Ubiquitination and Endothelial Fibrosis in Early Pulmonary Arterial Hypertension

项目摘要/摘要 这个K08临床科学家导师研究职业发展奖描述了一个为期五年的 研究和培训方案，以建立首席调查员(PI)作为独立的、由R01资助的 内科医生--肺血管医学领域的科学家。共同导师布拉德利·马龙，医学博士， 半胱氨酸硫醇生物化学、血管纤维化、多环芳烃病理生物学和病理生理学的专家，以及约瑟夫 Loscalzo，医学博士，氧化还原生物化学，网络医学和蛋白质组方面的专家，将负责PI的 布里格姆妇女医院和哈佛医学院的研究和专业发展活动 学校。科学出版物，咨询委员会的指导，说教教育，并在 科学会议将支持该项目的科学目标，并在三个关键方面提供职业发展 培训领域：PAH起始的病理生物学、激酶泛素化和实验性PAH模型。《少年派》 保证80%受保护的学术时间用于建议的K08课程。 PAH的特点是内皮功能障碍和氧化应激促进纤维化重塑 肺小动脉，导致右心衰竭和死亡。死亡率和住院率上升 从低于先前认为的值的平均肺动脉压(MPAP)开始递增 致病的。因此，早期治疗PAH可能会改善临床结果，但有效的治疗方法 不存在是因为早期PAH的内皮功能障碍和血管重构的病理生物学不存在 为人所知。我们提供了新的数据，早期PAH的特征是肺小动脉纤维化，受损 右室-肺动脉(RV-PA)偶联和内皮细胞C末端src激酶(CSK)增加 在体内mPAP升高之前就有表达。计算模型预测 CSK结合伙伴和E3泛素连接酶Casitas b系原癌基因(Cb1)易感 半胱氨酸硫醇396(Cys396)的氧化修饰。我们假设内皮细胞Cbl-Cys396的氧化 防止CSK泛素化以增加CSK依赖的纤维化，并削弱早期PAH中的RV-PA偶联。 为了验证这一假设，我们提出了以下具体目标：1)确定Cbl-Cys396氧化的影响 CSK泛素化及其在培养的人肺内皮细胞中的表达2)评估内皮CSK增加的作用 对培养的人肺血管内皮细胞纤维性胶原表达的影响；3)检测CSK抑制对RV-PA的影响 体内早、晚期多环芳烃的偶联作用。我们预计这次K08的调查结果将确定CSK- 靶向治疗是预防或逆转内皮纤维化和血管重塑的新策略 在PAH中出现mPAP升高和右心衰竭之前。此外，这项研究计划 将在早期PAH中建立激酶-泛素连接酶失调，作为与PI的分离平面 现任联合导师。

PROJECT SUMMARY/ABSTRACT This K08 Mentored Clinical Scientist Research Career Development Award describes a five-year research and training program to establish the principal investigator (PI) as an independent, R01-funded physician-scientist in the field of pulmonary vascular medicine. Co-mentorship by Bradley Maron, M.D., an expert in cysteinyl thiol biochemistry, vascular fibrosis, and PAH pathobiology and pathophysiology, and Joseph Loscalzo, M.D., Ph.D., an expert in redox biochemistry, network medicine, and proteomics, will oversee the PI’s research and professional development activities at Brigham and Women’s Hospital and Harvard Medical School. Scientific publication, guidance from an advisory committee, didactic education, and presentation at scientific conferences will support the scientific aims of the project and provide career development in three key training areas: the pathobiology of PAH inception, kinase ubiquitination, and experimental PAH models. The PI is guaranteed >80% protected academic time to dedicate to the proposed K08 program. PAH is characterized by endothelial dysfunction and oxidant stress that promotes fibrotic remodeling of the pulmonary arterioles, leading to right heart failure, and death. Mortality and hospitalization increase incrementally beginning at a mean pulmonary artery pressure (mPAP) below values that were previously thought to be pathogenic. Therefore, treatment of early-stage PAH may improve clinical outcome, but effective therapies do not exist because the pathobiology of endothelial dysfunction and vascular remodeling in early PAH is not known. We provide novel data that early-stage PAH is characterized by pulmonary arteriolar fibrosis, impaired right ventricle-pulmonary artery (RV-PA) coupling, and increased endothelial C-terminal src kinase (Csk) expression occurring prior to the development of elevated mPAP in vivo. Computational modeling predicts that the Csk binding-partner and E3 ubiquitin ligase Casitas b-lineage proto-oncogene (Cbl) is susceptible to oxidative modification at cysteinyl thiol 396 (Cys396). We hypothesize that oxidation of endothelial Cbl-Cys396 prevents Csk ubiquitination to increase Csk-dependent fibrosis and impair RV-PA coupling in early-stage PAH. To test this hypothesis, we propose the following specific aims: 1) Determine the impact of Cbl-Cys396 oxidation on Csk ubiquitination and expression in cultured human PAECs, 2) Assess the role of increased endothelial Csk on fibrillar collagen expression in cultured human PAECs, and 3) Test the effect of Csk inhibition on RV-PA coupling in early- and advanced-stage PAH in vivo. We anticipate that findings from this K08 will establish Csk- targeted therapy as a novel strategy by which to prevent or reverse endothelial fibrosis and vascular remodeling prior to the development of elevated mPAP and right heart failure in PAH. Furthermore, this research program will establish kinase-ubiquitin ligase dysregulation in early-stage PAH as a plane of separation from the PI’s current co-mentors.