Connexin 43 mediates LPS‐induced permeability in lung microvascular endothelial cells (LB785)

Abstract

Increased microvessel permeability is a major characteristic of lung inflammation. We showed inhibiting connexin 43 (Cx43)‐containing gap junctions blunts permeability increases in microvessels due to an inflammatory insult. However, it is unclear whether Cx43 levels directly influence the lung endothelial barrier. Toward this, we cultured rat lung microvascular endothelial cells (RLMVEC) on transwell inserts and determined permeability via flux of FITC dextran 40 kd (FDx40) across the endothelial monolayer. In vehicle‐treated monolayers, FDx40 fluorescence in the bottom chamber was 92±2 gray levels (mean±SEM; n=3; measurements in duplicate). Treating RLMVEC with LPS (100 ng/ml) for 4 hours increased FDx40 permeability to 99±3 gray levels. To determine responses to reductions in Cx43 levels, we treated RLMVEC monolayers with Cx43 shRNA lentiviral vector for 24 hours. FDx40 permeability in Cx43 shRNA‐treated monolayers was 13% lower than control shRNA‐treated cells. Further, LPS‐treatment of Cx43 shRNA‐treated monolayers induced a more than 27% decrease in permeability compared to control (P<0.05). To determine responses to elevated levels of Cx43, we transfected RLMVEC monolayers with Cx43‐cDNA. FDx40 permeability in Cx43 cDNA‐treated monolayers was 76% higher than control cDNA‐treated monolayers. Further, LPS‐treatment of Cx43 cDNA‐treated monolayers induced a more than 45% increase in permeability compared to control. Together, these data show for the first time that reduction and increase in Cx43 levels,respectively blunts and augments endothelial monolayer permeability. Thus we interpret, endothelial Cx43 levels directly influence endothelial barrier leak.Grant Funding Source: HL75503