Cell Biochem Biophys (2014) 70:1803–1806 DOI 10.1007/s12013-014-0132-0
ORIGINAL PAPER
Investigation on Etiology of Hepatic Venous Obstruction Budd-Chiari Syndrome Zhi-Long Tian • Gao-Lei Jia • Hai-Lin Xi Su Feng • Xiao-Kai Wang • Rui Li
•
Published online: 6 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Budd-Chiari syndrome (BCS) is an uncommon clinical condition with a complex etiology. Pathogenesis of BCS is still poorly understood. We included hepatic venoocclusive lesion tissues of 20 patients (patients group) with hepatic venous obstruction BCS and compared with 20 similar tissues with other etiologies (control group). Morphological changes in hepatic veno-occlusive lesion tissues and the positive expression of proliferating cell nuclear antigen (PCNA), C-myc, and P-53 were observed by the pathological examination (H&E staining) and immunohistochemistry assay. Our results showed that PCNA and C-myc positive cell densities were significantly higher in patient group than control group. P-53 positive cell density showed increasing trends in patients than control group. Moreover, we observed irregular hyperplasia in intimal tissue, fibrous connective tissue, and smooth muscle cell, accompanied by tissue degeneration (hyaloid degeneration and fibrinoid degeneration) and a large quantity of inflammatory cell infiltration. In conclusion, an overexpression of PCNA, C-myc, and a weak positive expression of P53 might launch the extremely irregular hepatic venous intimal hyperplasia, which is probably one of the etiologies of hepatic venous obstruction BCS.
Keywords Budd-Chiari syndrome Hepatic venous occlusion Intimal proliferation
Introduction Budd-Chiari syndrome (BCS) is an uncommon clinical condition induced by thrombotic or non-thrombotic obstruction to hepatic venous outflow. It presents with the classical triad of abdominal pain, ascites, and hepatomegaly. The etiology of BCS is very complex, and varies with different region, nationality, gender, and age. Its prevalence is 1 out of a million individuals [1]. Although traditional management of BCS involves thrombolysis or surgical portosystemic shunt creation, an increasing number of successful reports of BCS therapy have involved endovascular techniques, including angioplasty and stent placement [2]. Therefore, clinical treatment and improvement in diagnosis of BCS is very much needed. We have also conducted a preliminary study and reported an overexpression of PCNA, C-myc, and a weak positive expression of P53 in BCS patients.
Materials and Methods Z.-L. Tian G.-L. Jia H.-L. Xi (&) S. Feng X.-K. Wang Department of General Surgery, Xuzhou Central Hospital, Xuzhou Clinical School of Xuzhou Medical College, Affiliated Hospital of Southeast University, Xuzhou 221009, Jiangsu, China e-mail: [email protected]; [email protected] R. Li Department of Central Laboratory, Xuzhou Central Hospital, Xuzhou Clinical School of Xuzhou Medical College, Affiliated Hospital of Southeast University, Xuzhou 221009, Jiangsu, China
Patients In this study, from October 1993 to May 2006, the occlusive hepatic vein and membranous tissue of 20 cases with hepatic venous obstruction BCS have been excised. Patients were all farmers, including 16 males and 1 female with an average age of 34 years (rage 22–48 years). The control group was recruited from the Pathology Anatomy Room of People’s Liberation Army General Hospital, to obtain 20
123
1804
cases with normal hepatic vein from fresh corpses by pathological anatomy, including same gender and age with that of the patients. All individuals in this study were enrolled after giving their written informed consent. Diagnostic Criteria Wang Zhonggao classification was commonly used as diagnostic criteria of hepatic venous obstruction BCS, namely (1) inferior vena cava obstruction (type I), (2) hepatic venous obstruction (type II), and (3) mixed type, i.e., obstruction in varying degrees of the inferior vena cava and hepatic vein (type III). The selected hepatic venous obstruction BCS in this study was included of type II and III. Patients had clinical symptoms of portal hypertension or combination with inferior vena cava hypertension symptoms, which were diagnosed as BCS with color ultrasound, CT, and MRI or tests of interventional ultrasound, interventional angiography, and contrast-enhanced hepatic vein percutaneous liver puncture. All patients were diagnosed before operation, then confirmed intraoperatively, and then final confirmation was carried out by postoperative pathological examination of paraffin sections. Immunohistochemical Staining Immunohistochemical staining for proliferating cell nuclear antigen (PCNA), C-myc, and P53 was performed on paraffin-embedded, formalin-fixed liver tissues. Briefly, 4 lm-thick consecutive tissue sections were deparaffinized in xylene and rehydrated in graded alcohols. Antigen retrieval was done by heating slides in adequate buffer using water bath or microwave oven. Non-specific protein binding was blocked by 30-min incubation in phosphatebuffered saline (PBS) pH 7.4, with 2% goat serum. Endogenous peroxidase activity was blocked by 1% hydrogen peroxide (H2O2) for 5 min. Slides were alternatively rinsed with PBS (pH 7.4) followed by incubation with primary monoclonal antibodies, PCNA, C-myc, and P53 antibody (all were from Beijing Zhongshan Biological Technology Co., LTD). Slides were fixed with 0.05% glutaraldehyde in PBS pH 8.6, followed by incubation with biotinylated secondary antibody (labeled polymer HorseRadish Peroxidase anti-mouse antibody, K4000, Dako, Glostrup, Denmark), and finally stained with DAB chromogen and counterstained with hematoxylin and mounted. Immuno-Positive Cell Identification and Counting Proliferating cell nuclear antigen was localized in the nucleus, presenting with the yellow granule sediments in
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Cell Biochem Biophys (2014) 70:1803–1806
staining picture as the positive expression. C-myc antigen positive staining was also shown with brown nucleus, sometimes with brown cytoplasm, and P53 antigen located in the nucleus was shown with the stained brown nuclei as a positive expression. 10 fields of each slice were randomly chosen, and the total of 1,000 intimal cells were counted to calculate the percentage number of positive cells. Cumulative count of all the slices were averaged as the positive rate and processed by the statistics with the control group. Statistical Analysis Rank sum test and v2 test (Chi-squared test) were used to compare the quantitative variables between groups. P values less than 0.05 were considered significant. Statistical analysis was performed by using SPSS 15.0 software (SPSS Inc., Chicago, IL, USA).
Results Pathological Observations Irregular hyperplasia of intimal tissue, local excessive hyperplasia, and extreme thickening of intima, incomplete elastic fibers, and scattered smooth muscle cells were observed in the patient group as compared to the control group. Moreover, a large number of fibrous connective tissue hyperplasia, partial denaturation mainly on fibrosis, hyaloid degeneration, hyaline degeneration and fibrinoid degeneration, partially calcified necrosis accompanied by a large number of inflammatory cell infiltration were found in patients’ samples. In addition, pathological section of organized thrombosis and stasis cirrhosis were also observed in some cases. Immunohistochemical Detection of PCNA, C-myc and P-53 Our results indicated that more PCNA, C-myc, and P-53 positive cells were present in the hepatic vein occlusive tissue in the patient group compared to that of the control group. Specially they were higher in numbers in the intimal tissues indicating their proliferative actively. Our quantitative analysis of these markers demonstrated that PCNA and C-myc positive cell densities were three or more folds higher in patient group than control group which made the difference very significant (P \ 0.001) between both the study groups. Moreover, P-53 positive cell density showed increasing trends in patients than control group but the difference did not reach to the significant level (Table 1).
Cell Biochem Biophys (2014) 70:1803–1806
1805
Table 1 Quantitative positive expression of PCNA, C-myc, and P-53 in pathological tissues Groups
N
PCNA
C-myc
P-53
Patient group
20
25 ± 2.78*
15 ± 2.38*
6 ± 2.79
Control group
20
8 ± 1.35
5 ± 2.23
4 ± 0.178
*P \ 0.001
Discussion Budd-Chiari syndrome can cause partial stenosis or complete occlusion of hepatic vein or hepatic segment of inferior vena cava, leading to obstruction of hepatic venous reflux and inferior vena cava reflux, and thus resulting in portal hypertension (PHT) and inferior vena caval hypertension (IVCHT). Etiology of BCS is very complex, varying with different regions, nationality, gender, and age. Studies have shown that the etiology of hepatic venous stenosis or occlusion are closely associated with the following conditions: (1) Thrombosis: hypercoagulable states and anti-prothrombin gene deficiency is the basis for venous thrombosis, proved by a large number of clinical practices. (2) Septum formation: hepatic venous stenosis and membranous obstruction caused by congenital heteroplasia. This doctrine is mainly supported by the onset in more infants and young while also in the twin sisters. (3) Other issues such as mechanical injury, vascular local inflammation, and primary tumor compression [3–5]. In recent years, studies on cell proliferation have attracted widespread attention, with more focuses on intimal proliferation, apoptosis, and related gene expression. Studies have shown that vascular smooth muscle cell proliferation and imbalance between cell proliferation and apoptosis are the main reasons of vascular restenosis after vascular grafting and percutaneous transluminal angioplasty (PTA). With the improvement of medical technology and extensive development of occlusive BCS radical resection under direct vision, a variety of macroscopic and microscopic tissue structures can be observed systematically and in detail, such as visible inferior vena cava thrombosis or tumor thrombus, local stenosis, membranous tissue, scar tissue, staggered, long segmental, or skipping hepatic venous stenosis. However, there are less available clinical data of research on cell pathology of BCS local lesions, especially hepatic veno-occlusive lesions of BCS. Overexpression of PCNA and C-myc in patient’s lesions can act as the initiating factor causing irregular and extreme intimal hyperplasia. PCNA is a kind of nucleoprotein required for synthesis of eukaryotic DNA, which are auxiliary proteins of DNA polymerase d. A small amount of PCNA in quiescent cells make them enter into the proliferation cycle, so, PCNA is an important indicator
for detecting the cell proliferation [6, 7]. The vascular smooth muscle cells’ PCNA expression after vascular grafting has also been studied. PCNA expression appears immediately during anastomotic cell proliferation, repairing, and reconstruction after vascular grafting, especially the most intense around the anastomosis. As PCNA quantitative expression can objectively reflect the degree and sites of cell proliferation, therefore, there are no PCNA positive expressions in vast majority of normal vascular tissue [8]. In this study, we used PCNA as to quantify and to localize the proliferative sites. Our results showed a significantly higher PCNA expression in the lesions of patient group than that of the control group, indicating the presence of severe cell proliferation in hepatic vein lesions. C-myc is an important gene that regulates cell proliferation, and in human it is located on chromosome 8 along with N-myc and L-myc. They are transcription regulatory factors located in the nucleus. C-myc gene belongs to the immediate early gene, which can be stimulated by the specific growth signals to enter into the cell proliferation. For instance, platelet-derived growth factor (PDGF) and ConA can cause C-myc expression increased by 20–40 times in lymphocyte and quiescent fibroblasts [9, 10]. Therefore, C-myc expression is the foundation to make the resting cells into the proliferative state. In vitro experiments have proved that the important function of C-myc is to promote cell division. Overexpression of C-myc makes cells no longer respond to the growth retention signal, and allows the cells to come out of the dormancy stage into continued proliferation status. Currently, it is considered that the activation of C-myc transcription simultaneously initiates the cell proliferation and apoptosis [9, 10]. Bennett et al. [9] found that, in proliferating cells, intracellular C-myc gene expression increased and P53 expression decreased. Tao et al. [10] found that while proliferation of pulmonary vascular wall cells in chronic hypoxic rats, C-myc gene expression increased and expression of P53 gene decreased. Our results also showed similar correlation between C-myc and P-53. As a transcription factor, P53 is combined with specific DNA sequences in the form of a tetramer to regulate the expression of the target gene. Wild-type P53 is a growth inhibitory protein, and its overexpression can inhibit the growth of normal and transformed cells, leading to G1 arrest in the cell cycle. When the cell DNA damages, P53 negatively regulates the cell cycle and inhibits late G1 gene transcription, in order to ensure the stability of gene expression and fidelity of cell division [11, 12]. In this study, a strong positive expression of C-myc and a weak positive expression of P53 is also a trustworthy evidence for cell intimal reactive hyperplasia. Extremely irregular local intimal hyperplasia of lesions is the fundamental pathological feature of BCS. We
123
1806
observed that there are irregular hyperplasia in intimal tissue, fibrous connective tissue, and smooth muscle cell, accompanied by tissue degeneration (hyaloid degeneration and fibrinoid degeneration) and a large quantity of inflammatory cell infiltration. The hyperplastic intimal tissue contains more vascular endothelial cells and fibroblasts, while the hyperplastic smooth muscle cells in varying amounts can be observed. It is believed that the irregular local intimal hyperplasia in lesions is the fundamental pathological feature of BCS with hepatic venous obstruction. Long-term heavy physical labor or trauma may be a predisposing factor in the formation of BCS. Data show that the second hepatic portal is the position in which inferior vena cava passes through the diaphragm into the chest [13, 14]. The contrast between intra-abdominal positive pressure and intra-thoracic negative pressure, plus constant movement of the diaphragm, will lead to intimal damage of hepatic vein and inferior vena cava. Imbalance in gene regulation occurs in the process of damaged cell repairing and proliferation, thereby resulting in hepatic venous intimal proliferation, secondary hepatic venous stenosis, or occlusion and obstruction of hepatic venous blood flow. All the cases of this group are young farmers, during engaging in long-term heavy physical labor, breathing deeply and fast will cause the damage of second hepatic portal vascular intima and the formation of secondary BCS, in which a variety of mechanisms remain to be further discussed and investigated.
References 1. Rajani, R., Melin, T., Bjornsson, T., et al. (2009). Budd-Chiari syndrome in Sweden: epidemiology, clinical characteristics and survival—an 18-year experience. Liver International, 29, 253–259. 2. Bozorgmanesh, A., Selvam, D. A., & Caridi, J. G. (2007). BuddChiari syndrome: Hepatic venous web outflow obstruction treated
123
Cell Biochem Biophys (2014) 70:1803–1806
3.
4.
5.
6.
7.
8. 9.
10.
11.
12.
13.
14.
by percutaneous placement of hepatic vein stent. Seminars in Interventional Radiology, 24, 100–105. Rha, S. E., Lee, M. G., Lee, Y. S., et al. (2002). Nodular regenerative hyperplasia of the liver in Budd-Chiari syndrome. CT and MR features. Abdominal Imaging, 25, 255–258. Nakase, H., Kawasaki, T., Itani, T., et al. (2001). Budd-Chiari syndrome and extrahepatic portal obstruction associated with congenital antithrombin III deficiency. Journal of Gastroenterology, 36, 341–345. Sipahi, T., Duru, F., Yarah, N., et al. (2001). Compound heterozygosity for factor V Leiden and prothrombin G20210A mutations in a child with Budd-Chiari syndrome. European Journal of Pediatrics, 160, 198. Liu, Y. Y., Zhang, Y., Huang, Y. G., et al. (2003). Relationship between expression of p53, nm23, PCNA and invasion and metastasis of colon carcinoma. Guangdong Medical Journal, 4, 381. Bian, J. F., Zhang, B. G., & Chen, S. S. (1999). Antisense oligonucleotides inhibit c-myc and PCNA expression in the vascular smooth muscle cells. Zhonghua Yi Xue Za Zhi, 79, 10–14. Bioulac-Sage, P., & Balabaud, C. (2001). Liver nodules resembling focal nodular hyperplasia. Journal of Hepatology, 34, 781. Bennett, M. R., Evan, G. I., & Newby, A. C. (1994). Deregulated expression of the c-myc oncogene abolishes inhibition of proliferation of rat vascular smooth muscle cells by serum reduction, interferon-gamma, heparin, and cyclic nucleotide analogues and induces apoptosis. Circulation Research, 74, 525–536. Tao, Q. G., Zhang, Z. X., & Xu, Y. J. (1999). Investigation on c-myc and p53 gene expression and proliferation, apoptosis of pulmonary vascular wall cells in chronic hypoxic rats. Chinese Journal of Applied Physiology, 15, 169. Huang, X. R., Lai, R. Q., Wang, Z. C., et al. (2000). The relationship between the expression of p16, p53, Rb gene protein or proliferating nuclear antigen and clinicopathologic features of human gastric cancer. Chinese Journal of Cancer, 19, 899. Riedel, F., Gotte, K., Schwalb, J., et al. (2002). Vascular endothelial growth factor expression correlates with P53 mutation and angiogenesis in squamous cell carcinoma of the head and neck. Acta Otolaryngologica, 120, 105–111. Kage, M., Arakawa, M., Kojiro, M., et al. (1992). Histopathology of membranous obstruction of the inferior vena cava in the BuddChiars’s syndrome. Gastroenterology, 102, 2081–2090. Aguirrebarrena, G., Pulcinelli, S., Giovannoni, A. G., & Gidekel, L. (2001). Celiac disease and Budd-Chiari syndrome: Infrequent association. Revista Espanola de Enfermedades Digestivas, 93, 611–612.
ORIGINAL PAPER
Investigation on Etiology of Hepatic Venous Obstruction Budd-Chiari Syndrome Zhi-Long Tian • Gao-Lei Jia • Hai-Lin Xi Su Feng • Xiao-Kai Wang • Rui Li
•
Published online: 6 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Budd-Chiari syndrome (BCS) is an uncommon clinical condition with a complex etiology. Pathogenesis of BCS is still poorly understood. We included hepatic venoocclusive lesion tissues of 20 patients (patients group) with hepatic venous obstruction BCS and compared with 20 similar tissues with other etiologies (control group). Morphological changes in hepatic veno-occlusive lesion tissues and the positive expression of proliferating cell nuclear antigen (PCNA), C-myc, and P-53 were observed by the pathological examination (H&E staining) and immunohistochemistry assay. Our results showed that PCNA and C-myc positive cell densities were significantly higher in patient group than control group. P-53 positive cell density showed increasing trends in patients than control group. Moreover, we observed irregular hyperplasia in intimal tissue, fibrous connective tissue, and smooth muscle cell, accompanied by tissue degeneration (hyaloid degeneration and fibrinoid degeneration) and a large quantity of inflammatory cell infiltration. In conclusion, an overexpression of PCNA, C-myc, and a weak positive expression of P53 might launch the extremely irregular hepatic venous intimal hyperplasia, which is probably one of the etiologies of hepatic venous obstruction BCS.
Keywords Budd-Chiari syndrome Hepatic venous occlusion Intimal proliferation
Introduction Budd-Chiari syndrome (BCS) is an uncommon clinical condition induced by thrombotic or non-thrombotic obstruction to hepatic venous outflow. It presents with the classical triad of abdominal pain, ascites, and hepatomegaly. The etiology of BCS is very complex, and varies with different region, nationality, gender, and age. Its prevalence is 1 out of a million individuals [1]. Although traditional management of BCS involves thrombolysis or surgical portosystemic shunt creation, an increasing number of successful reports of BCS therapy have involved endovascular techniques, including angioplasty and stent placement [2]. Therefore, clinical treatment and improvement in diagnosis of BCS is very much needed. We have also conducted a preliminary study and reported an overexpression of PCNA, C-myc, and a weak positive expression of P53 in BCS patients.
Materials and Methods Z.-L. Tian G.-L. Jia H.-L. Xi (&) S. Feng X.-K. Wang Department of General Surgery, Xuzhou Central Hospital, Xuzhou Clinical School of Xuzhou Medical College, Affiliated Hospital of Southeast University, Xuzhou 221009, Jiangsu, China e-mail: [email protected]; [email protected] R. Li Department of Central Laboratory, Xuzhou Central Hospital, Xuzhou Clinical School of Xuzhou Medical College, Affiliated Hospital of Southeast University, Xuzhou 221009, Jiangsu, China
Patients In this study, from October 1993 to May 2006, the occlusive hepatic vein and membranous tissue of 20 cases with hepatic venous obstruction BCS have been excised. Patients were all farmers, including 16 males and 1 female with an average age of 34 years (rage 22–48 years). The control group was recruited from the Pathology Anatomy Room of People’s Liberation Army General Hospital, to obtain 20
123
1804
cases with normal hepatic vein from fresh corpses by pathological anatomy, including same gender and age with that of the patients. All individuals in this study were enrolled after giving their written informed consent. Diagnostic Criteria Wang Zhonggao classification was commonly used as diagnostic criteria of hepatic venous obstruction BCS, namely (1) inferior vena cava obstruction (type I), (2) hepatic venous obstruction (type II), and (3) mixed type, i.e., obstruction in varying degrees of the inferior vena cava and hepatic vein (type III). The selected hepatic venous obstruction BCS in this study was included of type II and III. Patients had clinical symptoms of portal hypertension or combination with inferior vena cava hypertension symptoms, which were diagnosed as BCS with color ultrasound, CT, and MRI or tests of interventional ultrasound, interventional angiography, and contrast-enhanced hepatic vein percutaneous liver puncture. All patients were diagnosed before operation, then confirmed intraoperatively, and then final confirmation was carried out by postoperative pathological examination of paraffin sections. Immunohistochemical Staining Immunohistochemical staining for proliferating cell nuclear antigen (PCNA), C-myc, and P53 was performed on paraffin-embedded, formalin-fixed liver tissues. Briefly, 4 lm-thick consecutive tissue sections were deparaffinized in xylene and rehydrated in graded alcohols. Antigen retrieval was done by heating slides in adequate buffer using water bath or microwave oven. Non-specific protein binding was blocked by 30-min incubation in phosphatebuffered saline (PBS) pH 7.4, with 2% goat serum. Endogenous peroxidase activity was blocked by 1% hydrogen peroxide (H2O2) for 5 min. Slides were alternatively rinsed with PBS (pH 7.4) followed by incubation with primary monoclonal antibodies, PCNA, C-myc, and P53 antibody (all were from Beijing Zhongshan Biological Technology Co., LTD). Slides were fixed with 0.05% glutaraldehyde in PBS pH 8.6, followed by incubation with biotinylated secondary antibody (labeled polymer HorseRadish Peroxidase anti-mouse antibody, K4000, Dako, Glostrup, Denmark), and finally stained with DAB chromogen and counterstained with hematoxylin and mounted. Immuno-Positive Cell Identification and Counting Proliferating cell nuclear antigen was localized in the nucleus, presenting with the yellow granule sediments in
123
Cell Biochem Biophys (2014) 70:1803–1806
staining picture as the positive expression. C-myc antigen positive staining was also shown with brown nucleus, sometimes with brown cytoplasm, and P53 antigen located in the nucleus was shown with the stained brown nuclei as a positive expression. 10 fields of each slice were randomly chosen, and the total of 1,000 intimal cells were counted to calculate the percentage number of positive cells. Cumulative count of all the slices were averaged as the positive rate and processed by the statistics with the control group. Statistical Analysis Rank sum test and v2 test (Chi-squared test) were used to compare the quantitative variables between groups. P values less than 0.05 were considered significant. Statistical analysis was performed by using SPSS 15.0 software (SPSS Inc., Chicago, IL, USA).
Results Pathological Observations Irregular hyperplasia of intimal tissue, local excessive hyperplasia, and extreme thickening of intima, incomplete elastic fibers, and scattered smooth muscle cells were observed in the patient group as compared to the control group. Moreover, a large number of fibrous connective tissue hyperplasia, partial denaturation mainly on fibrosis, hyaloid degeneration, hyaline degeneration and fibrinoid degeneration, partially calcified necrosis accompanied by a large number of inflammatory cell infiltration were found in patients’ samples. In addition, pathological section of organized thrombosis and stasis cirrhosis were also observed in some cases. Immunohistochemical Detection of PCNA, C-myc and P-53 Our results indicated that more PCNA, C-myc, and P-53 positive cells were present in the hepatic vein occlusive tissue in the patient group compared to that of the control group. Specially they were higher in numbers in the intimal tissues indicating their proliferative actively. Our quantitative analysis of these markers demonstrated that PCNA and C-myc positive cell densities were three or more folds higher in patient group than control group which made the difference very significant (P \ 0.001) between both the study groups. Moreover, P-53 positive cell density showed increasing trends in patients than control group but the difference did not reach to the significant level (Table 1).
Cell Biochem Biophys (2014) 70:1803–1806
1805
Table 1 Quantitative positive expression of PCNA, C-myc, and P-53 in pathological tissues Groups
N
PCNA
C-myc
P-53
Patient group
20
25 ± 2.78*
15 ± 2.38*
6 ± 2.79
Control group
20
8 ± 1.35
5 ± 2.23
4 ± 0.178
*P \ 0.001
Discussion Budd-Chiari syndrome can cause partial stenosis or complete occlusion of hepatic vein or hepatic segment of inferior vena cava, leading to obstruction of hepatic venous reflux and inferior vena cava reflux, and thus resulting in portal hypertension (PHT) and inferior vena caval hypertension (IVCHT). Etiology of BCS is very complex, varying with different regions, nationality, gender, and age. Studies have shown that the etiology of hepatic venous stenosis or occlusion are closely associated with the following conditions: (1) Thrombosis: hypercoagulable states and anti-prothrombin gene deficiency is the basis for venous thrombosis, proved by a large number of clinical practices. (2) Septum formation: hepatic venous stenosis and membranous obstruction caused by congenital heteroplasia. This doctrine is mainly supported by the onset in more infants and young while also in the twin sisters. (3) Other issues such as mechanical injury, vascular local inflammation, and primary tumor compression [3–5]. In recent years, studies on cell proliferation have attracted widespread attention, with more focuses on intimal proliferation, apoptosis, and related gene expression. Studies have shown that vascular smooth muscle cell proliferation and imbalance between cell proliferation and apoptosis are the main reasons of vascular restenosis after vascular grafting and percutaneous transluminal angioplasty (PTA). With the improvement of medical technology and extensive development of occlusive BCS radical resection under direct vision, a variety of macroscopic and microscopic tissue structures can be observed systematically and in detail, such as visible inferior vena cava thrombosis or tumor thrombus, local stenosis, membranous tissue, scar tissue, staggered, long segmental, or skipping hepatic venous stenosis. However, there are less available clinical data of research on cell pathology of BCS local lesions, especially hepatic veno-occlusive lesions of BCS. Overexpression of PCNA and C-myc in patient’s lesions can act as the initiating factor causing irregular and extreme intimal hyperplasia. PCNA is a kind of nucleoprotein required for synthesis of eukaryotic DNA, which are auxiliary proteins of DNA polymerase d. A small amount of PCNA in quiescent cells make them enter into the proliferation cycle, so, PCNA is an important indicator
for detecting the cell proliferation [6, 7]. The vascular smooth muscle cells’ PCNA expression after vascular grafting has also been studied. PCNA expression appears immediately during anastomotic cell proliferation, repairing, and reconstruction after vascular grafting, especially the most intense around the anastomosis. As PCNA quantitative expression can objectively reflect the degree and sites of cell proliferation, therefore, there are no PCNA positive expressions in vast majority of normal vascular tissue [8]. In this study, we used PCNA as to quantify and to localize the proliferative sites. Our results showed a significantly higher PCNA expression in the lesions of patient group than that of the control group, indicating the presence of severe cell proliferation in hepatic vein lesions. C-myc is an important gene that regulates cell proliferation, and in human it is located on chromosome 8 along with N-myc and L-myc. They are transcription regulatory factors located in the nucleus. C-myc gene belongs to the immediate early gene, which can be stimulated by the specific growth signals to enter into the cell proliferation. For instance, platelet-derived growth factor (PDGF) and ConA can cause C-myc expression increased by 20–40 times in lymphocyte and quiescent fibroblasts [9, 10]. Therefore, C-myc expression is the foundation to make the resting cells into the proliferative state. In vitro experiments have proved that the important function of C-myc is to promote cell division. Overexpression of C-myc makes cells no longer respond to the growth retention signal, and allows the cells to come out of the dormancy stage into continued proliferation status. Currently, it is considered that the activation of C-myc transcription simultaneously initiates the cell proliferation and apoptosis [9, 10]. Bennett et al. [9] found that, in proliferating cells, intracellular C-myc gene expression increased and P53 expression decreased. Tao et al. [10] found that while proliferation of pulmonary vascular wall cells in chronic hypoxic rats, C-myc gene expression increased and expression of P53 gene decreased. Our results also showed similar correlation between C-myc and P-53. As a transcription factor, P53 is combined with specific DNA sequences in the form of a tetramer to regulate the expression of the target gene. Wild-type P53 is a growth inhibitory protein, and its overexpression can inhibit the growth of normal and transformed cells, leading to G1 arrest in the cell cycle. When the cell DNA damages, P53 negatively regulates the cell cycle and inhibits late G1 gene transcription, in order to ensure the stability of gene expression and fidelity of cell division [11, 12]. In this study, a strong positive expression of C-myc and a weak positive expression of P53 is also a trustworthy evidence for cell intimal reactive hyperplasia. Extremely irregular local intimal hyperplasia of lesions is the fundamental pathological feature of BCS. We
123
1806
observed that there are irregular hyperplasia in intimal tissue, fibrous connective tissue, and smooth muscle cell, accompanied by tissue degeneration (hyaloid degeneration and fibrinoid degeneration) and a large quantity of inflammatory cell infiltration. The hyperplastic intimal tissue contains more vascular endothelial cells and fibroblasts, while the hyperplastic smooth muscle cells in varying amounts can be observed. It is believed that the irregular local intimal hyperplasia in lesions is the fundamental pathological feature of BCS with hepatic venous obstruction. Long-term heavy physical labor or trauma may be a predisposing factor in the formation of BCS. Data show that the second hepatic portal is the position in which inferior vena cava passes through the diaphragm into the chest [13, 14]. The contrast between intra-abdominal positive pressure and intra-thoracic negative pressure, plus constant movement of the diaphragm, will lead to intimal damage of hepatic vein and inferior vena cava. Imbalance in gene regulation occurs in the process of damaged cell repairing and proliferation, thereby resulting in hepatic venous intimal proliferation, secondary hepatic venous stenosis, or occlusion and obstruction of hepatic venous blood flow. All the cases of this group are young farmers, during engaging in long-term heavy physical labor, breathing deeply and fast will cause the damage of second hepatic portal vascular intima and the formation of secondary BCS, in which a variety of mechanisms remain to be further discussed and investigated.
References 1. Rajani, R., Melin, T., Bjornsson, T., et al. (2009). Budd-Chiari syndrome in Sweden: epidemiology, clinical characteristics and survival—an 18-year experience. Liver International, 29, 253–259. 2. Bozorgmanesh, A., Selvam, D. A., & Caridi, J. G. (2007). BuddChiari syndrome: Hepatic venous web outflow obstruction treated
123
Cell Biochem Biophys (2014) 70:1803–1806
3.
4.
5.
6.
7.
8. 9.
10.
11.
12.
13.
14.
by percutaneous placement of hepatic vein stent. Seminars in Interventional Radiology, 24, 100–105. Rha, S. E., Lee, M. G., Lee, Y. S., et al. (2002). Nodular regenerative hyperplasia of the liver in Budd-Chiari syndrome. CT and MR features. Abdominal Imaging, 25, 255–258. Nakase, H., Kawasaki, T., Itani, T., et al. (2001). Budd-Chiari syndrome and extrahepatic portal obstruction associated with congenital antithrombin III deficiency. Journal of Gastroenterology, 36, 341–345. Sipahi, T., Duru, F., Yarah, N., et al. (2001). Compound heterozygosity for factor V Leiden and prothrombin G20210A mutations in a child with Budd-Chiari syndrome. European Journal of Pediatrics, 160, 198. Liu, Y. Y., Zhang, Y., Huang, Y. G., et al. (2003). Relationship between expression of p53, nm23, PCNA and invasion and metastasis of colon carcinoma. Guangdong Medical Journal, 4, 381. Bian, J. F., Zhang, B. G., & Chen, S. S. (1999). Antisense oligonucleotides inhibit c-myc and PCNA expression in the vascular smooth muscle cells. Zhonghua Yi Xue Za Zhi, 79, 10–14. Bioulac-Sage, P., & Balabaud, C. (2001). Liver nodules resembling focal nodular hyperplasia. Journal of Hepatology, 34, 781. Bennett, M. R., Evan, G. I., & Newby, A. C. (1994). Deregulated expression of the c-myc oncogene abolishes inhibition of proliferation of rat vascular smooth muscle cells by serum reduction, interferon-gamma, heparin, and cyclic nucleotide analogues and induces apoptosis. Circulation Research, 74, 525–536. Tao, Q. G., Zhang, Z. X., & Xu, Y. J. (1999). Investigation on c-myc and p53 gene expression and proliferation, apoptosis of pulmonary vascular wall cells in chronic hypoxic rats. Chinese Journal of Applied Physiology, 15, 169. Huang, X. R., Lai, R. Q., Wang, Z. C., et al. (2000). The relationship between the expression of p16, p53, Rb gene protein or proliferating nuclear antigen and clinicopathologic features of human gastric cancer. Chinese Journal of Cancer, 19, 899. Riedel, F., Gotte, K., Schwalb, J., et al. (2002). Vascular endothelial growth factor expression correlates with P53 mutation and angiogenesis in squamous cell carcinoma of the head and neck. Acta Otolaryngologica, 120, 105–111. Kage, M., Arakawa, M., Kojiro, M., et al. (1992). Histopathology of membranous obstruction of the inferior vena cava in the BuddChiars’s syndrome. Gastroenterology, 102, 2081–2090. Aguirrebarrena, G., Pulcinelli, S., Giovannoni, A. G., & Gidekel, L. (2001). Celiac disease and Budd-Chiari syndrome: Infrequent association. Revista Espanola de Enfermedades Digestivas, 93, 611–612.
