How does DHEA improve diabetic microvascular dysfunction

描述(申请人提供)：糖尿病的长期并发症对数百万美国人的生活有毁灭性的影响。虽然微血管功能障碍在并发症的发展中起着关键作用，但对糖尿病微血管改变的病理生理学还知之甚少。衰老调节的肾上腺类固醇激素和营养补充剂脱氢表雄酮(DHEA)可以改善糖尿病患者的微血管功能障碍。这一作用的分子机制尚不清楚。我们的长期目标是通过开发针对糖尿病血管功能障碍的新药来降低糖尿病并发症的发病率。我们的短期目标是确定脱氢表雄酮血管作用的细胞机制。我们假设DHEA通过激活血管内皮细胞受体来影响血管功能。这一假设是基于我们发现了一种血管内皮细胞，G蛋白偶联，DHEA特异性受体，与一氧化氮的产生有关。分离这种受体是检验我们假设的关键的第一步。我们计划实现以下具体目标：1血管内皮细胞DHEA受体的分离、测序和克隆。评估瞬时转基因COS-7细胞中克隆受体的功能。 我们的研究设计包括两个策略：(1)亲和层析从溶解的牛主动脉质膜中分离纯化DHEA结合蛋白；(2)基因芯片鉴定表达于血管内皮细胞上的候选孤儿G蛋白偶联受体。候选的cDNA将在细胞表达和DHEA结合分析中进行测试。我们希望明确地展示DHEA是如何与血管内皮细胞相互作用的，并首次开始为DHEA声称的一些广泛的健康影响提供分子基础。这一结果将使我们能够确定受体-DHEA相互作用在血管功能、发育、衰老和疾病中的作用。我们将能够筛选高效受体激活分子来评估人类血管疾病的治疗效果，包括糖尿病微血管功能障碍。这些结果将有助于研究重要血管功能的信号通路，如一氧化氮的产生、细胞增殖和凋亡。因此，我们项目的成功完成将推进我们开发新型治疗性血管活性药物的长期目标，降低与糖尿病血管功能障碍相关的发病率。

DESCRIPTION (provided by applicant): Long-term complications of diabetes have a devastating impact on the lives of millions of Americans. While microvascular dysfunction plays a key role in the development of complications, the pathophysiology of microvascular changes in diabetes is poorly understood. The aging-regulated, adrenal steroid hormone and nutritional supplement, dehydroepiandrosterone (DHEA), improves microvascular dysfunction in diabetes. The molecular mechanism of this action is unknown. Our long-term objective is to decrease the morbidity of diabetic complications, by developing novel drugs that target diabetic vascular dysfunction. Our short-term goal is to define the cellular mechanism for the vascular action of DHEA. We hypothesize that DHEA affects vascular function by activating an endothelial receptor. This hypothesis is based on our discovery of a vascular endothelial, G-protein coupled, DHEA-specific receptor, linked to nitric oxide production. Isolating this receptor is a crucial first step in testing our hypothesis. We plan to achieve the following specific aims: 1 Isolate, sequence and clone the vascular endothelial DHEA receptor. Evaluate functional aspects of the cloned receptor in transiently transfected COS-7 cells. Our research design involves two strategies:(1) affinity chromatographic purification and isolation of a DHEA binding protein from solubilized bovine aortic plasma membranes and (2) cDNA microarray to identify candidate orphan G-protein coupled receptors, expressed on vascular endothelial cells. The candidate cDNAs will be tested in cellular expression and DHEA binding assays. We expect to show definitively how DHEA interacts with vascular endothelial cells and, for the first time, begin to provide a molecular basis for some of the widespread health effects claimed for DHEA. The result will allow us to determine the role of the receptor-DHEA interaction in vascular function, development, aging and disease. We will be able to screen for high potency receptor activating molecules to evaluate therapeutically in human vascular disease, including diabetic microvascular dysfunction. The results will facilitate the study of signaling pathways to important vascular functions such as nitric oxide production, cell proliferation and apoptosis. Thus, the successful completion of our project will advance our long-term goal of developing novel therapeutic vasoactive agents, to decrease the morbidity related to diabetic vascular dysfunction.