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.

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.

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.

Mesenchymal stem cell-derived extracellular vesicles attenuate pulmonary vascular permeability and lung injury induced by hemorrhagic shock and trauma.

BACKGROUND: Mesenchymal stem cells (MSCs) have been shown to mitigate vascular permeability in hemorrhagic shock (HS) and trauma-induced brain and lung injury. Mechanistically, paracrine factors secreted from MSCs have been identified that can recapitulate many of the potent biologic effects of MSCs in animal models of disease. Interestingly, MSC-derived extracellular vesicles (EVs), contain many of these key soluble factors, and have therapeutic potential independent of the parent cells. In this study we sought to determine whether MSC-derived EVs (MSC EVs) could recapitulate the beneficial therapeutic effects of MSCs on lung vascular permeability induced by HS in mice. METHODS: Mesenchymal stem cell EVs were isolated from human bone marrow-derived MSCs by ultracentrifugation. A mouse model of fixed pressure HS was used to study the effects of shock, shock + MSCs and shock + MSC EVs on lung vascular endothelial permeability. Mice were administered MSCs, MSC EVs, or saline IV. Lung tissue was harvested and assayed for permeability, RhoA/Rac1 activation, and for differential phosphoprotein expression. In vitro, human lung microvascular cells junctional integrity was evaluated by immunocytochemistry and endothelial cell impedance assays. RESULTS: Hemorrhagic shock-induced lung vascular permeability was significantly decreased by both MSC and MSC EV infusion. Phosphoprotein profiling of lung tissue revealed differential activation of proteins and pathways related to cytoskeletal rearrangement and regulation of vascular permeability by MSCs and MSC EVs. Lung tissue from treatment groups demonstrated decreased activation of the cytoskeletal GTPase RhoA. In vitro, human lung microvascular cells, MSC CM but not MSC-EVs prevented thrombin-induced endothelial cell permeability as measured by electrical cell-substrate impedance sensing system and immunocytochemistry of VE-cadherin and actin. CONCLUSION: 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.

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.

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.

Modulating the endotheliopathy of trauma: Factor concentrate versus fresh frozen plasma.

BACKGROUND: Transfusion of balanced ratios of plasma to platelets and red blood cells has been shown to reduce early death from exsanguination in trauma patients. Aside from hemostasis, recent work has shown that plasma reduces vascular endothelial permeability, inflammation, and organ edema after hemorrhagic shock (HS), all components of the endotheliopathy of trauma. We hypothesized that Kcentra could have protective effects on the endotheliopathy of trauma comparable with fresh frozen plasma (FFP). METHODS: In vitro, endothelial cell (EC) barrier function was assessed by measuring changes in transendothelial electrical resistance for Kcentra, FFP, and albumin. In vivo, a modified Miles assay was used on mice to study the effects of Kcentra, FFP, and albumin on vascular permeability induced by VEGF-A. The same groups were studied in a second in vivo model of pulmonary vascular leak induced by HS and laparotomy. The identification of proteins in Kcentra was assessed by liquid chromatography/mass spectrometry. RESULTS: We found that FFP and Kcentra inhibit EC permeability. We also found that Kcentra and FFP have equivalent capacity to restore EC adherens junction breakdown induced by VEGF-A. In vivo, we found that Kcentra and FFP, but not albumin, significantly inhibited vascular permeability induced by VEGF-A and HS-induced vascular permeability in mice. Investigation of the protein content of Kcentra by mass spectroscopy revealed that there are a number of proteins in Kcentra, derived from plasma that may have contributory roles in the noted effects of Kcentra on vascular leak. CONCLUSION: Taken together, we have demonstrated that FFP and Kcentra inhibit vascular permeability in vivo and in vitro. These beneficial effects of Kcentra may be due in part to the modulation of vascular function by soluble factors present in Kcentra aside from the known clotting factors II, VII, IX, and X. The clinical implications of these findings are unknown and warrant further investigation.

Perturbed mechanotransduction by endothelial surface glycocalyx modification greatly impairs the arteriogenic process.

The mechanisms that trigger initiation of arteriogenesis in response to pathogenic obstruction of arterial flow are not fully understood. Our objective is to determine whether glycocalyx mediated mechanotransduction of fluid shear stress to the endothelial layer is an essential first step in inducing arteriogenesis. Mice were implanted with an osmotic minipump containing saline or hyaluronan synthase inhibitor 4-methylesculetin (4ME) 2 wk before femoral artery ligation. 4ME was effective in modifying the endothelial glycocalyx as measured by dextran exclusion and perfused boundary region changes. Glycocalyx modification resulted in a 52% (P = 0.002) reduction in perfusion restoration through the 21-day follow-up [area under the curve, 4.9 ± 1.1 (n = 11) vs. 10.2 ± 3.2 (n = 10), 4ME vs. control (Ctrl)]. Upon femoral artery ligation, no change in collateral vessel diameter in 4ME treated mice (49.8 ± 26.3 vs. 47.1 ± 14.0 µm, ligated vs unligated) was observed (Ctrl, 88.5 ± 18.8 vs. 35.1 ± 3.0 µm, ligated vs unligated, P < 0.05). This impaired arteriogenic process was accompanied by lack of local induction of both endothelial and smooth muscle cell activation (Ki67, endothelial nitric oxide synthase, and ICAM-1), as well as a failure to recruit CD11b-positive cells in 4ME-treated collateral vessels (0.012 ± 0.003 vs. 0.010 ± 0.003 cells/µm vessel perimeter, ligated vs. unligated), whereas in Ctrls, the number of CD11b cells was increased (0.024 ± 0.002 vs. 0.010 ± 0.004 cells/µm vessel perimeter, P < 0.05). Modification of the glycocalyx by inhibition of hyaluronan synthesis renders the endothelium unresponsive to altered hemodynamic conditions resulting from femoral artery ligation, which results in a hampered restoration of distal perfusion.

Fresh frozen plasma and spray-dried plasma mitigate pulmonary vascular permeability and inflammation in hemorrhagic shock.

BACKGROUND: In retrospective and prospective observational studies, fresh frozen plasma (FFP) has been associated with a survival benefit in massively transfused trauma patients. A dry plasma product, such as spray-dried plasma (SDP), offers logistical advantages over FFP. Recent studies on FFP have demonstrated that FFP modulates systemic vascular stability and inflammation. The effect of SDP on these measures has not been previously examined. This study compares SDP with FFP using in vitro assays of endothelial function and in vivo assays of lung injury using a mouse model of hemorrhagic shock (HS) and trauma. METHODS: FFP, SDP, and lactated Ringer's (LR) solution were compared in vitro using assays of endothelial cell (EC) permeability, cytokine production and content, gene expression, as well as tight and adherens junction stability. All resuscitation products were also compared in a murine model of HS. Mean arterial pressures and physiologic measures were assessed. Pulmonary vascular permeability was measured using tagged dextran. Lung tissues were stained for CD68, VE-cadherin, and occludin. RESULTS: Treatment of ECs with FFP and SDP, but not LR, preserved the integrity of EC monolayers in vitro and resulted in similar EC gene expression patterns and cytokine/growth factor production. FFP and SDP also reduced HS-induced pulmonary vascular permeability in vivo to the same extent. In mice with HS, mean arterial pressures and base excess were corrected by both FFP and SDP to levels observed in sham-treated mice. Treatment after HS with FFP and SDP but not LR solution reduce alveolar wall thickening, leukocyte infiltration, and the breakdown of EC junctions, as measured by staining for VE-cadherin, and occludin. CONCLUSION: Both FFP and SDP similarly modulate pulmonary vascular integrity, permeability, and inflammation in vitro and in vivo in a murine model of HS and trauma.

The recovery time course of the endothelial cell glycocalyx in vivo and its implications in vitro.

Compelling evidence continues to emerge suggesting that the glycocalyx surface layer on vascular endothelial cells plays a determining role in numerous physiological processes including inflammation, microvascular permeability, and endothelial mechanotransduction. Previous research has shown that enzymes degrade the glycocalyx, whereas inflammation causes shedding of the layer. To track the endogenous recovery of the glycocalyx in vivo, we used fluorescent microparticle image velocimetry (micro-PIV) in mouse cremaster muscle venules to estimate the hydrodynamically relevant glycocalyx thickness 1, 3, 5, and 7 days after enzymatic or cytokine-mediated degradation of the layer. Results indicate that after acute degradation of the glycocalyx, 5 to 7 days are required for the layer to endogenously restore itself to its native hydrodynamically relevant thickness in vivo. In light of these findings, and because demonstrable evidence has emerged that standard cell culture conditions are not conducive to providing the environment and/or cellular conditions necessary to produce and maintain a physiologically relevant cell surface glycocalyx in vitro, we sought to determine whether merely the passage of time would be sufficient to promote the production of a hydrodynamically relevant glycocalyx on a confluent monolayer of human umbilical vein endothelial cells (HUVECs). Using micro-PIV, we found that the hydrodynamically relevant glycocalyx was substantially absent 7 days postconfluence on HUVEC-lined cylindrical collagen microchannels maintained under standard culture conditions. Thus, it remains to be determined how a hydrodynamically relevant glycocalyx surface layer can be synthesized and maintained in culture before the endothelial cell culture model can be used to elucidate glycocalyx-mediated mechanisms of endothelial cell function.

The hydrodynamically relevant endothelial cell glycocalyx observed in vivo is absent in vitro.

In recent years, the endothelial cell surface glycocalyx has emerged as a structure of fundamental importance to a broad range of phenomena that determine cardiovascular health and disease. This new understanding of the functional significance of the glycocalyx has been made possible through recently developed experimental techniques using intravital microscopy that are capable of directly probing the glycocalyx in vivo. Using fluorescent microparticle image velocimetry in venules and endothelialized cylindrical collagen microchannels, we show that the hydrodynamically relevant endothelial cell glycocalyx surface layer observed in microvessels in vivo (0.52+/-0.28 microm thickness), which is a fundamental determinant of the hydrodynamic and mechanical environment at the endothelial cell surface, is absent from human umbilical vein (0.03+/-0.04 microm thickness) and bovine aortic (0.02+/-0.04 microm thickness) endothelial cells grown and maintained under standard cell culture conditions in vitro. An endothelial surface-bound glycosaminoglycan layer, not necessarily indicative of but having similar hydrodynamic properties to the endothelial glycocalyx observed in vivo, was detected (0.21+/-0.27 microm thickness) only after hyaluronan and chondroitin sulfate were added to the cell culture media at hyperphysiological concentrations (0.2 mg/mL perfused for 75 minutes). The implications of this glycocalyx deficiency under standard cell culture conditions in these pervasive in vitro models broadly impact a myriad of studies involving endothelial cell monolayers in which inferences are made that may depend on endothelial cell surface chemistry. In light of these findings, conclusions drawn from such studies in the areas of microvascular permeability, inflammation, mechanotransduction, and atherosclerosis must be carefully reconsidered.

Formation of perfused, functional microvascular tubes in vitro.

This work describes the formation, perfusion, and maturation of three-dimensional microvascular tubes in vitro. These tubes consisted of confluent monolayers of human endothelial cells that lined open, cylindrical channels within collagen gels. Perivascular cells could be directly embedded within the gels or added after endothelial cells grew to confluence. The tubes spanned the entire 5-7 mm extent of the gels; their diameters initially ranged from 55 to 120 microm and increased to 75-150 microm after maturation. Endothelial tubes displayed a strong barrier function over 5 days, resisted adhesion of leukocytes, and reacted quickly to inflammatory stimuli by breakdown of the barrier and support of leukocyte adhesion. These tubes resembled venules and "giant" capillaries in both their cellular organization and function, and we believe that they will serve as useful in vitro models of inflammation under constant perfusion.