0319 : Non muscular myosin light chain kinase (nmMLCK) and NF-κB pathway interaction in endothelium is critical for lipopolysaccharide-induced vascular hyporeactivity

The lipopolysaccharide (LPS) is a major component of the Gram-negative bacteria affects cells of the vasculature causing hypotension by the initiation of smooth muscle cell hyporeactivity and endothelial dysfunction. LPS through the Toll-like receptor 4 activates the NF-κB pathway initiating the transcription of several inflammatory genes as cytokines or the inducible nitric oxide synthase (iNOS) and finally the overproduction of nitric oxide (NO). We and other have demonstrated that the deletion of non muscular myosin light chain kinase (nmMLCK), a kinase implicated in the regulation of endothelial permeability, enhances survival after intraperitoneal injection of LPS and prevents the LPS-induced hyporeactivity and inflammatory activation. This study was aimed to better understand the molecular mechanism of the protection of deletion nmMLCK against LPS-induced vascular alterations. Aortic rings were harvested from untreated wild-type or nmMLCK deficient mice after injection of LPS (100 μg/kg) for 20 hours and then vascular reactivity, NO production and iNOS expression in aorta were assessed. Human aortic endothelial cells were incubated with LPS (10 μg/ml) for 3 hours in absence or presence of ML-7 (5μM), an nmMLCK inhibitor, and NF-κB activation and interaction of nmMLCK and NF-κB were analyzed. We provide evidence that nmMLCK deficiency prevents LPS-induced vascular hyporeactivity but not endothelial dysfunction in the aorta. Deletion of nmMLCK inhibits both NF-κB activation and the increase of NO release via induction of iNOS within the vascular wall. In human aortic endothelial cells, LPSinduced NF-κB activation occurs via increase of nmMLCK activity sensitive to the MLCK inhibitor, ML-7. In addition, nmMLCK co-immunoprecipitates with NF-κB and vice versa, suggesting physical interactions between both proteins. We report that NF-κB as a novel partner of nmMLCK within endothelial cells.