Browsing by Subject "trabecular meshwork"
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Item A Novel Mouse Model of TGFbeta2-Induced Ocular Hypertension Using Lentiviral Gene Delivery(MDPI, 2022-06-21) Patil, Shruti V.; Kasetti, Ramesh B.; Millar, J. Cameron; Zode, Gulab S.Glaucoma is a multifactorial disease leading to irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form and is associated with the elevation of intraocular pressure (IOP). Reduced aqueous humor (AH) outflow due to trabecular meshwork (TM) dysfunction is responsible for IOP elevation in POAG. Extracellular matrix (ECM) accumulation, actin cytoskeletal reorganization, and stiffening of the TM are associated with increased outflow resistance. Transforming growth factor (TGF) beta2, a profibrotic cytokine, is known to play an important role in the development of ocular hypertension (OHT) in POAG. An appropriate mouse model is critical in understanding the underlying molecular mechanism of TGFbeta2-induced OHT. To achieve this, TM can be targeted with recombinant viral vectors to express a gene of interest. Lentiviruses (LV) are known for their tropism towards TM with stable transgene expression and low immunogenicity. We, therefore, developed a novel mouse model of IOP elevation using LV gene transfer of active human TGFbeta2 in the TM. We developed an LV vector-encoding active hTGFbeta2(C226,228S) under the control of a cytomegalovirus (CMV) promoter. Adult C57BL/6J mice were injected intravitreally with LV expressing null or hTGFbeta2(C226,228S). We observed a significant increase in IOP 3 weeks post-injection compared to control eyes with an average delta change of 3.3 mmHg. IOP stayed elevated up to 7 weeks post-injection, which correlated with a significant drop in the AH outflow facility (40.36%). Increased expression of active TGFbeta2 was observed in both AH and anterior segment samples of injected mice. The morphological assessment of the mouse TM region via hematoxylin and eosin (H&E) staining and direct ophthalmoscopy examination revealed no visible signs of inflammation or other ocular abnormalities in the injected eyes. Furthermore, transduction of primary human TM cells with LV_hTGFbeta2(C226,228S) exhibited alterations in actin cytoskeleton structures, including the formation of F-actin stress fibers and crossed-linked actin networks (CLANs), which are signature arrangements of actin cytoskeleton observed in the stiffer fibrotic-like TM. Our study demonstrated a mouse model of sustained IOP elevation via lentiviral gene delivery of active hTGFbeta2(C226,228S) that induces TM dysfunction and outflow resistance.Item Characterization and Function of Follistatin in Human Trabecular Meshwork Cell and Tissues(2013-05-01) Fitzgerald, Ashley M.; Robert WordingerPrimary Open Angle Glaucoma (POAG) is a leading cause of blindness affecting over 70 million people worldwide. The most important risk factor for developing POAG is elevated intraocular pressure (IOP), which results from increased resistance of aqueous humor (AH) through the trabecular meshwork (TM) outflow pathway. Transforming growth factor- beta II (TGF-β2) is elevated in the AH and TM of glaucoma patients. Recent evidence indicate an extracellular BMP antagonist, gremlin, regulates BMP signaling and TGF-β2 activity. Follistatin (FST), another secreted BMP antagonist is recognized for its ability to bind BMPs and their type I receptor, sequestering BMP signaling. The purpose is to evaluate the presence and relevant activity of follistatin in TM tissues and cells. We hypothesize expression of follistatin in human trabecular meshwork cells alters the expression of extracellular matrix (ECM) deposition seen in the pathogenesis of glaucoma. First, we examined differential FST expression in human trabecular meshwork cells and tissues. We observed a significant increase in expression of FST in glaucomatous as compared to normal protein and mRNA expression. Next, we determined if FST could be induced upon treatment of exogenous TGF-ß2 protein in human TM cells. Studies showed TGF-ß2 up-regulated FST mRNA transcript in a time dependent manner. FST protein secretion was increased in a time and does dependent manner. Third, we assessed FST effects on induction or inhibition of ECM proteins in human TM cells. ECM protein and mRNA expression was time dependent; nevertheless the response of ECM protein to FST treatment is different depending on isoform presence. Additional studies will be done to further elucidate these findings. Lastly, we evaluated FST-288 and FST-315 inhibition of BMP4 attenuation of TGF-ß2 induced ECM expression. Data suggest FST-315 to suppress BMP-4 effects on TGF-ß2 induced ECM and FST-288 enhanced BMP-4 effects on TGF-ß2 induced ECM. The goal is to evaluate additional factors that contribute to the pathogenesis of POAG and assess how these factors can provide possible therapeutic mechanisms for the treatment of glaucoma.Item CNS axonal degeneration and transport deficits at the optic nerve head precede structural and functional loss of retinal ganglion cells in a mouse model of glaucoma(BioMed Central Ltd., 2020-08-27) Maddineni, Prabhavathi; Kasetti, Ramesh B.; Patel, Pinkal D.; Millar, J. Cameron; Kiehlbauch, Charles; Clark, Abbot F.; Zode, Gulab S.BACKGROUND: Glaucoma is a leading neurodegenerative disease affecting over 70 million individuals worldwide. Early pathological events of axonal degeneration and retinopathy in response to elevated intraocular pressure (IOP) are limited and not well-defined due to the lack of appropriate animal models that faithfully replicate all the phenotypes of primary open angle glaucoma (POAG), the most common form of glaucoma. Glucocorticoid (GC)-induced ocular hypertension (OHT) and its associated iatrogenic open-angle glaucoma share many features with POAG. Here, we characterized a novel mouse model of GC-induced OHT for glaucomatous neurodegeneration and further explored early pathological events of axonal degeneration in response to elevated IOP. METHODS: C57BL/6 J mice were periocularly injected with either vehicle or the potent GC, dexamethasone 21-acetate (Dex) once a week for 10 weeks. Glaucoma phenotypes including IOP, outflow facility, structural and functional loss of retinal ganglion cells (RGCs), optic nerve (ON) degeneration, gliosis, and anterograde axonal transport deficits were examined at various stages of OHT. RESULTS: Prolonged treatment with Dex leads to glaucoma in mice similar to POAG patients including IOP elevation due to reduced outflow facility and dysfunction of trabecular meshwork, progressive ON degeneration and structural and functional loss of RGCs. Lowering of IOP rescued Dex-induced ON degeneration and RGC loss, suggesting that glaucomatous neurodegeneration is IOP dependent. Also, Dex-induced neurodegeneration was associated with activation of astrocytes, axonal transport deficits, ON demyelination, mitochondrial accumulation and immune cell infiltration in the optic nerve head (ONH) region. Our studies further show that ON degeneration precedes structural and functional loss of RGCs in Dex-treated mice. Axonal damage and transport deficits initiate at the ONH and progress toward the distal end of ON and target regions in the brain (i.e. superior colliculus). Most of anterograde transport was preserved during initial stages of axonal degeneration (30% loss) and complete transport deficits were only observed at the ONH during later stages of severe axonal degeneration (50% loss). CONCLUSIONS: These findings indicate that ON degeneration and transport deficits at the ONH precede RGC structural and functional loss and provide a new potential therapeutic window for rescuing neuronal loss and restoring health of damaged axons in glaucoma.Item Crosstalk Between Transforming Growth Factor Beta-2 and Toll-Like Receptor 4 in the Trabecular Meshwork(2017-12-01) Hernandez, Humberto; McDowell, Colleen; Clark, Abbot F.; Pang, Iok-HouThe trabecular meshwork (TM) is the main site of outflow resistance in primary-open angle glaucoma (POAG) patients. In these patients, aqueous humor outflow resistance increases, subsequently leading to a rise in intraocular pressure (IOP). The rise in IOP ultimately damages the optic nerve and leads to blindness. Accumulation of extracellular matrix (ECM) at the TM has been shown by our laboratory and many others to be responsible for the increase in outflow resistance. The molecular mechanisms underlying the pathology are beginning to be elucidated. The pro-fibrotic cytokine, transforming growth factor beta-2 (TGFβ2), has been shown to be elevated in the aqueous humor of POAG patients. Mice injected with adenovirus encoding active TGFβ2 develop ocular hypertension and ECM deposition at the TM. Recently, toll-like receptor 4 (TLR4) signaling has been linked to the development of fibrosis. In our studies, we evaluated the crosstalk between TGFβ2 and TLR4 in the TM. We utilized in vitro and in vivo models to evaluate the role of TLR4 on the production of ECM and development of ocular hypertension. We also utilized a conditional knockout in vitro and in vivo adenovirus delivery system to study BMP and Activin Membrane Bound Inhibitor (BAMBI), a critical molecule in the crosstalk between TGFβ2 and TLR4. Our studies reveal a novel pathway involved in the development of TM damage and potential targets to lower IOP.Item EFFECT OF TRANSFORMING GROWTH FACTOR BETA-2 SIGNALING AND GREMLIN INDUCTION ON FIBRONECTIN, OCULAR HYPERTENSION, AND OPTIC NERVE DAMAGE(2013-04-12) McDowell, ColleenPurpose: Transforming growth factor β2 (TGFβ2) induces extracellular matrix (ECM) remodeling and alters the cytoskeleton, which likely contribute to the inefficient function of the trabecular meshwork (TM) tissue leading to glaucomatous phenotypes. Bone morphogenetic proteins (BMPs) inhibit these profibrotic effects of TGFβ2. The BMP antagonist gremlin is elevated in glaucomatous TM cells and increases intraocular pressure (IOP) in an ex vivo perfusion culture model. The purpose of this study was to determine whether TGFβ2 and gremlin regulate ECM proteins in the TM, induce ocular hypertension, and cause optic nerve damage in mice. Methods: Ad5.hTGFβ226/228 or Ad5.Gremlin (2 uL, 2 X 10^7 pfu) was injected intravitreally into one eye of A/J mice (n=7-13 mice per group), with the uninjected contralateral eye serving as the control eye. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Optic nerve damage was assessed using the optic nerve damage score of PPD stained optic nerve cross sections. TGFβ2, fibronectin, and gremlin protein expression in the TM was determined by immunofluorescence and immunohistochemistry. Transduction of the TM with viral vector Ad5.hTGFβ2226/228 caused a prolonged, reproducible, and statistically significant IOP elevation. IOPs increased to approximately 25 mm Hg for 8 weeks (p<0.001). IOPs were stable (12-15 mm Hg) in the uninjected control eyes. The TGFP2 induced ocular hypertension also caused significant optic nerve damage with optic nerve damage scores (ONDS) > 3 (p<0.001) in the injected eye. Intraocular administration of viral vector Ad5.Gremlin also caused significant IOP elevation in A/J mice for 3 weeks (n=9, injected eye 23.2 +/- 5.6 mmmHg, uninjected eye 15.5 +/- 2.4; p<0.01). In addition, immunofluorescence and immunohistochemistry demonstrated that intraocular injection of Ad5.hTGFβ226/228 and Ad5.Gremlin increased TGFβ2 and fibronectin expression in the TM. Conclusions: These results demonstrate that intravitreal injections of Ad5.hTGFβ226/228 and Ad5.Gremlin in A/J mice elevate IOP and upregulate the ECM protein fibronectin. In addition, Ad5.hTGFβ226/228 expression induced significant optic nerve damage. These data demonstrate for the first time gremlin's role in inducing ocular hypertension in an in vivo model system and emphasize the importance of the TGFβ2 signaling pathway in ocular hypertension.Item Exploring Trabecular Meshwork Molecular Pathogenic Mechanisms In Primary Open Angle Glaucoma And Glucocorticoid Induced Glaucoma(2016-08-01) Bermudez, Jaclyn Y.; Clark, Abbot F.; Mao, Weiming; Singh, MeharvanIn a normal functioning eye, the aqueous humor, a fluid secreted by the ciliary body, drains through the trabecular meshwork (TM), a multilayered tissue in the anterior segment of the eye. The TM is the initial site of damage in glaucoma. Damaged TM results in higher aqueous humor outflow resistance and causes elevated IOP, the latter of which leads to optic nerve damage. Numerous clinical studies have shown that lowering IOP can prevent neuronal damage and slow/stop the progression of the disease. In the glaucomatous TM (GTM), there is excessive extracellular matrix protein deposition, cytoskeletal changes and altered cell function. The transforming growth factor β (TGFβ) pathway is activated by TGFβ2 which has been found to be more abundant in the GTM. Additionally, formation of cross-linked actin networks (CLANs) in the GTM is increased compared to non-glaucoma TM. Primary open angle glaucoma (POAG), glucocorticoid-induced glaucoma (GIG) and glucocorticoid-induced ocular hypertension (GCOHT), share similar pathophysiologies. GC-OHT differs from POAG in that about 40% of the population develops GC-OHT after topical treatment with glucocorticoids however, the mechanism that differentiates steroid responders from non-responders is unknown. In our studies we have explored trabecular meshwork molecular pathogenic mechanisms that are responsible for the disease pathology. We have studied epigenetics as a regulatory mechanism for increasing TGFβ2 expression. We have also used proteomics to determine proteins that are associated with CLANs. Lastly, we studied genes that are differentially expressed in glucocorticoid responders versus non-responders in our bovine model of GC-OHT. Overall, our research has enhanced our understanding of the TM and the molecular mechanisms that play a role in glaucoma. We hope to use this information to find new disease modifying therapies.Item Expression of Mutant Myocilin Induces Abnormal Intracellular Accumulation of Selected Extracellular Matrix Proteins in the Trabecular Meshwork(Association for Research in Vision and Ophthalmology, 2016-11-01) Kasetti, Ramesh B.; Phan, Tien N.; Millar, J. Cameron; Zode, Gulab S.PURPOSE: Abnormal accumulation of extracellular matrix (ECM) in the trabecular meshwork (TM) is associated with decreased aqueous humor outflow facility and IOP elevation in POAG. Previously, we have developed a transgenic mouse model of POAG (Tg-MYOCY437H) by expressing human mutant myocilin (MYOC), a known genetic cause of POAG. The purpose of this study is to examine whether expression of mutant myocilin leads to reduced outflow facility and abnormal ECM accumulation in Tg-MYOCY437H mice and in cultured human TM cells. METHODS: Conscious IOP was measured at various ages of Tg-MYOCY437H mice using a rebound tonometer. Outflow facility was measured in 10-month-old Tg-MYOCY437H mice. Selected ECM proteins were examined in human TM-3 cells stably expressing mutant myocilin and primary human TM cells (n = 4) as well as in the TM of Tg-MYOCY437H mice by real-time PCR, Western blotting, and immunostaining. Furthermore, TM cells expressing WT or mutant myocilin were treated with 5 mM sodium 4-phenylbutyrate (PBA), and ECM proteins were examined by Western blot and immunostaining. RESULTS: Starting from 3 months of age, Tg-MYOCY437H mice exhibited significant IOP elevation compared with wild-type (WT) littermates. Outflow facility was significantly reduced in Tg-MYOCY437H mice (0.0195 mul/min/mm Hg in Tg-MYOCY437H vs. 0.0332 mul/min/mm Hg in WT littermates). Increased accumulation of fibronectin, elastin, and collagen type IV and I was observed in the TM of Tg-MYOCY437H mice compared with WT littermates. Furthermore, increased ECM proteins were also associated with induction of endoplasmic reticulum (ER) stress markers, GRP78 and CHOP in the TM of Tg-MYOCY437H mice. Human TM-3 cells stably expressing DsRed-tagged Y437H mutant MYOC exhibited inhibition of myocilin secretion and its intracellular accumulation compared with TM cells expressing WT MYOC. Expression of mutant MYOC in TM-3 cells or human primary TM cells induced ER stress and also increased intracellular protein levels of fibronectin, elastin, laminin, and collagen IV and I. In addition, TM-3 cells expressing mutant myocilin exhibited reduced active forms of matrix metalloproteinase (MMP)-2 and MMP-9 in conditioned medium compared with TM-3 cells expressing WT myocilin. Interestingly, both intracellularly accumulated fibronectin and collagen I colocalized with mutant myocilin and also with ER marker KDEL further suggesting intracellular accumulation of these proteins in the ER of TM cells. Furthermore, reduction of ER stress via PBA decreased selected ECM proteins in primary TM cells. CONCLUSIONS: These studies demonstrate that mutant myocilin induces abnormal ECM accumulation in the ER of TM cells, which may be responsible for reduced outflow facility and IOP elevation in myocilin-associated glaucoma.Item Expression of Procollagen C Proteinase Enhancer Proteins in Trabecular Cells and Tissues(2013-05-01) Naik, Monal; Robert WordingerPrimary open angle glaucoma (POAG) is the most common form of glaucoma. Ocular hypertension is a major risk factor for POAG and is caused by increased aqueous humor (AH) outflow resistance in the trabecular meshwork (TM). Increased extracellular matrix (ECM) deposition within the TM is correlated with ocular hypertension. Transforming Growth Factor beta 2 (TGFb2) levels are elevated in the AH and TM of POAG patients, and TGFb2 increases ECM protein expression, aqueous outflow resistance, and intraocular pressure (IOP). Recently, TGFβ2 was found to induce bone morphogenetic protein 1 (BMP1) expression in TM cells suggesting that BMP1 activity might be involved in glaucoma pathogenesis. Procollagen C proteinase enhancers (PCOLCE 1 and PCOLCE 2) regulate BMP1 activity. Therefore, PCOLCE1 and PCOLCE2 may play an important role(s) in regulating ECM structural changes in the TM, and contribute to AH outflow resistance and elevated IOP in glaucoma. The purpose of this study was to determine if human TM cells and tissues express PCOLCE1 and PCOLCE2 and whether TGFb2 induces their expression. This is the first documentation that PCOLCE1 and PCOLCE2 are expressed in TM cells and tissues and that TGFb2 does induce expression of PCOLCE1.Item Glucororticoid Receptor Alternative Splicing: Key Players and Role in TM and Glaucoma(2012-12-01) Jain, Ankur; Clark, Abbot F.Elevated intraocular pressure (IOP) is the primary risk factor in glaucoma, a leading cause of irreversible blindness. Various morphological and biochemical changes in the trabecular meshwork (TM) appear to be responsible for blocking aqueous humor outflow, thereby elevating IOP. Glucocorticoids (GCs) are known to induce ocular hypertension and other biochemical changes associated with glaucoma. Interestingly, there are differences in steroid responsiveness among the population, with 40% people known as responders who significantly elevate IOP upon GC treatment and others being classified as nonresponders. The steroid-responders are at higher risk of developing primary open angle glaucoma (POAG) as compared to the steroid nonresponders. At the same time, almost all POAG patients are moderate to high steroid responders. GC responsiveness is regulated by the relative ratios of the GC activated transcription factor GC receptor alpha (GRα) and the alternatively spliced dominant negative regulator isoform of this receptor (GRβ). Glaucomatous TM cell strains have a higher GRα/GRβ ratio compared to normal TM cells making them more sensitive to GCs. Regulation of the GRα/GRβ splicing is not very well documented. The role of splicing factors that regulate spliceosome assembly seems to be one of the key factors regulating the process of alternative splicing. We have shown that the relative levels of the different serine-arginine (SR) proteins (SRps) in the TM regulate the differential expression of the two alternatively spliced isoforms of GR, GRα and GRβ and that expression of these SR proteins regulates GC responsiveness in TM cells. In addition, we evaluated a special class of compounds (thailanstatins or TSTs) and found them to modulate this splicing process to enhance GRβ levels in TM cells. These splicing modulators increased GRβ/GRα in TM, decreased GC response and provide potential glaucoma therapeutic agents.Item Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma(Springer Nature Limited, 2024-03-24) Patil, Shruti V.; Kaipa, Balasankara R.; Ranshing, Sujata; Sundaresan, Yogapriya; Millar, J. Cameron; Nagarajan, Bhavani; Kiehlbauch, Charles; Zhang, Qihong; Jain, Ankur; Searby, Charles C.; Scheetz, Todd E.; Clark, Abbot F.; Sheffield, Val C.; Zode, Gulab S.Mutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target MYOC using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_GFP expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOC(Y437H) mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.Item Modulation of Mitochondrial Metabolic Parameters and Antioxidant Enzymes in Healthy and Glaucomatous Trabecular Meshwork Cells with Hybrid Small Molecule SA-2(MDPI, 2023-07-29) Amankwa, Charles E.; Young, Olivia; DebNath, Biddut; Gondi, Sudershan R.; Rangan, Rajiv; Ellis, Dorette Z.; Zode, Gulab S.; Stankowska, Dorota L.; Acharya, SuchismitaOxidative stress (OS)-induced mitochondrial damage is a risk factor for primary open-angle glaucoma (POAG). Mitochondria-targeted novel antioxidant therapies could unearth promising drug candidates for the management of POAG. Previously, our dual-acting hybrid molecule SA-2 with nitric oxide-donating and antioxidant activity reduced intraocular pressure and improved aqueous humor outflow in rodent eyes. Here, we examined the mechanistic role of SA-2 in trabecular meshwork (TM) cells in vitro and measured the activity of intracellular antioxidant enzymes during OS. Primary human TM cells isolated from normal (hNTM) or glaucomatous (hGTM) post-mortem donors and transformed glaucomatous TM cells (GTM-3) were used for in vitro assays. We examined the effect of SA-2 on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro using Seahorse Analyzer with or without the oxidant, tert-butyl hydroperoxide (TBHP) treatment. Concentrations of total antioxidant enzymes, catalase (CAT), malondialdehyde (MDA), and glutathione peroxidase (GPx) were measured. We observed significant protection of both hNTM and hGTM cells from TBHP-induced cell death by SA-2. Antioxidant enzymes were elevated in SA-2-treated cells compared to TBHP-treated cells. In addition, SA-2 demonstrated an increase in mitochondrial metabolic parameters. Altogether, SA-2 protected both normal and glaucomatous TM cells from OS via increasing mitochondrial energy parameters and the activity of antioxidant enzymes.Item TGFβ Signaling and the Formation of Cross-Linked Actin Networks (CLANs) in Human Trabecular Meshwork Cells(2016-05-01) Montecchi-Palmer, Michela; Clark, Abbot F.; Mao, Weiming; Ghorpade, AnujaGlaucoma is the leading cause of irreversible vision loss and blindness worldwide. One of the major risk factors for glaucoma is increased intraocular pressure (IOP); however there is little understanding of the initial causes of abnormal IOP. Actin cytoskeletal rearrangements known as cross-linked actin networks (CLANs) form at a higher incidence in the glaucomatous trabecular meshwork (TM) cells compared to non-glaucomatous TM cells. The incidence of CLANs is believed to increase the stiffness of TM cells and TM tissue, thereby increasing aqueous humor (AH) outflow resistance and IOP. Even though these actin formations are known to be present, the actual cause of their formation has not yet been elucidated. CLANs can be induced by either transforming growth factor-β2 (TGFβ2) or glucocorticoids such as Dexamethasone (Dex). The primary focus of this research is the TGFβ2 induced CLAN formation and the TGFβ pathways leading to these cytoskeletal rearrangements. Primary human TM cell culture was used to identify whether the Smad or non-Smad TGFβ pathways are the primary path to TGFβ2 induced CLAN formation by testing the ability of various inhibitors to prevent and resolve TGFβ2 induced CLANs. The results of this research will be a novel and critical source of information in the development of therapeutic strategies for the treatment of elevated IOP and glaucoma, and possibly a better understanding of additional factors contributing to the onset of this sight threatening disease.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 Therapeutic Potential of Antioxidants and Hybrid TEMPOL Derivatives in Ocular Neurodegenerative Diseases: A Glimpse into the Future(MDPI, 2023-11-25) Amankwa, Charles E.; Kodati, Bindu; Donkor, Nina; Acharya, SuchismitaReactive oxygen species play a significant role in the pathogenesis of various ocular neurodegenerative diseases especially glaucoma, age-related macular degeneration (AMD), and ocular ischemic stroke. Increased oxidative stress and the accumulation of ROS have been implicated in the progression of these diseases. As a result, there has been growing interest in exploring potential therapeutic and prophylactic strategies involving exogenous antioxidants. In recent years, there have been significant advancements in the development of synthetic therapeutic antioxidants for targeting reactive oxygen species (ROS) in neurodegenerative diseases. One area of focus has been the development of hybrid TEMPOL derivatives. In the context of ocular diseases, the application of next-generation hybrid TEMPOL antioxidants may offer new avenues for neuroprotection. By targeting ROS and reducing oxidative stress in the retina and optic nerve, these compounds have the potential to preserve retinal ganglion cells and trabecular meshwork and protect against optic nerve damage, mitigating irreversible blindness associated with these diseases. This review seeks to highlight the potential impact of hybrid TEMPOL antioxidants and their derivatives on ocular neurodegenerative disorders.