Interleukin-10 deficiency increases atherosclerosis, thrombosis, and low-density lipoproteins in apolipoprotein E knockout mice

Abstract

Interleukin (IL)-10 is an anti-inflammatory cytokine that may play a protective role in atherosclerosis. The aim of this study was to assess the effect of IL-10 deficiency in the apolipoprotein E knockout mouse. Apolipoprotein E deficient (E-/-) and IL-10 deficient (-/-) mice were crossed to generate E-/- x IL-10-/- double knockout mice. By 16 wk, cholesterol and triglycerides were similar in double and single knockouts but the lack of IL-10 led to increased low-density lipoprotein cholesterol whereas very-low-density lipoprotein was reduced. In parallel, T-helper 1 responses and lesion size were dramatically increased in double knockout compared with E-/- controls. At 48 wk, matrix metalloproteinases and tissue factor activities were increased in lesions of double-knockout mice. Furthermore, markers of systemic coagulation were increased, and vascular thrombosis in response to i.v. thrombin occurred more frequently in E-/- x IL-10-/- than in E-/- mice. Our findings suggest that IL-10 deficiency plays a deleterious role in atherosclerosis. The early phase of lesion development was increased, and the proteolytic and procoagulant activity was elevated in advanced lesions. These data show that IL-10 may reduce atherogenesis and improve the stability of plaques.

Figures

Figure 1

Computer-assisted morphometry was applied to aortic cryosections, stained with oil red O, harvested at the level of the aortic cusps. Data show lesion size (surface area of lesions/surface area of vessel, %) and were averaged from 4 sections per mouse (400, 500, 600, and 700 μm from the appearance of the 1st cusp). —, mean; *P < 0.05, female compared with male mice; †P < 0.05, E−/− × IL-10−/− compared with E−/− × IL-10+/+ mice.

Figure 2

Plasma lipoprotein patterns of 16-wk-old male (A) and female (B) mice following separation by FPLC. Lines represent means and gray sections represent SEM. A: Thin line, 7 male E−/− × IL-10+/+ mice; thick line, 9 male E−/− × IL-10−/− mice. B: Thin line, 4 female E−/− × IL-10+/+ mice; thick line, 4 female E−/− × IL-10−/− mice. In both panels, light gray areas show the results of 2 different human samples, after 4 repetitive injections (before, after, and between mouse samples) to monitor assay drift over time and to show elution profile for human.

Figure 3

MMP 9 localization in plaques from E−/− × IL-10−/− (A) and E−/− × IL-10+/+ (B) mice. Original magnification ×200; c, core; f, fibrous cap; m, media; s, shoulder region. Note MMP 9 accumulates in the shoulder regions in E−/− × IL-10−/− and not in E−/− × IL-10+/+ mice. Quantification was performed by computer-assisted morphometry. Plaques were divided into 3 portions as indicated in the insert (s, shoulder). The optical density within shoulder regions and the optical density within the total surface area of the plaque were computed.

Figure 4

Gelatinolytic activities (zymography) of aortas and spleen mononuclear cells from 4 E−/− × IL-10+/+ and 4 E−/− × IL-10−/− mice. Samples were prepared as described in Materials and Methods. Gels were submitted to scanning densitometry analysis (see Table 2).

Figure 5

Platelet arterial thrombi in thrombin-injected E−/− × IL-10−/−mice. Masson’s trichrome, magnification ×200 (A,B) and ×100 (C). A: Platelet thrombi occupy the lumen of 2 small epicardial branches of the left anterior descending coronary artery. An atherosclerotic plaque is evident in 1 of 2 thrombosed arteries. Insert: A model depicts the thrombus (gray) and the plaque (light blue). B: A thrombus occluding the lumen of an atherosclerotic intramyocardial coronary branch. Insert: A model depicts the platelet-rich part of the thrombus (gray), the erythrocytes (red), and the plaque (light blue). C: An occlusive red clot in a pulmonary artery of a mouse that died within 5 min of intravenous thrombin injection.