Abdominal Aortic Endothelial Dysfunction Occurs in Female Mice With Dextran Sodium Sulfate-Induced Chronic Colitis Independently of Reactive Oxygen Species Formation

Abstract

Background and objective: Inflammatory bowel disease (IBD) produces significant local and systemic inflammation with increased reactive oxygen species (ROS) formation. IBD Patients are at an increased risk for developing endothelial dysfunction and cardiovascular diseases. The present study tested the hypothesis that IBD impairs aortic endothelial function via ROS formation and investigate potential sex-related differences.

Methods and results: Acute and chronic colitis models were induced in male and female C57BL/6 mice with dextran sodium sulfate (DSS) treatment. Aortic wall stiffness, endothelial function, and ROS levels, as well as serum levels of pro-inflammatory cytokines were evaluated. Acetylcholine (Ach)-induced endothelium-dependent relaxation of abdominal aorta without perivascular adipose tissue (PVAT) was significantly reduced in female mice, not males, with chronic colitis without a change in nitroglycerin-induced endothelium-independent relaxation. PVAT effectively preserved Ach-induced relaxation in abdominal aorta of female mice with chronic colitis. Aortic peak velocity, maximal intraluminal diameters, pulse wave velocity, distensibility and radial strain were preserved in mice with both acute and chronic colitis. Although pro-inflammatory cytokines levels were increased in mice with acute and chronic colitis, aortic ROS levels were not increased.

Conclusion: The data demonstrate that abdominal aortic endothelial function was attenuated selectively in female mice with chronic colitis independent of ROS formation. Further, PVAT played an important role in preserving endothelial function in female mice with chronic colitis.

Keywords: ROS; aorta; colitis; endothelial dysfunction; inflammatory bowel disease.

FIGURE 1

Both female and male mice with acute DSS-induced colitis displayed significant inflammation in the colon and systemic inflammation. (A) Experimental scheme illustrating DSS treatment protocol for acute models. Changes in (B) body weight and (D) disease activity index (DAI) during DSS treatment. (C) Colon length and representative photographs for colon tissue in DSS-treated and control mice. (E) Representative images of H&E staining of colon tissue (×200; scale bar, 50μm), and summary of histological score. (F) Levels of plasma cytokines in male and female mice with acute colitis model. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001, in unpaired 2-tailed Student’s t-test (B–F), n = 5–8 mice each group. F and M, female and male mice, respectively. G-CSF, granulocyte colony-stimulating factor; TNF-α, tumor necrosis factor alpha; IFN-γ, interferon gamma; IL, interleukin.

FIGURE 2

Both female and male mice with chronic DSS-induced colitis displayed significant inflammation in the colon and systemic inflammation. (A) Experimental scheme illustrating the experimental protocol with treatment of DSS and water for chronic models. Changes in (B) body weight and (D) disease activity index (DAI) during DSS treatment. (C) Colon length and representative photographs for colon tissue in DSS-treated and control mice. (E) Representative images of H&E staining of colon tissue (×200; scale bar, 50 μm), and summary of histological score. (F) Levels of plasma cytokines in male and female mice with chronic colitis model. Results are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, in unpaired 2-tailed Student’s t-test (B–F), n = 5–8 mice each group. F and M, female and male mice, respectively. G-CSF, granulocyte colony-stimulating factor; TNF-α, tumor necrosis factor alpha; IFN-γ, interferon gamma; IL, interleukin.

FIGURE 3

No significant changes in hemodynamics and arterial stiffness of abdominal aorta were observed in both male and female mice with DSS-induced acute and chronic colitis. (A–C) Analysis of PW doppler mode images for peak velocity. (D–F) Analysis of M-mode images for MILD. (G–I) Analysis of EKV images for PWV. (J–N) Analysis of EKV images for distensibility and radial strain. Results are expressed as mean ± SEM. *p < 0.05, in two-way ANOVA followed by Bonferroni correction (E–N), n = 6–8 mice each group. F and M, female and male mice, respectively. MILD, maximal intraluminal diameter; PWV, pulse wave velocity.

FIGURE 4

Aortic endothelial function was preserved in both male and female mice with DSS-induced acute colitis. (A–D) Ach-induced endothelium-dependent relaxation of thoracic aorta with or without PVAT. (E–H) Ach-induced endothelium-dependent relaxation of abdominal aorta with or without PVAT. (I) Maximal relaxation and EC₅₀ for Ach were calculated in the table. Results are expressed as means ± SEM. *p < 0.05, in two-way ANOVA followed by Bonferroni correction (A–H), n = 5–8 mice in each group. PVAT, perivascular adipose tissue; F and M, female and male mice, respectively. Ach, acetylcholine.

FIGURE 5

Ach-induced endothelium-dependent relaxation of abdominal aorta was selectively impaired in female mice with DSS-induced chronic colitis. (A–D) Ach-induced endothelium-dependent relaxation of thoracic aorta with or without PVAT. (E–H) Ach-induced endothelium-dependent relaxation of abdominal aorta with or without PVAT. (I) Maximal relaxation and EC₅₀ for Ach were calculated in the table. Results are expressed as means ± SEM. *p < 0.05, in two-way ANOVA followed by Bonferroni correction (A–H), n = 5–8 mice in each group. PVAT, perivascular adipose tissue; F and M, female and male mice, respectively. Ach, acetylcholine.

FIGURE 6

Reactive oxygen species (ROS) levels remained unchanged in thoracic and abdominal aorta in mice with acute and chronic colitis in both female and male mice. Representative images of ROS in acute colitis model (A,B) and chronic colitis model (C,D) for thoracic and abdominal aortic rings using DHE staining (×100) and graph for quantification of ROS levels in the aortic rings. Results are expressed as means ± SEM. *p < 0.05, in unpaired 2-tailed Student’s t-test (A–D), n = 5 mice in each group. DHE, dihydroethidium. AU, arbitrary units. F and M, female and male mice, respectively.