title: Molecular mechanisms mediating protective effect of cAMP on lipopolysaccharide (LPS)-induced human lung microvascular endothelial cells (HLMVEC) hyperpermeability.
abstract: 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 filamin1 in barrier maintenance and LPS-induced barrier compromise. Depletion of both VASP and filamin1 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 filamin1-depleted cells reacted to LPS with more robust stress fiber induction and more profound changes in ZO-1 and VE-cadherin peripheral organization. Both VASP and filamin1 phosphorylation was significantly increased as a result of PKA activation. We next analyzed the effect of VASP and filamin1 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 filamin1 depletion. Our data indicate that while PKA-dependent VASP phosphorylation contributes to the protective effect of cAMP elicited on LPS-compromised monolayers, filamin1 phosphorylation is unlikely to play a significant role in this process.
authors.affiliation: Vascular Biology Center, Medical College of Georgia, Augusta, Georgia 30912, USA. nbogatcheva@mcg.edu
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Bogatcheva
authors.forename: Marina A
authors.initials: MA
authors.lastname: Zemskova
authors.forename: Yevgeniy
authors.initials: Y
authors.lastname: Kovalenkov
authors.forename: Christophe
authors.initials: C
authors.lastname: Poirier
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Verin
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000199:Actins / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D002199:Capillary Permeability / Q000187:drug effects*; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D003285:Contractile Proteins / Q000235:genetics / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017097:Electric Impedance; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000503:physiopathology; D064448:Filamins; D020662:Guanine Nucleotide Exchange Factors / Q000819:agonists / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008168:Lung / Q000166:cytology*; D008565:Membrane Proteins / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics; D062826:Zonula Occludens-1 Protein; D000098662:Vasodilator-Stimulated Phosphoprotein; D000099152:Cadherin 5
chemical_list: D015056:1-Methyl-3-isobutylxanthine; D000199:Actins; D015703:Antigens, CD; D015820:Cadherins; D015815:Cell Adhesion Molecules; D005576:Colforsin; D003285:Contractile Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D064448:Filamins; D020662:Guanine Nucleotide Exchange Factors; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D062826:Zonula Occludens-1 Protein; D000098662:Vasodilator-Stimulated Phosphoprotein; D000099152:Cadherin 5; C116122:RAPGEF3 protein, human; C569042:TJP1 protein, human