Eye / Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21683
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Browsing Eye / Vision by Author "Kasetti, Ramesh"
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Item A Small Chemical Chaperone, Sodium 4 Phenylbutyrate Inhibits TGFβ2-Induced ECM Remodeling in Human TM Cells(2017-03-14) Kasetti, Ramesh; Phan, Tien; Maddineni, Prabhavathi; Zode, Gulab; Patel, PinkalPurpose: The pathological mechanisms underlying increased outflow resistance at the trabecular meshwork (TM) that is responsible for elevating intraocular pressure (IOP) have not been fully delineated. We have previously shown that progressive accumulation of unfolded proteins and endoplasmic reticulum (ER) stress play an important role in the pathophysiology of glaucomatous TM damage in myocilin-associated POAG. However, it is not understood whether other glaucoma factors lead to similar pathological ER stress in the TM, leading to IOP elevation. Transforming growth factor β2 (TGFβ2) is known to induce abnormal extracellular matrix (ECM) deposition in the TM cells, which may be responsible for IOP elevation. Here, we examined whether TGFβ2 induces ER stress and whether reducing ER stress via a small chemical chaperone, sodium 4-phenylbutyrate (PBA) reduces TGFβ2–induced ECM remodeling. Methods: Human GTM-3 cells or primary TM cells (n=2) were treated with TGFβ2 (5ng/ml) with or without 5mM PBA for 48 hours. Total cellular lysates, conditioned medium, and fixed cells were examined for ECM and ER stress markers by Western blotting and immunostaining. We also used RT-PCR to demonstrate XBP1 splicing in response to TGFβ2 treatment. Results: TGFβ2 increased synthesis and deposition of ECM proteins in GTM-3 and primary TM cells. TGFβ2 induced ER stress as evident from increased ER chaperones and splicing of XBP-1. Treatment of TM cells with TGFβ2 and PBA demonstrated reduced synthesis and deposition of ECM and ER stress markers as evident from reduced fibronectin, GRP78, GRP94 and CHOP. Conclusions: Our studies suggest that TGFβ2 induces abnormal ECM accumulation and ER stress, and PBA may reduce TGFβ2-induced IOP elevation by decreasing abnormal ECM accumulation and reducing ER stress.Item Inhibition of Transforming Growth Factor-β2 Signaling Prevents ECM Remodeling, Endoplasmic Reticulum Stress and Ocular Hypertension in Steroid-induced Glaucoma(2017-03-14) Maddineni, Prabhavathi; Patel, Pinkal; Millar, Cameron; Phan, Tien; Searby, Charles; Clark, Abbot; Sheffield, Val; Zode, Gulab; Kasetti, RameshPurpose: Ocular hypertension is a serious side effect of glucocorticoid (GC) therapy. Abnormal accumulation of extracellular matrix (ECM) and chronic endoplasmic reticulum (ER) stress in the trabecular meshwork (TM) is associated with GC-induced ocular hypertension. In the present study, we examined the role of TGFβ2 signaling in dexamethasone (Dex)-induced ECM remodeling, ER stress and ocular hypertension. Methods: Conscious IOP and outflow facility was measured in C57 mice treated with vehicle or Dex eye drops up to 7-weeks. TGFβ2 & fibronectin levels in the aqueous humor (AH) were analyzed by Western blotting. Effect of inhibition of TGFβ signaling on Dex-induced ER stress and ECM accumulation was examined by Western blot, immunostaining & Smad reporter assay in TM cells treated with or without TGFβ signaling inhibitors (SIS3 and LY364947). To further examine the role of TGF-β2 signaling, IOP, ECM and ER stress was examined in WT or Smad3-/- mice treated with Dex. Results: Dexamethasone (Dex) mediated reduction of outflow facility and IOP elevation is associated with increased abnormal extracellular matrix (ECM) accumulation in the TM, inducing ER stress. Biochemical analysis of the aqueous humor samples from Dex-treated eyes revealed significantly increased bioactive form of transforming growth factor-β2 (TGF-β2), a major regulator of ECM in the TM. Dex treatment increased both precursor and bioactive form of TGF-β2 in the conditioned medium and activated TGF-β2-induced Smad signaling pathway in primary human TM cells as evident from increased phosphorylation of Smad3 and increased Smad luciferase activity. Inhibition of TGF-β2 signaling significantly reduced Dex-induced abnormal intracellular ECM accumulation and ER stress in human TM cells. Smad3-/- mice, which are required for TGF-β2 signaling, protected from Dex-induced ocular hypertension, ER stress and abnormal ECM accumulation further indicating the role of TGF-β2 signaling in GC-induced glaucoma. Interestingly, knock out of ER stress-induced transcriptional factors, ATF4 and CHOP prevented activation of TGF-β2 signaling and also reduced intracellular ECM accumulation in the TM, thus preventing Dex-induced ER stress and IOP elevation. Conclusions: These studies indicate that Dex-induced TGF-β2 signaling is responsible for ECM remodeling, ER stress and elevation of IOP in GC-induced glaucoma.Item Overexpression of ATF-4 in trabecular meshwork causes elevation of intra ocular pressure and reduction of outflow facility in a CHOP dependent manner(2017-03-14) Kasetti, Ramesh; Zode, Gulab; Maddineni, PrabhavathiPurpose: Primary open-angle glaucoma (POAG) has been primarily associated with reduced aqueous humor outflow facility through trabecular meshwork (TM) and elevated intraocular pressure (IOP). Studies based on both human as well as mice models revealed that chronic endoplasmic reticulum (ER) stress in TM is one of the causative factors responsible for TM dysfunction and ocular hypertension. The purpose of this study is to examine whether forced expression of UPR downstream pro apoptotic molecules ATF4 and CHOP leads to reduced outflow facility and IOP elevation in normal C57 mice. Methods: Ad5.ATF-4/Ad5.CHOP/Ad5.empty virus (pfu=2x107) were injected intravitreally into C57BL/6J or CHOP KO (CHOP-/-) mice. Conscious IOP of both the eyes was monitored once in a week until 7 weeks using rebound tonometer. Outflow facility was measured by constant-flow infusion technique. Also, we examined the expression levels of ATF-4 in the TM of age-matched normal and POAG donors by immunohistochemistry. Results: Forced expression of ATF-4 but not CHOP caused significant IOP elevation (23.97 mmHg in Ad5.ATF4 v/s 14.6 mmHg in Ad5.null mice) and reduced outflow facility (0.022µL/min/mmHg in Ad5.ATF4 v/s 0.04 in Ad5.null mice) in C57BL/6J mice. Elevation of IOP in C57BL/6J was prominent from 3 weeks post injection and sustained until 7 weeks. Interestingly Ad5.ATF-4 did not elevate IOP (17.7 mmHg) in CHOP-/- mice, indicating that ATF-4 interaction with CHOP is the prerequisite for ATF-4-induced IOP elevation. Also, ER stress-induced pro death marker, ATF-4 was significantly increased in human post-mortem glaucomatous TM tissues compared to normal TM tissues. Expression of ATF4 in primary TM cells induced oxidative and ER stress and also upregulated pro-apoptotic markers. Conclusions: This data indicates that chronic ER marker ATF4 is increased in the glaucomatous TM tissues, which may be associated with TM dysfunction, reduction of outflow facility and IOP elevation via induction of ER and oxidative stress. Furthermore, interaction of ATF-4 and CHOP is essential to carry out downstream signal transduction pathways.