PROJECT SUMMARY Idiopathic Pulmonary Fibrosis (IPF) is a progressive scarring lung disease that very often leads to respiratory failure. There remains a large unmet need for effective therapies. IPF is thought to be driven by injury to a vulnerable epithelium, sensitive to dysfunction either by genetic mutations or by the dose or type of injury. Epithelial dysfunction and senescence lead to diffuse tissue injury and activation of myofibroblasts. Dysregulated wound repair mechanisms may play a role in amplification of the tissue injury, tipping the balance from repair to fibrosis. Data is emerging that there are abnormalities in the pulmonary endothelium of patients who develop pulmonary fibrosis, although the cause/effect of these changes on the process of fibrogenesis is not well understood. Increased vascular permeability has been shown in the lungs of IPF patients and predicts mortality. In addition, several recent single-cell RNA sequencing studies have demonstrated loss of pulmonary capillaries and an increase in a bronchial vessel population. The full mechanistic implications of these changes have not been defined. Rho-associated kinase (ROCK) signaling has been shown to play a major role in several endothelial cell functions, including cell contractility and subsequent alterations in vascular permeability. I have shown in preliminary data that loss of ROCK2 specifically in endothelial cells in a mouse model (EC ROCK2 KO) prevents the development of pulmonary fibrosis induced by the delivery of intratracheal bleomycin. In addition to less pulmonary fibrosis, these mice had less vascular permeability in the early time period after bleomycin, before fibrosis develops. This finding suggests a link between endothelial ROCK2, vascular function, and pulmonary fibrosis, and hints at the potential for an opportunity for early intervention, when fibrosis may be preventable or reversible. In this current proposal, I propose to explore the protective phenotype in EC ROCK2 KO mice further by using these mice in a repetitive bleomycin model of pulmonary fibrosis, which has been shown to produce progressive fibrosis and is more similar to IPF. I will characterize the endothelial changes in this repetitive bleomycin model through staining and permeability assays. I will also perform single-cell RNA sequencing of mouse lungs with or without ROCK2, in the repetitive bleomycin model of pulmonary fibrosis. I will look for mechanistic links between endothelial ROCK2 and angiogenesis, senescence, endothelial to mesenchymal transition and apoptosis, all in the context of fibrogenesis.