Endothelial Poldip2 regulates sepsis-induced lung injury via Rho pathway activation

Aims Sepsis-induced lung injury is associated with significant morbidity and mortality. Previously, we showed that heterozygous deletion of polymerase delta-interacting protein 2 (Poldip2) was protective against sepsis-induced lung injury. Since endothelial barrier disruption is thought to be the main mechanism of sepsis-induced lung injury, we sought to determine if the observed protection was specifically due to the effect of reduced endothelial Poldip2. Methods and results Endothelial-specific Poldip2 knock-out mice (EC-/-) and their wild-type littermates (EC+/+) were injected with saline or lipopolysaccharide (18 mg/kg) to model sepsis-induced lung injury. At 18 h post-injection mice, were euthanized and bronchoalveolar lavage (BAL) fluid and lung tissue were collected to assess leucocyte infiltration. Poldip2 EC-/- mice showed reduced lung leucocyte infiltration in BAL (0.21 +/- 0.9x10(6) vs. 1.29 +/- 1.8x10(6) cells/mL) and lung tissue (12.7 +/- 1.8 vs. 23 +/- 3.7% neutrophils of total number of cells) compared to Poldip2 EC+/+ mice. qPCR analysis of the lung tissue revealed a significantly dampened induction of inflammatory gene expression (TNF alpha 2.23 +/- 0.39 vs. 4.15 +/- 0.5-fold, I kappa B alpha 4.32 +/- 1.53 vs. 8.97 +/- 1.59-fold), neutrophil chemoattractant gene expression (CXCL1 68.8 +/- 29.6 vs. 147 +/- 25.7-fold, CXCL2 65 +/- 25.6 vs. 215 +/- 27.3-fold) and a marker of endothelial activation (VCAM1 1.25 +/- 0.25 vs. 3.8 +/- 0.38-fold) in Poldip2 EC-/- compared to Poldip2 EC+/+ lungs. An in vitro model using human pulmonary microvascular endothelial cells was used to assess the effect of Poldip2 knock-down on endothelial activation and permeability. TNF alpha-induced endothelial permeability and VE-cadherin disruption were significantly reduced with siRNA-mediated knock-down of Poldip2 (5 +/- 0.5 vs. 17.5 +/- 3-fold for permeability, 1.5 +/- 0.4 vs. 10.9 +/- 1.3-fold for proportion of disrupted VE-cadherin). Poldip2 knock-down altered expression of Rho-GTPase-related genes, which correlated with reduced RhoA activation by TNF alpha (0.94 +/- 0.05 vs. 1.29 +/- 0.01 of relative RhoA activity) accompanied by redistribution of active-RhoA staining to the centre of the cell. Conclusion Poldip2 is a potent regulator of endothelial dysfunction during sepsis-induced lung injury, and its endothelium-specific inhibition may provide clinical benefit.