Nonmuscle Myosin Light-Chain Kinase Deficiency Attenuates Atherosclerosis in Apolipoprotein E–Deficient Mice via Reduced Endothelial Barrier Dysfunction and Monocyte Migration

Abstract

Background Endothelial dysfunction and monocyte migration are key events in the pathogenesis of atherosclerosis. Non-muscle myosin light-chain kinase (nmMLCK), the predominant MLCK isoform in endothelial cells, has been shown to contribute to vascular inflammation by altering endothelial barrier function. However, its impact on atherogenesis remains unknown. Methods and Results We investigated the role of nmMLCK in the development of atherosclerotic lesions in apolipoprotein E-deficient (apoE−/−) mice fed an atherogenic diet for 12 weeks. Histopathological examination demonstrated that nmMLCK deficiency (apoE−/− nmmlck−/−) reduced the size of aortic lesions by 53%, lipid contents by 44% and macrophage deposition by 40%. Western blotting and reverse-transcription polymerase chain reaction revealed the expression of nmMLCK in aortic endothelial cells and peripheral blood monocytes. Measurements of transendothelial electric resistance indicated that nmMLCK deficiency attenuated endothelial barrier dysfunction caused by thrombin, oxLDL and TNF-α. In monocytes, nmMLCK deficiency reduced their migration in response to the chemokine MCP-1. Further mechanistic studies showed that nmMLCK acted through both myosin light chain (MLC) phosphorylation-coupled and -uncoupled pathways; the latter involved Src signaling. Moreover, depletion of Src via gene silencing, site-specific mutagenesis or pharmacological inhibition of Src greatly attenuated nmMLCK-dependent endothelial barrier dysfunction and monocyte migration. Conclusions nmMLCK contributes to atherosclerosis by regulating endothelial barrier function and monocyte migration via mechanisms involving not only kinase-mediated MLC phosphorylation but also Src activation.

Non-muscle Mlck is required for β-catenin- and FoxO1-dependent downregulation of Cldn5 in IL-1β-mediated barrier dysfunction in brain endothelial cells

“…Src-mediated phosphorylation of b-catenin and/or VE-cadherin has been shown to be an important signaling event leading to adherens junction uncoupling (Cohen et al, 1999;Dejana et al, 2008;McEwen et al, 2012). We recently reported that Src-activation in response to thrombin was dependent on the presence of nmMlck (Sun et al, 2011), suggesting that nmMlck could be upstream of Src-mediated tyrosine phosphorylation of the adherens junction and subsequent uncoupling. However, it is also thought that b-catenin that is dissociated from the adherens junction needs to be phosphorylated at serine residues in order to be stable in the cytoplasm (Daugherty and Gottardi, 2007).…”

“…Although the role of nmMlck in mediating both the opening of endothelial cell-cell junctions and the paracellular hyperpermeability response to proinflammatory agents (including histamine, thrombin, ROS and activated neutrophils) has been well documented (reviewed in Yuan and Rigor, 2010), the role of nmMlck in IL-1b-mediated dysfunction of the BBB has not been investigated. Because nmMlck has been shown to be involved in dissociating b-catenin from VEcadherin at the adherens junction (Sun et al, 2011), in this study, we hypothesized that nmMlck modulates IL-1b-mediated downregulation of Cldn5 and dysfunction of the BMVEC barrier in a manner that involves b-catenin activation, nuclear translocation and repression of the Cldn5 gene (Fig. 1).…”

“…Experiments using genetically modified animals have demonstrated the significance of nmMlck in mediating endothelial contractility and hyperpermeability under proinflammatory conditions. Importantly, nmMlck-isoform-specific knockout (nmmlck 2/2 ) mice demonstrate a phenotype devoid of cardiovascular defects, yet these mice display an attenuated vascular inflammatory response to acute lung injury (Wainwright et al, 2003), severe burns (Reynoso et al, 2007), atherosclerosis (Sun et al, 2011) and improved survival in endotoxic shock (Ranaivo et al, 2007). Although the role of nmMlck in mediating both the opening of endothelial cell-cell junctions and the paracellular hyperpermeability response to proinflammatory agents (including histamine, thrombin, ROS and activated neutrophils) has been well documented (reviewed in Yuan and Rigor, 2010), the role of nmMlck in IL-1b-mediated dysfunction of the BBB has not been investigated.…”

Nonmuscle Myosin Light Chain Kinase: A Key Player in Intermittent Hypoxia‐Induced Vascular Alterations

“…In endothelial cells, this retraction is linked to endothelial permeability enhancement and vascular leakage under proinflammatory conditions. In this context, an activation of the nmMLCK has been linked to several pathophysiological conditions, such as vascular inflammation and atherosclerosis,18 acute lung injury,19 endotoxic shock,20 and severe burns 21…”

Vessels With Cingulin Are Leakproof

“…4 Finally, barrier function plays a role in diseases such as atherosclerosis, where poor barrier maintenance allows increased monocyte extravasation. 5 The increase in monocyte extravasation leads to macrophage colonization and the eventual buildup of plaques.…”