Cytoprotective 3K3A-activated protein C and plasma: A comparison of therapeutics for the endotheliopathy of trauma.

BACKGROUND: Both healthy plasma and cytoprotective aPC (3K3A-aPC) have been shown to mitigate the endotheliopathy of trauma (EoT), but optimal therapeutics remain unknown. Our aim was therefore to determine optimal therapies to mitigate EoT by investigating the effectiveness of 3K3A-aPC with and without plasma-based resuscitation strategies. METHODS: Electric cell-substrate impedance sensing (ECIS) was used to measure real-time permeability changes in endothelial cells. Cells were treated with a 2 µg/mL solution of aPC 30 minutes prior to stimulation with plasma taken from severely injured trauma patients (ISS > 15 and BD < -6) (TP). Healthy plasma, or plasma frozen within 24 hours (FP24), was added concomitantly with TP. Cells treated with thrombin and untreated cells were included in this study as control groups. RESULTS: A dose-dependent difference was found between the 5% and 10% plasma-treated groups when HUVECs were simultaneously stimulated with TP (µd 7.346 95%CI 4.574 to 10.12). There was no difference when compared to TP alone in the 5% (µd 5.713 95%CI -1.751 to 13.18) or 10% group (µd -1.633 95%CI -9.097 to 5.832). When 3K3A-aPC was added to plasma and TP, the 5% group showed improvement in permeability compared to TP alone (µd 10.11 95%CI 2.642 to 17.57), but there was no difference in the 10% group (µd -1.394 95%CI -8.859 to 6.070). The combination of 3K3A-aPC, plasma, and TP at both the 5% plasma (µd -28.52 95%CI-34.72 to -22.32) and 10% plasma concentrations (µd -40.02 95%CI -46.22 to -33.82) had higher inter-cellular permeability than the 3K3A-aPC pre-incubation group. CONCLUSION: Our data shows that FP24, in a post-trauma environment, pre-treatment with 3K3A-aPC can potentially mitigate the EoT to a greater degree than FP24 with or without 3K3A-aPC. Although further exploration is needed, this represents a potentially ideal and perhaps superior therapeutic treatment for the dysregulated thromboinflammation of injured patients. LEVEL OF EVIDENCE: Prognostic/Epidemiological, Therapeutic/Care Management, Level III.

Platelet releasates mitigate the endotheliopathy of trauma.

BACKGROUND: Platelets are well known for their roles in hemostasis, but they also play a key role in thromboinflammatory pathways by regulating endothelial health, stimulating angiogenesis, and mediating host defense through both contact dependent and independent signaling. When activated, platelets degranulate releasing multiple active substances. We hypothesized that the soluble environment formed by trauma platelet releasates attenuates thromboinflammation via mitigation of trauma induced endothelial permeability and metabolomic reprogramming. METHODS: Blood was collected from injured and healthy patients to generate platelet releasates and plasma in parallel. Permeability of endothelial cells when exposed to trauma platelet releasates (TPR) and plasma (TP) was assessed via resistance measurement by Electric Cell-substrate Impedance Sensing (ECIS). Endothelial cells treated with TPR and TP were subjected to mass spectrometry-based metabolomics. RESULTS: TP increased endothelial permeability, whereas TPR decreased endothelial permeability when compared to untreated cells. When TP and TPR were mixed ex vivo, TPR mitigated TP-induced permeability, with significant increase in AUC compared to TP alone. Metabolomics of TPR and TP demonstrated disrupted redox reactions and anti-inflammatory mechanisms. CONCLUSION: TPRs provide endothelial barrier protection against TP-induced endothelial permeability. Our findings highlight a potential beneficial action of activated platelets on the endothelium in injured patients through disrupted redox reactions and increased antioxidants. Our findings support that soluble signaling from platelet degranulation may mitigate the endotheliopathy of trauma. The clinical implications of this are that activated platelets may prove a promising therapeutic target in the complex integration of thrombosis, endotheliopathy, and inflammation in trauma. LEVEL OF EVIDENCE: Prognostic/Epidemiological, Level III.

Trauma patients with type O blood exhibit unique multi-omics signature with decreased lectin pathway of complement levels.

BACKGROUND: Patients with type O blood may have an increased risk of hemorrhagic complications due to lower baseline levels of von Willebrand Factor (vWF) and factor VIII, but the transition to a mortality difference in trauma is less clear. We hypothesized that type O trauma patients will have differential proteomic and metabolomic signatures in response to trauma beyond vWF and FVIII alone. METHODS: Patients meeting the highest level of trauma activation criteria were prospectively enrolled. Blood samples were collected upon arrival to the emergency department. Proteomic and metabolomic (multi-omics) analyses of these samples were performed using liquid chromatography-mass spectrometry. Demographic, clinical, and multi-omics data were compared between patients with type O blood versus all other patients. RESULTS: There were 288 patients with multi-omics data; 146 (51%) had type O blood. Demographics, injury patterns, and initial vital signs and laboratory measurements were not different between groups. Type O patients had increased lengths of stay (7 vs. 6 days, p = 0.041) and a trend towards decreased mortality secondary to traumatic brain injury compared to other causes (TBI, 44.4 % vs. 87.5%, p = 0.055). Type O patients had decreased levels of mannose-binding lectin (MBL) and MBL associated serine proteases 1 and 2 which are required for the initiation of the lectin pathway of complement activation. Type O patients also had metabolite differences signifying energy metabolism and mitochondrial dysfunction. CONCLUSION: Blood type O patients have a unique multi-omics signature, including decreased levels of proteins required to activate the lectin complement pathway. This may lead to overall decreased levels of complement activation and decreased systemic inflammation in the acute phase possibly leading to a survival advantage, especially in TBI. However, this may later impair healing. Future work will need to confirm these associations, and animal studies are needed to test therapeutic targets. LEVEL OF EVIDENCE: Retrospective Comparative Study, Level IV.

Smoking primes the metabolomic response in trauma.

INTRODUCTION: Smoking is a public health threat because of its well-described link to increased oxidative stress-related diseases including peripheral vascular disease and coronary artery disease. Tobacco use has been linked to risk of inpatient trauma morbidity including acute respiratory distress syndrome; however, its mechanistic effect on comprehensive metabolic heterogeneity has yet to be examined. METHODS: Plasma was obtained on arrival from injured patients at a Level 1 trauma center and analyzed with modern mass spectrometry-based metabolomics. Patients were stratified by nonsmoker, passive smoker, and active smoker by lower, interquartile, and upper quartile ranges of cotinine intensity peaks. Patients were substratified by high injury/high shock (Injury Severity Score, ≥15; base excess, <-6) and compared with healthy controls. p Value of <0.05 following false discovery rate correction of t test was considered significant. RESULTS: Forty-eight patients with high injury/high shock (7 nonsmokers [15%], 25 passive smokers [52%], and 16 active smokers [33%]) and 95 healthy patients who served as controls (30 nonsmokers [32%], 43 passive smokers [45%], and 22 active smokers [23%]) were included. Elevated metabolites in our controls who were active smokers include enrichment in chronic inflammatory and oxidative processes. Elevated metabolites in active smokers in high injury/high shock include enrichment in the malate-aspartate shuttle, tyrosine metabolism, carnitine synthesis, and oxidation of very long-chain fatty acids. CONCLUSION: Smoking promotes a state of oxidative stress leading to mitochondrial dysfunction, which is additive to the inflammatory milieu of trauma. Smoking is associated with impaired mitochondrial substrate utilization of long-chain fatty acids, aspartate, and tyrosine, all of which accentuate oxidative stress following injury. This altered expression represents an ideal target for therapies to reduce oxidative damage toward the goal of personalized treatment of trauma patients. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level IV.

Mitigation of trauma-induced endotheliopathy by activated protein C: A potential therapeutic for postinjury thromboinflammation.

BACKGROUND: Activated Protein C (aPC) plays dual roles after injury, driving both trauma-induced coagulopathy (TIC) by cleaving, and thus inactivating, factors Va and VIIIa and depressing fibrinolysis while also mediating an inflammomodulatory milieu via protease activated receptor-1 (PAR-1) cytoprotective signaling. Because of this dual role, it represents and ideal target for study and therapeutics after trauma. A known aPC variant, 3K3A-aPC, has been engineered to preserve cytoprotective activity while retaining minimal anticoagulant activity rendering it potentially ideal as a cytoprotective therapeutic after trauma. We hypothesized that 3K3A-aPC would mitigate the endotheliopathy of trauma by protecting against endothelial permeability. METHODS: We used electric cell-substrate impedance sensing to measure permeability changes in real time in primary endothelial cells. These were cultured, grown to confluence, and treated with a 2 µg/mL solution of 3K3A-aPC at 180 minutes, 120 minutes, 60 minutes, 30 minutes prior to stimulation with ex vivo plasma taken from severely injured trauma patients (Injury Severity Score > 15 and BD < -6) (trauma plasma [TP]). Cells treated with thrombin and untreated cells were included in this study as control groups. Permeability changes were recorded in real time via electric cell-substrate impedance sensing for 30 minutes after treatment with TP. We quantified permeability changes in the control and treatment groups as area under the curve (AUC). Rac1/RhoA activity was also compared between these groups. Statistical significance was determined by one-way ANOVA followed by a post hoc analysis using Tukey's multiple comparison's test. RESULTS: Treatment with aPC mitigated endothelial permeability induced by ex vivo trauma plasma at all pre-treatment time points. The AUC of the 30-minute 3K3A-aPC pretreatment group was higher than TP alone (mean diff. 22.12 95% CI [13.75, 30.49], p < 0.0001) (Figure). Moreover, the AUC of the 60-minute, 120-minute, and 180-minute pretreatment groups was also higher than TP alone (mean diff., 16.30; 95% confidence interval [CI], 7.93-24.67; 19.43; 95% CI, 11.06-27.80, and 18.65; 95% CI, 10.28-27.02;, all p < 0.0001, respectively). Rac1/RhoA activity was higher in the aPC pretreatment group when compared with all other groups ( p < 0.01). CONCLUSION: Pretreatment with 3K3A-aPC, which retains its cytoprotective function but has only ~5% of its anticoagulant function, abrogates the effects of trauma-induced endotheliopathy. This represents a potential therapeutic treatment for dysregulated thromboinflammation for injured patients by minimizing aPC's role in trauma-induced coagulopathy while concurrently amplifying its essential cytoprotective function. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level III.

Endothelial Cell Calcium Influx Mediates Trauma-induced Endothelial Permeability.

OBJECTIVE: We aimed to investigate if ex vivo plasma from injured patients causes endothelial calcium (Ca2+) influx as a mechanism of trauma-induced endothelial permeability. SUMMARY BACKGROUND DATA: Endothelial permeability after trauma contributes to post-injury organ dysfunction. While the mechanisms remain unclear, emerging evidence suggests intracellular Ca2+ signaling may play a role. METHODS: Ex vivo plasma from injured patients with "Low Injury/Low Shock" (injury severity score [ISS]<15, base excess [BE])≥-6mEq/L) and "High Injury/High Shock" (ISS≥15, BE<-6mEq/L) were used to treat endothelial cells. Experimental conditions included Ca2+ removal from the extracellular buffer, cyclopiazonic acid pre-treatment to deplete intracellular Ca2+ stores, and GSK2193874 pre-treatment to block the TRPV4 Ca2+ channel. Live cell fluorescence microscopy and ECIS were used to assess cytosolic Ca2+ increases and permeability, respectively. Western blot and live cell actin staining were used to assess myosin light chain (MLC) phosphorylation and actomyosin contraction. RESULTS: Compared to Low Injury/Low Shock plasma, High Injury/High Shock induced greater cytosolic Ca2+ increase. Cytosolic Ca2+ increase, MLC phosphorylation, and actin cytoskeletal contraction were lower without extracellular Ca2+ present. High Injury/High Shock plasma did not induce endothelial permeability without extracellular Ca2+ present. TRPV4 inhibition lowered trauma plasma-induced endothelial Ca2+ influx and permeability. CONCLUSIONS: This study illuminates a novel mechanism of post-injury endotheliopathy involving Ca2+ influx via the TRPV4 channel. TRPV4 inhibition mitigates trauma-induced endothelial permeability. Moreover, widespread endothelial Ca2+ influx may contribute to trauma-induced hypocalcemia. This study provides the mechanistic basis for the development of Ca2+-targeted therapies and interventions in the care of severely injured patients.

Silencing Glypican-1 enhances the antitumor effects of Pictilisib via downregulating PI3K/Akt/ERK signaling in chemo-resistant esophageal adenocarcinoma.

Poorly differentiated esophageal adenocarcinoma (PDEAC) has a dismal prognosis. Glypican-1(GPC-1) is known to be upregulated in several cancer types in contrast to healthy tissues, rendering it as a biomarker. Nevertheless, the potential therapeutic targeting of GPC-1 has not been explored in PDEAC. There is accumulating evidence that GPC-1, via upregulation of PI3K/Akt/ERK signaling, plays a crucial role in the progression and chemoresistance in cancer. Pictilisib, a class I pan PI3K inhibitor, has shown promising antitumor results in clinical trials, however, has not gained widespread success due to acquired drug resistance. This study investigated the role of GPC-1 in chemo-resistant PDEAC and appraises the impact of targeted silencing of GPC-1 on the antitumor effects of Pictilisib in PDEAC cell lines. Immunohistochemistry assays in PDEAC tissue specimens demonstrated a pronounced intensity of staining with GPC-1. Upregulation of GPC-1 was found to be correlated with advanced stage and poor prognosis. In-vitro studies examined the influence of GPC-1 knockdown and Pictilisib, both as individual agents and in combination, on cytotoxicity, cell cycle distribution, apoptosis, and gene expression profiles. Silencing GPC-1 alone showed significantly reduced cell viability, migration, colony formation, epithelial-mesenchymal transition, and stemness in PDEAC cells. Significantly, knockdown of GPC-1 combined with low-dose Pictilisib led to enhancement of cytotoxicity, cell cycle arrest, and apoptosis in ESO-26 and OE-33 cells. In the xenograft mouse model, the combination of Pictilisib and GPC-1 knockdown exhibited synergy. These findings suggest that GPC-1 represents a promising target to augment chemosensitivity in esophageal adenocarcinoma.

The proteomic and metabolomic signatures of isolated and polytrauma traumatic brain injury.

BACKGROUND: The interactions of polytrauma, shock, and traumatic brain injury (TBI) on thromboinflammatory responses remain unclear and warrant investigation as we strive towards personalized medicine in trauma. We hypothesized that comprehensive omics characterization of plasma would identify unique metabolic and thromboinflammatory pathways following TBI. METHODS: Patients were categorized as TBI vs Non-TBI, and stratified into Polytrauma or minimally injured. Discovery 'omics was employed to quantify the top differently expressed proteins and metabolites of TBI and Non-TBI patient groups. RESULTS: TBI compared to Non-TBI showed gene enrichment in coagulation/complement cascades and neuronal markers. TBI was associated with elevation in glycolytic metabolites and conjugated bile acids. Division into isolated TBI vs polytrauma showed further distinction of proteomic and metabolomic signatures. CONCLUSION: Identified mediators involving in neural inflammation, blood brain barrier disruption, and bile acid building leading to TBI associated coagulopathy offer suggestions for follow up mechanistic studies to target personalized interventions.

A metabolomic and proteomic analysis of pathologic hypercoagulability in traumatic brain injury patients after dura violation.

BACKGROUND: The coagulopathy of traumatic brain injury (TBI) remains poorly understood. Contradictory descriptions highlight the distinction between systemic and local coagulation, with descriptions of systemic hypercoagulability despite intracranial hypocoagulopathy. This perplexing coagulation profile has been hypothesized to be due to tissue factor release. The objective of this study was to assess the coagulation profile of TBI patients undergoing neurosurgical procedures. We hypothesize that dura violation is associated with higher tissue factor and conversion to a hypercoagulable profile and unique metabolomic and proteomic phenotype. METHODS: This is a prospective, observational cohort study of all adult TBI patients at an urban, Level I trauma center who underwent a neurosurgical procedure from 2019 to 2021. Whole blood samples were collected before and then 1 hour following dura violation. Citrated rapid and tissue plasminogen activator (tPA) thrombelastography (TEG) were performed, in addition to measurement of tissue factory activity, metabolomics, and proteomics. RESULTS: Overall, 57 patients were included. The majority (61%) were male, the median age was 52 years, 70% presented after blunt trauma, and the median Glasgow Coma Score was 7. Compared with pre-dura violation, post-dura violation blood demonstrated systemic hypercoagulability, with a significant increase in clot strength (maximum amplitude of 74.4 mm vs. 63.5 mm; p < 0.0001) and a significant decrease in fibrinolysis (LY30 on tPAchallenged TEG of 1.4% vs. 2.6%; p = 0.04). There were no statistically significant differences in tissue factor. Metabolomics revealed notable increases in metabolites involved in late glycolysis, cysteine, and one-carbon metabolites, and metabolites involved in endothelial dysfunction/arginine metabolism/responses to hypoxia. Proteomics revealed notable increase in proteins related to platelet activation and fibrinolysis inhibition. CONCLUSION: A systemic hypercoagulability is observed in TBI patients, characterized by increased clot strength and decreased fibrinolysis and a unique metabolomic and proteomics phenotype independent of tissue factor levels.

Omics Signatures of Tissue Injury and Hemorrhagic Shock in Swine.

OBJECTIVE: Advanced mass spectrometry methods were leveraged to analyze both proteomics and metabolomics signatures in plasma upon controlled tissue injury (TI) and hemorrhagic shock (HS)-isolated or combined-in a swine model, followed by correlation to viscoelastic measurements of coagulopathy via thrombelastography. BACKGROUND: TI and HS cause distinct molecular changes in plasma in both animal models and trauma patients. However, the contribution to coagulopathy of trauma, the leading cause of preventable mortality in this patient population remains unclear. The recent development of a swine model for isolated or combined TI+HS facilitated the current study. METHODS: Male swine (n=17) were randomized to either isolated or combined TI and HS. Coagulation status was analyzed by thrombelastography during the monitored time course. The plasma fractions of the blood draws (at baseline; end of shock; and at 30 minutes, 1, 2, and 4 hours after shock) were analyzed by mass spectrometry-based proteomics and metabolomics workflows. RESULTS: HS-isolated or combined with TI-caused the most severe omic alterations during the monitored time course. While isolated TI delayed the activation of coagulation cascades. Correlation to thrombelastography parameters of clot strength (maximum amplitude) and breakdown (LY30) revealed signatures of coagulopathy which were supported by analysis of gene ontology-enriched biological pathways. CONCLUSION: The current study provides a comprehensive characterization of proteomic and metabolomic alterations to combined or isolated TI and HS in a swine model and identifies early and late omics correlates to viscoelastic measurements in this system.

A proteomic analysis of NETosis in trauma: Emergence of serpinB1 as a key player.

BACKGROUND: Release of neutrophil extracellular traps (NETosis) may mediate postinjury organ dysfunction, but mechanisms remain unclear. The intracellular serine protease inhibitor (serpin) B1 is vital to neutrophil function and has been shown to restrict NETosis in inflammatory settings. In this study, we used discovery proteomics to identify the proteomic signature of trauma-induced NETosis. We hypothesized that serpinB1 would be a major component of this NET protein profile and associated with adverse outcomes. METHODS: This was a post hoc analysis of data collected as part of the COMBAT randomized clinical trial. Blood was collected from injured patients at a single Level I Trauma Center. Proteomic analyses were performed through targeted liquid chromatography coupled with mass spectrometry. Abundances of serpinB1 and known NETosis markers were analyzed with patient and injury characteristics, clinical data, and outcomes. RESULTS: SerpinB1 levels on emergency department (ED) arrival were significantly correlated with proteomic markers of NETosis, including core histones, transketolase, and S100A8/A9 proteins. More severely injured patients had elevated serpinB1 and NETosis markers on ED arrival. Levels of serpinB1 and top NETosis markers were significantly elevated on ED arrival in nonsurvivors and patients with fewer ventilator- and ICU-free days. In proteome-wide receiver operating characteristic analysis, serpinB1 was consistently among the top proteins associated with adverse outcomes. Among NETosis markers, levels of serpinB1 early in the patient's course exhibited the greatest separation between patients with fewer and greater ventilator- and ICU-free days. Gene Ontology analysis of top predictors of adverse outcomes further supports NETosis as a potential mediator of postinjury organ dysfunction. CONCLUSION: We have identified a proteomic signature of trauma-induced NETosis, and NETosis is an early process following severe injury that may mediate organ dysfunction. In addition, serpinB1 is a major component of this NET protein profile that may serve as an early marker of excessive NETosis after injury.

Hemorrhagic shock and tissue injury provoke distinct components of trauma-induced coagulopathy in a swine model.

INTRODUCTION: Tissue injury (TI) and hemorrhagic shock (HS) are the major contributors to trauma-induced coagulopathy (TIC). However, the individual contributions of these insults are difficult to discern clinically because they typically coexist. TI has been reported to release procoagulants, while HS has been associated with bleeding. We developed a large animal model to isolate TI and HS and characterize their individual mechanistic pathways. We hypothesized that while TI and HS are both drivers of TIC, they provoke different pathways; specifically, TI reduces time to clotting, whereas, HS decreases clot strength stimulates hyperfibrinolysis. METHODS: After induction of general anesthesia, 50 kg male, Yorkshire swine underwent isolated TI (bilateral muscle cutdown of quadriceps, bilateral femur fractures) or isolated HS (controlled bleeding to a base excess target of - 5 mmol/l) and observed for 240 min. Thrombelastography (TEG), calcium levels, thrombin activatable fibrinolysis inhibitor (TAFI), protein C, plasminogen activator inhibitor 1 (PAI-1), and plasminogen activator inhibitor 1/tissue-type plasminogen activator complex (PAI-1-tPA) were analyzed at pre-selected timepoints. Linear mixed models for repeated measures were used to compare results throughout the model. RESULTS: TI resulted in elevated histone release which peaked at 120 min (p = 0.02), and this was associated with reduced time to clot formation (R time) by 240 min (p = 0.006). HS decreased clot strength at time 30 min (p = 0.003), with a significant decline in calcium (p = 0.001). At study completion, HS animals had elevated PAI-1 (p = 0.01) and PAI-1-tPA (p = 0.04), showing a trend toward hyperfibrinolysis, while TI animals had suppressed fibrinolysis. Protein C, TAFI and skeletal myosin were not different among the groups. CONCLUSION: Isolated injury in animal models can help elucidate the mechanistic pathways leading to TIC. Our results suggest that isolated TI leads to early histone release and a hypercoagulable state, with suppressed fibrinolysis. In contrast, HS promotes poor clot strength and hyperfibrinolysis resulting in hypocoagulability.

Glypican 1 promotes proliferation and migration in esophagogastric adenocarcinoma via activating AKT/GSK/ß-catenin pathway.

Background: Glypican 1 (GPC1) is a heparan sulphate proteoglycan cell membrane protein. It is implicated in driving cancers of the breast, brain, pancreas, and prostate; however, its role in esophagogastric cancer (EGAC) remains unexplored. The aim of the study was to investigate and elucidate the molecular mechanistic of GPC1 in human EGAC. Methods: Thirty tissue and 120 microarray sections of EGAC were evaluated with Anti-GPC1 immunohistochemistry. Loss and gain of GPC1 function were performed using lentivirus transfection in EGAC cell lines. Mechanistically, AKT/GSK/ß-catenin pathway was evaluated using AKT inhibitor MK-2206 and Wnt/ß-catenin stimulant LiCl. Results: GPC1 overexpression was found in 102 cases (68%). Overexpression of GPC1 correlated with lymph node metastasis, poor differentiation and decreased overall survival. Lentivirus mediated GPC1 knockdown resulted in decreased cell proliferation, migration, invasion, and colony formation. Knockdown caused G0/G1 cell cycle arrest, increased apoptosis, and reduced epithelial mesenchymal transition (EMT). GPC1 mediated its effects by activation of AKT/GSK/ß-catenin pathway. Conclusions: This is the first descriptive study to decipher the role of GPC1 in EGAC. Our results suggest that GPC1 regulates cell proliferation and growth and may serve as an attractive oncotarget in EGAC.

Zone 1 REBOA in a combat DCBI swine model does not worsen brain injury.

BACKGROUND: Zone 1 resuscitative endovascular balloon occlusion of the aorta has been recommended for refractory shock after a dismounted complex blast injury for the austere combat scenario. While resuscitative endovascular balloon occlusion of the aorta should enhance coronary perfusion, there is a potential risk of secondary brain injury due to loss of cerebral autoregulation. We developed a combat casualty relevant dismounted complex blast injury swine model to evaluate the effects of resuscitative endovascular balloon occlusion of the aorta zone I on intracranial pressure and cerebral edema. We hypothesized that zone 1 aortic occlusion with resuscitative endovascular balloon occlusion of the aorta would increase mean arterial pressure transmitted in excessive intracranial pressure, thereby worsening brain injury. METHODS: 50 kg male Yorkshire swine were subjected to a combination dismounted complex blast injury model consisting of blast traumatic brain injury (50 psi, ARA Mobile Shock Laboratory), tissue injury (bilateral femur fractures), and hemorrhagic shock (controlled bleeding to a base deficit goal of 10 mEq/L). During the shock phase, pigs were randomized to no aortic occlusion (n = 8) or to 30 minutes of zone 1 resuscitative endovascular balloon occlusion of the aorta (zone 1 aortic occlusion group, n = 6). After shock, pigs in both groups received a modified Tactical Combat Casualty Care-based resuscitation and were monitored for an additional 240 minutes until euthanasia/death for a total of 6 hours. Intracranial pressure was monitored throughout, and brains were harvested for water content. Linear mixed models for repeated measures were used to compare mean arterial pressure and intracranial pressure between zone 1 aortic occlusion and no aortic occlusion groups. RESULTS: After dismounted complex blast injury, the zone 1 group had a significantly higher mean arterial pressure during hemorrhagic shock compared to the control group (41.2 mm Hg vs 16.7 mm Hg, P = .002). During balloon occlusion, intracranial pressure was not significantly elevated in the zone 1 aortic occlusion group vs control, but intracranial pressure was significantly lower in the zone 1 group at the end of the observation period. In addition, the zone 1 aortic occlusion group did not have increased brain water content (zone 1 aortic occlusion: 3.95 ± 0.1g vs no aortic occlusion: 3.95 ± 0.3 g, P = .87). Troponin levels significantly increased in the no aortic occlusion group but did not in the zone 1 aortic occlusion group. CONCLUSION: Zone 1 aortic occlusion using resuscitative endovascular balloon occlusion of the aorta in a large animal dismounted complex blast injury model improved proximal mean arterial pressure while not significantly increasing intracranial pressure during balloon inflation. Observation up to 240 minutes postresuscitation did not show clinical signs of worsening brain injury or cardiac injury. These data suggest that in a dismounted complex blast injury swine model, resuscitative endovascular balloon occlusion of the aorta in zone 1 may provide neuro- and cardioprotection in the setting of blast traumatic brain injury. However, longer monitoring periods may be needed to confirm that the neuroprotection is lasting.

Postinjury complement C4 activation is associated with adverse outcomes and is potentially influenced by plasma resuscitation.

BACKGROUND: Complement activation after trauma promotes hemostasis but is associated with increased morbidity and mortality. However, the specific pathways and downstream mediators remain unclear. Recently, the anaphylatoxin C4a has been shown to bind to thrombin receptors. While plasma-based resuscitation has been shown to modify the endotheliopathy of trauma, it may provide complement zymogens that fuel ongoing inflammatory cascades. We sought to characterize the activation of complement after injury and the effect of fresh frozen plasma (FFP) on this inflammatory response. We hypothesized that trauma induces C4 activation, which is associated with worse outcomes and influenced by FFP resuscitation. METHODS: Blood was collected from injured patients at a single level I trauma center enrolled in the Control of Major Bleeding after Trauma (COMBAT) randomized clinical trial. Proteomic analyses were performed through targeted liquid chromatography coupled with mass spectrometry. For the present observational study, concentrations of complement proteins were analyzed at multiple time points, compared between treatment groups, and correlated with outcomes. RESULTS: C4 activation occurred over the first 6 hours postinjury with peak activation 6 to 24 hours. Tissue hypoperfusion, defined as base deficit >10 mEq/L, and requirement for massive transfusion were associated with greater C4 activation. C4 activation was associated with mortality, multiple organ failure, and longer ventilator requirement. In addition, temporal trends of C1q, factor B, and C3 by outcome groups support the prevailing theory of primary classical pathway activation with alternative pathway amplification. Resuscitation with FFP over the first 6 hours was associated with increased C4 activation at 12 and 24 hours. CONCLUSION: C4 activation has an important inflammatory role postinjury, and FFP has the potential to augment this complement activation during resuscitation. LEVEL OF EVIDENCE: Prognostic/epidemiological, level III.

Hypertonic saline attenuates the cytokine-induced pro-inflammatory signature in primary human lung epithelia.

Trauma/hemorrhagic shock is a complex physiological phenomenon that leads to dysregulation of many molecular pathways. For over a decade, hypertonic saline (HTS) has been used as an alternative resuscitation fluid in the setting of trauma/hemorrhagic shock. In addition to restoring circulating volume within the vascular space, studies have shown a positive immunomodulatory effect of HTS. Targeted studies have shown that HTS affects the transcription of several pro-inflammatory cytokines by inhibiting the NF-κB-IκB pathway in model cell lines and rats. However, few studies have been undertaken to assess the unbiased effects of HTS on the whole transcriptome. This study was designed to interrogate the global transcriptional responses induced by HTS and provides insight into the underlying molecular mechanisms and pathways affected by HTS. In this study, RNA sequencing was employed to explore early changes in transcriptional response, identify key mediators, signaling pathways, and transcriptional modules that are affected by HTS in the presence of a strong inflammatory stimulus. Our results suggest that primary human small airway lung epithelial cells (SAECS) exposed to HTS in the presence and absence of a strong pro-inflammatory stimulus exhibit very distinct effects on cellular response, where HTS is highly effective in attenuating cytokine-induced pro-inflammatory responses via mechanisms that involve transcriptional regulation of inflammation which is cell type and stimulus specific. HTS is a highly effective anti-inflammatory agent that inhibits the chemotaxis of leucocytes towards a pro-inflammatory gradient and may attenuate the progression of both the innate and adaptive immune response.

LysoPCs induce Hck- and PKCδ-mediated activation of PKCγ causing p47phox phosphorylation and membrane translocation in neutrophils.

Lysophosphatidylcholines (lysoPCs) are effective polymorphonuclear neutrophil (PMN) priming agents implicated in transfusion-related acute lung injury (TRALI). LysoPCs cause ligation of the G2A receptor, cytosolic Ca2+ flux, and activation of Hck. We hypothesize that lysoPCs induce Hck-dependent activation of protein kinase C (PKC), resulting in phosphorylation and membrane translocation of 47 kDa phagocyte oxidase protein (p47phox). PMNs, human or murine, were primed with lysoPCs and were smeared onto slides and examined by digital microscopy or separated into subcellular fractions or whole-cell lysates. Proteins were immunoprecipitated or separated by polyacrylamide gel electrophoresis and immunoblotted for proteins of interest. Wild-type (WT) and PKCγ knockout (KO) mice were used in a 2-event model of TRALI. LysoPCs induced Hck coprecipitation with PKCδ and PKCγ and the PKCδ:PKCγ complex also had a fluorescence resonance energy transfer (FRET)+ interaction with lipid rafts and Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2 (WAVE2). PKCγ then coprecipitated with p47phox Immunoblotting, immunoprecipitation (IP), specific inhibitors, intracellular depletion of PKC isoforms, and PMNs from PKCγ KO mice demonstrated that Hck elicited activation/Tyr phosphorylation (Tyr311 and Tyr525) of PKCδ, which became Thr phosphorylated (Thr507). Activated PKCδ then caused activation of PKCγ, both by Tyr phosphorylation (Τyr514) and Ser phosphorylation, which induced phosphorylation and membrane translocation of p47phox In PKCγ KO PMNs, lysoPCs induced Hck translocation but did not evidence a FRET+ interaction between PKCδ and PKCγ nor prime PMNs. In WT mice, lysoPCs served as the second event in a 2-event in vivo model of TRALI but did not induce TRALI in PKCγ KO mice. We conclude that lysoPCs prime PMNs through Hck-dependent activation of PKCδ, which stimulates PKCγ, resulting in translocation of phosphorylated p47phox.

Hypertonic Saline Primes Activation of the p53-p21 Signaling Axis in Human Small Airway Epithelial Cells That Prevents Inflammation Induced by Pro-inflammatory Cytokines.

Uncontrolled inflammatory responses underlie the etiology of acute lung injury and acute distress respiratory syndrome, the most common late complications in trauma, the leading cause of death under the age of 59. Treatment with HTS decreases lung injury in clinical trials, rat models of trauma and hemorrhagic shock and inflammation in lung cell lines, although the mechanisms underlying these responses are still incompletely understood. Transcriptomics (RNaseq), proteomics, and U-13C-glucose tracing metabolomics experiments were performed to investigate the mechanisms of cellular responses to HTS treatment in primary small airway epithelial cells in the presence or absence of inflammatory injury mediated by a cocktail of cytokines (10 ng/mL of IFNγ, IL-1ß, and TNFα). Modestly hyperosmolar HTS has an anti-inflammatory effect, triggers the p53-p21 signaling axis, and deregulates mitochondrial metabolism while inducing minimal apoptosis in response to a second hit by cytokines. Decreased transcription of pro-inflammatory cytokines suggested a role for the tumor suppressor protein p53 in mediating the beneficial effects of the HTS treatment. The anti-inflammatory mechanisms induced by HTS involves p53 gene regulation, promotes cell cycle arrest, and prevents ROS formation and mitochondria depolarization. Pharmaceutical targeting of the p53-p21 axis may mimic or reinforce the beneficial effects mediated by HTS when sustained hypertonicity cannot be maintained.

Overwhelming tPA release, not PAI-1 degradation, is responsible for hyperfibrinolysis in severely injured trauma patients.

BACKGROUND: Trauma-induced coagulopathy (TIC) is associated with a fourfold increased risk of mortality. Hyperfibrinolysis is a component of TIC, but its mechanism is poorly understood. Plasminogen activation inhibitor (PAI-1) degradation by activated protein C has been proposed as a mechanism for deregulation of the plasmin system in hemorrhagic shock, but in other settings of ischemia, tissue plasminogen activator (tPA) has been shown to be elevated. We hypothesized that the hyperfibrinolysis in TIC is not the result of PAI-1 degradation but is driven by an increase in tPA, with resultant loss of PAI-1 activity through complexation with tPA. METHODS: Eighty-six consecutive trauma activation patients had blood collected at the earliest time after injury and were screened for hyperfibrinolysis using thrombelastography (TEG). Twenty-five hyperfibrinolytic patients were compared with 14 healthy controls using enzyme-linked immunosorbent assays for active tPA, active PAI-1, and PAI-1/tPA complex. Blood was also subjected to TEG with exogenous tPA challenge as a functional assay for PAI-1 reserve. RESULTS: Total levels of PAI-1 (the sum of the active PAI-1 species and its covalent complex with tPA) are not significantly different between hyperfibrinolytic trauma patients and healthy controls: median, 104 pM (interquartile range [IQR], 48-201 pM) versus 115 pM (IQR, 54-202 pM). The ratio of active to complexed PAI-1, however, was two orders of magnitude lower in hyperfibrinolytic patients than in controls. Conversely, total tPA levels (active + complex) were significantly higher in hyperfibrinolytic patients than in controls: 139 pM (IQR, 68-237 pM) versus 32 pM (IQR, 16-37 pM). Hyperfibrinolytic trauma patients displayed increased sensitivity to exogenous challenge with tPA (median LY30 of 66.8% compared with 9.6% for controls). CONCLUSION: Depletion of PAI-1 in TIC is driven by an increase in tPA, not PAI-1 degradation. The tPA-challenged TEG, based on this principle, is a functional test for PAI-1 reserves. Exploration of the mechanism of up-regulation of tPA is critical to an understanding of hyperfibrinolysis in trauma. LEVEL OF EVIDENCE: Prognostic and epidemiologic study, level II.

Hyperosmolarity invokes distinct anti-inflammatory mechanisms in pulmonary epithelial cells: evidence from signaling and transcription layers.

Hypertonic saline (HTS) has been used intravenously to reduce organ dysfunction following injury and as an inhaled therapy for cystic fibrosis lung disease. The role and mechanism of HTS inhibition was explored in the TNFα and IL-1ß stimulation of pulmonary epithelial cells. Hyperosmolar (HOsm) media (400 mOsm) inhibited the production of select cytokines stimulated by TNFα and IL-1ß at the level of mRNA translation, synthesis and release. In TNFα stimulated A549 cells, HOsm media inhibited I-κBα phosphorylation, NF-κB translocation into the nucleus and NF-κB nuclear binding. In IL-1ß stimulated cells HOsm inhibited I-κBα phosphorylation without affecting NF-κB translocation or nuclear binding. Incubation in HOsm conditions inhibited both TNFα and IL-1ß stimulated nuclear localization of interferon response factor 1 (IRF-1). Additional transcription factors such as AP-1, Erk-1/2, JNK and STAT-1 were unaffected by HOsm. HTS and sorbitol supplemented media produced comparable outcomes in all experiments, indicating that the effects of HTS were mediated by osmolarity, not by sodium. While not affecting MAPK modules discernibly in A549 cells, both HOsm conditions inhibit IRF-1 against TNFα or IL-1ß, but inhibit p65 NF-kB translocation only against TNFα but not IL-1ß. Thus, anti-inflammatory mechanisms of HTS/HOsm appear to disrupt cytokine signals at distinct intracellular steps.