Angiogenesis in hyperoxic lung fibrosis

描述(由申请人提供)： 这项为期5年的培训计划提出了作为肺损伤和血管生成领域的独立生物医学研究人员的职业发展计划。首席研究员，通过ABIM研究途径完成了肺和重症监护研究员培训。在赞助商和经验丰富的合作者的帮助下，他将扩展自己的科学技能，为成为一名独立的内科科学家的职业发展做准备。该计划将强调使用小鼠高氧模型研究血管生成和巨噬细胞调节纤维化的分子生物学方面的技能。为了增进他在计算生物学方面的知识，他将参加应用统计学的课程。巨噬细胞生物学家史蒂文·格林伯格医学博士和细胞因子信号分子调控领域的著名免疫学家保罗·罗斯曼医学博士将提供赞助。该计划将受益于血管生成专家Jan Kitajesski博士和Patty Lee医学博士的合作专业知识，后者将为高氧血症研究提供咨询支持。此外，世界知名研究员乔治·扬科普洛斯将每8周与赞助商Kitajeski博士和Lee博士合作并在一个咨询委员会中服务。该委员会将审查进展情况，并提供密切的科学支持和职业建议。长期的高氧会导致人类和小鼠的肺纤维化。伴随而来的血管重塑导致了肺动脉高压、右心衰竭和过早死亡。该计划解决的问题是：巨噬细胞衍生的血管生成调节因子，特别是血管生成素-2(Ang-2)，是否有助于在长期亚致死性高氧条件下进行血管重构。这些血管改变是否独立地促进了肺纤维化的发展？巨噬细胞来源的血管紧张素-2是否直接抑制内皮细胞对高氧的生存？巨噬细胞衍生的介质在高氧性肺损伤中的作用尚不清楚。我们在初步实验中证明，在RAW 264.7和亚致死性高氧性肺纤维化的小鼠模型中，血管紧张素-2基因和蛋白在高氧暴露下增加。这些小鼠出现以巨噬细胞为主的细胞渗透、胶原沉积和肺血管退行性变，概括了人类亚急性肺纤维化的特征。1)通过免疫组织化学、Western和Northern印迹和ELISA法对高氧暴露小鼠肺纤维化和血管重塑反应进行研究，以量化肺纤维化、血管生成和血管生成调节因子及其受体表达的变化。对肺血管的影响将通过共聚焦显微镜进行评估。2)通过血管生成实验检测巨噬细胞衍生的血管生成调节因子表达变化时的功能变化。巨噬细胞耗尽的小鼠将被用来评估肺巨噬细胞的贡献。3)在持续亚致死性高氧肺组织中，通过腺病毒转染法或阻断抗体靶向过表达Ang-1或Ang-2，以确定Ang-2在亚致死性高氧肺血管重塑和纤维化中的作用。4)确定Ang-2介导的内皮细胞存活抑制是否依赖于PI3K/Akt。哥伦比亚大学为K08职业获奖者提供成功的指导和培训，帮助他们为独立的职业生涯做准备，这方面已经有了很好的记录。

DESCRIPTION (provided by applicant): This 5-year training program proposes the development plan for a career as an independent biomedical researcher in the area of lung injury and angiogenesis. The principal investigator, has completed Pulmonary and Critical Care Fellowship training through the ABIM Research Pathway. With the sponsors and experienced collaborators he will expand on his scientific skills in preparation for career progression as an independent physician-scientist. The program will emphasize skills in molecular biology of angiogenesis and macrophage regulation of fibrosis using a murine hyperoxia model. To advance his knowledge in computational biology he will attend courses in applied statistics. Steven Greenberg M.D. a macrophage biologist, and Paul Rothman M.D., a renowned immunologist in the area of molecular regulation of cytokine signaling, will provide sponsorship. The program will benefit from collaborative expertise of Jan Kitajewski PhD, an expert in angiogenesis and Patty Lee, M.D. who will provide consultative support for the hyperoxia studies. Additionally, George Yancopoulos, a world renowned investigator, will collaborate and serve with the sponsors, Drs. Kitajewski and Lee on an advisory committee every 8 weeks. This committee will review progress and provide close scientific support and career advice. Prolonged hyperoxia results in lung fibrosis in humans and mice. The accompanying vascular remodeling contributes to pulmonary hypertension, right heart failure, and premature death. This program addresses the questions: Do macrophage-derived angiogenic regulatory factors, particularly angiopoietin-2 (Ang-2), contribute to vascular remodeling in response to prolonged sublethal hyperoxia. Do these vascular alterations contribute, independently, to the development of lung fibrosis? And does macrophage-derived Ang-2 directly inhibit endothelial cell survival in response to hyperoxia? The role of macrophage-derived mediators in hyperoxic lung injury remains unclear. We demonstrate in preliminary experiments, increased Ang-2 mRNA and protein in response to hyperoxic exposure in RAW 264.7 and in a mouse model of sublethal hyperoxic lung fibrosis. These mice develop macrophage-predominant cellular infiltration, collagen deposition and pulmonary vascular regression that recapitulates features of subacute lung fibrosis in humans. The following specific aims will be accomplished: 1) The characterization of fibrotic and vascular remodeling responses in hyperoxia-exposed mice by immuno-histochemistry, Western and Northern blotting and ELISA to quantify changes in markers of lung fibrosis, angiogenesis and expression of angiogenic regulators and their receptors. The effects on pulmonary vasculature will be evaluated by confocal microscopy. 2) Functional changes in response to altered expression of macrophage-derived angiogenic regulators by angiogenesis assays. Macrophage-depleted mice will be used to assess the contribution of lung macrophages. 3) Lung targeted overexpression of Ang-1 or 2 by adenovirus transfection or blocking antibodies prior to hyperoxia to determine the contribution of Ang-2 to pulmonary vascular remodeling and fibrosis during prolonged sublethal hyperoxia. 4) Determine if Ang-2 mediated endothelial survival inhibition is PI3K/Akt dependent. Columbia University has an established record of successful mentorship and training for K08 Career awardees in their preparation for careers as independent.