Hypoxic Activation Research Articles

Role of Circulating S -Nitrosothiols in Control of Blood Pressure

The biological effects of nitric oxide (NO) are in large part mediated by S -nitrosylation of peptides and proteins to produce bioactive S -nitrosothiols (SNOs).1–3 The observation of abnormal SNO levels in numerous pathophysiological states2 suggests that dysregulation of SNO homeostasis may contribute to disease pathogenesis. For example, the hypotension of human sepsis is accompanied by increases in circulating levels of vasodilatory SNOs.3 Although such altered SNO levels may simply mirror NO production (eg, induction of inducible NO synthase in sepsis), they may also reflect changes specific to SNO biosynthesis and metabolism. Indeed, mice lacking a SNO-metabolizing enzyme are profoundly hypotensive under anesthesia.3 Thus, blood pressure is evidently regulated by both synthesis and turnover of SNOs. In this issue of Hypertension , Gandley et al4 extend this paradigm by proposing that a defect in SNO turnover contributes to the hypertension of preeclampsia. In the blood, S- nitrosoalbumin (SNO-albumin) and S -nitrosohemoglobin (SNO-Hb) constitute the major conduits for circulating NO bioactivity. Although both SNOs may influence blood pressure, they operate within distinct signaling circuits. SNO-Hb can be viewed as a principal regulator of SNO homeostasis, adaptively modulating NO chemistry to control NO bioactivity. SNO-Hb is formed by transfer of NO from heme-iron to Cysβ93 thiol on T to R structural transition (oxygenation) of the hemoglobin tetramer.5 SNO-Hb associates with the red blood cell (RBC) membrane via an interaction with the cytoplasmic domain of anion-exchanger 1 protein (CDAE1, Band 3); on deoxygenation (R→T) transfer of the NO group from SNO-Hb to a cysteine thiol within CDAE1 supports export of RBC vasodilatory activity.6 SNO-Hb thus serves as an O2 sensor and O2-dependent transducer of NO bioactivity. In contrast, it appears that rather than transducing a specific signal, albumin operates as a buffer to …

Red Blood Cell S-Nitrosohemoglobin Deficiency in Pulmonary Arterial Hypertension.

Recent studies have suggested that binding of oxygen to hemoglobin (Hb) facilitates reactions of nitric oxide (NO) that lead to production of S-nitrosohemoglobin (SNO-Hb), and that vasodilator S-nitrosthiol (SNO) is dispensed by red blood cells (RBCs) at low oxygen tension (pO2) to dilate blood vessels. In human lungs, NO bioactivity serves to attenuate hypoxic pulmonary vasoconstriction (HPV). We therefore considered the possibility that RBC-SNO may oppose HPV and that defective vasodilation by RBCs may contribute to the pathophysiology of pulmonary arterial hypertension (PAH). Here we report that RBCs from patients with PAH exhibit substantial depletion of SNO-Hb and consequent impairment in hypoxia-mediated vasodilation. Furthermore, levels of RBC-NO correlated inversely with pulmonary artery pressures. A SNO-Hb deficiency characteristic of PAH was reproduced in control RBCs by hypoxia: loss of SNO-Hb was accompanied by a buildup of heme-NO species that are deficient in the pO2-governed intramolecular transfer of NO to cysteine thiol, yielding RBCs deficient in NO bioactivity. SNO-deficient RBCs produced exaggerated HPV responses as compared to SNO-replete RBCs. In PAH patients, SNO-Hb repletion fully restored the hypoxic vasodilator activity of RBCs. Our results suggest that a deficiency in RBC-SNO contributes to pulmonary hypertension and hypoxemia, and that repletion of RBC-SNO represents a rational strategy for treating PAH patients.

Hypoxia-inducible factor 1: regulation by hypoxic and non-hypoxic activators

Oxygen availability is crucial for cellular metabolism. Hypoxia-inducible factor 1 (HIF-1) is the major oxygen homeostasis regulator. Under normoxic conditions, HIF-1 is rapidly degraded by the proteasome. However, under hypoxic conditions, HIF-1 is stabilized and permits the activation of genes essential to cellular adaptation to low oxygen conditions. These genes include the vascular endothelial growth factor (VEGF), erythropoietin and glucose transporter-1. There is increasing evidence showing that HIF-1 is also implicated in biological functions requiring its activation under normoxic conditions. Amongst others, growth factors and vascular hormones are implicated in this normoxic activation. In this review, we will focus on differences between hypoxic and non-hypoxic induction and activation of HIF-1. We will also discuss the biological functions of HIF-1 associated with these two induction pathways. The clear understanding of both HIF-1 activation mechanisms could have a major impact in cancer and vascular disease.

The gene encoding human retinoic acid-receptor-related orphan receptor alpha is a target for hypoxia-inducible factor 1.

Retinoic acid-receptor-related orphan receptor (ROR) alpha is a nuclear receptor involved in many pathophysiological processes such as cerebellar ataxia, inflammation, atherosclerosis and angiogenesis. In the present study we first demonstrate that hypoxia increases the amount of Rora transcripts in a wide panel of cell lines derived from diverse tissues. In addition, we identified a functional promoter sequence upstream of the first exon of the human Rora gene, spanning -487 and -45 from the translation initiation site of RORalpha1. When cloned in a luciferase reporter vector, this sequence allowed the efficient transcription of the luciferase gene in several cell lines. Interestingly, the activity of the Rora promoter was enhanced by hypoxia in HepG2 human hepatoma cells, and this effect was dependent on an HRE (hypoxia response element) spanning from -229 to -225. Using electrophoretic-mobility-shift assays, we showed that HIF-1 (hypoxia-inducible factor 1), which plays a key role in the transcriptional response to hypoxia, bound to this HRE. Overexpression of HIF-1alpha increased the activity of the Rora promoter through the HRE. Overexpression of a dominant-negative form of HIF-1alpha producing transcriptionally inactive HIF-1alpha/HIF-1beta dimers abolished hypoxic activation of the Rora promoter. This indicated that HIF-1 is involved in the response of RORalpha to hypoxia. Taken together, our data reveal Rora as a new HIF-1 target gene. This illustrates, at the molecular level, the existence of cross-talk between signalling pathways mediated by HIF-1 and those mediated by nuclear receptors.

Neuropeptide PACAP inhibits hypoxic activation of brain microglia: a protective mechanism against microglial neurotoxicity in ischemia

Hypoxia is one of the important physiological stimuli that are often associated with a variety of pathological states such as ischemia, respiratory diseases, and tumorigenesis. In the central nervous system, hypoxia that is accompanied by cerebral ischemia not only causes neuronal cell injury, but may also induce pathological microglial activation. We have previously shown that hypoxia induces inflammatory activation of cultured microglia and their inducible nitric oxide synthase induction via p38 mitogen-activated protein kinase (MAPK) pathway, and a neuropeptide PACAP selectively inhibits microglial signal transduction. Based on these findings, we hypothesized that the neuropeptide may inhibit the hypoxic activation of microglia, and this may provide a neuroprotection against inflammation-induced neuronal injury. When this possibility was tested using cultured microglia and PC12 cells, we found that PACAP attenuates inflammatory activation of microglia under hypoxic condition, and protects cocultured PC12 cells from microglial neurotoxicity. In addition, the neuropeptide reduced the hypoxia-induced activation of p38 MAPK, indicating that the p38 MAPK is a molecular target of the PACAP action in microglia. The neuroprotective effects of PACAP in animal models of cerebral hypoxia/ischemia may be partly due to its direct actions on brain microglia and neurotoxic inflammation.

Hypoxia-induced Synthesis of Hemoglobin in the Crustacean Daphnia magna Is Hypoxia-inducible Factor-dependent

Of the four known globin genes that exist in the fresh-water crustacean Daphnia magna, several are individually induced by hypoxia, lending pale normoxic animals a visible red color when challenged by oxygen deprivation. The promoter regions of the Daphnia globin genes each contain numerous hypoxia response elements (HREs) as potential binding sites for hypoxia-inducible factors (HIFs). Daphnia HIF, bound to human HRE sequences, was detected in extracts from hypoxic (red), but not normoxic (pale), animals. Taking advantage of the phylogenetically conserved HIF/HRE recognition, we employed heterologous transfections of HIF-expressing human and Drosophila cells to model HIF signaling in Daphnia. These experiments revealed that three functional HREs within the promoter of the D. magna globin-2 gene cooperate for maximal hypoxic induction of a downstream luciferase reporter gene. Two of these three cis-elements, at promoter positions -258 and -107, are able to specifically bind human, Drosophila, or Daphnia HIF complexes in vitro. The same two sites are also necessary for maximal induction of reporter transcription under low oxygen tension in the presence of either endogenous human or overexpressed Drosophila HIF proteins. The third motif of the globin-2 gene promoter, a CACGTG palindrome at position -146, functions as a docking site for an unknown constitutive transcription factor. In human cells, this -146 complex interferes with HIF occupancy at the adjacent -107 HRE and thus controls the extent of HIF-mediated hypoxic activation of the downstream target.

Superoxide dismutase-3 promotes full expression of the EPO response to hypoxia

AbstractExtracellular superoxide dismutase (SOD3) is the primary extracellular enzymatic scavenger of superoxide (O2). SOD3’s expression is highest in the kidney, but its distribution and biologic functions there are unknown. To investigate the function of renal SOD3, we colocalized it with erythropoietin (EPO) to proximal tubules using in situ hybridization and immunohistochemistry. We then exposed wild-type (Wt) and SOD3 knock-out (KO) mice to hypoxia and found a late hematocrit response in the KO strain. EPO mRNA expression was attenuated in KO mice during the first 6 hours of hypoxia preceded at 2 hours by less accumulation of nuclear hypoxia-inducible transcription factor 1 α (HIF-1α) protein. Meanwhile KO mice exposed to hypoxia showed increases in renal mRNA for superoxide-producing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX4) and early significant increases in glutathione disulfide (GSSG)/glutathione (GSH), a marker of oxidative stress, compared with Wt mice. Plasma nitrite/nitrate and renal 3-nitrotyrosine (3-NTyr), indicating peroxynitrite formation, increased later in hypoxia, and renal endothelial nitric oxide synthase protein induction was similar in both strains. These data show that hypoxic activation of HIF-1α and its target gene EPO in mouse kidney is regulated closely by the oxidant/antioxidant equilibrium involving SOD3, thus identifying renal SOD3 as a regulatory element in the body’s innate adaptation to hypoxia.

Minocycline suppresses hypoxic activation of rodent microglia in culture

Hypoxia is one of the important physiological stimuli that are often associated with a variety of pathological states such as ischemia, respiratory diseases, and tumorigenesis. In the central nervous system, hypoxia that is accompanied by cerebral ischemia not only causes neuronal cell injury, but may also induce pathological microglial activation. We have previously shown that hypoxia induces inflammatory activation of cultured microglia, and the hypoxic induction of nitric oxide production in microglia is mediated through p38 mitogen-activated protein kinase pathway. Now, we present evidence that minocycline, a tetracycline derivative, suppresses the hypoxic activation of cultured microglia by inhibiting p38 mitogen-activated protein kinase pathway. The drug markedly inhibited hypoxia-induced production of inflammatory mediators such as nitric oxide, TNFα, and IL-1β as well as iNOS protein expression. The signal transduction pathway that leads to the activation of p38 mitogen-activated protein kinase was the molecular target of minocycline. Thus, the known neuroprotective effects of minocycline in animal models of cerebral ischemia may be partly due to its direct actions on brain microglia.

Association of DNA-dependent protein kinase with hypoxia inducible factor-1 and its implication in resistance to anticancer drugs in hypoxic tumor cells.

Tumor hypoxia contributes to the progression of a malignant phenotype and resistance to ionizing radiation and anticancer drug therapy. Many of these effects in hypoxic tumor cells are mediated by expression of specific set of genes whose relation to therapy resistance is poorly understood. In this study, we revealed that DNA-dependent protein kinase (DNA-PK), which plays a crucial role in DNA double strand break repair, would be involved in regulation of hypoxia inducible factor-1 (HIF-1). HIF-1beta-deficient cells showed constitutively reduced expression and DNA-binding activity of Ku, the regulatory subunit of DNA-PK. Under hypoxic condition, the expression and activity of DNA- PK were markedly induced with a concurrent increase in HIF-1alpha expression. Our result also demonstrated that DNA-PK could directly interact with HIF-1, and especially DNA-PKcs, the catalytic subunit of DNA-PK, could be involved in phosphorylation of HIF-1alpha, suggesting the possibility that the enhanced expression of DNA- PK under hypoxic condition might attribute to modulate HIF-1alpha stabilization. Thus, the correlated regulation of DNA-PK with HIF-1 could contribute to therapy resistance in hypoxic tumor cells, and it provides new evidence for developing therapeutic strategies enhancing the efficacy of cancer therapy in hypoxic tumor cells.

Hypoxic gene activation by lipopolysaccharide in macrophages: implication of hypoxia-inducible factor 1α

AbstractHypoxia-inducible factor 1 (HIF-1) regulates many genes induced by low oxygen conditions. The expression of important hypoxic genes such as glucose transporter 1 and vascular endothelial growth factor are increased in macrophages during wound healing and in the presence of the endotoxin, lipopolysaccharide (LPS). Recent studies have demonstrated that nonhypoxic stimuli can also activate HIF-1 in a cell-specific manner. Here, we demonstrate that in macrophages, LPS can control the activation of hypoxia-regulated genes through the HIF-1 pathway. We show that in these cells, protein expression levels of HIF-1α are strongly increased to levels comparable to hypoxic induction. HIF-1α mRNA levels are markedly increased following LPS stimulation, suggesting a transcriptional induction. In functional studies, the LPS-induced HIF-1 complex could specifically bind to the HIF-1 DNA-binding motif. Additionally, when cells were transfected with an HIF-1-specific reporter construct, LPS could strongly activate the expression of the reporter to levels that surpassed those observed after hypoxic induction. This induction was blocked by the cotransfection of a dominant-negative form of HIF-1α. These results indicate that the HIF-1 complex is involved in macrophage gene activation following LPS exposure and identify a novel pathway that could play a determinant role during inflammation and wound healing. (Blood. 2004;103:1124-1130)

Antiangiogenic activity of genistein in pancreatic carcinoma cells is mediated by the inhibition of hypoxia-inducible factor-1 and the down-regulation of VEGF gene expression.

Previous reports indicate that Genistein, a naturally occurring isoflavonoid, exhibits strong antiangiogenic activity. The underlying mechanism of inhibition, however, remains unclear. Among the biologic effects of Genistein are the inhibition of tyrosine kinases and the inhibition of hypoxic activation of hypoxia-inducible factor-1 (HIF-1), one of the main regulators of VEGF gene expression. Hypoxic cell culture was performed in a modular incubator chamber. Vascular endothelial growth factor (VEGF) protein secretion was measured using the enzyme-linked immunosorbent assay, binding of DNA by HIF-1 was measured using the electrophoretic mobility shift assay, and mRNA quantification was performed using Northern blot analysis. Pancreatic carcinoma was studied in an orthotopic murine model. Angiogenesis in vivo was quantified by staining xenograft tumors for endothelial cell markers. VEGF protein secretion was dose-dependently suppressed with increasing doses of Genistein. Furthermore, treatment of pancreatic carcinoma cells with Genistein led to impaired activation of HIF-1 under hypoxic culture conditions. Northern blot analysis indicated that VEGF mRNA expression decreased upon treatment with Genistein, both under normoxic and hypoxic culture conditions. In vivo, Genistein inhibited tumor growth for xenograft pancreatic carcinoma cells, whereas extensive hypoxia was observed in xenograft tumors and was not influenced by Genistein therapy. Similarly, decreased VEGF mRNA levels were observed in Genistein-treated Capan-1 xenograft tumors. The current study indicates that the previously reported antiangiogenic activity of Genistein probably is mediated by the inhibition of HIF-1, an important regulator of VEGF gene homeostasis, particularly under low-oxygen conditions. Therefore, this bioactive compound may well be beneficial to patients with pancreatic carcinoma.

Hypoxic activation of the atrial natriuretic peptide gene promoter through direct and indirect actions of hypoxia-inducible factor-1.

Atrial natriuretic peptide (ANP) is a cardiac peptide, the transcription of which is up-regulated in the ischaemic ventricle. However, the molecular mechanism of ANP induction is unclear. This study demonstrated that ANP mRNA expression in rat ventricular myocardium is induced in an early phase of ischaemia, preceded by hypoxia-inducible factor-1 (HIF-1) alpha expression. The ANP gene was also induced by hypoxia or HIF-1 inducers such as CoCl2 and desferrioxamine in H9c2 and neonatal cardiomyocytes. The 2307 bp 5'-flanking region of the rat ANP gene was cloned and fused to the luciferase gene. Evidence of the promoter activity was only apparent in the myocytes and was induced by hypoxia and HIF-1 inducers. The overexpression of HIF-1alpha markedly enhanced ANP promoter activity, and a dominant-negative isoform completely suppressed it. We demonstrated that the promoter regions are essential for hypoxic ANP induction. One promoter region, containing the HIF-1-binding sequence, is regulated directly by HIF-1. The other region is also activated by HIF-1 despite having no HIF-1-binding sequence. These results suggest that HIF-1 enhances the transactivation of the ANP gene in hypoxic myocytes, implying that stimulation of the ANP promoter by HIF-1 may in fact be responsible for the induction of the ANP gene in ischaemic ventricular myocardium.

Pleiotropic functions of erythropoietin.

Erythropoietin (EPO) produced by the fetal liver and adult kidney is an essential stimulator of erythropoiesis. EPO production is regulated through hypoxic activation of gene transcription and possibly hypoxia-induced stabilization of its mRNA. In the liver of early embryos in which EPO production poorly responds to hypoxia, retinoic acid may be an important stimulator. In this decade, new sites of EPO production have been found: central nervous system and reproductive organs. These tissues have a paracrine and/or autocrine system of EPO, which is independent of the endocrine system (kidney/bone marrow) in adult erythropoiesis. In the central nervous system, astrocytes are the main producers of EPO, while EPO receptor is expressed in neurons. EPO protects neurons from a various types of damage. The uterine EPO is likely involved in the estrogen-dependent angiogenesis of the endometrial layer. The possible functions of EPO in other tissues and tissue-characteristic regulation of EPO production are also discussed in this review.

Ischemic reperfusion injury to donor lung endothelium is mediated through gene promoter NF-KB and upstream IKK kinase

Ischemic repefusion injury sustained by a donor lung limits its post transplant function. Efforts at inhibiting reperfusion have met with limited success due to inability to inhibit the large number of overlapping inflammatory pathways activated. The inflammatory cascade needs to be inhibited at its origin. The purpose of this study was to define the intracellular signal pathways leading to inflammatory gene activation in endotlelial cells during hypoxia prior to reperfusion. Inhibition of key central promoters and kinases in these pathways would better inhibit downstream reperfusion amplification. Endothelial cells cultured in vitro on flow chambers were exposed to increasing time periods of hypoxia to replicate the ischemic state of the donor lung during transport. We demonstrated that hypoxic activation of inflammatory genes was mediated through a central rate limiting kinase, IκB kinase [IKK] in a rapid and transient manner. Once activated, IKK then phosphorylated IκBα-NFκB complex leading to degradation of IκBα and the release of promoter NFκB. NFκB then translocated into the nucleus and activated transcription of multiple inflammatory genes including, MCP-1, IL-1, and IL-6. In follow up experiments we successfully inhibited inflammatory gene activation using dominant negative mutants of IKK. Pharmaceutical agents are being developed to specifically inhibit IKK and NFκB. Clinical applications would be limitless and provide improved outcomes in diverse areas affected by reperfusion injury. See Figure 1 for the signal pathway proposed.

Hypoxia-inducible factor 1 regulates vascular endothelial growth factor expression in human pancreatic cancer.

The microenvironment of low oxygen that is present in human pancreatic cancer in vivo may actively influence tumor growth as well as neovascularization. To determine whether hypoxia-inducible factor 1 (HIF-1) is specifically activated by hypoxia in vitro in pancreatic cancer cells and correlated these findings with tumor specimens. Hypoxic regulation of vascular endothelial growth factor (VEGF) was studied by northern blot analysis and enzyme-linked immunosorbent assay. Electrophoretic mobility shift assays and western blot analysis were used to demonstrate hypoxic activation of HIF-1. The relationship between HIF-1 and VEGF in human pancreatic cancer specimens was studied by immunohistochemical analysis, northern blot analysis, and in situ hybridization. Studies in vivo of human pancreatic cancer tissue showed co-localization of VEGF mRNA, which is produced in ductal cancer cells, and HIF-1alpha protein, which was detectable in cell nuclei of the same cells. HIF-1alpha mRNA expression was dramatically upregulated (approximately 13-fold) in these specimens as well. In vitro, all pancreatic cancer cell lines increased VEGF production when exposed to low oxygen levels, by highly specific activation of HIF-1 DNA binding activity to the VEGF promoter. Cancer cell lines with high constitutive levels of HIF-1alpha protein were found to produce higher basal levels of VEGF. We conclude that HIF-1 is the regulatory link between tumor hypoxia and VEGF production in pancreatic cancer, thus establishing a biochemical pathway between tumor hypoxia and neoangiogenesis in this highly aggressive neoplasm.

Hypoxic Activation of Adventitial Fibroblasts : Role in Vascular Remodeling

Substantial experimental evidence supports the idea that the fibroblast may play a significant role in the vascular response to injury, especially under hypoxic conditions. Fibroblasts have the ability to rapidly respond to hypoxic stress and to modulate their function to adapt rapidly to local vascular needs. Fibroblasts appear to be uniquely equipped to proliferate, transdifferentiate, and migrate under hypoxic conditions. Proliferative responses to hypoxia depend on the activation of Galpha(i) and Gq kinase family members, and on the subsequent stimulation of protein kinase C and mitogen-activated protein kinase family members. Extracellular nucleotides (eg, adenosine triphosphate [ATP]) are likely to be increased in the hypoxic adventitial compartment and can act as autocrine/paracrine modifiers of the hypoxia-induced proliferative response. The proliferative effects of ATP appear to be mediated largely through G-protein-coupled P2Y receptors in fetal and neonatal fibroblasts. Hypoxia, acting through Galpha(iota)-coupled pathways, also can directly up-regulate alpha-smooth muscle actin expression in fibroblast subpopulations, suggesting that hypoxia may play a direct role in mediating the "transdifferentiation" of fibroblasts into myofibroblasts in the vessel wall. In addition, chronic hypoxia causes stable (at least in vitro) phenotypic changes in fibroblasts that appear to be associated with changes in the signaling pathways used to elicit proliferation. However, it is also becoming clear that, similar to the heterogeneity described for vascular smooth muscle cells, numerous fibroblast subtypes exist in the vessel wall, and that each may respond in unique ways to hypoxia and other stimuli and thus serve special functions in response to injury. In fact, adventitia may be considered to be compartments in which cells with "stem-cell-like" characteristics reside. Future work is needed to determine more precisely the role of the fibroblast in the wide variety of vascular complications observed in many humans diseases, and in the genes and gene products that confer unique properties to this important vascular cell.

Functional activation of heat shock factor and hypoxia-inducible factor in the kidney.

Renal ischemia is the result of a complex series of events, including decreases in oxygen supply (hypoxia) and the availability of cellular energy (ATP depletion). In this study, the functional activation of two stress-responsive transcription factors, i.e., heat shock factor-1 (HSF-1) and hypoxia-inducible factor-1 (HIF-1), in the kidney was assessed. When rats were subjected to 45 min of renal ischemia, electrophoretic mobility shift assays of kidney nuclear extracts revealed rapid activation of both HIF-1 and HSF. Western blot analyses further demonstrated that this activation resulted in increased expression of the HSF and HIF-1 target genes heat shock protein-72 and heme oxygenase-1, respectively. Whether hypoxia or ATP depletion alone could produce similar activation patterns in vitro was then investigated. Renal epithelial LLC-PK(1) cells were subjected to either ATP depletion (0.1 microM antimycin A and glucose deprivation) or hypoxia (1% O(2)). After ATP depletion, HSF was rapidly activated (within 30 min), whereas HIF-1 was unaffected. In contrast, hypoxia led to the activation of HIF-1 but not HSF. Hypoxic activation of HIF-1 was observed within 30 min and persisted for 4 h, whereas no HSF activation was detected even with prolonged periods of hypoxia. HIF-1 was transcriptionally active in LLC-PK(1) cells, as demonstrated by luciferase reporter gene assays using the vascular endothelial growth factor promoter or a synthetic promoter construct containing three hypoxia-inducible elements. Interestingly, intracellular ATP levels were not affected by hypoxia but were significantly reduced by ATP depletion. These findings suggest that HIF-1 is activated specifically by decreased O(2) concentrations and not by reduced ATP levels alone. In contrast, HSF is activated primarily by metabolic stresses associated with ATP depletion and not by isolated O(2) deprivation. In vivo, the two transcription factors are simultaneously activated during renal ischemia, which might account for observed differences between in vivo and in vitro epithelial cell injury and repair. Selective modulation of either pathway might therefore be of potential interest for modification of the response of the kidney to ischemia, as well as the processes involved in recovery from ischemia.

Hypoxia Up-regulates Expression of Peroxisome Proliferator-activated Receptor γ Angiopoietin-related Gene (PGAR) in Cardiomyocytes: Role of Hypoxia Inducible Factor 1α

A. J. Belanger, H. Lu, T. Date, L. X. Liu, K. A. Vincent, G. Y. Akita, S. H. Cheng, R. J. Gregory and C. Jiang. Hypoxia Up-regulates Expression of Peroxisome Proliferator-activated Receptor γ Angiopoietin-related Gene (PGAR) in Cardiomyocytes: Role of Hypoxia Inducible Factor 1α. Journal of Molecular and Cellular Cardiology (2002)34 , 765–774. Peroxisome proliferator-activated receptors (PPAR), especially the PPARα and PPARγ, are associated with an extraordinary diverse spectrum of cardiovascular diseases including hypertension, angiogenesis, cardiac hypertrophy, and atherosclerosis. PGAR (for PPARγ angiopoietin-related gene) is a recently identified PPAR target gene which is associated with adipose differentiation, systemic lipid metabolism, energy homeostasis, and possibly angiogenesis. We report here that WY-14643, a selective PPARα ligand up-regulated PGAR expression in neonatal rat cardiomyocytes. In parallel to activating the expression of vascular endothelial growth factor and glucose transporter-4, hypoxia increased PGAR mRNA levels. PGAR expression was also increased by desferrioxamine and CoCl2, but not by sodium cyanide, results consistent with the pharmacological features of hypoxia-responsive genes. These studies are the first to demonstrate that hypoxia increases the mRNA levels of a PPAR target gene in cardiomyocytes. Furthermore, infection with adenoviral vectors encoding the wild-type or a hybrid form of HIF-1α highly increased PGAR mRNA levels. In contrast, neither hypoxia nor overexpression of HIF-1α affected the mRNA levels of PPARα, PPARγ, and muscle carnitine palmitoyltransferase, a known PPARα target gene. These results suggest that hypoxic activation of PGAR expression is likely mediated by HIF-1 but not the PPARα/RXR pathway.

HIF-1α-prolyl hydroxylase: molecular target of nitric oxide in the hypoxic signal transduction pathway

We have investigated inhibitory mechanisms of hypoxic activation of HIF-1α by nitric oxide (NO). Using a Hep3B cell-derived cell line, HRE7 cells, we found that the inhibition of HIF-1α activity by NO requires a substantial amount of oxygen, albeit at a lower level. We further investigated the effect of NO on the binding activity of the von Hippel–Lindau tumor suppressor protein (pVHL) to the N-terminal activation domain (NAD) overlapping the oxygen-dependent degradation domain (ODD) of HIF-1α, because this reaction involves prolyl hydroxylation in NAD that requires oxygen. Although we could not detect any binding activity when NAD was incubated with whole cell extracts from cells treated with CoCl 2 or desferrioxamine, the binding capacity was manifested when Hep3B cells were treated together with NO. This activation was also observed when whole cell extracts from CoCl 2-treated cells were incubated with NO. The prolyl hydroxylase from Hep3B cells treated with CoCl 2 was partially purified about 80-fold, and several enzymatic properties were examined. The enzyme required ferrous ion and 2-oxoglutaric acid. Strong activation of the prolyl hydroxylase by NO was observed without further addition of ferrous ion.

Hypoxia activates a background conductance in freshly isolated pulmonary arterial endothelial cells of the rat

Utilising the patch-clamp recording technique we have demonstrated for the first time the effects of hypoxia on the background current in pulmonary arterial endothelial cells. Electrophysiological studies revealed the presence of a novel oxygen-sensitive, non-selective cation conductance ( I NSC) in these cells. The inward component of I NSC was significantly potentiated by hypoxia. Both the inward and outward components of I NSC were inhibited by both La 3+ and Gd 3+. Hypoxic activation of I NSC may provide an important Ca 2+ influx pathway essential for the release of a pulmonary-selective vasoconstrictor pivotal to the sustained phase of hypoxic pulmonary vasoconstriction.