| Systemic Sclerosis (SSc) is a progressive autoimmune disease characterized by vasculopathy, immune dysfunction and fibrosis of skin and internal organs. Pulmonary fibrosis is the most severe complication. Although pathogenesis of SSc is not completely understood, it is believed to be initiated by early and persistent damage to endothelial cells (ECs), which may initiate inflammation and activation of fibroblasts. In this study we used subcutaneous instillation of bleomycin (BLM), which induces skin and lung fibrosis similar to SSc. ECs are targeted by BLM, however, the extent of EC contribution to pulmonary inflammation and fibrosis, and the degree of vasculopathy in this model are not fully understood. The core hypothesis of this work is that BLM induced injury to ECs contributes to pathogenic processes, resulting in pulmonary fibrosis. Through a unique method of cell sorting and gene expression analysis, we demonstrated EC injury (Matrix Metalloproteinase 12, von Willebrand Factor) and activation in response to BLM. Activation of ECs was demonstrated by expression of selectins, chemokines, and cytokines, contributing to inflammation and macrophage recruitment. Direct contribution of ECs to fibrosis was demonstrated by up-regulation of pro-fibrotic cytokines (Osteopontin, Connective Tissue Growth Factor, Plasminogen Activation Inhibitor-1), activation of collagen production by fibroblasts, and expression of Endothelial to Mesenchymal Transition (EndoMT) markers (Fibronectin, Fibroblast Specific Protein-1). Importantly, responses were sustained, suggesting that ECs perpetuate damage and repair mechanisms that promote fibrosis.;Previous work from our lab has shown that transcription factor Friend Leukemia Virus Integration-1 (Fli1) is down-regulated in fibroblasts and ECs in SSc, contributing to fibrosis and vasculopathy in the skin. Intriguingly, Fli1 is also down-regulated in the lung during BLM induced fibrosis, but not in ECs. Further studies demonstrated that Fli1 was down-regulated in macrophages, where it is regulated by inflammatory signals. Fli1 was also down-regulated in mesenchymal cells, which includes fibroblasts. Previous in vitro studies have shown that Fli1 regulates fibrosis through regulation of collagen and other extracellular matrix proteins. In this study, we identified Tissue Inhibitor of Metalloproteinase-3, an important molecule in collagen degradation, as a novel Fli 1 target, revealing another mechanism by which Fli1 contributes to development of fibrosis. |
