Aortic VCAM-1: an early marker of vascular inflammation in collagen-induced arthritis

Abstract

Cardiovascular disease (CVD) is a major cause of morbidity and mortality in rheumatoid arthritis (RA). There are limited experimental data on vascular involvement in arthritis models. To study the link between CVD and inflammation in RA, we developed a model of vascular dysfunction and articular inflammation by collagen-induced arthritis (CIA) in C57Bl/6 (B6) mice. We studied the expression of vascular inflammatory markers in CIA with and without concomitant hyperlipidic diet (HD). Collagen-induced arthritis was induced with intradermal injection of chicken type-II collagen followed by a boost 21 days later. Mice with and without CIA were fed a standard diet or an HD for 12 weeks starting from the day of the boost. Arthritis severity was evaluated with a validated clinical score. Aortic mRNA levels of vascular cell adhesion molecule-1 (VCAM-1), inducible nitric oxide synthase (iNOS) and interleukin-17 were analysed by quantitative RT-PCR. Vascular cell adhesion molecule-1 localization in the aortic sinus was determined by immunohistochemistry. Atherosclerotic plaque presence was assessed in aortas. Collagen-induced arthritis was associated with increased expression of VCAM-1, independent of diet. VCAM-1 overexpression was detectable as early as 4 weeks after collagen immunization and persisted after 15 weeks. The HD induced atheroma plaque formation and aortic iNOS expression regardless of CIA. Concomitant CIA and HD had no additive effect on atheroma or VCAM-1 or iNOS expression. CIA and an HD diet induced a distinct and independent expression of large-vessel inflammation markers in B6 mice. This model may be relevant for the study of CVD in RA.

Keywords: arthritis rheumatoid; atherosclerosis; collagen-induced arthritis; hyperlipidic diet; inflammation; mice; vasculature.

Figure 1

Effect of hypercholesterolemic diet (HD) on collagen‐induced arthritis (CIA), anti‐collagen antibody production and aortic lipid accumulation in mice. B6 mice were immunized with two injections of chicken CII (cCII). HD was started from the day of the second immunization (day 21). (A) Arthritis scores in immunized mice receiving HD (CIA+HD) (n = 12) or standard chow diet (CIA alone) (n = 12). Data are mean ± S.E.M. (B) Anti‐cCII antibody levels detected in mice at 15 weeks after the first immunization in CIA, CIA+HD, nonimmunized (NI) mice fed an HD or a standard chow diet. Horizontal bar is median Ab level. (C) Aortic lipid accumulation detected on thoracic sections by Oil‐red O staining (magnification ×16).

Figure 2

mRNA expression of pro‐inflammatory molecules in aortas (A–C) and synovium (D–F) of immunized and nonimmunized mice fed an HD or standard chow diet. Quantitative RT‐PCR (qRT‐PCR) analysis of mRNA levels of vascular cell adhesion molecule‐1 (VCAM‐1) (A and D), inducible nitric oxide synthase (iNOS) (B and E) and interleukin 17 (IL‐17) (C and F). Data are mean ± S.E.M. ^£ P < 0.05 versus HD and NI; *P < 0.05 versus CIA and NI, ^$ P < 0.05 versus CIA+HD, HD and NI.

Figure 3

Early (4 weeks) and late (15 weeks) aortic mRNA expression of VCAM‐1, iNOS, and IL‐17 in CIA mice. Aortas from CIA mice were removed at 4 weeks (n = 12) and 15 weeks (n = 6) after immunization. Controls were: mice immunized with only CFA (n = 6) and nonimmunized mice (NI) (n = 12 or n = 6 for aorta removed at 4 or 15 weeks, respectively). qRT‐PCR analysis of mRNA levels of VCAM‐1 (A), iNOS (B) and IL‐17 (C). Data are mean ± S.E.M.; ^$ P < 0.01 versus CFA and NI; ^£ P < 0.05 versus CFA.

Figure 4

VCAM‐1 staining in aortic sinus from mice fed an HD or standard chow diet. (A) Representative histological sections of the aortic sinus stained with blue‐haematoxylin for VCAM‐1 in NI mice fed a standard chow diet or an HD and in immunized mice fed a standard diet (CIA) or an HD (CIA+HD). Magnification is 50 × (a, d, g, j) or 400 × (b, e, h, k). Staining for isotype control of VCAM‐1 antibody (IgG2a, κ antibody) is shown in c, f, i and l (×400 magnification). Black arrows show VCAM‐1 endothelial staining; white arrows show VCAM‐1 staining in the aortic sinus wall. (B) Quantification of VCAM‐1–positive staining by use of Image J Fiji. ^$ P < 0.05 versus NI.

Figure 5

Early (4 weeks) and late (15 weeks) VCAM‐1 staining in aortic sinus of mice. Aortic sinuses from CIA mice were removed at 4 and 15 weeks after immunization. Controls were immunized with only CFA or were NI mice. (A) Representative microphotographs of VCAM‐1 staining in aortic sinus from cCII/CFA‐immunized (CIA: a, d), CFA‐immunized (CFA: b, e) and NI(c, f) mice. Black arrows show endothelial staining for VCAM‐1; white arrows show VCAM‐1 staining in the aortic sinus wall. (B) Quantification of VCAM‐1–staining at 4 and 15 weeks in CIA versus control mice (NI and CFA mice pooled) by use of Image J Fiji. ^£ P < 0.05 versus CT.