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Padmanabhan Pattabiraman, Rupalatha Maddala, Vasanth Rao, ; Regulation of Cell Plasticity and Fibrogenic Activity in Trabecular Meshwork by Rho/Rho kinase Signaling in the Context of Aqueous Humor Outflow Homeostasis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):378.
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© ARVO (1962-2015); The Authors (2016-present)
To test the hypothesis that abnormal activation of Rho/Rho kinase signaling in cells of the aqueous humor outflow pathway may trigger an endothelial to mesenchymal transition (EndMT)-dependent formation of matrix producing myofibroblasts and fibrogenic activity, events which in turn result in impairment of aqueous humor outflow.
Human primary trabecular meshwork (TM) cell cultures were transfected with plasmids expressing constitutively active RhoA (RhoAV14) or transcription factor MRTF-A, or transduced with adenoviral RhoAV14 and GFP. TM cells were treated separately with lysophosphatidic acid (LPA), TGF-β2, connective tissue growth factor (CTGF), Rho kinase inhibitor (Y-27632) or anti-fibrotic agent- Pirfenidone. Q-PCR analysis was performed to evaluate fibrogenic activity related changes in gene expression. Quantification of SMA, collagen 1 (Col1), fibroblast-specific protein (FSP1) and secreted total collagen was performed by immunoblotting, immunofluorescence and the Sircol assay.
Overexpression of RhoAV14 or MRTF-A in serum starved human TM cells resulted in significant increases (>2 fold) in expression of genes involved in fibrosis including Twist1, Slug, FSP1, Col1, miRNA29a and SMA. A significant increase in SMA, FSP1, Col1 and secreted collagen was recorded in TM cells expressing RhoAV14 and cells treated with LPA, TGFβ2 or CTGF (n=4; P<0.05). Inhibition of the transcriptional activity of serum response factor (SRF) using CCG-1423 or knocking down SRF expression, led to a significant decrease in the RhoAV14, LPA and TGF beta-induced increases in the protein levels of SMA, FSP1 and Col1 in TM cells. Finally, Y27632 and Pirfenidone significantly inhibited TGFβ2 induced increases in the protein levels of SMA, FSP1, Col1 and secreted Collagen in TM cells.
Collectively, these observations reveal an important role for the Rho/Rho kinase pathway in induction of TM cell plasticity/ transdifferentiation into matrix and SMA producing myofibroblasts, and identify several significant external cues (TGF-beta, CTGF, LPA) and transcriptional regulators (MRTF, SRF, miRNA-29, Slug, Twist) which control the fibrogenic activity of TM cells via Rho/Rho kinase signaling. These molecular events could be partly responsible for the RhoAV14-induced ocular hypertension in the rat model.
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