Preventive effects of autologous bone marrow mononuclear cell implantation on intrahepatic ischemic-type biliary lesion in rabbits

Zhao-Wei Qu, Da-Zhi Chen, Qin-Song Sheng, Ren Lang, Qiang He and Ming-Feng Wang

Beijing, China

Original Article / Transplantation

Preventive effects of autologous bone marrow mononuclear cell implantation on intrahepatic ischemic-type biliary lesion in rabbits

Zhao-Wei Qu, Da-Zhi Chen, Qin-Song Sheng, Ren Lang, Qiang He and Ming-Feng Wang

Beijing, China

BACKGROUND:The ischemic-type biliary lesion (ITBL) is one of the most serious biliary complications of liver transplantation. This study aimed to investigate the effects of autologous bone marrow mononuclear cell (BM-MNC) implantation on neovascularization and the prevention of intrahepatic ITBL in a rabbit model.

METHODS:The rabbits were divided into control, experimental model, and cell implantation groups, with 10 in each group. The model of intrahepatic ITBL was established by clamping the hepatic artery and common bile duct. Autologous BMMNCs were isolated from the tibial plateau by density gradient centrifugation and were implanted through the common hepatic artery. Changes in such biochemical markers as aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyltranspeptidase, total bilirubin and direct bilirubin were measured. Four weeks after operation, cholangiography, histopathological manifestations, differentiation of BM-MNCs, microvessel density and the expression of vascular endothelial growth factor were assessed.

RESULTS:Compared with the experimental model group, the BM-MNC implantation group showed superiority in the time to recover normal biochemistry. The microvessel density and vascular endothelial growth factor expression of the implantation group were significantly higher than those of the control and experimental model groups. The ITBL in the experimental model group was more severe than that in the implantation group and fewer new capillary blood vessels occurred around it.

CONCLUSIONS:Implanted autologous BM-MNCs can differentiate into vascular endothelial cells, promote neovascularization and improve the blood supply to the ischemic bile duct, and this provides a new way to diminish or prevent intrahepatic ITBL after liver transplantation.

(Hepatobiliary Pancreat Dis Int 2010; 9: 593-599)

bone marrow mononuclear cell; intrahepatic ischemic-type biliary lesion; liver transplantation; bile duct; angiogenesis

Introduction

Ischemic-type biliary lesion (ITBL) is one of the most serious biliary complications of liver transplantation, with an incidence varying between 5% and 15%.[1]It may lead to intra- and extra-hepatic biliary stricture, bile duct injury and late graft loss, and is one of the reasons for retransplantation.[1,2]Bone marrow mononuclear cells (BM-MNCs) are one of the components in bone marrow, which contains various cell lineages, such as hematopoietic stem cells, mesenchymal stem cells and endothelial progenitor cells. The therapeutic availability of BM-MNCs has been reported in animal models and humans with ischemic disease.[3]

We have established an animal model of intrahepatic ITBL in rabbits.[4]In the present study, the effects of autologous BM-MNC implantation on neovascularization and intrahepatic ITBL in this model were explored.

Methods

Animals and groups

Thirty male or female Japanese white rabbits (weighing 2.0-2.5 kg) were selected from the Institute of Laboratory Animal Science, Capital Medical University (Beijing, China). All of the rabbits were kept in a temperature-controlled environment (18-23 ℃) with a 12-hour lightdark cycle and provided food and waterad libitum. Animal care and experimental procedures were carried out strictly in accordance with the Guide for the Care and Use of Laboratory Animals (NIH, 1996) and the ethical regulations of our university. After a 1-week period of adaptation to the facilities, the rabbits were divided randomly into three groups (10 rabbits per group): control (group A), experimental model (group B) and BM-MNC implantation (group C). In group A, the liver was isolated from all vascular supply except for the main hepatic artery, the extra-hepatic peribiliary plexus and the portal vein. In group B, the models were established according to our recently described method:[4]the hepatic artery and common bile duct were clamped with microvascular clips for 2 hours, and conventional therapy was provided after operation. The animals in group C were treated by transplanting BM-MNCs (108cells) through the common hepatic artery after reperfusion as in group B.

Preparation of BM-MNCs for autologous transplantation

Under general anesthesia, bone marrow cells (6-8 ml) were aspirated from the tibial plateau. BM-MNCs were isolated by density gradient centrifugation over Lymphoprep (Nycomed, Oslo, Norway) and labeled with a red fluorescent cell linker (PKH26, Sigma-Aldrich, USA). More than 95% of the cells were viable according to the trypan blue exclusion test and the fluorescence intensity was observed under an epi-fluorescence microscope.

Observation indices

The animals in each group were maintained for 4 weeks after operation, and the survival rates in each group were recorded.

Liver function was monitored at different time points up to 4 weeks: pre-operatively, and on day 1 and at weeks 1, 2, 3 and 4 post-operation. Serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), total bilirubin (TBIL) and direct bilirubin (DBIL) were measured using standard analytical methods.

Four weeks after operation, all the rabbits in the 3 groups underwent cholangiography. Under routine anesthesia, the abdominal cavity was opened at the former incision. Three to five ml of 30% meglumine diatrizoate solution was injected slowly through the distal common bile duct, and an X-ray was taken.

After cholangiography, liver tissue samples from the hepatic hilum were collected and stained with hematoxylin and eosin for histopathological examination. Bile duct injury in biopsies at week 4 was semiquantified by calculating the bile duct injury severity score (BDISS),[5]based on the following three components: bile duct damage (graded as 0, absent; 1, mild; 2, moderate; or 3, severe, modified from the Banff criteria for acute rejection); ductular proliferation (graded 0-3, using a similar scale as above); and cholestasis (graded 0-3, using a similar scale as above). To detect the BM-MNC-derived cells labeled with red fluorescence by epi-fluorescence microscopy, serial 4-μm sections of formalin-fixed, paraffin-embedded liver tissues were treated with mouse anti-factor VIII antibody (Beijing Biosynthesis Biotechnology, Beijing, China), followed by incubation with FITC-conjugated secondary antisera (Beijing Biosynthesis Biotechnology, Beijing, China). New vessels were stained by immunohistochemistry using an SP kit (Beijing Biosynthesis Biotechnology, Beijing, China). Mouse anti-factor VIII antibody was used as the first antibody. Microvessel density (MVD) was measured by 2 pathologists who were unaware of the experiment. After the endothelial cells lining the microvessels had been highlighted with anti-factor VIII-related antigen/von Willebrand's factor (F8RA/vWF), the five most intense vascular areas (hotspots) were subjectively selected from each section at the original magnification ×40. The capillaries were counted at the magnification ×200[6]and the mean value was taken for further analysis. To assess the expression of vascular endothelial growth factor (VEGF), mouse anti-VEGF antibody (Beijing Biosynthesis Biotechnology, Beijing, China) was used as the first antibody for SP immunohistochemical staining. The expression was evaluated by Image-Pro Plus 6.0 software. Five typical areas were selected and their integrated optical density (IOD) values were measured.

Statistical analysis

Quantitative data are shown as mean±SD. Onefactor analysis of variance was used for statistical analysis. All statistical analysis was performed with SPSS 13.00 software (Chicago, IL., USA). APvalue less than 0.05 was considered statistically significant.

Results

Survival rates

The animals in group A lived throughout the period of the experiment with a survival rate of 100%. One animal died from intraperitoneal infection on day 7 after operation in group B, and one from abdominal hemorrhage on day 2 after operation in group C. The survival rates were each 90%.

Biochemical parameters

No biochemical abnormality was found in group A. All the biochemical parameters (ALT, AST, ALP, GGT, TBIL and DBIL) in groups B and C increased to different degrees after operation. They reached a peak on day 1, and then decreased gradually. At week 3 , all parameters returned to normal in group C, but, in group B, the ALP and GGT were significantly higher than in group C at weeks 3 and 4 (P<0.05) (Table 1).

Cholangiography

The cholangiograms of group A showed that the intrahepatic bile duct was normal and there was no intrahepatic biliary injury. However, biliary injury was found in group B, 80% of the cholangiograms showing lesions. In group C, 60% of the intrahepatic bile duct lesions were alleviated (Fig. 1).

Histopathology

Pathological examination revealed that the morphology of the intrahepatic bile duct epithelial cells was normal and there was no cell necrosis in group A. Epithelial cells were clearly damaged and sloughed into the bile duct lumen in group B, but in group C, the damage was alleviated and only a few cells sloughed; the BDISS was significantly lower than that in group B (Fig. 2, Table 2).

Differentiation of BM-MNCs

No fluorescence was observed in groups A and B at the excitation wavelength of 551 nm, but red fluorescence was found in group C. When the excitation wavelength was 525 nm, green fluorescence was observed in groups A, B and C. Color images of merged green (fluorescein isothiocyanate, FITC) and red (PKH26) fluorescence signals showed significant colocalization (yellow), indicating that some cells were positive for both (Fig. 3).

Angiogenesis

New vessels clearly increased in groups B and C, and most of them were distributed around the bile duct. Thenew vessels were more numerous in groups B and C than in group A, and there were significantly more in group C than in group B (Table 3, Fig. 4).

Table 1. Biochemical parameters in all groups at each observation time (mean±SD)

Fig. 1. Cholangiograms from the 3 groups at week 4 after operation. A (group A): The intrahepatic bile duct was normal. No biliary stricture or dilation was observed; B (group B): Some branches of the intrahepatic bile duct appeared loose, with the "pearl-bunch shape" manifestation in part of them; C (group C): Intrahepatic bile duct lesions were not apparent. The intrahepatic bile duct was similar to normal.

Fig. 2. Histopathological changes of the bile duct in each group at week 4 after operation (HE, original magnification ×200). A (group A): The morphology of the intrahepatic bile duct was normal. No epithelial cell necrosis was observed; B (group B): The epithelial cells were damaged and some were necrotic and sloughed into the lumen; C (group C): The morphology of the intrahepatic bile duct was almost normal and better than in group B; the epithelial cell damage was slight.

Fig. 3. Differentiation of BM-MNCs in group C. A: PKH26 emits red fluorescence at 551 nm; B: Fluorescein isothiocyanate emits green fluorescence at 525 nm; C: The merged image showed cells positive for both.

Table 2. Bile duct injury severity score of each group

Table 3. MVD of each group

Fig. 4. MVD of each group at week 4 after operation (SP, original magnification ×200). A (group A): The neovascularization was less; B (group B): The new blood vessels were increased, and the cavities were irregular; C (group C): Many new blood vessels surrounded the bile duct, and the numbers clearly increased compared with the other groups.

Fig. 5. Expression of VEGF in each group at week 4 after operation (SP, original magnification ×200). A: In group A, VEGF was expressed around the biliary tract and the staining was light; B: In group B, the expression was increased and the staining was deeper; C: In group C, besides the biliary tract, the VEGF had distributed to hepatic tissue.

Table 4. IOD value of VEGF in each group

Expression of VEGF

VEGF expression progressively increased from group A to group C. It was significantly higher in groups B and C than in group A, and significantly higher in group C than in group B. The expression in group C was more broadly distributed than in the other groups (Table 4, Fig. 5).

Discussion

ITBL is one of the most common biliary complications after liver transplantation, and its incidence is mainly associated with prolonged ischemic times, reperfusion injury, disturbance in blood flow through the peribiliary vascular plexus, immunologically induced injury, and cytotoxic injury.[7,8]So, ischemic injury is of vital importance for the occurrence of ITBL.[9,10]The patient prognosis and graft survival are poor for type II (intrahepatic) and type III (intra- and extrahepatic),[2]and especially for multiple and diffuse ITBL. Therefore, a significant number of patients need a re-transplant.[11]With the development of studies on the bile ducts and peribiliary microcirculation, several reports show that the intrahepatic bile duct is supplied by the arterial rete formed by hepatic arterial branches and peribiliary blood plexus.[12,13]When the hemodynamics of blood vessels is abnormal, ischemic necrosis of biliary ductal cells is associated with ITBL.[14]

Angiogenesis, the formation of new blood vessels from the existing vascular network, is a complicated process, which may be affected by many elements such as the secretion of vascular growth factor and proliferation of vascular endothelial cells. The BMMNCs are a population of primary cells with multiple differentiation potential. In recent years, implantation of BM-MNCs has been widely used in the treatment of ischemic diseases.[15-18]In our research, the PKH26-labeled autologous BM-MNCs distributed in liver tissues, and 4 weeks later, some cells expressed vWF. These data suggest that BM-MNCs can differentiate into vascular endothelial cells. In addition, the MVD value in group C was significantly higher than that in group B, suggesting that the implanted BM-MNCs promoted new capillary hyperplasia, improving biliary microcirculation ischemia and alleviating ITBL.

VEGF is a specific angiogenic factor, also known as vascular permeability factor and vasculotropin. It is closely associated with ischemic diseases.[19]Many factors are associated with VEGF expression and anoxia is the most effective. Meanwhile, a previous study[20]showed that expression of the VEGF receptors KDR/ Flk and Flt are up-regulated under hypoxia, but the exact pathophysiological mechanism is not clear. Our study showed that, in group A, the VEGF expression was mainly concentrated around the biliary tract, and it was significantly higher in group B than in group A, indicating that ischemia-hypoxia may promote its expression, thus promoting capillary proliferation in the ischemic area. In group C, VEGF expression increased significantly, which may have been a result of BM-MNC implantation. In this condition, BM-MNCs can secrete a broad spectrum of cytokines (including VEGF) to improve collateral vessel formation[21]or promote other cells to secrete it by a paracrine mechanism.[3,22,23]

Accordingly, the results indicated that the mechanism of promoting neovascularization and improving microcirculation mainly consists of two parts: 1) BM-MNCs are mainly mesenchymal stem cells, hematopoietic stem cells and endothelial progenitor cells. In situations such as ischemia, the cells can differentiate into vascular endothelial cells and vascular smooth muscle cells and promote angiogenesis. 2) The capacity of BM-MNCs to release cytokines and growth factors is probably by autocrine and paracrine mechanisms, thereby activating vascular endothelial cells to induce neovascularization.

The results of liver function tests in group B showed both serum ALP and GGT recovered slowly after operation and the values were higher than in group A at week 4, suggesting that the effects of ITBL are mainly focused on biliary epithelia and have little effect on hepatocytes. In group C, the indices recovered to normal levels at week 4, suggesting that the implanted BM-MNCs effectively prevented ITBL and the onset might be at week 3 after implantation. In addition, compared with the imaging and histopathological findings in group B, the lesion and the manifestations in group C were similar to those in group A. These features confirmed the preventive effects of BMMNCs implantation on rabbit intrahepatic ITBL at the macroscopic and microscopic levels. Furthermore, the implanted cells began to exert effects at weeks 3-4 after operation, which is earlier than the appearance of biliary stricture.[9]Besides that, the implanted cells in this experiment were autologous BM-MNCs, which can prevent transplant rejection.

In conclusion, the implantation of autologous BMMNCs can promote neovascularization and improve blood supply to the ischemic bile duct to diminish or prevent intrahepatic ITBL. This method may constitute a novel strategy to prevent and treat intrahepatic ITBL after liver transplantation.

Funding:None.

Ethical approval:This experimental animal study was approved by the Animal Care Ethics Committee of Capital Medical University and conducted in accordance with the Guide for the Care and Use of Laboratory Animals.

Contributors:QZW proposed the study and wrote the first draft. CDZ and SQS participated in the design of the study and performed the operations. LR, HQ and WMF participated adequately in the study by substantial contributions to the reference search and data analysis. QZW is the guarantor.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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February 5, 2010

Accepted after revision June 24, 2010

Author Affiliations: Department of Hepatobiliary and Pancreaticosplenic Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China (Qu ZW, Chen DZ, Sheng QS, Lang R, He Q and Wang MF); Department of Hepatobiliary Surgery, Tumor Hospital of Harbin Medical University, Harbin 150086, China (Qu ZW); Department of Colorectal and Anal Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Sheng QS)

Da-Zhi Chen, MD, Department of Hepatobiliary and Pancreaticosplenic Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China (Tel: 86-10-85231503; Fax: 86-10-85231503; Email: chen-dazhi@hotmail.com)

? 2010, Hepatobiliary Pancreat Dis Int. All rights reserved.