Endothelin-1 attenuates omega3 fatty acid-induced apoptosis by inhibition of caspase 3.
Endothelin-1 (ET-1) may be involved in the induction of vascular hypertrophy in hypertension. ET-1 may also modulate vascular growth through the exertion of antiapoptotic effects. The omega3 fatty acids (omega3 FAs), which have antiproliferative effects in various cell types, may have a beneficial role in hypertension. We tested the hypothesis that ET-1 could act as a survival factor against omega3 FA-induced apoptosis and attempted to elucidate possible molecular mechanisms underlying the protective action of ET-1 on docosahexaenoic acid (DHA)-induced apoptosis. Mesenteric vascular smooth muscle cells were stimulated with DHA, a representative omega3 FA. Dose-response curves of DHA at different apoptotic stages were assessed with the use of flow cytometry: (1) very early: plasma membrane phosphatidylserine (PS) translocation; (2) early: change in mitochondrial transmembrane potential (DeltaPsim); and (3) late: cell cycle analysis. Expression of the proapoptotic protein bax and the antiapoptotic protein bcl-2 was determined with Western blot assay. The activity and the expression of caspase 3, which is a critical proteolytic enzyme involved in the death-signaling pathway, were evaluated with a fluorometric immunosorbent enzyme assay and Western blot analysis, respectively. Apoptosis, which was detected with PS translocation, DeltaPsim disruption, and cell cycle analysis, was increased dose dependently by DHA. DHA-induced apoptosis was attenuated through exposure to ET-1 for 1 hour before DHA in cell cycle analysis. The interference of ET-1 with DHA-induced apoptosis, as detected with cell cycle analysis, was not apparent at the membrane (PS translocation) or the mitochondrial (DeltaPsim) level. The increase in bax/bcl-2 ratio in DHA-stimulated cells was not affected by ET-1. However, DHA increased both caspase 3 activity and the active forms of caspase 3 (20 and 17 kDa), resulting in enhanced DNA fragmentation as shown through Hoechst staining and fluorescence microscopy, which were attenuated by ET-1 pretreatment. In conclusion, DHA, an omega3 FA, induced apoptosis in vascular smooth muscle cells in a dose-dependent manner. ET-1 exerted important protective effects through the attenuation of DHA-induced caspase 3 activation and subsequent DNA fragmentation in the late stages of apoptosis.
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MRC Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal, Montreal, Quebec, Canada.
Intengan
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Schiffrin
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D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D053938:DNA Fragmentation / Q000187:drug effects; D004281:Docosahexaenoic Acids / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D019332:Endothelin-1 / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008638:Mesenteric Arteries / Q000166:cytology; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D010718:Phosphatidylserines / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000096:biosynthesis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D061945:Caspase Inhibitors; D019332:Endothelin-1; D010718:Phosphatidylserines; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D004281:Docosahexaenoic Acids; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases