Blood Brain Barrier (BBB) breakdown is a secondary form of brain injury which has yet to be fully elucidated mechanistically. Existing research suggests that breakdown of tight junction proteins ...between endothelial cells is a primary driver of increased BBB permeability following injury, and intercellular signaling between primary cells of the neurovascular unit: endothelial cells, astrocytes, and pericytes; contribute to tight junction restoration. To expound upon this body of research, we analyzed the effects of severely injured patient plasma on each of the cell types in monoculture and together in a triculture model for the transcriptional and translational expression of the tight junction proteins Claudins 3 and 5, (CLDN3, CLDN5) and Zona Occludens 1 (ZO-1). Conditioned media transfer studies were performed to illuminate the cell type responsible for differential tight junction expression. Our data show that incubation with 5% human ex vivo severely injured patient plasma is sufficient to produce a differential response in endothelial cell tight junction mRNA and protein expression. Endothelial cells in monoculture produced a significant increase of CLDN3 and CLDN5 mRNA expression, (3.98 and 3.51 fold increase vs. control respectively, p<0.01) and CLDN5 protein expression, (2.58 fold change vs. control, p<0.01), whereas in triculture, this increase was attenuated. Our triculture model and conditioned media experiments suggest that conditioned media from astrocytes and pericytes and a triculture of astrocytes, pericytes and endothelial cells are sufficient in attenuating the transcriptional increases of tight junction proteins CLDN3 and CLDN5 observed in endothelial monocultures following incubation with severely injured trauma plasma. This data suggests that inhibitory molecular signals from astrocytes and pericytes contributes to prolonged BBB breakdown following injury via tight junction transcriptional and translational downregulation of CLDN5.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Atrial fibrillation (AF) is the most common arrhythmia treated in the world. While medical treatment with antiarrhythmic drugs remains the primary treatment modality, symptomatic refractory AF often ...requires treatment with a catheter or surgical ablation. One minimally invasive therapy is the Mini-Maze procedure, which utilizes epicardial radiofrequency ablation via a subxiphoid approach to rid the heart of arrhythmogenic atrial foci without a median sternotomy or cardiopulmonary bypass. The goal of this retrospective cohort study was to identify clinical factors associated with metabolic acidosis following the Mini-Maze procedure.
After Institutional Review Board approval, we studied patients undergoing the Mini-Maze procedure, off-pump coronary artery bypass grafting or patients conventional Cox-Maze on cardiopulmonary bypass. The first base deficit value obtained in the Intensive Care Unit was used as a measure of metabolic acidosis. Using logistic regression with Akaike information criteria, we analyzed preoperative, intraoperative, and postoperative data to determine the factors associated with changes in base deficit.
A multivariable model using stepwise selection demonstrated that diabetes mellitus and weight were associated with a decrease in the base deficit by 2.87 mEq/L (95% CI: -5.55--0.19) and 0.04 mEq/L (95%CI: -0.08, 0.004), respectively. Furthermore, creatinine was associated with a 1.57 mEq/L (95% CI: 0.14, 2.99) increase in the base deficit.
The Mini-Maze procedure was not associated with postoperative metabolic acidosis. Instead, nondiabetic patients and patients with higher creatinine were associated with greater base deficits after undergoing cardiac surgery.
OBJECTIVES:Identify the metabolites that are increased in the plasma of severely injured patients that developed ARDS versus severely injured patients that did not, and assay if these increased ...metabolites prime PMNs and induce pulmonary sequestration in an animal model of ARDS. We hypothesize that metabolic derangement due to advanced shock in critically injured patients leads to the pulmonary sequestration of neutrophils (PMNs), which serves as the first event in the acute respiratory distress syndrome (ARDS).
SUMMARY BACKGROUND DATA:Intracellular metabolites accumulate in the plasma of severely injured patients.
METHODS:Untargeted metabolomics profiling of 67 critically injured patients was completed to establish a metabolic signature associated with ARDS development. Metabolites that signficantly increased were assayed for PMN priming activity in vitro. The metabolites that primed PMNs were tested in a two-event animal model of ARDS to identify a molecular link between circulating metabolites and clinical risk for ARDS.
RESULTS:After controlling for confounders, four metabolites significantly increasedcreatine, dehydroascorbate, fumarate, and succinate in trauma patients who developed ARDS (p<0.05). Succinate alone primed the PMN oxidase in vitro at physiologically relevant levels. Intravenous (IV) succinate-induced PMN sequestration in the lung, a first event, and followed by IV lipopolysaccharide, a second event, resulted in ARDS in vivo requiring PMNs. Succinate receptor (SUCNR1) inhibition abrogated PMN priming, PMN sequestration, and ARDS.
CONCLUSION:Significant increases in plasma succinate post-injury may serve as the first event in ARDS. Targeted inhibition of the SUCNR1 may decrease ARDS development from other disease states to prevent ARDS globally.
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.
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.
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.
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.
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.
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.
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.
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.
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.
•Omic techniques accelerate understanding of the molecular pathways triggered by TBI.•TBI bile acid accumulation may contribute to coagulopathy.•Key markers blood brain barrier disruption are detected in early plasma samples in TBI patients.
Introduction: Severely injured patients develop a dysregulated inflammatory state characterized by vascular endothelial permeability, which contributes to multiple organ failure. To date, however, ...the mediators of and mechanisms for this permeability are not well established. Endothelial permeability in other inflammatory states such as sepsis is driven primarily by overactivation of the RhoA GTPase. We hypothesized that tissue injury and shock drive endothelial permeability after trauma by increased RhoA activation leading to break down of endothelial tight and adherens junctions. Methods: Human umbilical vein endothelial cells (HUVECs) were grown to confluence, whereas continuous resistance was measured using electrical cell-substrate impedance sensing (ECIS) Z-Theta technology, 10% ex vivo plasma from severely injured trauma patients was added, and resistance measurements continued for 2 hours. Areas under the curve (AUCs) were calculated from resistance curves. For GTPase activity analysis, HUVECs were grown to confluence and incubated with 10% trauma plasma for 5 minutes before harvesting of cell lysates. Rho and Rac activity were determined using a G-LISA assay. Significance was determined using Mann-Whitney tests or Kruskal-Wallis test, and Spearman ρ was calculated for correlations. Results: Plasma from severely injured patients induces endothelial permeability with plasma from patients with both severe injury and shock contributing most to this increased permeability. Surprisingly, Injury Severity Score (ISS) does not correlate with in vitro trauma-induced permeability (-0.05, P > 0.05), whereas base excess (BE) does correlate with permeability (-0.47, P = 0.0001). The combined impact of shock and injury resulted in a significantly smaller AUC in the injury + shock group (ISS > 15, BE < -9) compared with the injury only (ISS > 15, BE > -9; P = 0.04) or minimally injured (ISS < 15, BE > -9; P = 0.005) groups. In addition, incubation with injury + shock plasma resulted in higher RhoA activation ( P = 0.002) and a trend toward decreased Rac1 activation ( P = 0.07) compared with minimally injured control. Conclusions: Over the past decade, improved early survival in patients with severe trauma and hemorrhagic shock has led to a renewed focus on the endotheliopathy of trauma. This study presents the largest study to date measuring endothelial permeability in vitro using plasma collected from patients after traumatic injury. Here, we demonstrate that plasma from patients who develop shock after severe traumatic injury induces endothelial permeability and increased RhoA activation in vitro . Our ECIS model of trauma-induced permeability using ex vivo plasma has potential as a high throughput screening tool to phenotype endothelial dysfunction, study mediators of trauma-induced permeability, and screen potential interventions.
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.
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.
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.
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.
Objective: We sought to identify potential drivers behind resuscitative endovascular balloon occlusion of the aorta (REBOA) induced reperfusion coagulopathy using novel proteomic methods. Background: ...Coagulopathy associated with REBOA is poorly defined. The REBOA Zone 1 provokes hepatic and intestinal ischemia that may alter coagulation factor production and lead to molecular pathway alterations that compromises hemostasis. We hypothesized that REBOA Zone 1 would lead to reperfusion coagulopathy driven by mediators of fibrinolysis, loss of coagulation factors, and potential endothelial dysfunction. Methods: Yorkshire swine were subjected to a polytrauma injury (blast traumatic brain injury, tissue injury, and hemorrhagic shock). Pigs were randomized to observation only (controls, n = 6) or to 30 min of REBOA Zone 1 (n = 6) or REBOA Zone 3 (n = 4) as part of their resuscitation. Thromboelastography was used to detect coagulopathy. ELISA assays and mass spectrometry proteomics were used to measure plasma protein levels related to coagulation and systemic inflammation. Results: After the polytrauma phase, balloon deflation of REBOA Zone 1 was associated with significant hyperfibrinolysis (TEG results: REBOA Zone 1 35.50% versus control 9.5% vs. Zone 3 2.4%, P < 0.05). In the proteomics and ELISA results, REBOA Zone 1 was associated with significant decreases in coagulation factor XI and coagulation factor II, and significant elevations of active tissue plasminogen activator, plasmin-antiplasmin complex complexes, and syndecan-1 (P < 0.05). Conclusion: REBOA Zone 1 alters circulating mediators of clot formation, clot lysis, and increases plasma levels of known markers of endotheliopathy, leading to a reperfusion-induced coagulopathy compared with REBOA Zone 3 and no REBOA.
OBJECTIVE—The purpose of these studies is to investigate the mechanism by which transforming growth factor (TGF)β1 regulates the synthesis of the extracellular matrix protein fibronectin (FN).
...METHODS AND RESULTS—TGFβ1 elicited a time-dependent induction of FN protein and mRNA in A10 rat aortic smooth muscle cells (SMCs). Ectopic expression of Smad3 in A10 cells stimulated both basal and TGFβ1-induced FN expression, whereas expression of Smad7 eliminated the TGFβ response. Because TGFβ activated PKCδ in SMCs, we tested the role of PKCδ in regulation of FN expression. Inhibition of PKCδ activity by rottlerin or dominant-negative adenovirus (AdPKCδ DN) blocked TGFβ1’s induction of FN, whereas overexpression of PKCδ enhanced TGFβ’s effect. Moreover, aortic SMCs isolated from PKCδ mice exhibited diminished FN induction in response to TGFβ. Furthermore, we found that Smad3 protein and mRNA were markedly reduced in AdPKCδ DN-treated A10 cells and in PKCδ null cells. Finally, restoring Smad3 in rottlerin-treated A10 and PKCδ null cells rescues the ability of TGFβ to upregulate FN protein and mRNA expression.
CONCLUSION—Our data suggest that TGFβ-activated PKCδ is critical to maintain normal expression of Smad3, which in turn is required for the induction of fibronectin. PKCδ represents a promising target for treating the fibroproliferative response after arterial injury.
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