Cela1 Mediates Stretch-regulated Elastin Remodeling During Alveolar Septation

 描述(由申请人提供)：这份建议书描述了一项为期31年半的培训计划，旨在发展一项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生的实习，并在阿拉巴马大学伯明翰分校完成了儿科重症监护医学的奖学金。自从接受辛辛那提儿童医院的职位以来，我已经从讲师晋升为助理教授，并开发了一个新的研究计划，定义了拉伸调节的基质重塑在肺发育和再生中的机制。杰弗里·惠特塞特博士是一位成就卓著的新生儿学家和发育生物学家，他曾指导过30多位成功的内科科学家，自从我来到这里以来，他一直是我的导师。他和一个成熟的奖学金监督委员会将在我过渡到R01资助的成功的、独立的研究生涯时为我提供建议。建议的研究集中在牵张调节的基质重塑在间隔中的作用。虽然肺拉伸和基质重塑是影响远端肺形态发生的关键因素，但我们不知道这些过程是如何协调的，也不知道它们是如何调节肺泡间隔的。在发育和代偿性肺生长过程中，拉伸可诱导肺弹性蛋白重塑，并调节成纤维细胞胰凝乳酶样弹力酶1(Cela1)的表达。随着应变的增加，Cela1与肺基质的结合呈指数增加，并且Cela1主要与细胞外基质中的Serpina3结合。这 该方案验证了CELA1活动的拉伸调节对肺泡间隔至关重要的总体假说。这项建议的目的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的假定抑制物。为了确定Cela1与肺原弹性蛋白结合的拉伸依赖机制，目标2测量了随着应变的增加，特定的原弹性蛋白多肽抑制Cela1与肺基质结合的程度。然后，它通过定点突变来测试介导这种相互作用的关键CELA1氨基酸。目的3首先定义了Cela1基因敲除小鼠的肺表型，并量化了Cela1基因敲除小鼠的S对肺弹性蛋白酶活性的贡献。随后，它测试了 CELA1在肺气肿再生的生理性基质重构和病理基质重构中的作用。这项提议中的职业发展部分是我向研究独立过渡的基础。它提供生物力学模型生成和测试方面的培训。当结合体外肺切片的动态成像和量化不同的细胞和基质重塑过程的方法时，这些知识将使人们能够从机械上深入了解拉伸和应变如何调节远端肺的形态发生。在分子层面上，这项提议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开发多学科、变革性研究计划的关键，该研究计划侧重于改善患有BPD的儿童的肺健康。

 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.