Tuesday, May 14, 2024
5/14/2024
PROJECT SUMMARY Idiopathic Pulmonary Fibrosis (IPF) is a progressive and fatal disease with limited therapies. IPF patient lungs display hallmarks of accelerated aging, including overabundance of senescent cells. In particular, senescent lung fibroblasts (sLFs) are key drivers of lung fibrosis. However, the signaling mechanisms that promote sLF accumulation and persistence in lung fibrosis remain poorly understood. This proposal seeks to fill these gaps with mechanistic studies that will enhance our capabilities to clear sLFs in IPF. Flavonoids are naturally occurring molecules which show potential to treat idiopathic pulmonary fibrosis (IPF). Their rise to prominence in recent years has been driven by the discovery that several of these compounds are can selectively induce apoptosis in senescent cells. In order to develop a better tool to investigate sLFs we designed and synthesized a novel, potent, senolytic flavonoid (F-4N). We present preliminary data shows that F-4N promotes senescence clearance and resolution of lung fibrosis in bleomycin-injured, aged mice with chronic fibrosis. We further observed F-4N selectively inhibits NF-¿B signaling in sLFs without affecting resident pulmonary leukocytes. NF-¿B activation is a hallmark of senescent cells, however its role in their apoptosis-resistance has not been explored, and senescence-unique mechanisms for targeting NF-¿B have not been identified. NF-¿B is critical for host defense, and systemic inhibition has very limited application. Based on these observations we propose to test the central hypothesis that sLF apoptosis resistance depends on NF-¿B activity and clearance of sLFs with F-4N represents a viable strategy to treat IPF. This hypothesis will be tested with three specific aims: First, we will establish the molecular mechanism of action essential to F-4N senolytic activity. We will perform apoptosis quantification, ChIP-seq, and RNA-seq from cultured sLFs to identify changes in response to gain and loss-of-function studies. We will use proteomic analyses to identify global phosphorylation changes in sLFs under these conditions. Second, we will characterize the cellular specific impact of F-4N on NF-¿B activity and apoptosis by quantifying the effect of F-4N across fibroblast, epithelial, endothelial, and leukocyte populations after acute administration to mice with bleomycin induced lung fibrosis. We will culture in vivo- derived low and high NF-¿B activity fibroblasts and epithelial cells in a 3D co-culture model to investigate paracrine signaling and senescence propagation. Lastly, will determine the antifibrotic efficacy of F-4N in chronic experimental fibrosis using one-time bleomycin lung injury in aged mice (18 months) and repeated bleomycin injury in young mice (2 months). F-4N will be benchmarked against the antifibrotic efficacy of pirfenidone and nintedanib, and previously established senolytic strategies. We will also measure the clearance of senescence burden and collagen deposition in precision-cut lung slice (PCLS) ex vivo cultures derived from IPF patient lungs. Together these proposed studies will enhance our understanding of the molecular and cellular mechanisms essential to sLF accumulation and persistence.
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