Abstract
OBJECTIVE: Aqueous humour (AH) contains relatively high concentrations of vitamin C (ascorbate). AH drains out of the anterior chamber through the trabecular meshwork (TM) and, therefore, TM cells in vivo are routinely bathed in this antioxidant. Yet, most TM cells are cultured in vitro in media without ascorbate. In this study, we investigated molecules expressed by TM cells cultured with and without ascorbate. METHODS AND ANALYSIS: Non-glaucomatous TM cell strains were grown to confluence and placed in serum-free media with or without 0.1 mM ascorbate for 3 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were used to investigate protein levels, and some bands were analysed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) mass spectrometry. RNA was isolated and gene expression was quantified using fluorescent barcode technology. Following data normalisation, bioinformatics software was used to identify differentially expressed genes (DEGs). Quantitative PCR was used to validate results. RESULTS: In ascorbate-treated cells, western immunoblotting showed that several collagen chains were properly modified and secreted and LC-MS/MS identified proteins. Gene expression showed that 76 genes were significantly upregulated, and 20 genes were downregulated. Not surprisingly, antioxidative stress genes (SOD1, PRDX1, TXN2) were upregulated. Several genes involved in collagen synthesis, extracellular matrix assembly and remodelling (HSP47, MMP2, TIMP2, SERPINE1, CDH5, NCAM1, ITGA4) were also upregulated, but ITGB3 was downregulated. Genes involved with vascular endothelium growth factor (VEGF) (VEGFA, VEGFB), transforming growth factor β (SMAD2, SMAD4) and Hedgehog (PTCH1, GLI1) signalling pathways were also significantly altered by ascorbate treatment. Interestingly, many DEGs have no currently assigned role in ascorbate response. CONCLUSIONS: This study revealed several collagens and 96 genes that are regulated by ascorbate in TM cells. Our results establish the importance of including ascorbate in culture media during the in vitro culture of TM cells. Further analysis of the function of these genes may improve our understanding of the importance of ascorbate for TM cell health.