**Matrix Restructuring During Liver Regeneration is Regulated by Glycosylation of the Matrix Glycoprotein Vitronectin**

Haruko Ogawa1, Kotone Sano2,

Naomi Sobukawa1 and Kimie Asanuma-Date1 *1Graduate School of Advanced Sciences and Humanities, and Glycoscience Institute, Ochanomizu University, 2Faculty of World Heritage, Department of Liberal Arts, Cyber University, Tokyo, Japan* 

#### **1. Introduction**

78 Liver Regeneration

York, J.D. & Majerus, P.W. (1994). *Nuclear phosphatidylinositols decrease during S-phase of the* 

There are three major approaches for regenerative medicine. The most innovative approach among them is: induction of target cells from various stem cells such as induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells) and implantation of them to regenerate the organ. The second approach is: in vitro tissue regeneration that involves preparation of artificial tissue by combining human cells with scaffolding biomaterials and growth factors. The third is: promotion of self-regeneration through controlling the repair activity of each tissue, which most organisms do naturally, is a more fundamental approach, but it will also be important in cell therapy to regulate tissue organization after induction of differentiation.

Because tissue homeostasis depends on spatially and temporally controlled expression of multifunctional adhesive glycoproteins and receptors, many studies have examined the changes of expression of extracellular matrix (ECM) molecules during tissue remodeling, inflammation and invasion by cancer cells (DeClerck, Y.A., et al. 2004; Seiffert, D. 1997; Kato, S., et al. 1992; Hughes, R.C. 1997) on the one hand. On the other hand, there is increasing evidence that glycosylations post-translationally modulate various biological phenomena by altering the activity and specificity or the stability of glycoproteins through the biosignaling functions of oligosaccharides (Varki, A. 1993; Varki, A., et al., 2009). During tissue remodeling, the glycosylated ECM molecules are different from those of normal tissue owing to the changes in the expression of many proteins that are responsible for glycan synthesis (Dalziel, M., et al. 1999). However, the glycan modulation of most glycoproteins that are involved in tissue remodeling has remained unknown.

When the three big lobes of a liver are excised, the remaining liver recovers its former mass and function within about two weeks in humans or 7 to 10 days in rats (Diehl, A.M. and Rai, R.M. 1996). ECM degradation occurs in the early stage of this process, followed by biosynthesis of the matrix, cell proliferation, and cell differentiation. During this process, many glycosyl transferases (Bauer, C.H., et al. 1976; Serafini-Cessi, F. 1977; Okamoto, Y., et

Matrix Restructuring During Liver Regeneration

to play a central role in matrix remodeling.

K., et al. 2010).

is Regulated by Glycosylation of the Matrix Glycoprotein Vitronectin 81

The ligand-binding sites on the domain structure of vitronectins are shown in Fig. 1. Vitronectin regulates the proteolytic degradation of matrix and fibrinolysis through binding with urokinase-type plasminogen activator, PAI-1, and urokinase receptor (Seiffert, D. 1997; Schvartz, I., et al. 1999; Preissner, K.T. 1991; Pretzlaff, R.K., et al. 2000). Besides this function, vitronectin plays a key role in cell adhesion and cellular motility during tissue remodeling through binding to major ECM receptors, integrins such as αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, and other ECM components like collagen and proteoglycans (Schvartz, I., et al. 1999). In plasma, vitronectin has been shown to regulate coagulation and thrombolytic and complement systems (Preissner, K.T. 1991). By providing a link between plasmin-regulated matrix proteolysis and integrin-mediated cell migration, activated vitronectin is considered

Fig. 1 also shows the glycosylation structures of vitronectins and ligand-binding sites. *N*glycosylation sites of vitronectins are well conserved among mammals, and vitronectin contains almost fully sialylated complex-type *N*-linked glycans at these sites. Most vitronectins, except normal human, additionally contain various amounts of *O*-linked glycans, which differ by species: bovine, rabbit, and especially chicken vitronectins possess more *O*-glycans than *N*-glycans. Human vitronectin does not contain *O*-glycans in the normal state, while rat vitronectin contain several *O*-glycans in the connecting region (Sano,

Fig. 1. Glycosylation and domain structure models of vitronectins. Human, rat/mouse, and porcine vitronectin contain three, four, and two *N*-glycosylation sites, respectively (Ogawa, H., Yoneda, A., et al. 1995) (Yoneda, A., Ogawa, H., et al. 1993) (Sano, K., Miyamoto, Y., et al. 2010). The numbers of *O*-glycosylation sites differ by animal species. The major ligandbinding sites that have been identified are indicated at the top of the figure (Yoneda, A., Ogawa, H., et al. 1998) (Preissner, K.T. 1991) (Preissner, K.T. and Seiffert, D. 1998).

al. 1978; Ip, C. 1979; Oda-Tamai, S., et al. 1985; Miyoshi, E., et al. 1995) and total glycoconjugates in the liver have been reported to change (Okamoto, Y. and Akamatsu, N. 1977; Kato, S. and Akamatsu, N. 1984; Kato, S. and Akamatsu, N. 1985; Ishii, I., et al. 1985). However, the nature of the links between such glycans changes, and the process of tissue remodeling has remained unclear. We consider it important to identify which molecules play important roles in the tissue remodeling during liver regeneration, and we will discuss the glycan modulation of one extracellular molecule, vitronectin, in this chapter. Vitronectin is a multifunctional adhesive glycoprotein that plays a central role in tissue remodeling by connecting pericellular tissue lysis with cell adhesion and motility.

We found that the glycans of vitronectin drastically change during liver regeneration after partial hepatectomy. In our studies to determine the glycan structures during the initial stage of the liver regeneration after partial hepatectomy of rats, we found that alterations in glycosylation, especially decreased sialylation of vitronectin, modulate the biological activities of vitronectin during tissue-remodeling processes by multiple steps (Uchibori-Iwaki, H., et al. 2000; Sano, K., et al. 2010). Liver regeneration is a normal repair process, while fibrosis and cirrhosis are considered to be excessive and abnormal repair processes that often give rise to cancer. In this context, elucidating how alterations of glycans occur and understanding how glycans modulate the glycans on vitronectin is useful in order to develop a strategy to regulate matrix remodeling in regeneration and deposition in liver cirrhosis. Therefore, we aimed to elucidate glycan modulation during liver regeneration after partial hepatectomy. We focused on the changes in vitronectin during liver regeneration, especially the changes of the glycan moiety, which plays a crucial role in controlling survival of hepatic stellate cells.

#### **1.1 Structure and function of vitronectin**

Vitronectin is a multifunctional adhesive glycoprotein that originates mainly in hepatocytes and circulates in the blood at high concentrations (0.2 mg/ml in human and no more than 0.1 mg/ml in rats). Vitronectin was first isolated from human serum by Holmes in 1967 as an 'α-1 protein' (Holmes, R. 1967), and has been referred to as 'serum spreading factor', 'epibolin', or 'S-protein'. It induces cell growth in vitro and is known as a major celladhesive component in cell culture mediums (Hayman, E.G., et al. 1983). Vitronectin is present as an ECM component in the liver, as well as various other organs, including skeletal muscle, kidney, and brain (Seiffert, D. 1997; Seiffert, D., et al. 1991). Most vitronectin in normal plasma is present as an inactive monomer form that does not bind to various ligands in the plasma (Gebb, C., et al. 1986; Izumi, M., et al. 1989). *In vivo*, vitronectin is activated in the presence of certain ligands such as heparin, type-1 plasminogen activator inhibitor (PAI-1), thrombin-AT-III, membrane attack complex of complements, and through a partial conformational change and multimerization process (Preissner, K.T. and Muller-Berghaus, G. 1987). Tissue vitronectin is considered to be present as an active multimeric form. Conformation-dependent binding of vitronectin was also observed for sulfatide (Gal(3-SO4)β1-1ceramide), cholesterol 3-sulfate, and various phospholipids including phosphatidylserine, but not gangliosides, while vitronectin bound to cholesterol 3-sulfate regardless of its conformational state (Yoneda, A., et al. 1998). The binding of vitronectin to the membrane lipids and β-endorphin-binding activities were found to be attributable to hemopexin domain 2 and hemopexin domain 1, as well as type I collagen and heparin (Yoneda, A., et al. 1998).

al. 1978; Ip, C. 1979; Oda-Tamai, S., et al. 1985; Miyoshi, E., et al. 1995) and total glycoconjugates in the liver have been reported to change (Okamoto, Y. and Akamatsu, N. 1977; Kato, S. and Akamatsu, N. 1984; Kato, S. and Akamatsu, N. 1985; Ishii, I., et al. 1985). However, the nature of the links between such glycans changes, and the process of tissue remodeling has remained unclear. We consider it important to identify which molecules play important roles in the tissue remodeling during liver regeneration, and we will discuss the glycan modulation of one extracellular molecule, vitronectin, in this chapter. Vitronectin is a multifunctional adhesive glycoprotein that plays a central role in tissue remodeling by

We found that the glycans of vitronectin drastically change during liver regeneration after partial hepatectomy. In our studies to determine the glycan structures during the initial stage of the liver regeneration after partial hepatectomy of rats, we found that alterations in glycosylation, especially decreased sialylation of vitronectin, modulate the biological activities of vitronectin during tissue-remodeling processes by multiple steps (Uchibori-Iwaki, H., et al. 2000; Sano, K., et al. 2010). Liver regeneration is a normal repair process, while fibrosis and cirrhosis are considered to be excessive and abnormal repair processes that often give rise to cancer. In this context, elucidating how alterations of glycans occur and understanding how glycans modulate the glycans on vitronectin is useful in order to develop a strategy to regulate matrix remodeling in regeneration and deposition in liver cirrhosis. Therefore, we aimed to elucidate glycan modulation during liver regeneration after partial hepatectomy. We focused on the changes in vitronectin during liver regeneration, especially the changes of the glycan moiety, which plays a crucial role in

Vitronectin is a multifunctional adhesive glycoprotein that originates mainly in hepatocytes and circulates in the blood at high concentrations (0.2 mg/ml in human and no more than 0.1 mg/ml in rats). Vitronectin was first isolated from human serum by Holmes in 1967 as an 'α-1 protein' (Holmes, R. 1967), and has been referred to as 'serum spreading factor', 'epibolin', or 'S-protein'. It induces cell growth in vitro and is known as a major celladhesive component in cell culture mediums (Hayman, E.G., et al. 1983). Vitronectin is present as an ECM component in the liver, as well as various other organs, including skeletal muscle, kidney, and brain (Seiffert, D. 1997; Seiffert, D., et al. 1991). Most vitronectin in normal plasma is present as an inactive monomer form that does not bind to various ligands in the plasma (Gebb, C., et al. 1986; Izumi, M., et al. 1989). *In vivo*, vitronectin is activated in the presence of certain ligands such as heparin, type-1 plasminogen activator inhibitor (PAI-1), thrombin-AT-III, membrane attack complex of complements, and through a partial conformational change and multimerization process (Preissner, K.T. and Muller-Berghaus, G. 1987). Tissue vitronectin is considered to be present as an active multimeric form. Conformation-dependent binding of vitronectin was also observed for sulfatide (Gal(3-SO4)β1-1ceramide), cholesterol 3-sulfate, and various phospholipids including phosphatidylserine, but not gangliosides, while vitronectin bound to cholesterol 3-sulfate regardless of its conformational state (Yoneda, A., et al. 1998). The binding of vitronectin to the membrane lipids and β-endorphin-binding activities were found to be attributable to hemopexin domain 2 and hemopexin domain 1, as well as type I collagen and heparin

connecting pericellular tissue lysis with cell adhesion and motility.

controlling survival of hepatic stellate cells.

**1.1 Structure and function of vitronectin** 

(Yoneda, A., et al. 1998).

The ligand-binding sites on the domain structure of vitronectins are shown in Fig. 1. Vitronectin regulates the proteolytic degradation of matrix and fibrinolysis through binding with urokinase-type plasminogen activator, PAI-1, and urokinase receptor (Seiffert, D. 1997; Schvartz, I., et al. 1999; Preissner, K.T. 1991; Pretzlaff, R.K., et al. 2000). Besides this function, vitronectin plays a key role in cell adhesion and cellular motility during tissue remodeling through binding to major ECM receptors, integrins such as αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, and other ECM components like collagen and proteoglycans (Schvartz, I., et al. 1999). In plasma, vitronectin has been shown to regulate coagulation and thrombolytic and complement systems (Preissner, K.T. 1991). By providing a link between plasmin-regulated matrix proteolysis and integrin-mediated cell migration, activated vitronectin is considered to play a central role in matrix remodeling.

Fig. 1 also shows the glycosylation structures of vitronectins and ligand-binding sites. *N*glycosylation sites of vitronectins are well conserved among mammals, and vitronectin contains almost fully sialylated complex-type *N*-linked glycans at these sites. Most vitronectins, except normal human, additionally contain various amounts of *O*-linked glycans, which differ by species: bovine, rabbit, and especially chicken vitronectins possess more *O*-glycans than *N*-glycans. Human vitronectin does not contain *O*-glycans in the normal state, while rat vitronectin contain several *O*-glycans in the connecting region (Sano, K., et al. 2010).

Fig. 1. Glycosylation and domain structure models of vitronectins. Human, rat/mouse, and porcine vitronectin contain three, four, and two *N*-glycosylation sites, respectively (Ogawa, H., Yoneda, A., et al. 1995) (Yoneda, A., Ogawa, H., et al. 1993) (Sano, K., Miyamoto, Y., et al. 2010). The numbers of *O*-glycosylation sites differ by animal species. The major ligandbinding sites that have been identified are indicated at the top of the figure (Yoneda, A., Ogawa, H., et al. 1998) (Preissner, K.T. 1991) (Preissner, K.T. and Seiffert, D. 1998).

Matrix Restructuring During Liver Regeneration

value on the BSA-immobilized reference cell.

**early stage of liver regeneration** 

is Regulated by Glycosylation of the Matrix Glycoprotein Vitronectin 83

Fig. 2. Changes in electrophoretic mobility and collagen-binding of rat vitronectin at 24 h after partial hepatectomy. (A) SDS-PAGE of vitronectins from non-operated rats (N), partially hepatectomized (PH) rats at 6-240h after operation, and sham-operated (SH) rats at 24 h after operation. (B) Type I collagen (1 μg/100 μL) was coated onto wells of microtiter plates. After blocking with 5% BSA, various concentrations of purified vitronectins were added to each well. The bound vitronectin was measured using HRP-conjugated rabbit antihuman vitronectin IgGs and ELISA. The absorbance of collagen-bound vitronectin was corrected for the antibody reactivity of each vitronectin. (C) Collagen was immobilized on a CM5 sensor chip, and each vitronectin in PBS was injected onto the sensor chip at a flow rate of 20 μL/min at 20°C. The change of resonance units (RU) was corrected by subtracting the

**2.1.2 Changes in glycosylation and carbohydrate concentration of vitronectin during** 

As shown in Fig. 3, the carbohydrate analyses of the three vitronectins indicated that total carbohydrate contents of PH-VN and SH-VN decreased to one-third and one-half of that of NO-VN, respectively, and that a remarkable decrease in sialic acids and amounts of glycans occurred due to partial hepatectomy. The lectin reactivity of the three vitronectins indicated that these vitronectins contain complex-type *N*-linked oligosaccharides. The reactivity toward *Phaseolus vulgaris* lectin L4 (L-PHA) varied remarkably among vitronectins, and PH-VN showed marked reactivity with L-PHA, but SH- and NO-VNs reacted only slightly, suggesting that tri- or tetraantennary lactosamine-branched structures multiplied dramatically after partial hepatectomy. The specificity of PVL toward clustered sialyl residues (Ueda, H., Kojima, K., et al. 1999) (Ueda, H., Matsumoto, H., et al. 2002), the
