Study of the molecular mechanism and clinical implication of a novel apoptosis gene

血管平滑肌细胞（SMC）的凋亡（程序性细胞死亡）最近在动脉粥样硬化和血管成形术后再狭窄等疾病状态下的血管壁以及生理性动脉重构中被认识到。动脉粥样硬化斑块中细胞的凋亡减少导致不稳定斑块的形成，不稳定斑块容易破裂并引发心血管事件。我们在小鼠动脉粥样硬化平滑肌细胞中发现了一个新的基因PL-3。瞬时转染PL-3表达载体诱导培养细胞凋亡。为了阐明pl -3诱导凋亡的机制，我们进行了Western blotting分析，发现细胞色素c从线粒体释放到胞质空间，随后激活caspase-9和caspase-3。通透性过渡孔（PTP）抑制剂、1-肉碱或环孢素A均能抑制线粒体细胞色素c的释放，但抗凋亡bcl-2家族蛋白bcl-2或bcl-xL不能抑制细胞色素c的释放。亲环蛋白D （PTP的一种成分）缺乏也能阻止PL-3诱导的细胞凋亡。这些数据表明，由PL-3表达诱导的线粒体细胞色素c释放依赖于PTP而不是bcl-2家族相关通道。为了阐明PL-3的生理功能，我们建立了利用反义质粒或RNAi抑制PL-3的实验体系。凋亡诱导试剂溶血磷脂酰氯、硝普钠或缺氧（1%氧）诱导的细胞死亡可通过抑制PL-3的表达而得以避免。这一结果表明在生理条件下，PL-3的表达与细胞死亡有关。我们已经生成了一种靶向PL-3的胚胎干细胞，并将其导入囊胚中，制造出具有PL-3基因的敲除小鼠。此外，我们建立了PL-3的腺病毒表达系统，用于研究颈动脉球囊损伤后肥大大鼠模型的基因治疗。

Apoptosis (programmed cell death) of vascular smooth muscle cells (SMC) has been recognised recently in the vessel wall in disease states such as atherosclerosis and restenosis after angioplasty, and also in physiological arterial remodelling. The decrease of cells in atherosclerotic plaques by apoptosis contributes to the formation of unstable plaques, which are prone to rupture and trigger cardiovascular events. We have identified a novel gene, PL-3,in atherosclerotic smooth muscle cells of mice. Transient transfection of PL-3 expression vector induced apoptosis of cultured cells. To clarify the mechanism of PL-3-induced apoptosis, we performed Western blotting analysis, and found that cytochrome c is released from mitochondria into cytosolic space, followed by the activating caspase-9 and caspase-3. The cytochrome c release from mitochondria was inhibited by permeability transition pore (PTP) inhibitors, 1-carnitine or cyclosporin A, but not by bcl-2 or bcl-xL, anti-apoptotic Bcl-2 family proteins. Cyclophilin D (a component of PTP)-deficiency also prevented the apoptosis induced by PL-3. These data indicate that cytochrome c release from mitochondria induced by PL-3 expression is dependent on PTP rather than bcl-2 family-related channels. In order to clarify the physiological function of PL-3,we established experimental system of PL-3 inhibition using antisense plasmid or RNAi. Cell death induced by apoptosis-inducing reagents including lysophosphatidyl chorine, sodium nitroprusside or by hypoxia (1% oxygen), was prevented by the inhibition of PL-3 expression. This result indicates that PL-3 expression is implicated in cell death under physiological conditions. We have generated a PL-3-targeted ES cells to and introduced into the blastcysts to make a knockout mice of PL-3 gene. Furthermore, we have established an adenoviral expression system of PL-3 for the study gene therapy using rat models of carotid artery hypertrophy after balloon injury.