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M. N. Mehta, E. Chou, E. Ahmed, W. Pfeifer, N. Rahimi, D. Husain; Novel Role of Phospholipase C 1 in Choroidal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3346.
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Phospholipase Cγ1 (PLCγ1) is considered a major substrate of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) and has been found to mediate endothelial cell proliferation and tube formation in cell culture systems. Recent study demonstrates that Casitas B-lineage lymphoma (c-Cbl) ubiquitinates PLCγ1 and suppresses its activation. The purpose of this study is to address the biological importance of c-Cbl dependent inhibition of PLCγ1 in choroidal neovascularization.
Experimental Choroidal Neovascularization (CNV) was created using a laser injury model (Argon laser, 50 micron spot size, 200 mW, 0.1 sec) in wild and c-Cbl knockout mice eyes. CNV formation was recorded using serial fundus fluorecein angiography with a Topcon digital angiography camera and Imagenet system. The eyes were enucleated, frozen in liquid nitrogen and embedded in OCT. Wild type mice lung, kidney and heart tissue were used as positive controls, frozen sections of which were stained with hematoxylin-eosin (HE), anti- CD 31 (staining endothelial cells) and anti- phospho PLCγ1. Three dilutions of anti-CD31 antibody and anti-phospho PLCγ1 antibody (1:200, 1:500, 1:750) were used to obtain an optimal dilution for histochemical staining. Frozen sections of wild mice eyes, c-Cbl knockout mice eyes (both with CNV) and wild mice normal eyes without CNV were then stained with HE, anti-CD31 (1:750) and anti-phospho PLCγ1 antibody (1:750). The histological characteristics on HE staining and staining patterns with anti-CD 31 and anti-phospho PLCγ1 were compared between normal wild mice eyes, wild mice eyes with CNV and c-Cbl knockout mice eyes with CNV.
On HE staining, we observed histologic features of focal pigmentary hypertrophy extending into the retina at the site of laser induced neovascularization in both wild type mice and c-Cbl knockout mice. We observed phospho PLCγ1 positive staining in experimental CNV in both wild and c-Cbl knockout mice eyes. There was significant up-regulation of anti-phospho PLCγ1 positivity in c-Cbl knockout mice eyes with CNV.
PLCγ1 plays an important role in laser-induced choroidal neovascularization. Targeting this molecule may provide a novel method for treatment of diseases caused by CNV such as wet macular degeneration.
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