Fibrinogen Fragment X Mediates Endothelial Barrier Disruption via Suppression of VE-Cadherin.

INTRODUCTION: Major traumatic injury is associated with early hemorrhage-related and late-stage deaths due to multiple organ failure (MOF). While improvements to hemostatic resuscitation have significantly reduced hemorrhage-related deaths, the incidence of MOF among trauma patients remains high. Dysregulation of vascular endothelial cell (EC) barrier function is a central mechanism in the development of MOF; however, the mechanistic triggers remain unknown. Accelerated fibrinolysis occurs in a majority of trauma patients, resulting in high circulating levels of fibrin(ogen) degradation products, such as fragment X. To date, the relationship between fragment X and EC dysregulation and barrier disruption is unknown. The goal of this study was to determine the effects of fragment X on EC barrier integrity and expression of paracellular junctional proteins that regulate barrier function. METHODS: Human lung microvascular endothelial cells (HLMVECs) were treated with increasing concentrations of fragment X (1, 10, and 100 µg/mL), and barrier function was monitored using the xCELLigence live-cell monitoring system. Quantitative PCR (qPCR) was performed to measure changes in EC expression of 84 genes. Immunofluorescent (IF) cytostaining was performed to validate qPCR findings. RESULTS: Fragment X treatment significantly increased endothelial permeability over time (P < 0.05). There was also a significant reduction in VE-cadherin mRNA expression in fragment X-treated HLMVECs compared to control (P = 0.01), which was confirmed by IF staining. CONCLUSIONS: Fragment X may induce EC hyperpermeability by reducing VE-cadherin expression. This suggests that a targeted approach to disrupting EC-fragment X interactions could mitigate EC barrier disruption, organ edema, and MOF associated with major trauma.

A 3-O-sulfated heparan sulfate dodecasaccharide (12-mer) suppresses thromboinflammation and attenuates early organ injury following trauma and hemorrhagic shock.

Introduction: Dysregulated inflammation and coagulation are underlying mechanisms driving organ injury after trauma and hemorrhagic shock. Heparan sulfates, cell surface glycosaminoglycans abundantly expressed on the endothelial surface, regulate a variety of cellular processes. Endothelial heparan sulfate containing a rare 3-O-sulfate modification on a glucosamine residue is anticoagulant and anti-inflammatory through high-affinity antithrombin binding and sequestering of circulating damage-associated molecular pattern molecules. Our goal was to evaluate therapeutic potential of a synthetic 3-O-sulfated heparan sulfate dodecasaccharide (12-mer, or dekaparin) to attenuate thromboinflammation and prevent organ injury. Methods: Male Sprague-Dawley rats were pre-treated subcutaneously with vehicle (saline) or dekaparin (2 mg/kg) and subjected to a trauma/hemorrhagic shock model through laparotomy, gut distention, and fixed-pressure hemorrhage. Vehicle and dekaparin-treated rats were resuscitated with Lactated Ringer's solution (LR) and compared to vehicle-treated fresh-frozen-plasma-(FFP)-resuscitated rats. Serial blood samples were collected at baseline, after induction of shock, and 3 hours after fluid resuscitation to measure hemodynamic and metabolic shock indicators, inflammatory mediators, and thrombin-antithrombin complex formation. Lungs and kidneys were processed for organ injury scoring and immunohistochemical analysis to quantify presence of neutrophils. Results: Induction of trauma and hemorrhagic shock resulted in significant increases in thrombin-antithrombin complex, inflammatory markers, and lung and kidney injury scores. Compared to vehicle, dekaparin treatment did not affect induction, severity, or recovery of shock as indicated by hemodynamics, metabolic indicators of shock (lactate and base excess), or metrics of bleeding, including overall blood loss, resuscitation volume, or hematocrit. While LR-vehicle-resuscitated rodents exhibited increased lung and kidney injury, administration of dekaparin significantly reduced organ injury scores and was similar to organ protection conferred by FFP resuscitation. This was associated with a significant reduction in neutrophil infiltration in lungs and kidneys and reduced lung fibrin deposition among dekaparin-treated rats compared to vehicle. No differences in organ injury, neutrophil infiltrates, or fibrin staining between dekaparin and FFP groups were observed. Finally, dekaparin treatment attenuated induction of thrombin-antithrombin complex and inflammatory mediators in plasma following trauma and hemorrhagic shock. Conclusion: Anti-thromboinflammatory properties of a synthetic 3-O-sulfated heparan sulfate 12-mer, dekaparin, could provide therapeutic benefit for mitigating organ injury following major trauma and hemorrhagic shock.

Influence of TRPM4 rs8104571 genotype on intracranial pressure and outcomes in African Americans with traumatic brain injury.

The TRPM4 gene codes for a membrane ion channel subunit related to inflammation in the central nervous system. Recent investigation has identified an association between TRPM4 single nucleotide polymorphisms (SNPs) rs8104571 and rs150391806 and increased intracranial (ICP) pressure following traumatic brain injury (TBI). We assessed the influence of these genotypes on clinical outcomes and ICP in TBI patients. We included 292 trauma patients with TBI. DNA extraction and real-time PCR were used for TRPM4 rs8104571 and rs150391806 allele discrimination. Five participants were determined to have the rs8104571 homozygous variant genotype, and 20 participants were identified as heterozygotes; 24 of these 25 participants were African American. No participants had rs150391806 variant alleles, preventing further analysis of this SNP. Genotypes containing the rs8104571 variant allele were associated with decreased Glasgow outcome scale-extended (GOSE) score (P = 0.0231), which was also consistent within our African-American subpopulation (P = 0.0324). Regression analysis identified an association between rs8104571 variant homozygotes and mortality within our overall population (P = 0.0230) and among African Americans (P = 0.0244). Participants with rs8104571 variant genotypes exhibited an overall increase in ICP (P = 0.0077), although a greater frequency of ICP measurements > 25 mmHg was observed in wild-type participants (P = < 0.0001). We report an association between the TRPM4 rs8104571 variant allele and poor outcomes following TBI. These findings can potentially be translated into a precision medicine approach for African Americans following TBI utilizing TRPM4-specific pharmaceutical interventions. Validation through larger cohorts is warranted.

Radiofrequency Ablation Remodels the Tumor Microenvironment and Promotes Neutrophil-Mediated Abscopal Immunomodulation in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) presents a 5-year overall survival rate of 11%, despite efforts to improve clinical outcomes in the past two decades. Therapeutic resistance is a hallmark of this disease, due to its dense and suppressive tumor microenvironment (TME). Endoscopic ultrasound-guided radiofrequency ablation (EUS-RFA) is a promising local ablative and potential immunomodulatory therapy for PDAC. In this study, we performed RFA in a preclinical tumor-bearing KrasG12D; Trp53R172H/+; Pdx1:Cre (KPC) syngeneic model, analyzed local and abscopal affects after RFA and compared our findings with resected PDAC specimens. We found that RFA reduced PDAC tumor progression in vivo and promoted strong TME remodeling. In addition, we discovered tumor-infiltrating neutrophils determined abscopal effects. Using imaging mass cytometry, we showed that RFA elevated dendritic cell numbers in RFA-treated tumors and promoted a significant CD4+ and CD8+ T-cell abscopal response. In addition, RFA elevated levels of programmed death-ligand 1 (PD-L1) and checkpoint blockade inhibition targeting PD-L1 sustained tumor growth reduction in the context of RFA. This study indicates RFA treatment, which has been shown to increase tumor antigen shedding, promotes antitumor immunity. This is critical in PDAC where recent clinical immunotherapy trials have not resulted in substantial changes in overall survival.