Extracellular matrix regulation of metalloproteinase and antiproteinase in human heart fibroblast cells.
Following myocardial infarction, extracellular matrix (ECM) is disrupted, which leads to the generation of collagen- and elastin-derived peptides (CDPs and EDPs, respectively). To investigate whether ECM-derived peptides (i.e., CDPs and EDPs) induce extracellular proteinases in human heart fibroblast (HHF) cells, we isolated CDP and EDP using gelfiltration and antibody affinity column chromatography. The CDP and EDP were characterized by their intrinsic fluorescence due to crosslink structure (pyridinoline and desmosine, respectively) and by immunoblot analysis using anti-desmosine antibody. Neutrophil elastase and cathepsin G were identified using selective chromogenic substrates and by their specific inhibition with alpha1-proteinase inhibitor and alpha1-antichymotrypsin, respectively. Elastase and cathepsin G were elevated in the infarcted tissue. Selective inhibition of matrix metalloproteinase (MMP) by a higher concentration of tetracycline or doxycycline in zymographic gels elicited an inhibition constant (IC50) of 278 +/- 10 microM and indicated that majority of MMP in the infarcted tissue is from fibroblast cells. The HHF proliferation was measured using an acid-phosphatase assay. The EDP and CDP induce HHF cell proliferation. After EDP treatment phenotypic (formation of pseudopodia) changes were observed in HHF cells. To measure whether phenotypic changes by EDP or CDP are associated with MMP and tissue inhibitor of metalloproteinase (TIMP) expression in HHF cells, we measured MMP and TIMP expression by zymographic and Northern blot (mRNA) analyses. The expression of MMP and TIMP were upregulated at both the protein and gene transcription levels. These results suggested that during ischemic cardiomyopathy, initially neutrophil proteinase activates latent myocardial MMP which can degrade ECM, which continuously degrades if not controlled by TIMP, leading to ventricular dilatation and dysfunction.
Tyagi
S C
SC
Department of Internal Medicine, University of Missouri-Health Sciences Center, Columbia 65212, USA.
Kumar
S G
SG
Alla
S R
SR
Reddy
H K
HK
Voelker
D J
DJ
Janicki
J S
JS
D004791:Enzyme Inhibitors / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009203:Myocardial Infarction / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*
D004791:Enzyme Inhibitors; D016326:Extracellular Matrix Proteins; D008666:Metalloendopeptidases