Abstract 18761: Non-Muscle Myosin Light Chain Kinase Mediates Vascular Endothelial Growth Factor Induced Vascular Hyperpermeability via Specificity Protein 1 During Asthmatic Inflammation

Abstract

INTRODUCTION: Increased vascular permeability and accumulation of alveolar fluid are cardinal features of lung inflammation and asthma. Two targets for these responses are vascular endothelial growth factor (VEGF) and the non-muscle isoform of myosin light chain kinase (nmMLCK), a key regulator of vascular barrier function via actin-myosin mediated cell contraction. Both VEGF and nmMLCK mRNA and protein expression are upregulated in asthmatic patients. Supported by preliminary in vitro studies that demonstrate increased VEGF-induced nmMLCK expression, we propose a linkage between these two asthma targets and hypothesize that nmMLCK mediates VEGF induced vascular hyperpermeability in asthmatic inflammation. Methods & Results: mRNA quantified by qPCR showed increases in nmMLCK mRNA in lung biopsies from human asthmatic patients and ovalbumin (OVA, 0.4 mg/kg) challenged murine asthma model. OVA-challenged mice overexpressing nmMLCK in the endothelium (nmMLCKec/ec) exhibit increased airway pressure time index (APTI) and BAL protein levels compared to WT, whereas OVA-challenged nmMLCK-/- mice demonstrated significant attenuation of increases in APTI and BAL protein levels. Human pulmonary artery endothelial cells (HPAEC) treated with recombinant hVEGF (100ng/ml) increased nmMLCK mRNA and protein expression. In silico analysis of nmMYLK 5’ UTR 2.4 kb region and in vitro luciferase promoter assay in HPAEC revealed transcription factor binding sites of downstream effectors of VEGF signaling, with -400bp upstream of TSS being the critical region, which included the binding sites for the transcription factor, Sp1. Consistent with this, reduced Sp1 expression by siRNA attenuated VEGF-induced decrease in transendothelial electrical resistance (TER) as measured by electrical cell-substrate impedance sensing system. CONCLUSIONS: Our data demonstrate that nmMLCK induced by VEGF is mediated through Sp1 in lung EC. nmMLCK is a novel asthmatic target and represents a potential candidate for therapeutic modulation of asthmatic inflammation.