Proangiogenic Cytokines as Hypoxia-Dependent Factors Stimulating Migration of Human Hepatic Stellate Cells
2007; Elsevier BV; Volume: 170; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2007.060887
ISSN1525-2191
AutoresErica Novo, Stefania Cannito, E. Zamara, L. Valfrè di Bonzo, Alessandra Caligiuri, Carlo Cravanzola, Alessandra Compagnone, Sebastiano Colombatto, Fabio Marra, Massimo Pinzani, Maurizio Parola,
Tópico(s)Hepatocellular Carcinoma Treatment and Prognosis
ResumoPathological angiogenesis is associated with the fibrogenic progression of chronic liver diseases. Experimental data suggest that hypoxia and vascular endothelial growth factor (VEGF) may stimulate proliferation and synthesis of type I collagen in activated, myofibroblast-like rat hepatic stellate cells (HSC/MFs). In this study, we investigated whether hypoxia, recombinant VEGF, or angiopoietin 1 (Ang-1) may affect other crucial profibrogenic features. In human HSC/MFs, which constitutively express VEGF receptor-1 and -2 (VEGFR-1, VEGFR-2) and the Ang-1 receptor Tie-2, exposure to hypoxia, VEGF, or Ang-1 resulted in a Ras/Erk-dependent stimulation of chemokinesis and chemotaxis. Migration of human HSC/MFs under hypoxic conditions involved up-regulation of VEGF-A, Ang-1, and related receptors and was mainly dependent on VEGFR-2 (Flk-1). In specimens from either cirrhotic rat livers or from patients with hepatitis C virus-related cirrhosis, HSC/MFs expressed proangiogenic factors and related receptors in areas of active fibrogenesis (ie, at the leading or lateral edge of developing incomplete fibrotic septa). Data presented herein suggest that VEGF and Ang-1 may contribute to fibrogenesis by acting as hypoxia-inducible, autocrine, and paracrine factors able to recruit myofibroblast-like cells. Moreover, HSC/MFs, in addition to their established profibrogenic role, may also contribute to neoangiogenesis during chronic hepatic wound healing. Pathological angiogenesis is associated with the fibrogenic progression of chronic liver diseases. Experimental data suggest that hypoxia and vascular endothelial growth factor (VEGF) may stimulate proliferation and synthesis of type I collagen in activated, myofibroblast-like rat hepatic stellate cells (HSC/MFs). In this study, we investigated whether hypoxia, recombinant VEGF, or angiopoietin 1 (Ang-1) may affect other crucial profibrogenic features. In human HSC/MFs, which constitutively express VEGF receptor-1 and -2 (VEGFR-1, VEGFR-2) and the Ang-1 receptor Tie-2, exposure to hypoxia, VEGF, or Ang-1 resulted in a Ras/Erk-dependent stimulation of chemokinesis and chemotaxis. Migration of human HSC/MFs under hypoxic conditions involved up-regulation of VEGF-A, Ang-1, and related receptors and was mainly dependent on VEGFR-2 (Flk-1). In specimens from either cirrhotic rat livers or from patients with hepatitis C virus-related cirrhosis, HSC/MFs expressed proangiogenic factors and related receptors in areas of active fibrogenesis (ie, at the leading or lateral edge of developing incomplete fibrotic septa). Data presented herein suggest that VEGF and Ang-1 may contribute to fibrogenesis by acting as hypoxia-inducible, autocrine, and paracrine factors able to recruit myofibroblast-like cells. Moreover, HSC/MFs, in addition to their established profibrogenic role, may also contribute to neoangiogenesis during chronic hepatic wound healing. Angiogenesis is a hypoxia-stimulated and growth factor-dependent process consisting in the formation of new vascular structures from pre-existing blood vessels. Formation of new vessels is known to occur in several organs and to be critical for both growth and repair of tissues in several pathophysiological conditions.1Yancopoulos GD Davis S Gale N Rudge J Wiegand S Holash J Vascular specific growth factors and blood vessel formation.Nature. 2000; 407: 242-248Crossref PubMed Scopus (3308) Google Scholar, 2Carmeliet P Mechanisms of angiogenesis and arteriogenesis.Nat Med. 2000; 6: 389-395Crossref PubMed Scopus (3516) Google Scholar, 3Ferrara N Gerber H LeCouter J The biology of VEGF and its receptors.Nat Med. 2003; 9: 669-676Crossref PubMed Scopus (7989) Google Scholar, 4Pugh CW Ratcliffe P Regulation of angiogenesis by hypoxia: role of the HIF system.Nat Med. 2003; 9: 677-684Crossref PubMed Scopus (2036) Google Scholar, 5Jain RK Molecular regulation of vessel maturation.Nat Med. 2003; 9: 685-693Crossref PubMed Scopus (2074) Google Scholar, 6Carmeliet P Angiogenesis in health and disease.Nat Med. 2003; 9: 653-660Crossref PubMed Scopus (3529) Google Scholar However, it has become increasingly clear that angiogenesis occurring during chronic wound healing and fibrogenesis provides a key contribution to disease progression. 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130: 1807-1821Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar Possible interplay and/or association between fibrogenesis and angiogenesis in CLDs is now suggested and supported by several findings: 1) angiogenesis and up-regulation of vascular endothelial growth factor (VEGF) expression has been documented in different models of acute and chronic liver injury7Medina J Arroyo AG Sanchez-Madrid F Moreno-Otero R Angiogenesis in chronic inflammatory liver disease.Hepatology. 2004; 39: 1185-1195Crossref PubMed Scopus (211) Google Scholar, 17Ankoma-Sey V Matli M Chang KB Lalazar A Donner DB Wong L Warren RS Friedman SL Coordinated induction of VEGF receptors in mesenchymal cell types during rat hepatic wound healing.Oncogene. 1998; 17: 115-121Crossref PubMed Scopus (115) Google Scholar, 18Ishikawa K Mochida S Mashiba S Inao M Matsui A Ikeda H Ohno A Shibuya M Fujiwara K Expression of vascular endothelial growth factor in nonparenchymal as well as parenchymal cells in rat liver after necrosis.Biochem Biophys Res Commun. 1999; 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and 4) paracrine expression of VEGF by rat HSCs as well as by hepatocytes has been shown to regulate the phenotype (ie, fenestration and CD-31 expression) of liver sinusoidal endothelial cells,28DeLeve LD Wang X Hu L McCuskey MK McCuskey RS Rat liver sinusoidal endothelial cell phenotype is maintained by paracrine and autocrine regulation.Am J Physiol. 2004; 287: G757-G763Crossref PubMed Scopus (226) Google Scholar a feature of possible relevance in CLDs. Data concerning expression of angiopoietins are, at present, much more limited.7Medina J Arroyo AG Sanchez-Madrid F Moreno-Otero R Angiogenesis in chronic inflammatory liver disease.Hepatology. 2004; 39: 1185-1195Crossref PubMed Scopus (211) Google Scholar, 22Medina J Caveda L Sanz-Cameno P Arroyo A Martin-Vilchez S Majano P Garcia-Buey L Sanchez-Madrid F Moreno-Otero R Hepatocyte growth factor activates endothelial pro-angiogenic mechanisms relevant in chronic hepatitis C-associated neoangiogenesis.J Hepatol. 2003; 38: 660-667Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar Recent work has demonstrated expression of angiopoietin 1 (Ang-1) in human activated HSC/MFs and its up-regulation by hypoxia.27Aleffi S Petrai I Bertolani C Parola M Colombatto S Novo E Vizzutti F Anania FA Milani S Rombouts K Laffi G Pinzani M Marra F Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells.Hepatology. 2005; 42: 1339-1348Crossref PubMed Scopus (302) Google Scholar In the present study, we report that VEGF-A and Ang-1 can stimulate migration and chemotaxis of human HSC/MFs and that, in liver tissue obtained either from cirrhotic rats or from patients with hepatitis C virus (HCV)-related cirrhosis, α-smooth muscle actin (α-SMA)-positive cells in areas of active fibrogenesis express VEGF-A and Ang-1 and their related receptors. These novel data suggest that hypoxia-dependent synthesis and release of VEGF and Ang-1 by activated HSC/MFs may contribute to both fibrogenesis and neovascularization by their actions on MF-like cells and sinusoidal endothelial cells. Enhanced chemiluminescence reagents, nitrocellulose membranes (Hybond-C extra), and secondary Cy3-conjugated antibodies were from Amersham Pharmacia Biotech (Cologno Monzese, Milano, Italy). Human recombinant growth factors and cytokines, including VEGF and Angiopoietin-1, were from PeproTech Inc. (Rocky Hill, NJ). Antibodies against phosphorylated and unphosphorylated Erk1/2 were from Upstate Biotechnology (Lake Placid, NY). Monoclonal and polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), except those against α-SMA and fluorescein isothiocyanate-conjugated antibodies (obtained from Sigma Aldrich Spa, Milano, Italy) and against CD-31 (BD Pharmingen, Erembodegem, Belgium). The monoclonal neutralizing antibody against Flk-1 was obtained from ImClone (New York, NY); although originally raised against mouse epitope, this antibody was found to also cross-react with human Flk-1, as confirmed by the block of Erk1/2 phosphorylation in human HSC/MFs treated with VEGF (data not shown). All of the other reagents were of analytical grade and obtained from Sigma Chemical Co. (Sigma Aldrich Spa). The use of human material was approved by Human Research Review Committee of the Università di Firenze, where cells were isolated and characterized from surgical wedge sections of human livers not suitable for transplantation, as extensively described elsewhere.29Casini A Pinzani M Milani S Grappone C Galli G Jezequel AM Schuppan D Rotella CM Surrenti C Regulation of extracellular matrix synthesis by transforming growth factor β1 in human fat-storing cells.Gastroenterology. 1993; 105: 245-253Abstract Full Text PDF PubMed Google Scholar Cells obtained by at least three different human livers were cultured in Iscove's medium supplemented with 20% fetal bovine serum and, unless otherwise stated, used between passages 4 and 7 as fully activated HSC/MFs with a marker profile identical to that of interface myofibroblasts described in fibrotic and cirrhotic human livers.13Cassiman D Libbrecht L Desmet V Denef C Roskams T Hepatic stellate cell/myofibroblast subpopulations in fibrotic human and rat livers.J Hepatol. 2002; 36: 200-209Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 30Cassiman D Roskams T Beauty is in the eye of the beholder: emerging concepts and pitfalls in hepatic stellate cell research.J Hepatol. 2002; 37: 527-535Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar HSC/MFs were plated in normoxic conditions to obtain the desired subconfluence level (65 to 70%) in a relatively short time (ie, to prevent significant synthesis of extracellular matrix components) and then left for 24 hours in serum-free Iscove's medium to obtain cells at the lowest level of spontaneous proliferation before the addition of the different stimuli. This procedure minimizes differences between cells coming from different individuals, as detailed in previous studies.31Failli P De Franco RM Caligiuri A Gentilini A Romanelli RG Marra F Batignani G Guerra CT Laffi G Gentilini P Pinzani M Nitrovasodilators inhibit platelet-derived growth factor-induced proliferation and migration of activated human hepatic stellate cells.Gastroenterology. 2000; 119: 479-492Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 32Caligiuri A De Franco RM Romanelli RG Gentilini A Meucci M Failli P Mazzetti L Rombouts K Geerts A Vanesia M Gentilini P Marra F Pinzani M Antifibrogenic effects of canrenone, an antialdosteronic drug, on human hepatic stellate cells.Gastroenterology. 2003; 124: 504-520Abstract Full Text PDF PubMed Scopus (56) Google Scholar, 33Novo E Marra F Zamara E Valfrè di Bonzo L Monitillo L Cannito S Petrai I Mazzocca A Bonacchi A De Franco RSM Colombatto S Autelli R Pinzani Parola M Overexpression of Bcl-2 by activated human hepatic stellate cells: resistance to apoptosis as a mechanism of progressive hepatic fibrogenesis in humans.Gut. 2006; 55: 1174-1182Crossref PubMed Scopus (153) Google Scholar Mean distribution for the cell cycle in cells serum-starved for 24 hours and then cultured for an additional 20 hours is ∼50% in G0/G1 phase, 35% in S phase, and the remaining in G2/M phase.31Failli P De Franco RM Caligiuri A Gentilini A Romanelli RG Marra F Batignani G Guerra CT Laffi G Gentilini P Pinzani M Nitrovasodilators inhibit platelet-derived growth factor-induced proliferation and migration of activated human hepatic stellate cells.Gastroenterology. 2000; 119: 479-492Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar Moreover, 24-hour serum-deprived cells continue to increase in cell number in serum-free medium for at least an additional 96 hours, at which time the number of cells is doubled.32Caligiuri A De Franco RM Romanelli RG Gentilini A Meucci M Failli P Mazzetti L Rombouts K Geerts A Vanesia M Gentilini P Marra F Pinzani M Antifibrogenic effects of canrenone, an antialdosteronic drug, on human hepatic stellate cells.Gastroenterology. 2003; 124: 504-520Abstract Full Text PDF PubMed Scopus (56) Google Scholar In experiments designed to evaluate the role of hypoxia, as previously detailed,27Aleffi S Petrai I Bertolani C Parola M Colombatto S Novo E Vizzutti F Anania FA Milani S Rombouts K Laffi G Pinzani M Marra F Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells.Hepatology. 2005; 42: 1339-1348Crossref PubMed Scopus (302) Google Scholar serum-deprived and subconfluent human HSC/MFs (65 to 70%) were incubated in strictly controlled hypoxic conditions (3% O2) for up to 24 hours. In these studies cells were tested for nonoriented migration in the wound-healing assay (WHA) (see later), and the culture medium of cells exposed to hypoxia was collected at different time points (hypoxia-conditioned medium). Cell lysates obtained by HSC/MFs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% or 7.5% acrylamide gels. The blots were incubated with desired primary antibodies and then incubated with peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulins in Tris-buffered saline-Tween containing 2% (w/v) nonfat dry milk as previously described33Novo E Marra F Zamara E Valfrè di Bonzo L Monitillo L Cannito S Petrai I Mazzocca A Bonacchi A De Franco RSM Colombatto S Autelli R Pinzani Parola M Overexpression of Bcl-2 by activated human hepatic stellate cells: resistance to apoptosis as a mechanism of progressive hepatic fibrogenesis in humans.Gut. 2006; 55: 1174-1182Crossref PubMed Scopus (153) Google Scholar, 34Zamara E Novo E Marra F Gentilini A Romanelli RG Caligiuri A Robino G Tamagno E Aragno M Danni O Autelli R Colombatto S Dianzani MU Pinzani M Parola M 4-Hydroxynonenal as a selective pro-fibrogenic stimulus for activated human hepatic stellate cells.J Hepatol. 2004; 40: 60-68Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 35Parola M Robino G Marra F Pinzani M Bellomo G Leonarduzzi G Chiarugi P Camandola S Poli G Gentilini P Dianzani MU HNE interacts directly with JNK isoforms in human hepatic stellate cells.J Clin Invest. 1998; 102: 1942-1950Crossref PubMed Scopus (276) Google Scholar, 36Robino G Parola M Marra F Caligiuri A De Franco RM Zamara E Bellomo G Gentilini P Pinzani M Dianzani MU Interaction between 4-hydroxy-2,3-alkenals and the platelet-derived growth factor-beta receptor. Reduced tyrosine phosphorylation and downstream signaling in hepatic stellate cells.J Biol Chem. 2000; 275: 40561-40567Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar and developed with the enhanced chemiluminescence reagents according to the manufacturer's instructions. To evaluate signaling of VEGF receptors (Flt-1 and Flk-1) and Ang-1 receptor (Tie-2), the state of phosphorylation of Erk1/2 and c-Akt (to evaluate involvement of Ras/Erk signaling and of PI 3-K activity, respectively), was analyzed as reported elsewhere.33Novo E Marra F Zamara E Valfrè di Bonzo L Monitillo L Cannito S Petrai I Mazzocca A Bonacchi A De Franco RSM Colombatto S Autelli R Pinzani Parola M Overexpression of Bcl-2 by activated human hepatic stellate cells: resistance to apoptosis as a mechanism of progressive hepatic fibrogenesis in humans.Gut. 2006; 55: 1174-1182Crossref PubMed Scopus (153) Google Scholar, 34Zamara E Novo E Marra F Gentilini A Romanelli RG Caligiuri A Robino G Tamagno E Aragno M Danni O Autelli R Colombatto S Dianzani MU Pinzani M Parola M 4-Hydroxynonenal as a selective pro-fibrogenic stimulus for activated human hepatic stellate cells.J Hepatol. 2004; 40: 60-68Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 35Parola M Robino G Marra F Pinzani M Bellomo G Leonarduzzi G Chiarugi P Camandola S Poli G Gentilini P Dianzani MU HNE interacts directly with JNK isoforms in human hepatic stellate cells.J Clin Invest. 1998; 102: 1942-1950Crossref PubMed Scopus (276) Google Scholar, 36Robino G Parola M Marra F Caligiuri A De Franco RM Zamara E Bellomo G Gentilini P Pinzani M Dianzani MU Interaction between 4-hydroxy-2,3-alkenals and the platelet-derived growth factor-beta receptor. 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Proliferation was evaluated by means of incorporation of radiolabeled [3H]thymidine and by cell counting using platelet-derived growth factor (PDGF)-BB as a positive control, as previously described.34Zamara E Novo E Marra F Gentilini A Romanelli RG Caligiuri A Robino G Tamagno E Aragno M Danni O Autelli R Colombatto S Dianzani MU Pinzani M Parola M 4-Hydroxynonenal as a selective pro-fibrogenic stimulus for activated human hepatic stellate cells.J Hepatol. 2004; 40: 60-68Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 35Parola M Robino G Marra F Pinzani M Bellomo G Leonarduzzi G Chiarugi P Camandola S Poli G Gentilini P Dianzani MU HNE interacts directly with JNK isoforms in human hepatic stellate cells.J Clin Invest. 1998; 102: 1942-1950Crossref PubMed Scopus (276) Google Scholar, 36Robino G Parola M Marra F Caligiuri A De Franco RM Zamara E Bellomo G Gentilini P Pinzani M Dianzani MU Interaction between 4-hydroxy-2,3-alkenals and the platelet-derived growth factor-beta receptor. Reduced tyrosine phosphorylation and downstream signaling in hepatic stellate cells.J Biol Chem. 2000; 275: 40561-40567Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar Synthesis and release in the culture medium of MCP-1 (as a parameter of proinflammatory responses) and of procollagen type I (as a parameter of synthesis of extracellular matrix components) was evaluated by enzyme-linked immunosorbent assay as previously described27Aleffi S Petrai I Bertolani C Parola M Colombatto S Novo E Vizzutti F Anania FA Milani S Rombouts K Laffi G Pinzani M Marra F Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells.Hepatology. 2005; 42: 1339-1348Crossref PubMed Scopus (302) Google Scholar, 34Zamara E Novo E Marra F Gentilini A Romanelli RG Caligiuri A Robino G Tamagno E Aragno M Danni O Autelli R Colombatto S Dianzani MU Pinzani M Parola M 4-Hydroxynonenal as a selective pro-fibrogenic stimulus for activated human hepatic stellate cells.J Hepatol. 2004; 40: 60-68Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar and using interleukin-1 and transforming growth factor-β1, respectively, as positive controls. All animals received humane care, and experimental protocols were conducted according to national and local guidelines. Male adult Wistar rats (Harlan-Nossan, Correnzana, Italy), initial weight 200 to 220 g, were fed with a standard pelleted diet and water ad libitum. Advanced fibrosis was induced by chronic treatment with CCl4 administered by gavage twice a week for 9 weeks.38Proctor E Chatamra K High yield micronodular cirrhosis in the rat.Gastroenterology. 1982; 83: 1183-1190Abstract Full Text PDF PubMed Scopus (268) Google Scholar, 39Sansoè G Aragno M Mastrocola R Restivo F Mengozzi G Smedile A Rosina F Danni O Parola M Rizzetto M Neutral endopeptidase (EC 3.4.24.11) in cirrhotic liver: a new target to treat portal hypertension?.J Hepatol. 2005; 43: 791-798Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Control animals received an equal volume of vehicle. 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