Decreased Glycocalyx Shedding on Presentation in Hemorrhaging Geriatric Trauma Patients.

INTRODUCTION: Plasma levels of syndecan-1 (Sdc-1), a biomarker of endothelial glycocalyx (EG) damage, correlate with worse outcomes in trauma patients. However, EG injury is not well characterized in injured older adults (OA). The aims of this study were to characterize Sdc-1 shedding in OA trauma patients relative to younger adults (YA) and determine associations with putative regulators of EG sheddases. METHODS: We performed a secondary analysis of data from the Pragmatic, Randomized Optimal Platelet, and Plasma Ratios (PROPPR) trial, stratifying bluntly injured subjects into OA and YA groups based on upper age quartile (57 y). Plasma Sdc-1 levels were compared in OA and YA at hospital arrival through postinjury day 3, and the independent association between age and Sdc-1 level at arrival was determined after adjusting for differences in gender, shock index (SI), and pre-existing comorbidities. In a follow-up analysis, case-control matching was used to create populations of OA and YA with equivalent SI and injury severity score. Levels of Sdc-1 were compared between these matched groups, and the relationships with candidate regulators of EG shedding were assessed. RESULTS: Of 680 subjects in the Pragmatic, Randomized Optimal Platelet, and Plasma Ratios trial, 350 (51%) had blunt injuries, and 92 (26.3%) of these were OA. Plasma Sdc-1 levels at arrival, 2 h, and 6 h were significantly lower in OA compared to YA (all P < 0.05). After adjusting for sex, pre-existing morbidities and SI, age was associated with decreased Sdc-1 levels at arrival. In the matched analyses, Sdc-1, high-mobility group box 1 and tissue inhibitor of metalloproteinase-2 levels were lower in OA compared to YA. Both high-mobility group box-1 and tissue inhibitor of metalloproteinase-2 significantly correlated with arrival Sdc-1 and were inversely associated with age. CONCLUSIONS: This study indicates that increased age is independently associated with decreased Sdc-1 levels among patients with blunt injuries. Suppressed plasma levels of sheddases in relation to diminished Sdc-1 shedding suggest that mechanisms regulating EG cleavage may be impaired in injured older adults. These findings provide novel insight into the age-dependent impact of injury on the vascular endothelium, which could have important implications for the clinical management of older adults following trauma.

A 3-O-sulfated heparan sulfate dodecasaccharide (12-mer) suppresses thromboinflammation and attenuates early organ injury following trauma and hemorrhagic shock.

Introduction: Dysregulated inflammation and coagulation are underlying mechanisms driving organ injury after trauma and hemorrhagic shock. Heparan sulfates, cell surface glycosaminoglycans abundantly expressed on the endothelial surface, regulate a variety of cellular processes. Endothelial heparan sulfate containing a rare 3-O-sulfate modification on a glucosamine residue is anticoagulant and anti-inflammatory through high-affinity antithrombin binding and sequestering of circulating damage-associated molecular pattern molecules. Our goal was to evaluate therapeutic potential of a synthetic 3-O-sulfated heparan sulfate dodecasaccharide (12-mer, or dekaparin) to attenuate thromboinflammation and prevent organ injury. Methods: Male Sprague-Dawley rats were pre-treated subcutaneously with vehicle (saline) or dekaparin (2 mg/kg) and subjected to a trauma/hemorrhagic shock model through laparotomy, gut distention, and fixed-pressure hemorrhage. Vehicle and dekaparin-treated rats were resuscitated with Lactated Ringer's solution (LR) and compared to vehicle-treated fresh-frozen-plasma-(FFP)-resuscitated rats. Serial blood samples were collected at baseline, after induction of shock, and 3 hours after fluid resuscitation to measure hemodynamic and metabolic shock indicators, inflammatory mediators, and thrombin-antithrombin complex formation. Lungs and kidneys were processed for organ injury scoring and immunohistochemical analysis to quantify presence of neutrophils. Results: Induction of trauma and hemorrhagic shock resulted in significant increases in thrombin-antithrombin complex, inflammatory markers, and lung and kidney injury scores. Compared to vehicle, dekaparin treatment did not affect induction, severity, or recovery of shock as indicated by hemodynamics, metabolic indicators of shock (lactate and base excess), or metrics of bleeding, including overall blood loss, resuscitation volume, or hematocrit. While LR-vehicle-resuscitated rodents exhibited increased lung and kidney injury, administration of dekaparin significantly reduced organ injury scores and was similar to organ protection conferred by FFP resuscitation. This was associated with a significant reduction in neutrophil infiltration in lungs and kidneys and reduced lung fibrin deposition among dekaparin-treated rats compared to vehicle. No differences in organ injury, neutrophil infiltrates, or fibrin staining between dekaparin and FFP groups were observed. Finally, dekaparin treatment attenuated induction of thrombin-antithrombin complex and inflammatory mediators in plasma following trauma and hemorrhagic shock. Conclusion: Anti-thromboinflammatory properties of a synthetic 3-O-sulfated heparan sulfate 12-mer, dekaparin, could provide therapeutic benefit for mitigating organ injury following major trauma and hemorrhagic shock.

Alterations in heparan sulfate proteoglycan synthesis and sulfation and the impact on vascular endothelial function.

The glycocalyx attached to the apical surface of vascular endothelial cells is a rich network of proteoglycans, glycosaminoglycans, and glycoproteins with instrumental roles in vascular homeostasis. Given their molecular complexity and ability to interact with the intra- and extracellular environment, heparan sulfate proteoglycans uniquely contribute to the glycocalyx's role in regulating endothelial permeability, mechanosignaling, and ligand recognition by cognate cell surface receptors. Much attention has recently been devoted to the enzymatic shedding of heparan sulfate proteoglycans from the endothelial glycocalyx and its impact on vascular function. However, other molecular modifications to heparan sulfate proteoglycans are possible and may have equal or complementary clinical significance. In this narrative review, we focus on putative mechanisms driving non-proteolytic changes in heparan sulfate proteoglycan expression and alterations in the sulfation of heparan sulfate side chains within the endothelial glycocalyx. We then discuss how these specific changes to the endothelial glycocalyx impact endothelial cell function and highlight therapeutic strategies to target or potentially reverse these pathologic changes.

Glycocalyx heparan sulfate cleavage promotes endothelial cell angiopoietin-2 expression by impairing shear stress-related AMPK/FoxO1 signaling.

Angiopoietin-2 (Ang-2) is a key mediator of vascular disease during sepsis, and elevated plasma levels of Ang-2 are associated with organ injury scores and poor clinical outcomes. We have previously observed that biomarkers of endothelial glycocalyx (EG) damage correlate with plasma Ang-2 levels, suggesting a potential mechanistic linkage between EG injury and Ang-2 expression during states of systemic inflammation. However, the cell signaling mechanisms regulating Ang-2 expression following EG damage are unknown. In the current study, we determined the temporal associations between plasma heparan sulfate (HS) levels as a marker of EG erosion and plasma Ang-2 levels in children with sepsis and in mouse models of sepsis. Second, we evaluated the role of shear stress-mediated 5'-adenosine monophosphate-activated protein kinase (AMPK) signaling in Ang-2 expression following enzymatic HS cleavage from the surface of human primary lung microvascular endothelial cells (HLMVECs). We found that plasma HS levels peaked before plasma Ang-2 levels in children and mice with sepsis. Further, we discovered that impaired AMPK signaling contributed to increased Ang-2 expression following HS cleavage from flow-conditioned HLMVECs, establishing a paradigm by which Ang-2 may be upregulated during sepsis.

Endotheliopathy is Associated with a 24-hour Fibrinolysis Phenotype Described by Low TEG Lysis and High D-Dimer after Trauma: a Secondary Analysis of the PROPPR Study.

Objectives: Determine associations between biomarkers of endotheliopathy, 24-hour fibrinolysis phenotypes and clinical outcomes after trauma. Background: The vascular endothelium is a critical regulator of hemostasis and organ function. The relationship between markers of endotheliopathy and fibrinolysis following trauma has not been evaluated. Methods: We performed a secondary analysis of prospectively collected biomarker data in the Pragmatic Randomized Optimal Platelet and Plasma Ratios (PROPPR) randomized controlled trial. We stratified subjects by 24-hour thromboelastography (TEG) percent clot lysis (LY30) and plasma D-dimer (DD) levels and evaluated differences in endotheliopathy biomarkers and clinical outcomes between subjects with one of four 24-hour fibrinolysis phenotypes: LY30 0.9-2.9% (LY30norm), LY30 >2.9% (LY30high), LY30 <0.9% and low DD (LY30low+DDlow), and LY30 <0.9% and high DD (LY30low+DDhigh). Results: The analysis included 168 subjects with LY30norm, 32 with LY30high, 147 with LY30low+DDlow and 124 with LY30low+DDhigh. LY30low+DDhigh subjects had greater injury severity and a higher incidence of severe head injury, multiorgan failure (MOF), and mortality than the other phenotypes. All endotheliopathy biomarkers were significantly higher in the LY30low+DDhigh phenotype. Adjusting for injury severity, mechanism and head trauma, 24-hour angiopoietin-2 and soluble thrombomodulin were independently associated with the LY30low+DDhigh phenotype. Both endothelial biomarkers were discriminating for MOF. Subjects with thrombomodulin level >9.5 ng/mL and angiopoietin-2 level >3.6 ng/mL accounted for 64% of subjects who developed MOF. Conclusions: In a multicenter trauma cohort, subjects with a fibrinolysis phenotype characterized by low TEG lysis and elevated DD 24 hours after injury have significantly worse endotheliopathy and clinical outcomes. Our findings support mechanistic evaluations of the role of the endothelium in fibrinolysis dysregulation that may drive late-stage organ injury.

The endothelial glycocalyx in critical illness: A pediatric perspective.

The vascular endothelium is the interface between circulating blood and end organs and thus has a critical role in preserving organ function. The endothelium is lined by a glycan-rich glycocalyx that uniquely contributes to endothelial function through its regulation of leukocyte and platelet interactions with the vessel wall, vascular permeability, coagulation, and vasoreactivity. Degradation of the endothelial glycocalyx can thus promote vascular dysfunction, inflammation propagation, and organ injury. The endothelial glycocalyx and its role in vascular pathophysiology has gained increasing attention over the last decade. While studies characterizing vascular glycocalyx injury and its downstream consequences in a host of adult human diseases and in animal models has burgeoned, studies evaluating glycocalyx damage in pediatric diseases are relatively few. As children have unique physiology that differs from adults, significant knowledge gaps remain in our understanding of the causes and effects of endothelial glycocalyx disintegrity in pediatric critical illness. In this narrative literature overview, we offer a unique perspective on the role of the endothelial glycocalyx in pediatric critical illness, drawing from adult and preclinical data in addition to pediatric clinical experience to elucidate how marked derangement of the endothelial surface layer may contribute to aberrant vascular biology in children. By calling attention to this nascent field, we hope to increase research efforts to address important knowledge gaps in pediatric vascular biology that may inform the development of novel therapeutic strategies.

Increased Plasma Hyaluronan Levels are Associated With Acute Traumatic Coagulopathy.

INTRODUCTION: Acute traumatic coagulopathy (ATC) is an endogenous impairment in hemostasis that often contributes to early mortality after trauma. Endothelial glycocalyx damage is associated with trauma-induced coagulation abnormalities; however, the specific relationship between hyaluronan (HA), a key glycocalyx constituent, and ATC has not been evaluated. METHODS: We performed a secondary analysis of prospectively collected data from a recent study in which trauma patients (>18 years) admitted to our Level I trauma center with an ABC Score≥2 were enrolled. Partial thromboplastin time (PTT), international normalized ratio (INR), and thromboelastography (TEG) parameters were recorded at arrival. Injury characteristics and clinical outcomes were obtained. Plasma HA levels were measured in healthy controls (HC) and in trauma subjects at arrival (t = 0 h) and 12, 24, and 48 h. ATC was defined as admission INR>1.2 or PTT≥36.5 s. Comparisons of HA levels were assessed, and Spearman's correlations were performed between 0 h and 24 h HA levels, coagulation measures and clinical outcomes. P values < 0.05 were considered significant. RESULTS: Forty-eight trauma patients and 22 controls were enrolled for study. Sixteen trauma subjects were coagulopathic at admission. HA levels in subjects with ATC were higher than non-coagulopathic subjects at all time points and elevated above HC levels at 24 and 48 h. At arrival, HA levels correlated with TEG R-time, PTT, and INR. HA levels at 24 h correlated with increased transfusion requirements and intensive care unit and hospital lengths of stay. CONCLUSION: Shed HA is associated with early coagulation abnormalities in trauma patients, which may contribute to worse outcomes. These findings highlight the need for additional studies to evaluate the mechanistic role of HA in ATC.

Associations of Plasma Angiopoietins-1 and -2 and Angiopoietin-2/-1 Ratios With Measures of Organ Injury and Clinical Outcomes in Children With Sepsis: A Preliminary Report.

OBJECTIVES: Results from preclinical and adult sepsis studies suggest that the balance of circulating angiopoietin-1 and -2 levels, represented as angiopoietin-2/-1 ratios, plays a pivotal role in mediating vascular dysfunction and organ injury during sepsis. However, the relationship of plasma angiopoietins with organ injury and clinical outcomes in children with sepsis remains unknown. We sought to determine whether plasma angiopoietin-1 and -2 levels and angiopoietin-2/-1 ratios in the acute phase of sepsis correlated with measures of organ injury and clinical outcomes in children with sepsis. DESIGN: Prospective observational cohort study. SETTING: PICU within a tertiary freestanding children's hospital. PATIENTS: Children 18 years old or less and greater than 3 kg admitted to the PICU for sepsis. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Plasma angiopoietin-1 and -2 levels were measured in 38 children with sepsis 0-6, 24, 48, and 72 hours following PICU admission. Children with elevated pediatric Sequential Organ Failure Assessment scores on the third day after PICU admission demonstrated significantly higher 24-72-hour angiopoietin-2/-1 ratios predominantly as a function of higher angiopoietin-2 levels. In children with sepsis-induced organ dysfunction, angiopoietin-2/-1 ratios correlated with oxygenation indices and serum levels of creatinine and bilirubin. Forty-eight- and 72-hour angiopoietin-2/-1 ratios correlated with PICU length of stay (Spearman rho = 0.485, p = 0.004 and rho = 0.440, p = 0.015, respectively). CONCLUSIONS: In the acute phase of sepsis in children, plasma angiopoietin-2/-1 ratios rise significantly above control levels and correlate with measures of organ injury and worse clinical outcomes after 24 hours. Our findings suggest that angiopoietin dysregulation begins early in sepsis and, if sustained, may promote greater organ injury that can lead to worse clinical outcomes.

Heparanase-enhanced Shedding of Syndecan-1 and Its Role in Driving Disease Pathogenesis and Progression.

Both heparanase and syndecan-1 are known to be present and active in disease pathobiology. An important feature of syndecan-1 related to its role in pathologies is that it can be shed from the surface of cells as an intact ectodomain composed of the extracellular core protein and attached heparan sulfate and chondroitin sulfate chains. Shed syndecan-1 remains functional and impacts cell behavior both locally and distally from its cell of origin. Shedding of syndecan-1 is initiated by a variety of stimuli and accomplished predominantly by the action of matrix metalloproteinases. The accessibility of these proteases to the core protein of syndecan-1 is enhanced, and shedding facilitated, when the heparan sulfate chains of syndecan-1 have been shortened by the enzymatic activity of heparanase. Interestingly, heparanase also enhances shedding by upregulating the expression of matrix metalloproteinases. Recent studies have revealed that heparanase-induced syndecan-1 shedding contributes to the pathogenesis and progression of cancer and viral infection, as well as other septic and non-septic inflammatory states. This review discusses the heparanase/shed syndecan-1 axis in disease pathogenesis and progression, the potential of targeting this axis therapeutically, and the possibility that this axis is widespread and of influence in many diseases.

Temporal Dysregulation of the Angiopoietin-2/-1 Ratio After Trauma and Associations With Injury Characteristics and Outcomes.

Traumatic injury and hemorrhagic shock result in endothelial cell activation and vascular dysfunction that, if not corrected, can propagate multiorgan failure. Angiopoietin-1 and angiopoietin-2 are important regulators of endothelial cell function, and the ratio of plasma angiopoietin-2-to-1 is a useful indicator of overall vascular health. We therefore characterized plasma angiopoietin-2/-1 ratios over time after trauma in adults in an effort to gain insight into the pathophysiology that may drive post-traumatic vasculopathy and organ injury. We performed a single-center prospective observational study to measure plasma angiopoietin-1 and -2 levels and determine angiopoietin-2/-1 ratios in adult trauma patients upon hospital arrival and after 12, 24, and 48 h. Compared with levels in healthy adults, angiopoietin-1 levels were significantly elevated at hospital arrival, and angiopoietin-2 levels were significantly elevated at 12, 24, and 48 h. These kinetics translated in angiopoietin-2/-1 ratios that were significantly greater than controls at 24 and 48 h. After regression analysis, elevated angiopoietin-2 levels were independently associated with blunt injuries at admission, with coagulopathy at admission and 12 h, and with hemorrhagic shock at 24 and 48 h. Significant correlations were observed between both angiopoietins and 24-h transfusion requirements. Angiopoietin-2/-1 ratios correlated with mechanical ventilation duration and intensive care unit and hospital lengths of stay. In this study, we demonstrate novel temporal associations between angiopoietin dysregulation and blunt injuries, acute coagulopathy, and hemorrhagic shock. Moreover, our findings highlight the presence of endothelial activation following traumatic insults in adults that may contribute to worse clinical outcomes.

Correction: Role of heme in lung bacterial infection after trauma hemorrhage and stored red blood cell transfusion: A preclinical experimental study.

[This corrects the article DOI: 10.1371/journal.pmed.1002522.].

Genetic strategy to decrease complement activation with adenoviral therapies.

BACKGROUND: A major obstacle to using recombinant adenoviral vectors in gene therapy is the natural ability of human adenovirus to activate the classical and alternate complement pathways. These innate immune responses contribute to hepatic adenoviral uptake following systemic delivery and enhance the humoral immune responses associated with adenoviral infection. METHODS: A recombinant Ad5 vector was genetically modified to display a peptide sequence ("rH17d'"), a known inhibitor of the classical complement pathway. The replication-defective vectors Ad5.HVR2-rH17d' and Ad5.HVR5-rH17d' were constructed by engineering the rH17d' peptide into the hypervariable region (HVR)-2 or HVR5 of their major capsid protein hexon. Control Ad5 vectors were created by incorporation of a 6-histidine (His6)-insert in either HVR2 or HVR5 (Ad5.HVR2-His6 and Ad5.HVR5-His6, respectively). All vectors encoded CMV promoter-controlled firefly luciferase (Luc). The four vectors were evaluated in TIB76 mouse liver cells and immunocompetent mice to compare infectivity and liver sequestration, respectively. RESULTS: In vitro studies demonstrated that preincubation of all the Ad5 vectors with fresh serum significantly increased their gene transfer relative to preincubation with PBS except Ad5.HVR5-rH17d', whose infectivity of liver cells showed no serum-mediated enhancement. In line with that, mice injected with Ad5.HVR2-rH17d' or Ad5.HVR5-rH17d' showed significantly lower luciferase expression levels in the liver as compared to the respective control vectors, whereas efficiency of tumor transduction by rH17d' and His6 vectors following their intratumoral injection was similar. CONCLUSIONS: Displaying a complement-inhibiting peptide on the Ad5 capsid surface by genetic modification of the hexon protein could be a suitable strategy for reducing Ad5 liver tropism (Ad5 sequestration by liver), which may be applicable to other gene therapy vectors with natural liver tropism.

Plasma Angiopoietin-2/-1 Ratio is Elevated and Angiopoietin-2 Levels Correlate With Plasma Syndecan-1 Following Pediatric Trauma.

BACKGROUND: Angiopoietin-1 (Agpt-1) and Agpt-2 are cytokine regulators of vascular endothelial integrity. Elevated plasma Agpt-2 levels and ratios of Agpt-2:Agpt-1 are associated with adverse outcomes in adult trauma and pediatric sepsis populations. However, the behavior of the angiopoietins after pediatric trauma has not been characterized, and their relationship to endothelial glycocalyx damage, indicated by plasma syndecan-1 (Syn-1) levels, has not been established. METHODS: We performed a secondary analysis of prospectively collected data from 52 pediatric trauma patients and 12 control patients at a level one pediatric trauma center from 2013 to 2016. We measured Agpt-1, Agpt-2, and Syn-1 levels from plasma taken upon hospital arrival and 24 h after admission. Angiopoietin levels were compared to controls, and the correlation between Agpt-2 and Syn-1 was assessed. RESULTS: Plasma Agpt-1 and Agpt-2 levels are elevated immediately after pediatric trauma compared with controls. At 24 h, trauma patients demonstrated significantly elevated plasma Agpt-2:Agpt-1 ratios relative to controls due to decline of Agpt-1 levels to near that of controls. Higher 24-h Agpt-2 levels are associated with more hypoperfusion, and elevated 24-h Agpt-2:Agpt-1 ratios are associated with adverse clinical outcomes. Significant positive correlations between Agpt-2 and Syn-1 upon admission and at 24 h after injury were identified. CONCLUSION: Our findings suggest dysregulation of circulating angiopoietins after pediatric trauma that may be linked to endothelial glycocalyx injury. Larger prospective studies are needed to validate these findings and determine the relationship of Agpt-2 with other markers of endotheliopathy.

Role of heme in lung bacterial infection after trauma hemorrhage and stored red blood cell transfusion: A preclinical experimental study.

BACKGROUND: Trauma is the leading cause of death and disability in patients aged 1-46 y. Severely injured patients experience considerable blood loss and hemorrhagic shock requiring treatment with massive transfusion of red blood cells (RBCs). Preclinical and retrospective human studies in trauma patients have suggested that poorer therapeutic efficacy, increased severity of organ injury, and increased bacterial infection are associated with transfusion of large volumes of stored RBCs, although the mechanisms are not fully understood. METHODS AND FINDINGS: We developed a murine model of trauma hemorrhage (TH) followed by resuscitation with plasma and leukoreduced RBCs (in a 1:1 ratio) that were banked for 0 (fresh) or 14 (stored) days. Two days later, lungs were infected with Pseudomonas aeruginosa K-strain (PAK). Resuscitation with stored RBCs significantly increased the severity of lung injury caused by P. aeruginosa, as demonstrated by higher mortality (median survival 35 h for fresh RBC group and 8 h for stored RBC group; p < 0.001), increased pulmonary edema (mean [95% CI] 106.4 µl [88.5-124.3] for fresh RBCs and 192.5 µl [140.9-244.0] for stored RBCs; p = 0.003), and higher bacterial numbers in the lung (mean [95% CI] 1.2 × 10(7) [-1.0 × 10(7) to 2.5 × 10(7)] for fresh RBCs and 3.6 × 10(7) [2.5 × 10(7) to 4.7 × 10(7)] for stored RBCs; p = 0.014). The mechanism underlying this increased infection susceptibility and severity was free-heme-dependent, as recombinant hemopexin or pharmacological inhibition or genetic deletion of toll-like receptor 4 (TLR4) during TH and resuscitation completely prevented P. aeruginosa-induced mortality after stored RBC transfusion (p < 0.001 for all groups relative to stored RBC group). Evidence from studies transfusing fresh and stored RBCs mixed with stored and fresh RBC supernatants, respectively, indicated that heme arising both during storage and from RBC hemolysis post-resuscitation plays a role in increased mortality after PAK (p < 0.001). Heme also increased endothelial permeability and inhibited macrophage-dependent phagocytosis in cultured cells. Stored RBCs also increased circulating high mobility group box 1 (HMGB1; mean [95% CI] 15.4 ng/ml [6.7-24.0] for fresh RBCs and 50.3 ng/ml [12.3-88.2] for stored RBCs), and anti-HMGB1 blocking antibody protected against PAK-induced mortality in vivo (p = 0.001) and restored macrophage-dependent phagocytosis of P. aeruginosa in vitro. Finally, we showed that TH patients, admitted to the University of Alabama at Birmingham ER between 1 January 2015 and 30 April 2016 (n = 50), received high micromolar-millimolar levels of heme proportional to the number of units transfused, sufficient to overwhelm endogenous hemopexin levels early after TH and resuscitation. Limitations of the study include lack of assessment of temporal changes in different products of hemolysis after resuscitation and the small sample size precluding testing of associations between heme levels and adverse outcomes in resuscitated TH patients. CONCLUSIONS: We provide evidence that large volume resuscitation with stored blood, compared to fresh blood, in mice increases mortality from subsequent pneumonia, which occurs via mechanisms sensitive to hemopexin and TLR4 and HMGB1 inhibition.

Leukoreduction of packed red blood cells attenuates proinflammatory properties of storage-derived microvesicles.

BACKGROUND: Leukoreduction prior to packed red blood cell (pRBC) storage is not a universally accepted practice. Our laboratory has previously shown that microvesicles (MVs) accumulate in pRBC units during storage and play an important role in lung injury after resuscitation. Currently, the effect of leukoreduction on MV formation in stored pRBC units is unknown. In the present study, we investigated the hypothesis that leukoreduction of pRBC units prior to storage would attenuate the production of MVs and decrease pulmonary inflammation after hemorrhage and resuscitation. METHODS: Leukoreduced and nonleukoreduced pRBC units were prepared from human donors and C57/Bl6 mice and stored for up to 42 d and 14 d, respectively. At intervals during storage, MVs were isolated from pRBC units, quantified and characterized based on size, morphology, and levels of proinflammatory cytokines. In additional experiments, mice underwent controlled hemorrhage followed by resuscitation with normal saline (NS) with or without equal numbers of MVs isolated from leukoreduced or nonleukoreduced stored mouse pRBC. Histologic lung sections were evaluated for the presence of tissue edema and inflammatory cells. RESULTS: For both human and mouse pRBCs, the number of MVs significantly increased throughout the storage period. There were significantly fewer MVs present in leukoreduced units. The average MV size significantly increased over time and was similar between groups. Levels of interleukin 1α (IL-1α), regulated on activation, normal T cell expressed and secreted (RANTES), and macrophage-derived chemokine (MDC) were lower in MVs from leukoreduced pRBC units as compared with MVs from nonleukoreduced units. Hemorrhaged mice resuscitated with NS with the addition of MV from leukoreduced pRBC demonstrated significantly less pulmonary edema and inflammatory cell recruitment as compared to those resuscitated with NS with the addition of MV from nonleukoreduced pRBC. CONCLUSIONS: Prestorage leukoreduction of pRBC units reduces the formation and proinflammatory properties of MV, which in turn decreases lung injury secondary to MV from stored pRBC units after hemorrhage and resuscitation.

Methods to Evaluate Cell Growth, Viability, and Response to Treatment in a Tissue Engineered Breast Cancer Model.

The use of in vitro, engineered surrogates in the field of cancer research is of interest for studies involving mechanisms of growth and metastasis, and response to therapeutic intervention. While biomimetic surrogates better model human disease, their complex composition and dimensionality make them challenging to evaluate in a real-time manner. This feature has hindered the broad implementation of these models, particularly in drug discovery. Herein, several methods and approaches for the real-time, non-invasive analysis of cell growth and response to treatment in tissue-engineered, three-dimensional models of breast cancer are presented. The tissue-engineered surrogates used to demonstrate these methods consist of breast cancer epithelial cells and fibroblasts within a three dimensional volume of extracellular matrix and are continuously perfused with nutrients via a bioreactor system. Growth of the surrogates over time was measured using optical in vivo (IVIS) imaging. Morphologic changes in specific cell populations were evaluated by multi-photon confocal microscopy. Response of the surrogates to treatment with paclitaxel was measured by optical imaging and by analysis of lactate dehydrogenase and caspase-cleaved cytokeratin 18 in the perfused medium. Each method described can be repeatedly performed during culture, allowing for real-time, longitudinal analysis of cell populations within engineered tumor models.

Macrophage-derived chemokine (CCL22) is a novel mediator of lung inflammation following hemorrhage and resuscitation.

Resuscitation of patients after hemorrhage often results in pulmonary inflammation and places them at risk for the development of acute respiratory distress syndrome. Our previous data indicate that macrophage-derived chemokine (MDC/CCL22) is elevated after resuscitation, but its direct role in this inflammatory response is unknown. Macrophage-derived chemokine signaling through the C-C chemokine receptor type 4 (CCR4) is implicated in other pulmonary proinflammatory conditions, leading us to hypothesize that MDC may also play a role in the pathogenesis of lung inflammation following hemorrhage and resuscitation. To test this, C57BL/6 mice underwent pressure-controlled hemorrhage followed by resuscitation with lactated Ringer's solution. Pulmonary inflammation and inflammatory cell recruitment were analyzed with histological staining, and serum- and tissue-level cytokines were measured by enzyme-linked immunosorbent assay. Pulmonary inflammation and cell recruitment following hemorrhage and resuscitation were associated with systemic MDC levels. Inhibition of MDC via injection of a specific neutralizing antibody prior to hemorrhage and resuscitation significantly reduced pulmonary levels of the chemotactic cytokines keratinocyte-derived chemokine and macrophage inflammatory proteins 2 and 1α, as well as inflammatory cell recruitment to the lungs. Intravenous administration of recombinant MDC prior to resuscitation augmented pulmonary inflammation and cell recruitment. Histological evaluation revealed the expression of CCR4 within the bronchial epithelium, and in vitro treatment of activated bronchial epithelial cells with MDC resulted in production and secretion of neutrophil chemokines. The present study identifies MDC as a novel mediator of lung inflammation after hemorrhage and resuscitation. Macrophage-derived chemokine neutralization may provide a therapeutic strategy to mitigate this inflammatory response.

Hemostatic properties and the role of cell receptor recognition in human hair keratin protein hydrogels.

Driven by new discoveries in stem-cell biology and regenerative medicine, there is broad interest in biomaterials that go beyond basic interactions with cells and tissues to actively direct and sustain cellular behavior. Keratin biomaterials have the potential to achieve these goals but have been inadequately described in terms of composition, structure, and cell-instructive characteristics. In this manuscript we describe and characterize a keratin-based biomaterial, demonstrate self-assembly of cross-linked hydrogels, investigate a cell-specific interaction that is dependent on the hydrogel structure and mediated by specific biomaterial-receptor interactions, and show one potential medical application that relies on receptor binding - the ability to achieve hemostasis in a lethal liver injury model. Keratin biomaterials represent a significant advance in biotechnology as they combine the compatibility of natural materials with the chemical flexibility of synthetic materials. These characteristics allow for a system that can be formulated into several varieties of cell-instructive biomaterials with potential uses in tissue engineering, regenerative medicine, drug and cell delivery, and trauma.

Microparticles from stored red blood cells activate neutrophils and cause lung injury after hemorrhage and resuscitation.

BACKGROUND: Transfusion of stored blood is associated with increased complications. Microparticles (MPs) are small vesicles released from RBCs that can induce cellular dysfunction, but the role of RBC-derived MPs in resuscitation from hemorrhagic shock is unknown. In the current study, we examined the effects of RBC-derived MPs on the host response to hemorrhage and resuscitation. STUDY DESIGN: MPs were isolated from murine packed RBC units, quantified using flow cytometry, and injected into healthy mice. Separate groups of mice underwent hemorrhage and resuscitation with and without packed RBC-derived MPs. Lungs were harvested for histology and neutrophil accumulation and assessed by myeloperoxidase content. Human neutrophils were treated with human RBC-derived MPs and CD11b expression, superoxide production, and phagocytic activity were determined. RESULTS: Stored murine packed RBC units contained increased numbers of RBC-derived MPs compared with fresh units. Hemorrhaged mice resuscitated with MPs demonstrated substantially increased pulmonary neutrophil accumulation and altered lung histology compared with mice resuscitated without MPs. Intravenous injection of MPs into normal mice resulted in neutrophil priming, evidenced by increased neutrophil CD11b expression. Human neutrophils treated with RBC-derived MPs demonstrated increased CD11b expression, increased superoxide production, and enhanced phagocytic ability compared with untreated neutrophils. CONCLUSIONS: Stored packed RBC units contain increased numbers of RBC-derived MPs. These MPs appear to contribute to neutrophil priming and activation. The presence of MPs in stored units can be associated with adverse effects, including lung injury, after transfusion.