Evaluation of Endothelial Hyperglycemia-Driven Alterations During Type 2 Diabetes

 描述(申请人提供)：糖尿病目前困扰着美国约2580万人和全球2.2亿人。在美国，2型糖尿病(T2 DM)约占所有确诊病例的95%，其中心血管疾病是导致死亡的首要原因。血管功能障碍导致冠状动脉疾病和外周血管病变的风险增加，仍然是糖尿病患者的一个重要临床问题。高血糖是T2 DM导致血管功能障碍的主要致病因素，但在这方面的机制研究还存在很大差距。该方案评估了高血糖引起的内皮糖蛋白组的潜在变化，这些变化导致重要的动态平衡信号通路的变化，如AT1R和HTR2A信号通路，并导致血管功能障碍。这项建议的目的是利用创新的糖蛋白质组和磷酸蛋白质组浓缩、灵敏的质谱学技术和生理相关的功能测试来探索高血糖诱导的细胞表面蛋白质糖基化(目标1)、细胞内O-GlcN酰化和磷酸化协同平衡的改变(目标2)，以及随后导致血管功能障碍的重要稳态信号通路的改变(目标3)。为了做到这一点，申请人需要在生物信息学、应用于生理系统的蛋白质组学、血管生物学、临床相关的人类队列实验以及在安德鲁·格林博士(生理学-MCW教授)和共同导师Michael Widlansky博士(医学和药理学-MCW副教授)的指导下进行职业发展的受保护时间。这项研究提案和职业发展模块将有助于培养首席调查员的独立性，使他能够领导一项旨在检查T2 DM因果因素的研究计划。导师职业发展奖将进一步加强候选人在信号通路本体分析的生物信息学处理方面的培训，以及在应用于复杂系统的蛋白质组学分析方面的继续培训。申请者还将接受与T2 DM大鼠模型和T2 DM患者血管的功能研究相关的血管生物学方面的广泛培训。培训议程包括基于实验室的培训(MCW)、临床培训(MCW/Froedtert和CTSI)、外部实验室的专业培训、课程和专业发展研讨会/研讨会。这种多学科的培训将确保申请者独立设计、执行、排除故障和解释实验的能力。这里描述的研究方案的结果将提供直接的证据来证实或驳斥这一基本假设，即高血糖诱导的糖基化正在驱动导致T2 DM血管功能障碍的特定改变，并结合职业生涯发展将促进候选人顺利过渡到独立资助的研究人员。

 DESCRIPTION (provided by applicant): Diabetes currently afflicts approximately 25.8 million people in the United States (US) and 220 million people worldwide. Type 2 diabetes mellitus (T2DM) accounts for ~95% of all diagnosed cases in the US, among which cardiovascular disease is the leading cause of mortality. Vascular dysfunction, leading to increased risk of coronary artery disease and peripheral vascular disease, remains an important clinical problem in diabetic patients. Hyperglycemia is a major causative factor of T2DM contributing to vascular dysfunction, however, there is a large gap of mechanistic studies in this area. This proposal evaluates the underlying hyperglycemia- induced alterations in the endothelium glycoproteome that lead to changes in important homeostatic signaling pathways, such as the AT1R and HTR2A signaling pathways, and result in vascular dysfunction. The aims of this proposal utilize innovative glycoproteome and phosphoproteome enrichment, sensitive mass spectrometry technology, and physiologically relevant functional tests to explore hyperglycemia-induced protein glycosylation at the cell surface (Aim 1), alterations of the synergistic balance of intracellular O-GlcNAcylation and phosphorylation (Aim 2), and subsequent alterations in important homeostatic signaling pathways leading to vascular dysfunction (Aim 3). In order to do this, the applicant requires protected time for supervised career development in bioinformatics, proteomics as applied to physiological systems, vascular biology, clinically relevant human cohort experimentation, and professional development under the direction of Dr. Andrew Greene (Professor of Physiology-MCW) and co-mentor Dr. Michael Widlansky (Associate Professor of Medicine and Pharmacology-MCW). This research proposal and career development modules will assist in fostering the Principal Investigator in his independence such that he can lead a research program aimed at examining causal factors in T2DM. The mentored career development award will further the candidate's training in bioinformatics processing for signal pathway ontology analyses, along with continued training in proteomics analysis applied to complex systems. The applicant will also receive extensive training in vascular biology as it relates to functional studies of T2DM rat models and vessels from T2DM patients. The training agenda includes lab-based training (MCW), clinical training (MCW/Froedtert & CTSI), specialty training in external labs, coursework, and professional development seminars/workshops. This multi-disciplinary training will ensure the ability of the applicant to design, perform, troubleshot, and interpret experiments independently. Outcomes of research proposal described here will provide direct evidence to confirm or refute the fundamental hypothesis that hyperglycemia-induced glycosylation is driving specific alterations leading to vascular dysfunction in T2DM and in combination with the professional career development will foster a smooth transition of the candidate to an independently funded investigator.