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Abstract Background Upon cellular injury, damage-associated molecular patterns (DAMPs) are released into the extracellular space and evoke proinflammatory and prothrombotic responses in animal models of sterile inflammation. However, in clinical settings, the dynamics of DAMP levels after trauma and links between DAMPs and trauma-associated coagulopathy remain largely undetermined. Methods Thirty-one patients with severe trauma, who were transferred to Kagoshima City Hospital between June 2018 and December 2019, were consecutively enrolled in this study. Blood samples were taken at the time of delivery, and 6 and 12 h after the injury, and once daily thereafter. The time-dependent changes of coagulation/fibrinolysis markers, including thrombin-antithrombin complex, α2-plasmin inhibitor (α2-PI), plasmin-α2-PI complex, and plasminogen activator inhibitor-1 (PAI-1), and DAMPs, including high mobility group box 1 and histone H3, were analyzed. The relationship between coagulation/fibrinolysis markers, DAMPs, Injury Severity Score, in-hospital death, and amount of blood transfusion were analyzed. Results The activation of coagulation/fibrinolysis pathways was evident at the time of delivery. In contrast, PAI-1 levels remained low at the time of delivery, and then were elevated at 6–12 h after traumatic injury. Histone H3 and high mobility group box 1 levels were elevated at admission, and gradually subsided over time. PAI-1 levels at 6 h were associated with serum histone H3 levels at admission. Increased histone H3 levels and plasmin-α2-PI complex levels were associated with in-hospital mortality. α2-PI levels at admission showed the strongest negative correlation with the amount of blood transfusion. Conclusion The elevation of histone H3 levels and fibrinolysis perturbation are associated with fatal outcomes in patients with traumatic injury. Patients with low α2-PI levels at admission tend to require blood transfusion.

IntroductionCerebral white matter hyperintensities (WMHs) are commonly found in the aging brain and have been implicated in the initiation and severity of many central nervous system diseases. Furthermore, an increased WMH volume indicates reduced brain health in older adults. This study investigated the association between WMH volume and physical activity in older adults with depressive symptoms (DS) and mild memory impairment (MMI). Factors associated with the WMH volume were also investigated.MethodsA total of 57 individuals aged over 65 years with DS and MMI were included in this study. The participants underwent magnetic resonance imaging to quantify WMH volumes. After WMH volume was accumulated, normalized to the total intracranial volume (TIV), the percentage of WMH volume was calculated. In addition, all participants wore a triaxial accelerometer for 2 weeks, and the average daily physical activity and number of steps were measured. The levels of blood biomarkers including cortisol, interleukin-6 (IL-6), brain-derived insulin-like growth factor-1, and brain-derived neurotrophic factor were measured. Motor and cognitive functions were also assessed.ResultsFaster maximum walking speed and longer time spent engaged in moderate physical activity were associated with a smaller percent of WMH volume, whereas higher serum IL-6 levels were associated with a larger percent of WMH volume. The number of steps per day, time spent engaged in low levels of physical activity, cognitive function, and all other measured biomarkers were not significantly associated with percent of WMH volume.DiscussionHigher blood inflammatory cytokine levels, shorter duration of moderate physical activity, and lower maximum walking speed were associated with a higher percent of WMH volume. Our results provide useful information for maintaining brain health in older adults at a high risk of developing dementia and may contribute to the development of preventive medicine for brain health.

Fatigue can lead to several health issues and is particularly prevalent among elderly individuals with chronic inflammatory conditions. Ninjin'yoeito, a traditional Japanese herbal medicine, is used to address fatigue and malaise, anorexia, and anemia. This study aimed to examine whether relieving inflammation in the brain and skeletal muscle of senescence-accelerated mice prone 8 (SAMP8) could reduce fatigue-like conditions associated with aging. First, SAMP8 mice were divided into two groups, with and without ninjin'yoeito treatment. The ninjin'yoeito-treated group received a diet containing 3% ninjin'yoeito for a period of 4 months starting at 3 months of age. At 7 months of age, all mice underwent motor function, treadmill fatigue, and behavioral tests. They were then euthanized and the skeletal muscle weight, muscle cross-sectional area, and concentration of interleukin (IL)-1β and IL-1 receptor antagonist (IL-1RA) in both the brain and skeletal muscle were measured. The results showed that the ninjin'yoeito-treated group had higher motor function and spontaneous locomotor activity than the untreated group did and ran for significantly longer in the treadmill fatigue test. Moreover, larger muscle cross-sectional area, lower IL-1β concentrations, and higher IL-1RA concentrations were observed in both the brain and skeletal muscle tissues of the ninjin'yoeito-treated group than in the untreated group. The results suggest that ninjin'yoeito improves age-related inflammatory conditions in both the central and peripheral tissues and reduces fatigue.

Abstract Remote ischemic perconditioning (RIPerC) is a novel neuroprotective method against cerebral infarction that has shown efficacy in animal studies but has not been consistently neuroprotective in clinical trials. We focused on the temporal regulation of ischemia–reperfusion by RIPerC to establish an optimal method for RIPerC. Rats were assigned to four groups: 10 min ischemia, 5 min reperfusion; 10 min ischemia, 10 min reperfusion; 5 min ischemia, 10 min reperfusion; and no RIPerC. RIPerC interventions were performed during ischemic stroke, which was induced by a 60-min left middle cerebral artery occlusion. Infarct volume, sensorimotor function, neurological deficits, and cellular expressions of brain-derived neurotrophic factor (BDNF), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and caspase 3 were evaluated 48 h after the induction of ischemia. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) was also performed. RIPerC of 10 min ischemia/10 min reperfusion, and 5 min ischemia/10 min reperfusion decreased infarct volume, improved sensorimotor function, decreased Bax, caspase 3, and TUNEL-positive cells, and increased BDNF and Bcl-2 expressions. Our findings suggest RIPerC with a reperfusion time of approximately 10 min exerts its neuroprotective effects via an anti-apoptotic mechanism. This study provides important preliminary data to establish more effective RIPerC interventions.

Abstract Background Administration of recombinant human soluble thrombomodulin (rTM) is often used in Japan to treat septic disseminated intravascular coagulation (DIC). Thrombin-activatable fibrinolysis inhibitor (TAFI) is a fibrinolysis inhibitor activated by the thrombin-thrombomodulin complex, however, it is unknown whether circulating activated TAFI is increased after rTM administration in patients with DIC. Furthermore, the relationship between TAFI activation and the prognosis of septic DIC is not defined yet. Case presentation We report a series of 8 patient’s TAFI activation with septic DIC treated by rTM. We sought to investigate the effect of rTM on TAFI activation and the association of plasma activated TAFI (TAFIa/ai) levels with the prognosis of septic DIC. Using plasma samples from clinical studies conducted from May 2016–March 2017 on eight patients with septic DIC at Kagoshima University Hospital, we measured plasma levels of total TAFI, TAFIa/ai, thrombin-antithrombin complex (TAT), prothrombin fragment 1 + 2 (F1 + 2), soluble fibrin (SF), antithrombin (AT), protein C (PC), protein S (PS), and plasminogen activator inhibitor-1 (PAI-1) before and after intravenous rTM administration. Then, we evaluated the relationship of these marker levels to prognosis. The thrombin-rTM complex activated TAFI in vitro in plasma from a healthy volunteer. However, TAFIa/ai levels did not significantly increase over baseline in the septic DIC patients after intravenous rTM administration. Baseline TAFIa/ai levels in non-survivors were significantly higher than those in survivors. Conclusions Plasma TAFIa/ai did not increase with rTM administration. Elevated baseline TAFIa/ai concentration may be a negative prognostic indicator in septic DIC. Larger studies are needed to confirm the in vivo effect of rTM on TAFI activation.

Abstract Background Rupture of an atherosclerotic plaque and subsequent exposure of the subendothelial prothrombotic matrix to blood cause arterial thrombosis. Circulating platelets play an indispensable role in the growth of arterial thrombi partially owing to their unique ability to adhere to the subendothelial matrix and to aggregate to each other under flow conditions. Recently, the Total Thrombus-formation Analysis System (T-TAS) was developed for ex vivo analysis of the thrombogenic potential of whole blood samples under flow conditions. Despite the potential clinical utility of the T-TAS in assessing the risk for thrombosis and bleeding, reference intervals for T-TAS analysis in healthy individuals have not been determined. Methods In total, 122 whole blood samples were collected from healthy volunteers ranging in age from 25 to 45 years. T-TAS analysis and hematological, physiological, and lifestyle assessments were conducted in these subjects. Whole blood samples anticoagulated with hirudin were perfused into a collagen-coated microchip (PL chip). The time to 10 kPa and the area under the flow pressure curve up to 10 min (AUC10) were analyzed as representative variables for thrombogenic potential. Reference intervals, which were defined as 2.5–97.5 percentiles, were determined. Additionally, univariate and multivariate analyses were performed to identify factors associated with the AUC10 in the T-TAS. Results The time to 10 kPa and the AUC10 widely varied, even in healthy volunteers. The reference intervals were 1.50–4.02 min and 223.4–456.8, respectively, at a shear rate of 1500 s− 1. Univariate and multivariate analyses showed that platelet counts were most significantly associated with the AUC10 of the T-TAS. The presence of one or more cardiovascular risk factors of a high body mass index, a high pulse pressure, high fasting serum glucose levels, high low-density lipoprotein-cholesterol levels, a history of smoking, and no habitual exercise, had the second largest effect on the AUC10 of the T-TAS. Conclusions Healthy volunteers who had any cardiovascular risk factors showed augmented thrombogenicity, even in artificial uniform capillaries, compared with those without any risk factors in the T-TAS.

Abstract The gut microbiota has tremendous potential to affect the host’s health, in part by synthesizing vitamins and generating nutrients from food that is otherwise indigestible by the host. 1,5-Anhydro-d-fructose (1,5-AF) is a monosaccharide with a wide range of bioactive potentials, including anti-oxidant, anti-inflammatory, and anti-microbial effects. Based on its potential benefits and minimal toxicity, it is anticipated that 1,5-AF will be used as a dietary supplement to support general health. However, the effects of 1,5-AF on the gut microbiota are yet to be clarified. Here, using an unbiased metagenomic approach, we profiled the bacterial taxa and functional genes in the caecal microbiota of mice fed a diet containing either 2% 1,5-AF or a reference sweetener. Supplementation with 1,5-AF altered the composition of the gut microbiota, enriching the proportion of Faecalibacterium prausnitzii. 1,5-AF also altered the metabolomic profile of the gut microbiota, enriching genes associated with nicotinamide adenine dinucleotide biosynthesis. These findings support the potential benefits of 1,5-AF, but further studies are required to clarify the impact of 1,5-AF on health and disease.

Abstract Background During sepsis or sterile tissue injury, the nuclear protein high mobility group box 1 (HMGB1) can be released to the extracellular space and ultimately into systemic circulation, where it mediates systemic inflammation and remote organ failure. The proinflammatory effects of HMGB1 can be suppressed by recombinant thrombomodulin (rTM), in part through a mechanism involving thrombin–rTM-mediated degradation of HMGB1. Given that HMGB1 is proinflammatory but the HMGB1 degradation product (desHMGB1) is not, an analytical method that discriminates between these two molecules may provide a more in-depth understanding of HMGB1-induced pathogenicity as well as rTM-mediated therapeutic efficiency. Methods A peptide that has a shared amino-terminal structure with desHMGB1 was synthesized. C3H/lpr mice were immunized with the desHMGB1 peptide conjugate, and antibody-secreting hybridoma cells were developed using conventional methods. The reactivity and specificity of the antibodies were then analyzed using antigen-coated enzyme-linked immunosorbent assay (ELISA) as well as antibody-coated ELISA. Next, plasma desHMGB1 levels were examined in a cecal ligation and puncture (CLP)-induced septic mouse model treated with rTM. Results Through a series of screening steps, we obtained a monoclonal antibody that recognized desHMGB1 but did not recognize intact HMGB1. ELISA using this antibody specifically detected desHMGB1, which was significantly increased in CLP-induced septic mice treated with rTM compared with those treated with saline. Conclusions In this study, we obtained a desHMGB1-specific monoclonal antibody. ELISA using the novel monoclonal antibody may be an option for the in-depth analysis of HMGB1-induced pathogenicity as well as rTM-mediated therapeutic efficiency.

Abstract Endothelial cells play a key role in maintaining intravascular patency through their anticoagulant properties. They provide a favorable environment for plasma anticoagulant proteins, including antithrombin, tissue factor pathway inhibitor, and protein C. Under septic conditions, however, the anticoagulant properties of endothelial cells are compromised. Rather, activated/injured endothelial cells can provide a scaffold for intravascular coagulation. For example, the expression of tissue factor, an important initiator of the coagulation pathway, is induced on the surface of activated endothelial cells. Phosphatidylserine, a high-affinity scaffold for gamma-carboxyglutamate domain containing coagulation factors, including FII, FVII, FIX, and FX, is externalized to the outer leaflet of the plasma membrane of injured endothelial cells. Hemodilution decreases not only coagulation factors but also plasma anticoagulant proteins, resulting in unleashed activation of coagulation on the surface of activated/injured endothelial cells. The aberrant activation of coagulation can be suppressed in part by the supplementation of recombinant antithrombin and recombinant thrombomodulin. This review aims to overview the physiological and pathological functions of endothelial cells along with proof-of-concept in vitro studies. The pathophysiology of COVID-19-associated thrombosis is also discussed.

Abstract Background The efficacy and safety of thrombomodulin alfa (TM‐α), a cofactor protein promoting thrombin‐mediated protein C activation, have been examined in a phase 3 randomized, double‐blinded, parallel‐group trial in Japan. We have previously reported that TM‐α is noninferior to heparin for the resolution of disseminated intravascular coagulation (DIC). Objective To investigate the basis for the efficacy of TM‐α in the phase 3 clinical trial in Japan through post hoc analysis of coagulation and fibrinolysis parameters. Patients/Methods The 227 patients of the full analysis set population described in the original phase 3 trial in Japan were included in this analysis. Changes in parameters between before and after TM‐α or heparin administration in each of the two patient groups, with underlying diseases of either hematologic malignancy or infection, were studied separately and results were compared between TM‐α and heparin treatment groups in a post hoc manner. Results TM‐α administration did not prolong activated partial thromboplastin time but significantly decreased thrombin‐antithrombin complex levels compared with heparin treatment. TM‐α administration reduced consumption of endogenous anticoagulants such as antithrombin and protein C by DIC, compared with the heparin group. DIC scores were decreased in both TM‐α and heparin groups during the 6‐day treatment. Conclusion TM‐α can alleviate intravascular coagulation and consumption of anticoagulants without extending coagulation times. This may be associated with the relatively low risk of bleeding during TM‐α treatment.

Abstract Background In the intensive care unit (ICU), patients with thrombocytopenia are at high risk for bleeding and should be assessed for their thrombogenic potential. However, the analytical conditions of conventional hemostatic tests are unsuitable for the evaluation of low-platelet samples. Here we aimed to establish suitable analytical conditions with the Total Thrombus-formation Analysis System (T-TAS) for quantitative assessment of thrombogenic potential in patients with thrombocytopenia and to investigate how T-TAS values relate to bleeding symptoms and the effects of platelet transfusion. Methods Modified chips with a different chamber depth were developed for the analysis of low-platelet samples in the T-TAS. We included 10 adult patients admitted to the ICU of Kagoshima University Hospital who required platelet transfusion. Patients were divided into major and minor bleeding groups according to their bleeding scale before platelet transfusion. The thrombogenic potential of these patients before and after platelet transfusion was assessed with hemostatic function tests, including rotational thromboelastometry, multiplate aggregometry, and the T-TAS. Results Analysis of low-platelet samples revealed that, compared with the conventional chip (80-μm-deep chamber), the modified chip (50-μm-deep chamber) achieved higher sensitivity in detecting elevation of flow pressure caused by growth of an occlusive thrombus in the T-TAS analytical chamber. All patients in the minor bleeding group retained thrombogenic potential that occluded the modified chip (occlusion time 16.3 ± 3.3 min), whereas most patients in the major bleeding group were unable to occlude the modified chip during the 30-min measurement (P

Background: Sepsis is defined as life-threatening organ dysfunction caused by dysregulated host responses to infection. Recent studies have suggested that endotheliopathy may be the common basis for multiple organ failure in sepsis. Under septic conditions, accumulation of proteases accelerates shedding of proteoglycans, such as syndecan-1, from the endothelial surface, resulting in augmented leukocyte adhesion to the vascular wall, enhanced vascular permeability, and intravascular coagulation. The purpose of this study was to determine the potential utility of syndecan-1 as a biomarker linking endotheliopathy to organ failure.Methods: One hundred patients with suspected infections who were admitted to the intensive care unit (ICU) at Kagoshima University Hospital were consecutively enrolled in the study. Serum syndecan-1 levels were measured using an in-house enzyme-linked immunosorbent assay. The difference between serum syndecan-1 levels in 28-day survivors and non-survivors was analyzed by the Mann–Whitney U-test. Receiver-operating characteristics curve analysis with area under the curve calculation was used to quantify the predictive performance of serum syndecan-1 for 28-day mortality. The correlations between serum syndecan-1 and coagulation markers were analyzed by Spearman's rank correlation test.Results: Serum syndecan-1 levels in non-survivors were significantly higher than those in survivors on Day 1 and Day 3 (P < 0.01). Among multiple organ failures, coagulation failure and renal failure were significantly correlated with serum syndecan-1. Spearman's rank correlation test indicated that serum syndecan-1 was weakly but significantly correlated with disseminated intravascular coagulation score (rho = 0.33, P < 0.01). Patients with serum syndecan-1 ≥21.4 ng/mL showed delayed recovery from thrombocytopenia relative to patients with serum syndecan-1

BackgroundSkeletal muscle ischemia/reperfusion (I/R) injury is an important clinical issue that can cause remote organ injury. Although its pathogenesis has not been fully elucidated, recent studies have suggested that damage-associated molecular patterns (DAMPs) are mediators of remote organ injury in sterile inflammation. The purpose of this study was to investigate the possible involvement of DAMPs, including the nuclear proteins high-mobility group box 1 (HMGB1) and histone H3, in the pathogenesis of skeletal muscle I/R injury in mice.MethodsHindlimb ischemia was induced in mice through bilateral ligation of inguinal regions using rubber grommets. Reperfusion was induced by cutting the rubber grommets after 2–12 h of ischemic period. Survival rates, localization of HMGB1 and histone H3 in the gastrocnemius muscle, and circulating HMGB1 and histone H3 levels were analyzed. The effect of anti-HMGB1 and anti-histone H3 antibodies on survival was analyzed in mice with I/R injury.ResultsAll mice with hindlimb ischemia survived for at least 36 h, while all mice died within 24 h if the hindlimbs were reperfused after ischemia for 4–12 h. Immunohistochemical analysis revealed that HMGB1 translocated from the nucleus to the cytoplasm in the ischemic gastrocnemius muscle, while histone H3 was confined to the nucleus. Accordingly, serum HMGB1 levels were significantly elevated in mice with hindlimb I/R compared with normal mice or mice with hindlimb ischemia (P < 0.05). Serum histone H3 levels were not elevated after I/R. Treatment with anti-HMGB1 antibodies significantly improved survival of mice with hindlimb I/R injury compared with control antibodies (P < 0.05).ConclusionsHMGB1, but not histone H3, translocated to the cytoplasm during skeletal muscle ischemia, and was released into the systemic circulation after reperfusion in mice with I/R injury. Treatment with anti-HMGB1 antibodies partially improved survival.

Abstract Background Recent studies have suggested that anticoagulant therapy does not confer a survival benefit overall in sepsis, but might be beneficial in sepsis-associated disseminated intravascular coagulation (DIC). In particular, those with high Sequential Organ Failure Assessment (SOFA) scores might be the optimal target for anticoagulant therapy. However, both DIC and SOFA scores require the measurement of multiple markers. The purpose of this study was to explore a minimal marker set for determining coagulopathy at high risk of death in septic patients, wherein histone H3 levels were evaluated as indicators of both organ failure and coagulation activation. Methods We analyzed correlations among levels of serum histone H3 and other coagulation markers in 85 cases of sepsis using Spearman’s rank correlation test. We then compared the utility of histone H3 to that of other coagulation markers in predicting the traditional DIC state or 28-day mortality by receiver-operating characteristics analysis. Finally, we suggested cut-off values for determining coagulopathy with high risk of death, and evaluated their prognostic utility. Results Serum histone H3 levels significantly correlated with thrombin-antithrombin complex (TAT) levels (Spearman’s ρ = 0.46, p < 0.001), and weakly correlated with platelet counts (Spearman’s ρ = − 0.26, p < 0.05). Compared to other coagulation markers, histone H3 levels showed better performance in predicting 28-day mortality. When combining serum histone H3 levels with platelet counts, our new scoring system showed a concordance rate of 69% with the traditional four-factor criteria of DIC established by the Japanese Association for Acute Medicine. The 28-day mortality rates of the new and the traditional criteria-positive patients were 43% and 21%, respectively. Those of the new and the traditional criteria-negative patients were 5.7% and 9.4%, respectively. Conclusions Serum histone H3 levels and platelet counts are potential markers for determining coagulopathy with high risk of death in septic patients. Further studies are needed to clarify the utility of serum histone H3 levels in the diagnostic of coagulopathy/DIC.

Abstract Background Thrombus formation is an important factor affecting cardiovascular events and venous thromboembolism in type 2 diabetes. However, it is unclear whether glycemic control reduces thrombogenicity. We investigated the effect of short-term glycemic control (STUDY 1) and hypoglycemia (STUDY 2) on thrombus formation using an automated microchip flow chamber system. Methods For STUDY 1, we recruited 10 patients with type 2 diabetes. Before and after 2 weeks of treatment, blood glucose was analyzed with a continuous glucose monitoring system, and thrombogenicity was analyzed with an automated microchip flow chamber system. For STUDY 2, we recruited 10 subjects without diabetes who underwent an insulin tolerance test. We evaluated the change in thrombogenic potential with hypoglycemia. Results STUDY1: The mean blood glucose level reduced from 10.1 ± 2.6 to 6.9 ± 0.97 mM (P

Abstract Background Nuclear histone proteins are released into the extracellular space, and act as major mediators of coagulopathy and remote organ failure in septic animals. However, the circulating histone levels in septic patients have not been precisely quantified. Methods Using a novel enzyme-linked immunosorbent assay for histone H3 detection, we measured the serum histone H3 levels in 85 patients admitted to the intensive care unit because of infectious diseases. We then evaluated the associations of circulating histone H3 levels with organ failure, coagulopathy, and mortality. Results Circulating histone H3 levels were significantly higher in patients with coagulopathy, and were positively correlated with numbers of organ failures. Circulating histone H3 levels were also associated with fatal outcome. Receiver-operating characteristic analyses revealed that the predictive performance of circulating histone H3 levels for mortality was higher than that of conventional inflammatory markers, including white blood cell count, C-reactive protein, and cell-free DNA. Conclusions Circulating histone H3 levels are associated with coagulopathy, multiple organ failure, and death in patients requiring intensive care because of infectious diseases.

Abstract Background Nuclear histone proteins are released into the extracellular space during sepsis and act as major mediators of death. However, circulating histone levels have not been precisely quantified. Methods We developed a novel enzyme-linked immunosorbent assay (ELISA) for detection of circulating histone H3 levels and evaluated its performance. Using the ELISA, we measured plasma histone H3 levels in C57BL/6 J mice subjected to cecal ligation and puncture (CLP)-induced sepsis. Results The newly developed ELISA enabled reproducible measurement of histone H3 levels with a working range up to 250 ng/mL. Using the ELISA, we found that plasma histone H3 levels were elevated in septic mice compared with sham-operated mice (p

Abstract Background Recombinant human soluble thrombomodulin (rTM) has been used for the treatment of disseminated intravascular coagulation in Japan, and an international phase III clinical trial for rTM is currently in progress. rTM mainly exerts its anticoagulant effects through an activated protein C (APC)-dependent mechanism, but the circulating APC levels after rTM treatment have not been clarified. This prospective observational study investigated plasma APC levels after rTM treatment. Methods Plasma levels of soluble thrombomodulin, thrombin-antithrombin complex (TAT), protein C, and APC were measured in eight septic patients treated with rTM. APC generation in vitro was assessed in the presence or absence of rTM. Results rTM significantly increased thrombin-mediated APC generation in vitro. In septic patients, soluble thrombomodulin levels were significantly increased during a 30–60-min period of rTM treatment and TAT levels were decreased. However, APC activity was not increased during the treatment period. Conclusions Plasma APC activity is not increased in septic patients treated with rTM. It is possible that APC acts locally and does not circulate systemically.

Histones, the major protein components of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and subsequently mediate organ failure. Extracellular histones can promote endothelial damage and platelet aggregation, which can be suppressed by administration of recombinant thrombomodulin (rTM). The present study aimed to clarify whether histones can activate neutrophils to induce NET formation and whether rTM can prevent histone-induced NET formation. NET formation was analyzed in vitro by stimulating human neutrophils with histones in the absence or presence of rTM. NET formation was further analyzed in vivo by intravenous infusion of histones into rats with or without rTM. Histones induced NET release in a dose-dependent manner in vitro and NET release was induced as early as 1 h after stimulation. Histone-induced NET release was independent of NADPH oxidase. rTM suppressed histone-induced NET release in vitro as well as in vivo. The suppression might be mediated by rTM binding to histones, as suggested by analysis using a quartz crystal microbalance system. The present findings suggest that histones can activate neutrophils to form NETs and that rTM can inhibit histone-induced NET formation.

Atrial fibrillation (AF) is the most common cardiac arrhythmia in the world and has a high risk of thromboembolism. The most effective approach, catheter ablation, requires evaluation by electrocardiography. The aim of our study was to investigate novel clinical markers that predict restoration of sinus rhythm (SR) after catheter ablation. Seventy-eight consecutive patients with AF underwent catheter ablation and were separated into 2 groups: restored SR and recurrent AF. The levels of 4 blood proteins (serum or plasma) and 3 mature microRNAs (miRNAs) and their primary miRNAs (pri-miRNAs) in serum were measured before and after ablation, and the associations between each parameter were analyzed statistically. Soluble thrombomodulin (s-TM) and plasminogen activator inhibitor-1 (PAI-1) levels increased above baseline after ablation in both the restored SR (s-TM 11.55 [2.92] vs 13.75 [3.38], P < .001; PAI-1 25.74 [15.25] vs 37.79 [19.56], P < .001) and recurrent AF (s-TM 10.28 [2.78] vs 11.67 [3.37], P < .001; PAI-1 26.16 [15.70] vs 40.74 [22.55], P < .001) groups. Levels of C-reactive protein and asymmetric dimethylarginine were not significantly changed. Pri-miR-126 levels significantly decreased after ablation in the recurrent AF group, but the other miRNAs and pri-miRNAs did not. The measurement of s-TM and pri-miR-126 in blood was a useful tool to reflect the condition of AF patients with catheter ablation.

Environmental tobacco smoke (ETS) exposure is linked to carcinogenic, oxidative and inflammatory cellular reactions. Green tea polyphenol reportedly plays a role in the prevention of inflammation-related diseases. To evaluate the effects of green tea extract (GTE) on cellular location of High Mobility Group Box-1 (HMGB1) protein, we studied the lung tissue in rats exposed to cigarette smoke (CS). Rats were divided into three groups; CS, CSG, and C, which were groups of CS-treated only, CS-treated with GTE dietary supplement, and the control, respectively. Our findings by immunocytochemistry showed that abundant HMGB1 translocated from the nucleus to the cytoplasm in the lung tissues of rats that were exposed to CS, whereas HMGB1 was localized to the nuclei of CSG and C group. For in vitro studies, cotinine stimulated the secretion of HMGB1 in a dose and time dependent manner and the HMGB1 level was suppressed by GTE in murine macrophage cell lines. Our results could suggest that GTE supplementation which could suppress HMGB1 may offer a beneficial effect against diseases.

BACKGROUND/PURPOSE:Lysine-specific gingipain (Kgp) is a virulence factor secreted from Porphyromonas gingivalis (P. gingivalis), a major etiological bacterium of periodontal disease. Keratin intermediate filaments maintain the structural integrity of gingival epithelial cells, but are targeted by Kgp to produce a novel cytokeratin 6 fragment (K6F). We investigated the release of K6F and its induction of cytokine secretion. METHODS:K6F present in the gingival crevicular fluid of periodontal disease patients and in gingipain-treated rat gingival epithelial cell culture supernatants was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer-based rapid quantitative peptide analysis using BLOTCHIP. K6F in gingival tissues was immunostained, and cytokeratin 6 protein was analyzed by immunofluorescence staining and flow cytometry. Activation of MAPK in gingival epithelial cells was evaluated by immunoblotting. ELISA was used to measure K6F and the cytokines release induced by K6F. Human gingival fibroblast migration was assessed using a Matrigel invasion chamber assay. RESULTS:We identified K6F, corresponding to the C-terminus region of human cytokeratin 6 (amino acids 359-378), in the gingival crevicular fluid of periodontal disease patients and in the supernatant from gingival epithelial cells cultured with Kgp. K6F antigen was distributed from the basal to the spinous epithelial layers in gingivae from periodontal disease patients. Cytokeratin 6 on gingival epithelial cells was degraded by Kgp, but not by Arg-gingipain, P. gingivalis lipopolysaccharide or Actinobacillus actinomycetemcomitans lipopolysaccharide. K6F, but not a scrambled K6F peptide, induced human gingival fibroblast migration and secretion of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1. These effects of K6F were mediated by activation of p38 MAPK and Jun N-terminal kinase, but not p42/44 MAPK or p-Akt. CONCLUSION:Kgp degrades gingival epithelial cell cytokeratin 6 to K6F that, on release, induces invasion and cytokine secretion by human gingival fibroblasts. Thus, Kgp may contribute to the development of periodontal disease.

Dabigatran and rivaroxaban are novel oral anticoagulants that specifically inhibit thrombin and factor Xa, respectively. The aim of this study is to elucidate antithrombotic properties of these anticoagulant agents under arterial and venous shear conditions. Whole blood samples treated with dabigatran or rivaroxaban at 250, 500, and 1000 nM, with/without aspirin and AR-C66096, a P2Y12 antagonist, were perfused over a microchip coated with collagen and tissue thromboplastin at shear rates of 240 and 600 s(-1). Fibrin-rich platelet thrombus formation was quantified by monitoring flow pressure changes. Dabigatran at higher concentrations (500 and 1000 nM) potently inhibited thrombus formation at both shear rates, whereas 1000 nM of rivaroxaban delayed, but did not completely inhibit, thrombus formation. Dual antiplatelet agents weakly suppressed thrombus formation at both shear rates, but intensified the anticoagulant effects of dabigatran and rivaroxaban. The anticoagulant effects of dabigatran and rivaroxaban were also evaluated under static conditions using thrombin generation (TG) assay. In platelet-poor plasma, dabigatran at 250 and 500 nM efficiently prolonged the lag time (LT) and moderately reduce peak height (PH) of TG, whereas rivaroxaban at 250 nM efficiently prolonged LT and reduced PH of TG. In platelet-rich plasma, however, both anticoagulants efficiently delayed LT and reduced PH of TG. Our results suggest that dabigatran and rivaroxaban may exert distinct antithrombotic effects under flow conditions, particularly in combination with dual antiplatelet therapy.

Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance leading to right ventricular failure and death. Recent studies have suggested that chronic inflammatory processes are involved in the pathogenesis of PAH. However, the molecular and cellular mechanisms driving inflammation have not been fully elucidated.To elucidate the roles of high mobility group box 1 protein (HMGB1), a ubiquitous DNA-binding protein with extracellular pro-inflammatory activity, in a rat model of PAH.Male Sprague-Dawley rats were administered monocrotaline (MCT). Concentrations of HMGB1 in bronchoalveolar lavage fluid (BALF) and serum, and localization of HMGB1 in the lung were examined over time. The protective effects of anti-HMGB1 neutralizing antibody against MCT-induced PAH were tested.HMGB1 levels in BALF were elevated 1 week after MCT injection, and this elevation preceded increases of other pro-inflammatory cytokines, such as TNF-α, and the development of PAH. In contrast, serum HMGB1 levels were elevated 4 weeks after MCT injection, at which time the rats began to die. Immunohistochemical analyses indicated that HMGB1 was translocated to the extranuclear space in periarterial infiltrating cells, alveolar macrophages, and bronchial epithelial cells of MCT-injected rats. Anti-HMGB1 neutralizing antibody protected rats against MCT-induced lung inflammation, thickening of the pulmonary artery wall, and elevation of right ventricular systolic pressure, and significantly improved the survival of the MCT-induced PAH rats.Our results identify extracellular HMGB1 as a promoting factor for MCT-induced PAH. The blockade of HMGB1 activity improved survival of MCT-induced PAH rats, and thus might be a promising therapy for the treatment of PAH.

INTRODUCTION: Recent studies have shown that histones, the chief protein component of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and act as major mediators of the death of an organism. This study was designed to elucidate the cellular and molecular basis of histone-induced lethality and to assess the protective effects of recombinant thrombomodulin (rTM). rTM has been approved for the treatment of disseminated intravascular coagulation (DIC) in Japan, and is currently undergoing a phase III clinical trial in the United States. METHODS: Histone H3 levels in plasma of healthy volunteers and patients with sepsis and DIC were measured using enzyme-linked immunosorbent assay. Male C57BL/6 mice were injected intravenously with purified histones, and pathological examinations were performed. The protective effects of rTM against histone toxicity were analyzed both in vitro and in mice. RESULTS: Histone H3 was not detectable in plasma of healthy volunteers, but significant levels were observed in patients with sepsis and DIC. These levels were higher in non-survivors than in survivors. Extracellular histones triggered platelet aggregation, leading to thrombotic occlusion of pulmonary capillaries and subsequent right-sided heart failure in mice. These mice displayed symptoms of DIC, including thrombocytopenia, prolonged prothrombin time, decreased fibrinogen, fibrin deposition in capillaries, and bleeding. Platelet depletion protected mice from histone-induced death in the first 30 minutes, suggesting that vessel occlusion by platelet-rich thrombi might be responsible for death during the early phase. Furthermore, rTM bound to extracellular histones, suppressed histone-induced platelet aggregation, thrombotic occlusion of pulmonary capillaries, and dilatation of the right ventricle, and rescued mice from lethal thromboembolism. CONCLUSIONS: Extracellular histones cause massive thromboembolism associated with consumptive coagulopathy, which is diagnostically indistinguishable from DIC. rTM binds to histones and neutralizes the prothrombotic action of histones. This may contribute to the effectiveness of rTM against DIC.

The pathogenesis of acute exacerbation of idiopathic pulmonary fibrosis (IPF) remains to be elucidated. To evaluate the roles of inflammatory mediators in acute exacerbation, the concentrations of high mobility group protein B1 (HMGB1), a chief mediator of acute lung injury, and 18 inflammatory cytokines were measured in the bronchoalveolar lavage fluid, serially sampled from seven IPF patients after the onset of acute exacerbation. HMGB1 gradually increased in the alveolar fluid after the onset of acute exacerbation, in positive correlation with monocytes chemotactic protein-1 (MCP-1), a potent fibrogenic mediator. In the lung tissues of eight IPF patients autopsied after acute exacerbation, intense cytoplasmic staining for HMGB1 was observed in the alveolar epithelial cells in alveolar capillary augmented lesions, where the capillary endothelial cells remarkably reduced the expression of thrombomodulin, an intrinsic antagonist of HMGB1. These results suggest pathogenic roles for HMGB1 and MCP-1 in the late phase of acute exacerbation of IPF.