血管平滑肌細胞(vascular smooth muscle cell，簡稱VSMC)的存活在血管的完整性上扮演極重要的角色，當血管受到損傷所導致VSMC的細胞凋亡(apoptosis)，除了會引發其他VSMC的移動(migration)與增生(proliferation)外，更會誘導血管內膜增生(intimal hyperplasia)及血管重新塑造(vascular remodeling)，在氧化壓力(oxidative stress)下VSMC的存活機制到目前為止仍未完全被了解。我們假設在血管內的前列腺素(prostacyclin，簡稱PGI2)主要由血管內皮細胞(vascular endothelial cell，簡稱EC)所產生而不是VSMC，而此由EC所產生之PGI2保護VSMC的存活，主要是藉由恢復過氧化體增殖物活化α受體(peroxisome proliferator-activated receptor α，簡稱PPARα)依靠之途徑中的14-3-3蛋白。我們使用能同時大量表現環氧化酶-1(cyclooxygenase-1)與PGI合成酶(prostacyclin synthase)的腺病毒載體(adenoviral vector簡稱Ad-COPI)轉染大鼠VSMC，使得VSMC自行生產大量PGI2，Ad-COPI轉染的VSMC對於H2O2誘導的細胞凋亡有較強的耐受性，同時carbaprostacyclin (簡稱cPGI2)為一種PGI2的穩定類似物，亦會降低H2O2誘導的細胞凋亡。cPGI2的抗細胞凋亡的作用會被PPARα抑制劑MK886所破壞，此外PPARα專一性啟動劑Wy14643除了能抑制H2O2誘導的細胞凋亡，由此可知此抗細胞凋亡的作用是經由PPARα。同時在VSMC大量表現PPARα會降低H2O2誘導的Caspase 3活化，由此可知PPARα在抵抗細胞凋亡中扮演一個關鍵的角色。緊接著要來確定cPGI2及PPARα啟動劑保護VSMC是經由轉錄調控14-3-3的表現，我們首先分析了7個不同的14-3-3 isoforms，除了σ之外，其餘6個isoform在VSMC內皆持續性的表現，一旦VSMC遭遇氧化壓力，H2O2會降低14-3-3β和θ的蛋白表現量。當使用Caspase 3抑制劑z-DEVD-FMK抑制Caspase 3活性時只有14-3-3β蛋白量恢復，同時在基礎狀態與H2O2作用之下，Ad-COPI及cPGI2亦能增加與恢復14-3-3β蛋白量，使用PPARα啟動劑Wy14643及GW9578大量表現PPARα也能達到相同的效果。當我們大量表現14-3-3β、θ和ε，只有表現14-3-3β具有降低apoptosis的效果，同時使用小分子RNA干擾(small interference RNA)減少14-3-3β的表現量能夠增加Caspase 3的活性。當14-3-3β蛋白量的增加伴隨著促進與Bad(Bcl-2-associated death promoter)蛋白的交互作用，使得Bad蛋白被隔離於細胞質而無法進入粒線體，因此降低apoptosis機制的啟動。綜合以上研究結果得知PPARα所引起14-3-3β蛋白量的增加，在保護細胞抵抗H2O2引起細胞凋亡的機制中扮演一個不可或缺的關鍵角色。

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 PPARα14-3-3β pathway is a target for developing vascular drugs.

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