Hypoxia stimulates insulin-like growth factor binding protein 1 (IGFBP-1) gene expression in HepG2 cells: a possible model for IGFBP-1 expression in fetal hypoxia

IGFBP-1 is elevated in fetuses with long-term, chronic hypoxia and intrauterine growth restriction. We investigated the hypothesis that hypoxia regulates IGFBP-1 in the human fetus in vivo and IGFBP-1 gene expression and protein in vitro. Umbilical artery IGFBP-1 levels (mean +/- SEM) from term babies with respiratory acidosis (acute hypoxia), normal babies, and those with mixed respiratory/metabolic acidosis (more profound and prolonged hypoxia) were measured using an immunoradiometric assay. IGFBP-1 levels were similar in normal (n = 12) and acutely hypoxic (n = 6) babies (189.1 +/- 71.8 vs. 175.8 +/- 45.9 ng /ml, respectively, P = 0.789). However, with more profound and prolonged hypoxia (n = 19), IGFBP-1 levels were markedly elevated (470.6 +/- 80.0 ng /ml, P = 0.044). To investigate IGFBP-1 regulation by hypoxia in vitro, HepG2 cells were incubated under hypoxia (pO2 = 2%) and normoxia (pO2 = 20%). IGFBP-1 protein and mRNA increased 8- and 12-fold, respectively, under hypoxic conditions. Hypoxia did not affect protein or mRNA levels of IGFBP-2 or -4. IGFBP-5 and -6 mRNAs, undetectable in control cells, were not induced by hypoxia, whereas minimally expressed IGFBP-3 mRNA increased twofold. Investigation into IGFBP-1 gene structure revealed three potential consensus sequences for the hypoxia response element (HRE) in the first intron. To investigate functionality, a 372-bp fragment of IGFBP-1 intron 1, containing putative HREs, was placed 5' to a heterologous hsp70 promoter in a plasmid using luciferase as a reporter gene. Under hypoxia, reporter gene activity increased up to 30-fold. Mutations in the middle HRE abolished reporter activity in response to hypoxia, suggesting that this HRE is functional in the IGFBP-1 hypoxia response. Cotransfection of HRE reporter genes with a constitutively expressing hypoxia-inducible factor 1 plasmid in HepG2 cells resulted in a fourfold induction of reporter activity, suggesting a role for hypoxia-inducible factor 1 in hypoxia induction of IGFBP-1 gene expression. These data support the hypothesis that hypoxia regulation of IGFBP-1 may be a mechanism operating in the human fetus to restrict insulin-like growth factor-mediated growth in utero under conditions of chronic hypoxia and limited substrate availability.     

Up-regulation of flk-1/vascular endothelial growth factor receptor 2 by its ligand in a cerebral slice culture system

Vascular endothelial growth factor (VEGF) and its tyrosine kinase receptors VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR) are key mediators of physiological and pathological angiogenesis. They are expressed in most tissues during embryonic development but are down-regulated in the adult, when angiogenesis ceases. Up-regulation of VEGFR-2 and of VEGF are observed in many pathological conditions under which angiogenesis is reinduced. A major regulator of VEGF expression is hypoxia. Although the temporal expression pattern of VEGFR-2 parallels VEGF expression to a high extent, little is known about its regulation. Here, we show that VEGFR-2 is highly expressed in early postnatal mouse brain but is down-regulated commencing at postnatal day 15 (P15) of mouse brain development and is hardly detectable in P30 mouse brain. Using P30 mouse brain slices, we observed that hypoxia up-regulates VEGFR-2 in the slices but not in human umbilical vein endothelial cells, suggesting the presence of a hypoxia-inducible factor in the murine neuroectoderm that up-regulates VEGFR-2. To identify the factors involved, normoxic P30 cerebral slices were cultured with growth factors that are either hypoxia-inducible (e.g., PDGF-BB, erythropoietin, and VEGF) and/or are known to act on endothelial cells (e.g., PDGF-BB, VEGF, and PIGF). Exogenously added recombinant VEGF led to an up-regulation of VEGFR-2 expression, which could be inhibited by preincubation with a neutralizing anti-VEGF antibody. Addition of PDGF-BB, PIGF, and erythropoietin had no effect on VEGFR-2 expression. Our results suggest a differential but synergistic regulation by hypoxia of VEGF and VEGFR-2: a direct induction of VEGF that subsequently up-regulates VEGFR-2 in endothelial cells. This autoenhancing system may represent an important mechanism of tumor angiogenesis.     

Mouse hypoxia-inducible factor-1alpha is encoded by two different mRNA isoforms: expression from a tissue-specific and a housekeeping-type promoter

Hypoxic induction of erythropoietin (Epo) and other oxygen-dependent genes is mediated by the hypoxia-inducible factor-1 (HIF-1), a heterodimeric transactivator consisting of an alpha and a beta subunit. We previously found that the mouse gene encoding HIF-1alpha harbors two alternative first exons (I.1 and I.2), giving rise to two different HIF-1alpha mRNA isoforms. Here, we show by RNase protection analysis that the exon I.1-derived mRNA isoform is differentially expressed in mouse tissues, being highest in kidney, tongue, stomach, and testis, but undetectable in liver, whereas the exon I.2 mRNA isoform is ubiquitously expressed. Sequence and methylation analysis showed that, in contrast to exon I.1, exon I.2 resides within a region showing typical features of a CpG island, known to be associated with the 5' end of housekeeping genes. We identified a 232-bp minimal exon I.2 promoter that strongly induced reporter gene expression in mouse L929 fibroblasts and Hepa1 hepatoma cells. In contrast to L929 cells, the exon I.1 promoter was inactive in Hepa1 cells and hypoxic exposure (1% O2) markedly reduced exon I.2 promoter activity in Hepa1 cells. Prolonged exposure of mice to hypoxia (7.5% O2 for up to 72 hours) also caused a decrease in liver HIF-1alpha mRNA, whereas aldolase mRNA levels increased. These findings might be related to the relatively low Epo levels in the adult liver.     

Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor and its receptors

Vascular endothelial growth factor (VEGF) plays a key role in physiological blood vessel formation and pathological angiogenesis such as tumor growth and ischemic diseases. Hypoxia is a potent inducer of VEGF in vitro. Here we demonstrate that VEGF is induced in vivo by exposing mice to systemic hypoxia. VEGF induction was highest in brain, but also occurred in kidney, testis, lung, heart, and liver. In situ hybridization analysis revealed that a distinct subset of cells within a given organ, such as glial cells and neurons in brain, tubular cells in kidney, and Sertoli cells in testis, responded to the hypoxic stimulus with an increase in VEGF expression. Surprisingly, however, other cells at sites of constitutive VEGF expression in normal adult tissues, such as epithelial cells in the choroid plexus and kidney glomeruli, decreased VEGF expression in response to the hypoxic stimulus. Furthermore, in addition to VEGF itself, expression of VEGF receptor-1 (VEGFR-1), but not VEGFR-2, was induced by hypoxia in endothelial cells of lung, heart, brain, kidney, and liver. VEGF itself was never found to be up-regulated in endothelial cells under hypoxic conditions, consistent with its paracrine action during normoxia. Our results show that the response to hypoxia in vivo is differentially regulated at the level of specific cell types or layers in certain organs. In these tissues, up- or down-regulation of VEGF and VEGFR-1 during hypoxia may influence their oxygenation after angiogenesis or modulate vascular permeability.     

Increased vascular endothelial growth factor production in the lungs of rats with hypoxia-induced pulmonary hypertension

Vascular endothelial growth factor (VEGF) is a potent mitogenic and permeability factor targeting predominantly endothelial cells. At least two tyrosine kinase receptors, Flk-1 and Flt-1, mediate its action and are mostly expressed by endothelial cells. VEGF and VEGF receptor expression are upregulated by hypoxia in vivo and the role of VEGF in hypoxia-induced angiogenesis has been extensively studied in a variety of disease entities. Although VEGF and its receptors are abundantly expressed in the lung, their role in hypoxic pulmonary hypertension and the accompanying vascular remodeling are incompletely understood. We report in this in vivo study that hypoxia increases mRNA levels for both VEGF and Flk-1 in the rat lung. The kinetics of the hypoxic response differ between receptor and ligand: Flk-1 mRNA showed a biphasic response to hypoxia with a significant, but transient, rise in mRNA levels observed after 9-15 h of hypoxic exposure and the highest levels noted after 3 wk. In contrast, VEGF mRNA levels did not show a significant increase with acute hypoxia, but increased progressively after 1-3 wk of hypoxia. By in situ hybridization, VEGF mRNA was localized predominantly in alveolar epithelial cells with increased signal in the lungs of hypoxic animals compared with controls. Immunohistochemical staining with anti-VEGF antibodies localized VEGF peptide throughout the lung parenchyma and was increased in hypoxic compared with normoxic animals. Furthermore, hypoxic animals had significantly higher circulating VEGF concentrations compared with normoxic controls. Lung vascular permeability as measured by extravasation of Evans Blue dye was not significantly different between normoxic and hypoxic animals, although a tendency for increased permeability was seen in the hypoxic animals. These findings suggest a possible role for VEGF in the pulmonary response to hypoxia.     

Prolonged hypoxia increases vascular endothelial growth factor mRNA and protein in adult mouse brain

Brain hypoxia induces an increase in brain vascularity, presumably mediated by vascular endothelial growth factor (VEGF), but it is unclear whether VEGF is required to maintain the increase. In these studies, brain VEGF mRNA and protein levels were measured in adult mice kept in hypobaric chambers at 0.5 atm for 0, 0.5, 1, 2, 4, 7, and 21 days. Hypoxia was accompanied by a transient increase of VEGF mRNA expression: twofold by 0.5 day and a maximum of fivefold by 2 days; these were followed by a decrease at 4 days and a return to basal levels by 7-21 days. VEGF protein expression induced by hypoxia was bimodal, initially paralleling VEGF mRNA. There was an initial small increase at 12 h that reached a maximum by day 2, and, after a transient decrease on day 4, the protein expression increased again on day 7 before it returned to normoxic levels after 21 days. Thus, despite continued hypoxia, both VEGF mRNA and protein levels returned to basal after 7 days. These data suggest a metabolic negative-feedback system for VEGF expression during prolonged hypoxia in the brain.     

Autoxidation of pyridoxalated hemoglobin polyoxyethylene conjugate

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen which stimulates angiogenesis. VEGF is regulated by multiple factors such as hypoxia, phorbol esters, and growth factors. However, data concerning the expression of VEGF in the different vascular cell types and its regulation by cAMP are not available. In the present study, we have investigated the effect of adenylate cyclase activation on VEGF mRNA expression in rat vascular cells in primary culture. Basal VEGF expression is greater in smooth muscle cells than in endothelial cells and fibroblasts. A 4-h treatment with forskolin (10(-5) M) induced a 2-fold stimulation of VEGF mRNA expression in smooth muscle cells and fibroblasts, but, in contrast, did not affect VEGF expression in endothelial cells. In smooth muscle cells, a pharmacologically induced increase in intracellular cAMP levels using iloprost or isoprenaline led to a rise in VEGF mRNA expression comparable to that induced by forskolin. Adenosine, which increases cAMP levels in smooth muscle cells, also increases VEGF expression. Moreover, the 2.2-fold stimulation of VEGF expression by adenosine was enhanced following a cotreatment with cobalt chloride (a hypoxia miming agent). The observed additive effect (4.3-fold increase) suggests that these two factors, hypoxia and adenosine, regulate VEGF mRNA expression in smooth muscle cells by independent mechanisms.