Role of APEG-1 in Vascular Smooth Muscle Biology

描述(来自应用程序的逐字)：血管平滑的增殖 肌细胞(VSMC)是动脉硬化的标志。因为没有基因有 已经被发现专门抑制VSMC的增殖，它已经被 很难为VSMC过度生长开发有效的治疗方法。我们有 分离到编码APEG的基因--L，这是一种新的在 分化的VSMC。在体外和体内VSMC去分化过程中， APEG-L基因的表达水平迅速下调。重组病毒感染血管平滑肌细胞的研究 组成性活性cGMP反应蛋白激酶(PKG)腺病毒 逆转了这一下调监管的趋势。APEG-L表达载体的显微注射 INTO可抑制VSMC的DNA合成，提示APEG-L是一种生长抑制因子。 因此，我们假设APEG-L是一种VSMC特异性生长抑制因子， 在cGMP/PKG信号通路下游发挥作用。2.7kb的片段 小鼠APEG-L 5-侧翼基因引导的高水平血管平滑肌细胞特异性启动子 活性可被位于5‘端的0.8kb的APEG-L DNA片段所抑制。 到2.7kb的片段。我们提议的工作的目标是调查 介导APEG-L血管内皮细胞特异性启动子活性的分子机制 检测APEG-L是否为VSMC特异性生长抑制物，并确定 L调节血管平滑肌细胞生长分化的分子机制。 为了实现这些目标，我们提出了四个具体目标。目标1.确定具体的 DNA序列(顺式作用元件)及其同源DNA结合蛋白 (反式作用蛋白)对APEG-L S在血管内皮细胞中的限制性表达至关重要 在试管中。此外，我们还将确定cGMP反应元件(S)和 它们在APEG-L启动子中的结合蛋白。目的2.测试特异性和 AIM1中APEG-L VSMC特异性顺式作用元件的效力 使用重组腺病毒或含有由 老鼠体内的这些元素。目的3.研究APEG-L对小鼠生长发育和生长发育的影响。 VSMC的分化。目的4.分离APEG-L的互动伙伴以 阐明APEG-1调控VSMC生长的分子机制 差异化。从这些拟议的实验中获得的信息应该 提供关于APEG生物学的基础知识--L和 VSMC的分化，以及对VSMC治疗的关键洞察 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特异性的鉴定 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.