Rationale Acute respiratory distress syndrome (ARDS) is a life-threatening disease process in which overwhelming inflammation causes disruption of the pulmonary endothelial cell (EC) barrier, leading to leakage of fluid and inflammatory cells from the blood stream into the airspaces. Current research aims to identify agents that could both decrease inflammation and increase pulmonary vascular barrier integrity. Recently published work suggests that imatinib, an FDA-approved Abl family kinase inhibitor, may attenuate vascular leak and inflammation; however the mechanism underlying these effects is not completely understood. In the present study we explored the effects of LPS on the expression of the Abl family kinases, c-Abl and Arg, as well as the effects of Abl family kinases on LPS-induced vascular permeability and inflammation.
Methods In silico analyses of the promoter regions of Abl1 (encodes c-Abl) and Abl2 (encodes Arg) were analyzed for potential responsive elements using the online programs Genomatix, TFsearch, and Jaspar. Cultured human pulmonary artery ECs were challenged with LPS (100 ng/mL, 24 hrs), harvested using RNeasy kit and reverse transcribed to cDNA. RT-PCR was performed to assess alterations in the expression of Abl1 and Abl2 after LPS challenge. In separate studies, siRNA was used to selectively silence either c-Abl or Arg and inter-endothelial gap formation was assessed by measuring FITC-dextran binding to a biotinylated avidin substrate. Complementary immunofluorescence studies were carried out to assess effects on adherens junction distributions. Western blotting was used to assess the effects of c-Abl and Arg silencing on NFkB phosphorylation.
Results In silico analyses revealed that c-Abl contains two antioxidant responsive elements, whereas Arg contains two mechanical stress responsive elements. LPS treatment caused an increase in the mRNA expression of c-Abl (1.5 fold, p<0.05), without effecting Arg expression. Silencing c-Abl, but not Arg, attenuated LPS induced NFkB phosphorylation. However, silencing Arg, but not c-Abl attenuated inter-endothelial gap formation (41%, p<0.05) and adherens junction dissociation (figure 1).
Conclusions The Abl family kinases c-Abl and Arg play complementary but distinct roles in mediating vascular permeability and inflammation following LPS challenge. The promoter of Abl1 (c-Abl) contains antioxidant response elements and LPS causes an increase in c-Abl expression. Additionally, LPS increases the mRNA expression of c-Abl, but not Arg. C-Abl contributes to LPS-induced NFκB signaling; whereas Arg contributes to inter-endothelial gap formation and adherens junction stability. Inhibition of both of these kinases may be of benefit in patients with ARDS.
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