PURPOSE. Lysophosphatidic acid (LPA) and soluble interleukin-6 receptor (sIL6R) are elevated in primary open angle glaucoma (POAG). LPA and IL6 modulate in response to biomechanical stimuli and converge on similar fibrotic phenotypes. Thus, we determined whether LPA and IL6 trans-signaling (IL6/sIL6R) interact via Yes-associated protein (YAP)/Transcriptional coactivator with a PDZ-binding motif (TAZ) or Signal transducer and activator of transcription 3 (STAT3) pathways in human trabecular meshwork (hTM) cells. METHODS. Confluent primary hTM cells were serum starved for 24 hours, and treated with vehicle, LPA (20 μM), IL6 (100 ng/mL)/sIL6R (200 ng/mL), or both (LPA + IL6/sIL6R) for 24 hours, with or without a YAP inhibitor (verteporfin; 2 μM) or STAT3 inhibitor (2 μM). Expression of key receptors and ligands, signaling mediators, actomyosin machinery, cell contractility, and extracellular matrix (ECM) targets of both signaling pathways was determined by immunocytochemistry, RT-qPCR, and Western blotting. RESULTS. LPA and IL6 trans-signaling coupling overexpressed/activated receptors and ligands, glycoprotein-130, IL6, and autotaxin; signaling mediators, YAP, TAZ, Pan-TEAD, and phosphorylated STAT3 (pSTAT3); actomyosin and contractile machinery components, myosin light chain 2 (MLC2), phosphorylated MLC2, rho-associated protein kinase 1, filamentous actin, and α-smooth muscle actin; and fibrotic ECM proteins, collagen I and IV, fibronectin, laminin, cysteine-rich angiogenic inducer 61, and connective tissue growth factor in hTM cells; mostly beyond LPA or IL6 trans-signaling alone. Verteporfin inhibited YAP, TAZ, and pSTAT3, with concomitant abrogation of aforementioned fibrotic targets; the STAT3 inhibitor was only partially effective. CONCLUSIONS. These data suggest synergistic crosstalk between LPA and IL6 transsignaling, mediated by YAP, TAZ, and pSTAT3. By completely inhibiting these mediators, verteporfin may be more efficacious in ameliorating LPA and/or IL6 trans-signalinginduced ocular hypertensive phenotypes in hTM cells.

BackgroundThere seems a preponderance of hospital-based studies on the prevalence of Allergic Conjunctivitis (AC) compared to community-based ones, particularly among children in Ghana and Africa as a whole. Meanwhile, literature supports the possibility of underdiagnosing AC in the hospital setting; exponentially so when males generally have poor hospital-attending behavior. This may lead to underestimation of the true burden of AC. Consequently, the purpose of the current community-based study was to determine the prevalence of AC among basic school children in the Kumasi Metropolis, while identifying its associated symptoms.MethodsA cross-sectional community-based study involving 1571 students from 11 basic schools (Primary and JHS) participated in the study. Data collection started in November 2011 and was completed in March 2014. After history taking, subjects underwent a battery of tests; visual acuity, objective refraction, anterior and posterior segments examination with a slit-lamp and a direct ophthalmoscope respectively.ResultsThe prevalence of AC was 39.9 %. The mean (±SD) age of participants was 8 ± 0.65 years. AC was significantly associated with gender (p < 0.05), but not with age (p > 0.05). A total of 70 % of the students with AC never had any form of treatment.ConclusionsAC is an endemic ocular disease among basic schools in the Kumasi metropolis and therefore calls for pragmatic and proactive measures to reduce its burden and effects on its victims. Public health measures may be required to help reduce the burden associated with this condition.

Purpose The purpose of this study was to determine whether genipin-induced crosslinked cell-derived matrix (XCDM) precipitates fibrotic phenotypes in human trabecular meshwork (hTM) cells by dysregulating β-catenin and Yes-associated protein (YAP)/ transcriptional coactivator with PDZ-binding motif (TAZ) signaling pathways. Methods Cell-derived matrices were treated with control or genipin for 5 hours to obtain respective uncrosslinked (CDM) and XCDMs and characterized. hTM cells were seeded on these matrices with/without Wnt pathway modulators in serum-free media for 24 hours. Elastic modulus, gene, and protein (whole cell and subcellular fractions) expressions of signaling mediators and targets of Wnt/β-catenin and YAP/TAZ pathways were determined. Results At the highest genipin concentration (10% XCDM), XCDM had increased immunostaining of N-ε(γ-glutamyl)-lysine crosslinks, appeared morphologically fused, and was stiffer (5.3-fold, P < 0.001). On 10% XCDM, hTM cells were 7.8-fold ( P < 0.001) stiffer, total β-catenin was unchanged, pβ-catenin was elevated, and pGSK3β was suppressed. Although 10% XCDM had no effect on cytoplasmic β-catenin levels, it reduced nuclear β-catenin, cadherin 11, and key Wnt target genes/proteins. The 10% XCDM increased total TAZ, decreased pTAZ, and increased cytoplasmic TAZ levels in hTM cells. The 10% XCDM increased total YAP, reduced nuclear YAP levels, and critical YAP/TAZ target genes/proteins. Wnt activation rescued hTM cells from 10% XCDM-induced stiffening associated with increased nuclear β-catenin. Conclusions Increased cytoplasmic TAZ may inhibit β-catenin from its nuclear shuttling or regulating cadherin 11 important for aqueous homeostasis. Elevated cytoplasmic TAZ may inhibit YAP's probable homeostatic function in the nucleus. Together, TAZ's cytoplasmic localization may be an important downstream event of how increased TM extracellular matrix (ECM) crosslinking may cause increased stiffness and ocular hypertension in vivo. However, Wnt pathway activation may ameliorate ocular hypertensive phenotypes induced by crosslinked ECM.

Ocular hypertension has been attributed to increased resistance to aqueous outflow often as a result of changes in trabecular meshwork (TM) extracellular matrix (ECM) using in vivo animal models (for example, by genetic manipulation) and ex vivo anterior segment perfusion organ cultures. These are, however, complex and difficult in dissecting molecular mechanisms and interactions. In vitro approaches to mimic the underlying substrate exist by manipulating either ECM topography, mechanics, or chemistry. These models best investigate the role of individual ECM protein(s) and/or substrate property, and thus do not recapitulate the multi-factorial extracellular microenvironment; hence, mitigating its physiological relevance for mechanistic studies. Cell-derived matrices (CDMs), however, are capable of presenting a 3Dmicroenvironment rich in topography, chemistry, and whose mechanics can be tuned to better represent the network of native ECM constituents in vivo. Critically, the composition of CDMs may also be fine-tuned by addition of small molecules or relevant bioactive factors to mimic homeostasis or pathology. Here, we first provide a streamlined protocol for generating CDMs from TM cell cultures from normal or glaucomatous donor tissues. Second, we document how TM cells can be pharmacologically manipulated to obtain glucocorticoid-induced CDMs and how generated pristine CDMs can be manipulated with reagents like genipin. Finally, we summarize how CDMs may be used in mechanistic studies and discuss their probable application in future TM regenerative studies.

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