Injury-induced endotheliopathy: What you need to know.

ABSTRACT: The endotheliopathy of trauma involves a complex interplay between the glycocalyx, von Willebrand factor, and platelets that leads to abnormalities in coagulation, inflammation, and endothelial cell (EC) function. The current review presents a synopsis of EC function under homeostatic conditions, the structure and function of the endothelial glycocalyx; mechanisms of EC injury and activation after trauma; pathological consequences of the EoT at the cellular level; and clinical implications of the EoT. Recent evidence is presented that links the EoT to extracellular vesicles and hyperadhesive ultralarge von Willebrand factor multimers through their roles in coagulopathy. Lastly, potential therapeutics to mitigate the EoT are discussed. Most research to date has focused on blood products, primarily plasma, and its contribution to restoring postinjury EC dysfunction. Additional therapeutic adjuvants that target the glycocalyx, ultralarge von Willebrand factor, low ADAMTS-13, and pathologic extracellular vesicles are reviewed. Much of the pathobiology of EoT is known, but a better mechanistic understanding can help guide therapeutics to further repair the EoT and improve patient outcomes.

Immediate use cryoprecipitate products provide lasting organ protection in a rodent model of trauma/hemorrhagic shock and prolonged hypotensive resuscitation.

BACKGROUND: Cryoprecipitate (CP) can augment hemostasis after hemorrhagic shock (HS). Similar to fresh frozen plasma (FFP), CP may provide short-term endothelial protection. We tested a new 5-day postthaw CP (5-day pathogen-reduced cryoprecipitate [5PRC]) and lyophilized pathogen-reduced cryoprecipitate (LPRC) to overcome challenges of early administration and hypothesized that 5PRC and LPRC would provide lasting organ protection in a rodent model of HS. METHODS: Mice underwent trauma/HS (laparotomy then HS), mean arterial pressure (MAP) 35 × 90 minutes, and then 6 hours of hypotensive resuscitation (MAP, 55-60 mm Hg) with lactated Ringer's solution (LR), FFP, CP, 5PRC, or LPRC and compared with shams. Animals were followed for 72 hours. Organs and blood were collected. Data are presented as mean ± SD and analysis of variance with Bonferroni post hoc. RESULTS: Mean arterial pressure was comparable between experimental groups at baseline, preresuscitation, and 6 hours per protocol. However, volume needed to resuscitate to target MAP over 6 hours was less than half for CP, 5PRC, LPRC, and FFP compared with LR, suggesting that CP products can serve as effective resuscitative agents. Mean arterial pressure at 72 hours was also significantly higher in the CP, 5PRC, and FFP groups compared with LR. Resuscitation with CP, 5PRC, and LPRC provided lasting protection from gut injury and enhanced syndecan immunostaining comparable with FFP, while LR mice demonstrated persistent organ dysfunction. Sustained endothelial protection was demonstrated by lessened lung permeability, while cystatin C was an indicator of kidney function, and liver aspartate aminotransferase and alanine transaminase returned to sham levels in all groups. CONCLUSION: Cryoprecipitate products can provide lasting organ protection comparable with FFP in a sustained rodent model of trauma/HS and hypotensive resuscitation. The availability of 5PRC and LPRC will allow for investigation into the immediate use of cryoprecipitate for severely injured patients. As lyophilized products such as cryoprecipitate become available clinically, their use has important implications for prehospital, rural, and battlefield usage.

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.

Fibrinogen Activates PAK1/Cofilin Signaling Pathway to Protect Endothelial Barrier Integrity.

INTRODUCTION: We recently demonstrated that fibrinogen stabilizes syndecan-1 on the endothelial cell (EC) surface and contributes to EC barrier protection, though the intracellular signaling pathway remains unclear. P21 (Rac1) activated kinase 1 (PAK1) is a protein kinase involved in intracellular signaling leading to actin cytoskeleton rearrangement and plays an important role in maintaining endothelial barrier integrity. We therefore hypothesized that fibrinogen binding to syndecan-1 activated the PAK1 pathway. METHODS: Primary human lung microvascular endothelial cells were incubated in 10% lactated Ringers (LR) solution or 10% fibrinogen saline solution (5 mg/mL). Protein phosphorylation was determined by Western blot analysis and endothelial permeability measured by fluorescein isothiocyanate (FITC)-dextran. Cells were silenced by siRNA transfection. Protein concentration was measured in the lung lavages of mice. RESULTS: Fibrinogen treatment resulted in increased syndecan-1, PAK1 activation (phosphorylation), cofilin activation (dephosphorylation), as well as decreased stress fibers and permeability when compared with LR treatment. Cofilin is an actin-binding protein that depolymerizes F-actin to decrease stress fiber formation. Notably, fibrinogen did not influence myosin light chain activation (phosphorylation), a mediator of EC tension. Silencing of PAK1 prevented fibrinogen-induced dephosphorylation of cofilin and barrier integrity. Moreover, to confirm the in vitro findings, mice underwent hemorrhagic shock and were resuscitated with either LR or fibrinogen. Hemorrhage shock decreased lung p-PAK1 levels and caused significant lung vascular leakage. However, fibrinogen administration increased p-PAK1 expression to near sham levels and remarkably prevented the lung leakage. CONCLUSION: We have identified a novel pathway by which fibrinogen activates PAK1 signaling to stimulate/dephosphorylate cofilin, leading to disassembly of stress fibers and reduction of endothelial permeability.

miR-19b targets pulmonary endothelial syndecan-1 following hemorrhagic shock.

Hemorrhagic shock results in systemic injury to the endothelium contributing to post-shock morbidity and mortality. The mechanism involves syndecan-1, the backbone of the endothelial glycocalyx. We have shown in a rodent model that lung syndecan-1 mRNA is reduced following hemorrhage, whereas the molecular mechanism underlying the mRNA reduction is not clear. In this study, we present evidence that miR-19b targets syndecan-1 mRNA to downregulate its expression. Our results demonstrate that miR-19b was increased in hemorrhagic shock patients and in-vitro specifically bound to syndecan-1 mRNA and caused its degradation. Further, hypoxia/reoxygenation (H/R), our in vitro hemorrhage model, increased miR-19b expression in human lung microvascular endothelial cells, leading to a decrease in syndecan-1 mRNA and protein. H/R insult and miR-19b mimic overexpression comparably exaggerated permeability and enhanced endothelial barrier breakdown. The detrimental role of miR-19b in inducing endothelial dysfunction was confirmed in vivo. Lungs from mice undergoing hemorrhagic shock exhibited a significant increase in miR-19b and a concomitant decrease in syndecan-1 mRNA. Pretreatment with miR-19b oligo inhibitor significantly decreased lung injury, inflammation, and permeability and improved hemodynamics. These findings suggest that inhibition of miR-19b may be a putative therapeutic avenue for mitigating post shock pulmonary endothelial dysfunction in hemorrhage shock.

Delayed splenic hemorrhage: Myth or mystery? A Western Trauma Association multicenter study.

BACKGROUND: Multi-detector computed tomography imaging is now the reference standard for identifying solid organ injuries, with a high sensitivity and specificity. However, delayed splenic hemorrhage (DSH), defined as no identified injury to the spleen on the index scan but delayed bleeding from a splenic injury, has been reported. We hypothesized that the occurrence of DSH would be minimized by utilization of modern imaging techniques. METHODS: Data was retrospectively collected from 2006 to 2016 in 12 adult Level I and II trauma centers. All patients had an initial CT scan demonstrating no splenic injury but subsequently were diagnosed with splenic bleeding. Demographic, injury characteristics, imaging parameters and results, interventions and outcomes were collected. RESULTS: Of 6867 patients with splenic injuries, 32 cases (0.4%) of blunt splenic hemorrage were identified. Patients were primarily male, had blunt trauma, severely injured (ISS 32 (9-57) and with associated injuries. Injuries of all grades were identified up to 16 days following admission. Overall, half of patients required splenectomy. All index images were obtained using multi-detector CT (16-320 slice). Secondary review of imaging by two trauma radiologists judged 72% (n = 23) of scans as suboptimal. This was due to poor scan quality primary from artifact(23), single phase contrast imaging (16), and/or poor contrast bolus timing or volume (6). Notably, only 28% of scans in patients with DSH were performed with optimal scanning techniques. CONCLUSION: This is the largest reported series of DSH in the era of modern imaging. Although the incidence of DSH is low, it still occurs despite the use of multi-detector imaging and when present, is associated with a high rate of splenectomy. Most cases of DSH can be attributed to missed diagnosis from suboptimal index imaging and ultimately be avoided.