Hypoxia and hypoxia-inducible factor 1α: potential links between angiogenesis and fibrogenesis in hepatic stellate cells
Year: 2010
Type: article
Abstract: The activation of hepatic stellate cells (HSCs) has been proposed to be a key event in chronic liver disease (1, 2). Activated HSCs are believed to be the main extracellular matrix (ECM)-producing cell population in the liver and thus responsible for the development of hepatic fibrosis (1, 2). Hepatic accumulation of ECM and changes in the hepatic vasculature are believed to cause the majority of complications of chronic liver disease, namely fibrosis and portal hypertension. Interestingly, hypoxia stimulates vascular endothelial growth factor (VEGF) expression in HSCs (3, 4). Moreover, HSCs have been shown to also contribute to angiogenesis secreting angiopoietin 1 (5). Notably, excessive ECM accumulation and changes in the liver architecture change blood flow and promote tissue hypoxia (6). Thus, it is likely that HSCs are at the crossroads of fibrosis and angiogenesis, and that hypoxia in HSCs and other cell types contributes to the vascular changes observed in the chronically injured liver. In their current study, Copple and colleagues investigate the impact of hypoxia on gene expression in culture-activated HSCs with a particular focus on the expression of the hypoxia-inducible factor (HIF)-1α. HIFs are heterodimeric transcription factors, and have been subdivided into an oxygen-regulated α subunit and a constitutively expressed β subunit. In addition to the ubiquitously expressed HIF-1α, two other members, HIF-2α and HIF-3α, have been identified. HIF-1α is known to be a master regulator in the adaptive response to hypoxia. Under normoxic conditions, HIF-1α undergoes oxygen-dependent hydroxylation of proline and asparagine residues leading to immediate degradation, keeping the concentration of HIF-1α low. When cells become hypoxic, this degradation pathway is weakened, leading to an increase in HIF-1α protein levels. HIF-1α dimerizes with β subunits such as HIF1β/aryl hydrocarbon receptor nuclear translocator (ARNT1), and enters the nucleus where it binds to general transcriptional co-activators in the regulatory region of hypoxia-activated genes (7). The most common HIF-1α target genes are involved in erythropoiesis and iron metabolism (erythropoietin, transferrin), angiogenesis (VEGF), and glucose metabolism (phosphofructokinase) (7). While HIF-1α and HIF-2α have been well characterized as oxygen-dependent transcription factors, the role of HIF-3α is less well understood. It has been suggested that a splice variant of HIF-3α exerts an inhibitory effect on the HIF-2α-inducible pathways (8). Using a tissue culture system in which HSCs were maintained either in 0.5% oxygen or room air (20–21% oxygen), Copple et al. (9) observed a strong upregulation of HIF-1α protein levels. Moreover, hypoxia induced the expression of 175 genes as determined by microarray analysis. Among these 175 genes, the authors identified several known targets of HIF-1α including VEGF, placental growth factor (PGF), angiopoietin-like 4 (AngL4) and macrophage migration inhibitory factor (MIF). Notably, the authors identified a number of genes that were not previously known to be regulated by hypoxia, which have been suggested to play a role in HSC activation and fibrogenesis, such as chemokine receptor (CCR) 1, CCR5, interleukin-13 receptor α1 (IL-13ra1) and α2b adrenergic receptor (Adra2b). Previous studies have demonstrated that CCR1, CCR5 and their ligands play an important role in hepatic fibrogenesis as demonstrated by reduced liver fibrosis in response to injury in CCR1-, CCR5- and RANTES-deficient mice (10, 11). IL-13 is a Th2 cytokine that plays a crucial role in promoting fibrosis in various organs including Schistosoma mansoni-induced liver fibrosis (12). Moreover, IL-13 levels are increased in the liver and serum of patients with hepatitis C, and tightly associated with the stage of fibrosis (13). In addition, Copple and colleagues uncovered an upregulation of the prolyl hydrolases, P4ha1 and P4ha2, two key enzymes necessary for the generation of stable collagen triple helices (14, 15), in hypoxic HSC. Using inducible Mx1-Cre to delete HIF-1α in all hepatic cell populations and subsequent isolation of HIF-1α-deleted HSCs, Copple and colleagues proceeded to demonstrate that several of the above-described hypoxia-regulated genes such as VEGF, PGF, AngL4, MIF, CCR1 and CCR5 were HIF-1α regulated. In addition, a number of hypoxia-induced genes including AngL4, Adra2b and P4ha2 were not regulated by HIF-1α. The authors suggest that these genes are regulated by HIF-2α and provide evidence that HIF-2α is upregulated in primary culture activated HSCs from adult mice exposed to 0.5% oxygen. Hypoxia affects various cell populations in the liver. Hepatocytes are particularly susceptible to hypoxia because of their high metabolic rate and the large amount of mitochondria they contain. In an earlier study, Copple et al. (16) found that hepatocytes isolated from mouse liver express both HIF-1α and HIF-2α under hypoxic conditions. A subsequent study by the same group showed that under hypoxia, HIF-1α is also upregulated in primary mouse Kupffer cells, the resident macrophages of the liver (17). These cells accumulate in the injured liver and express fibrogenic mediators such as PDGF-B and TGFβ to activate HSCs and stimulate collagen production. Interestingly in this study, PDGF, not TGF-β, was upregulated in hypoxic Kupffer cells in an HIF-dependent manner (17). Thus, it is likely that hypoxia regulates injury and the subsequent wound-healing response in different cell populations, and that HIF-1α in HSCs is only one component of a complex HIF-regulated network. Moreover, there is an oxygen gradient between different zones of the liver with higher oxygenation in the periportal areas and lower oxygenation in the perivenous area. It would be relevant to sort out the contribution of hypoxia not only in different cell types, but also determine whether there is a role in localization. Localization may not only determine gene expression and sensitivity of hepatocytes, but could also lead to differences in the response to hypoxia in different fibrogenic cell populations such as HSCs and portal fibroblasts. Accordingly, it has been suggested that intralobular HSCs might be more sensitive to hypoxia than periportal fibroblasts, and promote angiogenesis in response to hypoxic stimulation (6). It is important to be point out limitations of the study by Copple and colleagues. For one, cell culture studies under atmospheric conditions (20–21% O2) are usually considered normoxia when, in fact, the oxygen concentration is significantly lower in most tissues. Thus, an O2 concentration of 5% would be much closer to the situation of most cells in vivo (18). Therefore, it may be that the studies by Copple and colleagues, while certainly following standard conditions of cell culture, compare a hyperoxic state with a hypoxic state. Secondly, to understand the relevance of HIF-1α and hypoxia in liver fibrosis, the current studies need to be extended to in vivo conditions to (A) confirm the upregulation of HIF-1α in HSCs and myofibroblasts during chronic injury and (B) the role of HIF-1α in the activation of these cell types in vivo. Culture activation of HSCs lacks the typical environment of the injured liver including the interaction with cell types such as macrophages, lymphocytes and injured hepatocytes as well as the exposure to systemic inflammatory mediators and products from the intestinal microbiota, and at the same time puts the cells into contact with rigid plastic surfaces and fetal bovine serum. Accordingly, culture activation leads to nonphysiological gene expression patterns in human and murine HSCs (19, 20). Additionally, most of them demonstrated that increases in hypoxia-regulated pro-fibrotic gene expression were at a modest level, e.g. an about two-fold induction of CCR1 and CCR5 mRNA, and a three-fold induction of IL-13ra1 mRNA. It remains to be determined whether such a moderate upregulation has a major impact on HSC activation and fibrosis, and whether a similar upregulation is seen in vivo. In summary, the study by Copple and colleagues provides evidence that many genes in culture-activated HSCs are regulated by hypoxia and HIF-1α, and represents an important step in understanding how HIF-1α regulates gene expression in HSCs. Future studies need to study the role of HIF-1α in HSC activation and fibrogenesis in vivo, by using the floxed HIF-1α mouse model to selectively delete HIF-1α in HSCs or myofibroblasts.
Source: Liver International
Authors: Juliane S. Troeger, Robert F. Schwabe
Institution: Columbia University
Language: English
Subfield: Hepatology
Field: Medicine
Domain: Health Sciences
Open Access status: bronze
OpenAlex ID: W2093473169