Hypoxia-inducible factor may induce the development of liver fibrosis in Budd–Chiari syndrome by regulating CD248/endosialin expression: A hypothesis

Open access

Abstract

Budd-Chiari syndrome (BCS) leads to the development of liver fibrosis in most of the cases. However, the mechanism of BCS-related liver fibrosis is unclear, and it may be largely different from that induced by chronic viral hepatitis. Hepatic stellate cell (HSC) and its specific marker CD248/endosialin are known to play an important regulatory role in the development of liver fibrosis. Additionally, hypoxia microenvironment and hypoxia-inducible factor (HIF) are involved in the regulation of CD248/endosialin. Therefore, we hypothesize that hypoxia microenvironment which develops due to BCS can regulate the expression of CD248/endosialin in HSC via HIF signaling pathway, which then affects the function of HSC and development of liver fibrosis. To confirm the hypothesis, two major investigations are necessary: (1) in the BCS animal model and clinical studies, the relationship between the severity of liver fibrosis and the expression of HIF and CD248/endosialin in HSC will be explored; and (2) in the in vitro cell system, the effect of hypoxic microenvironment, HIF-1α or HIF-2α, on the expression of CD248/endosialin in HSC will be explored. It will be important to elucidate whether HIF signaling pathway regulates the expression of CD248/endosialin, thereby inducing the development of BCS-related liver fibrosis.

Abstract

Budd-Chiari syndrome (BCS) leads to the development of liver fibrosis in most of the cases. However, the mechanism of BCS-related liver fibrosis is unclear, and it may be largely different from that induced by chronic viral hepatitis. Hepatic stellate cell (HSC) and its specific marker CD248/endosialin are known to play an important regulatory role in the development of liver fibrosis. Additionally, hypoxia microenvironment and hypoxia-inducible factor (HIF) are involved in the regulation of CD248/endosialin. Therefore, we hypothesize that hypoxia microenvironment which develops due to BCS can regulate the expression of CD248/endosialin in HSC via HIF signaling pathway, which then affects the function of HSC and development of liver fibrosis. To confirm the hypothesis, two major investigations are necessary: (1) in the BCS animal model and clinical studies, the relationship between the severity of liver fibrosis and the expression of HIF and CD248/endosialin in HSC will be explored; and (2) in the in vitro cell system, the effect of hypoxic microenvironment, HIF-1α or HIF-2α, on the expression of CD248/endosialin in HSC will be explored. It will be important to elucidate whether HIF signaling pathway regulates the expression of CD248/endosialin, thereby inducing the development of BCS-related liver fibrosis.

Background

Budd–Chiari syndrome

Budd–Chiari syndrome (BCS) is a vascular disorder of the liver, characterized by hepatic venous outflow obstruction from the major hepatic vein to the confluence between suprahepatic portion of inferior vena cava and right atrium.[1, 2, 3] According to the epidemiological data from Denmark[4], France[5], Korea[6], northwestern Italy[7], and Sweden[8], the annual incidence of BCS ranges from 0.13 to 3.5 per million in the mentioned countries. Epidemiological data from Japan[9] and Sweden[8] reveal that the prevalence of BCS in those countries is between 1.4 and 4.02 per million. Acute or fluminant BCS can result in acute life-threatening liver failure. Chronic BCS can progress to liver fibrosis and cirrhosis, resulting in cirrhosis-related complications, such as ascites and variceal bleeding[10, 11], and even hepatocellular carcinoma.[12, 13] Histologically, veno-portal cirrhosis has been found to be the major pattern of fibrosis developing in some common liver diseases, such as viral hepatitis and alcohol abuse.[14, 15] By comparison, the histological patterns of liver fibrosis in BCS are different from those in common chronic liver diseases. Veno-centric cirrhosis, also called as reversed nodulation cirrhosis, is more specific to BCS and is characterized by bridging fibrosis between hepatic veins without septa to portal tracts.[15]

CD248/endosialin

Searching for “CD248” or “endosialin” in the PubMed database, we retrieved a total of 108 relevant papers. In the first paper published in 1992[16], Rettig et al. identified the expression of endosialin for the first time in the vascular endothelial cells of human malignant tumors, including sarcomas, carcinomas, and neuroectodermal tumors. So CD248/endosialin is also called as a tumor endothelial marker 1[17]. Recently, a monoclonal antibody to endosialin, also called as MORAb-004 or ontuxizumab, has been developed and its safety in the treatment of advanced solid tumors has been explored in a phase I clinical trial.[18] More recently, two phase II clinical trials from US for the use of ontuxizumab have been published.[19, 20] The first phase II trial randomized the patients with chemorefractory metastatic colorectal cancer into ontuxizumab (8 mg/kg) or placebo plus best supportive care groups. The second phase II trial randomized the patients with metastatic melanoma receiving at least one prior systemic treatment but developing disease progression into ontuxizumab (2 mg/kg) or ontuxizumab (4 mg/kg) groups. These preliminary findings suggested the potential antitumor efficacy of ontuxizumab by inhibiting CD248/endosialin.

Association of CD248/endosialin with hepatic stellate cell and liver fibrosis

In addition to the expression of CD248/endosialin in tumors, some studies also suggested its expression in liver fibrosis. In qRT-PCR analyses performed by Mogler and colleagues[21], a weak expression of CD248/endosialin was restricted to hepatic stellate cells and liver sinusoidal endothelial cells in healthy adult murine, but CD248/endosialin expression was not observed in Kupffer cells or hepatocytes. Similarly, a weak expression of CD248/endosialin was restricted to hepatic stellate cells in healthy adult human livers, but CD248/endosialin expression was not observed in hepatocytes. Therefore, CD248/endosialin might be a specific marker for hepatic stellate cells. Furthermore, CD248/endosialin expression was significantly higher in human fibrotic and cirrhotic liver than in normal human liver. In another study, Wihelm and colleagues measured the expression of CD248/endosialin at gene and protein levels in human and mouse liver tissues.[22] qPCR analyses also showed that the expression of CD248/endosialin mRNA were higher in cirrhotic patients due to alcohol abuse, nonalcoholic steatohepatitis, and autoimmune-related liver diseases than healthy controls. Similarly, the expression of CD248/endosialin mRNA was higher in carbon tetrachloride-induced liver fibrosis mice than in normal mice. In addition, immunofluorescent staining analysis demonstrated that the number of CD248+ cells was higher in cirrhotic patient liver tissues than in healthy human liver tissues. Similarly, the number of CD248+ cells was also higher in carbon tetrachloride-induced liver fibrosis mouse liver tissues than in normal mouse liver tissues. Taken together, both studies suggested a close relationship between CD248/endosialin and development of liver fibrosis.

Hypoxia and hypoxia-inducible factor (HIF)

The critical role of hypoxia and HIF in tumor development, progression, metastasis, and prognosis has been widely recognized.[23, 24, 25] Recently, our group also reviewed the evidence regarding the association of expression of HIFs and gene polymorphism with risk, stage, and survival of various human cancers.[26] Furthermore, HIFs could be involved in the development of various liver diseases.[27, 28] Targeting HIFs may be promising for the treatment of liver diseases.

Association of hypoxia/HIF with hepatic stellate cell and liver fibrosis

Experimental evidence supports the association of hypoxia with liver fibrosis. Corpechot et al. found that the level of collagen I, a marker of fibrosis, in activated hepatic stellate cells was increased under hypoxic conditions, but not the level of collagen IV.[29] Additionally, the expression of pimonidazole adducts in the hepatocytes, a marker of cellular hypoxia, was significantly higher in the rats with cirrhotic or fibrotic livers than those with normal livers. In another experimental study, Moon et al. found that the proportion of hypoxia area, which was characterized by the expression of pimonidazole–sulfur adducts, was gradually increased with the time of bile duct ligation in mice.[30] HIF-1 alpha levels were also increased with the time of bile duct ligation in mice.

On the other hand, experimental studies suggested that the severity of liver fibrosis should be decreased in HIF-1-alpha-deficient mice. Moon et al. showed that the collagen I mRNA expression level was significantly lower in HIF-1-alpha-deficient mice subjected to bile duct ligation than control mice subjected to bile duct ligation. Similarly, Copple et al. demonstrated that the expression of collagen I mRNA was significantly increased in wild-type mice after bile duct ligation, but decreased in HIF-1-alpha-deficient mice after bile duct ligation.[31] Roychowdhury et al. provided moderate chronic ethanol feeding to the carbon tetrachloride–induced mice and found that the area of Sirius Red staining was lower in hepatocyte-specific HIF-1-alpha-deficient mice.[32]

Association of CD248/endosialin with hypoxia/HIF

Only one experimental study by Ohradanova et al. investigated the regulatory role of hypoxia on endosialin expression. It showed that CD248/endosialin mRNA and protein levels in human cell lines were significantly higher in hypoxic conditions than normoxic conditions.[33] HIF-2 alpha binds hypoxia-responsive element at position–976/–969, thereby regulating the endosialin promoter and hypoxic induction of endosialin.

Hypothesis

Based on the existing knowledge, we hypothesize that hypoxia microenvironment induced by BCS can regulate the expression of CD248/endosialin in HSC via HIF signaling pathway, which then affects the function of HSC, leading to the development of liver fibrosis.

Evaluation of hypothesis

At least two major investigations are necessary to evaluate the hypothesis. In the first investigation, we will conduct the BCS animal model and clinical studies to explore the relationship between the severity of liver fibrosis and the expression of HIF and CD248/endosialin in HSC. In the second investigation, we will conduct the in vitro cell experiments to explore the effect of hypoxic microenvironment, HIF-1α or HIF-2α, on the expression of CD248/endosialin in HSC. Besides, we will explore the potential regulatory mechanisms of CD248/endosialin. For example, we may explore the effect of endosialin on the MAP kinase ERK-1/2 phosphorylation and early transcription factor c-Fos.

Conclusions

To our knowledge, few experimental studies have been performed regarding the mechanisms of liver fibrosis in BCS. Therefore, this hypothesis is important to elucidate whether HIF signaling pathway regulates endosialin expression, thereby inducing the development of BCS-related liver fibrosis.

Source of Foundation: This study was partially supported by the grant from the National Natural Science Foundation of China (no. 81500474) for Dr. Xingshun Qi.

Conflict of Interest: None.

References

  • 1

    Valla DC. Primary Budd-Chiari syndrome. J Hepatol 2009; 50:195-203.

  • 2

    De Stefano V, Martinelli I. Splanchnic vein thrombosis: clinical presentation, risk factors and treatment. Intern Emerg Med 2010; 5:487-94.

  • 3

    Riva N, Ageno W. Approach to thrombosis at unusual sites: Splanchnic and cerebral vein thrombosis. Vasc Med 2017; 22:529-40.

  • 4

    Almdal TP, Sorensen TI. Incidence of parenchymal liver diseases in Denmark, 1981 to 1985: analysis of hospitalization registry data. The Danish Association for the Study of the Liver. Hepatology 1991; 13:650-55.

  • 5

    Valla DC. Hepatic venous outflow tract obstruction etiopathogenesis: Asia versus the West. J Gastroenterol Hepatol 2004; 19:S204-11.

  • 6

    Ki M, Choi HY, Kim KA, Kim BH, Jang ES, Jeong SH. Incidence, prevalence and complications of Budd-Chiari syndrome in South Korea: a nationwide, population-based study. Liver Int 2016; 36:1067-73.

  • 7

    Ageno W, Dentali F, Pomero F, Fenoglio L, Squizzato A, Pagani G, et al. Incidence rates and case fatality rates of portal vein thrombosis and Budd-Chiari Syndrome. Thromb Haemost 2017; 117:794-800.

  • 8

    Rajani R, Melin T, Bjornsson E, Broome U, Sangfelt P, Danielsson A, et al. Budd-Chiari syndrome in Sweden: epidemiology, clinical characteristics and survival-an 18-year experience. Liver Int 2009; 29:253-9.

  • 9

    Okuda H, Yamagata H, Obata H, Iwata H, Sasaki R, Imai F, et al. Epidemiological and clinical features of Budd-Chiari syndrome in Japan. J Hepatol 1995; 22:1-9.

  • 10

    Liu L, Qi XS, Zhao Y, Chen H, Meng XC, Han GH. Budd-Chiari syndrome: current perspectives and controversies. Eur Rev Med Pharmacol Sci 2016; 20:3273-81.

  • 11

    Mancuso A. An update on management of Budd-Chiari syndrome. Ann Hepatol 2014; 13:323-6.

  • 12

    Ren W, Qi X, Jia J, Yang M, Han G. Prevalence of hepatocellular carcinoma in Chinese Budd-Chiari syndrome patients: an extended systematic review using Chinese-language databases. Eur J Gastroenterol Hepatol 2013; 25:1241-3.

  • 13

    Ren W, Qi X, Yang Z, Han G, Fan D. Prevalence and risk factors of hepatocellular carcinoma in Budd-Chiari syndrome: a systematic review. Eur J Gastroenterol Hepatol 2013; 25:830-41.

  • 14

    Tsochatzis EA, Bosch J, Burroughs AK. Liver cirrhosis. Lancet 2014; 383:1749-61.

  • 15

    Tanaka M, Wanless IR. Pathology of the liver in Budd-Chiari syndrome: portal vein thrombosis and the histogenesis of veno-centric cirrhosis, veno-portal cirrhosis, and large regenerative nodules. Hepatology 1998; 27:488-96.

  • 16

    Rettig WJ, Garin-Chesa P, Healey JH, Su SL, Jaffe EA, Old LJ. Identification of endosialin, a cell surface glycoprotein of vascular endothelial cells in human cancer. Proc Natl Acad Sci U S A 1992; 89:10832-6.

  • 17

    Kontsekova S, Polcicova K, Takacova M, Pastorekova S. Endosialin: molecular and functional links to tumor angiogenesis. Neoplasma 2016; 63:183-92.

  • 18

    Diaz LA Jr, Coughlin CM, Weil SC, Fishel J, Gounder MM, Lawrence S, et al. A first-in-human phase I study of MORAb-004, a monoclonal antibody to endosialin in patients with advanced solid tumors. Clin Cancer Res 2015; 21:1281-8.

  • 19

    Grothey A, Strosberg JR, Renfro LA, Hurwitz HI, Marshall JL, Safran H, et al. A Randomized, Double-Blind, Placebo-Controlled Phase II Study of the Efficacy and Safety of Monotherapy Ontuxizumab (MORAb-004) Plus Best Supportive Care in Patients with Chemorefractory Metastatic Colorectal Cancer. Clin Cancer Res 2018, 24:316-25.

  • 20

    D’Angelo SP, Hamid OA, Tarhini A, Schadendorf D, Chmielowski B, Collichio FA, et al. A phase 2 study of ontuxizumab, a monoclonal antibody targeting endosialin, in metastatic melanoma. Invest New Drugs 2018 36:103-13.

  • 21

    Mogler C, Wieland M, Konig C, Hu J, Runge A, Korn C, et al. Hepatic stellate cell-expressed endosialin balances fibrogenesis and hepatocyte proliferation during liver damage. EMBO Mol Med 2015; 7:332-8.

  • 22

    Wilhelm A, Aldridge V, Haldar D, Naylor AJ, Weston CJ, Hedegaard D, et al. CD248/endosialin critically regulates hepatic stellate cell proliferation during chronic liver injury via a PDGF-regulated mechanism. Gut 2016; 65:1175-85.

  • 23

    Dhani N, Fyles A, Hedley D, Milosevic M. The clinical significance of hypoxia in human cancers. Semin Nucl Med 2015; 45:110-21.

  • 24

    Vaupel P. The role of hypoxia-induced factors in tumor progression. Oncologist 2004; 9Suppl 5:10-7.

  • 25

    Vaupel P, Mayer A. Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev 2007; 26:225-39.

  • 26

    Zou D, Han T, Deng H, Shao X, Guo X, Qi X. Hypoxia-inducible factors in cancer: an overview of major findings from meta-analyses. AME Med J 2017; 2:48.

  • 27

    Wilson GK, Tennant DA, McKeating JA. Hypoxia inducible factors in liver disease and hepatocellular carcinoma: current understanding and future directions. J Hepatol 2014; 61:1397-406.

  • 28

    Ju C, Colgan SP, Eltzschig HK. Hypoxia-inducible factors as molecular targets for liver diseases. J Mol Med (Berl) 2016; 94:613-27.

  • 29

    Corpechot C, Barbu V, Wendum D, Kinnman N, Rey C, Poupon R, et al. Hypoxia-induced VEGF and collagen I expressions are associated with angiogenesis and fibrogenesis in experimental cirrhosis. Hepatology 2002; 35:1010-21.

  • 30

    Moon JO, Welch TP, Gonzalez FJ, Copple BL. Reduced liver fibrosis in hypoxia-inducible factor-1alpha-deficient mice. Am J Physiol Gastrointest Liver Physiol 2009; 296:G582-92.

  • 31

    Copple BL, Kaska S, Wentling C. Hypoxia-inducible factor activation in myeloid cells contributes to the development of liver fibrosis in cholestatic mice. J Pharmacol Exp Ther 2012; 341:307-16.

  • 32

    Roychowdhury S, Chiang DJ, McMullen MR, Nagy LE. Moderate, chronic ethanol feeding exacerbates carbon-tetrachloride-inducedhepatic fibrosis via hepatocyte-specific hypoxia inducible factor 1a. Pharmacol Res Perspect 2014; 2:e00061.

  • 33

    Ohradanova A, Gradin K, Barathova M, Zatovicova M, Holotnakova T, Kopacek J, et al. Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2. Br J Cancer 2008; 99:1348-56.

1

Valla DC. Primary Budd-Chiari syndrome. J Hepatol 2009; 50:195-203.

2

De Stefano V, Martinelli I. Splanchnic vein thrombosis: clinical presentation, risk factors and treatment. Intern Emerg Med 2010; 5:487-94.

3

Riva N, Ageno W. Approach to thrombosis at unusual sites: Splanchnic and cerebral vein thrombosis. Vasc Med 2017; 22:529-40.

4

Almdal TP, Sorensen TI. Incidence of parenchymal liver diseases in Denmark, 1981 to 1985: analysis of hospitalization registry data. The Danish Association for the Study of the Liver. Hepatology 1991; 13:650-55.

5

Valla DC. Hepatic venous outflow tract obstruction etiopathogenesis: Asia versus the West. J Gastroenterol Hepatol 2004; 19:S204-11.

6

Ki M, Choi HY, Kim KA, Kim BH, Jang ES, Jeong SH. Incidence, prevalence and complications of Budd-Chiari syndrome in South Korea: a nationwide, population-based study. Liver Int 2016; 36:1067-73.

7

Ageno W, Dentali F, Pomero F, Fenoglio L, Squizzato A, Pagani G, et al. Incidence rates and case fatality rates of portal vein thrombosis and Budd-Chiari Syndrome. Thromb Haemost 2017; 117:794-800.

8

Rajani R, Melin T, Bjornsson E, Broome U, Sangfelt P, Danielsson A, et al. Budd-Chiari syndrome in Sweden: epidemiology, clinical characteristics and survival-an 18-year experience. Liver Int 2009; 29:253-9.

9

Okuda H, Yamagata H, Obata H, Iwata H, Sasaki R, Imai F, et al. Epidemiological and clinical features of Budd-Chiari syndrome in Japan. J Hepatol 1995; 22:1-9.

10

Liu L, Qi XS, Zhao Y, Chen H, Meng XC, Han GH. Budd-Chiari syndrome: current perspectives and controversies. Eur Rev Med Pharmacol Sci 2016; 20:3273-81.

11

Mancuso A. An update on management of Budd-Chiari syndrome. Ann Hepatol 2014; 13:323-6.

12

Ren W, Qi X, Jia J, Yang M, Han G. Prevalence of hepatocellular carcinoma in Chinese Budd-Chiari syndrome patients: an extended systematic review using Chinese-language databases. Eur J Gastroenterol Hepatol 2013; 25:1241-3.

13

Ren W, Qi X, Yang Z, Han G, Fan D. Prevalence and risk factors of hepatocellular carcinoma in Budd-Chiari syndrome: a systematic review. Eur J Gastroenterol Hepatol 2013; 25:830-41.

14

Tsochatzis EA, Bosch J, Burroughs AK. Liver cirrhosis. Lancet 2014; 383:1749-61.

15

Tanaka M, Wanless IR. Pathology of the liver in Budd-Chiari syndrome: portal vein thrombosis and the histogenesis of veno-centric cirrhosis, veno-portal cirrhosis, and large regenerative nodules. Hepatology 1998; 27:488-96.

16

Rettig WJ, Garin-Chesa P, Healey JH, Su SL, Jaffe EA, Old LJ. Identification of endosialin, a cell surface glycoprotein of vascular endothelial cells in human cancer. Proc Natl Acad Sci U S A 1992; 89:10832-6.

17

Kontsekova S, Polcicova K, Takacova M, Pastorekova S. Endosialin: molecular and functional links to tumor angiogenesis. Neoplasma 2016; 63:183-92.

18

Diaz LA Jr, Coughlin CM, Weil SC, Fishel J, Gounder MM, Lawrence S, et al. A first-in-human phase I study of MORAb-004, a monoclonal antibody to endosialin in patients with advanced solid tumors. Clin Cancer Res 2015; 21:1281-8.

19

Grothey A, Strosberg JR, Renfro LA, Hurwitz HI, Marshall JL, Safran H, et al. A Randomized, Double-Blind, Placebo-Controlled Phase II Study of the Efficacy and Safety of Monotherapy Ontuxizumab (MORAb-004) Plus Best Supportive Care in Patients with Chemorefractory Metastatic Colorectal Cancer. Clin Cancer Res 2018, 24:316-25.

20

D’Angelo SP, Hamid OA, Tarhini A, Schadendorf D, Chmielowski B, Collichio FA, et al. A phase 2 study of ontuxizumab, a monoclonal antibody targeting endosialin, in metastatic melanoma. Invest New Drugs 2018 36:103-13.

21

Mogler C, Wieland M, Konig C, Hu J, Runge A, Korn C, et al. Hepatic stellate cell-expressed endosialin balances fibrogenesis and hepatocyte proliferation during liver damage. EMBO Mol Med 2015; 7:332-8.

22

Wilhelm A, Aldridge V, Haldar D, Naylor AJ, Weston CJ, Hedegaard D, et al. CD248/endosialin critically regulates hepatic stellate cell proliferation during chronic liver injury via a PDGF-regulated mechanism. Gut 2016; 65:1175-85.

23

Dhani N, Fyles A, Hedley D, Milosevic M. The clinical significance of hypoxia in human cancers. Semin Nucl Med 2015; 45:110-21.

24

Vaupel P. The role of hypoxia-induced factors in tumor progression. Oncologist 2004; 9Suppl 5:10-7.

25

Vaupel P, Mayer A. Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev 2007; 26:225-39.

26

Zou D, Han T, Deng H, Shao X, Guo X, Qi X. Hypoxia-inducible factors in cancer: an overview of major findings from meta-analyses. AME Med J 2017; 2:48.

27

Wilson GK, Tennant DA, McKeating JA. Hypoxia inducible factors in liver disease and hepatocellular carcinoma: current understanding and future directions. J Hepatol 2014; 61:1397-406.

28

Ju C, Colgan SP, Eltzschig HK. Hypoxia-inducible factors as molecular targets for liver diseases. J Mol Med (Berl) 2016; 94:613-27.

29

Corpechot C, Barbu V, Wendum D, Kinnman N, Rey C, Poupon R, et al. Hypoxia-induced VEGF and collagen I expressions are associated with angiogenesis and fibrogenesis in experimental cirrhosis. Hepatology 2002; 35:1010-21.

30

Moon JO, Welch TP, Gonzalez FJ, Copple BL. Reduced liver fibrosis in hypoxia-inducible factor-1alpha-deficient mice. Am J Physiol Gastrointest Liver Physiol 2009; 296:G582-92.

31

Copple BL, Kaska S, Wentling C. Hypoxia-inducible factor activation in myeloid cells contributes to the development of liver fibrosis in cholestatic mice. J Pharmacol Exp Ther 2012; 341:307-16.

32

Roychowdhury S, Chiang DJ, McMullen MR, Nagy LE. Moderate, chronic ethanol feeding exacerbates carbon-tetrachloride-inducedhepatic fibrosis via hepatocyte-specific hypoxia inducible factor 1a. Pharmacol Res Perspect 2014; 2:e00061.

33

Ohradanova A, Gradin K, Barathova M, Zatovicova M, Holotnakova T, Kopacek J, et al. Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2. Br J Cancer 2008; 99:1348-56.