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Yuzhen Pan, Binoy Appukuttan, Kathleen Mohs, Justine R. Smith; UCHL1 Promotes Proliferation of Human Retinal and Choroidal Endothelial Cells In Vitro. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3007.
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In a published gene expression microarray study (JR Smith et al, Invest Ophthalmol Vis Sci, 2007), we identified UCHL1 expression by cultured human choroidal and retinal endothelial cells. Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) is a deubiquitinating enzyme that counteracts VHL gene-driven ubiquitination of hypoxia-inducible factor (HIF)-1alpha, stabilizing HIF-1alpha. These observations led us to investigate the potential role of UCHL1 in human ocular angiogenesis.
70% ethanol-fixed human retina and choroid from 3 human cadaver eyes were indirectly immunostained with rabbit anti-human UCHL1 antibody (Cedar Lanes, 1:5000 dilution) to examine in vivo expression of UCHL1. Endothelial proliferation assays were performed using retinal and choroidal endothelial cells, isolated from additional human cadaveric eyes by established methods and immortalized by transduction with the murine recombinant amphotropic retrovirus, LXSN16E6E7, which encodes HPV E6 and E7 oncogenes. When no less than 80% confluent, cells were transfected with UCHL1-targeted or non-targeted small interfering (si)RNA, designed in-house and synthesized by Invitrogen, using Targefect (Targeting Systems), according to the manufacturer’s instructions. Subsequently transfected endothelial cells were incubated at 37 °C and 3.5% CO2 in MCDB-131 medium with 5-10% FBS and endothelial growth factors, and growth was evaluated by CyQUANT Cell Proliferation Assay (Invitrogen) 96 hours later.
Immunohistochemistry confirmed expression of UCHL1 by vascular endothelium in intact retina and choroid. siRNA treatment targeting UCHL1 resulted in significant reduction in endothelial proliferation when compared to treatment with non-targeted siRNA for choroidal (p ≤ 0.008, n= 3 independent donors) and retinal (p ≤ 0.004, n= 3 independent donors) endothelial cells. Results were confirmed for both cell types on one additional donor using siRNA targeting a different section of transcript sequence. Successful UCHL1 knock-down with UCHL1-targeted siRNA was confirmed by Western blot for all experiments, using protein harvested 48 hours after transfection.
UCHL1 protein is expressed by retinal and choroidal endothelium in vivo. Targeted knock-down of UCHL1 has an anti-proliferative effect on retinal and choroidal endothelial cells in vitro. Our results suggest that targeting UCHL1 may have therapeutic benefit for neovascular eye diseases, including diabetic retinopathy and age-related macular degeneration.
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