Regulation of the Endothelial Citrulline-Nitric Oxide Cycle

描述(申请人提供)：我们的实验室提供了内皮健康的独特视角，表明瓜氨酸-一氧化氮循环的精氨酸琥珀酸合成酶(AS)必须具有维持一氧化氮(NO)产生和维持细胞活力的功能。由于几乎所有功能正常的内皮细胞的表型特性都与NO的生物活性有关，因此NO产生的障碍是糖尿病等心血管危险因素介导血管壁有害效应的常见机制。基于我们的证据，胰岛素调节AS的表达和丝氨酸-苏氨酸的磷酸化，本方案的第一个目的是探索胰岛素通过AS依赖机制调节血管健康和NO产生的机制。我们还将通过检测在TNFa介导的致病条件下AS胰岛素调节的变化来探索疾病中损害胰岛素功能的机制。用于研究这一特定目的的方法学将包括：1)翻译后修饰分析；2)信号转导抑制剂与siRNA结合；3)通过酶活性和NO分析进行功能研究；4)荧光显微镜；5)实时聚合酶链式反应和蛋白质印迹；以及6)利用突变、荧光素酶分析和EMSA研究进行启动子分析。第二个目的是基于我们的工作，证明AS mRNA的内皮特异性上游开放阅读框架编码一种小蛋白-精氨酸琥珀酸合成酶调节蛋白(ARP)，它调节AS mRNA的翻译。我们假设ARP抑制AS的表达，可能是对致病刺激的反应，通过停滞作为mRNA颗粒，将它们路由到离散的细胞质焦点，称为应激颗粒。为了验证这一假说，我们将使用：1)体外翻译；2)RNA免疫沉淀；3)RNA EMSA；4)多聚体谱分析；5)应激颗粒形成和抑制试验；以及6)免疫荧光和原位荧光染色。在第三个具体目标中，我们将研究胰岛素对AS表达和活性的心血管作用的分子机制，胰岛素抵抗和AS功能之间的相互关系，以及利用大鼠糖尿病模型系统开发同时针对代谢和心血管疾病的有益治疗策略的意义。我们将利用两种已知的糖尿病动物模型，它们证明了一氧化氮依赖的内皮功能障碍，这将使我们的组织培养和体外系统中定义的机制转化为完整的动物系统的过程成为可能。实验方法包括：1)动物模型鉴定；2)实时荧光聚合酶链式反应；3)免疫印迹；4)无测定和AS活性测定；5)免疫荧光和原位荧光染色。总体而言，拟议的工作有望为控制血管内皮细胞健康的特定机制提供重要和新颖的理解。这项研究的结果有可能区分新的治疗靶点，用于预防或抑制糖尿病、肥胖症和其他相关血管疾病中发现的血管内皮功能障碍。 与公共健康相关的血管壁功能受损被认为是糖尿病的早期致病事件，导致动脉粥样硬化。因此，破译维持血管功能的细胞机制对于了解生理影响者如何调节其对血管壁的保护作用或有害作用至关重要。我们相信，对维持血管壁功能所需的一种关键酶的功能和调节的检查对于理解这一点是必不可少的，并有可能区分糖尿病、高血压和相关心血管疾病的治疗新策略。

DESCRIPTION (provided by applicant): Our laboratory has provided a unique perspective of endothelial health, showing that argininosuccinate synthase (AS) of the citrulline-nitric oxide cycle must be functional to sustain nitric oxide (NO) production and to maintain cell viability. Since virtually all phenotypic properties of normally functioning endothelial cells are related to the bioactivity of NO, impairment of NO production is a common mechanism by which cardiovascular risk factors, such as diabetes, mediate deleterious effects on the vascular wall. Based on our evidence that insulin regulates AS expression and serine-threonine phosphorylation, the first aim of this proposal is designed to explore the mechanisms by which insulin regulates vascular health and NO production through AS-dependent mechanisms. We will also explore the mechanisms that impair insulin function in disease by examining alterations to insulin regulation of AS under pathogenic conditions mediated by TNFa. Methodology used to investigate this specific aim will include: 1) post-translational modification analyses; 2) signal transduction inhibitors in conjunction with siRNA; 3) functional studies via enzyme activity and NO assays; 4) fluorescence microscopy; 5) real time PCR and western blotting; and 6) promoter analysis using mutagenesis, luciferase assays and EMSA studies. The second aim is based on our work demonstrating that endothelium-specific upstream open reading frame of AS mRNA encodes a small protein, Argininosuccinate Synthase Regulatory Protein (ARP), which regulates AS mRNA translation. We hypothesize that ARP represses AS expression, perhaps in response to pathogenic stimuli, by stalling AS mRNA particles, routing them into discrete cytoplasmic foci known as stress granules. To test this hypothesis, we will employ: 1) in vitro translation; 2) RNA immunoprecipitation; 3) RNA EMSA; 4) polysome profiling analyses; 5) stress granule formation and inhibition assays; and 6) immunofluorescence and in situ fluorescent staining. In the third specific aim, we will examine the molecular mechanisms underlying cardiovascular actions of insulin on AS expression and activity, the reciprocal relationships between insulin resistance and AS function, and implications for developing beneficial therapeutic strategies that simultaneously target metabolic and cardiovascular diseases using the rat diabetic model system. We will utilize two animal models of diabetes that are known to demonstrate NO-dependent endothelial dysfunction that will permit the process of translating the mechanisms defined in our tissue culture and in vitro systems into an whole animal system. Experimental approacheswillinclude:1) animal model characterization; 2)real time PCR; 3) westernblot;4) NO measurements and AS activity assay; 5) immunofluorescence and in situ fluorescent staining. Overall, the work proposed is anticipated to provide an important and novel understanding of specific mechanisms that control vascular endothelial health. Results from this study have the potential to distinguish new therapeutic targets to be used to prevent or inhibit vascular endothelial dysfunction found in diabetes, obesity and other related vascular diseases. PUBLIC HEALTH RELEVANCE Impairment of vascular wall function has been suggested to be an early, causative event in diabetes, leading to atherosclerosis. Therefore, deciphering the cellular mechanisms that maintain vascular function is vital to understanding how physiologic affecters mediate their protective effects or their deleterious effects on the vascular wall. We believe examination of the function and regulation of a critical enzyme that is required to maintain vascular wall function is essential to this understanding, and will potentially distinguish new therapeutic strategies for the treatment of diabetes, hypertension and related cardiovascular disease