Investigating the Relationship between Bleeding, Clotting, and Coagulopathy During Automated Partial REBOA Strategies in a Highly Lethal Porcine Hemorrhage Model.

BACKGROUND: Death due to hemorrhagic shock, particularly, non-compressible truncal hemorrhage (NCTH), remains one of the leading causes of potentially preventable deaths. Automated partial and intermittent resuscitative endovascular balloon occlusion of the aorta (i.e., pREBOA and iREBOA, respectively) are lifesaving endovascular strategies aimed to achieve quick hemostatic control while mitigating distal ischemia. In iREBOA, the balloon is titrated from full occlusion to no occlusion intermittently whereas in pREBOA, a partial occlusion is maintained. Therefore, these two interventions impose different hemodynamic conditions, which may impact coagulation and the endothelial glycocalyx layer (EGL). In this study, we aimed to characterize the clotting kinetics and coagulopathy associated with iREBOA and pREBOA, using thromboelastography (TEG). We hypothesized that iREBOA would be associated with a more hypercoagulopathic response compared to pREBOA due to more oscillatory flow. METHODS: Yorkshire swine (n = 8/group) were subjected to an uncontrolled hemorrhage by liver transection, followed by 90 minutes of automated partial REBOA (pREBOA), intermittent REBOA (iREBOA), or no balloon support (Control). Hemodynamic parameters were continuously recorded, and blood samples were serially collected during the experiment (i.e., 8 key time points: baseline (BL), T0, T10, T30, T60, T90, T120, T210 minutes). Citrated kaolin heparinase (CKH) assays were run on a TEG 5000 (Haemonetics, Niles, IL). General linear mixed models were employed to compare differences in TEG parameters between groups and over time using STATA (v17; College Station, TX), while adjusting for sex and weight. RESULTS: As expected, iREBOA was associated with more oscillations in proximal pressure (and greater magnitudes of peak pressure) because of the intermittent periods of full aortic occlusion and complete balloon deflation, compared to pREBOA. Despite these differences in acute hemodynamics, there were no significant differences in any of the TEG parameters between iREBOA and pREBOA groups. However, animals in both groups experienced a significant reduction in clotting times (R-time: p < 0.001; K-time: p < 0.001) and clot strength (MA: p = 0.01; G: p = 0.02) over the duration of the experiment. CONCLUSIONS: Despite observing acute differences in peak proximal pressures between iREBOA and pREBOA groups, we did not observe any significant differences in TEG parameters between iREBOA and pREBOA. The changes in TEG profiles were significant over time, indicating that a severe hemorrhage followed by both pREBOA and iREBOA can result in faster clotting reaction times (i.e., R-times). Nevertheless, when considering the significant reduction in transfusion requirements and more stable hemodynamic response in the pREBOA group, there may be some evidence favoring pREBOA usage over iREBOA.

MAN VERSUS MACHINE: PROVIDER DIRECTED VERSUS PRECISION AUTOMATED CRITICAL CARE MANAGEMENT IN A PORCINE MODEL OF DISTRIBUTIVE SHOCK.

ABSTRACT: Background: Critical care management of shock is a labor-intensive process. Precision Automated Critical Care Management (PACC-MAN) is an automated closed-loop system incorporating physiologic and hemodynamic inputs to deliver interventions while avoiding excessive fluid or vasopressor administration. To understand PACC-MAN efficacy, we compared PACC-MAN to provider-directed management (PDM). We hypothesized that PACC-MAN would achieve equivalent resuscitation outcomes to PDM while maintaining normotension with lower fluid and vasopressor requirements. Methods : Twelve swine underwent 30% controlled hemorrhage over 30 min, followed by 45 min of aortic occlusion to generate a vasoplegic shock state, transfusion to euvolemia, and randomization to PACC-MAN or PDM for 4.25 h. Primary outcomes were total crystalloid volume, vasopressor administration, total time spent at hypotension (mean arterial blood pressure <60 mm Hg), and total number of interventions. Results : Weight-based fluid volumes were similar between PACC-MAN and PDM; median and IQR are reported (73.1 mL/kg [59.0-78.7] vs. 87.1 mL/kg [79.4-91.8], P = 0.07). There was no statistical difference in cumulative norepinephrine (PACC-MAN: 33.4 µg/kg [27.1-44.6] vs. PDM: 7.5 [3.3-24.2] µg/kg, P = 0.09). The median percentage of time spent at hypotension was equivalent (PACC-MAN: 6.2% [3.6-7.4] and PDM: 3.1% [1.3-6.6], P = 0.23). Urine outputs were similar between PACC-MAN and PDM (14.0 mL/kg vs. 21.5 mL/kg, P = 0.13). Conclusion : Automated resuscitation achieves equivalent resuscitation outcomes to direct human intervention in this shock model. This study provides the first translational experience with the PACC-MAN system versus PDM.

Targeted Analysis of the Size Distribution of Heavy Chain-Modified Hyaluronan with Solid-State Nanopores.

The glycosaminoglycan hyaluronan (HA) plays important roles in diverse physiological functions where the distribution of its molecular weight (MW) can influence its behavior and is known to change in response to disease conditions. During inflammation, HA undergoes a covalent modification in which heavy chain subunits of the inter-alpha-inhibitor family of proteins are transferred to its structure, forming heavy chain-HA (HC•HA) complexes. While limited assessments of HC•HA have been performed previously, determining the size distribution of its HA component remains a challenge. Here, we describe a selective method for extracting HC•HA from mixtures that yields material amenable to MW analysis with a solid-state nanopore sensor. After demonstrating the approach in vitro, we validate extraction of HC•HA from osteoarthritic human synovial fluid as a model complex biological matrix. Finally, we apply our technique to pathophysiology by measuring the size distributions of HC•HA and total HA in an equine model of synovitis.

Investigating the variability in pressure-volume relationships during hemorrhage and aortic occlusion.

Introduction: The pressure-volume (P-V) relationships of the left ventricle are the classical benchmark for studying cardiac mechanics and pumping function. Perturbations in the P-V relationship (or P-V loop) can be informative and guide the management of heart failure, hypovolemia, and aortic occlusion. Traditionally, P-V loop analyses have been limited to a single-beat P-V loop or an average of consecutive P-V loops (e.g., 10 cardiac cycles). While there are several algorithms to obtain single-beat estimations of the end-systolic and end-diastolic pressure-volume relations (i.e., ESPVR and EDPVR, respectively), there remains a need to better evaluate the variations in P-V relationships longitudinally over time. This is particularly important when studying acute and transient hemodynamic and cardiac events, such as active hemorrhage or aortic occlusion. In this study, we aim to investigate the variability in P-V relationships during hemorrhagic shock and aortic occlusion, by leveraging on a previously published porcine hemorrhage model. Methods: Briefly, swine were instrumented with a P-V catheter in the left ventricle of the heart and underwent a 25% total blood volume hemorrhage over 30 min, followed by either Zone 1 complete aortic occlusion (i.e., REBOA), Zone 1 endovascular variable aortic control (EVAC), or no occlusion as a control, for 45 min. Preload-independent metrics of cardiac performance were obtained at predetermined time points by performing inferior vena cava occlusion during a ventilatory pause. Continuous P-V loop data and other hemodynamic flow and pressure measurements were collected in real-time using a multi-channel data acquisition system. Results: We developed a custom algorithm to quantify the time-dependent variance in both load-dependent and independent cardiac parameters from each P-V loop. As expected, all pigs displayed a significant decrease in the end-systolic pressures and volumes (i.e., ESP, ESV) after hemorrhage. The variability in response to hemorrhage was consistent across all three groups. However, upon introduction of REBOA, we observed significantly high levels of variability in both load-dependent and independent cardiac metrics such as ESP, ESV, and the slope of ESPVR (Ees). For instance, pigs receiving REBOA experienced a 342% increase in ESP from hemorrhage, while pigs receiving EVAC experienced only a 188% increase. The level of variability within the EVAC group was consistently less than that of the REBOA group, which suggests that the EVAC group may be more supportive of maintaining healthier cardiac performance than complete occlusion with REBOA. Discussion: In conclusion, we successfully developed a novel algorithm to reliably quantify the single-beat and longitudinal P-V relations during hemorrhage and aortic occlusion. As expected, hemorrhage resulted in smaller P-V loops, reflective of decreased preload and afterload conditions; however, the cardiac output and heart rate were preserved. The use of REBOA and EVAC for 44 min resulted in the restoration of baseline afterload and preload conditions, but often REBOA exceeded baseline pressure conditions to an alarming level. The level of variability in response to REBOA was significant and could be potentially associated to cardiac injury. By quantifying each P-V loop, we were able to capture the variability in all P-V loops, including those that were irregular in shape and believe that this can help us identify critical time points associated with declining cardiac performance during hemorrhage and REBOA use.

Automated partial resuscitative endovascular balloon occlusion of the aorta reduces blood loss and hypotension in a highly lethal porcine liver injury model.

BACKGROUND: Partial and intermittent resuscitative endovascular balloon occlusion of the aorta (pREBOA and iREBOA, respectively) are lifesaving techniques designed to extend therapeutic duration, mitigate ischemia, and bridge patients to definitive hemorrhage control. We hypothesized that automated pREBOA balloon titration compared with automated iREBOA would reduce blood loss and hypotensive episodes over a 90-minute intervention phase compared with iREBOA in an uncontrolled liver hemorrhage swine model. METHODS: Twenty-four pigs underwent an uncontrolled hemorrhage by liver transection and were randomized to automated pREBOA (n = 8), iREBOA (n = 8), or control (n = 8). Once hemorrhagic shock criteria were met, controls had the REBOA catheter removed and received transfusions only for hypotension. The REBOA groups received 90 minutes of either iREBOA or pREBOA therapy. Surgical hemostasis was obtained, hemorrhage volume was quantified, and animals were transfused to euvolemia and then underwent 1.5 hours of automated critical care. RESULTS: The control group had significantly higher mortality rate (5 of 8) compared with no deaths in both REBOA groups, demonstrating that the liver injury is highly lethal ( p = 0.03). During the intervention phase, animals in the iREBOA group spent a greater proportion of time in hypotension than the pREBOA group (20.7% [16.2-24.8%] vs. 0.76% [0.43-1.14%]; p < 0.001). The iREBOA group required significantly more transfusions than pREBOA (21.0 [20.0-24.9] mL/kg vs. 12.1 [9.5-13.9] mL/kg; p = 0.01). At surgical hemostasis, iREBOA had significantly higher hemorrhage volumes compared with pREBOA (39.2 [29.7-44.95] mL/kg vs. 24.7 [21.6-30.8] mL/kg; p = 0.04). CONCLUSION: Partial REBOA animals spent significantly less time at hypotension and had decreased transfusions and blood loss. Both pREBOA and iREBOA prevented immediate death compared with controls. Further refinement of automated pREBOA is necessary, and controller algorithms may serve as vital control inputs for automated transfusion. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level III.

ENDOTHELIAL GLYCOCALYX SHEDDING IN INTRA-ABDOMINAL SEPSIS: A FEASIBILITY STUDY.

ABSTRACT: Background: The endothelial glycocalyx layer (EGL) is a complex meshwork of glycosaminoglycans and proteoglycans that protect the vascular endothelium. Cleavage or shedding of EGL-specific biomarkers, such as hyaluronic acid (HA) and syndecan-1 (SDC-1, CD138) in plasma, have been shown to be associated with poor clinical outcomes. However, it is unclear whether levels of circulating EGL biomarkers are representative of the EGL injury within the tissues. The objective of the present feasibility study was to describe a pathway for plasma and tissue procurement to quantify EGL components in a cohort of surgical patients with intra-abdominal sepsis. We sought to compare differences between tissue and plasma EGL biomarkers and to determine whether EGL shedding within the circulation and/or tissues correlated with clinical outcomes. Methods: This was a prospective, observational, single-center feasibility study of adult patients (N = 15) with intra-abdominal sepsis, conducted under an approved institutional review boards. Blood and resected tissue (pathologic specimen and unaffected peritoneum) samples were collected from consented subjects at the time of operation and 24-48 hours after surgery. Endothelial glycocalyx layer biomarkers (i.e., HA and SDC-1) were quantified in both tissue and plasma samples using a CD138 stain and ELISA kit, respectively. Pairwise comparisons were made between plasma and tissue levels. In addition, we tested the relationships between measured EGL biomarkers and clinical status and patient outcomes. Results: Fifteen patients with intra-abdominal sepsis were enrolled in the study. Elevations in EGL-specific circulating biomarkers (HA, SDC-1) were positively correlated with postoperative SOFA scores and weakly associated with resuscitative volumes at 24 hours. Syndecan-1 levels from resected pathologic tissue significantly correlated with SOFA scores at all time points ( R = 0.69 and P < 0.0001) and positively correlated with resuscitation volumes at 24 hours ( R = 0.41 and P = 0.15 for t = 24 hours). Tissue and circulating HA and SDC-1 positively correlated with SOFA >6. Conclusions: Elevations in both circulating and tissue EGL biomarkers were positively correlated with postoperative SOFA scores at 24 hours, with resected pathologic tissue EGL levels displaying significant correlations with SOFA scores at all time points. Tissue and circulating EGL biomarkers were positively correlated at higher SOFA scores (SOFA > 6) and could be used as indicators of resuscitative needs within 24 hours of surgery. The present study demonstrates the feasibility of tissue and plasma procurement in the operating room, although larger studies are needed to evaluate the predictive value of these EGL biomarkers for patients with intra-abdominal sepsis.

Development of a computational fluid dynamic model to investigate the hemodynamic impact of REBOA.

Background: Resuscitative endovascular balloon occlusion of the aorta (REBOA) is a lifesaving intervention for major truncal hemorrhage. Balloon-tipped arterial catheters are inserted via the femoral artery to create a temporary occlusion of the aorta, which minimizes the rate of internal bleeding until definitive surgery can be conducted. There is growing concern over the resultant hypoperfusion and potential damage to tissues and organs downstream of REBOA. To better understand the acute hemodynamic changes imposed by REBOA, we developed a three-dimensional computational fluid dynamic (CFD) model under normal, hemorrhage, and aortic occlusion conditions. The goal was to characterize the acute hemodynamic changes and identify regions within the aortic vascular tree susceptible to abnormal flow and shear stress. Methods: Hemodynamic data from established porcine hemorrhage models were used to build a CFD model. Swine underwent 20% controlled hemorrhage and were randomized to receive a full or partial aortic occlusion. Using CT scans, we generated a pig-specific aortic geometry and imposed physiologically relevant inlet flow and outlet pressure boundary conditions to match in vivo data. By assuming non-Newtonian fluid properties, pressure, velocity, and shear stresses were quantified over a cardiac cycle. Results: We observed a significant rise in blood pressure (∼147 mmHg) proximal to REBOA, which resulted in increased flow and shear stress within the ascending aorta. Specifically, we observed high levels of shear stress within the subclavian arteries (22.75 Pa). Alternatively, at the site of full REBOA, wall shear stress was low (0.04 ± 9.07E-4 Pa), but flow oscillations were high (oscillatory shear index of 0.31). Comparatively, partial REBOA elevated shear levels to 84.14 ± 19.50 Pa and reduced flow oscillations. Our numerical simulations were congruent within 5% of averaged porcine experimental data over a cardiac cycle. Conclusion: This CFD model is the first to our knowledge to quantify the acute hemodynamic changes imposed by REBOA. We identified areas of low shear stress near the site of occlusion and high shear stress in the subclavian arteries. Future studies are needed to determine the optimal design parameters of endovascular hemorrhage control devices that can minimize flow perturbations and areas of high shear.

Looking Back on Graduate BME Admissions Data: Lessons Learned and Implications for Holistic Review and Diversity.

Graduate school applications in Biomedical Engineering (BME) are steadily rising, making competition stiffer, applications more complex, and reviews more resource intensive. Holistic reviews are being increasingly adopted to support increased diversity, equity, and inclusion in graduate student BME admissions, but which application metrics are the strongest predictors of admission and enrollment into BME programs remains unclear. In this perspectives article, we aim to shed light on some of the key predictors of student acceptance in graduate school. We share data from a three-year retrospective review of our own institution's graduate BME applications and admission rates and review the influence of grade point averages (GPA), standardized test scores (e.g., GRE), and prior research experience on graduate school admission rates. We also examine how the waiver of GRE requirements has changed the landscape of BME graduate applications in recent years. Finally, we discuss efforts taken by our institution and others to develop and implement holistic reviews of graduate applications that encourage students from underrepresented backgrounds to apply and successfully gain admission to graduate school. We share five key lessons we learned by performing the retrospective review and encourage other institutions to "self-reflect" and examine their historical graduate admissions data and past practices. Efforts aimed at engaging faculty to overcome their own implicit biases, engaging with underrepresented students in hands-on, research-intensive programs, and networking with diverse student populations have strong potential to enhance the diversity of BME graduate programs and our STEM workforce. Supplementary Information: The online version contains supplementary material available at 10.1007/s43683-022-00080-5.

Unmasking the Confounder: The Inherent Physiologic Variability of Swine During an Automated Experimental Model of Ischemia-Reperfusion Injury.

BACKGROUND: We sought to determine the magnitude of the inherent inter-animal physiologic variability by automating a porcine Resuscitative Endovascular Balloon Occlusion of the Aorta (REBOA) protocol to minimize external influences that might alter physiology and confound experimental results. METHODS: Swine (n = 42) underwent a controlled 30% blood volume hemorrhage followed by 30 minutes of REBOA (ie, ischemic phase). The animals were weaned from REBOA autonomously over 15 minutes, beginning the reperfusion phase, while continuing to provide partial flow balloon support to maintain a target proximal mean arterial pressure (pMAP) of 65 mmHg. Simultaneously, shed blood was re-transfused as part of the resuscitation efforts. Physiologic data were continuously recorded, and serum samples were serially collected. Baseline characteristics, variance in vital signs, and 8-isoprostane levels were quantified during hemorrhage, REBOA, and reperfusion phases. RESULTS: There was no significant difference in baseline physiology across animals (P > .05). Hemodynamic variability was highest for pMAP during the ischemic phase (P = .001) and for distal mean arterial pressure (dMAP) during the weaning/reperfusion phase (P = .001). The latter finding indicated the variable physiologic response to ischemia-reperfusion injury, as the automated balloon support required by each animal to maintain pMAP was highly variable. Circulating 8-isoprostane variance was significantly higher following the start of reperfusion compared to baseline levels (P = .001). DISCUSSION: Despite subjecting animals to a highly consistent ischemia-reperfusion injury through automation, we noted significant variability in the hemodynamic and biochemical response. These findings illustrate the inherent physiologic variability and potential limitations of porcine large animal models for the study of shock.

Optimizing the sensitivity and resolution of hyaluronan analysis with solid-state nanopores.

Hyaluronan (HA) is an essential carbohydrate in vertebrates that is a potentially robust bioindicator due to its critical roles in diverse physiological functions in health and disease. The intricate size-dependent function that exists for HA and its low abundance in most biological fluids have highlighted the need for sensitive technologies to provide accurate and quantitative assessments of polysaccharide molecular weight and concentration. We have demonstrated that solid state (SS-) nanopore technology can be exploited for this purpose, given its molecular sensitivity and analytical capacity, but there remains a need to further understand the impacts of experimental variables on the SS-nanopore signal for optimal interpretation of results. Here, we use model quasi-monodisperse HA polymers to determine the dependence of HA signal characteristics on a range of SS-nanopore measurement conditions, including applied voltage, pore diameter, and ionic buffer asymmetry. Our results identify important factors for improving the signal-to-noise ratio, resolution, and sensitivity of HA analysis with SS-nanopores.

Methods for isolating and analyzing physiological hyaluronan: a review.

The carbohydrate hyaluronan (or hyaluronic acid, HA) is found in all human tissues and biofluids where it has wide-ranging functions in health and disease that are dictated by both its abundance and size. Consequently, hyaluronan evaluation in physiological samples has significant translational potential. Although the analytical tools and techniques for probing other biomolecules such as proteins and nucleic acids have become standard approaches in biochemistry, those available for investigating hyaluronan are less well established. In this review, we survey methods related to the assessment of native hyaluronan in biological specimens, including protocols for separating it from biological matrices and technologies for determining its concentration and molecular weight.

Exploiting three-dimensional human hepatic constructs to investigate the impact of rs174537 on fatty acid metabolism.

The Modern Western Diet has been associated with the rise in metabolic and inflammatory diseases, including obesity, diabetes, and cardiovascular disease. This has been attributed, in part, to the increase in dietary omega-6 polyunsaturated fatty acid (PUFA) consumption, specifically linoleic acid (LA), arachidonic acid (ARA), and their subsequent metabolism to pro-inflammatory metabolites which may be driving human disease. Conversion of dietary LA to ARA is regulated by genetic variants near and within the fatty acid desaturase (FADS) haplotype block, most notably single nucleotide polymorphism rs174537 is strongly associated with FADS1 activity and expression. This variant and others within high linkage disequilibrium may potentially explain the diversity in both diet and inflammatory mediators that drive chronic inflammatory disease in human populations. Mechanistic exploration into this phenomenon using human hepatocytes is limited by current two-dimensional culture models that poorly replicate in vivo functionality. Therefore, we aimed to develop and characterize a three-dimensional hepatic construct for the study of human PUFA metabolism. Primary human hepatocytes cultured in 3D hydrogels were characterized for their capacity to represent basic lipid processing functions, including lipid esterification, de novo lipogenesis, and cholesterol efflux. They were then exposed to control and LA-enriched media and reproducibly displayed allele-specific metabolic activity of FADS1, based on genotype at rs174537. Hepatocytes derived from individuals homozygous with the minor allele at rs174537 (i.e., TT) displayed the slowest metabolic conversion of LA to ARA and significantly reduced FADS1 and FADS2 expression. These results support the feasibility of using 3D human hepatic cultures for the study of human PUFA and lipid metabolism and relevant gene-diet interactions, thereby enabling future nutrition targets in humans.

Exploiting maleimide-functionalized hyaluronan hydrogels to test cellular responses to physical and biochemical stimuli.

Currentin vitrothree-dimensional (3D) models of liver tissue have been limited by the inability to study the effects of specific extracellular matrix (ECM) components on cell phenotypes. This is in part due to limitations in the availability of chemical modifications appropriate for this purpose. For example, hyaluronic acid (HA), which is a natural ECM component within the liver, lacks key ECM motifs (e.g. arginine-glycine-aspartic acid (RGD) peptides) that support cell adhesion. However, the addition of maleimide (Mal) groups to HA could facilitate the conjugation of ECM biomimetic peptides with thiol-containing end groups. In this study, we characterized a new crosslinkable hydrogel (i.e. HA-Mal) that yielded a simplified ECM-mimicking microenvironment supportive of 3D liver cell culture. We then performed a series of experiments to assess the impact of physical and biochemical signaling in the form of RGD peptide incorporation and transforming growth factorß(TGF-ß) supplementation, respectively, on hepatic functionality. Hepatic stellate cells (i.e. LX-2) exhibited increased cell-matrix interactions in the form of cell spreading and elongation within HA-Mal matrices containing RGD peptides, enabling physical adhesions, whereas hepatocyte-like cells (HepG2) had reduced albumin and urea production. We further exposed the encapsulated cells to soluble TGF-ßto elicit a fibrosis-like state. In the presence of TGF-ßbiochemical signals, LX-2 cells became activated and HepG2 functionality significantly decreased in both RGD-containing and RGD-free hydrogels. Altogether, in this study we have developed a hydrogel biomaterial platform that allows for discrete manipulation of specific ECM motifs within the hydrogel to better understand the roles of cell-matrix interactions on cell phenotype and overall liver functionality.

Hyaluronic acid synthesis, degradation, and crosslinking in equine osteoarthritis: TNF-α-TSG-6-mediated HC-HA formation.

BACKGROUND: TNF-α-stimulated gene 6 (TSG-6) protein, a TNF-α-responsive hyaladherin, possesses enzymatic activity that can catalyze covalent crosslinks of the polysaccharide hyaluronic acid (HA) to another protein to form heavy chain-hyaluronic acid (HC-HA) complexes in pathological conditions such as osteoarthritis (OA). Here, we examined HA synthase and inflammatory gene expression; synovial fluid HA, TNF-α, and viscosity; and TSG-6-mediated HC-HA complex formation in an equine OA model. The objectives of this study were to (1) evaluate the TNF-α-TSG-6-HC-HA signaling pathway across multiple joint tissues, including synovial membrane, cartilage, and synovial fluid, and (2) determine the impact of OA on synovial fluid composition and biophysical properties. METHODS: HA and inflammatory cytokine concentrations (TNF-α, IL-1ß, CCL2, 3, 5, and 11) were analyzed in synovial fluid from 63 OA and 25 control joints, and HA synthase (HAS1-3), TSG-6, and hyaluronan-degrading enzyme (HYAL2, HEXA) gene expression was measured in synovial membrane and cartilage. HA molecular weight (MW) distributions were determined using agarose gel electrophoresis and solid-state nanopore measurements, and HC-HA complex formation was detected via immunoblotting and immunofluorescence. SEC-MALS was used to evaluate TSG-6-mediated HA crosslinking, and synovial fluid and HA solution viscosities were analyzed using multiple particle-tracking microrheology and microfluidic measurements, respectively. RESULTS: TNF-α concentrations were greater in OA synovial fluid, and TSG6 expression was upregulated in OA synovial membrane and cartilage. TSG-6-mediated HC-HA complex formation was greater in OA synovial fluid and tissues than controls, and HC-HA was localized to both synovial membrane and superficial zone chondrocytes in OA joints. SEC-MALS demonstrated macromolecular aggregation of low MW HA in the presence of TSG-6 and inter-α-inhibitor with concurrent increases in viscosity. CONCLUSIONS: Synovial fluid TNF-α concentrations, synovial membrane and cartilage TSG6 gene expression, and HC-HA complex formation were increased in equine OA. Despite the ability of TSG-6 to induce macromolecular aggregation of low MW HA with resultant increases in the viscosity of low MW HA solutions in vitro, HA concentration was the primary determinant of synovial fluid viscosity rather than HA MW or HC-HA crosslinking. The TNF-α-TSG-6-HC-HA pathway may represent a potential therapeutic target in OA.

Acquired antithrombin deficiency is a risk factor for venous thromboembolism after major trauma.

BACKGROUND: Up to 30% of severely injured patients on prophylactic anticoagulation experience venous thromboembolism (VTE). Our previous work shows that acquired antithrombin (AT) deficiency [AT<80%] occurs in approximately 20% of trauma patients upon admission and drives poor responsiveness to enoxaparin. However, changes in AT over time and its association with VTE remain unknown. The aim of this study was to determine the relationship between acquired AT deficiency and VTE in severely injured patients. METHODS: A secondary analysis of the Pragmatic, Randomized Optimal Platelet and Plasma Ratios (PROPPR) clinical trial was performed. Patients who died within 24 h of hemorrhage were excluded from analysis. Demographics, mechanism and severity of injury, transfusions volumes, and outcomes were compared between patients who did and did not develop VTE. Non-parametric statistical tests were used to compare patients with and without VTE. Logistic regression analyses were performed to identify predictors of VTE risk, controlling for AT deficiency (over first 72 h), age, gender, race, body mass index, study site, randomization group and injury severity. A Cox proportional hazards model was used to assess the contribution of AT deficiency to the risk of VTE, while censoring for early deaths. RESULTS: Of the 680 patients enrolled in PROPPR, 101 died of hemorrhage. Of the remaining 579 patients, 86 (14.9%) developed VTE. The median time to VTE was 6 days (IQR 3, 13). No differences in demographics, injuries, or transfusion volumes were identified between VTE cases and controls. AT deficiency at 72 h post-admission was independently associated with VTE. Patients who experienced AT deficiency at 72 h had a 3.3 fold increased risk of VTE [p < 0.01; 95% CI 1.56, 6.98]. Lastly, patients who developed VTE had worse outcomes as displayed by significantly fewer hospital-free days compared to non-VTE patients [0 (0, 8) vs. 4 (0, 18), p < 0.01, respectively]. CONCLUSIONS: Acquired AT deficiency (AT<80%) is an important risk factor for VTE in severely injured patients. These data indicate that intervening, perhaps through AT supplementation, in the first three days after injury could mitigate the risk of VTE and improve patient outcomes.

Impact of rs174537 on Critically Ill Patients with Acute Lung Injury: A Secondary Analysis of the OMEGA Randomized Clinical Trial.

BACKGROUND: Nutrition in the intensive care unit is vital for patient care; however, immunomodulatory diets rich in PUFAs like γ-linolenic acid (GLA), EPA, and DHA remain controversial for patients with acute respiratory distress syndrome. We postulate that genetic variants impacting PUFA metabolism contribute to mixed responses to PUFA-rich diets. OBJECTIVES: In this study, we aimed to test the effects of single nucleotide polymorphism (SNP) rs174537 on differential responses to PUFA-rich diets. METHODS: We performed a secondary analysis of the OMEGA trial (NCT00609180) where 129 subjects received placebo control diets and 143 received omega-oil. DNA was extracted from buffy coats and used to genotype rs174537; plasma was used to quantitate PUFAs. We tested for SNP-diet interactions on PUFA concentrations, inflammatory biomarkers, and patient outcomes. RESULTS: We observed that all individuals receiving omega-oil displayed significantly higher concentrations of GLA, EPA, and DHA (all P < 0.0001), but they did not vary by genotype at rs174537. Statistically significant SNP-diet interactions were observed on circulating DHA concentrations in African Americans. Specifically, African American T-allele carriers on placebo illustrated elevated DHA concentrations. Additionally, all individuals receiving omega-oil had higher concentrations of EPA-derived urinary F3-isoprostane (Caucasians: P = 0.0011; African Americans: P = 0.0002). Despite these findings, we did not detect any significant SNP-diet interactions on pulmonary functional metrics, clinical outcomes, and mortality. CONCLUSIONS: This study highlights the importance of genetic and racial contributions to PUFA metabolism and inflammation. In particular, rs174537 had a significant impact on circulating DHA and urinary isoprostane concentrations. Given our relatively small sample size, further investigations in larger multiethnic cohorts are needed to evaluate the impact of rs174537 on fatty acid metabolism and downstream inflammation.

A Pilot Study Assessing the Impact of rs174537 on Circulating Polyunsaturated Fatty Acids and the Inflammatory Response in Patients with Traumatic Brain Injury.

Traumatic brain injury (TBI) is a leading cause of death and disability in persons under age 45. The hallmark secondary injury profile after TBI involves dynamic interactions between inflammatory and metabolic pathways including fatty acids. Omega-3 polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) have been shown to provide neuroprotective benefits by minimizing neuroinflammation in rodents. These effects have been less conclusive in humans, however. We postulate genetic variants influencing PUFA metabolism in humans could contribute to these disparate findings. Therefore, we sought to (1) characterize the circulating PUFA response and (2) evaluate the impact of rs174537 on inflammation after TBI. A prospective, single-center, observational pilot study was conducted to collect blood samples from Level-1 trauma patients (N = 130) on admission and 24 h post-admission. Plasma was used to quantify PUFA levels and inflammatory cytokines. Deoxyribonucleic acid was extracted and genotyped at rs174537. Associations between PUFAs and inflammatory cytokines were analyzed for all trauma cases and stratified by race (Caucasians only), TBI (TBI: N = 47; non-TBI = 83) and rs174537 genotype (GG: N = 33, GT/TT: N = 44). Patients with TBI had higher plasma DHA levels compared with non-TBI at 24 h post-injury (p = 0.013). The SNP rs174537 was associated with both PUFA levels and inflammatory cytokines (p < 0.05). Specifically, TBI patients with GG genotype exhibited the highest plasma levels of DHA (1.33%) and interleukin-8 (121.5 ± 43.3 pg/mL), which were in turn associated with poorer outcomes. These data illustrate the impact of rs174537 on the post-TBI response. Further work is needed to ascertain how this genetic variant directly influences inflammation after trauma.

Self-regenerating giant hyaluronan polymer brushes.

Tailoring interfaces with polymer brushes is a commonly used strategy to create functional materials for numerous applications. Existing methods are limited in brush thickness, the ability to generate high-density brushes of biopolymers, and the potential for regeneration. Here we introduce a scheme to synthesize ultra-thick regenerating hyaluronan polymer brushes using hyaluronan synthase. The platform provides a dynamic interface with tunable brush heights that extend up to 20 microns - two orders of magnitude thicker than standard brushes. The brushes are easily sculpted into micropatterned landscapes by photo-deactivation of the enzyme. Further, they provide a continuous source of megadalton hyaluronan or they can be covalently-stabilized to the surface. Stabilized brushes exhibit superb resistance to biofilms, yet are locally digested by fibroblasts. This brush technology provides opportunities in a range of arenas including regenerating tailorable biointerfaces for implants, wound healing or lubrication as well as fundamental studies of the glycocalyx and polymer physics.

Development of Zinc Chelating Resin Polymer Beads for the Removal of Cell-Free Hemoglobin.

Red blood cell (RBC) hemolysis is one of the most common storage lesions in packed RBCs (pRBC). Older units of pRBCs, especially those > 21 days old, have increasing levels of hemolysis leading to increased oxidative stress and premature platelet activation. This effect can mostly be attributed to the increase of cell-free hemoglobin (Hb). Therefore, removal of cell-free Hb from pRBCs prior to transfusion could mitigate these deleterious effects. We propose a new method for the removal of Hb from pRBCs using zinc beads. Prepared Hb solutions and pRBCs were treated with zinc beads using two different protocols. UV-Vis spectrophotometry was used to determine Hb concentrations, before and after treatment. Experiments were run in triplicate and paired t tests were used to determine significant differences between groups. Zinc beads removed on average 94% of cell-free Hb within 15 min and 78% Hb from pRBCs (p < 0.0001), demonstrating a maximum binding capacity ~ 66.2 ± 0.7 mg Hb/mL beads. No differences in RBC morphology or deformability were observed after treatment. This study demonstrates the feasibility of using zinc beads for the rapid and targeted removal of Hb from pRBC units. Further investigation is needed to scale this method for large volume removal.

Erythrocytic bioactivation of nitrite and its potentiation by far-red light.

BACKGROUND: Nitrite is reduced by heme-proteins and molybdenum-containing enzymes to form the important signaling molecule nitric oxide (NO), mediating NO signaling. Substantial evidence suggests that deoxygenated hemoglobin within red blood cells (RBCs) is the main erythrocytic protein responsible for mediating nitrite-dependent NO signaling. In other work, infrared and far red light have been shown to have therapeutic potential that some attribute to production of NO. Here we explore whether a combination of nitrite and far red light treatment has an additive effect in NO-dependent processes, and whether this effect is mediated by RBCs. METHODS AND RESULTS: Using photoacoustic imaging in a rat model as a function of varying inspired oxygen, we found that far red light (660 nm, five min. exposure) and nitrite feeding (three weeks in drinking water at 100 mg/L) each separately increased tissue oxygenation and vessel diameter, and the combined treatment was additive. We also employed inhibition of human platelet activation measured by flow cytometry to assess RBC-dependent nitrite bioactivation and found that far red light dramatically potentiates platelet inhibition by nitrite. Blocking RBC-surface thiols abrogated these effects of nitrite and far-red light. RBC-dependent production of NO was also shown to be enhanced by far red light using a chemiluminescence-based nitric oxide analyzer. In addition, RBC-dependent bioactivation of nitrite led to prolonged lag times for clotting in platelet poor plasma that was enhanced by exposure to far red light. CONCLUSIONS: Our results suggest that nitrite leads to the formation of a photolabile RBC surface thiol-bound species such as an S-nitrosothiol or heme-nitrosyl (NO-bound heme) for which far red light enhances NO signaling. These findings expand our understanding of RBC-mediated NO production from nitrite. This pathway of NO production may have therapeutic potential in several applications including thrombosis, and, thus, warrants further study.