Rationale Our prior genomic and genetic studies identified pre-B-cell colony-enhancing factor (PBEF) as a potential novel biomarker in acute lung injury (ALI) (Ye et al, 2005). To elucidate the molecular mechanism underlying PBEF in the pathogenesis of ALI, we assessed the role of PBEF in in vitro vascular barrier regulation using confluent human pulmonary artery endothelial cell (HPAEC) or microvascular cells (HMVEC-L) monolayers since increased vascular permeability is a cardinal feature of ALI.
Methods Inhibition of PBEF expression was achieved by the PBEF siRNAs. Overexpression of the PBEF gene was accomplished by adeno-PBEF. Endothelial cells (EC) were transfected with PBEF siRNA or infected with adeno-PBEF for 48 hours before treated with thrombin (0.1 U/mL) or S1P (1 mm) for various time. Parameters to evaluate endothelial cell barrier functions include transendothelial electric resistance (TER), actin staining, myosin light chain phosphorylation, calcium influx.
Results Reductions in PBEF protein expression (> 70%) by siRNA significantly attenuated EC barrier dysfunction induced by the potent edemagenic agent, thrombin reflected by reductions in TER (60 to 70% reduction). Furthermore, PBEF siRNA blunted thrombin-mediated increases in increases in Ca2+ entry, polymerized actin formation, and myosin light chain phosphorylation, events critical to the thrombin-mediated permeability response. Overexpression of PBEF by adeno-PBEF vector significantly increases EC permeability reflected by decreased TER in HMVEC-L by ≈30%, indicating that overexpression of PBEF results in dysfunction.
Conclusions These in vitro observations strongly support that PBEF is critically involved in the endothelial cell barrier regulation. It encourages us to further explore the mechanistic insight into PBEF in the murine model of ALI.
Funded by NHLBI SCCOR-Molecular Approaches to Ventilator-Associated Lung Injury (U01HL 073994).
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.