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P.S. Mettu, P.F. Deng, J. Kumar, D.L. Epstein, P.V. Rao; Role of Lysophospholipid Growth Factors and Edg Receptors in the Regulation of Aqueous Humor Outflow Facility . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1179.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate the role of the lysophospholipid growth factors, lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P), in the regulation of aqueous humor outflow through the trabecular meshwork (TM). Methods: Expression profile of Endothelial differentiation gene (Edg) family of G-protein coupled receptors was determined by primer-specific PCR analysis of cDNA libraries and by RT-PCR for Edg isoforms 1-5. Effects of LPA and S1P on the human trabecular meshwork (HTM) cell actin cytoskeleton were determined under serum starvation. LPA- and S1P-induced effects on HTM cell myosin light chain (MLC) phosphorylation were evaluated by Western blot analysis. Pull-down assays were used to evaluate activation of Rho GTPase by LPA and S1P. Effects of each factor on aqueous humor outflow facility were evaluated by perfusion of enucleated porcine eyes with LPA or S1P using the standard constant-pressure Grant system. Results: Each of the Edg isoforms 1, 2, 3, and 4 were readily detectable in libraries prepared from SC cells and HTM cells. Expression of Edg-2 was also confirmed in HTM tissue. Both LPA (20 µM) and S1P (1 µM) caused increased actin stress fiber and focal adhesion formation, but S1P appeared to cause a marked increase in cortical actin fibers and cell contraction. While LPA-induced cytoskeletal alterations in HTM cells were markedly inhibited by the Rho kinase-specific inhibitor Y-27632 (5 µM), S1P-mediated cytoskeletal effects were partially inhibited. Furthermore, S1P-induced cytoskeletal alterations were also only partially sensitive to inhibitors of PKC (GF-109203X) and MLC kinase (ML-7). Both LPA (20 µM) and S1P (1 µM) increased MLC phosphorylation and demonstrated marked activation of Rho GTPase in HTM cells. Perfusion of LPA (20 µM) and S1P (5 µM) in a thus far limited number of porcine eyes appears to lower the aqueous humor outflow facility by 14% (n=10) and 30% (n=2), respectively. The effects of LPA and S1P on calcium mobilization in HTM cells and on HTM tissue contractility are being investigated. Conclusions: These studies confirm the expression of Edg receptors in HTM and SC cells and in HTM tissue and demonstrate that activation of these receptors by LPA and S1P, their physiologic agonists, influences cytoskeletal organization, cell-ECM interactions, and contractile properties of HTM cells through MLC phosphorylation. Importantly, activation of Edg receptors by LPA and S1P in perfused porcine eyes seems to decrease outflow facility suggesting a potential role for modulation of aqueous humor outflow facility.
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