Ubiquitination and Endothelial Fibrosis in Early Pulmonary Arterial Hypertension

项目总结/摘要 这个K 08指导临床科学家研究职业发展奖描述了一个为期五年的 研究和培训计划，以建立一个独立的，R 01资助的主要研究者（PI） 肺血管医学领域的医生兼科学家。由医学博士布拉德利马龙共同指导，一个 半胱氨酰巯基生物化学、血管纤维化、PAH病理生物学和病理生理学方面的专家，Joseph 洛斯卡尔佐，医学博士，博士，一位氧化还原生物化学、网络医学和蛋白质组学方面的专家，将监督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，教学教育和演讲 科学会议将支持该项目的科学目标，并在三个关键领域提供职业发展机会。 培训领域：PAH发病的病理生物学、激酶泛素化和PAH实验模型。的PI 保证>80%受保护的学术时间致力于拟议的K 08计划。 PAH的特征是内皮功能障碍和氧化应激，促进肺纤维化重塑。 导致右心衰竭和死亡死亡率和住院率增加 从平均肺动脉压（mPAP）开始逐渐低于以前认为的值 是致病的因此，治疗早期PAH可改善临床结局，但有效的治疗方法 不存在，因为早期PAH中内皮功能障碍和血管重塑的病理生物学不存在 知道的我们提供了新的数据，即早期PAH的特征是肺小动脉纤维化，受损， 右心室-肺动脉（RV-PA）偶联，内皮细胞C-末端src激酶（Csk）增加 在体内发生升高的mPAP之前表达。计算模型预测， Csk结合配偶体和E3泛素连接酶Casitas b系原癌基因（Cbl）易受 在半胱氨酰硫醇396（Cys 396）处的氧化修饰。我们假设内皮细胞Cbl-Cys 396的氧化 阻止Csk泛素化，从而增加Csk依赖性纤维化并损害早期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-Cys 396氧化的影响 对培养的人PAEC中Csk泛素化和表达的影响，2）评估增加的内皮Csk 3）测试Csk抑制对RV-PA的影响 体内早期和晚期PAH的偶联。我们预计，从这个K 08的调查结果将建立Csk- 靶向治疗作为预防或逆转内皮纤维化和血管重塑的新策略 在PAH发生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分离的平面 现任导师

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.