OXIDIZED LOW-DENSITY LIPOPROTEIN-MEDIATED ACTIVATION OF PHOSPHOLIPASE-D IN SMOOTH-MUSCLE CELLS - A POSSIBLE ROLE IN CELL-PROLIFERATION AND ATHEROGENESIS

Low density lipoproteins (LDL) are risk factors in atherosclerosis and oxidative modification of LDL to oxidized LDL (OX-LDL) increases its atherogenicity. Development of atherosclerosis likely involves OX-LDL- mediated smooth muscle cell (SMC) proliferation. However, the mechanis m(s) of SMC proliferation by OX-LDL is unknown. We hypothesized that O X-LDL may mediate SMC proliferation by activation of phospholipase D ( PLD) through the generation of the second-messenger, phosphatidic acid (PA). To test this hypothesis, activation of PLD by OX-LDL was invest igated in [H-3]myristic acid- or [P-32]orthophosphate-labeled rabbit f emoral artery smooth muscle cells (RFASMC) in the presence of 0.5% eth anol or 0.05% butanol. Phospholipase D activation, as measured by labe led phosphatidylethanol (PEt) or phosphatidylbutanol (PBt) formation, was enhanced (3- to 5-fold) by OX-LDL. This activation of PLD was spec ific for OX-LDL, as native LDL or acetylated LDL had no effect. Furthe r, OX-LDL-mediated [P-32]PEt formation was dose- and time-dependent. T o determine the mechanism(s) of OX-LDL-induced PLD activation, the rol e of protein kinase C (PKC) and Ca2+ was investigated. Pretreatment of [P-32]orthophosphate-labeled RFASMC with known inhibitors of PKC such as staurosporine, calphostin-C, or H-7, had no effect on OX-LDL-induc ed PLD activation. Also, down-regulation of PKC by 12-O-tetradecanoylp horbol 13-acetate (TPA) (100 nM, 18 h) did not alter the OX-LDL-mediat ed [P-32]PEt formation. However, pretreatment of RFASMC with genistein , a putative inhibitor of tyrosine kinases, attenuated the OX-LDL-medi ated [P-32]PEt formation. In addition, exposure of RFASMC to sodium or thovanadate, an inhibitor of phosphatases, enhanced the OX-LDL-mediate d PLD activation. The effects of genistein and vanadate on PLD activat ion were specific for OX-LDL as these agents did not alter the TPA-ind uced [P-32]PEt formation. Treatment of quiescent RFASMC with OX-LDL in creased [H-3]thymidine incorporation into DNA. This enhanced incorpora tion of [H-3]thymidine into DNA was also mimicked by exogenously added phosphatidic acid (PA) or lysophosphatidic acid (LPA). These findings suggest that OX-LDL is a potent activator of the PLD pathway in SMC. The activation of PLD by OX-LDL generates second-messengers like PA an d/or LPA which modulate mitogenesis. Thus, these results indicate that OX-LDL, in atherosclerotic lesions, may enhance SMC proliferation thr ough the modulation of signal transduction pathways including activati on of PLD.