Src mediates 15(S)-HETE–induced Egr-1 expression in HDMVECs, leading to their migration and tube-like structure formation. (A) Quiescent HDMVECs were treated with and without 15(S)-HETE (0.1μM) for the indicated time periods, and cell extracts were prepared and analyzed by Western blotting for pSrc with the use of its phosphospecific antibodies. The blot was reprobed with anti-Src antibodies for normalization. (B) HDMVECs were transduced with Ad-GFP or Ad-dnSrc at a moi of 40, quiesced, and treated with and without 15(S)-HETE (0.1μM) for either 30 minutes for RNA isolation or 30 minutes and 60 minutes for cell extract preparation. RNA was analyzed for Egr-1 and β- actin mRNA levels by QRT-PCR, and cell extracts were analyzed by Western blotting for Egr-1 with the use of its specific antibodies. The blot was reprobed sequentially with anti–β-tubulin antibodies and anti-Src antibodies for normalization and to show the overexpression of dnSrc, respectively. (C-D) HDMVECs that were transduced with Ad-GFP or Ad-dnSrc at a moi of 40 and quiesced were subjected to 15(S)-HETE (0.1μM)–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 in combination with and without Ad-GFP or Ad-dnSrc (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 vWF and CD31 expression by double immunofluorescence staining with their specific antibodies or analyzed for hemoglobin content using Drabkin reagent. The bar graphs in panels B, 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 Ad-GFP; **P < .01 vs Ad-GFP + 15(S)-HETE.
