Thursday, December 26, 2024
12/26/2024
Project Summary/Abstract This proposal outlines a two-year research training plan to provide the applicant the technical skills, experience with both in vivo and in vitro models, and analytic skills necessary for earning a K08 award on alveolar epithelial repair mechanisms and fibrotic responses following lung injury. Research Plan: Interstitial lung diseases such as idiopathic pulmonary fibrosis (IPF) remain a major source of morbidity and mortality worldwide, and available therapies are limited in their efficacy - despite decades of research, clinical management revolves around supportive care, avoidance of further injurious stimuli, and lung transplantation in select cases. Our lab made the seminal discovery that after injury, progenitor cells proliferate and pass through a distinct transitional cell state during regeneration of alveolar type 1 (AT1) cells. This state is transient during physiologic regeneration but appears to persist in non-resolving injury, suggesting that persistence of the transitional state may be a key defect contributing to disordered repair and fibrosis. This transitional state activates TGF-β signaling pathways known to be involved in IPF pathogenesis and exhibits markers of cellular senescence. Senescence is a state of cell cycle arrest which has been strongly implicated in the pathogenesis of IPF and other fibrotic lung diseases. However, the mechanisms by which these transitional cells acquire senescence remain unknown. Cellular communication network factor 1 (CCN1, also known as Cyr61) is a secreted matricellular protein that is highly upregulated in the transitional state and known to mediate senescence in other tissues. Some evidence suggests that its function is profibrotic in lung injury. However, the mechanisms that regulate its expression in transitional epithelial cells and by which it promotes fibrosis are unknown. This proposal seeks to examine the effects of CCN1 on transitional state senescence and the regulation of CCN1 by TGF-β signaling. I hypothesize that in the transitional state, CCN1 is driven by TGF-β signaling and promotes transitional cell senescence. In Aim 1, we will assess whether CCN1 induces senescence using murine models of bleomycin-induced lung injury. To test this in vivo, we will use RNA interference-based and genetic strategies to knock down CCN1 in lung epithelial cells. In vitro, we will use primary murine alveolar type 2 (AT2) cell culture assays to understand the mechanisms by which CCN1 affects transitional cell senescence. In Aim 2, we will inhibit TGF-β signaling by multiple methods in vitro to understand whether and how CCN1 expression is regulated by canonical TGF-β signaling. We will validate our findings in vivo using TGF-β receptor loss-of-function in the bleomycin model. We will validate key in vitro experiments in both aims in human induced pluripotent stem cell (iPSC)-derived AT2 cells and in fixed tissue from IPF patients.
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