Molecular Mechanisms Mediating Protective Effect of cAMP on Lipopolysaccharide (LPS)-Induced Human Lung Microvascular Endothelial Cells (HLMVEC) Hyperpermeability

Up to date, the nature of the sepsis-induced vascular leakage is understood only partially, which limits pharmacological approaches for its management. Here we studied the protective effect of cAMP using endotoxin-induced hyperpermeability as a model for barrier dysfunction observed in gram-negative sepsis. We demonstrated that the alleviation of lipopolysaccharide (LPS)-induced barrier compromise could be achieved by the specific activation of either protein kinase A (PKA) or Epac with cAMP analogs Bnz-cAMP or O-Me-cAMP, respectively. We next studied the involvement of PKA substrates VASP and filamin I in barrier maintenance and LPS-induced barrier compromise. Depletion of both VASP and filamin I with the specific siRNAs significantly exacerbated both the quiescent cells barrier and LPS-induced barrier dysfunction, suggesting barrier-protective role of these proteins. VASP depletion was associated with the more severe loss of ZO-1 peripheral staining in response to LPS, whereas filamin I-depleted cells reacted to LIPS with more robust stress fiber induction and more profound changes in ZO-1 and VE-cadherin peripheral organization. Both VASP and filamin I phosphorylation was significantly increased as a result of PKA activation. We next analyzed the effect of VASP and filamin I depletion on the PKA-dependent alleviation of LPS-induced barrier compromise. We observed that Bnz-cAMP ability to counteract LPS-induced hyperpermeability was attenuated only by VASP, but not filamin I depletion. Our data indicate that while PKA-dependent VASP phosphorylation contributes to the protective effect of cAMP elicited on LPS-compromised monolayers, filamin I phosphorylation is unlikely to play a significant role in this process. J. Cell. Physiol. 221: 750-759, 2009. (C) 2009 Wiley-Liss, Inc.