Browsing by Subject "Glaucoma, Open-Angle / metabolism"
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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 ATF4 leads to glaucoma by promoting protein synthesis and ER client protein load(Springer Nature, 2020-11-05) Kasetti, Ramesh B.; Patel, Pinkal D.; Maddineni, Prabhavathi; Patil, Shruti; Kiehlbauch, Charles; Millar, J. Cameron; Searby, Charles C.; Raghunathan, Vijaykrishna; Sheffield, Val C.; Zode, Gulab S.The underlying pathological mechanisms of glaucomatous trabecular meshwork (TM) damage and elevation of intraocular pressure (IOP) are poorly understood. Here, we report that the chronic endoplasmic reticulum (ER) stress-induced ATF4-CHOP-GADD34 pathway is activated in TM of human and mouse glaucoma. Expression of ATF4 in TM promotes aberrant protein synthesis and ER client protein load, leading to TM dysfunction and cell death. These events lead to IOP elevation and glaucomatous neurodegeneration. ATF4 interacts with CHOP and this interaction is essential for IOP elevation. Notably, genetic depletion or pharmacological inhibition of ATF4-CHOP-GADD34 pathway prevents TM cell death and rescues mouse models of glaucoma by reducing protein synthesis and ER client protein load in TM cells. Importantly, glaucomatous TM cells exhibit significantly increased protein synthesis along with induction of ATF4-CHOP-GADD34 pathway. These studies indicate a pathological role of ATF4-CHOP-GADD34 pathway in glaucoma and provide a possible treatment for glaucoma by targeting this pathway.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 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.