Purpose: This research was conducted to make a primary culture of human retinal capillary endothelial cells (HRCEC) and to study the cytotoxic effect of human immunodeficiency virus-1 (envelope) glycoprotein 120 (HIV-1 gp120) on cultured HRCEC.
Methods: HRCEC were isolated and primarily cultured as dissociated single cell cultures. Immunohistochemistry and immunofluorescence were used to identify specific markers of HRCEC and to reveal HIV-1 gp120 related receptors (cluster of differentiation 4 [CD4], C-X-C chemokine receptor type 4 [CXCR4], and C-C chemokine receptor type 5 [CCR5]). The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay was used to demonstrate the effect of HIV-1 gp120 on cell viability at seven different concentrations (0.01-0.15 mg/l) for 24 h or at a fixed concentration of 0.08 mg/l for varying time intervals (4-72 h). After 0.08, 0.1, 0.12, and 0.15 mg/l HIV-1 gp120 were applied to HRCEC for 24 h, cell apoptotic rates and the mitochondrial membrane potential were measured with flow cytometry; pro-caspase-9 and cleaved caspase-9 were evaluated with immunoblotting. Under each research condition, 0.15 mg/l of HIV-1 gp120 mutated proteins (423 I/P) were used as controls.
Results: Primary cultures of pure HRCEC were established, and the cells were characterized with their specific markers. HIV-1 gp120 receptors CXCR4 and CCR5 were found on the cell surface of HRCEC; however, CD4 was negative. Treatment of HRCEC with HIV-1 gp120 at concentrations <0.08 mg/l did not influence cell viability. However, a concentration- and time-dependent increase of HIV-1 gp120-induced cell inhibition was demonstrated with MTT, when the concentration of HIV-1 gp120 was more than 0.08 mg/l (r=-0.763, p<0.01). With increasing concentrations of HIV-1 gp120, the numbers of apoptotic cells and expression of cleaved caspase-9 protein increased, but Rho123 staining mitochondrial membrane potential decreased.
Conclusions: HIV-1 gp120 assistant receptors CXCR4 and CCR5 are expressed on the cell surface of HRCEC, and HIV-1 gp120 can inhibit cell viability and induce apoptosis of HRCEC. The mitochondrial pathway is probably involved in HIV-1 gp120-induced apoptosis of HRCEC, but the specific mechanisms remain to be uncovered.