the trabecular meshwork (TM). The preclinical characterization of netarsudil, a new ROCK/norepinephrine transporter (NET) inhibitor currently in clinical development, is presented herein. METHODS: The kinase inhibitory activity of netarsudil was compared to its esterase metabolite, netarsudil-M1, and 3 other ROCK inhibitors using a commercially available kinase assay kit. Disruption of actin stress fibers was measured in primary porcine TM cells and disruption of focal adhesions in transformed human TM (HTM) cells. Induction of fibrosis markers after exposure to transforming growth factor-ß2 (TGF-ß2) was conducted in primary HTM cells. Ocular hypotensive activity and tolerability of topical formulations were evaluated in normotensive Dutch Belted rabbits and Formosan Rock monkeys. In vitro corneal metabolism assays were conducted using dog, pig, rabbit, monkey, and human corneas. In vivo ocular pharmacokinetics was studied in Dutch Belted rabbits. RESULTS: Netarsudil inhibited kinases ROCK1 and ROCK2 with a Ki of 1 nM each, disrupted actin stress fibers and focal adhesions in TM cells with IC50s of 79 and 16 nM, respectively, and blocked the profibrotic effects of TGF-ß2 in HTM cells. Netarsudil produced large reductions in IOP in rabbits and monkeys that were sustained for at least 24 h after once daily dosing, with transient, mild hyperemia observed as the only adverse effect. CONCLUSION: Netarsudil is a novel ROCK/NET inhibitor with high potency in biochemical and cell-based assays, an ability to produce large and durable IOP reductions in animal models, and favorable pharmacokinetic and ocular tolerability profiles.

GADD34 Function in Protein Trafficking Promotes Adaptation to Hyperosmotic Stress in Human Corneal Cells.

GADD34, a stress-induced regulatory subunit of the phosphatase PP1, is known to function in hyperosmotic stress through its well-known role in the integrated stress response (ISR) pathway. Adaptation to hyperosmotic stress is important for the health of corneal epithelial cells exposed to changes in extracellular osmolarity, with maladaptation leading to dry eye syndrome. This adaptation includes induction of SNAT2, an endoplasmic reticulum (ER)-Golgi-processed protein, which helps to reverse the stress-induced loss of cell volume and promote homeostasis through amino acid uptake. Here, we show that GADD34 promotes the processing of proteins synthesized on the ER during hyperosmotic stress independent of its action in the ISR. We show that GADD34/PP1 phosphatase activity reverses hyperosmotic-stress-induced Golgi fragmentation and is important for cis- to trans-Golgi trafficking of SNAT2, thereby promoting SNAT2 plasma membrane localization and function. These results suggest that GADD34 is a protective molecule for ocular diseases such as dry eye syndrome.

Role of the Rho GTPase/Rho kinase signaling pathway in pathogenesis and treatment of glaucoma: Bench to bedside research.

Glaucoma is a leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP) is considered to be a predominant risk factor for primary open angle glaucoma, the most prevalent form of glaucoma. Although the etiological mechanisms responsible for increased IOP are not completely clear, impairment in aqueous humor (AH) drainage through the conventional or trabecular pathway is recognized to be a primary cause in glaucoma patients. Importantly, lowering of IOP has been demonstrated to reduce progression of vision loss and is a mainstay of treatment for all types of glaucoma. Currently however, there are limited therapeutic options available for lowering IOP especially as it relates to enhancement of AH outflow through the trabecular pathway. Towards addressing this challenge, bench and bedside research conducted over the course of the last decade and a half has identified the significance of inhibiting Rho kinase for lowering IOP. Rho kinase is a downstream effector of Rho GTPase signaling that regulates actomyosin dynamics in numerous cell types. Studies from several laboratories have demonstrated that inhibition of Rho kinase lowers IOP via relaxation of the trabecular meshwork which enhances AH outflow. By contrast, activation of Rho GTPase/Rho kinase signaling in the trabecular outflow pathway increases IOP by altering the contractile, cell adhesive and permeability barrier characteristics of the trabecular meshwork and Schlemm's canal tissues, and by influencing extracellular matrix production and fibrotic activity. This article, written in honor of the late David Epstein, MD, summarizes findings from both basic and clinical studies that have been instrumental for recognition of the importance of the Rho/Rho kinase signaling pathway in regulation of AH outflow, and in the development of Rho kinase inhibitors as promising IOP- lowering agents for glaucoma treatment.

The exit strategy: Pharmacological modulation of extracellular matrix production and deposition for better aqueous humor drainage.

Primary open angle glaucoma (POAG) is an optic neuropathy and an irreversible blinding disease. The etiology of glaucoma is not known but numerous risk factors are associated with this disease including aging, elevated intraocular pressure (IOP), race, myopia, family history and use of steroids. In POAG, the resistance to the aqueous humor drainage is increased leading to elevated IOP. Lowering the resistance and ultimately the IOP has been the only way to slow disease progression and prevent vision loss. The primary drainage pathway comprising of the trabecular meshwork (TM) is made up of relatively large porous beams surrounded by extracellular matrix (ECM). Its juxtacanalicular tissue (JCT) or the cribriform meshwork is made up of cells embedded in dense ECM. The JCT is considered to offer the major resistance to the aqueous humor outflow. This layer is adjacent to the endothelial cells forming Schlemm's canal, which provides approximately 10% of the outflow resistance. The ECM in the TM and the JCT undergoes continual remodeling to maintain normal resistance to aqueous humor outflow. It is believed that the TM is a major contributor of ECM proteins and evidence points towards increased ECM deposition in the outflow pathway in POAG. It is not clear how and from where the ECM components emerge to hinder the normal aqueous humor drainage. This review focuses on the involvement of the ECM in ocular hypertension and glaucoma and the mechanisms by which various ocular hypotensive drugs, both current and emerging, target ECM production, remodeling, and deposition.

Hic-5 Regulates Actin Cytoskeletal Reorganization and Expression of Fibrogenic Markers and Myocilin in Trabecular Meshwork Cells.

PURPOSE: To explore the role of inducible focal adhesion (FA) protein Hic-5 in actin cytoskeletal reorganization, FA formation, fibrogenic activity, and expression of myocilin in trabecular meshwork (TM) cells. METHODS: Using primary cultures of human TM (HTM) cells, the effects of various external factors on Hic-5 protein levels, as well as the effects of recombinant Hic-5 and Hic-5 small interfering RNA (siRNA) on actin cytoskeleton, FAs, myocilin, α-smooth muscle actin (αSMA), and collagen-1 were determined by immunofluorescence and immunoblot analyses. RESULTS: Hic-5 distributes discretely to the FAs in HTM cells and throughout the TM and Schlemm's canal of the human aqueous humor (AH) outflow pathway. Transforming growth factor-ß2 (TGF-ß2), endothelin-1, lysophosphatidic acid, hydrogen peroxide, and RhoA significantly increased Hic-5 protein levels in HTM cells in association with reorganization of actin cytoskeleton and FAs. While recombinant Hic-5 induced actin stress fibers, FAs, αv integrin redistribution to the FAs, increased levels of αSMA, collagen-1, and myocilin, Hic-5 siRNA suppressed most of these responses in HTM cells. Hic-5 siRNA also suppressed TGF-ß2-induced fibrogenic activity and dexamethasone-induced myocilin expression in HTM cells. CONCLUSIONS: Taken together, these results reveal that Hic-5, whose levels were increased by various external factors implicated in elevated intraocular pressure, induces actin cytoskeletal reorganization, FAs, expression of fibrogenic markers, and myocilin in HTM cells. These characteristics of Hic-5 in TM cells indicate its importance in regulation of AH outflow through the TM in both normal and glaucomatous eyes.