Infectious Diseases, 2015; Early Online: 1–6
ORIGINAL ARTICLE
Clinical significance of circulating endothelial cells in patients with severe sepsis or septic shock
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JUNG-WAN YOO1, JAE-YOUNG MOON2, SANG-BUM HONG1, CHAE-MAN LIM1, YOUNSUCK KOH1 & JIN-WON HUH1 From the 1Department of Pulmonary and Critical Care Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea, and 2Department of Internal Medicine, Chungnam National University Hospital, Chungnam National University School of Medicine, Daejeon, Republic of Korea
Abstract Background: Endothelial damage developing in severe sepsis or septic shock results in multiorgan dysfunction. An increased circulating endothelial cell (CEC) count represents a novel marker of endothelial damage, which has been reported in cases of severe sepsis or septic shock. The aim of this study was to evaluate the clinical significance of CECs in patients with severe sepsis or septic shock. Methods: CECs were evaluated and quantified by flow cytometry using plasma collected from patients with severe sepsis or septic shock who were admitted to a medical intensive care unit from February 2011 to August 2011. Results: During the study period, 77 patients were enrolled. The median CEC count was 350 cells/ml (range 0–15 984 cells/ml). There was no significant difference between cases of severe sepsis and septic shock [163 cells/ml (0–15 984 cells/ml) vs 363 cells/ml (0–7884 cells/ml), p ⫽ 0.507]. There were no correlations between the number of CECs and inflammatory markers. CEC counts were significantly increased in non-survivors compared to survivors [588.5 cells/ml (1–15 984 cells/ml) vs 292 cells/ml (0–12 882 cells/ml), p ⫽ 0.044] within 28 days. The sequential-related organ failure assessment score, CEC counts ⱖ 500 cells/ml and blood lactate levels were significantly associated with 28 day mortality. Conclusions: CEC counts were higher in non-survivors of severe sepsis or septic shock and could be used as a biomarker to predict the prognosis in these patients.
Keywords: Circulating endothelial cells, mortality, septic shock, severe sepsis
Introduction Severe sepsis and septic shock are critical conditions that account for high mortality in the intensive care unit (ICU) [1,2]. Several pathogenic mechanisms, including innate immunity, the immune response, coagulopathy and microcirculatory dysfunction, affect the development of severe sepsis and septic shock [3–5]. Vascular alterations, including vascular tone or permeability, are known to be associated with organ dysfunction and clinical outcomes [5]. Endothelial cells play crucial roles in maintaining the integrity of the microvascular system. Endothelial damage results in disruption of the microvascular system and hemodynamic alterations, which can lead to organ dysfunction in patients with sepsis [6]. Circulating endothelial cells (CECs) include mature endothelial cells derived from vessel walls
and a subpopulation of endothelial precursors that originate from monocytic cells. Thus, CECs have been recognized as a novel biomarker of endothelial damage as they are purely endothelial in origin and represent cells that have detached in response to vascular damage [7,8]. An increased frequency of CECs in plasma has been detected in non-infectious conditions associated with endothelial damage, including cardiovascular disease [9], connective tissue diseases [10] and malignancies [11,12]. Some studies have demonstrated that increased circulating endothelial progenitor cells in septic patients are associated with survival [13] and correlate inversely with organ dysfunction [14]. A few studies have shown that mature CECs are more highly elevated in patients with severe sepsis or septic shock than in non-septic patients or healthy
Correspondence: Jin-Won Huh, Department of Pulmonary and Critical Care Medicine, University of Ulsan College of Medicine, Asan Medical Center, 88, Olympic-Ro 43-Gil, Songpa-gu, Seoul, South Korea. Tel: ⫹ 82 2 3010 3985. Fax: ⫹ 82 2 3010 6968. E-mail: [email protected] (Received 5 October 2014 ; accepted 16 December 2014 ) ISSN 2374-4235 print/ISSN 2374-4243 online © 2015 Informa Healthcare DOI: 10.3109/00365548.2014.1001999
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individuals [15,16]. Although increased mature CECs in patients with severe sepsis or septic shock have been identified, little is known about the association between CECs and mortality. The aim of this study was to evaluate whether CECs are associated with prognosis in patients with severe sepsis or septic shock.
Materials and methods
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Study population From February 2011 to August 2011, patients who met the American College of Chest Physicians/Society for Critical Care Medicine criteria for severe sepsis and septic shock [17] were enrolled. Adult patients older than 18 years were admitted to the medical ICU at the Asan Medical Center, a universityaffiliated hospital. At enrollment, demographic data and sequential-related organ failure assessment (SOFA) scores were collected. Sources of infection were evaluated and laboratory data were measured. This study complied with the principles of the Helsinki Declaration. Informed consent was obtained from each subject or a designated surrogate before enrollment. This study was approved by the Institutional Review Board at Asan Medical Center. Flow-cytometric analysis of circulating endothelial cells CECs were evaluated by flow-cytometric analysis according to a previously described procedure [18,19]. Peripheral blood samples were collected in a 3 ml vacutainer tube containing liquid tripotassium ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Red blood cell lysis was performed to obtain white blood cells (WBCs) with 10 ml of lysing solution (Sigma-Aldrich, USA). The
following directly conjugated mouse anti-human monoclonal antibodies were used: phycoerythrin (PE)-labeled anti-CD146 (BD Biosciences, USA), fluorescein isothiocyanate (FITC)-labeled antiCD45 (BD Biosciences, USA) and PE-Cy 5-labeled anti-CD 34 (BD Biosciences, USA). Mononuclear cell gating from WBCs was carried out using a FACScalibur flow cytometer (BD Biosciences, USA) and data were analyzed using BD CellQuest Pro software. CECs were defined as CD45–, CD146⫹ and CD34⫹ (Figure 1). Clinical outcomes The primary aim was to evaluate the difference in CEC counts between non-survivors and survivors over the 28 day study period. The secondary aim was to investigate CEC counts that were associated with 28 day mortality in patients with severe sepsis or septic shock. Statistical analysis Data are expressed as number (%) for categorical variables or as median (range). Fisher’s exact test was used to compare categorical data and the Mann– Whitney U test was used to compare continuous data. Logistic regression analyses were performed to evaluate factors associated with mortality. Factors associated with mortality with p values less than 0.05 by univariate analysis were included in the multivariate analysis. The Kaplan–Meier method and logrank test were used to estimate 28 day mortality and compare survival curves according to CEC counts. Prism 5 was used to draw the plots. All statistical analyses were performed using SPSS version 18 (SPSS, Chicago, IL, USA). A p value less than 0.05 was considered statistically significant.
Figure 1. Flow cytometric dot-plot panels to identify mature circulating endothelial cells (CECs): (a) cell subpopulation based on forward scatter (FSC) vs side scatter (SSC) (R1); (b) gate to exclude the CD45-positive population (R2); (c) mature CEC (R3) population expressing the CD34 and CD146 antigens. FITC, fluorescein isothiocyanate; PE, phycoerythrin.
Circulating endothelial cells in sepsis Results
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Baseline characteristics and laboratory findings of the enrolled patients In total, 77 patients were enrolled in this study: 20 (26%) exhibited severe sepsis and 57 (74%) had septic shock. The characteristics of the enrolled patients are listed in Table I. The median age of the patients was 69 years, and most (74%) were men. Bacteremia was identified in 28 patients (36.4%), and Gramnegative bacteremia was predominant. The respiratory tract was the most common site of infection. Patients with septic shock had more chronic liver disease and Gram-negative bacteremia, and higher SOFA scores and arterial lactate levels than those with severe sepsis. Circulating endothelial cell count and clinical outcomes Table II shows a comparison of the characteristics between survivors and non-survivors over the 28 day period. The median CEC count was 350 cells/ml
(range 0–15 984 cells/ml). There was a significantly higher CEC count in non-survivors than in survivors. SOFA scores and arterial lactate levels on days 1, 3 and 7 were significantly higher in non-survivors than in survivors. During follow-up, only survivors showed significant improvements in SOFA score (p ⬍ 0.001) and arterial lactate (p ⬍ 0.001). Relationships between circulating endothelial cell counts, clinical characteristics and inflammatory markers There was no correlation between CEC counts and known inflammatory markers (C-reactive protein, procalcitonin and WBC counts), or between CEC counts and SOFA score (r ⫽ –0.065, p ⫽ 0.575). There was no significant difference between cases of severe sepsis and septic shock [163 cells/ml (0–15984 cells/ml) vs 363 cells/ml (0–7884 cells/ml), respectively; p ⫽ 0.507]. There was also no significant difference in the median CEC counts for patients with and those without bacteremia [356.5 cells/ml (0–7884 cells/ml) vs 339 cells/ml (0–15 984 cells/ml), respectively, p ⫽ 0.996].
Table I. Baseline characteristics and laboratory findings in patients with severe sepsis or septic shock.
Characteristic Age (years) Gender, male Underlying disease Diabetes mellitus Malignancy Cardiovascular disease Chronic lung disease Chronic liver disease Chronic kidney disease Rheumatological disease SOFA score Bacteremia Gram-positive pathogen Gram-negative pathogen Site of infection Respiratory tract Gastrointestinal and biliary Urinary tract Central nervous system Soft tissue or skin Intravascular catheter Primary bacteremia Others or unknown Laboratory findings WBC (⫻ 103/μl) CRP (mg/dl) Procalcitonin (ng/ml) Lactate, arterial (mmol/l) 14 day mortality 28 day mortality
Total (N ⫽ 77) 69 (29–87) 57 (74)
Severe sepsis (N ⫽ 20) 68.5 (31–83) 18 (90)
Septic shock (N ⫽ 57) 69 (29–87) 39 (68.4)
26 35 7 18 12 19 3 11 28 5 23
(33.3) (45.5) (9.1) (23.4) (15.6) (24.7) (3.9) (4–19) (36.4) (17.9) (82.1)
9 12 1 7 0 9 0 9 4 3 1
(45) (60) (5) (35) (0) (45) (0) (4–17) (20) (75) (25)
17 30 6 11 12 10 3 12 24 2 22
(29.8) (52.6) (10.5) (19.3) (21.1) (17.5) (5.3) (4–19) (42.1) (8.3) (91.7)
45 17 2 1 3 2 4 3
(58.4) (22.1) (2.6) (1.3) (3.9) (2.6) (5.2) (3.9)
15 0 0 1 1 2 0 1
(75) (0) (0) (5) (5) (10) (0) (5)
30 17 2 0 2 0 4 2
(52.6) (29.8) (3.5) (0) (3.5) (0) (0) (3.6)
p 0.749 0.058 0.217 0.569 0.669 0.218 0.03 0.032 0.564 ⬍ 0.001 0.011
0.946
12.8 17.1 5.7 2.6 20 28
(0.1–62.7) (0.7–67.4) (0.1–200) (0.6–15) (26) (36.4)
3
11.3 17.6 3.8 1.5 4 6
(2.9–38.5) (0.7–32.1) (0.1–180) (0.6–9.2) (20) (30)
13.6 17.1 6.3 3.1 16 22
(0.1–62.7) (1.5–67.4) (0.1–200) (0.8–15) (28.1) (38.6)
0.150 0.593 0.269 0.001 0.479 0.492
SOFA, sequential organ failure assessment; WBC, white blood cell; CRP, C-reactive protein. Data are expressed as n (%) for categorical variables and median (range) for non-categorical variables.
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J.-W. Yoo et al. Table II. Comparison of characteristics between survivors and non-survivors over 28 days. Survivors (n ⫽ 49)
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Variable Age (years) Gender, male Diabetes mellitus Chronic lung disease Cardiovascular disease Chronic liver disease Chronic kidney disease Malignancy Severe sepsis Septic shock CEC counts (cells/ml) CRP (mg/dl) Procalcitonin (ng/ml) WBC (⫻ 103/μl) Lactate, arterial (day 1) (mmol/l) Lactate, arterial (day 3) (mmol/l) Lactate, arterial (day 7) (mmol/l) Bacteremia SOFA score (day 1) SOFA score (day 3) SOFA score (day 7)
67 40 17 15 5 5 14 22 14 35 340 18.2 6.24 13 2.25 1.6 1.3 17 11 8 6
Non-survivors (n ⫽ 28)
(29–87) (81.6) (34.7) (30.6) (10.2) (10.2) (28.6) (44.9) (28.6) (71.4) (0–23 458.8) (0.7–67.4) (0.05–200) (0.6–44.5) (0.6–12.8) (0.5–7.9) (0.5–3.8) (34.7) (4–17) (2–19) (0–18)
70 17 9 3 2 7 5 13 6 22 1047.95 16.4 5.56 11.9 5.25 2.8 1.9 11 14 13 12.5
(48–83) (60.7) (32.1) (10.7) (7.1) (25.0) (17.9) (46.4) (21.4) (78.6) (10–26 330.4) (1.5–35.6) (0.16–78.73) (0.1–62.7) (1.1–15) (0.9–15) (0.8–15) (39.3) (5–19) (3–24) (0–23)
p 0.418 0.044 0.820 0.047 1.000 0.108 0.294 0.897 0.492 0.016 0.179 0.527 0.304 0.005 0.003 0.023 0.687 0.003 0.001 0.005
CEC, circulating endothelial cell; CRP, C-reactive protein; WBC, white blood cell; SOFA, sequential organ failure assessment. Data are expressed as number (%) for categorical variables and median (range) for non-categorical variables.
Univariate and multivariate analyses of factors associated with 28 day mortality We dichotomized CEC counts at a level of 500 cells/ml to evaluate factors associated with 28 day mortality. Female gender, SOFA score, CEC counts greater than or equal to 500 cells/ml and blood lactate levels were associated with 28 day mortality in the univariate analysis. In the multivariate analysis, SOFA score, CEC counts greater than or equal to 500 cells/ml and blood lactate were associated with
28 day mortality (Table III). Patients with CEC counts less than 500 cells/ml showed a better prognosis than patients with CEC counts of 500 cells/ml and above (p ⫽ 0.027) (Figure 2).
Discussion The current findings showed that among patients with severe sepsis or septic shock who were admitted to the ICU, CEC counts were higher in non-survivors
Table III. Univariate and multivariate analysis for factors associated with 28 day mortality. Univariate Variable Age Female gender Diabetes mellitus Malignancy Cardiovascular disease Rheumatological disease SOFA CEC counts ⱖ 500 cells/ml Blood lactate CRP Procalcitonin
Multivariate
OR
95% CI
p-
OR
95% CI
p-
1.020 2.876 0.892 1.064 0.677 0.870 1.275 4.000 1.257 0.971 0.987
0.980–1.062 1.008–8.201 0.332–2.393 0.419–2.701 0.122–3.743 0.075–10.053 1.097–1.481 1.434–11.155 1.083–1.459 0.927–1.016 0.968–1.007
0.326 0.048 0.820 0.897 0.655 0.911 0.002 0.008 0.003 0.203 0.198
1.032 2.718
0.979–1.0088 0.745–9.924
0.236 0.30
1.203 4.123 1.212
1.002–1.443 1.189–14.297 1.032–1.423
0.048 0.026 0.019
OR, odds ratio; CI, confidence interval; SOFA, sequential organ failure assessment; CEC, circulating endothelial cell; CRP, C-reactive protein. A Hosmer–Lemeshow goodness-of-fit test showed χ2 ⫽ 5.998 and p ⫽ 0.647.
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Circulating endothelial cells in sepsis
Figure 2. Kaplan–Meier survival curves according to circulating endothelial cell (CEC) counts (p ⫽ 0.027). The survival rate was 70.5 ⫾ 7.4% when the CEC count was ⬍ 500 cells/ml, whereas it was 39.4 ⫾ 10.3% when the CEC count was ⱖ 500 cells/ml.
than in survivors over the 28 day study period. CEC counts greater than or equal to 500 cells/ml were associated with higher 28 day mortality, suggesting that levels of CECs could be used as be a predictive biomarker for mortality in patients with severe sepsis or septic shock. The endothelium plays a key role in mediating vasomotor tone, permeability and hemostasis [20]. It has been shown that endothelial damage or dysfunction plays an important role in the development of sepsis-associated organ dysfunction [6]. Microparticles, known as proinflammatory and procoagulant fragments originating from plasma membranes, are another surrogate for cell activation analysis during sepsis and septic shock. They play a role in altering vascular tone and contribute to spreading inflammatory and prothrombotic vascular status in septic conditions [21]. Furthermore, Delabranche et al. demonstrated that endothelial-derived microparticles are biomarkers of septic shock-induced disseminated intravascular coagulopathy [22]. The frequency of CECs represents a novel marker of endothelial damage that has been associated with various diseases [8]. The presence of CECs in the peripheral blood of healthy individuals is rare. The mean values of the percentage and numbers of CEC, measured by flow cytometry, have been suggested to be 0.005 ⫾ 0.004% and 306 ⫾ 243 cells/ml, respectively, in healthy subjects [18]. Boos et al. showed that the number of CECs is elevated when lipopolysaccharide is infused into healthy male subjects (endotoxinic condition) [23]. Moreover, two other studies have reported a relationship between CECs and sepsis [15,16]. All of these reports found that CEC counts are elevated in septic patients compared to non-septic or healthy subjects. By demonstrating increased CEC counts, they suggested that endothelial damage occurs
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in patients with severe sepsis or septic shock. Furthermore, Mutunga et al. found that CEC counts in nonsurvivors of septic shock are higher than in survivors, supporting the concept that the number of CECs in sepsis is associated with the severity of vascular injury [15]. However, small numbers of cases with sepsis were enrolled in previous studies. This limits explanation of the correlation between CEC counts and prognosis of patients with sepsis. Whether CECs are associated with prognosis in patients with sepsis thus remains unclear. CECs have previously been detected by various methods, including immunofluorescence, immunohistochemistry and flow cytometry [8,15,19]. Flow cytometry has been used most widely to detect CECs because it can simultaneously distinguish between stem cell and endothelial markers. In the current study, CECs were detected by flow-cytometric analysis using three cell-surface markers. We defined CECs as CD45– (to exclude hematopoietic cells), CD34⫹ (a stem cell marker) and CD146⫹ (an endothelial cell marker). In agreement with other studies, we found that CEC counts were higher in non-survivors than in survivors of severe sepsis or septic shock over the 28 day study period. When we divided the 77 patients into two groups (CEC counts ⬍ 500 cells/ml vs ⱖ 500 cells/ml), patients with CEC counts of 500 cells/ml and above had a higher 28 day mortality rate than patients with CEC counts below 500 cells/ml. This result suggests that CECs could be used to predict the prognosis of patients with severe sepsis or septic shock. There were some limitations to our study. First, the study was conducted in a single center, making it difficult to generalize the findings to all cases of severe sepsis or septic shock. Secondly, we used a flow-cytometric method as an analysis modality, but no standardized and easily utilized protocol for the measurement of CECs in a clinical setting has been established to date. Thirdly, we did not evaluate CEC counts in healthy controls for comparison to patients with severe sepsis or septic shock. Compared to 24 non-septic patients admitted to the ICU, CEC counts were significantly higher in those with severe sepsis or septic shock [350 cells/ml (range 0–15984 cells/ml) vs 107.9 cells/ml (range 0–8000 cells/ml), p ⫽ 0.008)]. In conclusion, CEC counts in the blood were elevated in cases of both severe sepsis and septic shock. The number of CECs was higher in nonsurvivors of sepsis than in survivors, and CEC counts of 500 cells/ml and higher were associated with an increased 28 day mortality. Thus, the CECs may be a biomarker of severe sepsis or septic shock, although validation in a multicenter trial is needed.
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Acknowledgement We thank Eun-young Jang for technical support. Declaration of interest: The authors have no conflicts of interest to declare. This study was supported by the Asan Institute for Life Science, Asan Medical Center, Seoul, Korea [grant 11–502].
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References [1] Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001;29:1303–10. [2] Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med 2003;348:1546–54. [3] Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med 2003;348:138–50. [4] Hotchkiss RS, Monneret G, Payen D. Immunosuppression in sepsis: a novel understanding of the disorder and a new therapeutic approach. Lancet Infect Dis 2013;13:260–8. [5] Bateman R, Sharpe M, Ellis C. Bench-to-bedside review: microvascular dysfunction in sepsis – hemodynamics, oxygen transport, and nitric oxide. Crit Care 2003;7:359–73. [6] Aird WC. The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood 2003;101: 3765–77. [7] Blann AD, Woywodt A, Bertolini F, Bull TM, Buyon JP, Clancy RM, et al. Circulating endothelial cells. Biomarker of vascular disease. Thromb Haemost 2005;93:228–35. [8] Erdbruegger U, Haubitz M,Woywodt A. Circulating endothelial cells: a novel marker of endothelial damage. Clin Chim Acta 2006;373:17–26. [9] Boos CJ, Soor SK, Kang D, Lip GY. Relationship between circulating endothelial cells and the predicted risk of cardiovascular events in acute coronary syndromes. Eur Heart J 2007;28:1092–101. [10] Woywodt A, Streiber F, de Groot K, Regelsberger H, Haller H, Haubitz M. Circulating endothelial cells as markers for ANCAassociated small-vessel vasculitis. Lancet 2003;361:206–10. [11] Wierzbowska A, Robak T, Krawczynska A, Wrzesien-Kus A, Pluta A, Cebula B, et al. Circulating endothelial cells in patients with acute myeloid leukemia. Eur J Haematol 2005; 75:492–7.
[12] Ronzoni M, Manzoni M, Mariucci S, Loupakis F, Brugnatelli S, Bencardino K, et al. Circulating endothelial cells and endothelial progenitors as predictive markers of clinical response to bevacizumab-based first-line treatment in advanced colorectal cancer patients. Ann Oncol 2010; 21:2382–9. [13] Rafat N, Hanusch C, Brinkkoetter PT, Schulte J, Brade J, Zijlstra JG, et al. Increased circulating endothelial progenitor cells in septic patients: correlation with survival. Crit Care Med 2007;35:1677–84. [14] Cribbs S, Sutcliffe D, Taylor W, Rojas M, Easley K, Tang L, et al. Circulating endothelial progenitor cells inversely associate with organ dysfunction in sepsis. Intensive Care Med 2012;38:429–36. [15] Mutunga M, Fulton B, Bullock R, Batchelor A, Gascoigne A, Gillespie JI, et al. Circulating endothelial cells in patients with septic shock. Am J Respir Crit Care Med 2001;163: 195–200. [16] Schlichting DE, Waxman AB, O’Brien LA, Wang T, Naum CC, Rubeiz GJ, et al. Circulating endothelial and endothelial progenitor cells in patients with severe sepsis. Microvasc Res 2011;81:216–21. [17] Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, et al. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. the ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine. Chest 1992;101:1644–55. [18] Mariucci S, Rovati B, Bencardino K, Manzoni M, Danova M. Flow cytometric detection of circulating endothelial cells and endothelial progenitor cells in healthy subjects. Int J Lab Hematol 2010;32:e40–8. [19] Masouleh BK, Baraniskin A, Schmiegel W, Schroers R. Quantification of circulating endothelial progenitor cells in human peripheral blood: establishing a reliable flow cytometry protocol. J Immunol Methods 2010;357: 38–42. [20] Aird WC. Spatial and temporal dynamics of the endothelium. J Thromb Haemost 2005;3:1392–406. [21] Meziani F, Delabranche X, Asfar P, Toti F. Bench-to-bedside review: circulating microparticles – a new player in sepsis? Crit Care 2010;14:236. [22] Delabranche X, Boisrame-Helms J, Asfar P, Berger A, Mootien Y, Lavigne T, et al. Microparticles are new biomarkers of septic shock-induced disseminated intravascular coagulopathy. Intensive Care Med 2013;39:1695–703. [23] Boos CJ, Mayr FB, Lip GYH, Jilma B. Endotoxemia enhances circulating endothelial cells in humans. J Thromb Haemost 2006;4:2509–11.
ORIGINAL ARTICLE
Clinical significance of circulating endothelial cells in patients with severe sepsis or septic shock
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JUNG-WAN YOO1, JAE-YOUNG MOON2, SANG-BUM HONG1, CHAE-MAN LIM1, YOUNSUCK KOH1 & JIN-WON HUH1 From the 1Department of Pulmonary and Critical Care Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea, and 2Department of Internal Medicine, Chungnam National University Hospital, Chungnam National University School of Medicine, Daejeon, Republic of Korea
Abstract Background: Endothelial damage developing in severe sepsis or septic shock results in multiorgan dysfunction. An increased circulating endothelial cell (CEC) count represents a novel marker of endothelial damage, which has been reported in cases of severe sepsis or septic shock. The aim of this study was to evaluate the clinical significance of CECs in patients with severe sepsis or septic shock. Methods: CECs were evaluated and quantified by flow cytometry using plasma collected from patients with severe sepsis or septic shock who were admitted to a medical intensive care unit from February 2011 to August 2011. Results: During the study period, 77 patients were enrolled. The median CEC count was 350 cells/ml (range 0–15 984 cells/ml). There was no significant difference between cases of severe sepsis and septic shock [163 cells/ml (0–15 984 cells/ml) vs 363 cells/ml (0–7884 cells/ml), p ⫽ 0.507]. There were no correlations between the number of CECs and inflammatory markers. CEC counts were significantly increased in non-survivors compared to survivors [588.5 cells/ml (1–15 984 cells/ml) vs 292 cells/ml (0–12 882 cells/ml), p ⫽ 0.044] within 28 days. The sequential-related organ failure assessment score, CEC counts ⱖ 500 cells/ml and blood lactate levels were significantly associated with 28 day mortality. Conclusions: CEC counts were higher in non-survivors of severe sepsis or septic shock and could be used as a biomarker to predict the prognosis in these patients.
Keywords: Circulating endothelial cells, mortality, septic shock, severe sepsis
Introduction Severe sepsis and septic shock are critical conditions that account for high mortality in the intensive care unit (ICU) [1,2]. Several pathogenic mechanisms, including innate immunity, the immune response, coagulopathy and microcirculatory dysfunction, affect the development of severe sepsis and septic shock [3–5]. Vascular alterations, including vascular tone or permeability, are known to be associated with organ dysfunction and clinical outcomes [5]. Endothelial cells play crucial roles in maintaining the integrity of the microvascular system. Endothelial damage results in disruption of the microvascular system and hemodynamic alterations, which can lead to organ dysfunction in patients with sepsis [6]. Circulating endothelial cells (CECs) include mature endothelial cells derived from vessel walls
and a subpopulation of endothelial precursors that originate from monocytic cells. Thus, CECs have been recognized as a novel biomarker of endothelial damage as they are purely endothelial in origin and represent cells that have detached in response to vascular damage [7,8]. An increased frequency of CECs in plasma has been detected in non-infectious conditions associated with endothelial damage, including cardiovascular disease [9], connective tissue diseases [10] and malignancies [11,12]. Some studies have demonstrated that increased circulating endothelial progenitor cells in septic patients are associated with survival [13] and correlate inversely with organ dysfunction [14]. A few studies have shown that mature CECs are more highly elevated in patients with severe sepsis or septic shock than in non-septic patients or healthy
Correspondence: Jin-Won Huh, Department of Pulmonary and Critical Care Medicine, University of Ulsan College of Medicine, Asan Medical Center, 88, Olympic-Ro 43-Gil, Songpa-gu, Seoul, South Korea. Tel: ⫹ 82 2 3010 3985. Fax: ⫹ 82 2 3010 6968. E-mail: [email protected] (Received 5 October 2014 ; accepted 16 December 2014 ) ISSN 2374-4235 print/ISSN 2374-4243 online © 2015 Informa Healthcare DOI: 10.3109/00365548.2014.1001999
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J.-W. Yoo et al.
individuals [15,16]. Although increased mature CECs in patients with severe sepsis or septic shock have been identified, little is known about the association between CECs and mortality. The aim of this study was to evaluate whether CECs are associated with prognosis in patients with severe sepsis or septic shock.
Materials and methods
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Study population From February 2011 to August 2011, patients who met the American College of Chest Physicians/Society for Critical Care Medicine criteria for severe sepsis and septic shock [17] were enrolled. Adult patients older than 18 years were admitted to the medical ICU at the Asan Medical Center, a universityaffiliated hospital. At enrollment, demographic data and sequential-related organ failure assessment (SOFA) scores were collected. Sources of infection were evaluated and laboratory data were measured. This study complied with the principles of the Helsinki Declaration. Informed consent was obtained from each subject or a designated surrogate before enrollment. This study was approved by the Institutional Review Board at Asan Medical Center. Flow-cytometric analysis of circulating endothelial cells CECs were evaluated by flow-cytometric analysis according to a previously described procedure [18,19]. Peripheral blood samples were collected in a 3 ml vacutainer tube containing liquid tripotassium ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Red blood cell lysis was performed to obtain white blood cells (WBCs) with 10 ml of lysing solution (Sigma-Aldrich, USA). The
following directly conjugated mouse anti-human monoclonal antibodies were used: phycoerythrin (PE)-labeled anti-CD146 (BD Biosciences, USA), fluorescein isothiocyanate (FITC)-labeled antiCD45 (BD Biosciences, USA) and PE-Cy 5-labeled anti-CD 34 (BD Biosciences, USA). Mononuclear cell gating from WBCs was carried out using a FACScalibur flow cytometer (BD Biosciences, USA) and data were analyzed using BD CellQuest Pro software. CECs were defined as CD45–, CD146⫹ and CD34⫹ (Figure 1). Clinical outcomes The primary aim was to evaluate the difference in CEC counts between non-survivors and survivors over the 28 day study period. The secondary aim was to investigate CEC counts that were associated with 28 day mortality in patients with severe sepsis or septic shock. Statistical analysis Data are expressed as number (%) for categorical variables or as median (range). Fisher’s exact test was used to compare categorical data and the Mann– Whitney U test was used to compare continuous data. Logistic regression analyses were performed to evaluate factors associated with mortality. Factors associated with mortality with p values less than 0.05 by univariate analysis were included in the multivariate analysis. The Kaplan–Meier method and logrank test were used to estimate 28 day mortality and compare survival curves according to CEC counts. Prism 5 was used to draw the plots. All statistical analyses were performed using SPSS version 18 (SPSS, Chicago, IL, USA). A p value less than 0.05 was considered statistically significant.
Figure 1. Flow cytometric dot-plot panels to identify mature circulating endothelial cells (CECs): (a) cell subpopulation based on forward scatter (FSC) vs side scatter (SSC) (R1); (b) gate to exclude the CD45-positive population (R2); (c) mature CEC (R3) population expressing the CD34 and CD146 antigens. FITC, fluorescein isothiocyanate; PE, phycoerythrin.
Circulating endothelial cells in sepsis Results
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Baseline characteristics and laboratory findings of the enrolled patients In total, 77 patients were enrolled in this study: 20 (26%) exhibited severe sepsis and 57 (74%) had septic shock. The characteristics of the enrolled patients are listed in Table I. The median age of the patients was 69 years, and most (74%) were men. Bacteremia was identified in 28 patients (36.4%), and Gramnegative bacteremia was predominant. The respiratory tract was the most common site of infection. Patients with septic shock had more chronic liver disease and Gram-negative bacteremia, and higher SOFA scores and arterial lactate levels than those with severe sepsis. Circulating endothelial cell count and clinical outcomes Table II shows a comparison of the characteristics between survivors and non-survivors over the 28 day period. The median CEC count was 350 cells/ml
(range 0–15 984 cells/ml). There was a significantly higher CEC count in non-survivors than in survivors. SOFA scores and arterial lactate levels on days 1, 3 and 7 were significantly higher in non-survivors than in survivors. During follow-up, only survivors showed significant improvements in SOFA score (p ⬍ 0.001) and arterial lactate (p ⬍ 0.001). Relationships between circulating endothelial cell counts, clinical characteristics and inflammatory markers There was no correlation between CEC counts and known inflammatory markers (C-reactive protein, procalcitonin and WBC counts), or between CEC counts and SOFA score (r ⫽ –0.065, p ⫽ 0.575). There was no significant difference between cases of severe sepsis and septic shock [163 cells/ml (0–15984 cells/ml) vs 363 cells/ml (0–7884 cells/ml), respectively; p ⫽ 0.507]. There was also no significant difference in the median CEC counts for patients with and those without bacteremia [356.5 cells/ml (0–7884 cells/ml) vs 339 cells/ml (0–15 984 cells/ml), respectively, p ⫽ 0.996].
Table I. Baseline characteristics and laboratory findings in patients with severe sepsis or septic shock.
Characteristic Age (years) Gender, male Underlying disease Diabetes mellitus Malignancy Cardiovascular disease Chronic lung disease Chronic liver disease Chronic kidney disease Rheumatological disease SOFA score Bacteremia Gram-positive pathogen Gram-negative pathogen Site of infection Respiratory tract Gastrointestinal and biliary Urinary tract Central nervous system Soft tissue or skin Intravascular catheter Primary bacteremia Others or unknown Laboratory findings WBC (⫻ 103/μl) CRP (mg/dl) Procalcitonin (ng/ml) Lactate, arterial (mmol/l) 14 day mortality 28 day mortality
Total (N ⫽ 77) 69 (29–87) 57 (74)
Severe sepsis (N ⫽ 20) 68.5 (31–83) 18 (90)
Septic shock (N ⫽ 57) 69 (29–87) 39 (68.4)
26 35 7 18 12 19 3 11 28 5 23
(33.3) (45.5) (9.1) (23.4) (15.6) (24.7) (3.9) (4–19) (36.4) (17.9) (82.1)
9 12 1 7 0 9 0 9 4 3 1
(45) (60) (5) (35) (0) (45) (0) (4–17) (20) (75) (25)
17 30 6 11 12 10 3 12 24 2 22
(29.8) (52.6) (10.5) (19.3) (21.1) (17.5) (5.3) (4–19) (42.1) (8.3) (91.7)
45 17 2 1 3 2 4 3
(58.4) (22.1) (2.6) (1.3) (3.9) (2.6) (5.2) (3.9)
15 0 0 1 1 2 0 1
(75) (0) (0) (5) (5) (10) (0) (5)
30 17 2 0 2 0 4 2
(52.6) (29.8) (3.5) (0) (3.5) (0) (0) (3.6)
p 0.749 0.058 0.217 0.569 0.669 0.218 0.03 0.032 0.564 ⬍ 0.001 0.011
0.946
12.8 17.1 5.7 2.6 20 28
(0.1–62.7) (0.7–67.4) (0.1–200) (0.6–15) (26) (36.4)
3
11.3 17.6 3.8 1.5 4 6
(2.9–38.5) (0.7–32.1) (0.1–180) (0.6–9.2) (20) (30)
13.6 17.1 6.3 3.1 16 22
(0.1–62.7) (1.5–67.4) (0.1–200) (0.8–15) (28.1) (38.6)
0.150 0.593 0.269 0.001 0.479 0.492
SOFA, sequential organ failure assessment; WBC, white blood cell; CRP, C-reactive protein. Data are expressed as n (%) for categorical variables and median (range) for non-categorical variables.
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J.-W. Yoo et al. Table II. Comparison of characteristics between survivors and non-survivors over 28 days. Survivors (n ⫽ 49)
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Variable Age (years) Gender, male Diabetes mellitus Chronic lung disease Cardiovascular disease Chronic liver disease Chronic kidney disease Malignancy Severe sepsis Septic shock CEC counts (cells/ml) CRP (mg/dl) Procalcitonin (ng/ml) WBC (⫻ 103/μl) Lactate, arterial (day 1) (mmol/l) Lactate, arterial (day 3) (mmol/l) Lactate, arterial (day 7) (mmol/l) Bacteremia SOFA score (day 1) SOFA score (day 3) SOFA score (day 7)
67 40 17 15 5 5 14 22 14 35 340 18.2 6.24 13 2.25 1.6 1.3 17 11 8 6
Non-survivors (n ⫽ 28)
(29–87) (81.6) (34.7) (30.6) (10.2) (10.2) (28.6) (44.9) (28.6) (71.4) (0–23 458.8) (0.7–67.4) (0.05–200) (0.6–44.5) (0.6–12.8) (0.5–7.9) (0.5–3.8) (34.7) (4–17) (2–19) (0–18)
70 17 9 3 2 7 5 13 6 22 1047.95 16.4 5.56 11.9 5.25 2.8 1.9 11 14 13 12.5
(48–83) (60.7) (32.1) (10.7) (7.1) (25.0) (17.9) (46.4) (21.4) (78.6) (10–26 330.4) (1.5–35.6) (0.16–78.73) (0.1–62.7) (1.1–15) (0.9–15) (0.8–15) (39.3) (5–19) (3–24) (0–23)
p 0.418 0.044 0.820 0.047 1.000 0.108 0.294 0.897 0.492 0.016 0.179 0.527 0.304 0.005 0.003 0.023 0.687 0.003 0.001 0.005
CEC, circulating endothelial cell; CRP, C-reactive protein; WBC, white blood cell; SOFA, sequential organ failure assessment. Data are expressed as number (%) for categorical variables and median (range) for non-categorical variables.
Univariate and multivariate analyses of factors associated with 28 day mortality We dichotomized CEC counts at a level of 500 cells/ml to evaluate factors associated with 28 day mortality. Female gender, SOFA score, CEC counts greater than or equal to 500 cells/ml and blood lactate levels were associated with 28 day mortality in the univariate analysis. In the multivariate analysis, SOFA score, CEC counts greater than or equal to 500 cells/ml and blood lactate were associated with
28 day mortality (Table III). Patients with CEC counts less than 500 cells/ml showed a better prognosis than patients with CEC counts of 500 cells/ml and above (p ⫽ 0.027) (Figure 2).
Discussion The current findings showed that among patients with severe sepsis or septic shock who were admitted to the ICU, CEC counts were higher in non-survivors
Table III. Univariate and multivariate analysis for factors associated with 28 day mortality. Univariate Variable Age Female gender Diabetes mellitus Malignancy Cardiovascular disease Rheumatological disease SOFA CEC counts ⱖ 500 cells/ml Blood lactate CRP Procalcitonin
Multivariate
OR
95% CI
p-
OR
95% CI
p-
1.020 2.876 0.892 1.064 0.677 0.870 1.275 4.000 1.257 0.971 0.987
0.980–1.062 1.008–8.201 0.332–2.393 0.419–2.701 0.122–3.743 0.075–10.053 1.097–1.481 1.434–11.155 1.083–1.459 0.927–1.016 0.968–1.007
0.326 0.048 0.820 0.897 0.655 0.911 0.002 0.008 0.003 0.203 0.198
1.032 2.718
0.979–1.0088 0.745–9.924
0.236 0.30
1.203 4.123 1.212
1.002–1.443 1.189–14.297 1.032–1.423
0.048 0.026 0.019
OR, odds ratio; CI, confidence interval; SOFA, sequential organ failure assessment; CEC, circulating endothelial cell; CRP, C-reactive protein. A Hosmer–Lemeshow goodness-of-fit test showed χ2 ⫽ 5.998 and p ⫽ 0.647.
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Circulating endothelial cells in sepsis
Figure 2. Kaplan–Meier survival curves according to circulating endothelial cell (CEC) counts (p ⫽ 0.027). The survival rate was 70.5 ⫾ 7.4% when the CEC count was ⬍ 500 cells/ml, whereas it was 39.4 ⫾ 10.3% when the CEC count was ⱖ 500 cells/ml.
than in survivors over the 28 day study period. CEC counts greater than or equal to 500 cells/ml were associated with higher 28 day mortality, suggesting that levels of CECs could be used as be a predictive biomarker for mortality in patients with severe sepsis or septic shock. The endothelium plays a key role in mediating vasomotor tone, permeability and hemostasis [20]. It has been shown that endothelial damage or dysfunction plays an important role in the development of sepsis-associated organ dysfunction [6]. Microparticles, known as proinflammatory and procoagulant fragments originating from plasma membranes, are another surrogate for cell activation analysis during sepsis and septic shock. They play a role in altering vascular tone and contribute to spreading inflammatory and prothrombotic vascular status in septic conditions [21]. Furthermore, Delabranche et al. demonstrated that endothelial-derived microparticles are biomarkers of septic shock-induced disseminated intravascular coagulopathy [22]. The frequency of CECs represents a novel marker of endothelial damage that has been associated with various diseases [8]. The presence of CECs in the peripheral blood of healthy individuals is rare. The mean values of the percentage and numbers of CEC, measured by flow cytometry, have been suggested to be 0.005 ⫾ 0.004% and 306 ⫾ 243 cells/ml, respectively, in healthy subjects [18]. Boos et al. showed that the number of CECs is elevated when lipopolysaccharide is infused into healthy male subjects (endotoxinic condition) [23]. Moreover, two other studies have reported a relationship between CECs and sepsis [15,16]. All of these reports found that CEC counts are elevated in septic patients compared to non-septic or healthy subjects. By demonstrating increased CEC counts, they suggested that endothelial damage occurs
5
in patients with severe sepsis or septic shock. Furthermore, Mutunga et al. found that CEC counts in nonsurvivors of septic shock are higher than in survivors, supporting the concept that the number of CECs in sepsis is associated with the severity of vascular injury [15]. However, small numbers of cases with sepsis were enrolled in previous studies. This limits explanation of the correlation between CEC counts and prognosis of patients with sepsis. Whether CECs are associated with prognosis in patients with sepsis thus remains unclear. CECs have previously been detected by various methods, including immunofluorescence, immunohistochemistry and flow cytometry [8,15,19]. Flow cytometry has been used most widely to detect CECs because it can simultaneously distinguish between stem cell and endothelial markers. In the current study, CECs were detected by flow-cytometric analysis using three cell-surface markers. We defined CECs as CD45– (to exclude hematopoietic cells), CD34⫹ (a stem cell marker) and CD146⫹ (an endothelial cell marker). In agreement with other studies, we found that CEC counts were higher in non-survivors than in survivors of severe sepsis or septic shock over the 28 day study period. When we divided the 77 patients into two groups (CEC counts ⬍ 500 cells/ml vs ⱖ 500 cells/ml), patients with CEC counts of 500 cells/ml and above had a higher 28 day mortality rate than patients with CEC counts below 500 cells/ml. This result suggests that CECs could be used to predict the prognosis of patients with severe sepsis or septic shock. There were some limitations to our study. First, the study was conducted in a single center, making it difficult to generalize the findings to all cases of severe sepsis or septic shock. Secondly, we used a flow-cytometric method as an analysis modality, but no standardized and easily utilized protocol for the measurement of CECs in a clinical setting has been established to date. Thirdly, we did not evaluate CEC counts in healthy controls for comparison to patients with severe sepsis or septic shock. Compared to 24 non-septic patients admitted to the ICU, CEC counts were significantly higher in those with severe sepsis or septic shock [350 cells/ml (range 0–15984 cells/ml) vs 107.9 cells/ml (range 0–8000 cells/ml), p ⫽ 0.008)]. In conclusion, CEC counts in the blood were elevated in cases of both severe sepsis and septic shock. The number of CECs was higher in nonsurvivors of sepsis than in survivors, and CEC counts of 500 cells/ml and higher were associated with an increased 28 day mortality. Thus, the CECs may be a biomarker of severe sepsis or septic shock, although validation in a multicenter trial is needed.
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J.-W. Yoo et al.
Acknowledgement We thank Eun-young Jang for technical support. Declaration of interest: The authors have no conflicts of interest to declare. This study was supported by the Asan Institute for Life Science, Asan Medical Center, Seoul, Korea [grant 11–502].
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