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RhGH Attenuates Ischemia Injury of Intrahepatic Bile Ducts Relating to Liver Transplantation
Journal of Surgical Research 171, 300–310 (2011) doi:10.1016/j.jss.2010.02.003
RhGH Attenuates Ischemia Injury of Intrahepatic Bile Ducts Relating to Liver Transplantation Zheng Wang, M.D., Jie Zhou, M.M.,1 Jianhua Lin, M.D., Yu Wang, M.D., Yixiong Lin, M.D., and Xianghong Li, M.D. Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China Submitted for publication September 11, 2009
Background. To study the effect of rhGH administration on intrahepatic cholangiocytes relating to liver transplantation with ischemia of hepatic artery, and ultimately, clarify pathologic mechanism of the injury. Methods. Rat orthotopic autologous liver transplantation was performed first. Three hours later, the rats were grouped as followed: HAL (hepatic artery ligation) group; HAL D rhGH (hepatic artery ligation followed by rhGH administration) group; CON (without hepatic artery ligation) group. Specimen was collected after 7 d. ALT and ALP of serum were measured. The pathologic changes of bile ducts of liver tissue were observed. The number of bile ducts and blood vessels in portal area were counted. Immunochemistry for VEGF, VEGFR-2, VEGFR-3, GHR, and IGF-1R of intrahepatic cholangiocytes was performed. Cholangiocytes apoptosis was evaluated by TUNEL analysis. Cholangiocytes proliferation was evaluated by PCNA immunolabeling. Results. ALT and ALP of HAL D rhGH group were significantly ameliorated compared with untreated animals (P < 0.05). ALT and ALP of HAL group were significantly higher compared with CON group (P < 0.05). In HAL group, the main injury of bile ducts was not reversible, whereas it was reversible in CON and rhGH groups. In HAL group, the number of bile ducts in portal area decreased, while the number of bile ducts not accompanying blood vessels increased (P < 0.05). In rhGH group, the number of bile ducts in portal area increased, while the number of bile ducts accompanying blood vessels increased compared with HAL group (P < 0.05). The expression of VEGF, VEGFR-2, VEGFR-3, GHR, and IGF-1R was significantly lower 1 To whom correspondence and reprint requests should be addressed at Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou 510515, China. E-mail: [email protected].
0022-4804/$36.00 Ó 2011 Elsevier Inc. All rights reserved.
in HAL group than in CON group (P < 0.05). Following administration of rhGH to HAL rats, the expression of VEGF, VEGFR-2, VEGFR-3, IGF-1R, and GHR was significantly higher (P < 0.05). Administration of rhGH prevented increase in cholangiocytes apoptosis induced by HAL (P < 0.05). Administration of rhGH promoted increase in cholangiocytes proliferation held by HAL (P < 0.05). Conclusions. Administration of rhGH appears to attenuate ischemia injury of intrahepatic bile ducts relating to liver transplantation. This function is partly related to the capacity that rhGH inhibits the apoptosis of intrahepatic cholangiocytes and prompts the proliferation and angiogenesis by increasing the expression of VEGF, VEGFR2, VEGFR3, GHR, and IGF1-R. Ó 2011 Elsevier Inc. All rights reserved. Key Words: liver transplantation; hepatic artery; ischemia; intrahepatic bile duct; rhGH.
INTRODUCTION
In the past 40 y or more, liver transplantation has been an established treatment for many end-stage liver diseases. Although surgical techniques, postoperative management, and especially graft preservation methods have greatly improved in recent years, clinical research has demonstrated that biliary tract complication remain highly, 7% to 30% of patients have complication after transplantation, and mortality is 6% to 12.5%, which remains one of the main obstacles in successful transplantation. It should be pointed out that with the improvement in surgical techniques, the incidence of anastomotic biliary strictures decreased remarkably, whereas the nonanastomotic biliary strictures and dilatations became the major type of biliary complications of liver allograft, which
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had been called the Achilles’ heel revisited [1]. Nonanastomotic biliary strictures, which have also been called ischemic cholangiopathy (IC) [2], is characterized by biliary strictures and dilatations that involve the whole graft biliary tree and is defined as a focal or extensive changes to bile ducts because of impaired blood supply, ischemia-reperfusion injury, infection, or acute/chronic rejections[3]. IC is a complication that appears early during the immediate postoperative period. The incidence ranges from 11% to 19% and is related to a substantial cause of morbidity (50%–75%), characterized by the presence of repeated episodes of cholangitis and the necessity for retransplantation (35%–40%). A significant rate of mortality (25%–40%) and increased cost are also associated with this complication [4, 5]. The causes of nonanastomotic biliary stricture are multifactorial. Impaired blood supply of the biliary epithelia is considered to be one of the major causes. It is well-known that cholangiocytes proliferation is a compensation for bile duct injury. GH has direct effect on different kinds of tissues or organs, and also stimulates IGF-1 hepatic production to induce protein synthesis and cell mitosis [6]. Actually, evidence has been accumulated that GH is involved in liver regeneration [7]. Recently, Alvaro et al. pointed out, VEGF modulated cholangiocytes proliferation through the same signal pathway as IGF-1 did [8]. In light of the above-mentioned findings, the present study aimed to prove the hypothesis that administration of rhGH (recombinant human growth hormone) offers intrahepatic biliary epithelia protective effect against IC injury in rats and explore its mechanism. MATERIALS AND METHODS Animals, Drug, and Materials Adult male Wistar rats weighing 200 6 20 g were obtained from Southern Medical University Animal Center, Guangzhou,China. All animals received humane care in compliance with European Convention on Animal Care. The following experimental protocol was approved by the Southern Medical University Animal Care and Use Committee. The rats were housed and fed at the Animal Center of Nanfang Hospital at least 7 d before surgery to acclimate them to the environment. The rhGH was purchased from Shanghai United Cell Biotechnology Co. Ltd. (Shanghai, China). Infrahepatic venacaval endo-canal was made up of joining tube of no. 9 venous puncture needle. The length was 3.3 cm, and the diameter was 1.8 mm. Portalcervical bypass cannula was made up of joining tube of no. 7 venous puncture needle. The length was 10 cm, and the diameter was 1.5 mm. The canales were irrigated with 0.5% heparin before the operation.
Surgical Procedures An orthotopic autologous liver transplantation model was established, imitating the models created by Zhao and Zhou [9] and Luo et al. [10], which simulates the whole process of liver transplantation, simple and with high successful rate, better reflects the pathophysiologic process of bile ducts, provides an approach for investigation of
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bile ducts injury concerned with liver transplantation. It was described as follows: The rats were anesthetized by intraperitoneal injection of sodium pentobarbital 30 mg/kg body weight. The animal was fixed with a self-made operation cushion, placed supinely. An incision of right cervix was performed. The right external jugular vein was dissociated; thereafter the self-made portal-cervical bypass cannula was inserted into the external jugular vein and blocked temporarily with a vascular clamp. A middle incision of inverse ‘‘T’’ shape was made on the abdomen. The ligaments around the liver were dissociated and severed. The left diaphragm vein, hepatoesophageal ligament vein and right renal vein were separated, ligated and severed. The liver was turned left, the suprahepatic venae cavae (SVC) was anatomized; then the common bile duct, hepatic artery, and portal vein were anatomized over the margin of the duodenal bulb. The infrahepatic venae cavae (IVC) was dissociated downwards about 6–8 mm. The liver was dissociated completely, except the hepatic blood vessels in and out, and the common bile duct. An incision of about 3 mm was made in the IVC above right renal vein for inserting venacaval cannula completely embedded into the vena cava. The upper end of the cannula went beyond over the upper margin of the liver nearly 3 mm. The upper and lower ends of the incision in the IVC were ligated temporarily. Thereafter, the blood of the inferior vena cava flowed over into the heart through the cannula. The portal vein was dissociated. Either the portal vein or the hepatic artery was blocked temporarily with a vascular clamp. The other end of the portal-cervical bypass cannula was inserted into the centrifugal end of the portal vein, and the vascular clamp on the cannula was relaxed at once. The portal-cervical bypass came into operation to make blood of the portal vein backflow into the heart. Five u/mL heparin saline of 2–3 mL about 0–4 C was injected into the centripetal end of the portal vein to drive the blood of the liver into the systematic circulation. The SVC was ligated temporarily on the suprahepatic venacaval cannula. The ligation silk of the upper end of the incision in the IVC was relaxed. Subsequently, the liver was irrigated with combination Ringer’s solution of 0–4 C thus preserved, and the solution effused outside the cannula through the incision in the IVC. The liver had been irrigated and preserved for 2.5 h in vivo. The velocity of flow was 150 mL/h. After finishing irrigation, the inferior venacaval cannula was removed at once, and the incision in the IVC was oversewn with 11-0 suture. The portal-cervical bypass cannula was removed, and the pinhole in the portal vein was repaired. The blockage of the portal vein was relieved, followed by the blockage of the hepatic artery 30 min later. Subsequently, the circulation of the liver was resumed, while the circulation of the inferior vena cava was also resumed. At the same time, the surface of the liver was irrigated with 20 mL normal saline solution of 38 C for rapidly rewarming the liver. The end of the portal-cervical bypass cannula in the cervix was removed, and the right external jugular vein was ligated. The incision in the cervix was sutured. According to clinical practice, the time duration between the opening of portal vein and the hepatic artery was 30 min; 3 h after the operation, the hepatic artery was ligated; 7 d later, the tissues and blood were sampled.
Experimental Groups Three groups were designed as the follows: CON (control) group (n ¼ 6). The animals had the relevant operation of orthotopic autologous liver transplantation without hepatic artery ligation. HAL group (n ¼ 6). The hepatic artery was ligated after the operation. Subsequently 0.3 mL/kg/d water for injection was administered subcutaneously for 1 wk. HAL þ rhGH group (n ¼ 6). The hepatic artery was ligated after the operation. Subsequently, rhGH had been administered subcutaneously 0.3 U/kg diluted in 0.3 mL water for injection for 1 wk.
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There was no procedure-related mortality in any of the groups. After 7 d, the animals underwent re-laparotomy. Blood was collected from the inferior vena cava, and the tissue of bile ducts from the hepatic left lateral lobe was rapidly excised and preserved in 10% paraformaldehyde for further analysis, either was the tissue of the liver.
TUNEL-positive nuclei in brown were identified from negatively unstained nuclei in blue. Apoptotic cells were counted for each tissue sample under high-power magnification (3400) in a blinded fashion. Apoptotic index (AI) was used as a quantitative indicator, indicating the percentage of TUNEL-positive cholangiocytes by counting the number of positive cells in 30 random microscopic high-power fields.
Analysis of Serum Serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were measured in the serum in duplicate using commercial kits, according to the manufacturers’ protocols with Olympus AU5421 biochemistry analyzer.
Pathological Examination The tissue of bile ducts preserved in 10% paraformaldehyde was taken, cropped and made into wax pattern, thereafter sliced up, dyed with hematoxylin-eosin. Edema of cholangiocytes, infiltration of inflammatory cells, necrosis, and exfoliation of cholangiocytes were observed with high power field microscope.
Bile Ducts and Blood Vessels in Portal Area The wax pattern of liver tissue was cut into slices of 2.5 mm. continuously. The blood vessels were tagged with rabbit anti-CD34 polyclonal antibody (Beijing Biosynthesis Biotechnology Co. Ltd). In portal area, the number of bile ducts, blood vessels, bile ducts accompanying blood vessels, bile ducts not accompanying blood vessels, and isolated blood vessels were counted, respectively.
Immunohistochemical Examination for VEGF, VEGFR2, VEGFR3, GHR, and IGF-1R Immunohistochemical staining for VEGF (VEGFR2, VEGFR3, GHR, and IGF-1R) was performed with anti-VEGF (VEGFR2, VEGFR3, GHR, and IGF-1R) rabbit polyclonal antibodies (Beijing Biosynthesis Biotechnology Co. Ltd) at a dilution of 1:200 overnight at 4 C. A two-step Envision Plus Sunpoly-H III HRP rabbit/mouse kit (Shanghai Sun Biotech Co. Ltd.) was used according to the manufacturer’s instructions. DAB was used as chromogen and all sections were counterstained with hematoxylin. Following staining, sections were analyzed in a coded fashion with an Olympus U-25ND6 T2 (Diagnostic Instrument, Tokyo, Japan) and processed with an IAS image analysis system (Delta Sistemi, Delta Sistemi, Rome, Italy). The intensity and distribution of immunostaining were assessed in a coded fashion.
TUNEL Staining Part of the specimens were immediately fixed at 4 C buffered formalin (10% paraformaldehyde) and embedded in paraffin. In situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) of fragmented DNA was performed on 2.5 mm thick sections of paraffin-embeded tissue using the in situ TUNEL assay (Roche Diagnostics, Basel, Switzerland) according to the supplier’s instructions. The sections were deparaffinized in xylene and rehydrated before analysis. After treatment with proteinase K (20 mg/mL in 10 mL Tris-HCl, PH 7.6) at 37 C for 30 min, sections were washed in PBS and endogenous peroxidase activity was inhibited by immersing the slides in 3% hydrogen peroxide in methanol for 30 min. Then, the slides were treated with permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 15 min. For the labeling reaction, a TUNEL mixture containing terminal deoxynucleotidyl transferase (TdT), its buffer solution, and biotinylated dUTP was added to cover the sections, which were then incubated at 37 C for 60 min in a humidified chamber. Washed sections were incubated with peroxidase-labeled streptavidin for 30 min and then stained with diaminobenzidine, followed by counterstaining with hematoxylin.
PCNA Immunolabeling After rehydration, the silane-coated slides were treated with 0.3% H2O2 in methyl alcohol for 15 min and then briefly washed in phosphate buffer saline. They were then incubated overnight at 4 C, with a 1:100 dilution of anti-proliferating-cell nuclear antigen (PCNA) monoclonal antibody (BOSTER), and subsequently incubated with Envision Plus Sunpoly-H III HRP rabbit/mouse kit (Shanghai Sun Biotech Co. Ltd.) for 30min at 37 C. Finally, the sections were counterstained with hematoxylin and coverslipped. PCNA labeling of cholangiocytes were semi-quantitatively evaluated by counting at least 500 bile-duct cells in 20 randomly selected high-power fields (3400) and by determining the percentages of PCNA-reactive cells. Thus, PCNA labeling index was evaluated. Only bile-duct cells that exhibited strong nuclear immunostaining were considered for reactive cell counting.
Statistical Analysis All the data were expressed as mean value 6 SD. Difference between groups was analyzed by Student’s unpaired t-test when two groups were analyzed, and analysis of variance (ANOVA) when more than two groups were analyzed, followed by an appropriate post hoc test. The analysis was performed by SPSS 13.0 and P-values of less than 0.05 were accepted as statistically significant.
RESULTS Serum Analysis
ALT and ALP of HAL group were significantly higher compared with CON group (674.50 6 145.55 verus 176.50 6 33.62U/L, 790.67 6 77.96 verus 278.50 6 30.03 U/L, P < 0.05). When animals were administrated rhGH, ALT and ALP were significantly ameliorated compared with untreated animals (308.67 6 87.13 verus 674.50 6 145.55U/L, 356.50 6 30.20 verus 790.67 6 77.96U/L, P < 0.05). However, they remained higher compared with CON group (308.67 6 87.13 verus 176.50 6 33.62U/L, 356.50 6 30.20 verus TABLE 1 ALT and ALP in Each Group Group
CON
HAL
HAL þ rhGH
ALT (U/L) ALP (U/L)
176.50 6 33.62y 278.50 6 30.03y
674.50 6 145.55* 790.67 6 77.96*
308.67 6 87.13*,y 356.50 6 30.20*,y
ALT and ALP of HAL group were significantly higher compared with CON group. When animals were administered with rhGH, ALT and ALP were significantly ameliorated compared with untreated animals, however, remained significantly higher compared with CON group. * P < 0.05 versus CON group. y P < 0.05 versus HAL group.
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TABLE 2 The Number of Bile Ducts and Blood Vessels in Portal Area Group
CON
HAL
HAL þ rhGH
Bile ducts Blood vessels Bile ducts with blood vessels Bile ducts without blood vessels Isolated blood vessels
11.17 6 3.37 8.50 6 1.05 6.00 6 0.89
4.83 6 1.17* 2.67 6 1.03* 2.17 6 0.75*
7.67 6 0.52y 5.83 6 0.75*,y 4.67 6 0.52*,y
1.17 6 0.41
5.50 6 2.43*
2.50 6 0.84y
2.50 6 1.38
0.67 6 0.52
2.83 6 1.47
In HAL group, the number of bile ducts and bile ducts accompanying by blood vessels in portal area decreased, whereas they increased in HAL þ rhGH group. * P < 0.05 versus CON group. y P < 0.05 versus HAL group.
damage, which was still worse in comparison with CON group. Pathologic Changes with Hematoxylin-Eosin Dyeing
In CON group, the main injury was reversible such as edema of cholangiocytes and infiltration of inflammatory cells. In HAL group, the main injury was not reversible such as necrosis and exfoliation of cholangiocytes. In HAL þ rhGH group, the main injury was edema of cholangiocytes and infiltration of inflammatory cells, while necrosis and exfoliation of cholangiocytes were remarkably decreased (Fig. 1). Bile Ducts and Blood Vessels in Portal Area
In HAL group, the bile ducts in portal area were injured, and the number decreased compared with CON group (P < 0.05). In HAL þ rhGH group, the number of bile ducts in portal area clearly increased compared with HAL group (P < 0.05). In HAL group, the number of bile ducts accompanying blood vessels significantly decreased, while those not accompanying blood vessels significantly increased (P < 0.05). In HAL þ rhGH group, the number of bile ducts accompanying blood vessels significantly increased, while those not accompanying blood vessels significantly decreased (P < 0.05; Table 2). FIG. 1. In CON and HAL þ rhGH group, the main injury of bile ducts was edema of cholangiocytes and infiltration of inflammatory cells. In HAL group, the main injury was necrosis and exfoliation of cholangiocytes. (Color version of figure is available online.)
278.50 6 30.03 U/L, P < 0.05; Table 1). Liver function and cholestasis were evaluated with ALT and ALP, respectively. In most cases, the damage was most evident in HAL groups. Administration of rhGH alleviated the
Effects of HAL and rhGH on VEGF, VEGFR2, and VEGFR3 Expression
Immunohistochemistry in CON liver sections showed that bile ducts expressed VEGF, VEGFR2, and VEGFR3. HAL induced a decrease in the number of cholangiocytes positive for VEGF, VEGFR2, and VEGFR3 compared with liver sections from 1-wk CON rats (5.15 6 0.41 versus 14.02 6 1.62, 3.58 6 0.63 versus
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FIG. 2. Immunohistochemistry in CON liver sections showed that bile ducts expressed VEGF. HAL induced a decrease in the number of cholangiocytes positive for VEGF compared with liver sections from 1-wk CON rats. Following administration of rhGH to HAL rats, expression of VEGF was significantly higher compared with untreated animals, however, remained lower compared with CON group (3400) (see Table 3). (Color version of figure is available online.)
FIG. 3. Immunohistochemistry in CON liver sections showed that bile ducts expressed VEGFR2. HAL induced a decrease in the number of cholangiocytes positive for VEGFR2 compared with liver sections from 1-wk CON rats. Following administration of rhGH to HAL rats, expression of VEGFR2 was significantly higher compared with untreated animals, however, remained lower compared with CON group (3400) (see Table 3). (Color version of figure is available online.)
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TABLE 3 VEGF, VEGFR2, VEGFR3 Expression % Positive Cholangiocytes in Each Group Group
CON
HAL
HAL þ rhGH
VEGF VEGFR2 VEGFR3s
14.02 6 1.62y 11.22 6 1.23y 12.07 6 0.92y
5.15 6 0.41* 3.58 6 0.63* 4.75 6 0.59*
10.70 6 1.46*,y 9.08 6 1.17*,y 9.55 6 0.64*,y
Immunohistochemistry in CON liver sections showed that bile ducts expressed VEGF, VEGFR2 and VEGFR3. HAL induced a decrease in the number of cholangiocytes positive for VEGF, VEGFR2, and VEGFR3 compared with liver sections from 1-wk CON rats. Following administration of rhGH to HAL rats, expression of VEGF, VEGFR2, and VEGFR3 was significantly higher compared with untreated animals, however, remained lower compared with CON group. * P < 0.05 versus CON group. y P < 0.05 versus HAL group.
11.22 6 1.23, 4.75 6 0.59 versus 12.07 6 0.92, P < 0.05). Following administration of rhGH to HAL rats, the expression of VEGF, VEGFR2, and VEGFR3 was significantly higher compared with untreated animals (10.70 6 1.46 versus 5.15 6 0.41, 9.08 6 1.17 versus 3.58 6 0.63, 9.55 6 0.64 versus 4.75 6 0.59, P < 0.05), however, remained lower compared with CON group (10.70 6 1.46 versus 14.02 6 1.62, 9.08 6 1.17 versus 11.22 6 1.23, 9.55 6 0.64 versus 12.07 6 0.92, P < 0.05; Figs. 2, 3, and 4 and Table 3). Effects of HAL and rhGH on GHR and IGF-1R Expression
Immunohistochemistry in CON liver sections showed that bile ducts expressed GHR and IGF-1R. HAL induced a decrease in the number of cholangiocytes positive for GHR and IGF-1R compared with liver sections from 1-week CON rats (2.06 6 0.43 versus 9.94 6 0.63, 5.26 6 0.37 versus 8.99 6 0.69, P < 0.05). Following administration of rhGH to HAL rats, the expression of GHR and IGF-1R was significantly higher compared with untreated animals (7.01 6 1.61 versus 2.06 6 0.43, 7.61 6 0.25 versus 5.26 6 0.37, P < 0.05), however, they remained lower compared with CON group (7.01 6 1.61 versus 9.94 6 0.63, 7.61 6 0.25 versus 8.99 6 0.69, P < 0.05; Figs. 5, and 6 and Table 4). Cholangiocytes Apoptosis and Proliferation
FIG. 4. Immunohistochemistry in CON liver sections showed that bile ducts expressed VEGFR3. HAL induced a decrease in the number of cholangiocytes positive for VEGFR3 compared with liver sections from 1-wk CON rats. Following administration of rhGH to HAL rats, expression of VEGFR3 was significantly higher compared with untreated animals, however, remained lower when compared with CON group (3400) (see Table 3). (Color version of figure is available online.)
TUNEL analysis showed a few apoptotic bodies in the liver sections of CON rats. The number of cholangiocytes undergoing apoptosis increased in liver sections from HAL rats compared with CON rats (4.91 6 0.26 versus 1.63 6 0.52, P < 0.05). Administration of rhGH prevented the increase in cholangiocytes apoptosis induced by HAL (2.61 6 0.58 versus 4.91 6 0.26, P < 0.05; Fig. 7).
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FIG. 5. Immunohistochemistry in CON liver sections showed that bile ducts expressed GHR. HAL induced a decrease in the number of cholangiocytes positive for GHR compared with CON rats. Following administration of rhGH to HAL rats, expression of GHR was significantly higher, but remained lower compared with CON group (3400) (see Table 4). (Color version of figure is available online.)
FIG. 6. Immunohistochemistry in CON liver sections showed that bile ducts expressed IGF-1R. HAL induced a decrease in the number of cholangicytes positive for IGF-1R compared with liver sections from 1-wk CON rats. Following administration of rhGH to HAL rats, expression of IGF-1R was significantly higher compared with untreated animals, however, remained lower compared with CON group (3400) (see Table 4). (Color version of figure is available online.)
WANG ET AL.: RhGH FOR LIVER TRANSPLANTATION
TABLE 4 GHR, IGF1-R Expression % Positive Cholangiocytes in Each Group Group
CON
HAL
HAL þrhGH
GHR IGF-1R
9.94 6 0.63y 8.99 6 0.69y
2.06 6 0.43* 5.26 6 0.37*
7.01 6 1.61*,y 7.61 6 0.25*,y
Immunohistochemistry in CON liver sections showed that bile ducts expressed GHR and IGF-1R. HAL induced a decrease in the number of cholangiocytes positive for GHR and IGF-1R compared with liver sections from 1-wk CON rats. Following administration of rhGH to HAL rats, expression of GHR and IGF-1R was significantly higher compared with untreated animals, however, remained lower compared with CON group. * P < 0.05 versus CON group. y P < 0.05 versus HAL group.
Following HAL, the number of PCNA-positive cholangiocytes decreased compared with liver sections from CON rats (10.87 6 1.85 versus 22.95 6 2.08, P < 0.05). Administration of rhGH prevented inhibitory effect of HAL on the number of PCNA-positive cholangiocytes (19.03 6 1.58 versus 10.87 6 1.85, P < 0.05; Fig. 8). DISCUSSION
In the field of hepatic surgery, liver transplantation, organ preservation, or embolism and interventional treatment might destroy hepatic artery system, affect blood supply of bile ducts, and thereby damnify cholangiocytes. In the liver, the terminal arteriole of hepatic artery branches off into the peribiliary plexus (PBP), which supplies the intrahepatic bile ducts. Thereby, the changes of PBP usually result in changes of intrahepatic bile duct structure. Post-transplantational hepatic artery ischemia is the vital reason leading to bile duct complication, which induces ischemia and occlusion of PBP, ulteriorly aggravates ischemia of intrahepatic bile ducts, resulting in intrahepatic bile ducts injury [11]. In our laboratory, after the pathophysiologic progression of cold perfusion and reperfusion, the cholangiocytes were exposed to damnification of different degree. So the injury of intrahepatic cholangiocytes became worse subsequent to hepatic artery ligation. Cholangiocytes proliferation is the vital compensatory feedback for bile duct injury, because of its benefit to restore biliary drainage and ameliorate intrahepatic biliary cholestasis [12]. However, little is known about the possible growth factors that modulate these proliferative responses. In a review by Strazzabosco et al., cholangiocytes were described to have multi-faceted functions including secretion of a range of different proinflammatory
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mediators, and paracrine secretion of growth factors and peptide which mediate crosstalk with other cells of the liver. It was noted that cholangiopathies share a number of pathogenetic mechanisms, including inflammation, cholestasis, fibrosis, apoptosis, altered development, and neoplastic transformation [13]. Cholangiocytes are increasingly recognized as biologically important epithelia because of the diverse array of cellular processes in which they participate. The cholangiocyte is a dynamic participant in normal liver function and not simply a component of passive conduits for delivery of hepatic bile. It was shown in our experiment that GH and their receptors play a crucial role in modulating cholangiocyte proliferation and this probably occurs by synergizing the effects of growth factors. IGF-1 is an index of GH action. Insulin-like growth factor 1 (IGF-1) is a circulating peptide hormone and locally acting growth factor with endocrine, paracrine, and autocrine functions. The liver is the main source of circulating IGF-1. The IGF-1 produced by the liver is under the control of GH (growth hormone) which, by acting on specific receptors (GHR), induces the synthesis and release of IGF-1. The proliferative effect of IGF-1 requires functioning IGF1-R as well as intact ERK and PI3-kinase/AKT pathways, the same intracellular transduction pathways that were activated in cholangiocytes exposed to GH or to IG-1 [14]. In our research, GHR and IGF1-R were expressed in cholangiocytes, which responded to GH with enhanced expression and release of IGF-1, suggesting that the biliary epithelium is a components of GH/IGF-1 liver axis. There is evidence that signaling activated by IGF-1 and IGF-1 receptor (IGFI-R) is important in cell survival and prevention of apoptotic cell death, which is documented in different cell types. Binding of IGF-1 to its receptor induces the stimulation of tyrosine kinase activity, autophosphorylation of tyrosine residues, and other sites in the receptor and associated substrate protein which, in turn, activate downstream signaling including the PI3K/AKT pathway, by which IGF-1 prevents apoptosis and favors cell survival [15]. Little information exists on the expression and function of IGF-1 in the liver. We investigated IGF-1R in rat cholangiocytes and evaluated their involvement in cell proliferation or damage. Rat cholangiocytes express locally acting IGF-1 isoforms, which decreased during cell damage and increased during cell proliferation. The locally acting IGF-1 is active in protecting cholangiocytes. In addition, our experiment found that although after undergoing injury of different degree during orthotopic autologous liver transplantation, intrahepatic cholangiocytes secreted and overexpressed VEGF and its receptors-VEGFR2 and VEGFR3. However, the
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FIG. 7. TUNEL analysis showed a few apoptotic bodies in the liver sections of CON rats. The number of cholangiocytes undergoing apoptosis increased in liver sections from HAL rats compared with CON rats. Administration of rhGH prevented the increase in cholangiocytes apoptosis induced by HAL (3400). (Color version of figure is available online.)
increased cholangiocytes apoptosis and decreased VEGF happened, followed by ischemia of the graft hepatic artery, which acted on its main injured targetintrahepatic cholangiocytes. After administration of rhGH, cholangiocytes apoptosis decreased and autosecreted VEGF and its receptors, thereby inducing chol-
angiocytes proliferating, which confronted functional disorder of the bile ducts. Increased VEGF autosecreted by cholangiocytes was compensation for injured bile ducts. Increased VEGF also promoted PBP proliferating, thus increased blood supply of injured bile ducts, ultimately promoted compensary small bile ducts
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FIG. 8. Following HAL, the number of PCNA-positive cholangiocytes decreased compared with liver sections from CON rats. Administration of rhGH prevented inhibitory effect of HAL on the number of PCNA-positive cholangiocytes (3400). (Color version of figure is available online.)
proliferating. Overexpressed VEGF could activate vascular endothelia proliferating, subsequently new-natal PBP could increase blood supply of intrahepatic bile ducts, consequently promoting compensation for injured intrahepatic bile ducts [11].
VEGF and their related receptors appeared immunopositive in proliferating cholangiocytes. VEGF has autocrine proliferative effect on cholangiocyte growth and paracrine effect on portal vasculature, thus promoting the growth of bile ducts and their vascular
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supply [16]. VEGF potently blocks cell apoptosis. It had been originally thought that VEGF was secreted in a paracrine manner to attract and stimulate proliferation. It has since been shown that VEGF can also act in an autocrine manner, having a protective/survival effect on a number of cell types [17]. New evidence demonstrated that by interacting with specific receptors (VEGFR2 and VEGFR3), VEGF stimulates choalngiocytes proliferation, both directly and playing synergistic effects with estrogens and IGF-1 on modulation of cholangiocyte proliferation with convergence on common signaling pathways, including ERK system [8, 18]. After reviewing literatures, we concluded from our research that rhGH promoted transcription of IGF, thereafter up-regulated VEGF indirectly via IGF. Then, proliferation of vascular endothelia and epithelia of bile ducts, and angiogenesis were promoted because of the function of VEGF in paracrine/autocrine manners. It was implied in our experiment that exogenous rhGH administration promoted increased IGF-1 and GHR of the liver undergoing orthotopic autologous liver transplantation, which influenced the cholangiocytes through increased IGF-1R and GHR; furthermore, promoted overexpression of VEGF, VEGFR2, and VEGFR3 of intrahepatic cholangiocytes, thus in common, promoted cholangiocytes proliferation confronting injured intrahepatic bile ducts. So exogenous rhGH administration might be a therapeutic strategy for pathologic conditions characterized by damage of the hepatic artery or biliary tree, especially ischemic cholangiopathy, which is a complication of liver transplantation. However, the mechanism should be clarified further. Collectively based on the finding above, it is reasonable to conclude that rhGH could attenuate ischemia injury of intrahepatic bile ducts relating to liver transplantation. This function is partly related to the capacity that rhGH inhibits apoptosis of intrahepatic cholangiocytes and accelerates proliferation and angiogenesis, by increasing expression of VEGF, VEGFR2, VEGFR3, GHR, and IGF1-R. Therefore, rhGH may be valuable as a clinical candidate for such disorders. Although the clinical application of rhGH was reported to be associated with subsequent toxicities, especially metabolic disorders, years of clinical use have shown that rhGH is safe and well tolerated, suggesting that rhGH can fulfill the role as a potential ideal protective agent. In addition to the attempts to reduce potential toxicity during GH administration, future strategies also must seek to optimize the timing of GH administration.
ACKNOWLEDGMENTS This research was supported by the Research Center of Clinical Medicine of Nanfang Hospital affiliated with Southern Medical University.
REFERENCES 1. Pascher A, Neuhaus P. Bile duct complications after liver transplantation. Transpl Int 2005;18:627. 2. Cameron AM, Busuttil RW. Ischemic cholangiopathy after liver transplantation. Hepatobiliary Pancreat Dis Int 2005; 4:495. 3. Demetris AJ, Fontes P, Lunz JG, III, et al. Wound healing in the biliary tree of liver allografts. J Cell Transplant 2006; 15:S57. 4. Nishida S, Nakamura N, Kadono J, et al. Intrahepatic biliary strictures after liver transplantation. J Hepatobiliary Pancreas Surg 2006;13:511. 5. Rull R, Garcia Valdecasas JC, Grande L, et al. Intrahepatic biliary lesions after orthotopic liver transplantation. Transpl Int 2001;14:129. 6. Carroll PV, Jackson NC, Russell Jones DL, et al. Combined growth hormone/insulin-like growth factor-I in addition to glutamine supplemented TPN results in net protein anabolism in critical illness. Am J Physiol Endocrinol Metab 2004; 286:E151. 7. Krupczak-Hollis K, Wang X, Dennewitz MB, et al. Growth hormone stimulates proliferation of old-aged regenerating liver through forkhead box m1b. Hepatology 2003;38:1552. 8. Alvaro D, Mancino MG, Glaser S, et al. Proliferating cholangiocytes: A neuroendocrine compartment in the diseased liver. Gastroenterology 2006;132:415. 9. Zhao H, Zhou J. Establishment of an orthotopic autologous liver transplantation model with bile ducts ischemia-reperfusion injury in rats. Acad J Sec Milit Med Univ 2006;27:429. 10. Luo Z, Zhang Y, Han B, Wang H, et al. Establishment of orthotopic liver autotransplantation in rats with portal-cervical bypass. J Dig Surg 2005;4:219. 11. Li H, Dong J, Zhang Y. The effect of autocrine VEGF of intrahepatic cholangiocytes after liver transplantation with ischemia of hepatic artery. Chin J Curr Adv Gen Surg 2007; 10:124. 12. Wu T, Yang G, Zhao J, et al. Rapamycin inhibits compensatory bile duct growth after ischemia injury. Acad J Sec Milit Med Univ 2008;29:800. 13. Strazzabosco M, Fabris L, Spirli C. Pathophysiology of cholangiopathies. J Clin Gastroenterol 2005;S90. 14. Alvaro D, Drudi-Metalli V, Gianfranco Alpini G, et al. The intrahepatic biliary epithelium is a target of the growth hormone/insulin-like growth factor 1 axis. J Hepatol 2005; 43:875. 15. Gatto M, Drudi-Metalli V, Torrice A, et al. Insulin-like growth factor-1 isoforms in rat hepatocytes and cholangiocytes and their involvement in protection against cholestatic injury. Lab Invest 2008;88:986. 16. Bogert PT, LaRusso NF. Cholangiocyte biology. Curr Opinion Gastroenterol 2007;23:299. 17. Brusselmans K, Bono F, Collen D, et al. A novel role for vascular endothelial growth factor as an autocrine survival factor for embryonic stem cells during hypoxia. J Biol Chem 2005; 80:3493. 18. Gaudio E, Barbaro B, Alvaro D, et al. Vascular endothelial growth factor stimulates rat cholangiocyte proliferation via an autocrine mechanism. Gastroenterology 2006;130:1270.
RhGH Attenuates Ischemia Injury of Intrahepatic Bile Ducts Relating to Liver Transplantation Zheng Wang, M.D., Jie Zhou, M.M.,1 Jianhua Lin, M.D., Yu Wang, M.D., Yixiong Lin, M.D., and Xianghong Li, M.D. Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China Submitted for publication September 11, 2009
Background. To study the effect of rhGH administration on intrahepatic cholangiocytes relating to liver transplantation with ischemia of hepatic artery, and ultimately, clarify pathologic mechanism of the injury. Methods. Rat orthotopic autologous liver transplantation was performed first. Three hours later, the rats were grouped as followed: HAL (hepatic artery ligation) group; HAL D rhGH (hepatic artery ligation followed by rhGH administration) group; CON (without hepatic artery ligation) group. Specimen was collected after 7 d. ALT and ALP of serum were measured. The pathologic changes of bile ducts of liver tissue were observed. The number of bile ducts and blood vessels in portal area were counted. Immunochemistry for VEGF, VEGFR-2, VEGFR-3, GHR, and IGF-1R of intrahepatic cholangiocytes was performed. Cholangiocytes apoptosis was evaluated by TUNEL analysis. Cholangiocytes proliferation was evaluated by PCNA immunolabeling. Results. ALT and ALP of HAL D rhGH group were significantly ameliorated compared with untreated animals (P < 0.05). ALT and ALP of HAL group were significantly higher compared with CON group (P < 0.05). In HAL group, the main injury of bile ducts was not reversible, whereas it was reversible in CON and rhGH groups. In HAL group, the number of bile ducts in portal area decreased, while the number of bile ducts not accompanying blood vessels increased (P < 0.05). In rhGH group, the number of bile ducts in portal area increased, while the number of bile ducts accompanying blood vessels increased compared with HAL group (P < 0.05). The expression of VEGF, VEGFR-2, VEGFR-3, GHR, and IGF-1R was significantly lower 1 To whom correspondence and reprint requests should be addressed at Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou 510515, China. E-mail: [email protected].
0022-4804/$36.00 Ó 2011 Elsevier Inc. All rights reserved.
in HAL group than in CON group (P < 0.05). Following administration of rhGH to HAL rats, the expression of VEGF, VEGFR-2, VEGFR-3, IGF-1R, and GHR was significantly higher (P < 0.05). Administration of rhGH prevented increase in cholangiocytes apoptosis induced by HAL (P < 0.05). Administration of rhGH promoted increase in cholangiocytes proliferation held by HAL (P < 0.05). Conclusions. Administration of rhGH appears to attenuate ischemia injury of intrahepatic bile ducts relating to liver transplantation. This function is partly related to the capacity that rhGH inhibits the apoptosis of intrahepatic cholangiocytes and prompts the proliferation and angiogenesis by increasing the expression of VEGF, VEGFR2, VEGFR3, GHR, and IGF1-R. Ó 2011 Elsevier Inc. All rights reserved. Key Words: liver transplantation; hepatic artery; ischemia; intrahepatic bile duct; rhGH.
INTRODUCTION
In the past 40 y or more, liver transplantation has been an established treatment for many end-stage liver diseases. Although surgical techniques, postoperative management, and especially graft preservation methods have greatly improved in recent years, clinical research has demonstrated that biliary tract complication remain highly, 7% to 30% of patients have complication after transplantation, and mortality is 6% to 12.5%, which remains one of the main obstacles in successful transplantation. It should be pointed out that with the improvement in surgical techniques, the incidence of anastomotic biliary strictures decreased remarkably, whereas the nonanastomotic biliary strictures and dilatations became the major type of biliary complications of liver allograft, which
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had been called the Achilles’ heel revisited [1]. Nonanastomotic biliary strictures, which have also been called ischemic cholangiopathy (IC) [2], is characterized by biliary strictures and dilatations that involve the whole graft biliary tree and is defined as a focal or extensive changes to bile ducts because of impaired blood supply, ischemia-reperfusion injury, infection, or acute/chronic rejections[3]. IC is a complication that appears early during the immediate postoperative period. The incidence ranges from 11% to 19% and is related to a substantial cause of morbidity (50%–75%), characterized by the presence of repeated episodes of cholangitis and the necessity for retransplantation (35%–40%). A significant rate of mortality (25%–40%) and increased cost are also associated with this complication [4, 5]. The causes of nonanastomotic biliary stricture are multifactorial. Impaired blood supply of the biliary epithelia is considered to be one of the major causes. It is well-known that cholangiocytes proliferation is a compensation for bile duct injury. GH has direct effect on different kinds of tissues or organs, and also stimulates IGF-1 hepatic production to induce protein synthesis and cell mitosis [6]. Actually, evidence has been accumulated that GH is involved in liver regeneration [7]. Recently, Alvaro et al. pointed out, VEGF modulated cholangiocytes proliferation through the same signal pathway as IGF-1 did [8]. In light of the above-mentioned findings, the present study aimed to prove the hypothesis that administration of rhGH (recombinant human growth hormone) offers intrahepatic biliary epithelia protective effect against IC injury in rats and explore its mechanism. MATERIALS AND METHODS Animals, Drug, and Materials Adult male Wistar rats weighing 200 6 20 g were obtained from Southern Medical University Animal Center, Guangzhou,China. All animals received humane care in compliance with European Convention on Animal Care. The following experimental protocol was approved by the Southern Medical University Animal Care and Use Committee. The rats were housed and fed at the Animal Center of Nanfang Hospital at least 7 d before surgery to acclimate them to the environment. The rhGH was purchased from Shanghai United Cell Biotechnology Co. Ltd. (Shanghai, China). Infrahepatic venacaval endo-canal was made up of joining tube of no. 9 venous puncture needle. The length was 3.3 cm, and the diameter was 1.8 mm. Portalcervical bypass cannula was made up of joining tube of no. 7 venous puncture needle. The length was 10 cm, and the diameter was 1.5 mm. The canales were irrigated with 0.5% heparin before the operation.
Surgical Procedures An orthotopic autologous liver transplantation model was established, imitating the models created by Zhao and Zhou [9] and Luo et al. [10], which simulates the whole process of liver transplantation, simple and with high successful rate, better reflects the pathophysiologic process of bile ducts, provides an approach for investigation of
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bile ducts injury concerned with liver transplantation. It was described as follows: The rats were anesthetized by intraperitoneal injection of sodium pentobarbital 30 mg/kg body weight. The animal was fixed with a self-made operation cushion, placed supinely. An incision of right cervix was performed. The right external jugular vein was dissociated; thereafter the self-made portal-cervical bypass cannula was inserted into the external jugular vein and blocked temporarily with a vascular clamp. A middle incision of inverse ‘‘T’’ shape was made on the abdomen. The ligaments around the liver were dissociated and severed. The left diaphragm vein, hepatoesophageal ligament vein and right renal vein were separated, ligated and severed. The liver was turned left, the suprahepatic venae cavae (SVC) was anatomized; then the common bile duct, hepatic artery, and portal vein were anatomized over the margin of the duodenal bulb. The infrahepatic venae cavae (IVC) was dissociated downwards about 6–8 mm. The liver was dissociated completely, except the hepatic blood vessels in and out, and the common bile duct. An incision of about 3 mm was made in the IVC above right renal vein for inserting venacaval cannula completely embedded into the vena cava. The upper end of the cannula went beyond over the upper margin of the liver nearly 3 mm. The upper and lower ends of the incision in the IVC were ligated temporarily. Thereafter, the blood of the inferior vena cava flowed over into the heart through the cannula. The portal vein was dissociated. Either the portal vein or the hepatic artery was blocked temporarily with a vascular clamp. The other end of the portal-cervical bypass cannula was inserted into the centrifugal end of the portal vein, and the vascular clamp on the cannula was relaxed at once. The portal-cervical bypass came into operation to make blood of the portal vein backflow into the heart. Five u/mL heparin saline of 2–3 mL about 0–4 C was injected into the centripetal end of the portal vein to drive the blood of the liver into the systematic circulation. The SVC was ligated temporarily on the suprahepatic venacaval cannula. The ligation silk of the upper end of the incision in the IVC was relaxed. Subsequently, the liver was irrigated with combination Ringer’s solution of 0–4 C thus preserved, and the solution effused outside the cannula through the incision in the IVC. The liver had been irrigated and preserved for 2.5 h in vivo. The velocity of flow was 150 mL/h. After finishing irrigation, the inferior venacaval cannula was removed at once, and the incision in the IVC was oversewn with 11-0 suture. The portal-cervical bypass cannula was removed, and the pinhole in the portal vein was repaired. The blockage of the portal vein was relieved, followed by the blockage of the hepatic artery 30 min later. Subsequently, the circulation of the liver was resumed, while the circulation of the inferior vena cava was also resumed. At the same time, the surface of the liver was irrigated with 20 mL normal saline solution of 38 C for rapidly rewarming the liver. The end of the portal-cervical bypass cannula in the cervix was removed, and the right external jugular vein was ligated. The incision in the cervix was sutured. According to clinical practice, the time duration between the opening of portal vein and the hepatic artery was 30 min; 3 h after the operation, the hepatic artery was ligated; 7 d later, the tissues and blood were sampled.
Experimental Groups Three groups were designed as the follows: CON (control) group (n ¼ 6). The animals had the relevant operation of orthotopic autologous liver transplantation without hepatic artery ligation. HAL group (n ¼ 6). The hepatic artery was ligated after the operation. Subsequently 0.3 mL/kg/d water for injection was administered subcutaneously for 1 wk. HAL þ rhGH group (n ¼ 6). The hepatic artery was ligated after the operation. Subsequently, rhGH had been administered subcutaneously 0.3 U/kg diluted in 0.3 mL water for injection for 1 wk.
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There was no procedure-related mortality in any of the groups. After 7 d, the animals underwent re-laparotomy. Blood was collected from the inferior vena cava, and the tissue of bile ducts from the hepatic left lateral lobe was rapidly excised and preserved in 10% paraformaldehyde for further analysis, either was the tissue of the liver.
TUNEL-positive nuclei in brown were identified from negatively unstained nuclei in blue. Apoptotic cells were counted for each tissue sample under high-power magnification (3400) in a blinded fashion. Apoptotic index (AI) was used as a quantitative indicator, indicating the percentage of TUNEL-positive cholangiocytes by counting the number of positive cells in 30 random microscopic high-power fields.
Analysis of Serum Serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were measured in the serum in duplicate using commercial kits, according to the manufacturers’ protocols with Olympus AU5421 biochemistry analyzer.
Pathological Examination The tissue of bile ducts preserved in 10% paraformaldehyde was taken, cropped and made into wax pattern, thereafter sliced up, dyed with hematoxylin-eosin. Edema of cholangiocytes, infiltration of inflammatory cells, necrosis, and exfoliation of cholangiocytes were observed with high power field microscope.
Bile Ducts and Blood Vessels in Portal Area The wax pattern of liver tissue was cut into slices of 2.5 mm. continuously. The blood vessels were tagged with rabbit anti-CD34 polyclonal antibody (Beijing Biosynthesis Biotechnology Co. Ltd). In portal area, the number of bile ducts, blood vessels, bile ducts accompanying blood vessels, bile ducts not accompanying blood vessels, and isolated blood vessels were counted, respectively.
Immunohistochemical Examination for VEGF, VEGFR2, VEGFR3, GHR, and IGF-1R Immunohistochemical staining for VEGF (VEGFR2, VEGFR3, GHR, and IGF-1R) was performed with anti-VEGF (VEGFR2, VEGFR3, GHR, and IGF-1R) rabbit polyclonal antibodies (Beijing Biosynthesis Biotechnology Co. Ltd) at a dilution of 1:200 overnight at 4 C. A two-step Envision Plus Sunpoly-H III HRP rabbit/mouse kit (Shanghai Sun Biotech Co. Ltd.) was used according to the manufacturer’s instructions. DAB was used as chromogen and all sections were counterstained with hematoxylin. Following staining, sections were analyzed in a coded fashion with an Olympus U-25ND6 T2 (Diagnostic Instrument, Tokyo, Japan) and processed with an IAS image analysis system (Delta Sistemi, Delta Sistemi, Rome, Italy). The intensity and distribution of immunostaining were assessed in a coded fashion.
TUNEL Staining Part of the specimens were immediately fixed at 4 C buffered formalin (10% paraformaldehyde) and embedded in paraffin. In situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) of fragmented DNA was performed on 2.5 mm thick sections of paraffin-embeded tissue using the in situ TUNEL assay (Roche Diagnostics, Basel, Switzerland) according to the supplier’s instructions. The sections were deparaffinized in xylene and rehydrated before analysis. After treatment with proteinase K (20 mg/mL in 10 mL Tris-HCl, PH 7.6) at 37 C for 30 min, sections were washed in PBS and endogenous peroxidase activity was inhibited by immersing the slides in 3% hydrogen peroxide in methanol for 30 min. Then, the slides were treated with permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 15 min. For the labeling reaction, a TUNEL mixture containing terminal deoxynucleotidyl transferase (TdT), its buffer solution, and biotinylated dUTP was added to cover the sections, which were then incubated at 37 C for 60 min in a humidified chamber. Washed sections were incubated with peroxidase-labeled streptavidin for 30 min and then stained with diaminobenzidine, followed by counterstaining with hematoxylin.
PCNA Immunolabeling After rehydration, the silane-coated slides were treated with 0.3% H2O2 in methyl alcohol for 15 min and then briefly washed in phosphate buffer saline. They were then incubated overnight at 4 C, with a 1:100 dilution of anti-proliferating-cell nuclear antigen (PCNA) monoclonal antibody (BOSTER), and subsequently incubated with Envision Plus Sunpoly-H III HRP rabbit/mouse kit (Shanghai Sun Biotech Co. Ltd.) for 30min at 37 C. Finally, the sections were counterstained with hematoxylin and coverslipped. PCNA labeling of cholangiocytes were semi-quantitatively evaluated by counting at least 500 bile-duct cells in 20 randomly selected high-power fields (3400) and by determining the percentages of PCNA-reactive cells. Thus, PCNA labeling index was evaluated. Only bile-duct cells that exhibited strong nuclear immunostaining were considered for reactive cell counting.
Statistical Analysis All the data were expressed as mean value 6 SD. Difference between groups was analyzed by Student’s unpaired t-test when two groups were analyzed, and analysis of variance (ANOVA) when more than two groups were analyzed, followed by an appropriate post hoc test. The analysis was performed by SPSS 13.0 and P-values of less than 0.05 were accepted as statistically significant.
RESULTS Serum Analysis
ALT and ALP of HAL group were significantly higher compared with CON group (674.50 6 145.55 verus 176.50 6 33.62U/L, 790.67 6 77.96 verus 278.50 6 30.03 U/L, P < 0.05). When animals were administrated rhGH, ALT and ALP were significantly ameliorated compared with untreated animals (308.67 6 87.13 verus 674.50 6 145.55U/L, 356.50 6 30.20 verus 790.67 6 77.96U/L, P < 0.05). However, they remained higher compared with CON group (308.67 6 87.13 verus 176.50 6 33.62U/L, 356.50 6 30.20 verus TABLE 1 ALT and ALP in Each Group Group
CON
HAL
HAL þ rhGH
ALT (U/L) ALP (U/L)
176.50 6 33.62y 278.50 6 30.03y
674.50 6 145.55* 790.67 6 77.96*
308.67 6 87.13*,y 356.50 6 30.20*,y
ALT and ALP of HAL group were significantly higher compared with CON group. When animals were administered with rhGH, ALT and ALP were significantly ameliorated compared with untreated animals, however, remained significantly higher compared with CON group. * P < 0.05 versus CON group. y P < 0.05 versus HAL group.
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TABLE 2 The Number of Bile Ducts and Blood Vessels in Portal Area Group
CON
HAL
HAL þ rhGH
Bile ducts Blood vessels Bile ducts with blood vessels Bile ducts without blood vessels Isolated blood vessels
11.17 6 3.37 8.50 6 1.05 6.00 6 0.89
4.83 6 1.17* 2.67 6 1.03* 2.17 6 0.75*
7.67 6 0.52y 5.83 6 0.75*,y 4.67 6 0.52*,y
1.17 6 0.41
5.50 6 2.43*
2.50 6 0.84y
2.50 6 1.38
0.67 6 0.52
2.83 6 1.47
In HAL group, the number of bile ducts and bile ducts accompanying by blood vessels in portal area decreased, whereas they increased in HAL þ rhGH group. * P < 0.05 versus CON group. y P < 0.05 versus HAL group.
damage, which was still worse in comparison with CON group. Pathologic Changes with Hematoxylin-Eosin Dyeing
In CON group, the main injury was reversible such as edema of cholangiocytes and infiltration of inflammatory cells. In HAL group, the main injury was not reversible such as necrosis and exfoliation of cholangiocytes. In HAL þ rhGH group, the main injury was edema of cholangiocytes and infiltration of inflammatory cells, while necrosis and exfoliation of cholangiocytes were remarkably decreased (Fig. 1). Bile Ducts and Blood Vessels in Portal Area
In HAL group, the bile ducts in portal area were injured, and the number decreased compared with CON group (P < 0.05). In HAL þ rhGH group, the number of bile ducts in portal area clearly increased compared with HAL group (P < 0.05). In HAL group, the number of bile ducts accompanying blood vessels significantly decreased, while those not accompanying blood vessels significantly increased (P < 0.05). In HAL þ rhGH group, the number of bile ducts accompanying blood vessels significantly increased, while those not accompanying blood vessels significantly decreased (P < 0.05; Table 2). FIG. 1. In CON and HAL þ rhGH group, the main injury of bile ducts was edema of cholangiocytes and infiltration of inflammatory cells. In HAL group, the main injury was necrosis and exfoliation of cholangiocytes. (Color version of figure is available online.)
278.50 6 30.03 U/L, P < 0.05; Table 1). Liver function and cholestasis were evaluated with ALT and ALP, respectively. In most cases, the damage was most evident in HAL groups. Administration of rhGH alleviated the
Effects of HAL and rhGH on VEGF, VEGFR2, and VEGFR3 Expression
Immunohistochemistry in CON liver sections showed that bile ducts expressed VEGF, VEGFR2, and VEGFR3. HAL induced a decrease in the number of cholangiocytes positive for VEGF, VEGFR2, and VEGFR3 compared with liver sections from 1-wk CON rats (5.15 6 0.41 versus 14.02 6 1.62, 3.58 6 0.63 versus
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FIG. 2. Immunohistochemistry in CON liver sections showed that bile ducts expressed VEGF. HAL induced a decrease in the number of cholangiocytes positive for VEGF compared with liver sections from 1-wk CON rats. Following administration of rhGH to HAL rats, expression of VEGF was significantly higher compared with untreated animals, however, remained lower compared with CON group (3400) (see Table 3). (Color version of figure is available online.)
FIG. 3. Immunohistochemistry in CON liver sections showed that bile ducts expressed VEGFR2. HAL induced a decrease in the number of cholangiocytes positive for VEGFR2 compared with liver sections from 1-wk CON rats. Following administration of rhGH to HAL rats, expression of VEGFR2 was significantly higher compared with untreated animals, however, remained lower compared with CON group (3400) (see Table 3). (Color version of figure is available online.)
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TABLE 3 VEGF, VEGFR2, VEGFR3 Expression % Positive Cholangiocytes in Each Group Group
CON
HAL
HAL þ rhGH
VEGF VEGFR2 VEGFR3s
14.02 6 1.62y 11.22 6 1.23y 12.07 6 0.92y
5.15 6 0.41* 3.58 6 0.63* 4.75 6 0.59*
10.70 6 1.46*,y 9.08 6 1.17*,y 9.55 6 0.64*,y
Immunohistochemistry in CON liver sections showed that bile ducts expressed VEGF, VEGFR2 and VEGFR3. HAL induced a decrease in the number of cholangiocytes positive for VEGF, VEGFR2, and VEGFR3 compared with liver sections from 1-wk CON rats. Following administration of rhGH to HAL rats, expression of VEGF, VEGFR2, and VEGFR3 was significantly higher compared with untreated animals, however, remained lower compared with CON group. * P < 0.05 versus CON group. y P < 0.05 versus HAL group.
11.22 6 1.23, 4.75 6 0.59 versus 12.07 6 0.92, P < 0.05). Following administration of rhGH to HAL rats, the expression of VEGF, VEGFR2, and VEGFR3 was significantly higher compared with untreated animals (10.70 6 1.46 versus 5.15 6 0.41, 9.08 6 1.17 versus 3.58 6 0.63, 9.55 6 0.64 versus 4.75 6 0.59, P < 0.05), however, remained lower compared with CON group (10.70 6 1.46 versus 14.02 6 1.62, 9.08 6 1.17 versus 11.22 6 1.23, 9.55 6 0.64 versus 12.07 6 0.92, P < 0.05; Figs. 2, 3, and 4 and Table 3). Effects of HAL and rhGH on GHR and IGF-1R Expression
Immunohistochemistry in CON liver sections showed that bile ducts expressed GHR and IGF-1R. HAL induced a decrease in the number of cholangiocytes positive for GHR and IGF-1R compared with liver sections from 1-week CON rats (2.06 6 0.43 versus 9.94 6 0.63, 5.26 6 0.37 versus 8.99 6 0.69, P < 0.05). Following administration of rhGH to HAL rats, the expression of GHR and IGF-1R was significantly higher compared with untreated animals (7.01 6 1.61 versus 2.06 6 0.43, 7.61 6 0.25 versus 5.26 6 0.37, P < 0.05), however, they remained lower compared with CON group (7.01 6 1.61 versus 9.94 6 0.63, 7.61 6 0.25 versus 8.99 6 0.69, P < 0.05; Figs. 5, and 6 and Table 4). Cholangiocytes Apoptosis and Proliferation
FIG. 4. Immunohistochemistry in CON liver sections showed that bile ducts expressed VEGFR3. HAL induced a decrease in the number of cholangiocytes positive for VEGFR3 compared with liver sections from 1-wk CON rats. Following administration of rhGH to HAL rats, expression of VEGFR3 was significantly higher compared with untreated animals, however, remained lower when compared with CON group (3400) (see Table 3). (Color version of figure is available online.)
TUNEL analysis showed a few apoptotic bodies in the liver sections of CON rats. The number of cholangiocytes undergoing apoptosis increased in liver sections from HAL rats compared with CON rats (4.91 6 0.26 versus 1.63 6 0.52, P < 0.05). Administration of rhGH prevented the increase in cholangiocytes apoptosis induced by HAL (2.61 6 0.58 versus 4.91 6 0.26, P < 0.05; Fig. 7).
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FIG. 5. Immunohistochemistry in CON liver sections showed that bile ducts expressed GHR. HAL induced a decrease in the number of cholangiocytes positive for GHR compared with CON rats. Following administration of rhGH to HAL rats, expression of GHR was significantly higher, but remained lower compared with CON group (3400) (see Table 4). (Color version of figure is available online.)
FIG. 6. Immunohistochemistry in CON liver sections showed that bile ducts expressed IGF-1R. HAL induced a decrease in the number of cholangicytes positive for IGF-1R compared with liver sections from 1-wk CON rats. Following administration of rhGH to HAL rats, expression of IGF-1R was significantly higher compared with untreated animals, however, remained lower compared with CON group (3400) (see Table 4). (Color version of figure is available online.)
WANG ET AL.: RhGH FOR LIVER TRANSPLANTATION
TABLE 4 GHR, IGF1-R Expression % Positive Cholangiocytes in Each Group Group
CON
HAL
HAL þrhGH
GHR IGF-1R
9.94 6 0.63y 8.99 6 0.69y
2.06 6 0.43* 5.26 6 0.37*
7.01 6 1.61*,y 7.61 6 0.25*,y
Immunohistochemistry in CON liver sections showed that bile ducts expressed GHR and IGF-1R. HAL induced a decrease in the number of cholangiocytes positive for GHR and IGF-1R compared with liver sections from 1-wk CON rats. Following administration of rhGH to HAL rats, expression of GHR and IGF-1R was significantly higher compared with untreated animals, however, remained lower compared with CON group. * P < 0.05 versus CON group. y P < 0.05 versus HAL group.
Following HAL, the number of PCNA-positive cholangiocytes decreased compared with liver sections from CON rats (10.87 6 1.85 versus 22.95 6 2.08, P < 0.05). Administration of rhGH prevented inhibitory effect of HAL on the number of PCNA-positive cholangiocytes (19.03 6 1.58 versus 10.87 6 1.85, P < 0.05; Fig. 8). DISCUSSION
In the field of hepatic surgery, liver transplantation, organ preservation, or embolism and interventional treatment might destroy hepatic artery system, affect blood supply of bile ducts, and thereby damnify cholangiocytes. In the liver, the terminal arteriole of hepatic artery branches off into the peribiliary plexus (PBP), which supplies the intrahepatic bile ducts. Thereby, the changes of PBP usually result in changes of intrahepatic bile duct structure. Post-transplantational hepatic artery ischemia is the vital reason leading to bile duct complication, which induces ischemia and occlusion of PBP, ulteriorly aggravates ischemia of intrahepatic bile ducts, resulting in intrahepatic bile ducts injury [11]. In our laboratory, after the pathophysiologic progression of cold perfusion and reperfusion, the cholangiocytes were exposed to damnification of different degree. So the injury of intrahepatic cholangiocytes became worse subsequent to hepatic artery ligation. Cholangiocytes proliferation is the vital compensatory feedback for bile duct injury, because of its benefit to restore biliary drainage and ameliorate intrahepatic biliary cholestasis [12]. However, little is known about the possible growth factors that modulate these proliferative responses. In a review by Strazzabosco et al., cholangiocytes were described to have multi-faceted functions including secretion of a range of different proinflammatory
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mediators, and paracrine secretion of growth factors and peptide which mediate crosstalk with other cells of the liver. It was noted that cholangiopathies share a number of pathogenetic mechanisms, including inflammation, cholestasis, fibrosis, apoptosis, altered development, and neoplastic transformation [13]. Cholangiocytes are increasingly recognized as biologically important epithelia because of the diverse array of cellular processes in which they participate. The cholangiocyte is a dynamic participant in normal liver function and not simply a component of passive conduits for delivery of hepatic bile. It was shown in our experiment that GH and their receptors play a crucial role in modulating cholangiocyte proliferation and this probably occurs by synergizing the effects of growth factors. IGF-1 is an index of GH action. Insulin-like growth factor 1 (IGF-1) is a circulating peptide hormone and locally acting growth factor with endocrine, paracrine, and autocrine functions. The liver is the main source of circulating IGF-1. The IGF-1 produced by the liver is under the control of GH (growth hormone) which, by acting on specific receptors (GHR), induces the synthesis and release of IGF-1. The proliferative effect of IGF-1 requires functioning IGF1-R as well as intact ERK and PI3-kinase/AKT pathways, the same intracellular transduction pathways that were activated in cholangiocytes exposed to GH or to IG-1 [14]. In our research, GHR and IGF1-R were expressed in cholangiocytes, which responded to GH with enhanced expression and release of IGF-1, suggesting that the biliary epithelium is a components of GH/IGF-1 liver axis. There is evidence that signaling activated by IGF-1 and IGF-1 receptor (IGFI-R) is important in cell survival and prevention of apoptotic cell death, which is documented in different cell types. Binding of IGF-1 to its receptor induces the stimulation of tyrosine kinase activity, autophosphorylation of tyrosine residues, and other sites in the receptor and associated substrate protein which, in turn, activate downstream signaling including the PI3K/AKT pathway, by which IGF-1 prevents apoptosis and favors cell survival [15]. Little information exists on the expression and function of IGF-1 in the liver. We investigated IGF-1R in rat cholangiocytes and evaluated their involvement in cell proliferation or damage. Rat cholangiocytes express locally acting IGF-1 isoforms, which decreased during cell damage and increased during cell proliferation. The locally acting IGF-1 is active in protecting cholangiocytes. In addition, our experiment found that although after undergoing injury of different degree during orthotopic autologous liver transplantation, intrahepatic cholangiocytes secreted and overexpressed VEGF and its receptors-VEGFR2 and VEGFR3. However, the
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FIG. 7. TUNEL analysis showed a few apoptotic bodies in the liver sections of CON rats. The number of cholangiocytes undergoing apoptosis increased in liver sections from HAL rats compared with CON rats. Administration of rhGH prevented the increase in cholangiocytes apoptosis induced by HAL (3400). (Color version of figure is available online.)
increased cholangiocytes apoptosis and decreased VEGF happened, followed by ischemia of the graft hepatic artery, which acted on its main injured targetintrahepatic cholangiocytes. After administration of rhGH, cholangiocytes apoptosis decreased and autosecreted VEGF and its receptors, thereby inducing chol-
angiocytes proliferating, which confronted functional disorder of the bile ducts. Increased VEGF autosecreted by cholangiocytes was compensation for injured bile ducts. Increased VEGF also promoted PBP proliferating, thus increased blood supply of injured bile ducts, ultimately promoted compensary small bile ducts
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FIG. 8. Following HAL, the number of PCNA-positive cholangiocytes decreased compared with liver sections from CON rats. Administration of rhGH prevented inhibitory effect of HAL on the number of PCNA-positive cholangiocytes (3400). (Color version of figure is available online.)
proliferating. Overexpressed VEGF could activate vascular endothelia proliferating, subsequently new-natal PBP could increase blood supply of intrahepatic bile ducts, consequently promoting compensation for injured intrahepatic bile ducts [11].
VEGF and their related receptors appeared immunopositive in proliferating cholangiocytes. VEGF has autocrine proliferative effect on cholangiocyte growth and paracrine effect on portal vasculature, thus promoting the growth of bile ducts and their vascular
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supply [16]. VEGF potently blocks cell apoptosis. It had been originally thought that VEGF was secreted in a paracrine manner to attract and stimulate proliferation. It has since been shown that VEGF can also act in an autocrine manner, having a protective/survival effect on a number of cell types [17]. New evidence demonstrated that by interacting with specific receptors (VEGFR2 and VEGFR3), VEGF stimulates choalngiocytes proliferation, both directly and playing synergistic effects with estrogens and IGF-1 on modulation of cholangiocyte proliferation with convergence on common signaling pathways, including ERK system [8, 18]. After reviewing literatures, we concluded from our research that rhGH promoted transcription of IGF, thereafter up-regulated VEGF indirectly via IGF. Then, proliferation of vascular endothelia and epithelia of bile ducts, and angiogenesis were promoted because of the function of VEGF in paracrine/autocrine manners. It was implied in our experiment that exogenous rhGH administration promoted increased IGF-1 and GHR of the liver undergoing orthotopic autologous liver transplantation, which influenced the cholangiocytes through increased IGF-1R and GHR; furthermore, promoted overexpression of VEGF, VEGFR2, and VEGFR3 of intrahepatic cholangiocytes, thus in common, promoted cholangiocytes proliferation confronting injured intrahepatic bile ducts. So exogenous rhGH administration might be a therapeutic strategy for pathologic conditions characterized by damage of the hepatic artery or biliary tree, especially ischemic cholangiopathy, which is a complication of liver transplantation. However, the mechanism should be clarified further. Collectively based on the finding above, it is reasonable to conclude that rhGH could attenuate ischemia injury of intrahepatic bile ducts relating to liver transplantation. This function is partly related to the capacity that rhGH inhibits apoptosis of intrahepatic cholangiocytes and accelerates proliferation and angiogenesis, by increasing expression of VEGF, VEGFR2, VEGFR3, GHR, and IGF1-R. Therefore, rhGH may be valuable as a clinical candidate for such disorders. Although the clinical application of rhGH was reported to be associated with subsequent toxicities, especially metabolic disorders, years of clinical use have shown that rhGH is safe and well tolerated, suggesting that rhGH can fulfill the role as a potential ideal protective agent. In addition to the attempts to reduce potential toxicity during GH administration, future strategies also must seek to optimize the timing of GH administration.
ACKNOWLEDGMENTS This research was supported by the Research Center of Clinical Medicine of Nanfang Hospital affiliated with Southern Medical University.
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