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

• 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
• 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; 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

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.

项目摘要/摘要 该项目旨在了解和控制成纤维细胞逃避的抗凋亡机制 纤维化过程中的凋亡。肺纤维化是一种毁灭性的疾病，影响了全球数百万。 纤维细胞纤维细胞是纤维化中的关键效应细胞，它们铺设胶原蛋白并可以收缩以变形 组织。在正常的伤口愈合中，成纤维细胞会发生凋亡并清除，但在病理纤维化中 疾病对细胞凋亡的抗纤维细胞耐药性使其能够持续存在并有助于进行性和非 - 解决纤维化。凋亡的固有途径由Bcl-2蛋白质家族控制。众所周知 在肺纤维化中，成纤维细胞具有抗凋亡蛋白Bcl-2的水平。但是，有 超过15个Bcl-2家庭成员有些是促凋亡的，有些是抗凋亡。所以， 了解细胞的凋亡环境不仅需要简单地测量一个Bcl-2家族的水平 蛋白质。在此提案中，我们将使用尖端实验室技术来描述抗凋亡 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 和肺纤维化的分辨率。我们将使用体外培养的原发性肺成纤维细胞来探索Bcl- 2使用从健康和纤维化肺部分离的人和鼠的成纤维细胞中的成纤维细胞中的家庭相互作用。 在特定目标1中，我们将阐明健康的Bcl-2家族蛋白水平，表达和相互作用 和纤维化状态，并确定纤维化肺的成纤维细胞是否对固有途径更具耐药性 凋亡。然后，我们将评估抑制和抗凋亡蛋白对成纤维细胞的影响 凋亡。在特定的目标2中，我们将在小鼠（二氧化硅和 重复的博霉素）探讨用药物ABT-263抑制Bcl-2对成纤维细胞细胞凋亡和 肺纤维化。我们将使用微CT，组织学和羟基水平测量纤维化。与小鼠一起使用 在ASMA启动子的控制下，红昔芬诱导的红色荧光蛋白番茄的表达 在治疗期间和之后，我们将跟踪和量化成纤维细胞。我们的工作将提供更多的理解 成纤维细胞采用的抗凋亡机制，并探索可行的治疗选择来诱导成纤维细胞 凋亡。我们的工作将大大增加我们对凋亡的内在途径如何控制的理解 纤维化的分辨率和持久性，并有可能确定治疗干预的靶标。 我和我的导师制定了一个结构化的培训计划，该计划将在以下领域提供培训： 问题和假设建设，实验方法的发展，基本和先进的尖端 实验室技术，数据评估和解释，数据表现和手稿准备，口头 演示技巧，并授予准备和授予技巧。该项目将在高度进行 国家犹太人健康的协作环境，它是一个杰出的平台 作为独立资助的医师科学家的职业。