Dissection of the biphasic nature of hypoxia-induced motogenic action in bone marrow-derived human mesenchymal stem cells.

Hypoxic conditions have been reported to facilitate preservation of undifferentiated mesenchymal stem cell (MSC) phenotype and positively affect their colony-forming potential, proliferation, and migration/mobilization. In this study, designed to dissect mechanisms underlying hypoxia-dependent migration of bone marrow-derived human MSC (hMSC), signal transduction, and molecular mechanisms were evaluated by integrating morphological, molecular, and cell biology techniques, including the wound healing assay (WHA) and modified Boyden's chamber assay (BCA) to monitor migration. Exposure of hMSCs to moderate hypoxia resulted in a significant increase of migration of hMSCs in both WHA (from 6 to 20 hours) and BCA (within 6 hours). Mechanistic experiments outlined the following sequence of hypoxia-dependent events: (a) very early (15 minutes) increased generation of intracellular reactive oxygen species (ROS), which (b) was sufficient to switch on activation of extracellular regulated kinase 1/2 and c-Jun N-terminal protein kinase 1/2, found to be relevant for the early phase of hMSC migration; (c) hypoxia inducible factor-1 (HIF-1)-dependent increased expression of vascular endothelial growth factor (VEGF) (facilitated by ROS) and its progressive release that was responsible for (d) a delayed and sustained migration of hMSCs. These results suggest that hypoxia-dependent migration relies on a previously unrecognized biphasic scenario involving an early phase, requiring generation of ROS, and a delayed phase sustained by HIF-1-dependent expression and release of VEGF.

Angiogenesis and liver fibrogenesis.

Angiogenesis is a dynamic, hypoxia-stimulated and growth factor-dependent process, eventually leading to the formation of new vessels from pre-existing blood vessels. In the last decade experimental and clinical studies have described the occurrence of hepatic angiogenesis in a number of different pathophysiological conditions, including those involving inflammatory, fibrotic and ischemic features. In particular, the literature evidence indicates that hepatic angiogenesis is strictly associated with, and may even favour fibrogenic progression of chronic inflammatory liver diseases of different aetiology. In this review, current "in vivo" and "in vitro" evidence supporting the potential pathogenetic role of angiogenesis in chronic liver diseases will be reviewed in an attempt to outline cellular and molecular mechanisms involved, with a specific emphasis on the crucial role of hypoxic conditions and hepatic stellate cells (HSCs), particularly when activated to the myofibroblast-like pro-fibrogenic phenotype.

Redox mechanisms switch on hypoxia-dependent epithelial-mesenchymal transition in cancer cells.

Epithelial-mesenchymal transition (EMT) and hypoxia are considered as crucial events favouring invasion and metastasis of many cancer cells. In this study, different human neoplastic cell lines of epithelial origin were exposed to hypoxic conditions in order to investigate whether hypoxia per se may trigger EMT programme as well as to mechanistically elucidate signal transduction mechanisms involved. The following human cancer cell lines were used: HepG2 (from human hepatoblastoma), PANC-1 (from pancreatic carcinoma), HT-29 (from colon carcinoma) and MCF-7 (from breast carcinoma). Cancer cells were exposed to carefully controlled hypoxic conditions and investigated for EMT changes and signal transduction by using morphological, cell and molecular biology techniques. All cancer cells responded to hypoxia within 72 h by classic EMT changes (fibroblastoid phenotype, SNAIL and beta-catenin nuclear translocation and changes in E-cadherin) and by increased migration and invasiveness. This was involving very early inhibition of glycogen synthase kinase-3beta (GSK-3beta), early SNAIL translocation as well as later and long-lasting activation of Wnt/beta-catenin-signalling machinery. Experimental manipulation, including silencing of hypoxia-inducible factor (HIF)-1alpha and the specific inhibition of mitochondrial generation of reactive oxygen species (ROS), revealed that early EMT-related events induced by hypoxia (GSK-3beta inhibition and SNAIL translocation) were dependent on transient intracellular increased generation of ROS whereas late migration and invasiveness were sustained by HIF-1alpha- and vascular endothelial growth factor (VEGF)-dependent mechanisms. These findings indicate that in cancer cells, early redox mechanisms can switch on hypoxia-dependent EMT programme whereas increased invasiveness is sustained by late and HIF-1alpha-dependent release of VEGF.

Beta-catenin triggers nuclear factor kappaB-dependent up-regulation of hepatocyte inducible nitric oxide synthase.

Disruption of cell-to-cell contacts, as observed in many pathophysiological conditions, prime hepatocytes for compensatory hyperplastic response that involves induction of several genes, including proto-oncogenes and other gene targets of beta-catenin signaling pathway. By using cultured hepatocytes and experimental models of adherens junction disruption we have investigated changes in beta-catenin subcellular localization and their relationships with inducible nitric oxide synthase (iNOS) expression. Two experimental models were employed: (a) rat hepatocytes obtained by collagenase liver perfusion within the first 48 h of culture; (b) 48-h old cultured hepatocytes, transiently transfected or not with a plasmid encoding for dominant/negative inhibitory kappa B-alpha, exposed to ethylene glycol-bis-(2-aminoethylether)-N,N,N',N'-tetraacetic acid/LiCl treatment. beta-Catenin signaling and cellular localization, iNOS expression and nuclear factor kappaB involvement, were investigated using morphological, cell and molecular biology techniques. E-cadherin-mediated disruption of cell-to-cell contacts induces early beta-catenin translocation from membrane to cytoplasm and nuclear compartments, events that are followed by up-regulation of c-myc, cyclin D1 and beta-transducin repeat-containing protein expression. This, in turn, resulted eventually in iNOS induction that was mechanistically related to nuclear factor kappaB activation, as unequivocally shown in cells expressing dominant negative inhibitory kappa B-alpha. Our data indicate that E-cadherin disassembly and concomitant inactivation of glycogen synthase kinase-3beta result in nuclear factor kappaB-dependent induction of iNOS in hepatocytes.

Proangiogenic cytokines as hypoxia-dependent factors stimulating migration of human hepatic stellate cells.

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.