Regulation of vascular endothelial integrity by mesenchymal stem cell extracellular vesicles after hemorrhagic shock and trauma.

BACKGROUND: Patients with hemorrhagic shock and trauma (HS/T) are vulnerable to the endotheliopathy of trauma (EOT), characterized by vascular barrier dysfunction, inflammation, and coagulopathy. Cellular therapies such as mesenchymal stem cells (MSCs) and MSC extracellular vesicles (EVs) have been proposed as potential therapies targeting the EOT. In this study we investigated the effects of MSCs and MSC EVs on endothelial and epithelial barrier integrity in vitro and in vivo in a mouse model of HS/T. This study addresses the systemic effects of HS/T on multiorgan EOT. METHODS: In vitro, pulmonary endothelial cell (PEC) and Caco-2 intestinal epithelial cell monolayers were treated with control media, MSC conditioned media (CM), or MSC EVs in varying doses and subjected to a thrombin or hydrogen peroxide (H2O2) challenge, respectively. Monolayer permeability was evaluated with a cell impedance assay, and intercellular junction integrity was evaluated with immunofluorescent staining. In vivo, a mouse model of HS/T was used to evaluate the effects of lactated Ringer's (LR), MSCs, and MSC EVs on endothelial and epithelial intercellular junctions in the lung and small intestine as well as on plasma inflammatory biomarkers. RESULTS: MSC EVs and MSC CM attenuated permeability and preserved intercellular junctions of the PEC monolayer in vitro, whereas only MSC CM was protective of the Caco-2 epithelial monolayer. In vivo, both MSC EVs and MSCs mitigated the loss of endothelial adherens junctions in the lung and small intestine, though only MSCs had a protective effect on epithelial tight junctions in the lung. Several plasma biomarkers including MMP8 and VEGF were elevated in LR- and EV-treated but not MSC-treated mice. CONCLUSIONS: In conclusion, MSC EVs could be a potential cell-free therapy targeting endotheliopathy after HS/T via preservation of the vascular endothelial barrier in multiple organs early after injury. Further research is needed to better understand the immunomodulatory effects of these products following HS/T and to move toward translating these therapies into clinical studies.

Inter- and Intra-donor variability in bone marrow-derived mesenchymal stromal cells: implications for clinical applications.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are attractive as a therapeutic modality in multiple disease conditions characterized by inflammation and vascular compromise. Logistically they are advantageous because they can be isolated from adult tissue sources, such as bone marrow (BM). The phase 2a START clinical trial determined BM-MSCs to be safe in patients with moderate-to-severe acute respiratory distress syndrome (ARDS). Herein, we examine a subset of the clinical doses of MSCs generated for the phase 2a START trial from three unique donors (1-3), where one of the donors' donated BM on two separate occasions (donor 3 and 3W). METHODS: The main objective of this study was to correlate properties of the cells from the four lots with plasma biomarkers from treated patients and relevant to ARDS outcomes. To do this we evaluated MSC donor lots for (i) post-thaw viability, (ii) growth kinetics, (iii) metabolism, (iv) surface marker expression, (v) protein expression, (vi) immunomodulatory ability and (vii) their functional effects on regulating endothelial cell permeability. RESULTS: MSC-specific marker expression and protection of thrombin-challenged endothelial barrier permeability was similar among all four donor lots. Inter and intra-donor variability was observed in all the other in vitro assays. Furthermore, patient plasma ANG-2 and protein C levels at 6 hours post-transfusion were correlated to cell viability in an inter- and intra-donor dependent manner. CONCLUSIONS: These findings highlight the potential of donor dependent (inter-) and collection dependent (intra-) effects in patient biomarker expression.

Bone marrow donor selection and characterization of MSCs is critical for pre-clinical and clinical cell dose production.

BACKGROUND: Cell based therapies, such as bone marrow derived mesenchymal stem cells (BM-MSCs; also known as mesenchymal stromal cells), are currently under investigation for a number of disease applications. The current challenge facing the field is maintaining the consistency and quality of cells especially for cell dose production for pre-clinical testing and clinical trials. Here we determine how BM-donor variability and thus the derived MSCs factor into selection of the optimal primary cell lineage for cell production and testing in a pre-clinical swine model of trauma induced acute respiratory distress syndrome. METHODS: We harvested bone marrow and generated three different primary BM-MSCs from Yorkshire swine. Cells from these three donors were characterized based on (a) phenotype (morphology, differentiation capacity and flow cytometry), (b) in vitro growth kinetics and metabolic activity, and (c) functional analysis based on inhibition of lung endothelial cell permeability. RESULTS: Cells from each swine donor exhibited varied morphology, growth rate, and doubling times. All expressed the same magnitude of standard MSC cell surface markers by flow cytometry and had similar differentiation potential. Metabolic activity and growth potential at each of the passages varied between the three primary cell cultures. More importantly, the functional potency of the MSCs on inhibition of endothelial permeability was also cell donor dependent. CONCLUSION: This study suggests that for production of MSCs for cell-based therapy, it is imperative to examine donor variability and characterize derived MSCs for marker expression, growth and differentiation characteristics and testing potency in application dependent assays prior to selection of the optimal cell lineage for large scale expansion and dose production.

Platelets regulate vascular endothelial stability: assessing the storage lesion and donor variability of apheresis platelets.

BACKGROUND: In current blood banking practices, platelets (PLTs) are stored in plasma at 22°C, with gentle agitation for up to 5 days. To date, the effects of storage and donor variability on PLT regulation of vascular integrity are not known. STUDY DESIGN AND METHODS: In this study, we examined the donor variability of leukoreduced fresh (Day 1) or stored (Day 5) PLTs on vascular endothelial barrier function in vitro and in vivo. In vitro, PLT effects on endothelial cell (EC) monolayer permeability were assessed by analyzing transendothelial electrical resistances (TEER). PLT aggregation, a measure of hemostatic potential, was analyzed by impedance aggregometry. In vivo, PLTs were investigated in a vascular endothelial growth factor A (VEGF-A)-induced vascular permeability model in NSG mice, and PLT circulation was measured by flow cytometry. RESULTS: Treatment of endothelial monolayers with fresh Day 1 PLTs resulted in an increase in EC barrier resistance and decreased permeability in a dose-dependent manner. Subsequent treatment of EC monolayers with Day 5 PLTs demonstrated diminished vasculoprotective effects. Donor variability was noted in all measures of PLT function. Day 1 PLT donors were more variable in their effects on TEER than Day 5 PLTs. In mice, while all PLTs regardless of storage time demonstrated significant protection against VEGF-A-induced vascular leakage, Day 5 PLTs exhibited reduced protection when compared to Day 1 PLTs. Day 1 PLTs demonstrated significant donor variability against VEGF-A-challenged vascular leakage in vivo. Systemic circulating levels of Day 1 PLTs were higher than those of Day 5 PLTs CONCLUSIONS: In vitro and in vivo, Day 1 PLTs are protective in measures of vascular endothelial permeability. Donor variability is most prominent in Day 1 PLTs. A decrease in the protective effects is found with storage of the PLT units between Day 1 and Day 5 at 22°C, thereby suggesting that Day 5 PLTs are diminished in their ability to attenuate vascular endothelial permeability.

The effects of 22°C and 4°C storage of platelets on vascular endothelial integrity and function.

BACKGROUND: Although a majority of the studies conducted to date on platelet (PLT) storage have been focused on PLT hemostatic function, the effects of 4°C PLTs on regulation of endothelial barrier permeability are still not known. In this study, we compared the effects of room temperature (22°C) stored and (4°C) stored PLTs on the regulation of vascular endothelial cell (EC) permeability in vitro and in vivo. STUDY DESIGN AND METHODS: Day 1, Day 5, and Day 7 leukoreduced apheresis PLTs stored at 4 or 22°C were studied in vitro and in vivo. In vitro, PLT effects on EC permeability and barrier function, adhesion, and impedance aggregometry were investigated. In vivo, using a mouse model of vascular leak, attenuation of vascular leak and circulating PLT numbers were measured. RESULTS: Treatment of EC monolayers with Day 5 or Day 7 PLTs, stored at both 22°C and 4°C, resulted in similar decreases in EC permeability on average. However, analysis of individual samples revealed significant variation that was donor dependent. Additional in vitro measurements revealed a decrease in inflammatory mediators, nonspecific PLT-endothelial aggregation and attenuated loss of aggregation over time to TRAP, ASPI, ADP, and collagen with 4°C storage. In mice, while 22°C and 4°C PLTs both demonstrated significant protection against vascular endothelial growth factor A (VEGF-A)-induced vascular leak 22°C PLTs exhibited increased protection compared to 4°C PLTs. Systemic circulating levels of 4°C PLTs were decreased compared to 22°C PLTs. CONCLUSIONS: In vitro, 4°C-stored PLTs exhibit a greater capacity to inhibit EC permeability than 22°C-stored PLTs. In vivo, 22°C PLTs provide superior control of vascular leak induced by VEGF-A. This discrepancy may be due to increased clearance of 4°C PLTs from the systemic circulation.

Regulation of Vascular Endothelial Integrity by Mesenchymal Stem Cell Extracellular Vesicles after Hemorrhagic Shock and Trauma.

BACKGROUND: Patients with hemorrhagic shock and trauma (HS/T) are vulnerable to the endotheliopathy of trauma (EOT), characterized by vascular barrier dysfunction, inflammation, and coagulopathy. Cellular therapies such as mesenchymal stem cells (MSCs) and MSC extracellular vesicles (EVs) have been proposed as potential therapies targeting the EOT. In this study we investigated the effects of MSCs and MSC EVs on endothelial and epithelial barrier integrity in vitro and in vivo in a mouse model of HS/T. This study addresses systemic effects of HS/T on multiorgan EOT in HS/T model. METHODS: In vitro, pulmonary endothelial cell (PEC) and Caco-2 intestinal epithelial cell monolayers were treated with control media, MSC conditioned media (CM), or MSC EVs in varying doses and subjected to a thrombin or hydrogen peroxide (H2O2) challenge, respectively. Monolayer permeability was evaluated with a cell impedance assay, and intercellular junction integrity was evaluated with immunofluorescent staining. In vivo, a mouse model of HS/T was used to evaluate the effects of lactated Ringer's (LR), MSCs, and MSC EVs on endothelial and epithelial intercellular junctions in the lung and small intestine as well as on plasma inflammatory biomarkers. RESULTS: MSC EVs and MSC CM attenuated permeability and preserved intercellular junctions of the PEC monolayer in vitro, whereas only MSC CM was protective of the Caco-2 epithelial monolayer. In vivo, both MSC EVs and MSCs mitigated the loss of endothelial adherens junctions in the lung and small intestine, though only MSCs had a protective effect on epithelial tight junctions in the lung. Several plasma biomarkers including MMP8 and VEGF were elevated in LR- and EV-treated but not MSC-treated mice. CONCLUSIONS: In conclusion, MSC EVs could be a potential cell-free therapy targeting endotheliopathy after HS/T via preservation of the vascular endothelial barrier in multiple organs early after injury. Further research is needed to better understand the immunomodulatory effects of these products following HS/T and to move toward translating these therapies into clinical studies.

Pilot study of frozen platelet extracellular vesicles as a therapeutic agent in hemorrhagic shock in rats.

BACKGROUND: Hemorrhage accounts for the most preventable deaths after trauma. Resuscitation is guided by studies that demonstrate improved outcomes in patients receiving whole blood or balanced administration of blood products. Platelets present a logistical challenge due to short shelf life and need for refrigeration. Platelet-derived extracellular vesicles (PEVs) are a possible platelet alternative. Platelet-derived extracellular vesicles are secreted from platelets, have hemostatic effects and mitigate inflammation and vascular injury, similar to platelets. This pilot study aimed to elucidate the therapeutic effects of PEVs in a rat model of uncontrolled hemorrhage. METHODS: Male rats were anesthetized and femoral vessels cannulated. Vital signs (MAP, HR, and RR) were monitored. Electrolytes, lactate and ABG were obtained at baseline, 1-hour and 3-hours post injury. Laparotomy was performed, 50% of the middle hepatic lobe excised and the abdomen packed with gauze. Rats received 2 mL PEVs or lactated Ringers (LR) over 6 minutes immediately after injury. Peritoneal blood loss was quantified using preweighed gauze at 5 minutes, 15 minutes, 30 minutes, 45 minutes, and 60 minutes. Laparotomy was closed 1-hour postinjury. Animals were monitored for 3 hours postinjury then euthanized. Generalized Linear Mixed Effects models were performed to assess effects of treatment and time on lactate and MAP. RESULTS: Twenty-one rats were included (11 LR, 10 PEV). Overall blood loss was between 6 mL and 10 mL and not significantly different between groups. There was a 36% mortality rate in the LR group and 0% mortality in the PEV group ( p = 0.03). The LR group had significantly higher lactates at 1 hour ( p = 0.025). At 15 minutes, 45 minutes, 60 minutes, and 180 minutes, the MAP of the PEV group was significantly higher than the LR group. CONCLUSION: Early studies are encouraging regarding the potential use of PEVs in uncontrolled hemorrhagic shock based on improved survival and hemodynamics.

Histone deacetylase-6 modulates the effects of 4°C platelets on vascular endothelial permeability.

Platelets (PLTs) stored at 4°C exhibit equivalent or superior hemostatic function compared with 22°C PLTs, but have shorter circulation times and a decreased ability to modulate vascular permeability. These differences may be due to morphological changes and storage-induced activation. Using a proteomics-based approach, we found that 4°C-stored PLTs express decreased α-tubulin, a key PLT structural protein. PLT activation is characterized by α-tubulin deacetylation, which is regulated by histone deacetylase-6 (HDAC-6). We hypothesized that inhibition of HDAC-6 in stored PLTs will improve their ability to regulate vascular permeability through reduced activation and α-tubulin deacetylation. In an in vivo model of vascular permeability, treatment of 4°C PLTs with the HDAC-6 inhibitor tubacin enhanced the vasculoprotective properties of untreated 4°C PLTs. 4°C PLT circulation, however, was unchanged by tubacin treatment, suggesting that circulation time may not be a critical factor in determining the vasculoprotective effects of PLTs. Assessing the factor content of stored PLTs revealed that angiopoietin-1 (Ang-1) increased in 4°C PLTs over time, which was further enhanced by tubacin treatment. In addition, angiopoietin-2, an inducer of vascular leak and antagonist of Ang-1, inhibited PLT barrier protection, suggesting involvement of the Tie-2 pathway. This study demonstrates that HDAC-6 inhibition with tubacin attenuates the diminished vasculo-protective properties of 4°C PLTs, and these properties may be independent of PLT circulation time.

RECOVERY OF ENDOTHELIOPATHY AT 24 HOURS IN AN ESTABLISHED MOUSE MODEL OF HEMORRHAGIC SHOCK AND TRAUMA.

ABSTRACT: Introduction: The endotheliopathy of trauma develops early after injury and consists of increased vascular permeability, inflammation, and dysfunctional coagulation. Persistence of these abnormalities ultimately leads to multiorgan failure. We hypothesized that extending an established 3-hour acute mouse model of hemorrhagic shock and trauma (HS/T) to a 24-hour survival model would allow for evaluation of persistent endotheliopathy and organ injury after HS/T. Methods: Adult male C57BL/6J mice underwent laparotomy, femoral artery cannulation, and blood withdrawal to induce HS to a MAP of 35 mm Hg for 90 minutes. Mice were resuscitated with either lactated Ringer's (LR) or fresh frozen plasma (FFP). Vascular permeability in the lung and gut was assessed by measuring extravasation of a fluorescent dextran dye. Lungs were evaluated for histopathologic injury, and immunofluorescent staining was used to evaluate intercellular junction integrity. Pulmonary inflammatory gene expression was evaluated using NanoString (Seattle, WA). All endpoints were evaluated at both 3 and 24 hours after initiation of shock. Results: Lactated Ringer's- and FFP-treated mice had an equal mortality rate of 17% in the 24-hour model. Lactated Ringer's-treated mice demonstrated increased vascular permeability in the lung and gut at 3 hours compared with sham mice (lung, P < 0.01; gut, P < 0.001), which was mitigated by FFP treatment (lung, P < 0.05; gut, P < 0.001). Twenty-four hours after shock, however, there were no differences in vascular permeability between groups. Similarly, although at 3 hours, the lungs of LR-treated mice demonstrated significant histopathologic injury, loss of tight and adherens junctions, and a pro-inflammatory gene expression profile at 3 hours, these endpoints in LR mice were similar to sham mice by 24 hours. Conclusions: In an established mouse model of HS/T, endotheliopathy and lung injury are evident at 3 hours but recover by 24 hours. Polytrauma models or larger animal models allowing for more severe injury coupled with supportive care are likely necessary to evaluate endotheliopathy and organ injury outside of the acute period.

An engineered activated factor V for the prevention and treatment of acute traumatic coagulopathy and bleeding in mice.

Acute traumatic coagulopathy (ATC) occurs in approximately 30% of patients with trauma and is associated with increased mortality. Excessive generation of activated protein C (APC) and hyperfibrinolysis are believed to be driving forces for ATC. Two mouse models were used to investigate whether an engineered activated FV variant (superFVa) that is resistant to inactivation by APC and contains a stabilizing A2-A3 domain disulfide bond can reduce traumatic bleeding and normalize hemostasis parameters in ATC. First, ATC was induced by the combination of trauma and shock. ATC was characterized by activated partial thromboplastin time (APTT) prolongation and reductions of factor V (FV), factor VIII (FVIII), and fibrinogen but not factor II and factor X. Administration of superFVa normalized the APTT, returned FV and FVIII clotting activity levels to their normal range, and reduced APC and thrombin-antithrombin (TAT) levels, indicating improved hemostasis. Next, a liver laceration model was used where ATC develops as a consequence of severe bleeding. superFVa prophylaxis before liver laceration reduced bleeding and prevented APTT prolongation, depletion of FV and FVIII, and excessive generation of APC. Thus, prophylactic administration of superFVa prevented the development of ATC. superFVa intervention started after the development of ATC stabilized bleeding, reversed prolonged APTT, returned FV and FVIII levels to their normal range, and reduced TAT levels that were increased by ATC. In summary, superFVa prevented ATC and traumatic bleeding when administered prophylactically, and superFVa stabilized bleeding and reversed abnormal hemostasis parameters when administered while ATC was in progress. Thus, superFVa may be an attractive strategy to intercept ATC and mitigate traumatic bleeding.

Mesenchymal stem cell extracellular vesicles mitigate vascular permeability and injury in the small intestine and lung in a mouse model of hemorrhagic shock and trauma.

BACKGROUND: Hemorrhagic shock and trauma (HS/T)-induced gut injury may play a critical role in the development of multi-organ failure. Novel therapies that target gut injury and vascular permeability early after HS/T could have substantial impacts on trauma patients. In this study, we investigate the therapeutic potential of human mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles (MSC EVs) in vivo in HS/T in mice and in vitro in Caco-2 human intestinal epithelial cells. METHODS: In vivo, using a mouse model of HS/T, vascular permeability to a 10-kDa dextran dye and histopathologic injury in the small intestine and lungs were measured among mice. Groups were (1) sham, (2) HS/T + lactated Ringer's (LR), (3) HS/T + MSCs, and (4) HS/T + MSC EVs. In vitro, Caco-2 cell monolayer integrity was evaluated by an epithelial cell impedance assay. Caco-2 cells were pretreated with control media, MSC conditioned media (CM), or MSC EVs, then challenged with hydrogen peroxide (H2O2). RESULTS: In vivo, both MSCs and MSC EVs significantly reduced vascular permeability in the small intestine (fluorescence units: sham, 456 ± 88; LR, 1067 ± 295; MSC, 765 ± 258; MSC EV, 715 ± 200) and lung (sham, 297 ± 155; LR, 791 ± 331; MSC, 331 ± 172; MSC EV, 303 ± 88). Histopathologic injury in the small intestine and lung was also attenuated by MSCs and MSC EVs. In vitro, MSC CM but not MSC EVs attenuated the increased permeability among Caco-2 cell monolayers challenged with H2O2. CONCLUSION: Mesenchymal stem cell EVs recapitulate the effects of MSCs in reducing vascular permeability and injury in the small intestine and lungs in vivo, suggesting MSC EVs may be a potential cell-free therapy targeting multi-organ dysfunction in HS/T. This is the first study to demonstrate that MSC EVs improve both gut and lung injury in an animal model of HS/T.

PAI-1 is an essential component of the pulmonary host response during Pseudomonas aeruginosa pneumonia in mice.

RATIONALE: Elevated plasma and bronchoalveolar lavage fluid plasminogen activator inhibitor 1 (PAI-1) levels are associated with adverse clinical outcome in patients with pneumonia caused by Pseudomonas aeruginosa. However, whether PAI-1 plays a pathogenic role in the breakdown of the alveolar-capillary barrier caused by P aeruginosa is unknown. OBJECTIVES: The role of PAI-1 in pulmonary host defence and survival during P aeruginosa pneumonia in mice was tested. The in vitro mechanisms by which P aeruginosa causes PAI-1 gene and protein expression in lung endothelial and epithelial cells were also examined. METHODS AND RESULTS: PAI-1 null and wild-type mice that were pretreated with the PAI-1 inhibitor Tiplaxtinin had a significantly lower increase in lung vascular permeability than wild-type littermates after the airspace instillation of 1×10(7) colony-forming units (CFU) of P aeruginosa bacteria. Furthermore, P aeruginosa in vitro induced the expression of the PAI-1 gene and protein in a TLR4/p38/RhoA/NF-κB (Toll-like receptor 4/p38/RhoA/nuclear factor-κB) manner in lung endothelial and alveolar epithelial cells. However, in vivo disruption of PAI-1 signalling was associated with higher mortality at 24 h (p<0.03) and higher bacterial burden in the lungs secondary to decreased neutrophil migration into the distal airspace in response to P aeruginosa. CONCLUSIONS: The results indicate that PAI-1 is a critical mediator that controls the development of the early lung inflammation that is required for the activation of the later innate immune response necessary for the eradication of P aeruginosa from the distal airspaces of the lung.

Cryoprecipitate attenuates the endotheliopathy of trauma in mice subjected to hemorrhagic shock and trauma.

BACKGROUND: Plasma has been shown to mitigate the endotheliopathy of trauma. Protection of the endothelium may be due in part to fibrinogen and other plasma-derived proteins found in cryoprecipitate; however, the exact mechanisms remain unknown. Clinical trials are underway investigating early cryoprecipitate administration in trauma. In this study, we hypothesize that cryoprecipitate will inhibit endothelial cell (EC) permeability in vitro and will replicate the ability of plasma to attenuate pulmonary vascular permeability and inflammation induced by hemorrhagic shock and trauma (HS/T) in mice. METHODS: In vitro, barrier permeability of ECs subjected to thrombin challenge was measured by transendothelial electrical resistance. In vivo, using an established mouse model of HS/T, we compared pulmonary vascular permeability among mice resuscitated with (1) lactated Ringer's solution (LR), (2) fresh frozen plasma (FFP), or (3) cryoprecipitate. Lung tissue from the mice in all groups was analyzed for markers of vascular integrity, inflammation, and inflammatory gene expression via NanoString messenger RNA quantification. RESULTS: Cryoprecipitate attenuates EC permeability and EC junctional compromise induced by thrombin in vitro in a dose-dependent fashion. In vivo, resuscitation of HS/T mice with either FFP or cryoprecipitate attenuates pulmonary vascular permeability (sham, 297 ± 155; LR, 848 ± 331; FFP, 379 ± 275; cryoprecipitate, 405 ± 207; p < 0.01, sham vs. LR; p < 0.01, LR vs. FFP; and p < 0.05, LR vs. cryoprecipitate). Lungs from cryoprecipitate- and FFP-treated mice demonstrate decreased lung injury, decreased infiltration of neutrophils and activation of macrophages, and preserved pericyte-endothelial interaction compared with LR-treated mice. Gene analysis of lung tissue from cryoprecipitate- and FFP-treated mice demonstrates decreased inflammatory gene expression, in particular, IL-1ß and NLRP3, compared with LR-treated mice. CONCLUSION: Our data suggest that cryoprecipitate attenuates the endotheliopathy of trauma in HS/T similar to FFP. Further investigation is warranted on active components and their mechanisms of action.

Freeze-dried platelets promote clot formation, attenuate endothelial cell permeability, and decrease pulmonary vascular leak in a murine model of hemorrhagic shock.

BACKGROUND: Hemorrhagic shock (HS) and trauma induce endothelial barrier compromise, inflammation, and aberrant clotting. We have shown that fresh human platelets (Plts) and Plt extracellular vesicles mitigate vascular leak in murine models of injury. Here, we investigate the potential of freeze-dried platelets (FDPlts) to attenuate pulmonary vascular permeability, decrease inflammation, and promote clotting in a murine model of HS. METHODS: Human FDPlts were characterized using in vitro assays of Plt marker expression, aggregation, coagulation, and endothelial cell permeability. An intravital model of vascular injury in the mouse cremaster muscle was used to assess the ability of FDPlts to incorporate into clots. Mouse groups subjected to controlled hemorrhage for 90 minutes were (1) lactated Ringer solution (LR), (2) FDPlts, (3) fresh human Plts, (4) murine whole blood (WB), and (5) shams (only instrumented). Hemorrhagic shock mouse endpoints included coagulation, pulmonary vascular permeability, and lung injury. RESULTS: Freeze-dried Plts expressed Plt-specific markers and retained functionality similar to fresh Plts. In in vitro assays of Plt aggregation, differences were noted. In vivo, FDPlts and Plts were found to incorporate into clots in postcapillary venules in the mouse cremaster muscle. Hemorrhagic shock mice resuscitated with LR displayed increased pulmonary vascular permeability compared with sham (sham, 686.6 ± 359.7; shock-LR, 2,637 ± 954.7; p = 0.001), and treatment with FDPlts or WB attenuated permeability compared with shock: shock-FDPlts, 1,328 ± 462.6 (p = 0.05), and shock-WB, 1,024 ± 370.5 (p = 0.0108). However, human Plts (Days 1-3) did not attenuate vascular leak in HS mice compared with shock-LR (shock-Plts, 3,601 ± 1,581; p = 0.33). CONCLUSION: FDPlts contribute to clot formation similar to fresh human Plts. FDPlts also attenuated vascular permeability in vitro and in vivo. Mouse WB resuscitation but not fresh human Plts attenuated vascular permeability after HS. These data suggest that the effect of FDPlts may be a suitable alternative to fresh Plts in modulating hemostasis and the endotheliopathy associated with injury.

Activation of the stress protein response inhibits the STAT1 signalling pathway and iNOS function in alveolar macrophages: role of Hsp90 and Hsp70.

BACKGROUND AND AIM: Alveolar fluid clearance is impaired by inducible nitric oxide synthase (iNOS)/nitric oxide (NO)-dependent mechanisms in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). The activation of the stress protein response (SPR) in alveolar macrophages on iNOS-dependent NO production in response to interferon gamma (IFNgamma), a major cytokine present in the airspace of patients with ALI, was investigated. METHODS: The SPR was activated in murine and primary human alveolar macrophages prior to analysis of signal transducer and activator of transcription factor 1 (STAT1) activation, iNOS mRNA and protein synthesis, and NO production. RESULTS: SPR activation resulted in inhibition of IFNgamma-mediated NO production (p=0.001) with >95% detergent insolubilisation of the STAT1 protein. Its subsequent proteasomal degradation was partially reversed with pretreatment of cells with the chemical chaperone glycerol. This early effect of the SPR was caused by the complete disruption of heat shock protein 90 (Hsp90)-STAT1 binding, as shown by immunoprecipitation. Recovery of STAT1 activation and recovery of iNOS synthesis occurred within 12 h after SPR activation (p=0.02). NO production (as compared with non-SPR controls) did not occur until 48 h later (p=0.02). SPR-induced Hsp70 (Hsp70i) expression caused a late inhibition of NO production (p=0.02). Inhibiting >50% Hsp70i expression recovered NO production to control levels whereas overexpressing Hsp70i in the absence of the SPR inhibited NO production (p=0.02). CONCLUSION: Early inhibition of STAT1 following its dissociation from Hsp90, and later inhibition of iNOS activity by Hsp70i, represent novel mechanisms by which SPR activation modulates the IFNgamma signalling in alveolar macrophages. These results highlight a potential clinical application for Hsp90 inhibitors in modulating NO signalling during the early phase of acute lung injury.

Interleukin-1beta causes acute lung injury via alphavbeta5 and alphavbeta6 integrin-dependent mechanisms.

Interleukin (IL)-1beta has previously been shown to be among the most biologically active cytokines in the lungs of patients with acute lung injury (ALI). Furthermore, there is experimental evidence that lung vascular permeability increases after short-term exposure to IL-1 protein, although the exact mechanism is unknown. Therefore, the objective of this study was to determine the mechanisms of IL-1beta-mediated increase in lung vascular permeability and pulmonary edema following transient overexpression of this cytokine in the lungs by adenoviral gene transfer. Lung vascular permeability increased with intrapulmonary IL-1beta production with a maximal effect 7 days after instillation of the adenovirus. Furthermore, inhibition of the alphavbeta6 integrin and/or transforming growth factor-beta attenuated the IL-1beta-induced ALI. The results of in vitro studies indicated that IL-1beta caused the activation of transforming growth factor-beta via RhoA/alphavbeta6 integrin-dependent mechanisms and the inhibition of the alphavbeta6 integrin and/or transforming growth factor-beta signaling completely blocked the IL-1beta-mediated protein permeability across alveolar epithelial cell monolayers. In addition, IL-1beta increased protein permeability across lung endothelial cell monolayers via RhoA- and alphavbeta5 integrin-dependent mechanisms. The final series of in vivo experiments demonstrated that pretreatment with blocking antibodies to both the alphavbeta5 and alphavbeta6 integrins had an additive protective effect against IL-1beta-induced ALI. In summary, these results demonstrate a critical role for the alphavbeta5/beta6 integrins in mediating the IL-1beta-induced ALI and indicate that these integrins could be a potentially attractive therapeutic target in ALI.

The effects of human prothrombin complex concentrate on hemorrhagic shock-induced lung injury in rats: Implications for testing human blood products in rodents.

BACKGROUND: Hemorrhagic shock (HS) and trauma can result in an endotheliopathy of trauma, characterized by endothelial compromise, inflammation, and aberrant coagulation. Kcentra, a prothrombin concentrate, has been demonstrated to mitigate pulmonary vascular leak in a murine model of HS. We investigated the effects of Kcentra in a rat model of HS, to achieve physiologic endpoints of relevance. METHODS: Rats subjected to a grade intravenous splenic injury and controlled hemorrhage for 60 minutes were resuscitated with shed volumes of (1) Lactated Ringer's (LR) solution, (2) LR + 20 IU/kg Kcentra, (3) LR + 50 IU/kg Kcentra, (4) rat fresh frozen plasma (RFFP), or (5) human fresh frozen plasma (HFFP). Blood was harvested for monitoring metabolic and coagulation function. Rat lungs were evaluated for lung injury and permeability. RESULTS: Animals resuscitated with LR displayed a significant increase in pulmonary vascular permeability (sham, 407.9 ± 122.4; shock + LR, 2040 ± 1462). Resuscitation with RFFP (606.5 ± 169.3) reduced leak; however, treatment with Kcentra (HS + Kcentra [20 IU/kg]: 1792 ± 903.4, HS + Kcentra [50 IU/kg]: 1876 ± 1103), and HFFP (1450 ± 533.2) had no significant effect on permeability. Kcentra modestly altered clotting parameters. Metabolic measures, such as lactate, pH, and base deficit, were restored to baseline levels by both RFFP and HFFP, but not Kcentra or LR. CONCLUSION: Kcentra did not alter pulmonary vascular permeability, but modestly increased clotting potential in injured rats. This suggests that there may be a xenogenic reaction of human products in rats and that the effects of Kcentra on vascular stability may be distinct from its ability to modulate clotting. Our data indicate that the species chosen and utilized for in vivo preclinical testing of human derived blood products is of critical importance in determining their efficacy in animal models and is the primary impetus to communicate these results.

Platelet-derived- Extracellular Vesicles Promote Hemostasis and Prevent the Development of Hemorrhagic Shock.

Every year more than 500,000 deaths are attributed to trauma worldwide and severe hemorrhage is present in most of them. Transfused platelets have been shown to improve survival in trauma patients, although its mechanism is only partially known. Platelet derived-extracellular vesicles (PEVs) are small vesicles released from platelets upon activation and/or mechanical stimulation and many of the benefits attributed to platelets could be mediated through PEVs. Based on the available literature, we hypothesized that transfusion of human PEVs would promote hemostasis, reduce blood loss and attenuate the progression to hemorrhagic shock following severe trauma. In this study, platelet units from four different donors were centrifuged to separate platelets and PEVs. The pellets were washed to obtain plasma-free platelets to use in the rodent model. The supernatant was subjected to tangential flow filtration for isolation and purification of PEVs. PEVs were assessed by total count and particle size distribution by Nanoparticle Tracking Analysis (NTA) and characterized for cells of origin and expression of EV specific-surface and cytosolic markers by flow cytometry. The coagulation profile from PEVs was assessed by calibrated automated thrombography (CAT) and thromboelastography (TEG). A rat model of uncontrolled hemorrhage was used to compare the therapeutic effects of 8.7 × 108 fresh platelets (FPLT group, n = 8), 7.8 × 109 PEVs (PEV group, n = 8) or Vehicle (Control, n = 16) following severe trauma. The obtained pool of PEVs from 4 donors had a mean size of 101 ± 47 nm and expressed the platelet-specific surface marker CD41 and the EV specific markers CD9, CD61, CD63, CD81 and HSP90. All PEV isolates demonstrated a dose-dependent increase in the rate and amount of thrombin generated and overall clot strength. In vivo experiments demonstrated a 24% reduction in abdominal blood loss following liver trauma in the PEVs group when compared with the control group (9.9 ± 0.4 vs. 7.5 ± 0.5 mL, p < 0.001>). The PEV group also exhibited improved outcomes in blood pressure, lactate level, base excess and plasma protein concentration compared to the Control group. Fresh platelets failed to improve these endpoints when compared to Controls. Altogether, these results indicate that human PEVs provide pro-hemostatic support following uncontrolled bleeding. As an additional therapeutic effect, PEVs improve the outcome following severe trauma by maintaining hemodynamic stability and attenuating the development of ischemia, base deficit, and cardiovascular shock.

Regulation of endothelial cell permeability by platelet-derived extracellular vesicles.

BACKGROUND: Platelet (Plt)-derived extracellular vesicles (Plt-EVs) have hemostatic properties similar to Plts. In addition to hemostasis, Plts also function to stabilize the vasculature and maintain endothelial cell (EC) barrier integrity. We hypothesized that Plt-EVs would inhibit vascular EC permeability, similar to fresh Plts. To investigate this hypothesis, we used in vitro and in vivo models of vascular endothelial compromise and bleeding. METHODS: In the vitro model, Plt-EVs were isolated by ultracentrifugation and characterized for Plt markers and particle size distribution. Effects of Plts and Plt-EVs on endothelial barrier function were assessed by transendothelial electrical resistance measurements and histological analysis of endothelial junction proteins. Hemostatic potential of Plt-EVs and Plts was assessed by multiple electrode Plt aggregometry. Using an in vivo model, the effects of Plts and Plt-EVs on vascular permeability and bleeding were assessed in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice by an established Miles assay of vascular permeability and a tail snip bleeding assay. RESULTS: In the in vitro model, Plt-EVs displayed exosomal size distribution and expressed Plt-specific surface markers. Platelets and Plt-EVs decreased EC permeability and restored EC junctions after thrombin challenge. Multiplate aggregometry revealed that Plt-EVs enhanced thrombin receptor-activating peptide-mediated aggregation of whole blood, whereas Plts enhanced thrombin receptor-activating peptide-, arachidonic acid-, collagen-, and adenosine diphosphate-mediated aggregation. In the in vivo model, Plt-EVs are equivalent to Plts in attenuating vascular endothelial growth factor (VEGF)-A-induced vascular permeability and uncontrolled blood loss in a tail snip hemorrhage model. CONCLUSION: Our study is the first to report that Plt-EVs might provide a feasible product for transfusion in trauma patients to attenuate bleeding, inhibit vascular permeability, and mitigate the endotheliopathy of trauma.

Lyophilized plasma attenuates vascular permeability, inflammation and lung injury in hemorrhagic shock.

In severe trauma and hemorrhage the early and empiric use of fresh frozen plasma (FFP) is associated with decreased morbidity and mortality. However, utilization of FFP comes with the significant burden of shipping and storage of frozen blood products. Dried or lyophilized plasma (LP) can be stored at room temperature, transported easily, reconstituted rapidly with ready availability in remote and austere environments. We have previously demonstrated that FFP mitigates the endothelial injury that ensues after hemorrhagic shock (HS). In the current study, we sought to determine whether LP has similar properties to FFP in its ability to modulate endothelial dysfunction in vitro and in vivo. Single donor LP was compared to single donor FFP using the following measures of endothelial cell (EC) function in vitro: permeability and transendothelial monolayer resistance; adherens junction preservation; and leukocyte-EC adhesion. In vivo, using a model of murine HS, LP and FFP were compared in measures of HS- induced pulmonary vascular inflammation and edema. Both in vitro and in vivo in all measures of EC function, LP demonstrated similar effects to FFP. Both FFP and LP similarly reduced EC permeability, increased transendothelial resistance, decreased leukocyte-EC binding and persevered adherens junctions. In vivo, LP and FFP both comparably reduced pulmonary injury, inflammation and vascular leak. Both FFP and LP have similar potent protective effects on the vascular endothelium in vitro and in lung function in vivo following hemorrhagic shock. These data support the further development of LP as an effective plasma product for human use after trauma and hemorrhagic shock.