A Novel Mouse Model of TGFß2-Induced Ocular Hypertension Using Lentiviral Gene Delivery.

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) ß2, 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 TGFß2-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 TGFß2 in the TM. We developed an LV vector-encoding active hTGFß2C226,228S under the control of a cytomegalovirus (CMV) promoter. Adult C57BL/6J mice were injected intravitreally with LV expressing null or hTGFß2C226,228S. 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 TGFß2 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_hTGFß2C226,228S 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 hTGFß2C226,228S that induces TM dysfunction and outflow resistance.

Neogenin pathway positively regulates fibronectin production by glomerular mesangial cells.

Neogenin, a transmembrane receptor, was recently found in kidney cells and immune cells. However, the function of neogenin signaling in kidney is not clear. Mesangial cells (MCs) are a major source of extracellular matrix (ECM) proteins in glomerulus. In many kidney diseases, MCs are impaired and manifest myofibroblast phenotype. Overproduction of ECM by the injured MCs promotes renal injury and accelerates the progression of kidney diseases. The present study aimed to determine if neogenin receptor was expressed in MCs and if the receptor signaling regulated ECM protein production by MCs. We showed that neogenin was expressed in the glomerular MCs. Deletion of neogenin using CRISPR/Cas9 lentivirus system significantly reduced the abundance of fibronectin, an ECM protein. Netrin-1, a ligand for neogenin, also significantly decreased fibronectin production by MCs and decreased neogenin protein expression in MCs. Furthermore, treatment of human MCs with high glucose (HG, 25 mM) significantly increased the protein abundance of neogenin as early as 8 h. Consistently, neogenin expression in glomerulus significantly increased in the eNOS-/-db/db diabetic mice starting as early as the age of 8 wk and this increase sustained at least to the age of 24 wk. We further found that the HG-induced increase in neogenin abundance was blunted by antioxidant PEG-catalase and N-acetyl cysteine. Taken together, our results suggest a new mechanism of regulation of fibronectin production by MCs. This previously unrecognized neogenin-fibronectin pathway may contribute to glomerular injury responses during the course of diabetic nephropathy.

Enhanced Orai1-mediated store-operated Ca2+ channel/calpain signaling contributes to high glucose-induced podocyte injury.

Podocyte injury induced by hyperglycemia is the main cause of kidney dysfunction in diabetic nephropathy. However, the underlying mechanism is unclear. Store-operated Ca2+ entry (SOCE) regulates a diversity of cellular processes in a variety of cell types. Calpain, a Ca2+-dependent cysteine protease, was recently shown to be involved in podocyte injury. In the present study, we sought to determine whether increased SOCE contributed to high glucose (HG)-induced podocyte injury through activation of the calpain pathway. In cultured human podocytes, whole-cell patch clamp indicated the presence of functional store-operated Ca2+ channels, which are composed of Orai1 proteins and mediate SOCE. Western blots showed that HG treatment increased the protein abundance of Orai1 in a dose-dependent manner. Consistently, calcium imaging experiments revealed that SOCE was significantly enhanced in podocytes following HG treatment. Furthermore, HG treatment caused overt podocyte F-actin disorganization as well as a significant decrease in nephrin protein abundance, both of which are indications of podocyte injury. These podocyte injury responses were significantly blunted by both pharmacological inhibition of Orai1 using the small molecule inhibitor BTP2 or by genetic deletion of Orai1 using CRISPR-Cas9 lentivirus. Moreover, activation of SOCE by thapsigargin, an inhibitor of Ca2+ pump on the endoplasmic/sarcoplasmic reticulum membrane, significantly increased the activity of calpain, which was inhibited by BTP2. Finally, the calpain-1/calpain-2 inhibitor calpeptin significantly blunted the nephrin protein reduction induced by HG treatment. Taken together, our results suggest that enhanced signaling via an Orai1/SOCE/Calpain axis contributes to HG-induced podocyte injury.

Astragaloside IV Attenuates Ocular Hypertension in a Mouse Model of TGFß2 Induced Primary Open Angle Glaucoma.

Elevated intraocular pressure (IOP) is a major risk factor in developing primary open angle glaucoma (POAG), which is the most common form of glaucoma. Transforming growth factor-beta 2 (TGFß2) is a pro-fibrotic cytokine that plays an important role in POAG pathogenesis. TGFß2 induced extracellular matrix (ECM) production, deposition and endoplasmic reticulum (ER) stress in the trabecular meshwork (TM) contribute to increased aqueous humor (AH) outflow resistance and IOP elevation. Drugs which alter the glaucomatous fibrotic changes and ER stress in the TM may be effective in reducing ocular hypertension. Astragaloside IV (AS.IV), a novel saponin isolated from the roots of Astragalus membranaceus, has demonstrated antifibrotic and ER stress lowering effects in various tissues during disease conditions. However, the effect of AS.IV on glaucomatous TM fibrosis, ER stress and ocular hypertension has not been studied. Primary human TM cells treated with AS.IV decreased TGFß2 induced ECM (FN, Col-I) deposition and ER stress (KDEL, ATF4 and CHOP). Moreover, AS.IV treatment reduced TGFß2 induced NF-κB activation and αSMA expression in TM cells. We found that AS.IV treatment significantly increased levels of matrix metalloproteases (MMP9 and MMP2) and MMP2 enzymatic activity, indicating that the antifibrotic effects of AS.IV are mediated via inhibition of NF-κB and activation of MMPs. AS.IV treatment also reduced ER stress in TM3 cells stably expressing mutant myocilin. Interestingly, the topical ocular AS.IV eye drops (1 mM) significantly decreased TGFß2 induced ocular hypertension in mice, and this was associated with a decrease in FN, Col-1 (ECM), KDEL (ER stress) and αSMA in mouse TM tissues. Taken together, the results suggest that AS.IV prevents TGFß2 induced ocular hypertension by modulating ECM deposition and ER stress in the TM.

Sodium 4-Phenylbutyrate Reduces Ocular Hypertension by Degrading Extracellular Matrix Deposition via Activation of MMP9.

Ocular hypertension (OHT) is a serious adverse effect of the widely prescribed glucocorticoid (GC) therapy and, if left undiagnosed, it can lead to glaucoma and complete blindness. Previously, we have shown that the small chemical chaperone, sodium-4-phenylbutyrate (PBA), rescues GC-induced OHT by reducing ocular endoplasmic reticulum (ER) stress. However, the exact mechanism of how PBA rescues GC-induced OHT is not completely understood. The trabecular meshwork (TM) is a filter-like specialized contractile tissue consisting of TM cells embedded within extracellular matrix (ECM) that controls intraocular pressure (IOP) by constantly regulating aqueous humor (AH) outflow. Induction of abnormal ECM deposition in TM is a hallmark of GC-induced OHT. Here, we investigated whether PBA reduces GC-induced OHT by degrading abnormal ECM deposition in TM using mouse model of GC-induced OHT, ex vivo cultured human TM tissues and primary human TM cells. We show that topical ocular eye drops of PBA (1%) significantly lowers elevated IOP in mouse model of GC-induced OHT. Importantly, PBA prevents synthesis and deposition of GC-induced ECM in TM. We report for the first time that PBA can degrade existing abnormal ECM in normal human TM cells/tissues by inducing matrix metalloproteinase (MMP)9 expression and activity. Furthermore, inhibition of MMPs activity by chemical-inhibitor (minocycline) abrogated PBA's effect on ECM reduction and its associated ER stress. Our study indicates a non-chaperone activity of PBA via activation of MMP9 that degrades abnormal ECM accumulation in TM.

Impaired TRPV4-eNOS signaling in trabecular meshwork elevates intraocular pressure in glaucoma.

Primary Open Angle Glaucoma (POAG) is the most common form of glaucoma that leads to irreversible vision loss. Dysfunction of trabecular meshwork (TM) tissue, a major regulator of aqueous humor (AH) outflow resistance, is associated with intraocular pressure (IOP) elevation in POAG. However, the underlying pathological mechanisms of TM dysfunction in POAG remain elusive. In this regard, transient receptor potential vanilloid 4 (TRPV4) cation channels are known to be important Ca2+ entry pathways in multiple cell types. Here, we provide direct evidence supporting Ca2+ entry through TRPV4 channels in human TM cells and show that TRPV4 channels in TM cells can be activated by increased fluid flow/shear stress. TM-specific TRPV4 channel knockout in mice elevated IOP, supporting a crucial role for TRPV4 channels in IOP regulation. Pharmacological activation of TRPV4 channels in mouse eyes also improved AH outflow facility and lowered IOP. Importantly, TRPV4 channels activated endothelial nitric oxide synthase (eNOS) in TM cells, and loss of eNOS abrogated TRPV4-induced lowering of IOP. Remarkably, TRPV4-eNOS signaling was significantly more pronounced in TM cells compared to Schlemm's canal cells. Furthermore, glaucomatous human TM cells show impaired activity of TRPV4 channels and disrupted TRPV4-eNOS signaling. Flow/shear stress activation of TRPV4 channels and subsequent NO release were also impaired in glaucomatous primary human TM cells. Together, our studies demonstrate a central role for TRPV4-eNOS signaling in IOP regulation. Our results also provide evidence that impaired TRPV4 channel activity in TM cells contributes to TM dysfunction and elevated IOP in glaucoma.

Caveolar peroxynitrite formation impairs endothelial TRPV4 channels and elevates pulmonary arterial pressure in pulmonary hypertension.

Recent studies have focused on the contribution of capillary endothelial TRPV4 channels to pulmonary pathologies, including lung edema and lung injury. However, in pulmonary hypertension (PH), small pulmonary arteries are the focus of the pathology, and endothelial TRPV4 channels in this crucial anatomy remain unexplored in PH. Here, we provide evidence that TRPV4 channels in endothelial cell caveolae maintain a low pulmonary arterial pressure under normal conditions. Moreover, the activity of caveolar TRPV4 channels is impaired in pulmonary arteries from mouse models of PH and PH patients. In PH, up-regulation of iNOS and NOX1 enzymes at endothelial cell caveolae results in the formation of the oxidant molecule peroxynitrite. Peroxynitrite, in turn, targets the structural protein caveolin-1 to reduce the activity of TRPV4 channels. These results suggest that endothelial caveolin-1-TRPV4 channel signaling lowers pulmonary arterial pressure, and impairment of endothelial caveolin-1-TRPV4 channel signaling contributes to elevated pulmonary arterial pressure in PH. Thus, inhibiting NOX1 or iNOS activity, or lowering endothelial peroxynitrite levels, may represent strategies for restoring vasodilation and pulmonary arterial pressure in PH.

Autophagy stimulation reduces ocular hypertension in a murine glaucoma model via autophagic degradation of mutant myocilin.

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 causes of POAG. We have previously shown that mutant myocilin accumulates in the 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 activated autophagy but was functionally impaired in cultured human TM cells and in a mouse model of myocilin-associated POAG (Tg-MYOCY437H). Genetic and pharmacological inhibition of autophagy worsened mutant myocilin accumulation and exacerbated IOP elevation in Tg-MYOCY437H mice. Remarkably, impaired autophagy was associated with chronic ER stress-induced transcriptional factor CHOP. Deletion of CHOP corrected impaired autophagy, enhanced recognition and degradation of mutant myocilin by autophagy, and reduced glaucoma in Tg-MYOCY437H mice. Stimulating autophagic flux via tat-beclin 1 peptide or torin 2 promoted autophagic degradation of mutant myocilin and reduced elevated IOP in Tg-MYOCY437H mice. Our study provides an alternate treatment strategy for myocilin-associated POAG by correcting impaired autophagy in the TM.

ATF4 leads to glaucoma by promoting protein synthesis and ER client protein load.

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.

Heterozygous Loss of Yap1 in Mice Causes Progressive Cataracts.

Purpose: Yap1 encodes an evolutionarily conserved transcriptional coactivator and functions as a down-stream effector of the Hippo signaling pathway that controls tissue size and cell growth. Yap1 contributes to lens epithelial development. However, the effect of Yap1 haplodeficiency on the lens epithelium and its role in the development of cataracts has not been reported. The aim of the current study is to investigate Yap1 function and its regulatory mechanisms in lens epithelial cells (LECs). Methods: Lens phenotypes were investigated in Yap1 heterozygous mutant mice by visual observation and histological and biochemical methods. Primary LEC cultures were used to study regulatory molecular mechanism. Results: The heterozygous inactivation of Yap1 in mice caused cataracts during adulthood with defective LEC phenotypes. Despite a normal early development of the eye including the lens, the majority of Yap1 heterozygotes developed cataracts in the first six months of age. Cataract was preceded by multiple morphological defects in the lens epithelium, including decreased cell density and abnormal cell junctions. The low LEC density was coincident with reduced LEC proliferation. In addition, expression of the Yap1 target gene Crim1 was reduced in the Yap1+/- LEC, and overexpression of Crim1 restored Yap1+/- LEC cell proliferation in vitro. Conclusions: Homozygosity of the Yap1 gene was critical for adequate Crim1 expression needed to maintain the constant proliferation of LEC and to maintain a normal-sized lens. Yap1 haplodeficiency leads to cataracts.

Correction: Ex-vivo cultured human corneoscleral segment model to study the effects of glaucoma factors on trabecular meshwork.

[This corrects the article DOI: 10.1371/journal.pone.0232111.].

CNS axonal degeneration and transport deficits at the optic nerve head precede structural and functional loss of retinal ganglion cells in a mouse model of glaucoma.

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 21-acetate (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. RESULTS: Prolonged treatment with Dex leads to glaucoma in mice similar to POAG patients including IOP elevation due to reduced outflow facility and dysfunction of trabecular meshwork, progressive ON degeneration and structural and functional loss of RGCs. Lowering of IOP rescued Dex-induced ON degeneration and RGC loss, suggesting that glaucomatous neurodegeneration is IOP dependent. Also, Dex-induced neurodegeneration was associated with activation of astrocytes, axonal transport deficits, ON demyelination, mitochondrial accumulation and immune cell infiltration in the optic nerve head (ONH) region. Our studies further show that ON degeneration precedes structural and functional loss of RGCs in Dex-treated mice. Axonal damage and transport deficits initiate at the ONH and progress toward the distal end of ON and target regions in the brain (i.e. superior colliculus). Most of anterograde transport was preserved during initial stages of axonal degeneration (30% loss) and complete transport deficits were only observed at the ONH during later stages of severe axonal degeneration (50% loss). CONCLUSIONS: These findings indicate that ON degeneration and transport deficits at the ONH precede RGC structural and functional loss and provide a new potential therapeutic window for rescuing neuronal loss and restoring health of damaged axons in glaucoma.

Technical brief: Direct, real-time electrochemical measurement of nitric oxide in ex vivo cultured human corneoscleral segments.

Chronic elevation of intraocular pressure (IOP) is a major risk factor associated with primary open angle glaucoma (POAG), a common form of progressive optic neuropathy that can lead to debilitating loss of vision. Recent studies have identified the role of nitric oxide (NO) in the regulation of IOP, and as a result, several therapeutic ventures are currently targeting enhancement of NO signaling in the eye. Although a low level of NO is important for ocular physiology, excess exogenous NO can be detrimental. Therefore, the ability to directly measure NO in real time is essential for determining the role of NO signaling in glaucomatous pathophysiology. Historically, NO activity in human tissues has been determined by indirect methods that measure levels of NO metabolites (nitrate/nitrite) or downstream components of the NO signaling pathway (cGMP). In this proof-of-concept work, we assess the feasibility of direct, real-time measurement of NO in ex vivo cultured human corneoscleral segments using electrochemistry. A NO-selective electrode (ISO-NOPF200) paired to a free radical analyzer (TBR1025) was placed on the trabecular meshwork (TM) rim for real-time measurement of NO released from cells. Exogenous NO produced within cells was measured after treatment of corneoscleral segments with esterase-dependent NO-donor O2-acetoxymethylated diazeniumdiolate (DETA-NONOate/AM; 20 µM) and latanoprostene bunod (5-20 µM). A fluorescent NO-binding dye DAF-FM (4-Amino-5-methylamino- 2',7'-difluorofluorescein diacetate) was used for validation. A linear relationship was observed between the electric currents measured by the NO-sensing electrode and the NO standard concentrations, establishing a robust calibration curve. Treatment of ex vivo cultured human donor corneoscleral segments with DETA-NONOate/AM and latanoprostene bunod led to a significant increase in NO production compared with vehicle-treated controls, as detected electrochemically. Furthermore, the DAF-FM fluorescence intensity was higher in outflow pathway tissues of corneoscleral segments treated with DETA-NONOate/AM and latanoprostene bunod compared with vehicle-treated controls. In conclusion, these results demonstrate that NO-sensing electrodes can be used to directly measure NO levels in real time from the tissues of the outflow pathway.

Ex-vivo cultured human corneoscleral segment model to study the effects of glaucoma factors on trabecular meshwork.

Glaucoma is the second leading cause of irreversible blindness worldwide. Primary open angle glaucoma (POAG), the most common form of glaucoma, is often associated with elevation of intraocular pressure (IOP) due to the dysfunction of trabecular meshwork (TM) tissues. Currently, an ex vivo human anterior segment perfusion cultured system is widely used to study the effects of glaucoma factors and disease modifying drugs on physiological parameters like aqueous humor (AH) dynamics and IOP homeostasis. This system requires the use of freshly enucleated intact human eyes, which are sparsely available at very high cost. In this study, we explored the feasibility of using human donor corneoscleral segments for modeling morphological and biochemical changes associated with POAG. Among the number of corneas donated each year, many are deemed ineligible for transplantation due to stringent acceptance criteria. These ineligible corneoscleral segments were obtained from the Lions Eye Bank, Tampa, Florida. Each human donor anterior corneoscleral segment was dissected into four equal quadrants and cultured for 7 days by treating with the glaucoma factors dexamethasone (Dex) or recombinant transforming growth factor (TGF) ß2 or transduced with lentiviral expression vectors containing wild type (WT) and mutant myocilin. Hematoxylin and Eosin (H&E) staining analysis revealed that the TM structural integrity is maintained after 7 days in culture. Increased TUNEL positive TM cells were observed in corneoscleral quadrants treated with glaucoma factors compared to their respective controls. However, these TUNEL positive cells were mainly confined to the scleral region adjacent to the TM. Treatment of corneoscleral quadrants with Dex or TGFß2 resulted in glaucomatous changes at the TM, which included increased extracellular matrix (ECM) proteins and induction of endoplasmic reticulum (ER) stress. Western blot analysis of the conditioned medium showed an increase in ECM (fibronectin and collagen IV) levels in Dex- or TGFß2-treated samples compared to control. Lentiviral transduction of quadrants resulted in expression of WT and mutant myocilin in TM tissues. Western blot analysis of conditioned medium revealed decreased secretion of mutant myocilin compared to WT myocilin. Moreover, increased ECM deposition and ER stress induction was observed in the TM of mutant myocilin transduced quadrants. Our findings suggest that the ex-vivo cultured human corneoscleral segment model is cost-effective and can be used as a pre-screening tool to study the effects of glaucoma factors and anti-glaucoma therapeutics on the TM.

Mechanisms underlying selective coupling of endothelial Ca2+ signals with eNOS vs. IK/SK channels in systemic and pulmonary arteries.

KEY POINTS: Endothelial cell TRPV4 (TRPV4EC ) channels exert a dilatory effect on the resting diameter of resistance mesenteric and pulmonary arteries. Functional intermediate- and small-conductance K+ (IK and SK) channels and endothelial nitric oxide synthase (eNOS) are present in the endothelium of mesenteric and pulmonary arteries. TRPV4EC sparklets preferentially couple with IK/SK channels in mesenteric arteries and with eNOS in pulmonary arteries. TRPV4EC channels co-localize with IK/SK channels in mesenteric arteries but not in pulmonary arteries, which may explain TRPV4EC -IK/SK channel coupling in mesenteric arteries and its absence in pulmonary arteries. The presence of the nitric oxide-scavenging protein, haemoglobin α, limits TRPV4EC -eNOS signalling in mesenteric arteries. Spatial proximity of TRPV4EC channels with eNOS and the absence of haemoglobin α favour TRPV4EC -eNOS signalling in pulmonary arteries. ABSTRACT: Spatially localized Ca2+ signals activate Ca2+ -sensitive intermediate- and small-conductance K+ (IK and SK) channels in some vascular beds and endothelial nitric oxide synthase (eNOS) in others. The present study aimed to uncover the signalling organization that determines selective Ca2+ signal to vasodilatory target coupling in the endothelium. Resistance-sized mesenteric arteries (MAs) and pulmonary arteries (PAs) were used as prototypes for arteries with predominantly IK/SK channel- and eNOS-dependent vasodilatation, respectively. Ca2+ influx signals through endothelial transient receptor potential vanilloid 4 (TRPV4EC ) channels played an important role in controlling the baseline diameter of both MAs and PAs. TRPV4EC channel activity was similar in MAs and PAs. However, the TRPV4 channel agonist GSK1016790A (10 nm) selectively activated IK/SK channels in MAs and eNOS in PAs, revealing preferential TRPV4EC -IK/SK channel coupling in MAs and TRPV4EC -eNOS coupling in PAs. IK/SK channels co-localized with TRPV4EC channels at myoendothelial projections (MEPs) in MAs, although they lacked the spatial proximity necessary for their activation by TRPV4EC channels in PAs. Additionally, the presence of the NO scavenging protein haemoglobin α (Hbα) within nanometer proximity to eNOS limits TRPV4EC -eNOS signalling in MAs. By contrast, co-localization of TRPV4EC channels and eNOS at MEPs, and the absence of Hbα, favour TRPV4EC -eNOS coupling in PAs. Thus, our results reveal that differential spatial organization of signalling elements determines TRPV4EC -IK/SK vs. TRPV4EC -eNOS coupling in resistance arteries.

Zika Virus Infects Trabecular Meshwork and Causes Trabeculitis and Glaucomatous Pathology in Mouse Eyes.

Zika virus (ZIKV) infection during pregnancy leads to devastating fetal outcomes, including neurological (microcephaly) and ocular pathologies such as retinal lesions, optic nerve abnormalities, chorioretinal atrophy, and congenital glaucoma. Only clinical case reports have linked ZIKV infection to causing glaucoma, a major blinding eye disease. In the present study, we have investigated the role of ZIKV in glaucoma pathophysiology using in vitro and in vivo experimental models. We showed that human primary trabecular meshwork (Pr. TM) cells, as well as a human GTM3 cell line, were permissive to ZIKV infection. ZIKV induced the transcription of various genes expressing pattern recognition receptors (TLR2, TLR3, and RIG-I), cytokines/chemokines (TNF-α, IL-1ß, CCL5, and CXCL10), interferons (IFN-α2, IFN-ß1, and IFN-γ), and interferon-stimulated genes (ISG15 and OAS2) in Pr. TM cells. ZIKV infection in IFNAR1-/- and wild-type (WT) mouse eyes resulted in increased intraocular pressure (IOP) and the development of chorioretinal atrophy. Anterior chamber (AC) inoculation of ZIKV caused infectivity in iridocorneal angle and TM, leading to the death of TM cells in the mouse eyes. Moreover, anterior segment tissue of infected eyes exhibited increased expression of inflammatory mediators and interferons. Furthermore, ZIKV infection in IFNAR1-/- mice resulted in retinal ganglion cell (RGC) death and loss, coinciding with optic nerve infectivity and disruption of anterograde axonal transport. Because of similarity in glaucomatous pathologies in our study and other experimental glaucoma models, ZIKV infection can be used to study infectious triggers of glaucoma, currently an understudied area of investigation.IMPORTANCE Ocular complications due to ZIKV infection remains a major public health concern because of their ability to cause visual impairment or blindness. Most of the previous studies have shown ZIKV-induced ocular pathology in the posterior segment (i.e., retina) of the eye. However, some recent clinical reports from affected countries highlighted the importance of ZIKV in affecting the anterior segment of the eye and causing congenital glaucoma. Because glaucoma is the second leading cause of blindness worldwide, it is imperative to study ZIKV infection in causing glaucoma to identify potential targets for therapeutic intervention. In this study, we discovered that ZIKV permissively infects human TM cells and evokes inflammatory responses causing trabeculitis. Using a mouse model, we demonstrated that ZIKV infection resulted in higher IOP, increased RGC loss, and optic nerve abnormalities, the classical hallmarks of glaucoma. Collectively, our study provides new insights into ocular ZIKV infection resulting in glaucomatous pathology.

Transforming growth factor ß2 (TGFß2) signaling plays a key role in glucocorticoid-induced ocular hypertension.

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 ß (TGFß) signaling, leading to GC-induced ECM accumulation, ER stress, and IOP elevation. Dex increased both the precursor and bioactive forms of TGFß2 in conditioned medium and activated TGFß-induced SMAD signaling in primary human TM cells. Dex also activated TGFß2 in the aqueous humor and TM of a mouse model of Dex-induced ocular hypertension. We further show that Smad3-/- mice are protected from Dex-induced ocular hypertension, ER stress, and ECM accumulation. Moreover, treating WT mice with a selective TGFß 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 TGFß2 activation and ECM accumulation in TM cells. These observations suggested that chronic ER stress promotes Dex-induced ocular hypertension via TGFß signaling. Our results indicate that TGFß2 signaling plays a central role in GC-induced ocular hypertension and provides therapeutic targets for GC-induced ocular hypertension.

Glaucomatous cell derived matrices differentially modulate non-glaucomatous trabecular meshwork cellular behavior.

Ocular hypertension is a causal risk-factor to developing glaucoma. This is associated with stiffening of the trabecular meshwork (TM), the primary site of resistance to aqueous-humor-outflow. The mechanisms underlying this stiffening or how pathologic extracellular matrix (ECM) affects cell function are poorly understood. It is recognized that mechanotransduction systems allow cells to sense and translate the intrinsic biophysical properties of ECM into intracellular signals to control gene transcription, protein expression, and cell behavior. Using an anterior segment perfusion model, we document that there are significantly more low flow regions that are much stiffer, and fewer high flow regions that are less stiff in glaucomatous TM (GTM) when compared to non-glaucomatous TMs (NTM). GTM tissue also has fewer cells overall when compared with NTM tissue. In order to study the role of pathologic ECM in glaucoma disease progression, we conducted studies using cell derived matrices (CDM). First, we characterized the mechanics, composition and organization of fibronectin in ECM deposited by GTM and NTM cells treated with glucocorticosteroids. Then, we determined that these GTM-derived ECM are able to induce stiffening of normal NTM cells, and alter their gene/protein expression to resemble that of a glaucomatous phenotype. Further, we demonstrate that GTM-derived ECM causes endoplasmic reticular stress in NTM. They also became resistant to being reorganized by these NTM cells. These phenomena were exacerbated by ECMs obtained from steroid treated glaucoma model groups. Collectively, our data demonstrates that CDMs represent a novel tool for the study of bidirectional interactions between TM cells and their immediate microenvironment. STATEMENT OF SIGNIFICANCE: Extracellular matrix (ECM) changes are prevalent in a number of diseases. The precise mechanisms by which changes in the ECM contribute to disease progression is unclear, primarily due to absence of appropriate models. Here, using glaucoma as a disease model, we document changes in cell derived matrix (CDM) and tissue mechanics that contribute to the pathology. Subsequently, we determine the effect that ECMs from diseased and healthy individuals have on healthy cell behaviors. Data emanating from this study demonstrate that CDMs are a potent tool for the study of cell-ECM interactions.

Methods for Analyzing Endoplasmic Reticulum Stress in the Trabecular Meshwork of Glaucoma Models.

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. Recent studies have shown that progressive accumulation of misfolded proteins and induction of endoplasmic reticulum (ER) stress is associated with the pathophysiology of glaucomatous TM damage and IOP elevation. We have shown that known causes of human glaucoma, including expression of mutant myocilin or dexamethasone treatment induce abnormal protein accumulation and ER stress in the TM in vitro and in vivo models. To cope up with abnormal protein accumulation, TM cells activate a cytoprotective pathway of unfolded protein response (UPR). However, chronic ER stress can lead to TM dysfunction and IOP elevation. Using cell culture, mouse models, and human postmortem tissues as well as genetic and pharmacological manipulations, we have analyzed ER stress and UPR mediators in the glaucomatous TM damage and IOP elevation. In this chapter, we have described a detailed protocol for the analysis of protein misfolding and ER stress in TM cells and tissues and its association with glaucomatous TM damage and IOP elevation.

Increased synthesis and deposition of extracellular matrix proteins leads to endoplasmic reticulum stress in the trabecular meshwork.

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.