Degradation Of Hypoxia-inducible Factor Research Articles

Deubiquitinating enzyme mutagenesis screens identify a USP43-dependent HIF-1 transcriptional response.

The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1α isoform, and while USP43 does not alter HIF-1α stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia and phosphorylation dependent manner to increase the nuclear pool of HIF-1. Together, our results highlight the multifunctionality of DUBs, illustrating that they can provide important signalling functions alongside their catalytic roles.

Cancer-associated fibroblast-secreted FGF7 as an ovarian cancer progression promoter.

Ovarian cancer (OC) is distinguished by its aggressive nature and the limited efficacy of current treatment strategies. Recent studies have emphasized the significant role of cancer-associated fibroblasts (CAFs) in OC development and progression. Employing sophisticated machine learning techniques on bulk transcriptomic datasets, we identified fibroblast growth factor 7 (FGF7), derived from CAFs, as a potential oncogenic factor. We investigated the relationship between FGF7 expression and various clinical parameters. A series of in vitro experiments were undertaken to evaluate the effect of CAFs-derived FGF7 on OC cell activities, such as proliferation, migration, and invasion. Single-cell transcriptomic analysis was also conducted to elucidate the interaction between FGF7 and its receptor. Detailed mechanistic investigations sought to clarify the pathways through which FGF7 fosters OC progression. Our findings indicate that higher FGF7 levels correlate with advanced tumor stages, increased vascular invasion, and poorer prognosis. CAFs-derived FGF7 significantly enhanced OC cell proliferation, migration, and invasion. Single-cell analysis and in vitro studies revealed that CAFs-derived FGF7 inhibits the ubiquitination and degradation of hypoxia-inducible factor 1 alpha (HIF-1α) via FGFR2 interaction. Activation of the FGF7/HIF-1α pathway resulted in the upregulation of mesenchymal markers and downregulation of epithelial markers. Importantly, in vivo treatment with neutralizing antibodies targeting CAFs-derived FGF7 substantially reduced tumor growth. Neutralizing FGF7 in the medium or inhibiting HIF-1α signaling reversed the effects of FGF7-mediated EMT, emphasizing the dependence of FGF7-mediated EMT on HIF-1α activation. These findings suggest that targeting the FGF7/HIF-1α/EMT axis may offer new therapeutic opportunities to intervene in OC progression.

USP13 promotes deubiquitination of ZHX2 and tumorigenesis in kidney cancer

Clear cell renal cell carcinoma (ccRCC) is characterized by the loss of tumor suppressor Von Hippel Lindau (VHL) function. VHL is the component of an E3 ligase complex that promotes the ubiquitination and degradation of hypoxia inducible factor α (HIF-α) (including HIF1α and HIF2α) and Zinc Fingers And Homeoboxes 2 (ZHX2). Our recent research showed that ZHX2 contributed to ccRCC tumorigenesis in a HIF-independent manner. However, it is still unknown whether ZHX2 could be modified through deubiquitination even in the absence of pVHL. Here, we performed a deubiquitinase (DUB) complementary DNA (cDNA) library binding screen and identified USP13 as a DUB that bound ZHX2 and promoted ZHX2 deubiquitination. As a result, USP13 promoted ZHX2 protein stability in an enzymatically dependent manner, and depletion of USP13 led to ZHX2 down-regulation in ccRCC. Functionally, USP13 depletion led to decreased cell proliferation measured by two-dimensional (2D) colony formation and three-dimensional (3D) anchorage-independent growth. Furthermore, USP13 was essential for ccRCC tumor growth invivo, and the effect was partially mediated by its regulation on ZHX2. Our findings support that USP13 may be a key effector in ccRCC tumorigenesis.

Modeling the role of HIF in the regulation of metabolic key genes LDH and PDH: Emergence of Warburg phenotype

AbstractOxygenation of tumors and the effect of hypoxia on cancer cell metabolism is a widely studied subject. Hypoxia‐inducible factor (HIF), the main actor in the cell response to hypoxia, represents a potential target in cancer therapy. HIF is involved in many biological processes such as cell proliferation, survival, apoptosis, angiogenesis, iron metabolism, and glucose metabolism. This protein regulates the expressions of lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH), both essential for the conversion of pyruvate to be used in aerobic and anaerobic pathways. HIF upregulates LDH, increasing the conversion of pyruvate into lactate which leads to higher secretion of lactic acid by the cell and reduced pH in the microenvironment. HIF indirectly downregulates PDH, decreasing the conversion of pyruvate into acetyl coenzyme A, which leads to reduced usage of the tricarboxylic acid (TCA) cycle in aerobic pathways. Upregulation of HIF may promote the use of anaerobic pathways for energy production even in normal extracellular oxygen conditions. Higher use of glycolysis even in normal oxygen conditions is called the Warburg effect. In this paper, we focus on HIF variations during tumor growth and study, through a mathematical model, its impact on the two metabolic key genes PDH and LDH, to investigate its role in the emergence of the Warburg effect. Mathematical equations describing the enzyme regulation pathways were solved for each cell of the tumor represented in an agent‐based model to best capture the spatio‐temporal oxygen variations during tumor development caused by cell consumption and reduced diffusion inside the tumor. Simulation results show that reduced HIF degradation in normoxia can induce higher lactic acid production. The emergence of the Warburg effect appears after the first period of hypoxia before oxygen conditions return to a normal level. The results also show that targeting the upregulation of LDH and the downregulation of PDH could be relevant in therapy.

Cancer associated fibroblast–derived CCL5 promotes hepatocellular carcinoma metastasis through activating HIF1α/ZEB1 axis

Cancer-associated fibroblasts (CAFs) are one of the most enriched components of Hepatocellular carcinoma (HCC) microenvironment, which are tightly related to the metastasis and invasion of HCC. We identified a mechanism by which CAF-derived chemokine CCL5 enhanced HCC metastasis by triggering the HIF1α/ZEB1 axis. We demonstrated that CAFs derived from HCC tissues promoted the migration and invasion of HCC cells and facilitated metastasis to the lung of NOD/SCID mice. Then the chemokine antibody array elucidated the higher chemokine CCL5 level secreted by CAFs than by paracancerous tissue fibroblasts (PTFs). Mechanistically, we found that CAF-derived CCL5 inhibited the ubiquitination and degradation of hypoxia-inducible factor 1 alpha (HIF1α) by binding to specific receptors, maintained HIF1α under normoxia, thereby up-regulated the downstream gene zinc finger enhancer-binding protein 1 (ZEB1) and induced epithelial-mesenchymal transition (EMT), ultimately validating its ability to promote lung metastasis of HCC. And this novel mechanism may have association with poor prognosis. Taken together, targeting CAF-derived CCL5 mediated HIF1α/ZEB1 cascade possibly propose a new therapeutic route for HCC.

Butyrate Analogues Mimicking Hypoxia by the Chemical Stabilization of Hypoxia Inducible Factor (HIF)

Short chain fatty acids and specifically butyrate (BA) have been well documented to show multiple beneficial influences on health. Typically, the concentration of colonic butyrate can range from 10‐20 mM and 95‐99% is metabolized into energy by the mucosa. BA plays a key role in epithelial barrier regulation, ameliorates inflammation and regulates cell growth and differentiation. There are multiple mechanisms on how it plays such an important role in gut health, many related to its regulatory capacity for gene expression. Our laboratory recently reported a direct influence of BA on the stabilization of the transcription factor hypoxia‐inducible factor (HIF). BA acts directly and non‐competitively inhibits HIF prolyl hydroxylase 2 (PHD2), responsible for the rapid degradation of HIF in normoxia (NX). However, the efficacy of BA on HIF stabilization is limited due to its rapid metabolism as fuel. Based on these observations, we hypothesize that analogues structurally related to butyrate stabilize HIF with a longer biological half‐life. A small library of BA derivatives was screened in search of a non‐endogenous analogue with higher potency and/or half‐life for HIF stabilization. In vitro screenings at a physiologically‐relevant concentration (5 mM) were performed in intestinal epithelial cell lines T84 and CaCo2 in NX and HIF‐1a ELISA was used to determine protein abundance. Several hits were observed, with 4‐mercapto‐butyrate (SHBA) being the most promising candidate. The best BA analogues were validated through the induction of classic HIF gene targets, including BNIP3 and CaIX by q‐PCR. SHBA exhibited higher induction of these targets compared to BA. Dose response and time‐course studies were performed to compare SHBA and BA. Notably, SHBA exhibits a much longer half‐life as observed in the stabilization of HIF and induction of gene targets at least up to 72 h compared to BA (24 h). In vivo studies in C57BL/6 mice explored the induction of classic HIF targets and specifically erythropoietin (EPO) in circulation following administration of BA and SHBA at similar doses. These studies revealed that induction of EPO with a single dose of SHBA exceeded that of native BA. Thus, a non‐endogenous BA derivative (SHBA) stabilizes HIF at a higher potency and longer half‐life than BA and will be useful as a template for the development of a HIF stabilizing agent.

Zafirlukast Induces VHL- and HIF-2α-Dependent Oxidative Cell Death in 786-O Clear Cell Renal Carcinoma Cells.

Mutations in the Von Hippel–Lindau (VHL) gene are the driving force in many forms of clear cell renal cell carcinoma (ccRCC) and promote hypoxia-inducible factor (HIF)-dependent tumor proliferation, metastasis and angiogenesis. Despite the progress that has already been made, ccRCC generally remain resistant to conventional therapies and ccRCC patients suffer from metastasis and acquired resistance, highlighting the need for novel therapeutic options. Cysteinyl leukotriene receptor 1 (CysLTR1) antagonists, like zafirlukast, are administered in bronchial asthma to control eicosanoid signaling. Intriguingly, long-term use of zafirlukast decreases cancer risk and leukotriene receptor antagonists inhibit tumor growth, but the mechanisms still remain unexplored. Therefore, we aim to understand the mechanisms of zafirlukast-mediated cell death in ccRCC cells. We show that zafirlukast induces VHL-dependent and TNFα-independent non-apoptotic and non-necroptotic cell death in ccRCC cells. Cell death triggered by zafirlukast could be rescued with antioxidants and the PARP-1 inhibitor Olaparib, and additionally relies on HIF-2α. Finally, MG-132-mediated proteasome inhibition sensitized VHL wild-type cells to zafirlukast-induced cell death and inhibition of HIF-2α rescued zafirlukast- and MG-132-triggered cell death. Together, these results highlight the importance of VHL, HIF and proteasomal degradation in zafirlukast-induced oxidative cell death with potentially novel therapeutic implications for ccRCC.

Elongin C (ELOC/TCEB1)-associated von Hippel-Lindau disease.

Around 95% of patients with clinical features that meet the diagnostic criteria for von Hippel–Lindau disease (VHL) have a detectable inactivating germline variant in VHL. The VHL protein (pVHL) functions as part of the E3 ubiquitin ligase complex comprising pVHL, elongin C, elongin B, cullin 2 and ring box 1 (VCB-CR complex), which plays a key role in oxygen sensing and degradation of hypoxia-inducible factors. To date, only variants in VHL have been shown to cause VHL disease. We undertook trio analysis by whole-exome sequencing in a proband with VHL disease but without a detectable VHL mutation. Molecular studies were also performed on paired DNA extracted from the proband’s kidney tumour and blood and bioinformatics analysis of sporadic renal cell carcinoma (RCC) dataset was undertaken. A de novo pathogenic variant in ELOC NM_005648.4(ELOC):c.236A>G (p.Tyr79Cys) gene was identified in the proband. ELOC encodes elongin C, a key component [C] of the VCB-CR complex. The p.Tyr79Cys substitution is a mutational hotspot in sporadic VHL-competent RCC and has previously been shown to mimic the effects of pVHL deficiency on hypoxic signalling. Analysis of an RCC from the proband showed similar findings to that in somatically ELOC-mutated RCC (expression of hypoxia-responsive proteins, no somatic VHL variants and chromosome 8 loss). These findings are consistent with pathogenic ELOC variants being a novel cause for VHL disease and suggest that genetic testing for ELOC variants should be performed in individuals with suspected VHL disease with no detectable VHL variant.

РОЛЬ ГИПОКСИЯ-ИНДУЦИБЕЛЬНОГО ФАКТОРА В НОРМЕ И ПРИ ПАТОЛОГИИ

Hypoxia inducible factors (HIFs) are important intracellular regulators of transcription. In normoxia, these proteins undergo posttranslational modifications (hydroxylation) that prevent interaction with coactivators (CBP/p300) and pro- mote HIF degradation. The two main systems that hydroxylate HIF are prolylhydroxylases and factor-inhibiting HIF-1 (Factor-inhibiting HIF-1, FIH-1). Under hypoxia, these inhibitory mechanisms stop working. This leads to dimerization of HIF-1α, HIF-2α, and HIF-3α with constitutively synthesized oxygen-independent HIF-1β. The main vector of HIF operation is adaptation to hypoxia. To perform this task, oxygen capacity of the blood (HIF stimulates the synthesis of erythropoietin and its receptors, iron absorption and transport), angiogenesis, anaerobic ATP production, compensa- tion of intracellular acidosis, etc.). Hyperfunction of HIF underlies carcinogenesis. Tumor cells are characterized by enhanced angiogenesis, proliferation, evasion of apoptosis, immune surveillance, and atypical metabolism (e.g., the Warburg effect). These processes are under the control of HIF. Herminative mutations of HIF genes or other genes involved in the regulation of this transcription factor lead to von Hippel-Lindau disease, congenital erythrocytosis, and other pathologies. Suppression of HIF is being studied as a promising anti-tumor strategy. Hypofunction of HIF contrib- utes to the pathogenesis of anemia in chronic kidney disease and vascular rarity in pathological myocardial hypertro- phy. Stimulation of HIF, by inhibition of prolyl hydroxylases, has been investigated in phase 3 trials and is used to treat anemia in chronic kidney disease. This therapy appears to be comparable in safety profile to the drug erythropoietins. The risk of thrombosis is slightly higher with prolylhydroxylase inhibitors.

HIF2 Regulates Intestinal Wnt5a Expression.

Radiation therapy for abdominal tumors is challenging because the small intestine is exquisitely radiosensitive. Unfortunately, there are no FDA-approved therapies to prevent or mitigate GI radiotoxicity. The EGLN protein family are oxygen sensors that regulate cell survival and metabolism through the degradation of hypoxia-inducible factors (HIFs). Our group has previously shown that stabilization of HIF2 through genetic deletion or pharmacologic inhibition of the EGLNs mitigates and protects against GI radiotoxicity in mice by improving intestinal crypt stem cell survival. Here we aimed to elucidate the molecular mechanisms by which HIF2 confers GI radioprotection. We developed duodenal organoids from mice, transiently overexpressed non-degradable HIF2, and performed bulk RNA sequencing. Interestingly, HIF2 upregulated known radiation modulators and genes involved in GI homeostasis, including Wnt5a. Non-canonical Wnt5a signaling has been shown by other groups to improve intestinal crypt regeneration in response to injury. Here we show that HIF2 drives Wnt5a expression in multiple duodenal organoid models. Luciferase reporter assays performed in human cells showed that HIF2 directly activates the WNT5A promoter via a hypoxia response element. We then evaluated crypt regeneration using spheroid formation assays. Duodenal organoids that were pre-treated with recombinant Wnt5a had a higher cryptogenic capacity after irradiation, compared to vehicle-treated organoids. Conversely, we found that Wnt5a knockout decreased the cryptogenic potential of intestinal stem cells following irradiation. Treatment with recombinant Wnt5a prior to irradiation rescued the cryptogenic capacity of Wnt5a knockout organoids, indicating that Wnt5a is necessary and sufficient for duodenal radioprotection. Taken together, our results suggest that HIF2 radioprotects the GI tract by inducing Wnt5a expression.

Selective HIF stabilization alleviates hepatocellular steatosis and ballooning in a rodent model of 70% liver resection.

Small-for-size syndrome (SFSS) looms over patients needing liver resection or living-donor transplantation. Hypoxia has been shown to be crucial for the successful outcome of liver resection in the very early postoperative phase. While poorly acceptable as such in real-world clinical practice, hypoxia responses can still be simulated by pharmacologically raising levels of its transducers, the hypoxia-inducible factors (HIFs). We aimed to assess the potential role of a selective inhibitor of HIF degradation in 70% hepatectomy (70%Hx). In a pilot study, we tested the required dose of roxadustat to stabilize liver HIF1α. We then performed 70%Hx in 8-week-old male Lewis rats and administered 25 mg/kg of roxadustat (RXD25) at the end of the procedure. Regeneration was assessed: ki67 and 5-ethynyl-2'-deoxyuridine (EdU) immunofluorescent labeling, and histological parameters. We also assessed liver function via a blood panel and functional gadoxetate-enhanced magnetic resonance imaging (MRI), up to 47 h after the procedure. Metabolic results were analyzed by means of RNA sequencing (RNAseq). Roxadustat effectively increased early HIF1α transactivity. Liver function did not appear to be improved nor liver regeneration to be accelerated by the experimental compound. However, treated livers showed a mitigation in hepatocellular steatosis and ballooning, known markers of cellular stress after liver resection. RNAseq confirmed that roxadustat unexpectedly increases lipid breakdown and cellular respiration. Selective HIF stabilization did not result in an enhanced liver function after standard liver resection, but it induced interesting metabolic changes that are worth studying for their possible role in extended liver resections and fatty liver diseases.

Prolyl Hydroxylase 3 Knockdown Accelerates VHL-Mutant Kidney Cancer Growth In Vivo.

Von Hippel Lindau (VHL) inactivation, which is common in clear cell renal cell carcinoma (ccRCC), leads directly to the disruption of oxygen homoeostasis. VHL works through hypoxia-inducible factors (HIFs). Within this VHL-HIF system, prolyl hydroxylases (PHDs) are the intermediary proteins that initiate the degradation of HIFs. PHD isoform 3′s (PHD3) role in ccRCC growth in vivo is poorly understood. Using viral transduction, we knocked down the expression of PHD3 in the human ccRCC cell line UMRC3. Compared with control cells transduced with scrambled vector (UMRC3-SC cells), PHD3-knockdown cells (UMRC3-PHD3KD cells) showed increased cell invasion, tumor growth, and response to sunitinib. PHD3 knockdown reduced HIF2α expression and increased phosphorylated epidermal growth factor (EGFR) expression in untreated tumor models. However, following sunitinib treatment, expression of HIF2α and phosphorylated EGFR were equivalent in both PHD3 knockdown and control tumors. PHD3 knockdown changed the overall redox state of the cell as seen by the increased concentration of glutathione in PHD3 knockdown tumors relative to control tumors. UMRC3-PHD3KD cells had increased proliferation in cell culture when grown in the presence of hydrogen peroxide compared to UMRC3-SC control cells. Our findings illustrate (1) the variable effect of PHD3 on HIF2α expression, (2) an inverse relationship between PHD3 expression and tumor growth in ccRCC animal models, and (3) the role of PHD3 in maintaining the redox state of UMRC3 cells and their proliferative rate under oxidative stress.

Development of BODIPY FL VH032 as a High-Affinity and Selective von Hippel-Lindau E3 Ligase Fluorescent Probe and Its Application in a Time-Resolved Fluorescence Resonance Energy-Transfer Assay.

The von Hippel–Lindau (VHL) tumor suppressor associates with transcription factors elongin-C and elongin-B to form the VHL–elongin-C–elongin-B protein complex and carry out its functions, such as degradation of hypoxia-inducible factors. VHL ligands are used not only to modulate hypoxia-signaling pathways and potentially treat chronic anemia or ischemia but also to form bivalent ligands as proteolysis-targeting chimeras to degrade proteins for potential therapeutic applications. Sensitive and selective VHL-based binding assays are critical for identifying and characterizing VHL ligands with high-throughput screening approaches. VHL ligand-binding assays, such as isothermal titration calorimetry, surface plasmon resonance, and fluorescence polarization assays, are reported but with limitations. Isothermal titration calorimetry requires higher protein concentrations with a lower throughput than fluorescence-based assays do. Surface plasmon resonance requires protein immobilization, which introduces variation and is not suitable for testing a large number of ligands. Fluorescence polarization can be sensitive with high-throughput capability but is susceptible to assay interference, and small-molecule-based fluorescent probes are not available. We developed the first small-molecule-based high-affinity VHL fluorescent probe BODIPY FL VH032 (5), with a Kd of 3.01 nM, for a time-resolved fluorescence resonance energy-transfer assay. This new assay is sensitive, selective, resistant to assay interference, and capable of characterizing VHL ligands with a wide range of affinities. It is also suitable for VHL ligand identification and characterization with high-throughput screening.

Hypoxia-inducible factors not only regulate but also are myeloid-cell treatment targets.

Hypoxia describes limited oxygen availability at the cellular level. Myeloid cells are exposed to hypoxia at various bodily sites and even contribute to hypoxia by consuming large amounts of oxygen during respiratory burst. Hypoxia-inducible factors (HIFs) are ubiquitously expressed heterodimeric transcription factors, composed of an oxygen-dependent α and a constitutive β subunit. The stability of HIF-1α and HIF-2α is regulated by oxygen-sensing prolyl-hydroxylases (PHD). HIF-1αand HIF-2α modify the innate immune response and are context dependent. We provide a historic perspective of HIF discovery, discuss the molecular components of the HIF pathway, and how HIF-dependent mechanisms modify myeloid cell functions. HIFs enable myeloid-cell adaptation to hypoxia by up-regulating anaerobic glycolysis. In addition to effects on metabolism, HIFs control chemotaxis, phagocytosis, degranulation, oxidative burst, and apoptosis. HIF-1α enables efficient infection defense by myeloid cells. HIF-2αdelays inflammation resolution and decreases antitumor effects by promoting tumor-associated myeloid-cell hibernation. PHDs not only control HIF degradation, but also regulate the crosstalk between innate and adaptive immune cells thereby suppressing autoimmunity. HIF-modifying pharmacologic compounds are entering clinical practice. Current indications include renal anemia and certain cancers. Beneficial and adverse effects on myeloid cells should be considered and could possibly lead to drug repurposing for inflammatory disorders.

Interaction with p53 explains a pro-proliferative function for VHL in cancer.

The von Hippel-Lindau (VHL) protein binds and degrades hypoxia-inducible factors (HIF) hydroxylated by prolyl-hydroxylases under normoxia. Although originally described as a tumor suppressor, there is growing evidence that VHL may paradoxically promote tumor growth. The significance of its described interactions with many other proteins remains unclear. We found that VHL interacts with p53, preventing its tetramerization, promoterbinding and expression of its target genes p21, PUMA, and Bax. VHL limited the decrease in proliferation and increase in apoptosis caused by p53 activation, independent of prolyl-hydroxylation and HIF activity, and its presence in tumors caused a resistance to p53-inducing chemotherapy in vivo. We propose that VHL has both anti-tumor function, via HIF degradation, and a new pro-tumor function via p53 target (p21, PUMA, Bax) inhibition. Because p53 plays a critical role in tumor biology, isactivated by many chemotherapies, and because VHL levels vary among different tumors and its function can even be lost by mutations in some tumors, our results have important clinical applications. KEY MESSAGES: VHL and p53 physically interact and VHL inhibits p53 activity by limiting the formation of p53 tetramers. VHL attenuates the expression of p53 target genes in response to p53 stimuli. The inhibition of p53 by VHL is independent of HIF and prolyl-hydroxylation.

Nonclinical Characterization of the Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor Roxadustat, a Novel Treatment of Anemia of Chronic Kidney Disease.

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.

NO placental inflammation.

NO placental inflammation.

Molecular Mechanisms of Hypoxia‐Induced Extracellular Vesicle Release

BackgroundExtracellular vesicles (EVs) are released by many cells and provide a mechanism for intercellular communication in autocrine and paracrine fashion. EVs of various sizes, namely microvesicles (ranging from 100–1000 nm) or exosomes (with sizes between 30–150 nm) are released by most eukaryotic cells. EVs are known to contain bioactive molecules including proteins lipids and nucleic acids including miRNAs and LNC RNAs that regulate gene expression and cell functions in recipient cells. For example, tumour released exosomes remodel the microenvironment allowing cancer cells to survive and thrive. Low oxygen (hypoxia) is known to increase the release of exosomes in cancer cells. Hypoxia leads to the activation of hypoxia inducible factor (HIF) transcription factors, considered master regulators of the hypoxic response. The objective of this study was to investigate whether hypoxia induced EV (exosome) release is dependent on hypoxia inducible factor (HIF) proteins.MethodsWe isolated exosomes and microvesicles by differential centrifugation of conditioned media of cells exposed to normoxia or hypoxia (1% Oxygen) for different lengths of time ranging from 1–24hrs. We determined, size distribution and concentration of vesicles by nanoparticle tracking analysis (NTA). Expression of HIF1 and HIF2 was monitored by western blot of cellular lysates. We expressed oxygen‐stable mutated HIF1 and HIF2 isoforms either alone or in combination and determined EV release by NTA. We used chemical inhibitors of dioxygenase (PHD) and von Hippel–Lindau (VHL) proteins, the two proteins involved in HIF degradation under oxygen conditions, to stabilize HIF expression in normoxic cells and analysed EV release and HIF expression. We also analysed EV release in a cell line lacking VHL and after a VHL knock‐in. Western blotting of cellular lysates was used to monitor HIFs, VHL, and VEGF proteins. NTA was used to determine EV concentration and size. ANOVA statistical analysis was performed to analyse the data.ResultsHypoxia increased the amount extracellular vesicles in the conditioned media of all cells tested. The release of small vesicles (exosomes) was most increased following the exposure to hypoxia. HIF protein expression increased steadily during the time course of hypoxia, reaching a plateau after 2hrs following exposure. Expression of mutated (oxygen stable) HIF1 and HIF2 proteins in normoxic cells, either alone or in combination, did not increase exosome release. Chemical inhibition of PHD or VHL did not affect EV release. Exosome release was not different in VHL KD or KI cells.Conclusionthese data suggest that hypoxia driven exosome release is HIF independent.Support or Funding InformationSponsored by Fundacion Ramon Areces

How does Cell Machinery Sense and Deal with Hypoxia? The 2019 Nobel Prize for Medicine

The Nobel Prize in Physiology or Medicine for the year 2019 has been awarded to three scientists, namely William G Kaelin, Gregg Semenza and Peter J Ratcliffe, for their research work leading to an understanding of the mechanism used by living cells to sense the level of O2. They discovered the occurrence of a molecule called hypoxia-inducible factor (HIF) within the cells and demonstrated that under normoxic condition, HIF undergoes hydroxylation and ubiquitination followed by its degradation in proteasome. Under conditions of hypoxia, HIF becomes active and binds to hypoxia response element available within hundreds of target genes leading to their activation to overcome the problems due to hypoxia. Any aberration in this process of degradation and activation of HIF may cause a variety of diseases, so drugs based on BIF stabilizers/inhibitors have been developed and are under different stages of clinical trials.

Xiaoyukang Jiaonang Promotes the Degradation of Hypoxia-Inducible Factor 1α and Antiangiogenesis and Anti-Inflammation in Chronic Subdural Hematoma Rat Model.

Xiaoyukang Jiaonang (XYK) is a Chinese patent medicine approved by the National Medical Product Administration which is used to treat intracranial hematoma in China. In this study, we observed the molecular mechanism of XYK in hypoxia-inducible factor 1α (HIF-1α), inflammation and angiogenesis of chronic subdural hematoma (CSDH). The CSDH model was made by using internal iliac vein blood of Wister rats, and rats were divided into sham group, CSDH group and XYK group. The rats in the XYK group were gavaged with Xiaoyukang Jiaonang (185 mg/kg) for 7 days, and rats in the CSDH group and sham group were gavaged with the same amount of physiological saline for 7 days. In the CSHD rat model, active inflammation and angiogenesis were observed around the hematoma. XYK promoted the ubiquitination and degradation of HIF-1α, and reduced the concentration of VEGF and the ratio of angiopoietin-1/angiopoietin-2. XYK reduced proinflammatory cytokines and increased anti-inflammatory cytokine. In tissue section, XYK reduced the size of the hematoma and membrane, and reduced vWF positive cells in membrane. Furthermore, the endothelial progenitor cells in blood decreased as well. Overall, XYK shows anti-inflammatory and antiangiogenesis effects which may relate to the degradation of HIF-1α.