Autologous Peripheral Blood Stem Cell Transplantation Improves Portal Hemodynamics in Patients with Hepatitis B Virus-related Decompensated Cirrhosis

1. Background

Chronic hepatitis B virus (HBV) infection may eventually lead to decompensated liver cirrhosis, which is a terminal illness. Decompensated cirrhosis is characterized by a number of complications, including ascites, hepatorenal syndrome, and upper gastrointestinal bleeding. These complications develop when the subject suffers from liver dysfunction that causes portal hypertension. Since the incidence of these complications is high, the disease is associated with a poor prognosis (1). Thus, cirrhosis requires medical therapy, which is mostly directed at relief of its complications. However, the efficacy of medical treatment, particularly for cirrhotic portal hypertension, is poor (2, 3). One of the major treatments for cirrhosis is the use of the nucleoside analogues of anti-HBV to suppress viral replication (anti-viral therapy). However, this medical approach cannot effectively reverse cirrhosis and portal hypertension in a short span of time. Thus, alternative effective treatments are needed for reducing portal hypertension and/or even reversing cirrhosis.

Recently, stem cell transplantation has been developed for the treatment of cirrhosis, which has achieved good improvement of liver function in some patients (4). The most commonly used stem cells are obtained from the umbilical cord, bone marrow, the liver itself, or peripheral blood (5-7). Peripheral blood stem cells exhibit numerous advantages, including convenient source, absence of rejection, simplicity of procedure, and low cost. A previous study showed that autologous peripheral blood stem cell (APBSC) transplantation improved liver function in HBV-linked liver cirrhosis patients.6 However, whether APBSCs could attenuate portal hypertension, which is one of the major complications of liver cirrhosis, has not been investigated. The majority of studies on stem cell transplantation for the treatment of cirrhosis have observed liver function and Alb changes after transplantation.

2. Objectives

In the present study, we used APBSC transplantation to treat patients with decompensated HBV-linked cirrhosis, and explored the transplantation’s impact on portal vein hemodynamics.

3. Patients and Methods

3.1. Patients

A total of 635 patients with HBV-related decompensated cirrhosis were treated at the Department of Liver Disease, Ningbo No. 2 Hospital, Zheijiang Province, China. This experimental study was conducted from July 2011 to December 2013. The diagnosis of cirrhosis was based on the 2009 version of the Acute-on-chronic Liver Failure: Consensus Recommendations of the Asia Pacific Association for the Study of Liver (APASL) (8) and Chronic Hepatitis B: Update 2009 by the American Association for the Study of Liver Diseases.1 The study was approved by the hospital’s ethics committee.

The inclusion criteria were as follows: 1) age of 18 – 65 years; 2) treatment for more than six months with nucleoside analog anti-HBV therapy, so that HBV DNA levels were < 10³ IU/mL; and 3) no treatment with plasma, albumin, or other blood components for one month prior to enrolling in the study. The exclusion criteria included a medical history of any of the following: 1) cirrhosis caused by factors other than HBV (including hepatitis C/D virus infection, autoimmune liver disease, fatty liver, alcoholic liver disease, and genetic metabolic liver disease); 2) tumor; 3) primary or secondary renal disease or receiving renal replacement therapy; 4) severe cardiopulmonary disease or hemodynamic instability; 5) severe infection; 6) active bleeding; 7) coma; or 8) blood diseases. After applying the inclusion and exclusion criteria, 70 patients were included in the study. Before participating, all patients signed informed consent forms.

3.2. Treatment Methods

Seventy patients that met the inclusion criteria and agreed to enter the trial were assigned serial numbers. Among these patients, 35 agreed to undergo APBSC transplantation and were placed in the transplantation group. The remaining 35 received only conventional medical therapy, forming the control group. All patients received no other drugs or other treatments affecting portal vein hemodynamics, including propranolol, and HBV DNA levels were < 10³ IU/mL during the study. In the transplantation group, two patients were lost to follow-up during the 48-week follow-up period. Therefore, data analyses were performed on only 33 subjects from the transplantation group. Three days after receiving written consent from the patients, we began conducting this experimental study, and that time-point was used as the baseline.

Before undergoing APBSC transplantation, the patients received subcutaneous injections of granulocyte colony-stimulating factor (G-CSF) for four days at a dosage of 400 μg/day. The APBSCs were harvested on the second day after the fourth daily G-CSF injection by isolating 50 mL of stem cells from the peripheral blood using a blood cell separator. The CD34⁺ cell count was determined using flow cytometry. Purified stem cells (2 – 4 × 10⁷) were administered to the hepatic artery via transfemoral catheterization within 2 h of purification. Both groups of patients received conventional medical treatment with nucleoside analogs administered in the form of entecavir (0.5 mg qd). All patients had received antiviral therapy for more than six months before they were enrolled in the study. The patients were also given silymarin and polyene phosphatidylcholine during the study to ensure liver protection. Thus, they received symptomatic and supportive treatment; however, no plasma, albumin, or other blood products were used during this period.

3.3. Detection Methods, Instruments, and Reagents

G-CSF was purchased from Kyowa Hakko Kirin Co., Ltd. (Shanghai, China). The COM.TEC blood cell separation instrument was purchased from Fresenius Kabi AG (Shanghai, China). The automatic biochemical analyzer and reagents were purchased from Beckman Coulter Commercial Enterprise Co., Ltd. (Beijing, China). The X300PE color Doppler ultrasound diagnostic apparatus was purchased from Siemens, Ltd. (Beijing, China). Before performing the test to assess portal hemodynamics, patients observed a fasting period of 8 hours. They were then placed in the supine position and asked to breathe quietly. Using the Siemens ×300PE color Doppler ultrasonic diagnostic apparatus, we measured the indices of portal hemodynamics, including the diameter of the portal vein (DPV), portal vein peak blood flow (PV_max), and mean portal vein blood flow velocity (PV_mean). While carrying out this procedure, the apparatus was operated at a probe frequency of 3.5 MHz. To determine liver and kidney function, blood coagulation, and HBV DNA levels, all of the patients underwent routine blood tests. The patients in both groups were observed during a follow-up period of at least 48 weeks. Before (time 0, baseline) performing the transplantation, we observed the patients’ clinical symptoms, and at 12, 24, 36, and 48 weeks after undergoing APBSC transplantation, we recorded any changes in these symptoms. We also noted changes in total bilirubin (TBil), albumin (Alb), prothrombin activity (PTA), spleen length and thickness, DPV, PV_max, and PV_mean.

3.4. Statistical Analysis

EPI DATA software (China Center for Disease Control and Prevention, Chinese version, Beijing, China) was used for data entry and verification. SPSS statistical software version 13.0 (IBM Corp., Beijing, China) was used for data processing and analysis. The measured data was expressed in terms of mean ± standard error of the mean (SEM). The paired-sample t test was used for performing intragroup comparisons, and the independent-sample t test was conducted for comparisons among groups. Homoscedasticity and normality tests were conducted; t test was performed to assess homoscedasticity, while a non-parametric test was performed for identifying any abnormal distributions of the data. The x² test was used to perform ratio comparisons. P < 0.05 was considered to be statistically significant.

4. Results

4.1. Comparison Between Groups of Demographic and Basic Clinical Characteristics at Baseline

Two patients were lost to follow-up because they moved to other areas, so only their 0-week data was collected during the 48-week follow-up period. Consequently, 68 patients were included in the study, with a median age of 49.85 ± 10.77 years (range 29 – 69 years). The 33 patients in the treatment group (20 males and 13 females) had a median age of 49.48 ± 11.07 years, and the 35 patients in the control group (23 males and 12 females) had a median age of 50.20 ± 10.64 years. Between the two groups, there were no significant differences before treatment in their demographic and basic clinical characteristics, such as age, sex, weight, TBil, Alb, PTA, spleen length and thickness, DPV, PV_max, PV_mean, or other indicators (P > 0.05) (Table 1).

4.2. Biochemical Index and MELD Score Changes after APBSC Transplantation

There was no significant difference in TBil before and after transplantation or between the transplantation and control groups. Before transplantation, Alb was 30.10 ± 3.75 g/L, which significantly increased to 32.82 ± 5.10 g/L, 34.02 ± 3.60 g/L, and 34.70 ± 3.80 g/L at 24, 36, and 48 weeks after transplantation, respectively (t = -2.79, -5.63, and -6.66, respectively; P < 0.05). The differences in Alb were also significant compared with the control group at the same time-points (t = 2.68, 2.93, and 3.61, respectively; P < 0.01). PTA also increased significantly to 53.39 ± 9.20%, 55.58 ± 9.53%, 57.39±7.44%, and 58.15 ± 8.11% at 12, 24, 36, and 48 weeks after transplantation, respectively (t = -2.53, -3.22, -5.73, and -6.30; P < 0.05). In the transplantation group, the increased parameters at each time-point were significantly different from the control group (t = -2.17, 2.95 and 3.78, respectively; P < 0.05). At 48 weeks after transplantation, the Model for End-stage Liver Disease (MELD) score was 11.30 ± 3.08 in the transplantation group. These values were significantly different from those observed before transplantation in the experimental group (t = -2.99, P < 0.01). The MELD scores for the transplantation group were also significantly different from those of the control group (t = 2.43, P < 0.05) (Table 2).

4.3. Reduced Spleen Length After APBSC Transplantation

Before transplantation, there was no significant difference in spleen thickness between the transplantation and control groups (P > 0.05). However, eight weeks after the transplantation, spleen length decreased from 149.36 ± 24.56 mm to 139.55 ± 24.12 mm in the transplantation group (t = -2.21, P < 0.05), which was significantly different from the spleen length of 151.66 ± 19.76 mm that was observed in the control group (t = -2.27, P < 0.05) (Table 3).

4.4. Improved Portal Vein Hemodynamics after APBSC Transplantation

At 36 and 48 weeks after transplantation, DPV measurements in the transplant group were 11.42 ± 1.30 mm and 11.21 ± 1.05 mm, respectively. These values were significantly different from those observed before transplantation in this group (t = 2.26 and 3.44, P < 0.05). The DPV values were also significantly different from those of the control group (t = 2.70 and 3.25, P < 0.05). At 36 and 48 weeks after transplantation, PV_max values were 20.43 ± 2.42 cm/s and 20.03 ± 2.51 cm/s, respectively, which were significantly different from 18.61 ± 3.22 cm/s, observed before transplantation (t = -3.24, P < 0.01; t = -2.35, P < 0.05); these DPV values also significantly differed from those of the control group (t = 2.12, P < 0.05; t=2.37, P < 0.05). At 36 and 48 weeks after transplantation, PV_mean values were 17.54 ± 3.16 cm/s and 17.84 ± 3.40 cm/s, respectively, differing significantly from the PV_mean value of 15.78 ± 3.41 cm/s that was observed before transplantation in the experimental group (t = -2.28, P < 0.05; t = -2.54, P < 0.05). These values were also different from those observed in the control group (t = 2.57, P < 0.05; t = 2.60, P < 0.05) (Table 4).

4.5. Changes in Clinical Symptoms, Signs, and Child-Turcotte-Pugh (CTP) Score

There were no deaths during this experiment, and no fever, bleeding, or other adverse events were detected in these patients. Moreover, according to the results of the B-ultrasound examinations, no signs of hepatic carcinoma were detected in any patient after successfully undergoing transplantation. There were no statistical differences in CTP scores before transplantation between the two groups (P > 0.05) (Table 1). At 48 weeks after the transplantation, CTP scores decreased from 8.88 ± 1.27 to 7.67 ± 0.99 in the transplantation group (t = 6.80, P < 0.01), differing significantly from the CTP scores of 9.09 ± 2.59 observed in the control group (t = -3.00, P < 0.01).

5. Discussion

In this study, we performed APBSC transplantation to treat patients with HBV-associated liver cirrhosis. After the operation, the patients were followed for 48 weeks. During the follow-up sessions, we found improvements in liver function and portal vein hemodynamics in the patients who had undergone transplantation. The spleen size and portal vein diameter reduced after transplantation; the blood flow in the portal vein also improved gradually, a remarkable achievement that we consistently observed during the follow-up period of 48 weeks. Thus, our findings indicate that it is advantageous to carry out APBSC transplantation in patients diagnosed with HBV-linked decompensated cirrhosis. Although several previous studies have described how liver cirrhosis can be treated with stem cell transplantation, those patients showed elevated levels of serum albumin after undergoing transplantation (9-11). However, one of the limitations of these previous studies was that the follow-up period was short (usually 12 – 24 weeks), and the transplantation was performed on a small number of patients. In our study, we also found that the level of circulating albumin increased significantly in patients subjected to APBSC treatment; however, these elevated levels lasted for up to 48 weeks postoperatively. In addition, in the APBSC treatment group, the prothrombin levels were significantly higher than in the control group. Since circulating albumin and prothrombin are sensitive indicators of hepatic function, an increase in the levels of these two proteins indicates an improvement in liver function. This finding agreed with the results of a previous report that described the use of APBSCs to treat liver cirrhosis (6).

Very few studies have investigated portal vein hemodynamics. Moreover, these studies were conducted for shorter time durations and used samples of smaller sizes (12). However, we have not come across any report that described the hemodynamic parameters of patients who underwent APBSC transplantation.

Activated hepatic stellate cells play an important role in the occurrence and development of liver fibrosis, which causes increased resistance to blood flow in the portal vein. In addition, increased serum NO, glucagon, H₂S, and other factors may also induce portal vein vasoconstriction, thus contributing to the development of portal hypertension (13, 14). Portal hypertension is also associated with a higher incidence of complications and a poor prognosis. Thus, it is a major challenge that needs to be addressed when treating decompensated cirrhosis with our current approach. We have proven that APBSC transplantation improves portal vein hemodynamics, as we achieved decreased spleen size and DPV postoperatively. Thus, the blood flow rate in the portal vein was increased postoperatively. The potential mechanisms involved in this improvement are elaborated as follows: 1) the transplanted stem cells differentiate into liver cells, which replace/repair the damaged dysfunctional cells; 2) a paracrine mechanism promotes endogenous regeneration of hepatocytes and enhances immunomodulatory and antifibrotic activity (15, 16); 3) stem cell transplantation triggers apoptosis of hepatic stellate cells and ameliorates liver fibrosis (17); and 4) as shown in a previous study, APBSC transplantation can regulate serum NO, glucagon, H₂S, and other factors (need reference). Thus, APBSC transplantation is beneficial for portal venous hemodynamics via multiple mechanisms.

In this study, after performing stem cell transplantation, we observed that serum levels of PTA and Alb were significantly increased at 12 and 24 weeks, respectively. Subsequently, within 36 weeks after transplantation, portal hemodynamics showed substantial improvement, with reduced spleen length at 48 weeks. Thus, decreased spleen length and improved portal hemodynamics were observed at later stages, while improved liver function was observed within a shorter time-interval postoperatively because degradation of the extracellular matrix takes a long time (i.e., reversing liver fibrosis is a slow process). Therefore, our observations indicate that stem cell transplantation diminishes liver fibrosis and probably improves portal hemodynamics as a consequence. In our previous study, we observed an improvement in liver reserve function following the recovery of liver synthetic function (6). Although we postulate that there are several potential mechanisms through which APBSC transplantation can benefit portal hemodynamics, we do not rule out the possibility of other mechanism(s). In addition, we need to investigate how long the improved portal hemodynamics are sustained in patients who have undergone APBSC transplantation.

Pathological examination of the liver is the gold standard for determining the degree of liver fibrosis. To precisely explore whether APBSC transplantation improves liver function by reversing fibrosis, we planned to examine the liver pathology before and after treatment in both of the groups. However, since the coagulation function of patients with decompensated cirrhosis is poor, liver biopsies probably would have caused bleeding. Therefore, we did not conduct liver biopsies in this study. Similarly, although the wedged hepatic vein pressure (WHVP) of portal blood flow is the best tool for identifying improvements in portal pressure, the risk of bleeding is quite high (18-20). Therefore, we used an indirect method (B-ultrasound) to assess portal hemodynamics in this study. Furthermore, B-ultrasound is able to accurately evaluate changes in the blood flow of the portal vein system, including DPV and PV_mean, so it is widely used as a simple and reliable means for the appraisal of portal hemodynamics.10 However, a limitation of our study was the fact that we neither performed liver biopsy nor did we measure WHVP.

Currently, poor efficacy is observed with the drugs used for the treatment of portal hemodynamic abnormalities arising from liver cirrhosis. In this scenario, APBSC transplantation offers an alternative and efficient way to improve portal hemodynamics, relieve portal hypertension, and reduce the complications associated with portal hypertension. Thus, given its numerous benefits and easy operability, APBSC transplantation may be used for treating liver cirrhosis, and it is a promising candidate that can be adapted in clinical practice.