- Email: [email protected]
Expression of Discoidin Domain Receptors (DDR2) in Alcoholic Liver Fibrosis in Rats
Archives of Medical Research 41 (2010) 586e592
ORIGINAL ARTICLE
Expression of Discoidin Domain Receptors (DDR2) in Alcoholic Liver Fibrosis in Rats Xi-Hong Zhang,a Ming Yan,b Li Liu,c Tie-Jun Wu,a Long-Le Ma,d and Le-Xin Wange a
Intensive Care Unit, Liaocheng People’s Hospital, Liaocheng, P.R. China Department of Gastroenterology, Qilu Hospital, Shandong University, Jinan, P.R. China c Jinan Infectious Diseases Hospital, Jinan, P.R. China d Department of Cardiology, Liaocheng People’s Hospital, Liaocheng, P.R. China e School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, Australia
b
Received for publication June 26, 2010; accepted October 18, 2010 (ARCMED-D-10-00327).
Background and Aims. We undertook this study to evaluate the expression of discoidin domain receptor 2 (DDR2) and its relationship with alcoholic liver fibrosis. Methods. Liver fibrosis was induced by intragastric administration of alcohol in 30 rats. Pathological changes and ultrastructure of the liver were studied. The expression of DDR2 mRNA and protein was detected by RT-PCR and Western blot, respectively, at weeks 12, 16 and 20 during the alcohol administration. Results. In the control group (con) (n 5 10), DDR2 mRNA expression and DDR2 protein were 1.02 0.13 (con ratio x103) and 0.32 0.03, respectively. In the study groups there was a progressive increase in DDR2 mRNA expression from weeks 12, 16 and 20 (3.64 1.69, 8.34 2.39, 15.73 4.57 con ratio x103, p !0.05). There was also a progressive increase in DDR2 protein from weeks 12e20 (0.48 0.05, 0.74 0.06 and 0.99 0.05, p !0.05). The mean DDR2 mRNA and protein in the three study groups was higher than in the control group ( p !0.01). The expressions of DDR2 mRNA and protein were positively correlated with collagen type I, III and IV in liver tissues as well as with the serum biomarkers of liver fibrosis, collagen type III, hyaluronic acid, collagen type IV and laminin ( p !0.01). Conclusions. The expression of DDR2 in this alcohol-induced liver fibrosis rat model is enhanced. The expression of DDR2 is closely associated with collagens in the fibrotic liver tissues. Ó 2010 IMSS. Published by Elsevier Inc. Key Words: Hepatic fibrosis, Discoidin domain receptor, Collagen, Procollagen.
Introduction Liver fibrosis is a common chronic disease and is related to excessive intrahepatic deposition of extracellular matrix (ECM). Hepatic stellate cell (HSC) is the main source cell of ECM in the liver (1), and its activation is the key factor for liver fibrosis (2e4). Discoidin domain receptors (DDR) are a new subgroup of the receptor tyrosine kinase family recently discovered (5,6). DDR can be activated by collagen type I and III of ECM (7e10). Previous studies
showed that the activated HSCs express DDR2 mRNA (11,12), suggesting that DDR2 may further activate HSC by combining with collagenous fibers, thus participating in the development of liver fibrosis. Little is known about the relationship between DDR2 and liver fibrosis. The purpose of this study was to investigate the role of DDR2 in the pathogenesis of liver fibrosis in a rat model. Materials and Methods Materials
Address reprint requests to: Ming Yan, PhD, Department of Gastroenterology, Qilu Hospital, Shandong University, Jinan 251200, Shandong Province, P.R. China; Phone: þ8613708937968; FAX: þ86 53182169171; E-mail: [email protected]
The origin of the research materials are as follows: olive oil and Beijing starking erguotou liquor (Wal-Mart, Jinan, China); Pyrazole (Kangda Chemical, Changzhou, China);
0188-4409/$ - see front matter. Copyright Ó 2010 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2010.10.010
DDR2 in Alcoholic Liver Fibrosis
587
Table 1. Serum markers for liver fibrosis Group Control (n 5 10) Study 1 (n 5 10) Study 2 (n 5 10) Study 3 (n 5 10)
HA (ng/mL) 68.48 123.67 148.08 253.91
26.58 10.96a 13.32a 13.26a
Laminin (ng/mL) 20.73 24.91 30.74 38.87
3.48 3.66b 2.48b 3.58b
PC III (ng/mL) 14.86 21.71 26.24 35.43
1.75 1.37a 2.37a 2.32a
CIV (ng/mL) 93.08 142.01 220.22 289.77
7.77 12.22a 10.98a 11.54a
HA, hyaluronic acid; PC III, procollagen type III; CIV, collagen type IV. a p !0.01. b p !0.05 compared with control group.
hyaluronic acid, procollagen type III and collagen type IV radioimmunoassay kits (Haiyan Medical Biotech, Shanghai, China); collagen type I, III and IV rabbit anti-mouse polyclonal antibody (Boshide Bioengineering, Wuhan, China); goat anti-mouse DDR2 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA); and PCR primers (Yingjun Biotech, Shanghai, China). Animals This study was approved by the institutional review board of Liaocheng People’s Hospital. Forty male Wistar rats (6 weeks old, body weight 180 10 g) were obtained from the Experimental Animal Center of Shandong University. Rats were randomly divided into study and control groups. Both groups were fed with standard food adding olive oil. The study group was given intragastric alcohol infusion to induce liver fibrosis. The concentration and amount
of alcohol was increased gradually as follows: 4.5 g/kg/ day during weeks 1e4, 6.5 g/kg/day during weeks 5e8, 9 g/kg/day during weeks 9e12, and 9.6 g/kg/day during weeks 13e20. The daily dose of alcohol was administered in two divided doses, 12 h apart. Control group was given the same volume of saline. Study group animals were euthanized by on the 12th (study group 1, n 5 10), 16th (study group 2, n 5 10), and 20th week (study group 3, n 5 10), respectively. Control group (n 5 10) was euthanized at the 12th week. Euthanasia was achieved by IP injection of sodium pentobarbital to the right lower abdomen in order to avoid causing pain by hitting the liver or any other organs. Serological Test Whole blood was obtained by cardiac puncture and serum was separated to test the amount of hyaluronic acid (HA),
Figure 1. (a) Control group showing a normal hepatic lobule structure. (b) Inflammatory cell infiltration in portal areas with fibrous tissue proliferation. (c) Wide hepatocyte ballooning degeneration around the central veins. (d) A large amount of fibrous tissue around central veins and in portal areas. (A color figure can be found in the online version of this article.)
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laminin, procollagen type III and collagen type IV with type GC-9 g-radiation immunity arithmometer. All measurements were conducted according to the manufacturer’s instructions. Histopathology and Immunohistochemistry Liver tissue samples were harvested from the right hepatic lobe and were fixed with 4% paraformaldehyde solution. Paraffin-embedded sections were stained with HE and Masson. Paraffin-embedded sections were added with 3% hydrogen peroxide and incubated in room temperature to block endogenous peroxidase activity. The sections were washed three times with distilled water followed by citrate buffer microwave repair antigen. The first antibodies (1:120 diluted concentration of I, III, IV) were added and kept at 4 C overnight. The second antibodies were added and sections were incubated at room temperature for 1 h before staining with hematoxylin. The contents of collagen I, III and IV were also determined in the three study groups. Hepatic tissue sections were viewed with JD801 image analysis system (Jiangsu JEDA Science-Technology, Nanjing, China). For each type of collagen, areas of the collagen were measured from five randomly selected fields in each section, and the mean area of the five measurements was used.
dehyde. After 2e3 min, samples were trimmed into 1 mm3 pieces and added with fresh 3% glutaraldehyde. After rinsing, tissue samples were fixed in OsO4, followed by further rinsing and dehydration. The samples were then embedded in Epon 812 epoxy resin before ultrathin sections were obtained. Ultrathin sections were stained with lead citrate and uranyl acetate. Ultrastructures of these sections were visualized by JEM-1200EX electron microscope. DDR2 mRNA Expression DDR2 mRNA expression was detected by fluorescence quantitative PCR. Total RNA in liver tissues was extracted according to Trizol instructions, with reverse transcription after the quantitative by UV spectrophotometer. DDR2 primer sequence: upstream primer 50 TAACCGACATGGG AAACA 30 ; downstream primer 50 TAACCGACATGGG AAACA 30 , amplified fragment 106 bp. b-actin primer sequence: upstream primer 50 AAGATCCTGACCGAGC GTGG 30 ; downstream primer 50 CAGCACTGTGTTGG CATAGAGG 30 , amplified fragment 327 bp. All primers were designed by Primer 5.0 and the specific products were verified by NCBI Blast. The melting curve was analyzed after amplification, and corresponding copies were calculated according to standard curves.
Specimen Preparation of Electron Microscope
DDR2 Protein Expression by Western Blot
Tissue samples of the right hepatic lobe were washed rapidly in phosphate buffer and were fixed in 3% glutaral-
Liver tissue (150 mg) was cut into pieces and mixed with 300 ml of protein extracts and 10 mL of proteinase inhibitor.
Figure 2. (a) Control group showing a small amount of collagen fibers located in sinusoid wall around the central veins. (bed) Progressive increase in fibrous tissues. (A color figure can be found in the online version of this article.)
DDR2 in Alcoholic Liver Fibrosis
After ice bath for 30 min and centrifugation, supernatant was collected for analysis. Equal amounts of protein and loading buffer were mixed before SDS-PAGE electrophoresis (120 V, 0.5 h) and transblotting (200 mA, 3 h) at room temperature for 1 h. The samples were incubated, respectively, with anti-DDR2 antibody (1:250 at 4 C) and horseradish peroxidase-labeled secondary antibody (1:1250, vibrating and shaking at room temperature for 1 h). The membrane was washed three times before diaminobenzidine coloring for 3e10 min. Finally, photographs were taken with Fluorchem 9900 imaging system. DDR2 protein was quantified by first scanning the gel with JIEDA 801 imaging system (JIEDA, Nanjing, China). The intensity of each band on Western blot was quantified by densitometry, and the ratio of DDR2 protein and internal control was calculated. Statistical Analysis Data are expressed as mean SE. All data were processed by SPSS for Windows (v.11.0) software. One-way ANOVA was used to compare means of multiple groups, and leastsignificant difference test was used in multiple comparisons. Pearson’s correlation was used to analyze the relationship between DDR2 mRNA or DDR2 protein and hepatic collagen I, III and IV, as well as serum biomarkers for collagen. Statistical significance was set at p !0.05.
589
Results Serum Indices for Hepatic Fibrosis Serum hyaluronic acid, procollagen III, collagen type IV and laminin concentration in the study groups were higher than in the control group (Table 1, p !0.01 and 0.05, respectively).
Histopathological Findings The control group showed a normal hepatic lobule structure (Figure 1a). In study group 1 (Figure 1b), there was inflammatory cell infiltration in portal areas with fibrous tissue proliferation. In study group 2 (Figure 1c), there was wide hepatocyte ballooning degeneration around the central veins accompanied by necrosis, inflammatory cell infiltration and fibrous tissue proliferation. In study group 3 (Figure 1d), a large amount of fibrous tissues was present in the periportal area. Masson staining displayed collagen fibers in blue, muscle fibers in red and red blood cells in orange. There was only a small amount of collagen fibers in the control group (Figure 2a), located in the sinusoid wall around the central veins. There was a gradual increase in fibrous tissue over time in the study group, with fibrous tissue mainly located in hepatic acinus (Figures 2be2d).
Figure 3. (a) Control group showing weak expression of collagen fibers. (b) Type III collagen mainly found in liver cytoplasm around central veins and fibrous septum. (c) Type I collagen in periportal arterial and venous wall and sinusoid. (d) Type IV collagen located in the basement membrane of central vein, portal area, hepatic artery and bile duct. (A color figure can be found in the online version of this article.)
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Immunohistochemistry staining showed a weak expression of collagen fibers in control group, with type I collagen mainly located in portal areas and around the veins. Type III collagen was found along the sinusoidal walls, and type IV collagen in bile duct and basement membrane of vascular walls (Figure 3a). In the study groups there was a strong expression of all types of collagen, with type III collagen mainly found in liver cytoplasm around the central veins and fibrous septum (Figure 3b). Type I collagen was located in periportal arterial and venous wall, and in the sinusoid (Figure 3c). Type IV collagen was located in the basement membrane of the central vein, portal area, hepatic artery and the bile duct (Figure 3d). Ultrastructure In the control group the connection between liver cells was close, with visible bile canaliculi and a small amount of collagen fibers. Organelles were abundant and well developed, endoplasmic reticulum and mitochondria distributed normally, and nuclei were round and large (Figure 4a). Study Group 1 Interhepatocytes and perisinusoid appeared fasciculus arranged interlacing, and the Disse space was filled with coarse hepatocyte microvilli, which caused stenosis and densifica-
tion of the Disse space (Figure 4b). The boundary between liver cells and Disse space was unclear. The cell matrix had many petal-shaped glycogen granules (Figure 4b). Study Group 2 Intranuclear heterochromatin showed massive diffuse distribution without obvious nucleolar structure (Figure 4c). The perinuclear gaps were widened and mitochondria were not visible. Cytoplasmic mitochondria and endoplasmic reticulum were decreased, with bullous fat droplets (Figure 4c). Study Group 3 The size of the liver cells was reduced with increased intercellular space (Figure 4d). Liver parenchymal cells and matrix were electron dense, with few microvilli in the Disse space and sinusoid. Compared to blood sinus cells, collagen tissue was at a lower functional state, such as apoptotic changes. Their nuclei were pyknotic to form clumps, with increased heterochromatin and decreased euchromatin and without the nucleolus structure. Cytoplasmic mitochondria were intensive and irregular, whereas cristae became vague and lost (Figure 4d). Expression of DDR2 mRNA and Protein The expression of DDR2 mRNA and protein in the control and three study groups is shown in Figure 5. DDR2 mRNA
Figure 4. (a) Normal liver cell ultrastructure in the control group. (b) Interhepatocytes and perisinusoid appeared fasciculus arranged interlacing (arrow 1), the Disse space was filled with coarse hepatocyte microvilli (arrow 2). The cell matrix had many petal-shaped glycogen granules (arrow 3). (c) Intranuclear heterochromatin showed massive diffuse distribution without obvious nucleolar structure. Perinuclear gaps were widened (arrow 1), cytoplasmic mitochondria and endoplasmic reticulum was decreased (arrow 2). (d) The size of the liver cells was reduced with increased cell space. Few microvilli in the Disse space and sinusoid; collagen tissue was in function of the lower state. Their nuclei were pyknotic to form into clumps (arrow 2), with increased heterochromatin and decreased euchromatin, and without the nucleolus structure. The cytoplasmic mitochondria were intensive and irregular, whereas cristae became vague and lost (arrow 1).
DDR2 in Alcoholic Liver Fibrosis
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Figure 5. Western blotting of DDR2 protein (a) and b-actin protein. (b) 1: control group; 2: study group 1; 3: study group 2; 4: study group 3.
and protein expression in the study groups was higher than in the control group ( p !0.01, Table 2). The expression of DDR2 mRNA and protein in study groups 2 and 3 were higher than in study group 1 ( p !0.01, Table 2). The expression of DDR2 mRNA and protein was correlated with hepatic type I, III and IV collagen and serum biomarkers procollagen type III, collagen type IV, hyaluronic acid and laminin (Table 3, p !0.01). Discussion Alcoholic liver fibrosis, a result of alcoholic liver injury, is characterized by excessive accumulation of ECM in the liver. It is long believed that alcoholic liver fibrosis is a passively and irreversibly pathological process induced by the necrosis of liver parenchymal cells. In the present study, liver tissue examination at three time points of the alcohol-induced liver fibrosis showed progressive increase in collagen tissues in the liver, suggesting connective tissue proliferation plays a key role in initiating alcoholic liver fibrosis. HSCs have been identified as major collagen-producing cells in the injured liver (1), and activation of HSCs has become the central link for liver fibrosis (2). The factors that regulate the biological behavior of HSCs include soluble regulatory factors and nonsoluble regulatory factors, the latter mainly refer to ECM itself. The interaction between HSCs and ECM alters the ability of HSCs to create ECM, which means ECM can directly regulate the activation of HSCs. A previous study has shown that HSCs have no active proliferative activity in the matrix culture simulating the environment of Disse space and produce only small amounts of ECM (13). In the plastic Table 2. Expression of DDR2 mRNA and protein in liver tissues DDR2 mRNA (con ratio 103) Control (n 5 10) Study group 1 (n 5 10) Study group 2 (n 5 10) Study group 3 (n 5 10)
1.02 3.64 8.334 15.73
p !0.01 compared with control group.
a
0.13 1.69a 2.39a 4.57a
DDR2 protein 0.32 0.48 0.74 0.99
0.03 0.05a 0.06a 0.05a
culture dish without any packets, HSCs show active proliferation and may gradually transform into myofibroblasts with activation state, generating a large amount of collagen (13). HSCs produce different ECM components when cultured in different types of single collagen matrix. This ECM-HSC interaction was previously attributed to integrin receptors that can upregulate MMP-2 (14), but there is no evidence that the integrin receptors can mediate activation of HSCs. The discovery of DDR has great significance for explaining the phenomenon that HSCs have different biological activities under different conditions of ECM. DDRs, a novel subfamily of receptor tyrosine kinases, were discovered during the search for tyrosine kinase proteins expressed in human malignancies (5,6). DDRs can modulate cell responses such as adhesion, migration, differentiation, survival and proliferation, in response to the changes in the amount and configuration of collagen in microenvironment. In in vitro primary culture, activated HSCs induce DDR2 expression, whereas HSCs in period of quiescence restored by matrix gel decreased DDR2 expression (15). Type I collagen can upregulate DDR2 mRNA expression of HSCs and promote HSC tyrosinephosphorylation (15). HSCs with DDR2 overexpression showed stronger cell proliferation and migration activity (15). As a result, a positive feedback mechanism between collagen and HSCs can be formed: collagen induces HSCs activation and DDR2 expression, and HSCs with the overexpression of DDR2 produce more collagen by proliferation and Table 3. Correlations between DDR2 mRNA and liver tissue collagen and serum collagen markers in the study group
Collagen Type I Type III Type IV PCIII CIV HA Laminin
DDR mRNA
DDR protein
0.733a 0.799b 0.799a 0.618a 0.794b 0.676b 0.609a
0.833a 0.899b 0.861a 0.570a 0.861b 0.649b 0.531a
PCIII, procollagen type III; CIV, collagen type IV; HA, hyaluronic acid. a p !0.05. b p !0.01.
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activation. The in vitro results are in line with our in vivo study on liver fibrosis, indicating DDR2 may regulate the biological activation of HSCs, leading to liver fibrosis in response to the changes in collagen contents after alcohol-induced liver injury. In the present study, real-time fluorescence quantitative PCR and Western blot were used to detect the expression of DDR2 mRNA and protein of rat liver tissues. We found that the expression of DDR2 in the study groups increased gradually with time. Further analysis on DDR2 and liver fibrosis found that DDR2 expression level was closely related to the degrees of fibrosis. Correlation analysis showed that the expression of DDR2 mRNA was positively related to the areas of type I, III and IV collagen, as well as serum concentration of HA, laminin, procollagen II and collagen IV. The pathological imaging analysis of collagen and immunohistochemistry staining of liver tissues showed that type III collagen increased mainly in the early stage of liver fibrosis and distributed along the sinusoidal walls. Whereas laminin and collagen type IV produced by HSCs were the main components of basement membrane of the sinusoidal walls, they may have prompted DDR2regulated HSC activity, possibly by combining with type III collagen and releasing more type IV collagen and laminin in Disse space. The increase in type III collage and laminin may have caused reconstitution of basement membrane and formation of basement membrane under the sinusoidal endothelium. Comparative analysis between DDR2 expression and ultrastructural changes of liver tissues found that with increased DDR2 expression, the hepatocellular injury was aggravated, and the fiber bundles in the Disse space and cell compartment were increased. These results suggest that DDR2 has an intrinsic link to liver cell injury and the collagen deposition in ECM. In summary, this study in rats has demonstrated that there is an enhanced expression of DDR2 in the fibrotic liver tissues. The expression of DDR2 mRNA and protein is closely associated with the liver collagens and their serum biomarkers. It is likely that that during the development of alcoholic liver fibrosis, liver injury and the changes of matrix elements in Disse space induced
DDR2 expression. Whether therapeutic intervention on collagen-DDR2 pathway is able to reduce or prevent alcoholic liver fibrosis warrants further investigation.
References 1. Li D, Friedman SL. Liver fibrogenesis and the role of hepatic stellate cells: new insights and prospects for therapy. J Gastroenterol Hepatol 1999;14:618e633. 2. Kisseleva T, Brenner DA. Role of hepatic stellate cells in fibrogenesis and the reversal of fibrosis. J Gastroenterol Hepatol 2007;22(suppl 1): S73eS78. 3. Schuppan D, Ruehl M, Somasundaram R, et al. Matrix as a modulator of hepatic fibrogenesis. Semin Liver Dis 2001;21:351e372. 4. Labrador JP, Azcoitia V, Tuckermann J, et al. The collagen receptor DDR2 regulates proliferation and its elimination leads to dwarfism. EMBO Rep 2001;2:446e452. 5. Ichikawa O, Osawa M, Nishida N, et al. Structural basis of the collagen-binding mode of discoidin domain receptor 2. EMBO J 2007;26:4168e4176. 6. Springer WR, Cooper DNW, Barondes SH. Discoidin I is implicated in cell-substratum attachment and ordered cell migration of dictyostelium discoideum and resembles fibronectin. Cell 1984;39:557e564. 7. Vogel W, Gish GD, Alves F, et al. The Discoidin domain receptor tyrosine kinases are activated by collagen. Mol Cell 1997;1:13e123. 8. Shrivastava A, Radziejewski C, Campbell E, et al. An orphan receptor tyrosine kinase family whose members serve as nonintegrin collagen receptors. Mol Cell 1997;1:25e34. 9. Alves F, Vogel W, Mossie K, et al. Distinct structural characteristics of discoidin I subfamily receptor tyrosine kinases and complementary expression in human cancer. Oncogene 1995;10:609e618. 10. Schlessinger J. Direct binding and activation of receptor tyrosine kinases by collagen. Cell 1997;91:869e872. 11. Li D, Friedman SL. Liver fibrogenesis and the role of hepatic stellate cells: New insights and prospects for therapy. J Gastroenterol Hepatol 1999;14:618e633. 12. Ankoma-Sey V, Matli M, Chang KB, et al. Coordinated induction of VEGF receptors in mesenchymal cell types during rat hepatic wound healing. Oncogene 1998;17:115e121. 13. Gac¸a MD, Zhou X, Issa R, et al. Basement membrane-like matrix inhibits proliferation and collagen synthesis by activated rat hepatic stellate cells: evidence for matrix-dependent deactivation of stellate cells. Matrix Biol 2003;22:229e239. 14. Vogel WF. Collagen-receptor signaling in health and disease. Eur J Dermatol 2001;11:506e514. 15. Olaso E, Ikeda K, Eng FJ, et al. DDR2 receptor promotes MMP2-mediated proliferation and invasion by hepatic stellate cells. J Clin Invest 2001;1369e1378.
ORIGINAL ARTICLE
Expression of Discoidin Domain Receptors (DDR2) in Alcoholic Liver Fibrosis in Rats Xi-Hong Zhang,a Ming Yan,b Li Liu,c Tie-Jun Wu,a Long-Le Ma,d and Le-Xin Wange a
Intensive Care Unit, Liaocheng People’s Hospital, Liaocheng, P.R. China Department of Gastroenterology, Qilu Hospital, Shandong University, Jinan, P.R. China c Jinan Infectious Diseases Hospital, Jinan, P.R. China d Department of Cardiology, Liaocheng People’s Hospital, Liaocheng, P.R. China e School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, Australia
b
Received for publication June 26, 2010; accepted October 18, 2010 (ARCMED-D-10-00327).
Background and Aims. We undertook this study to evaluate the expression of discoidin domain receptor 2 (DDR2) and its relationship with alcoholic liver fibrosis. Methods. Liver fibrosis was induced by intragastric administration of alcohol in 30 rats. Pathological changes and ultrastructure of the liver were studied. The expression of DDR2 mRNA and protein was detected by RT-PCR and Western blot, respectively, at weeks 12, 16 and 20 during the alcohol administration. Results. In the control group (con) (n 5 10), DDR2 mRNA expression and DDR2 protein were 1.02 0.13 (con ratio x103) and 0.32 0.03, respectively. In the study groups there was a progressive increase in DDR2 mRNA expression from weeks 12, 16 and 20 (3.64 1.69, 8.34 2.39, 15.73 4.57 con ratio x103, p !0.05). There was also a progressive increase in DDR2 protein from weeks 12e20 (0.48 0.05, 0.74 0.06 and 0.99 0.05, p !0.05). The mean DDR2 mRNA and protein in the three study groups was higher than in the control group ( p !0.01). The expressions of DDR2 mRNA and protein were positively correlated with collagen type I, III and IV in liver tissues as well as with the serum biomarkers of liver fibrosis, collagen type III, hyaluronic acid, collagen type IV and laminin ( p !0.01). Conclusions. The expression of DDR2 in this alcohol-induced liver fibrosis rat model is enhanced. The expression of DDR2 is closely associated with collagens in the fibrotic liver tissues. Ó 2010 IMSS. Published by Elsevier Inc. Key Words: Hepatic fibrosis, Discoidin domain receptor, Collagen, Procollagen.
Introduction Liver fibrosis is a common chronic disease and is related to excessive intrahepatic deposition of extracellular matrix (ECM). Hepatic stellate cell (HSC) is the main source cell of ECM in the liver (1), and its activation is the key factor for liver fibrosis (2e4). Discoidin domain receptors (DDR) are a new subgroup of the receptor tyrosine kinase family recently discovered (5,6). DDR can be activated by collagen type I and III of ECM (7e10). Previous studies
showed that the activated HSCs express DDR2 mRNA (11,12), suggesting that DDR2 may further activate HSC by combining with collagenous fibers, thus participating in the development of liver fibrosis. Little is known about the relationship between DDR2 and liver fibrosis. The purpose of this study was to investigate the role of DDR2 in the pathogenesis of liver fibrosis in a rat model. Materials and Methods Materials
Address reprint requests to: Ming Yan, PhD, Department of Gastroenterology, Qilu Hospital, Shandong University, Jinan 251200, Shandong Province, P.R. China; Phone: þ8613708937968; FAX: þ86 53182169171; E-mail: [email protected]
The origin of the research materials are as follows: olive oil and Beijing starking erguotou liquor (Wal-Mart, Jinan, China); Pyrazole (Kangda Chemical, Changzhou, China);
0188-4409/$ - see front matter. Copyright Ó 2010 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2010.10.010
DDR2 in Alcoholic Liver Fibrosis
587
Table 1. Serum markers for liver fibrosis Group Control (n 5 10) Study 1 (n 5 10) Study 2 (n 5 10) Study 3 (n 5 10)
HA (ng/mL) 68.48 123.67 148.08 253.91
26.58 10.96a 13.32a 13.26a
Laminin (ng/mL) 20.73 24.91 30.74 38.87
3.48 3.66b 2.48b 3.58b
PC III (ng/mL) 14.86 21.71 26.24 35.43
1.75 1.37a 2.37a 2.32a
CIV (ng/mL) 93.08 142.01 220.22 289.77
7.77 12.22a 10.98a 11.54a
HA, hyaluronic acid; PC III, procollagen type III; CIV, collagen type IV. a p !0.01. b p !0.05 compared with control group.
hyaluronic acid, procollagen type III and collagen type IV radioimmunoassay kits (Haiyan Medical Biotech, Shanghai, China); collagen type I, III and IV rabbit anti-mouse polyclonal antibody (Boshide Bioengineering, Wuhan, China); goat anti-mouse DDR2 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA); and PCR primers (Yingjun Biotech, Shanghai, China). Animals This study was approved by the institutional review board of Liaocheng People’s Hospital. Forty male Wistar rats (6 weeks old, body weight 180 10 g) were obtained from the Experimental Animal Center of Shandong University. Rats were randomly divided into study and control groups. Both groups were fed with standard food adding olive oil. The study group was given intragastric alcohol infusion to induce liver fibrosis. The concentration and amount
of alcohol was increased gradually as follows: 4.5 g/kg/ day during weeks 1e4, 6.5 g/kg/day during weeks 5e8, 9 g/kg/day during weeks 9e12, and 9.6 g/kg/day during weeks 13e20. The daily dose of alcohol was administered in two divided doses, 12 h apart. Control group was given the same volume of saline. Study group animals were euthanized by on the 12th (study group 1, n 5 10), 16th (study group 2, n 5 10), and 20th week (study group 3, n 5 10), respectively. Control group (n 5 10) was euthanized at the 12th week. Euthanasia was achieved by IP injection of sodium pentobarbital to the right lower abdomen in order to avoid causing pain by hitting the liver or any other organs. Serological Test Whole blood was obtained by cardiac puncture and serum was separated to test the amount of hyaluronic acid (HA),
Figure 1. (a) Control group showing a normal hepatic lobule structure. (b) Inflammatory cell infiltration in portal areas with fibrous tissue proliferation. (c) Wide hepatocyte ballooning degeneration around the central veins. (d) A large amount of fibrous tissue around central veins and in portal areas. (A color figure can be found in the online version of this article.)
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Zhang et al./ Archives of Medical Research 41 (2010) 586e592
laminin, procollagen type III and collagen type IV with type GC-9 g-radiation immunity arithmometer. All measurements were conducted according to the manufacturer’s instructions. Histopathology and Immunohistochemistry Liver tissue samples were harvested from the right hepatic lobe and were fixed with 4% paraformaldehyde solution. Paraffin-embedded sections were stained with HE and Masson. Paraffin-embedded sections were added with 3% hydrogen peroxide and incubated in room temperature to block endogenous peroxidase activity. The sections were washed three times with distilled water followed by citrate buffer microwave repair antigen. The first antibodies (1:120 diluted concentration of I, III, IV) were added and kept at 4 C overnight. The second antibodies were added and sections were incubated at room temperature for 1 h before staining with hematoxylin. The contents of collagen I, III and IV were also determined in the three study groups. Hepatic tissue sections were viewed with JD801 image analysis system (Jiangsu JEDA Science-Technology, Nanjing, China). For each type of collagen, areas of the collagen were measured from five randomly selected fields in each section, and the mean area of the five measurements was used.
dehyde. After 2e3 min, samples were trimmed into 1 mm3 pieces and added with fresh 3% glutaraldehyde. After rinsing, tissue samples were fixed in OsO4, followed by further rinsing and dehydration. The samples were then embedded in Epon 812 epoxy resin before ultrathin sections were obtained. Ultrathin sections were stained with lead citrate and uranyl acetate. Ultrastructures of these sections were visualized by JEM-1200EX electron microscope. DDR2 mRNA Expression DDR2 mRNA expression was detected by fluorescence quantitative PCR. Total RNA in liver tissues was extracted according to Trizol instructions, with reverse transcription after the quantitative by UV spectrophotometer. DDR2 primer sequence: upstream primer 50 TAACCGACATGGG AAACA 30 ; downstream primer 50 TAACCGACATGGG AAACA 30 , amplified fragment 106 bp. b-actin primer sequence: upstream primer 50 AAGATCCTGACCGAGC GTGG 30 ; downstream primer 50 CAGCACTGTGTTGG CATAGAGG 30 , amplified fragment 327 bp. All primers were designed by Primer 5.0 and the specific products were verified by NCBI Blast. The melting curve was analyzed after amplification, and corresponding copies were calculated according to standard curves.
Specimen Preparation of Electron Microscope
DDR2 Protein Expression by Western Blot
Tissue samples of the right hepatic lobe were washed rapidly in phosphate buffer and were fixed in 3% glutaral-
Liver tissue (150 mg) was cut into pieces and mixed with 300 ml of protein extracts and 10 mL of proteinase inhibitor.
Figure 2. (a) Control group showing a small amount of collagen fibers located in sinusoid wall around the central veins. (bed) Progressive increase in fibrous tissues. (A color figure can be found in the online version of this article.)
DDR2 in Alcoholic Liver Fibrosis
After ice bath for 30 min and centrifugation, supernatant was collected for analysis. Equal amounts of protein and loading buffer were mixed before SDS-PAGE electrophoresis (120 V, 0.5 h) and transblotting (200 mA, 3 h) at room temperature for 1 h. The samples were incubated, respectively, with anti-DDR2 antibody (1:250 at 4 C) and horseradish peroxidase-labeled secondary antibody (1:1250, vibrating and shaking at room temperature for 1 h). The membrane was washed three times before diaminobenzidine coloring for 3e10 min. Finally, photographs were taken with Fluorchem 9900 imaging system. DDR2 protein was quantified by first scanning the gel with JIEDA 801 imaging system (JIEDA, Nanjing, China). The intensity of each band on Western blot was quantified by densitometry, and the ratio of DDR2 protein and internal control was calculated. Statistical Analysis Data are expressed as mean SE. All data were processed by SPSS for Windows (v.11.0) software. One-way ANOVA was used to compare means of multiple groups, and leastsignificant difference test was used in multiple comparisons. Pearson’s correlation was used to analyze the relationship between DDR2 mRNA or DDR2 protein and hepatic collagen I, III and IV, as well as serum biomarkers for collagen. Statistical significance was set at p !0.05.
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Results Serum Indices for Hepatic Fibrosis Serum hyaluronic acid, procollagen III, collagen type IV and laminin concentration in the study groups were higher than in the control group (Table 1, p !0.01 and 0.05, respectively).
Histopathological Findings The control group showed a normal hepatic lobule structure (Figure 1a). In study group 1 (Figure 1b), there was inflammatory cell infiltration in portal areas with fibrous tissue proliferation. In study group 2 (Figure 1c), there was wide hepatocyte ballooning degeneration around the central veins accompanied by necrosis, inflammatory cell infiltration and fibrous tissue proliferation. In study group 3 (Figure 1d), a large amount of fibrous tissues was present in the periportal area. Masson staining displayed collagen fibers in blue, muscle fibers in red and red blood cells in orange. There was only a small amount of collagen fibers in the control group (Figure 2a), located in the sinusoid wall around the central veins. There was a gradual increase in fibrous tissue over time in the study group, with fibrous tissue mainly located in hepatic acinus (Figures 2be2d).
Figure 3. (a) Control group showing weak expression of collagen fibers. (b) Type III collagen mainly found in liver cytoplasm around central veins and fibrous septum. (c) Type I collagen in periportal arterial and venous wall and sinusoid. (d) Type IV collagen located in the basement membrane of central vein, portal area, hepatic artery and bile duct. (A color figure can be found in the online version of this article.)
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Immunohistochemistry staining showed a weak expression of collagen fibers in control group, with type I collagen mainly located in portal areas and around the veins. Type III collagen was found along the sinusoidal walls, and type IV collagen in bile duct and basement membrane of vascular walls (Figure 3a). In the study groups there was a strong expression of all types of collagen, with type III collagen mainly found in liver cytoplasm around the central veins and fibrous septum (Figure 3b). Type I collagen was located in periportal arterial and venous wall, and in the sinusoid (Figure 3c). Type IV collagen was located in the basement membrane of the central vein, portal area, hepatic artery and the bile duct (Figure 3d). Ultrastructure In the control group the connection between liver cells was close, with visible bile canaliculi and a small amount of collagen fibers. Organelles were abundant and well developed, endoplasmic reticulum and mitochondria distributed normally, and nuclei were round and large (Figure 4a). Study Group 1 Interhepatocytes and perisinusoid appeared fasciculus arranged interlacing, and the Disse space was filled with coarse hepatocyte microvilli, which caused stenosis and densifica-
tion of the Disse space (Figure 4b). The boundary between liver cells and Disse space was unclear. The cell matrix had many petal-shaped glycogen granules (Figure 4b). Study Group 2 Intranuclear heterochromatin showed massive diffuse distribution without obvious nucleolar structure (Figure 4c). The perinuclear gaps were widened and mitochondria were not visible. Cytoplasmic mitochondria and endoplasmic reticulum were decreased, with bullous fat droplets (Figure 4c). Study Group 3 The size of the liver cells was reduced with increased intercellular space (Figure 4d). Liver parenchymal cells and matrix were electron dense, with few microvilli in the Disse space and sinusoid. Compared to blood sinus cells, collagen tissue was at a lower functional state, such as apoptotic changes. Their nuclei were pyknotic to form clumps, with increased heterochromatin and decreased euchromatin and without the nucleolus structure. Cytoplasmic mitochondria were intensive and irregular, whereas cristae became vague and lost (Figure 4d). Expression of DDR2 mRNA and Protein The expression of DDR2 mRNA and protein in the control and three study groups is shown in Figure 5. DDR2 mRNA
Figure 4. (a) Normal liver cell ultrastructure in the control group. (b) Interhepatocytes and perisinusoid appeared fasciculus arranged interlacing (arrow 1), the Disse space was filled with coarse hepatocyte microvilli (arrow 2). The cell matrix had many petal-shaped glycogen granules (arrow 3). (c) Intranuclear heterochromatin showed massive diffuse distribution without obvious nucleolar structure. Perinuclear gaps were widened (arrow 1), cytoplasmic mitochondria and endoplasmic reticulum was decreased (arrow 2). (d) The size of the liver cells was reduced with increased cell space. Few microvilli in the Disse space and sinusoid; collagen tissue was in function of the lower state. Their nuclei were pyknotic to form into clumps (arrow 2), with increased heterochromatin and decreased euchromatin, and without the nucleolus structure. The cytoplasmic mitochondria were intensive and irregular, whereas cristae became vague and lost (arrow 1).
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Figure 5. Western blotting of DDR2 protein (a) and b-actin protein. (b) 1: control group; 2: study group 1; 3: study group 2; 4: study group 3.
and protein expression in the study groups was higher than in the control group ( p !0.01, Table 2). The expression of DDR2 mRNA and protein in study groups 2 and 3 were higher than in study group 1 ( p !0.01, Table 2). The expression of DDR2 mRNA and protein was correlated with hepatic type I, III and IV collagen and serum biomarkers procollagen type III, collagen type IV, hyaluronic acid and laminin (Table 3, p !0.01). Discussion Alcoholic liver fibrosis, a result of alcoholic liver injury, is characterized by excessive accumulation of ECM in the liver. It is long believed that alcoholic liver fibrosis is a passively and irreversibly pathological process induced by the necrosis of liver parenchymal cells. In the present study, liver tissue examination at three time points of the alcohol-induced liver fibrosis showed progressive increase in collagen tissues in the liver, suggesting connective tissue proliferation plays a key role in initiating alcoholic liver fibrosis. HSCs have been identified as major collagen-producing cells in the injured liver (1), and activation of HSCs has become the central link for liver fibrosis (2). The factors that regulate the biological behavior of HSCs include soluble regulatory factors and nonsoluble regulatory factors, the latter mainly refer to ECM itself. The interaction between HSCs and ECM alters the ability of HSCs to create ECM, which means ECM can directly regulate the activation of HSCs. A previous study has shown that HSCs have no active proliferative activity in the matrix culture simulating the environment of Disse space and produce only small amounts of ECM (13). In the plastic Table 2. Expression of DDR2 mRNA and protein in liver tissues DDR2 mRNA (con ratio 103) Control (n 5 10) Study group 1 (n 5 10) Study group 2 (n 5 10) Study group 3 (n 5 10)
1.02 3.64 8.334 15.73
p !0.01 compared with control group.
a
0.13 1.69a 2.39a 4.57a
DDR2 protein 0.32 0.48 0.74 0.99
0.03 0.05a 0.06a 0.05a
culture dish without any packets, HSCs show active proliferation and may gradually transform into myofibroblasts with activation state, generating a large amount of collagen (13). HSCs produce different ECM components when cultured in different types of single collagen matrix. This ECM-HSC interaction was previously attributed to integrin receptors that can upregulate MMP-2 (14), but there is no evidence that the integrin receptors can mediate activation of HSCs. The discovery of DDR has great significance for explaining the phenomenon that HSCs have different biological activities under different conditions of ECM. DDRs, a novel subfamily of receptor tyrosine kinases, were discovered during the search for tyrosine kinase proteins expressed in human malignancies (5,6). DDRs can modulate cell responses such as adhesion, migration, differentiation, survival and proliferation, in response to the changes in the amount and configuration of collagen in microenvironment. In in vitro primary culture, activated HSCs induce DDR2 expression, whereas HSCs in period of quiescence restored by matrix gel decreased DDR2 expression (15). Type I collagen can upregulate DDR2 mRNA expression of HSCs and promote HSC tyrosinephosphorylation (15). HSCs with DDR2 overexpression showed stronger cell proliferation and migration activity (15). As a result, a positive feedback mechanism between collagen and HSCs can be formed: collagen induces HSCs activation and DDR2 expression, and HSCs with the overexpression of DDR2 produce more collagen by proliferation and Table 3. Correlations between DDR2 mRNA and liver tissue collagen and serum collagen markers in the study group
Collagen Type I Type III Type IV PCIII CIV HA Laminin
DDR mRNA
DDR protein
0.733a 0.799b 0.799a 0.618a 0.794b 0.676b 0.609a
0.833a 0.899b 0.861a 0.570a 0.861b 0.649b 0.531a
PCIII, procollagen type III; CIV, collagen type IV; HA, hyaluronic acid. a p !0.05. b p !0.01.
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activation. The in vitro results are in line with our in vivo study on liver fibrosis, indicating DDR2 may regulate the biological activation of HSCs, leading to liver fibrosis in response to the changes in collagen contents after alcohol-induced liver injury. In the present study, real-time fluorescence quantitative PCR and Western blot were used to detect the expression of DDR2 mRNA and protein of rat liver tissues. We found that the expression of DDR2 in the study groups increased gradually with time. Further analysis on DDR2 and liver fibrosis found that DDR2 expression level was closely related to the degrees of fibrosis. Correlation analysis showed that the expression of DDR2 mRNA was positively related to the areas of type I, III and IV collagen, as well as serum concentration of HA, laminin, procollagen II and collagen IV. The pathological imaging analysis of collagen and immunohistochemistry staining of liver tissues showed that type III collagen increased mainly in the early stage of liver fibrosis and distributed along the sinusoidal walls. Whereas laminin and collagen type IV produced by HSCs were the main components of basement membrane of the sinusoidal walls, they may have prompted DDR2regulated HSC activity, possibly by combining with type III collagen and releasing more type IV collagen and laminin in Disse space. The increase in type III collage and laminin may have caused reconstitution of basement membrane and formation of basement membrane under the sinusoidal endothelium. Comparative analysis between DDR2 expression and ultrastructural changes of liver tissues found that with increased DDR2 expression, the hepatocellular injury was aggravated, and the fiber bundles in the Disse space and cell compartment were increased. These results suggest that DDR2 has an intrinsic link to liver cell injury and the collagen deposition in ECM. In summary, this study in rats has demonstrated that there is an enhanced expression of DDR2 in the fibrotic liver tissues. The expression of DDR2 mRNA and protein is closely associated with the liver collagens and their serum biomarkers. It is likely that that during the development of alcoholic liver fibrosis, liver injury and the changes of matrix elements in Disse space induced
DDR2 expression. Whether therapeutic intervention on collagen-DDR2 pathway is able to reduce or prevent alcoholic liver fibrosis warrants further investigation.
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