Cela1 Mediates Stretch-regulated Elastin Remodeling During Alveolar Septation

 描述（由申请人提供）：该提案描述了一个为期31/2年的培训计划，旨在发展一个专注于调节出生后远端肺形态发生机制的职业生涯。我在科罗拉多大学健康科学中心完成了儿科住院医师实习，并在伯明翰的亚拉巴马大学获得了儿科重症监护医学奖学金。自从接受了辛辛那提儿童医院的职位，我已经从讲师升为助理教授，我已经开发了一个新的研究项目，定义了肺发育和再生中拉伸调节基质重塑的机制.杰弗里惠特塞特博士是一位有成就的微生物学家和发育生物学家，他有指导30多位成功的内科医生-科学家的记录，自从我到来以来，他一直担任我的导师。他沿着一个已建立的奖学金监督委员会将建议我在我的过渡到一个成功的，独立的研究生涯与R01的资金。拟议的研究重点是拉伸调节的基质重塑在分隔中的作用。虽然肺伸展和基质重塑是影响远端肺形态发生的关键因素，但我们不了解这些过程是如何协调的，也不知道它们是如何调节肺泡分隔的。在发育和代偿性肺生长期间，牵张诱导肺弹性蛋白重塑并调节成纤维细胞糜蛋白酶样弹性蛋白酶1（Cela1）的表达。Cela1与肺基质的结合随应变呈指数增加，并且Cela1主要与细胞外基质中的Serpina3结合。这 该提案测试了Cela1活性的拉伸调节对肺泡分隔至关重要的总体假设。该提议的目的1通过Serpina3反应中心环的下拉和突变来测试Serpina3是否是肺基质中Cela1的推定抑制剂。为了定义Cela1结合肺弹性蛋白原的拉伸依赖性机制，目的2测量特定弹性蛋白原肽随着应变增加而淬灭Cela1与肺基质结合的程度。然后通过定点诱变测试介导这种相互作用的关键Cela1氨基酸。目的3首先定义Cela1敲除小鼠的肺表型，并量化Cela1对肺弹性蛋白酶活性的贡献。随后，它测试了 Cela1在肺气肿病理性基质重塑和再生的生理性基质重塑中的作用这个建议的职业发展部分是我过渡到研究独立的基础。它提供生物力学模型生成和测试方面的培训。当与离体肺切片的动态成像和用于量化不同细胞和基质重塑过程的方法相结合时，这种知识将允许对拉伸和应变如何调节远端肺形态发生的机理性洞察。在分子水平上，该提案还将提供分子模型生成和测试这些模型的方法方面的培训。这种培训和相关的合作关系将是制定多学科，变革性研究计划的关键，重点是改善BPD儿童的肺部健康。

 DESCRIPTION (provided by applicant): This proposal describes a 31/2 year training program for developing a career focused on mechanisms regulating postnatal distal lung morphogenesis. I completed a residency in pediatrics at the University of Colorado Health Sciences Center and a fellowship in pediatric critical care medicine at the University of Alabama at Birmingham. Since accepting a position at Cincinnati Children's Hospital, I have risen from Instructor to Assistant Professor, and I have developed a nascent research program defining the mechanisms of stretch- regulated matrix remodeling in lung development and regeneration. Dr. Jeffrey Whitsett, an accomplished neonatologist and developmental biologist, has a track record of mentoring over 30 successful physician- scientists and has served as my mentor since my arrival. He along with an established scholarship oversight committee will advise me in my transition to a successful, independent research career with R01 funding. The proposed research focuses on the role of stretch-regulated matrix remodeling in septation. Although lung stretch and matrix remodeling are critical factors impacting distal lung morphogenesis, we do not understand how these processes are coordinated or how they regulate alveolar septation. During development and compensatory lung growth stretch induces lung elastin remodeling and regulates fibroblast expression of chymotrypsin-like elastase 1 (Cela1). Cela1 binding to lung matrix increase exponentially with strain, and Cela1 is largely bound to Serpina3 in the extracellular matrix. This proposal tests the overall hypothesis that stretch-regulation of Cela1 activity is critical to alvelar septation. Aim 1 of this proposal tests whether Serpina3 is the putative inhibitor of Cela1 in the lung matrix by pull-down and mutation of the Serpina3 reactive center loop. To define the stretch-dependent mechanism by which Cela1 binds lung tropoelastin, Aim 2 measures the extent to which specific tropoelastin peptides quench the binding of Cela1 to lung matrix with increasing strain. It then tests the critical Cela1 amino acids mediating this interaction by site directed mutagenesis. Aim 3 first defines the lung phenotype of the Cela1 knockout mouse and quantifies Cela1's contribution to lung elastase activity. Subsequently, it tests the importance of Cela1 in the physiologic matrix remodeling of regeneration and the pathologic matrix remodeling of emphysema. The career development portion of this proposal is foundational for my transition to research independence. It provides training in bio-mechanical model generation and testing. When combined with dynamic imaging of ex vivo lung sections and methods for quantifying different cellular and matrix-remodeling processes, this knowledge will permit mechanistic insight into how stretch and strain regulate distal lung morphogenesis. On a molecular level, this proposal also will provide training in molecular model generation and the methods testing these models. This training and the associated collaborative relationships will be key in developing a multidisciplinary, transformative research program focused on improving lung health in children with BPD.