Fibrinogen Inhibits Metalloproteinase-9 Activation and Syndecan-1 Cleavage to Protect Lung Function in ApoE Null Mice After Hemorrhagic Shock.

INTRODUCTION: Obesity is associated with higher mortality following trauma, although the pathogenesis is unclear. Both obesity and trauma are associated with syndecan-1 shedding and metalloproteinase-9 (MMP-9) activation, which can adversely affect endothelial cell function. We recently demonstrated that fibrinogen stabilizes endothelial cell surface syndecan-1 to reduce shedding and maintain endothelial barrier integrity. We thus hypothesized that MMP-9 activation and syndecan-1 shedding would be exacerbated by obesity after trauma but attenuated by fibrinogen-based resuscitation. MATERIALS AND METHODS: ApoE null (-/-) mice were fed a Western diet to induce obesity. Mice were subjected to hemorrhage shock and laparotomy then resuscitated with Lactated Ranger's (LR) or LR containing fibrinogen and compared to null and lean sham wild type mice. Mean arterial pressure (MAP) was monitored. Bronchial alveolar lavage protein as an indicator of permeability and lung histopathologic injury were assessed. Syndecan-1 protein and active MMP-9 protein were measured. RESULTS: MAP was similar between lean sham and ApoE-/- sham mice. However, following hemorrhage, ApoE-/- mice resuscitated with fibrinogen had significantly higher MAP than LR mice. Lung histopathologic injury and permeability were increased in LR compared to fibrinogen resuscitated animals. Compared with lean sham mice, both active MMP-9 and cleaved syndecan-1 level were significantly higher in ApoE-/- sham mice. Resuscitation with fibrinogen but not lactated Ringers largely reduced these changes. CONCLUSIONS: Fibrinogen as a resuscitative adjunct in ApoE-/- mice after hemorrhage shock augmented MAP and reduced histopathologic injury and lung permeability, suggesting fibrinogen protects the endothelium by inhibiting MMP-9-mediated syndecan-1 cleavage in obese mice.

c-Jun-mediated miR-19b expression induces endothelial barrier dysfunction in an in vitro model of hemorrhagic shock.

BACKGROUND: Our previous data demonstrated that miR-19b expression was increased in human lung microvascular endothelial cells in-vitro-, in-vivo and in patients with hemorrhagic shock, leading to a decrease in syndecan-1 mRNA and protein and resulting in loss of endothelial barrier function. However, the mechanism underlying increased miR-19b expression remains unclear. The objective of the current study was to determine if c-Jun mediates the early responsive microRNA, miR-19b, to cause endothelial barrier dysfunction. METHOD: Human lung microvascular endothelial cells (HLMEC) or HEK293T cells were transfected with c-Jun overexpressing vector, c-Jun siRNA, miR-19b promoter vector, miR-19b mutated promoter vector, miR-19b oligo inhibitor, then subjected to hypoxia/reoxygenation as in-vitro model of hemorrhagic shock. Levels of protein, miRNA, and luciferase activity were measured. Transwell permeability of endothelial monolayers were also determined. Plasma levels of c-Jun were measured in injured patients with hemorrhagic shock. RESULT: Hypoxia/reoxygenation induced primary (pri-)miR-19b, mature miR-19b, and c-Jun expression over time in a comparable timeframe. c-Jun silencing by transfection with its specific siRNA reduced both pri-miR-19b and mature miR-19b levels. Conversely, c-Jun overexpression enhanced H/R-induced pri-miR-19b. Studies using a luciferase reporter assay revealed that in cells transfected with vectors containing the wild-type miR-19b promoter and luciferase reporter, c-Jun overexpression or hypoxia/ reoxygenation significantly increased luciferase activity. c-Jun knockdown reduced the luciferase activity in these cells, suggesting that the miR-19b promoter is directly activated by c-Jun. Further, chromatin immunoprecipitation assay confirmed that c-Jun directly bound to the promoter DNA of miR-19b and hypoxia/reoxygenation significantly increased this interaction. Additionally, c-Jun silencing prevented cell surface syndecan-1 loss and endothelial barrier dysfunction in HLMECs after hypoxia/reoxygenation. Lastly, c-Jun was significantly elevated in patients with hemorrhagic shock compared to healthy controls. CONCLUSION: Transcription factor c-Jun is inducible by hypoxia/reoxygenation, binds to and activates the miR-19b promoter. Using an in-vitro model of hemorrhagic shock, our findings identified a novel cellular mechanism whereby hypoxia/ reoxygenation increases miR-19b transcription by inducing c-Jun and leads to syndecan-1 decrease and endothelial cell barrier dysfunction. This finding supports that miR-19b could be a potential therapeutic target for hemorrhage shock.

TRAUMA-DERIVED EXTRACELLULAR VESICLES ARE SUFFICIENT TO INDUCE ENDOTHELIAL DYSFUNCTION AND COAGULOPATHY.

ABSTRACTINTRODUCTION: Although a number of studies have demonstrated increased release of extracellular vesicles (EVs) and changes in their origin differentials after trauma, the biologic significance of EVs is not well understood. We hypothesized that EVs released after trauma/hemorrhagic shock (HS) contribute to endotheliopathy and coagulopathy. To test this hypothesis, adoptive transfer experiments were performed to determine whether EVs derived from severely injured patients in shock were sufficient to induce endothelial dysfunction and coagulopathy. Methods: Total EVs were enriched from plasma of severely injured trauma/HS patients or minimally injured patients by ultracentrifugation and characterized for size and numbers. Under isoflurane anesthesia, noninjured naive C57BL/6J mice were administered EVs at varying concentrations and compared with mice receiving equal volume vehicle (phosphate-buffered saline (PBS)) or to mice receiving EVs from minimally injured patients. Thirty minutes after injection, mice were sacrificed, and blood was collected for thrombin generation (thrombin-antithrombin, thrombin-antithrombin complex [TAT] assay) and syndecan-1 by enzyme-linked immunoabsorbent assay (ELISA). Lungs were harvested for examination of histopathologic injury and costained with von Willebrand factor and fibrin to identify intravascular coagulation. Bronchial alveolar lavage fluid was aspirated from lungs for protein measurement as an indicator of the endothelial permeability. Data are presented as mean ± SD, P < 0.05 was considered significant, and t test was used. Results: An initial proof-of-concept experiment was performed in naive mice receiving EVs purified from severely injured trauma/HS patients (Injury Severity Score [ISS], 34 ± 7) at different concentrations (5 × 106 to 3.1 × 109/100 µL/mouse) and compared with PBS (control) mice. Neither TAT nor syndecan-1 levels were significantly different between groups at 30 minutes after EV infusion. However, lung vascular permeability and histopathologic injury were significantly higher in the EV group, and lung tissues demonstrated intravascular fibrin deposition. Based on these data, EVs from severely injured trauma/HS patients (ISS, 32 ± 6) or EVs from minimally injured patients (ISS, 8 ± 3) were administered to naive mice at higher concentrations (1 × 109 to 1 × 1010 EV/100 µL/mouse). Compared with mice receiving EVs from minimally injured patients, plasma TAT and syndecan-1 levels were significantly higher in the trauma/HS EV group. Similarly, bronchial alveolar lavage protein and lung histopathologic injury were higher in the trauma/HS EV group, and lung tissues demonstrated enhanced intravascular fibrin deposition. Conclusion: These data demonstrate that trauma/HS results in the systemic release of EVs, which are capable of inducing endotheliopathy as demonstrated by elevated syndecan-1 and increased permeability and coagulopathy as demonstrated by increased TAT and intravascular fibrin deposition. Targeting trauma-induced EVs may represent a novel therapeutic strategy.