Browsing by Subject "ECM"
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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 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 Store operated calcium entry in glomerular mesangial cells and diabetic nephropathy(2016-08-01) Chaudhari, Sarika; Rong Ma; J. Thomas Cunningham; Robert T. MalletGlomerular mesangial cells (MCs) are the major source of extracellular matrix (ECM). One of the early pathological changes in diabetic nephropathy (DN) is accumulation of ECM in glomeruli. Multifunctional store-operated Ca2+ entry (SOCE) regulates MC function. However, whether and how SOCE in MCs contributes to pathophysiology of DN remains unknown. The aim of the study was to investigate association of SOCE in MCs with ECM protein expression and the underlying mechanism using both in vitro and in vivo systems. Study I was to determine the effect of diabetes on SOCE. In cultured human MCs, we found that prolonged high glucose (HG) treatment (7 days) significantly increased SOCE and membrane currents through store-operated channels (SOC). These responses were abolished by SOC inhibitors. Consistently, prolonged HG treatment also increased the abundance of SOC proteins STIM1 and Orai1. HG also increased STIM1, but not Orai1 mRNA expression. Furthermore, both STIM1 and Orai1 proteins were also increased in the glomeruli/renal cortices of diabetic rats. Study II determined the influence of SOCE in MCs on ECM protein expression. We found that activation of SOC by thapsigargin reduced the abundance of fibronectin and collagen IV while inhibitors of SOC had opposite effects. Knockdown of Orai1 in human MCs increased fibronectin abundance. The HG induced increase in fibronectin was attenuated by SOCE. Using a nanoparticle siRNA delivery system, specific knockdown of Orai1 in MCs in mice increased glomerular fibronectin and collagen IV protein content and mesangial expansion. Study III determined the mechanism for inhibition of ECM protein expression by SOCE. We found that activation of SOC attenuated TGFβ1 mediated phosphorylation and translocation of Smad3, a known fibrotic pathway in MCs. However, there was no change in the production or secretion of TGFβ1 by MCs. Orai1 knockdown in MCs in mice increased the activation of Smad3. Taken together, our results indicate that SOCE in MCs may be increased in the late stage of diabetes, which suppresses ECM protein expression by inhibiting TGFβ1-smad3 pathway.