Fig. 4.

Laminar flow increases EETs and down-regulates sEH in ECs. BAECs in A, D, and E and HUVECs in C were kept as static controls or subjected to a laminar flow at 12 dyne/cm² for the indicated times. (A) Total lipids were extracted from static BAECs or those exposed to laminar flow in the absence of FBS. EETs were quantified by LC/MS/MS. The amounts of EETs were normalized to the mass of the cell pellets (in grams). (B) BAECs in 12-well plates were cotransfected with MH100×4-TK-Luc, GAL-mPPARγ-LBD, and CMV-Renilla-Luc. The transfected cells were then incubated with the condition media collected from the flow experiments in A for 8 h and lysed for luciferase activity assays. The results represent the relative luciferase activity defined as the normalized luciferase activity of various experiments in reference to that of static medium controls as 1. (C) Total RNA was isolated from HUVECs, and the level of sEH mRNA was determined by quantitative RT-PCR with β-actin used as an internal control. (D) BAECs were lysed for immunoblotting with the use of polyclonal anti-sEH Ab. (E) Cytosolic supernatants obtained from BAECs were incubated with trans-[³H]stilbene oxide for sEH activity assays. ^*, P < 0.05, compared with static controls.