Original articleMuscle-derived extracellular superoxide dismutase inhibits endothelial activation and protects against multiple organ dysfunction syndrome in mice
Graphical abstract
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.
Section snippets
Animals
Mice were housed in temperature-controlled (22 °C) quarters with a 12:12-h light-dark cycle and free access to water and normal chow (Harlan). Muscle-specific EcSOD transgenic mouse line was generated at the University of Virginia Gene Targeting and Transgenic Facility as described [21] and backcrossed into pure C57BL/6J background (≥ 10 generations). CX3CR1-GFP mice were purchased from the Jackson Laboratory. To induce endotoxemia, mice received a single injection of LPS (20 mg/kg i.p.)(Sigma,
Enhanced EcSOD expression in skeletal muscle profoundly protects against MODS with reduced mortality under endotoxemia
TG mice had significantly increased EcSOD in all peripheral organs except for the brain (Figs. 1A and S4) probably due to the blood-brain barrier, and a 6-fold increase of serum EcSOD assessed by western blot (p < 0.001; Fig. S1A) [21] as well as colorimetric measurement of the enzyme activity (4.27 ± 1.96 U/ml in WT mice vs 25.2 ± 1.61 U/ml in TG mice; p < 0.001) (Fig. S1A). This finding confirms that the transgene-encoded EcSOD is biologically active with antioxidant enzyme activity. TG mice
Discussion
MODS is a major cause of mortality in intensive care units, and accumulating evidence suggests that oxidative stress and endothelial activation are major contributing factors to the pathology [5]. Although general antioxidants have been shown to be effective in various MODS models in animals, clinical trials have failed, probably due to lack of targeting specificity of the antioxidants [5]. We therefore need antioxidants that target the vital organs to break the vicious cycle of endothelial
Conclusions
We have obtained novel findings to show that EcSOD overexpressed in skeletal muscle redistributes through the circulation to peripheral organs and provides potent protection against MODS under endotoxemia. On the contrary, loss of binding of EcSOD binding to the peripheral organs due to mutation mimicking a human SNP leads to exacerbated organ damages. This EcSOD-mediated protection is a result of decreased oxidative stress and reduced endothelial activation. Importantly, systemic delivery of
Acknowledgements
We would like to thank Drs. Jonathan Kipnis and Igor Smirnov for helping setting up the surgical procedure for parabiosis. This work was supported by the National Institutes of Health (R01GM114840 to ZY) and American Heart Association (12POST12030231 Postdoctoral Fellowship to JAC). JAC, JD, ZY were involved in designing research studies, conducting experiments, acquiring data, analyzing data and writing the manuscript. KSM, AKS, XC, JZ, JC, CAG, MO, ZD, VAL, MZ were involved in conducting
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Contributed equally to this work.