Regulation of the Endothelial Citrulline-Nitric Oxide Cycle

描述（由申请人提供）：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）生成和维持细胞活力的功能。由于正常功能内皮细胞的几乎所有表型特性都与一氧化氮的生物活性有关，因此一氧化氮产生的损害是心血管危险因素（如糖尿病）介导血管壁有害作用的常见机制。基于我们的证据，胰岛素调节AS表达和丝氨酸-苏氨酸磷酸化，本建议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。我们还将通过检查由TNFa介导的致病性条件下AS胰岛素调节的改变来探索损害疾病中胰岛素功能的机制。用于研究这一特定目标的方法将包括：1)翻译后修饰分析；2)与siRNA结合的信号转导抑制剂；3)通过酶活性和NO测定进行功能研究；4)荧光显微镜；5)实时PCR和western blotting；6)利用诱变、荧光素酶测定和EMSA研究进行启动子分析。第二个目标是基于我们的工作，证明AS mRNA的内皮特异性上游开放阅读框编码一个小蛋白，精氨酸琥珀酸合成酶调节蛋白（ARP），该蛋白调节AS mRNA的翻译。我们假设ARP抑制AS表达，可能是对致病刺激的反应，通过阻止AS mRNA颗粒，将其引导到称为应激颗粒的离散细胞质灶中。为了验证这一假设，我们将采用：1)体外翻译；2) RNA免疫沉淀；3) RNA emsa；4)多聚体分析；5)应力颗粒形成及抑制试验；6)免疫荧光和原位荧光染色。在第三个特定目标中，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证明no依赖性内皮功能障碍的糖尿病动物模型，这将允许将组织培养和体外系统中定义的机制转化为整个动物系统的过程。实验方法包括：1)动物模型表征；2)实时PCR；3) westernblot;4) NO测定和AS活性测定；5)免疫荧光和原位荧光染色。总的来说，这项工作有望为控制血管内皮健康的特定机制提供重要而新颖的理解。本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、肥胖和其他相关血管疾病的血管内皮功能障碍。

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