P2253Intake of the omega 3 PUFAs formulation EPA:DHA 6:1 by aged rats reduced shedding of microvesicles from spleen-derived cultured leukocytes and their ability to promote senescence in endothelial cells

Abstract Introduction Ageing is associated with the appearance of endothelial senescence promoting endothelial dysfunction and, ultimately, cardiovascular events. Circulating microvesicles (MVs) of patients with acute coronary syndrome promoted premature endothelial senescence by stimulating the local angiotensin system. Omega 3 PUFAs have been shown to reduce the risk of cardiovascular disease in patients at high risk. Purpose This study investigated whether a 7-day intake of the omega 3 formulation EPA:DHA 6:1 by rats affects the level of MVs released by spleen-derived cultured leukocytes as well as their ability to promote premature senescence in target endothelial cells (ECs), and, if so, to clarify the underlying mechanism. Methods Middle-aged male Wistar rats (M, 48-week old) received 500 mg/kg/d of either EPA:DHA 6:1, EPA:DHA 1:1, or vehicle (CTL) for 7 days. Thereafter, spleen-derived leukocytes, a rich source of MVs, were prepared and cultured for 24 h. Cultured ECs were prepared from porcine coronary arteries. Senescence-associated β-galactosidase activity (SA-β-gal) was assessed by C12FDG, protein expression level by Western blot analysis, oxidative stress by dihydroethidium using confocal microscopy, and procoagulant MVs by prothrombinase assay. Spleen-derived leukocytes from untreated young (Y, 12-week) and old (O, 72-week) rats were also studied. Results Shedding of MVs by spleen-derived leukocytes significantly increased with increasing age. Incubation of ECs with leukocyte-derived MVs (10 nM Phtd Ser eq.) from M and O but not those from Y induced premature senescence after 48 h. The stimulatory effect of M-MVs was prevented by losartan and associated with oxidative stress. M-MVs induced an upregulation of senescence markers (p16, p21, p53), pro-atherothrombotic markers (VCAM-1, ICAM-1, tissue factor), the pro-inflammatory marker cyclooxygenase-2 (COX-2) but not COX-1, and of the angiotensin system (angiotensin-converting enzyme and type 1 angiotensin receptor), whereas endothelial NO synthase was down-regulated. A one-week intake of EPA:DHA 1:1 and 6:1 by M rats decreased the leukocyte-derived MVs shedding by about 14% and 24%, and EPA:DHA 6:1 reduced their ability to induce ECs senescence by 38%. The stimulatory effect of M-MVs on the expression of target proteins was also observed with those from the EPA:DHA 1:1 but not with those from the 6:1 group. Conclusion These findings indicate that ingestion of EPA:DHA 6:1 by middle-aged rats reduces not only the shedding of MVs by spleen-derived leukocytes but also their ability to induce pro-senescent, pro-thrombotic and pro-inflammatory responses in endothelial cells most likely by decreasing the local angiotensin system. They further suggest that EPA:DHA 6:1 may help to delay ageing-related endothelial dysfunction. Acknowledgement/Funding Unrestricted research grant from PIVOTAL Therapeutics Inc.