Abstract
Vascular smooth muscle cell survival is vital to blood vessel integrity. Vascular injury leads to VSMC apoptosis which triggers VSMC migration and proliferation to induce intimal hyperplasia and vascular remodeling. The mechanism by which VSMCs survives under oxidative stress is not entirely clear. We postulated that prostacyclin (PGI2) which is produced by vascular endothelial cells and to a lesser extent by VSMC, protects VSMC against apoptosis by restoring 14-3-3 through a peroxisome proliferator-activated receptor (PPAR)-dependent pathway. We transfected rat VSMCs with an adenoviral vector containing a bicistronic COX-1/PGI synthase construct (Ad-COPI) which co-overexpresses the essential synthetic enzymes (COX-1 and PGI synthase) resulting in robust authentic PGI2 production in VSMC. Ad-COPI transfected cells were less susceptible to apoptosis induced by H2O2. Carbaprostacyclin (cPGI2), a stable analog of PGI2 also attenuated H¬2O2-induced apoptosis. The anti-apoptotic effect of cPGI2 was abrogated by MK 886, a PPARα antagonist and not GSK 3787, a PPARδ antagonist, suggesting that its action is mediated via PPARα. PPARα agonists such as Wy14643 and GW9578 suppressed H2O2-induced apoptosis. Furthermore, PPARα overexpression reduced H2O2-induced caspase 3 activation. These results confirm that PPARα is pivotal in conferring resistance to apoptosis. To determine that PGI2 and PPARα agonists protect VSMC via 14-3-3, we analyzed the protein level of 7 mammalian 14-3-3 isoforms. All isoforms except 14-3-3σ were constitutively expressed. H2O2 treatment resulted in reduction of 14-3- 3β and 14-3-3θ isoforms. 14-3-3β but not 14-3-3θ reduction was rescued by caspase 3 inhibitor, z-DEVD-FMK. Ad-COPI and cPGI2 increased basal 14-3-3β and restored 14-3-3β in H2O2-treated cells. Similarly, Wy14643 as well as PPARα overexpression increased 14-3-3β expression and restored 14-3-3β in H2O2 treated cells. PGI2 and PPARα agonists increase 14-3-3β by transcriptional upregulation. 14-3-3β overexpression attenuated H2O2-induced apoptosis whereas 14-3-3θ or 14-3-3ε overexpression had no effect. Knockdown of 14-3-3β increased VSMC caspase 3 activation. 14-3-3β upregulation was accompanied by enhanced Bad sequestration and reduced Bad translocation to mitochondria. These results reveal that 14-3-3β occupies a central position in oxidant induced apoptosis in VSMCs. The PPARα14-3-3β pathway is a target for developing vascular drugs.