Browsing by Subject "Trabecular Meshwork"
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Item EFFECT OF CELLULAR AND PLASMA FIBRONECTIN ISOFORMS ON NORMAL HUMAN TRABECULAR MESHWORK CELLS(2013-04-12) Medina-Ortiz, Wanda E.Purpose: The expression of cellular (cFN) and plasma (pFN) fibronectin isoforms are induced by TGF-β2 in human trabecular meshwork (HTM) cultured cells. Expression of specific FN isoforms can alter ECM homeostasis, ECM-cell interactions, and gene expression. Our purpose is to determine cFN levels in HTM tissues and to explore the impact of FN isoforms on HTM cells by studying changes in adhesion, cytoskeletal organization and gene expression. Methods: Differences between cFN levels in normal (NTM) and glaucomatous (GTM) tissues were obtained by immunohistochemistry. NTM cell strains were cultured for 24-48 hrs on surfaces coated with cFN or pFN, and the responses were compared to PBS controls. Changes in formation and redistribution of F-actin fibers and adhesion proteins were analyzed by phalloidin staining, Western immunoblots, and immunocytochemistry. Gene expression changes were analyzed using PCR arrays. Results: GTM tissues exhibited significantly greater cFN levels (1.7-fold, p<0.05). NTM strains exposed to both FN isoforms showed increased F-actin formation and redistribution; however, the F-actin pattern and distribution was different between cFN and pFN. Similarly, adhesion molecules such as talin, vinculin, paxillin and integrin beta 1 were increased and redistributed. Both FN isoforms changed gene expression, including alpha-smooth muscle actin-2, metalloproteases and their inhibitors, inflammatory cytokines, and TGF-β related genes. Conclusions: Our results show that GTM tissues expressed more cFN and that NTM cells respond differently depending on the FN isoform. The relationship between TGF-β2 modulation of FN isoform expression and the effect of FN isoforms on NTM cells suggests that this type of ECM remodeling may contribute to the TM changes associated with glaucoma.Item EFFECTS OF TGF-BETA2, FOLLISTATIN AND ACTIVIN A ON EXTRACELLULAR MATRIX IN NORMAL HUMAN TRABECULAR MESHWORK CELLS AND TISSUES.(2013-04-12) Fisher, AndrewPurpose: Primary open angle glaucoma (POAG) is characterized as a group of eye diseases resulting in optic nerve head damage and irreversible blindness. A major risk factor for developing POAG is increased intraocular pressure (IOP) leading to decreased outflow of aqueous humor (AH) through the trabecular meshwork (TM). Transforming growth factor-beta2 (TGF-β2) is increased in the AH of glaucoma patients, and causes increased extracellular matrix (ECM) protein synthesis in the TM. Bone morphogenetic protein-4 (BMP-4) has been shown to inhibit TGF-β2 actions. Follistatin (FST) is an antagonist of BMP-4 and is elevated in the glaucomatous TM. Elevated levels of FST in the TM may block BMP-4 ability to attenuate TGF-β2 induction of ECM proteins. FST may also have a direct role in regulating ECM protein expression in human TM (HTM) cells. HTM cells also express Activin A (Act A). The purpose of this study was to assess the role of FST and Act A in HTM cells as related to TGF-β2/BMP-4 signaling. Understanding these interactions may provide possible new therapeutic targets for the treatment of glaucoma. Methods: Normal HTM cell lines were cultured and treated with TGF-β2 (5ng/ml), FST-315, FST-288, or Act A (each at 50ng/ml) alone and/or simultaneously for 24 and 48 hrs. Western blot analysis was used to evaluate the effects of FST-315/288, Act A, and TGF-β2 on ECM protein synthesis including fibronectin (FN), PAI-1, and collagen1A. Results: TGF-β2 induced expression of PAI-1 and FN.. ACT-A mildly induced PAI-1 and FN proteins as compared to TGF-β2. TGF-β2 and Act A treatment appeared to have a synergistic effect on the expression of PAI-1 and FN protein as compared to individual treatment (Tx) of TGF-β2 or Act A. FST 288 does not seem to change the Act A + TGF-β2 synergism. FST 315 inhibits the synergism of Act A + TGF-β2 showing a decrease in PAI-1 and FN protein. Conclusions: FST-315 decreased the induction of ECM proteins by TGF-β2 and Act-A. FST-288 increased induction of ECM proteins in cells treated with TGF-β2 and Act A. FST-288 treatment for 24 hours induced increased ECM proteins PAI-1 and FN, but there was no induction at 48 hours. FST-315 treatment for 24 hours slightly induced ECM PAI-1 and FN, but there was no induction at 48 hours. Act A treatment for 24 and 48 hours increased induction of PAI-1 or FN. These results further our knowledge of the potential role of BMP antagonists in the human TM and their potential roles in the pathogenesis of glaucoma.Item EPIGENETIC REGULATION OF GLAUCOMA-ASSOCIATED GROWTH FACTORS IN THE TRABECULAR MESHWORK(2014-03) Bermudez, Jaclyn Y.; Webber, Hannah; Cheng, Yi-Qiang; Clark, Abbot F.; Mao, WeimingGlaucoma is a leading cause of blindness in the U.S. and worldwide. The primary risk factor of primary open angle glaucoma (POAG), the major type of glaucoma, is elevated intraocular pressure (IOP). IOP elevation in glaucoma patients is due to glaucomatous insults to the trabecular meshwork (TM) and compromised TM function. Therefore, it is important to study how glaucoma-associated growth factors in the TM are regulated. We investigated how heritable changes in gene activity regulate the TM without altering the DNA sequence. Purpose (a): Glaucoma is a leading cause of blindness in the U.S. and worldwide. This disease leads to progressive, irreversible damage to the optic nerve and visual function. The primary risk factor of primary open angle glaucoma (POAG), the major type of glaucoma, is elevated intraocular pressure (IOP). IOP elevation in glaucoma patients is due to glaucomatous insults to the trabecular meshwork (TM) and compromised TM function, which increase aqueous humor outflow resistance. In the glaucomatous TM (GTM), there is excessive extracellular matrix (ECM) protein deposition. Many studies have suggested that cell signaling pathways, such as the transforming growth factor beta (TGF-β) and Wnt signaling pathways, play key roles in TM homeostasis.The growth factors that are associated with these pathways, including TGFβ2, Gremlin and sFRP1, are found to be at higher levels in the GTM cells compared to normal TM cells. Little is known about the role of epigenetics in regulating glaucoma-associated growth factors in the TM. One of the major epigenetic regulatory mechanisms is histone acetylation.We hypothesize that histone acetylation is responsible for the increased expression of glaucoma associated factors in the TM. Methods (b): Primary human TM cell cultures were treated with 10nM Thailandepsin (TDP-A), a histone deacetylase inhibitor (HDACi), or 1% DMSO as vehicle control for 4 days. Cells were harvested for qPCR to compare gene expression levels or for ChIP assays to compare promoter associated histone acetylation status. We also treated paired perfusion cultured bovine anterior segments with DMSO or TDP-A for 7 to 10 days. The IOP change of the treated bovine eyes was monitored and recorded. Data were analyzed by using Student’s t-test or one-way ANOVA. P values less than 0.05 were considered significant. Results (c): TDP-A significantly elevated the expression of sFRP-1 and TGFβ2 (n=3, p2 as well as elevated IOP. Conclusions (d): Histone acetylation may play an important role in the dysregulation of growth factors in the TM. This mechanism provides a unique opportunity to elucidate the etiology of POAG. Also, TDP-A is a potent HDACi that can be used as a powerful tool in glaucoma research.Item Histological Investigation of Human Glaucomatous Eyes: Extracellular Fibrotic Changes and Galectin3 Expression in the Trabecular Meshwork and Optic Nerve Head(2018-05) Belmares, Ricardo; Clark, Abbot F.; Rosales, Armando; Lovely, Rehana S.; Reeves, Rustin E.; Jung, Marianna; Yao, HaiGlaucoma is a leading cause of vision loss and is associated with fibrotic changes in two ocular tissues: the optic nerve head (ONH) and trabecular meshwork (TM). We investigated the differences of extracellular components of the two ocular tissues in human glaucomatous eyes to determine fibrotic changes. The extracellular components studied included: collagen, elastin, Galectin 3 (Gal3), and Transforming Growth Factor beta-2 Type II receptor (TGFβ-2 RII). We hypothesized that these components will be increased in glaucomatous eyes using chemical staining and immunohistochemistry. Chemical staining included: Masson's Trichrome and Sirius Red stains (collagen) and Vernhoeff-Van Giesen (elastin). Immunohistochemistry was used to determine expression of Gal3 and TGFβ-2 RII. Data was analyzed using Image J software to quantify expression of the extracellular components. The results from Image J analysis of extracellular components demonstrated an overall increase in glaucomatous tissue. TM studies showed an increase of collagen (P=0.0469), and Gal3 (P [less than] 0.0001), and TGFβ-2 RII (P=0.0005) in glaucomatous eyes. Collagen was apparently increased in ONH ((P=0.0517) and Galectin3 (P=0.041) was increased in myelin transition zone of the glaucomatous optic nerve. Vernhoeff-Van Giesen stain showed increased thickness and irregular arrangement of elastic fibers in ONH. Vernoeff-van Giesen and Sirius Red stains also showed increased staining in glaucomatous tissue, but were not quantifiable with Image J software. Analysis showed a correlation of TGFβ-2 RII with Gal3 in TM (P [less than ]0.0001) and myelin transition zone of optic nerve (P=0.0003). Analysis of extracellular components of TM and ONH showed that glaucomatous eyes demonstrate a fibrotic state. Increased collagen deposition and thickened elastic fibers are recognized features of fibrosis and both TM and ONH revealed these features through chemical staining. Galectin3, another known marker for fibrosis, was also elevated in TM and optic nerve. Moreover, Gal3 co-localization with TGFβ-2 RII suggests that it may be involved with the pro-fibrotic TGFβ-2 signaling pathway.Item TGF Beta 2 and Gremlin Signaling Pathways Regulate Extracellular Matrix Changes in TM Cells: Implications for Glaucoma(2011-05-01) Sethi, Anirudh; Clark, Abbot F.Purpose: Glaucoma is a leading cause of blindness worldwide. The leading risk factor is elevated intraocular pressure (IOP) due to fibrotic changes in the trabecular meshwork (TM) tissue. The profibrotic cytokine TGFβ2 is elevated in the aqueous humor and TM of glaucomatous eyes. TGFβ2-mediated extracellular matrix (ECM) deposition in the TM appears to be responsible for increased IOP in ex vivo and in vivo models. Bone morphogenetic proteins (BMPs) inhibit TGFβ regulation of ECM, and elevated levels of the BMP antagonist gremlin in the glaucomatous TM restores the fibrotic response of TGFβ2. Lysyl oxidase (LOX) is a collagen and elastin polymer crosslinking enzyme, and recent genome wide association studies showed that SNPs in LOXL1, a LOX family member, significantly increased the risk of developing exfoliation glaucoma. The overall aim of my work is to delineate the signaling pathways involved in TGFβ2 and gremlin alteration of the TM ECM and to evaluate the potential role of the LOX family of cross-linking enzymes in this TM ECM remodeling. Methods: Human TM cells were cultured in the presence or absence of recombinant human TGFβ (0.1-10 ng/ml) or mouse gremlin (100-5000 ng/ml) for 1-72 hours, and total RNA or protein lysates and conditioned medium were harvested from the cells. Effects of gremlin treatment on ECM gene and protein expression were assayed by qRT-PCR and western immunoblotting, respectively. TGFβ2- and gremlin-treated TM cells were also examined for Smad2/3, p38, and JNK activation using western immunoblotting. Cells were treated with ii Smad3 inhibitor SIS3 (5 uM), TGFβ receptor inhibitors LY364947 and SB431542 (5 uM), JNK inhibitor SP600125 (10 uM), and AP-1 inhibitor SR11342 (5 uM) with or without TGFβ2/gremlin, to examine the involvement of the TGFβ/Smad signaling pathway. Results: TGFβ2 and gremlin induced expression of each other in TM cells. TGFβ2 activated both Smad and non-Smad pathways and strongly induced mRNA and protein expression of all 5 LOX genes. Using a novel LOX activity assay, we observed greater ECM crosslinking in TGFβ- treated cells. Gremlin elevated ECM gene and protein expression via the Smad signaling pathway. Conclusions: The TGFβ induction of LOXs and gremlin-induction of ECM proteins highlight the complex interplay of Smad and the non-Smad signaling in regulating the TGFβ response. TGFβ2 and gremlin are profibrotic in a feed forward loop. The TGFβ response in TM cells involves evasion of BMP inhibition by gremlin induction and also ECM crosslinking through the LOX enzymes.Item The Role of Mechanosensory TRPV4 Channels and Nitric Oxide Signaling in Intraocular Pressure Homeostasis and its Impairment in Glaucoma(2020-08) Patel, Pinkal D.; Zode, Gulab S.; Clark, Abbot F.; Pang, Iok-Hou; Krishnamoorthy, Raghu R.; Rickards, Caroline A.Several population-based studies have identified elevated intraocular pressure (IOP) as a major causative risk factor associated with primary open angle glaucoma (POAG), the most common form of glaucoma that affects millions of people worldwide. Moreover, multi-ethnic clinical trials in several different countries over the last few decades have provided overwhelming evidence showing correlation between lowering of IOP and reduced progression of vision loss. As a result, IOP reducing therapeutic interventions are the gold standard in glaucoma therapy. Although the role of IOP is evident in pathology of POAG, very few studies have delved into the complex physiological mechanisms that regulate IOP homeostasis. From continuous telemetry recordings in nonhuman primates, we now know that IOP is a dynamic variable that fluctuates throughout the day. However, despite the fluctuations, the mean IOP is still maintained within a narrow physiological range. The level of IOP elevation at any given time depends on the resistance to aqueous humor outflow encountered in the conventional outflow pathway consisting of the trabecular meshwork (TM), Schlemm's canal (SC), and the distal episcleral vessels. Recent studies have suggested that the cells of the outflow pathway have intrinsic ability to detect biomechanical stimuli in their environment (like shear stress) and convert these stimuli into biochemical signals to elicit specific cellular responses. Although mechanotransduction at the TM is deemed critical for IOP homeostasis, we are yet to conclusively identify the exact signaling pathway involved. In this study, we identify the role of transient receptor potential vanilloid IV channels (TRPV4) in sensing mechanical stress on the TM. We show that shear stress activates TRPV4 channels in human primary TM cells, which leads to endothelial nitric oxide synthase (eNOS)-dependent nitric oxide (NO) production. NO, itself has been identified as a key regulator of IOP. Exogenous NO delivery to the eye has been shown to reduce IOP in humans. However, the underlying mechanism that regulates endogenous levels of NO still remains unknown. To this end, we demonstrate that TRPV4 channels regulate eNOS-dependent NO production in primary human TM cells and ex vivo cultured human TM tissues. We show that TRPV4 activation by mechanical shear leads to activation of eNOS signaling and NO production. Furthermore, pharmacological activation of TRPV4 channels via a selective agonist GSK1016790A (GSK101) leads to eNOS phosphorylation and NO production. In animal models, we demonstrate a role of TRPV4 channels in regulating physiological IOP. Treatment of C57BL/6J mouse eyes with TRPV4 agonist GSK101 leads to reduction in baseline night-time IOP and nominal improvement in outflow facility. We also show that conditional knockout of TRPV4 channels in Ad5-Cre injected TRPV4f/f mice leads to increase in IOP. We use the NOS3-/- (eNOS) to further show that TRPV4 mediated lowering of IOP is eNOS dependent. Dysregulation of the TM cells leads to increase in resistance and IOP elevation. Furthermore, glaucomatous human TM cells show impaired activity of TRPV4 channels and disrupted TRPV4-eNOS signaling. Flow/shear stress activation of TRPV4 channels and subsequent NO release were also impaired in glaucomatous primary human TM cells. Together, our studies demonstrate a central role for TRPV4-eNOS signaling in lowering the resting IOP. Our results also provide evidence that impaired TRPV4 channel activity in TM cells contributes to TM dysfunction and elevated IOP in glaucoma.Item TRANSFORMING GROWTH FACTOR-β2 EFFECTS EXPRESSION OF PERIOSTIN AND PROCOLLAGEN C-ENDOPEPTIDASE ENHANCER 1 IN HUMAN TRABECULAR MESHWORK CELLS(2014-03) Tovar, Tara; Naik, Monal; Clark, Abbot F.; Wordinger, Robert J.Transforming growth factor-beta 2 (TGF-β2) has been implicated in the development of elevated intraocular pressure (IOP) in primary open-angle glaucoma (POAG). Glaucoma patients have increased levels of TGF-β2 in their aqueous humor and trabecular meshwork (TM), and TGF-β2 increases extracellular matrix (ECM) proteins. Thus, we are interested in growth factors that are associated with glaucoma and the ECM. Purpose (a): Transforming growth beta-2 (TGF-β2) has been associated with increased extracellular matrix (ECM) deposition, which is attributed to increased aqueous humor outflow resistance through the trabecular meshwork (TM). We have previously demonstrated that bone morphogenetic protein 1 (BMP1) (an enzyme responsible for the cleavage and maturation of ECM proteins) is expressed and regulated by TGF-β2 in the human TM and that BMP1 regulates lysyl oxidase activity. Also, other factors associated with the ECM remodeling include periostin (POSTN) and procollagen c-endopeptidase enhancer 1 (PCOLCE1). The purpose of this study was to determine whether human TM cells (a) express POSTN and PCOLCE1 (b) whether expression of POSTN and PCOLCE1 are regulated by TGF-β2. Methods (b): Primary human normal (NTM) and glaucomatous (GTM) cells were isolated and subjected to qPCR and Western immunoblotting (WB) for POSTN and PCOLCE1 expression. qPCR was used to determine POSTN and PCOLCE1 expression between control and TGF-β2 treated (5ng/ml for 24 hours) TM cells. WBs of cell lysates and conditioned medium were used to compare POSTN and PCOLCE1 protein expression between control and TGF-β2 treated NTM and GTM cells. Results (c): Human TM cells expressed POSTN and PCOLCE1 mRNA and protein. Exogenous TGF-β2 increased POSTN mRNA expression (p<0.05) and decreased PCOLCE1 expression (p<0.0005) compared to control cells. WB analysis showed increased POSTN secretion in NTM compared to GTM cells (p<0.05). TGF-β2 induced POSTN in NTM cells (p<0.05). However, no POSTN was detected in cell lysates of TM cells. WB analysis showed decreased PCOLCE1 secretion in NTM cells compared to GTM cells (p<0.05). Conclusions (d): POSTN and PCOLCE1 are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of POSTN and PCOLCE1 may lead to structural and functional changes in the ECM within the TM.