ENOS Location is a Fundamental Mechanism for Inactivation of Inflammation‐induced Hyperpermeability

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Inflammation disrupts the endothelial barrier and increases microvascular permeability (hyperpermeability), leading to tissue edema. Mechanisms for the onset of hyperpermeability have been the focus of many studies. However, the major pathological consequences are related to impairment in terminating hyperpermeability, rather than its onset, and to sustained inflammation. Therefore, we are innovatively studying mechanisms involved in the inactivation of hyperpermeability and restoration of endothelial barrier and normal microvascular permeability. We demonstrated that agonist‐induced translocation of eNOS (endothelial nitric oxide synthase) from cell membrane to cytosol initiates hyperpermeability. We hypothesize that agonist signaling for hyperpermeability also initiates a delayed increase in [cAMP], causing translocation of eNOS and Epac1 (exchange protein activated by cAMP) from cytosol to the cell membrane, which inactivates hyperpermeability. We tested whether platelet‐activating factor (PAF) induced hyperpermeability first and in delayed manner increased cAMP concentration to selectively stimulate Epac1 leading to translocation of eNOS from cytosol back to the cell membrane. In addition, we studied whether vasodilator‐stimulated phosphoprotein (VASP) is involved in the inactivation of hyperpermeability since PAF‐induced hyperpermeability in VASP‐KO endothelial cells remains elevated after Epac1 stimulation. We used human microvascular endothelial cells (HMVEC) and ECV‐304 cells transfected with green fluorescent protein (GFP)‐conjugated eNOS or the constructs GFPeNOSG2A (which anchors eNOS to the cytoplasm, ECV‐GFPeNOS‐G2A) and GFPeNOS‐CAAX (which anchors eNOS to the plasma membrane, ECV‐GFPeNOS‐CAAX) to study the restoration of endothelial barrier after hyperpermeability induced by PAF or vascular endothelial growth factor (VEGF). The time‐correlation between [NO] and [cAMP] in PAF or VEGF‐stimulated HMVEC and ECV‐eNOSGFP indicates that NO increase precedes [cAMP] increase. The increase in [cAMP] in HMVEC and ECV‐eNOSGFP correlates with a close proximity between eNOS, Epac1 and VASP, and both eNOS and Epac1 translocate to the cell membrane. None of these observations occur when eNOS is anchored to the cell membrane or cytosol. Furthermore, stimulation of Epac1 in HMVEC and ECV‐eNOSGFP inactivates their hyperpermeability response to PAF or VEGF. Stimulation of Epac1 in ECV‐GFPeNOS‐CAAX, after PAF application, inhibits hyperpermeability, while Epac1 stimulation does not reduce PAF‐elicited hyperpermeability in ECV‐GFPeNOS‐G2A. Stimulation of Epac1, after application of PAF, in the in vivo male and female hamster cheek pouch prevents hyperpermeability. In conclusion, our results support the concept that eNOS, cAMP, Epac1 and VASP work in synchrony to terminate hyperpermeability by translocating eNOS back to the cell membrane, and the overarching concept that location of eNOS is an important determinant in the regulation of microvascular permeability.