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Anemia of chronic kidney disease (CKD) is a multifactorial disorder caused by impaired erythropoietin (EPO) production and altered iron homeostasis associated with inflammation. Hypoxia-inducible factor (HIF) is a transcription factor that stimulates erythropoiesis via a coordinated response involving increased EPO production and enhanced iron availability for Hb synthesis. HIF degradation is regulated by HIF-prolyl hydroxylase (HIF-PH) enzymes. We hypothesized that roxadustat, an orally available small-molecule inhibitor of HIF-PH, would increase EPO production and promote erythropoiesis in animal models of anemia. In cells, roxadustat increased both HIF-1α and HIF-2α proteins, leading to an increase in EPO production, even in the presence of EPO-suppressing inflammatory cytokines. Roxadustat administered intermittently to healthy rats and cynomolgus monkeys increased circulating EPO levels, reticulocytes, blood Hb, and hematocrit in a dose-dependent manner. Roxadustat corrected anemia in a rat model of CKD after five-sixth nephrectomy and in a rat model of anemia of inflammation with impaired iron metabolism induced by peptidoglycan-polysaccharide (PG-PS). In the PG-PS model, roxadustat significantly decreased hepatic expression of hepcidin, a hormone responsible for iron sequestration and functional iron deficiency, and increased expression of two genes involved in duodenal iron absorption: divalent metal transporter 1 and duodenal cytochrome b. In conclusion, by activating the HIF pathway, roxadustat increased EPO production, elevated Hb, corrected anemia, and improved iron homeostasis. The coordinated erythropoietic response stimulated by roxadustat, involving both EPO production and mobilization of iron stores, makes this compound a promising treatment of anemia of CKD and anemia associated with functional iron deficiency. SIGNIFICANCE STATEMENT: Roxadustat is a novel orally available small-molecule inhibitor of HIF prolyl hydroxylase enzymes that reversibly stabilizes HIF-α, thus activating transcription of HIF-dependent genes, including EPO and regulators of iron homeostasis. Activation of the HIF pathway by roxadustat induces erythropoiesis in healthy rats and monkeys and corrects experimentally induced anemia in rats. The coordinated erythropoietic response that increases EPO production and mobilizes iron stores makes roxadustat a promising treatment for anemia of chronic kidney disease and anemia associated with functional iron deficiency.

Abstract Non-alcoholic fatty liver disease (NAFLD) parallels the global obesity epidemic with unmet therapeutic needs. We investigated whether inhibition of hypoxia-inducible factor prolyl 4-hydroxylase-2 (HIF-P4H-2), a key cellular oxygen sensor whose inhibition stabilizes HIF, would protect from NAFLD by subjecting HIF-P4H-2-deficient (Hif-p4h-2gt/gt) mice to a high-fat, high-fructose (HFHF) or high-fat, methionine-choline-deficient (HF-MCD) diet. On both diets, the Hif-p4h-2gt/gt mice gained less weight and had less white adipose tissue (WAT) and its inflammation, lower serum cholesterol levels, and lighter livers with less steatosis and lower serum ALT levels than the wild type (WT). The intake of fructose in majority of the Hif-p4h-2gt/gt tissues, including the liver, was 15–35% less than in the WT. We found upregulation of the key fructose transporter and metabolizing enzyme mRNAs, Slc2a2, Khka, and Khkc, and higher ketohexokinase activity in the Hif-p4h-2gt/gt small intestine relative to the WT, suggesting enhanced metabolism of fructose in the former. On the HF-MCD diet, the Hif-p4h-2gt/gt mice showed more browning of the WAT and increased thermogenesis. A pharmacological pan-HIF-P4H inhibitor protected WT mice on both diets against obesity, metabolic dysfunction, and liver damage. These data suggest that HIF-P4H-2 inhibition could be studied as a novel, comprehensive treatment strategy for NAFLD. Key messages • HIF-P4H-2 inhibition enhances intestinal fructose metabolism protecting the liver. • HIF-P4H-2 inhibition downregulates hepatic lipogenesis. • Induced browning of WAT and increased thermogenesis can also mediate protection. • HIF-P4H-2 inhibition offers a novel, comprehensive treatment strategy for NAFLD.

Graphical abstract Image 1, Alcoholic fatty liver disease (AFLD) is a growing health problem for which no targeted therapy is available. We set out to study whether systemic inactivation of the main hypoxia-inducible factor prolyl 4-hydroxylase, HIF-P4H-2 (PHD2/EglN1), whose inactivation has been associated with protection against metabolic dysfunction, could ameliorate it. HIF-P4H-2-deficient and wild-type (WT) mice or HIF-P4H inhibitor-treated WT mice were subjected to an ethanol diet for 3–4 weeks and their metabolic health, liver and white adipose tissue (WAT) were analyzed. Primary hepatocytes from the mice were used to study cellular ethanol metabolism. The HIF-P4H-2-deficient mice retained a healthier metabolic profile, including less adiposity, better lipoprotein profile and restored insulin sensitivity, while on the ethanol diet than the WT. They also demonstrated protection from alcohol-induced steatosis and liver damage and had less WAT inflammation. In liver and WAT the expression of the key lipogenic and adipocytokine mRNAs, such as Fas and Ccl2, were downregulated, respectively. The upregulation of metabolic and antioxidant hypoxia-inducible factor (HIF) target genes, such as Slcs 16a1 and 16a3 and Gclc, respectively, and a higher catalytic activity of ALDH2 in the HIF-P4H-2-deficient hepatocytes improved handling of the toxic ethanol metabolites and oxidative stress. Pharmacological HIF-P4H inhibition in the WT mice phenocopied the protection against AFLD. Our data show that global genetic inactivation of HIF-P4H-2 and pharmacological HIF-P4H inhibition can protect mice from alcohol-induced steatosis and liver injury, suggesting that HIF-P4H inhibitors, now in clinical trials for renal anemia, could also be studied in randomized clinical trials for treatment of AFLD.

An endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm−/− mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm−/− mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497–treated Hif-p4h-2 hypomorphic (Hif-p4h-2gt/gt) and Hif-p4h-3−/− mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm−/− and wild-type mice, but caused higher increases in both values in the Hif-p4h-2gt/gt mice and in hematocrit value in the Hif-p4h-3−/− mice than in the wild-type. Hif-p4h-2gt/gt/P4h-tm−/− double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2gt/gt or P4h-tm−/− mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

Among endogenous adaptive systems to hypoxia, neuroglobin, a recently discovered heme protein, was suggested as a novel oxygen-dependent neuroprotectant. We aimed to characterize i) maturational age-related regulation of neuroglobin in the developing mouse brain under normoxic and hypoxic conditions, and ii) the role of hypoxia-inducible transcription factors (HIFs) as possible mediators of O(2)-dependent regulation of neuroglobin in vitro and in vivo. During early stages of postnatal brain maturation (P0-P14) neuroglobin mRNA levels significantly increased in developing mouse forebrains. By immunohistochemical analysis we confirmed expression of neuroglobin protein in the cytoplasm of developing neurons but not glial cells under normoxic conditions. Exposure of the immature brains (P0, P7) to acute (8% O(2), 6h) and chronic systemic hypoxia (10% O(2), 7 days) led to differential activation of neuroglobin varying with maturational stage (P0, P7) and severity of hypoxia. This observation may indicate that neuroglobin is involved in adaptive responses of immature neurons to acute hypoxia during an early stage of mouse brain maturation (P0). In response to activation of the HIF system by prolyl-4-hydroxylase inhibitor (FG-4497), neuroglobin mRNA expression was significantly up-regulated in primary mouse cortical neurons (DIV6) exposed to normoxia and hypoxia (1% O(2)) compared to non-treated controls. In conclusion, present results strongly indicate that cerebral regulation of neuroglobin is related to maturational stage and that hypoxia-induced neuroglobin up-regulation is modified by the HIF system.

Hypoxia-inducible transcription factors (HIFs) are crucially involved in brain development and cellular adaptation to hypoxia and ischemia. Degradation of HIF is regulated under normoxia by oxygen-dependent hydroxylation of specific prolyl residues on the labile alpha-subunit by HIF prolyl hydroxylases (PHD). Prolyl-4-hydroxylase inhibitors (PHI) have shown protective effects in vitro and in vivo in adult kidney and brain. The aim of the present study was to investigate in vivo short-term effects of a novel low molecular weight PHI, FG-4497, on HIF-regulated cytotrophic and vasoactive factors in developing mouse brain. Neonatal (P7, n=26) C57/BL6 mice were treated with PHI FG-4497 (30-100 mg/kg, i.p., duration 6 h). Gene expression was analyzed by TaqMan RT-PCR in kidney and developing brain in comparison to controls (NaCl 0.9% and non-treated animals). HIF-1alpha protein was quantified by Western blot analysis. Dose-response studies revealed prominent effects of FG-4497 at a dose of 100 mg/kg as assessed by significant up-regulation of mRNA in both kidney and brain of the following HIF-dependent genes: vascular endothelial growth factor, adrenomedullin and erythropoietin. Organ-specific transcriptional regulation was evident from analysis of hexokinase 2, inducible NO synthase and PHD3 mRNA concentrations. In the brain, HIF-1alpha and HIF-2alpha protein markedly accumulated in response to FG-4497. Besides vasoactive factors, PHI significantly increased cerebral chemokine receptor CXCR-4 mRNA levels. In conclusion, the novel PHI FG-4497 activates HIFs at an early stage of brain maturation and modulates neurotrophic processes known to be crucially involved in brain development and hypoxia-induced brain pathology.

Objective— Small-molecule hypoxia-inducible factor prolyl 4-hydroxylase (HIF-P4H) inhibitors are being explored in clinical studies for the treatment of anemia. HIF-P4H-2 (also known as PHD2 or EglN1) inhibition improves glucose and lipid metabolism and protects against obesity and metabolic dysfunction. We studied here whether HIF-P4H-2 inhibition could also protect against atherosclerosis. Approach and Results— Atherosclerosis development was studied in low-density lipoprotein (LDL) receptor–deficient mice treated with an oral HIF-P4H inhibitor, FG-4497, and in HIF-P4H-2-hypomorphic/C699Y-LDL receptor–mutant mice, all mice being fed a high-fat diet. FG-4497 administration to LDL receptor–deficient mice reduced the area of atherosclerotic plaques by ≈50% when compared with vehicle-treated controls and also reduced their weight gain, insulin resistance, liver and white adipose tissue (WAT) weights, adipocyte size, number of inflammation-associated WAT macrophage aggregates and the high-fat diet–induced increases in serum cholesterol levels. The levels of atherosclerosis-protecting circulating autoantibodies against copper-oxidized LDL were increased. The decrease in atherosclerotic plaque areas correlated with the reductions in weight, serum cholesterol levels, and WAT macrophage aggregates and the autoantibody increase. FG-4497 treatment stabilized HIF-1α and HIF-2α and altered the expression of glucose and lipid metabolism and inflammation-associated genes in liver and WAT. The HIF-P4H-2-hypomorphic/C699Y-LDL receptor–mutant mice likewise had a ≈50% reduction in atherosclerotic plaque areas, reduced WAT macrophage aggregate numbers, and increased autoantibodies against oxidized LDL, but did not have reduced serum cholesterol levels. Conclusions— HIF-P4H-2 inhibition may be a novel strategy for protecting against the development of atherosclerosis. The mechanisms involve beneficial modulation of the serum lipid profile and innate immune system and reduced inflammation.

Obesity is a major public health problem, predisposing subjects to metabolic syndrome, type 2 diabetes, and cardiovascular diseases. Specific prolyl 4-hydroxylases (P4Hs) regulate the stability of the hypoxia-inducible factor (HIF), a potent governor of metabolism, with isoenzyme 2 being the main regulator. We investigated whether HIF-P4H-2 inhibition could be used to treat obesity and its consequences. Hif-p4h-2–deficient mice, whether fed normal chow or a high-fat diet, had less adipose tissue, smaller adipocytes, and less adipose tissue inflammation than their littermates. They also had improved glucose tolerance and insulin sensitivity. Furthermore, the mRNA levels of the HIF-1 targets glucose transporters, glycolytic enzymes, and pyruvate dehydrogenase kinase-1 were increased in their tissues, whereas acetyl-CoA concentration was decreased. The hepatic mRNA level of the HIF-2 target insulin receptor substrate-2 was higher, whereas that of two key enzymes of fatty acid synthesis was lower. Serum cholesterol levels and de novo lipid synthesis were decreased, and the mice were protected against hepatic steatosis. Oral administration of an HIF-P4H inhibitor, FG-4497, to wild-type mice with metabolic dysfunction phenocopied these beneficial effects. HIF-P4H-2 inhibition may be a novel therapy that not only protects against the development of obesity and its consequences but also reverses these conditions.

Ischemic stroke results in disruption of the blood-brain barrier (BBB), edema formation and neuronal cell loss. Some neuroprotective factors such as vascular endothelial growth factor (VEGF) favor edema formation, while others such as erythropoietin (Epo) can mitigate it. Both factors are controlled by hypoxia inducible transcription factors (HIF) and the activity of prolyl hydroxylase domain proteins (PHD). We hypothesize that activation of the adaptive hypoxic response by inhibition of PHD results in neuroprotection and prevention of vascular leakage. Mice, subjected to cerebral ischemia, were pre- or post-treated with the novel PHD inhibitor FG-4497. Inhibition of PHD activity resulted in HIF-1α stabilization, increased expression of VEGF and Epo, improved outcome from ischemic stroke and reduced edema formation by maintaining BBB integrity. Additional in vitro studies using brain endothelial cells and primary astrocytes confirmed that FG-4497 induces the HIF signaling pathway, leading to increased VEGF and Epo expression. In an in vitro ischemia model, using combined oxygen and glucose deprivation, FG-4497 promoted the survival of neurons. Furthermore, FG-4497 prevented the ischemia-induced rearrangement and gap formation of the tight junction proteins zonula occludens 1 and occludin, both in cultured endothelial cells and in infarcted brain tissue in vivo. These results indicate that FG-4497 has the potential to prevent cerebral ischemic damage by neuroprotection and prevention of vascular leakage.

Activin A is a multifunctional growth and differentiation factor with pronounced neuroprotective properties that is strongly up-regulated in various forms of acute brain disorders and injuries including epilepsy, stroke and trauma. In a pediatric context, activin A has been advanced as a potential marker for the severity of perinatal hypoxic-ischemic brain injury. Here we investigated the regulation of activin A under global hypoxia without ischemia in primary cultures of cortical neurons and in neonatal and adult mice of two strains (C57BL/6 and CD-1). From birth to adulthood, activin βA subunit, activin receptors, and functional activin antagonists were all expressed at roughly similar mRNA levels in the brain of C57BL/6 mice. Independent of mouse line and age, we found both moderate (11% O2, 2h) and severe hypoxia (8%, 6h) to be consistently associated with normal or even reduced levels of activin βA (Inhba) mRNA. The surprising unresponsiveness of Inhba expression to hypoxia was confirmed at the protein level. In situ hybridization did not indicate regional, hypoxia-related differences in Inhba expression. Pharmacologic stabilization of hypoxia inducible factors with the prolyl hydroxylase inhibitor FG-4497 did not influence Inhba mRNA levels in neonatal mice. Our data indicate that pure hypoxia differs from other, more complex types of brain damage in that it appears not to recruit activin A as an endogenous neuroprotective agent.

Ischemic heart disease (IHD) is the leading cause of death worldwide. Novel cardioprotective strategies are therefore required to improve clinical outcomes in patients with IHD. Although a large number of novel cardioprotective strategies have been discovered in the research laboratory, their translation to the clinical setting has been largely disappointing. The reason for this failure can be attributed to a number of factors including the inadequacy of the animal ischemia-reperfusion injury models used in the preclinical cardioprotection studies and the inappropriate design and execution of the clinical cardioprotection studies. This important issue was the main topic of discussion of the UCL-Hatter Cardiovascular Institute 6th International Cardioprotection Workshop, the outcome of which has been published in this article as the "Hatter Workshop Recommendations". These have been proposed to provide guidance on the design and execution of both preclinical and clinical cardioprotection studies in order to facilitate the translation of future novel cardioprotective strategies for patient benefit. © 2010 The Author(s).

We have previously shown that high-dose erythropoietin (EPO) treatment improves hippocampal plasticity and cognitive performance in rodents and in patients suffering from neuropsychiatric diseases. It was therefore of interest to explore whether upregulation of endogenous EPO in brain by hypoxia inducible factor (HIF) stabilization would increase hippocampal memory similar to exogenous EPO. HIFs are transcription factors involved in the cellular response to low oxygen, including upregulation of transcripts like vascular endothelial growth factor (VEGF) and EPO. Under normal oxygen, prolylhydroxylases decrease HIF-alpha stability. This is banned by prolylhydroxylase inhibitors, which prevent oxygen dependent degradation and thus prolong HIF-alpha half life. In an experimental set-up identical to the one yielding strong cognitive effects with EPO, healthy male 28-day-old mice received FG-4497, a HIF prolylhydroxylase inhibitor, or placebo intraperitoneally every other day for 3 weeks. Behavioral testing and hematocrit determinations were conducted in independent cohorts at 1, 3, or 4 weeks after treatment completion. Increased EPO and VEGF mRNA expression in hippocampus or primary hippocampal neurons 6h after the application of FG-4497 confirmed its ability to stabilize HIF and upregulate HIF dependent transcription in brain. At 3 and 4 weeks after the last injection, respectively, FG-4497 treated mice compared to placebo mice had improved hippocampal memory in fear conditioning without change in hematocrit. In contrast, no improvement in memory was detected at 1 week, when the hematocrit was increased, indicating that cognitive improvement and hematocrit are not directly related. FG-4497 application for 3 weeks leads to delayed but lasting enhancement of hippocampal memory, making HIF stabilization an attractive target for pharmacological manipulation of cognition.