APOPTOSIS OF CARDIAC MUSCLE AS A TARGET FOR GENE THERAPY

细胞程序性死亡(细胞凋亡)日益被认为是一种 心肌缺血/再灌流后心肌细胞丢失的原因 损伤、心肌梗死和长期心力衰竭。而当 在这些环境中启动细胞凋亡的分子机制仍然存在 在未经证实的情况下，有研究表明，包括 BCL-2本身和Bax是细胞凋亡的一般调节因子 神经酰胺途径、DNA损伤等多种原因。vbl.使用 腺病毒基因转移到体外培养的心肌细胞的研究 证实了Bcl2和Bcl2预期的抗细胞凋亡作用 腺病毒E1B(Bax的一种抑制剂)，在这种细胞背景下。其他 至少在培养的细胞中建立了覆盖凋亡信号的手段 哺乳动物细胞包括：白细胞介素1β转化的抑制物 酶(ICE)/CED-3家族蛋白；细胞因子受体突变 Fas/APO-1及相关死亡结构域蛋白；哺乳动物家族 或杆状病毒凋亡抑制蛋白(IAPs)；以及一种显性- 阴性形式的肝白血病因子(HLF)，一种哺乳动物同源物 对于转录因子CES-2，保守的细胞死亡规范 秀丽隐杆线虫的蛋白质。然而，目前几乎没有或 关于保护哺乳动物的可能性，我们一无所知 通过一种或多种途径抑制心肌细胞的凋亡 机械装置。在目前的建议中，腺病毒基因转移到成人 小鼠心肌将被用来测试以下假设： (1)Bcl2和Bclxl，(2)ICE/CED-3酶抑制剂，(3)优势- 干扰死亡结构域蛋白，(4)IAP，和(5)显性- 干扰CES-2转录因子将对 活体心脏细胞凋亡。心脏限制性转基因将是 并行使用的。因此，这项建议将系统地测试基因 冠状动脉结扎后转移以保护细胞免于凋亡， 对于五种互补的蛋白质类，具有已证实的抑制能力 细胞凋亡，至少在体外是这样。到目前为止，只有E1B和Bcl-2被认为是 抑制心肌细胞的凋亡，即使在培养中也是如此。给定 细胞凋亡是导致心肌死亡的原因的证据程度， 尤其是在急性脑梗塞和再灌注损伤的情况下， 迫切需要进行研究来定义分子 在这些环境中驱动细胞凋亡的途径 体内阻断细胞凋亡途径的干预措施。

Programmed cell death (apoptosis) is recognized, increasingly, as a contributing cause of cardiac myocyte loss with ischemia/reperfusion injury, myocardial infarction, and long-standing heart failure. While the molecular mechanisms initiating apoptosis in these settings remain unproven, it has been suggested that Bcl-2 family members including Bcl-2 itself and Bax are general regulators of apoptosis arising from the ceramide pathway, DNA damage, and other diverse causes. Using adenoviral gene transfer to cardiac myocytes in vitro, the Investigator has substantiated the predicted anti-apopotic effect of Bcl-2 and adenoviral E1B (an inhibitor of Bax), in this cell background. Other established means to override apoptotic signals at least in cultured mammalian cells include: inhibitors of interleukin-1beta converting enzyme (ICE)/CED-3 family proteases; mutations of the cytokine receptor Fas/APO-1 and related death domain proteins; the family of mammalian or baculovirus inhibitor of Apoptosis Proteins (IAPs); and a dominant- negative form of hepatic leukemia factor (HLF), a mammalian homologue for the transcription factor CES-2, a conserved cell death specification protein in Caenorhabditis elegans. At present, however, little or nothing is known concerning the potential to protect mammalian ventricular muscle from apoptosis, through one or more of these mechanisms. In the present proposal, adenoviral gene transfer to adult mouse myocardium will be used to test the hypotheses that delivery of: (1) Bcl-2 and Bcl-Xl, (2) ICE/CED-3 protease inhibitors, (3) dominant- interfering death domain proteins, (4) IAPs, and (5) dominant- interfering CES-2 transcription factors will be protective against cardiac apoptosis in vivo. Cardiac-restricted transgenes will be utilized in parallel. Thus, this proposal will systematically test gene transfer for protection from apoptosis after coronary artery ligation, for five complementary classes of protein with proven ability to inhibit apoptosis, at least in vitro. To date, only E1B and Bcl-2 are known to inhibit apoptosis of cardiac myocytes, even in culture. Given the extent of evidence for apoptosis as a cause of cardiac muscle death, especially in the acute settings of infarction and reperfusion injury, there is a compelling need for research that would define the molecular pathways driving apoptosis in these settings and for potential interventions to arrest the apoptotic pathway in vivo.