VASOACTIVE HORMONE INDUCED GENES IN DIABETIC VASCULATURE

糖尿病和高血压密切相关，是主要的危险因素。 血管疾病发展中的因素。不断发展的试验性和 临床证据表明，肾素-血管紧张素系统 (RAS)在这些血管并发症的发病机制中起作用。 这些研究表明，血管紧张素转换酶(ACE) 抑制剂通过以下途径减少糖尿病血管并发症的进展 这是一种部分独立于血压控制的机制。 最近，在筛选血管活性激素的新作用时， 差异显示，我发现血管紧张素II(AII) 是纤溶酶原激活物抑制物-2(PAI-2)的有效刺激因子 大鼠微血管内皮细胞和大鼠主动脉中的表达 平滑肌细胞(RASMC)。这些研究已经扩展到表明 在这些血管细胞中，PAI-1的表达也同样被诱导。 对AII刺激PAI-2表达机制的研究揭示 依赖于PKC和依赖于PKC的信号通路都有助于 44.8+/-12.5(+/-S.E.M)使RME细胞PAI-2mRNA表达增加。这个 与AII刺激的PAI-2偶联的PKC非依赖性通路 表达，被酪氨酸激酶抑制剂完全阻断 染料木素。相比之下，AII刺激了12.4+/-1.6倍的增长 PAI-2mRNA在RASMC中的表达，这种增加完全与PKC有关 激活。我们已经开始寻找一部受AII刺激的小说 酪氨酸激酶反应转录元件与一系列13 PAI-2/氯霉素乙酰转移酶(CAT)质粒，包括 3.3kb的5‘侧翼启动子序列。 血管组织纤溶酶原激活物-1和纤溶酶原激活物-2基因表达的Northern印迹分析 来自对照组和糖尿病大鼠的研究表明，这些基因的表达 在糖尿病动物的心脏和主动脉中，基因水平升高。这些 糖尿病大鼠的mRNA水平升高与许多 最近的临床研究报道，PAI-1在高血压患者中升高。 糖尿病患者的血浆和组织中。 这项提议的目标是审查关键的作用，以及 可能是速率限制，所有的信号转导元件- 调节RME细胞PAI-2表达的刺激信号通路。 这些信号通路在PAI-2转录中的作用将是 我们将使用一系列PAI-2/CAT构造来关联 PAI-2中所有反应元件的这些信号机制 推动者。AII介导的PAI-2和PAI-1在血管内皮细胞的表达 培养的血管细胞也将被用来检验这样的假设 所有这些作用都有助于糖尿病患者基因表达的改变。 血管系统，血管紧张素转换酶抑制剂的作用机制之一是使 这种异常的基因表达。

Diabetes mellitus and hypertension are closely linked, and are major risk factors in the development of vascular disease. Growing experimental and clinical evidence has demonstrated that the renin-angiotensin system (RAS) contributes to the pathogenesis of these vascular complications. These studies have demonstrated that angiotensin-converting-enzyme (ACE) inhibitors reduce the progression of diabetic vascular complications by a mechanism which is, in part, independent of blood pressure control. Recently, while screening for novel actions of vasoactive hormones by differential mRNA display, I have discovered that angiotensin II (AII) is a potent stimulator of plasminogen activator inhibitor-2 (PAI-2) expression in rat microvessel endothelial (RME) cells and rat aortic smooth muscle cells (RASMC). These studies have been extended to show that PAI-1 expression is similarly induced in these vascular cells. Studies on the mechanism of AII-stimulated PAI-2 expression reveal that both PKC-dependent and PKC-independent signaling pathways contribute to the 44.8+/-12.5 (+/-S.E.M) fold increase in PAI-2 mRNA in RME cells. The PKC independent pathway which is coupled to AII-stimulated PAI-2 expression, is completely blocked with the tyrosine kinase inhibitor genistein. In contrast, AII stimulates a 12.4+/-1.6 fold increase in PAI-2 mRNA in RASMC, and this increase is entirely associated with PKC activation. We have initiated a search for a novel AII-stimulated tyrosine kinase-responsive transcriptional element with a series of 13 PAI-2/Chloramphenicol acetyltransferase (CAT) plasmids, including up to 3.3 Kb of the 5' flanking promoter sequence. Northern blot analysis of PAI-1 and PAI-2 mRNA levels in vascular tissues from control and diabetic rats demonstrated that the expression of these genes was elevated in the heart and aorta of diabetic animals. These elevated mRNA levels in diabetic rats are consistent with a number of recent clinical studies which have reported that PAI-1 is elevated in the plasma and in the tissues from diabetic individuals. The objective of this proposal is to examine the role of key, and possibly rate limiting, signal transduction elements in the AII- stimulated signaling pathway that regulate PAI-2 expression in RME cells. The role of these signaling pathways in PAI-2 transcription will be examined and we will use a series of PAI-2/CAT constructs to associate these signaling mechanisms with AII-responsive elements in the PAI-2 promoter. The striking AII-mediated induction of PAI-2 and PAI-1 in cultured vascular cells will also be used to examine the hypothesis that AII action contributes to alterations in gene expression in the diabetic vasculature, and a mechanism of ACE inhibitors action is to normalize this aberrant gene expression.