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Angiogenesis in liver disease

  • Mercedes Fernández
    Affiliations
    Hepatic Hemodynamic Laboratory, Liver Unit, Hospital Clı´nic-IDIBAPS, University of Barcelona, Villarroel 170, 08036 Barcelona, Spain

    Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain
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  • David Semela
    Affiliations
    Division of Gastroenterology and Hepatology, University Hospital Basel, Basel, Switzerland
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  • Jordi Bruix
    Affiliations
    Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain

    Barcelona Clı´nic Liver Cancer (BCLC) Group, Liver Unit, Hospital Clı´nic-IDIBAPS, University of Barcelona, Spain
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  • Isabelle Colle
    Affiliations
    Department of Hepatology and Gastroenterology, Ghent University Hospital, Ghent, Belgium
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  • Massimo Pinzani
    Affiliations
    Dipartimento di Medicina Interna – Center for Research, High Education and Transfer “DENOThe”- University of Florence, Florence, Italy
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  • Jaume Bosch
    Correspondence
    Corresponding author. Address: Hepatic Hemodynamic Laboratory, Liver Unit, Hospital Clı´nic-IDIBAPS, University of Barcelona, Villarroel 170, 08036 Barcelona, Spain. Tel.: +34 932275790; fax: +34 932279348.
    Affiliations
    Hepatic Hemodynamic Laboratory, Liver Unit, Hospital Clı´nic-IDIBAPS, University of Barcelona, Villarroel 170, 08036 Barcelona, Spain

    Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain
    Search for articles by this author
Open AccessPublished:December 31, 2008DOI:https://doi.org/10.1016/j.jhep.2008.12.011
      Angiogenesis and disruption of liver vascular architecture have been linked to progression to cirrhosis and liver cancer (HCC) in chronic liver diseases, which contributes both to increased hepatic vascular resistance and portal hypertension and to decreased hepatocyte perfusion. On the other hand, recent evidence shows that angiogenesis modulates the formation of portal-systemic collaterals and the increased splanchnic blood flow which are involved in the life threatening complications of cirrhosis. Finally, angiogenesis plays a key role in the growth of tumours, suggesting that interference with angiogenesis may prevent or delay the development of HCC. This review summarizes current knowledge on the molecular mechanisms of liver angiogenesis and on the consequences of angiogenesis in chronic liver disease. On the other hand, it presents the different strategies that have been used in experimental models to counteract excessive angiogenesis and its potential role in preventing transition to cirrhosis, development of portal hypertension and its consequences, and its application in the treatment of hepatocellular carcinoma.

      Abbreviations:

      CLDs (chronic liver diseases), PDGF (platelet-derived growth factor), FGF (fibroblast growth factor), ECM (extra-cellular matrix), VEGF (vascular endothelial growth factor), HSC (hepatic stellate cells), MFs (myofibroblasts), PVL (portal vein ligation), LSECs (liver sinusoidal endothelial cells), HGF (hepatocyte growth factor), HCC (hepatocellular carcinoma)

      Keywords

      1. Introduction

      Cirrhosis and hepatocellular carcinoma (HCC) are common lethal diseases in European countries, together representing the third cause of death in adults over 50 years old, as well as the indication for over 90% of the 5.000 liver transplants that are performed every year within the EU. These features are increasing due to the consequences of the hepatitis C epidemic in the 70’s. Thus, its socioeconomic impact is extraordinary.
      The formation of new vessels (angiogenesis) and the establishment of an abnormal angioarchitecture of the liver is a process strictly related to the progressive fibrogenesis leading to cirrhosis and liver cancer. Investigation into these aspects is complex and certainly requires a joint effort of a multidisciplinary team of basic investigators, pathologists, and hepatologists in the areas of liver fibrosis, hepatic circulation, and portal hypertension and its complications.
      Established evidence clearly indicates that chronic liver diseases are characterized by intrahepatic vascular remodelling with capillarization of sinusoids, fibrogenesis and development of intrahepatic shunts, which would lead to increased hepatic resistance (and hence to increased portal pressure) and decreased effective hepatocyte perfusion (and hence to liver failure). In addition, new original data obtained by the authors of this review suggest that vascular endothelial growth factor (VEGF)/ platelet-derived growth factor (PDGF) driven angiogenesis is of paramount importance in the formation of portal-systemic collaterals and of the hyperdynamic circulation which are responsible for the main complications of cirrhosis often leading to death: gastroesophageal varices, massive upper gastrointestinal bleeding, ascites, spontaneous bacterial peritonitis and hepatic encephalopathy. Finally, angiogenesis is known to play a critical role in the growth of tumours, which makes it plausible to hypothesize that early interference with angiogenesis signalling may prevent the transition from hepatic dysplasia to HCC.
      This article reviews the translational research effort that has been made recently on both the molecular mechanisms and signal transduction cascade of liver angiogenesis, and the consequences of angiogenesis in chronic liver disease, emphasizing studies exploring different strategies to counteract excessive angiogenesis to prevent progression of liver fibrosis and transition to cirrhosis in chronic hepatitis, to prevent the development of portal hypertension and its consequences, and finally to prevent the formation and growth of hepatocellular carcinoma often occurring in patients with cirrhosis.

      2. Angiogenesis and fibrogenesis

      Pathological angiogenesis, irrespective of the aetiology, has been indeed extensively described in chronic liver diseases (CLDs) characterized by an extensive and prolonged necro-inflammatory and fibrogenic process, including chronic HBV, HCV and autoimmune hepatitis [
      • Garcia-Monzon C.
      • Sanchez-Madrid F.
      • Garcia-Buey L.
      • Garcia-Arroyo A.
      • Garcia-Sanchez A.
      • Moreno-Otero R.
      Vascular adhesion molecule expression in viral chronic hepatitis: evidence of neoangiogenesis in portal tracts.
      ,
      • Medina J.
      • Arroyo A.G.
      • Sanchez-Madrid F.
      • Moreno-Otero R.
      Angiogenesis in chronic inflammatory liver disease.
      ], and primary biliary cirrhosis [
      • Medina J.
      • Sanz-Cameno P.
      • Garcia-Buey L.
      • Martin-Vilchez S.
      • Lopez-Cabrera M.
      • Moreno-Otero R.
      Evidence of angiogenesis in primary biliary cirrhosis: an immunohistochemical descriptive study.
      ]. The formation of new vessels, which is closely associated with the pattern of fibrosis development typical of the different CLDs [
      • Pinzani M.
      • Rombouts K.
      Liver fibrosis: from the bench to clinical targets.
      ], leads to the progressive formation of the abnormal angio-architecture distinctive of cirrhosis, i.e. the common end-point of fibrogenic CLDs. Accordingly, the association of fibrogenesis and angiogenesis should be regarded as crucial in the modern evaluation of disease progression and in the search for therapeutic targets. In addition, depending on the different pattern of fibrogenic evolution (i.e. post-necrotic, biliary, centrolobular, pericellular/perisinusoidal), the extent of neo-angiogenesis may have profound consequences on the rate of disease progression to cirrhosis and represents a key determinant affecting reversibility of fibrosis (Table 1).
      Table 1Reversibility of liver fibrosis according to the pattern.
      Fibrosis patternEarly portal to central septaNeo angiogenesisReversibility
      Post-necrotic+++++++++
      Biliary+++++++
      Centrolobular±++++
      Pericellular-perisinusoidal+++++
      From a mechanistic point of view, angiogenesis in fibrogenic CLDs can be interpreted according to two main pathways. First, the process of liver chronic wound healing typical of fibrogenic CLDs is characterized by an over-expression of several growth factors, cytokines and metalloproteinases (MMPs) with an inherent pro-angiogenic action [
      • Pinzani M.
      • Marra F.
      Cytokine receptors and signalling in hepatic stellate cells.
      ]. In particular, platelet-derived growth factor (PDGF), transforming growth factor-β1 (TGF-β1), fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) have been shown to exert a potent pro-fibrogenic and pro-angiogenic role. In addition, an increased gene expression of integrins, β-catenin, ephrins, and other adhesion molecules involved in extra-cellular matrix (ECM) remodelling and angiogenesis has been clearly demonstrated in CLDs [
      • Shackel N.A.
      • McGuinness P.H.
      • Abbott C.A.
      • Gorrell M.D.
      • McCaughan G.W.
      Insights into the pathobiology of hepatitis C virus-associated cirrhosis: analysis of intrahepatic differential gene expression.
      ,
      • Shackel N.A.
      • McGuinness P.H.
      • Abbott C.A.
      • Gorrell M.D.
      • McCaughan G.W.
      Identification of novel molecules and pathogenic pathways in primary biliary cirrhosis: cDNA array analysis of intrahepatic differential gene expression.
      ]. Second, neo-angiogenesis is stimulated in hepatic tissue by the progressive increase of tissue hypoxia. This mechanism is strictly linked to the anatomical modifications following the establishment of periportal fibrosis with an increased contribution of the hepatic artery to the formation of sinusoidal blood [
      • Hoofring A.
      • Boitnott J.
      • Torbenson M.
      Three-dimensional reconstruction of hepatic bridging fibrosis in chronic hepatitis C viral infection.
      ]. Accordingly, sinusoidal blood flow becomes increasingly arterialized with hepatocytes adjusting to an abnormally high oxygen concentration. Subsequently, the progressive capillarization of sinusoids leads to an impairment of oxygen diffusion from the sinusoids to hepatocytes with the consequent up-regulation of pro-angiogenic pathways [
      • 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.
      ,
      • DeLeve L.D.
      Hepatic microvasculature in liver injury.
      ,
      • Rosmorduc O.
      • Wendum D.
      • Corpechot C.
      • Galy B.
      • Sebbagh N.
      • Raleigh J.
      • et al.
      Hepatocellular hypoxia-induced vascular endothelial growth factor expression and angiogenesis in experimental biliary cirrhosis.
      ].
      Although neo-angiogenesis is a common feature of most chronic inflammatory and fibrogenic disorders [
      • Carmeliet P.
      • Jain R.K.
      Angiogenesis in cancer and other diseases.
      ,
      • Carmeliet P.
      Angiogenesis in life, disease and medicine.
      ], hepatic angiogenesis may substantially differ from homologous processes in other organs or tissue on the basis of: (a) the rather unique phenotypic profile and functional role of activated hepatic stellate cells (HSC) and of other liver myofibroblasts (MFs) [
      • Cassiman D.
      • Libbrecht L.
      • Desmet V.
      • Denef C.
      • Roskams T.
      Hepatic stellate cell/myofibroblast subpopulations in fibrotic human and rat livers.
      ,
      • Friedman S.L.
      Liver fibrosis – from bench to bedside.
      ,
      • Lee J.S.
      • Semela D.
      • Iredale J.
      • Shah V.H.
      Sinusoidal remodelling and angiogenesis: a new function for the liver-specific pericyte?.
      ,
      • Novo E.
      • Cannito S.
      • Zamara E.
      • Valfre di B.L.
      • Caligiuri A.
      • Cravanzola C.
      • et al.
      Proangiogenic cytokines as hypoxia-dependent factors stimulating migration of human hepatic stellate cells.
      ,
      • Parola M.
      • Marra F.
      • Pinzani M.
      Myofibroblast – like cells and liver fibrogenesis: emerging concepts in a rapidly moving scenario.
      ,
      • Ramadori G.
      • Saile B.
      Mesenchymal cells in the liver – one cell type or two?.
      ,
      • Bataller R.
      • Brenner D.A.
      Liver fibrosis.
      ] (b) the presence of two different microvascular structures described (i.e., sinusoids lined by fenestrated endothelium versus large vessels lined by a continuous one); (c) the existence of ANGPTL3, a liver specific angiogenic factor [
      • Camenisch G.
      • Pisabarro M.T.
      • Sherman D.
      • Kowalski J.
      • Nagel M.
      • Hass P.
      • et al.
      ANGPTL3 stimulates endothelial cell adhesion and migration via integrin alpha vbeta 3 and induces blood vessel formation in vivo.
      ]. Evidence obtained from morphological studies suggests that angiogenesis occurring in hepatic tissue undergoing chronic wound healing is characterized by branching of neo-vessels from the existing vasculature. The large majority of these neo-vessels originate from the fine portal vein branches and tend to establish a connection between the portal system and the hepatic veins [
      • Hoofring A.
      • Boitnott J.
      • Torbenson M.
      Three-dimensional reconstruction of hepatic bridging fibrosis in chronic hepatitis C viral infection.
      ,
      • Onori P.
      • Morini S.
      • Franchitto A.
      • Sferra R.
      • Alvaro D.
      • Gaudio E.
      Hepatic microvascular features in experimental cirrhosis: a structural and morphometrical study in CCl4-treated rats.
      ]. The role of bone marrow-derived endothelial precursors (vasculogenesis) in hepatic angiogenesis has been suggested by studies employing animal models of hepatic fibrogenesis [
      • Jin Y.L.
      • Enzan H.
      • Kuroda N.
      • Hayashi Y.
      • Toi M.
      • Miyazaki E.
      • et al.
      Vascularization in tissue remodelling after rat hepatic necrosis induced by dimethylnitrosamine.
      ,
      • Nakamura T.
      • Torimura T.
      • Sakamoto M.
      • Hashimoto O.
      • Taniguchi E.
      • Inoue K.
      • et al.
      Significance and therapeutic potential of endothelial progenitor cell transplantation in a cirrhotic liver rat model.
      ] and needs to be substantiated in human CLDs.
      A key area in the study of the cellular and molecular relationships existing between fibrogenesis and angiogenesis concerns the pro-angiogenic role of activated HSC and other ECM-producing cells such as portal fibroblasts and myofibroblasts. Hypoxic conditions, through the involvement of the transcription factor HIF-1α, are able to up-regulate expression of VEGF [
      • Medina J.
      • Arroyo A.G.
      • Sanchez-Madrid F.
      • Moreno-Otero R.
      Angiogenesis in chronic inflammatory liver disease.
      ,
      • Novo E.
      • Cannito S.
      • Zamara E.
      • Valfre di B.L.
      • Caligiuri A.
      • Cravanzola C.
      • et al.
      Proangiogenic cytokines as hypoxia-dependent factors stimulating migration of human hepatic stellate cells.
      ,
      • Ankoma-Sey V.
      • Matli M.
      • Chang K.B.
      • Lalazar A.
      • Donner D.B.
      • Wong L.
      • et al.
      Coordinated induction of VEGF receptors in mesenchymal cell types during rat hepatic wound healing.
      ,
      • Aleffi S.
      • Petrai I.
      • Bertolani C.
      • Parola M.
      • Colombatto S.
      • Novo E.
      • et al.
      Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells.
      ,
      • Wang Y.Q.
      • Luk J.M.
      • Ikeda K.
      • Man K.
      • Chu A.C.
      • Kaneda K.
      • et al.
      Regulatory role of vHL/HIF-1alpha in hypoxia-induced VEGF production in hepatic stellate cells.
      ] and angiopoietin I [
      • Novo E.
      • Cannito S.
      • Zamara E.
      • Valfre di B.L.
      • Caligiuri A.
      • Cravanzola C.
      • et al.
      Proangiogenic cytokines as hypoxia-dependent factors stimulating migration of human hepatic stellate cells.
      ,
      • Aleffi S.
      • Petrai I.
      • Bertolani C.
      • Parola M.
      • Colombatto S.
      • Novo E.
      • et al.
      Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells.
      ] in rat or human HSC. Moreover, exposure to hypoxia results in up-regulation of VEGF receptors type I (Flt-1) and type II (Flk-1) as well as of Tie-2 (i.e., the receptor for angiopoietin I) in the same cell type [
      • Rosmorduc O.
      • Wendum D.
      • Corpechot C.
      • Galy B.
      • Sebbagh N.
      • Raleigh J.
      • et al.
      Hepatocellular hypoxia-induced vascular endothelial growth factor expression and angiogenesis in experimental biliary cirrhosis.
      ,
      • Novo E.
      • Cannito S.
      • Zamara E.
      • Valfre di B.L.
      • Caligiuri A.
      • Cravanzola C.
      • et al.
      Proangiogenic cytokines as hypoxia-dependent factors stimulating migration of human hepatic stellate cells.
      ,
      • Ankoma-Sey V.
      • Matli M.
      • Chang K.B.
      • Lalazar A.
      • Donner D.B.
      • Wong L.
      • et al.
      Coordinated induction of VEGF receptors in mesenchymal cell types during rat hepatic wound healing.
      ]. Hypoxia-dependent up-regulation and release of VEGF by human HSC/MFs can stimulate, in a paracrine and/or autocrine manner, non-oriented migration and chemotaxis of human HSC/MFs [
      • Novo E.
      • Cannito S.
      • Zamara E.
      • Valfre di B.L.
      • Caligiuri A.
      • Cravanzola C.
      • et al.
      Proangiogenic cytokines as hypoxia-dependent factors stimulating migration of human hepatic stellate cells.
      ]. This feature depends mainly on the interaction between VEGF and Flk-1 and may explain the significant “in vivo” anti-fibrotic effect reported in an experimental model in which animals were treated with neutralizing anti-Flk-1 antibodies [
      • Yoshiji H.
      • Kuriyama S.
      • Yoshii J.
      • Ikenaka Y.
      • Noguchi R.
      • Hicklin D.J.
      • et al.
      Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis.
      ]. Recent “in vivo” data obtained in human and rat fibrotic/cirrhotic livers, indicate that α-SMA-positive cells (i.e., myofibroblast-like phenotype) expressing VEGF, Ang-1 or the related receptors Flk-1 and Tie-2, are consistently localized at the leading edge of tiny and incomplete developing septa, but not in larger bridging septa [
      • Novo E.
      • Cannito S.
      • Zamara E.
      • Valfre di B.L.
      • Caligiuri A.
      • Cravanzola C.
      • et al.
      Proangiogenic cytokines as hypoxia-dependent factors stimulating migration of human hepatic stellate cells.
      ]. This distribution may reflect two different phases of angiogenic process during chronic wound healing: an early phase, occurring in developing septa, in which fibrogenesis and angiogenesis may be driven/modulated by ECM-producing cells, and a later phase occurring in larger and more mature fibrotic septa where the chronic wound healing is less active and fibrogenic transformation more established. In this latter setting pro-angiogenic factors are expressed only by endothelial cells, a scenario that is likely to favour the stabilization of the newly formed vessels. In this context, it is relevant that the promotion of a pro-angiogenic phenotype in activated HSC is stimulated also by non-hypoxic conditions and particularly by the exposure to the pro-fibrogenic adipokine leptin [
      • Aleffi S.
      • Petrai I.
      • Bertolani C.
      • Parola M.
      • Colombatto S.
      • Novo E.
      • et al.
      Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells.
      ].
      An elegant and convincing demonstration of the interplay between inflammatory response, angiogenesis, and fibrogenesis has been recently provided by an experimental study in which all these features have been significantly reduced by the treatment with the multitargeted receptor tyrosine kinase inhibitor sunitinib [
      • Tugues S.
      • Fernandez-Varo G.
      • Munoz-Luque J.
      • Ros J.
      • Arroyo V.
      • Rodes J.
      • et al.
      Antiangiogenic treatment with sunitinib ameliorates inflammatory infiltrate, fibrosis, and portal pressure in cirrhotic rats.
      ]. This study is of relevance because it provides evidence for a possible dual and converging pharmacological action (i.e. anti-fibrogenic and anti-angiogenic) able to interfere directly with liver myofibroblasts, presumably by negatively affecting PDGF-dependent signalling.

      3. Role of angiogenesis in portal hypertension

      Portal hypertension is a major complication of cirrhosis of the liver, which represents a leading cause of death and liver transplantation [
      • Bosch J.
      • Pizcueta P.
      • Feu F.
      • Fernandez M.
      • Garcia-Pagan J.C.
      Pathophysiology of portal hypertension.
      ,
      • Bosch J.
      • Garcia-Pagan J.C.
      The splanchnic circulation in cirrhosis.
      ,
      • Bosch J.
      • Berzigotti A.
      • Garcia-Pagan J.C.
      • Abraldes J.G.
      The management of portal hypertension: rational basis, available treatments and future options.
      ]. A salient feature of portal hypertension is the formation of an extensive network of portosystemic collateral vessels, which include the oesophageal and gastric varices, responsible for variceal bleeding, associated with a high mortality rate [
      • Bosch J.
      • Pizcueta P.
      • Feu F.
      • Fernandez M.
      • Garcia-Pagan J.C.
      Pathophysiology of portal hypertension.
      ,
      • Bosch J.
      • Garcia-Pagan J.C.
      The splanchnic circulation in cirrhosis.
      ,
      • Bosch J.
      • Berzigotti A.
      • Garcia-Pagan J.C.
      • Abraldes J.G.
      The management of portal hypertension: rational basis, available treatments and future options.
      ]. In addition, collateral vessels result in shunting of portal blood into the systemic circulation, causing high systemic concentrations of several substances normally metabolized by the liver, such as drugs, toxins, hormones, and bacteria. These in turn contribute to severe complications of cirrhosis, including portosystemic encephalopathy and sepsis [
      • Bosch J.
      • Pizcueta P.
      • Feu F.
      • Fernandez M.
      • Garcia-Pagan J.C.
      Pathophysiology of portal hypertension.
      ,
      • Bosch J.
      • Garcia-Pagan J.C.
      The splanchnic circulation in cirrhosis.
      ,
      • Bosch J.
      • Berzigotti A.
      • Garcia-Pagan J.C.
      • Abraldes J.G.
      The management of portal hypertension: rational basis, available treatments and future options.
      ]. Therefore, successful design of medical treatment for portal hypertension requires a better understanding of the mechanisms underlying the formation of portosystemic collateral vessels, an issue that has remained largely unexplored. Traditionally, formation of collaterals was considered to be a mechanical consequence of the increased portal pressure that will result in the opening of these vascular channels. Accordingly, therapeutic strategies are mainly aimed at decreasing portal pressure [
      • Bosch J.
      • Pizcueta P.
      • Feu F.
      • Fernandez M.
      • Garcia-Pagan J.C.
      Pathophysiology of portal hypertension.
      ,
      • Bosch J.
      • Garcia-Pagan J.C.
      The splanchnic circulation in cirrhosis.
      ,
      • Bosch J.
      • Berzigotti A.
      • Garcia-Pagan J.C.
      • Abraldes J.G.
      The management of portal hypertension: rational basis, available treatments and future options.
      ]. However, as discussed in this article, recent studies have examined another approach, based on the potential involvement of angiogenesis in the development of these collateral vessels.
      Another characteristic feature of the portal hypertensive syndrome is the development of a hyperdynamic circulatory state, with an increase in blood flow in splanchnic organs draining into the portal vein and a subsequent increase in portal venous inflow [
      • Bosch J.
      • Pizcueta P.
      • Feu F.
      • Fernandez M.
      • Garcia-Pagan J.C.
      Pathophysiology of portal hypertension.
      ,
      • Bosch J.
      • Garcia-Pagan J.C.
      The splanchnic circulation in cirrhosis.
      ,
      • Bosch J.
      • Berzigotti A.
      • Garcia-Pagan J.C.
      • Abraldes J.G.
      The management of portal hypertension: rational basis, available treatments and future options.
      ]. Such an increased portal venous inflow represents a significant factor maintaining and worsening portal hypertension [
      • Bosch J.
      • Pizcueta P.
      • Feu F.
      • Fernandez M.
      • Garcia-Pagan J.C.
      Pathophysiology of portal hypertension.
      ,
      • Bosch J.
      • Garcia-Pagan J.C.
      The splanchnic circulation in cirrhosis.
      ,
      • Bosch J.
      • Berzigotti A.
      • Garcia-Pagan J.C.
      • Abraldes J.G.
      The management of portal hypertension: rational basis, available treatments and future options.
      ]. The mechanisms underlying this splanchnic hyperemia are not fully understood, but involve overproduction of endogenous vasodilators and decreased vascular reactivity to vasoconstrictors [
      • Bosch J.
      • Pizcueta P.
      • Feu F.
      • Fernandez M.
      • Garcia-Pagan J.C.
      Pathophysiology of portal hypertension.
      ,
      • Bosch J.
      • Garcia-Pagan J.C.
      The splanchnic circulation in cirrhosis.
      ,
      • Bosch J.
      • Berzigotti A.
      • Garcia-Pagan J.C.
      • Abraldes J.G.
      The management of portal hypertension: rational basis, available treatments and future options.
      ]. An intriguing possibility is that an increased formation of splanchnic blood vessels through active angiogenesis could also be involved in the maintenance of a hyperdynamic splanchnic circulation in portal hypertension.
      In the last few years, these possibilities have been addressed by studying the effects of different anti-angiogenic strategies aimed at inhibiting the signalling pathways of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and placental growth factor (PLGF), which are essential mediators of angiogenesis [
      • Carmeliet P.
      Mechanisms of angiogenesis and arteriogenesis.
      ,
      • Folkman J.
      • D’Amore P.A.
      Blood vessel formation: what is its molecular basis?.
      ,
      • Ferrara N.
      • Gerber H.P.
      • LeCouter J.
      The biology of VEGF and its receptors.
      ], on the development and maintenance of hyperdynamic splanchnic circulation and portosystemic collateralization in experimental models of portal hypertension.

      3.1 Increased angiogenesis in portal hypertension

      Evidence supporting a role for angiogenesis in the pathogenesis of portal hypertension includes recent investigations demonstrating that VEGF, a potent angiogenic factor [
      • Ferrara N.
      • Gerber H.P.
      • LeCouter J.
      The biology of VEGF and its receptors.
      ], is overexpressed in splanchnic organs from portal hypertensive animals (Fig. 1) [
      • Fernandez M.
      • Vizzutti F.
      • Garcia-Pagan J.C.
      • Rodes J.
      • Bosch J.
      Anti-VEGF receptor-2 monoclonal antibody prevents portal-systemic collateral vessel formation in portal hypertensive mice.
      ,
      • Fernandez M.
      • Mejias M.
      • Angermayr B.
      • Garcia-Pagan J.C.
      • Rodes J.
      • Bosch J.
      Inhibition of VEGF receptor-2 decreases the development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in portal hypertensive rats.
      ]. The expression of VEGF receptor-2 (VEGFR-2) and the endothelial cell marker CD31 [
      • Newman P.J.
      • Berndt M.C.
      • Gorski J.
      • White G.C.
      • Lyman S.
      • Paddock C.
      • et al.
      PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily.
      ] is also increased in the splanchnic territory in experimental models of portal hypertension [
      • Fernandez M.
      • Vizzutti F.
      • Garcia-Pagan J.C.
      • Rodes J.
      • Bosch J.
      Anti-VEGF receptor-2 monoclonal antibody prevents portal-systemic collateral vessel formation in portal hypertensive mice.
      ,
      • Fernandez M.
      • Mejias M.
      • Angermayr B.
      • Garcia-Pagan J.C.
      • Rodes J.
      • Bosch J.
      Inhibition of VEGF receptor-2 decreases the development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in portal hypertensive rats.
      ]. These and other studies provided evidence of increased VEGF-driven splanchnic angiogenesis in portal hypertensive animals and in cirrhotic patients [
      • Cejudo-Martin P.
      • Ros J.
      • Navasa M.
      • Fernandez J.
      • Fernandez-Varo G.
      • Ruiz-del-Arbol L.
      • et al.
      Increased production of vascular endothelial growth factor in peritoneal macrophages of cirrhotic patients with spontaneous bacterial peritonitis.
      ,
      • Tsugawa K.
      • Hashizume M.
      • Tomikawa M.
      • Migou S.
      • Kawanaka H.
      • Shiraishi S.
      • et al.
      Immunohistochemical localization of vascular endothelial growth factor in the rat portal hypertensive gastropathy.
      ,
      • Sieber C.C.
      • Sumanovski L.T.
      • Stumm M.
      • van der K.M.
      • Battegay E.
      In vivo angiogenesis in normal and portal hypertensive rats: role of basic fibroblast growth factor and nitric oxide.
      ,
      • Sumanovski L.T.
      • Battegay E.
      • Stumm M.
      • van der K.M.
      • Sieber C.C.
      Increased angiogenesis in portal hypertensive rats: role of nitric oxide.
      ].
      Figure thumbnail gr1
      Fig. 1Overexpression of VEGF, VEGF receptor-2, and CD31 in portal hypertensive mice. (Left) Protein expressions in splanchnic organs from partial portal vein-ligated (PPVL) mice and sham-operated (SO) control animals, seven days after the initial surgery. (Right) Protein expressions at days 1, 5, and 7 after the induction of portal hypertension in mice. Representative western blots are shown at the top and densitometric quantification normalized to α-tubulin is shown at the bottom of each panel. P < 0.05 vs. SO mice (left) or vs. day 1 (right).
      The precise mechanism triggering VEGF-dependent angiogenesis in portal hypertension remains speculative, but it is likely to be multifactorial. Indeed, several factors relevant to the pathogenesis of portal hypertension, such as tissue hypoxia, cytokines, and mechanical stress, have been shown to promote VEGF expression in various cell types and tissues [
      • Bosch J.
      • Pizcueta P.
      • Feu F.
      • Fernandez M.
      • Garcia-Pagan J.C.
      Pathophysiology of portal hypertension.
      ,
      • Bosch J.
      • Garcia-Pagan J.C.
      The splanchnic circulation in cirrhosis.
      ,
      • Bosch J.
      • Berzigotti A.
      • Garcia-Pagan J.C.
      • Abraldes J.G.
      The management of portal hypertension: rational basis, available treatments and future options.
      ,
      • Ferrara N.
      • Gerber H.P.
      • LeCouter J.
      The biology of VEGF and its receptors.
      ].

      3.2 VEGF signalling blockade in portal hypertension

      Recent studies have determined the effects of several angiogenesis inhibitors, with different modes of action, in experimental models of portal hypertension. These studies demonstrated that treatment with an anti-VEGFR-2 monoclonal antibody (DC101) [
      • Witte L.
      • Hicklin D.J.
      • Zhu Z.
      • Pytowski B.
      • Kotanides H.
      • Rockwell P.
      • et al.
      Monoclonal antibodies targeting the VEGF receptor-2 (Flk1/KDR) as an anti-angiogenic therapeutic strategy.
      ] from the induction of portal hypertension markedly decreased the formation of portosystemic collateral vessels and reduced splanchnic vascularization in portal hypertensive mice [
      • Fernandez M.
      • Vizzutti F.
      • Garcia-Pagan J.C.
      • Rodes J.
      • Bosch J.
      Anti-VEGF receptor-2 monoclonal antibody prevents portal-systemic collateral vessel formation in portal hypertensive mice.
      ]. Similar results were obtained using SU5416, a specific inhibitor of the tyrosine kinase domain of VEGFR-2 [
      • Fernandez M.
      • Mejias M.
      • Angermayr B.
      • Garcia-Pagan J.C.
      • Rodes J.
      • Bosch J.
      Inhibition of VEGF receptor-2 decreases the development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in portal hypertensive rats.
      ,
      • Fong T.A.
      • Shawver L.K.
      • Sun L.
      • Tang C.
      • App H.
      • Powell T.J.
      • et al.
      SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types.
      ] as an anti-angiogenic strategy, which caused a significant 52% decrease in the extent of portosystemic collateral formation in rats with partial portal vein ligation (PPVL) [
      • Fernandez M.
      • Mejias M.
      • Angermayr B.
      • Garcia-Pagan J.C.
      • Rodes J.
      • Bosch J.
      Inhibition of VEGF receptor-2 decreases the development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in portal hypertensive rats.
      ]. In addition, SU5416 also markedly reduced the splanchnic hyperdynamic circulation in these animals, indicating that increased splanchnic arteriolar bed size mediated by a VEGF-dependent angiogenic process significantly contributes to the increased portal blood inflow. Portal pressure however was not modified, most likely because of the concomitant inhibition in the formation of portosystemic collateral vessels [
      • Fernandez M.
      • Mejias M.
      • Angermayr B.
      • Garcia-Pagan J.C.
      • Rodes J.
      • Bosch J.
      Inhibition of VEGF receptor-2 decreases the development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in portal hypertensive rats.
      ]. These findings were further confirmed using rapamycin, which inhibits VEGF production and reduces portosystemic collateral vessel formation by 67% in portal hypertensive rats, in parallel with a significant attenuation of the hyperdynamic splanchnic circulation [
      • Fernandez M.
      • Mejias M.
      • Garcia-Pras E.
      • Mendez R.
      • Garcia-Pagan J.C.
      • Bosch J.
      Reversal of portal hypertension and hyperdynamic splanchnic circulation by combined vascular endothelial growth factor and platelet-derived growth factor blockade in rats.
      ].
      Taken together, these data indicate that the development of hyperdynamic splanchnic circulation and splanchnic neovasculature as well as the formation of portosystemic collateral vessels in portal hypertensive animals are in part VEGF-dependent angiogenic processes that can be significantly prevented by inhibitors of the VEGF/VEGF receptor-2 signalling pathway, when its administration was started at the time of portal hypertension initiation [
      • Fernandez M.
      • Vizzutti F.
      • Garcia-Pagan J.C.
      • Rodes J.
      • Bosch J.
      Anti-VEGF receptor-2 monoclonal antibody prevents portal-systemic collateral vessel formation in portal hypertensive mice.
      ,
      • Fernandez M.
      • Mejias M.
      • Angermayr B.
      • Garcia-Pagan J.C.
      • Rodes J.
      • Bosch J.
      Inhibition of VEGF receptor-2 decreases the development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in portal hypertensive rats.
      ,
      • Fernandez M.
      • Mejias M.
      • Garcia-Pras E.
      • Mendez R.
      • Garcia-Pagan J.C.
      • Bosch J.
      Reversal of portal hypertension and hyperdynamic splanchnic circulation by combined vascular endothelial growth factor and platelet-derived growth factor blockade in rats.
      ].

      3.3 Combined VEGF and PDGF signalling blockade

      The studies described so far highlight the importance of angiogenesis in the pathogenesis of portal hypertension and suggest that anti-angiogenic treatment might be a promising therapeutic strategy to prevent the progression of the portal hypertensive syndrome [
      • Fernandez M.
      • Vizzutti F.
      • Garcia-Pagan J.C.
      • Rodes J.
      • Bosch J.
      Anti-VEGF receptor-2 monoclonal antibody prevents portal-systemic collateral vessel formation in portal hypertensive mice.
      ,
      • Fernandez M.
      • Mejias M.
      • Angermayr B.
      • Garcia-Pagan J.C.
      • Rodes J.
      • Bosch J.
      Inhibition of VEGF receptor-2 decreases the development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in portal hypertensive rats.
      ,
      • Fernandez M.
      • Mejias M.
      • Garcia-Pras E.
      • Mendez R.
      • Garcia-Pagan J.C.
      • Bosch J.
      Reversal of portal hypertension and hyperdynamic splanchnic circulation by combined vascular endothelial growth factor and platelet-derived growth factor blockade in rats.
      ]. In clinical practice, however, portal hypertension does not represent a significant problem until it is quite advanced and associated with clinical manifestations. Thus, for anti-angiogenic treatment strategies to become of great clinical relevance these should be able to revert the circulatory abnormalities associated with portal hypertension once these are fully developed [
      • Fernandez M.
      • Mejias M.
      • Garcia-Pras E.
      • Mendez R.
      • Garcia-Pagan J.C.
      • Bosch J.
      Reversal of portal hypertension and hyperdynamic splanchnic circulation by combined vascular endothelial growth factor and platelet-derived growth factor blockade in rats.
      ].
      In this regard, it should be noted that in the process of neovascularization, VEGF plays a predominant role in the initial stages of formation of new blood vessels, activating the proliferation of endothelial cells and the subsequent formation of an endothelial tubule, while maturation of the newly formed vessels is mainly modulated by the proangiogenic growth factor platelet-derived growth factor (PDGF), which regulates the investiture of the endothelial tubule with mural cell and pericyte populations, thereby stabilizing the vascular architecture of the nascent vessel [
      • Carmeliet P.
      Mechanisms of angiogenesis and arteriogenesis.
      ,
      • Jain R.K.
      Molecular regulation of vessel maturation.
      ]. Based on these considerations, it was hypothesized that the simultaneous targeting of the VEGF and PDGF signalling pathways, that is the simultaneous targeting of endothelial cells and pericytes, could provide a greater vascular destabilization and a better vascular regression than targeting either alone.
      It was first demonstrated that development of portal hypertension in PPVL rats was associated with a progressive overexpression of PDGF, which reached its peak later in the course of portal hypertension than VEGF overexpression [
      • Fernandez M.
      • Mejias M.
      • Garcia-Pras E.
      • Mendez R.
      • Garcia-Pagan J.C.
      • Bosch J.
      Reversal of portal hypertension and hyperdynamic splanchnic circulation by combined vascular endothelial growth factor and platelet-derived growth factor blockade in rats.
      ]. In accordance with the working hypothesis, the continued administration of the VEGF signalling inhibitor rapamycin plus the PDGF signalling inhibitor Gleevec® markedly decreased the splanchnic neovascularization and the pericyte coverage of neovessels in portal hypertensive rats (Fig. 2) [
      • Fernandez M.
      • Mejias M.
      • Garcia-Pras E.
      • Mendez R.
      • Garcia-Pagan J.C.
      • Bosch J.
      Reversal of portal hypertension and hyperdynamic splanchnic circulation by combined vascular endothelial growth factor and platelet-derived growth factor blockade in rats.
      ]. This combined treatment also resulted in a virtually complete reversal of the increased portal pressure (40% reduction) and the increased portal venous blood inflow of these animals (Fig. 3). This is important since clinical studies have shown a dramatic reduction of the risk of portal hypertensive complications and improved survival in patients achieving a decrease in portal pressure of at least 20% under drug therapy. Notably, the magnitude of the effects of the combination treatment was superior than the addition of the effects of either drug alone, suggesting a synergistic regulatory interaction between the VEGF and PDGF signalling pathways in mediating the maintenance of the vascular and hemodynamic abnormalities observed in portal hypertensive rats [
      • Fernandez M.
      • Mejias M.
      • Garcia-Pras E.
      • Mendez R.
      • Garcia-Pagan J.C.
      • Bosch J.
      Reversal of portal hypertension and hyperdynamic splanchnic circulation by combined vascular endothelial growth factor and platelet-derived growth factor blockade in rats.
      ]. These findings further suggest that in the absence of proliferating perivascular cells (ie, after PDGF signalling inhibition), the endothelium is more vulnerable to anti-angiogenic therapies targeting endothelial cells, such as VEGF signalling blockade [
      • Bergers G.
      • Song S.
      • Meyer-Morse N.
      • Bergsland E.
      • Hanahan D.
      Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors.
      ].
      Figure thumbnail gr2
      Fig. 2Effects of rapamycin (RAPA), Gleevec or RAPA + Gleevec on angiogenesis mediators when portal hypertension was completely established. (A) Expression of CD31, VEGF, VEGFR-2, PDGF, PDGFR-beta, and α-smooth muscle actin (α-SMA) in the intestine of PPVL rats after treatment with RAPA, Gleevec, RAPA + Gleevec or vehicle. Representative blots are shown at the left and quantification of expression normalized to α-tubulin is shown at the right. (a) P < 0.05 vs. PPVL-vehicle. (b) P < 0.05 vs. PPVL-RAPA. (B) Representative histological images of mesentery sections stained with H&E from PPVL and SO rats treated with RAPA or vehicle. Original magnification 40×. (C) Quantitative analysis of neovascularization in the mesentery from PPVL and SO rats treated with RAPA or vehicle. (a) P < 0.05 vs. PPVL-vehicle.
      Figure thumbnail gr3
      Fig. 3Effects of RAPA, Gleevec or RAPA + Gleevec on splanchnic hemodynamics when portal hypertension was completely established. (a) P < 0.05 vs. SO-vehicle; (b) P < 0.05 vs. SO-RAPA; (c) P < 0.05 vs. SO-Gleevec; (d) P < 0.05 vs. PPVL-vehicle.
      Overall, these findings demonstrate that angiogenesis is a pathological hallmark of portal hypertension, and have made the control of new blood vessel formation a promising therapeutic target to prevent the progression and to promote the regression of portal hypertension-related complications in clinical scenarios.

      3.4 Role of PLGF-derived angiogenesis in portal hypertension

      Placental growth factor (PLGF) was originally discovered in human placenta in 1991 and is a member of the VEGF family. Alternative splicing of the human PLGF gene generates three isoforms (PLGF-1, PLGF-2, and PLGF-3), while in mice only PLGF-2 is present [
      • Tjwa M.
      • Luttun A.
      • Autiero M.
      • Carmeliet P.
      VEGF and PlGF: two pleiotropic growth factors with distinct roles in development and homeostasis.
      ]. The 3-dimensional structures of VEGF and PLGF are similar; however they have only 42% identical amino-acids [
      • Tjwa M.
      • Luttun A.
      • Autiero M.
      • Carmeliet P.
      VEGF and PlGF: two pleiotropic growth factors with distinct roles in development and homeostasis.
      ,
      • De Falco S.
      • Gigante B.
      • Persico M.G.
      Structure and function of placental growth factor.
      ].
      PLGF is not highly expressed in normal tissue and during embryogenesis, as PLGF-deficient mice are viable and fertile and do not display major abnormalities [
      • De Falco S.
      • Gigante B.
      • Persico M.G.
      Structure and function of placental growth factor.
      ,
      • Carmeliet P.
      • Moons L.
      • Luttun A.
      • Vincenti V.
      • Compernolle V.
      • De Mol M.
      • et al.
      Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.
      ,
      • Luttun A.
      • Tjwa M.
      • Moons L.
      • Wu Y.
      • ngelillo-Scherrer A.
      • Liao F.
      • et al.
      Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1.
      ,
      • Nagy J.A.
      • Dvorak A.M.
      • Dvorak H.F.
      VEGF-A(164/165) and PlGF: roles in angiogenesis and arteriogenesis.
      ,
      • Oura H.
      • Bertoncini J.
      • Velasco P.
      • Brown L.F.
      • Carmeliet P.
      • Detmar M.
      A critical role of placental growth factor in the induction of inflammation and edema formation.
      ]. In the healthy state, PLGF plays a role during pregnancy and during the ovarian cycle, without affecting quiescent vessels. However, loss of PLGF impairs angiogenesis in the wounded skin, ischemic retina, limb, heart and in cancer, whereas administration of recombinant PLGF (rPLGF) promotes collateral vessel growth in models of limb and myocardial ischemia [
      • Carmeliet P.
      • Moons L.
      • Luttun A.
      • Vincenti V.
      • Compernolle V.
      • De Mol M.
      • et al.
      Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.
      ,
      • Autiero M.
      • Waltenberger J.
      • Communi D.
      • Kranz A.
      • Moons L.
      • Lambrechts D.
      • et al.
      Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Flt1 and Flk1.
      ].
      Placental growth factor binds only to VEGFR-1 and neuropilin-1 and enhances the effects of VEGF and thus angiogenesis only under pathological conditions [
      • Autiero M.
      • Waltenberger J.
      • Communi D.
      • Kranz A.
      • Moons L.
      • Lambrechts D.
      • et al.
      Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Flt1 and Flk1.
      ,
      • Otrock Z.K.
      • Makarem J.A.
      • Shamseddine A.I.
      Vascular endothelial growth factor family of ligands and receptors: review.
      ,
      • Adini A.
      • Kornaga T.
      • Firoozbakht F.
      • Benjamin L.E.
      Placental growth factor is a survival factor for tumor endothelial cells and macrophages.
      ]. VEGFR1 is minimally expressed in adult quiescent vessels but membranous localisation is markedly up-regulated during pathological conditions, stimulating the PLGF-dependent angiogenic signals [
      • Otrock Z.K.
      • Makarem J.A.
      • Shamseddine A.I.
      Vascular endothelial growth factor family of ligands and receptors: review.
      ]. This makes PLGF attractive as a therapeutic target [
      • De Falco S.
      • Gigante B.
      • Persico M.G.
      Structure and function of placental growth factor.
      ,
      • Otrock Z.K.
      • Makarem J.A.
      • Shamseddine A.I.
      Vascular endothelial growth factor family of ligands and receptors: review.
      ]. The synergistic effects between PLGF and VEGF are shown in Fig. 4.
      Figure thumbnail gr4
      Fig. 4Diagramm illustrating the synergism between vascular endothelial growth factor (VEGF) and placental growth factor (PLGF). (VEGF receptor 1, VEGFR1; VEGF receptor 2, VEGFR2).

      3.5 Effects of the PLGF/VEGFR1 signalling pathway

      PLGF reconstitutes haematopoiesis by recruiting VEGFR1 positive stem cells from the bone marrow, stimulates the survival of endothelial cells and monocytes [
      • Adini A.
      • Kornaga T.
      • Firoozbakht F.
      • Benjamin L.E.
      Placental growth factor is a survival factor for tumor endothelial cells and macrophages.
      ,
      • Hattori K.
      • Heissig B.
      • Wu Y.
      • Dias S.
      • Tejada R.
      • Ferris B.
      • et al.
      Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1(+) stem cells from bone-marrow microenvironment.
      ,
      • Odorisio T.
      • Schietroma C.
      • Zaccaria M.L.
      • Cianfarani F.
      • Tiveron C.
      • Tatangelo L.
      • et al.
      Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability.
      ], and plays an important role in inflammation by recruiting endothelial cells, monocytes, progenitor cells and functional natural killer cells and by stimulating migration of inflammatory cells and chemotaxis. Leukocytes and inflammation can cause the release of multiple angiogenic factors such as VEGF, platelet-derived growth factor (PDGF), PLGF and basic fibroblast growth factor [
      • Carmeliet P.
      Angiogenesis in health and disease.
      ].
      PLGF is also a major player in arteriogenesis (and mature vessel formation) via recruitment of bone marrow cells, smooth muscle cells, pericytes, endothelial cells and monocytes [
      • Carmeliet P.
      Manipulating angiogenesis in medicine.
      ]. PLGF enhances collateral growth by stimulating endothelial and smooth muscle cell growth. Other angiogenic factors such as VEGF, PDGF, and angiopoetin 1 enhance the formation of collaterals [
      • Luttun A.
      • Tjwa M.
      • Moons L.
      • Wu Y.
      • ngelillo-Scherrer A.
      • Liao F.
      • et al.
      Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1.
      ,
      • Carmeliet P.
      Angiogenesis in health and disease.
      ,
      • Carmeliet P.
      Manipulating angiogenesis in medicine.
      ,
      • Heil M.
      • Ziegelhoeffer T.
      • Pipp F.
      • Kostin S.
      • Martin S.
      • Clauss M.
      • et al.
      Blood monocyte concentration is critical for enhancement of collateral artery growth.
      ,
      • Kamihata H.
      • Matsubara H.
      • Nishiue T.
      • Fujiyama S.
      • Amano K.
      • Iba O.
      • et al.
      Improvement of collateral perfusion and regional function by implantation of peripheral blood mononuclear cells into ischemic hibernating myocardium.
      ,
      • Pipp F.
      • Heil M.
      • Issbrucker K.
      • Ziegelhoeffer T.
      • Martin S.
      • van den H.J.
      • et al.
      VEGFR-1-selective VEGF homologue PlGF is arteriogenic: evidence for a monocyte-mediated mechanism.
      ].

      3.6 PLGF in portal hypertension

      Geerts et al. have recently demonstrated that neo-angiogenesis in the mesentery of portal hypertensive mice is associated with an up-regulation of VEGF, PLGF protein levels, and CD31 (marker for endothelial cells and thus angiogenesis) [
      • Geerts A.
      • Vanheule E.
      • Carmeliet P.
      • Van Vlierberghe H.
      • Leybaert L.
      • De Vos M.
      • et al.
      Placental growth factor plays a role in the mesenteric neo-angiogenesis of portal hypertensive mice.
      ,
      • Geerts A.M.
      • De Vriese A.S.
      • Vanheule E.
      • Van V.H.
      • Mortier S.
      • Cheung K.J.
      • et al.
      Increased angiogenesis and permeability in the mesenteric microvasculature of rats with cirrhosis and portal hypertension: an in vivo study.
      ].
      Recent studies using PLGF knockout mice (PLGF−/−) have shown that PLGF(−/−) portal hypertensive mice do not develop mesenteric neo-angiogenesis. CD31 protein expression in portal hypertensive PLGF(−/−) mice was significantly lower than in portal hypertensive wild- type mice, and similar to those of sham operated mice [
      • Geerts A.
      • Vanheule E.
      • Carmeliet P.
      • Van Vlierberghe H.
      • Leybaert L.
      • De Vos M.
      • et al.
      Placental growth factor plays a role in the mesenteric neo-angiogenesis of portal hypertensive mice.
      ,
      • Geerts A.M.
      • De Vriese A.S.
      • Vanheule E.
      • Van V.H.
      • Mortier S.
      • Cheung K.J.
      • et al.
      Increased angiogenesis and permeability in the mesenteric microvasculature of rats with cirrhosis and portal hypertension: an in vivo study.
      ]. This was also confirmed by immunohistochemistry. Portal hypertensive PLGF(−/−) mice showed a tendency towards a lower portal pressure and a significant decrease (but not normalization) in spleen weight [
      • Geerts A.
      • Vanheule E.
      • Carmeliet P.
      • Van Vlierberghe H.
      • Leybaert L.
      • De Vos M.
      • et al.
      Placental growth factor plays a role in the mesenteric neo-angiogenesis of portal hypertensive mice.
      ,
      • Geerts A.M.
      • De Vriese A.S.
      • Vanheule E.
      • Van V.H.
      • Mortier S.
      • Cheung K.J.
      • et al.
      Increased angiogenesis and permeability in the mesenteric microvasculature of rats with cirrhosis and portal hypertension: an in vivo study.
      ]. These findings confirm that portal hypertension induces neo-angiogenesis and that this plays a role in the maintenance of the splanchnic hyperdynamic state. The lack of reduction in portal pressure in PLGF(−/−) mice is in accordance with previous observations inhibiting VEGF [
      • Fernandez M.
      • Vizzutti F.
      • Garcia-Pagan J.C.
      • Rodes J.
      • Bosch J.
      Anti-VEGF receptor-2 monoclonal antibody prevents portal-systemic collateral vessel formation in portal hypertensive mice.
      ,
      • Fernandez M.
      • Mejias M.
      • Angermayr B.
      • Garcia-Pagan J.C.
      • Rodes J.
      • Bosch J.
      Inhibition of VEGF receptor-2 decreases the development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in portal hypertensive rats.
      ,
      • Fernandez M.
      • Mejias M.
      • Garcia-Pras E.
      • Mendez R.
      • Garcia-Pagan J.C.
      • Bosch J.
      Reversal of portal hypertension and hyperdynamic splanchnic circulation by combined vascular endothelial growth factor and platelet-derived growth factor blockade in rats.
      ]. Further studies with anti-PLGF antibodies in the prevention and treatment of portal hypertension are underway. As the role of PLGF is especially restricted to pathological conditions it is a good target for therapy with potentially less severe side effects than the blockade of VEGF/PDGF.

      4. Angiogenesis in liver regeneration

      Much of our knowledge about vascular biology of the liver has come from studying angiogenesis during regeneration after partial hepatectomy. The different phases of physiological angiogenesis and sinusoidal remodelling during liver regeneration have been analyzed extensively. Dysregulation of the same pathways and processes in chronic liver disease leads to endothelial dysfunction, pathological angiogenesis, with formation of collaterals and eventually to portal hypertension. This section summarizes the timely, limited, highly orchestrated cellular and molecular interactions related to physiological angiogenesis during liver regeneration.

      4.1 Role of liver sinusoidal endothelial cells in liver regeneration

      The liver receives 25% of the total cardiac output, which arrives via the hepatic artery (1/3 of hepatic blood flow) and the portal vein (2/3 of hepatic blood flow). Blood flows through liver sinusoids, a unique microvasculature which consists of plates of liver sinusoidal endothelial cells (LSECs) between plates of hepatocytes, before coming in contact with the liver parenchyma. LSECs account for 20% of total liver cells (an estimated 1 × 108 cells) whereas hepatocytes represent the majority of liver cells (estimated 60% or 3 × 108 cells). LSECs have a unique phenotype in comparison to other organs characterized by a discontinuous, fenestrated endothelium, which lacks an organized basement membrane. Cellular cross-talk between LSECs and hepatocytes plays an important role in sinusoidal homeostasis and physiologic angiogenesis during liver regeneration and hepatic organogenesis.
      Following partial hepatectomy (up to 70% removal), the liver will completely regenerate and reach its original functional mass with normal microscopic architecture, which is a unique capacity compared to all other organs with none or only limited regeneration. Experimental partial hepatectomy (PHx) involves the removal of two-thirds of the liver [
      • Higgins M.
      • Anderson R.
      Experimental pathology of the liver. I. Restoration of the liver of the white rat following partial surgical removal.
      ]. Within 8–10 days, the liver remnant enlarges, until the previous functional liver mass is restored. LSECs and hepatocytes in the normal liver are quiescent, with only 0.01–0.001% undergoing mitosis at any given time. PHx activates numerous transcription factors, induces the expression of more than 70 genes, and promotes cell cycle entry with synchronous proliferation of almost all hepatocytes by 24–36 h post-PHx. The initial wave of hepatocyte proliferation is followed by a second wave of replication of non-parenchymal liver cells, including LSECs and HSC, which start to proliferate 48–72 h after resection peaking at day 4–5 [
      • Ross M.A.
      • Sander C.M.
      • Kleeb T.B.
      • Watkins S.C.
      • Stolz D.B.
      Spatiotemporal expression of angiogenesis growth factor receptors during the revascularization of regenerating rat liver.
      ].
      Hemodynamically seen, PHx leads to an increased sinusoidal blood flow since the portal influx to the liver remnant remains unchanged [
      • Marubashi S.
      • Sakon M.
      • Nagano H.
      • Gotoh K.
      • Hashimoto K.
      • Kubota M.
      • et al.
      Effect of portal hemodynamics on liver regeneration studied in a novel portohepatic shunt rat model.
      ]. Hemodynamic changes and shear stress might induce hepatocyte proliferation through activation of urokinase plasminogen activator, matrix remodelling, and subsequent release of preformed hepatocyte growth factor (HGF) [
      • Michalopoulos G.K.
      Liver regeneration.
      ]. The initial proliferation of hepatocytes leads to the formation of avascular clusters of hepatocytes, where the central cells reside outside the oxygen diffusion distance of 200 μm of a capillary [
      • Ross M.A.
      • Sander C.M.
      • Kleeb T.B.
      • Watkins S.C.
      • Stolz D.B.
      Spatiotemporal expression of angiogenesis growth factor receptors during the revascularization of regenerating rat liver.
      ,
      • Greene A.K.
      • Wiener S.
      • Puder M.
      • Yoshida A.
      • Shi B.
      • Perez-Atayde A.R.
      • et al.
      Endothelial-directed hepatic regeneration after partial hepatectomy.
      ,
      • Wack K.E.
      • Ross M.A.
      • Zegarra V.
      • Sysko L.R.
      • Watkins S.C.
      • Stolz D.B.
      Sinusoidal ultrastructure evaluated during the revascularization of regenerating rat liver.
      ]. This is accompanied by ultrastructural changes of the sinusoids, with external compression of the sinusoids by proliferating hepatocytes, decrease of sinusoidal fenestrations and endothelial porosity 72 h post-PHx [
      • Wack K.E.
      • Ross M.A.
      • Zegarra V.
      • Sysko L.R.
      • Watkins S.C.
      • Stolz D.B.
      Sinusoidal ultrastructure evaluated during the revascularization of regenerating rat liver.
      ]. In a next stage, hypoxia and hepatocyte signalling induce LSEC activation, proliferation, and migration of neighbouring LSECs into the avascular clusters [
      • Martinez-Hernandez A.
      • Amenta P.S.
      The extracellular matrix in hepatic regeneration.
      ,
      • 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.
      ]. This leads to separation of hepatocytes with subsequent recanalization and formation of patent sinusoids. Around day 6 after PHx (when the regenerating liver is approaching its preoperative functional mass), a wave of apoptosis in LSEC can be detected with a maximum at day 8 [
      • Greene A.K.
      • Wiener S.
      • Puder M.
      • Yoshida A.
      • Shi B.
      • Perez-Atayde A.R.
      • et al.
      Endothelial-directed hepatic regeneration after partial hepatectomy.
      ,
      • Folkman J.
      Angiogenesis: an organizing principle for drug discovery?.
      ,
      • Shimizu H.
      • Mitsuhashi N.
      • Ohtsuka M.
      • Ito H.
      • Kimura F.
      • Ambiru S.
      • et al.
      Vascular endothelial growth factor and angiopoietins regulate sinusoidal regeneration and remodeling after partial hepatectomy in rats.
      ]. This is in contrast to hepatocytes which do not show increased apoptotic rates during the regenerative process [
      • Greene A.K.
      • Wiener S.
      • Puder M.
      • Yoshida A.
      • Shi B.
      • Perez-Atayde A.R.
      • et al.
      Endothelial-directed hepatic regeneration after partial hepatectomy.
      ]. Eight to 10 days following PHx, liver mass is fully reconstituted, and the sinusoidal architecture restored to normal.
      Circulating endothelial progenitor cells (characterized by stem cell markers such as CD117 or CD133) have also been shown to play a role in angiogenesis during liver regeneration. Circulating endothelial cells are mobilized from bone marrow by systemically circulating growth factors and chemokines produced in the regenerating liver and participate in this process by homing to sites of neovascularization in the liver and by committing to LSECs [
      • Beaudry P.
      • Hida Y.
      • Udagawa T.
      • Alwayn I.P.
      • Greene A.K.
      • Arsenault D.
      • et al.
      Endothelial progenitor cells contribute to accelerated liver regeneration.
      ,
      • Fujii H.
      • Hirose T.
      • Oe S.
      • Yasuchika K.
      • Azuma H.
      • Fujikawa T.
      • et al.
      Contribution of bone marrow cells to liver regeneration after partial hepatectomy in mice.
      ]. Treatment with exogenous VEGF increased mobilization of circulating endothelial progenitor cells and accelerated liver regeneration in mice after PHx [
      • Beaudry P.
      • Hida Y.
      • Udagawa T.
      • Alwayn I.P.
      • Greene A.K.
      • Arsenault D.
      • et al.
      Endothelial progenitor cells contribute to accelerated liver regeneration.
      ].

      4.2 Sinusoidal remodelling in the regenerating liver

      Recent evidence suggests that an alternative mode of angiogenesis, called intussusceptive angiogenesis contributes to the angiogenic process in addition to the classical form of sprouting angiogenesis described above. Sprouting (angiogenesis from a pre-existing vascular bed) is dependent on endothelial cell proliferation with subsequent lumen formation in the sprout. In contrast to sprouting, angiogenesis by intussusception consists of microvascular remodelling by transcapillary pillar formation and relies much less on endothelial cell proliferation [
      • Djonov V.
      • Schmid M.
      • Tschanz S.A.
      • Burri P.H.
      Intussusceptive angiogenesis: its role in embryonic vascular network formation.
      ,
      • Semela D.
      • Piguet A.C.
      • Kolev M.
      • Schmitter K.
      • Hlushchuk R.
      • Djonov V.
      • et al.
      Vascular remodeling and antitumoral effects of mTOR inhibition in a rat model of hepatocellular carcinoma.
      ]. Growth of these endothelial pillars leads to sinusoidal multiplication by successive fusion and partitioning of the existing vascular lumen. Intussusceptive angiogenesis has been found to occur as early as 12 h after PHx in mice [

      Stroka D, Trochlser M, Hlushchuk R, Baum O, Candinas D, Dufour JF. Sinusoidal duplication by intussusception is an early event in liver regeneration after parcial hepatectomy. J Hepatol 2007;46(Suppl. 1).

      ]. The split sinusoids increase in girth and undergo augmentation by day 3 to 4, concomitant with endothelial cell proliferation [

      Stroka D, Trochlser M, Hlushchuk R, Baum O, Candinas D, Dufour JF. Sinusoidal duplication by intussusception is an early event in liver regeneration after parcial hepatectomy. J Hepatol 2007;46(Suppl. 1).

      ].
      The role of HSC in liver regeneration and sinusoidal remodelling is less well studied. HSC are closely associated with LSEC and function as liver-specific pericytes (vascular mural cells of the hepatic sinusoids) [
      • Lee J.S.
      • Semela D.
      • Iredale J.
      • Shah V.H.
      Sinusoidal remodelling and angiogenesis: a new function for the liver-specific pericyte?.
      ,
      • Semela D.
      • Das A.
      • Langer D.
      • Kang N.
      • Leof E.
      • Shah V.
      Platelet-derived growth factor signaling through ephrin-b2 regulates hepatic vascular structure and function.
      ]. They regulate vessel stabilization, maturation and sinusoidal remodelling by direct contact and paracrine interaction with LSECs such as secretion of VEGF [
      • Lee J.S.
      • Semela D.
      • Iredale J.
      • Shah V.H.
      Sinusoidal remodelling and angiogenesis: a new function for the liver-specific pericyte?.
      ]. Mediated by platelet-derived growth factor (PDGF) produced by hepatocytes and LSECs, resting HSC start to proliferate (peaking at 48–72 h) and become activated within the first 72 h after PHx [
      • Michalopoulos G.K.
      Liver regeneration.
      ,
      • Balabaud C.
      • Bioulac-Sage P.
      • Desmouliere A.
      The role of hepatic stellate cells in liver regeneration.
      ,
      • Budny T.
      • Palmes D.
      • Stratmann U.
      • Minin E.
      • Herbst H.
      • Spiegel H.U.
      Morphologic features in the regenerating liver–a comparative intravital, lightmicroscopical and ultrastructural analysis with focus on hepatic stellate cells.
      ,
      • Mabuchi A.
      • Mullaney I.
      • Sheard P.W.
      • Hessian P.A.
      • Mallard B.L.
      • Tawadrous M.N.
      • et al.
      Role of hepatic stellate cell/hepatocyte interaction and activation of hepatic stellate cells in the early phase of liver regeneration in the rat.
      ]. In addition, HSC are thought to control remodelling of the extracellular matrix in the space of Disse during regeneration (i.e. deposition of collagen IV, fibronectin and laminin) which will influence function of LSECs [
      • Michalopoulos G.K.
      Liver regeneration.
      ,
      • Wack K.E.
      • Ross M.A.
      • Zegarra V.
      • Sysko L.R.
      • Watkins S.C.
      • Stolz D.B.
      Sinusoidal ultrastructure evaluated during the revascularization of regenerating rat liver.
      ,
      • Balabaud C.
      • Bioulac-Sage P.
      • Desmouliere A.
      The role of hepatic stellate cells in liver regeneration.
      ]. Similar to circulating endothelial progenitor cells, bone marrow-derived cells have also been shown to commit to HSC during liver regeneration [
      • Baba S.
      • Fujii H.
      • Hirose T.
      • Yasuchika K.
      • Azuma H.
      • Hoppo T.
      • et al.
      Commitment of bone marrow cells to hepatic stellate cells in mouse.
      ].

      4.3 Hypoxia and vascular factors regulate angiogenesis during liver regeneration

      VEGF is the most important growth and survival factor for endothelium. VEGF promotes proliferation of endothelial cells, acts as an anti-apoptotic factor and regulates vascular permeability by inducing fenestration in LSECs [
      • Gerber H.P.
      • Dixit V.
      • Ferrara N.
      Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells.
      ,
      • Yokomori H.
      • Oda M.
      • Yoshimura K.
      • Nagai T.
      • Ogi M.
      • Nomura M.
      • et al.
      Vascular endothelial growth factor increases fenestral permeability in hepatic sinusoidal endothelial cells.
      ]. Furthermore, VEGF induces the expression of proteases like collagenase [
      • Unemori E.N.
      • Ferrara N.
      • Bauer E.A.
      • Amento E.P.
      Vascular endothelial growth factor induces interstitial collagenase expression in human endothelial cells.
      ], matrix metalloproteinases [
      • Zucker S.
      • Mirza H.
      • Conner C.E.
      • Lorenz A.F.
      • Drews M.H.
      • Bahou W.F.
      • et al.
      Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation.
      ], urokinase- and tissue-type plasminogen activators [
      • Pepper M.S.
      • Ferrara N.
      • Orci L.
      • Montesano R.
      Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasminogen activator inhibitor-1 in microvascular endothelial cells.
      ], which enable endothelial cells to breakdown the surrounding extracellular matrix in order to migrate and form new blood vessels. VEGF is constitutively expressed in hepatocytes at low levels [
      • Taniguchi E.
      • Sakisaka S.
      • Matsuo K.
      • Tanikawa K.
      • Sata M.
      Expression and role of vascular endothelial growth factor in liver regeneration after partial hepatectomy in rats.
      ]. VEGF production by hepatocytes and HSC rapidly increases during liver regeneration. of VEGF is induced by cytokines (i.e. IL-6) and hypoxia in the centre of the regenerative and avascular hepatocyte clusters [
      • Ross M.A.
      • Sander C.M.
      • Kleeb T.B.
      • Watkins S.C.
      • Stolz D.B.
      Spatiotemporal expression of angiogenesis growth factor receptors during the revascularization of regenerating rat liver.
      ]. Hypoxia activates the transcription factor hypoxia-inducible factor (HIF)-1, which in turn induces the expression of downstream target genes including cMet, EPO (erythropoietin), VEGF and VEGFR-1 (VEGF receptor 1) [
      • Maeno H.
      • Ono T.
      • Dhar D.K.
      • Sato T.
      • Yamanoi A.
      • Nagasue N.
      Expression of hypoxia inducible factor-1alpha during liver regeneration induced by partial hepatectomy in rats.
      ,
      • Redaelli C.A.
      • Semela D.
      • Carrick F.E.
      • Ledermann M.
      • Candinas D.
      • Sauter B.
      • et al.
      Effect of vascular endothelial growth factor on functional recovery after hepatectomy in lean and obese mice.
      ,
      • Yim S.H.
      • Shah Y.
      • Tomita S.
      • Morris H.D.
      • Gavrilova O.
      • Lambert G.
      • et al.
      Disruption of the Arnt gene in endothelial cells causes hepatic vascular defects and partial embryonic lethality in mice.
      ]. Hepatocyte production of VEGF peaks 48–72 h after PHx and is detected mainly in periportal hepatocytes [
      • Martinez-Hernandez A.
      • Amenta P.S.
      The extracellular matrix in hepatic regeneration.
      ,
      • Taniguchi E.
      • Sakisaka S.
      • Matsuo K.
      • Tanikawa K.
      • Sata M.
      Expression and role of vascular endothelial growth factor in liver regeneration after partial hepatectomy in rats.
      ]. Administration of VEGF in hepatectomized rodents increases LSEC and hepatocyte proliferation [
      • Taniguchi E.
      • Sakisaka S.
      • Matsuo K.
      • Tanikawa K.
      • Sata M.
      Expression and role of vascular endothelial growth factor in liver regeneration after partial hepatectomy in rats.
      ,
      • Assy N.
      • Spira G.
      • Paizi M.
      • Shenkar L.
      • Kraizer Y.
      • Cohen T.
      • et al.
      Effect of vascular endothelial growth factor on hepatic regenerative activity following partial hepatectomy in rats.
      ], accelerates gain in liver mass [
      • LeCouter J.
      • Moritz D.R.
      • Li B.
      • Phillips G.L.
      • Liang X.H.
      • Gerber H.P.
      • et al.
      Angiogenesis-independent endothelial protection of liver: role of VEGFR-1.
      ] and improves functional hepatic recovery [
      • Redaelli C.A.
      • Semela D.
      • Carrick F.E.
      • Ledermann M.
      • Candinas D.
      • Sauter B.
      • et al.
      Effect of vascular endothelial growth factor on functional recovery after hepatectomy in lean and obese mice.
      ]. Transduction of VEGF before hepatic resection also hastens functional hepatic recovery in mice with fatty liver, which is known for its impaired regenerative capacity [
      • Redaelli C.A.
      • Semela D.
      • Carrick F.E.
      • Ledermann M.
      • Candinas D.
      • Sauter B.
      • et al.
      Effect of vascular endothelial growth factor on functional recovery after hepatectomy in lean and obese mice.
      ]. Neutralizing antibodies against VEGF inhibit hepatocyte and endothelial cell proliferation after PHx [
      • Taniguchi E.
      • Sakisaka S.
      • Matsuo K.
      • Tanikawa K.
      • Sata M.
      Expression and role of vascular endothelial growth factor in liver regeneration after partial hepatectomy in rats.
      ].
      VEGF production is accompanied by an increase in the expression of the VEGFR-1 on hepatocytes and of VEGFR-1 and VEGFR-2 on LSECs [
      • Ross M.A.
      • Sander C.M.
      • Kleeb T.B.
      • Watkins S.C.
      • Stolz D.B.
      Spatiotemporal expression of angiogenesis growth factor receptors during the revascularization of regenerating rat liver.
      ,
      • 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.
      ,
      • Redaelli C.A.
      • Semela D.
      • Carrick F.E.
      • Ledermann M.
      • Candinas D.
      • Sauter B.
      • et al.
      Effect of vascular endothelial growth factor on functional recovery after hepatectomy in lean and obese mice.
      ,
      • Mochida S.
      • Ishikawa K.
      • Inao M.
      • Shibuya M.
      • Fujiwara K.
      Increased expressions of vascular endothelial growth factor and its receptors, flt-1 and KDR/flk-1, in regenerating rat liver.
      ]. Activation of VEGFR-2 stimulates LSEC proliferation. On the other hand, binding of VEGF to VEGFR-1 on LSEC induces secretion of growth and survival factors such as HGF and interleukin-6 (IL-6), which in turn stimulate hepatocyte proliferation and reduce liver damage in mice exposed to CCl4 [
      • LeCouter J.
      • Moritz D.R.
      • Li B.
      • Phillips G.L.
      • Liang X.H.
      • Gerber H.P.
      • et al.
      Angiogenesis-independent endothelial protection of liver: role of VEGFR-1.
      ]. This suggests an angiogenesis-independent endothelial protection of hepatocytes through VEGFR-1.
      In addition to VEGF and HGF, other angiogenic growth factors and their receptors are upregulated during liver regeneration, including PDGF, FGF and angiopoietins. Angiopoietins Ang1 and Ang2 regulate vessel stability by activating (Ang1) or antagonizing (Ang2) signalling via the receptor tyrosine kinase Tie2 [
      • Lee J.S.
      • Semela D.
      • Iredale J.
      • Shah V.H.
      Sinusoidal remodelling and angiogenesis: a new function for the liver-specific pericyte?.
      ]. Ang1 stabilizes vessels by promoting pericyte recruitment. Tie2 which is expressed by LSECs and its ligands Ang1 and Ang2 increases during liver regeneration [
      • Ross M.A.
      • Sander C.M.
      • Kleeb T.B.
      • Watkins S.C.
      • Stolz D.B.
      Spatiotemporal expression of angiogenesis growth factor receptors during the revascularization of regenerating rat liver.
      ,
      • 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.
      ,
      • Shimizu H.
      • Mitsuhashi N.
      • Ohtsuka M.
      • Ito H.
      • Kimura F.
      • Ambiru S.
      • et al.
      Vascular endothelial growth factor and angiopoietins regulate sinusoidal regeneration and remodeling after partial hepatectomy in rats.
      ,
      • Kraizer Y.
      • Mawasi N.
      • Seagal J.
      • Paizi M.
      • Assy N.
      • Spira G.
      Vascular endothelial growth factor and angiopoietin in liver regeneration.
      ]. The orphan receptor Tie1 was found to be expressed on LSECs surrounding avascular hepatocyte clusters [
      • Ross M.A.
      • Sander C.M.
      • Kleeb T.B.
      • Watkins S.C.
      • Stolz D.B.
      Spatiotemporal expression of angiogenesis growth factor receptors during the revascularization of regenerating rat liver.
      ]. Neuropilin-1 and Neuropilin-2 are recently discovered VEGF co-receptors unrelated to VEGFR-1 and -2, which have no intrinsic signalling but enhance binding of VEGF to VEGFR-2 [
      • Soker S.
      • Takashima S.
      • Miao H.Q.
      • Neufeld G.
      • Klagsbrun M.
      Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor.
      ,
      • Neufeld G.
      • Kessler O.
      • Herzog Y.
      The interaction of Neuropilin-1 and Neuropilin-2 with tyrosine-kinase receptors for VEGF.
      ]. Neuropilin-1 has been shown to be upregulated regenerating livers [
      • Kraizer Y.
      • Mawasi N.
      • Seagal J.
      • Paizi M.
      • Assy N.
      • Spira G.
      Vascular endothelial growth factor and angiopoietin in liver regeneration.
      ,
      • Braet F.
      • Shleper M.
      • Paizi M.
      • Brodsky S.
      • Kopeiko N.
      • Resnick N.
      • et al.
      Liver sinusoidal endothelial cell modulation upon resection and shear stress in vitro.
      ].
      Thrombocytes play an important role in liver regeneration [
      • Lesurtel M.
      • Graf R.
      • Aleil B.
      • Walther D.J.
      • Tian Y.
      • Jochum W.
      • et al.
      Platelet-derived serotonin mediates liver regeneration.
      ]. Thrombocytes contain high concentrations of angiogenesis stimulators (i.e. VEGF, bFGF, PDGF) as well as inhibitors (i.e. endostatin, thrombospondin 1) packaged in distinct populations of α-granules which can be released selectively [
      • Folkman J.
      Angiogenesis: an organizing principle for drug discovery?.
      ,
      • Italiano Jr., J.E.
      • Richardson J.L.
      • Patel-Hett S.
      • Battinelli E.
      • Zaslavsky A.
      • Short S.
      • et al.
      Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released.
      ]. Although unexplored, one can speculate that as thrombocytes adhere to activated endothelium their action can enhance or inhibit local angiogenesis and thereby influence liver regeneration.
      In summary, intense research using partial hepatectomy in animal models leads to identification of mechanisms and pathways which regulate angiogenesis in the liver. Emerging antiangiogenic drugs are under investigation to counterbalance dysregulated angiogenic pathways in chronic liver disease and HCC and are discussed in the other sections.

      5. Angiogenesis and liver cancer

      The growing incidence of hepatocellular carcinoma (HCC) on a worldwide scale [
      • Parkin D.M.
      • Bray F.
      • Ferlay J.
      • Pisani P.
      Global cancer statistics, 2002.
      ] and the current capacity to diagnose and treat this cancer at an early stage has raised interest in this neoplasm [
      • Bruix J.
      • Sherman M.
      • Llovet J.M.
      • Beaugrand M.
      • Lencioni R.
      • Burroughs A.K.
      • et al.
      Clinical management of hepatocellular carcinoma: conclusions of the Barcelona-2000 EASL conference.
      ,
      • Bruix J.
      • Sherman M.
      Management of hepatocellular carcinoma.
      ]. It is well known that in most patients, this malignancy emerges in a liver with long-standing cell damage that has resulted in extensive fibrosis or cirrhosis [
      • Bruix J.
      • Sherman M.
      Management of hepatocellular carcinoma.
      ]. In fact, HCC development is now a leading cause of death in cirrhotic patients. This has prompted major research activity into all aspects related to the pathogenesis and clinical translation of laboratory data into clinical practice.
      There are several in-depth reviews that expand into the genomic abnormalities that might be found in experimental liver tumours as well as in cell lines and human tumour tissue banks [
      • Thorgeirsson S.S.
      • Grisham J.W.
      Molecular pathogenesis of human hepatocellular carcinoma.
      ,
      • Lee J.S.
      • Thorgeirsson S.S.
      Genome-scale profiling of gene expression in hepatocellular carcinoma: classification, survival prediction, and identification of therapeutic targets.
      ,
      • Buendia M.A.
      Genetics of hepatocellular carcinoma.
      ,
      • Lee J.S.
      • Thorgeirsson S.S.
      Comparative and integrative functional genomics of HCC.
      ]. An enormous amount of data describing chromosome abnormalities and gene expression has been published in recent years and gene expression has also been tentatively correlated with prognosis both with and without treatment application. Furthermore, analysis of tumours obtained at different evolutionary stages have allowed to propose gene events that herald the transformation of premalignant clones into overt HCC [
      • Llovet J.M.
      • Chen Y.
      • Wurmbach E.
      • Roayaie S.
      • Fiel M.I.
      • Schwartz M.
      • et al.
      A molecular signature to discriminate dysplastic nodules from early hepatocellular carcinoma in HCV cirrhosis.
      ] and at the same time, the recognition of different gene expression patterns have triggered the proposal of a molecular classification of HCC [
      • Laurent-Puig P.
      • Legoix P.
      • Bluteau O.
      • Belghiti J.
      • Franco D.
      • Binot F.
      • et al.
      Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis.
      ,
      • Smith M.W.
      • Yue Z.N.
      • Korth M.J.
      • Do H.A.
      • Boix L.
      • Fausto N.
      • et al.
      Hepatitis C virus and liver disease: global transcriptional profiling and identification of potential markers.
      ,
      • Boyault S.
      • Rickman D.S.
      • de Reynies A.
      • Balabaud C.
      • Rebouissou S.
      • Jeannot E.
      • et al.
      Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.
      ]. Furthermore, the dissection of the derangement of several signal transduction pathways that govern cell proliferation, invasion and survival [
      • Hanahan D.
      • Weinberg R.A.
      The hallmarks of cancer.
      ] has fuelled the development of new agents aimed at targeting the specific event that is responsible for cancer evolution [
      • Llovet J.M.
      • Bruix J.
      Novel advancements in the management of hepatocellular carcinoma in 2008.
      ]. Blockade of the effective signalling by growth factors has been generated in the benches and some of them have reached human testing. The same applies for the pathways that are abrogated and permit malignant cell survival, and also for the different molecules that are involved in the active neoangiogenic process that characterize all cancer types [
      • Roberts L.R.
      • Gores G.J.
      Hepatocellular carcinoma: molecular pathways and new therapeutic targets.
      ].
      It is important to note that HCC is a highly vascularised tumour. Hence, profiling and targeting of the steps leading from a premalignant poorly vascularised nodule to the transition into an overt malignant phenotype with enhanced arterial blood supply has become a landmark event both for diagnosis, current treatment and novel therapeutic approaches [
      • Roberts L.R.
      • Gores G.J.
      Hepatocellular carcinoma: molecular pathways and new therapeutic targets.
      ,
      • Forner A.
      • Vilana R.
      • Ayuso C.
      • Bianchi L.
      • Sole M.
      • Ayuso J.R.
      • et al.
      Diagnosis of hepatic nodules 20 mm or smaller in cirrhosis: prospective validation of the noninvasive diagnostic criteria for hepatocellular carcinoma.
      ].

      5.1 Clinical relevance of the vascularization profile in HCC

      The cell origin of HCC is not well-established. It may derive from hepatic stem cells or from the transformation from dysplastic hepatocytes into malignant cells. This last evolution has been quite well characterized in recent years [
      • Takayama T.
      • Makuuchi M.
      • Hirohashi S.
      • Sakamoto M.
      • Okazaki N.
      • Takayasu K.
      • et al.
      Malignant transformation of adenomatous hyperplasia to hepatocellular carcinoma.
      ] when imaging techniques have permitted the monitoring of the evolution from a small (<1 cm) hypoechoic nodule within a cirrhotic liver into typical HCC [
      • Borzio M.
      • Fargion S.
      • Borzio F.
      • Fracanzani A.L.
      • Croce A.M.
      • Stroffolini T.
      • et al.
      Impact of large regenerative, low grade and high grade dysplastic nodules in hepatocellular carcinoma development.
      ]. Small hypoechoic nodules have been given different names that include adenomatous hyperplasia, dysplastic nodule, macroregenerative nodule with dysplastic changes, but recently have been divided into regenerative nodules and low or high-grade dysplastic nodules [

      Terminology of nodular hepatocellular lesions. International Working Party. Hepatology 1995;22:983-993.

      ,
      • Kojiro M.
      • Roskams T.
      Early hepatocellular carcinoma and dysplastic nodules.
      ]. The risk of malignant transformation of the first type is nil, it is minimal in the low-grade category and is really high in the nodules classified as high-grade dysplastic nodules [
      • Kojiro M.
      • Roskams T.
      Early hepatocellular carcinoma and dysplastic nodules.
      ]. All of them have a blood supply dependent from the portal vein, the hepatic artery supply being absent [
      • Nakashima Y.
      • Nakashima O.
      • Tanaka M.
      • Okuda K.
      • Nakashima M.
      • Kojiro M.
      Portal vein invasion and intrahepatic micrometastasis in small hepatocellular carcinoma by gross type.
      ]. Because of this characteristic, the nodules are recognised as hypoechoic at plain ultrasonography and characteristically lack contrast uptake in the arterial phase if explored by contrast enhanced-US, dynamic CT or MRI [
      • Lencioni R.
      • Cioni D.
      • Della Pina C.
      • Crocetti L.
      • Bartolozzi C.
      Imaging diagnosis.
      ]. In some cases the progressive transition into early HCC is associated with fat accumulation that turns the nodule into hyperechoic. If the nodule is first detected at that stage, it might be wrongly suspected to correspond to a small angioma, while it indeed corresponds to a transformed clone that it is likely to have experienced oxidative stress due to impaired blood supply. If stress is excessive some apoptotic death will take place, but ultimately, the malignant profile is established and overt cancer is in place. At this point, the vessel pattern has sharply changed and arterial supply is predominant. This feature is the basis for the imaging diagnosis of HCC within a cirrhotic liver [
      • Forner A.
      • Vilana R.
      • Ayuso C.
      • Bianchi L.
      • Sole M.
      • Ayuso J.R.
      • et al.
      Diagnosis of hepatic nodules 20 mm or smaller in cirrhosis: prospective validation of the noninvasive diagnostic criteria for hepatocellular carcinoma.
      ,
      • Lencioni R.
      • Cioni D.
      • Della Pina C.
      • Crocetti L.
      • Bartolozzi C.
      Imaging diagnosis.
      ]. Confident diagnosis is easy in large HCC as the vascular bed is fully established. However, at an early stage the arterial net is not extensive enough and only one-third of the nodules ultimately corresponding to an HCC can be confidently diagnosed by imaging characterization, the rest requiring a diagnostic biopsy.
      Recent studies have tried to correlate gene expression in tumour tissue with imaging characterization [
      • Segal E.
      • Sirlin C.B.
      • Ooi C.
      • Adler A.S.
      • Gollub J.
      • Chen X.
      • et al.
      Decoding global gene expression programs in liver cancer by noninvasive imaging.
      ]. Encouraging results have been reported but it is expected that a more sensitive assessment will be obtained through metabolic/molecular characterization by MRI, rather than with the mere evaluation of contrast behaviour. In that sense, the clinical need and urgent research challenge is not to classify already advanced cancer, but to characterize small nodules. It is crucial to confidently establish if they have not yet reached the oncogenic capacity or if they are already entering the neoangiogenic stage that precedes malignant evolution.
      Detailed pathology studies carried out by the team of M Kojiro in Kurume, Japan have further divided small HCC into two different types: distinctly nodular vs. vaguely nodular [
      • Nakashima Y.
      • Nakashima O.
      • Tanaka M.
      • Okuda K.
      • Nakashima M.
      • Kojiro M.
      Portal vein invasion and intrahepatic micrometastasis in small hepatocellular carcinoma by gross type.
      ]. The latter is assumed to correspond to the carcinoma in situ entity and lacks any invasive feature. By contrast, the distinctly nodular HCC type is clearly more evolved. A surrounding fibrotic capsule is more frequently observed and it exhibits a higher rate of microscopic vascular invasion. Interestingly, the transition from carcinoma in situ to established cancer is associated with the appearance of the prominent arterial blood supply that might be recognised by dynamic imaging techniques. Immunohistochemical staining is able to capture this neovessel formation and thus, staining for endothelial cells to display isolated arteries within a nodule is used as a diagnostic characteristic by pathologists. Hence, new blood vessel formation by either angiogenesis or vasculogenesis is a critical step not only for cancer progression as it occurs with all cancer types, but is recognised as the event that marks the frontier between the potential to achieve complete cure [
      • Takayama T.
      • Makuuchi M.
      • Hirohashi S.
      • Sakamoto M.
      • Yamamoto J.
      • Shimada K.
      • et al.
      Early hepatocellular carcinoma as an entity with a high rate of surgical cure.
      ] and the stage where removal is feasible but the risk of dissemination is already acquired and long-term disease-free status is less likely.
      The relevance of angiogenesis as a key event in the emergence and recognition of early HCC does not detract the relevance of intense arterial blood supply both for the development of recurrence after potentially curative therapy and also for the progression of advanced cancer. Growth of metastatic nests requires an extensive net of new arterial vessels to be formed and not surprisingly, progression from early to advanced cancer also requires this activity. Manoeuvres like chemoembolization employed to impair blood supply and induce ischemic necrosis have been one of the effective treatments for advanced HCC. Despite being in place for years, it has just been confirmed recently that data from randomised controlled trials have demonstrated that chemoembolization (a combination of arterial obstruction and selective chemotherapy administration) improves survival of patients that have reached this evolutionary stage [
      • Llovet J.M.
      • Bruix J.
      Systematic review of randomized trials for unresectable hepatocellular carcinoma: chemoembolization improves survival.
      ]. Hence, acting against blood supply has become a validated target and thus the backbone of treatment of HCC diagnosed at an intermediate stage.
      The relevance of vascularization in the evolution of HCC is also reinforced by several studies that have correlated the intensity of arterial blood supply or vessel formation inside the tumour with prognosis [
      • El Assal O.N.
      • Yamanoi A.
      • Soda Y.
      • Yamaguchi M.
      • Igarashi M.
      • Yamamoto A.
      • et al.
      Clinical significance of microvessel density and vascular endothelial growth factor expression in hepatocellular carcinoma and surrounding liver: possible involvement of vascular endothelial growth factor in the angiogenesis of cirrhotic liver.
      ]. The same applies for markers of angiogenic activity in peripheral blood. Increased concentration of VEGF in serum has been correlated with outcome after surgical resection [
      • Poon R.T.
      • Ho J.W.
      • Tong C.S.
      • Lau C.
      • Ng I.O.
      • Fan S.T.
      Prognostic significance of serum vascular endothelial growth factor and endostatin in patients with hepatocellular carcinoma.
      ] or ablation [
      • Poon R.T.
      • Lau C.
      • Pang R.
      • Ng K.K.
      • Yuen J.
      • Fan S.T.
      High serum vascular endothelial growth factor levels predict poor prognosis after radiofrequency ablation of hepatocellular carcinoma: importance of tumor biomarker in ablative therapies.
      ]. Interestingly, HCC cell lines (Fig. 5) derived from human tumours may produce VEGF by themselves and this demonstrates their pivotal role in inducing vessel development [
      • Armengol C.
      • Tarafa G.
      • Boix L.
      • Sole M.
      • Queralt R.
      • Costa D.
      • et al.
      Orthotopic implantation of human hepatocellular carcinoma in mice: analysis of tumor progression and establishment of the BCLC-9 cell line.
      ].
      Figure thumbnail gr5
      Fig. 5BCLC-9 cell line derived from a human HCC. It has been shown to produce VEGF that is further enhanced under hypoxic conditions
      [
      • Armengol C.
      • Tarafa G.
      • Boix L.
      • Sole M.
      • Queralt R.
      • Costa D.
      • et al.
      Orthotopic implantation of human hepatocellular carcinoma in mice: analysis of tumor progression and establishment of the BCLC-9 cell line.
      ]
      .
      All these data have provided the rationale to develop molecular targeted therapies that could provide an effective (biology) based treatment of HCC.

      5.2 Targeted anti-angiogenic therapy

      For years the treatment of HCC has been largely based on local procedures. Effective options have comprised blades, needles and devices aimed at removing or necrosing tumor cells. However, since cancer is a process governed by biologic events, the optimal approach should target the signals and events that make a malignant cell proliferate, invade, disseminate and survive. Angiogenesis and vasculogenesis are complex processes that involve many factors, the best characterized are fibroblast growth factors (FGFs), vascular endothelial growth factors (VEGFs) and angiopoietins [
      • Rossant J.
      • Howard L.
      Signaling pathways in vascular development.
      ,
      • Thurston G.
      Role of angiopoietins and Tie receptor tyrosine kinases in angiogenesis and lymphangiogenesis.
      ]. Recently, other factors such are semaphorin, ephrins [
      • Cheng N.
      • Brantley D.M.
      • Liu H.
      • Lin Q.
      • Enriquez M.
      • Gale N.
      • et al.
      Blockade of EphA receptor tyrosine kinase activation inhibits vascular endothelial cell growth factor-induced angiogenesis.
      ], TGF-β/BMP [
      • Miyazono K.
      • Kusanagi K.
      • Inoue H.
      Divergence and convergence of TGF-beta/BMP signaling.
      ] and Notch/Delta have been added to the list [
      • Lawson N.D.
      • Scheer N.
      • Pham V.N.
      • Kim C.H.
      • Chitnis A.B.
      • Campos-Ortega J.A.
      • et al.
      Notch signaling is required for arterial-venous differentiation during embryonic vascular development.
      ,
      • Mailhos C.
      • Modlich U.
      • Lewis J.
      • Harris A.
      • Bicknell R.
      • Ish-Horowicz D.
      Delta4, an endothelial specific notch ligand expressed at sites of physiological and tumor angiogenesis.
      ].
      The VEGF growth factors are a family of homodimeric glycoproteins that are encoded by 4 VEGF genes (A–D), and a related growth factor, placental growth factor (PLGF) encoded by placenta growth factor gene PlGF [
      • Neufeld G.
      • Cohen T.
      • Gengrinovitch S.
      • Poltorak Z.
      Vascular endothelial growth factor (VEGF) and its receptors.
      ]. VEGF-A gene produces five isoforms of VEGF-A protein, being that of 164 amino acids the isoform acting as the strongest mitogen. It is well known that VEGF-A expression is regulated by hypoxia, glucose concentration, pH and several oncogenes and all VEGF family members elicit their biological function by their binding to cell-surface VEGF receptors (VEGF-Rs). The binding initiates a cellular response via activation of the intracellular tyrosine kinase domain of the receptor. VEGF-R2 (also called KDR) is the key receptor that induces both angiogenesis and vascular permeability [
      • Kanno S.
      • Oda N.
      • Abe M.
      • Terai Y.
      • Ito M.
      • Shitara K.
      • et al.
      Roles of two VEGF receptors, Flt-1 and KDR, in the signal transduction of VEGF effects in human vascular endothelial cells.
      ].
      Inhibitors of the VEGF pathway are the best characterized and consequently, the most clinically advanced agents to inhibit tumour angiogenesis.
      At present, these include:
      • (a) monoclonal antibodies targeting VEGF-A or the VEGF receptors (mAb)
      • (b) a variety of small specific molecule that inhibit ligand-dependent receptor autophosphorylation of VEGF-R1 and VEGF-R2 (TKI)
      • (c) antisense and siRNA targeting VEGF-A or its receptors
      • (d) targeting microRNAs can block endothelial cell migration, proliferation and angiogenesis [
        • Kuehbacher A.
        • Urbich C.
        • Dimmeler S.
        Targeting microRNA expression to regulate angiogenesis.
        ].
      VEGF inhibitors induce the arrest of endothelial cell proliferation, regression of the existing vessels (increasing endothelial cell death), suppress the mobilization of endothelial progenitor cells from bone marrow (vasculogenesis), and are reported to be cytotoxic for some malignant cells.
      Table 2 lists some agents that have undergone intensive investigation. For most, it is assumed that the mechanism of action is highly selective, but probably there are other unknown targets that are affected at the same time. It is likely that some agents that appear to be effective are so because of a combination of biological actions, while agents that would appear similar because they share the main target, may be useless or deleterious as a result of the additional molecules that may be affected. In that sense, clinical studies using VEGF inhibitors have provided a lot of relevant information but also, a disparity of results. The increasing evidence of the potential roles of VEGF in other cell types different than those implying the vasculature, make the side effects of anti-VEGF therapies a matter of concern [
      • Verheul H.M.
      • Pinedo H.M.
      Possible molecular mechanisms involved in the toxicity of angiogenesis inhibition.
      ].
      Table 2Some of the drugs that have shown anti-angiogenic activity and that have been investigated in experimental models and/or in humans.
      AgentClassTarget(s)
      Bevacizumab (Avastin)mAbVEGF-A
      Imatinib (Gleevec)TKIABL1/2, PDGFR a/b, c-Kit
      Dasatinib (Sprycel)TKIABL1/2, PDGFR a/b, c-Kit, Src family
      Nilotinib (Tasigna)TKIABL1/2, PDGFR a/b, c-Kit
      Sunitinib (Sutent)TKIVEGFR1-3,c-Kit,PDGFRa/b,RET,CSF1R, FLT3
      Sorafenib (Nexavar)TKIVEGFR2,PDGFRb,c-Kit,FLT3,RAF1,BRAF
      AG-013736 (Pfizer)VEGFR, PDGFR
      AMG706 (Amgen)VEGFR,PDGFR,c-Kit, RET
      AP23573 (Ariad Pharmaceuticals)mTOR, VEGFR
      AZD2171 (AstraZeneca)VEGFR1-3, PDGFR
      CCI-779 (Wyeth)mTOR, VEGFR
      CDP-791 (Imclone Systems)VEGFR2
      Everolimus (Novartis)mTOR, VEGFR
      XL184 (Exelixis)MMET,VEGFR,RTK,FLT3,TIE2
      XL880 (Exelixis)c-Met, RTK
      XL999 (Exelixis)VEGFR,PDGFR, EGFR,FLT3, Src
      Since safety of agents does not appear to be homogeneous careful adjustment of dosage is needed (a relevant aspect in patients with liver failure) or consider the extra-VEGF activity of each. In that sense, the safety profile and the phase 2 data available with sorafenib [
      • Abou-Alfa G.K.
      • Schwartz L.
      • Ricci S.
      • Amadori D.
      • Santoro A.
      • Figer A.
      • et al.
      Phase II study of sorafenib in patients with advanced hepatocellular carcinoma.
      ] allowed to conduct a phase 3 placebo controlled randomised clinical trial in patients with advanced HCC. The results have been unequivocal. Sorafenib delays tumour progression as evaluated by conventional radiology criteria and this translates into a highly significant improvement in survival [
      • Llovet J.M.
      • Ricci S.
      • Mazzafero V.
      • Hilgard P.
      • Gane E.
      • Blanc J.F.
      • et al.
      Sorafenib in advanced hepatocellular carcinoma.
      ]. Hence, nowadays sorafenib should be considered the first-line systemic therapy for patients with advanced HCC. Until now, these patients had no therapeutic option with established efficacy and thus, this is a landmark outcome. However, in addition to the benefits of an effective therapy for the patients with this malignancy, the positive findings of sorafenib have major consequences. They validate antiangiogenics as a useful therapeutic tool and at the same time, demonstrate that the search and hope for molecular targeted agents is viable and effective. Accordingly, clinical trials have to incorporate bullets for additional pathways and/or combine these new drugs with the one that has been shown to be effective. Sorafenib is not only an inhibitor of the receptor of vascular endothelial growth factor receptors (VEGFR1-3) but also of the platelet-derived growth factor receptor-beta (PDGFR-β), and of the Raf-1 and B-Raf pathway [
      • Wilhelm S.
      • Carter C.
      • Lynch M.
      • Lowinger T.
      • Dumas J.
      • Smith R.A.
      • et al.
      Discovery and development of sorafenib: a multikinase inhibitor for treating cancer.
      ]. Raf kinases are MAPK kinases, key components of pro-survival pathway [
      • Chen J.
      • Fujii K.
      • Zhang L.
      • Roberts T.
      • Fu H.
      Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK-ERK independent mechanism.
      ]. Raf-1 inhibits two pro-apoptotic kinases both of them implicated in oxidative stress-induced injury. c-Kit, the receptor for stem cell factor, is also inhibited by sorafenib. The role of this receptor in normal tissues is to ensure the mobilization of endothelial progenitor cells to the sites of injury. Thus, c-Kit inhibition could provide a means of preventing vasculogenesis in tumours [
      • Wang C.H.
      • Anderson N.
      • Li S.H.
      • Szmitko P.E.
      • Cherng W.J.
      • Fedak P.W.
      • et al.
      Stem cell factor deficiency is vasculoprotective: unraveling a new therapeutic potential of imatinib mesylate.
      ]. c-Kit and other elements are also inhibited at a lower intensity by sorafenib, and this collateral profile is a key characteristic to be taken into account when thinking of potential combinations as while efficacy may be increased as expected, the side effects may also be increased. This is especially important if it is recalled that in most HCC there is an underlying liver disease that should not be further aggravated by the therapy for HCC.

      5.3 Summary and future perspectives

      There is overwhelming evidence that angiogenesis is of paramount relevance in the field of liver cancer. It is key in all evolutionary stages of the disease and its evaluation is important for diagnosis and prognostic evaluation. At the same time, vessel development and function has become a therapeutic target for which there are agents that have shown therapeutic efficacy and impact in survival.
      The advent of sorafenib for advanced HCC is an excellent example of the potential of research moving from bench to bedside, not only because of the benefit for patients, but also importantly since it confirms the expectations placed in molecular targeted therapies. Earlier stages of the disease may also benefit from sorafenib either as an adjuvant or even at preventing transition from premalignant to malignant stage. Obviously, combination of agents to increase the efficacy offered by sorafenib as a single agent is an easy to propose aim. However, a note of caution has to be raised, since combination of agents requires a careful preclinical evaluation of the mix and the conduction of phase 1-2 exploratory trials to define dosage and safety prior to going for efficacy, as well as a better knowledge of molecular pathophysiology.
      As in anything in life, expertise and caution permit advancements, while the opposite may put patients at risk and misuse valuable resources. Collaborative teams including cell biologists and physicians should be encouraged. Only a candid and honest exchange of concepts and data among these multidisciplinary consortia which in turn will allow the proper design of effective research plans, based on our current knowledge will expand and permit a better future for patients with HCC.

      Acknowledgements

      Declarations of funding interests: This work was supported in part by grants from: Ministerio de Eduación y Ciencia (SAF 2005-05825) and Fondo de Investigaciones Sanitarias (FIS 06/0623, FIS PI 05/150); Swiss Association for the Study of the Liver and the Swiss Association of Gastroenterology; Fund for Scientific Research – Flanders (1.5.083.03) and (1.1.466.07.N00); Flemish Society of Gastroenterology, and Instituto Toscano Tumori (ITT), Florence, Italy. Ciberehd is funded by Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación.
      We are indebted to Ms Clara Esteva for her expert management of the manuscript. Supported in part by Acción Transversal en Cáncer.

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