Targeting Bcl-2 Family Proteins to Facilitate Fibroblast Apoptosis and Promote the Resolution of Pulmonary Fibrosis

靶向Bcl-2家族蛋白促进成纤维细胞凋亡并促进肺纤维化消退

基本信息

批准号：
10701452
负责人：
Joseph Charles Cooley
金额：
$ 0.25万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-09 至 2022-06-08
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10701452
关键词：
Address Affect Aftercare Apoptosis Apoptotic Autoimmune Diseases Automobile Driving BCL2 gene Behavior Bleomycin Cells Cicatrix Clinical Collagen Contracts Data Dental Care Deposition Development Disease Effector Cell Evaluation Exposure to Extracellular Matrix Family Family member Fibroblasts Fibrosis Flow Cytometry Funding Gold Grant Health Histology Human Hydroxyproline In Vitro Induction of Apoptosis Infection Laboratories Life Expectancy Lung Lung diseases Malignant Neoplasms Manuscripts Measures Mentors Methods Mining Modeling Mus Natural Gas Occupational Occupational Exposure Oral Outcome Pathologic Pathway interactions Patients Pharmaceutical Preparations Pharmacology Physicians Preparation Process Prognosis Protein Family Proteins Pulmonary Fibrosis Reaction Resistance Resolution Risk Factors Role Scientist Signal Transduction Silicon Dioxide Silicosis Skin Small Interfering RNA South Africa Structure Tamoxifen Techniques Testing Therapeutic Therapeutic Intervention Tissues Tomatoes Training United States Work career clinically relevant collaborative environment crystallinity fibrotic lung fibrotic lung disease follower of religion Jewish hydraulic fracturing idiopathic pulmonary fibrosis improved in vivo Model inducible gene expression knock-down microCT mouse model prevent profibrotic fibroblast promoter red fluorescent protein skills targeted treatment therapeutic target wound healing

项目摘要

Project Summary/Abstract This project aims to understand and control the anti-apoptotic mechanisms employed by fibroblasts to evade apoptosis during the fibrotic process. Pulmonary fibrosis is a devastating disease affecting millions worldwide. Profibrotic fibroblasts are key effector cells in fibrosis, which lay down collagen and can contract to deform tissues. In normal wound healing, fibroblasts undergo apoptosis and are cleared, but in pathologic fibrotic diseases fibroblast resistance to apoptosis allows them to persist and contribute to progressive and non- resolving fibrosis. The intrinsic pathway of apoptosis is controlled by the Bcl-2 family of proteins. It is known that in pulmonary fibrosis, fibroblasts have increased levels of the anti-apoptotic protein Bcl-2. However, there are more than 15 Bcl-2 family members some of which are pro-apoptotic and some anti-apoptotic. Therefore, understanding the apoptotic milieu of a cell requires more than simply measuring the levels of one Bcl-2 family protein. In this proposal we will use cutting edge laboratory techniques to delineate the anti-apoptotic strategy employed by profibrotic fibroblasts and leverage this behavior to induce fibroblast apoptosis and resolution of pulmonary fibrosis. We will use primary lung fibroblasts cultured in vitro to explore the Bcl- 2 family interactions in fibroblasts using human and murine fibroblasts isolated from healthy and fibrotic lungs. In Specific Aim 1, we will elucidate the Bcl-2 family protein levels, expression, and interactions in both the healthy and fibrotic state, and determine if fibroblasts from fibrotic lungs are more resistant to intrinsic pathway driven apoptosis. We will then evaluate the effects of inhibition and knockdown of anti-apoptotic proteins on fibroblast apoptosis. In Specific Aim 2, we will use two in vivo models of persistent pulmonary fibrosis in mice (silica and repetitive bleomycin) to explore the effects of Bcl-2 inhibition with the drug ABT-263 on fibroblast apoptosis and pulmonary fibrosis. We will measure fibrosis with micro-CT, histology, and hydroxyproline levels. Using mice with tamoxifen-inducible expression of the red fluorescent protein tomato under the control of the aSMA promoter, we will track and quantify fibroblasts during and after treatment. Our work will provide a greater understanding of anti-apoptotic mechanisms employed by fibroblasts and explore a viable therapeutic option to induce fibroblast apoptosis. Our work will significantly add to our understanding of how the intrinsic pathway of apoptosis controls the resolution and persistence of fibrosis and will potentially identify targets for therapeutic intervention. My mentors and I have created a structured training plan that will provide training in the following domains: question and hypothesis building, development of experimental approaches, basic and advanced cutting-edge lab techniques, data evaluation and interpretation, data presentation and manuscript preparation, oral presentation skills, and grant preparation and grantsmanship. This project will be carried out in the highly collaborative environment at National Jewish Health, which serves as an outstanding platform on which to build a career as an independently funded physician scientist.

项目概要/摘要该项目旨在了解和控制成纤维细胞逃避细胞凋亡的抗凋亡机制纤维化过程中的细胞凋亡。肺纤维化是一种毁灭性的疾病，影响着全世界数百万人。促纤维化成纤维细胞是纤维化的关键效应细胞，它们沉积胶原蛋白并可以收缩变形组织。在正常伤口愈合中，成纤维细胞发生凋亡并被清除，但在病理性纤维化中成纤维细胞对细胞凋亡的抵抗使它们能够持续存在并导致进行性和非解决纤维化。细胞凋亡的内在途径由 Bcl-2 蛋白家族控制。据了解，在肺纤维化中，成纤维细胞的抗凋亡蛋白 Bcl-2 水平升高。然而，有超过 15 个 Bcl-2 家族成员，其中一些是促凋亡的，一些是抗凋亡的。所以，了解细胞的凋亡环境需要的不仅仅是测量一个 Bcl-2 家族的水平蛋白质。在本提案中，我们将使用尖端的实验室技术来描述抗细胞凋亡促纤维化成纤维细胞采用的策略，并利用这种行为诱导成纤维细胞凋亡以及肺纤维化的消退。我们将使用体外培养的原代肺成纤维细胞来探索 Bcl- 使用从健康和纤维化肺部分离的人类和小鼠成纤维细胞，观察成纤维细胞中的 2 个家族相互作用。在具体目标 1 中，我们将阐明 Bcl-2 家族蛋白在健康人体内的水平、表达和相互作用。和纤维化状态，并确定来自纤维化肺的成纤维细胞是否对内在途径驱动更具抵抗力细胞凋亡。然后我们将评估抗凋亡蛋白的抑制和敲低对成纤维细胞的影响细胞凋亡。在具体目标 2 中，我们将使用小鼠持续性肺纤维化的两种体内模型（二氧化硅和重复博来霉素）探索药物 ABT-263 抑制 Bcl-2 对成纤维细胞凋亡的影响肺纤维化。我们将通过显微 CT、组织学和羟脯氨酸水平来测量纤维化。使用鼠标在 aSMA 启动子的控制下，番茄红色荧光蛋白的他莫昔芬诱导表达，我们将在治疗期间和治疗后跟踪和量化成纤维细胞。我们的工作将提供更好的理解成纤维细胞采用的抗凋亡机制，并探索诱导成纤维细胞的可行治疗选择细胞凋亡。我们的工作将显着增进我们对细胞凋亡的内在途径如何控制的理解纤维化的消退和持续性，并将有可能确定治疗干预的目标。我和我的导师制定了一个结构化的培训计划，将提供以下领域的培训：问题和假设的建立、实验方法的开发、基础和先进的前沿实验室技术、数据评估和解释、数据呈现和手稿准备、口头演讲技巧、赠款准备和赠款技巧。该项目将在高度国家犹太健康中心的协作环境，这是一个杰出的平台，可以在此基础上建立独立资助的医师科学家的职业生涯。