Role of APEG-1 in Vascular Smooth Muscle Biology

描述（申请书中的逐字描述）：血管平滑肌增殖 肌肉细胞（VSMC）是动脉硬化的标志。因为没有基因 已经鉴定出特异性抑制VSMC增殖， 难以开发出有效的治疗VSMC过度生长的方法。我们有 分离了编码APEG-1的cDNA，APEG-1是一种新的蛋白质，优先表达于 分化的VSMC。在体内外VSMC脱分化过程中， APEG-1 mRNA表达迅速下调。用重组体感染VSMC 组成型活性cGMP应答蛋白激酶腺病毒 逆转了这种下调。APEG-I表达质粒的显微注射 APEG-1抑制VSMC DNA合成，提示APEG-1是VSMC的生长抑制剂。 因此，我们假设APEG-1是VSMC特异性生长抑制剂， 在cGMP/PKG信号通路的下游起作用。一个2.7kb的 小鼠APEG-15-侧翼DNA指导的高水平VSMC特异性启动子 活性，该活性可被位于5'端的0.8kb的APEG-1DNA片段抑制。 2.7kb的片段。我们提出的工作目标是调查 介导APEG-1的VSMC特异性启动子活性的分子机制， 测试APEG-I是否是VSMC特异性生长抑制剂，并确定APEG-I是否是VSMC特异性生长抑制剂。 APEG-I调节VSMC生长和分化的分子机制。 为了实现这些目标，我们提出了四个具体目标。AIM 1.确定具体 DNA序列（顺式作用元件）及其同源DNA结合蛋白 对APEG-I在VSMC中的限制性表达重要的（反式作用蛋白） 体外此外，我们将鉴定cGMP响应元件， 它们在APEG-1启动子中的结合蛋白。AIM 2.检测特异性， AIM 1中鉴定的APEG-1 VSMC特异性顺式作用元件的效力 使用重组腺病毒或含有由 这些元素在老鼠身上。AIM 3.研究APEG-I对生长和代谢的影响。 VSMC的分化。目标4.分离APEG-1的相互作用伙伴， 阐明APEG- 1调节VSMC生长的分子机制， 分化从这些拟议的实验中获得的信息应 提供有关APEG-I生物学的基础知识， VSMC的分化，以及对VSMC治疗的重要见解。 冠状动脉搭桥术后动脉粥样硬化和再狭窄， 血管成形术/支架置入。此外，VSMC特异性 启动子将是未来使用基因治疗来引导外源基因的关键 在血管壁中的表达。

DESCRIPTION (Verbatim from the application): Proliferation of vascular smooth muscle cells (VSMC) is a hallmark of arteriosclerosis. Because no genes have been identified that specifically inhibit VSMC proliferation, it has been difficult to develop an effective therapy for excessive VSMC growth. We have isolated the cDNA encoding APEG-l, a novel protein preferentially expressed in differentiated VSMC. During dedifferentiation of VSMC in vitro and in vivo, APEG-l mRNA underwent a rapid downregulation. Infecting VSMC with a recombinant adenovirus of constitutively active cGMP-responsive protein kinase (PKG) reversed this downregulation. Microinjection of an APEG-l expression plasmid into VSMC inhibited DNA synthesis, implicating APEG-l as a growth inhibitor. Thus, we hypothesize that APEG-l is a VSMC-specific growth inhibitor that functions downstream of the cGMP/PKG-signaling pathway. A 2.7-kb fragment of mouse APEG-l 5-flanking DNA directed a high level of VSMC-specific promoter activity, which could be repressed by a 0.8-kb APEG-l DNA fragment located 5' to the 2.7-kb fragment. The goals of our proposed work are to investigate the molecular mechanisms mediating APEG-l's VSMC-specific promoter activity, to test whether APEG-l is a VSMC-specific growth inhibitor, and to determine the molecular mechanisms by which APEG-l regulates VSMC growth and differentiation. To achieve these goals, we propose four specific aims. AIM 1. Identify specific DNA sequences (cis-acting elements) and their cognate DNA-binding proteins (trans-acting proteins) important for APEG-l 's restricted expression in VSMC in vitro. In addition, we will identify the cGMP-responsive element(s) and their binding proteins in the APEG-l promoter. AIM 2. Test the specificity and potency of the APEG-l VSMC-specific cis-acting elements identified in AIM 1 by using recombinant adenoviruses or transgenes containing reporters driven by these elements in mice. AIM 3. Study the effect of APEG-l on growth and differentiation in VSMC. AIM 4. Isolate APEG- l's interaction partners to elucidate the molecular mechanisms by which APEG- 1 regulates VSMC growth and differentiation. information obtained from these proposed experiments should provide fundamental knowledge about the biology of APEG-l and the differentiation of VSMC, and also critical insight into the treatment of atherosclerosis and restenosis after coronary artery bypass or angioplasty/stent placement. Furthermore, the identification of VSMC-specific promoters will be crucial for future use of gene therapy to direct foreign gene expression in the blood vessel wall.