VASOACTIVE HORMONE INDUCED GENES IN DIABETIC VASCULATURE

糖尿病和高血压密切相关，是主要风险 血管疾病的发展因素。 不断增长的实验和 临床证据表明， (RAS)有助于这些血管并发症的发病机制。 这些研究表明，血管紧张素转换酶（ACE） 抑制剂通过以下方式减少糖尿病血管并发症的进展： 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mRNA显示，我发现血管紧张素II（AII） 是纤溶酶原激活物抑制剂-2（派-2）的强效刺激剂 在大鼠微血管内皮（RME）细胞和大鼠主动脉 平滑肌细胞（RASMC）。 这些研究已经扩展到显示 派-1的表达在这些血管细胞中同样被诱导。 对AII刺激派-2表达机制的研究表明， PKC依赖性和PKC非依赖性信号通路都有助于 RME细胞中派-2 mRNA增加44.8+/-12.5（+/-S.E.M）倍。 的 与AII刺激的派-2偶联的PKC非依赖性途径 表达，被酪氨酸激酶抑制剂完全阻断 染料木黄酮 相比之下，AII刺激12.4+/-1.6倍的增加， 派-2 mRNA在RASMC中的表达增加，这种增加完全与PKC有关 activation. 我们已经开始寻找一种新的人工智能刺激的 酪氨酸激酶应答转录元件与一系列的13 派-2/氯霉素乙酰转移酶（CAT）质粒，包括高达 3.3 5'侧翼启动子序列的Kb。 北方印迹法检测血管组织中派-1和派-2 mRNA的表达 从对照组和糖尿病大鼠中证实， 基因在糖尿病动物的心脏和主动脉中升高。 这些 糖尿病大鼠中mRNA水平升高与一些 最近的临床研究报道派-1在糖尿病患者中升高， 血浆和糖尿病个体的组织中。 本建议的目的是审查关键的作用， AII中可能的速率限制信号转导元件， 刺激调节RME细胞中派-2表达信号通路。 这些信号通路在派-2转录中的作用将被进一步研究。 检查，我们将使用一系列派-2/CAT结构， 派-2中AII反应元件的这些信号传导机制 启动子 AII介导的派-2和派-1的显著诱导作用， 培养的血管细胞也将用于检验假设， 所有这些作用都有助于糖尿病患者基因表达的改变。 血管，ACE抑制剂的作用机制是正常化 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.