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.

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...

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.

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 21acetate (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.

BackgroundTuberculosis remains the foremost cause of morbidity and mortality, more than any other single infectious disease in the world. Cell mediated immune response plays a crucial role in the control of tuberculosis. Therefore, measuring cell mediated immune response against the antigens is having a vital role in understanding the pathogenesis of tuberculosis, which will also help in the diagnosis of and vaccination for tuberculosis.FindingsThe aim of the present study was to compare and optimize the assay conditions to measure the cell mediated immune response against M. tuberculosis specific antigens. Because the conventional PBMC assays (due to requirement of large volume of blood sample) are unable to screen more number of antigens within the same blood sample. So, here we have compared 6 days culture supernatants of 1:5 and 1:10 diluted blood and PBMCs from healthy laboratory volunteers, to assess the proliferative response of T lymphocytes and secreted IFN-γ levels against purified recombinant antigen of M. tuberculosis (MPT51, Rv3803c), crude antigens of M. tuberculosis (PPD) and mitogen (PHA).ConclusionsWe have observed good correlation between each assay and also the mean difference of these assays did not reach the statistical significance (p > 0.05). From these results, we conclude that 1:10 diluted whole-blood cultures can be well-suited as an alternative assay to measure cytokine production and lymphocyte proliferation in comparison to the conventional PBMC assays. Moreover, 1:10 diluted blood assays require less volume of blood when compared to PBMC assays which will be useful particularly in paediatric and field studies in endemic countries, where blood volume is a limiting factor.

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Elevation of intraocular pressure (IOP) due to trabecular meshwork (TM) damage is associated with Primary Open Angle Glaucoma (POAG). Myocilin mutations resulting in elevated IOP are the most common genetic cause of POAG. We have previously shown that mutant myocilin accumulates in the endoplasmic reticulum (ER) and induces chronic ER stress, leading to TM damage and IOP elevation. However, it is not understood how chronic ER stress leads to TM dysfunction and loss. Here, we report that mutant myocilin activates autophagy but it is functionally impairecd in cultured human trabecular meshwork (TM) cells and in a mouse model of myocilin-associated POAG (Tg-MYOC Y437H). Genetic and pharmacological inhibition of autophagy worsens mutant myocilin accumulation and exacerbates IOP elevation in Tg-MYOC Y437H mice. Remarkably, impaired autophagy is associated with chronic ER stress-induced transcriptional factor, CHOP. Deletion of CHOP corrects impaired autophagy, enhances recognition and degradation of mutant myocilin by autophagy,and reduces glaucoma in Tg-MYOC Y437H mice. Stimulating autophagic flux via Tat-beclin 1 peptide or torin 2, promotes autophagic degradation of mutant myocilin and reduces elevated IOP in Tg-MYOC Y437H mice. Together, our studies provide an alternate treatment strategy for myocilin-associated POAG by correcting impaired autophagy in the TM.