Hypoxia increases α_v-integrin mRNA expression.

(A) hMVECs were cultured for 16 hours in normoxic (N) and hypoxic (H) conditions in M199 supplemented with 10% HS and not stimulated (control) or stimulated with 10 ng/mL each FGF-2+TNF-α. After the incubation period, total RNA was isolated and analyzed by Northern blotting using α-³²P CTP-labeled probes for α_v-integrin, β₅-integrin, and actin. (B) The cell adhesion assay was performed as described in “Materials and methods.” hMVECs were allowed to adhere to vitronectin for 90 minutes in the presence of control antibody 10 μg/mL anti-FITC (fluorescein isothiocyanate), 10 μg/mL LM609, 10 μg/mL P1F6, or a combination of 10 μg/mL each LM609 and P1F6. The data are expressed as the mean percentage of the control plus or minus SD. Each condition was performed 4-fold. The different groups were compared by an analysis of variance (ANOVA). The asterisk indicates P < .001, which is significantly different from control, and the number sign indicatesP = .005, which is significantly different from P1F6. (C) hMVECs were cultured on top of a 3-dimensional fibrin matrix in M199 supplemented with 10% HS and 10% NBCS under normoxic or hypoxic culture conditions and were stimulated with 10 ng/mL each FGF-2+TNF-α (control) alone or in the presence of 10 μg/mL of the α_vβ₃-blocking mAb LM609, the α_vβ₅-blocking mAb P1F6, or a combination of these antibodies (LM609/P1F6). After 3 days of culture, the mean tube length (mm/cm²) was measured as described. The effect of LM609 and P1F6 is expressed as the mean percentage of FGF-2+TNF-α–stimulated cells plus or minus the range of 2 independent experiments performed in duplicate wells.