Histone-complexed DNA Fragments Research Articles

GIP_HUMAN [22-51] Peptide Encoded by the Glucose-Dependent Insulinotropic Polypeptide (GIP) Gene Suppresses Insulin Expression and Secretion in INS-1E Cells and Rat Pancreatic Islets.

GIP_HUMAN [22-51] is a recently discovered peptide that shares the same precursor molecule with glucose-dependent insulinotropic polypeptide (GIP). In vivo, chronic infusion of GIP_HUMAN [22-51] in ApoE-/- mice enhanced the development of aortic atherosclerotic lesions and upregulated inflammatory and proatherogenic proteins. In the present study, we evaluate the effects of GIP_HUMAN [22-51] on insulin mRNA expression and secretion in insulin-producing INS-1E cells and isolated rat pancreatic islets. Furthermore, we characterize the influence of GIP_HUMAN [22-51] on cell proliferation and death and on Nf-kB nuclear translocation. Rat insulin-producing INS-1E cells and pancreatic islets, isolated from male Wistar rats, were used in this study. Gene expression was evaluated using real-time PCR. Cell proliferation was studied using a BrdU incorporation assay. Cell death was quantified by evaluating histone-complexed DNA fragments. Insulin secretion was determined using an ELISA test. Nf-kB nuclear translocation was detected using immunofluorescence. GIP_HUMAN [22-51] suppressed insulin (Ins1 and Ins2) in INS-1E cells and pancreatic islets. Moreover, GIP_HUMAN [22-51] promoted the translocation of NF-κB from cytoplasm to the nucleus. In the presence of a pharmacological inhibitor of NF-κB, GIP_HUMAN [22-51] was unable to suppress Ins2 mRNA expression. Moreover, GIP_HUMAN [22-51] downregulated insulin secretion at low (2.8 mmol/L) but not high (16.7 mmol/L) glucose concentration. By contrast, GIP_HUMAN [22-51] failed to affect cell proliferation and apoptosis. We conclude that GIP_HUMAN [22-51] suppresses insulin expression and secretion in pancreatic β cells without affecting β cell proliferation or apoptosis. Notably, the effects of GIP_HUMAN [22-51] on insulin secretion are glucose-dependent.

Early Interferon-Gamma Response in Nonhuman Primate Recipients of Solid-Organ Xenotransplantation

To understand changes in biological responses in nonhuman primate (NHP) recipients of xenotransplantation (XTP), we retrospectively investigated chronological changes in cytokine profiles of NHP recipients after solid-organ XTP. Plasma samples were collected from 7 NHP recipients of pig heart or kidney XTP with α-1,3-galactosyltransferase gene knockout (GTKO)under anti-CD154-based immune suppression at the following time points: immediately before; 2 hours, 3 days, and 7 days after XTP; and weekly thereafter until the graft failed. The plasma levels of the following cytokines were measured: interleukin (IL)-1α, IL-1β, IL-6, IL-12p70, IL-8, IL-10, IL-15, tumor necrosis factor, interferon gamma (IFN-γ), D-dimer, C3a, and histone-complexed DNA fragments. For in vitro experiments, human natural killer (NK) cells were cocultured with wild-type porcine endothelial cells (PECs), GTKO-PECs, and human umbilical vein endothelial cells, with or without anti-CD154 antibody. IFN-γ levels in the culture supernatants were compared. IFN-γ levels peaked on day 7 or 10 of XTP and then decreased to basal levels, whereas proinflammatory cytokine levels increased along with the elevation of histone-complexed DNA fragments and were sustained until xenograft failure. In vitro, human NK cells produced more IFN-γ when in contact with wild-type PECs than with human umbilical vein endothelial cells, which was not reduced by the use of GTKO-PECs or addition of anti-CD154 antibody to the mixture. In NHP recipients of XTP, the early peak of IFN-γ priming subsequent inflammatory responses may be attributed to NK cell activation in response to xenografts.

Tailoring of silica-based nanoporous pod by spermidine multi-activity

Ubiquitous in nature, polyamines (PAs) are a class of low-molecular aliphatic amines critically involved in cell growth, survival and differentiation. The polycation behavior is validated as a successful strategy in delivery systems to enhance oligonucleotide loading and cellular uptake. In this study, the chemical features and the functional roles of the PA spermidine are synergistically exploited in the synthesis and bioactive functionalization of SiO2-based structures. Inspired by biosilicification, the role of spermidine is assessed both as catalyst and template in a biomimetic one-pot synthesis of dense silica-based particles (SPs) and as a competitive agent in an interfacial reassembly strategy, to empty out SPs and generate spermidine-decorated hollow silica nanoporous pods (spd-SNPs). Spermidine bioactivity is then employed for targeting tumor cell over-expressed polyamine transport system (PTS) and for effective delivery of functional miRNA into melanoma cells. Spermidine decoration promotes spd-SNP cell internalization mediated by PTS and along with hollow structure enhances oligonucleotide loading. Accordingly, the functional delivery of the tumor suppressor miR-34a 3p resulted in intracellular accumulation of histone-complexed DNA fragments associated with apoptosis. Overall, the results highlight the potential of spd-SNP as a multi-agent anticancer therapy.

Acute Traumatic Coagulopathy: The Value of Histone in Pediatric Trauma Patients.

Objective:Acute traumatic coagulopathy occurs after trauma with impairment of hemostasis and activation of fibrinolysis. Some endogenous substances may play roles in this failure of the coagulation system. Extracellular histone is one such molecule that has recently attracted attention. This study investigated the association between plasma histone-complexed DNA (hcDNA) fragments and coagulation abnormalities in pediatric trauma patients.Materials and Methods:This prospective case-control study was conducted in pediatric patients with trauma. Fifty trauma patients and 30 healthy controls were enrolled. Demographic data, anatomic injury characteristics, coagulation parameters, computerized tomography findings, trauma, and International Society on Thrombosis and Haemostasis disseminated intravascular coagulation (ISTH DIC) scores were recorded. Blood samples for hcDNA were collected and assessed by enzyme-linked immunosorbent assay.Results:Thirty-two patients had multiple trauma, while 18 patients had isolated brain injury. hcDNA levels were significantly higher in trauma patients than healthy controls (0.474 AU and 0.145 AU, respectively). There was an association between plasma hcDNA levels and trauma severity. Thirteen patients had acute coagulopathy of trauma shock (ACoTS). ACoTS patients had higher plasma histone levels than those without ACoTS (0.703 AU and 0.398 AU, respectively). Plasma hcDNA levels were positively correlated with the ISTH DIC score and length of stay in the intensive care unit and were negatively correlated with fibrinogen level.Conclusion:This study indicated that hcDNA levels were increased in pediatric trauma patients and associated with the early phase of coagulopathy. Further studies are needed to clarify the role of hcDNA levels in mortality and disseminated intravascular coagulation.

Effects of propranolol and clonidine on brain edema, blood-brain barrier permeability, and endothelial glycocalyx disruption after fluid percussion brain injury in the rat.

Traumatic brain injury causes a disruption of the vascular endothelial glycocalyx layer that is associated with an overactivation of the sympathoadrenal system. We hypothesized that early and unselective beta-blockade with propranolol alone or in combination with the alfa2-agonist clonidine would decrease brain edema, blood-brain barrier permeability, and glycocalyx disruption at 24 hours after trauma. We subjected 53 adult male Sprague-Dawley rats to lateral fluid percussion brain injury and randomized infusion with propranolol (n = 16), propranolol + clonidine (n = 16), vehicle (n = 16), or sham (n = 5) for 24 hours. Primary outcome was brain water content at 24 hours. Secondary outcomes were blood-brain barrier permeability and plasma levels of syndecan-1 (glycocalyx disruption), cell damage (histone-complexed DNA fragments), epinephrine, norepinephrine, and animal motor function. We found no difference in brain water content (mean ± SD) between propranolol (80.8 ± 0.3%; 95% confidence interval [CI], 80.7-81.0) and vehicle (81.1 ± 0.6%; 95% CI, 80.8-81.4) (p = 0.668) or between propranolol/clonidine (80.8 ± 0.3%; 95% CI, 80.7-81.0) and vehicle (p = 0.555). We found no effect of propranolol and propranolol/clonidine on blood-brain barrier permeability and animal motor scores. Unexpectedly, propranolol and propranolol/clonidine caused an increase in epinephrine and syndecan-1 levels. This study does not provide any support for unselective beta-blockade with propranolol or the combination of propranolol and the alfa2-agonist clonidine on brain water content. The novel finding of an increase in plasma concentrations of epinephrine and syndecan-1 after propranolol treatment in traumatic brain injury is of unclear significance and should be investigated further.

Histone-Complexed DNA Fragments Levels are Associated with Coagulopathy, Endothelial Cell Damage, and Increased Mortality after Severe Pediatric Trauma.

The release of damage-associated molecular pattern molecules in the extracellular space secondary to injury has been shown to cause systemic activation of the coagulation system and endothelial cell damage. We hypothesized that pediatric trauma patients with increased levels of histone-complexed DNA fragments (hcDNA) would have evidence of coagulopathy and endothelial damage that would be associated with poor outcomes. We conducted a prospective observational study of 149 pediatric trauma patients and 62 control patients at two level 1 pediatric trauma centers from 2013 to 2016. Blood samples were collected upon arrival and at 24 h, analyzed for hcDNA, coagulation abnormalities, endothelial damage, and clinical outcome. Platelet aggregation was assessed with impedance aggregometry (Multiplate) and coagulation parameters were assessed by measuring prothrombin time ratio in plasma and the use of viscoelastic techniques (Rotational Thromboelastometry) in whole blood. The median age was 8.3 years, the median injury severity score (ISS) was 20, and overall mortality was 10%. Significantly higher levels of hcDNA were found on admission in patients with severe injury (ISS > 25), coagulopathy, and/or abnormal platelet aggregation. Patients with high hcDNA levels also had significant elevations in plasma levels of syndecan-1, suggesting damage to the endothelial glycocalyx. Finally, significantly higher hcDNA levels were found in non-survivors. hcDNA is released following injury and correlates with coagulopathy, endothelial glycocalyx damage, and poor clinical outcome early after severe pediatric trauma. These results indicate that hcDNA may play an important role in development of coagulation abnormalities and endothelial glycocalyx damage in children following trauma.

APOPTOSIS INDEX BETWEEN FEMALES AND MALES IN REGULAR HEMODIALYSIS

Many reports have documented apoptosis index in hemodialysis patients, but to date, no single study has directly compared the apoptosis index of males to females. Data on mortality rate among hemodialytic patients in the hemodialysis center at the Department of Internal Medicine Dr Soetomo General Hospital, Surabaya, Indonesia show a high number predominated by female patients. Therefore, to answer the question of whether there is a gender difference in apoptosis index, the researcher studied leukocyte responses in male and female hemodialysis patients. The apoptosis index of the sample was measured by indirect immunoassay method. Cell lyses, followed by immunochemical determination of histone-complexed DNA fragments in a microtiter plate wells. The apoptosis quantization was obtained by determining the amount of colored product spectrophotometrically. One hundred and four non-diabetic subjects who received hemodialysis (HD), and 24 normal controls (NC), were evaluated. The apoptosis index in ESRD patients group and control group showed no significant difference (0.6172 vs 0.4008, p=0.114), neither did it vary in both sexes and age groups. When the sex factor was analyzed (after exclusion from the diabetic ESRD patients), females apoptosis index was significantly higher than that of the males (0.7325 vs 0.55175, p<0.05). In conclusion, apoptosis index in females among non-diabetic patients undergoing hemodialysis is higher than that occur in males and controls.

Coagulopathy in patients with acute pulmonary embolism: a pilot study of whole blood coagulation and markers of endothelial damage

Whole blood coagulation and markers of endothelial damage were studied in patients with acute pulmonary embolism (PE), and evaluated in relation to PE severity. Twenty-five patients were enrolled prospectively each having viscoelastical analysis of whole blood done using thrombelastography (TEG) and Multiplate aggregometry. Fourteen of these patients were investigated for endothelial damage by ELISA measurements of Syndecan-1 (endothelial glycocalyx degradation), soluble endothelial Selectin (endothelial cell activation), soluble Thrombomodulin (endothelial cell injury) and Histone Complexed DNA fragments (endothelial cytotoxic histones). The mean values of TEG and Multiplate parameters were all within the reference levels, but a significant difference between patients with high and intermediate risk PE was observed for Ly30 (lytic activity) 1.5% [0–10] vs. 0.2% [0–2.2] p = .04, and ADP (platelet reactivity) 92 U [20–145] vs. 59 U [20–111] p = .03. A similar difference was indicated for functional fibrinogen 21 mm [17–29] vs. 18 mm [3–23] p = .05. Analysis of endothelial markers identified a significant difference in circulating levels between high and intermediate risk PE patients for Syndecan-1 118.6 ng/mL [76–133] vs. 36.3 ng/mL [11.8–102.9] p = .008. In conclusion, patients with acute PE had normal whole blood coagulation, but high risk PE patients had signs of increased activity of the haemostatic system and significantly increased level of endothelial glycocalyx degradation.

Advanced Glycation End-Products Induce Apoptosis of Vascular Smooth Muscle Cells: A Mechanism for Vascular Calcification.

Vascular calcification, especially medial artery calcification, is associated with cardiovascular death in patients with diabetes mellitus and chronic kidney disease (CKD). To determine the underlying mechanism of vascular calcification, we have demonstrated in our previous report that advanced glycation end-products (AGEs) stimulated calcium deposition in vascular smooth muscle cells (VSMCs) through excessive oxidative stress and phenotypic transition into osteoblastic cells. Since AGEs can induce apoptosis, in this study we investigated its role on VSMC apoptosis, focusing mainly on the underlying mechanisms. A rat VSMC line (A7r5) was cultured, and treated with glycolaldehyde-derived AGE-bovine serum albumin (AGE3-BSA). Apoptotic cells were identified by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. To quantify apoptosis, an enzyme-linked immunosorbent assay (ELISA) for histone-complexed DNA fragments was employed. Real-time PCR was performed to determine the mRNA levels. Treatment of A7r5 cells with AGE3-BSA from 100 µg/mL concentration markedly increased apoptosis, which was suppressed by Nox inhibitors. AGE3-BSA significantly increased the mRNA expression of NAD(P)H oxidase components including Nox4 and p22phox, and these findings were confirmed by protein levels using immunofluorescence. Dihydroethidisum assay showed that compared with cBSA, AGE3-BSA increased reactive oxygen species level in A7r5 cells. Furthermore, AGE3-induced apoptosis was significantly inhibited by siRNA-mediated knockdown of Nox4 or p22phox. Double knockdown of Nox4 and p22phox showed a similar inhibitory effect on apoptosis as single gene silencing. Thus, our results demonstrated that NAD(P)H oxidase-derived oxidative stress are involved in AGEs-induced apoptosis of VSMCs. These findings might be important to understand the pathogenesis of vascular calcification in diabetes and CKD.

Theranostic properties of a survivin-directed molecular beacon in human melanoma cells.

Survivin is an inhibitor of apoptosis overexpressed in different types of tumors and undetectable in most terminally differentiated normal tissues. In the current study, we sought to evaluate the in vitro theranostic properties of a molecular beacon-oligodeoxynucleotide (MB) that targets survivin mRNA. We used laser scanning confocal microscopy to study MB delivery in living cells and real-time PCR and western blot to assess selective survivin-targeting in human malignant melanoma cells. We further assess the pro-apoptotic effect of MB by measuring internucleosomal DNA fragmentation, dissipation of mitochondrial membrane potential (MMP) and changes in nuclear morphology. Transfection of MB into A375 and 501 Mel cells generated high signal intensity from the cytoplasm, while no signal was detected in the extracellular environment and in survivin-negative cells (i.e., human melanocytes and monocytes). MB time dependently decreased survivin mRNA and protein expression in melanoma cells with the maximum effect reached at 72 h. Treatment of melanoma cells with MB induced apoptosis by significant changes in MMP, accumulation of histone-complexed DNA fragments in the cytoplasm and nuclear condensation. MB also enhanced the pro-apoptotic effect of standard chemotherapeutic drugs tested at clinically relevant concentrations. The MB tested in the current study conjugates the ability of imaging with the pharmacological silencing activity against survivin mRNA in human melanoma cells and may represent an innovative approach for cancer diagnosis and treatment.

Effect of acute hypobaric hypoxia on the endothelial glycocalyx and digital reactive hyperemia in humans

Introduction: Hypoxia is associated with increased capillary permeability. This study tested whether acute hypobaric hypoxia involves degradation of the endothelial glycocalyx.Methods: We exposed 12 subjects to acute hypobaric hypoxia (equivalent to 4500 m for 2–4 h) and measured venous blood concentrations of biomarkers reflecting endothelial and glycocalyx degradation (catecholamines, syndecan-1, soluble CD40 ligand, protein C, soluble thrombomodulin, tissue-type plasminogen activators, histone-complexed DNA fragments, and nitrite/nitrate). Endothelial function was assessed by the hyperemic response to brachial artery occlusion by peripheral arterial tonometry.Results: Compared with normoxic baseline levels, hypoxia increased concentrations of syndecan-1 from 22 (95% confidence interval: 17–27) to 25 (19–30) ng/ml (p < 0.02) and protein C from 76 (70–83)% to 81 (74–88)% (p < 0.02). Nitrite/nitrate decreased from 23 (18–27) μM at baseline to 19 (14–24) μM and 18 (14–21) μM in hypoxia and recovery, respectively (p < 0.05). Other biomarkers remained unchanged. The post-occlusion/pre-occlusion ratio (reactive hyperemia index, RHI) decreased from 1.80 (1.52–2.07) in normoxia to 1.62 (1.28–1.96) after 2–4 h of hypobaric hypoxia and thereafter increased to 2.43 (1.99–2.86) during normoxic recovery (p < 0.01).Conclusions: The increase in syndecan-1 and protein C suggests that acute hypobaric hypoxia produces a minor degree of glycocalyx degradation and overall cellular damage. After hypoxia RHI rebounded to higher than baseline levels suggesting improved endothelial functionality.

Coagulopathy, catecholamines, and biomarkers of endothelial damage in experimental human endotoxemia and in patients with severe sepsis: A prospective study

PurposeThe aim of this study was to investigate associations between circulating catecholamines, endothelial damage, and coagulopathy in experimental human endotoxemia and septic patients. Materials and MethodsNine healthy male volunteers undergoing endotoxemia (4-hour 0.5 ng/kg/hour infusion of E. coli lipopolysaccharide, blood sampling at 0, 4, and 6 hours) and 20 patients with severe sepsis. Analysis of plasma biomarkers (adrenaline, noradrenaline, thrombomodulin, syndecan-1, soluble vascular endothelial cadherin, histone-complexed DNA fragments, soluble CD40 ligand [sCD40L], protein C, tissue-type plasminogen activator, plasminogen activator inhibitor 1) and routine coagulation tests. ResultsEndotoxemia increased heart rate, temperature, white blood cell count, C-reactive protein and procalcitonin, decreased blood pressure and induced a hemostatic response with platelet consumption, reduced protein C and sCD40L levels and enhanced tissue-type plasminogen activator release (all P < .05). Septic patients had increased levels of noradrenaline, syndecan-1, thrombomodulin, histone-complexed DNA and sCD40L but reduced soluble vascular endothelial cadherin and plasminogen activator inhibitor 1 (all P < .05) and plasma catecholamines correlated positively with syndecan-1 (adrenaline and noradrenaline) and sTM (only noradrenaline) (all P < .05), biomarkers reflecting endothelial damage. Furthermore, noradrenaline, syndecan-1 and thrombomodulin levels correlated with INR and disease severity scores (noradrenaline and thrombomodulin) (all P < .05). ConclusionsExperimental endotoxemia induced a discrete hemostatic response without sympathoadrenal activation or endothelial damage. Septic patients had high levels of catecholamines and endothelial damage biomarkers that correlated with each other and with markers of hypocoagulability and disease severity.

Blood levels of histone-complexed DNA fragments are associated with coagulopathy, inflammation and endothelial damage early after trauma

Background:Tissue injury increases blood levels of extracellular histones and nucleic acids, and these may influence hemostasis, promote inflammation and damage the endothelium. Trauma-induced coagulopathy (TIC) may result from an endogenous response to the injury that involves the neurohumoral, inflammatory and hemostatic systems.Aims:To study the contribution of extracellular nucleic constituents to TIC, inflammation and endothelial damage.Setting and Design:Prospective observational study.Materials and Methods:We investigated histone-complexed DNA fragments (hcDNA) along with biomarkers of coagulopathy, inflammation and endothelial damage in plasma from 80 trauma patients admitted directly to the Trauma Centre from the scene of the accident. Blood was sampled a median of 68 min (IQR 48-88) post injury. Trauma patients with hcDNA levels >median or ≤median were compared.Results:Trauma patients with high plasma hcDNA had higher Injury Severity Score (ISS) and level of sympathoadrenal activation (higher adrenaline and noradrenaline) and a higher proportion of prolonged activated partial thromboplastin time (APTT) and higher D-dimer, tissue-type plasminogen activator (tPA), Annexin V and soluble CD40 ligand (sCD40L) concurrent with lower plasminogen activator inhibitor (PAI)-1) and prothrombin fragment (PF) 1 + 2 (all P < 0.05), all indicative of impaired thrombin generation, hyperfibrinolysis and platelet activation. Furthermore, patients with high hcDNA had enhanced inflammation and endothelial damage evidenced by higher plasma levels of terminal complement complex (sC5b-9), IL-6, syndecan-1, thrombomodulin and tissue factor pathway inhibitor (all P < 0.05).Conclusions:Excessive release of extracellular histones and nucleic acids seems to contribute to the hypocoagulability, inflammation and endothelial damage observed early after trauma.

Thromboembolic Complications in Fontan Patients: Population-Based Prevalence and Exploration of the Etiology

After the Fontan procedure, patients face an increased risk for thromboembolic events (TE). The etiology for this increased thrombogenecity is incompletely understood. This study aimed to determine the prevalence of TE in Danish Fontan patients and to bring new insights into the etiology of TE. Using a population-based design, we retrospectively identified all TEs in 210 Fontan patients. Whole blood assays (thromboelastography, thromboelastography functional fibrinogen and Multiplate) reflecting global hemostasis, clot strength and platelet aggregation were analyzed prospectively in 112 patients and plasma was analyzed in 76 patients for biomarkers reflecting endothelial-, glycocalyx-, platelet-, and fibrinolysis function (histone-complexed DNA fragments, Protein C, soluble CD40 ligand, soluble thrombomodulin, syndecan-1, tissue-type plasminogen activator). The results were compared in groups stratified according to age, antithrombotic therapy, TE, and glycocalyx degradation (syndecan-1 < or ≥ median). Correlation between biomarkers and demographic-, anatomical-, clinical- and biochemical parameters was investigated. The prevalence of TE was 8.1 % after a mean follow-up of 8.4 years. None of the stratified groups demonstrated evidence of hypercoagulability in the whole blood assays and no unexpected significant differences were found between the groups. All biomarkers, except protein C, correlated with one another and after stratification of glycocalyx degradation only syndecan-1 levels ≥ median correlated with other biomarkers. The prevalence of TEs was 8.1 % after mean follow-up of 8.4 years. Overall, the hemostatic profile appeared normal, however, in a subset of patients, evidence of some endothelial activation/damage including glycocalyx degradation and fibrinolysis was found, identifying a potentially more thrombogenic group.

Endothelial glycocalyx degradation induces endogenous heparinization in patients with severe injury and early traumatic coagulopathy

There is emerging evidence that early trauma-induced coagulopathy (TIC) is mechanistically linked to disruption of the vascular endothelium and its glycocalyx, assessed by thrombomodulin and syndecan 1, respectively. This study evaluated if degradation of the endothelial glycocalyx and ensuing release of its heparin-like substances induce autoheparinization and thereby contributes to TIC. Prospective observational study of 77 trauma patients admitted to a Level I trauma center having blood sampled at admission. Data on demography, hematology, Injury Severity Score, transfusion requirements, 30-day mortality, and thrombelastography (TEG, concurrent kaolin-activated/kaolin-heparinase-activated) were recorded. Retrospective analysis of plasma/serum for biomarkers reflecting endothelial glycocalyx and cell damage (syndecan 1, thrombomodulin), tissue injury (histone-complexed DNA fragments), sympathoadrenal activation (adrenaline, noradrenaline), coagulation activation/anticoagulation (prothrombin fragment 1+2, fibrinogen, von Willebrand factor, factor XIII, antithrombin, protein C, activated protein C, tissue factor pathway inhibitor), fibrinolysis (tissue-type plasminogen activator, plasminogen activator inhibitor 1) and inflammation (interleukin 6, terminal complement complex). Stratification of patients was according to the degree of TEG-measured heparinization. Four patients (5.2%) displayed evidence of high-degree autoheparinization, and these patients had higher Injury Severity Score (median [interquartile range], 31 [26-37] vs. 17 [10-26]), increased glucose (median, 13.6 vs. 8.0 mmol/L), and lower hemoglobin level (median, 5.8 vs. 8.4 mmol/L) and received more transfusions during the first 1 hour (median, 5 vs. 0) and 24 hours (median, 10 vs. 0) (all p < 0.05). Importantly, patients with autoheparinization had fourfold higher syndecan 1 levels (median [interquartile range], 116 ng/mL [78-140 ng/mL] vs. 31 ng/mL [18-49 ng/mL]), and they had higher international normalized ratio (median, 1.4 vs. 1.1), thrombomodulin (median, 4.1 vs. 1.7 ng/mL) and interleukin 6 (median, 129 vs. 71 pg/mL) but lower protein C (85% vs. 109%) (all p < 0.05), indicating profound endothelial damage, coagulopathy and inflammation. Five percent of the patients with trauma in the present study had evidence of acute endogenous coagulopathy with autoheparinization by TEG, which appeared mechanistically linked to endothelial glycocalyx degradation. Acute endogenous autoheparinization may contribute to TIC. Prognostic study, level III.

Elderly trauma patients have high circulating noradrenaline levels but attenuated release of adrenaline, platelets, and leukocytes in response to increasing injury severity

High patient age is a strong predictor of poor outcome in trauma patients. The present study investigated the effect of age on mortality and biomarkers of sympathoadrenal activation, tissue, endothelial, and glycocalyx damage, coagulation activation/inhibition, fibrinolysis, and inflammation in trauma patients at admission. Prospective observational study. Single level I trauma center. Eighty adult trauma patients (≥18 yrs) who met criteria for full trauma team activation and had an arterial cannula. Blood sampling a median of 68 min (interquartile range 48-88) post injury. Data on demography, biochemistry, Injury Severity Score, and 30-day mortality were recorded and plasma/serum was analyzed for biomarkers reflecting sympathoadrenal activation (adrenaline, noradrenaline), tissue/endothelial cell/glycocalyx damage (histone-complexed DNA fragments, annexin V, thrombomodulin, syndecan-1), platelet activation (soluble CD40 ligand), coagulation activation/inhibition (prothrombin fragment 1.2, thrombin/antithrombin complex, antithrombin, protein C, activated protein C, protein S, soluble endothelial protein C receptor, tissue factor pathway inhibitor, von Willebrand factor, fibrinogen, factor XIII), fibrinolysis (D-dimer, tissue-type plasminogen activator, plasminogen activator inhibitor-1), and inflammation (interleukin-6, terminal complement complex). Patients were stratified according to the median age (46 yrs) of the full cohort. Older trauma patients had markedly higher noradrenaline (p < .001) but an attenuated increase in adrenaline with increasing Injury Severity Score and lower platelets and leukocytes (both p < .05) compared to the younger patients. Older patients displayed a biomarker profile suggestive of enhanced release, activation, and consumption of the natural anticoagulants (low antithrombin, high activated protein C, protein S, and tissue factor pathway inhibitor) and hyperfibrinolysis (high tissue-type plasminogen activator) (all p < .05 vs. younger patients). Age was an independent predictor of mortality (hazard ratio 1.04 [95% confidence interval 1.01-1.07], p = .005) after adjusting for Injury Severity Score, prehospital Glasgow Coma Scale, and plasma catecholamines. In trauma patients, the association between age and mortality was confirmed. Older patients had high plasma noradrenaline but attenuated adrenaline release with higher Injury Severity Score, impaired platelet and leukocyte mobilization, enhanced consumption of anticoagulants, and hyperfibrinolysis, which may all contribute to the poor outcome in these patients.

High sCD40L levels early after trauma are associated with enhanced shock, sympathoadrenal activation, tissue and endothelial damage, coagulopathy and mortality

Severe injury activates the sympathoadrenal, hemostatic and inflammatory systems, but a maladapted response may contribute to a poor outcome. Soluble CD40L is a platelet-derived mediator that links inflammation, hemostasis and vascular dysfunction. To investigate the association between the sCD40L level and tissue injury, shock, coagulopathy and mortality in trauma patients. A prospective, observational study of 80 trauma patients admitted to a Level I Trauma Center. Data on demography, biochemistry, Injury Severity Score (ISS) and 30-day mortality were recorded and admission plasma/serum analyzed for sCD40L and biomarkers reflecting sympathoadrenal activation (adrenaline, noradrenaline), tissue/endothelial cell/glycocalyx damage (histone-complexed DNA fragments [hcDNA], Annexin V, thrombomodulin and syndecan-1), coagulation activation/inhibition (PF1.2, TAT-complex, antithrombin, protein C, activated protein C, sEPCR, TFPI, von Willebrand factor [VWF], fibrinogen and factor [F] XIII), fibrinolysis (D-dimer, tissue plasminogen activator [tPA] and plasminogen activator inhibitor-1 [PAI-1]) and inflammation (interleukin-6 [IL-6] and sC5b-9). We compared patients stratified by median sCD40L level and investigated predictive values of sCD40L for mortality. High circulating sCD40L was associated with enhanced tissue and endothelial damage (ISS, hcDNA, Annexin V, syndecan-1 and sTM), shock (pH, standard base excess), sympathoadrenal activation (adrenaline) and coagulopathy evidenced by reduced thrombin generation (PF1.2), hyperfibrinolysis (D-dimer), increased activated partial thromboplastin time (APTT) and inflammation (IL-6) (all P < 0.05). A higher ISS (P = 0.017), adrenaline (P = 0.049) and platelet count (P = 0.012) and lower pH (P =0.002) were associated with higher sCD40L by multivariate linear regression analysis. High circulating sCD40L (odds ratio [OR] 1.84 [95% CI 1.05-3.23], P = 0.034), high age (P = 0.002) and low Glasgow Coma Score (GCS) pre-hospital (P = 0.002) were independent predictors of increased mortality. High early sCD40L levels in trauma patients reflect tissue injury, shock, coagulopathy and sympathoadrenal activation and predict mortality. As sCD40L has pro-inflammatory activity and activates the endothelium, sCD40L may be involved in trauma-induced endothelial damage and coagulopathy.

High circulating adrenaline levels at admission predict increased mortality after trauma

Trauma-induced acute coagulopathy predicts a poor outcome. Although its pathophysiology is unclear, severe injury and shock(hypoperfusion) are proposed drivers. This study investigated the association between sympathoadrenal activation (circulating catecholamines) and biomarkers of coagulopathy. Prospective study of 75 adult trauma patients admitted to a Level I trauma center directly from the scene of accident. Patients were selected blinded post hoc from three predefined Injury Severity Score groups (<16, 16-27, and >27) and had available blood samples on arrival. We measured activated partial thromboplastin time, international normalized ratio, hematology, biochemistry, circulating adrenaline and noradrenaline, 11 biomarkers of tissue and endothelial damage, glycocalyx degradation, natural anticoagulation and fibrinolysis (histone-complexed DNA fragments, high-mobility group box 1, syndecan-1, von Willebrand factor, soluble thrombomodulin, protein C, tissue factor pathway inhibitor, antithrombin, tissue-type plasminogen activator, plasminogen activator inhibitor-1,D-dimer) and registered 30-day mortality. Biomarkers were compared between survivors and nonsurvivors. The adrenaline level was increased in nonsurvivors (p = 0.026), it was independently associated with increased activated partial thromboplastin time (p = 0.034) and syndecan-1 (p = 0.007), a marker of glycocalyx degradation, and it correlated with biomarkers of tissue and endothelial damage (histone-complexed DNA, high-mobility group box 1, soluble thrombomodulin) and hyperfibrinolysis (tissue-type plasminogen activator, D-dimer). Furthermore, nonsurvivors had higher syndecan-1, tissue factor pathway inhibitor, and D-dimer levels (all p < 0.05). Circulating adrenaline was independently associated with 30-day mortality(OR, 5.92 [95% CI, 1.48 –23.73]; p = 0.012) together with age (p = 0.001) and severe head injury (Abbreviated Injury Scale head >3; p = 0.011). The trauma-induced catecholamine surge is closely associated with biomarkers of tissue and endothelial damage, glycocalyx degradation,coagulopathy including hyperfibrinolysis and independently predicts mortality.

High levels of soluble VEGF receptor 1 early after trauma are associated with shock, sympathoadrenal activation, glycocalyx degradation and inflammation in severely injured patients: a prospective study

BackgroundThe level of soluble vascular endothelial growth factor receptor 1 (sVEGFR1) is increased in sepsis and strongly associated with disease severity and mortality. Endothelial activation and damage contribute to both sepsis and trauma pathology. Therefore, this study measured sVEGFR1 levels in trauma patients upon hospital admission hypothesizing that sVEGFR1 would increase with higher injury severity and predict a poor outcome.MethodsProspective observational study of 80 trauma patients admitted to a Level I Trauma Centre. Data on demography, biochemistry, Injury Severity Score (ISS), transfusions and 30-day mortality were recorded and plasma/serum (sampled a median of 68 min (IQR 48-88) post-injury) was analyzed for sVEGFR1 and biomarkers reflecting sympathoadrenal activation (adrenaline, noradrenaline), tissue injury (histone-complexed DNA fragments, hcDNA), endothelial activation and damage (von Willebrand Factor Antigen, Angiopoietin-2, soluble endothelial protein C receptor, syndecan-1, soluble thrombomodulin (sTM)), coagulation activation/inhibition and fibrinolysis (prothrombinfragment 1 + 2, protein C, activated Protein C, tissue-type plasminogen activator, plasminogen activator inhibitor-1, D-dimer) and inflammation (interleukin-6). Spearman correlations and regression analyses to identify variables associated with sVEGFR1 and its predictive value.ResultsCirculating sVEGFR1 correlated with injury severity (ISS, rho = 0.46), shock (SBE, rho = -0.38; adrenaline, rho = 0.47), tissue injury (hcDNA, rho = 0.44) and inflammation (IL-6, rho = 0.54) (all p < 0.01) but by multivariate linear regression analysis only lower SBE and higher adrenaline and IL-6 were independent predictors of higher sVEGFR1. sVEGFR1 also correlated with biomarkers indicative of endothelial glycocalyx degradation (syndecan-1, rho = 0.67), endothelial cell damage (sTM, rho = 0.66) and activation (Ang-2, rho = 0.31) and hyperfibrinolysis (tPA, rho = 0.39; D-dimer, rho = 0.58) and with activated protein C (rho = 0.31) (all p < 0.01). High circulating sVEGFR1 correlated with high early and late transfusion requirements (number of packed red blood cells (RBC) at 1 h (rho = 0.27, p = 0.016), 6 h (rho = 0.27, p = 0.017) and 24 h (rho = 0.31, p = 0.004) but was not associated with mortality.ConclusionssVEGFR1 increased with increasing injury severity, shock and inflammation early after trauma but only sympathoadrenal activation, hypoperfusion, and inflammation were independent predictors of sVEGFR1 levels. sVEGFR1 correlated strongly with other biomarkers of endothelial activation and damage and with RBC transfusion requirements. Sympathoadrenal activation, shock and inflammation may be critical drivers of endothelial activation and damage early after trauma.

A High Admission Syndecan-1 Level, A Marker of Endothelial Glycocalyx Degradation, Is Associated With Inflammation, Protein C Depletion, Fibrinolysis, and Increased Mortality in Trauma Patients

To investigate the association between markers of acute endothelial glycocalyx degradation, inflammation, coagulopathy, and mortality after trauma. Hyperinflammation and acute coagulopathy of trauma predict increased mortality. High catecholamine levels can directly damage the endothelium and may be associated with enhanced endothelial glycocalyx degradation, evidenced by high circulating syndecan-1. Prospective cohort study of trauma patients admitted to a Level 1 Trauma Centre in 2003 to 2005. Seventy-five patients were selected blindly post hoc from 3 predefined injury severity score (ISS) groups (<16, 16-27, >27). In all patients, we measured 17 markers of glycocalyx degradation, inflammation, tissue and endothelial damage, natural anticoagulation, and fibrinolysis (syndecan-1, IL-6, IL-10, histone-complexed DNA fragments, high-mobility group box 1 (HMGB1), thrombomodulin, von Willebrand factor, intercellular adhesion molecule-1, E-selectin, protein C, tissue factor pathway inhibitor (TFPI), antithrombin, D-dimer, tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), soluble uPA receptor, and plasminogen activator inhibitor-1), hematology, coagulation, catecholamines, and assessed 30-day mortality. Variables were compared in patients stratified according to syndecan-1 median. Patients with high circulating syndecan-1 had higher catecholamines, IL-6, IL-10, histone-complexed DNA fragments, HMGB1, thrombomodulin, D-dimer, tPA, uPA (all P < 0.05), and 3-fold increased mortality (42% vs. 14%, P = 0.006) despite comparable ISS (P = 0.351). Only in patients with high glycocalyx degradation was higher ISS correlated with higher adrenaline, IL-6, histone-complexed DNA fragments, HMGB1, thrombomodulin, and APTT, lower protein C (all P < 0.05), unchanged TFPI and blunted D-dimer response (P < 0.001) because D-dimer was profoundly increased even at low ISS. After adjusting for age and ISS, syndecan-1 was an independent predictor of mortality (OR: 1.01 [95%CI, 1.00-1.02]; P = 0.043). In trauma patients, high circulating syndecan-1, a marker of endothelial glycocalyx degradation, is associated with inflammation, coagulopathy and increased mortality.