*Contributed equally
It has been reported that angiopoietin 2 (Ang-2) plays an integral role in the pathophysiology of sepsis and many other inflammatory diseases. However, the specific role of Ang-2 in septic shock has not been defined. The aim of the present study was to assess the predictive value of serum Ang-2 in patients with septic shock. Clinical data of 85 patients with septic shock and 10 healthy controls admitted to the intensive care unit with a diagnosis of septic shock were collected between January 2020 and October 2020 at Tongji Hospital (Wuhan, China). The serum levels of Ang-2 mRNA were quantified using a quantitative real-time PCR assay. Ang-2, SOFA and APACHE II scores were retrospectively analyzed in relation to 28-day mortality. The area under the receiver operating characteristic (ROC) curve (AUC) was used to discriminate the accuracy of the prediction. Mean Ang-2 mRNA levels in the patients with septic shock were significantly higher than those in the healthy controls (P<0.05), and the Ang-2 levels showed a downwards trend over time following treatment. The three indicators (AUCs, SEMs, P-values) were Ang-2 (0.82, 0.03, P<0.01), SOFA score (0.76, 0.04, P<0.01), and APACHE II score (0.73, 0.04, P<0.01). The present study confirmed that Ang-2 mRNA levels were significantly elevated in septic shock. The Ang-2 mRNA level at ICU admission in a patient with septic shock could be a predictive biomarker for mortality.
Septic shock is defined as a subset of sepsis in which specific and profound circulatory, cellular, coagulation and metabolic abnormalities are associated with a greater risk of mortality than with sepsis alone (
The Sequential Organ Failure Assessment (SOFA) and Acute Physiology And Chronic Health Evaluation II (APACHE II) scores are the most widely applied prognostic markers, owing primarily to their simplicity, accuracy, and integrity of use in clinical practice (
In this retrospective study, clinical data of patients admitted to the intensive care unit (ICU) with a diagnosis of septic shock were collected between January 2020 and October 2020 at Tongji Hospital (Wuhan, Hubei, China). Patients underwent laboratory tests, including but not limited to, routine blood tests, biochemical items (including liver and renal function tests), coagulation factors and inflammation indicators such as C-reactive protein (CRP) and procalcitonin (PCT). To measure Ang-2 mRNA levels, citrated whole blood samples were collected upon ICU admission at baseline (day 0), as well as on days 3, 5 and 7 for those remaining in the ICU at those times. Plasma samples were stored at -80˚C prior to analysis. The baseline clinical characteristics of the patients are provided in
Septic shock was diagnosed according to the consensus of Sepsis-3 published in February 2016(
Patients with advanced cancer, serious cardiovascular disease, trauma with haemorrhagic shock and other severe illnesses with a high risk of short-term mortality were excluded from the study.
Cell-free RNA (cf.-RNA) was isolated from 2 ml blood samples using a commercially available RNA simple kit (Tiangen) according to the manufacturer's instructions. The amount of total RNA was quantified by the absorbance at 260 nm using an N-5000 spectrophotometer (Yoke Technologies). RNA integrity was analyzed by electrophoresis on a 1% agarose gel and stained with ethidium bromide.
Complementary DNA (cDNA) was synthesized from cf.-RNA with mRNA-specific stem-loop reverse transcription (RT) primers by the First-Strand cDNA Synthesis Kit (Tiangen) using the oligo(dT) primers included in the kit according to the manufacturer's protocol. A 20-µl reaction mixture for each sample was prepared containing 5 µl MgCl2, 2 µl 10X PCR buffer, 2 µl dNTPs, 2 µl oligo-deoxythymidine primer, 5 µl RNase-free dH2O, 2 µl ribonuclease inhibitor, 1 µl of Moloney murine leukaemia virus reverse transcriptase and 1 µg of total RNA diluted in 1 µl and placed into a 0.2 ml thin-wall PCR tube. The reverse transcription (RT) reaction was performed at 42˚C for 50 min and inactivated by heating to 90˚C for 10 min. The first-strand cDNA was cooled on ice and then stored at 0˚C until PCR was performed.
qPCR was performed on an ABI Prism® 7000 PCR instrument (Applied Biosystems China Inc.) using program parameters provided by the manufacturer. GAPDH was used as an internal control for normalization. A total of 100 µl of PCR mixture was prepared containing 0.2 mmol/l dNTPs and 2.5 U
All statistical analyses were performed using GraphPad Prism (version 7.0 for Windows, GraphPad Software). Quantitative data are expressed as mean ± standard deviation (SD). Mann-Whitney U test was used to compare the medians between survivors and non-survivors. One-way ANOVA was used to assess the differences in Ang-2 levels between baseline (Day 0), and Day 3, Day 5 and Day 7. The Tukey-Kramer post hoc test was performed for pairwise comparison between groups. The t-tests were applied to compare the means between healthy controls and patients and the means between survivors and non-survivors. A χ2 test or Fisher's exact test was performed for comparison of qualitative data. Receiver operating characteristic (ROC) procedures were used to predict mortality outcomes, and the areas under the ROC curves (AUCs) were calculated. The DeLong method was used to test the statistical significance of the difference between ROC curves (Ang-2, SOFA score and APACHE II score). All analyses were 2-tailed, with P-values <0.05 considered to indicate a statistically significant difference.
Clinical data of 85 patients with septic shock and 10 healthy controls were collected for the study. Differences in sex and age were not significant between the patients and the control group. The serum lactate and prothrombin time (PT) were significantly higher in the non-survivors (6.1±2.9 mmol/l vs. 3.4±1.6 mmol/l; and 20.5±5.6 sec vs. 14.3±3.0 sec, P<0.001, respectively). However, serum albumin and fibrinogen levels were significantly higher in the survivors (26.3±3.3 g/l vs. 24.1±3.9 g/l; and 2.0±0.7 g/l vs. 1.1±0.7 g/l, P=0.03 and P<0.001, respectively). Serum procalcitonin (PCT) and C-reactive protein (CRP) are generally accepted indicators of inflammation, but only PCT differed significantly between the survivors and the non-survivors (10.92±8.13 ng/ml vs. 21.86±15.95 ng/ml, P=0.007). The non-survivors had higher SOFA (11.0±2.9 vs. 6.7±2.4) and APACHE II (22.2±6.5 vs. 12.1±4.5) scores (P<0.001). Overall, the survivors had a shorter length of ICU stay (11.2±6.8 days vs. 17.3±10.8 days, P=0.025). Data are presented in
Both surgical and nonsurgical diseases can result in septic shock. However, no significant difference was found in regards to the mortality between the two groups (P=0.25). Among sequential sites of infection, the lung, abdomen and urinary tract were the most frequent sources of infection, with proportions of 30.6, 23.5 and 20%, respectively. There were no significant differences in mortality in each category (P=0.410, 0.540, and 0.750, respectively) (
The mean Ang-2 mRNA levels in all patients with septic shock at baseline (Day 0), Day 3, Day 5, and Day 7 were compared to those in the normal controls. Mean Ang-2 levels in patients with septic shock at Day 0, Day 3, Day 5, and Day 7 were significantly higher than those in the controls (
Of the 85 patients for which mortality data were reported, 65 survived and 20 died, giving a 28-day mortality rate of 23.57%. The mean Ang-2 mRNA levels at admission and baseline clinical measures of illness severity, including SOFA and APACHE II scores, were significantly lower in survivors than in non-survivors (
In the critical setting, the upregulation of angiopoietin 2 (Ang-2) is a physiologic and potentially lifesaving response and is significantly associated with the severity of disease. Ang-2 has been proven to increase in the context of sepsis (
Sepsis is defined as a life-threatening multiple organ dysfunction caused by a dysregulated host response to infection (
Genetic and molecular studies spanning thousands of human subjects have linked imbalances of Ang-2 levels to major adverse clinical events arising from bacterial sepsis and other severe infections (
In recent years, increasing empirical evidence has shown that extensive cross-talk between inflammation and coagulation systems plays a pivotal role in the pathogenesis of microvascular failure and subsequent multiple organ failure as a result of severe infection (
The key pathophysiology underlying this life-threatening disease is the preponderant inflammatory host response to pathogen infections leading to the overexpression of inflammatory mediators. Sepsis-induced acute lung injury (ALI) is characterized by injury and dysfunction of the pulmonary microvasculature and pulmonary microvascular endothelial cells (
As septic shock can occur across a spectrum of severities, it is of great importance to quantify disease severity in any study population. Our data showed that among the 85 septic patients involved in surgical and nonsurgical diseases, the lung, abdomen and urinary tract were the most frequent sites of infection, with proportions of 30.6, 23.5 and 20%, respectively. However, there were no significant differences in the mortality rate among the infection sites. Surgical intervention had no impact on mortality. Sequential Organ Failure Assessment (SOFA) scores were calculated daily in the ICU and are recommended as clinical criteria, combining the axes of the respiratory, hematologic, hepatic, cardiovascular, and central nervous systems, and renal function, to describe the clinical status. Organ dysfunction can be indicated by an increase in the SOFA score of 2 points or more, associated with an in-hospital mortality greater than 10% (
In a prospective study, Ang-2 expression was found to be significantly upregulated in sepsis-associated coagulopathy, and this biomarker was used to stratify patients with sepsis into non-overt DIC and overt DIC (
However, the inflammatory indicator of C-reactive protein (CRP) did not differ between the survivors and the non-survivors in this study. A possible explanation for this phenomenon is that prolonged septic shock leads to severe endothelial dysfunction and extensive leukocyte insufficiency as well as consumption of inflammatory factors. In general, the indeterminacy of clinical applications of synthetic human Ang-2 requires more clinical evidence.
In summary, the present study confirmed that the serum Ang-2 mRNA level is a prognostic indicator that is superior to SOFA and APACHE II scores currently used in the ICU. Since sepsis is not a specific illness but rather a syndrome encompassing a still uncertain pathology, additional studies to explore therapeutic interventions in regards to serum angiopoietin 2 are of great importance for the clinical management of septic shock.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JF and LW designed the study and reviewed the manuscript, and share first authorship. YF and GY performed the laboratory examinations, acquired the majority of the data and drafted the manuscript. DZ and JW confirmed the authenticity of all the raw data, analyzed the data and revised the manuscript. All authors have read and approved the final manuscript.
Data collection and analysis were undertaken in compliance with the approval and supervision of the Tongji Hospital Research Institutional Review Board (approval no. TJ-IRB20211252; Wuhan, China). The local ethics committee classified this study as a retrospective study based on data analysis, and patient consent was not required.
Patient consent for publication was not necessary because this retrospective study did not contain any personal information that could lead to the identification of the patient.
The authors declare that they have no competing interests.
(A) Significant increase of Ang-2 levels in septic shock patients and (B) the downward trend over time. *P<0.001. Ang-2, angiopoietin 2.
Septic shock is correlated with significantly higher (A) Ang-2 levels and (B) SOFA and (C) APACHE II scores in the non-survivors than in the survivors. *P<0.001. Ang-2, angiopoietin 2; SOFA, Sequential Organ Failure Assessment; APACHE, Acute Physiology and Chronic Health Evaluation.
ROC curves of three predictors (Ang-2, SOFA and APACHE II scores) for mortality outcome. Ang-2, angiopoietin 2; SOFA, Sequential Organ Failure Assessment; APACHE, Acute Physiology and Chronic Health Evaluation.
Main clinical characteristics of the patients with sepsis.
Parameters | Healthy controls (n=10) | All patients (n=85) | Survivors (n=65) | Non-survivors (n=20) | P-value |
---|---|---|---|---|---|
Mean age (range) (years) |
53 (30-68) | 55 (30-80) | 55 (30-80) | 57 (33-69) | 0.820 |
Sex (male/female) |
7/3 | 55/30 | 40/25 | 15/5 | 0.300 |
Ang II level (ratio) |
1±0.2 | 10.5±3.7 | 7.8±2.9 | 12.6±2.7 | <0.001 |
PCT (ng/ml) |
0.3±0.1 | 16.39±13.1 | 10.92±8.13 | 21.86±15.95 | 0.007 |
CRP (ng/ml) |
5±2.1 | 135.7±59.58 | 139.6±63.69 | 127.85±44.71 | 0.360 |
Albumin (g/l) |
43.3±3.2 | 26.3±3.7 | 26.3±3.3 | 24.1±3.9 | 0.030 |
Lactate (mmol/l) |
0.8±0.5 | 4.8±2.5 | 3.4±1.6 | 6.1±2.9 | <0.001 |
PT (sec) |
12±1.1 | 13.5±6.2 | 14.3±3.0 | 20.5±5.6 | <0.001 |
Fibrinogen (g/l) |
2.8±1.5 | 1.6±0.8 | 2.0±0.7 | 1.1±0.7 | <0.001 |
SOFA score |
- | 8.6±3.7 | 6.7±2.4 | 11.0±2.9 | <0.001 |
APACHE II score |
- | 16.3±7.4 | 12.1±4.5 | 22.2±6.5 | <0.001 |
Length of ICU stay (mean ± SD, days) |
- | 14.6±8.6 | 11.2±6.8 | 17.3±10.8 | 0.025 |
aData are expressed as median (range) or number;
bData are expressed as mean ± SD. PCT procalcitonin; CRP C-reactive protein; PT, prothrombin time; SOFA, Sequential Organ Failure Assessment; APACHE II, Acute Physiology And Chronic Health Evaluation II; Ang II, Angiopoietin II. P-value is calculated between survivors and non-survivors (significant for P<0.05).
Infection localization of septic shock.
Parameters | All patients (n=85) | Survivors, (n=65) | Non-survivors (n=20) | P-value |
---|---|---|---|---|
Surgical intervention | 22 (25.9) | 17 (26.2) | 5 (22.7) | 0.250 |
Infection location | ||||
Pulmonary | 26 (30.6) | 18 (27.7) | 8(40) | 0.410 |
Abdominal | 20 (23.5) | 15 (23.1) | 3(15) | 0.540 |
Liver abscess | 3 (3.5) | 2 (3.1) | 1(5) | 0.560 |
Biliary tract | 3 (3.5) | 2 (3.1) | 1(5) | 0.560 |
Urinary tract | 17(20) | 14 (21.5) | 3(15) | 0.750 |
Trauma | 8 (9.4) | 7 (10.8) | 1(5) | 0.670 |
Central nervous system | 5 (5.9) | 3 (4.6) | 2(10) | 0.590 |
Undefined | 3 (3.5) | 2 (3.1) | 1(5) | 0.560 |
Data are expressed as n (%). P-value is calculated between survivors and non-survivors (significant for P<0.05).