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Jonathan Lautz, Cynthia Lynn Pervan, Evan B Stubbs; TGF-β2 Perturbation of the Kallikrein-Kinin System in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4698. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In healthy eyes, intraocular pressure (IOP) is maintained through balanced production and outflow of aqueous humor (AH). Increased resistance to AH outflow is a major contributor of aberrant elevation of IOP. TGF-β2, a pro-fibrotic cytokine, is markedly elevated in AH of patients with POAG. The mechanism by which TGF-β2 promotes pathological elevation of IOP remains elusive, but may involve perturbation of the kallikrein-kinin system. Bradykinin (BK), a key mediator of the kinin system and a potent vasodilator, is produced locally within the eye. BK receptors subtypes have similarly been localized to ocular tissues. Activation of the BK B2-receptor lowers IOP in ocular hypertensive non-human primates. Here, we investigated the relationship between TGF-β2 and BK 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. Rho GTPase signaling was blocked by pretreatment with exoenzyme C3 transferase. De novo transcription was inhibited by co-treatment with actinomycin D. Relative changes in BK receptor mRNA and protein content were quantified by qRT-PCR and immunoblot, respectively.
Porcine anterior segments chronically perfused with TGF-β2 exhibited a sustained increase in IOP compared to vehicle-treated matched segments. Quiescent TM cells expressed measurable levels of bradykinin receptor mRNA and protein. TM cells cultured in the presence of TGF-β2 exhibited a marked reduction in BK receptor expression in a time dependent manner. siRNA-targeted knockdown of Smad3, but not Smad2, prevented TGF-β2 mediated attenuation of BK receptor mRNA and protein expression. In contrast, disrupting the non-canonical Rho GTPase signaling pathway with exoenzyme C3 transferase had no effect on TGF-β2 mediated attenuation of BK receptor expression. TGF-β2 mediated decreases in BK mRNA content were not altered in the presence of actinomycin D.
Constitutive expression of bradykinin receptors in human TM cells is markedly attenuated by 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.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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