Scandinavian Journal of Gastroenterology. 2015; Early Online, 1–10
ORIGINAL ARTICLE
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von Willebrand factor as a novel noninvasive predictor of portal hypertension and esophageal varices in hepatitis B patients with cirrhosis
HAO WU*, SHIPING YAN*, GUANGCHUAN WANG, SHAOBO CUI, CHUNQING ZHANG & QIANG ZHU Department of Gastroenterology, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, China
Abstract Objective. At present, there is no perfect noninvasive method to assess portal hypertension and esophageal varices. Early predicting esophageal varices can provide evidence for managing cirrhotic patients. We aimed to further investigate von Willebrand factor (vWF) as a noninvasive predictor of portal hypertension, especially of esophageal varices. Material and Methods. A total of 60 hepatitis B patients with cirrhosis and 45 healthy subjects were enrolled in this study. Levels of six markers were examined. All patients underwent hepatic venous pressure gradient (HVPG) and esophagogastroduodenoscopy. We evaluated the performance of six factors for diagnosis of portal hypertension and esophageal varices. The vWF levels in liver tissues were observed by immunohistochemistry. Correlations between the level of vWF in liver tissues and HVPG and between levels of vWF in tissues and plasma were examined. Results. Cutoff values of plasma vWF (1510.5 mU/mL and 1701 mU/mL) showed high positive predictive value (PPV, 90.2% and 87.5%) in predicting clinically significant portal hypertension and severe portal hypertension. Cutoff values of vWF (1414 mU/ml and 1990 mU/mL, PPV 90.3% and 86.3%, respectively) were provided to detect the presence and degree of esophageal varices. Linear correlations were observed between levels of vWF in liver tissues and HVPG (r2 = 0.552, p < 0.001) and between the level of vWF in liver tissues and in plasma (r2 = 0.461, p < 0.001). Conclusion. The vWF is a noninvasive predictor of portal hypertension and esophageal varices in hepatitis B patients with cirrhosis. Increased levels of vWF in liver tissues may induce the elevated plasma vWF levels, but molecular mechanism is needed for further study.
Key Words: Esophageal varices, liver cirrhosis, noninvasive predictor, portal hypertension, von Willebrand factor
Introduction Liver cirrhosis, the most common cause of portal hypertension (PH), is almost caused by chronic hepatitis B virus infection in Asia and most of Africa [1,2]. PH induces the severe complications of cirrhosis, such as esophageal varices (EV), bleeding, ascites, and decompensation [3]. Studies indicated that early diagnosis of PH could help in starting timely treatment and in reducing the mortality of its complications [4,5].
Nowadays, guidelines preferred hepatic venous pressure gradient (HVPG) as the diagnosis, prognostic, and therapeutic indications of PH [6]. A lot of studies indicated HVPG to have good ability to predict liver-related variceal hemorrhage and assess fibrosis or cirrhosis despite etiology [7–9]. However, it was considered as invasive procedure and only available in centers with adequate resources and expertise. Nowadays, esophagogastroduodenoscopy (EGD) was the gold standard for determining varices that should be screened in all cirrhotic patients at
Correspondence: Qiang Zhu, Department of Gastroenterology, Provincial Hospital Affiliated to Shandong University, 324, Jing 5 Rd, Ji’nan, 250021, Shandong Province, China. Tel: +86-0531-68777012. Fax: +86-0531-87902348. E-mail: [email protected] *These authors contributed to the work equally and should be regarded as co-first authors.
(Received 27 February 2015; revised 28 March 2015; accepted 29 March 2015) ISSN 0036-5521 print/ISSN 1502-7708 online 2015 Informa Healthcare DOI: 10.3109/00365521.2015.1037346
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diagnosis [10]. But this technique was invasive, time-consuming, and costly. Up to now, there is no effective and perfect noninvasive method to predict PH and EV. Therefore, novel noninvasive markers were required. von Willebrand factor (vWF) is a multimeric glycoprotein mainly synthesized by endothelial cells [11]. It was released by activated endothelial cells and recognized as an index of endothelial dysfunction [12–15]. Endothelium plays a crucial role in many vascular diseases and endothelial dysfunction is a fundamental component of the increased hepatic vascular tone [16], which induces the development of PH. The vWF also regulated angiogenesis [17,18], the formation of new blood vessels that leads to the progressive formation of the abnormal angio-architecture distinctive in cirrhotic livers [19,20]. A vicious circle appears between liver fibrosis and pathological angiogenesis, and this oppression induces venous pressure and portal venous resistance to increase significantly [21]. Evidence clearly reported that angiogenesis and disruption of liver vascular architecture have been linked to the progression to cirrhosis, which leads both to increased hepatic vascular resistance and PH [20]. Elevated levels of vWF were frequently found and were reported as the noninvasive marker in predicting PH and the mortality in cirrhotic patients [22–26]. However, no study reported that vWF could directly predict EV, the mechanisms why vWF reflects portal vein pressure and why vWF increases in patients’ plasma remain unclear. In this study, we examined the level of plasma vWF in hepatitis B patients with cirrhosis and evaluated the diagnostic performance of vWF as a new noninvasive predictor not only of PH but also EV, compared to HVPG and EGD in cirrhotic patients. At the same time, we tested the expression of vWF in patients’ liver tissues and analyzed the possible mechanism of PH and vWF levels increasing in cirrhotic patients’ plasma. Methods Patients Sixty hepatitis B viral cirrhotic patients were recruited in this study. Liver cirrhosis with hepatitis B infection were diagnosed by clinical data, laboratory and imaging examinations according to American Association for the Study of Liver Disease practice guideline [27]. Exclusion criteria were the presence of pre- and posthepatic causes of PH, variceal bleeding, refractory ascites, severe cardiopulmonary or renal impairment, and ongoing treatment of PH. Patients with other causes of cirrhosis and presence of hepatocellular
carcinoma and patients with the other fibrosis diseases must be excluded. Child–Pugh stage, transjugular HVPG measurement, EGD, liver biopsy, and blood biomarkers including plasma vWF were examined for each patient. Blood samples from 45 health subjects with no evidence of liver disease were used to establish control group in this study. Forty-five normal liver tissues from hepatic hemangioma and liver trauma were included as control. All procedures were approved by the Ethics Committee of Provincial Hospital Affiliated to Shandong University. Assessment and scoring of laboratory parameters Laboratory tests included platelet count, alanine aminotransferase, aspartate aminotransferase (AST), total bilirubin, serum albumin, and international normalized ratio (INR). AST/platelet ratio index (APRI) was also calculated according to published formulas. Measurement of plasma vWF About 2 ml of vein blood was sampled from patients by HVPG measurement in ethylene diamine tetraacetic acid tubes and centrifuged. Plasma was stored at 80 C and thawed for 12 h at 4 C before being assayed. Levels of vWF were measured strictly using enzyme-linked immunosorbent assays kits (Abcam, Cambride, UK) according to the instructions. Immunohistochemistry A total of 60 liver tissues of liver cirrhosis were obtained via biopsy, and 45 normal liver tissues from hepatic hemangioma and liver trauma were included as control. Thin sections were prepared by embedding in paraffin. Sections were deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval in 0.01 M citrate for 5 min. Endogenous peroxidase activity was quenched with 3% H2O2 for 5 min. Thereafter, sections were blocked with 5% goat serum, and then incubated for 2 h at room temperature with anti-vWF antibodies (Abcam, Cambride, UK). Sections were incubated with horseradish peroxidase-conjugated secondary antibodies and reacted with a diaminobenzidine solution and counterstained with hematoxylin. Liver specimens were stained for vWF. Positively stained portal vessels and sinusoids appeared as brown linear deposits, observed by light microscopy. Four most intensely stained highpower microscopic fields (400) were assessed in each sample. The vWF staining extension was calculated by the percentage of stained area per high-power field, and the average vWF scoring of each sample was calculated.
vWF as a noninvasive predictor of PH and EV The vWF staining scoring was measured using ImagePro Plus 6 software (Baltimore, USA).
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Esophagogastroduodenoscopy The same operator performed EGD for each patient. EVs are classified into three sizes, small, medium, and large, by a semi-quantitative morphological assessment in most centers and consensus. The small EV defined as minimally elevated veins above the esophageal mucosal surface. Medium EV is defined as tortuous veins that occupy less than one-third of the esophageal lumen and large EV is defined as veins occupying more than one-third of the esophageal lumen [28]. Since recommendations for mediumsized EV are the same as for large EV, we took the medium and large EV in the same group. Measurement of HVPG Methods for accurate HVPG measurement are according to the previously study [29]. Before the measurement of HVPG, the measurement operator should obtain permission and ask the patient to fast for 8 h. The operator places balloon catheters in the right hepatic vein through a right jugular vein puncture for measurement of the free hepatic venous pressures (FHVPs). The wedged hepatic venous pressure (WHVP) is measured by inflation of the balloon catheter at the right hepatic vein. Last, the HVPG is determined by subtracting the FHVP from the WHVP. Measurements were performed at least three times to get the average and for minimizing the error. All data are obtained from the same measurement operator. Normal portal pressure was defined as the range of 1 to 5 mmHg according to HVPG. When the HVPG ‡10 mmHg, PH was defined as clinically significant PH (CSPH), whereas HVPG ‡12 mm Hg was diagnosed as severe PH (SPH). Statistic analysis Statistical analysis was performed with Statistical Analysis Software, SPSS version 16.0 (SPSS Inc., Chicago, IL, USA) and MedCalc package v.10.0 (Mariakerke, Belgium). Categorical variables are reported as numbers (percentage); continuous variables were reported as mean and standard deviation or in median and interquartile range on the basis of their distribution analyzed by the means of Kolmogorov–Smirnov’s test. Differences between groups were analyzed using Mann–Whitney U test. The correlation between vWF in plasma and HVPG, between expression levels of vWF in tissues and HVPG and levels of plasma vWF were used as a
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linear regression analysis. Receiver operator characteristic (ROC) curve was constructed to determinate test performance for prediction of CSPH, SPH, EV, and medium/large EV, and each area under the curve (AUC) was calculated. The cutoff value of the variable was determined at the point of highest sensitivity and specificity. Positive predictive value (PPV), negative predictable value (NPV), the positive likelihood ratio (LR+) and the negative likelihood ratio (LR ) were calculated for the cutoff value. All p-Values reported are two-sided, and p-Values < 0.05 are considered statistically significant. Results Patients’ characteristics In this study, 48 patients (80%) had CSPH and 40 patients (66.7%) had SPH. All patients were tested through EGD, 49 (81.7%) and showed the presence of EV, 19 (38.8%) of whom had the small EV and 30 (61.2%) had the medium or large EV. Demographical, clinical, and laboratory characteristics of patients group and control group were shown in Table I. Six examined variables including vWF were associated with the degree of PH, with the presence and grade of EV Many markers are significantly associated with PH and EV in liver cirrhosis. To identify the best parameter, we examined the blood levels of the following markers: platelet count, the albumin, bilirubin, INR, APRI, and vWF. We measured patients’ HVPG and divided them into CSPH and SPH groups. Our study Table I. Characteristics of subjects. Characteristic
Control group
Patients
Number (n) Age (years) Sex (M:F) Child–Pugh A/B/C ALT (U/L) AST (U/L) Bilirubin (mg/dL) Albumin (g/dL) INR Platelet count (n 109/L) APRI HVPG (mmHg) vWF (mU/mL)
45 42 (36–48) 31:14 NA 19.4 (7.0–36.2) 20.1 (11.2–38.6) 0.47 (0.21–1.29) 4.78 (3.90–5.19) 0.99 ± 0.17 298 ± 28.73 0.49 ± 0.78 NA 655 ± 106.4
60 49 (45–52) 43:17 28/19/13 47 (37.0–74.8) 56 (40.3–67.5) 1.69 (1.18–2.24) 3.44 (3.08–3.81) 1.21 ± 0.12 79.5 ± 32.41 0.91 ± 0.67 14.39 ± 4.65 2190 ± 855.3
Data expressed as median (interquartile range) or mean ± standard deviation. Abbreviations: M = male; F = female; ALT = alanine aminotransferase; AST = aspartate aminotransferase; INR = international normalized ratio; APRI = aspartate aminotransferase/platelet ratio index; HVPG = hepatic venous pressure gradient; vWF = von Willebrand factor; NA = not applicable.
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Figure 1. Levels of vWF were substantially elevated in patients’ plasma. Levels of plasma vWF were measured using enzyme-linked immunosorbent assays from all 105 plasma samples. (A) The level of plasma vWF was significantly higher in patients compared to control group (*p < 0.01). (B) Levels of plasma vWF were significantly higher in patients with CSPH compared to patients without CSPH. Plasma vWF levels were also higher in patients with SPH compared to patients who did not have SPH (*p < 0.01). (C) The level of vWF in patients’ plasma was significantly higher in patients who had EV compared to patients who did not have EV. Levels of plasma vWF were also higher in patients with medium/large EV compared to patients with small EV (*p < 0.01). Abbreviations: vWF = von Willebrand factor; CSPH = clinically significant portal hypertension; SPH = severe portal hypertension; EV = esophageal varices.
indicated that all of these six biomarkers were significantly associated with the presence of CSPH and SPH (all p < 0.05). The EV’s degree of each patient was measured by EGD and was divided into three groups: without EV, small EV, and medium/large EV. Results showed that six examined variables including vWF were related with the presence of EV and medium/large EV (all p < 0.05).
respectively (Figure 1B). In addition, plasma vWF levels were higher in patients who had EV (2350 ± 824.0 mU/mL) compared to patients who did not have EV (1490 ± 627.2 mU/mL, p < 0.01), and higher in patients with medium/large EV (2730 ± 644.1 mU/ mL, p < 0.01) compared to patients with small EV (1750 ± 723.7 mU/mL, p < 0.01) (Figure 1C).
Levels of vWF were substantially elevated in patients’ plasma
Plasma vWF was an effective noninvasive predictor for the diagnosis of CSPH and SPH in hepatitis B patients with cirrhosis
The vWF is a marker for endothelial dysfunction and regulates the angiogenesis. In order to confirm the role of vWF in predicting PH and EV, we examined the concentration of vWF in patients’ plasma. Upon examination, the average vWF level in liver cirrhosis group was 2190 ± 855.3 mU/mL, which was significantly higher than the healthy controls (655 ± 106.4 mU/mL, p < 0.01) (Figure 1A). Moreover, vWF levels (2430 ± 760.3 mU/mL) were higher in patients with CSPH and in patients with SPH (2560 ± 705.6 mU/mL), compared to patients with no CSPH (1240 ± 470.3 mU/mL, p < 0.01) and no SPH (1440 ± 612.3 mU/mL, p < 0.01),
Platelet count, albumin, bilirubin, INR, APRI, and vWF were related with PH. In the next step, we addressed the question of whether the level of vWF in patients’ plasma could be used for predicting the degree of PH. According to ROC analysis, the AUC was calculated for each biomarker (Figure 2A and 2B). Among six blood biomarkers, vWF showed the best diagnostic performance in predicting not only CSPH but also SPH. AUC of vWF for the diagnosis of CSPH was 0.885 (95% confidence interval [CI]: 0.796–0.975) and 0.871 (95% CI: 0.780–0.963) for the diagnosis of SPH. Based on the diagnostic performance observed, cutoff values of plasma vWF,
vWF as a noninvasive predictor of PH and EV A
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Figure 2. Plasma vWF was an effective noninvasive predictor for the diagnosis of CSPH and SPH in hepatitis B patients with cirrhosis. (A) ROC curves showing the prediction of CSPH with six blood markers. The AUC for vWF, platelet, albumin, bilirubin, INR, and APRI was 0.885 (95% CI: 0.796–0.975), 0.803 (95% CI: 0.678–0.928), 0.707 (95% CI: 0.524–0.889), 0.694 (95% CI: 0.531–0.858), 0.690 (95% CI: 0.504–0.876), and 0.793 (95% CI: 0.648–0.938), respectively. (B) ROC curves for six blood markers in prediction of SPH are shown. The AUC for vWF, platelet, albumin, bilirubin, INR, and APRI was 0.871 (95% CI: 0.780–0.963), 0.746 (95% CI: 0.619–0.873), 0.675 (95% CI: 0.525–0.825), 0.726 (95% CI: 0.597–0.854), 0.723 (95% CI: 0.583–0.863), and 0.747 (95% CI: 0.619–0.876), respectively. (C) Correlation between levels of vWF in patients’ plasma and HVPG is shown according to linear regression analysis. Abbreviations: AUC = area under the curve; APRI = aspartate aminotransferase/platelet ratio index; CI = confidence interval; CSPH = clinically significant portal hypertension; INR = international normalized ratio; SPH = severe portal hypertension; vWF = von Willebrand factor; HVPG = hepatic venous pressure gradient; ROC = receiver operating characteristic.
1510.5 mU/mL and 1701 mU/mL, provided optimal sensitivity and specificity to discriminate patients with CSPH or with SPH, respectively. PPV, NPV, LR+ and LR were calculated according to both cutoff points to show the diagnostic efficiency of plasma vWF (Table II). Furthermore, a linear correlation was observed between plasma vWF and HVPG (r2 = 0.485, p < 0.001) (Figure 2C). In conclusion, plasma vWF was correlated positively with HVPG and could be a credible noninvasive predictor for diagnosing the degree of PH. Plasma vWF was a valid noninvasive predictor for assessment of the presence and degree of EV in hepatitis B patients with cirrhosis According to the above study, levels of vWF had significant relationship with the degree of EV. In
addition, plasma vWF exhibited the highest accuracy in the diagnosis of CSPH and SPH. We next focused our attention to identify whether plasma vWF could also be a noninvasive candidate to predict EV. The diagnostic performance of each noninvasive parameter for detecting the presence and grade of EV was showed in our study (Figure 3). Among six variables, AUC of plasma vWF was 0.785 (95% CI: 0.649– 0.921), which demonstrated excellent performance in predicting the presence of EV. The cutoff values of plasma vWF (1414 mU/ml) provided optimal sensitivity and specificity for predicting the presence of EV. Furthermore, plasma vWF level also presented the best distinguishing value of small EV from medium/ large EV (AUC = 0.830, 95% CI: 0.714–0.945), according to ROC curve analysis. The cutoff values of plasma vWF (1990 mU/mL) could differentiate small EV from medium/large EV. PPV, NPV, LR+
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Table II. Performance of vWF for prediction of CSPH, SPH, EV, and medium/large EV.
CSPH SPH EV Medium/large EV
Cutoff value of vWF (mU/mL)
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
LR+
LR-
1510.5 1701 1414 1990
93.8 87.5 93.7 88.2
59.3 75 54.8 77.9
90.2 87.5 90.3 86.3
70.5 75.0 66.1 80.7
2.31 3.50 2.07 3.91
0.11 0.17 0.13 0.15
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Abbreviations: vWF = von Willebrand factor; CSPH = clinically significant portal hypertension; SPH = severe portal hypertension; EV = esophageal varices; PPV = positive predictive value; NPV = negative predictive value; LR+ = Positive likelihood ratio; LR- = Negative likelihood ratio.
and LR were also calculated for these two vWF cutoff points to prove its diagnostic efficiency (Table II). In conclusion, plasma vWF was an effective noninvasive marker in predicting the presence and grade of EV in patients with hepatitis B-related cirrhosis. The vWF levels were obviously increased in patients’ liver tissues, positively correlated with plasma vWF levels and PH Since pathological angiogenesis plays a crucial role in liver cirrhosis, and vWF is usually used as a marker for angiogenesis to reflect the degree of angiogenesis, we collected liver tissues from control group and 60 patients. We observed the expression of vWF in these liver tissues by immunohistochemistry. Immunostaining in normal liver tissues with antibody was positive mainly in the great vessels within the portal tract area. The vWF was positively stained not only in vessels of the portal tracts and central veins but also A
along the sinusoids in cirrhotic patients’ liver tissues (Figure 4A). The expression levels of vWF (8.42 ± 2.8%) that were reflected by the staining scores in patients’ tissues were higher than vWF expression levels (2.25 ± 0.56%, p < 0.01) in normal liver tissues (Figure 4B). To study the relationship between the degree of intrahepatic angiogenesis in liver tissue and PH, we analyzed the expression levels of vWF in liver tissues and PH. The expression level of vWF in patients’ liver tissues was significantly associated with CSPH and SPH (p < 0.01). The linear correlation was observed between the expression level of vWF in liver tissues and HVPG (r2 = 0.552, p < 0.001) (Figure 4C). We speculated that the degree of intrahepatic angiogenesis in liver tissue results in increased portal pressure, and the level of angiogenesis can reflect the degree of PH. A linear regression showed the correlation between levels of vWF in liver tissues and the level of plasma vWF (r2 = 0.461, p < 0.001) (Figure 4D). We presumed that the increased expression of vWF in B
Figure 3. Plasma vWF was a valid noninvasive predictor for assessment of the presence and degree of EV in hepatitis B patients with cirrhosis. (A) ROC curves of six blood markers for prediction of the presence of EV are shown. The AUC for vWF, platelet, albumin, bilirubin, INR and APRI was 0.785 (95% CI: 0.649–0.921), 0.759 (95% CI: 0.625–0.893), 0.736 (95% CI: 0.562–0.909), 0.762 (95% CI: 0.640–0.883), 0.721 (95% CI: 0.543–0.898), and 0.748 (95% CI: 0.596–0.899), respectively. (B) ROC curves showing the prediction of small EV or medium/large EV with six blood markers. The AUC for vWF, platelet, albumin, bilirubin, INR, and APRI was 0.830 (95% CI: 0.714–0.945), 0.782 (95% CI: 0.654–0.909), 0.716 (95% CI: 0.564–0.867), 0.714 (95% CI: 0.570–0.858), 0.777 (95% CI: 0.646–0.908), and 0.788 (95% CI: 0.657–0.919), respectively. Abbreviations: AUC = area under the curve; APRI = aspartate aminotransferase/platelet ratio index; CI = confidence interval; EV = esophageal varices; INR = international normalized ratio; vWF = von Willebrand factor; ROC = receiver operating characteristic.
vWF as a noninvasive predictor of PH and EV
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Figure 4. The vWF levels were obviously increased in patients’ liver tissue, positively correlated with plasma vWF levels and PH. (A) Representative immunohistochemistry images for vWF with hematoxylin counterstaining are shown. In normal patients’ liver tissues, vWF immunostaining is positive in intimal lining of the great vessels, but sparing of the sinusoidal microvasculature. vWF immunostaining is positive in intimal lining of the great vessels, as well as staining along the sinusoids in patients’ cirrhotic liver tissues. (B) The vWF staining score in patients’ liver tissues were significantly higher than its staining score in control group (*p < 0.01). (C) Linear regression analysis showed the correlation between immunohistochemistry staining score of vWF in patients’ liver tissues and HVPG. (D) Correlation between the vWF staining score in patients’ liver tissues and levels of vWF in patients’ plasma are shown according to linear regression analysis. Abbreviations: vWF = von Willebrand factor; HVPG = hepatic venous pressure gradient; PH = portal hypertension.
liver tissues might induce the increasing levels of vWF in patients’ plasma. Discussion Liver cirrhosis is a clinical syndrome that can lead to the increased PH, which is responsible for EV and variceal bleeding, associated with a high mortality rate. In order to find one noninvasive predictor for identifying PH and EV, we studied the vWF levels in plasma and in liver tissues of the patients with hepatitis B-related liver cirrhosis. We demonstrated that vWF is not only a creditable, novel noninvasive method to predict PH but also a direct predictor to diagnose the presence and degree of EV in hepatitis B patients with cirrhosis. The expression levels of vWF in patients’ liver tissues were positively correlated with vWF levels in plasma and with the HVPG. We
provided a possible pathophysiology mechanism that intrahepatic angiogenesis in liver tissue results in PH and can reflect the degree of PH, and increased vWF expression in liver tissues results in elevated vWF levels in patients’ plasma. At present, a variety of noninvasive techniques have been proposed to measure PH in cirrhotic patients. Different blood markers of liver fibrosis and vascular resistance were easily tested. But these markers in predicting PH need further clinical investigations [30–34]. Liver stiffness testing by transient elastography, the new noninvasive method, having good performance for predicting PH but limited operator experience and high costs has led to its limitation [35–37]. However, there is no perfect noninvasive technique for assessing PH. In this study, we chose the following markers: platelet count, which was proved to independently
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predict the prevalence of EV in several studies [38– 40]; the albumin, bilirubin, and INR, which were significantly associated with the presence of CSPH, SPH, and EV in compensated liver cirrhosis [41,42]; APRI, which was proved as a noninvasive marker to predict CSPH in many studies [43–45]; vWF as a marker for assessment of liver fibrosis and cirrhosis in patients with chronic hepatitis C, and could be proved that it could diagnose PH with excellent diagnostic value and predict clinical outcome in patient with liver cirrhosis [24–26]. We also demonstrated vWF as a creditable novel noninvasive predictor of PH in patients with liver cirrhosis. Moreover, among noninvasive blood factors evaluated in our study, the plasma vWF presents the best diagnostic performance for assessment of CSPH and SPH in hepatitis B patients with cirrhosis. In line with our finding, Ferlitsch et al. reported that vWF could predict the PH in cirrhotic patient [25]. AUC for the diagnosis of CSPH was 0.884 (95% CI: 0.841–0.928) and 0.88 (95% CI: 0.84–0.92) for the diagnosis of SPH. In our study, AUC of plasma vWF for the diagnosis of CSPH and SPH is 0.885 (95% CI: 0.796–0.975) and 0.871 (95% CI: 0.780–0.963), respectively, which are similar to Ferlitsch et al.’s results. Ferlitsch et al. found a cutoff value of 241% to discriminate between patients with and without CSPH. In this paper, we used the cutoff value of 1510.5 mU/mL and the control plasma vWF (655 ± 106.4 mU/mL) to approximately calculate the cutoff value percentage, which was 231%. Furthermore, we also reported the cutoff value (1701 mU/mL) to discriminate the presence or absence of SPH in cirrhotic patients. Cirrhotic patients in our study were all infected with chronic hepatitis B infection. Ferlitsch et al. did not clarify the etiology of cirrhosis. The vWF cutoff value to predict PH may be different in various causes of cirrhosis. La Mura et al.’s study suggested that vWF levels correlated with HVPG in cirrhosis [24]. Maieron et al. reported vWF as a marker to evaluate the stage of fibrosis to diagnose subclinical cirrhosis in patients with chronic hepatitis C [26]. However, La Mura et al. and Maieron et al. did not report the AUC and cutoff value of plasma vWF in predicting PH. Numerous noninvasive methods can be used to evaluate the presence and degree of EV in hepatic cirrhosis. For example, the models of combining several variables in predicting EV were easily gotten, but the diagnostic efficiency differ among studies [46,47]; computerized tomographic scanning was shown to be safe and tolerated in patients but experience of endoscopists played a major role in detecting
EV [48,49]. However, none of reliable noninvasive technique can predict EV in patients with liver cirrhosis; therefore, finding valuable noninvasive methods to predict EV are required. To identify the best parameter, we examined the blood levels of six markers: platelet count, albumin, bilirubin, INR, APRI, and vWF. Among these factors, vWF showed the best diagnostic performance in predicting the presence and degree of EV in patients with hepatitis B-related cirrhosis. AUCs of plasma vWF were 0.785 (95% CI: 0.649–0.921) and 0.830 (95% CI: 0.714–0.945) in the diagnosis of EV and medium/large EV. Two cutoff values of plasma vWF (1414 mU/mL and 1990 mU/ mL) showed PPV (90.3% and 86.3%) for the detection of EV and medium/large EV. Detecting the presence and degree of EV can provide the important directly clinical basis for clinician in the management of patients with liver cirrhosis to identify patients who can benefit from b-blocker therapy and those at risk of bleeding. Our study provides a possible simple, quick, reproducible, and costeffective method for testing the level of plasma vWF and for predicting the presence and grade of EV in patients with liver cirrhosis. Up to now, there is no paper published about vWF predicting the presence of EV in liver cirrhosis. We put forward a possible pathophysiology mechanism to explain why vWF is increased in patients’ plasma. We found that the expression of vWF also increased in patients’ liver tissues. Furthermore, vWF levels in cirrhotic tissues were correlated positively with the HVPG and the levels of plasma vWF. When vWF positively stains along large vessels in portal tract area, the central vein and sinusoidal endothelial cells is believed to express capillarization of the sinusoids in liver cirrhosis [50,51]. The vWF, as a marker to reflect the degree of intrahepatic angiogenesis, may reflect the degree of PH. We speculate that the increased synthesization of vWF from large vessels’ endothelial cells and capillarity of the sinusoids in liver tissue was released into patients’ plasma and therefore induce the increased levels of plasma vWF. However, early study reported that elevated vWF levels might be a consequence of a reduced clearance of vWF, resulting from decreased expression or activity of the vWF cleaving protease (ADAMTS13) in cirrhotic patients [52]. At the same time, investigators indicated that ADAMTS13 activity and concentration were highly variable in patients with liver disease [53]. In brief, increased plasma vWF in cirrhosis maybe not only come from decreased vWF clearance, but also from increased vWF production. Further works are needed to study the mechanism of evaluated levels of vWF in liver tissues and in plasma in cirrhotic patients.
vWF as a noninvasive predictor of PH and EV Declaration of interest: The authors do not have a commercial or other association with pharmaceutical companies or other parties that might pose a conflict of interest.
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[18] Randi AM, Laffan MA, Starke RD. Von Willebrand factor, angiodysplasia and angiogenesis. Mediterr J Hematol Infect Dis 2013;5:e2013060. [19] Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med 2000;6:389–95. [20] Fernández M, Semela D, Bruix J, Colle I, Pinzani M, Bosch J. Angiogenesis in liver disease. J Hepatol 2009;50: 604–20. [21] Zhang Z, Zhang F, Lu Y, Zheng S. Update on implications and mechanisms of angiogenesis in liver fibrosis. Hepatol Res 2015;45:162–78. [22] Albornoz L, Alvarez D, Otaso JC, Gadano A, Salviú J, Gerona S, et al. Von Willebrand factor could be an index of endothelial dysfunction in patients with cirrhosis: relationship to degree of liver failure and nitric oxide levels. J Hepatol 1999;30:451–5. [23] Lisman T, Bongers TN, Adelmeijer J, Janssen HL, de Maat MP, de Groot PG, et al. Elevated levels of von Willebrand Factor in cirrhosis support platelet adhesion despite reduced functional capacity. Hepatology 2006;44: 53–61. [24] La Mura V, Reverter JC, Flores-Arroyo A, Raffa S, Reverter E, Seijo S, et al. Von Willebrand factor levels predict clinical outcome in patients with cirrhosis and portal hypertension. Gut 2011;60:1133–8. [25] Ferlitsch M, Reiberger T, Hoke M, Salzl P, Schwengerer B, Ulbrich G, et al. von Willebrand factor as new noninvasive predictor of portal hypertension, decompensation and mortality in patients with liver cirrhosis. Hepatology 2012;56: 1439–47. [26] Maieron A, Salzl P, Peck-Radosavljevic M, Trauner M, Hametner S, Schöfl R, et al. Von Willebrand Factor as a new marker for non-invasive assessment of liver fibrosis and cirrhosis in patients with chronic hepatitis C. Aliment Pharmacol Ther 2014;39:331–8. [27] Lok AS, McMahon BJ. Chronic hepatitis B: update 2009. Hepatology 2009;50:661–2. [28] North Italian Endoscopic Club for the Study and Treatment of Esophageal Varices. Prediction of the first variceal hemorrhage in patients with cirrhosis of the liver and esophageal varices. A prospective multicenter study. N Engl J Med 1988; 319:983–9. [29] Groszmann RJ, Wongcharatrawee S. The hepatic venous pressure gradient: anything worth doing should be done right. Hepatology 2004;39:280–2. [30] Abdelmoneim SS, Talwalkar J, Sethi S, Kamath P, Fathalla MM, Kipp BR, et al. A prospective pilot study of circulating endothelial cells as a potential new biomarker in portal hypertension. Liver Int 2010;30:191–7. [31] Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 2005; 115:209–18. [32] Kondo M, Miszputen SJ, Leite-mor MM, Parise ER. The predictive value of serum laminin for the risk of variceal bleeding related to portal pressure levels. Hepatogastroenterology 1995;42:542–5. [33] Thabut D, Imbert-Bismut F, Cazals-Hatem D, Messous D, Muntenau M, Valla DC, et al. Relationship between the Fibrotest and portalhypertension in patients with liver disease. Aliment Pharmacol Ther 2007;26:359–68. [34] Calès P, Oberti F, Michalak S, Hubert-Fouchard I, Rousselet MC, Konaté A, et al. A novel panel of blood markers to assess the degree of liver fibrosis. Hepatology 2005;42:1373–81. [35] Vizzutti F, Arena U, Romanelli RG, Rega L, Foschi M, Colagrande S, et al. Liver stiffness measurement predicts
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H. Wu et al. severe portal hypertension in patients with HCV-related cirrhosis. Hepatology 2007;45:1290–7. Castera L, Pinzani M, Bosch J. Noninvasive evaluation of portal hypertension using transient elastography. J Hepatol 2012;56:696–703. Hong WK, Kim MY, Baik SK, Shin SY, Kim JM, Kang YS, et al. The usefulness of non-invasive liver stiffness measurements in predicting clinically significant portal hypertension in cirrhotic patients: Korean data. Clin Mol Hepatol 2013;19:370–5. Cherian JV, Deepak N, Ponnusamy RP, Somasundaram A, Jayanthi V. Non-invasive predictors of esophageal varices. Saudi J Gastroenterol 2011;17:64–8. Sharma SK, Aggarwal R. Prediction of large esophageal varices in patients with cirrhosis of the liver using clinical, laboratory and imaging parameters. J Gastroenterol Hepatol 2007;22:1909–15. Qamar AA, Grace ND, Groszmann RJ, Garcia-Tsao G, Bosch J, Burroughs AK, et al. Portal Hypertension Collaborative Group. Platelet count is not a predictor of the presence or development of gastroesophageal varices in cirrhosis. Hepatology 2008;47:153–9. Bressler B, Pinto R, El-Ashry D, Heathcote EJ. Which patients with primary biliary cirrhosis or primary sclerosing cholangitis should undergo endoscopic screening for oesophageal varices detection? Gut 2005;54:407–10. Berzigotti A, Gilabert R, Abraldes JG, Nicolau C, Bru C, Bosch J, et al. Noninvasive prediction of clinically significant portal hypertension and esophageal varices in patients with compensated liver cirrhosis. Am J Gastroenterol 2008;103: 1159–67. Leroy V, Hilleret MN, Sturm N, Trocme C, Renversez JC, Faure P, et al. Prospective comparison of six non-invasive scores for the diagnosis of liver fibrosis in chronic hepatitis C. J Hepatol 2007;46:775–82. Güzelbulut F, Sezıklı M, Akkan-Cetınkaya ¸ Z, Yas¸ar B, Özkara S, Kurdas¸-Övünç AO. AST-platelet ratio index in
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the prediction of significant fibrosis and cirrhosis in patients with chronic hepatitis B. Turk J Gastroenterol 2012;23:353–8. Burton JR Jr, Liangpunsakul S, Lapidus J, Giannini E, Chalasani N, Zaman A. Validation of a multivariate model predicting presence and size of varices. J Clin Gastroenterol 2007;41:609–15. Sarangapani A, Shanmugam C, Kalyanasundaram M, Rangachari B, Thangavelu P, Subbarayan JK. Noninvasive prediction of large esophageal varices in chronic liver disease patients. Saudi J Gastroenterol 2010;16:38–42. Thomopoulos KC. Non-invasive prediction of esophageal varices: is it possible? Saudi J Gastroenterol 2011;17: 1–3. Kim YJ, Raman SS, Yu NC, To’o KJ, Jutabha R, Lu DS. Esophageal varices in cirrhotic patients: evaluation with liver CT. AJR Am J Roentgenol 2007;188:139–44. Perri RE, Chiorean MV, Fidler JL, Fletcher JG, Talwalkar JA, Stadheim L, et al. A prospective evaluation of computerized tomographic (CT) scanning as a screening modality for esophageal varices. Hepatology 2008;47:1587– 94. Hattori M, Fukuda Y, Imoto M, Koyama Y, Nakano I, Urano F. Histochemical properties of vascular and sinusoidal endothelial cells in liver diseases. Gastroenterol Jpn 1991;26: 336–43. Beer JH, Clerici N, Baillod P, von Felten A, Schlappritzi E, Büchi L. Quantitative and qualitative analysis of platelet GPIb and von Willebrand factor in liver cirrhosis. Thromb Haemost 1995;73:601–9. Moake JL. Thrombotic microangiopathies. N Engl J Med 2002;347:589–600. Lisman T, Bongers TN, Adelmeijer J, Janssen HL, de Maat MP, de Groot PG, et al. Elevated levels of von Willebrand Factor in cirrhosis support platelet adhesion despite reduced functional capacity. Hepatology 2006;44: 53–61.
ORIGINAL ARTICLE
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von Willebrand factor as a novel noninvasive predictor of portal hypertension and esophageal varices in hepatitis B patients with cirrhosis
HAO WU*, SHIPING YAN*, GUANGCHUAN WANG, SHAOBO CUI, CHUNQING ZHANG & QIANG ZHU Department of Gastroenterology, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong, China
Abstract Objective. At present, there is no perfect noninvasive method to assess portal hypertension and esophageal varices. Early predicting esophageal varices can provide evidence for managing cirrhotic patients. We aimed to further investigate von Willebrand factor (vWF) as a noninvasive predictor of portal hypertension, especially of esophageal varices. Material and Methods. A total of 60 hepatitis B patients with cirrhosis and 45 healthy subjects were enrolled in this study. Levels of six markers were examined. All patients underwent hepatic venous pressure gradient (HVPG) and esophagogastroduodenoscopy. We evaluated the performance of six factors for diagnosis of portal hypertension and esophageal varices. The vWF levels in liver tissues were observed by immunohistochemistry. Correlations between the level of vWF in liver tissues and HVPG and between levels of vWF in tissues and plasma were examined. Results. Cutoff values of plasma vWF (1510.5 mU/mL and 1701 mU/mL) showed high positive predictive value (PPV, 90.2% and 87.5%) in predicting clinically significant portal hypertension and severe portal hypertension. Cutoff values of vWF (1414 mU/ml and 1990 mU/mL, PPV 90.3% and 86.3%, respectively) were provided to detect the presence and degree of esophageal varices. Linear correlations were observed between levels of vWF in liver tissues and HVPG (r2 = 0.552, p < 0.001) and between the level of vWF in liver tissues and in plasma (r2 = 0.461, p < 0.001). Conclusion. The vWF is a noninvasive predictor of portal hypertension and esophageal varices in hepatitis B patients with cirrhosis. Increased levels of vWF in liver tissues may induce the elevated plasma vWF levels, but molecular mechanism is needed for further study.
Key Words: Esophageal varices, liver cirrhosis, noninvasive predictor, portal hypertension, von Willebrand factor
Introduction Liver cirrhosis, the most common cause of portal hypertension (PH), is almost caused by chronic hepatitis B virus infection in Asia and most of Africa [1,2]. PH induces the severe complications of cirrhosis, such as esophageal varices (EV), bleeding, ascites, and decompensation [3]. Studies indicated that early diagnosis of PH could help in starting timely treatment and in reducing the mortality of its complications [4,5].
Nowadays, guidelines preferred hepatic venous pressure gradient (HVPG) as the diagnosis, prognostic, and therapeutic indications of PH [6]. A lot of studies indicated HVPG to have good ability to predict liver-related variceal hemorrhage and assess fibrosis or cirrhosis despite etiology [7–9]. However, it was considered as invasive procedure and only available in centers with adequate resources and expertise. Nowadays, esophagogastroduodenoscopy (EGD) was the gold standard for determining varices that should be screened in all cirrhotic patients at
Correspondence: Qiang Zhu, Department of Gastroenterology, Provincial Hospital Affiliated to Shandong University, 324, Jing 5 Rd, Ji’nan, 250021, Shandong Province, China. Tel: +86-0531-68777012. Fax: +86-0531-87902348. E-mail: [email protected] *These authors contributed to the work equally and should be regarded as co-first authors.
(Received 27 February 2015; revised 28 March 2015; accepted 29 March 2015) ISSN 0036-5521 print/ISSN 1502-7708 online 2015 Informa Healthcare DOI: 10.3109/00365521.2015.1037346
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diagnosis [10]. But this technique was invasive, time-consuming, and costly. Up to now, there is no effective and perfect noninvasive method to predict PH and EV. Therefore, novel noninvasive markers were required. von Willebrand factor (vWF) is a multimeric glycoprotein mainly synthesized by endothelial cells [11]. It was released by activated endothelial cells and recognized as an index of endothelial dysfunction [12–15]. Endothelium plays a crucial role in many vascular diseases and endothelial dysfunction is a fundamental component of the increased hepatic vascular tone [16], which induces the development of PH. The vWF also regulated angiogenesis [17,18], the formation of new blood vessels that leads to the progressive formation of the abnormal angio-architecture distinctive in cirrhotic livers [19,20]. A vicious circle appears between liver fibrosis and pathological angiogenesis, and this oppression induces venous pressure and portal venous resistance to increase significantly [21]. Evidence clearly reported that angiogenesis and disruption of liver vascular architecture have been linked to the progression to cirrhosis, which leads both to increased hepatic vascular resistance and PH [20]. Elevated levels of vWF were frequently found and were reported as the noninvasive marker in predicting PH and the mortality in cirrhotic patients [22–26]. However, no study reported that vWF could directly predict EV, the mechanisms why vWF reflects portal vein pressure and why vWF increases in patients’ plasma remain unclear. In this study, we examined the level of plasma vWF in hepatitis B patients with cirrhosis and evaluated the diagnostic performance of vWF as a new noninvasive predictor not only of PH but also EV, compared to HVPG and EGD in cirrhotic patients. At the same time, we tested the expression of vWF in patients’ liver tissues and analyzed the possible mechanism of PH and vWF levels increasing in cirrhotic patients’ plasma. Methods Patients Sixty hepatitis B viral cirrhotic patients were recruited in this study. Liver cirrhosis with hepatitis B infection were diagnosed by clinical data, laboratory and imaging examinations according to American Association for the Study of Liver Disease practice guideline [27]. Exclusion criteria were the presence of pre- and posthepatic causes of PH, variceal bleeding, refractory ascites, severe cardiopulmonary or renal impairment, and ongoing treatment of PH. Patients with other causes of cirrhosis and presence of hepatocellular
carcinoma and patients with the other fibrosis diseases must be excluded. Child–Pugh stage, transjugular HVPG measurement, EGD, liver biopsy, and blood biomarkers including plasma vWF were examined for each patient. Blood samples from 45 health subjects with no evidence of liver disease were used to establish control group in this study. Forty-five normal liver tissues from hepatic hemangioma and liver trauma were included as control. All procedures were approved by the Ethics Committee of Provincial Hospital Affiliated to Shandong University. Assessment and scoring of laboratory parameters Laboratory tests included platelet count, alanine aminotransferase, aspartate aminotransferase (AST), total bilirubin, serum albumin, and international normalized ratio (INR). AST/platelet ratio index (APRI) was also calculated according to published formulas. Measurement of plasma vWF About 2 ml of vein blood was sampled from patients by HVPG measurement in ethylene diamine tetraacetic acid tubes and centrifuged. Plasma was stored at 80 C and thawed for 12 h at 4 C before being assayed. Levels of vWF were measured strictly using enzyme-linked immunosorbent assays kits (Abcam, Cambride, UK) according to the instructions. Immunohistochemistry A total of 60 liver tissues of liver cirrhosis were obtained via biopsy, and 45 normal liver tissues from hepatic hemangioma and liver trauma were included as control. Thin sections were prepared by embedding in paraffin. Sections were deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval in 0.01 M citrate for 5 min. Endogenous peroxidase activity was quenched with 3% H2O2 for 5 min. Thereafter, sections were blocked with 5% goat serum, and then incubated for 2 h at room temperature with anti-vWF antibodies (Abcam, Cambride, UK). Sections were incubated with horseradish peroxidase-conjugated secondary antibodies and reacted with a diaminobenzidine solution and counterstained with hematoxylin. Liver specimens were stained for vWF. Positively stained portal vessels and sinusoids appeared as brown linear deposits, observed by light microscopy. Four most intensely stained highpower microscopic fields (400) were assessed in each sample. The vWF staining extension was calculated by the percentage of stained area per high-power field, and the average vWF scoring of each sample was calculated.
vWF as a noninvasive predictor of PH and EV The vWF staining scoring was measured using ImagePro Plus 6 software (Baltimore, USA).
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Esophagogastroduodenoscopy The same operator performed EGD for each patient. EVs are classified into three sizes, small, medium, and large, by a semi-quantitative morphological assessment in most centers and consensus. The small EV defined as minimally elevated veins above the esophageal mucosal surface. Medium EV is defined as tortuous veins that occupy less than one-third of the esophageal lumen and large EV is defined as veins occupying more than one-third of the esophageal lumen [28]. Since recommendations for mediumsized EV are the same as for large EV, we took the medium and large EV in the same group. Measurement of HVPG Methods for accurate HVPG measurement are according to the previously study [29]. Before the measurement of HVPG, the measurement operator should obtain permission and ask the patient to fast for 8 h. The operator places balloon catheters in the right hepatic vein through a right jugular vein puncture for measurement of the free hepatic venous pressures (FHVPs). The wedged hepatic venous pressure (WHVP) is measured by inflation of the balloon catheter at the right hepatic vein. Last, the HVPG is determined by subtracting the FHVP from the WHVP. Measurements were performed at least three times to get the average and for minimizing the error. All data are obtained from the same measurement operator. Normal portal pressure was defined as the range of 1 to 5 mmHg according to HVPG. When the HVPG ‡10 mmHg, PH was defined as clinically significant PH (CSPH), whereas HVPG ‡12 mm Hg was diagnosed as severe PH (SPH). Statistic analysis Statistical analysis was performed with Statistical Analysis Software, SPSS version 16.0 (SPSS Inc., Chicago, IL, USA) and MedCalc package v.10.0 (Mariakerke, Belgium). Categorical variables are reported as numbers (percentage); continuous variables were reported as mean and standard deviation or in median and interquartile range on the basis of their distribution analyzed by the means of Kolmogorov–Smirnov’s test. Differences between groups were analyzed using Mann–Whitney U test. The correlation between vWF in plasma and HVPG, between expression levels of vWF in tissues and HVPG and levels of plasma vWF were used as a
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linear regression analysis. Receiver operator characteristic (ROC) curve was constructed to determinate test performance for prediction of CSPH, SPH, EV, and medium/large EV, and each area under the curve (AUC) was calculated. The cutoff value of the variable was determined at the point of highest sensitivity and specificity. Positive predictive value (PPV), negative predictable value (NPV), the positive likelihood ratio (LR+) and the negative likelihood ratio (LR ) were calculated for the cutoff value. All p-Values reported are two-sided, and p-Values < 0.05 are considered statistically significant. Results Patients’ characteristics In this study, 48 patients (80%) had CSPH and 40 patients (66.7%) had SPH. All patients were tested through EGD, 49 (81.7%) and showed the presence of EV, 19 (38.8%) of whom had the small EV and 30 (61.2%) had the medium or large EV. Demographical, clinical, and laboratory characteristics of patients group and control group were shown in Table I. Six examined variables including vWF were associated with the degree of PH, with the presence and grade of EV Many markers are significantly associated with PH and EV in liver cirrhosis. To identify the best parameter, we examined the blood levels of the following markers: platelet count, the albumin, bilirubin, INR, APRI, and vWF. We measured patients’ HVPG and divided them into CSPH and SPH groups. Our study Table I. Characteristics of subjects. Characteristic
Control group
Patients
Number (n) Age (years) Sex (M:F) Child–Pugh A/B/C ALT (U/L) AST (U/L) Bilirubin (mg/dL) Albumin (g/dL) INR Platelet count (n 109/L) APRI HVPG (mmHg) vWF (mU/mL)
45 42 (36–48) 31:14 NA 19.4 (7.0–36.2) 20.1 (11.2–38.6) 0.47 (0.21–1.29) 4.78 (3.90–5.19) 0.99 ± 0.17 298 ± 28.73 0.49 ± 0.78 NA 655 ± 106.4
60 49 (45–52) 43:17 28/19/13 47 (37.0–74.8) 56 (40.3–67.5) 1.69 (1.18–2.24) 3.44 (3.08–3.81) 1.21 ± 0.12 79.5 ± 32.41 0.91 ± 0.67 14.39 ± 4.65 2190 ± 855.3
Data expressed as median (interquartile range) or mean ± standard deviation. Abbreviations: M = male; F = female; ALT = alanine aminotransferase; AST = aspartate aminotransferase; INR = international normalized ratio; APRI = aspartate aminotransferase/platelet ratio index; HVPG = hepatic venous pressure gradient; vWF = von Willebrand factor; NA = not applicable.
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Figure 1. Levels of vWF were substantially elevated in patients’ plasma. Levels of plasma vWF were measured using enzyme-linked immunosorbent assays from all 105 plasma samples. (A) The level of plasma vWF was significantly higher in patients compared to control group (*p < 0.01). (B) Levels of plasma vWF were significantly higher in patients with CSPH compared to patients without CSPH. Plasma vWF levels were also higher in patients with SPH compared to patients who did not have SPH (*p < 0.01). (C) The level of vWF in patients’ plasma was significantly higher in patients who had EV compared to patients who did not have EV. Levels of plasma vWF were also higher in patients with medium/large EV compared to patients with small EV (*p < 0.01). Abbreviations: vWF = von Willebrand factor; CSPH = clinically significant portal hypertension; SPH = severe portal hypertension; EV = esophageal varices.
indicated that all of these six biomarkers were significantly associated with the presence of CSPH and SPH (all p < 0.05). The EV’s degree of each patient was measured by EGD and was divided into three groups: without EV, small EV, and medium/large EV. Results showed that six examined variables including vWF were related with the presence of EV and medium/large EV (all p < 0.05).
respectively (Figure 1B). In addition, plasma vWF levels were higher in patients who had EV (2350 ± 824.0 mU/mL) compared to patients who did not have EV (1490 ± 627.2 mU/mL, p < 0.01), and higher in patients with medium/large EV (2730 ± 644.1 mU/ mL, p < 0.01) compared to patients with small EV (1750 ± 723.7 mU/mL, p < 0.01) (Figure 1C).
Levels of vWF were substantially elevated in patients’ plasma
Plasma vWF was an effective noninvasive predictor for the diagnosis of CSPH and SPH in hepatitis B patients with cirrhosis
The vWF is a marker for endothelial dysfunction and regulates the angiogenesis. In order to confirm the role of vWF in predicting PH and EV, we examined the concentration of vWF in patients’ plasma. Upon examination, the average vWF level in liver cirrhosis group was 2190 ± 855.3 mU/mL, which was significantly higher than the healthy controls (655 ± 106.4 mU/mL, p < 0.01) (Figure 1A). Moreover, vWF levels (2430 ± 760.3 mU/mL) were higher in patients with CSPH and in patients with SPH (2560 ± 705.6 mU/mL), compared to patients with no CSPH (1240 ± 470.3 mU/mL, p < 0.01) and no SPH (1440 ± 612.3 mU/mL, p < 0.01),
Platelet count, albumin, bilirubin, INR, APRI, and vWF were related with PH. In the next step, we addressed the question of whether the level of vWF in patients’ plasma could be used for predicting the degree of PH. According to ROC analysis, the AUC was calculated for each biomarker (Figure 2A and 2B). Among six blood biomarkers, vWF showed the best diagnostic performance in predicting not only CSPH but also SPH. AUC of vWF for the diagnosis of CSPH was 0.885 (95% confidence interval [CI]: 0.796–0.975) and 0.871 (95% CI: 0.780–0.963) for the diagnosis of SPH. Based on the diagnostic performance observed, cutoff values of plasma vWF,
vWF as a noninvasive predictor of PH and EV A
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Figure 2. Plasma vWF was an effective noninvasive predictor for the diagnosis of CSPH and SPH in hepatitis B patients with cirrhosis. (A) ROC curves showing the prediction of CSPH with six blood markers. The AUC for vWF, platelet, albumin, bilirubin, INR, and APRI was 0.885 (95% CI: 0.796–0.975), 0.803 (95% CI: 0.678–0.928), 0.707 (95% CI: 0.524–0.889), 0.694 (95% CI: 0.531–0.858), 0.690 (95% CI: 0.504–0.876), and 0.793 (95% CI: 0.648–0.938), respectively. (B) ROC curves for six blood markers in prediction of SPH are shown. The AUC for vWF, platelet, albumin, bilirubin, INR, and APRI was 0.871 (95% CI: 0.780–0.963), 0.746 (95% CI: 0.619–0.873), 0.675 (95% CI: 0.525–0.825), 0.726 (95% CI: 0.597–0.854), 0.723 (95% CI: 0.583–0.863), and 0.747 (95% CI: 0.619–0.876), respectively. (C) Correlation between levels of vWF in patients’ plasma and HVPG is shown according to linear regression analysis. Abbreviations: AUC = area under the curve; APRI = aspartate aminotransferase/platelet ratio index; CI = confidence interval; CSPH = clinically significant portal hypertension; INR = international normalized ratio; SPH = severe portal hypertension; vWF = von Willebrand factor; HVPG = hepatic venous pressure gradient; ROC = receiver operating characteristic.
1510.5 mU/mL and 1701 mU/mL, provided optimal sensitivity and specificity to discriminate patients with CSPH or with SPH, respectively. PPV, NPV, LR+ and LR were calculated according to both cutoff points to show the diagnostic efficiency of plasma vWF (Table II). Furthermore, a linear correlation was observed between plasma vWF and HVPG (r2 = 0.485, p < 0.001) (Figure 2C). In conclusion, plasma vWF was correlated positively with HVPG and could be a credible noninvasive predictor for diagnosing the degree of PH. Plasma vWF was a valid noninvasive predictor for assessment of the presence and degree of EV in hepatitis B patients with cirrhosis According to the above study, levels of vWF had significant relationship with the degree of EV. In
addition, plasma vWF exhibited the highest accuracy in the diagnosis of CSPH and SPH. We next focused our attention to identify whether plasma vWF could also be a noninvasive candidate to predict EV. The diagnostic performance of each noninvasive parameter for detecting the presence and grade of EV was showed in our study (Figure 3). Among six variables, AUC of plasma vWF was 0.785 (95% CI: 0.649– 0.921), which demonstrated excellent performance in predicting the presence of EV. The cutoff values of plasma vWF (1414 mU/ml) provided optimal sensitivity and specificity for predicting the presence of EV. Furthermore, plasma vWF level also presented the best distinguishing value of small EV from medium/ large EV (AUC = 0.830, 95% CI: 0.714–0.945), according to ROC curve analysis. The cutoff values of plasma vWF (1990 mU/mL) could differentiate small EV from medium/large EV. PPV, NPV, LR+
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Table II. Performance of vWF for prediction of CSPH, SPH, EV, and medium/large EV.
CSPH SPH EV Medium/large EV
Cutoff value of vWF (mU/mL)
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
LR+
LR-
1510.5 1701 1414 1990
93.8 87.5 93.7 88.2
59.3 75 54.8 77.9
90.2 87.5 90.3 86.3
70.5 75.0 66.1 80.7
2.31 3.50 2.07 3.91
0.11 0.17 0.13 0.15
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Abbreviations: vWF = von Willebrand factor; CSPH = clinically significant portal hypertension; SPH = severe portal hypertension; EV = esophageal varices; PPV = positive predictive value; NPV = negative predictive value; LR+ = Positive likelihood ratio; LR- = Negative likelihood ratio.
and LR were also calculated for these two vWF cutoff points to prove its diagnostic efficiency (Table II). In conclusion, plasma vWF was an effective noninvasive marker in predicting the presence and grade of EV in patients with hepatitis B-related cirrhosis. The vWF levels were obviously increased in patients’ liver tissues, positively correlated with plasma vWF levels and PH Since pathological angiogenesis plays a crucial role in liver cirrhosis, and vWF is usually used as a marker for angiogenesis to reflect the degree of angiogenesis, we collected liver tissues from control group and 60 patients. We observed the expression of vWF in these liver tissues by immunohistochemistry. Immunostaining in normal liver tissues with antibody was positive mainly in the great vessels within the portal tract area. The vWF was positively stained not only in vessels of the portal tracts and central veins but also A
along the sinusoids in cirrhotic patients’ liver tissues (Figure 4A). The expression levels of vWF (8.42 ± 2.8%) that were reflected by the staining scores in patients’ tissues were higher than vWF expression levels (2.25 ± 0.56%, p < 0.01) in normal liver tissues (Figure 4B). To study the relationship between the degree of intrahepatic angiogenesis in liver tissue and PH, we analyzed the expression levels of vWF in liver tissues and PH. The expression level of vWF in patients’ liver tissues was significantly associated with CSPH and SPH (p < 0.01). The linear correlation was observed between the expression level of vWF in liver tissues and HVPG (r2 = 0.552, p < 0.001) (Figure 4C). We speculated that the degree of intrahepatic angiogenesis in liver tissue results in increased portal pressure, and the level of angiogenesis can reflect the degree of PH. A linear regression showed the correlation between levels of vWF in liver tissues and the level of plasma vWF (r2 = 0.461, p < 0.001) (Figure 4D). We presumed that the increased expression of vWF in B
Figure 3. Plasma vWF was a valid noninvasive predictor for assessment of the presence and degree of EV in hepatitis B patients with cirrhosis. (A) ROC curves of six blood markers for prediction of the presence of EV are shown. The AUC for vWF, platelet, albumin, bilirubin, INR and APRI was 0.785 (95% CI: 0.649–0.921), 0.759 (95% CI: 0.625–0.893), 0.736 (95% CI: 0.562–0.909), 0.762 (95% CI: 0.640–0.883), 0.721 (95% CI: 0.543–0.898), and 0.748 (95% CI: 0.596–0.899), respectively. (B) ROC curves showing the prediction of small EV or medium/large EV with six blood markers. The AUC for vWF, platelet, albumin, bilirubin, INR, and APRI was 0.830 (95% CI: 0.714–0.945), 0.782 (95% CI: 0.654–0.909), 0.716 (95% CI: 0.564–0.867), 0.714 (95% CI: 0.570–0.858), 0.777 (95% CI: 0.646–0.908), and 0.788 (95% CI: 0.657–0.919), respectively. Abbreviations: AUC = area under the curve; APRI = aspartate aminotransferase/platelet ratio index; CI = confidence interval; EV = esophageal varices; INR = international normalized ratio; vWF = von Willebrand factor; ROC = receiver operating characteristic.
vWF as a noninvasive predictor of PH and EV
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Figure 4. The vWF levels were obviously increased in patients’ liver tissue, positively correlated with plasma vWF levels and PH. (A) Representative immunohistochemistry images for vWF with hematoxylin counterstaining are shown. In normal patients’ liver tissues, vWF immunostaining is positive in intimal lining of the great vessels, but sparing of the sinusoidal microvasculature. vWF immunostaining is positive in intimal lining of the great vessels, as well as staining along the sinusoids in patients’ cirrhotic liver tissues. (B) The vWF staining score in patients’ liver tissues were significantly higher than its staining score in control group (*p < 0.01). (C) Linear regression analysis showed the correlation between immunohistochemistry staining score of vWF in patients’ liver tissues and HVPG. (D) Correlation between the vWF staining score in patients’ liver tissues and levels of vWF in patients’ plasma are shown according to linear regression analysis. Abbreviations: vWF = von Willebrand factor; HVPG = hepatic venous pressure gradient; PH = portal hypertension.
liver tissues might induce the increasing levels of vWF in patients’ plasma. Discussion Liver cirrhosis is a clinical syndrome that can lead to the increased PH, which is responsible for EV and variceal bleeding, associated with a high mortality rate. In order to find one noninvasive predictor for identifying PH and EV, we studied the vWF levels in plasma and in liver tissues of the patients with hepatitis B-related liver cirrhosis. We demonstrated that vWF is not only a creditable, novel noninvasive method to predict PH but also a direct predictor to diagnose the presence and degree of EV in hepatitis B patients with cirrhosis. The expression levels of vWF in patients’ liver tissues were positively correlated with vWF levels in plasma and with the HVPG. We
provided a possible pathophysiology mechanism that intrahepatic angiogenesis in liver tissue results in PH and can reflect the degree of PH, and increased vWF expression in liver tissues results in elevated vWF levels in patients’ plasma. At present, a variety of noninvasive techniques have been proposed to measure PH in cirrhotic patients. Different blood markers of liver fibrosis and vascular resistance were easily tested. But these markers in predicting PH need further clinical investigations [30–34]. Liver stiffness testing by transient elastography, the new noninvasive method, having good performance for predicting PH but limited operator experience and high costs has led to its limitation [35–37]. However, there is no perfect noninvasive technique for assessing PH. In this study, we chose the following markers: platelet count, which was proved to independently
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predict the prevalence of EV in several studies [38– 40]; the albumin, bilirubin, and INR, which were significantly associated with the presence of CSPH, SPH, and EV in compensated liver cirrhosis [41,42]; APRI, which was proved as a noninvasive marker to predict CSPH in many studies [43–45]; vWF as a marker for assessment of liver fibrosis and cirrhosis in patients with chronic hepatitis C, and could be proved that it could diagnose PH with excellent diagnostic value and predict clinical outcome in patient with liver cirrhosis [24–26]. We also demonstrated vWF as a creditable novel noninvasive predictor of PH in patients with liver cirrhosis. Moreover, among noninvasive blood factors evaluated in our study, the plasma vWF presents the best diagnostic performance for assessment of CSPH and SPH in hepatitis B patients with cirrhosis. In line with our finding, Ferlitsch et al. reported that vWF could predict the PH in cirrhotic patient [25]. AUC for the diagnosis of CSPH was 0.884 (95% CI: 0.841–0.928) and 0.88 (95% CI: 0.84–0.92) for the diagnosis of SPH. In our study, AUC of plasma vWF for the diagnosis of CSPH and SPH is 0.885 (95% CI: 0.796–0.975) and 0.871 (95% CI: 0.780–0.963), respectively, which are similar to Ferlitsch et al.’s results. Ferlitsch et al. found a cutoff value of 241% to discriminate between patients with and without CSPH. In this paper, we used the cutoff value of 1510.5 mU/mL and the control plasma vWF (655 ± 106.4 mU/mL) to approximately calculate the cutoff value percentage, which was 231%. Furthermore, we also reported the cutoff value (1701 mU/mL) to discriminate the presence or absence of SPH in cirrhotic patients. Cirrhotic patients in our study were all infected with chronic hepatitis B infection. Ferlitsch et al. did not clarify the etiology of cirrhosis. The vWF cutoff value to predict PH may be different in various causes of cirrhosis. La Mura et al.’s study suggested that vWF levels correlated with HVPG in cirrhosis [24]. Maieron et al. reported vWF as a marker to evaluate the stage of fibrosis to diagnose subclinical cirrhosis in patients with chronic hepatitis C [26]. However, La Mura et al. and Maieron et al. did not report the AUC and cutoff value of plasma vWF in predicting PH. Numerous noninvasive methods can be used to evaluate the presence and degree of EV in hepatic cirrhosis. For example, the models of combining several variables in predicting EV were easily gotten, but the diagnostic efficiency differ among studies [46,47]; computerized tomographic scanning was shown to be safe and tolerated in patients but experience of endoscopists played a major role in detecting
EV [48,49]. However, none of reliable noninvasive technique can predict EV in patients with liver cirrhosis; therefore, finding valuable noninvasive methods to predict EV are required. To identify the best parameter, we examined the blood levels of six markers: platelet count, albumin, bilirubin, INR, APRI, and vWF. Among these factors, vWF showed the best diagnostic performance in predicting the presence and degree of EV in patients with hepatitis B-related cirrhosis. AUCs of plasma vWF were 0.785 (95% CI: 0.649–0.921) and 0.830 (95% CI: 0.714–0.945) in the diagnosis of EV and medium/large EV. Two cutoff values of plasma vWF (1414 mU/mL and 1990 mU/ mL) showed PPV (90.3% and 86.3%) for the detection of EV and medium/large EV. Detecting the presence and degree of EV can provide the important directly clinical basis for clinician in the management of patients with liver cirrhosis to identify patients who can benefit from b-blocker therapy and those at risk of bleeding. Our study provides a possible simple, quick, reproducible, and costeffective method for testing the level of plasma vWF and for predicting the presence and grade of EV in patients with liver cirrhosis. Up to now, there is no paper published about vWF predicting the presence of EV in liver cirrhosis. We put forward a possible pathophysiology mechanism to explain why vWF is increased in patients’ plasma. We found that the expression of vWF also increased in patients’ liver tissues. Furthermore, vWF levels in cirrhotic tissues were correlated positively with the HVPG and the levels of plasma vWF. When vWF positively stains along large vessels in portal tract area, the central vein and sinusoidal endothelial cells is believed to express capillarization of the sinusoids in liver cirrhosis [50,51]. The vWF, as a marker to reflect the degree of intrahepatic angiogenesis, may reflect the degree of PH. We speculate that the increased synthesization of vWF from large vessels’ endothelial cells and capillarity of the sinusoids in liver tissue was released into patients’ plasma and therefore induce the increased levels of plasma vWF. However, early study reported that elevated vWF levels might be a consequence of a reduced clearance of vWF, resulting from decreased expression or activity of the vWF cleaving protease (ADAMTS13) in cirrhotic patients [52]. At the same time, investigators indicated that ADAMTS13 activity and concentration were highly variable in patients with liver disease [53]. In brief, increased plasma vWF in cirrhosis maybe not only come from decreased vWF clearance, but also from increased vWF production. Further works are needed to study the mechanism of evaluated levels of vWF in liver tissues and in plasma in cirrhotic patients.
vWF as a noninvasive predictor of PH and EV Declaration of interest: The authors do not have a commercial or other association with pharmaceutical companies or other parties that might pose a conflict of interest.
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