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【摘要】
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.
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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.1-6 However, it has become increasingly clear that angiogenesis occurring during chronic wound healing and fibrogenesis provides a key contribution to disease progression. Pathological angiogenesis, as recently reviewed,7 has indeed been described in chronic inflammatory/fibrotic liver diseases of different etiology.
Hepatic angiogenesis differs from homologous processes in other tissues for a number of reasons, including the existence of two different types of microvascular structures in the liver (ie, large vessels lined by a continuous endothelium versus sinusoids lined by a fenestrated endothelium),8 the apparent production of the liver-specific angiogenic factor AN-GPTL3,9 and the unique but not homogenous phenotypic profile and functional role of hepatic stellate cells (HSCs).10-13 HSCs are also regarded as liver-specific pericytes, but their role in modulating angiogenesis, particularly in pathological conditions, may substantially differ from the role attributed to microcapillary pericytes.7 During the fibrotic progression of chronic liver diseases (CLDs), activated and myofibroblast-like HSCs (HSC/MFs) play a major profibrogenic role together with portal (myo)fibroblast and, possibly, bone marrow-derived stem cells, giving rise to hepatic populations of highly proliferative, profibrogenic, and contractile myofibroblast-like cells (MFs).10-16
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 injury7,17-21 as well as in specimens from human fibrotic/cirrhotic liver and hepatocellular carcinoma7,22-24 ; 2) in HSCs, hypoxia has been shown to up-regulate expression of VEGF,20,25-27 VEGF receptor type I (fms-like tyrosine kinase receptor or Flt-1),20,25 and collagen type I20 ; 3) VEGF has been proposed to directly stimulate proliferation and expression of 1(I)-procollagen mRNA in activated rat HSCs21 ; 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,28 a feature of possible relevance in CLDs. Data concerning expression of angiopoietins are, at present, much more limited.7,22 Recent work has demonstrated expression of angiopoietin 1 (Ang-1) in human activated HSC/MFs and its up-regulation by hypoxia.27
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, -smooth muscle actin (-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.
【关键词】 proangiogenic cytokines hypoxia-dependent stimulating migration stellate
Materials and Methods
Materials
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 -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).
Cell Isolation and Culture
The use of human material was approved by Human Research Review Committee of the Universit
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作者单位：From the Dipartimento Medicina e Oncologia Sperimentale,* Universit