Endothelial Barrier Restoration after Inflammation‐Induced Hyperpermeability

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Inflammation is characterized by an increase in endothelial barrier permeability (hyperpermeability) to macromolecules. Major efforts have focused on understanding the mechanisms involved in the hyperpermeability response of endothelial cells; however, less attention has been given to the restoration of vascular barrier integrity following hyperpermeability. Because many negative effects of hyperpermeability are due to its persistence beyond what is required for preserving organ function, we are investigating mechanisms that terminate hyperpermeability and thereby restore microvascular barrier properties. Understanding the mechanisms involved in the restoration of endothelial barrier integrity will have direct clinical impact because sustained inflammation is a hallmark in clinical disorders such as trauma and ischemia‐reperfusion. We demonstrated that eNOS translocation from plasma membrane to cytosol is required for the onset of hyperpermeability. We also demonstrated that stimulation of Epac1 (exchange protein activated by cAMP) contributes to restoration of basal permeability after ischemia‐reperfusion. In this study, we tested the hypothesis that 1) hyperpermeability induces delayed increase in cAMP concentration; and 2) cAMP stimulates Epac1 and VASP (vasodilator‐stimulated phosphoprotein) to promote translocation of eNOS back to the plasma membrane, and restores endothelial barrier properties. We use human microvascular (HMVEC) and mouse myocardial endothelial cells (Myend) to study the restoration of endothelial barrier after hyperpermeability induced by platelet‐activating factor (PAF). Myend VASP knockout (KO) endothelial cells (EC) have significantly greater baseline permeability to macromolecules as well as hyperpermeability response to PAF compared to wild type Myend cells. Stimulation of Epac1 in HMVEC inactivates their hyperpermeability response to PAF. However, Epac1 stimulation fails to inactivate PAF‐induced hyperpermeability in VASP KO Myend EC. PAF activates VASP by phosphorylation at Serine 157 and Serine 239. This phosphorylation is sensitive to eNOS inhibition indicating that PAF stimulates production of eNOS‐derived NO and VASP phosphorylation in a sequential manner. In addition, stimulation of Epac1 after the onset of PAF‐induced hyperpermeability translocates eNOS back from the cytosol to the plasma membrane in HMVEC and in control Myend. eNOS is preferentially located in the cytosol in Myend VASP KO cells. Lack of VASP prevents movement of eNOS associated with stimulation of Epac1. We conclude that VASP is a required protein for movement of eNOS and restoration of EC barrier.Support or Funding InformationRutgers School of Gradute StudiesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.