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Anesthesia & Analgesia:
doi: 10.1213/ANE.0000000000000170
Anesthetic Pharmacology: Research Report

Thrombomodulin Improved Liver Injury, Coagulopathy, and Mortality in an Experimental Heatstroke Model in Mice

Kawasaki, Takashi MD, PhD; Okamoto, Kohji MD, PhD; Kawasaki, Chika MD; Sata, Takeyoshi MD, PhD

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Author Information

From the Department of Anesthesiology, University of Occupational and Environmental Health, Kitakyushu, Japan.

Accepted for publication January 28, 2014.

Funding: This work was supported by University of Occupational and Environmental Health Grant for Advanced Research (to Dr. T. Kawasaki) and Grant-in-Aid for Scientific Research (C-22591755 to Dr. T. Kawasaki, C-24592321 to Dr. C. Kawasaki) from the Japan Society for the Promotion of Science.

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Takashi Kawasaki, MD, PhD, Department of Anesthesiology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807–8555 Japan. Address e-mail to takasi-k@med.uoeh-u.ac.jp.

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Abstract

BACKGROUND: Heatstroke is a life-threatening illness and causes high mortality due to multiple organ injuries. Thrombomodulin (TM) is an endothelial anticoagulant cofactor that plays an important role in the regulation of intravascular coagulation. In this study, we investigated the effect of TM on the inflammatory process, liver function, coagulation status, and mortality in experimental heatstroke.

METHODS: Male C3H/HeN (8–10 weeks) mice were randomly assigned to the TM-treated group (TG-Pre) or nontreated heatstroke group (HS). In group TG-Pre, mice were treated with recombinant soluble TM (1 mg/kg, intraperitoneally) before heat exposure. In some experiments, recombinant soluble TM was administrated during heat exposure (TG-Delay). Heatstroke was induced by exposure to ambient temperature of 38°C for 4 hours. After heat exposure, the levels of tumor necrosis factor-α, interleukin-6, and plasma high-mobility group box 1 (HMGB1), liver function, plasma aspartate aminotransferase and alanine aminotransferase concentrations, and immunohistochemical and histopathological characteristics of the livers were determined. The coagulation status, plasma protein C levels, and thrombin–antithrombin complex levels were also measured.

RESULTS: In group HS, plasma cytokines and HMGB1 concentrations increased after heat exposure. Plasma aspartate aminotransferase and alanine aminotransferase concentrations increased after heat exposure. In group HS livers, strong and extensive immunostaining for HMGB1 was observed. In addition, there was extensive hepatocellular necrosis and collapse of nuclei observed. In group HS, plasma protein C levels were suppressed and plasma thrombin–antithrombin complex levels increased. In group TG-Pre, plasma cytokines and HMGB1 concentrations were suppressed after heat exposure compared with group HS. Liver injury, coagulopathy, and mortality also improved in group TG-Pre. Furthermore, recombinant soluble TM treatment decreased mortality even with delayed treatment.

CONCLUSIONS: This study demonstrated that recombinant soluble TM suppressed plasma cytokines and HMGB1 concentrations after heat exposure. Recombinant soluble TM also improved liver injury and coagulopathy. Recombinant soluble TM treatment improved mortality even with delayed treatment. Recombinant soluble TM may be a beneficial treatment for heatstroke patients.

Heatstroke is a life-threatening illness caused by an extreme increase in core body temperature.1,2 Despite adequate lowering of the body temperature and aggressive treatment, heatstroke is often fatal, and specific and effective therapies are lacking. The pathophysiological responses to heatstroke resemble those from sepsis in many respects. Patients with heatstroke usually present clinically with systemic inflammatory response syndrome criteria.3 The plasma levels of inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8 are increased in heatstroke patients, and these elevated concentrations have been correlated with the severity of the disease.4–6

High-mobility group box 1 protein (HMGB1), identified as a lethal late-phase mediator, was closely correlated with the development of sepsis.7,8 Although heatstroke resembles sepsis in many aspects, the contribution of HMGB1 to organ system dysfunction in this setting has not been well investigated. The liver function and coagulation status often become the main targets of heatstroke-associated insult. Thus, heatstroke-induced liver injury and coagulopathy become the main factors compromising the life of these patients.

Thrombomodulin (TM) is a transmembrane glycoprotein that plays an important role in the regulation of intravascular coagulation.9 TM inhibits the procoagulant activity of thrombin, and the thrombin–TM complex accelerates the activation of protein C. Activated protein C (APC) inhibits the activation of monocytes and macrophages.10,11 Thus, TM suppresses the production of proinflammatory cytokines via activation of APC.11

This study was designed to investigate the effect of recombinant soluble TM on inflammatory mediator production, liver function, and coagulation status in experimental heatstroke and to explore how recombinant soluble TM improves the development of liver injury. We hypothesized that recombinant soluble TM treatment improves the development of the inflammatory process, liver injury, coagulopathy, and outcome after heatstroke.

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METHODS

Mice

All experiments were performed after approval from the Animal Experimental Ethics Committee of the University of Occupational and Environmental Health. Male C3H/HeN (8–10 weeks) mice were acclimatized to room temperature at 25°C, a relative humidity of 50% ± 10%, and a 12-hour light/dark cycle for 1 week before the start of experiments. Tap water and mouse chow were provided ad libitum.

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Murine Model of Heatstroke

Mice were randomly assigned to the TM-treated group (TG-Pre), nontreated heatstroke group (HS), or normothermic control group (NC). In group TG-Pre, mice were treated with recombinant human soluble TM (ART-123; Asahi Kasei Pharma Co., Tokyo, Japan; 1 mg/kg, intraperitoneally) before heat exposure. In some experiments, recombinant soluble TM (1 mg/kg, intraperitoneally) was administered during heat exposure (2 hours after the start of heat exposure, TG-Delay). Heatstroke was induced by exposure to an ambient temperature of 38°C for up to 4 hours. During heat exposure, mice had free access to water ad libitum. The heat-stressed mice were returned to room temperature (25°C) after the end of heat exposure. In the survival study, surviving mice were monitored for up to 24 hours after a 6-hour exposure to heatstroke.

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Sample Collection

After heat exposure, mice were killed by neck dislocation. Blood and livers were harvested for further studies. Plasma samples were obtained by centrifugation of blood at 3000g for 10 minutes and frozen at −80°C until used. Livers were fixed in 4% phosphate-buffered saline–formaldehyde for histopathology and immunohistopathology.

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Cytokines Determination

Commercial enzyme-linked immunosorbent assay (ELISA) kits were used for measurement of plasma level of TNF-α, IL-6 (R&D Systems, Minneapolis, MN) and HMGB1 (Shino-TEST, Kanazawa, Japan).

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Plasma Protein C and Thrombin–Antithrombin Complex Level

Commercial ELISA kits were used for measurement of plasma levels of protein C and thrombin–antithrombin complex (TAT; USCN Life Science Inc., Wuhan, China).

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Myeloperoxidase Assay

The accumulation of neutrophils in liver tissue was assessed by determination of myeloperoxidase (MPO) activity. Tissue samples were collected, frozen in liquid nitrogen, and stored at −80°C until used. The organs were homogenized in RIPA (Radio-Immunoprecipitation Assay) buffer (Thermo Fisher Scientific Pierce Biotechnology, Rockford, IL) using the Polytron PT 1600E (Kinematica, Lucerne, Switzerland). MPO antigen levels were measured using the murine MPO ELISA kit (Hycult Biotechnology, Uden, The Netherlands).

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Evaluation of Liver Function

The degree of liver dysfunction was evaluated through the measurement of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations (SRL, Kitakyushu, Japan).

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Immunohistochemical Staining of the Liver

Liver tissue specimens were embedded in paraffin and sectioned into 2-μm slices. Immunohistochemical staining for HMGB1 was performed using rabbit polyclonal antibody (Shino-TEST). After blocking the endogenous peroxidase activity, sections were preincubated with 10% normal goat serum for 20 minutes. The sections were incubated with a 1:500 dilution of the primary antibody HMGB1 overnight at 4°C. Then the sections were incubated with EnVision+ reagent (EnVision+ System, DAKO, Carpinteria, CA) for 30 minutes. 3,3′-Diaminobenzidine was used as the substrate, and the sections were counterstained with Mayer’s hematoxylin and mounted.

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Statistical Analysis

The data are presented as the mean ± SEM. The data were analyzed using 1-way analysis of variance and unpaired t test for single comparisons. For the survival study, the data were analyzed using Kaplan–Meier analysis. A P value of <0.05 was considered to be statistically significant.

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RESULTS

The Effect of Recombinant Soluble TM on Survival after Heat Exposure

Heat exposure induced death in 0% of the mice in group HS within 4 hours. However, 70% of the mice in group HS died 5 hours after heat exposure. Eighty percent of mice in group TG-Pre and 70% of mice in group TG-Delay survived for 5 hours after heat exposure. A Kaplan–Meier analysis revealed a significantly shorter time to death in group HS compared with both recombinant soluble TM treatment groups (Fig. 1).

Figure 1
Figure 1
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In the survival study, heat exposure up to 4 hours did not induce death in our experimental model. However, heat exposure for 5 hours induced death in >50% of the mice. Therefore, we used up to a 4-hour heat exposure for obtaining samples in our experiments. Rectal temperatures were increased from 36.8°C ± 0.3°C to 41.2°C ± 0.5°C after 4-hour exposure to heat stress.

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The Effect of Recombinant Soluble TM on Heat Exposure–Induced Inflammatory Mediator Production

In group HS, plasma TNF-α increased after heat exposure, reaching a plateau 2 hours after heat exposure. Plasma IL-6 and HMGB1 concentrations also increased in group HS, peaking at 4 hours after heat exposure. In group NC, plasma cytokines and HMGB1 did not increase. In group TG-Pre, recombinant soluble TM treatment before heat exposure suppressed increased plasma TNF-α, IL-6, and HMGB1 levels after heat exposure (Fig. 2, A–C). Furthermore, in group TG-Delay, recombinant soluble TM treatment during heat exposure attenuated heat exposure–induced plasma HMGB1 elevation (Fig. 3).

Figure 2
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Figure 3
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The Effect of Recombinant Soluble TM on Heat Exposure–Induced Neutrophil Infiltration in the Liver

In group HS, MPO activity as a measurement of liver neutrophil infiltration was significantly increased after heat exposure, peaking at 4 hours after heat exposure. Recombinant soluble TM treatment before heat exposure suppressed increased hepatic MPO activity after heat exposure (Fig. 4).

Figure 4
Figure 4
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The Effect of Recombinant Soluble TM on Heat Exposure–Induced Liver Dysfunction

After heat exposure, the degree of liver dysfunction was evaluated through the measurement of plasma AST and ALT concentrations. In group HS, plasma AST and ALT concentrations increased group heat exposure, peaking 4 hours after heat exposure. This result suggests that heat stress induces liver dysfunction in a mouse heatstroke model. Recombinant soluble TM treatment before heat exposure improved heat exposure–induced liver dysfunction (Fig. 5, A and B).

Figure 5
Figure 5
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Correlation Between Plasma HMGB1 Level and Liver Enzyme

We next evaluated whether liver dysfunction involves increased plasma HMGB1 level. As a result, there was a strong positive correlation between plasma HMGB1 levels and plasma AST levels (Fig. 6A). In addition, plasma HMGB1 levels and plasma ALT levels also showed a strong positive correlation (Fig. 6B).

Figure 6
Figure 6
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Immunohistochemical and Histopathological Examination of the Mouse Liver

After heat exposure, strong and extensive immunostaining for HMGB1 was observed in the hepatic macrophages and hepatic nuclei (Fig. 7C). This positive immunostaining for HMGB1 was less pronounced among recombinant TM-pretreated mice than in HS mice (Fig. 7B). In addition, hematoxylin–eosin staining of liver tissue sections performed taken 4 hours after heat exposure was used for detection of liver injury. There was hepatocellular necrosis and collapse of nuclei observed in group HS (Fig. 8C). Recombinant soluble TM treatment before heat exposure revealed markedly reduced necrosis of the hepatic cells and the collapse of the nuclei compared with group HS (Fig. 8B).

Figure 7
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Figure 8
Figure 8
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The Effect of Recombinant Soluble TM on Heat Exposure–Induced Coagulopathy

The coagulation status was evaluated through the measurement of plasma protein C and TAT concentrations. After heat exposure, plasma protein C levels were significantly suppressed compared with group NC, and recombinant soluble TM improved this suppression (Fig. 9A). TAT levels in group HS significantly increased compared with group NC. Recombinant soluble TM treatment before heat exposure suppressed increased TAT levels after heat exposure (Fig. 9B).

Figure 9
Figure 9
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DISCUSSION

In this study, we showed that plasma levels of inflammatory mediators, such as TNF-α, IL-6, and HMGB1, increased after heat exposure in an experimental mouse model. Heat exposure induces liver dysfunction and coagulopathy. Recombinant soluble TM treatment before heat exposure improved the inflammatory process, liver injury, coagulopathy, and mortality after heat exposure. Recombinant soluble TM treatment decreased the plasma HMGB1 levels and mortality even with delayed treatment.

Previous studies demonstrated that plasma inflammatory cytokine level increases in patients with heatstroke. Elevated cytokine concentrations were correlated with the severity of heatstroke.5,6,12 The plasma levels of inflammatory cytokines such as TNF-α, IL-6, and IL-8 are increased in heatstroke patients.13 In addition, plasma IL-1β and IL-6 levels increased in experimental heatstroke mice.14 Our results also showed that these cytokines increase after heat exposure. Furthermore, in our experimental heatstroke model, plasma HMGB1 levels increased after heat exposure. A previous study demonstrated that HMGB1 is released from macrophages and monocytes which are activated by inflammatory cytokines.7 HMGB1 is also leaked from necrotic or damaged cells.15–19 In our results, TNF-α and IL-6 levels had already increased by the time plasma HMGB1 levels increased. In addition, immunohistochemical and histopathological examination showed that HMGB1 was observed in the hepatic macrophages and hepatic nuclei in heat-exposed mice. These results suggested that the increase of plasma HMGB1 after heat exposure was caused by the active secretion from activated macrophages and monocytes. Recent studies showed elevated plasma HMGB1 levels in experimental animal heatstroke models and in patients with heat stroke.20–22 Tong et al.21 suggested that the HMGB1 level is an indicator of the severity of illness and a useful mortality predictor in heatstroke patients. In this study, we demonstrated that soluble TM treatment decreased the plasma HMGB1 levels even with delayed treatment. This result suggested that recombinant soluble TM treatment may be a beneficial treatment for heatstroke patients.

After heatstroke, multiple organ failure, such as respiratory dysfunction, cardiovascular failure, renal dysfunction, and coagulopathy, occurred and finally lead to death.1,2 There are some studies regarding heat exposure–induced liver dysfunction.23–25 Liver injury is characterized by the accumulation of activated neutrophils in the liver.26,27 These activated neutrophils produce proinflammatory cytokines, such as IL-1 and TNF-α, and release reactive oxygen species as well as other mediators contributing to tissue injury. Liver levels of HMGB1 are increased in the setting of liver injury, and HMGB1 itself can produce liver injury by the accumulation of activated neutrophils.28–30 Therefore, regulating HMGB1 levels may be a strategy to suppress liver injury from activated neutrophils. In this study, we demonstrated that TM suppresses plasma HMGB1 levels and MPO activity in the liver of a mouse heatstroke model. Our results suggest that heat exposure induces neutrophil accumulation which results in liver injury; however, recombinant soluble TM has a protective effect on liver injury via suppression of neutrophil accumulation in the liver. Our results also demonstrated the anti-HMGB1 mechanism of recombinant soluble TM. Recombinant soluble TM administration decreases immunostaining reaction in the hepatocyte nuclei of HMGB1. Therefore, recombinant soluble TM has an effect on the intranuclear expression of HMGB1. Our results are supported by recent studies by Tong et al.25 and Dehbi et al.23 which demonstrated that inhibition of HMGB1 activity with the HMGB1 antibody or DNA-binding A Box protects the liver from heatstroke.

A previous study by Bouchama et al.31 demonstrated that animals with severe heatstroke exhibited a marked increase in plasma TM levels. Huisse et al.13 also demonstrated that whole-blood tissue factor, TAT, and soluble TM levels increase significantly in heatstroke patients. Levels of the major physiologic anticoagulants, antithrombin, protein C, and protein S were markedly decreased in heatstroke patients. Coagulation and fibrinolysis are frequently activated during heatstroke and may progress to disseminated intravascular coagulation. The coagulation status is more potent than that of sepsis. APC has been used in a rat model of heatstroke and has been shown to improve survival by reducing systemic inflammation, the hypercoagulable state, and organ injury.32 Previous reports demonstrated that TM can bind thrombin to convert protein C to APC.33–39 TM also plays an important role in the attenuation of thrombin formation associated with the properties of APC,40 which reflects negative feedback of the thrombin generation.41 We previously demonstrated that TM suppressed TAT formation in a rat sepsis model.42 Moreover, in this study, TM also inhibited the generation of thrombin in heatstroke animals, suggesting that TM may be effective for treatment of disseminated intravascular coagulation in heatstroke patients.

Many treatments have been reported as the potential HMGB1-inhibiting therapeutic agents in experimental endotoxemia or sepsis models.43–45 However, most of these agents are not effective in the clinical setting for humans. These agents suppress the systemic HMGB1 accumulation by attenuating HMGB1 release from damaged cells. Therefore, these agents cannot regulate HMGB1 already in the extracellular space. Previous reports demonstrated that TM inhibits the active secretion of HMGB1 by suppressing the activation of macrophages and monocytes.11 In addition, human soluble TM (ART-123) binds to HMGB1 protein and inhibits HMGB1 activity at the level of D1 (N-terminal lectin-like domain) of TM.46 Our results showed recombinant soluble TM’s protective effect on heat exposure–induced plasma HMGB1 level elevation even with delayed treatment. Therefore, recombinant soluble TM seems to have the ability to suppress HMGB1 directly.

There are some limitations in this study. First, we evaluated the inflammatory process, liver function, and coagulant status. Therefore, other vital organs such as heart, lungs, kidneys, or vasculature could be affected after heat exposure and soluble TM treatment. We are planning future studies of these functions and outcomes after heat exposure. Second, we demonstrated that soluble TM can reduce the levels of plasma TNF-α and IL-6 as well as HMGB1, however, considering HMGB1 as the factor that induces liver injury associated with heat exposure. Therefore, it is possible that TNF-α and IL-6 also contribute to liver injury. In this study, we focused on HMGB1; however, further studies are required to identify the major factor in heat exposure–induced organ injuries and mortality. Third, we did not show the direct effects of HMGB1 on heat exposure. We need to perform more mechanistic studies to support the hypothesis that HMGB1 is the main contributor to the development of liver injury after heat stroke.

In conclusion, this study demonstrated that recombinant soluble TM treatment improves the development of inflammatory processes, liver injury, coagulopathy, and mortality after heatstroke. Recombinant soluble TM treatment decreases plasma HMGB1 levels and mortality even with delayed treatment. These results suggest that recombinant soluble TM treatment may be a beneficial treatment for heatstroke patients.

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DISCLOSURES

Name: Takashi Kawasaki, MD, PhD.

Contribution: This author helped design the study, conduct the study, analyze the data, and prepare the manuscript.

Attestation: Takashi Kawasaki has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study records.

Name: Kohji Okamoto, MD, PhD.

Contribution: This author helped design the study, conduct the study, analyze the data, and prepare the manuscript.

Attestation: Kohji Okamoto has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Chika Kawasaki, MD.

Contribution: This author helped design the study, conduct the study, analyze the data, and prepare the manuscript.

Attestation: Chika Kawasaki has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study records.

Name: Takeyoshi Sata, MD, PhD.

Contribution: This author helped analyze the data and prepare the manuscript.

Attestation: Takeyoshi Sata has reviewed the analysis of the data and approved the final manuscript.

This manuscript was handled by: Marcel E. Durieux, MD, PhD.

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