CARMIL1 and Endothelial Barrier Function Regulation

ABSTRACT The acute respiratory distress syndrome (ARDS) is characterized by pulmonary vascular leak and flooding of the normally air-filled alveolar space with protein rich edema fluid and causes severe hypoxemia, respiratory failure and death among critically ill patients. Identification of genetic factors which modify the risk of developing ARDS or influence clinical outcomes may improve our pathologic understanding of this disease. Human variants in the gene LRRC16A are implicated in improved ARDS outcomes but the mechanism remains unknown. LRRC16A encodes capping protein, Arp 2/3 and myosin-I linker (CARMIL1), a cytoskeletal regulatory protein which has been studied primarily in cell motility. CARMIL contributes to peripheral actin polymerization by antagonizing capping protein at the end of growing actin strands. Decreased CARMIL expression slows cell migration by attenuating protrusion of the cell membrane. Maintenance of pulmonary endothelial cell (EC) barrier function is critical to prevent lung vascular leak. Cytoskeletal rearrangement and force generation determine barrier integrity through dynamic changes to the plasma membrane and cell shape. Peripheral actin polymerization protrudes the endothelial cell membrane to increase contact with neighboring cells, reduce intercellular gaps and increase barrier function. We hypothesize that CARMIL1 is a key regulator of peripheral actin structure and branched actin polymerization which facilitate membrane protrusion to determine EC barrier function. This proposal will use complementary biochemical, imaging and functional studies to characterize the role of CARMIL1 in EC barrier function. We will investigate CARMIL1 in the context of known regulators of endothelial cytoskeletal and membrane dynamics to improve our understanding of the mechanisms responsible for pulmonary vascular leak. Specific Aim 1 will investigate the role of CARMIL1 in EC peripheral actin structures and dynamics by employing biochemical and advanced imaging techniques after CARMIL1 silencing and/or overexpression of wild-type or variant constructs. Specific Aim 2 will determine the effect of modified CARMIL1 expression on EC barrier integrity using multiple assays of local and global permeability. Specific Aim 3 will characterize the function of endothelial CARMIL1 in murine lung injury. Endothelial specific delivery of siRNA and lentiviral constructs will investigate the effect of CARMIL1 manipulation on the intact lung vasculature following inflammatory or infectious insults. These studies will provide novel mechanistic insights into the cellular mechanisms of ARDS and provide a link between CARMIL1 variants and clinical outcomes. This knowledge has the potential to identify new therapeutic targets and improve the care of future patients.