Genistein and cAMP signaling in vascular endothelium

描述（由申请人提供）：本研究的长期目标是开发新型血管活性药物，可能有利于降低美国老龄化人口中心血管发病率的高水平。最近的研究表明，摄入与异黄酮相关的大豆蛋白可能对血管系统有保护作用。染料木素是主要的大豆异黄酮，具有多种生物作用。然而，目前尚不清楚大豆蛋白的有益作用是由于染料木素还是其他成分。此外，豆制品的血管保护作用的细胞或分子机制尚不清楚。我们最近发现染料木素直接作用于血管内皮细胞（EC），导致cAMP积累、一氧化氮合酶（eNOS）的激活和表达。此外，我们证明染料木素可以诱导大鼠的全身血压（BP）迅速下降。因此，本应用的目的是确定染料木素如何触发cAMP信号通路，从而影响cAMP相关的血管功能。主要解决以下四个问题：1)。染料木素如何提高cAMP，激活eNOS，从而刺激EC中的一氧化氮？2)染料木素是否通过激活cAMP信号通路降低动物血压？3)。染料木黄酮引起的cAMP升高是否也会改变eNOS基因的表达？4)。染料木素是否通过cAMP信号保护内皮屏障功能？我们将采用细胞、分子和生化分析来确定染料木素是否通过激活EC中gs介导的腺苷酸环化酶来升高cAMP。通过核糖核酸酶保护实验和Real-Time PCR检测染料木素调控的eNOS基因表达。染料木素对内皮屏障功能的影响，通过细胞骨架重组和屏障通透性来确定，将通过免疫细胞化学、共聚焦显微镜和大分子渗漏方法来评估。药理学和分子干预方法将被用来确定在染料木黄酮的作用调解的特定途径的参与。这些研究结果将为染料木素在血管中的作用机制的建立奠定基础，从而为进一步研究染料木素对血管健康和高血压等疾病的影响奠定基础。大豆异黄酮在血管系统中的健康益处的证明预计将对公众健康产生重大影响，因为异黄酮被广泛用作膳食补充剂。

DESCRIPTION (provided by applicant): The long-range goal of this research is to develop novel vasoactive agents that may be of benefit in lowering the high level of cardiovascular morbidity in the aging American population. Recent studies have shown that ingestion of soy protein associated with isoflavones may have a protective role in vasculature. Genistein, the major soy isoflavone, has various biologgical actions. However, it is not clear whether the beneficial effect of soy protein is due to genistein or other components. In addition, the cellular or molecular mechanisms underlying the vessel protective effect of soy products remain unclear. We recently found that genistein directly acts on vascular dothelial cells (EC), leading to cAMP accumulation, nitric oxide synthase (eNOS) activation and expression. Further, We demonstrated that genistein induced a rapid decrease in systemic blood pressure (BP) in rats. The objective of this application, is therefore to determine how genistein triggers the cAMP signaling pathway and thereby influencing the cAMP-related vascular function. The four following questions will be addressed: 1). How does genistein elevate cAMP, activate eNOS, and consequently stimulate nitric oxide in EC? 2) Does genistein decrease BP in animals through activation of the cAMP signaling? 3). Does elevated cAMP by genistein also alter eNOS gene expression ? 4). Does genistein protect endothelial barrier function via the cAMP signaling? We will employ cellular, molecular and biochemical analysis to determine whether genistein elevates cAMP via activation of Gs-mediated adenylate cyclase in EC. Genistein-regulated eNOS gene expression will be evaluated by ribonuclease protection assay and Real-Time PCR. The effect of genistein on endothelial barrier function, as determined by cytoskeletal reorganization and barrier permeability, will be assessed using immunocytochemistry, confocal microscope and macromolecular leakage methods. Pharmacological and molecular intervention approaches will be used to detemine the involvement of a specific pathway in mediation of genistein action. The results from these studies will establish the mechanisms of the genistein action in vasculature and thereby provide foundation for further studies to determine the effects of genistein on vascular health and diseases such as hypertension. The demonstration of the health benefits of soy isoflavones in vasculature is expected to have significant effect on public health since isoflavones are widely used as a dietary supplements.