Diltiazem, a calcium antagonist, inhibits matrix metalloproteinase-1 (tissue collagenase) production and collagenolytic activity in human vascular smooth muscle cells

Calcium antagonists (CAs) are widely prescribed for patients with cardiovascular diseases. CAs have been reported to inhibit smooth muscle cell (SMC) proliferation in addition to their effects on vascular tone. To determine whether CAs potentially affect vascular remodeling, we measured the expression of matrix-degrading enzymes in growth factor-stimulated SMC. Human cultured SMC were stimulated with 10 ng/ml of platelet-derived growth factor (PDGF)-BB with or without a calcium antagonist, diltiazem. In the cell counting assay, diltiazem (10-5 M) alone had no effect on the proliferation of quiescent SMC, however 10-6-10-5 M of diltiazem dose-dependently inhibited PDGF-stimulated SMC proliferation. The inhibitory effects of diltiazem on SMC proliferation were further confirmed by a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay and flow cytometry. In Western blotting, matrix metalloproteinase (MMP)-1 (tissue collagenase) but not MMP-2 (72-kDa gelatinase) expression was upregulated by PDGF and phorbol ester (TPA), which were reduced by diltiazem in a dose-dependent manner. The downregulation of MMP-1 expression was consistent with the reduction of collagenolytic activity measured by a FITC-conjugated type I collagen breakdown assay. PDGF-stimulated c-Jun/AP-1 expression, a major transcriptional factor for MMP-1, was not affected by diltiazem. In contrast, intracellular calcium ions measured with a fluorometric assay of Fluo-3AM-loaded cells revealed that the PDGF-stimulated increase in the intracellular calcium content was dose-dependently reduced by diltiazem. Our data suggest that diltiazem inhibits not only proliferation but also MMP-1 expression and collagenolytic activity in PDGF-stimulated SMC. The administration of CAs potentially influences the process of vascular remodeling, and this possibility should be further verified in vivo.