Critical role for the lung endothelial nonmuscle myosin light‐chain kinase isoform in the severity of inflammatory murine lung injury

Abstract

Global knockout of the nonmuscle isoform of myosin light-chain kinase (nmMLCK), a primary cellular regulator of cytoskeletal machinery, is strongly protective in preclinical murine models of inflammatory lung injury. The current study was designed to assess the specific contribution of endothelial cell (EC) nmMLCK to the severity of murine inflammatory lung injury produced by lipopolysaccharide (LPS) and mechanical ventilation ventilatorinduced lung injury or ventilation (VILI). Responses to combined LPS/VILI exposure were assessed in: (i) wild-type (WT) C57BL/6J mice; (ii) transgenic mice with global deletion of nmMLCK (nmMylk −/− ); (iii) transgenic nmMylk −/− mice with overexpression of nmMLCK restricted to the endothelium (nmMylk −/−/ec-tg+ ). Lung inflammation indices included lung histology, bronchoalveolar lavage (BAL) polymorphonuclear leukocytes (PMNs), lung protein biochemistry, tissue albumin levels, Evans blue dye (EBD) lung extravasation, and plasma cytokines (interleukin-6 [IL-6],keratinocyte chemoattractant [KC]/IL-8, IL-1bβ, extracellular nicotinamide phosphoribosyltransferase, tumor necrosis factor-α). Compared to WT C57BL/ 6J mice, the severity of LPS/VILI-induced lung injury was markedly reduced in mice with global nmMLCK deletion reflected by reductions in histologic inflammatory lung injury, BAL PMN counts, mitogen-activated protein kinase, and NF-kB pathway activation in lung homogenates, plasma cytokine levels, and parameters of lung permeability (increased BAL protein, tissue albumin levels, EBD lung extravasation). In contrast, mice with restricted overexpression of nmMLCK in EC (nmMylk −/−/ec-tg+ ) showed significant persistence of LPS/VILI-induced lung injury severity compared to WT mice. In conclusion, these studies strongly endorse the role of EC nmMLCK in driving the severity of preclinical inflammatory lung injury. Precise targeting of EC

Myosin Light Chain Kinase Deletion Worsens Lung Permeability and Increases Mortality in Pneumonia-Induced Sepsis

“…A key role of MLCK is to increase paracellular permeability, and numerous studies have shown that MLCK deletion, inhibition, or decreased activation decreases paracellular permeability in multiple cell types (37–41), with the most robust evidence in the vascular endothelium and the intestinal epithelium. Notably, MLCK mediates increased lung vascular endothelial permeability in LPS-induced lung injury (42–45). Further, mice have a marked increase in lung neutrophil infiltration after an i.p.…”

Dexamethasone restores TNFα-induced epithelial barrier dysfunction in primary rat alveolar epithelial cells

“…In ALI/ARDS in a clinical setting, the disruption of both the epithelial and vascular endothelial barriers occurs, resulting in the leakage of inflammatory cells and protein-rich effluent into the alveolar space [ 3 , 8 , 9 ]. Recent evidence suggests that MLCK-mediated regulation of the barrier function in vascular endothelial cells is important as per an ARDS model in MLCK-knockout mice [ 41 ]. In contrast, in a model of lung injury caused by pneumoedematogenic gas inhalation, MLCK inhibitors improved lung injury by enhancing the alveolar epithelial cell barrier function [ 35 ].…”

A Razor's Edge: Vascular Responses to Acute Inflammatory Lung Injury/Acute Respiratory Distress Syndrome

“…These EC actin-binding cytoskeletal effectors are involved in (a) regulation of vascular barrier processes, including inflammatory leukocyte trafficking (43,44); (b) vascular responses to ventilator-generated mechanical stress (45); and (c) harboring of genetic variants that are overrepresented in Blacks with ARDS and contributing to ARDS susceptibility and mortality (46,47). nmMLCK exhibits dual functionality as a proinflammatory effector contributing to lung EC reactive oxygen species (ROS) formation (48) and barrier disruption (49), but also a barrier-protective molecule in response to specific environmental cues. Both nmMLCK and cortactin are intimately involved in maintaining barrier integrity in quiescent ECs and are essential to the formation of a cortical actomyosin band at the cell periphery.…”

“…Proinflammatory agonists, cytokines, DAMPs, PAMPS, and increased mechanical stress all significantly increase nmMLCK protein expression via hypoxia-inducible factors (HIF-1a/ HIF-2a) (58) with unique negative nmMYLK transcriptional regulation by nuclear factor erythroid 2-related factor 2 (NRF2)/antioxidant elements (59). These proinflammatory mediators robustly increase nmMLCK activity to elicit spatially localized central contraction, paracellular gap formation, and disruption of the lung vascular barrier in vitro and in vivo (45,49). The Ras homolog family member A (RhoA) guanosine triphosphatase (GTPase) directly binds and activates nmMLCK and is a significant promoter of EC permeability (60,61).…”

“…Despite this abysmal record of failure, current mechanistic advances indicate that lung EC barrier regulation/immune effector function may serve as attractive druggable targets to drive restoration of the integrity of the injured pulmonary circulation and to reduce ARDS mortality. For example, the nmMLCK isoform is an attractive ARDS/VILI therapeutic target, as nmMLCK inhibitory approaches [short interfering RNAs (siRNAs)] and nanoparticle delivery of peptide inhibitor of kinase (PIK), a nmMLCK kinase inhibitor, effectively reduce alveolar and vascular permeability and lung inflammation (45,49,135), suggesting the targeting of EC nmMLCK as a therapeutic strategy.…”