Muscle-derived extracellular superoxide dismutase inhibits endothelial activation and protects against multiple organ dysfunction syndrome in mice

Highlights

• Increased EcSOD in vital organs from skeletal muscle protects against MODS.

• EcSOD mutation mimicking human R213G SNP exacerbates MODS.

• Parabiosis and serum transfusion confirm causation of EcSOD in protection.

• The mechanism is inhibition of endothelial activation in endotoxemia.

Abstract

Multiple organ dysfunction syndrome (MODS) is a detrimental clinical complication in critically ill patients with high mortality. Emerging evidence suggests that oxidative stress and endothelial activation (induced expression of adhesion molecules) of vital organ vasculatures are key, early steps in the pathogenesis. We aimed to ascertain the role and mechanism(s) of enhanced extracellular superoxide dismutase (EcSOD) expression in skeletal muscle in protection against MODS induced by endotoxemia. We showed that EcSOD overexpressed in skeletal muscle-specific transgenic mice (TG) redistributes to other peripheral organs through the circulation and enriches at the endothelium of the vasculatures. TG mice are resistant to endotoxemia (induced by lipopolysaccharide [LPS] injection) in developing MODS with significantly reduced mortality and organ damages compared with the wild type littermates (WT). Heterogenic parabiosis between TG and WT mice conferred a significant protection to WT mice, whereas mice with R213G knock-in mutation, a human single nucleotide polymorphism leading to reduced binding EcSOD in peripheral organs, exacerbated the organ damages. Mechanistically, EcSOD inhibits vascular cell adhesion molecule 1 expression and inflammatory leukocyte adhesion to the vascular wall of vital organs, blocking an early step of the pathology in organ damage under endotoxemia. Therefore, enhanced expression of EcSOD in skeletal muscle profoundly protects against MODS by inhibiting endothelial activation and inflammatory cell adhesion, which could be a promising therapy for MODS.

Introduction

Multiple organ dysfunction syndrome (MODS) is a serious clinical syndrome as a result of trauma, hemorrhagic shock and sepsis (see Review [1]). Starting as an inflammatory response in the originally affected tissues/organs, it perpetuates to damage all organs if left unchecked. Despite extensive research, MODS remain the main cause of death in the intensive care units with an extremely high rate of mortality (30–80%) [2]. To date, there is no effective therapy for MODS [2], probably due to an incomplete understanding of the pathogenic process.

The pathogenesis of MODS involves cascades of events including systemic inflammatory responses, local cell activation, inflammatory cell infiltration, coagulation, ischemia and eventually failure of the vital organs [3]. Emerging evidence suggests that oxidative stress and activation of the endothelium are key, early steps in the etiology [3]. Free radicals produced by leukocytes and/or endothelial cells [4] activate endothelial cells to expression cell surface adhesion molecules and promote inflammatory cell adhesion and transmigration into the interstitial space. These will eventually cause organ damage, as well as platelet adhesion and the consequent coagulation in the vasculature, which further impairs the circulation and metabolism of the vital organs in a vicious cycle. At the center of the pathology is the induction of oxidant and free radicals by leukocytes and/or endothelial cells that trigger endothelial activation [4]. Although general antioxidants have been shown to have promising effects against MODS in preclinical studies, they failed in clinical trials [5] probably due to lack of target specificity, which leads to ineffectiveness and sometimes fatal side effects [5].

Extracellular superoxide dismutase (EcSOD) functions in scavenging biologically toxic superoxide in the extracellular space, and is widely expressed in all tissues with the highest expression in the lung and kidney [6], [7]. EcSOD expression decreases in affected tissues in a variety of chronic diseases [8], [9], [10], and genetic approaches in animal models have provided strong evidence for functional roles of ectopic expression of EcSOD in cardiovascular, pulmonary and neuronal disorders [11], [12], [13], [14], [15], [16], [17], [18], [19]. We have previously shown that EcSOD is expressed by myofibers in adult skeletal muscle, which can be enhanced by exercise training, leading to elevated EcSOD levels in the blood and peripheral organs [20], [21]. Using molecular genetics model in mice, we showed that EcSOD expressed in muscle-specific EcSOD transgenic mice (TG) also redistributes to other peripheral organs through the circulation [20], [21]. These TG mice are resistant to dexamethasone- and heart failure-induced muscle wasting [21] as well as diabetic cardiomyopathy [20], supporting a role of EcSOD in protecting peripheral organs against oxidative stress. Therefore, induced EcSOD expression in skeletal muscle may mediate the health benefits of exercise in protection against chronic disease. We speculated that enhanced EcSOD expression in skeletal muscle might provide protection against severe disease conditions, such as MODS. The premise is that enhanced expression of EcSOD in skeletal muscle enriches at and enters endothelium of the peripheral organs [21], [22], which can effectively mitigate oxidative stress and endothelial activation, the two nodal steps in MODS development.

Herein, we took advantage of gain- and loss of function molecular genetics approaches in endotoxemia-induced MODS model in mice in combination with powerful parabiosis and intraperitoneal serum transfusion to determine if enhanced expression of muscle-derived EcSOD protects against MODS and to elucidate the underlying mechanism(s) with a focus on endothelial activation and organ damage. Since skeletal muscle is the largest organ (~ 40% body mass), positive findings would justify using exercise training and/or muscle-mediated gene therapy to prevent and/or treat MODS.