Inhibition of CD40 signaling limits evolution of established atherosclerosis in mice

Abstract

Interruption of inflammatory pathways may provide a novel approach to the therapy of atherosclerosis. Recently, we and others have implicated the immune mediator dyad CD40/CD40L (CD40 ligand), which is expressed on endothelial and smooth muscle cells, macrophages, and T lymphocytes within human atherosclerotic lesions, in aspects of atherogenesis and the acute coronary syndromes, including regulation of matrix metalloproteinases, procoagulant activity, cytokines, etc. In vivo, interruption of CD40 signaling reduced the initiation and early phases of atheroma formation in hypercholesterolemic mice. However, whether interruption of CD40 signaling can retard the progression or even regress established lesions remains unknown. We report here that anti-CD40L antibody treatment of randomly assigned low-density lipoprotein receptor-deficient mice during the second half of a 26-week regimen of high-cholesterol diet did not regress, but did significantly reduce further evolution of established atherosclerotic lesions within the aortic arch and particularly the thoracic and abdominal aorta, as compared with control treatment (application of rat-IgG or saline; 13 weeks, continued high-cholesterol diet). In addition to limiting lesion progression, anti-CD40L treatment changed the composition of atheroma in manners thought to favor plaque stability, e.g., reduced relative content of macrophages and lipid, as well as increased relative content of smooth muscle cells and collagen. These data implicate CD40/CD40L as crucial mediators not only in the initial events of atherogenesis but also during the evolution of established atheroma. This study lends further support to the importance of this specific inflammatory signaling pathway in atherosclerosis and its complications.

Figure 1

Anti-CD40L antibody administration reduces evolution of established atherosclerotic lesions. Ldlr-deficient mice consumed a high-cholesterol diet for 13 weeks (diet alone; white bars) before treatment with either rat IgG (light gray bars), saline (dark gray bars), or anti-CD40L antibody (α-CD40L; black bars) while continuing a high-cholesterol diet (13 weeks). Photomicrographs of longitudinally cut tissue sections of mouse aortic arches were analyzed by computer-assisted image quantification. The maximal wall, medial and intimal area as well as the maximal thickness of the inner aortic arch wall for each mouse was measured extending 3 mm distal from the right carotid. Calculation of total areas, thickness, and percent positive areas was performed independently by two blinded observers. Data are presented as mean ± SD, and comparison between the respective study groups used Student's t test.

Figure 2

Evidence for enhancement of features associated with plaque stability by anti-CD40L antibody treatment. Ldlr-deficient mice consumed a high-cholesterol diet for 13 weeks before treatment with either rat IgG, saline, or anti-CD40L antibody (α-CD40L) for 13 weeks during continued high-cholesterol diet. Aortic arches were perfused with PBS and snap-frozen, and sequential (6 μm) tissue sections were examined for the expression of α-actin (SMCs), CD68 (macrophages, MØ), or interstitial collagen by Picrosirius red polarization. The carotid arteries are on top, and the aortic sinus on the right. Representative specimens are shown.

Figure 3

Evidence for enhancement of features associated with plaque stability by anti-CD40L antibody treatment. Photomicrographs of tissue sections of mouse aortic arches as shown in Fig. 2 were analyzed by computer-assisted image quantification. The percent positive area using staining for CD68 (macrophages, MØ), lipids, α-actin (SMCs), or collagen was determined within the area extending 3 mm distal from the right carotid. Data are mean ± SD for mice that consumed high-cholesterol diet for 13 weeks (diet alone, white bars), or 26 weeks with application of either irrelevant rat IgG (light gray bars), saline (dark gray bars), or anti-CD40L antibody (black bars) during the last 13 weeks. Calculation of percent positive areas was performed independently by two blinded observers, and comparison between the respective study groups used Student's t test.

Figure 4

Anti-CD40L antibody treatment reduces lipid deposition in the abdominal aorta of hypercholesterolemic mice. Ldlr-deficient mice consumed a high-cholesterol diet for 13 weeks (A and B; white bar, diet only) before treatment with either rat IgG (A and C; light gray bar), saline (A and C; dark gray bar), or anti-CD40L antibody (A and C; black bar, α-CD40L) for 13 weeks during continued regimen of the diet. Photomicrographs of tissue sections of longitudinally cut abdominal aortas pinned out on black wax surface and stained for lipid deposition with Sudan IV (B and C), were analyzed by computer-assisted image quantification (shown are mean ± SD analysis, calculation of percent positive areas was performed independently by two blinded observers, comparison between the respective study groups used Student's t test) (A). The thoracic section of the aorta is on the right. Representative specimens from each group are shown.

Figure 5

Anti-CD40L antibody treatment reduces the extent of atherosclerotic lesions in the abdominal aorta of hypercholesterolemic mice. Ldlr-deficient mice consumed a high-cholesterol diet for 13 weeks before treatment with either rat IgG, saline, or anti-CD40L antibody (α-CD40L) for 13 weeks during continued high-cholesterol diet. Cross sections of the longitudinally cut abdominal aortae from three mice (proximal to the renal artery bifurcation) are shown.

Figure 6

Reduced evolution of established atherosclerotic lesions of the abdominal aorta by anti-CD40L antibody treatment. Photomicrographs of cross sections of formalin-fixed and longitudinally opened abdominal aorta (as exemplified in Fig. 5) were analyzed by computer-assisted image quantification. The maximal wall, medial, and intimal area as well as the maximal thickness of the inner aortic arch wall for each mouse was measured. Data are mean ± SD for mice treated with either irrelevant rat IgG (light gray bars), saline (dark gray bars), or anti-CD40L antibody (black bars). These groups are compared with mice fed 13 weeks with high-cholesterol diet (white bars; diet alone). Comparison between the respective study groups used Student's t test.