Myosin light chain phosphorylation and pulmonary endothelial cell hyperpermeability in burns

Abstract

Major cutaneous burns result in not only localized tissue damage but broad systemic inflammation causing organ system damage distal to the burn site. It is well recognized that many problems result from the release of inflammatory mediators that target vascular endothelial cells, causing organ dysfunction. The pulmonary microvessels are particularly susceptible to functional abnormalities as a direct consequence of exposure to burn-induced inflammatory mediators. Traditional therapeutic intervention is quite often ineffective in treating burn patients suffering from systemic problems. A possible explanation for this ineffectiveness may be that because so many mediators are released, supposedly activating numerous signaling cascades that interact with each other, targeting of upstream factors in these cascades on an individual basis becomes futile. Therefore, if an end-point effector responsible for endothelial dysfunction following burn injury could be identified, it may present a target for intervention. In this study, we identified phosphorylation of myosin light chain (MLC) as a required element of burn plasma-induced hyperpermeability across rat lung microvascular endothelial cell monolayers. In addition, pharmacological inhibition of myosin light chain kinase (MLCK) and Rho kinase as well as transfection of MLCK-inhibiting peptide blocked actin stress fiber formation and MLC phosphorylation in response to burn plasma. The results suggest that blocking MLC phosphorylation may provide therapeutic intervention in burn patients with the goal of alleviating systemic inflammation-induced endothelial dysfunction.

Involvement of ROCK-mediated endothelial tension development in neutrophil-stimulated microvascular leakage

“…We also report that inhibition of ROCK attenuates the PMN-stimulated increases in endothelial isometric tension. These data are concordant with our previous reports that ROCK inhibition attenuates PMN-induced actin polymerization in endothelial cells (8) and dual phosphorylation of MLC caused by inflammatory stimuli (41). However, it is also worth noting that we observed a lag between ca-ROCKinduced MLC phosphorylation (noticeably increased at 30 min) and force development (starting at 10 min), indicating that the initial stage of ROCK-mediated contraction may involve other mechanisms that rapidly and transiently affect cell tension.…”

PKC-dependent, burn-induced adherens junction reorganization and barrier dysfunction in pulmonary microvascular endothelial cells

“…We have shown that activated neutrophils induce tyrosine phosphorylation of VE-cadherin and ␤-catenin followed by gap formation and hyperpermeability (25). Furthermore, we found that application of plasma from burned rats to cultured microvascular endothelial cells induced MLC phosphorylation-dependent permeability increases and actin stress fiber formation (23).…”

“…Characteristic phenomena resulting from endothelial cell exposure to various agonists are phosphorylation of myosin light chain (MLC), actomyosin contractile forces, and modification/reorganization of the AJ (23)(24)(25)27). We have shown that activated neutrophils induce tyrosine phosphorylation of VE-cadherin and ␤-catenin followed by gap formation and hyperpermeability (25).…”

Inhibition of long myosin light-chain kinase activation alleviates intestinal damage after binge ethanol exposure and burn injury

“…A rise in proinflammatory cytokines is associated with MLCK activation, and it has been shown that TNF-␣, IL-1␤, and lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry mediator on T cells (LIGHT) can upregulate MLCK transcription (2,12,25,29,30,36). Furthermore, increased MLCK activation has been linked to intestinal permeability after burn injury or chronic ethanol exposure alone (29,50). Traumatic injury, like burn, causes an overexuberant systemic inflammatory response characterized by a rise in systemic and tissue levels of IL-6, TNF-␣, and IL-1␤ (22,34,43,44).…”