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Jonathan Lautz, Evan B Stubbs; Elevated hydrostatic pressure selectively alters TGF-β2, ET-1, and CTGF gene expression in human trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3486.
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Primary open-angle glaucoma (POAG) is a progressive optic neuropathy characterized by loss of peripheral vision secondarily associated with elevated IOP. Transforming growth factor (TGF)-β2 is markedly elevated in the AH of patients with POAG. We and others have previously shown that TGF-β2 increases IOP, in part, by inducing expression and release of endothelin-1 (ET-1) and connective tissue growth factor (CTGF) within the trabecular meshwork (TM). ET-1 and CTGF may ultimately lead to increases in IOP by enhancing TM cell contractility and ECM deposition. Despite these advancements, the direct effect of elevated IOP on TM cell responsiveness remains unknown. Here we determined whether elevated hydrostatic pressure (HP) can selectively alter gene expression in a transformed human TM cell line.
Confluent cultures of transformed human TM cells were serum-starved x 24h and subsequently cultured in the absence (ambient) or presence of elevated HP (15, 30, or 45 mm Hg above ambient) for up to 24h. Changes in the expression and/or release of TGF-β2, ET-1, CTGF, and select ECM constituents were quantified by qRT-PCR, Western immunoblot, or ELISA. Cell viability and integrity was assessed by quantifying MTT reduction and LDH release.
Transformed human TM cells exposed to elevated HP exhibited a transient increase in TGF-β2 and ET-1 mRNA expression and protein secretion compared to ambient controls. Exposing cells to elevated HP for up to 12h did not significantly affect cell viability, as quantified by LDH release or MTT reduction. In contrast, elevated HP elicited a sustained increase in CTGF mRNA and protein secretion over 24h and elicited a time-dependent change in select ECM gene expression. By comparison, elevated HP had no effect on the expression of 14 other genes implicated in the pathogenesis of POAG. Modest changes in HPs, as low as 15 mm Hg, were found to elicit significant changes in TGF-β2, ET-1, and CTGF mRNA expression.
Cultured human TM cells exposed to elevated HP exhibit marked and selective changes in the expression of genes associated with the pathogenesis of POAG. We propose that pressure-dependent changes in TM cell gene expression represent a feed-forward mechanism that exacerbates TGF-β2 associated increases in TM cell contractility and altered ECM synthesis and deposition in affected POAG patients.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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