Vascular endothelial growth factor and liver regeneration

Abstract

Under normal conditions, hepatocytes are highly differentiated, quiescent cells with a long life span [[1]Fausto N. Liver regeneration.in: Arias I.M. Boyer J.L. Jakoby W.B. Fausto N. Schachter D. Shafritz D.A. The liver: biology and pathobiology. Raven, New York1994: 1059-1084Google Scholar]. In normal liver, mitosis is observed in approximately 1 per 20 000 hepatocytes. However, the liver has unique and remarkable abilities to restore itself following cell loss (toxic, viral or surgical). Strictly speaking, the liver does not regenerate, but undergoes hyperplasia or hypertrofy in order to reestablish optimal mass relative to body mass. Three mechanisms to restore liver mass have been identified: (i) proliferation of remaining differentiated cells, (ii) proliferation and differentiation of stem cells and (iii) hypertrophy of periportal hepatocytes. In conditions in which hepatocyte proliferation cannot compensate for cell loss, new hepatocytes will derive from differentiating liver stem cells [[2]Thorgeirsson S.S. Hepatic stem cells in liver regeneration.FASEB J. 1996; 10: 1249-1256Crossref PubMed Scopus (292) Google Scholar]. If also this second mechanism is inhibited, the liver will restore its original mass by hypertrophy of periportal hepatocytes [[3]Nagy P. Teramoto T. Factor V.M. Sanchez A. Schnur J. Paku S. et al.Reconstitution of liver mass via cellular hypertrophy in the rat.Hepatology. 2001; 33: 339-345Crossref PubMed Scopus (62) Google Scholar]. The articles by Shimizu et al. [[4]Shimizu H. Miyazaki M. Wakabayashi Y. Mitsuhashi N. Kato A. Ito H. et al.Vascular endothelial growth factor secreted by replicating hepatocytes induces sinusoidal endothelial cell proliferation during regeneration after partial hepatectomy in rats.J Hepatol. 2001; 34: 683-689Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar] and Sato et al. [[5]Sato T. El-Assal O.N. Ono T. Yamanoi A. Dhar D.K. Nagasue N. Sinusoidal endothelial cell proliferation and expression of angiopoietin/Tie family in regenerating rat liver.J Hepatol. 2001; 34: 690-698Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar] in this issue of the Journal of Hepatology, pertain to the first mechanism.Although all mature liver cell populations proliferate after cell loss, (hepatocytes, biliary epithelial cells, sinusoidal endothelial cells, Kupffer cells and hepatic stellate cells), hepatocytes are the first cells to do so. Within minutes following 70% hepatectomy, hepatocytes reenter the cell cycle accompanied by the expression of a large number of growth-response genes. Onset of DNA synthesis in hepatocytes occurs within 12 to 16 h after partial hepatectomy and reaches a peak level at 24–48 h [6Bucher N.L. Experimental aspects of liver regeneration.N Engl J Med. 1967; 277: 738-746Crossref PubMed Scopus (60) Google Scholar, 7Bucher N.L. Experimental aspects of liver regeneration.N Engl J Med. 1967; 277: 686-696Crossref PubMed Scopus (152) Google Scholar]. Hepatocyte proliferation starts in the periportal area and progresses to pericentral areas. Replication of non-hepatocytes is delayed by approximately 24 h, but follows a similar pattern of DNA synthesis and mitosis. In rats, liver mass is completely restored by day 7–10 after which proliferation stops [8Michalopoulos G.K. DeFrances M.C. Liver regeneration.Science. 1997; 276: 60-66Crossref PubMed Scopus (2865) Google Scholar, 9Steer C.J. Liver regeneration.FASEB J. 1995; 9: 1396-1400Crossref PubMed Scopus (185) Google Scholar].Over the last decade it has become obvious that liver regeneration is not a simple response to one growth factor, but rather a delicate and complex interplay of many cellular events. Several growth factors playing a role in liver regeneration have been identified. Among others, epidermal growth factor (EGF), transforming growth factor-α (TGFα), and Hepatocyte Growth Factor (HGF) are the most important ones to stimulate DNA synthesis in hepatocytes, whereas TGFβ1 and activin are potential inhibitors of proliferation [[10]Fausto N. Laird A.D. Webber E.M. Liver regeneration. 2. Role of growth factors and cytokines in hepatic regeneration.FASEB J. 1995; 9: 1527-1536Crossref PubMed Scopus (569) Google Scholar]. Furthermore, several cytokines are involved. Interleukin-6 (Il-6), interleukin-1 (Il-1) and tumor necrosis factor-α (TNF-α) are the crucial factors in early signalling leading to liver regeneration. TNF-α stimulates IL-6 secretion from Kupffer cells after partial hepatectomy. These cytokines induce at least two transcription factors: (1) ‘post hepatectomy factor/nuclear factor kappa B’ (PHF/NF-κB) and (2) ‘signal transducer and activator of transcription-3’ (STAT 3). It has been demonstrated that liver regeneration is greatly impaired and that STAT 3 activation does not occur in IL-6 and TNF-α receptor deficient mice [11Cressman D.E. Greenbaum L.E. DeAngelis R.A. Ciliberto G. Furth E.E. Poli V. et al.Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice.Science. 1996; 274: 1379-1383Crossref PubMed Scopus (1302) Google Scholar, 12Yamada Y. Kirillova I. Peschon J.J. Fausto N. Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor.Proc Natl Acad Sci USA. 1997; 94: 1441-1445Crossref PubMed Scopus (827) Google Scholar]. Activation of the above transcription factors causes transcription of immediate-early genes, which is independent of de novo protein synthesis. Delayed-early response genes are induced within hours after partial hepatectomy. In this way hepatocytes manage to divide and at the same time provide differentiated functions via liver associated transcription factors.Because of the imbalance in replication rate of hepatocytes and their surrounding extracellular matrix (ECM), normal liver architecture is lost. Newly formed hepatocytes are arranged in clusters without associated sinusoids. By day 4 after partial hepatectomy, cell processes of laminin containing hepatic stellate cells penetrate between hepatocyte islands. Fenestrated endothelial cells trail the hepatic stellate cells, thereby separating hepatocytes into cell plates and restoring normal vascular architecture [[13]Martinez-Hernandez A. Amenta P.S. The extracellular matrix in hepatic regeneration.FASEB J. 1995; 9: 1401-1410Crossref PubMed Scopus (274) Google Scholar].Even though extensive research has been performed on liver regeneration, the role of ECM and non-parenchymal cells (sinusoidal endothelial cells, hepatic stellate cells, Kupffer cells) has been relatively underexplored. In this issue of the Journal of Hepatology, Shimizu et al. [[4]Shimizu H. Miyazaki M. Wakabayashi Y. Mitsuhashi N. Kato A. Ito H. et al.Vascular endothelial growth factor secreted by replicating hepatocytes induces sinusoidal endothelial cell proliferation during regeneration after partial hepatectomy in rats.J Hepatol. 2001; 34: 683-689Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar] and Sato et al. [[5]Sato T. El-Assal O.N. Ono T. Yamanoi A. Dhar D.K. Nagasue N. Sinusoidal endothelial cell proliferation and expression of angiopoietin/Tie family in regenerating rat liver.J Hepatol. 2001; 34: 690-698Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar] report on sinusoidal endothelial cell proliferation induced by vascular endothelial growth factor (VEGF) during liver regeneration.Angiogenesis is a fundamental prerequisite for organ development, wound healing and reproductive functions, and plays an important role in tumor growth and metastasis development. Numerous non-specific growth factors acting on vascular endothelium have been described, including TGF-α and β, TNF-α, HGF and Il-8 [[14]Ferrara N. Molecular and biological properties of vascular endothelial growth factor.J Mol Med. 1999; 77: 527-543Crossref PubMed Scopus (1070) Google Scholar]. Vascular endothelial growth factor was shown to be a potent, diffusable and specific growth factor for endothelium. In recent years, several members of the VEGF family have been described, namely placenta growth factor (PlGF), VEGF-A, VEGF-B, VEGF-C and VEGF-D [[15]Yancopoulos G.D. Davis S. Gale N.W. Rudge J.S. Wiegand S.J. Holash J. Vascular-specific growth factors and blood vessel formation.Nature. 2000; 407: 242-248Crossref PubMed Scopus (3253) Google Scholar]. Vascular endothelial growth factors act through at least three different receptors VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4) of which VEGFR-2 seems to be the principal mediator of growth and permeability actions of VEGF. Angiopoietins, another family of four specific growth factors for vascular endothelial cells (Ang-1, Ang-2, Ang-3 and Ang-4), act through Tie-1 and Tie-2 tyrosin kinase receptors [[16]Senger D.R. Galli S.J. Dvorak A.M. Perruzzi C.A. Harvey V.S. Dvorak H.F. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid.Science. 1983; 219: 983-985Crossref PubMed Scopus (3380) Google Scholar]. The first described effect of VEGF (formerly termed vascular permeability factor) was the ability to induce vascular leakage [[14]Ferrara N. Molecular and biological properties of vascular endothelial growth factor.J Mol Med. 1999; 77: 527-543Crossref PubMed Scopus (1070) Google Scholar]. Vascular leakage of plasma proteins results in formation of extravascular fibrin gel, a substrate for endothelial cell growth. Later, multiple other biological effects have been attributed to VEGF. Without any doubt, its strong mitogenic activity on vascular endothelial cells in arteries, veins and lymphatic vessels is very important. VEGF also stimulates expression of metalloproteinases, urokinase-type and tissue-type plasminogen activators and plasminogen activator inhibitor 1 as well as in vitro and in vivo fenestrations in endothelial cells. It has also been reported that VEGF promotes expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 in endothelial cells. VEGF also induces nitric oxide mediated vasodilatation and promotes monocyte chemotaxis [[14]Ferrara N. Molecular and biological properties of vascular endothelial growth factor.J Mol Med. 1999; 77: 527-543Crossref PubMed Scopus (1070) Google Scholar]. Finally, VEGF was found to have an inhibitory effect on functional maturation of dendritic cells, suggesting that VEGF may facilitate tumor growth by attenuating immune response [[17]Gabrilovich D.I. Chen H.L. Girgis K.R. Cunningham H.T. Meny G.M. Nadaf S. et al.Production of vascular endothelial growth factor by human tumours inhibits the functional maturation of dendritic cells.Nat Med. 1996; 2: 1096-1103Crossref PubMed Scopus (1575) Google Scholar]. VEGF mRNA has been confirmed to be upregulated in the vast majority of human tumours, and both tumor and tumor-associated stroma are important sites of VEGF production [14Ferrara N. Molecular and biological properties of vascular endothelial growth factor.J Mol Med. 1999; 77: 527-543Crossref PubMed Scopus (1070) Google Scholar, 18Fukumura D. Xavier R. Sugiura T. Chen Y. Park E.C. Lu N. et al.Tumor induction of VEGF promoter activity in stromal cells.Cell. 1998; 94: 715-725Abstract Full Text Full Text PDF PubMed Scopus (828) Google Scholar].Only recently, the role of VEGF in liver necrosis and regeneration has been explored. Following liver necrosis, VEGF mRNA and protein was found in hepatocytes, but also in Kupffer cells, hepatic stellate cells and infiltrating inflammatory cells [19Ishikawa K. Mochida S. Mashiba S. Inao M. Matsui A. Ikeda H. et al.Expressions of vascular endothelial growth factor in nonparenchymal as well as parenchymal cells in rat liver after necrosis.Biochem Biophys Res Commun. 1999; 254: 587-593Crossref PubMed Scopus (78) Google Scholar, 20Mochida S. Ishikawa K. Inao M. Shibuya M. Fujiwara K. Increased expressions of vascular endothelial growth factor and its receptors, flt-1, KDR/flk-1, in regenerating liver.Biochem Biophys Res Commun. 1996; 226: 176-179Crossref PubMed Scopus (113) Google Scholar]. The studies by Shimizu and Sato and their colleagues in this issue of the Journal of Hepatology, [4Shimizu H. Miyazaki M. Wakabayashi Y. Mitsuhashi N. Kato A. Ito H. et al.Vascular endothelial growth factor secreted by replicating hepatocytes induces sinusoidal endothelial cell proliferation during regeneration after partial hepatectomy in rats.J Hepatol. 2001; 34: 683-689Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 5Sato T. El-Assal O.N. Ono T. Yamanoi A. Dhar D.K. Nagasue N. Sinusoidal endothelial cell proliferation and expression of angiopoietin/Tie family in regenerating rat liver.J Hepatol. 2001; 34: 690-698Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar] extended these observations. Shimizu et al. [[4]Shimizu H. Miyazaki M. Wakabayashi Y. Mitsuhashi N. Kato A. Ito H. et al.Vascular endothelial growth factor secreted by replicating hepatocytes induces sinusoidal endothelial cell proliferation during regeneration after partial hepatectomy in rats.J Hepatol. 2001; 34: 683-689Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar]. showed that remnant liver after partial hepatectomy increases the VEGF expression at both the mRNA and protein level. Serum levels of VEGF and its in vitro production by isolated hepatocytes increased 72 h after partial hepatectomy. Using immunohistochemistry, the authors demonstrated that hepatocytes located in the periportal area were the main source of VEGF production. Moreover, they were able to detect upregulation of VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) on sinusoidal endothelial cells. Finally, proliferation of sinusoidal endothelial cells was shown to follow hepatocyte proliferation by 24–48 h. In agreement with Shimizu's report, Sato and colleagues also showed a lag of 24 h between sinusoidal endothelial cells and hepatocyte proliferation [[5]Sato T. El-Assal O.N. Ono T. Yamanoi A. Dhar D.K. Nagasue N. Sinusoidal endothelial cell proliferation and expression of angiopoietin/Tie family in regenerating rat liver.J Hepatol. 2001; 34: 690-698Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar]. Both teams also report an increase in the mRNA level of VEGF peaking at 72 h after partial hepatectomy and an upregulation of the VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) receptors. Furthermore, mRNA expression of angiogenic growth factors Ang-1 and Ang-2, and Tie-1 and Tie-2 receptors increased significantly 96 h after partial hepatectomy. While mRNA expression of Ang-1, Tie-1 and Tie-2 decreased thereafter, Ang-2 mRNA continued to increase reaching a peak at 168 h after partial hepatectomy.Vascular endothelial growth factor is up-regulated by hypoxia and a number of cytokines including EGF, TGF-β, Il-1 and Il-6 [[14]Ferrara N. Molecular and biological properties of vascular endothelial growth factor.J Mol Med. 1999; 77: 527-543Crossref PubMed Scopus (1070) Google Scholar]. It has been demonstrated that activated rat hepatic stellate cells also express VEGF receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) [[21]Mashiba S. Mochida S. Ishikawa K. Inao M. Matsui A. Ohno A. et al.Inhibition of hepatic stellate cell contraction during activation in vitro by vascular endothelial growth factor in association with upregulation of FLT tyrosine kinase receptor family, FLT-1.Biochem Biophys Res Commun. 1999; 258: 674-678Crossref PubMed Scopus (19) Google Scholar] and that hypoxic stimulation induces VEGF and VEGFR-1 (Flt-1) mRNA and VEGF secretion in activated hepatic stellate cells [[22]Ankoma-Sey V. Wang Y. Dai Z. Hypoxic stimulation of vascular endothelial growth factor expression in activated rat hepatic stellate cells.Hepatology. 2000; 31: 141-148Crossref PubMed Scopus (160) Google Scholar]. Cellular hypoxia is known to occur in alcoholic liver injury, hepatic fibrosis and in tumours, three conditions in which hepatic stellate cells play an important role.Recently, the expression of VEGF in hepatocellular carcinoma was examined. In well differentiated hepatocellular carcinoma, VEGF was highly expressed, whereas it was weakly expressed in poorly differentiated hepatocellular carcinoma [[23]Yamaguchi R. Yano H. Iemura A. Ogasawara S. Haramaki M. Kojiro M. Expression of vascular endothelial growth factor in human hepatocellular carcinoma.Hepatology. 1998; 28: 68-77Crossref PubMed Scopus (354) Google Scholar]. Small, well differentiated hepatocellular carcinomas receive blood from portal veins. With increasing size and decreasing differentiation, the tumours receive more arterial blood supply. It was suggested that VEGF plays a role in the early stage of angiogenesis in hepatocellular carcinoma, but further studies are needed to clarify the role of VEGF in angiogenesis in hepatocellular carcinoma in vivo.In conclusion, the work performed by Shimizu, Takashi and their colleagues is of importance in unravelling the bio-ecological system formed by sinusoidal cells, hepatocytes and their surrounding ECM. It is clear that communication between different cell types is of the highest importance in restoring homeostasis following various types of liver injury. Furthermore, improved knowledge of the influence of VEGF on neovascularisation will lead to better understanding of progression of hepatocellular carcinoma. Under normal conditions, hepatocytes are highly differentiated, quiescent cells with a long life span [[1]Fausto N. Liver regeneration.in: Arias I.M. Boyer J.L. Jakoby W.B. Fausto N. Schachter D. Shafritz D.A. The liver: biology and pathobiology. Raven, New York1994: 1059-1084Google Scholar]. In normal liver, mitosis is observed in approximately 1 per 20 000 hepatocytes. However, the liver has unique and remarkable abilities to restore itself following cell loss (toxic, viral or surgical). Strictly speaking, the liver does not regenerate, but undergoes hyperplasia or hypertrofy in order to reestablish optimal mass relative to body mass. Three mechanisms to restore liver mass have been identified: (i) proliferation of remaining differentiated cells, (ii) proliferation and differentiation of stem cells and (iii) hypertrophy of periportal hepatocytes. In conditions in which hepatocyte proliferation cannot compensate for cell loss, new hepatocytes will derive from differentiating liver stem cells [[2]Thorgeirsson S.S. Hepatic stem cells in liver regeneration.FASEB J. 1996; 10: 1249-1256Crossref PubMed Scopus (292) Google Scholar]. If also this second mechanism is inhibited, the liver will restore its original mass by hypertrophy of periportal hepatocytes [[3]Nagy P. Teramoto T. Factor V.M. Sanchez A. Schnur J. Paku S. et al.Reconstitution of liver mass via cellular hypertrophy in the rat.Hepatology. 2001; 33: 339-345Crossref PubMed Scopus (62) Google Scholar]. The articles by Shimizu et al. [[4]Shimizu H. Miyazaki M. Wakabayashi Y. Mitsuhashi N. Kato A. Ito H. et al.Vascular endothelial growth factor secreted by replicating hepatocytes induces sinusoidal endothelial cell proliferation during regeneration after partial hepatectomy in rats.J Hepatol. 2001; 34: 683-689Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar] and Sato et al. [[5]Sato T. El-Assal O.N. Ono T. Yamanoi A. Dhar D.K. Nagasue N. Sinusoidal endothelial cell proliferation and expression of angiopoietin/Tie family in regenerating rat liver.J Hepatol. 2001; 34: 690-698Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar] in this issue of the Journal of Hepatology, pertain to the first mechanism. Although all mature liver cell populations proliferate after cell loss, (hepatocytes, biliary epithelial cells, sinusoidal endothelial cells, Kupffer cells and hepatic stellate cells), hepatocytes are the first cells to do so. Within minutes following 70% hepatectomy, hepatocytes reenter the cell cycle accompanied by the expression of a large number of growth-response genes. Onset of DNA synthesis in hepatocytes occurs within 12 to 16 h after partial hepatectomy and reaches a peak level at 24–48 h [6Bucher N.L. Experimental aspects of liver regeneration.N Engl J Med. 1967; 277: 738-746Crossref PubMed Scopus (60) Google Scholar, 7Bucher N.L. Experimental aspects of liver regeneration.N Engl J Med. 1967; 277: 686-696Crossref PubMed Scopus (152) Google Scholar]. Hepatocyte proliferation starts in the periportal area and progresses to pericentral areas. Replication of non-hepatocytes is delayed by approximately 24 h, but follows a similar pattern of DNA synthesis and mitosis. In rats, liver mass is completely restored by day 7–10 after which proliferation stops [8Michalopoulos G.K. DeFrances M.C. Liver regeneration.Science. 1997; 276: 60-66Crossref PubMed Scopus (2865) Google Scholar, 9Steer C.J. Liver regeneration.FASEB J. 1995; 9: 1396-1400Crossref PubMed Scopus (185) Google Scholar]. Over the last decade it has become obvious that liver regeneration is not a simple response to one growth factor, but rather a delicate and complex interplay of many cellular events. Several growth factors playing a role in liver regeneration have been identified. Among others, epidermal growth factor (EGF), transforming growth factor-α (TGFα), and Hepatocyte Growth Factor (HGF) are the most important ones to stimulate DNA synthesis in hepatocytes, whereas TGFβ1 and activin are potential inhibitors of proliferation [[10]Fausto N. Laird A.D. Webber E.M. Liver regeneration. 2. Role of growth factors and cytokines in hepatic regeneration.FASEB J. 1995; 9: 1527-1536Crossref PubMed Scopus (569) Google Scholar]. Furthermore, several cytokines are involved. Interleukin-6 (Il-6), interleukin-1 (Il-1) and tumor necrosis factor-α (TNF-α) are the crucial factors in early signalling leading to liver regeneration. TNF-α stimulates IL-6 secretion from Kupffer cells after partial hepatectomy. These cytokines induce at least two transcription factors: (1) ‘post hepatectomy factor/nuclear factor kappa B’ (PHF/NF-κB) and (2) ‘signal transducer and activator of transcription-3’ (STAT 3). It has been demonstrated that liver regeneration is greatly impaired and that STAT 3 activation does not occur in IL-6 and TNF-α receptor deficient mice [11Cressman D.E. Greenbaum L.E. DeAngelis R.A. Ciliberto G. Furth E.E. Poli V. et al.Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice.Science. 1996; 274: 1379-1383Crossref PubMed Scopus (1302) Google Scholar, 12Yamada Y. Kirillova I. Peschon J.J. Fausto N. Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor.Proc Natl Acad Sci USA. 1997; 94: 1441-1445Crossref PubMed Scopus (827) Google Scholar]. Activation of the above transcription factors causes transcription of immediate-early genes, which is independent of de novo protein synthesis. Delayed-early response genes are induced within hours after partial hepatectomy. In this way hepatocytes manage to divide and at the same time provide differentiated functions via liver associated transcription factors. Because of the imbalance in replication rate of hepatocytes and their surrounding extracellular matrix (ECM), normal liver architecture is lost. Newly formed hepatocytes are arranged in clusters without associated sinusoids. By day 4 after partial hepatectomy, cell processes of laminin containing hepatic stellate cells penetrate between hepatocyte islands. Fenestrated endothelial cells trail the hepatic stellate cells, thereby separating hepatocytes into cell plates and restoring normal vascular architecture [[13]Martinez-Hernandez A. Amenta P.S. The extracellular matrix in hepatic regeneration.FASEB J. 1995; 9: 1401-1410Crossref PubMed Scopus (274) Google Scholar]. Even though extensive research has been performed on liver regeneration, the role of ECM and non-parenchymal cells (sinusoidal endothelial cells, hepatic stellate cells, Kupffer cells) has been relatively underexplored. In this issue of the Journal of Hepatology, Shimizu et al. [[4]Shimizu H. Miyazaki M. Wakabayashi Y. Mitsuhashi N. Kato A. Ito H. et al.Vascular endothelial growth factor secreted by replicating hepatocytes induces sinusoidal endothelial cell proliferation during regeneration after partial hepatectomy in rats.J Hepatol. 2001; 34: 683-689Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar] and Sato et al. [[5]Sato T. El-Assal O.N. Ono T. Yamanoi A. Dhar D.K. Nagasue N. Sinusoidal endothelial cell proliferation and expression of angiopoietin/Tie family in regenerating rat liver.J Hepatol. 2001; 34: 690-698Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar] report on sinusoidal endothelial cell proliferation induced by vascular endothelial growth factor (VEGF) during liver regeneration. Angiogenesis is a fundamental prerequisite for organ development, wound healing and reproductive functions, and plays an important role in tumor growth and metastasis development. Numerous non-specific growth factors acting on vascular endothelium have been described, including TGF-α and β, TNF-α, HGF and Il-8 [[14]Ferrara N. Molecular and biological properties of vascular endothelial growth factor.J Mol Med. 1999; 77: 527-543Crossref PubMed Scopus (1070) Google Scholar]. Vascular endothelial growth factor was shown to be a potent, diffusable and specific growth factor for endothelium. In recent years, several members of the VEGF family have been described, namely placenta growth factor (PlGF), VEGF-A, VEGF-B, VEGF-C and VEGF-D [[15]Yancopoulos G.D. Davis S. Gale N.W. Rudge J.S. Wiegand S.J. Holash J. Vascular-specific growth factors and blood vessel formation.Nature. 2000; 407: 242-248Crossref PubMed Scopus (3253) Google Scholar]. Vascular endothelial growth factors act through at least three different receptors VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4) of which VEGFR-2 seems to be the principal mediator of growth and permeability actions of VEGF. Angiopoietins, another family of four specific growth factors for vascular endothelial cells (Ang-1, Ang-2, Ang-3 and Ang-4), act through Tie-1 and Tie-2 tyrosin kinase receptors [[16]Senger D.R. Galli S.J. Dvorak A.M. Perruzzi C.A. Harvey V.S. Dvorak H.F. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid.Science. 1983; 219: 983-985Crossref PubMed Scopus (3380) Google Scholar]. The first described effect of VEGF (formerly termed vascular permeability factor) was the ability to induce vascular leakage [[14]Ferrara N. Molecular and biological properties of vascular endothelial growth factor.J Mol Med. 1999; 77: 527-543Crossref PubMed Scopus (1070) Google Scholar]. Vascular leakage of plasma proteins results in formation of extravascular fibrin gel, a substrate for endothelial cell growth. Later, multiple other biological effects have been attributed to VEGF. Without any doubt, its strong mitogenic activity on vascular endothelial cells in arteries, veins and lymphatic vessels is very important. VEGF also stimulates expression of metalloproteinases, urokinase-type and tissue-type plasminogen activators and plasminogen activator inhibitor 1 as well as in vitro and in vivo fenestrations in endothelial cells. It has also been reported that VEGF promotes expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 in endothelial cells. VEGF also induces nitric oxide mediated vasodilatation and promotes monocyte chemotaxis [[14]Ferrara N. Molecular and biological properties of vascular endothelial growth factor.J Mol Med. 1999; 77: 527-543Crossref PubMed Scopus (1070) Google Scholar]. Finally, VEGF was found to have an inhibitory effect on functional maturation of dendritic cells, suggesting that VEGF may facilitate tumor growth by attenuating immune response [[17]Gabrilovich D.I. Chen H.L. Girgis K.R. Cunningham H.T. Meny G.M. Nadaf S. et al.Production of vascular endothelial growth factor by human tumours inhibits the functional maturation of dendritic cells.Nat Med. 1996; 2: 1096-1103Crossref PubMed Scopus (1575) Google Scholar]. VEGF mRNA has been confirmed to be upregulated in the vast majority of human tumours, and both tumor and tumor-associated stroma are important sites of VEGF production [14Ferrara N. Molecular and biological properties of vascular endothelial growth factor.J Mol Med. 1999; 77: 527-543Crossref PubMed Scopus (1070) Google Scholar, 18Fukumura D. Xavier R. Sugiura T. Chen Y. Park E.C. Lu N. et al.Tumor induction of VEGF promoter activity in stromal cells.Cell. 1998; 94: 715-725Abstract Full Text Full Text PDF PubMed Scopus (828) Google Scholar]. Only recently, the role of VEGF in liver necrosis and regeneration has been explored. Following liver necrosis, VEGF mRNA and protein was found in hepatocytes, but also in Kupffer cells, hepatic stellate cells and infiltrating inflammatory cells [19Ishikawa K. Mochida S. Mashiba S. Inao M. Matsui A. Ikeda H. et al.Expressions of vascular endothelial growth factor in nonparenchymal as well as parenchymal cells in rat liver after necrosis.Biochem Biophys Res Commun. 1999; 254: 587-593Crossref PubMed Scopus (78) Google Scholar, 20Mochida S. Ishikawa K. Inao M. Shibuya M. Fujiwara K. Increased expressions of vascular endothelial growth factor and its receptors, flt-1, KDR/flk-1, in regenerating liver.Biochem Biophys Res Commun. 1996; 226: 176-179Crossref PubMed Scopus (113) Google Scholar]. The studies by Shimizu and Sato and their colleagues in this issue of the Journal of Hepatology, [4Shimizu H. Miyazaki M. Wakabayashi Y. Mitsuhashi N. Kato A. Ito H. et al.Vascular endothelial growth factor secreted by replicating hepatocytes induces sinusoidal endothelial cell proliferation during regeneration after partial hepatectomy in rats.J Hepatol. 2001; 34: 683-689Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 5Sato T. El-Assal O.N. Ono T. Yamanoi A. Dhar D.K. Nagasue N. Sinusoidal endothelial cell proliferation and expression of angiopoietin/Tie family in regenerating rat liver.J Hepatol. 2001; 34: 690-698Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar] extended these observations. Shimizu et al. [[4]Shimizu H. Miyazaki M. Wakabayashi Y. Mitsuhashi N. Kato A. Ito H. et al.Vascular endothelial growth factor secreted by replicating hepatocytes induces sinusoidal endothelial cell proliferation during regeneration after partial hepatectomy in rats.J Hepatol. 2001; 34: 683-689Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar]. showed that remnant liver after partial hepatectomy increases the VEGF expression at both the mRNA and protein level. Serum levels of VEGF and its in vitro production by isolated hepatocytes increased 72 h after partial hepatectomy. Using immunohistochemistry, the authors demonstrated that hepatocytes located in the periportal area were the main source of VEGF production. Moreover, they were able to detect upregulation of VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) on sinusoidal endothelial cells. Finally, proliferation of sinusoidal endothelial cells was shown to follow hepatocyte proliferation by 24–48 h. In agreement with Shimizu's report, Sato and colleagues also showed a lag of 24 h between sinusoidal endothelial cells and hepatocyte proliferation [[5]Sato T. El-Assal O.N. Ono T. Yamanoi A. Dhar D.K. Nagasue N. Sinusoidal endothelial cell proliferation and expression of angiopoietin/Tie family in regenerating rat liver.J Hepatol. 2001; 34: 690-698Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar]. Both teams also report an increase in the mRNA level of VEGF peaking at 72 h after partial hepatectomy and an upregulation of the VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) receptors. Furthermore, mRNA expression of angiogenic growth factors Ang-1 and Ang-2, and Tie-1 and Tie-2 receptors increased significantly 96 h after partial hepatectomy. While mRNA expression of Ang-1, Tie-1 and Tie-2 decreased thereafter, Ang-2 mRNA continued to increase reaching a peak at 168 h after partial hepatectomy. Vascular endothelial growth factor is up-regulated by hypoxia and a number of cytokines including EGF, TGF-β, Il-1 and Il-6 [[14]Ferrara N. Molecular and biological properties of vascular endothelial growth factor.J Mol Med. 1999; 77: 527-543Crossref PubMed Scopus (1070) Google Scholar]. It has been demonstrated that activated rat hepatic stellate cells also express VEGF receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) [[21]Mashiba S. Mochida S. Ishikawa K. Inao M. Matsui A. Ohno A. et al.Inhibition of hepatic stellate cell contraction during activation in vitro by vascular endothelial growth factor in association with upregulation of FLT tyrosine kinase receptor family, FLT-1.Biochem Biophys Res Commun. 1999; 258: 674-678Crossref PubMed Scopus (19) Google Scholar] and that hypoxic stimulation induces VEGF and VEGFR-1 (Flt-1) mRNA and VEGF secretion in activated hepatic stellate cells [[22]Ankoma-Sey V. Wang Y. Dai Z. Hypoxic stimulation of vascular endothelial growth factor expression in activated rat hepatic stellate cells.Hepatology. 2000; 31: 141-148Crossref PubMed Scopus (160) Google Scholar]. Cellular hypoxia is known to occur in alcoholic liver injury, hepatic fibrosis and in tumours, three conditions in which hepatic stellate cells play an important role. Recently, the expression of VEGF in hepatocellular carcinoma was examined. In well differentiated hepatocellular carcinoma, VEGF was highly expressed, whereas it was weakly expressed in poorly differentiated hepatocellular carcinoma [[23]Yamaguchi R. Yano H. Iemura A. Ogasawara S. Haramaki M. Kojiro M. Expression of vascular endothelial growth factor in human hepatocellular carcinoma.Hepatology. 1998; 28: 68-77Crossref PubMed Scopus (354) Google Scholar]. Small, well differentiated hepatocellular carcinomas receive blood from portal veins. With increasing size and decreasing differentiation, the tumours receive more arterial blood supply. It was suggested that VEGF plays a role in the early stage of angiogenesis in hepatocellular carcinoma, but further studies are needed to clarify the role of VEGF in angiogenesis in hepatocellular carcinoma in vivo. In conclusion, the work performed by Shimizu, Takashi and their colleagues is of importance in unravelling the bio-ecological system formed by sinusoidal cells, hepatocytes and their surrounding ECM. It is clear that communication between different cell types is of the highest importance in restoring homeostasis following various types of liver injury. Furthermore, improved knowledge of the influence of VEGF on neovascularisation will lead to better understanding of progression of hepatocellular carcinoma.