Multiorgan failure following trauma remains a significant health-care issue, contributing to intensive care unit resource utilization and late-stage mortality (1). Organ dysfunction following trauma is likely mediated by an imbalanced inflammatory response that, as in sepsis (2), precipitates vascular endothelial cell (EC) activation and glycocalyx disruption, culminating in endotheliopathy (3). Endotheliopathy of trauma (EoT) manifests as capillary leak, pro-inflammatory propagation, and coagulopathy (4). EoT has been well characterized in adults (5); however, little is known about trauma-related endotheliopathy in children. Moreover, the mechanisms underlying EoT pathophysiology remain elusive.
Preclinical studies suggest that endothelial-derived angiopoietins are integral in the development of endotheliopathy (6, 7) and post-traumatic organ injury (8). Of the four circulating angiopoietins, angiopoietin-1 (Agpt-1) and angiopoietin-2 (Agpt-2) are best characterized and most widely studied (9). In health, Agpt-1 is released by periendothelial cells (10) and maintains endothelial integrity by activating the EC-surface tyrosine kinase Tie2 receptor (11). Conversely, Agpt-2 is released by ECs in response to injury and destabilizes endothelial adherens junctions by antagonizing Tie2 receptor signaling, promoting vascular leakage (12). Thus, the relative plasma concentrations of the two angiopoietins, quantified as the Agpt-2:Agpt-1 ratio, reflect overall endothelial health and stability (13). In adults, plasma Agpt-2 levels are elevated after trauma and are associated with adverse outcomes (14). A more recent study in adult trauma demonstrated that Agpt-2 was only significantly elevated in patients presenting with hemorrhagic shock (15). The Agpt-2:Agpt-1 ratio has been characterized in children with sepsis (16) and is associated with worse outcomes (17). However, the behavior of the angiopoietins after pediatric trauma has not been characterized.
In vitro data suggest a mechanistic linkage between endothelial glycocalyx injury and Agpt-2 upregulation (18, 19), which may be integral to the development of EoT. The glycocalyx is predominantly anchored to the EC surface through the transmembrane proteoglycan syndecan-1 (Syn-1). Circulating Syn-1 levels are used as a biomarker of glycocalyx damage to quantitate EoT after adult trauma (5) and are associated with the development of post-traumatic coagulopathy (20), inflammation propagation (21), and increased endothelial permeability (22). Similarly, plasma Agpt-2 levels are elevated after adult trauma and are associated with organ dysfunction and mortality (14). However, glycocalyx injury has yet to be directly linked to Agpt-2 upregulation, and the association of Agpt-2 with EoT remains undefined.
The objectives of the current study were to characterize plasma levels of Agpt-1 and Agpt-2 and the Agpt-2:Agpt-1 ratio and correlate Agpt-2 levels with plasma Syn-1 levels following pediatric trauma. We hypothesized that the angiopoietins become dysregulated following pediatric trauma, represented as an increased plasma Agpt-2 level and Agpt-2:Agpt-1 ratio at admission after trauma. We also anticipated a positive correlation between Agpt-2 and Syn-1.
Study design and participants
We performed a secondary analysis of prospectively collected data from a recently published study (23) performed between March 2013 and March 2016. Only pediatric trauma patients who met original study inclusion criteria (<18 years of age, time from injury to emergency department (ED) presentation <6 h, initial presentation to a Level 1 trauma center), were admitted to Children's of Alabama for trauma-related injuries, and had sufficient stored plasma available for Agpt-1, Agpt-2, and Syn-1 measurements from ED presentation and 24 h after hospital admission were included for analysis. Controls were selected from the original study control group of patients who underwent an elective surgical procedure at Children's of Alabama and had plasma available for Agpt-1 and Agpt-2 measurement. The University of Alabama at Birmingham Institutional Review Board approved this study.
Demographics, injury characteristics, physiologic variables, and outcome measures
Patient demographics, physiologic measurements on hospital arrival, and clinical outcomes for trauma patients were collected from the original study. Data not available during the original study were obtained from the medical record. Demographic variables included age (years), gender, race, and weight (kilograms). For trauma patients, we recorded Injury Severity Score (ISS), mechanism of injury (blunt versus penetrating), and time from injury to ED admission (hours). Admission physiologic variables included heart rate (beats per minute), systolic blood pressure (mmHg), and serum base excess (mmol/L). We also recorded admission serum international normalized ratio (INR) and partial thromboplastin time (PTT, seconds) as markers of coagulation. Outcome measures included in-hospital mortality, mechanical ventilation (MV) duration (hours), transfusion requirements (mL) within the first 24 h of hospitalization, and pediatric intensive care unit (PICU) and hospital length of stay (LOS) (days).
Sample collection and biomarkers
Blood samples were collected in sodium citrated tubes during the original study (23) from pediatric trauma patients within 20 min of ED admission and 24 h after hospital admission. For controls, plasma was collected from children immediately prior to the elective surgical procedure. Plasma was aliquoted from each sample and stored at −80°C. Agpt-1 and Agpt-2 were measured for the current study from trauma patients and controls using human enzyme-linked immunosorbent assays (DuoSet ELISA, R&D Systems Inc, Minneapolis, Minn). Agpt-2:Agpt-1 ratios were calculated for trauma patients at the respective time points and for controls. When available, Syn-1 levels measured using human ELISA (Cell Sciences, Newburyport, Mass) were recorded from the parent study for trauma patients included in the current analyses. For the remainder of the included trauma patients, Syn-1 was measured for the current study using the same human ELISA.
Data are presented as medians with interquartile ranges (IQR) for continuous variables or numbers with the percentage of total for categorical variables. To characterize the angiopoietins, trauma patients were compared to controls. Bivariate analyses were performed using the Pearson chi-square, Fisher exact, or Mann–Whitney U tests, as appropriate, for categorical or continuous variables. Plasma biomarkers were compared between controls, trauma patients at admission, and trauma patients 24 h after admission using the Kruskal–Wallis test. Dunn–Bonferroni multiple comparisons were performed for significant associations determined after Kruskal–Wallis testing. Simple correlations between plasma levels of Agpt-2 and Syn-1 at admission and 24 h were performed using Spearman correlation to evaluate the association between glycocalyx injury and Agpt-2 upregulation. A P value ≤0.05 was considered significant. All statistical analyses were performed using Statistical Package for Social Sciences, version 25 (SPSS Inc, Chicago, Ill).
We compared 52 pediatric trauma patients (median age 9.7 years (IQR, 6.2, 13.6)) to 12 pediatric controls (median age 5 years (IQR, 1.8, 15); P = 0.102). There were no significant differences in age, gender, or race between the trauma cohort and controls (Table 1). Trauma was predominantly from blunt injury (87%), and median ISS was 26 (IQR, 18, 34). Median time from injury to ED presentation was 1.9 h (IQR, 0.9, 3.5). There was 8% in-hospital mortality in the trauma cohort.
Plasma angiopoietin levels and association with injury severity
Admission plasma Agpt-1 levels were significantly higher in trauma patients (1,585 pg/mL (IQR, 767, 2,776)) compared with controls (516 pg/mL (IQR, 306, 989); P = 0.005) before declining at 24-h (425 pg/mL (IQR, 258, 664); P < 0.001) (Fig. 1). Twenty-four hour Agpt-1 levels did not differ from controls (Fig. 1A). Plasma Agpt-2 was significantly higher at admission (742 pg/mL (IQR, 443, 1,058)) compared with controls (135 pg/mL (IQR, 56, 356); P = 0.004) and remained elevated compared with controls at 24 h (722 pg/mL (IQR, 254, 1,463); P = 0.005) (Fig. 1B). Admission Agpt-2:Agpt-1 ratios were not different from controls (0.5 (IQR, 0.2, 1.1) vs 0.2 (IQR, 0.1, 1.2); P = 1.00). However, by 24 h, the Agpt-2:Agpt-1 ratio was significantly elevated in trauma patients (1.1 (IQR, 0.6, 4.6)) compared with controls (P < 0.001) (Fig. 1C).
We then stratified trauma patients according to ISS (≤25 versus >25) (24) to evaluate a potential association between injury severity and angiopoietin levels. See Supplemental Table 1 (http://links.lww.com/SHK/A820) for injury characteristic, physiologic, and outcome comparisons between injury severity groups. We did not see a difference in Agpt-1 between groups at either time point (Fig. 2). Agpt-2 was not significantly different between the groups at admission but was significantly higher at 24 h in those with ISS >25 (P = 0.029). Similarly, the Agpt-2:Agpt-1 ratio was not significantly different between groups at admission but was significantly higher in the severely injured group by 24 h (P = 0.048). Syn-1 was not significantly different between ISS groups at either time point.
Plasma angiopoietin associations with hypoperfusion and coagulopathy
Hypoperfusion caused by hemorrhagic shock results in tissue hypoxia and EC dysfunction (25). In adults, Agpt-2 is only elevated in polytrauma patients with concomitant hemorrhagic shock (15). To assess the association of plasma angiopoietin levels with hypoperfusion after pediatric trauma, we compared angiopoietin levels in trauma patients stratified by admission base excess (>-6 mmol/L versus ≤-6 mmol/L) (26). In patients with an admission serum base excess ≤-6 mmol/L, Agpt-2 levels and Agpt-2:Agpt-1 ratios were significantly elevated at 24 h compared with patients with higher base excess (Agpt-2, P = 0.050; Agpt-2:Agpt-1 ratio, P = 0.030) (Fig. 3). Agpt-1 and Syn-1 levels were not associated with base excess at either time point. As we saw a lower base excess in children with higher ISS, we performed a multivariate linear regression analysis to assess the independent association of admission base excess with Agpt-2 levels and Agpt-2:Agpt-1 ratios after controlling for ISS (Table 2). We found a significant association of base excess with 24-h Agpt-2 (P < 0.001) but not with Agpt-2:Agpt-1 ratio (P = 0.088).
Early coagulopathy following trauma is linked to systemic hypoperfusion (27) and glycocalyx disruption (20) and is associated with worse outcomes (28). As Agpt-2 has been shown to be elevated in adults with acute traumatic coagulopathy (14), we compared the angiopoietin levels between children with coagulopathy (INR >1.5 or PTT >36.5 s) (29) and those without coagulopathy at admission following trauma. Children with acute traumatic coagulopathy had significantly higher 24-h Agpt-2 levels and admission Syn-1 levels compared with children without coagulopathy (Agpt-2, P = 0.014; Syn-1, P = 0.038) (Fig. 4). As children with higher ISS presented with a higher INR, we performed a multivariate linear regression analysis to assess the independent association of admission INR with 24-h Agpt-2 and admission Syn-1 levels after controlling for ISS (Table 3). We saw significant associations between INR and both Agpt-2 at 24 h (P = 0.002) and admission Syn-1 (P = 0.024).
Plasma angiopoietin-2:angiopoietin-1 ratio association with clinical outcomes
Elevated Agpt-2:Agpt-1 ratios are associated with adverse clinical outcomes in pediatric sepsis (17). Presuming a similar endothelial activation occurs in trauma as in sepsis, we evaluated the association between Agpt-2:Agpt-1 ratios and outcomes following traumatic injury. Because we did not see an elevation in Agpt-2:Agpt-1 ratio until 24 h, we performed the evaluation using 24-h Agpt-2:Agpt-1 ratios. After stratifying the trauma cohort by 24-h Agpt-2:Agpt-1 ratio ≤1 versus >1 (Table 4), we found children with a 24-h Agpt-2:Agpt-1 ratio >1 experienced significantly longer MV duration (48 h (IQR, 24, 192) vs 20 h (IQR, 0, 72); P = 0.033) and PICU LOS (3 days (IQR, 1, 10) vs 1 day (IQR, 0, 4); P = 0.018). There was no significant difference in mortality, volume of blood transfused in the first 24 h of hospitalization, or hospital LOS between the groups.
Correlation between angiopoietin-2 and syndecan-1
To evaluate the association between plasma Agpt-2 levels and EoT, we evaluated the temporal relationship between plasma levels of Agpt-2 and Syn-1 by performing correlations of admission and 24-h Agpt-2 levels with Syn-1 at admission and Syn-1 at 24 h. All four comparisons resulted with significant correlations (Fig. 5).
To our knowledge, this study is the first to characterize angiopoietins after pediatric trauma and the first to correlate these cytokines with Syn-1 as a biomarker of EoT. We demonstrated that plasma Agpt-1 and Agpt-2 levels were significantly elevated in trauma patients at admission compared with controls. However, by 24 h, plasma Agpt-1 declined to near-control levels while Agpt-2 remained elevated, translating in significantly higher Agpt-2:Agpt-1 ratios compared with controls. More severely injured children had higher Agpt-2 levels and Agpt-2:Agpt-1 ratios 24 h after trauma. Moreover, children with elevated 24-h Agpt-2:Agpt-1 ratios experienced worse clinical outcomes. Agpt-2 levels also correlated with levels of Syn-1. Our findings suggest dysregulation of circulating angiopoietins after pediatric trauma that may correlate with pediatric EoT.
Though the angiopoietins have been well characterized following adult trauma, little is known regarding their behavior following pediatric trauma. Ganter et al. (14) reported that higher Agpt-2 levels are associated with increased injury severity in adults. However, in a more recent longitudinal study, Agpt-2 was only significantly elevated in adult polytrauma patients who had concomitant hemorrhagic shock and was not associated with injury severity (15). This suggests the presence of shock as a more important mediator of EC injury than injury severity alone. The study by Ganter et al. (14) described a similar elevation in Agpt-2 levels in patients with shock as indicated by base excess, but multivariate analysis was not performed that would discern independent associations between shock or injury severity with Agpt-2 levels. In each of the above-mentioned studies, Agpt-1 was not well characterized if at all. In our study of pediatric trauma, we found both Agpt-1 and Agpt-2 to be elevated at admission followed by a significant decrease in Agpt-1 and a persistent elevation of Agpt-2 at 24 h. Admission Agpt-1 levels in our pediatric trauma patients were similar to admission Agpt-1 levels described in adult trauma patients (14); however, in this adult study, only admission angiopoietin levels were measured, and control values of Agpt-1 were not reported for comparison. In our adjusted regression model, base excess, and not ISS, was associated with 24-h Agpt-2 levels, suggesting that, like Halbgebauer et al. (15), it is the presence of shock and not injury severity that is more strongly associated with Agpt-2 levels. In light of the angiopoietin kinetics after pediatric trauma, the immediate elevation in Agpt-1 could represent an initial protective and compensatory anti-inflammatory response that is attenuated over the first 24 h following the inflammatory insult. Thereafter, persistently elevated circulating Agpt-2 is available to act at the Tie2 receptor unopposed to drive endothelial dysfunction. Though pattern of injury (30), impact distribution, and resuscitation strategies may differ between children and adults, hemorrhagic shock after trauma may elicit a similar endothelial response. Further mechanistic and prospective clinical studies are needed to fully elucidate the relationship between perfusion injury and angiopoietin regulation after pediatric trauma.
In our pediatric trauma cohort, we demonstrated an association between mechanical ventilation duration and PICU length of stay and 24-h Agpt-2:Agpt-1 ratio. Ganter et al. (14) similarly showed an association between elevated plasma Agpt-2 levels and increased odds of prolonged mechanical ventilation, acute kidney injury, and mortality after adult trauma. In a heterogeneous cohort of adults receiving invasive mechanical ventilation in an ICU, the Agpt-2:Agpt-1 ratio was a useful biomarker to stratify risk of mortality (13). Elevated Agpt-2:Agpt-1 ratios in children presenting with sepsis, a disease process with an arguably similar angiopoietin dysregulation to trauma, are associated with worse survival (17). Taken together, these studies suggest that the Agpt-2:Agpt-1 ratio early after pediatric trauma may have utility in predicting risk of developing organ failure and ultimately mortality. Moreover, Agpt-2 measurements may be useful as a trend, informing clinicians of on-going shock or a developing inflammatory process like ARDS (31) or sepsis (32). However, at present, the diagnostic and prognostic potential of Agpt-2 and the Agpt-2:Agpt-1 ratio is limited by the need for ELISA-based detection. Additionally, clear prognostic cut-off values and age-specific norms for these biomarkers are needed.
The role of the angiopoietins in the post-traumatic inflammatory response remains elusive and the precise mechanisms driving angiopoietin dysregulation following a traumatic insult are not clear. There may be a mechanistic linkage between glycocalyx injury and EC Agpt-2 upregulation that facilitates endothelial dysfunction in the setting of inflammation. In vitro studies suggest that a loss of Syn-1 from the glycocalyx decreases EC PI3K/Akt cell-survival signaling (18). Inactivated Akt signaling activates Forkhead box protein 1 (FoxO1)-regulated transcription of Agpt-2 (33), packaging of Agpt-2 into Weibel–Palade bodies (34), and release of Agpt-2 into circulation by Weibel–Palade body exocytosis (19, 34). Agpt-2 may then act in an autocrine manner to exacerbate endothelial glycocalyx injury (6, 12) and reduce EC pro-survival and anti-inflammatory signaling downstream of Tie2 (35), generating further Agpt-2 synthesis and sustained endotheliopathy (7). Our study results suggest a positive correlation between Syn-1 at admission and Agpt-2 24 h after admission. We also demonstrated a significant elevation in 24-h Agpt-2 levels in patients with coagulopathy at admission. Understanding that glycocalyx damage may instigate the development of coagulopathy after trauma (20), our data support a potential mechanistic connection between glycocalyx injury and Agpt-2 release.
The results presented in this study have a number of limitations. First, the study was performed at a single center with a limited sample size and secondary analysis design, which significantly under-powered our ability to detect outcome differences in mortality and hospital LOS between angiopoietin ratio groups, increased the potential for type II error, and limits the external validity of our findings. Second, we were unable to age- and gender-match controls with trauma patients. In our trauma patients, median angiopoietin levels and Agpt-2:Agpt-1 ratio did not significantly differ between genders or races (data not shown). However, the effect of age and gender on circulating angiopoietin levels are not yet known, and these variables remain potential confounders. Third, only the first 24 h of hospital admission were observed in our study. As Halbgebauer et al. (15) have shown in a more longitudinal evaluation of post-traumatic plasma biomarkers, the kinetics of the angiopoietins require 24 to 48 h to fully evolve, which may have been truncated in our study. Fourth, our study lacked the data granularity needed to perform sensitivity analyses on the interaction between body parts injured and plasma angiopoietins. Head and chest injuries are the predominant body parts injured in trauma that result in death in children with single-system injuries (24). However, the individual body region abbreviated injury scale scores in single-system-injury and polytrauma patients were unavailable, limiting our ability to assess plasma angiopoietin variation with pattern of injury.
In summary, in a study of pediatric trauma patients, we demonstrate that the plasma Agpt-2:Agpt-1 ratio is elevated by 24 h after trauma, higher in patients with shock, and associated with worse outcomes. We also found that plasma Agpt-2 levels correlate with Syn-1 levels, suggesting a relationship between endothelial glycocalyx injury and Agpt-2 release. Larger prospective studies are needed to verify our findings. Additionally, further mechanistic studies are needed to fully elucidate the linkage between EC glycocalyx damage and angiopoietin regulation following pediatric trauma.
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