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Cynthia Lynn Pervan, Jonathan D Lautz, Andrea L Blitzer, Evan B Stubbs; Constitutive Expression and Release of TGF-β2 by Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3295.
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© ARVO (1962-2015); The Authors (2016-present)
Irreversible loss of peripheral vision in patients with primary open-angle glaucoma (POAG) is closely associated with increased intraocular pressure (IOP). Although the cause of POAG remains unclear, elevated levels of TGF-β2 in aqueous humor (AH) of affected patients is strongly implicated in the pathophysiology of enhanced AH outflow resistance. However, the mechanism responsible for, and the cellular origin of, elevated TGF-β2 in AH remains poorly defined. Here, we investigated whether human TM cells endogenously express and secrete biologically-active TGF-β2.
Primary or transformed (GTM3) human TM cells were incubated x24h in the absence or presence of chemically-activated lovastatin (10 µM), GGTI-298 (an inhibitor of geranylgeranyl transferase-I; 20 µM), or exoenzyme C3 transferase (10 µM). Constitutive TGF-β2 mRNA or biologically-active protein secretion was quantified by qRT-PCR or ELISA analysis, respectively. Stabilized porcine anterior segments (n = 5 pairs) were perfused in the absence or presence of SB-431542 (a TGFβRI/ALK-5 antagonist; 1 µM) and IOP quantified every 3 minutes for up to 48h.
Quiescent GTM3 cells expressed measurable quantities of TGF-β2 mRNA while constitutively secreting biologically-active TGF-β2 into the culture medium. Endogenous TGF-β2 expression was markedly reduced by lovastatin, as compared to vehicle controls. Incubation with GGTI-298 mimicked the effect of lovastatin in both primary and transformed human TM cells, eliciting a 60-70% decrease in TGF-β2 mRNA content and a 70-100% reduction in secreted active TGF-β2 protein. Incubation with C3 exoenzyme resulted in a 90% reduction of TGF-β2 mRNA content, and a 40% attenuation of active TGF-β2 secretion, strongly implicating a role for the Rho GTPase subfamily (RhoA/B/C) in regulating constitutive TGF-β2 expression. Importantly, perfusing hypertensive porcine anterior segments with SB-505124 reduced, by 50%, IOP by 15h post-infusion.
We demonstrate for the first time that human TM cells endogenously express and secrete biologically-active TGF-β2. Constitutive expression/release of TGF-β2 in human TM cells is regulated, in part, by the Rho subfamily GTPase signaling pathways. We propose that human TM cells serve as a novel local source of functionally active TGF-β2.
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