HIF-1α Stabilization Research Articles

Hypoxia mimetics restore bone biomineralisation in hyperglycaemic environments

Diabetic patients have an increased risk of fracture and an increased occurrence of impaired fracture healing. Diabetic and hyperglycaemic conditions have been shown to impair the cellular response to hypoxia, via an inhibited hypoxia inducible factor (HIF)-1α pathway. We investigated, using an in vitro hyperglycaemia bone tissue engineering model (and a multidisciplinary bone characterisation approach), the differing effects of glucose levels, hypoxia and chemicals known to stabilise HIF-1α (CoCl2 and DMOG) on bone formation. Hypoxia (1% O2) inhibited bone nodule formation and resulted in discrete biomineralisation as opposed to the mineralised extracellular collagen fibres found in normoxia (20% O2). Unlike hypoxia, the use of hypoxia mimetics did not prevent nodule formation in normal glucose level. Hyperglycaemic conditions (25 mM and 50 mM glucose) inhibited biomineralisation. Interestingly, both hypoxia mimetics (CoCl2 and DMOG) partly restored hyperglycaemia inhibited bone nodule formation. These results highlight the difference in osteoblast responses between hypoxia mimetics and actual hypoxia and suggests a role of HIF-1α stabilisation in bone biomineralisation that extends that of promoting neovascularisation, or other system effects associated with hypoxia and bone regeneration in vivo. This study demonstrates that targeting the HIF pathway may represent a promising strategy for bone regeneration in diabetic patients.

Binary SiO2–CoO spherical bioactive glass nanoparticles for tissue engineering applications

Pro-angiogenic capabilities are desired for biomaterials intended for tissue repair and tissue engineering applications, and bioactive glass (BG) nanoparticles are good vehicles for the delivery of ions with therapeutic properties, such as cobalt, which displays a well-known angiogenic effect by mimicking hypoxic conditions by HIF-1α stabilization. In this work, novel monodispersed spherical dense BG nanoparticles were obtained by the sol-gel process with the incorporation of cobalt into the glass structure as a potential platform for the delivery of pro-angiogenic ions, and particles with sizes ranging from 93 to 112 nm were obtained. Structural evaluation showed that cobalt (II,III) oxide were formed and deposited on the particles’ surface, while X-ray photoelectron spectroscopy (XPS) analysis pointed that Co2+ ions were also incorporated in the glass as a network modifier. Glasses also displayed a rapid and sustained Co release when immersed in phosphate buffered saline, and MTT assay showed good cell viability for BG concentrations of 100 μg mL−1 and 500 μg mL−1, confirming its potential to be applied for tissue engineering. Therefore, this study showed that Co containing nanoparticles, with great potential for tissue repair with the release of pro-angiogenic ions, can be obtained by a simple sol-gel based method.

Mitochondrial ROS drive resistance to chemotherapy and immune-killing in hypoxic non-small cell lung cancer

BackgroundSolid tumors subjected to intermittent hypoxia are characterized by resistance to chemotherapy and immune-killing by effector T-lymphocytes, particularly tumor-infiltrating Vγ9Vδ2 T-lymphocytes. The molecular circuitries determining this double resistance are not known.MethodsWe analyzed a panel of 28 human non-small cell lung cancer (NSCLC) lines, using an in vitro system simulating continuous and intermittent hypoxia. Chemosensitivity to cisplatin and docetaxel was evaluated by chemiluminescence, ex vivo Vγ9Vδ2 T-lymphocyte expansion and immune-killing by flow cytometry. Targeted transcriptomics identified efflux transporters and nuclear factors involved in this chemo-immuno-resistance. The molecular mechanism linking Hypoxia-inducible factor-1α (HIF-1α), CCAAT/Enhancer Binding Protein-β (C/EBP-β) isoforms LAP and LIP, ABCB1, ABCC1 and ABCA1 transporters were evaluated by immunoblotting, RT-PCR, RNA-IP, ChIP. Oxidative phosphorylation, mitochondrial ATP, ROS, depolarization, O2 consumption were monitored by spectrophotometer and electronic sensors. The role of ROS/HIF-1α/LAP axis was validated in knocked-out or overexpressing cells, and in humanized (Hu-CD34+NSG) mice bearing LAP-overexpressing tumors. The clinical meaning of LAP was assessed in 60 NSCLC patients prospectively enrolled, treated with chemotherapy.ResultsBy up-regulating ABCB1 and ABCC1, and down-regulating ABCA1, intermittent hypoxia induced a stronger chemo-immuno-resistance than continuous hypoxia in NSCLC cells. Intermittent hypoxia impaired the electron transport chain and reduced O2 consumption, increasing mitochondrial ROS that favor the stabilization of C/EBP-β mRNA mediated by HIF-1α. HIF-1α/C/EBP-β mRNA binding increases the splicing of C/EBP-β toward the production of LAP isoform that transcriptionally induces ABCB1 and ABCC1, promoting the efflux of cisplatin and docetaxel. LAP also decreases ABCA1, limiting the efflux of isopentenyl pyrophosphate, i.e. the endogenous activator of Vγ9Vδ2 T-cells, and reducing the immune-killing. In NSCLC patients subjected to cisplatin-based chemotherapy, C/EBP-β LAP was abundant in hypoxic tumors and was associated with lower response to treatment and survival. LAP-overexpressing tumors in Hu-CD34+NSG mice recapitulated the patients’ chemo-immuno-resistant phenotype. Interestingly, the ROS scavenger mitoquinol chemo-immuno-sensitized immuno-xenografts, by disrupting the ROS/HIF-1α/LAP cascade.ConclusionsThe impairment of mitochondrial metabolism induced by intermittent hypoxia increases the ROS-dependent stabilization of HIF-1α/LAP complex in NSCLC, producing chemo-immuno-resistance. Clinically used mitochondrial ROS scavengers may counteract such double resistance. Moreover, we suggest C/EBP-β LAP as a new predictive and prognostic factor in NSCLC patients.

Roxadustat alleviates nitroglycerin-induced migraine in mice by regulating HIF-1α/NF-κB/inflammation pathway.

Sensitization of central pain and inflammatory pathways play essential roles in migraine, a primary neurobiological headache disorder. Since hypoxia-inducible factor-1α (HIF-1α) is implicated in neuroprotection and inflammation inhibition, herein we investigated the role of HIF-1α in migraine. A chronic migraine model was established in mice by repeated injection of nitroglycerin (10 mg/kg, i.p.) every other day for 5 total injections. In the prevention and acute experiments, roxadustat, a HIF-1α stabilizer, was orally administered starting before or after nitroglycerin injection, respectively. Pressure application measurement, and tail flick and light-aversive behaviour tests were performed to determine the pressure pain threshold, thermal nociceptive sensitivity and migraine-related light sensitivity. At the end of experiments, mouse serum samples and brain tissues were collected for analyses. We showed that roxadustat administration significantly attenuated nitroglycerin-induced basal hypersensitivity and acute hyperalgesia by improving central sensitization. Roxadustat administration also decreased inflammatory cytokine levels in serum and trigeminal nucleus caudalis (TNC) through NF-κB pathway. Consistent with the in vivo results showing that roxadustat inhibited microglia activation, roxadustat (2, 10, and 20 μM) dose-dependently reduced ROS generation and inflammation in LPS-stimulated BV-2 cells, a mouse microglia cell line, by inhibiting HIF-1α/NF-κB pathway. Taken together, this study demonstrates that roxadustat administration ameliorates migraine-like behaviours and inhibits central pain sensitization in nitroglycerin-injected mice, which is mainly mediated by HIF-1α/NF-κB/inflammation pathway, suggesting the potential of HIF-1α activators as therapeutics for migraine.

Abstract P3079: Hypoxia Stimulates Neonatal Hypoxia-inducible Factor 1α Dependent Macrophage Secretion Of Cardiomyocyte Mitogens

Background: The neonatal murine heart can regenerate after injury. This regenerative capacity is dependent on macrophages and lost shortly after birth. How macrophages facilitate regeneration remains vague, and the neonatal myeloid response to hypoxia as well as the role of myeloid Hypoxia-Inducible Factors (HIFs) in cardiac regeneration are unknown. We hypothesized that post-ischemic regulation of regenerative programs are confined to neonatal cardiac macrophages and are lost in the adult. We also hypothesized hypoxia is a stimulus for neonatal macrophages to secrete cardiomyocyte mitogens in a HIF dependent manner. Methods: Single-cell sequencing was performed on cardiac myeloid cells post myocardial infarction (MI) in wildtype neonatal and adult mice to explore differences in regenerative vs reparative responses to injury. Neonatal cardiomyocytes harvested from wildtype mice were co-cultured with supernatant from hypoxia stimulated neonatal bone marrow-derived macrophages from myeloid HIF-1 α expressing vs HIF-1 α genetically depleted mice. Immunofluorescent staining with antibodies to Troponin T, Ki67, and DAPI was performed to quantify cardiomyocyte proliferation. Results: High-resolution clustering identified 3 distinct macrophage populations present in neonatal and adult mice post MI. Gene Ontology identified significantly upregulated genes in biological pathways associated with regeneration and HIF-1α stabilization in Timd4+Mrc1+ macrophages in the neonate but not the adult. Immunofluorescent staining of neonatal cardiomyocytes at steady state revealed an average of 6.0% Ki67+ cardiomyocytes compared to 35.4% Ki67+ cardiomyocytes exposed to supernatant from hypoxia stimulated HIF-1 α + macrophages (N=10/group; SEM + 1.96; p value <0.0001) and 10.1% Ki67+ cardiomyocytes exposed to supernatant from hypoxia stimulated HIF-1 α - macrophages (N=10/group; SEM + 1.54; p value 0.0162). Conclusions: We have newly identified enrichment of regenerative and HIF-1α stabilization pathways specific to neonatal resident cardiac macrophages after ischemic injury. We also newly conclude hypoxia is a stimulus for neonatal macrophages to secrete HIF-1 α dependent mitogenic factors that induce cardiomyocyte proliferation.

Preclinical Characterization of Vadadustat (AKB-6548), an Oral Small Molecule Hypoxia-Inducible Factor Prolyl-4-Hydroxylase Inhibitor, for the Potential Treatment of Renal Anemia.

Pharmacological inhibition of prolyl-4-hydroxylase domain (PHD) enzymes stabilizes hypoxia-inducible factors (HIFs), transcription factors that activate target genes that, among others, increase erythropoietin (EPO) synthesis, resulting in the production of new red blood cells (RBCs). Herein, we summarize the preclinical characteristics of the small molecule HIF prolyl-4-hydroxylase inhibitor vadadustat (AKB-6548), which is in development for the treatment of anemia in patients with chronic kidney disease (CKD). Vadadustat inhibits the enzyme activity of all three human PHD isozymes, PHD1, PHD2, and PHD3, with similar low nanomolar inhibitory constant values. PHD enzyme inhibition by vadadustat is competitive with endogenous cofactor 2-oxoglutarate and is insensitive to free iron concentration. In the human hepatocellular carcinoma cell line (Hep 3B) and human umbilical vein endothelial cells, PHD inhibition by vadadustat leads to the time- and concentration-dependent stabilization of HIF-1α and HIF-2α In Hep 3B cells, this in turn results in the synthesis and secretion of EPO; vascular endothelial growth factor is not measured at detectable levels. A single oral dose of vadadustat in rats potently increases circulating levels of EPO, and daily oral dosing for 14 days increases RBC indices in healthy rats and in the 5/6 nephrectomy model of CKD. In mice and dogs, once-daily repeat oral dosing increases hemoglobin and hematocrit. Vadadustat has a relatively short half-life in all nonclinical species evaluated and does not accumulate when administered as a single bolus dose (oral or intravenous) or upon repeat oral dosing. The pharmacological profile of vadadustat supports continued development for treatment of renal anemia. SIGNIFICANCE STATEMENT: Vadadustat (AKB-6548) is an orally bioavailable small molecule prolyl-4-hydroxylase inhibitor in development for anemia of chronic kidney disease. It is an equipotent inhibitor of the three human prolyl-4-hydroxylase domain isoforms, which activates erythropoiesis through stabilization of hypoxia-inducible factor (HIF)-1α and HIF-2α, increasing production of erythropoietin, without detectable stimulation of vascular endothelial growth factor.

The effect of alpha-ketoglutarate on phosphate and hypoxia induced calcification of vascular smooth muscle cells

Background and Aims : Recent studies revealed that hypoxia contributes to vascular calcification via the activation of hypoxia-inducible factor 1 (HIF-1) pathway. The alpha-ketoglutarate-dependent prolyl hydroxylase domain enzymes regulate the stability of HIF-1α in response to oxygen availability. As a rate-limiting substrate, alpha-ketoglutarate regulates the activity of prolyl hydroxylases, therefore we addressed the inhibitory effect of alpha-ketoglutarate on vascular smooth muscle cell calcification under hypoxic condition.

Normoxic HIF-1Α stabilization at the vascular barrier in atherosclerosis

Background and Aims : Not much is known about hypoxia-inducible factor (HIF)-1α stabilization under normoxic conditions. We hypothesize that micromilieu factors (MF) induce normoxic HIF-1α stabilization, which could represent a first trigger for the development of atherosclerosis through a loss of the endothelial barrier function (EBF).

Decreased expression of hypoxia-inducible factor 1α (HIF-1α) in cord blood monocytes under anoxia

BackgroundInfections are a major cause for morbidity and mortality in neonates; however, the underling mechanisms for increased infection susceptibility are incompletely understood. Hypoxia, which is present in inflamed tissues, has been identified as an important activation signal for innate immune cells in adults and is mainly mediated by hypoxia-inducible factor 1α (HIF-1α). Fetal tissue pO2 physiologically is low but rises immediately after birth.MethodsIn this study, the effect of low oxygen partial pressure (pO2) on HIF-1α expression and its targets phagocytosis, reactive oxygen species (ROS) production and vascular endothelial growth factor (VEGF) secretion was compared in vitro between immune cells from adult peripheral blood and cord blood using anoxia, HIF-1α stabilizer desferroxamin (DFO) and E. coli as stimuli.ResultsWe show that anoxia-induced HIF-1α protein accumulation, phagocytosis, ROS-production and VEGF-expression were greatly diminished in cord blood compared to adult cells. E. coli led to HIF-1α gene expression in adult and cord blood immune cells; however, cord blood cells failed to accumulate HIF-1α protein and VEGF upon E. coli stimulation.ConclusionsTaken together, our results show a diminished activation of cord blood immune cells by low pO2, which might contribute to impaired reactivity in the context of infection.ImpactNeonatal immune cells do not accumulate HIF-1α under low oxygen partial pressure leading to decreased phagocytosis and decreased ROS production.We demonstrate a previously unknown mechanism of reduced activation of neonatal immune cells in the context of an inflammatory response.This could contribute to the increased susceptibility of newborns and preterm infants to infection.

Campylobacter jejuni induces differentiation of human neutrophils to the CD16hi /CD62Llo subtype.

The discovery of neutrophil subtypes has expanded what is known about neutrophil functions, yet there is still much to learn about the role of these subtypes during bacterial infection. We investigated whether Campylobacter jejuni induced differentiation of human neutrophils into the hypersegmented, CD16hi /CD62Llo subtype. In addition, we investigated whether C. jejuni-dependent differentiation of this neutrophil subtype induced cancer-promoting activities of human T cells and colonocytes, which were observed in other studies of hypersegmented, CD16hi /CD62Llo neutrophils. We found that C. jejuni causes a significant shift in human neutrophil populations to the hypersegmented, CD16hi /CD62Llo subtype and that those populations exhibit delayed apoptosis, elevated arginase-1 expression, and increased reactive oxygen species production. Furthermore, incubation of C. jejuni-infected neutrophils with human T cells resulted in decreased expression of the ζ-chain of the TCR, which was restored upon supplementation with exogenous l-arginine. In addition, incubation of C. jejuni-infected neutrophils with human colonocytes resulted in increased HIF-1α stabilization and NF-κB activation in those colonocytes, which may result in the up-regulation of protumorigenic genes.

Mitochondrial oxidative phosphorylation became functional under aglycemic hypoxia conditions in A549 cells.

Normal cells produce energy (ATP) through mitochondrial oxidative phosphorylation in the presence of oxygen. However, many of the cancer cells produce energy with accelerated glycolysis and perform lactic acid production even under normoxic conditions called "The Warburg Effect". In this study, human lung carcinoma cells (A549) were incubated in either a normoxic or hypoxic environment containing 5 mM glucose (Glc 5), 25 mM glucose (Glc 25), or 10 mM galactose (OXPHOS/aglycemic), and then the bioenergetic pathway was anaylsed. HIF-1α stabilization of A549 cells with different metabolic conditions in normoxia and hypoxia (1% O2) was determined using the western blot method. After that, L-lactic acid analysis, p-PDH/PDH expression ratio, ATP analysis, and citrate synthase activity experiments were also performed. It was determined that HIF-1α stabilization reached the maximum level at the 4h. It has been found that glycolytic cells produce approximately five times more lactate than OXPHOS cells under both normoxia and hypoxia conditions and also have a higher p-PDH/PDH ratio. It has been determined that citrate synthase activity in hypoxia of all metabolic conditions is lower than normoxia. It has been determined that Glc 5 and Glc 25 cells have more ATP production under normoxia than Glc 5 and Glc 25 cells in hypoxia. OXPHOS cells have showed more ATP production in hypoxia. It has been determined that oxidative phosphorylation became functional in a hypoxic aglycemic environment despite the metabolic programming regulated by HIF-1α. This data is important in determining targets for therapeutic intervention.

HIF-1α induces glycolytic reprograming in tissue-resident alveolar macrophages to promote cell survival during acute lung injury.

Cellular metabolism is a critical regulator of macrophage effector function. Tissue-resident alveolar macrophages (TR-AMs) inhabit a unique niche marked by high oxygen and low glucose. We have recently shown that in contrast to bone marrow-derived macrophages (BMDMs), TR-AMs do not utilize glycolysis and instead predominantly rely on mitochondrial function for their effector response. It is not known how changes in local oxygen concentration that occur during conditions such as acute respiratory distress syndrome (ARDS) might affect TR-AM metabolism and function; however, ARDS is associated with progressive loss of TR-AMs, which correlates with the severity of disease and mortality. Here, we demonstrate that hypoxia robustly stabilizes HIF-1α in TR-AMs to promote a glycolytic phenotype. Hypoxia altered TR-AM metabolite signatures, cytokine production, and decreased their sensitivity to the inhibition of mitochondrial function. By contrast, hypoxia had minimal effects on BMDM metabolism. The effects of hypoxia on TR-AMs were mimicked by FG-4592, a HIF-1α stabilizer. Treatment with FG-4592 decreased TR-AM death and attenuated acute lung injury in mice. These findings reveal the importance of microenvironment in determining macrophage metabolic phenotype and highlight the therapeutic potential in targeting cellular metabolism to improve outcomes in diseases characterized by acute inflammation.

HIF-1α Stabilization Boosts Pulp Regeneration by Modulating Cell Metabolism

Stem cell–based therapeutics is a promising strategy in dental pulp regeneration. However, low cell viability after transplantation in vivo due to the ischemic microenvironment is still a critical challenge for future clinical application. With the aim of improving postimplantation cell survival and pulp tissue regeneration, stem cells from human exfoliated deciduous teeth (SHED) were preconditioned to a hypoxic condition by hypoxia-inducible factor 1α (HIF-1α) stabilization via knockdown of prolyl hydroxylase domain-containing protein 2 (PHD2) using lentiviral short hairpin RNA. HIF-1α–stabilized SHED were encapsulated in PuraMatrix hydrogel, injected into root canals of human tooth fragments, and implanted in the subcutaneous space of immunodeficient mice. After 28 d, enhanced dental pulp–like tissue formation was observed with a significantly higher level of vascularization, which could be attributed to both endothelial differentiation of SHED and recruitment of host blood vessels. Furthermore, dentin-like tissue formation in vivo and accelerated odontogenic/osteogenic differentiation both in vivo and in vitro were observed. At 7 d postimplantation, significantly less DNA damage and higher Ki67 expression were detected in the HIF-1α–stabilized SHED group compared with the control SHED. Accordingly, cell viability assay and staining for Ki67 and apoptotic cells in vitro showed that HIF-1α stabilization could decrease cell apoptosis and enhance cell survival significantly. We demonstrated that PI3K/AKT pathway activation had resulted in low caspase 3 expression in HIF-1α–stabilized SHED in hypoxic conditions. Furthermore, we found that HIF-1α–induced cell survival could also be attributed to the upregulated expression of PDK1, HK2, and Glut1, which contributes to the maintenance of reactive oxygen species homeostasis and metabolic adaptation in hypoxia. In addition, we identified Smad7 as 1 of the top 3 upregulated genes through RNA sequencing in HIF-1α–stabilized SHED and demonstrated its essential role in HK2 and Glut1 upregulation. Taken together, HIF-1α stabilization enhances cell survival of SHED through modulating various target genes and potential signaling pathways, as well as odontogenic tissue formation during dental pulp regeneration, which could benefit stem cell–based therapy in general.

ID2 and HIF-1α collaborate to protect quiescent hematopoietic stem cells from activation, differentiation, and exhaustion.

Defining mechanism(s) that maintain tissue stem quiescence is important for improving tissue regeneration, cell therapies, aging, and cancer. We report here that genetic ablation of Id2 in adult hematopoietic stem cells (HSCs) promotes increased HSC activation and differentiation, which results in HSC exhaustion and bone marrow failure over time. Id2Δ/Δ HSCs showed increased cycling, ROS production, mitochondrial activation, ATP production, and DNA damage compared with Id2+/+ HSCs, supporting the conclusion that Id2Δ/Δ HSCs are less quiescent. Mechanistically, HIF-1α expression was decreased in Id2Δ/Δ HSCs, and stabilization of HIF-1α in Id2Δ/Δ HSCs restored HSC quiescence and rescued HSC exhaustion. Inhibitor of DNA binding 2 (ID2) promoted HIF-1α expression by binding to the von Hippel-Lindau (VHL) protein and interfering with proteasomal degradation of HIF-1α. HIF-1α promoted Id2 expression and enforced a positive feedback loop between ID2 and HIF-1α to maintain HSC quiescence. Thus, sustained ID2 expression could protect HSCs during stress and improve HSC expansion for gene editing and cell therapies.

Homocysteine-targeting compounds as a new treatment strategy for diabetic wounds via inhibition of the histone methyltransferase SET7/9

In hypoxia and hyperglycemia, SET7/9 plays an important role in controlling HIF-1α methylation and regulating the transcription of HIF-1α target genes, which are responsible for angiogenesis and wound healing. Here, we report the Ir(III) complex Set7_1a bearing acetonitrile (ACN) ligands as a SET7/9 methyltransferase inhibitor and HIF-1α stabilizer. Interestingly, Set7_1a could engage SET7/9 and strongly inhibit SET7/9 activity, especially after preincubation with homocysteine (Hcy), which is elevated in diabetes. We hypothesize that Set7_1a exchanges ACN subunits for Hcy to disrupt the interaction between SET7/9 and SAM/SAH, which are structurally related to Hcy. Inhibition of SET7/9 methyltransferase activity by Set7_1a led to reduced HIF-1α methylation at the lysine 32 residue, causing increased HIF-1α level and recruitment of HIF-1α target genes that promote angiogenesis, such as VEGF, GLUT1, and EPO, in hypoxia and hyperglycemia. Significantly, Set7_1a improved wound healing in a type 2 diabetic mouse model by activating HIF-1α signaling and downstream proangiogenic factors. To our knowledge, this is the first Hcy-targeting iridium compound shown to be a SET7/9 antagonist that can accelerate diabetic wound healing. More importantly, this study opens a therapeutic avenue for the treatment of diabetic wounds by the inhibition of SET7/9 lysine methyltransferase activity.

Hypoxia-Inducible Factor 1-Alpha Stabilizers in the Treatment of Inflammatory Bowel Diseases: Oxygen as a Novel IBD Therapy?

Despite the significant advances in the medical armamentarium for inflammatory bowel diseases [IBD], current treatment options have notable limitations. Durable remission rates remain low, loss of response is common, administration routes are largely parenteral for novel biologics, and medication safety remains a concern. This explains an ongoing unmet need for safe medications with novel mechanisms of action that are administered orally. In line with these criteria, hypoxia-inducible factor [HIF]-1α stabilizers, acting via inhibition of prolyl hydroxylase enzymes, are emerging as an innovative therapeutic strategy. We herein review the mechanism of action and available clinical data for HIF-1α stabilizers and their potential place in the future IBD treatment algorithm.

The Molecular Role of HIF1α Is Elucidated in Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is potentially fatal blood cancer, but there is an unmet need to discover novel molecular biomarkers. The hypothesis of this study aimed to elucidate the relationship of HIF1α with the redox system, Krebs cycles, notch1, and other regulatory proteins to better understand the pathophysiology and clinical relevance in chronic myeloid leukemia (CML) patients, as the molecular mechanism of this axis is still not clear. This study included CML patient samples (n = 60; 60: blood; 10: bone marrow tissues) and compared them with healthy controls (n = 20; blood). Clinical diagnosis confirmed on bone marrow aspiration, marrow trephine biopsy, and BCR/ABL1 translocation. Cases were subclassified into chronic, accelerated, and blast crises as per WHO guidelines. Molecular experiments included redox parameters, DNA fragmentation, Krebs cycle metabolites, and gene expression by RT-PCR/Western blot/LC-MS, PPI (STRING), Pearson correlation, and ROC curve analysis. Here, our findings show that p210/p190BCR/ABL1 translocation is common in all blast crisis phases of CML. Redox factor/Krebs oncometabolite concentrations were high, leading to upregulation and stabilization of HIF1α. HIF1α leads to the pathogenesis in CML cells by upregulating their downstream genes (Notch 2/4/Ikaros/SIRT1/Foxo-3a/p53, etc.). Whereas, downregulated ubiquitin proteasomal and apoptotic factors in CML pateints, can trigger degradation of HIF1α through proline hydroxylation. However, HIF1α showed a negative corelation with the notch1 pathway. Notch1 plays a tumor-suppressive role in CML and might have the potential to be used as a diagnostic marker along with other factors in CML patients. The outcome also revealed that oxidant treatment could not be effective in augmentation with conventional therapy because CML cells can enhance the levels of antioxidants for their survival. HIF1α might be a novel therapeutic target other than BCR/ABL1 translocation.

SM22α cell-specific HIF stabilization mitigates hyperoxia-induced neonatal lung injury.

Though survival rates for preterm infants are improving, the incidence of chronic lung disease of infancy, or bronchopulmonary dysplasia (BPD), remains high. Histologically, BPD is characterized by larger and fewer alveoli. Hypoxia-inducible factors (HIFs) may be protective in the context of hyperoxia-induced lung injury, but the cell-specific effects of HIF expression in neonatal lung injury remain unknown. Thus, we sought to determine whether HIF stabilization in SM22α-expressing cells can limit hyperoxia-induced neonatal lung injury. We generated SM22α-specific HIF-1α-stabilized mice (SM22α-PHD1/2-/- mice) by cross-breeding SM22α-promotor-driven Cre recombinase mice with prolyl hydroxylase PHD1flox/flox and PHD2flox/flox mice. Neonatal mice were randomized to 21% O2 (normoxia) or 80% O2 (hyperoxia) exposure for 14 days. For the hyperoxia recovery studies, neonatal mice were recovered from normoxia for an additional 10 wk. SM22α-specific HIF-1α stabilization mitigated hyperoxia-induced lung injury and preserved microvessel density compared with control mice for both neonates and adults. In SM22α-PHD1/2-/- mice, pulmonary artery endothelial cells (PAECs) were more proliferative and pulmonary arteries expressed more collagen IV compared with control mice, even under hyperoxic conditions. Angiopoietin-2 (Ang2) mRNA expression in pulmonary artery smooth muscle cells (PASMC) was greater in SM22α-PHD1/2-/- compared with control mice in both normoxia and hyperoxia. Pulmonary endothelial cells (PECs) cocultured with PASMC isolated from SM22α-PHD1/2-/- mice formed more tubes and branches with greater tube length compared with PEC cocultured with PASMC isolated from SM22α-PHD1/2+/+ mice. Addition of Ang2 recombinant protein further augmented tube formation for both PHD1/2+/+ and PHD1/2-/- PASMC. Cell-specific deletion of PHD1 and 2 selectively increases HIF-1α expression in SM22α-expressing cells and protects neonatal lung development despite prolonged hyperoxia exposure. HIF stabilization in SM22α-expressing cells preserved endothelial cell proliferation, microvascular density, increased angiopoietin-2 expression, and lung structure, suggesting a role for cell-specific HIF-1α stabilization to prevent neonatal lung injury.

Differential HIF2α Protein Expression in Human Carotid Body and Adrenal Medulla under Physiologic and Tumorigenic Conditions.

Simple SummaryThoraco-abdominal paraganglioma and pheochromocytoma (PPGL) are pathogenically linked to mutations in SDH genes. Loss-of-function of SDH has been associated with stabilization of HIF2α, which otherwise would be degraded via oxygen-sensitive mechanisms. SDH dysfunction also predispose to the development of paragangliomas arising at the carotid body or other head and neck paraganglia (HNPGL). Although PPGL and HNPGL share similar features, they have certain clinical and genetic peculiarities. By comparison of HIF2α expression in HNPGL and PPGL, we found that functional HIF2α is overexpressed in 80% of PPGLs, including those with SDH mutations as compared with non-tumor tissue. However, HIF2α is already highly expressed in the carotid body under physiologic conditions, and it is not overexpressed in HNPGL. These data suggest that selective HIF2α inhibitors already in clinical trials may benefit a wide spectrum of PPGL.Hypoxia-inducible factors (HIF) 2α and 1α are the major oxygen-sensing molecules in eukaryotic cells. HIF2α has been pathogenically linked to paraganglioma and pheochromocytoma (PPGL) arising in sympathetic paraganglia or the adrenal medulla (AM), respectively. However, its involvement in the pathogenesis of paraganglioma arising in the carotid body (CB) or other parasympathetic ganglia in the head and neck (HNPGL) remains to be defined. Here, we retrospectively analyzed HIF2α by immunohistochemistry in 62 PPGL/HNPGL and human CB and AM, and comprehensively evaluated the HIF-related transcriptome of 202 published PPGL/HNPGL. We report that HIF2α is barely detected in the AM, but accumulates at high levels in PPGL, mostly (but not exclusively) in those with loss-of-function mutations in VHL and genes encoding components of the succinate dehydrogenase (SDH) complex. This is associated with upregulation of EPAS1 and the HIF2α-regulated genes COX4I2 and ADORA2A. In contrast, HIF2α and HIF2α-regulated genes are highly expressed in CB and HNPGL, irrespective of VHL and SDH dysfunctions. We also found that HIF2α and HIF1α protein expressions are not correlated in PPGL nor HNPGL. In addition, HIF1α-target genes are almost exclusively overexpressed in VHL-mutated HNPGL/PPGL. Collectively, the data suggest that involvement of HIF2α in the physiology and tumor pathology of human paraganglia is organ-of-origin-dependent and HIF1α-independent.

LPS stimulation stabilizes HIF-1α by enhancing HIF-1α acetylation via the PARP1-SIRT1 and ACLY-Tip60 pathways in macrophages.

Hypoxia and inflammatory mediators stabilize hypoxia-inducible factor (HIF)-1α through posttranslational modifications, such as phosphorylation and succinylation. Here, we identified sirtuin 1 (SIRT1) and 60 kDa Tat-interactive protein (Tip60)-mediated acetylation as another critical posttranslational modification that regulates HIF-1α protein stability under lipopolysaccharide (LPS) stimulation. Mechanistically, DNA damage induced by excessive reactive oxygen species (ROS) activated poly (ADP-ribose) polymerase 1 (PARP1) to consume oxidized nicotinamide adenine dinucleotide (NAD+ ). Correspondingly, SIRT1 activity was decreased with the decline in NAD+ levels, resulting in increased HIF-1α acetylation. LPS also activated the ATP-citrate lyase (ACLY)-Tip60 pathway to further enhance HIF-1α acetylation. Acetylation contributed to HIF-1α stability and exacerbated LPS-induced inflammation. Thus, inhibiting HIF-1α stability by decreasing its acetylation could partly alleviate LPS-induced inflammation. In conclusion, we revealed the mechanism by which LPS stabilized HIF-1α by increasing its acetylation via the PARP1-SIRT1 and ACLY-Tip60 pathways in fish macrophages. This study may provide novel insights for manipulation of HIF-1α acetylation as a therapeutic strategy against inflammation from the perspective of acetylation in vertebrates.