Mechanism of endoplasmic reticulum stress-induced vascular endothelial dysfunction

Abstract

Background: We recently reported that ER stress plays a key role in vascular endothelial dysfunction during hypertension. In this study we aimed to elucidate the mechanisms by which ER stress induction and oxidative stress impair vascular endothelial function.

Methodology/principal findings: We conducted in vitro studies with primary endothelial cells from coronary arteries stimulated with tunicamycin, 1μg/mL, in the presence or absence of two ER stress inhibitors: tauroursodeoxycholic acid (Tudca), 500μg/mL, and 4-phenylbutyric acid (PBA), 5mM. ER stress induction was assessed by enhanced phosphorylation of PERK and eIF2α, and increased expression of CHOP, ATF6 and Grp78/Bip. The ER stress induction increased p38 MAPK phosphorylation, Nox2/4 mRNA levels and NADPH oxidase activity, and decreased eNOS promoter activity, eNOS expression and phosphorylation, and nitrite levels. Interestingly, the inhibition of p38 MAPK pathway reduced CHOP and Bip expressions enhanced by tunicamycin and restored eNOS promoter activation as well as phosphorylation. To study the effects of ER stress induction in vivo, we used C57BL/6J mice and p47phox(-/-) mice injected with tunicamycin or saline. The ER stress induction in mice significantly impaired vascular endothelium-dependent and independent relaxation in C57BL/6J mice compared with p47phox(-/-) mice indicating NADPH oxidase activity as an intermediate for ER stress in vascular endothelial dysfunction.

Conclusion/significance: We conclude that chemically induced ER stress leads to a downstream enhancement of p38 MAPK and oxidative stress causing vascular endothelial dysfunction. Our results indicate that inhibition of ER stress could be a novel therapeutic strategy to attenuate vascular dysfunction during cardiovascular diseases.

Keywords: Endoplasmic reticulum; Endothelial dysfunction; MAPKinase; Oxidative stress.

Figure 1. ER stress markers expression in endothelial cells (EC). A-E

Representative Western blots and quantitative analysis of P-PERK, P-eIF2α, CHOP, ATF6 and grp78 or Bip, n=5, and; F, G. CHOP and ATF6 mRNA levels, normalized to 18S rRNA, n=6. The cells were incubated with 1 μg/ml tunicamycin (Tunica) for 6 hours and pre-treated or not with ER stress inhibitors (+Tudca / +PBA) 1 h prior to add the tunicamycin. Data are expressed as mean ± SEM. *P≤0.05 for Tunica vs. cells treated with vehicle (C) or cells pre-treated with Tudca or PBA (Tunica + Tudca/PBA); #P≤0.05 for Tunica + Tudca/PBA vs. C or C + Tudca/PBA.

Figure 2. NADPH oxidase expression / activity induction by ER stress in endothelial cells

A, B. Quantitative real Time PCR assessment of Nox2 and Nox4 gene expression, normalized to 18S rRNA, in cells treated with vehicle (C) or tunicamycin (Tunica) in the absence or presence of Tudca or PBA, n=6. C. NADPH oxidase activity measured by lucigenin chemiluminescence in cells treated with vehicle or tunicamycin in the absence or presence of Tudca or apocynin (Apo, 100 μmol/L), n=6. D. Representative images of cells stained with dihydroethidium and fluorescence signal quantification, n=4. E. NADPH oxidase activity measured inEC transfected with scrambled RNA (Scr), Nox2 siRNA or Nox4 siRNA, incubated with or without tunicamycin, n=5. F, G. Nox2 and Nox4 gene expression, normalized to 18S rRNA, in EC transfected with either Nox2 siRNA or Nox4 siRNA and incubated with or without tunicamycin, n=6. In all experiments cells were incubated with tunicamycin for 6 hours. Data are expressed as mean ± SEM. *P≤0.05 for Tunica, Tunica + Tudca/PBA or Tunica + Apo vs. C ± Tudca/PBA; $P≤0.05 for Tunica + PBA vs. Tunica; &P≤0.05 for Tunica vs. Tunica + Tudca/PBA or Tunica + Apo; #P<0.05 for Nox4siRNA / Nox2siRNA ± Tunica vs. Scr.

Figure 3. Effect of ER stress on eNOS activity / expression in endothelial cells

A, B. eNOS promoter activity in transfected EC with plasmid containing eNOS promoter in cells added with vehicle (C) or incubated with tunicamycin (Tunica) ± Tudca / PBA and in cells co-transfected with plasmids overexpressing CHOP and ATF6, respectively, n=5. C. Quantitative evaluation of eNOS mRNA levels, normalized to 18S rRNA, in cells with vehicle or incubated with tunicamycin ± Tudca / PBA, n=6. D. Quantitative evaluation of eNOS mRNA levels, normalized to 18S rRNA, in a time course assay with tunicamycin, n=5; E. Representative pictures of eNOS mRNA detected by RNA-FISH analysis with quantitative fluorescence evaluation, n=3; F. eNOS mRNA levels in a time course experiment with Actinomycin D after incubation with tunicamycin with or without Tudca, n=5; G. Representative Western blot for eNOS and quantitative analysis in a time course assay with tunicamycin, n=4; H. Representative Western blot for phosphorylated eNOS (P-eNOS), total eNOS and quantitative analysis in cells with vehicle (C) or incubated with tunicamycin (Tunica) ± Tudca / PBA, n=4, and; I. nitrites production measured in supernatants of cultured EC with vehicle (C) or incubated with tunicamycin (Tunica) in the absence or presence of Tudca, PBA, Apocynin or L-NAME (100 μmol/L), n=6. In these experiments cells were incubated with tunicamycin for 6 hours. *P≤0.05 for Tunica vs. C or C+Tudca; #P≤0.05 for Tunica vs. Tunica+Tudca; &P≤0.05 for Tunica+L-NAME vs. Control+L-NAME.

Figure 3. Effect of ER stress on eNOS activity / expression in endothelial cells

A, B. eNOS promoter activity in transfected EC with plasmid containing eNOS promoter in cells added with vehicle (C) or incubated with tunicamycin (Tunica) ± Tudca / PBA and in cells co-transfected with plasmids overexpressing CHOP and ATF6, respectively, n=5. C. Quantitative evaluation of eNOS mRNA levels, normalized to 18S rRNA, in cells with vehicle or incubated with tunicamycin ± Tudca / PBA, n=6. D. Quantitative evaluation of eNOS mRNA levels, normalized to 18S rRNA, in a time course assay with tunicamycin, n=5; E. Representative pictures of eNOS mRNA detected by RNA-FISH analysis with quantitative fluorescence evaluation, n=3; F. eNOS mRNA levels in a time course experiment with Actinomycin D after incubation with tunicamycin with or without Tudca, n=5; G. Representative Western blot for eNOS and quantitative analysis in a time course assay with tunicamycin, n=4; H. Representative Western blot for phosphorylated eNOS (P-eNOS), total eNOS and quantitative analysis in cells with vehicle (C) or incubated with tunicamycin (Tunica) ± Tudca / PBA, n=4, and; I. nitrites production measured in supernatants of cultured EC with vehicle (C) or incubated with tunicamycin (Tunica) in the absence or presence of Tudca, PBA, Apocynin or L-NAME (100 μmol/L), n=6. In these experiments cells were incubated with tunicamycin for 6 hours. *P≤0.05 for Tunica vs. C or C+Tudca; #P≤0.05 for Tunica vs. Tunica+Tudca; &P≤0.05 for Tunica+L-NAME vs. Control+L-NAME.

Figure 4. Effect of p38 MAPK activation by ER stress on eNOS

A, B. Representative Western blots and quantitative analysis for P-ERK 1/2 and P-p38 MAPK in EC with vehicle (C) or incubated with tunicamycin (Tunica) in the absence or presence of Tudca or PBA, n=5; C. Representative Western blots and quantitative analysis for P-p38 in EC treated with tunicamycin in the absence or presence of p38 inhibitor (± p38I), SB203580 (10 μmol/L), or apocynin (Apo), n=4; D. P-eIF2α and ATF6 and E, F. CHOP and BiP representative blots and quantitative analysis, n=4; G. Representative blot and quantitative data of phosphorylated eNOS, n=4, and H. eNOS promoter activity in transiently transfected EC incubated with or without tunicamycin ± p38I, n=5; I. eNOS promoter activity in cells co-transfected with the plasmid expressing ATF6 ± p38I, n=5. *P≤0.05 for Tunica or Tunica + Apo vs. C; #P≤0.05 for Tunica or Tunica + Apo vs. Tunica + Tudca or Tunica + p38I; &P≤0.05 for eNOSp + ATF6 vs. eNOSp + ATF6 + p38I or eNOSp.

Figure 5. ER stress markers expression in aorta and MRA

A, F. Western blot analysis and quantitative data for P-eIF2-α normalized to eIF2-α in aorta and MRA, n=4; B-C, G-H. CHOP mRNA levels, normalized to 18S rRNA, n=6, and representative blot for CHOP protein and quantitative data respectively in aorta and MRA, n=4; D-E, I-J. ATF6 mRNA levels, normalized to 18S rRNA, n=6, and representative blots for cleaved ATF6 and quantitative data respectively in aorta and MRA from control (C) and p47phox^−/− mice untreated or treated with tunicamycin (Tunica), n=4. *P<0.05 for C + Tunica or p47phox^−/− + Tunica vs. C and p47phox^−/−; # P<0.05 for p47phox^−/− + Tunica vs. C + Tunica.

Figure 6. Endothelium dependent relaxation, eNOS and NADPH oxidase in aortas from control and p47phox^−/− (p47^−/−) mice

A. Endothelium dependent relaxation (EDR) in response to ACh, n=6; B. EDR in response to ACh in the presence of apocynin (Apo), n=6; C, D. Western blot analysis for P-eNOS and T-eNOS, normalized to β-actin, n=4, and eNOS mRNA levels, normalized to 18S rRNA, n=6; D. E. Nox2 mRNA levels, normalized to 18S rRNA, n=6 and F. NADPH oxidase activity in tissue lysates of vessels from control and p47phox^−/− mice injected with saline or tunicamycin, n=5. *P<0.05 for Control+Tunica or p47^−/−+Tunica vs. Control and p47−/−; #P<0.05 for Control+Tunica vs. p47phox^−/−+Tunica; $P<0.05 for p47phox ^−/− vs. p47phox ^−/− +Tunica.

Figure 7. Endothelium dependent relaxation, eNOS and NADPH oxidase in MRA from control and p47phox^−/− (p47^−/−) mice

A. Endothelium dependent relaxation (EDR) in response to ACh, n=6; B. EDR in response to ACh in the presence of apocynin (Apo), n=6; C, D. Western blot analysis for P-eNOS and T-eNOS, normalized to β-actin, n=4, and eNOS mRNA levels, normalized to 18S rRNA, n=6; E. Nox2 mRNA levels, normalized to 18S rRNA, n=6; F. NADPH oxidase activity measured in tissue lysates of vessels from control and p47phox^−/− mice injected with saline or tunicamycin, n=5. *P<0.05 for Control+Tunica or p47 phox^−/− +Tunica vs. Control and p47phox^−/−; #P<0.05 for Control+Tunica vs. p47 phox^−/− +Tunica; $P<0.05 for p47phox^−/− vs. p47phox^−/− +Tunica.