HUMAN MONOCYTE-DERIVED MACROPHAGES INDUCE COLLAGEN BREAKDOWN IN FIBROUS CAPS OF ATHEROSCLEROTIC PLAQUES - POTENTIAL ROLE OF MATRIX-DEGRADING METALLOPROTEINASES AND IMPLICATIONS FOR PLAQUE RUPTURE

Background Rupture of the fibrous cap of the atherosclerotic plaque is a key event that predisposes to coronary thrombosis, leading to acute coronary syndromes. Recent studies have shown that the fibrous caps o f vulnerable and ruptured atherosclerotic plaques have reduced collage n and glycosaminoglycan content in association with an increased macro phage density and a reduced smooth muscle cell density. Since collagen breakdown in the fibrous caps may contribute to a thinning and weaken ing of the cap, increasing its vulnerability to rupture, we tested the hypothesis that monocyte-derived macrophages, by producing matrix-deg rading metalloproteinases (MMPs), could induce collagen breakdown in h uman atherosclerotic fibrous caps. Methods and Results Monocytes were isolated from human blood by Ficoll-Paque density gradient and allowed to grow in cell culture until phenotypic and staining characteristics indicated transformation into macrophages (4 to 7 days). Fibrous caps were dissected from human aortic or carotid plaques and incubated for 48 hours with macrophages in serum-free medium without (n=21) and wit h (n=10) an MMP inhibitor or with cell- and serum-free medium only (n= 9). Hydroxyproline released in the culture medium was measured by a sp ectrophotometric method and used as evidence of collagen breakdown in the fibrous caps. Immunocytochemistry with specific monoclonal antibod ies was used to identify expression of MMP-1 (interstitial collagenase ) and MMP-2 (72-kD gelatinase) in cell culture, and zymography was use d to detect MMP activity in the culture supernatant. The amount of hyd roxyproline released was significantly greater when fibrous caps were incubated with macrophages than when incubated with cell-free medium ( 0.4+/-0.16 mu g . mL(-1). min(-1) versus 0.02+/-0.03 mu g . mL(-1). mg (-1) of tissue; P<.04 by Mann-Whitney test). There was no hydroxyproli ne release when fibrous caps were incubated with macrophages in the pr esence of an MMP inhibitor. Immunocytochemistry demonstrated MMP-1 and MMP-2 expression by macrophages between days 4 and 7, and zymography confirmed the presence of MMP-2 activity in the supernatant. Conclusio ns In this study, human monocyte-derived macrophages were shown to ind uce collagen breakdown in fibrous caps of human atherosclerotic plaque s associated with cellular expression and zymographic evidence of MMP activity; no evidence of collagen breakdown was found in the presence of an MMP inhibitor. These findings support the hypothesis that increa sed macrophage density and/or activation in the atherosclerotic plaque may induce collagen breakdown in the fibrous cap by secreting MMPs an d possibly other proteases, thus contributing to vulnerability to plaq ue rupture.