Differential Regulation of Extracellular Matrix Metalloproteinase and Tissue Inhibitor by Heparin and Cholesterol in Fibroblast Cells

Abstract

Heparin has been shown to stimulate angiogenesis in the border zones surrounding infarcted myocardium. Matrix metalloproteinases (MMP), which are involved in extracellular matrix (ECM) organization, have also been shown to be activated. Cholesterol is required for receptor signaling in the plasma membrane, but a role of MMPs for cholesterol in ECM remodeling has not yet been shown. To examine whether heparin and cholesterol induce MMP and tissue inhibitor of metalloproteinase (TIMP) in human heart fibroblast (HHF) cells, confluent HHF cells were treated with cholesterol (100μm) or heparin (20μm). MMP activity was measured using zymography and TIMP was measured by Western blot analysis. The number of HHF cells, measured by a hemocytometer, increased after heparin or cholesterol treatment. Gelatinase A (MMP-2) activity increased in heparin treated cells, and the TIMP-1 level increased in cholesterol-treated cells. Based on Northern blot analysis, we observed that both MMP-1 and MMP-2 were induced at the gene transcription level by heparin and that TIMP-1 was induced by cholesterol. To examine whether the effects of heparin and cholesterol were due to Ca2+mobilization, we carried out Ca2+transient assays using FURA-2/AM as a fluorescence probe in HHF cells. Heparin induced a slow rise in the Ca2+transient with a slow decay, and cholesterol induced a rapid rise with a slow reversal to the baseline calcium level. This suggested that the effect of heparin on Ca2+release from HHF may be secondary to the receptor binding on the cell membrane but that cholesterol may have a direct effect. Protein kinase inhibitor and Ca2+-channel blocker have been shown to inhibit MMP expression. To examine whether the effect of heparin on MMP expression is mediated through the collagenase promoter activity, we carried out gel-shift assays using a 21-oligonucleotide analogue to the MMP-1 promoter sequence. Results suggested that the increase in MMP promoter activity by heparin is due to a specific transcription factor binding to MMP-1 promoter sequence. The effect of cholesterol on fibroblast cell proliferation is due in part to the tissue inhibitor. This study demonstrated the role of heparin and cholesterol in ECM remodeling and has implications for angiogenesis and athersclerosis, respectively.