Extracellular Matrix Modulates Macrophage Functions Characteristic to Atheroma

Collagen Type I Enhances Acquisition of Resident Macrophage Traits by Human Peripheral Blood Monocytes In Vitro

From Emory University School of Medicine, Department of Medicine, Division of Cardiology, Atlanta, Ga.

Correspondence to Zorina S. Galis, PhD, Emory University School of Medicine, Department of Medicine, Division of Cardiology, 1639 Pierce Dr, Woodruff Memorial Bldg, Room 311, Atlanta, GA 30322. E-mail zgalis{at}emory.edu

Abstract—Activated resident macrophages sustain atheroma, and a high macrophage content is associated with plaque vulnerability. Factors leading to differentiation and activation of these blood-derived cells remain largely uncharacterized. We investigated the contribution of interaction with collagen type I, the predominant component of atherosclerotic matrix, to differentiation and modulation of characteristic macrophage functions, including intracellular lipid accumulation and production of the typical matrix-degrading enzyme matrix metalloproteinase (MMP)-9. When used as an adhesion substrate for human peripheral blood monocytes in vitro, collagen type I increased monocyte differentiation, assessed by analysis of CD71 expression and cell spreading. Culturing on collagen type I doubled the number of differentiated monocytes at 24 hours (44.9±1.4% versus 18.4±1.7% on uncoated dishes, P<.001, n=3 independent experiments) and was a stronger stimulus for differentiation than phorbol myristate acetate, a known inducer of monocyte differentiation. The effect of substrate on intracellular accumulation of modified lipoproteins was assessed by quantitative confocal microscopy of monocytes incubated with fluorescent acetylated LDL. The collagen type I substrate also doubled the number of macrophages containing intracellular lipid and significantly increased the individual intracellular loading. Monocytes cultured on collagen type I also released more MMP-9 than did cells plated directly on plastic. The role of monocyte spreading was further assessed by treatment with colchicine, an inhibitor of cytoskeletal function, or with genistein, a nonspecific inhibitor of tyrosine kinases, shown to participate in cell adhesion. Cell spreading was inhibited in 72.3±6.7% of colchicine-treated and in 62.4±6.4% of genistein-treated monocytes (n=3, P<.01 in both cases). The same conditions also decreased secretion of MMP-9, and genistein reduced the number of acetylated LDL-containing cells (from 286±7 to 184±8 cells/mm² with genistein, n=3, P<.001). Data showed a strong correlation (r>.98) between monocyte spreading on collagen type I and intracellular lipid accumulation. Our results indicate that interaction with vascular matrix may play an important role in differentiation of peripheral blood monocytes into resident lipid-laden macrophages, which act as central stimulators throughout the natural history of atheroma.

Key Words: foam cell • atherosclerosis • matrix metalloproteinase

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