Regulation of Vascular Calcification by Adenosine Signaling

描述（由申请人提供）：此K22/R00应用程序描述了一个职业发展计划，旨在促进我过渡到研究独立。在这个奖项的指导阶段，我的目标是培养各种新的研究技能，以及接受必要的职业培训，以获得终身助理教授职位。为了拓宽我的研究专长，我的目标是掌握异位钙化、诱导多能干细胞和慢性肾脏疾病小鼠模型领域的新技术;获得这些领域的专业知识将为我提供成功过渡到并维持独立研究专业所需的背景，在指导阶段，我还打算继续发展职业技能，例如实习生指导，教学以及与同事和公众有效沟通我的工作。我计划参加NIH校内培训和教育办公室提供的各种研讨会和讲习班，以提高我的领导能力和实验室管理技能。我组建了一个咨询委员会，由我的主要导师、三名科学顾问和一名职业发展顾问组成，他们都承诺在本提案的整个授予期间提供支持和指导。在校外阶段，我计划招募我的独立实验室的分子和细胞生物学家，以及临床研究员感兴趣的是， 进行基于病人的研究。我的长期职业计划是将我在分子生物学，生物化学和遗传方法方面的专业知识与临床合作相结合，以推进血管钙化和血管完整性疾病的研究，最终目标是确定药物靶点，用于治疗受影响患者的治疗方法的开发。血管钙化通常表现为慢性肾病和糖尿病等疾病的继发性并发症，并且是过早死亡的预测因素。我所在的团队发现了一种新的单基因疾病，受影响的个体会在下肢动脉中出现中动脉钙化。受影响的动脉是曲折的，并有显着减少血流量，导致疼痛和缺乏患者的流动性。这种疾病-CD73缺乏引起的动脉钙化（ACDC）-是由编码CD73蛋白的基因中的双等位基因失活突变引起的。CD73将细胞外AMP转化为腺苷，我们发现ACDC中的血管钙化源于缺乏通过腺苷受体（AR）的细胞外腺苷信号传导。血管钙化和AR信号之间的联系是前所未有的。我在本申请中提出的研究将寻求阐明调节血管钙化和血管稳态的机制和过程。目的1将使用体外患者特异性成纤维细胞钙化模型以及体内患者特异性诱导多能干细胞（iPSC）疾病模型来确定ACDC细胞中调节钙化的机制。目的2将确定CD73在血管壁稳态中的作用，具体地1）CD73介导的腺苷信号传导在保持血管平滑肌细胞（VSMCs）处于完全分化状态中的作用，以及2）CD73介导的腺苷信号传导在调节细胞外基质交联蛋白中的作用。目标3将使用已建立的CD73敲除小鼠来评估该遗传系用作人类疾病模型的能力。此外，CD73和腺苷信号传导的作用将在慢性肾脏疾病的小鼠模型中进行评估，因为伴随这种疾病的下肢中膜钙化在组织学上与ACDC患者中观察到的相似，并且可能具有共同的机制并对共同的治疗策略有反应。这项研究的意义最好地体现在受血管钙化影响的人数上;除了ACDC，慢性肾脏疾病和糖尿病患者经常发生下肢内侧动脉钙化。这项研究的创新之处在于使用新的单基因疾病作为模型来研究调节血管钙化的过程，并创建了一种可用于药物筛选的基于iPSC的体内钙化模型。腺苷信号作为调节血管钙化的机制的鉴定为异位钙化领域的研究开辟了新的途径。阐明这种单基因疾病的潜在途径将为了解其他疾病中的中膜钙化过程提供重要见解，并可能带来更好的干预和治疗方法。

DESCRIPTION (provided by applicant): This K22/R00 application describes a career development program designed to facilitate my transition to research independence. My goals during the mentored phase of this award are to cultivate a variety of new research skills as well as receive the career training necessary to acquire a tenure-track assistant professorship. To broaden my research expertise I aim to master new techniques in the fields of ectopic calcification, induced- pluripotent stem cells, and murine models of chronic kidney disease; gaining expertise in these areas will pro- vide me with the background necessary to successfully transition to and sustain an independent research pro- gram. During the mentored phase I also intend to continue to develop career skills in such areas as trainee mentorship, teaching, and the effective communication of my work to colleagues and the general public. I plan on participating in a variety of seminars and workshops offered by the NIH Office of Intramural Training and Education to enhance my leadership and lab management skills. I have assembled an Advisory Committee made up of my primary mentor, three scientific consultants, and a career development advisor, all of whom have pledged their support and guidance throughout the award period of this proposal. During the extramural phase I plan to recruit to my independent laboratory molecular and cellular biologists, as well as clinical fellows interested in conducting patient-based research. My long-term career plan is to pair my expertise in molecular biology, biochemistry, and genetic approaches with clinical collaborations to advance the study of vascular calcification and vessel integrity disorders, with the ultimate goal of identifying drug targets for the development of therapeutics to treat affected patients. Vascular calcification often manifests as a secondary complication to diseases such as chronic kidney disease and diabetes, and is a predictor of premature death. I was part of a team that discovered a novel, monogenetic disease in which affected individuals develop medial arterial calcifications in their lower-extremity arteries. Affected arteries are tortuous and have significantly reduced blood flow, resulting in pain and lack of patient mobility. This disease-Arterial Calcification due to Deficiency of CD73 (ACDC)-results from biallelic inactivating mutations in the gene encoding for the CD73 protein. CD73 converts extracellular AMP to adenosine, and we showed that the vascular calcification in ACDC stems from a lack of extracellular adenosine signaling via adenosine receptors (ARs). The link between vascular calcification and AR signaling is unprecedented. The research I propose in this application will seek to elucidate the mechanisms and processes regulating vascular calcification and vascular homeostasis. Aim 1 will identify the mechanisms regulating calcification in ACDC cells using an in vitro patient-specific fibroblast calcification model as well as an in vivo patient-specific induced pluripotent stem cell (iPSC) disease model. Aim 2 will define the role of CD73 in vessel wall homeostasis, specifically 1) the role of CD73-mediated adenosine signaling in keeping vascular smooth muscle cells (VSMCs) in their fully differentiated state, and 2) the role of CD73-mediated adenosine signaling in regulating extracellular matrix cross-linking proteins. Aim 3 will use an established CD73 knockout mouse to assess the ability of this genetic line to be used as a model for the human disease. Additionally, the role of CD73 and adenosine signaling will be assessed in a murine model of chronic kidney disease, as the lower-extremity medial calcification accompanying this disease is histologically similar to that seen in ACDC patients, and may share common mechanisms and respond to common therapeutic strategies. The significance of this study is best exemplified by the number of people affected by vascular calcification; in addition to ACDC, individuals with chronic kidney disease and diabetes often develop lower-extremity medial arterial calcifications. The innovation of this study is the use of novel, monogenetic disease as a model to study processes regulating vascular calcification, and the creation of an in vivo iPSC-based calcification model that can be used in drug screening. The identification of adenosine signaling as a mechanism that regulates vascular calcification opens up a new avenue of research in the field of ectopic calcification. Elucidating the pathways underlying this monogenetic disease will provide important insight into the process of medial calcification in other disorders, and may lead to better interventions and therapeutics.