Primary open-angle glaucoma (POAG) is a leading cause of irreversible vision loss worldwide, with elevated intraocular pressure (IOP) a major risk factor. Myocilin (MYOC) dominant gain-offunction mutations have been reported in ∼4% of POAG cases. MYOC mutations result in protein misfolding, leading to endoplasmic reticulum (ER) stress in the trabecular meshwork (TM), the tissue that regulates IOP. We use CRISPR-Cas9-mediated genome editing in cultured human TM cells and in a MYOC mouse model of POAG to knock down expression of mutant MYOC, resulting in relief of ER stress. In vivo genome editing results in lower IOP and prevents further glaucomatous damage. Importantly, using an ex vivo human organ culture system, we demonstrate the feasibility of human genome editing in the eye for this important disease.myocilin | CRISPR | glaucoma | trabecular meshwork | genome editing

BackgroundThe available drugs for diabetes, Insulin or Oral hypoglycemic agents have one or more side effects. Search for new antidiabetic drugs with minimal or no side effects from medicinal plants is a challenge according to WHO recommendations. In this aspect, the present study was undertaken to evaluate the antihyperglycemic and antihyperlipidemic effects of Piper longum root aqueous extract (PlrAqe) in streptozotocin (STZ) induced diabetic rats.MethodsDiabetes was induced in male Wister albino rats by intraperitoneal administration of STZ (50 mg/kg.b.w). Fasting blood glucose (FBG) levels were measured by glucose-oxidase & peroxidase reactive strips. Serum biochemical parameters such as glycosylated hemoglobin (HbA1c), total cholesterol (TC), triglycerides (TG), very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol were estimated. The activities of liver and kidney functional markers were measured. The statistical analysis of results was carried out using Student t-test and one-way analysis (ANOVA) followed by DMRT.ResultsDuring the short term study the aqueous extract at a dosage of 200 mg/kg.b.w was found to possess significant antidiabetic activity after 6 h of the treatment. The administration of aqueous extract at the same dose for 30 days in STZ induced diabetic rats resulted in a significant decrease in FBG levels with the corrections of diabetic dyslipidemia compared to untreated diabetic rats. There was a significant decrease in the activities of liver and renal functional markers in diabetic treated rats compared to untreated diabetic rats indicating the protective role of the aqueous extract against liver and kidney damage and its non-toxic property.ConclusionsFrom the above results it is concluded that the plant extract is capable of managing hyperglycemia and complications of diabetes in STZ induced diabetic rats. Hence this plant may be considered as one of the potential sources for the isolation of new oral anti hypoglycemic agent(s).

Elevation of intraocular pressure (IOP) is a serious adverse effect of glucocorticoid (GC) therapy. Increased extracellular matrix (ECM) accumulation and endoplasmic reticulum (ER) stress in the trabecular meshwork (TM) is associated with GC-induced IOP elevation. However, the molecular mechanisms by which GCs induce ECM accumulation and ER stress in the TM have not been determined. Here, we show that a potent GC, dexamethasone (Dex), activates transforming growth factor β (TGFβ) signaling, leading to GC-induced ECM accumulation, ER stress, and IOP elevation. Dex increased both the precursor and bioactive forms of TGFβ2 in conditioned medium and activated TGFβ-induced SMAD signaling in primary human TM cells. Dex also activated TGFβ2 in the aqueous humor and TM of a mouse model of Dex-induced ocular hypertension. We further show that mice are protected from Dex-induced ocular hypertension, ER stress, and ECM accumulation. Moreover, treating WT mice with a selective TGFβ receptor kinase I inhibitor, LY364947, significantly decreased Dex-induced ocular hypertension. Of note, knockdown of the ER stress-induced activating transcription factor 4 (ATF4), or C/EBP homologous protein (CHOP), completely prevented Dex-induced TGFβ2 activation and ECM accumulation in TM cells. These observations suggested that chronic ER stress promotes Dex-induced ocular hypertension via TGFβ signaling. Our results indicate that TGFβ2 signaling plays a central role in GC-induced ocular hypertension and provides therapeutic targets for GC-induced ocular hypertension.

Increased synthesis and deposition of extracellular matrix (ECM) proteins in the trabecular meshwork (TM) is associated with TM dysfunction and intraocular pressure (IOP) elevation in glaucoma. However, it is not understood how ECM accumulation leads to TM dysfunction and IOP elevation. Using a mouse model of glucocorticoid (GC)-induced glaucoma, primary human TM cells and human post-mortem TM tissues, we show that increased ECM accumulation leads to endoplasmic reticulum (ER) stress in the TM. The potent GC, dexamethasone (Dex) increased the secretory protein load of ECM proteins in the ER of TM cells, inducing ER stress. Reduction of fibronectin, a major regulator of ECM structure, prevented ER stress in Dex-treated TM cells. Overexpression of fibronectin via treatment with cellular fibronectin also induced chronic ER stress in primary human TM cells. Primary human TM cells grown on ECM derived from Dex-treated TM cells induced ER stress markers. TM cells were more prone to ER stress from ECM accumulation compared to other ocular cell types. Moreover, increased co-localization of ECM proteins with ER stress markers was observed in human post-mortem glaucomatous TM tissues. These data indicate that ER stress is associated with increased ECM accumulation in mouse and human glaucomatous TM tissues.

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Glucocorticoid (GC)-induced ocular hypertension (OHT) is a serious adverse effect of prolonged GC therapy that can lead to iatrogenic glaucoma and permanent vision loss. An appropriate mouse model can help us understand precise molecular mechanisms and etiology of GC-induced OHT. We therefore developed a novel, simple, and reproducible mouse model of GC-induced OHT. GC-induced myocilin expression in the trabecular meshwork (TM) has been suggested to play an important role in GC-induced OHT. We further determined whether myocilin contributes to GC-OHT. C57BL/6J mice received weekly periocular conjunctival fornix injections of a dexamethasone-21-acetate (DEX-Ac) formulation. Intraocular pressure (IOP) elevation was relatively rapid and significant, and correlated with reduced conventional outflow facility. Nighttime IOPs were higher in ocular hypertensive eyes compared to daytime IOPs. DEX-Ac treatment led to increased expression of fibronectin, collagen I, and a-smooth muscle actin in the TM in mouse eyes. No changes in body weight indicated no systemic toxicity associated with DEX-Ac treatment. Wild-type mice showed increased myocilin expression in the TM on DEX-Ac treatment. Both wild-type and Myoc À/À mice had equivalent and significantly elevated IOP with DEX-Ac treatment every week. In conclusion, our mouse model mimics many aspects of GC-induced OHT in humans, and we further demonstrate that myocilin does not play a major role in DEX-induced OHT in mice. (Am J Pathol 2017, 187: 713e723; http://dx.doi.org/10.1016/j.ajpath.2016 Glucocorticoids (GCs) are one of the most commonly prescribed medications worldwide for the treatment of a plethora of diseases and conditions. Because of their broadspectrum anti-inflammatory and immunosuppressive properties, the worldwide market for GC use is estimated to be >$10 billion per year.1 Approximately 1.2% of US and 0.85% of UK populations are prescribed therapeutic GCs every year.2,3 GCs also remain the mainstay of treatment for a variety of ocular inflammatory diseases involving almost all tissues of the eye, such as eyelids, conjunctiva, cornea, sclera, uvea, retina, and optic nerve. 4 The routes of GC administration in treatment of these disorders can be topical ocular, oral, systemic, intravitreal injections and implants, and periocular injections (including subconjunctival, subtenon, retrobulbar, and peribulbar).5 However, prolonged GC therapy is associated with serious ocular adverse effects, including development of posterior subcapsular cataracts, and the development of GC-induced ocular hypertension (GC-OHT) and iatrogenic open-angle glaucoma.The clinical presentation of GC-induced glaucoma is similar to primary open-angle glaucoma (POAG), and for >50 years, reports have suggested a link between glaucoma and GCs. Development of GC-induced OHT depends on GC dose and duration of treatment, method of administration, potency of GC, and individual susceptibility to GCs. 6e8 There are varying degrees of steroid responsiveness (ie, development of GC-OHT) among individ...

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PurposeAbnormal 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.MethodsConscious 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.ResultsStarting 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 μl/min/mm Hg in Tg-MYOCY437H vs. 0.0332 μl/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.ConclusionsThese 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.