Role Of HES1 Regulating Extracellular Matrix In Human Trabecular Meshwork Cells Under Oxidative Stress

Purpose:In primary open-angle glaucoma（POAG）,elevated intraocular pressure（IOP）is regarded as the critical risk factor of disease progression.Trabecular meshwork（TM）is the main structure that maintains normal IOP by regulating the aqueous humor（AH）outflow.The excessive accumulation of extraceluar matrix（ECM）within the TM is a primary pathophysiological process associated with elevated IOP in POAG.Oxidative damage is considered as an important step in the pathogenesis of POAG and contributed to the ECM accumulation.Hairy and enhancer of split-1（HES1）is a protein that belongs to the basic helix–loop–helix family of transcription factors and is a direct downstream regulatory node of Notch signaling-mediated fibrotic diseases.Considering that HES1 exhibits several characteristics directly relevant to abnormal ECM deposition,we hypothesized that the activation of HES1 in the TM may accelerate the fibrotic ECM accumulation under oxidative stress.Methods:1.Primary HTMCs were incubated with different H2O2 concentrations（0,100,200,300,400,600,800,and 1000μM）.Cell viability was examined by CCK-8 counting assay.ECM（fibronectin,collagen I,and laminin）expression was assessed through Western blot after treated with 300μM H₂O₂ at different stimulation times.2.HTMCs were incubated with 300μM H₂O₂ for 2 h to induce oxidative stress.The expression of profibrotic ECM were analyzed through Western blot and Immunofluorescence.Cell migration and proliferation were examined through chemotaxis and CCK-8 counting assay.HES1 expression was analyzed through Western blot and qPCR.3.RNAi and Lentivirus Packaging were used to produce the HES1 shRNA lentivirus.The effects of HES1 knockdown on HES1 and ECM expression were assessed through qPCR and Western blot.Cell functions were examined as previous.4.A lentivirus expressing the HES1 was used to infected HTMCs.The expression of HES1 and ECM in HES1-overexpressing and control HTMCs was analyzed through qPCR and Western blot.Immunofluorescence was used to further detect the cytoplasmic profibrotic ECM.Cell functions were examined as previous.Results:1.Different H₂O₂ concentrations showed a different reduction in cell viability.Low concentration of H₂O₂ caused a moderate decrease,especially 300μM.While high concentration of H2O2 could cause a significant reduction.The expression level of ECM showed no gradually increased with increasing H₂O₂ stimulation times.Only treated HTMCs for 2 h led to a significant increase in the ECM expression.2.HTMCs were incubated with 300μM H₂O₂ for 2 h to induce oxidative stress.The H₂O₂-treated cells exhibited a significantly higher level of profibrotic ECM proteins than the control cells did.Immunofluorescence further revealed that the cytoplasmic ECM accumulated.H2O2 stimulation led to a decrease in the number of cells.And the HES1 expression was upregulated after treated with H2O2.3.The HTMCs stably infected with HES1shRNA/Scramble were treated with H₂O₂.The expression levels of HES1 and ECM of the H₂O₂-treated groups were higher than those of the H₂O₂-untreated groups.HTMCs pretreated with HES1 shRNA exhibited a great decrease in H₂O₂-induced increase in profibrotic ECM proteins.Immunofluorescence analysis further showed the same effects.And the HTMCs yielded high rates of chemotactic migration and proliferation.4.HTMCs were infected with the control/HES1 p.cDNA lentivirus to assess HES1 in ECM regulation.The HES1-overexpression significantly upregulated the HES1expression in the multiple profibrotic ECM proteins.The mRNA levels also varied.The same effects were also shown through immunofluorescence analysis.Furthermore,the number of proliferating and migrating HTMCs were decreased.Conclusions:1.HTMCs were incubated with 300μM H₂O₂ for 2 h to induce the oxidative stress.2.HES1 could serve as an important regulator of profibrotic ECM production.3.HES1 may provide a thought for researching the pathophysiological mechanism of glaucoma.