Targeting Cell-specific Functions of the Rho Kinase Pathway in Pulmonary Fibrosis

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Project Summary Lung fibrosis is thought to be driven by aberrant wound healing responses to repetitive alveolar epithelial cell (AEC) injury, culminating in excessive fibroblast accumulation and extracellular matrix production. The aberrant wound healing responses that drive fibrosis overlap substantially with physiologic responses that mediate tissue repair, however, creating a major challenge in drug development: anti-fibrotic therapies need to inhibit pathologic wound healing responses while preserving physiologic responses as much as possible. We hypothesize that cell-specific drug delivery will be able to help to meet this challenge. Here we will identify specific cell types in which deletion of a central pro-fibrotic pathway in those cells alone is adequate to reduce fibrosis, and then develop the ability to deliver inhibitors of that pathway exclusively to that specific cell type. RhoA‒Rho kinase signaling is emerging as nodal point in pulmonary fibrosis, through which many upstream signals induce pro-fibrotic downstream responses. Activation of the Rho kinase isoforms ROCK 1 and ROCK2 regulates the cytoskeleton through actin filament assembly, driving many pro-fibrotic wound healing responses, including gene expression: actin filament assembly promotes nuclear translocation of the myocardin-related transcription factors (MRTFs), which activate serum response factor (SRF)-induced transcription of pro-fibrotic mediators. Based on its position at the center of multiple pro-fibrotic pathways, inhibition of RhoA‒ROCK signaling may be a particularly potent strategy for pulmonary fibrosis. The pleitropic effects of this pathway, however, have raised concerns about on-target adverse effects of its inhibition. We aim to develop a novel strategy to effectively but safely inhibit RhoA-ROCK signaling in pulmonary fibrosis, by developing the capacity to deliver inhibitors of this pathway in a cell-specific manner. We will first identify cell types in which RhoA‒ROCK signaling is critical to fibrosis, focusing on the AEC and the fibroblast. We will define the cell-specific roles of RhoA‒ROCK signaling in pulmonary fibrosis using mice in which either ROCK1 or ROCK 2 is specifically deleted in AECs or fibroblasts. We then will develop nanomaterial-based drug delivery vehicles to target inhibitors of RhoA‒ROCK signaling specifically to AECs or fibroblasts, and test their ability to treat fibrosis. We will encapsulate ROCK, MRTF or SRF inhibitors in polymeric nanoparticles that will be targeted by peptide affinity ligands to AECs or fibroblasts. We will study the efficacy of these nanoagents in two fibrosis models: a standard bleomycin model and a model in which low-dose bleomycin produces fibrosis in the context of exaggerated AEC endoplasmic reticulum (ER) stress, capturing the “gene- by-environment” nature of pulmonary fibrosis. In addition to invasive assessments of fibrosis, we will assess fibrosis non-invasively using a near-infrared fluorescent imaging agent specific for collagen, allowing for longitudinal studies of nanoagent efficacy. If successful, our experiments will provide evidence for the potential of novel cell-specific targeting strategies to enhance our ability to treat pulmonary fibrosis.