Cytotoxic effect of HIV-1 gp120 on primary cultured human retinal capillary endothelial cells.
OBJECTIVE
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.
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Haotian
H
Lin
Weirong
W
Chen
Lixia
L
Luo
Changrui
C
Wu
Qilin
Q
Wang
Yizhi
Y
Liu
D000328:Adult; D017209:Apoptosis / Q000187:drug effects*; D053453:Caspase 9 / Q000096:biosynthesis; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000494:pharmacology*; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D061251:Primary Cell Culture; D019713:Receptors, CCR5 / Q000096:biosynthesis; D019718:Receptors, CXCR4 / Q000096:biosynthesis; D012160:Retina / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism
D015699:HIV Envelope Protein gp120; D019713:Receptors, CCR5; D019718:Receptors, CXCR4; D053453:Caspase 9