ROCK Dependent Endothelial Mechanotransduction Perpetuates Pulmonary Fibrosis

Abstract Introduction: Mechanotransduction in the lung plays a role in fibrogenesis, however the direct role of mechanical changes on microvascular endothelial cells (EC) in the context of pulmonary fibrosis is not well defined. EC sense injury through exposure to circulating mediators and changes in their physical environment, including stiffness. Many injuries cause transient mechanical alterations, but aberrant or sustained mechanical activation may lead to persistently altered endothelial cell states as seen in idiopathic pulmonary fibrosis (IPF). Rho kinase (ROCK), a well-documented mechanosensor, regulates EC barrier function, inflammatory and pro-fibrotic signaling. We and others have shown that persistent, sustained vascular leak exacerbates injury-induced pulmonary fibrosis regardless of injury. We hypothesize that EC ROCK inhibition will block early mechanotransduction events key to initiating a feedback loop of permeability and pro-fibrotic signaling, to preventing progressive fibrosis in IPF. Methods: For this study we utilized publicly available scRNAseq data sets of lung tissue collected from IPF and control subjects. Established pre-clinical models of bleomycin induced pulmonary fibrosis (single and repetitive dosing) were used to test the ability of genetic ROCK deletion to attenuate mortality and lung fibrosis (Hydroxyproline Assay, Ashcroft Scoring) after injury. Human lung microvascular endothelial cells and normal human lung fibroblasts were used in in vitro systems with commercially available hydrogels (1-25kPa) to further investigate the role of mechanical induction of endothelial ROCK-dependent signaling to promote EC cell state changes (ACKR1, IGFBP7, COL15) and paracrine fibroblast activation (CTHRC1, COL1A1 and POSTN). Results: Public scRNAseq datasets were interrogated for differential expression. Within a fibrosis associated EC cluster (COL15+, ACKR1+, IGFBP7+), we found increased expression of ROCK isoforms, as well as upregulation of mechanotransduction associated genes. Inducible EC specific ROCK2 deletion was protective in both a single dose and repetitive dosing model of bleomycin induced lung fibrosis. In vitro, we observed increased ROCK activity (p-cofilin) as well as markers of fibrosis associated endothelial cells with ECM stiffness that was inhibited with pharmacologic ROCK inhibition. Normal human lung fibroblasts (nHLFb) cultured on physiologic stiffness (1kPa) became activated upon exposure to conditioned media from stiffness activated EC. Conclusions: We find that lung ECs from IPF patients are highly mechanically activated, have increased ROCK activity, and that EC-specific ROCK2 isoform deletion is protective in bleomycin models of lung fibrosis. Our data position EC ROCK signaling at the center of a pathogenic feedforward loop of persistent endothelial barrier dysfunction that perpetuates fibroblast activation to drive the progression of IPF.