Abstract-Interleukin (IL)-1␤ 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-1␤-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-1␤ production with a maximal effect 7 days after instillation of the adenovirus. Furthermore, inhibition of the ␣v␤6 integrin and/or transforming growth factor-␤ attenuated the IL-1␤-induced ALI. The results of in vitro studies indicated that IL-1␤ caused the activation of transforming growth factor-␤ via RhoA/␣v␤6 integrin-dependent mechanisms and the inhibition of the ␣v␤6 integrin and/or transforming growth factor-␤ signaling completely blocked the IL-1␤-mediated protein permeability across alveolar epithelial cell monolayers. In addition, IL-1␤ increased protein permeability across lung endothelial cell monolayers via RhoA-and ␣v␤5 integrin-dependent mechanisms. The final series of in vivo experiments demonstrated that pretreatment with blocking antibodies to both the ␣v␤5 and ␣v␤6 integrins had an additive protective effect against IL-1␤-induced ALI. In summary, these results demonstrate a critical role for the ␣v␤5/␤6 integrins in mediating the IL-1␤-induced ALI and indicate that these integrins could be a potentially attractive therapeutic target in ALI. Key Words: lung Ⅲ cytokines Ⅲ inflammation Ⅲ endothelial cells Ⅲ epithelial cells Ⅲ rodents A cute lung injury (ALI) is a devastating clinical syndrome in critically ill patients with an overall mortality rate of 30% to 40%. 1 The syndrome is characterized by alveolar epithelial and lung endothelial injury leading to increased permeability across the alveolar-capillary barrier, pulmonary edema, and acute respiratory failure. 2 Despite an improved understanding of the pathogenesis of ALI in recent years, the molecular steps regulating the development of increased lung endothelial and epithelial permeability remain poorly understood, and no specific pharmacological therapies are currently available.During the early phase of ALI, a variety of inflammatory mediators are released into the distal air spaces. 2 Among those, interleukin (IL)-1␤ has been shown to be among the most biologically active cytokines in the lungs early after the onset of ALI. 3-5 Furthermore, IL-1␤ stimulates the production of a variety of chemokines (eg, IL-8, monocyte chemotactic protein [MCP]-1, and macrophage inflammatory protein [MIP]-1␣) 6 involved in epithelial wound repair 7,8 and is a potent inducer of lung fibrosis. 9,10 It has been previously shown in rats that lung vascular permeability increases after short-term exposure of IL-1␣ and IL-1␤ protein when ...

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.

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.

Mesenchymal stem cells and MSC EVs modulate cytoskeletal signaling and attenuate lung vascular permeability after HS. Mesenchymal stem cell EVs may potentially be used as a novel "stem cell free" therapeutic to treat HS-induced lung injury.