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Jonathan Lautz, Cynthia Lynn Pervan, Evan B Stubbs; Transforming Growth Factor-β2 Attenuates Bradykinin B2 Receptor Expression in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3293.
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Primary open-angle glaucoma (POAG) is a progressive optic neuropathy associated with elevated 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. Despite recent advancements, the mechanism(s) by which TGF-β2 elevates IOP remains unknown. Bradykinin, a potent vasodilator, is currently under investigation as an alternative therapeutic strategy for the management of IOP in affected POAG patients. Here, we investigated the effects of TGF-β2 on bradykinin B2 receptor expression and signaling in primary and transformed human trabecular meshwork (TM) cells.
Primary or transformed human TM cells were conditioned overnight in serum-free media and incubated in the absence or presence of TGF-β2 (5 ng/ml). Canonical TGF-β2 signaling was blocked by siRNA targeted knockdown of Smad2 or Smad3. Non-canonical (Rho GTPase) signaling was blocked by pretreatment with Y-27632 or with C3 exoenzyme. Relative changes in B2 receptor mRNA and protein content were quantified by qRT-PCR and immunoblot, respectively.
In agreement with previously reported findings, porcine anterior segments chronically perfused with TGF-β2 exhibited a sustained increase in IOP compared to vehicle-treated matched segments. TGF-β2 dependent changes in bradykinin signaling were subsequently determined using cultured primary or transformed human TM cells. Quiescent TM cells expressed measurable levels of bradykinin B2 receptor mRNA and protein. In contrast, TM cells cultured overnight in the presence of TGF-β2 exhibited a marked 40-70% reduction in B2 receptor mRNA and protein expression, respectively. Interestingly, siRNA-targeted disruption of the canonical (Smad2, Smad3) signaling pathway prevented TGF-β2 mediated attenuation of B2 receptor expression in TM cells. In contrast, disrupting the non-canonical signaling pathway with either Y-27632 or with C3 exoenzyme did not prevent TGF-β2 mediated attenuation of B2 receptor expression in TM cells.
Constitutive expression of bradykinin B2 receptors in human TM cells is markedly attenuated in the presence of TGF-β2. Elevated content of TGF-β2 in the AH of POAG patients may elevate IOP, in part, by attenuating constitutive B2 receptor expression within the conventional outflow pathway.
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