Peroxisome proliferators compete and ameliorate Hcy-mediated endocardial endothelial cell activation.
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.
Hunt
Matthew J
MJ
Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, USA.
Tyagi
Suresh C
SC
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002478:Cells, Cultured; D002995:Clofibric Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004699:Endocardium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D058607:Fibric Acids; D006710:Homocysteine / Q000378:metabolism* / Q000494:pharmacology; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008024:Ligands; D020782:Matrix Metalloproteinases / Q000378:metabolism; D051379:Mice; D009206:Myocardium / Q000737:chemistry; D015230:Prostaglandin D2 / Q000031:analogs & derivatives* / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists / Q000378:metabolism*; D014157:Transcription Factors / Q000819:agonists / Q000378:metabolism*
D058607:Fibric Acids; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D006710:Homocysteine; D018799:Intercellular Adhesion Molecule-1; D002995:Clofibric Acid; C037112:9-deoxy-delta-9-prostaglandin D2; D020782:Matrix Metalloproteinases; C019304:ciprofibrate; D015230:Prostaglandin D2