Effect of Hevin Deletion in Mice and Characterization in Trabecular Meshwork

The etiology of primary open angle glaucoma, a leading cause of age-related blindness, remains poorly defined, although elevated intraocular pressure (IOP) contributes to the disease progression. To better understand the mechanisms causing elevated IOP from aqueous humor circulation, we pursued proteomic analyses of trabecular meshwork (TM) from glaucoma and age-matched control donors. These analyses demonstrated that Cochlin, a protein associated with deafness disorder DFNA9, is present in glaucomatous but absent in normal TM. Cochlin was also detected in TM from the glaucomatous DBA/2J mouse preceding elevated IOP but found to be absent in three other mouse lines that do not develop elevated IOP. Histochemical analyses revealed co-deposits of Cochlin and mucopolysaccharide in human TM around Schlemm's canal, similar to that observed in the cochlea in DFNA9 deafness. Purified Cochlin was found to aggregate after sheer stress and to induce the aggregation of TM cells in vitro. Age-dependent in vivo increases in Cochlin were observed in glaucomatous TM, concomitant with a decrease in type II collagen, suggesting that Cochlin may disrupt the TM architecture and render components like collagen more susceptible to degradation and collapse. Overall, these observations suggest that Cochlin contributes to elevated IOP in primary open angle glaucoma through altered interactions within the TM extracellular matrix, resulting in cell aggregation, mucopolysaccharide deposition, and significant obstruction of the aqueous humor circulation.

Lentiviral Vector-Mediated Expression of Exoenzyme C3 Transferase Lowers Intraocular Pressure in Monkeys.

Primary open-angle glaucoma (POAG) is the most common form of glaucoma and is accompanied by elevated intraocular pressure (IOP) resulting from increased aqueous humor outflow resistance through the trabecular meshwork (TM). The pathological mechanisms underlying increased outflow resistance have not been fully delineated. We recently demonstrated that chronic endoplasmic reticulum (ER) stress in the TM is associated with ocular hypertension in mouse models of glaucoma. The purpose of this study was to determine whether ER stress is also increased in human glaucomatous TM cells and tissues. Endoplasmic reticulum stress markers including GRP78, GRP94, and C/EBP homologous protein (CHOP) were examined by immunohistochemistry in the TM of age-matched normal (n = 18) and open-angle glaucoma donors (n = 18). GRP78, GRP94, activating transcription factor (ATF)-4, endoplasmic oxidoreductin-1alpha (ERO-1α), phosphorylated eukaryotic translation initiation factor 2α (EIF-2α), and CHOP were examined by Western blot analysis in TM tissue lysates from age-matched normal (n = 4) and POAG donors (n = 5). In addition, ER stress markers were examined in primary TM cells isolated from normal (n = 4 NTM) and glaucoma (n = 4 GTM) human donors. Immunohistochemical analysis demonstrated a significant increase in GRP78 and GRP94 in the glaucomatous TM (n = 18) compared to normal TM (P < 0.0001, n = 18). Interestingly, there was minimum CHOP immunostaining observed in normal TM tissues. However, there was a 3-fold increase in CHOP levels in the glaucomatous TM (P < 0.0001; n = 18), indicating the presence of chronic ER stress in the glaucomatous TM. Western blot analysis of TM tissue lysates also demonstrated increased ER stress markers in the glaucomatous TM tissues including GRP78, GRP94, ATF-4, ERO-1α, and CHOP. Densitometric analysis of Western blots showed a significant increase in ATF-4, ERO-1α, and CHOP expression in the glaucomatous TM (n = 5) compared to age-matched normal TM (n = 4). In addition, primary TM cells obtained from glaucoma donors demonstrated increased ER stress markers including increased GRP78, GRP94, ATF-4, ERO-1α, and CHOP compared to normal TM cells. However, glaucomatous TM cells did not show splicing of XBP-1, a marker of unfolded protein response pathway. These studies indicate the presence of chronic ER stress in human glaucomatous TM tissues and cells and further suggest that ER stress pathway may provide a novel target for developing disease-modifying glaucoma treatments.

To compare the gene expression profile of trabecular meshwork (TM) and Schlemm's canal (SC) primary cultures and to identify promoters for targeting gene expression to specific cells in the outflow pathway. The differential gene expression profile of four human TM and three SC primary cultures was analyzed by gene microarrays (Affymetrix, Santa Clara, CA) and confirmed by quantitative real-time PCR. Based on the results, a recombinant adenovirus was constructed with the expression of the reporter gene LacZ driven by the 5' promoter region of the chitinase 3-like 1 (Ch3L1) gene (AdCh3L1-LacZ). The expression of the Ch3L1 promoter was analyzed in human TM and SC cells and in human perfused anterior segments infected with AdCh3L1-LacZ. gamma-Sarcoglycan, fibulin-2, and collagen XV were identified as the genes more highly expressed in SC than in TM cells. Ch3L1 showed the highest levels of differential expression in TM versus SC cells. Expression analysis of the Ch3L1 promoter demonstrated specific expression in a subset of the TM cells in cell culture and in perfused anterior segments. Comparative analysis of gene expression between SC and TM primary cultures identified several genes with promoters potentially capable of targeting gene expression to specific cells within the outflow pathway. Results with the Ch3L1 promoter indicated that two different cell subtypes may be present in the TM. This study provides a new potential tool to investigate the role of these different cell types in both normal and pathophysiological function of the outflow pathway, with implications for possible future glaucoma gene therapy.

PurposeInflammatory responses may be involved in the glaucomatous process. Our previous studies mapped a T104M mutation in interleukin-20 receptor beta (IL-20RB) in a family with primary open angle glaucoma (POAG). IL-20RB can heterodimerize with IL-20RA to propagate signals from IL-20 family cytokines, IL-19, IL-20, and IL-24 (the type I receptor complex), or it can heterodimerize with IL-22RA to propagate signals from IL-20 and IL-24 (type II receptor complex). In this study, we investigated IL-20 heterodimeric receptor complexes in the trabecular meshwork (TM) compared to dermal fibroblast cell cultures, and examined the phosphorylation of signal transducer and activator of transcription (STAT)-1, -3, and -5 following exposure to IL-20 family cytokines. Additionally, we determined the effects of IL-20 family cytokines on outflow rates in anterior segment perfusion culture, an in vitro model of intraocular pressure (IOP) regulation.MethodsPrimary human TM (HTM) cells were grown from dissected TM tissue, and IL-20 receptor expression was investigated with PCR. A Duolink assay was performed to investigate in situ IL-20 receptor protein interactions in HTM or dermal fibroblasts, and Imaris software was used to quantitate the association of the heterodimeric complexes. Phosphorylation of STAT-1, -3, and -5 were evaluated in HTM or dermal fibroblasts using Western immunoblotting after exposure to IL-10, IL-19, IL-20, IL-22, or IL-24. Anterior segment perfusion culture was performed in human cadaver and porcine eyes treated with IL-20, IL-19, or IL-24.ResultsAll of the IL-20 receptors, IL-20RA, IL-20RB, and IL-22RA1 were expressed in HTM cells. Two isoforms of IL-20RA were expressed: The V1 variant, which is the longest, is the predominant isoform, while the V3 isoform, which lacks exon 3, was also expressed. The Duolink assay demonstrated that the type I (IL-20RA–IL-20RB) and type II (IL-22RA1–IL-20RB) receptors were expressed in HTM cells and dermal fibroblasts. However, in the HTM cells, the type I receptor was present at significantly higher levels, while the type II receptor was preferentially used in the dermal fibroblasts. The HTM cells and the dermal fibroblasts predominantly phosphorylate the Ser727 site in STAT-3. The dermal fibroblasts had higher induction of phosphorylated STAT-1 compared to the HTM cells, while neither cell type had phosphorylated STAT-5 in the cell lysates. The outflow rates in the human anterior segment cultures were increased 2.3-fold by IL-20. However, IL-19 and IL-24 showed differential responses. For IL-19 and IL-24, 50% of the eyes responded with a 1.7- or 1.5-fold increase, respectively, while the other half did not respond. Similarly, perfused porcine anterior segments showed “responders” and “non-responders”: IL-20 responders (2.3-fold increase in outflow, n=12) and non-responders (n=11); IL-19 responders (2.1-fold increase, n=7) and non-responders (n=5); and IL-24 responders (1.8-fold increase, n=12) and non-responders (n=5).ConclusionsType I and type II IL-20 receptor complexes are expressed in human TM cells with predominant expression of the type I receptor (IL-20RA and IL-20RB), which propagates signals from all three IL-20 family cytokines. However, there was a variable response in the outflow rates following perfusion of cytokines in two different species. This may explain why some people are more susceptible to developing elevated IOP in response to inflammation.

In order to identify how differential gene expression in the trabecular meshwork (TM) contributes to racial disparities of caveolar protein expression, TM dysfunction and development of primary open angle glaucoma (POAG), RNA sequencing was performed to compare TM tissue obtained from White and Black POAG surgical (trabeculectomy) specimens. Healthy donor TM tissue from White and Black donors was analyzed by PCR, qPCR, immunohistochemistry staining, and Western blot to evaluate SDPR (serum deprivation protein response; Cavin 2) and CAV1/CAV2 (Caveolin 1/Caveolin 2). Standard transmission electron microscopy (TEM) and immunogold labeled studies were performed. RNA sequencing demonstrated reduced SDPR expression in TM from Black vs White POAG patients’ surgical specimens, with no significant expression differences in other caveolae-associated genes, confirmed by qPCR analysis. No racial differences in SDPR gene expression were noted in healthy donor tissue by PCR analysis, but there was greater expression as compared to specimens from patients with glaucoma. Analysis of SDPR protein expression confirmed specific expression in the TM regions, but not in adjacent tissues. TEM studies of TM specimens from healthy donors did not demonstrate any racial differences in caveolar morphology, but a significant reduction of caveolae with normal morphology and immuno-gold staining of SDPR were noted in glaucomatous TM as compared to TM from healthy donors. Linkage of SDPR expression levels in TM, POAG development, and caveolar ultrastructural morphology may provide the basis for a novel pathway of exploration of the pathologic mechanisms of glaucoma. Differential gene expression of SDPR in TM from Black vs White subjects with glaucoma may further our understanding of the important public health implications of the racial disparities of this blinding disease.

BackgroundA traditional Chinese medicine, Tetramethylpyrazine (TMP), has been prescribed as a complementary treatment for glaucoma to improve patient prognosis. However, the pharmacological mechanism of action of TMP is poorly understood. In previous studies, we demonstrated that TMP exerts potent inhibitory effects on neovascularization, suppresses the tumorigenic behavior of glioma cells, and protects neural cells by regulating CXCR4 expression. Here, we further investigated whether the SDF-1/CXCR4 pathway is also involved in the TMP-mediated activity in trabecular meshwork cells.Methodology/Principal FindingsCXCR4 expression was examined by quantitative real-time PCR in trabecular and iris specimens from 54 primary open-angle glaucoma (POAG) patients who required surgery and 19 non-glaucomatous donors. Our data revealed markedly elevated CXCR4 expression in the trabecular meshwork of POAG patients compared with that of controls. Consistently, CXCR4 expression was much higher in glaucomatous trabecular meshwork cells than in normal trabecular meshwork cells. Using RT-PCR and western blot assays, we determined that glaucoma-related cytokines and dexamethasone (DEX) also significantly up-regulated CXCR4 expression in primary human trabecular meshwork (PHTM) cells. Moreover, the TGF-β1-mediated induction of CXCR4 expression in PHTM cells was markedly down-regulated by TMP compared with control treatment (PBS) and the CXCR4 antagonist AMD3100. In addition, TMP could counteract the TGF-β1-induced effects on stress fiber accumulation and expansion of PHTM cells. TMP markedly suppressed the migration of PHTM cells stimulated by TGF-β1 in transwell and scratch wound assays. TMP also suppressed the extracellular matrix (ECM) accumulation induced by TGF-β2.ConclusionsOur findings demonstrate that CXCR4 might be involved in the pathogenetic changes in the trabecular meshwork of patients with POAG. Additionally, TMP might exert its beneficial effects in POAG patients by down-regulating CXCR4 expression.

To examine the applicability of TAT (the protein transduction domain of transactivating transcription polypeptide)-mediated protein-transduction technology, in introducing proteins of interest into trabecular meshwork (TM) cells in various culture systems. Normal human TM cell cultures, human tissues in organ cultures, and bovine eyes in perfusion organ cultures were incubated or perfused for various lengths of time with TAT- and hemagglutinin (HA)-tagged fusion proteins, TAT-HA-beta-galactosidase (TAT-HA-beta-gal), TAT-HA-myocilin, and TAT-HA-myocilin-EGFP. Transduction of TAT-HA-beta-gal was detected by X-gal staining. Transduction of myocilin or myocilin-EGFP was evaluated by immunostaining or fluorescence. beta-Gal and EGFP proteins were used as the negative control. Blue X-gal staining, signifying beta-gal activity resulting from transduction, was observed in cultured TM cells in a concentration- and time-dependent manner. TAT-HA-beta-gal was also transduced into cells in all regions of TM tissues in organ cultures. TM cell cultures, after TAT-HA-myocilin incubation, showed an enhanced myocilin staining compared with the control cultures. Stronger myocilin or HA staining was also noted in TM tissues of TAT-HA-myocilin-incubated or -perfused eyes. Myocilin transduction resulted in a loss of actin stress fibers and focal adhesions in TM cells in culture. The level of phosphorylated myosin light chain was reduced. Human and bovine TM tissues after TAT-myocilin transduction also exhibited a diminished actin and paxillin-vinculin staining. TAT fusion proteins can be efficiently transduced into TM cells and tissues. The TAT-mediated protein transduction technology may be valuable in studies of proteins such as myocilin in the TM.

A Myosin Light Chain Kinase Inhibitor, ML-9, Lowers the Intraocular Pressure in Rabbit Eyes

We examined ultrastructurally the localization of myocilin (formerly called trabecular meshwork inducible glucocorticoid response, or TIGR) protein in cultured human trabecular meshwork (TM) cells and in normal human TM tissues. The TM, a specialized tissue located at the chamber angle of the eye, is believed to be responsible for the development of glaucoma. The myocilin gene has been directly linked to both juvenile and primary open-angle glaucomas, and multiple mutations have been identified. Human TM cells were treated with 0.1 mM of dexamethasone (DEX) to induce myocilin expression. This protein was immunolocalized by colloidal gold electron microscopy using an anti-human myocilin polyclonal antibody. Double labeling with different sizes of gold particles was also performed with additional monoclonal antibodies specific for cell organelles and structures. In both DEX-treated and untreated cultured cells, myocilin was associated with mitochondria, cytoplasmic filaments, and vesicles. In TM tissues, myocilin was localized to mitochondria and cytoplasmic filaments of TM cells, elastic-like fibers in trabecular beams, and extracellular matrices in the juxtacanalicular region. These results indicate that myocilin is localized both intracellularly and extracellularly at multiple sites. This protein may exert diverse biological functions at different sites.

Expression of the kallikrein/kinin system in human anterior segment

IntroductionGlaucoma, a leading cause of irreversible blindness, is characterized by optic neuropathy and retinopathy, with primary open-angle glaucoma (POAG) being the most prevalent form. The primary pathogenic mechanism of POAG involves elevated intraocular pressure caused by chronic fibrosis of the trabecular meshwork (TM). Autophagy, a critical process for maintaining cellular homeostasis, has been implicated in fibrosis across various organs. However, its precise role in the fibrosis associated with POAG pathogenesis remains unclear. This study investigates the involvement of autophagy in TM fibrosis and explores its potential impact on POAG development, aiming to provide insights into new therapeutic targets.MethodsTo assess autophagy activity and its relationship with fibrosis, we analyzed TM tissues from POAG patients and healthy donors. Autophagic activity in human TM tissues was measured through immunohistochemical analyses. An in vitro aging model using chronic H2O2 treatment was established to investigate the change of fibrosis in TM cells. Additionally, we used dexamethasone-treated TM cells as a POAG model to explore the role of autophagy in fibrotic progression. The involvement of the TGF-β2/Smad signaling pathway was investigated through western blot analysis and quantitative real-time PCR.ResultsThis study reveals increased autophagic activity in tissues from POAG patients and an age-related upregulation of autophagy in healthy human TM tissues. In the H2O2-induced aging model, TM cells displayed both elevated autophagic activity and fibrosis. Further investigation showed that enhanced autophagy activity promoted fibrotic progression via activation of the TGF-β2/Smad signaling pathway. Similarly, in the dexamethasone-treated TM cell model, autophagy was found to exacerbate fibrosis, aligning with observations in the aging model.DiscussionIn this study, we uncover the interplay between autophagy and the TGF-β2/Smad pathway in the pathogenesis of POAG. We observed increased autophagic activity in TM tissues from POAG patients and in TM tissues of aging healthy individuals. In human primary TM cells, we confirmed that autophagy becomes activated in the context of cellular senescence and the development of POAG, which further facilitates fibrotic progression via the TGF-β2/Smad signaling pathway. These findings underscore the important role of autophagy in POAG pathogenesis and confirm senescence as a pivotal risk factor.

PurposeContinuous reductions in trabecular meshwork (TM) cellularity inhibit aqueous humor (AH) outflow, which is the main cause of primary open-angle glaucoma. Rho-associated protein kinase inhibitor (ROCKi) targets the TM to reduce intraocular pressure (IOP) and increase AH outflow facility. However, the underlying mechanisms are not entirely clear. Here, we aimed to investigate the effect of a ROCKi (Y-27632) on TM cell proliferation and phagocytosis.MethodsImmortalized human TM (iHTM) cells, glaucomatous TM (GTM3) cells, and primary human TM (pTM) cells were cultured and identified. The effects of various concentrations of Y-27632 on F-actin cytoskeleton were assessed using immunofluorescence. Cell proliferation effects were evaluated using a cell counting kit-8 (CCK8), cell counting, and Ki67 immunostaining. Cell phagocytosis was evaluated using immunofluorescence and flow cytometry in immortalized TM cells. C57BL/6J and Tg-MYOCY437H mice were used to investigate the proliferative effects of Y-27632 on TM cells in vivo. The effect of Y-27632 on IOP was monitored for 2 weeks, and the outflow facility was detected 2 weeks after IOP measurement. TM cells in mice were counted using immunohistochemistry.ResultsY-27632 (100 μM) significantly promoted the proliferation of both immortal TM cells and pTM cells. In GTM3 cells, phagocytosis was significantly greater in the Y-27632 group than in the control group, nearly reaching the level of phagocytosis in iHTM, as determined using immunofluorescence and flow cytometry. In Tg-MYOCY437H mice, treatment with Y-27632 significantly decreased IOP and increased outflow facility, which greatly influenced the long-term IOP-lowering effect. The number of TM cells in Tg-MYOCY437H mice was significantly improved after Y-27632 administration.ConclusionY-27632 promoted cell proliferation and phagocytosis of TM cells, and its proliferative effect was demonstrated in a transgenic mouse model. These results revealed a new IOP-lowering mechanism of Y-27632 through effects on TM cells, suggesting the potential for a correlation between TM cellularity and long-term recovery of IOP.

The matricellular protein SPARC is expressed in human trabecular meshwork

Intercellular adherens junctions and cell-extracellular matrix interactions are presumed to influence aqueous humor (AH) drainage via the conventional route, however, their direct role in modulation of intraocular pressure (IOP) is not well understood. Here, we investigated the role of Rac GTPase signaling in basal and growth factor-induced formation of adherens junctions in human trabecular meshwork (HTM) cells as compared to human umbilical vascular endothelial cells, and evaluated the effects of inhibition of Rac GTPase activity on IOP in rabbits. Expression of a constitutively active Rac1 GTPase or treatment with platelet derived growth factor (PDGF), a known activator of Rac GTPase, induced formation of β-catenin-based adherens junctions, actin cytoskeletal reorganization and membrane ruffle in HTM cells. In contrast, treatment of HTM cells with inhibitors of Rac GTPase caused cell-cell separation, a decrease in adherens junctions, and reorganization of actin stress fibers to the cell cortical regions and focal adhesion to the cell leading edges. Both, constitutively active Rac1 and PDGF stimulated generation of Reactive Oxygen Species (ROS) in HTM cells, and ROS were found to increase adherens junction formation and transendothelial electrical resistance (TEER) in HTM cells. Topical application of Rac GTPase inhibitors (EHT1864 and NSC23766), however, only marginally influenced IOP in rabbit eyes. Taken together, these data reveal that while Rac GTPase signaling plays a significant role in regulation of adherens junctions, ROS production and TEER in cells of the AH outflow pathway, Rac inhibitors showed only a marginal influence on IOP in live rabbits.