The biphasic nature of hypoxia‐induced directional migration of activated human hepatic stellate cells

Abstract

Liver fibrogenesis is sustained by pro-fibrogenic myofibroblast-like cells (MFs), mainly originating from activated hepatic stellate cells (HSC/MFs) or portal (myo)fibroblasts, and is favoured by hypoxia-dependent angiogenesis. Human HSC/MFs were reported to express vascular-endothelial growth factor (VEGF) and VEGF-receptor type 2 and to migrate under hypoxic conditions. This study was designed to investigate early and delayed signalling mechanisms involved in hypoxia-induced migration of human HSC/MFs. Signal transduction pathways and intracellular generation of reactive oxygen species (ROS) were evaluated by integrating morphological, cell, and molecular biology techniques. Non-oriented and oriented migration were evaluated by using wound healing assay and the modified Boyden's chamber assay, respectively. The data indicate that hypoxia-induced migration of HSC/MFs is a biphasic process characterized by the following sequence of events: (a) an early (15 min) and mitochondria-related increased generation of intracellular ROS which (b) was sufficient to switch on activation of ERK1/2 and JNK1/2 that were responsible for the early phase of oriented migration; (c) a delayed and HIF-1α-dependent increase in VEGF expression (facilitated by ROS) and its progressive, time-dependent release in the extracellular medium that (d) was mainly responsible for sustained migration of HSC/MFs. Finally, immunohistochemistry performed on HCV-related fibrotic/cirrhotic livers revealed HIF-2α and haem-oxygenase-1 positivity in hepatocytes and α-SMA-positive MFs, indicating that MFs were likely to be exposed in vivo to both hypoxia and oxidative stress. In conclusion, hypoxia-induced migration of HSC/MFs involves an early, mitochondrial-dependent ROS-mediated activation of ERK and JNK, followed by a delayed- and HIF-1α-dependent up-regulation and release of VEGF.

Cellular and molecular mechanisms in liver fibrogenesis

“…ROS, not HNE, have been reported to stimulate proliferation of HSC/MFs and ROS can also contribute to stimulate oriented migration of these pro-fibrogenic cells by involving activation of isoforms 1 and 2 of c-Jun-NH2-kinases (JNK1/2). This redox-sensitive pro-migratory action relies just on a significant increase of intracellular ROS, whatever their origin; HSC/MFs can migrate in response to i) ROS entering them from the chronically injured microenvironment, ii) ROS released by mitochondria following exposure to hypoxia or iii) ROS generated by NADPH-oxidase activation that parallels the activation of ligand-receptor interaction elicited by chemotactic polypeptide factors such as PDGF-BB, VEGF-A or MCP-1 [20,21]. 4.…”

“…Hepatic MFs, in particular HSC/MFs, can efficiently respond to conditions of hypoxia that are progressively increasing in chronically injured liver during CLD progression [81,82,84]. Major concepts can be summarized as follows: i) HSC and HSC/MFs respond to hypoxia in a HIF-1 related way by up-regulating expression of VEGF, Angiopoietin 1 as well as of their related receptor VEGFR-2 and Tie2 [83,86], then behaving as pro-angiogenic cells; ii) HSC/MFs and, likely, all hepatic MFs (including those originating from bone-marrow derived mesenchymal stem cells recruited in chronically injured liver) [4] represent an effective target for VEGF and angiopoietin 1; in these cells VEGF can stimulate proliferation and increased deposition of extracellular matrix components [81,82]), as well as increased migration and chemotaxis [83,87], the latter response being also significantly elicited in cells just exposed to hypoxia [87]. In particular, oriented migration of MFs in response to either hypoxia or VEGF (as other chemotactic peptides) has been described as a biphasic mechanism that is first switched on by ROS released by either mitochondria or through ligandreceptor related activation of NADPH-oxidase and proceeds through redox-dependent activation of Ras/ERK and c-Jun-NH2-terminal kinase isoforms (JNKs).…”

“…In particular, oriented migration of MFs in response to either hypoxia or VEGF (as other chemotactic peptides) has been described as a biphasic mechanism that is first switched on by ROS released by either mitochondria or through ligandreceptor related activation of NADPH-oxidase and proceeds through redox-dependent activation of Ras/ERK and c-Jun-NH2-terminal kinase isoforms (JNKs). This is followed by a delayed and sustained phase of migration depending on HIF-1-mediated, ROS-stabilized, up-regulation of VEGF expression, resulting in the subsequent chemotactic action of extracellularly released VEGF [87]. Immunohistochemistry analysis performed on either human or rodent fibrotic/cirrhotic liver has showed that positive staining for both HIF-2 and heme-oxygenase 1 (HO1, a redox-related marker) is mainly evident in MF-like cells in developing septa and at the border of more mature and larger fibrotic septa suggesting that the scenario is likely to operate also in vivo [87].…”

“…This is followed by a delayed and sustained phase of migration depending on HIF-1-mediated, ROS-stabilized, up-regulation of VEGF expression, resulting in the subsequent chemotactic action of extracellularly released VEGF [87]. Immunohistochemistry analysis performed on either human or rodent fibrotic/cirrhotic liver has showed that positive staining for both HIF-2 and heme-oxygenase 1 (HO1, a redox-related marker) is mainly evident in MF-like cells in developing septa and at the border of more mature and larger fibrotic septa suggesting that the scenario is likely to operate also in vivo [87]. The relevant point is that such a morphological evidence overlaps with those observed in human and rat fibrotic/cirrhotic livers [83] where -SMA positive hepatic MFs expressing VEGF, Ang-1 or the related receptors VEGFR-2 and Tie-2, were detected at the leading edge of tiny and incomplete developing septa, but not in larger bridging septa.…”

Mechanisms of adaptation of the hepatic vasculature to the deteriorating conditions of blood circulation in liver cirrhosis

“…In turn, SECs generate PDGF and TGFβ, helping to attract and migration of HSCs. This process includes ROSmediated activation of ERK and cJunNH2terminal kinase (JNK) followed by a delayed and HIF1αdependent up regulation and release of VEGF [51] .…”