SERUM ENDOCAN LEVEL IN DIABETES MELLITUS OF PATIENTS WITH CIRRHOSIS AND RISK OF SUBSEQUENT DEVELOPMENT
OF SPONTANEOUS BACTERIAL PERITONITIS

Endothelial dysfunction affects the protective balance and endothelial barrier permeability that is part of the inflammatory responses that occur in an inflammatory diseases and variety of infections such as sepsis and spontaneous bacterial peritonitis (SBP) (1). Identifying markers of inflammation and surrogates of endothelial cell activation and/or dysfunction is of clinical relevance. Endocan (endothelial-cell-specific molecule-1 (ESM-1)) is a soluble proteoglycan (50 kDa) (2), secreted by human vascular endothelial cells, and is involved in the regulation of major endothelial processes such as cell adhesion, migration, proliferation, and neovascularization (3). Its secretion is increased in a variety of endothelium-dependent pathological states, such as inflammation, infections, tumor progression, and atherosclerosis (3, 4). Growing evidence suggests that endocan is a potential endothelial cell biomarker representing the immunoinflammatory activation of endothelium (5, 6).

Liver diseases begin with chronic hepatitis and can evolve to cirrhosis decompensation, and the decompensation is often accompanied by spontaneous bacterial peritonitis (SBP), which refers to a series of pathological symptoms, such as intestinal abscess and intestinal perforation induced by infection of organs in the abdominal cavity (7). Progression of liver cirrhosis and endothelial dysfunction are linked, as the underlying important mechanism is possibly persistent inflammation. It may be considered that endothelial injury, activation and dysfunction are affected by oxidative stress explaining the relationship between liver injury and endothelial dysfunction. Our previous studies have shown an association between inflammatory markers and liver cirrhosis (8, 9).

Type 2 diabetes mellitus (DM) can cause endothelial injury. Several studies suggested that chronic, subclinical inflammation plays a role in the development of insulin resistance which may then proceed to overt DM (10). Risk factors for DM are the same as those for the progression of cirrhosis (i.e., older age and male gender, metabolic syndrome, alcohol abuse) (11). Diabetic patients with compensated cirrhosis have increased incidence of developing decompensated cirrhosis (12, 13). Liu et al. (14) suggest that cirrhotic patients with DM have an increased risk of each category of adverse events such as incident ascites, SBP, variceal bleeding, and hepatic encephalopathy. Elevated serum endocan levels have been found in patients with diabetes (15-17), and relatively high levels of endocan have also been observed in patients with chronic hepatitis and liver cirrhosis (18, 19).

The aim of this retrospective cohort study was to examine the relationship between both serum endocan and DM, and risk of developing spontaneous bacterial peritonitis in patients with cirrhosis.

PATIENTS AND METHODS

Patients

We conducted a retrospective observational study on a cohort of 458 patients treated for liver cirrhosis at the Department of Infectious Diseases and Hepatology at the Wroclaw Medical University from January 2007 to December 2016. For this study, we selected patients fulfilling following inclusion criteria: 1) biopsy-proven cirrhosis, 2) alcoholic liver disease defined by daily alcohol intake and absence of other etiologies, 3) absence of co-existing diseases like an HIV infection, chronic kidney disease, cardiovascular disease, cardiac decompensation, and presence of hepatocellular carcinoma at the time of serum collection, 4) adequate frozen serum sample available at inclusion. Four etiological factors were implicated in cirrhosis: alcohol, HBV (hepatitis B), HCV (hepatitis C) chronic infections and nonalcoholic fatty liver disease (NAFLD). Only a small number of patients were diagnosed with biopsy-proven NAFLD in the entire cohort. A total of 128 subjects had an NAFLD or an cryptogenic cirrhosis and were excluded from further analyses. Liver cirrhosis was called „cryptogenic“ when an additional extensive evaluation yielded no specific etiology. Three hundred and thirty patients with alcoholic and viral etiology of cirrhosis fulfilling the inclusion criteria were selected for the study. The first day of admission to our hospital was used as baseline. At baseline clinical history, physical exam, blood tests, and abdominal ultrasonography were performed. Data collected at this time were age, gender, current alcohol consumption, etiology of cirrhosis, a history of DM, presence of ascites and presence of esophageal varices. The diagnosis of HBV and HCV cirrhosis was based on raised serum transaminases for at least 6 months, positivity for HBsAg and anti-HB core, and anti-HCV antibodies, respectively. Patients with proven viral hepatitis and concomitant excessive alcohol consumption (3%) were classified as having viral cirrhosis. The patients who were HBV and HCV negative, with a history of daily alcohol intake ≥ 80 g in males or ≥ 40 g in females were defined as patients with alcoholic liver cirrhosis. The Child-Pugh score was assessed as an independent prognostic predictor in patients with cirrhosis. The model for end-stage liver disease (MELD) score was also calculated; it is the most commonly used alternative prognostic indicator for cirrhotic patients to the Child-Pugh score (20). The presence of DM was defined as a history of diagnosed type 2 diabetes mellitus (with or without medication), a random blood glucose > 11.1 mmol/L together with symptoms, or two fasting blood glucose levels > 6.9 mmol/L (21). Owing to frequent changes in therapy, we recorded the type of treatment most recently initiated for DM, categorized into no medical treatment, oral antihyperglycemic agents or insulin. A total of 28 patients with DM (19%) received no medical treatment, 42 (28%) received oral antihyperglycemic agents and 79 were treated with insulin (53%).

Control serum samples were collected from 75 age- and gender-matched healthy subjects in whom liver diseases were ruled out, recruited in other epidemiological study performed by the University of Physical Education at Wroclaw. These healthy subjects had normal serum concentrations of aminotransferases and γ-glutamyltransferase or alkaline phosphatase, and had no history of gastrointestinal or chronic liver disease, smoking and alcohol intake and systemic diseases.

All patients gave written consent for blood sampling at the baseline visit; Bioethics Committee of the Wroclaw Medical University approval for the protocol was obtained. The studies were conducted in compliance with the ethical standards formulated in the Helsinki Declaration of 1975 (revised in 1983).

Follow-up and spontaneous bacterial peritonitis diagnosis

Follow-up of the patients was performed until May 2017. The end point was the development of SBP. Diagnostic paracentesis was performed in all patients with cirrhosis and ascites, and in patients with signs of systemic infection, abdominal symptoms or acute kidney injury/liver failure. When the ascitic PMN (polymorphonuclear) count was > 250 cells/mm3, SBP was considered positive, and the patient was treated accordingly with intravenous antibiotics regardless of culture results, according to EASL (European Association for the Study of the Liver) guidelines (22). Asymptomatic patients with positive ascitic cultures, but without signs of infection or progressive renal/liver failure were considered SBP-free and were followed up clinically. Patients were censored at the time of death without SBP development, the last visit when lost to follow-up, or the end of the study period.

Laboratory determinations

Peripheral venous blood from fasted healthy subjects and fasted cirrhotic patients was collected into separate tubes, one containing the EDTA anticoagulant, and the other without serum anticoagulant. The blood was allowed to clot for 30 min at 25°C, and was 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 –80°C until they were used to study different parameters. Standard laboratory methods were used to measure hemoglobin, platelet count, non-fasting blood glucose, AST (aspartate aminotransferase), ALT (alanine aminotransferase), total bilirubin, albumin, creatinine and prothrombin activity expressed as INR (International Normalized Ratio) in all patients at the first contact. 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 0.08 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 interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) were assayed with enzyme-linked immunosorbent assay (ELISA) kits (Diaclone. Human IL-1β ELISA KIT, Catalogue Number 850006096, 2017: 1–19 and R&D Systems Inc., Minneapolis, MN, USA, respectively for IL-1β and TNF-α, IL-6). All analyses were performed in duplicate strictly according to the manufacturer’s instructions.

Size calculation and power of study

A pilot study was first carried out using 50 subjects per group. Based on these preliminary results, with a confidence interval of 95%, an estimated P value < 0.05, and a power of 80%, the present sample size was derived. Ten subjects were added in each group to account for the high level of variation seen in the levels of serum biomarkers.

Statistical analysis

The Kolmogorov-Smirnov test was used to identify parametric/non-parametric distribution, accordingly, the parameters were reported as mean ± standard deviation or median (interquartile range). Student’s t-test or the Mann-Whitney U-test were applied for group comparisons as appropriate. Categorical variables were reported as numbers (proportions) and analyzed with Chi-square test. Associations between variables were assessed using Spearman’s rank correlation coefficients for nonparametric correlations. To evaluate the diagnostic performance of serum endocan in predicting subsequent SBP development, receiver-operating characteristic (ROC) curve analysis was carried out. Diagnostic accuracy was expressed as the specificity and sensitivity, positive predictive value, negative predictive value, and area under the ROC curve (AUROC). The optimal cut-off point was obtained by maximizing the Youden’s index. The Kaplan-Meier method was used to estimate the cumulative SBP incidence, and the differences between groups were analyzed with the log-rank test. In the analysis of risk factors for spontaneous bacterial peritonitis, we tested the following variables, obtained at the time of entry in univariate and multivariate Cox proportional hazard regression analysis: age, type 2 diabetes mellitus, severity of cirrhosis (Child-Pugh or MELD score), platelet count, serum proinflammatory cytokines, procalcitonin, C-reactive protein, and endocan level. The predictive value of endocan levels were also analyzed in the subgroup of cirrhotic patients with serum stored for less than 5 years. All P values were two-tailed, and P-values < 0.05 were considered significant. Data processing and analysis were performed using Statistica, version 13.3 and R version 3.5.1.

RESULTS

Patients profile and serum endocan levels

A total of 330 patients with liver cirrhosis were consecutively analyzed. Baseline characteristics of patients with and without DM are shown in Table 1. The causes of liver cirrhosis were HCV infection (n = 145), HBV infection (n = 44) and alcohol abuse (n = 141). Viral infection was the predominant reason of cirrhosis (57.3%). The prevalence of DM (44%) tended to increase with the severity of liver disease (6.7%, 33.6% and 59.7%, respectively for Child-Pugh class A, B and C) (Table 1). DM was associated with a significantly higher WBC count and AST (P < 0.001). Furthermore, hemoglobin levels, platelet counts, and the Child-Pugh score as well as the MELD score were similar among groups (Table 1). Serum concentrations of endocan were statistically significantly higher in all patients with cirrhosis when compared with a fully age- and gender-matched control group of 75 healthy subjects (median 0.95 ng/mL; range, 0.0 – 1.5 ng/mL, P < 0.001). We could not find association between endocan and the patients’ gender. Furthermore, serum endocan did not differ between the etiology subgroups (data not shown). As to the contribution of systemic inflammation to endocan production, we investigated serum levels of proinflammatory cytokines including IL-1β, IL-6, and TNF-α, and two acute phase serum proteins (serum CRP and PCT) (Table 2). Serum TNF-α (P < 0.01), IL-1β levels (P < 0.01), and CRP (P < 0.001) were significantly higher in diabetic patients with cirrhosis than those in the patients with cirrhosis only. However, IL-6 and PCT levels in serum were similar between both groups of patients with cirrhosis (Table 2).

Table 1. Clinical and biochemical characteristics of patients with cirrhosis.

Continuous variables are expressed as median (interquartile range; IQR) and categorical variables as number (percentage). Significance between groups: +P < 0.01; ++P < 0.001 vs. cirrhosis only. Abbreviations: DM, diabetes mellitus; INR, normalized international ratio; MELD, model for end stage liver disease; WBC, white blood cells.

Table 2. Serum concentrations of endocan, proinflammatory cytokines, C-reactive protein and procalcitonin in a cohort of 330 patients with cirrhosis.

Variables are expressed as median (interquartile range; IQR). Significance between groups: +P < 0.01; ++P < 0.001 vs. cirrhosis only. Abbreviations: CRP, C-reactive protein; IL, interleukin; PCT, procalcitonin; TNF, tumour necrosis factor.

Correlation of serum endocan levels and clinical parameters

Clinical and biochemical factors correlated with serum endocan levels in a cohort of 330 patients with cirrhosis are reported in Table 3. In cirrhotic patients, the serum endocan level was correlated negatively with platelet count, and positively with age, and was higher in patients with DM. We found significant correlations of serum endocan levels with the Child-Pugh score as well as the MELD score (Table 3). However, serum endocan were not correlated with AST and ALT (r = 0.19; P = 0.215, r = 0.10; P = 0.712, respectively). Endocan showed significant correlation with the C-reactive protein (r = 0.42; P < 0.05) and TNF-α (r = 0.36, P < 0.01) levels (Table 3). Moreover, serum IL-1β level showed a very weak positive correlation with endocan (r = 0.18; P = 0.05). However, no correlation was observed of serum endocan levels with serum levels of WBC, PCT, and IL-6 (P > 0.05) (Table 3).

Table 3. Clinical and biochemical factors correlated with serum endocan levels in a cohort of 330 patients with cirrhosis.

Abbreviations: CRP, C-reactive protein; DM, diabetes mellitus; IL, interleukin; INR, normalized international ratio; MELD, model for end stage liver disease; PCT, procalcitonin; TNF, tumour necrosis factor; WBC, white blood cells.

Incidence of spontaneous bacterial peritonitis stratified based on serum endocan levels

The median follow-up period was 6 years (mean: 6.5 ± 4.2 years). 64 (19.5%) cirrhotic patients were lost upon follow-up. During the study period, 126 (69 males and 57 females) patients developed SBP. As endocan level was identified as an independent predictor of SBP development, we stratified patients into 2 groups using the cut-off point of 2.05 ng/mL that best predicted SBP development (sensitivity, 72.9%; specificity, 83.9%; area under ROC curve, 0.873 (95%CI: 0.763 – 0.985, P < 0.001). The overall 5-year SBP incidence was 28.6%. Overall, 168 (51%) of the 330 patients had a high endocan levels (> 2.05 ng/mL). The 5-year incidence of SBP was 22.2% and 35.1% for cirrhotic patients with low (endocan < 2.05 ng/mL) and high endocan levels (endocan > 2.05 ng/mL), respectively (hazard ratio (HR) 0.83; 95% CI, 0.42 – 1.64, P = 0.035) (Fig. 1).

Fig. 1. Cumulative incidence of spontaneous bacterial peritonitis according to a 2.05 ng/mL cut-off of serum endocan in patients with cirrhosis (P=0.035; log-rank test)

Risk factors of spontaneous bacterial peritonitis development

Independent factors related to the development of SBP were the Child-Pugh and MELD scores, DM and high endocan level (> 2.05 ng/mL). Serum levels of proinflammatory cytokines, PCT and CRP were not associated with SBP development (Table 4). The impact of serum endocan and levels of PCT and CRP on SBP development was similar in the subgroup of patients with serum stored for less than 5 years (endocan (HR) 2.402 (95% CI: 1.248 – 4.602; P < 0.001), PCT (HR) 1.164 (95% CI: 0.589 – 2.244) and CRP (HR) 1.015 (95% CI: 0.912 – 1.103). As endocan was correlated with parameters of liver function (prothrombin activity expressed as INR, serum albumin, bilirubin), we performed a Cox proportional hazard regression analysis adjusted with these variables. The HR for endocan was 1.644 (95% CI: 1.011 – 2.707). When restricted to the subgroup of cirrhotic patients without DM (n = 181), the endocan is also predictive of SBP development with an HR 1.673 (95% CI:1.031 – 2.806) P = 0.007 in univariate analysis. In multivariate analysis restricted to this subgroup, the same parameters were identified with HR of the endocan similar to that found in the whole study group (1.646 (95% CI: 1.032 – 2.638; P = 0.057) and 1.634 (95% CI: 1.012 – 2.638; P = 0.047), respectively).

Table 4. Univariate and multivariate analysis of factors associated with spontaneous bacterial peritonitis development in patients with cirrhosis.

Abbreviations: CI, confidence interval; CRP, C-reactive protein; DM, diabetes mellitus; HR, hazard ratio; IL, interleukin; INR, normalized international ratio; MELD, model for end stage liver disease; PCT, procalcitonin; TNF, tumour necrosis factor; WBC, white blood cells.

DISCUSSION

Endocan is a potential immunoinflammatory marker that may be linked to liver cirrhosis. Raised level of endocan is associated with liver diseases (18, 19) that often begin with chronic hepatitis and can evolve to liver decompensation, and the elevation in endocan levels has been reported to be correlated with type 2 diabetes mellitus (15-17). Remarkably, we found that cirrhotic patients with DM having high serum endocan levels had a higher risk of developing SBP. To our knowledge, this is the first study reporting a positive association between both serum endocan and DM and future SBP development.

Persistent inflammation may induce a massive loss of hepatocytes and hence exacerbate the severity of various hepatic conditions, including diabetes mellitus, de facto being associated with irreversible liver damage, fibrosis and/or cirrhosis, and cirrhosis decompensation (10, 23). Endocan is naturally expressed by endothelial cells, is highly regulated in the presence of proinflammatory cytokines (IL-1β and TNF-α) (6, 7), and may be considered an accurate marker of endothelial dysfunction (2). Since endothelial injury is pivotal in the development of liver failure (24) and in type 2 diabetes mellitus (15, 16), an endothelial marker, such as endocan might not only reflect the severity of liver disease but also represent a promising marker of developing SBP in patients dually diagnosed with cirrhosis and DM. We showed that endocan, IL-1β, and TNF-α and its secondary mediator (i.e. CRP) were elevated in cirrhotic patients with DM, and a higher endocan level was an independent risk factor for SBP development. The fact that the predictive value of endocan did not change when adjusted to liver function test (prothrombin activity, serum albumin, bilirubin) suggests that endocan have a specific role in the adverse outcome. Diabetic patients with cirrhosis may present increased endocan in response to factors such as inflammation and bacterial translocation which might induce production of endocan without infection. It is possible that in response to subsequent local inflammation, endocan may become elevated and may reflect the degree of endothelial cell injury induced by a systemic inflammatory response (25), a process that could modify the clinical course of advanced cirrhosis, particularly in diabetic subjects.

The physiological importance of endocan is imparted by its ability to bind growth factors and other molecules due to the hybrid nature of its structural components (chondroitin sulfate/dermatan sulfate). Importantly, regulation of fine structural features of chondroitin sulfate/dermatan sulfate has been observed by growth factor such as transforming growth factor β1 (TGF-β1) (26), which is a link between hepatic inflammatory and fibrosis processes. This is an interesting observation especially in the light of the fact that there is an increase in TGF-β1 production in the liver during diabetes, which promotes accumulation of extracellular matrix components (10). Experimental animal models confirmed that endocan itself elicit severe inflammatory responses (27). Furthermore, a recent study found that endocan induces expression of TNF-α, and also revealed that endocan stimulates nuclear factor kappa β expression (28). In addition, TNF-α and nuclear factor kappa β stimulate hepatic inflammation and promote fibrosis processes. (29, 30). Serum TNF-α levels in patients with advanced cirrhosis are elevated, as shown by our studies (9, 19). Thus, endocan may represent a surrogate serological indicator of cirrhosis progression, as it was positively correlated with both TNF-α level and disease severity in diabetic patients with cirrhosis. In this setting, other serological markers of cirrhosis in patients with metabolic disorders, sensitive to progression of this disease to more advanced stages, could have a substantial significance for clinical monitoring and medical treatment, as well as prevention against complications (e.g. bacterial infection) (31, 32). The early diagnosis of SBP would certainly help to initiate adequate and timely antibiotics and would possibly improve outcome of cirrhotic patients, particularly diabetic subjects. Antibiotics can successfully modify the sequence linking alterations in intestinal permeability with bacterial translocation and pro- inflammatory state, mainly through their effect on intestinal microbiota. As previous antibiotic administration is strongly related to the development of SBP, 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 (33).

Our retrospective cohort study found an increased risk of developing SBP incident among patients who were dually diagnosed with cirrhosis and DM. Few case-control studies on patients with ascites did show higher rates of DM in patients with SBP compared with those with sterile ascites (34, 35). The observed association between DM and SBP may represent an increased susceptibility to infections in patients with both cirrhosis and DM, in agreement with observations that DM was also associated with more urinary and respiratory tract infections in cirrhotic patients (36, 37). In this context, careful management of DM in patients with cirrhosis may reduce the risk of developing SBP in this population. With this in mind, screening for DM among those patients may prove to be worthwhile (38).

To understand our findings better, some limitations should be considered. One limitation of the present study is the relatively small number of patients in a single center. However, the numbers of subjects in each group were enough to get a sufficient power for data analysis. We obtained complete baseline data and a median follow-up of 6 years for most of patients, and only 64 patients were lost to follow-up. A major limitation of the study is its retrospective design which may lead to selection or information bias. However, all patients with cirrhosis must be coded with one of the used diagnosis-treatment combinations for financial purposes, and we have no reason to believe that patients with or without DM were differently registered. Another limitation of our study is the lack of data on hypertension for cirrhotic patients, as the levels of endocan have been reported as increased with blood pressure (39). The lack of proper data on episodes of variceal bleeding, a risk factor for SBP (22), is a further limitation of our research. Although Child-Pugh score, MELD score, hemoglobin levels and platelet counts were comparable between cirrhotic patients with and without DM, we can therefore not fully exclude residual confounding. For this reason it is necessary to have a plan for further multicenter studies in a larger population to confirm the present results.

Conclusion

When confirmed by other studies, our results might imply that a high endocan levels in patients dually diagnosed with cirrhosis and type 2 diabetes mellitus is a marker to identify subjects at increased risk for the subsequent development of spontaneous bacterial peritonitis.

Acknowledgments: The project described was supported in part by a grant from the Wroclaw Medical University No. ST-D020.16.004.

Conflict of interests: None declared.

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