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. 2017 Dec:113:212-223.
doi: 10.1016/j.freeradbiomed.2017.09.029. Epub 2017 Oct 2.

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

Jarrod A Call  1 , Jean Donet  1 , Kyle S Martin  2 , Ashish K Sharma  3 , Xiaobin Chen  4 , Jiuzhi Zhang  5 , Jie Cai  6 , Carolina A Galarreta  7 , Mitsuharu Okutsu  1 , Zhongmin Du  8 , Vitor A Lira  1 , Mei Zhang  1 , Borna Mehrad  8 , Brian H Annex  8 , Alexander L Klibanov  8 , Russell P Bowler  9 , Victor E Laubach  3 , Shayn M Peirce  2 , Zhen Yan  10
Affiliations

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

Jarrod A Call et al. Free Radic Biol Med. 2017 Dec.

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.

Keywords: EcSOD; Endothelial activation; Endotoxemia; Free radicals; MODS; Oxidative stress; Parabiosis; Skeletal muscle; VCAM-1.

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Figures

Fig. 1
Fig. 1. Skeletal muscle-specific EcSOD transgenic mice are protected from LPS-induced MODS
A) Immunoblot images showing that EcSOD levels are increased in various peripheral tissues and serum in muscle-specific EcSOD transgenic mice under the control of muscle creatine kinase promoter, but not in the brain, compared with WT littermate mice. The vertical lines separates images from different parts of the same membrane or different membranes with the exact same experimental condition (probed together) and exposure; B) EcSOD was detected at smooth muscles of blood vessel in WT lung, not so much at vascular endothelial cells (v) (arrows) bronchial (br) and alveoli (al). Significantly elevated EcSOD was detected at smooth muscles, vascular endothelial cells (arrows) and alveoli, but not at bronchial in TG mice. Experiments were repeated three times for each conditions; C) Mann-Whitney survival curves for TG and WT following LPS injection (20 mg/kg) (n = 10–11). *** denotes p < 0.001); D) Quantification of BUN, SCR, LDH and ALT (n = 7–12). ** and *** denote p < 0.01 and p < 0.001, respectively; and E) Quantitative measurements of lung physiological function in airway resistance, pulmonary compliance and pulmonary artery pressure (n = 6). ** and *** denote p < 0.01 and p < 0.001, respectively.
Fig. 2
Fig. 2. Heterogenic parabiosis confers protection of WT mice against MODS from TG mice and R213G KI mice are more vulnerable to organ damage
A) Graphical representation of WT, TG and isogenic (WT-WT) and heterogenic (WT-TG) parabiotic mice (top) and representative immunoblot of serum EcSOD protein demonstrating elevated serum EcSOD in WT mice of the WT-TG parabiotic pairs, but not in WT-WT parabiotic pairs (bottom); B) Representative image of lung in fixative (float or sink) demonstrating greater lung density in WT-WT compared with WT-TG pairs following LPS injection; C) Representative H&E histological images of the lungs; D) Representative pan-macrophage and iNOS immunohistochemistry images; E) Quantification of BUN, SCR, LDH, and ALT (n = 4). * and *** denote p < 0.05 and p < 0.001, respectively. F) PCR-based genotyping for KI and WT mice showing the detection of the KI and WT alleles (top) and immuoblot analysis of EcSOD proteins in the kidney and serum using GAPDH and Ponseau staining as controls for loading. The vertical lines separates images from different parts of the same membrane; and G) Quantification of BUN in homozygous KI and WT mice following LPS injection (6 hrs) (n = 4–5). * denotes p < 0.05.
Fig. 3
Fig. 3. TG mice are resistant to endotoxemia-induced endothelial activation
A) Representative VCAM-1 immunohistochemistry image of the lung demonstrating less endothelial cell activation in TG-LPS compared to WT-LPS. The experiment was done in 4–5 mice for each condition with similar findings; B) Representative immunoblot and quantitative representation of VCAM-1 protein in the lung (n = 5–11). The vertical lines separates images from different parts of the same membrane. * and *** denote p < 0.05 and p < 0.001, respectively; and C) Representative immunoblot and quantitative representation of VCAM-1 protein in the kidney (n = 5–9). *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively.
Fig. 4
Fig. 4. Serum EcSOD is sufficient to reduce endothelial cell activation in vitro and in vivo
A) Immunoblot of VCAM-1 protein in HUVEC cells pre-incubated with WT or TG serum followed by TNF-α treatment; B) Representative immunoblot for serum EcSOD protein in recipient WT mice before and after receiving WT or TG serum (Pre vs. Post serum, respectively), and following LPS injection (Post LPS) (n = 6–10); C) Representative ultrasound-based images of VCAM-1 target microbubbles in WT recipient mice that received WT or TG serum followed by LPS injection. Kidneys were imaged prior to microbubble injection (Baseline), immediately (Peak signal) and 5 minutes after injection (Stable signal), and after application of a high power destructive pulse (After burst); D) Quantification of stable signal (n = 6–10). * denotes p < 0.05; and E) Quantification of BUN (n = 4–7). * and *** denotes p < 0.05 and p < 0.001, respectively.
Fig. 5
Fig. 5. Muscle-specific EcSOD transgenic mice are resistant the endotoxemia-induced monocyte adhesion
A) CX3CR1-GFP (Monocyte-GPF reporter mice)-TG double transgenic mice or CX3CR1-GFP-WT control mice receiving LPS injection were fitted with a vessel-chamber window (left panel) at 6 hours post LPS injection to observe and record vascular monocyte adhesion and rolling (middle and right panels). Red arrows indicate GFP-monocytes adhered to the vessel walls; B) Quantification of monocyte adhesion per vessel wall surface area for CX3CR1-GFP-WT controls (WT) and CX3CR1-GFP-TG (TG) mice after LPS injection (n = 4–9); C) Quantification of rolling monocytes per vessel wall surface area (n = 5–9). * and *** denote p < 0.05 and p < 0.001, respectively; and D) A working model of EcSOD-mediated protection against endothelial cell activation and monocyte adhesion under endotoxemia. Endotoxemia induces expression of adhesion and chemokines in endothelial cells via NFκB mediated transcription to promote inflammatory cell interaction and adhesion. This process will in turn promote free radical generation from both inflammatory and endothelial cells, exacerbating the inflammatory responses. EcSOD could scavenge the free radicals at and inside the endothelial cells and attenuate the inflammatory cell-endothelial cell interaction.
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