Hypoxia promotes functional endothelial cell surface expression of A2. (A) Immunoblot of cell surface biotinylated A2 and p11 on HUVECs treated with hypoxia (0.5% O2, 16 hours). Pan-cadherin served as the loading control. (B) Flow cytometric analysis of HUVEC surface A2 and VE-cadherin 5 after normoxia (21% O2, 16 hours; blue histograms) or hypoxia (0.5% O2, 16 hours; red histograms). Isotype-matched IgG served as control (shaded histogram). (C) Immunoblot showing total cellular HIF-1α, total A2, Tyr 416 phospho-Src, total Src, p11, and actin after hypoxia (0.5% O2, 16 hours). Phospho-A2 was captured by immunoprecipitation, followed by immunoblot analysis. (D) Anxa2+/+ and Anxa2−/− CMEC-associated plasmin generation for cells maintained in 21% versus 0.5% O2 (0.22 ± 0.014 vs 0.404 ± 0.005 vs 0.13 ± 0.01 vs 0.11 ± 0.007; n = 6; **P < .01; NS indicates no significant difference). (E) Migration of Anxa2+/+ (8.35% ± 0.074% vs 18.3% ± 1.36%) and Anxa2−/− (3.9% ± 0.34% vs 4.4% ± 0.5%) CMECs maintained in 21% or 0.5% O2 (16 hours, n = 3; **P < .01). (F) Immunoblot of surface and intracellular A2 and p11 in Anxa2−/− CMECs 48 hours after infection with either empty or A2-encoding viruses. Pan-cadherin and actin served as surface and intracellular protein loading controls, respectively. (G) Anxa2−/− CMEC-associated plasmin generation after infection with empty (V; 0.195 ± 0.014 RFU/min2 ) or A2-encoding virus (A2; 0.312 ± 0.025 RFU/min2 ; 48 hours, n = 3; *P < .05, **P < .01). (H) Migration of Anxa2−/− CMECs infected with either empty (V; 2.6% ± 0.14%) or A2-encoding virus (A2; 5.8% ± 0.23%; 48 hours, n = 3; *P < .05, **P < .01). The data are representative of 3 independent experiments (A-H).
