Lisa A. Baer

Bone marrow derived mesenchymal stem cells inhibit inflammation and preserve vascular endothelial integrity in the lungs after hemorrhagic shock.

Hemorrhagic shock (HS) and trauma is currently the leading cause of death in young adults worldwide. Morbidity and mortality after HS and trauma is often the result of multi-organ failure such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), conditions with few therapeutic options. Bone marrow derived mesenchymal stem cells (MSCs) are a multipotent stem cell population that has shown therapeutic promise in numerous pre-clinical and clinical models of disease. In this paper, in vitro studies with pulmonary endothelial cells (PECs) reveal that conditioned media (CM) from MSCs and MSC-PEC co-cultures inhibits PEC permeability by preserving adherens junctions (VE-cadherin and β-catenin). Leukocyte adhesion and adhesion molecule expression (VCAM-1 and ICAM-1) are inhibited in PECs treated with CM from MSC-PEC co-cultures. Further support for the modulatory effects of MSCs on pulmonary endothelial function and inflammation is demonstrated in our in vivo studies on HS in the rat. In a rat “fixed volume” model of mild HS, we show that MSCs administered IV potently inhibit systemic levels of inflammatory cytokines and chemokines in the serum of treated animals. In vivo MSCs also inhibit pulmonary endothelial permeability and lung edema with concurrent preservation of the vascular endothelial barrier proteins: VE-cadherin, Claudin-1, and Occludin-1. Leukocyte infiltrates (CD68 and MPO positive cells) are also decreased in lungs with MSC treatment. Taken together, these data suggest that MSCs, acting directly and through soluble factors, are potent stabilizers of the vascular endothelium and inflammation. These data are the first to demonstrate the therapeutic potential of MSCs in HS and have implications for the potential use of MSCs as a cellular therapy in HS-induced lung injury.

Elevated tissue plasminogen activator and reduced plasminogen activator inhibitor promote hyperfibrinolysis in trauma patients

ABSTRACT Severe hyperfibrinolysis after trauma is a poorly understood phenomenon associated with profound shock, serious anatomic injuries, increased transfusions, and high mortality rates. Molecular mechanisms driving hyperfibrinolysis in trauma have not been completely delineated. The authors aimed to determine the relationship between severe hyperfibrinolysis and outcomes in trauma patients and characterize the role of the plasminogen activator (PA) system in this condition. A prospective observational study was performed in 163 adult level I trauma patients admitted between April and August 2012. Blood was collected on admission, and fibrinolysis was determined by plasmin-&agr;2 antiplasmin (PAP) levels. Tissue-derived and urokinase PA (tPA and uPA, respectively), PA inhibitor (PAI-1), fibrinogen, and antithrombin levels were also measured. Patient demographics, vital signs, laboratory values, mechanisms and severity of injuries, transfusions, and outcomes were collected at admission or from patient records. Moderate fibrinolysis was defined as PAP level 1,500 to 20,000 &mgr;g/L and severe hyperfibrinolysis as PAP level more than 20,000 &mgr;g/L. Severe hyperfibrinolysis was observed in 10% of patients and associated with increased injury severity, greater transfusions, fewer ventilator and hospital-free days, and higher mortality. Plasmin-&agr;2 antiplasmin level was directly correlated with tPA level and inversely correlated with PAI-1 level. Patients with both elevated tPA and reduced PAI-1 were more severely injured, received more transfusions, and experienced fewer ventilator and hospital-free days. In conclusion, Severe hyperfibrinolysis is observed in a small percentage of trauma patients and is associated with severe injuries, greater transfusions, and worse outcomes. This condition is mediated, in part, by excessive upregulation of profibrinolytic tPA in the absence of concomitant increases in antifibrinolytic PAI-1.

Measuring thrombin generation as a tool for predicting hemostatic potential and transfusion requirements following trauma.

BACKGROUND Thrombin is the central coagulation protease that activates clotting proteins, triggers platelet aggregation, and converts fibrinogen to fibrin. Relationships between thrombin generation (TG) and clinical outcomes have not been defined following trauma. We hypothesize that TG is predictive of transfusion requirements and patient outcomes. METHODS Plasma was collected from 406 highest-level activation trauma patients upon admission and 29 healthy donors. Standard coagulation tests were performed, and TG was measured by calibrated automated thrombogram. Mann-Whitney U-tests were used to compare healthy versus trauma patients, and subgroup analyses were used to compare hypocoagulable versus nonhypocoagulable patients. Hypocoagulability was determined by area under the receiver operating characteristic curve analysis and was defined as peak TG of less than 250 nM. Multiple logistic regressions were used to assess the ability of TG to predict massive transfusion and mortality. RESULTS The median (interquartile range) age was 39 years (28–52 years), with an Injury Severity Score (ISS) of 17 (9–26). The trauma patients had greater TG (peak, 316.2 nM [270.1–355.5 nM]) compared with the healthy controls (124.6 nM [91.1–156.2 nM]), p < 0.001. The overall rate of hypocoagulability was 17%. The patients with peak TG of less than 250 nM were more severely injured (ISS, 25 [13–30] vs. 16 [9–25], p = 0.003); required more transfusions of red blood cells (p = 0.02), plasma (p = 0.003), and platelets (p = 0.006); had fewer hospital-free days (p = 0.001); and had increased mortality (10% vs. 3% at 24 hours, p = 0.006, and 29% vs. 11% at 30 days, p = 0.0004). Peak TG of less than 250 nM was predictive of massive transfusion (odds ratio, 4.18; p = 0.01) and 30-day mortality (odds ratio, 2.78; p = 0.02). Finally, peak TG was inversely correlated with standard coagulation tests. CONCLUSION While the physiologic response to injury is to upregulate plasma procoagulant activity, the patients with reduced TG required more transfusions and had poorer outcomes. Measuring TG may provide an exquisitely sensitive tool for detecting disturbances in the enzymatic phases of coagulation in critically injured patients. LEVEL OF EVIDENCE Prognostic/epidemiologic study, level III.

Mechanical unloading impairs keratinocyte migration and angiogenesis during cutaneous wound healing

Although initially thought to improve an individuals ability to heal, mechanical unloading promoted by extended periods of bed rest has emerged as a contributing factor to delayed or aberrant tissue repair. Using a rat hindlimb unloading (HLU) model of hypogravity, we mimicked some aspects of physical inactivity by removing weight-bearing loads from the hindlimbs and producing a systemic cephalic fluid shift. This model simulates bed rest in that the animal undergoes physiological adaptations, resulting in a reduction in exercise capability, increased frequency of orthostatic intolerance, and a reduction in plasma volume. To investigate whether changes associated with prior prolonged bed rest correlate with impaired cutaneous wound healing, we examined wound closure, angiogenesis, and collagen content in day 2 to day 21 wounds from rats exposed to HLU 2 wk before excisional wounding. Wound closure was delayed in day 2 wounds from HLU rats compared with ambulatory controls. Although the levels of proangiogenic growth factors, fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor (VEGF) were similar between the two groups, wound vascularity was significantly reduced in day 7 wounds from HLU animals. To further examine this disparity, total collagen content was assessed but found to be similar between the two groups. Taken together, these results suggest that keratinocyte and endothelial cell function may be impaired during the wound healing process under periods of prolonged inactivity or bed rest.

Maternal reproductive experience enhances early postnatal outcome following gestation and birth of rats in hypergravity.

Abstract A major goal of space life sciences research is to broaden scientific knowledge of the influence of gravity on living systems. Recent spaceflight and centrifugation studies demonstrate that reproduction and ontogenesis in mammals are amenable to study under gravitational conditions that deviate considerably from those typically experienced on Earth (1 × g). In the present study, we tested the hypothesis that maternal reproductive experience determines neonatal outcome following gestation and birth under increased (hyper) gravity. Primigravid and bigravid female rats and their offspring were exposed to 1.5 × g centrifugation from Gestational Day 11 either through birth or through the first postnatal week. On the day of birth, litter sizes were identical across gravity and parity conditions, although significantly fewer live neonates were observed among hypergravity-reared litters born to primigravid dams than among those born to bigravid dams (82% and 94%, respectively; 1.0 × g controls, 99%). Within the hypergravity groups, neonatal mortality was comparable across parity conditions from Postnatal Day 1 through Day 7, at which time litter sizes stabilized. Maternal reproductive experience ameliorated neonatal losses during the first 24 h after birth but not on subsequent days, and neonatal mortality was associated with changes in maternal care patterns. These results indicate that repeated maternal reproductive experience affords protection against neonatal losses during exposure to increased gravity. Differential mortality of neonates born to primigravid versus bigravid dams denotes gravitational load as one environmental mechanism enabling the expression of parity-related variations in birth outcome.

Sympathoadrenal activation and endotheliopathy are drivers of hypocoagulability and hyperfibrinolysis in trauma: A prospective observational study of 404 severely injured patients.

BACKGROUND One third of severely injured patients present with a laboratory-based diagnosis of coagulopathy. This study investigated clinical and biomarker profile of patients with rapid thrombelastography (rTEG) coagulopathy, hypothesizing that sympathoadrenal activation and endothelial damage were drivers of this condition. METHODS Prospective observational study of 404 trauma patients admitted to a Level 1 US Trauma Center. Patients with admission rTEG and plasma measurements of catecholamines (adrenaline, noradrenaline) and biomarkers reflecting endothelial activation/damage (syndecan-1, thrombomodulin, sE-selectin, sVE-cadherin, nucleosomes) were included. Demography, injury type/severity, physiology, treatment, and inhospital mortality were recorded. RESULTS Patients had a median Injury Severity Score (ISS) of 17, 73% from blunt injury. One third (35%) of the patients had rTEG coagulopathy, which was associated with higher plasma adrenaline, syndecan-1, and nucleosomes (all <0.05), higher transfusion requirements and higher early (<24 hours, 9.3% vs. 2.5%) and late (28 days, 23.8% vs. 13.4%) mortality. By adjusted linear regression analyses, high plasma adrenaline, sVE-cadherin, and syndecan-1 (reflecting sympathoadrenal activation and endothelial cell junction and glycocalyx damage) along with male sex, high ISS, low platelet count and prehospital red blood cell transfusion were independently associated with hypocoagulable rTEG, whereas prehospital plasma and sE-selectin (reflecting endothelial activation) were independently associated with more hypercoagulable rTEG. CONCLUSION In this cohort of severely injured trauma patients, rTEG coagulopathy was associated with sympathoadrenal activation, endotheliopathy, and excess mortality. High adrenaline and biomarkers reflecting endothelial cell junction and glycocalyx damage were independently associated with hypocoagulability and hyperfibrinolysis. These findings support that sympathoadrenal activation and endotheliopathy contribute to trauma-induced coagulopathy and warrants further studies of endothelial repair management. LEVEL OF EVIDENCE Prognostic, Level III.

Effects of Hypergravity Exposure on the Developing Central Nervous System: Possible Involvement of Thyroid Hormone

The present study examined the effects of hypergravity exposure on the developing brain and specifically explored the possibility that these effects are mediated by altered thyroid status. Thirty-four timed-pregnant Sprague-Dawley rats were exposed to continuous centrifugation at 1.5 G (HG) from gestational Day 11 until one of three key developmental points: postnatal Day (P) 6, P15, or P21 (10 pups/dam: 5 males/5 females). During the 32-day centrifugation, stationary controls (SC, n = 25 dams) were housed in the same room as HG animals. Neonatal body, forebrain, and cerebellum mass and neonatal and maternal thyroid status were assessed at each time point. The body mass of centrifuged neonates was comparatively lower at each time point. The mass of the forebrain and the mass of the cerebellum were maximally reduced in hypergravity-exposed neonates at P6 by 15.9% and 25.6%, respectively. Analysis of neonatal plasma suggested a transient hypothyroid status, as indicated by increased thyroid stimulating hormone (TSH) level (38.6%) at P6, while maternal plasma TSH levels were maximally elevated at P15 (38.9%). Neither neonatal nor maternal plasma TH levels were altered, suggesting a moderate hypothyroid condition. Thus, continuous exposure of the developing rats to hypergravity during the embryonic and neonatal periods has a highly significant effect on the developing forebrain and cerebellum and neonatal thyroid status (P < 0.05, Bonferroni corrected). These data are consistent with the hypothesized role of the thyroid hormone in mediating the effect of hypergravity in the developing central nervous system and begin to define the role of TH in the overall response of the developing organism to altered gravity.

The impact of muscle disuse on muscle atrophy in severely burned rats.

BACKGROUND Severe burn induces a sustained hypermetabolic response, which causes long-term loss of muscle mass and decrease in muscle strength. In this study, we sought to determine whether muscle disuse has additional impact on muscle atrophy after severe burn using a rat model combining severe cutaneous burn and hindlimb unloading. METHODS Forty Sprague-Dawley rats (≈ 300 g) were randomly assigned to sham ambulatory (S/A), sham hindlimb unloading (S/HLU), burn ambulatory (B/A), or burn hindlimb unloading (B/HLU) groups. Rats received a 40% total body surface (TBSA) full thickness scald burn, and rats with hindlimb unloading were placed in a tail traction system. At d 14, lean body mass (LBM) was determined using DEXA scan, followed by measurement of the isometric mechanical properties in the predominantly fast-twitch plantaris muscle (PL) and the predominantly slow-twitch soleus muscle (SL). Muscle weight (wt), protein wt, and wet/dry wt were determined. RESULTS At d 14, body weight had decreased significantly in all treatment groups; B/HLU resulted in significantly greater loss compared with the B/A, S/HLU, and S/A. The losses could be attributed to loss of LBM. PL muscle wt and Po were lowest in the B/HLU group (<0.05 versus S/A, S/HLU, or B/A). SL muscle wt and Po were significantly less in both S/HLU and B/HLU compared with that of S/A and B/A; no significant difference was found between S/HLU and B/HLU. CONCLUSIONS Cutaneous burn and hindlimb unloading have an additive effect on muscle atrophy, characterized by loss of muscle mass and decrease in muscle strength in both fast (PL) and slow (SL) twitch muscles. Of the two, disuse appeared to be the dominant factor for continuous muscle wasting after acute burn in this model.

Traumatic Endotheliopathy: A Prospective Observational Study of 424 Severely Injured Patients

Objective: Investigate and confirm the association between sympathoadrenal activation, endotheliopathy and poor outcome in trauma patients. Background: The association between sympathoadrenal activation, endotheliopathy, and poor outcome in trauma has only been demonstrated in smaller patient cohorts and animal models but needs confirmation in a large independent patient cohort. Methods: Prospective observational study of 424 trauma patients admitted to a level 1 Trauma Center. Admission plasma levels of catecholamines (adrenaline, noradrenaline) and biomarkers reflecting endothelial damage (syndecan-1, thrombomodulin, and sE-selectin) were measured and demography, injury type and severity, physiology, treatment, and mortality up till 28 days were recorded. Results: Patients had a median ISS of 17 with 72% suffering from blunt injury. Adrenaline and noradrenaline correlated with syndecan-1 (r = 0.38, P < 0.001 and r = 0.23, P < 0.001, respectively) but adrenaline was the only independent predictor of syndecan-1 by multiple linear regression adjusted for age, injury severity score, Glascow Coma Scale, systolic blood pressure, base excess, platelet count, hemoglobin, prehospital plasma, and prehospital fluids (100 pg/mL higher adrenaline predicted 2.75 ng/mL higher syndecan-1, P < 0.001). By Cox analyses adjusted for age, sex, injury severity score, Glascow Coma Scale, base excess, platelet count and hemoglobin, adrenaline, and syndecan-1 were the only independent predictors of both <24-hours, 7-day and 28-day mortality (all P < 0.05). Furthermore, noradrenaline was an independent predictor of <24-hours mortality and thrombomodulin was an independent predictor of 7-day and 28-day mortality (all P < 0.05). Conclusions: We confirmed that sympathoadrenal activation was strongly and independently associated with endothelial glycocalyx and cell damage (ie, endotheliopathy) and furthermore that sympathoadrenal activation and endotheliopathy were independent predictors of mortality in trauma patients.

Plasma resuscitation promotes coagulation homeostasis following shock-induced hypercoagulability

Background: Increased thrombin generation in injured patients possibly contributes to early consumption of coagulation factors, exacerbating hemorrhage. Identifying optimal resuscitation products for restoring plasma homeostasis following injury is important for improving management of these patients. Objectives: To determine the effects of crystalloid versus plasma resuscitation on thrombin generation in a rat model of trauma and hemorrhagic shock (HS). Patients/Methods: Rats were subjected to trauma and HS followed by resuscitation with Lactated Ringers solution (LR) or fresh frozen plasma (FFP). Blood was collected at baseline, decompensation, and 3-h post-resuscitation. Thrombin generation was measured by calibrated automated thrombogram and antithrombin III (AT) by ELISA. In a prospective observational study, admission blood samples were collected on highest-level activation trauma patients and diluted with LR or FFP for thrombin generation analysis. Results: Resuscitation with LR resulted in persistent hypercoagulability; however, FFP resuscitation reversed this hypercoagulability to baseline thrombin generation or below. Plasma AT levels decreased following HS and remained low in rats receiving LR, but were corrected in rats receiving FFP. Similarly, in trauma patient plasma LR increased thrombin generation while FFP reduced it. However, results with AT-deficient plasma dilution were similar to LR. In patients with admission hypocoagulability, FFP slightly increased thrombin generation. Conclusions: HS in rats is associated with increased thrombin generation and resuscitation with FFP, not LR, reverses hypercoagulability. Dilution of trauma patient plasma with LR or FFP yielded similar results; however, the modulatory effects of FFP were attenuated when AT was absent. Importantly, FFP reduced thrombin generation in hypercoagulable patient plasma, but slightly increased thrombin generation in hypocoagulable patient plasma. Thus, FFP restores hemostatic balance following trauma and HS which is, in part, by delivering AT.