by Christina K. Ullrich, Jerome E. Groopman, and Ramesh K. Ganju HIV-1 gp120- and gp160-induced apoptosis in cultured endothelial cells is mediated by caspases by Christina K. Ullrich, Jerome E. Groopman, and Ramesh K. Ganju Blood Volume 96(4):1438-1442 August 15, 2000 ©2000 by American Society of Hematology

HIV-1 gp120/160 induce endothelial apoptosis in a time- and concentration-dependent manner.HUVEC were grown in LSM (0.5%), as described in “Materials and methods” and were treated with HIV-1 gp120 or gp160 for different time periods (A) or at different conc... HIV-1 gp120/160 induce endothelial apoptosis in a time- and concentration-dependent manner.HUVEC were grown in LSM (0.5%), as described in “Materials and methods” and were treated with HIV-1 gp120 or gp160 for different time periods (A) or at different concentrations (B). Cell samples were analyzed for apoptosis using the photometric sandwich ELISA to detect cytoplasmic nucleosome degradation. The fold increase in nucleosome degradation was calculated by comparing the optical density (OD) values of the gp120/160-treated cells with those of the untreated HUVEC. *P < .0005. Christina K. Ullrich et al. Blood 2000;96:1438-1442 ©2000 by American Society of Hematology

HIV-1 gp120/160-induced HUVEC apoptosis as detected by TUNEL assay HIV-1 gp120/160-induced HUVEC apoptosis as detected by TUNEL assay.HUVEC grown in LSM were untreated (A) or treated with gp120 (1000 ng/mL) (B) or gp160 (1000 ng/mL) (C) for 10 hours; gp120-treated (1000 ng/mL) HUVEC at a higher magnification are represente... HIV-1 gp120/160-induced HUVEC apoptosis as detected by TUNEL assay.HUVEC grown in LSM were untreated (A) or treated with gp120 (1000 ng/mL) (B) or gp160 (1000 ng/mL) (C) for 10 hours; gp120-treated (1000 ng/mL) HUVEC at a higher magnification are represented in (D). Samples were analyzed for apoptosis using the TUNEL method. Green fluorescent cells represent apoptotic cells. Christina K. Ullrich et al. Blood 2000;96:1438-1442 ©2000 by American Society of Hematology

HIV-1 gp120/160-induced HUVEC apoptosis is inhibited by anti-CXCR4 receptor antibody.HUVEC grown in LSM were untreated (control) or treated with gp120 (100 ng/mL) or gp160 (100 ng/mL) for 10 hours in the presence of the CXCR4 neutralizing antibody 12G5 (6 μ... HIV-1 gp120/160-induced HUVEC apoptosis is inhibited by anti-CXCR4 receptor antibody.HUVEC grown in LSM were untreated (control) or treated with gp120 (100 ng/mL) or gp160 (100 ng/mL) for 10 hours in the presence of the CXCR4 neutralizing antibody 12G5 (6 μg/mL) or the class-matched control IgG (6 μg/mL). Cell samples were analyzed for apoptosis by nucleosome ELISA. cAb, control antibody. *P < .036; **P < .003. Christina K. Ullrich et al. Blood 2000;96:1438-1442 ©2000 by American Society of Hematology

Inhibition of HUVEC apoptosis by caspase inhibitors Inhibition of HUVEC apoptosis by caspase inhibitors.HUVEC grown in LSM were untreated (control) or treated with gp120 or gp160 at 100 ng/mL each for 10 hours in the presence or absence of the caspase pathway inhibitor (I), Z-VAD-FMK, at a 40-μmol/L concentr... Inhibition of HUVEC apoptosis by caspase inhibitors.HUVEC grown in LSM were untreated (control) or treated with gp120 or gp160 at 100 ng/mL each for 10 hours in the presence or absence of the caspase pathway inhibitor (I), Z-VAD-FMK, at a 40-μmol/L concentration. In addition, gp120-treated cells were incubated with the inhibitor control (IC), Z-FA-FMK, at a 40-μmol/L concentration. Apoptosis was measured using ELISA. *P < .0007; **P < .011. Christina K. Ullrich et al. Blood 2000;96:1438-1442 ©2000 by American Society of Hematology

Activation of caspase-3 in HUVEC by HIV-1 gp120/160 Activation of caspase-3 in HUVEC by HIV-1 gp120/160.HUVEC were grown in LSM with gp120 or gp160 at a 100-ng/mL concentration for 6 hours. Activation of caspase-3 in HUVEC by HIV-1 gp120/160.HUVEC were grown in LSM with gp120 or gp160 at a 100-ng/mL concentration for 6 hours. Cells were lysed in low-detergent buffer, and the lysates were analyzed for caspase-3 activity using a specific substrate (AC-DEVD-AFC) as described in “Materials and methods.” The release of AFC was measured using a fluorometer setting of 450-nm excitation and 505-nm emission. *P < .0007. Christina K. Ullrich et al. Blood 2000;96:1438-1442 ©2000 by American Society of Hematology

Induction of Bax expression by HIV-1 gp120/160 treatment Induction of Bax expression by HIV-1 gp120/160 treatment.Cell lysates (100 μg) from HUVEC, prepared as described in “Materials and methods,” untreated or treated with gp120 or gp160 (100 ng/mL) for 1 hour or 3 hours, were resolved on 15% SDS-PAGE and blotte... Induction of Bax expression by HIV-1 gp120/160 treatment.Cell lysates (100 μg) from HUVEC, prepared as described in “Materials and methods,” untreated or treated with gp120 or gp160 (100 ng/mL) for 1 hour or 3 hours, were resolved on 15% SDS-PAGE and blotted with anti-Bax antibody. Christina K. Ullrich et al. Blood 2000;96:1438-1442 ©2000 by American Society of Hematology