title: Human monocyte-derived macrophages induce collagen breakdown in fibrous caps of atherosclerotic plaques. Potential role of matrix-degrading metalloproteinases and implications for plaque rupture.
abstract: 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 of vulnerable and ruptured atherosclerotic plaques have reduced collagen and glycosaminoglycan content in association with an increased macrophage density and a reduced smooth muscle cell density. Since collagen breakdown in the fibrous caps may contribute to a thinning and weakening of the cap, increasing its vulnerability to rupture, we tested the hypothesis that monocyte-derived macrophages, by producing matrix-degrading metalloproteinases (MMPs), could induce collagen breakdown in human atherosclerotic fibrous caps.
  
  
  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 with (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 spectrophotometric method and used as evidence of collagen breakdown in the fibrous caps. Immunocytochemistry with specific monoclonal antibodies was used to identify expression of MMP-1 (interstitial collagenase) and MMP-2 (72-kD gelatinase) in cell culture, and zymography was used to detect MMP activity in the culture supernatant. The amount of hydroxyproline released was significantly greater when fibrous caps were incubated with macrophages than when incubated with cell-free medium (0.4 +/- 0.16 micrograms.mL-1.mg-1 versus 0.02 +/- 0.03 micrograms.mL-1.mg-1 of tissue; P < .04 by Mann-Whitney test). There was no hydroxyproline release when fibrous caps were incubated with macrophages in the presence 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.
  
  
  CONCLUSIONS
  In this study, human monocyte-derived macrophages were shown to induce collagen breakdown in fibrous caps of human atherosclerotic plaques 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 increased macrophage density and/or activation in the atherosclerotic plaque may induce collagen breakdown in the fibrous cap by secreting MMPs and possibly other proteases, thus contributing to vulnerability to plaque rupture.
authors.affiliation: Atherosclerosis Research Center, Cedars-Sinai Medical Center, Los Angeles, CA 90077, USA.
authors.forename: P K
authors.initials: PK
authors.lastname: Shah
authors.forename: E
authors.initials: E
authors.lastname: Falk
authors.forename: J J
authors.initials: JJ
authors.lastname: Badimon
authors.forename: A
authors.initials: A
authors.lastname: Fernandez-Ortiz
authors.forename: A
authors.initials: A
authors.lastname: Mailhac
authors.forename: G
authors.initials: G
authors.lastname: Villareal-Levy
authors.forename: J T
authors.initials: JT
authors.lastname: Fallon
authors.forename: J
authors.initials: J
authors.lastname: Regnstrom
authors.forename: V
authors.initials: V
authors.lastname: Fuster
mesh_terms: D001161:Arteriosclerosis / Q000378:metabolism*; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D017364:Collagenases / Q000502:physiology*; D018093:Gelatinases / Q000502:physiology*; D006801:Humans; D006909:Hydroxyproline / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D020781:Matrix Metalloproteinase 1; D020778:Matrix Metalloproteinase 2; D008666:Metalloendopeptidases / Q000502:physiology*; D009000:Monocytes / Q000378:metabolism
chemical_list: D003094:Collagen; D017364:Collagenases; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D020781:Matrix Metalloproteinase 1; D006909:Hydroxyproline