STAT-5B mediates 15(S)-HETE–induced HRMVEC migration and tube formation in vitro and Matrigel plug angiogenesis in vivo. (A) Quiescent HRMVECs were treated with and without 15(S)-HETE (0.1 μM) for the indicated time periods, and cell extracts were prepared. An equal amount of protein from control and 15(S)-HETE–treated HRMVECs was immunoprecipitated with anti–STAT-5B antibodies, and the resulting immunocomplexes were analyzed by Western blotting using phosphospecific anti–STAT-5 antibodies. The blot was reprobed with anti–STAT-5B antibodies for normalization. (B,C) HRMVECs were transduced with Ad-GFP or Ad-dnSTAT-5B at 80 MOI, quiesced, and subjected to 15(S)-HETE–induced migration (B) or tube formation (C). (D) C57BL/6 mice were injected subcutaneously with 0.5 mL of Matrigel premixed with vehicle or 5 μM 15(S)-HETE in combination with Ad-GFP or Ad-dnSTAT-5B (5 × 109 pfu/mL). One week later, the animals were killed and the Matrigel plugs were harvested from underneath the skin, and cryosections were either made and immunostained for CD31 using anti-CD31 antibodies or analyzed for hemoglobin content using Drabkin reagent. The bar graphs in panels A through D represent the mean ± SD values of 3 independent experiments or 6 plugs from 6 animals. *P < .01 versus control or Ad-GFP; **P < .01 versus Ad-GFP + 15(S)-HETE.
