Vascular hyperpermeability is a hallmark of inflammation. Current therapy interferes with mechanisms involved in onset of hyperpermeability. We will focus on mechanisms that terminate hyperpermeability because negative effects of hyperpermeability are due to its persistence beyond the time required for preserving organ function. We will elucidate mechanisms that actively terminate hyperpermeability. This work is based on a) current knowledge of protein traffic mechanisms, b) the fundamental role of VASP (vasodilator stimulated phosphoprotein) in cell adhesion and endothelial barrier properties, c) our demonstration that eNOS translocation to cytosol is necessary for onset of hyperpermeability, d) our preliminary data demonstrating that selective stimulation of Epac1 (exchange protein activated by cAMP) returns eNOS to the cell membrane, and e) our preliminary data showing that VASP is required for returning eNOS to the plasma membrane. We will test the central hypothesis that the agonist signaling that leads to hyperpermeability initiates a delayed increase in [cAMP], which causes VASP-assisted translocation of eNOS and Epac1 to the cell membrane and triggers inactivation of hyperpermeability. This novel hypothesis, in which barrier restoration is an active process operating via signaling mechanisms distinct from those that cause hyperpermeability, will be tested through two Specific Aims (SA). In each Specific Aim, we will stimulate eNOS translocation to the cell membrane with 8-cPT-2-O-Me-cAMP, a selective activator of Epac1, and will measure permeability as an end-point. SA1: To test whether the activity of cytoplasmic eNOS during hyperpermeability causes an increase in cAMP that leads to inactivation of hyperpermeability. We will assess inactivation of hyperpermeability and measure [cAMP] as a function of NO and time under conditions that anchor eNOS to cytosol or plasma membrane and protocols that stimulate or inhibit [cAMP] in vivo and in vitro. SA2: To determine the role of phosphorylated VASP as a molecular partner in eNOS and Epac1 translocation to cell membrane. In wild-type endothelial cells (EC) and in EC VASP-KO cells, we will explore the relationships among [NO], VASP phosphorylation, eNOS translocation and inactivation of hyperpermeability. We will stimulate eNOS translocation to cell membrane with 8-cPT-2-O-Me-cAMP in vivo and in vitro. We will assess the relationship between VASP phosphorylation and co-localization of eNOS and Epac1 using proximity ligation assay (PLA). The elucidation of the processes that inactivate and terminate hyperpermeability will be novel advances in knowledge of regulatory mechanisms of microvascular permeability and will provide the basis for developing new therapies for treating vascular inflammation.
Inflammation-increased vascular permeability (hyperpermeability) causes significant tissue damage, including compartment syndrome in muscle. Our studies will elucidate mechanisms that terminate hyperpermeability, aiming to avoid the negative effects of prolonged hyperpermeability and retain its shorter-term beneficial effects. Our results will assist vascular surgeons and physicians to improve management of inflammation and vascular disease.