Interferon Alpha: The Link Between Lupus and Endothelial Dysfunction

描述（由申请人提供）：加速动脉粥样硬化是由严重的血管内皮功能障碍（VED）引发的，VED是系统性红斑狼疮（SLE）患者中常见的表型。虽然VED的原因是多因素的，但所有途径都集中在内皮型一氧化氮合酶（eNOS）表达活性降低和对血管保护重要的NO生物利用度丧失上。最近的研究表明，干扰素（IFN）-α通过损害内皮修复机制促进SLE受试者VED的发展，然而，缺乏表明IFN-α在血管损伤中作用的研究。我们最近发现用IFN-α处理人主动脉和脐静脉内皮细胞（HAECs/HUVECs）导致eNOS表达和磷酸化降低。因此，拟议的研究的目的是梳理出负责在体外HUVEC/HAEC细胞培养模型中的I型干扰素介导的eNOS表达和激活的抑制机制。我们打算检验我们的假设，即IFN-α通过1）降低转录因子与eNOS启动子区的相互作用和2）引起蛋白磷酸酶2A活化增加和/或Akt（蛋白激酶B）表达降低而引起eNOS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，我们将测量IFN-α治疗后NO和环磷酸鸟苷（cGMP）水平，以确定其对功能性eNOS输出的影响。这一系列的研究是有意义的，因为它们将提供充分的证据证明I型干扰素在损害内皮功能中的作用。拟议的研究伴随着强大的课程，科学会议和研讨会的介绍，并与其他科学家的互动将提供一个培训计划，这将导致申请人的成功作为一个院士。课程将涉及免疫学和血管疾病的高级主题，补充以前的课程，包括生物统计学，赠款写作，科学伦理学和药理学。菲利普·肖尔学习技术目前在实验室和目前的工作会议，包括流变学会议，自由基生物学和医学会议的美国学院，和许多戈登会议之一。最后，学生将提交工作发表在同行评审的期刊。

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.