A dysfunctional endothelium is increasingly recognized as a key component of a wide variety of pathologies, including hemorrhagic shock (HS). The endothelium is the platform on which adhesion of leukocytes and the activation of coagulation occur after shock. In addition, it is the surface to which membrane-bound syndecan 1 is attached. Syndecan 1 is a cell surface heparan sulfate proteoglycan that forms the structural backbone of a protective network of plasma components referred to as the glycocalyx. The glycocalyx is thought to consist primarily of membrane-bound proteoglycans with glycosaminoglycan side chains, membrane-bound glypicans, and adsorbed plasma proteins. Syndecan 1 is one of the proteoglycans found on the luminal surface of endothelial cells (1).
Syndecan 1 plays an important role in inflammation (2). In noninfectious states of inflammation, it facilitates resolution of inflammation by inhibiting leukocyte adhesion onto the endothelium, mitigating the expression and activity of proinflammatory mediators, and confining leukocytes to the site of injury (3). On the other hand, it serves primarily a pathologic role in infectious conditions by promoting attachment of bacteria and inhibiting host defense systems (4). Hemorrhagic shock is a proinflammatory state, but the function of syndecan 1 in the resolution of inflammation after HS is unknown.
Vascular hyperpermeability is being recognized as an increasingly important component of HS, although the “endotheliopathy” of HS is poorly characterized and not well understood (5, 6). Perturbations of the glycocalyx with shedding of syndecan 1 are postulated to alter endothelial integrity and lead to enhanced permeability and subsequent edema, although this is primarily based on clinical associations rather than mechanistic studies. We hypothesize that HS–induced shedding of the syndecan 1 ectodomain is injurious, resulting in the exposure of the injured endothelium to proinflammatory leukocytes and in alterations to the structural integrity of the endothelium with resultant hyperpermeability.
We have been interested in fresh frozen plasma (FFP) as a therapeutic modality to hasten repair of the injured endothelium after HS. Recent retrospective and prospective data suggest that the early and empiric use of FFP may decrease mortality in HS patients, although the mechanism of this protection is unknown (7, 8). Based on our prior finding that FFP aided in restoration of the glycocalyx after HS (9), we further hypothesized that FFP would decrease shedding of syndecan 1, thereby protecting the endothelium from inflammatory insults and vascular hyperpermeability. We tested this hypothesis both in vitro using pulmonary endothelial cells (PECs) and in vivo using a clinically relevant coagulopathic mouse model of HS and trauma.
Materials and Methods
In vitro studies
Human PECs were purchased from Lonza (Walkersville, Md). Cells were maintained in EGM-2MV media on 96-well plates at 37°C and 5% CO2 in a humidified incubator. For experiments, cells were treated with either 10% to 30% concentrations of lactated Ringer’s (LR) or FFP. Donor units of FFP used in both in vitro and in vivo studies were obtained from Gulf Coast Regional Blood Center (Houston, Tex), and as per standard procedure were frozen within 8 h of being drawn. It was kept frozen until the day of the experiment and was used within 1 to 2 h of thaw.
Endothelial cell permeability
Two different parameters of permeability were assessed, flux and transendothelial electrical resistance (TEER). Collagen-coated 0.4-μm-pore-size inserts were seeded with PECs at 104 cells/ insert. When confluency was reached, monolayers were pretreated for 1 h with either 10% FFP or LR and compared with media alone (10). Permeability was then induced with VEGF-A165 (50 ng/mL; R&D, Minneapolis, Minn), administered simultaneously with the addition of fluorescein isothiocyanate (FITC)–dextran 70 kd. Permeability was then assessed by adding 50 μL of 1 mg/mL 70-kd FITC-dextran to the upper chamber of each well (final concentration).
To calculate the baseline flux of FITC–dextran 70, we used Fick’s first law of diffusion (P = J / AΔC], where P is the permeability coefficient expressed as cm/min, J is the solute flux (or movement of FITC–dextran 70 per minute), and A is the area of the transwell at 0.3 cm2. ΔC the difference in concentration of FITC–dextran 70 between the transwell and the bottom well after FITC is added to the transwell. ΔC was assigned a value of 1 mg/mL, assuming that the concentration of FITC–dextran 70 in the bottom well is zero upon addition of FITC. Accumulated FITC–dextran 70 was regressed against time, using the quadratic regression method. Flux was calculated as the average slope from 5 to 120 min for each regression model (11).
In a separate set of experiments, TEER was measured using an electric cell-substrate impedance sensing (ECIS) (Ztheta; Applied Biophysics, Troy, NY) system. The ECIS system provides real-time quantitative measurements of vascular integrity using a change in impedance of the cell monolayer (12). Transendothelial electrical resistance is a measurement of the resistance of the endothelial monolayer to the flow of charged ions through it. The higher the resistance, the more energy it takes to propel an ion across it. Although the permeability to small ions is not the same as the movement of proteins, experimental data show that the inverse of TEER at 4,000 Hz is roughly equivalent to the permeability of an endothelial monolayer (13). Briefly, 8W10E+ plates (Applied Biophysics) were pretreated with cysteine and then media per the manufacturer’s instructions. Pulmonary endothelial cells were seeded at a density of 80,000 cells per well. Resistance and impedance of the cell monolayer were measured continuously from the time of seeding. After 24 h, half of media in each well was replaced with fresh media then 1 h later half was replaced with fresh media containing 10 U/mL heparin and either 10% FFP or LR and compared with media alone (controls). This treatment is considered time 0. Changes in TEER at 4,000 Hz were monitored as a measure permeability as demonstrated by Tiruppathi et al. (13). Resistance traces are normalized to a point −0.5 h before treatment, the top of the peak produced by the addition of fresh media.
Pulmonary endothelial cells (104 cells/well) were seeded and incubated at 37°C until confluent then treated for 1 h with 5%, 10%, or 30% FFP or LR and compared with controls. Adhesion molecule expression was then stimulated by the addition of tumor necrosis factor α (50 ng/mL) for 4 h, which increases binding of U937, a monocytoid cell line that we have previously used to study leukocyte-endothelial interactions (14). U937 cells were fluorescently labeled with calcein-AM (Invitrogen, Carlsbad, Calif) (1 μg/mL), then 104 cells were added to wells and allowed to adhere for 1 h. Nonadherent cells were washed in phosphate-buffered saline (PBS), and labeled cells that remained bound to the endothelial cells were quantified by fluorescent readings on the Biotek Analyzer (Biotek, Winooski, Vt) at 490-nm wavelength excitation and 520-nm emission.
In vivo studies
Coagulopathic mouse model of HS and trauma
All procedures performed were protocols approved by the University of Texas Houston Medical School Animal Welfare Committee. The experiments were conducted in compliance with the National Institutes of Health guidelines on the use of laboratory animals. All animals were housed at constant room temperature with a 12:12-h light-dark cycle with access to food and water ad libitum. Male C57BL/6J mice, 8 to 10 weeks of age, were used for all experiments. To mimic the clinical scenario of trauma-induced coagulopathy in patients in shock, the coagulopathic mouse model of trauma-HS described by Chesebro et al. (15) was used. In brief, under isoflurane anesthesia, a 2-cm midline laparotomy incision was made, organs inspected, and the incision closed. The bilateral femoral arteries were cannulated for continuous hemodynamic monitoring and blood withdrawal or resuscitation, respectively. After a 10-min period of equilibration, mice were bled to a mean arterial pressure (MAP) of 35 ± 5 mmHg and maintained for 90 min. Shams underwent anesthesia and placement of catheters but were not subjected to HS. Similar to Chesebro et al., mice were coagulopathic with a prothrombin time 12.1 ± 0.6 after HS vs. 7.5 ± 0.2 sham (P = 0.02). Mice were resuscitated over the next 15 min with either LR at 3× shed blood volume (16) or FFP at 1× shed blood volume and compared with animals that underwent shock alone. At the conclusion of resuscitation, vascular catheters were removed, incisions closed, and the animals were awoken from anesthesia. After 3 h, animals were killed by exsanguination under isoflurane anesthesia. Blood was obtained at the time the animals were killed, and lungs harvested for further analysis. The 3-h time point was chosen based on our previous investigation showing that the endothelial glycocalyx was being restored by 3 h of resuscitation (9).
Vascular permeability was assessed in intact organs by measuring intravenous dye extravasation into the lung using a Caliper Lumina XR imaging system (In Vivo Imaging System [IVIS]) (17). Animals received an intravenous bolus of the infrared-sensitive dye Alexa Fluor 680 (0.2 mL of 10 mg/mL; molecular weight, 10,000; D34689; Invitrogen) before the onset of shock (18). Upon completion of the 3-h shock resuscitation period, animals were perfused via right ventricle with 4°C PBS for 10 min to remove intravascular dye followed by 4% paraformaldehyde at 4°C. Then lungs were excised and placed in the Lumina XR, from which the images were taken and quantified. Dye extravasation was measured as a function of signal intensity in response to applied fluorescent light at 675 nm. Permeability was quantified by measuring overall fluorescence intensity via Caliper software (19).
In a separate set of animals, Evans blue dye extravasation was measured to confirm lung permeability. Animals received an intravenous injection of 3% Evans blue (4 mL/kg) 1 h before the animals were killed then were perfused via right ventricle with 4°C PBS for 10 min to remove intravascular dye followed by 4%paraformadehyde at 4°C. Lungs were incubated in N-methyl-formamide for 24 h at 55°C to allow for dye extraction. After centrifugation, absorbance was measured in the supernatant at 620 nm using the VersaMax plate reader (Molecular Devices Inc, Sunnyvale, Calif). Tissues were then dried at 50°C for 72 h to obtain dry weight to normalize the absorbance.
Infiltration of neutrophils into the lung tissue was assessed by myeloperoxidase (MPO) immunofluorescence staining and MPO activity. For immunofluorescence, paraffin-embedded tissue was cut into 5-μm-thick sections then incubated with MPO primary antibody (1:100 MPO mouse monoclonal antibody; Abcam, Cambridge, Mass) followed by incubation with secondary antibody (goat anti–mouse; Alexa Fluor 568; Invitrogen). Two random images were taken from each lung section with a fluorescent microscope (Nikon Eclipse Ti, Melville, NY) at 200× and quantified using ImageJ software (National Institutes of Health). Myeloperoxidase activity was measured using a commercially available kit per manufacturer’s instructions (ADI-907-029; Enzo Life Sciences, Ann Arbor, Mich).
Plasma was stored at −80°C until use for measurement of syndecan 1 shedding by enzyme-linked immunosorbent assay (ELISA Kit, UCL-E1966m; Hölzel Diagnostika, Cologne, Germany). Rat anti–mouse syndecan 1 primary antibody was used with rabbit anti–rat horseradish peroxidase–conjugated secondary antibody. Presence of secondary antibody was assessed with Magellen software to measure absorbance at 570 nm. Absorbance was corrected by comparison to wells without added plasma.
To detect syndecan 1, lungs were sectioned and stained with rat anti–mouse syndecan 1 antibody (BD Biosciences, San Diego, Calif) and green fluorescent goat anti–rat secondary antibody (Life Technologies, Grand Island, NY) following deparaffinization and antigen retrieval with citrate buffer. Two random images were taken from each lung section with a fluorescent microscope at 200× and quantified using ImageJ software (National Institutes of Health) (9).
Data were analyzed by one-way analysis of variance and individual group means compared using Tukey post hoc test or repeated-measure analysis of variance (ANOVA), followed by Tukey post hoc tests and Bonferroni correction for multiple comparisons; P < 0.05 was considered significant. Data are expressed as mean ± SEM, n = 8 per group for animal experiments.
FFP compared with LR reduces pulmonary endothelial hyperpermeability
Endothelial cell hyperpermeability is a hallmark characteristic of the injured vasculature. This was studied in an in vitro model using vascular endothelial growth factor A (165aa), a well-known inducer of hyperpermeability (20). In Figure 1A, FFP significantly decreased endothelial cell paracellular permeability at all time points measured compared with LR and control. Calculation of the flux and permeability coefficients indicates that overall FFP is superior to LR and control in inhibiting endothelial cell permeability (Fig. 1B). In a separate set of experiments, real-time quantitative measurements of vascular integrity were obtained over 24 h using an electric cell-substrate impedance sensing system (Fig. 1C). Fresh frozen plasma treatment resulted in a significant increase in endothelial resistance (TEER), which translates to decreased endothelial cell paracellular permeability when compared with LR- and control-treated wells.
Leukocyte binding is reduced by FFP but not LR in PECs
The purpose of these next studies was to determine and compare in vitro the effects of FFP to LR on inflammatory leukocyte-PEC adhesion. To stimulate inflammatory cell (U937) binding, endothelial cells were treated with tumor necrosis factor α and the relative binding of calcein-labeled cells to treated endothelial cells was quantified by fluorimetry. Leukocyte–endothelial cell binding studies revealed that treatment of PECs with 5% to 30% concentrations of FFP inhibited U937 binding to the endothelial cells compared with LR and controls (Fig. 2). Control- and LR-treated cells did not affect leukocyte binding at any concentration tested while there was a significant decrease in leukocyte binding in FFP-treated cells at all concentrations.
FFP and LR similarly restore MAP
To determine if our findings in vitro would translate to similar effects in vivo, we sought to determine if FFP would alter pulmonary inflammation and endothelial dysfunction in an established mouse model of HS and trauma. All mouse groups had a similar baseline weight and MAP as well as similar hemorrhage volume during the shock period (Table 1). By study design, the LR group received a larger volume of resuscitation, whereas the sham and shock-alone groups received no resuscitation. During the early resuscitation period, there was no significant difference in MAP between the LR and FFP groups, whereas the HS group had a significantly lower MAP, and the sham group a significantly higher MAP than the two resuscitation groups (Fig. 3).
FFP mitigates lung hyperpermeability compared with LR resuscitation
Using a novel technique, HS-induced hyperpermeability was quantified in intact organs by measuring extravasation of an intravenously delivered fluorescently conjugated dextran into the lung using an IVIS Lumina XR. Lung permeability was significantly increased after HS (9.5 × 1010 ± 1.9 × 1010) compared with shams (2.4 × 1010 ± 2.7 × 109). Permeability was reduced by 3× LR (6.7 × 1010 ± 5.0 × 109) compared with HS but further reduced by 1× FFP (4.1 × 1010 ± 3.2 × 109) (Fig. 4). To validate our IVIS imaging and quantification, permeability was also assessed by Evans blue extravasation, and findings were consistent. Evans blue extravasation as an indicator of lung permeability to circulating albumin (∼65 kd) was minimal in shams (4.1 ± 0.4) but was significantly increased after HS (15.1 ± 1.9), lessened by 3× LR (9.7 ± 1.1), but further significantly decreased by 1× FFP (5.7 ± 0.5).
Lung inflammation is reduced by FFP compared with LR resuscitation
Myeloperoxidase is a measure of neutrophil infiltration. Lung MPO immunofluorescence demonstrated a significant increase in MPO staining in lungs after HS (1,172 ± 207 relative fluorescence units [RFUs]) compared with shams (287 ± 32 RFUs) and 3× LR resuscitation (815 ± 126 RFUs) that was lessened by 1× plasma resuscitation (485 ± 65 RFUs) (Figs. 5A, B). Myeloperoxidase activity was consistent with changes in MPO immunofluorescence (Fig. 5C).
Pulmonary syndecan 1 is increased and shed syndecan 1 reduced by FFP compared with LR resuscitation
After HS alone (329 ± 48 RFUs) or 3× LR resuscitation (402 ± 44 RFUs), pulmonary syndecan 1 immunostaining was significantly decreased compared with 1× plasma resuscitation (688 ± 69 RFUs) and shams (724 ± 76 RFUs) (Figs. 6A, B). We have previously demonstrated similar results in pulmonary syndecan 1 biology using a rat model of pressure-controlled HS, thus highlighting the generalizability of these findings (9).
Pulmonary syndecan 1 inversely correlated with an increase in systemic syndecan 1 shedding after HS. Shams had low levels of detectable shed syndecan 1 (14 ± 4 ng/mL) that was significantly increased after HS (150 ± 23 ng/mL), reduced by 3× LR (103 ± 17 ng/mL), but further lessened by 1× plasma (55 ± 8 ng/mL) (Fig. 7).
In vitro and in vivo, we demonstrated that FFP-based resuscitation mitigated lung hyperpermeability and reduced lung inflammation compared with LR resuscitation. These findings were not due to differences in MAP in the early postresuscitation period but were associated with enhanced pulmonary syndecan 1 immunostaining and reduced systemic syndecan 1 ectodomain shedding.
We expanded on our previous in vitro findings of shock-induced hyperpermeability (10) by now assessing flux and extended permeability studies to examine changes over time with the use of ECIS. Electric cell-substrate impedance sensing can assess barrier function over time with measurements being performed directly in the cell culture medium (12). Fresh frozen plasma lessened flux and maintained reduced permeability compared with LR for the 24-h study period. In addition, FFP reduced leukocyte binding compared with LR-based resuscitation. This is important as FFP has been associated with transfusion-related acute lung injury (21) and suggests that when evaluating leukocyte-mediated transfusion-related complications all components, including crystalloid, be considered. As Middleburg et al. (22) have shown, there are additional factors that are not well understood. Ex vivo, we used an innovative technique to image and quantify hyperpermeability in intact organs by measuring extravasation of a fluorescent intravenous dye into the lung using IVIS. This technique will permit whole-animal imaging in vivo with serial visual and quantitative analysis of hyperpermeability (17). As a first step, we imaged intact lungs ex vivo and compared our results with the well-established technique of Evans blue extravasation. Results were consistent between assays.
We chose 3 h after the HS as our experimental end point. This time point was based on a prior study where we demonstrated partial restoration of the endothelial glycocalyx at 3 h (9). Importantly, at 3 h, we are not studying acute lung injury or adult respiratory distress syndrome as part of multiple organ dysfunction syndrome. Rather, we are looking at changes in pulmonary vascular hyperpermeability and inflammation as a result of HS, at a clinically relevant time (8).
Vascular hyperpermeability is being recognized as an increasingly important component of HS. Alterations of the adherens junction complex (via vascular endothelial-cadherin) with the underlying cytoskeleton (through the catenins) have been implicated as an etiology of hyperpermeability after HS (23, 24). We have shown that vascular endothelial (VE)–cadherin is disrupted in an in vitro model of HS and is associated with hyperpermeability (25). Although the clinical sequela of organ injury from hyperpermeability is well appreciated, the contribution of hyperpermeability to death after HS is not known. Interestingly, there is rare disorder termed systemic capillary leak syndrome that may provide some important clues (26). This disorder is characterized by transient episodes of hypotensive shock and death, which may be linked to alterations in VE-cadherin (27). This at least suggests that hyperpermeability alone may be contributing to mortality by reducing intravascular volume. If reductions in hyperpermeability by FFP demonstrated in the current study translate into the clinical arena, this may partially explain the decrease in mortality in hemorrhagic patients receiving early empiric FFP (7, 8, 28).
We have shown in a small pilot study that syndecan 1 is shed at the time of injury in severely injured patients in HS (25). Our data also suggest that patients with higher postresuscitation syndecan 1 levels had a higher mortality. More recently, Johansson et al. (29) demonstrated increased mortality in patients with high admission syndecan 1 shedding and that shedding correlated with inflammation and coagulopathy. The etiology of this association, however, is unclear. It is also uncertain as to the biologic role, if any, of circulating syndecan. Shedding is regulated by multiple intracellular signaling pathways converging on a diverse group of activated matrix metalloproteinases, known as sheddases (30). While shedding is known to be activated by pathologic stimuli, little is known about how ectodomain release from the cell surface is regulated, and there is no information on regulation after HS. The shed ectodomains have been shown to facilitate resolution of inflammation by removing chemokines from the lung in the mouse model of endotoxemia (3). We hypothesize the HS-induced shedding results in exposure of the injured endothelium to proinflammatory mediators and in alterations to the structural integrity of the endothelium with resultant hyperpermeability. We demonstrated this in the lung, showing that loss of syndecan 1 was associated with pulmonary permeability and inflammation. How we resuscitate the injured endothelium can either propagate injury or hasten repair. Our results suggest that FFP-based resuscitation can hasten repair by restoring syndecan 1 on the cell surface and reducing syndecan 1 shedding. Syndecan 1 provides the structural backbone for the endothelial glycocalyx, and the glycocalyx has been implicated as a key modulator of permeability and inflammation in a wide variety of models and clinical diseases. In models of cardiac ischemia, shedding of the glycocalyx was associated with vascular hyperpermeability. This effect was mitigated by pretreatment with antithrombin, which can lower susceptibility to enzymatic degradation, or hydrocortisone, which can inhibit mast cell degranulation and thus inflammation (31, 32). Grundmann et al. (33) have shown that alterations in the endothelial glycocalyx may be responsible for vascular leakage and leukocyte adhesion seen after cardiac arrest, and Rehm et al. (34) have demonstrated shedding in patients undergoing abdominal aortic aneurysm repair. Chappell et al. (35) have highlighted the importance of the microcirculation in patients with systemic inflammatory response syndrome and sepsis and suggested targeting the glycocalyx as a therapy to improve oxygen distribution in these patients. A dysfunctional glycocalyx has also been implicated in tissue edema seen with diabetes and is a key initiator of atherothrombosis (36).
Our data at least suggest that targeting the endothelial glycocalyx may be beneficial in severely injured patients in shock. Regulation of syndecan 1 postinjury could potentially serve as a viable therapeutic target for novel drug discovery. As FFP is composed of thousands of circulating proteins, investigation of the specific component(s) of FFP responsible for its protective effects may warrant further investigation. It would also be interesting to investigate the effects of FFP in other models of vascular instability such as pulmonary hypertension, venous insufficiency, and acute renal failure or in models of organ edema such as burns or pancreatitis.
There are several limitations to this study. One difficulty in evaluating systemic syndecan 1 levels is that ectodomain shedding may occur from multiple sites, making it possible that its loss from the lungs may not directly correlate with the levels detected in the plasma. However, our results suggest that there is an association between loss of syndecan 1 in the lungs and the corresponding increase in systemic levels. In addition, syndecan 1 is expressed on both epithelial and endothelial cells; thus, we cannot differentiate the cell type of shed ectodomain. This is a flaw of most published reports of systemic syndecan shedding. Shed syndecan is frequently attributed to the endothelial glycocalyx, but the contribution of epithelial syndecan is not discussed. To address this problem, we are planning on generating an endothelial overexpressing syndecan 1 null mouse. Although we assume that endothelial syndecan 1 is shed, we do know that loss of endothelial syndecan 1 and the glycocalyx is associated with vascular hyperpermeability (25). Lastly, we cannot rule out the possibility that enhanced perfusion by FFP may be an explanation for its beneficial effects. Our results in the early resuscitation period do not support this, as there was no difference in MAP between FFP and LR. However, as animals were awoken as soon as volume resuscitation was complete, we have only early hemodynamic data. A recent study by Jin et al. (37) in a large animal model of HS and traumatic brain injury reported MAP for 6 h after shock. They also found that FFP did not augment blood pressure or heart rate compared with crystalloid resuscitation. Interestingly, they also demonstrated decreased brain injury size and brain edema with FFP. A related limitation is that different volumes of resuscitation were used between experimental groups in vivo. Resuscitation with crystalloids at 3× shed blood volume or even 4× shed blood is common in models of HS. This practice is based on the laboratory findings that demonstrated that extracellular fluid was redistributed during shock into both the intravascular and intracellular spaces. To correct this extracellular fluid deficit, infusion of isotonic crystalloid fluid in a ratio of three volumes of crystalloid to one of blood was required (38). However, using crystalloid in excess of FFP may have affected our results, particularly permeability. Our in vitro experiments utilized equal volumes between groups, and results were consistent.
In conclusion, in vitro endothelial cell permeability and flux were decreased, TEER was increased, and leukocyte-endothelial binding was inhibited by FFP compared with LR-treated endothelial cells. In a coagulopathic mouse model of HS and trauma, FFP resuscitation inhibited endothelial cell hyperpermeability and inflammation and restored pulmonary syndecan 1 expression. Modulation of pulmonary syndecan 1 expression may mechanistically contribute to the beneficial effects FFP.
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