Interleukin-17 causes Rho-kinase-mediated endothelial dysfunction and hypertension

Abstract

Aims: Elevated levels of pro-inflammatory cytokine interleukin-17A (IL-17) are associated with hypertensive autoimmune diseases; however, the connection between IL-17 and hypertension is unknown. We hypothesized that IL-17 increases blood pressure by decreasing endothelial nitric oxide production.

Methods and results: Acute treatment of endothelial cells with IL-17 caused a significant increase in phosphorylation of the inhibitory endothelial nitric oxide (NO) synthase residue threonine 495 (eNOS Thr495). Of the kinases known to phosphorylate eNOS Thr495, only inhibition of Rho-kinase prevented the IL-17-induced increase. IL-17 caused a threefold increase in the Rho-kinase activator RhoA, and this was prevented by an IL-17 neutralizing antibody. In isolated mouse aortas, IL-17 significantly increased eNOS Thr495 phosphorylation, induced RhoA expression, and decreased NO-dependent relaxation responses, all of which were prevented by either an IL-17 neutralizing antibody or inhibition of Rho-kinase. In mice, IL-17 treatment for 1 week significantly increased systolic blood pressure and this was associated with decreased aortic NO-dependent relaxation responses, increased eNOS Thr495 phosphorylation, and increased RhoA expression. Inhibition of Rho-kinase prevented the hypertension caused by IL-17.

Conclusion: These data demonstrate that IL-17 activates RhoA/Rho-kinase leading to endothelial dysfunction and hypertension. Inhibitors of IL-17 or Rho-kinase may prove useful as anti-hypertensive drugs in IL-17-associated autoimmune diseases.

Figure 1

Effects of IL-17 on eNOS phosphorylation and RhoA in endothelial cells. (A) Endothelial cells were treated with vehicle, mouse recombinant IL-17 (1 µg/mL), and/or the Rho-kinase inhibitor Y-27632 (50 µmol/L) for 1 h and eNOS Thr495 phosphorylation and eNOS were measured by western blot. (B) Endothelial cells were treated with vehicle, mouse recombinant IL-17, and/or an IL-17 neutralizing antibody (20 ng/mL) for 1 h and RhoA was measured by western blot. Representative western blots and densitometry quantification. Results are expressed as mean + SEM. *P < 0.05 vs. control. N = 3–5 independent experiments.

Figure 2

Effects of IL-17 on eNOS phosphorylation and RhoA in isolated mouse aortas. Isolated, endothelium-intact mouse aortas were treated with vehicle, mouse recombinant IL-17, and/or the Rho-kinase inhibitor Y-27632 for 1 h and (A) eNOS Thr495 and (B) eNOS Ser1177 phosphorylation was measured by western blot following a pull-down with anti-eNOS. IgG was used as a loading control and for normalization as this corresponded directly to eNOS levels. (C) Isolated, endothelium-intact mouse aortas were treated with vehicle, mouse recombinant IL-17, and/or an IL-17 neutralizing antibody for 1 h and RhoA was measured by western blot. Representative western blot and densitometry quantification. (D) Nitrate production was measured in vehicle-treated and IL-17-treated aortas using the Griess assay. Results are expressed as mean + SEM. *P < 0.05 vs. control. N = 3–6 independent experiments.

Figure 3

Effects of IL-17 on endothelial function in isolated mouse aortas. (A) Isolated, endothelium-intact mouse aortas were treated with vehicle, mouse recombinant IL-17, the NOS inhibitor L-NAME, and/or the superoxide scavenger PEG-SOD for 1 h and relaxation responses to ACh were measured. (B) Relaxation responses to bradykinin were measured in vehicle-treated and IL-17-treated aortas. Aortas were treated with vehicle or mouse recombinant IL-17 in the absence and presence of an IL-17 neutralizing antibody with and without the NOS inhibitor L-NAME for 1 h and relaxation responses to ACh (C) and SNP (D) were measured. (E) Aortas were treated with vehicle or mouse recombinant IL-17 in the absence and presence of the Rho-kinase inhibitors Y-27632 or H-1152P or the cPKC inhibitor Gö6976 and relaxation responses to ACh were measured. (F) Isolated, endothelium-intact mouse aortas were treated with vehicle or mouse recombinant IL-17 in the absence and presence of the Rho-kinase inhibitor Y-27632 and contraction responses to PE were measured. Results are expressed as mean + SEM. *P < 0.05 vs. control. N for each group is given in parentheses.

Figure 4

Effects of daily IL-17 treatment on serum cytokines, blood pressure, and T cells in mice. (A) Serum levels of IL-17 and IL-6 were measured in mice treated with vehicle or mouse recombinant IL-17 daily for 1 week. (B) Tail-cuff systolic blood pressure was measured in mice at baseline and on Days 1, 4, and 7 of daily treatment with vehicle or mouse recombinant IL-17. (C) CD3+/CD4+ T cells were measured in the spleens of vehicle-treated and IL-17-treated mice using flow cytometry. (D) Quantification of splenic CD3+/CD4+ T cells as a per cent of lymphocytes in vehicle-treated and IL-17-treated mice. Results are expressed as mean ± SEM. *P < 0.05 vs. control, ⁺P < 0.05 vs. previous time point. N = 9 mice for each.

Figure 5

Effects of daily IL-17 treatment in mice on aortic endothelial function, eNOS phosphorylation, and RhoA expression. (A) Relaxation responses to ACh were measured in endothelium-intact aortas isolated from vehicle-treated and IL-17-treated mice in the absence and presence of the NOS inhibitor L-NAME or the superoxide scavenger PEG-SOD. (B) Endothelium-independent relaxation responses to SNP were measured in endothelium-intact aortas isolated from vehicle-treated and IL-17-treated mice. (C) eNOS Thr495 phosphorylation was measured in endothelium-intact aortas isolated from vehicle-treated and IL-17-treated mice by western blot following a pull-down with anti-eNOS. IgG was used as a loading control and for normalization as this corresponded directly to eNOS levels. (D) RhoA was measured in endothelium-intact aortas isolated from vehicle-treated and IL-17-treated mice by western blot. Representative western blot and densitometry quantification. Results are expressed as mean + SEM. *P < 0.05 vs. control. N = 9 mice in each group.

Figure 6

Effects of Rho-kinase inhibition on blood pressure and eNOS phosphorylation in daily IL-17-treated mice. (A) Tail-cuff systolic blood pressure was measured in mice at baseline and on Day 7 of daily treatment with vehicle, mouse recombinant IL-17, or IL-17 and the Rho-kinase inhibitor Y-27632. (B) RhoA was measured in endothelium-intact aortas isolated from IL-17-treated and IL-17 + Y-27632-treated mice by western blot. (C) eNOS Thr495 phosphorylation was measured in endothelium-intact aortas isolated from IL-17-treated and IL-17 + Y-27632-treated mice by western blot following a pull-down with anti-eNOS. IgG was used as a loading control and for normalization as this corresponded directly to eNOS levels. Representative western blot and densitometry quantification. Results are expressed as mean + SEM. *P < 0.05 vs. control. N = 9 mice for each.