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Navita Lopez, Abbot F Clark, Tara Tovar-Vidales; Epigenetic regulation of optic nerve head fibrosis in glaucoma. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5668. doi: https://doi.org/.
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Expression of the pro-fibrotic cytokine transforming growth factor β2 (TGFβ2) is elevated in the glaucomatous optic nerve head (ONH) and is therefore implicated in remodeling of extracellular matrix (ECM) proteins seen in the glaucomatous ONH. Previous research suggests that miR-29c-3p and miR-200b-3p are anti-fibrotic miRNAs and regulate the synthesis of ECM proteins. We hypothesize that increased TGFβ2 signaling downregulates anti-fibrotic miRNAs, stimulating a fibrotic response and ONH remodeling in glaucoma.
Non-glaucomatous primary human lamina cribrosa (LC) cells and ONH astrocytes (ONHA) were grown to 100% confluency and treated with TGFβ2 (5ng/ml) or control for 24 hours. Differences in expression of miRNAs were analyzed by miRNA qPCR array. LC cells and ONHA were transfected with miR-29c-3p and miR-200b-3p (mimics or inhibitors) and analyzed by qPCR to confirm overexpression or knockdown of these miRNAs. mRNA targets of miR-29c-3p and miR-200b-3p were determined through protein expression analysis by immunocytochemistry. The effects of miR-29c-3p and miR-200b-3p on TGFβ2 induced fibronectin (FN), collagen (COL) types I and IV protein expression were evaluated by immunocytochemistry.
miRNA PCR arrays showed that TGFβ2 treatment downregulated the expression of miR-29c-3p in LC cells (n=3) and downregulated miR-200b-3p in ONHA (n=3). Transfection of miR-29c-3p and miR-200b-3p mimics or inhibitors caused upregulation or downregulation of these miRNAs. Immunocytochemistry analysis showed that miR-29c-3p regulated the expression of COL I and IV in LC cells. miR200b-3p regulated the expression of FN and COL IV in ONHA. Overexpression of miR-29c-3p decreased TGFβ2 induced COL I and IV expression. Overexpression of miR-200b-3p decreased TGFβ2 induced FN and COL IV expression. Inhibition of miR-29c-3p and miR-200b-3p exacerbated the effects of TGFβ2 on collagen and FN expression.
TGFβ2 induced the downregulation of miR-29c-3p and miR-200b-3p, anti-fibrotic miRNAs, which may stimulate a pro-fibrotic environment and pathogenic remodeling of the ONH. The inhibitory effects of miR-29c-3p and miR-200b-3p on TGFβ2 induced FN, COL I and COL IV, suggest that these miRNAs regulate ECM protein synthesis.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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