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

Abstract

AIMS\nSepsis-induced lung injury is associated with significant morbidity and mortality. Previously, we showed that heterozygous deletion of polymerase δ-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.\n\n\nMETHODS AND RESULTS\nEndothelial-specific Poldip2 knock-out mice (EC-/-) and their wild type littermates (EC+/+) were injected with saline or lipopolysaccharide (LPS, 18\u2009mg/kg) to model sepsis-induced lung injury. At 18\u2009hours post-injection mice, were euthanized and bronchoalveolar lavage (BAL) fluid and lung tissue were collected to assess leukocyte infiltration. Poldip2 EC-/- mice showed reduced lung leukocyte infiltration in BAL (0.21\u2009±\u20090.9x106 vs 1.29\u2009±\u20091.8x106 cells/mL) and lung tissue (12.7\u2009±\u20091.8 vs 23\u2009±\u20093.7% 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 (TNFα 2.23\u2009±\u20090.39 vs 4.15\u2009±\u20090.5 fold, IκBα 4.32\u2009±\u20091.53 vs 8.97\u2009±\u20091.59 fold), neutrophil chemoattractant gene expression (CXCL1 68.8\u2009±\u200929.6 vs 147\u2009±\u200925.7 fold, CXCL2 65\u2009±\u200925.6 vs 215\u2009±\u200927.3 fold) and a marker of endothelial activation (VCAM1 1.25\u2009±\u20090.25 vs 3.8\u2009±\u20090.38 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. TNFα-induced endothelial permeability and VE-cadherin disruption were significantly reduced with siRNA-mediated knockdown of Poldip2 (5\u2009±\u20090.5 vs 17.5\u2009±\u20093 fold for permeability, 1.5\u2009±\u20090.4 vs 10.9\u2009±\u20091.3 fold for proportion of disrupted VE-cadherin). Poldip2 knockdown altered expression of Rho-GTPase-related genes, which correlated with reduced RhoA activation by TNFα (0.94\u2009±\u20090.05 vs 1.29\u2009±\u20090.01 of relative RhoA activity) accompanied by redistribution of active RhoA staining to the center of the cell.\n\n\nCONCLUSION\nPoldip2 is a potent regulator of endothelial dysfunction during sepsis-induced lung injury, and its endothelium-specific inhibition may provide clinical benefit.