EXTRACELLULAR-MATRIX REGULATION OF METALLOPROTEINASE AND ANTIPROTEINASE IN HUMAN HEART FIBROBLAST CELLS

Following myocardial infarction, extracellular matrix (ECM) is disrupt ed, which leads to the generation of collagen- and elastin-derived pep tides (CDPs and EDPs, respectively). To investigate whether ECM-derive d peptides (i.e., CDPs and EDPs) induce extracellular proteinases in h uman heart fibroblast (HHF) cells, we isolated CDP and EDP using gelfi ltration and antibody affinity column chromatography. The CDP and EDP were characterized by their intrinsic fluorescence due to crosslink st ructure (pyridinoline and desmosine, respectively) and by immunoblot a nalysis using anti-desmosine antibody. Neutrophil elastase and catheps in G were identified using selective chromogenic substrates and by the ir specific inhibition with alpha 1-proteinase inhibitor and alpha 1-a ntichymotrypsin, respectively. Elastase and cathepsin G were elevated in the infarcted tissue. Selective inhibition of matrix metalloprotein ase (MMP) by a higher concentration of tetracycline or doxycycline in zymographic gels elicited an inhibition constant (IC50) of 278 +/- 10 mu M and indicated that majority of MMP in the infarcted tissue is fro m fibroblast cells. The HHF proliferation was measured using an acid-p hosphatase assay. The EDP and CDP induce HHF cell proliferation. After EDP treatment phenotypic (formation of pseudopodia) changes were obse rved in HHF cells. To measure whether phenotypic changes by EDP or CDP are associated with MMP and tissue inhibitor of metalloproteinase (TI MP) expression in HHF cells, we measured MMP and TIMP expression by zy mographic and Northern blot (mRNA) analyses. The expression of MMP and TIMP were upregulated at both the protein and gene transcription leve ls. These results suggested that during ischemic cardiomyopathy, initi ally neutrophil proteinase activates latent myocardial MMP which can d egrade ECM, which continuously degrades if not controlled by TIMP, lea ding to ventricular dilatation and dysfunction. (C) 1996 Wiley-Liss, I nc.