Peroxisome proliferators compete and ameliorate Hcy-mediated endocardial endothelial cell activation |
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Authors: | Hunt Matthew J Tyagi Suresh C |
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Affiliation: | Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, USA. |
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Abstract: | To determine whether homocysteine(Hcy)-mediated activation of endocardial endothelial (EE) cells isameliorated by peroxisome proliferator-activated receptor (PPAR), weisolated EE cells from mouse endocardium. Matrix metalloproteinase(MMP) activity and intercellular adhesion molecule (ICAM)-1 in EE cellswere measured in the presence and absence of Hcy, and ciprofibrate (CF;PPAR- agonist) or 15-deoxy-12,14-prostaglandinJ2 (PGJ2; PPAR- agonist) by zymography andWestern blot analyses, respectively. Results suggest that Hcy-mediated MMP activation and ICAM-1 expression are ameliorated by CF and PGJ2. To test the hypothesis that Hcy competes with otherligands for binding to PPAR and -, we prepared cardiac nuclearextracts. Extracts were loaded onto an Hcy-cellulose affinity column.Bound proteins were eluted with CF and PGJ2. To determineconformational changes in PPAR upon binding to Hcy, we measured PPARfluorescence at 334 nm. Dose-dependent increase in PPAR fluorescencedemonstrated a primary binding affinity of 0.32 ± 0.06 µM. There wasdose-dependent quenching of PPAR fluorescence byfluorescamine-homocysteine (F-Hcy). PPAR- fluorescence quenching wasabrogated by the addition of CF but not by PGJ2. PPAR-fluorescence quenching was abrogated by the addition ofPGJ2 but not by CF. These results suggest that Hcy competeswith CF and PGJ2 for binding to PPAR- and -,respectively, indicating a role of PPAR in amelioration of Hcy-mediatedEE dysfunction. |
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