Abstract
Migration of cardiac fibroblasts is implicated in infarct healing and ventricular remodeling. Activation of matrix metalloproteinases induced by three-dimensional type I collagen, the principal component of the myocardial interstitium, is hypothesized to be essential for this migration. By utilizing primary cultures of cardiac fibroblasts and collagen lattice models, we demonstrated that type I collagen induced MMP-2 activation, and cells undergoing a change from isometric tension to mechanical unloading were associated with increased levels of total and active MMP-2 species. The collagen-induced MMP-2 activation coincided with up-regulated cellular levels of both membrane type 1-matrix metalloproteinase (MT1-MMP) and TIMP-2. A fraction of cellular membrane prepared from cells embedded in the collagen lattice containing active MT1-MMP and TIMP-2 was capable of activating pro-MMP-2, and exogenous TIMP-2 had a biphasic effect on this membrane-mediated MMP-2 activation. Interestingly, the presence of 43-kDa MT1-MMP species in a fraction of intracellular soluble proteins prepared from monolayer cells but not cells embedded in the lattices indicates that MT1-MMP metabolizes differently under the two different culture conditions. Treatment of cells embedded in the lattice with furin inhibitor attenuated pro-MT1-MMP processing and MMP-2 activation and impeded cell migration and invasion. These results suggest that the migration and invasion of cardiac fibroblasts is furin-dependent and that the active species of MT1-MMP and MMP-2 may be involved in both events.
Highlights
Cardiac fibrosis, a consequence of infarct healing and ventricular remodeling, is characterized by hyperplasia of ␣-smooth muscle actin expressing fibroblast-like cells and deposition of excessive extracellular matrix (ECM)1 proteins by these cells in the myocardium
In an earlier study using neonatal rat cardiac fibroblasts embedded in three-dimensional type I collagen lattice [4], a high level of active MMP-2 species was only detected in whole cell lysates prepared from the neonatal cells
Cardiac fibroblasts embedded in three-dimensional type I collagen lattices secrete, and are associated with, high levels of active MMP-2
Summary
Cell Culture—Cardiac fibroblasts were prepared by enzymatic dissociation of left ventricular tissue from adult male Sprague-Dawley rats (250 – 450 g), as described previously [34], and maintained in minimal essential medium (MEM) (Invitrogen) containing 10% (v/v) newborn bovine calf serum (Invitrogen) at 37 °C, in humidified air supplemented with 5% CO2 until the cells were almost confluent. Preparation of Whole Cell Lysates—Polymerized collagen lattices containing embedded cells were centrifuged at 400 ϫ g to remove as much water as possible, suspended in a large volume of ice-cold PBS, mixed well, and centrifuged again This wash procedure was performed 5 times. Following 24 h of culture, monolayer cells or cell-populated collagen lattices were washed three times with PBS and incubated with PBS (pH 7.2) containing 0.1% (w/v) saponin, 75 mM potassium acetate, and 25 mM Hepes for 30 min at room temperature. When migration or invasion experiments were performed, trypsinized cardiac fibroblasts were preincubated with 50 M furin inhibitor I (FI, decanoyl-RVKR-chloromethyl ketone, Calbiochem) for 20 min and seeded in type I collagen solution at a density of ϳ3.0 ϫ 105 cells/ml. All values are presented as the means Ϯ S.E. in n experiments as indicated. p values less than 0.05 (p Ͻ 0.05) were considered statistically significant
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