Soluble CD40 ligand induces endothelial dysfunction in human and porcine coronary artery endothelial cells

Abstract

The purpose of this study was to determine the effects and mechanisms of sCD40L on endothelial dysfunction in both human coronary artery endothelial cells (HCAECs) and porcine coronary artery rings. HCAECs treated with sCD40L showed significant reductions of endothelial nitric oxide synthase (eNOS) mRNA and protein levels, eNOS mRNA stability, eNOS enzyme activity, and cellular NO levels, whereas superoxide anion (O(2)(-)) production was significantly increased. sCD40L enhanced eNOS mRNA 3'UTR binding to cytoplasmic molecules and induced a unique expression pattern of 95 microRNAs. sCD40L significantly decreased mitochondrial membrane potential, and catalase and SOD activities, whereas it increased NADPH oxidase (NOX) activity. sCD40L increased phosphorylation of MAPKs p38 and ERK1/2 as well as IkappaBalpha and enhanced NF-kappaB nuclear translocation. In porcine coronary arteries, sCD40L significantly decreased endothelium-dependent vasorelaxation and eNOS mRNA levels, whereas it increased O(2)(-) levels. Antioxidant seleno-l-methionine; chemical inhibitors of p38, ERK1/2, and mitochondrial complex II; as well as dominant negative mutant forms of IkappaBalpha and NOX4 effectively blocked sCD40L-induced eNOS down-regulation in HCAECs. Thus, sCD40L reduces eNOS levels, whereas it increases oxidative stress through the unique molecular mechanisms involving eNOS mRNA stability, 3'UTR-binding molecules, microRNAs, mitochondrial function, ROS-related enzymes, p38, ERK1/2, and NF-kappaB signal pathways in endothelial cells.

Figures

Figure 1

Effects of sCD40L on eNOS mRNA levels in HCAECs. (A) Concentration-dependent study. HCAECs were treated with different concentrations of sCD40L for 24 hours. The eNOS levels were determined by real-time PCR. Heat-inactivated (HI) sCD40L was included as a negative control. (B) Time course study. HCAECs were treated with sCD40L (5 μg/mL) for different times. The eNOS levels were determined by real-time PCR. (C) Effect of anti-CD40L antibody and (D) effect of anti-CD40 antibody. HCAECs were pretreated with different concentrations of anti-CD40L antibody or anti-CD40 antibody for 30 minutes and followed with sCD40L treatment for 24 hours. The eNOS mRNA levels were determined by real-time PCR. Isotype IgG was used for a negative control. (E) eNOS mRNA stability. HCAECs were treated with actinomycin D (2.5 μg/mL) in the presence or absence of sCD40L (5 μg/mL) for different time points, and eNOS mRNA levels were determined by real-time PCR. (F) eNOS mRNA 3′UTR-binding molecules. Biotin-labeled human eNOS mRNA 3′UTR probe was incubated with cytoplasmic extracts of HCAECs treated with or without sCD40L for 24 hours. The binding reaction was electrophoresed on a native polyacrylamide gel. For the competition experiment, excess unlabeled RNA probe was preincubated with cytoplasmic protein prior to the addition of biotin-labeled RNA probe. *P < .05 and **P < .01, compared with the control. #P < .05 and ##P < .01, compared with sCD40L treatment. n = 3. Data are means and SE of multiple experiments (n).

Figure 2

Effects of sCD40L on eNOS protein levels and NO production in HCAECs. (A) Western blot analysis. HCAECs were treated with sCD40L for 24 hours and eNOS protein levels were determined by Western blot. (B) eNOS enzyme activity. HCAECs were treated with sCD50L for 24 hours. The eNOS enzyme activity was determined by a commercial eNOS fluorimetric assay kit. (C) Cellular NO levels. HCAECs were treated with sCD40L for 24 hours and cellular NO levels were determined by DAF-FM DA staining and flow cytometric analysis. (D) Effect of sCD40L trimer on eNOS mRNA and (E) protein levels. HCAECs were treated with sCD40L monomer form or sCD40L trimer form for 24 hours, and eNOS mRNA and protein levels were determined by real-time PCR analysis and Western blot, respectively. *P < .05 and **P < .01, compared with the control. n = 3. Data are means and SE of multiple experiments (n).

Figure 3

Effects of sCD40L on eNOS mRNA levels and endothelium-dependent vasorelaxation in porcine coronary arteries. (A) Porcine eNOS mRNA levels. Porcine coronary artery rings were treated with sCD40L (5 μg/mL) for 24 hours, and porcine eNOS mRNA levels were determined by real-time PCR. *P < .05 compared with the control. t test. n = 4. (B) Endothelium-dependent vasorelaxation. Porcine coronary artery rings were treated with sCD40L for 24 hours. Vasomotor reactivity was analyzed with a myograph device. The vessel ring was initially contracted with thromboxane A2 analog U46619, and then a relaxation concentration-response curve was generated by 4 cumulative additions of the endothelium-dependent vasodilator bradykinin. *P < .05 compared with the control. U test. n = 8. Data are means and SE of multiple experiments (n).

Figure 4

Effects of sCD40L on O2− production in HCAECs and porcine coronary arteries. (A) DHE staining in HCAECs. Cells were treated with sCD40L for 24 hours, and intracellular O2− levels were determined by DHE staining and flow cytometric analysis. U test. n = 3. (B) Porcine coronary artery rings. The vessel rings were treated with sCD40L for 24 hours and O2− levels in the endothelial layer of porcine coronary arteries were tested with lucigenin-enhanced chemiluminescence assay. *P < .05 compared with the control. t test. n = 6. (C) Mitochondrial membrane potential (representative histograms of flow cytometric analysis). HCAECs were treated with sCD40L for 24 hours, and mitochondrial membrane potential was determined by JC-1 staining and flow cytometric analysis. (D) Quantitative data of JC-1 staining showed a significant decrease in mitochondrial membrane potential after sCD40L treatment. Antioxidant SeMet effectively reversed these changes. U test. n = 3. (E) ATP content. HCAECs were treated with sCD40L for 24 hours, and ATP production was determined by a commercial ATPLite kit. Antioxidant SeMet was included. t test. n = 3. (F) Effect of mitochondrial complex II inhibitor TTFA on eNOS protein levels. HCAECs were treated with sCD40L in the presence or absence of mitochondrial complex II inhibitor TTFA (10 μM) for 24 hours, and eNOS protein levels were determined by Western blot analysis. *P < .05 and **P < .01, compared with the control. #P < .05 compared with sCD40L treatment. U test. n = 3. Data are means and SE of multiple experiments (n).

Figure 5

Effects of sCD40L and SeMet on activities of NOX, CAT, and SOD and eNOS mRNA levels in HCAECs. (A) NADPH oxidase (NOX) activity. HCAECs were treated with sCD40L for 24 hours, and NOX activities were determined by lucigenin-enhanced chemiluminescence with the presence of its substrate β-NADPH. O2− scavenger Tiron or flavoprotein inhibitor DPI was included in the assay to confirm the specificity of NOX activity. (B) CAT activity. HCAECs were treated with sCD40L for 24 hours and CAT activity was determined with a commercial kit. Antioxidant SeMet was included. (C) SOD activity. HCAECs were treated with sCD40L for 24 hours and SOD activity was determined with a commercial kit. Antioxidant SeMet was included. (D) eNOS mRNA levels. HCAECs were treated with sCD40L and/or SeMet for 24 hours, and eNOS mRNA levels were determined by real-time PCR analysis. *P < .05 and **P < .01, compared with the control. #P < .05 compared with sCD40L treatment. t test. n = 3. Data are means and SE of multiple experiments (n).

Figure 6

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).

Figure 7

Effects of sCD40L on NF-κB activation in HCAECs. (A) IκBα phosphorylation. HCAECs were treated with sCD40L for different time points, and phosphorylation levels of IκBα were determined by a Bio-Plex Luminex Immunoassay with a commercial kit. U test. (B) NF-κB p65 protein nuclear translocations. HCAECs were infected with recombinant adenovirus Ad-IκB DN (inhibition of IκBα) or Ad-GFP (a negative control). The cells were then treated with sCD40L for different time frames, and nuclear extract was prepared. Nuclear NF-κB protein levels were determined by Western blot analysis. Nuclear protein laminin was included as a loading control. (C) Effect of dominant negative mutant form of IκBα on eNOS mRNA levels. HCAECs were infected with recombinant adenovirus Ad-IκB DN or Ad-GFP for 72 hours, and then cells were treated with sCD40L for 24 hours. The NOS mRNA levels of eNOS were determined by real-time PCR analysis. U test. (D) Effect of dominant negative mutant form of IκBα on eNOS protein levels. HCAECs were infected with recombinant adenovirus Ad-IκB DN or Ad-GFP for 72 hours, and then cells were treated with sCD40L for 24 hours. The eNOS protein levels of eNOS were determined by Western blot analysis. *P < .05; **P < .01 compared with the control. n = 3. Data are means and SE of multiple experiments (n).