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. Author manuscript; available in PMC: 2023 Jul 19.
Published in final edited form as: Shock. 2022 Jul 19;58(1):38–44. doi: 10.1097/SHK.0000000000001950

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

Ahmad Zeineddin 1, Feng Wu 1, Jing-Fei Dong 2,3, Huang Huang 4, Lin Zou 4, Wei Chao 4, Brooke Dorman 1, Rosemary A Kozar 1
PMCID: PMC9750939  NIHMSID: NIHMS1854203  PMID: 35984759

Abstract

INTRODUCTION:

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 (trauma/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 or minimally-injured patients by ultracentrifugation and characterized for size and numbers. Under isoflurane anesthesia, non-injured naïve C57BL/6J mice were administered EVs at varying concentrations and compared to mice receiving equal volume vehicle (PBS) or to mice receiving EVs from minimally injured patients. Thirty minutes after injection, mice were sacrificed and blood collected for thrombin generation (thrombin-antithrombin, TAT assay) and syndecan-1 by ELISA. Lungs were harvested for examination of histopathologic injury and co-stained with von Willebrand Factor and fibrin to identify intravascular coagulation. Bronchoalveolar (BAL) fluid was aspirated from lungs for protein measurement as an indicator of the endothelial permeability. Data are presented as mean±SD, p<0.05 significant, t-test.

RESULTS:

An initial proof- of- concept experiment was performed in naïve mice receiving EVs purified from severely injured trauma/HS patients (injury severity score ISS 34±7) at different concentrations (5x106 to 3.1x109/100 μl/mouse) and compared to 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 intra-vascular fibrin deposition. Based on this data, EVs from severely injured trauma/HS patients (ISS 32±6) or EVs from minimally injured patients (ISS 8±3) were administered to naïve mice at higher concentrations (1x109 - 1x1010 EV/100 μl/mouse). Compared to mice receiving EVs from minimally injured patients, plasma TAT and syndecan-1 levels were significantly higher in the trauma/HS EV group. Similarly, BAL protein and lung histopathologic injury were higher in the trauma/HS EV group and lung tissues demonstrated enhanced intra-vascular 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 intra-vascular fibrin deposition. Targeting trauma-induced EVs may represent a novel therapeutic strategy.

Keywords: extracellular vesicles, endothelial cell dysfunction, trauma and hemorrhagic shock

INTRODUCTION:

Extracellular vesicles (EVs) are small vesicles that range in size from 10 nm - 1 μm which are released from all cell types during cell activation or injury or secreted in response to external stimuli. They include membrane fragments, intracellular organelles, shedding vesicles, and secreted exosomes and can be detected in the systemic circulation under both physiologic and pathologic conditions.1–6 EVs have been increasingly recognized as a new class of biological mediators or effectors that mediate intercellular communication and have a role in disease processes including sepsis7,8 and cancer.9,10

Following traumatic injury, multiple studies have demonstrated changes in EV levels and phenotypes with increase in procoagulant EVs and an increase in endothelial cell-derived EVs. These changes in EVs have been found to be associated with injury severity, and changes in their phenotypes associated with coagulopathy, transfusion requirements, and mortality in injured patients.11–16 However, these are associations only and do not show causation or the underlying pathways.

Traumatic injury remains the leading cause of death in patients 5-44 years of age17 and hemorrhagic shock remains the leading cause of early, potentially reversible death after trauma.18 Patients in hemorrhagic shock often present in a state of coagulopathy and endothelial activation and dysfunction, a phenomenon termed the Endotheliopathy of Trauma (EoT).19 This involves reduced clot formation, hyperfibrinolysis, and increased endothelial activation and permeability. The role EVs play in the propagation or amelioration of this phenomenon is not fully understood. We hypothesize that EVs released after trauma/hemorrhagic shock (HS) contribute to endotheliopathy and coagulopathy. To test this hypothesis, we developed an adoptive transfer model in mice to determine whether EVs derived from severely injured patients in shock were sufficient to induce endothelial dysfunction and coagulopathy as seen in patients with EoT.

METHODS

Patient Recruitment and blood collection

This study was part of an ongoing prospective observational study in severely injured patients in shock (HP00085892) approved by the Institutional Review Board of the University of Maryland School of Medicine. This study included severely injured trauma/HS patients, defined as patients presenting with hypotension (systolic blood pressure <90) who received 2 units of blood in the trauma bay, and minimally injured patients, defined as Injury Severity Score (ISS) ≤9. Blood was collected in the trauma bay in EDTA tubes and immediately processed. Plasma was separated and stored in −80°C until time of experiment.

Isolation of EVs

Plasma was thawed at 4 °C and EVs were isolated by differential ultracentrifugation as described previously20. In brief, 250 μl of patient plasma was combined and diluted with an equal volume of Ca2+/Mg2+–free Dulbecco’s PBS (DPBS) and then centrifuged at 12000xg at 4°C for 30 minutes to remove cell debris and protein aggregates. Supernatants were then transferred to polyallomer tubes and diluted with 3.5 ml of Ca2+/Mg2+–free DPBS and further centrifuged at 110,000xg (fixed-angle MLA-80 Rotor; Beckman Coulter) at 4°C for 60 minutes. EV pellets were resuspended in 100 μl of cold Ca2+/Mg2+–free DPBS, and EVs were quantified using a nano-tracking system (ViewSizer™3000, Horiba). All EVs used for cell and animal treatments were isolated under sterile conditions.

EV administration

Under isoflurane anesthesia, EVs were administered directly through the femoral vein of non-injured male naïve C57BL/6J mice (22-25 grams, approximately 10 weeks) at varying concentrations as a single bolus. Control mice were administered a similar volume of the vehicle PBS or EVs from minimally injured patients. Thirty minutes after injection, animals were euthanized and blood was collected via cardiac puncture. Bronchoalveolar fluid was aspirated from the lungs and the tissues were collected for histopathologic staining.

Sample Analysis

Total plasma was separated and analyzed for Thrombin-Antithrombin (TAT) complex levels (Novus Bio - NBP2-75962) as the surrogate marker of thrombin generation, and Syndecan-1 levels (Boster Bio - EK1554) as the marker of endothelial activation and glycocalyx shedding. Bronchial alveolar lavage (BAL) protein was assayed using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) as a marker of endothelial permeability. Lung tissues were stained with H&E staining for histopathologic scoring, and co-stained with a von Willebrand factor (VWF) polyclonal antibody (Thermo Fisher Scientific - PA5-16634) and an anti-fibrin mouse monoclonal antibody (MilliporeSigma - MABS2155) as a marker for intravascular coagulation.

Statistical Analysis

Statistical analysis was performed using STATA software (Stata, version. 13.0; StataCorp). The distributions of the continuous variables were expressed as mean±SD. The statistical significance of the difference between groups was measured by two-tailed unpaired Student t test, and a p-value < 0.05 was considered significant.

RESULTS:

Trauma/hemorrhagic shock EVs vs vehicle control in naïve mice demonstrate enhanced permeability and lung histopathologic injury.

We first performed a proof-of-concept and dose-titration experiment in naïve mice receiving EVs purified from severely injured trauma/HS patients (ISS 34±7) with different concentrations: 5x106 (n=3), 5x107 (n=3), 1.2x109 (n=1), 2.2x109 (n=1), and 3.1x109 (n=1) EV/100 μl/mouse (n=9 total) and compared to PBS mice (n=6). (Figure 1.) The EV concentrations for this pilot experiment were chosen based on previous work using brain-derived EVs.21

Figure 1. Dose response to EVs purified from patients with HS compared to control (PBS).

Figure 1.

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Naïve mice were injected intravenously with EVs purified from severely injured trauma/HS patients at a concentration of 5x106 (n=3), 5x107 (n=3), 1.2x109 (n=1), 2.2x109 (n=1), and 3.1x109 (n=1) EV/100 μl/mouse and compared to vehicle control (PBS) mice (n=6). Arrows indicate dose selected for further study. Assays were syndecan-1 (Sdc-1), Thrombin-antithrombin (TAT), bronchial alveolar lavage (BAL) protein and histopathologic injury as measured by hematoxylin and eosin (HE) staining.

When combined, we did not detect significant difference between the case and controls in plasma syndecan-1 (1.28±0.06 controls vs 1.29±0.24 EV; p=0.33) and TAT (0.65±0.28 control vs 0.81±0.33 EVs; p=0.23) (Figure 2A and B). However, the lung microvascular permeability was significantly increased in EV-infused mice compared to vehicle control mice, as measured by the amount of BAL protein (57±15 controls vs 103 ±34 EVs; p=0.016, Figure 2C). Additionally, the EV-infused mice presented with more severe histopathologic injury (1.2 ±0.4 controls vs 2.1 ±0.9 EVs; p=.03) was higher (Figure 2D and E) and more extensive fibrin deposition to the wall of pulmonary microvasculature (Figure 3) as compared to the control mice receiving an equal volume of PBS.

Figure 2. Lung permeability and histopathologic injury but not shed syndecan-1 nor thrombin-antithrombin were increased in mice receiving EVs from trauma/hemorrhagic shock patients.

Figure 2.

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Naïve mice were injected intravenously with EVs purified from severely injured trauma/HS patients as above (n=9) and compared to vehicle control (PBS, n=6). Blood was collected after 30 minutes for: A.) shed syndecan-1 (Sdc-1); B.) thrombin-antithrombin complexes (TAT); C.) protein in lung bronchial alveolar lavage (BAL) fluid as an indicator of permeability and D/E.) histopathologic injury. Results are displayed as mean+SD per group, difference assessed using two-tailed t-test.

Figure 3. Intravascular thrombosis in naïve mice receiving EVs from trauma/hemorrhagic shock EVs patients.

Figure 3.

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Naïve mice were injected intravenously with EVs purified from severely injured trauma/HS patients at varying concentrations as above (n=9) and compared to vehicle control (PBS) mice (n=6). Thirty minutes later, lungs were harvested, sectioned, then double fluorescent stained for von Willebrand Factor (green) and fibrin (red). Animals receiving EVs demonstrated intra-vascular thrombosis (white arrows, lower panel).

Higher concentrations of trauma/hemorrhagic shock EVs vs minimally injured EVs in naïve mice demonstrate both increased endothelial injury and coagulopathy.

Based on this pilot data, we hypothesized that higher concentrations of EVs, closer to those we found in our trauma patients, would induce more severe changes in the outcome measures. To test the hypothesis that trauma/HS-induced changes in EVs were responsible for the observed changes, we compared EVs isolated from trauma/HS patients to EVs isolated from minimally injured patients. We found that the mean numbers of EVs found in plasma from severely injured trauma/HS patients were similar to those from minimally injured patients. (Average number: 6.5x1010 vs. 5.5x1010 EVs/ml, and size: 152 ±51 vs 154 ±58 nM for trauma/HS and minimally injured patients, respectively). We therefore infused EVs to naïve mice at a higher concentration to approximate that found in patient: 1x109 (n=6), and 1x1010 (n=3) 100 μl/mouse; (n=9 total) and included minimally injured EVs as a control with similar concentrations (1x109 (n=6), and 1x1010 (n=3) 100 μl/mouse; n=9 total). (Figure 4).

Figure 4. Effects of different doses of EVs purified from patients with hemorrhagic shock compared to EVs purified from minimally injured patients.

Figure 4.

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Naïve mice were injected intravenously with EVs purified from severely injured trauma/HS and minimally injured patients at either a concentrations 1x109 (n=6) or 1x1010 (n=3) EV/100 μl/mouse. Assays were syndecan-1 (Sdc-1), Thrombin-antithrombin (TAT), bronchial alveolar lavage (BAL) protein and histopathologic injury as measured by hematoxylin and eosin (HE) staining.

EVs from trauma/HS patients now induced greater increase than those from minimally injured patients in plasma syndecan-1 (5.43±2.5 ng/ml vs. 2.24±0.8 ng/ml; p=0.002) and TAT (1.21±0.8 ng/ml vs. 0.61±0.2 ng/ml; p=0.03) (Figure 5 A and B). BAL was higher in the lungs from mice infused with EVs from trauma/HS patients than those infused with EVs from minimally injured patients (77±26 μg/ml vs. 39±18 μg/ml; p=0.005) (Figure 5C). The mice infused with EVs from trauma/HS patients also developed more severe lung histopathologic injury than those with EVs from minimally injured patients (1.8 ±0.2 vs. 1.2 ±0.3; p<0.01) (Figure 5 D and E). A similar difference was also detected in the fibrin deposition to the pulmonary microvasculature (Figure 6).

Figure 5. Plasma syndecan-1, Thrombin-antithrombin, Lung permeability, and histopathologic injury, were all increased in mice receiving EVs from trauma/HS patients compared to mice receiving EVs from minimally injured patients.

Figure 5.

Open in a new tab

Naïve mice were injected intravenously with EVs purified from severely injured trauma/HS patients as above (n=9) and compared to EVs from minimally injured patients (n=9). Blood was collected after 30 minutes for: A.) shed syndecan-1 (Sdc-1), B.) thrombin-antithrombin complexes (TAT); C.) protein was measured in lung bronchial alveolar lavage (BAL) fluid as an indicator of permeability and D/E.) histopathologic injury. Results are displayed as mean+SD per group, difference assessed using two-tailed t-test.

Figure 6. Intravascular thrombosis was increased in naïve mice receiving EVs from trauma/HS patients compared to minimally injured patients.

Figure 6.

Open in a new tab

Naïve mice were injected intravenously with EVs at a higher concentrations of either 1x109 (n=6) or 1x1010 (n=3) EV/100 μl/mouse purified from either severely injured trauma/HS patients (n=9) or minimally injured patients (n=9). Thirty minutes later, lungs were harvested, sectioned, then double fluorescent stained for von Willebrand Factor (green) and fibrin (red). Animals receiving EVs demonstrated intra-vascular thrombosis (white arrows, lower panel).

DISCUSSION

This study demonstrated that, while at comparable numbers, EVs isolated from severely injured hemorrhagic shock patients induced significantly more severe injury to endothelial cells, defined by the syndecan-1 shedding, which results in glycocalyx depletion, as compared to EVs from minimally injured patients. EVs from trauma/HS patients were also more active in causing intravascular coagulation, defined by fibrin deposition to the lungs. This finding combined with elevated levels of plasma TAT strongly demonstrate that EVs from trauma/HS patients caused a systemic hypercoagulable state that could potentially cause consumptive coagulopathy. Together, these results suggest that EVs from trauma/HS patients are functionally distinct from those found in minimally injured patients and healthy subjects.

While not specifically studied, there could be two likely explanations for the different activities of EVs from the two types of patients. First, there may be a dose effect as trauma/HS patients have a greater number of circulating EVs. This notion is supported by the finding that lower doses of EVs increased injury defined by endothelial permeability and fibrin deposition without datable difference in plasma levels of TAT and syndecan-1 (Figure 1). In contrast, EVs at higher doses, induced not only pulmonary pathologies observed in mice infused with lower dose of EV, but also increased levels of TAT complex and syndecan-1. These differential presentations suggest that injury induced by the low dose of EVs is not systemic, but rather may target vulnerable organs such as the lungs.22 However, this explanation is not supported by our findings because the total numbers and size of EVs are similar in plasma samples collected from patients with severe trauma/HS and those with minimal injuries.

Second, EVs from severe trauma/HS patients are structurally and/or functionally different from those from minimally injured patients. While EVs from trauma patients are known to originate from multiple cell types,11–13,15,23 EVs from the two groups of patients may have different compositions of EVs from parental cells. For examples, EVs from endothelial cells, smooth muscle cells, and monocytes contain tissue factors critical for injury-induced coagulation cascade, whereas those from platelets contain low or no tissue factor.24 Furthermore, levels of the membrane anionic phospholipids such as phosphatidylserine (PS) can also differ on EVs from different parental cells.25 Distinguishing cell type specific EVs is important because severe trauma and HS could cause larger and deeper tissue injuries, releasing more tissue factor-bearing EVs. HS causes secondary ischemia, leading to greater endothelial injury. Similarly, the amounts and types of EV-carrying cargos vary significantly. For example, we have previously identified that microRNA-19b is increased after trauma/HS and contributes to endothelial cell dysfunction.26 This and other microRNAs are likely to be EV-bound to reduce the rate of cleavage and clearance by the intrinsic RNases. Studying the structural and biochemical differences among EVs from different patients is ongoing and could provide new insight into the differential activities of EVs that we have observed. Results from this ongoing study could potentially allow us to develop an EV composite score that can be used for risk assessment of trauma patients for timing and severity trauma-induced endotheliopathy and coagulopathy.

The EV composite score could address issues related to how to use EVs for clinical risk assessment. For example, previous studies have shown varying results when examining the total levels of EVs following severe injury. Kuravi et al23 showed an increase in overall level of plasma EVs after trauma while Matijevic et al13 and Frohlich et al12 did not. Our study also did not demonstrate a difference in the levels of EVs between severely and minimally injured patients. Other investigators have shown that lower levels of PS-expressing EVs on admission were associated with higher transfusion rates and mortality.13–16,27 This may be due to lower levels of procoagulant EVs and especially of platelet-derived EVs (PEVs), being involved in a consumptive process such that PEVs cannot be accurately measured systemically. Similarly, previous adoptive transfer models of PEVs from healthy donors showed that EVs enhance hemostasis and decrease vascular permeability in murine models.28,29 PEVs are typically derived from normal platelets isolated from healthy donors.28 However, Dyer et al also demonstrated that PEVs released after both human and murine trauma were procoagulant and in fact contributed to deep venous thrombosis.30 On the other hand, Tian et al have shown in a rodent model of traumatic brain injury that brain-derived EVs induced a consumptive coagulopathy and EV-induced endothelial permeability is enhanced in the presence of life platelets in mouse models of traumatic brain injury.21 Whether such an EV-induced coagulopathy can occurs after trauma/HS has yet to be determined.

Previous studies have also shown an increase in endothelial cell derived-EVs (ECEV) in severe trauma compared to healthy controls, and that this increase correlates closely with injury severity and mortality.12 ECEVs have been shown to carry ultra-large von Willebrand factor31 which are significantly more adhesive compared to cleaved von Willebrand factor molecules and can mediate EV-EC interaction and activate platelets to propagate injury-induced coagulation, and endothelial activation.32 This increase in ECEVs relative to PEVs poses an important question regarding whether change in the EV phenotypes, such as an increase in ECEVs, is a driver/propagator of endothelial dysfunction, or whether it is a biomarker resulting from the depletion of PEVs after severe hemorrhagic shock. Wade et al., however, demonstrated in a small prospective observational study that the EOT occurred in the absence of changes in ECEVs33. Further studies are needed to clarify this discrepancy.

This study has several limitations. First, we utilized different concentrations of EVs in both sets of experiments rather than testing a single concentration and therefore cannot comment on a threshold above which an EV concentration may be deleterious. Second, our initial model utilized concentrations that were lower than those found in trauma patients and potentially caused the negative findings in the first cohort. These concentrations were based on previous work of concentrations of brain-derived EVs which may have higher procoagulant potential and were organ-specific.21 We also only examined a single timepoint following EV administration into the animal. Changes in coagulopathy seen in hemorrhagic shock are dynamic and range from hypo- to hypercoagulability. Our selected timepoint may have missed a consumptive hypocoagulable state had another timepoint been examined. We did not analyze hyperfibrinolysis, a key maker of the hypocoagulable state following trauma. We did not profile the EV contents and exam how trauma might have impacted on the EV cargo. Identifying the EV cargos that are responsible for the EV-induced endotheliopathy observed in the current study would be a natural extension for our future investigations. We also used EVs from all cell origins and did not isolate the effects of EVs from any specific cell type. Our aim, though, was to examine biologic changes induced by EVs approximating those in our trauma patients which include all phenotypes of EVs. Lastly, we did not compare the effects of EVs versus whole plasma so cannot comment on the specific contribution of EVs.

CONCLUSION:

These data demonstrate that severe injury/hemorrhagic shock 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 fibrin deposition. Targeting trauma induced EVs may represent a novel therapeutic strategy.

Acknowledgments

This work was supported by National Institute of Health R01GM140983 (R.A.K, J.F.D) and R35GM140822 (W.C).

Footnotes

This work was presented in part as a poster at the 44th annual (virtual) meeting of the Shock Society October 2021.

Disclosures: None of the authors have any conflicts of interest, financial or otherwise, to disclose.

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