12225971

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4

To determine whether homocysteine (Hcy)-mediated activation of endocardial endothelial (EE) cells is ameliorated by peroxisome proliferator-activated receptor (PPAR), we isolated EE cells from mouse endocardium. Matrix metalloproteinase (MMP) activity and intercellular adhesion molecule (ICAM)-1 in EE cells were measured in the presence and absence of Hcy, and ciprofibrate (CF; PPAR-alpha agonist) or 15-deoxy-Delta(12,14)-prostaglandin J(2) (PGJ(2); PPAR-gamma agonist) by zymography and Western blot analyses, respectively. Results suggest that Hcy-mediated MMP activation and ICAM-1 expression are ameliorated by CF and PGJ(2). To test the hypothesis that Hcy competes with other ligands for binding to PPARalpha and -gamma, we prepared cardiac nuclear extracts. Extracts were loaded onto an Hcy-cellulose affinity column. Bound proteins were eluted with CF and PGJ(2). To determine conformational changes in PPAR upon binding to Hcy, we measured PPAR fluorescence at 334 nm. Dose-dependent increase in PPAR fluorescence demonstrated a primary binding affinity of 0.32 +/- 0.06 microM. There was dose-dependent quenching of PPAR fluorescence by fluorescamine-homocysteine (F-Hcy). PPAR-alpha fluorescence quenching was abrogated by the addition of CF but not by PGJ(2). PPAR-gamma fluorescence quenching was abrogated by the addition of PGJ(2) but not by CF. These results suggest that Hcy competes with CF and PGJ(2) for binding to PPAR-alpha and -gamma, respectively, indicating a role of PPAR in amelioration of Hcy-mediated EE dysfunction.

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0363-6143

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C1073-9

pubmed-meshheading:12225971-Animals, pubmed-meshheading:12225971-Binding, Competitive, pubmed-meshheading:12225971-Cells, Cultured, pubmed-meshheading:12225971-Clofibric Acid, pubmed-meshheading:12225971-Dose-Response Relationship, Drug, pubmed-meshheading:12225971-Endocardium, pubmed-meshheading:12225971-Endothelium, Vascular, pubmed-meshheading:12225971-Fibric Acids, pubmed-meshheading:12225971-Homocysteine, pubmed-meshheading:12225971-Intercellular Adhesion Molecule-1, pubmed-meshheading:12225971-Ligands, pubmed-meshheading:12225971-Matrix Metalloproteinases, pubmed-meshheading:12225971-Mice, pubmed-meshheading:12225971-Myocardium, pubmed-meshheading:12225971-Prostaglandin D2, pubmed-meshheading:12225971-Protein Conformation, pubmed-meshheading:12225971-Receptors, Cytoplasmic and Nuclear, pubmed-meshheading:12225971-Transcription Factors

Peroxisome proliferators compete and ameliorate Hcy-mediated endocardial endothelial cell activation.

Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't