JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2017, 68, 2, 273-282

www.jpp.krakow.pl

J. ZUWALA-JAGIELLO1, K. SIMON2, M. KUKLA3, E. MURAWSKA-CIALOWICZ4

J. GORKA-DYNYSIEWICZ1, E. GRZEBYK1, M. PAZGAN-SIMON2

INCREASED CIRCULATING ENDOCAN IN PATIENTS WITH CIRRHOSIS:

RELATION TO BACTERIAL INFECTION AND SEVERITY OF DISEASE

1Department of Pharmaceutical Biochemistry, Wroclaw Medical University, Poland;

2Clinic of Infectious Diseases, Liver Diseases and Acquired Immune Deficiency, Wroclaw Medical University, Poland;
3Department of Gastroenterology and Hepatology, Medical University of Silesia, Poland;
4Department of Physiology and Biochemistry, University of Physical Education, Wroclaw, Poland

Life expectancy of patients with liver cirrhosis is closely linked to the degree of liver dysfunction and the occurrence of
bacterial infection. An early diagnosis of infection helps to initiate adequate and timely measures and improves outcome of
cirrhotic patients. Endocan is a newly recognized biomarker of sepsis. However, there have been no studies of the trends
in endocan levels in cirrhotic patients with bacterial infection and their associations with markers of infection and
inflammation. This study sought to assess the diagnostic value of serum levels of endocan, procalcitonin (PCT), C-reactive
protein (CRP), tumor necrosis factor- (TNF-), and interleukin-6 (IL-6) in 126 patients with cirrhosis: 51 with
decompensated infected cirrhosis, 56 with decompensated uninfected and 19 with compensated uninfected cirrhosis at
inclusion. We analyzed the association of endocan with clinical factors in cirrhosis by comparison with indicators of
infection and inflammation. Endocan, PCT, CRP, IL-6 and TNF- were assayed in serum samples by ELISA analyses.
Serum levels of endocan, PCT, CRP and TNF- were significantly higher in cirrhotic patients with clinically overt
infections. Endocan levels were correlated to neither PCT levels nor IL-6 levels in each group of patients with cirrhosis.
CRP and TNF- levels and Child-Pugh score correlated only in the infected group of patients with endocan levels, while
in the uninfected groups of cirrhotic patients no significant correlation could be detected. The diagnostic accuracy of
endocan increased in advanced stage of the disease. Serum endocan levels 2.05 ng/ml had a sensitivity of 76.1% and
specificity of 85% for the diagnosis bacterial infection in decompensated cirrhotic patients. The endocan measured at
admission is a good clinical parameter predicting the occurrence of infection in these patients. Elevated endocan may reflect
the degree of endothelial cell injury induced by a systemic inflammatory response, a pathologic process that could modify
the course of advanced cirrhosis.

K e y w o r d s : bacterial infection, chronic liver disease, cirrhosis, C-reactive protein, endocan, inflammation, interleukin-6,
procalcitonin, tumor necrosis factor-

INTRODUCTION a key player in the regulation of cell adhesion, inflammatory

Patients suffering from liver cirrhosis often die of life-
threatening bacterial infections. The course of advanced
cirrhosis, regardless of its etiology, is complicated by cirrhosis-
associated immune dysfunction and this constitutes the
pathophysiological hallmark of an increased susceptibility to
bacterial infection, distinctive of the disease (1). An early
diagnosis of infection helps to initiate adequate and timely
measures and improves outcome of cirrhotic patients (2-5). In
clinical practice, a large number of molecules secreted by the
endothelial cells have been investigated as potential biomarkers
for the early diagnosis of bacterial infection. These have
included regulators of endothelial activation, adhesion
molecules, as well as mediators of inflammation (6).
Endothelial cell-specific molecule-1 (ESM-1) - so-called
endocan - is a soluble 50-kDa proteoglycan that is mainly
produced by activated endothelial cells (7). This proteoglycan is
disorders, and tumor progression (8). It has been established as
a serum biomarker in cancer (9-15), bacteremia (16) and sepsis
(17-19). Additionally, the serum endocan levels are altered by
antileukemic chemotherapy (20) and leukapheresis procedure
(stem cell harvesting) in multiple myeloma patients (21) as well
as acute graft-versus-host disease after allogeneic stem cell
transplantation (22). Endocan gene expression levels may be due
to the inflammatory cytokines as well as the lipopolysaccharide
(LPS) of the gram-negative bacterial cell wall, and thus
increases (23, 24, 17). On the other hand, the endocan itself has
been shown to elicit severe inflammatory responses both in vitro
in human umbilical vein endothelial cells (HUVECs) and in vivo
in mice model (25).
Bacterial LPS is the main stimulus for the production of
endocan and proinflammatory cytokines such as IL-6 and TNF-
. The inflammatory response to infection as estimated by the
levels of TNF- and IL-6 in serum are increased in patients with
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liver cirrhosis. A few studies have shown that endocan can be
acknowledged as a good marker of endothelial dysfunction and
multiple organ failure in sepsis (18, 19). In clinical practice,
however, the usefulness and characteristics of endocan in
bacterial infection in cirrhotic patients, have not been
investigated.
In light of increasing evidence that endocan and
inflammatory response are closely linked, the purpose of the
present study was to investigate the association between
endocan, as a specific biomarker of bacterial infection in patients
with liver cirrhosis, and markers of infection and inflammation.
Furthermore, we evaluated the performance of endocan assay for
the early diagnosis of bacterial infection in decompensated
cirrhotic patients.
PATIENTS AND METHODS
Patients
This study was performed in 126 patients with liver cirrhosis
with and without decompensation admitted to the Clinic of
Infectious Diseases, Liver Diseases and Acquired Immune
Deficiency for evaluation. Patients were divided into three
groups based on morphological ad bacteriological results:
patients with decompensated cirrhosis and infection at
admission (n = 51), patients with decompensated cirrhosis
without infection (n = 56) and compensated patients without
infection at admission (n = 19). The control group (n = 25)
consisted of healthy blood donors (16 males / 9 females, median
age 53 years) with normal aminotransferases, normal blood
counts and negative markers for virus hepatitis and HIV. Blood
samples were collected in the Department of Physiology and
Biochemistry, University of Physical Education in Wroclaw.
Clinical and biochemical characteristics of the study group are
reported in detail in Table 1.
Inclusion criteria were: histological or clinical diagnosis of
cirrhosis, no evidence of metabolic, toxic or autoimmune liver
disease and at least 1 year of alcohol abstinence. Diagnosis of
cirrhosis was established according histological criteria when
liver biopsy was performed, or by the combination of clinical,
biochemical and ultrasound imaging data presence of irregular
margins on ultrasound, portal hypertension with laboratory
evidence of chronic liver disease consistent with such a
diagnosis. Patients were grouped according to Child-Pugh
classification. Three biochemical variables (serum albumin,
bilirubin, and prothrombin time (international normalized ratio,
INR)) in addition to the presence or absence of ascites and
clinical signs of encephalopathy determine the Child-Pugh score
(26). Patients were scored as follows: 5 6 as class (group) A, 7
9 as class (group) B and 10 5 as class (group) C. At the time
of the study no Child-Pugh A patients showed clinical features of
decompensated liver cirrhosis (ascites or hepatic
encephalopathy).
The model for end-stage liver disease (MELD) score was
also calculated which is the most commonly used alternative
prognostic indicator for cirrhotic patients to the Child-Pugh
score. At enrollment, variceal bleeding was detected by
endoscopy in 29% of patients, ascites and hepatic
encephalopathy were present by physical examination in 52
(41%) and 22 (17%) patients, respectively (Table 1). Presence of
ascites was assessed by ultrasonography. Bacterial infection was
confirmed by clinical history, physical examination, differential
and total white blood cell count, analysis and culture of urine,
thorax X-ray and by the culture and white blood cell count of
ascetic fluid in patients with ascites. Infections of pneumonia,
skin and urinary tract (cystitis, pyelonephritis) were diagnosed
on the basis of conventional criteria. Spontaneous bacterial
peritonitis was defined as an infection of the ascitic fluid in the
absence of any intra-abdominal source of infection with an
ascitic fluid polymorphonuclear cell (PMN) count higher than
250 cells/mm3 and/or positive culture. The onset of infection was
defined as the time of admission.
Exclusion criteria were co-existing diseases like chronic
kidney disease, diabetes mellitus, cardiovascular disease,
cardiac decompensation, and hepatocellular carcinoma.
Peripheral venous blood from fasted healthy subjects and
fasted cirrhotic patients was collected in separate tubes, one
containing the anticoagulant EDTA and the other without serum
anticoagulant. The blood was allowed to clot for 30 min at 25C
and centrifuged at 2000 g for 15 min at room temperature, and
the serum was then separated and aliquoted into tubes for
storage. The tubes were stored frozen at 80C until they were
used to study different parameters. Average storage time for
patients was 30 months and for controls 25 months. The consent
of the Bioethics Committee of the Wroclaw Medical University
was obtained and all patients were informed about the character
of analyses made. Studies were conducted in compliance with
the ethical standards formulated in the Helsinki Declaration of
1975 (revised in 1983).
Laboratory determinations
Biochemical parameters were measured before the use of
antibiotics at admission. Endocan levels were determined by
ELISA analyses (JDIEK H1) (Lunginnov SAS, Lille, France).
The assay range of the ELISA kit was 0.15 ng/ml to 10 ng/ml.
Procalcitonin (PCT) was analyzed using an
immunoluminometric assay (LUMI test R PCT; BRAHMS
Diagnostica, Berlin, Germany). Detection limit was 0.05 ng/ml.
Serum C-reactive protein (CRP) level was determined with a
high-sensitivity nephelometric method using the Beckman
Image Immunochemistry system (Beckman Instruments,
Fullerton, CA, USA), which has a minimum level of detection of
0.2 mg/L. Serum levels of TNF- and IL-6 were assayed with
enzyme-linked immunosorbent assay (ELISA) kits (R&D
Systems Inc., Minneapolis, MN, USA). All analyses were
performed in duplicate strictly according to the manufacturers
instructions.
Statistical analysis
Statistical analyses were performed using Statistica 12.5
software. Continuous variables are expressed as mean standard
deviation (S.D.) or as median - interquartile range (IQR) and
categorical variables as number (percentage). Frequency data
were compared using the 2 test or the Fischers exact test when
necessary. Because many of the variables analyzed did not have
a normal distribution as determined by the Kolmogorov-
Smirnov test, nonparametric tests were used for comparison of
data. The Mann-Whitney U test and the Kruskal-Wallis ANOVA
test were used to analyze differences among two or more groups,
respectively. Regression analysis to determine significant
correlations among different parameters was performed using
the Spearman correlation coefficient. Statistical significance was
established at P < 0.05.
To evaluate the diagnostic performance of endocan receiver
operator characteristics (ROC) analysis was performed for all
significant differences between groups. ROC curves were
generated by plotting the sensitivity against 1 - specificity, and
the area under the curve (AUC) with 95% confidence intervals
(95% CI) was calculated. The empirical non-parametric method
according to DeLong (27) was performed to make pairwise
comparisons of ROC curves. The optimum cut-off point based
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on the ROC analysis was established by selecting the value that
provides the greatest sum of the sensitivity and specificity, that
is, the point closest to the upper left point of the ROC plot. For
the optimum cut-off point provided by each ROC analysis, the
sensitivity, specificity, positive predictive value (PPV), and
negative predictive value (NPV) were calculated using standard
formulas.
RESULTS
A total of 126 patients with liver cirrhosis were
consecutively analyzed. Table 1 exhibits the characteristics of
included patients. The median age was 58 years (range 47 to 70
years) and a male predominance was observed (60%). The
causes of liver cirrhosis were HCV infection (n = 28), HBV
infection (n = 19) and alcohol abuse (n = 79). Alcohol was the
predominant reason of cirrhosis (63%). The most frequent
infection were spontaneous bacterial peritonitis (59%) and
urinary tract infection (22%).
Characteristics of decompensated cirrhotic patients with
infection at admission
The infected patients with liver cirrhosis were compared to
the uninfected ones (Table 2). The prevalence of infection
particularly tended to increase with the severity of liver disease
(21%, 35% and 44%, respectively for Child-Pugh class A, B and
C). Moreover, bacterial infection was associated with
significantly higher MELD scores (P < 0.001). Two further
factors tended to be related to bacterial infection: presence of
ascites (59%) and alcoholic cirrhosis (73%) (Table 2). No
differences between infected and uninfected cirrhotic patients
were noted regarding the presence of variceal bleeding and
encephalopathy. Albumin and total bilirubin levels were similar
among all groups. On the other hand, bacterial infection was
associated with higher median values of white blood cells
(WBC) counts, INR and creatinine (P < 0.001, P < 0.01, P <
0.001, respectively) (Table 2).
patients. Although serum CRP levels were significantly higher in
the decompensated patients with infection than in the uninfected
patients, this biomarker was not a good discriminator because of
the considerable overlap among all groups (Fig. 2). Meanwhile,
there was no overlap between the levels of TNF- in the infected
patients and that of TNF- in the uninfected groups of cirrhotic
patients, as can be seen from Fig. 4. However, the non-stability
at room temperature similar to that of other cytokines limits the
prognostic significance of TNF- determination in bacterial
infection. The patients with compensated cirrhosis showed high
circulating levels of endocan and cytokines but the median of
this group was not significantly greater than for the control
group (endocan; 1.98 ng/mL (0.3 2.78 ng/mL) versus 0.95
ng/mL (0.0 1.5 ng/mL). Although levels of endocan, CRP, PCT
and cytokines were higher in decompensated group without
infection when compared with compensated group, the
difference was not statistically significant (Table 3). At
admission 30 (59%) cirrhotic patients with infection had
spontaneous bacterial peritonitis (SBP) (Table 1). Endocan
concentrations in cirrhotic patients with SBP were not different
to endocan concentrations measured in patients with other types
of bacterial infections (urinary tract infection, pneumonia, skin
infection) (data not shown).
In patients with decompensated cirrhosis regardless whether
infection was present or not, endocan levels were not correlated
with WBC (P = 0.61), PCT (P = 0.28) or with IL-6 (P = 0.21). C-
reactive protein (r = 0.36; P < 0.05) and TNF- (r = 0.42, P <
0.01) levels and Child-Pugh score (r = 0.48, P < 0.01) correlated
only in the infected group of patients with endocan levels, while
in the uninfected groups of cirrhotic patients no significant
correlation could be detected. This was also true for
compensated patients without infections at admission were no
correlation of the investigated biomarkers with endocan could be
detected. These results indicate that the elevation in endocan
Table 1. Clinical and biochemical characteristics of the study
subjects.

Serum concentrations of endocan, procalcitonin, C-reactive All patients

protein and inflammatory cytokines in cirrhotic patients (n) 126
according to the presence of bacterial infection Male : Female ratio (n) 76:50
Age (years) 58 (47 70)
Serum endocan levels have been reported to vary in healthy
Etiology, n (%)
subjects, and our results (median, 0.95 ng/mL; range, 0.01.5
HCV 28 (22)
ng/mL) were somewhat higher than those reported in previous
HBV 19 (15)
studies (median, 0.3 0.77 ng/mL; range 0.0 1.0 ng/mL) (9,
Alcoholic 79 (63)
11, 25). The cut-off value for endocan was established as 2.05
ng/mL. The patients results are presented in Figs. 1-5 and the Child-Pugh grade,
statistical analyses are summarized in Table 3. Serum endocan A/B/C (n) 26/53/47
(Fig. 1), CRP (Fig 2), PCT (Fig. 3) and levels of TNF- (Fig. 4) Variceal bleeding n (%) 36 (29)
and IL-6 (Fig. 5) were statistically significantly higher in both Ascites, n (%) 52 (41)
decompensated cirrhotic patients with and without infection Encephalopathy, n (%) 22 (17)
when compared with healthy subjects. Infection at admission Infection source, n (%)
was associated with significantly higher median levels of Spontaneous bacterial peritonitis 30 (59)
endocan (P < 0.001), TNF- (P < 0.001), CRP (P < 0.0001) and Urinary tract infection 11 (22)
PCT (P < 0.0001) as compared to the uninfected patients with Pneumonia 7 (14)
both compensated and decompensated cirrhosis (Table 3). IL-6 Skin infection 3 (5)
levels in serum were similar between groups. All decompensated
Albumin (g/L) 28 (16 45)
patients with a bacterial infection had endocan concentrations >
2.05 ng/mL. Additionally, there was no overlap between the Bilirubin (mg/dL) 1.2 (1.0 3.6)
decompensated cirrhosis with bacterial infection and the WBC ( 109/L) 8.5 4.78
compensated cirrhosis and healthy controls (Fig. 1). There was INR (0.8 1.1) 1.4 (0.8 2.9)
only a small overlap between the levels of endocan (Fig. 1) and Creatinine (mg/dL) 1.5 (0.7 5.4)
PCT (Fig. 3) in the infected group of patients and that of
endocan and PCT in the uninfected group of decompensated INR, normalized international ratio; WBC, white blood cells.
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Table 2. Clinical and biochemical variables associated with bacterial infection at admission.
Healthy Compensated Decompensated Decompensated
Controls cirrhosis cirrhosis cirrhosis
Absence of bacterial Presence of
-infection bacterial infection
(n) 25 19 56 51
Male:Female ratio (n) 16:9 11:8 33:23 32:19
Age (years) 53 (39 56) 59 (49 67) 58 (47 70) 65 (57 70)
Etiology n (%)
Viral 3 (16) 30 (53) 14 (27)
Alcoholic 16 (84) 26 (47) 37 (73)+
Child-Pugh score 6.9 2.3 8.7 3.2 16.8 7.0++
Child-Pugh grade;
A/B/C (n) 12/7/0 3/28/25 11/18/22
MELD score 5.8 4.6 12.9 7.9 21.1 5.9++
Variceal bleeding n (%) 0 19 (34) 17 (33)
Ascites n (%) 22 (39) 30 (59)+
Encephalopathy n (%) 10 (18) 12 (24)
Albumin (g/L) 45 (36 57) 35 (28 45) 29 (20 40)* 25 (16 32)*
Bilirubin (mg/dL) 0.7 (0.6 0.9) 1.01(1.02 1.03) 1.5 (1.0 2.0)* 2.3 (1.1 3.6)*
WBC ( 109/L) 4.6 (4.2 5.0) 5.1 (1.1 8.6) 8.5 (3.0 12.4)** 10.4 (2.7 16.4)**++
INR (0.8 1.1) 0.9 (0.8 1.09) 1.2 (1.1 1.3) 2.3 (1.6 2.9)+
Creatinine (mg/dL) 0.8 (0.7 1.0) 0.9 (0.7 1.4) 1.32 (0.8 2.4)* 2.6 (0.8 5.4)**++
Continuous variables are expressed as median (interquartile range, IQR) and categorical variables as number (percentage).
Significance between groups: *P < 0.05; **P < 0.01 versus healthy controls; +P < 0.01; ++P < 0.001 versus decompensated cirrhosis
without infection.
INR, normalized international ratio; MELD, model for end stage liver disease; WBC, white blood cells.

Table 3. Comparison between cirrhotic patients classified according to the presence of bacterial infection.

Healthy Compensated Decompensated Decompensated

Controls cirrhosis cirrhosis Cirrhosis
Absence of bacterial Presence of bacterial
infection infection
(n) 25 19 56 51
Endocan 0.95 (0.0 1.5) 1.98 (0.3 2.78) 3.2 (0.55 5.9)* 6.2 (5.3 8.75)**+ #
(ng/mL)
TNF- 26.0 (22.0 35.0) 34.2 (30.8 41.3) 39.3 (35.6 46.5)* 55.7 (48.7 59.8)**+ #
(pg/mL)
IL-6 5.8 (2.5 10.7) 14.28 (3.0 20.4) 18,7 (3.0 24.8)** 24.8 (3.0 34.5)***
(pg/mL)
CRP 1.05 (0.58 2.5) 3.8 (1.75 7.01)* 6.8 (3.1 17.0)** 85.9 (4.9 110.0)***++ ##
(mg/L)
PCT 0.05 (0.0 0.09) 0.18 (0.0 1.1)*** 0.4 (0.0 1.1)*** 2.65 (1.00 4.0)***++ ##
(ng/mL)

Continuous variables are expressed as median (interquartile range; IQR).

Significance between groups: *P < 0.01; **P < 0.001; ***P < 0.0001 versus healthy controls; +P < 0.001; ++P < 0.0001 versus
decompensated cirrhosis without infection; #P < 0.001; ##P < 0.0001 versus compensated cirrhosis without infection.
CRP, C-reactive protein; IL, interleukin; PCT, procalcitonin; TNF-, tumor necrosis factor-.

levels was greater than that of PCT and IL-6, and endocan was
parameter that was independent of the PCT and IL-6 levels in
patients with decompensated cirrhosis.
The diagnostic performance of endocan for identification of
bacterial infection in decompensated cirrhotic patients
Using biomarkers at admission, receiver operator characteristic
(ROC) analysis showed that areas under the ROC curve (AUC) of
endocan, PCT, CRP and TNF- were 0.832, 0.734, 0.584, 0.599,
respectively. The serum endocan level had a specificity of 85%, a
sensitivity of 76.1%, positive predictive value of 95.9%, and
negative predictive value of 43.7% at the cut-off level of 2.05
ng/mL (Table 4). Furthermore, the serum PCT level had a
specificity of 81.2%, a sensitivity of 69.9%, positive predictive
value of 91.5%, and negative predictive value of 42% at the cut-off
level of 0.5 pg/mL. The endocan-assay was the most powerful assay
for early diagnosis of infection in decompensated cirrhotic patients.
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Fig. 1. Serum concentrations of endocan in cirrhotic patients according to the presence of bacterial infection. Horizontal bars represent
medians of the concentrations in the different figures.

Fig. 2. Serum concentrations of C-reactive protein (CRP) in cirrhotic patients according to the presence of bacterial infection.
Horizontal bars represent medians of the concentrations in the different figures.

Fig. 6 shows ROC curves of endocan for identification of infection in cirrhotic patients increased in advanced liver
patients with infection according to disease severity indicated by disease. The diagnostic accuracy of endocan for identifying
Child-Pugh stage. The accuracy of endocan for diagnosis of patients with infection was the best for Child C stage cirrhosis:
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Fig. 3. Serum concentrations of procalcitonin (PCT) in cirrhotic patients according to the presence of bacterial infection. Horizontal
bars represent medians of the concentrations in the different figures.

Fig. 4. Serum concentrations of tumor necrosis factor- (TNF-) in cirrhotic patients according to the presence of bacterial infection.
Horizontal bars represent medians of the concentrations in the different figures.

AUC, (95%CI): 0.917 (0.880 0.939). Endocan showed a
slightly lower accuracy in patients with Child B: AUC, (95%CI):
0.900 (0.857 0.929), whereas the performance of endocan in
patients with Child A: AUC, (95%CI): 0.794 (0.761 0.817),
was clearly inferior to the values of endocan for identification of
infected patients with Child B or C stage cirrhosis.
Using the DeLong method (27), pairwise comparison of
ROC curves was performed. There were significant differences
between the AUC of endocan in patients with Child A and that
of endocan in patients with Child C (P = 0.019) for identification
of bacterial infection. There was no difference between the area
under the ROC curves of endocan for identification of bacterial
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Fig. 5. Serum concentrations of interleukin-6 (IL-6) in cirrhotic patients according to the presence of bacterial infection. Horizontal
bars represent medians of the concentrations in the different figures.

Fig. 6. Receiver operating characteristics (ROC) curves of endocan for identification of cirrhotic patients with bacterial infection
according to disease severity indicated by Child-Pugh stage.

infection AUC = 0.794 (95%CI 0.761 0.817) versus 0.900 DISCUSSION

(95%CI 0.857 0.929; P = 0.592), in cirrhotic patients with
Child A and Child B, respectively. There were also no significant
differences between the AUC of endocan in patients with Child
B and that of endocan in patients with Child C (P = 0.094) (Fig.
6) for identification of infection, but the AUC of endocan in
patients with Child C was higher than that of endocan in patients
with Child B (0.917 versus 0.900).
Endocan is naturally expressed by endothelial cells, is highly
regulated in presence of proinflammatory cytokines and
proangiogenic molecules and may be considered an accurate
marker of endothelial activation (7). Since endothelial injury is
pivotal in the development of liver failure (28) and in sepsis (18,
19), an endothelial marker such as endocan might not only
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Table 4. The accuracy of endocan, PCT, CRP and TNF- for the diagnosis of bacterial infection in decompensated cirrhotic patients.
Infected Cut-off Sensitivity
vs. values (%)
Uninfected
Endocan 2.05 ng/mL 76.1
PCT 0.20 ng/mL 69.9
CRP 14.0 mg/L 53.4
TNF- 37.2 pg/mL 37.9
The optimal cut-off value was calculated from the ROC analysis for endocan, PCT, CRP, and TNF- and subsequently the sensitivity,
specificity, positive predictive
value (PPV) and negative predictive value (NPV) of the markers was calculated.
CRP, C-reactive protein; IL, interleukin; PCT, procalcitonin; TNF-, tumor necrosis factor-.
reflect the severity of liver disease but also represent a promising
diagnostic marker of bacterial infection in cirrhotic patients.
The present study provides several lines of evidence to
suggest that endocan acts as mediator of inflammatory state
associated with bacterial infection in liver cirrhosis. First, serum
endocan levels in decompensated cirrhotic patients with
infections were significantly higher than in uninfected patients.
Second, and more importantly, a significant positive correlation
between endocan levels and both Child-Pugh score and
inflammatory markers (TNF-, CRP) was observed in cirrhotic
patients with infection.
In cirrhosis, systemic inflammation, in form of activated
circulating immune cells and increased serum levels of both
proinflammatory cytokines (e.g. TNF-, IL-6) (29, 30) and cell
activation markers, is the result of persistent episodic activation of
circulating immune cells from damage-associated molecular
patterns, released from necrotic liver cells and, as infection occurs,
from pathogen associated molecular patterns, released from the
leaky gut (1). In our study, we found a correlation between serum
endocan level and disease severity and a high level of circulating
endocan was associated with TNF- and its secondary mediator
(i.e CRP) in infected cirrhotic patients. In a recent report, endocan
appeared to reflect the degree of endothelial cell injury (31).
Furthermore, endocan expression in primary cultured HUVECs is
regulated by TNF-, a cytokine that is known to stimulate
endothelial cell activation and injury (32), although the precise
mechanism of endocan expression in infection has not been
elucidated. TNF- is a known attractant for leukocytes, enhances
expression of adhesion molecules on endothelial cells, and,
therefore, may play an important role in hepatic inflammatory
responses and cirrhosis progress. In addition, the proinflammatory
activities of endocan on endothelial cells is mediated by in part by
local release of TNF- (25), which may induces a systemic release
of CRP by stimulation of IL-6. Even if there was no correlation
between endocan and IL-6, we demonstrated weak association
with inflammatory CRP in cirrhotic patients. Although endocan
has not been found to be specific for any systemic inflammatory
diseases, it is known to mediate recruitment of circulating
lymphocytes and monocytes to inflammatory sites (7). As a
consequence, these effects of endocan on inflammation status may
cause the deterioration of hepatic function in patients with
advanced cirrhosis and bacterial infection. Collectively, elevated
endocan may reflect degree of endothelial cell injury induced by a
systemic inflammatory response, a pathologic process that could
modify the course of advanced liver failure (33).
Several studies suggested a possible role of endocan in
vascular contribution to organ-specific inflammation and in
endothelium-dependent pathological disorders (26, 34, 35). Some
other studies also suggest that an elevated serum endocan level
can be used to predict future or worsening hepatic
decompensation and consequent mortality (36, 37, 38). As
Specificity PPV NPV
(%) (%) (%)
85.0 95.9 43.7
81.2 91.5 42.0
60.0 67.5 31.0
62.0 70.0 51.3
hepatocellular carcinoma often develops in cirrhotic liver, death
from the underlying disease constitutes a competitive risk of death
from the tumor. In this context, the combined use of the Child-
Pugh score and the serum endocan level enables better prognostic
stratification of patients (36). Meanwhile, in our patients with
cirrhosis, the MELD score was significantly related to the
occurrence of bacterial infection, indicating that the hepatic
decompensation was associated with an increased risk of
infection. Accordingly, the early diagnosis of bacterial infection in
decompensated patients with cirrhosis remain a major challenge,
where time plays a crucial role. Some unique characteristics of
these patients make the diagnosis of bacterial infections
challenging. For example, the presence of leukocyturia does not
always correlate with urinary tract infection and the diagnosis of
spontaneous bacteremia can only be established once the results of
blood cultures are received; since dyspnea, as the predominant
presenting symptom in a pulmonary complication of advanced
cirrhosis, is frequent in the subclinical forms of hepatopulmonary
syndrome (39), difficulties exists to diagnose pneumonia. Thus, an
early diagnosis of bacterial infection would certainly help to
initiate adequate and timely antibiotics and would possibly
improve outcome of decompensated patients with cirrhosis.
Antibiotics can successfully modify the sequence linking
alterations in gut microbiota and intestinal permeability with
bacterial translocation and pro-inflammatory state, mainly
through their effect on intestinal microbiota. However, infections
caused by multiresistant bacteria are a growing threat in patients
with advanced liver cirrhosis. As previous antibiotic
administration is strongly related to the development of such
infections, alternatives to antibiotics are urgently needed in the
prevention of bacterial translocation and its consequences. The
evidence is mounting that probiotic treatment through modulation
of intestinal microbiota have the potential to affect the course of
advanced liver disease (40). It has been observed that VSL#3
probiotic therapy was well tolerated and led to improvement of
several clinical parameters in patients with decompensated
cirrhosis (41). Interestingly, none of the cirrhotic patients
developed bacterial infection, which would require
administration of antibiotics. Therefore, the surrogate marker for
bacterial infection which also acts as a guide to the effectiveness
of therapy is needed. Thus, in the constant search for biomarkers
of bacterial infection, endocan has arisen as an attractive
candidate (16-19).
Although, increased levels of endocan in advanced liver
disease had been previously reported (36, 37, 42), its potential
value as a diagnostic tool has not been studied. To our
knowledge, ours is the first study in which endocan levels
revealed significant associations with the severity of liver
disease within the infected group, as demonstrated by significant
correlations with the Child-Pugh score. In addition, the accuracy
of endocan for diagnosis of infection in cirrhotic patients

increased in advanced liver disease. Diagnostic accuracy of
endocan for identifying infected patients was the best for Child
C stage cirrhosis (AUC, 0.917), reaching a sensitivity of 81.8%.
Recently Mosevoll et al. (43) demonstrated that the plasma
levels of endocan show a considerable overlap when comparing
healthy subjects, patients with suspected thrombosis and the
patients without verified thrombosis. Therefore, they suggested
that the plasma endocan alone do not seem to be useful in the
diagnostic evaluation of these patients, but it may become useful
in combination with other markers. In our study, a relatively
small overlap of endocan concentrations existed between the 8
decompensated patients without infection and the 18 patients
with bacterial infections, which had no a negative impact on the
specificity of the test. Additionally, the best cut-off level for
endocan in predicting infection in patients with decompensated
cirrhosis was superior in its diagnostic accuracy compared with
the optimum PCT, CRP and TNF- cut-off levels. These results
indicate that serum endocan alone may be useful marker for
diagnosing bacterial infection in cirrhotic patients with a high
level of specificity (85%) and sensitivity (76.1%). This
proteoglycan differs from other biomarkers of bacterial
infection. Serum endocan levels are detectable as early as 2
hours after an inflammatory response begins (23), which is
earlier than the increase in the PCT or CRP (44, 45). Endocan
shows kinetic properties that enable it to serve as an early as well
as a follow-up (72 hours and beyond) marker of inflammation
and infection (9). Endocan is still detectable when screened on a
daily basis, whereas CRP or PCT will have already disappeared
from the blood. Our results suggest that serum endocan is an
independent parameter capable of distinguishing infected from
uninfected decompensated patients with cirrhosis and that it is
useful biomarker for early diagnosis of infection in these patients
with a high level of specificity and sensitivity.
To understand our findings better, some limitations should
be considered. First, although the number of patients enrolled
might seem small, it adequately represents the sample size
estimated to provide the specific power. Second, this study was
not designed and powered to assess the ability of endocan to
predict the incidence of bacterial infections in patients without
overt infections. Lastly, we excluded some diseases that may
influence endocan levels; however, some diseases may be
unrecognized in our study group.
Life expectancy of patients with liver cirrhosis is closely
linked to the degree of liver dysfunction and the occurrence of
bacterial infection. Our study identified serum endocan as a
powerful diagnostic marker to assess the severity of liver disease
and cirrhotic patients with bacterial infection. It may be useful to
implement endocan in future diagnostic algorithms for gauging
the prognosis of patients with advanced liver disease, and larger
prospective studies should investigate the practical clinical value
of serum endocam measurements.
Acknowledgments: The project described was supported by
a grant from the Wroclaw Medical University No. ST-
D020.16.004.
Conflict of interests: None declared.
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R e c e i v e d : February, 7, 2017
A c c e p t e d : April 10, 2017
Authors address: Assoc. Prof. Jolanta Zuwala-Jagiello,
Department of Pharmaceutical Biochemistry, Wroclaw Medical
University, 211 Borowska Street, 50-556 Wroclaw, Poland.
E-mail: jolanta.zuwala-jagiello@umed.wroc.pl