Interferon Alpha: The Link Between Lupus and Endothelial Dysfunction

描述(申请人提供)：加速的动脉粥样硬化是由严重的血管内皮功能障碍(VED)引起的，这是系统性红斑狼疮(SLE)患者中常见的一种表型。虽然VED的病因是多因素的，但所有的通路都集中在内皮型一氧化氮合酶(ENOS)表达的活性降低和NO生物利用度的丧失上，这对血管保护至关重要。最近的研究表明，干扰素-α通过损害血管内皮细胞修复机制参与了系统性红斑狼疮患者VED的发展，然而，缺乏关于干扰素-α在血管损伤中的作用的研究。我们最近发现，干扰素-α处理人主动脉和脐静脉内皮细胞(HAECs/HUVECs)后，eNOS的表达和磷酸化水平降低。因此，本研究的目的是在体外HUVEC/HAEC细胞培养模型中梳理I型干扰素抑制eNOS表达和激活的机制。我们打算验证我们的假设，即干扰素-α通过1)减少转录因子与eNOS启动子区域的相互作用和2)通过增加蛋白磷酸酶2A的活性和/或降低Akt(蛋白激酶B)的表达来导致eNOS的表达/活性降低。为了验证这一假设，我们将解决以下具体目标：首先，分析eNOS启动子区域活性、mRNA稳定性和转录因子结合的变化。其次，我们将量化不同磷酸酶和激酶的变化，这些变化可能归因于eNOS激活的变化。最后，我们将检测干扰素-α治疗后的NO和环鸟苷一磷酸(CGMP)水平，以确定其对功能性eNOS输出的影响。这一系列研究意义重大，因为它们将为I型IFN在损害内皮功能中的作用提供充分的证据。拟议的研究伴随着强有力的课程作业、科学会议和研讨会演示以及与其他科学家的互动，将提供一个培训计划，使申请者成为一名成功的院士。课程作业将解决免疫学和血管疾病方面的高级主题，补充以前的课程，包括生物统计学、拨款撰写、科学伦理学和药理学。这名学生还将与没有专家Philip Shaul博士合作，学习实验室目前无法获得的技术，并在包括美国风湿病学会会议、自由基生物学和医学学会会议以及许多戈登会议之一的会议上展示工作。最后，学生将提交论文并发表在同行评议的期刊上。

DESCRIPTION (provided by applicant): Accelerated atherosclerosis is initiated by severe vascular endothelial dysfunction (VED), a phenotype common amongst systemic lupus erythematosus (SLE) patients. Although causes of VED are multifactorial, all pathways converge on the diminished activity of endothelial nitric oxide synthase (eNOS) expression and loss of NO bioavailability important for blood vessel protection. Recent studies indicate that interferon (IFN) -alpha contributes to the development of VED in SLE subjects by impairing endothelial repair mechanisms however, studies are missing that indicate a role for IFN-alpha in vascular damage. We recently showed that treatment of human aortic and umbilical vein endothelial cells (HAECs/HUVECs) with IFN-alpha leads to decreased eNOS expression and phosphorylation. Therefore, the purpose of the proposed study is to tease out mechanisms responsible for type I interferon-mediated inhibition of eNOS expression and activation in an in vitro HUVEC/HAEC cell culture model. We intend to test our hypothesis that IFN-alpha causes diminished eNOS expression/activity by 1) decreasing transcription factor interaction with the eNOS promoter region and by 2) causing increased protein phosphatase 2A activation and/or decreased Akt (protein kinase B) expression. To test this hypothesis we will address the following specific aims: first analyze changes in eNOS promoter region activity, mRNA stability, and transcription factor binding. Secondly, we will quantify changes in different phosphatases and kinases that may attribute to changes in eNOS activation. Finally, we will measure NO and cyclic guanosine monophosphate (cGMP) levels following IFN-alpha treatment to ascertain its impact on functional eNOS outputs. This series of studies are significant because they will provide ample evidence for the role of type I IFNs in compromising endothelial function. The proposed studies accompanied by robust coursework, scientific meeting and seminar presentations, and interactions with other scientist will provide a training plan that will lead tothe applicant's success as an academician. Coursework will address advanced topics in immunology and vascular disease, complementing previous courses including biostatistics, grant writing, scientific ethics, and pharmacology. The student will also work with NO expert, Dr. Philip Shaul to learn techniques currently not available in the laboratory and present work at conferences including the American College of Rheumatology Conference, Society for Free Radical Biology and Medicine Conference, and one of the many Gordon Conferences. Finally, the student will submit work for publication in peer-reviewed journals.