Genistein and cAMP signaling in vascular endothelium

描述(由申请人提供)：这项研究的长期目标是开发新的血管活性物质，这些药物可能有助于降低美国老龄化人口的高心血管发病率。最近的研究表明，摄入与异黄酮类相关的大豆蛋白可能对血管系统具有保护作用。染料木素是主要的大豆异黄酮类化合物，具有多种生物学作用。然而，目前还不清楚大豆蛋白的有益效果是由于染料木素还是其他成分。此外，大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，导致cAMP积聚，一氧化氮合酶(ENOS)激活和表达。此外，我们还证明金雀异黄素能使大鼠的全身血压迅速下降。因此，该应用的目的是确定金雀异黄素如何触发cAMP信号通路，从而影响cAMP相关的血管功能。将解决以下四个问题：1)。金雀异黄素如何升高cAMP，激活eNOS，从而在EC中刺激一氧化氮？2)金雀异黄素是否通过激活cAMP信号来降低动物的血压？金雀异黄素升高cAMP是否也改变eNOS基因表达？金雀异黄素是否通过cAMP信号保护内皮屏障功能？我们将使用细胞、分子和生化分析来确定金雀异黄素是否通过激活Gs介导的腺苷环化酶来提高EC中cAMP的水平。核糖核酸酶保护实验和实时定量聚合酶链式反应将检测染料木素对eNOS基因表达的调节作用。金雀异黄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确定，将使用免疫细胞化学、共聚焦显微镜和大分子渗漏方法进行评估。药理学和分子干预方法将被用来确定参与金雀异黄素作用的特定途径。这些研究结果将确立染料木素在血管系统中的作用机制，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提供基础。由于大豆异黄酮被广泛用作膳食补充剂，大豆异黄酮在血管系统中的健康益处的展示预计将对公众健康产生重大影响。

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.