Effects of sCD40L on activation of MAPKs in HCAECs. (A) MAPK p38 and ERK2 phosphorylation. HCAECs were treated with sCD40L for different time points and the phosphorylation levels of MAPK p38 and ERK2 were determined by Bio-Plex luminex immunoassay with a commercial kit. U test. (B) Effects of MAPK inhibitors on eNOS mRNA levels. HCAECs were treated with sCD40L in the presence or absence of p38 inhibitor (SB239036) or ERK1/2 inhibitor (PD98059) for 24 hours, and eNOS mRNA levels were determined by real-time PCR analysis. t test. (C) Effect of p38 inhibitor (SB239036) on eNOS protein levels. HCAECs were treated with sCD40L in the presence or absence of different concentrations of p38 inhibitor (SB239036) for 24 hours, and eNOS protein levels were determined by Western blot. (D) Effect of ERK1/2 inhibitor (PD98059) on eNOS protein levels. HCAECs were treated with sCD40L in the presence or absence of different concentrations of ERK1/2 inhibitor (PD98059) for 24 hours, and eNOS protein levels were determined by Western blot. (E) Effects of sCD40L, MAPK inhibitors, and mitochondrial complex II inhibitor TTFA on O₂⁻ production (DHE staining). HCAECs were treated with sCD40L monomer form or sCD40L trimer form in the presence or absence of MAPK inhibitors (p38 and ERK2) or TTFA for 24 hours. O₂⁻ production was determined by DHE staining and flow cytometric analysis. (F) Effects of recombinant adenovirus–based gene delivery on eNOS protein levels. HCAECs were infected with different recombinant adenoviruses for 48 hours, and followed by sCD40L treatment for 24 hours. Ad-LacZ was included as a negative control. eNOS mRNA levels were determined by Western blot analysis. **P < .01 compared with the control. #P < .05 compared with sCD40L treatment. n = 3. Data are means and SE of multiple experiments (n).