Figure 3
HIV gp120 stimulate expression of genes associated with endothelial cell activation and constrictive mediators. (a) HIV gp120 induces genetic changes in pulmonary endothelial cells. HPAEC were cultured with 500 ng/mL of recombinant gp120-X4IIIb for 16 h. Endothelial gene expression was analyzed by using the Endothelial Cell Biology PCR array (Qiagen). Only statistically significant fold changes are shown. Genes with > twofold regulation are highlighted in gray. Note the fivefold increased expression of vasoconstrictive arachidonate 5-lipoxygenase (ALOX5). (b) Confirmation of gp120 X4-induced increased expression of pulmonary ALOX5 by Western blot. HPAEC were treated with media containing 500 ng/mL of either R5 or X4 gp120. The cells were grown for 72 h with media replacement every 24 h. The increased expression of ALOX5 was confirmed by immunoblotting, using actin as internal control. Densitometry analyses are shown after normalization with actin and mock samples. (c) ALOX5 inhibitor Zileuton is not toxic to endothelial cells. We tested different concentrations of the ALOX5 inhibitor Zileuton in cultured HPAEC, using apoptosis as a readout for cellular toxicity. Adherent HPAEC were treated with media containing 5–20 µM of Zileuton, incubated for 48 h and analyzed for expression of active caspase 3. No statistically significant differences were observed in activation of apoptosis among all treatments, compared to vehicle control. (d) Zileuton decreases HIV gp120-induced proliferation in HPAEC. We analyzed HPAEC proliferation in the presence of HIV gp120 with and without Zileuton for 48 h, using nuclear counts as readout. For all datasets: *p < 0.05 when compared to vehicle/mock control.
