Introduction:
Vascular inflammation is an underlying causative factor in the progression of various ailments including acute lung injury. In the inflamed lung, chronic endothelial cell (EC) activation facilitates leukocyte recruitment and extravasation into tissue beds, leading to end organ damage. Delineating the mechanisms of EC activation may uncover novel areas of therapeutic opportunity. Our laboratory has identified polymerase delta-interacting protein 2 (Poldip2) as a novel regulator of endothelial activation and permeability.
Methods:
Poldip2 +/- and +/+ mice were injected with either saline or lipopolysaccharide (LPS, 18 mg/kg) for either 6 or 18 hours to induce acute lung injury. Bronchoalveolar lavage (BAL) fluid was collected to assess leukocyte infiltration via flow cytometry and cytokine expression via ELISA. Evans blue staining was used to evaluate lung permeability, and tissue lysates were analyzed for inflammatory marker expression. For
in vitro
analysis, inflammatory marker expression, leukocyte adherence and cell permeability were tested in human pulmonary microvascular endothelial cells (HPMVECs) treated with saline or LPS (1 μg/mL). To test the efficacy of Poldip2 silencing in HPMVECs, siRNA was used at 100 nM (siControl vs siPoldip2). Comparisons between groups were analyzed with a one-way ANOVA followed by Tukey’s post-hoc test.
Results:
Compared to Poldip2 +/+, Poldip2 +/- mice treated with LPS exhibited significantly reduced mortality (80% vs. 20% survival), Evans blue staining, and leukocyte infiltration. FACS analysis of BAL fluid revealed a decrease in LPS-induced monocyte and neutrophil infiltration in Poldip2 +/- mice compared to Poldip2 +/+ mice. RT-qPCR analysis indicated a reduction in inflammatory gene induction in lung tissue. In HPMVECs, LPS induced increases in VCAM-1 protein expression, THP-1 leukocyte adherence and endothelial permeability, which were attenuated with siRNA-mediated knockdown of Poldip2 (
P<0.05
for all).
Conclusion:
Poldip2 is an important regulator of EC activation, leukocyte adherence and EC permeability. In scenarios of persistent vascular inflammation and endothelial barrier dysfunction, such as in acute lung injury, inhibition of Poldip2 may provide clinical benefit.
Blocking p21-activated Kinase Reduces Lipopolysaccharide-induced Acute Lung Injury by Preventing Polymorphonuclear Leukocyte Infiltration