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

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.