Egr-1 mediates 15(S)-HETE–induced HDMVEC migration and tube-like structure formation in vitro and Matrigel plug angiogenesis in vivo. (A-B) Quiescent human dermal microvascular endothelial cells (HDMVECs) were treated with and without 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE; 0.1μM) for the indicated time periods, and either RNA was isolated or cell extracts were prepared. (A) RNA was analyzed by QRT-PCR for Egr-1 and β-actin mRNA levels. (B) Cell extracts were analyzed by Western blotting for Egr-1 levels by the use of its specific antibodies. The blot was reprobed with anti–β-tubulin antibodies for normalization. (C-D) HDMVECs were transduced with Ad-GFP or Ad-dnEgr-1 at a moi of 40, quiesced, and subjected to 15(S)-HETE–induced migration (C) or tube-like structure formation (D). (E) C57BL/6 mice were injected subcutaneously with 0.5 mL of Matrigel premixed with vehicle or 5μM 15(S)-HETE along with and without Ad-GFP or Ad-dnEgr-1 (5 × 109 pfu/mL). One week later, the animals were sacrificed, and the Matrigel plugs were harvested from underneath the skin and either processed for von Willebrand Factor (vWF) and CD31 expression by double immunofluorescence staining with their specific antibodies or analyzed for hemoglobin content with Drabkin reagent. The bar graphs in panels A, C, D, and E represent the quantitative analysis of 3 independent experiments or 6 plugs from 6 animals. The values are presented as the mean ± SD. *P < .01 vs control or Ad-GFP; **P < .01 vs Ad-GFP + 15(S)-HETE.
