Angiopoietin-1 Requires Oxidant Signaling through p47phox to Promote Endothelial Barrier Defense

Abstract

BackgroundReactive oxygen species (ROS) are largely considered to be pathogenic to normal endothelial function in disease states such as sepsis. We hypothesized that Angiopoietin-1 (Angpt-1), an endogenous agonist of the endothelial-specific receptor, Tie-2, promotes barrier defense by activating NADPH oxidase (NOX) signaling.Methods and FindingsUsing primary human microvascular endothelial cells (HMVECs), we found that Angpt-1 stimulation induces phosphorylation of p47phox and a brief oxidative burst that is lost when chemical inhibitors of NOX activity or siRNA against the NOX component p47phox were applied. As a result, there was attenuated ROS activity, disrupted junctional contacts, enhanced actin stress fiber accumulation, and induced gap formation between confluent HMVECs. All of these changes were associated with weakened barrier function. The ability of Angpt-1 to prevent identical changes induced by inflammatory permeability mediators, thrombin and lipopolysaccharides (LPS), was abrogated by p47phox knockdown. P47phox was required for Angpt-1 to activate Rac1 and inhibit mediator-induced activation of the small GTPase RhoA. Finally, Angpt-1 gene transfer prevented vascular leakage in wildtype mice exposed to systemically administered LPS, but not in p47phox knock out (p47−/−) littermates.ConclusionsThese results suggest an essential role for NOX signaling in Angpt-1-mediated endothelial barrier defense against mediators of systemic inflammation. More broadly, oxidants generated for signal transduction may have a barrier-promoting role in vascular endothelium.

The Angiopoietin-Tie2 Signaling Axis in Systemic Inflammation

“…69 Two subsequent studies have also implicated Foxo1 activation during endothelial inflammation. 60,72 This amplifier role of Angpt-2 downstream of acute phase reactants aligns well with the clinical results that implicate Angpt-2-and, indeed, more global endothelial activation-in the pathogenesis of adverse outcomes.…”

“…To achieve this remarkable defense against unrelated ligands acting on the endothelium, we and others have proposed that Angpt-1 achieves vascular barrier defense by signaling to conserved downstream regulators of endothelial cell shape and intercellular connectivity. [59][60][61][62][63][64] Angpt-1 application reorganizes the actin-myosin cytoskeleton of the endothelial cell such that its boundaries spread centrifugally and tensile strength develops at the cell's edges. The Rho family GTPases Rac1 and RhoA coordinate this cytoskeletal reorganization.…”

Idiopathic systemic capillary leak syndrome (Clarkson disease)

“…Hypomorphic genetic manipulations of p47phox, IQGAP1, or p190RhoGAP induce an EC phenotype similar to what we might predict in SCLS: minimal basal abnormalities, yet unusual susceptibility to otherwise ordinary inflammatory stressors. 6566, 67 …”

Calpain-14 and its association with eosinophilic esophagitis

“…This hypothesis is supported by correlative evidence that CAPN14 mRNA expression in pigs, a species with keratinized esophagus, 32 is virtually undetectable 7 and by evolution of cationic antimicrobial peptides, 35 particularly defensins, 36 that provide antifungal resistance of human esophageal surface. 37 CAPN14’s role in regulation of esophageal tissue barrier function can account for the fact that an imbalance in proteolytic function in the human esophagus caused by genetic 9 or environmental 38 factors can result in predisposition to tissue damage, particularly loss of esophageal tissue integrity. 26 …”