Hypoxia Inducible Factor 1A Signaling Research Articles

Nitroxide-HMP-Protects Human Trophoblast HTR-8/SVneo Cells from H2O2-Induced Oxidative Stress by Reducing the HIF1A Signaling Pathway.

Preeclampsia (PE) is a pregnancy-specific syndrome affecting 5-7% of patients. There is no effective treatment available. Early abnormal placental development is associated with oxidative stress (OS) and a release of reactive oxygen species (ROS) in the placenta. This phenomenon leads to downstream signaling, Hypoxia Inducible Factor 1A (HIF1A) stabilization and transcription of the anti-angiogenic factors soluble fms-like tyrosine kinase 1 (sFLT1) and soluble endoglin (sEng), which are known to cause endothelial and trophoblast dysfunction and cardinal features of PE: hypertension, proteinuria and, in severe cases, eclampsia. We tested whether 3-(Hydroxymethyl)-1-oxy-2,2,5,5-tetramethylpyrrolidine (HMP)-a nitroxide-type antioxidant molecule-can reduce placental OS and mitigate PE symptoms in vitro. We induced OS in human trophoblast (HTR-8/SVneo) cells with hydrogen peroxide (H2O2) and assessed whether modulating cell redox function with HMP reduces cell injury, mitochondrial stress and HIF1A and sFLT1 production. Pre-treatment with HMP reduced mitochondrial-derived ROS production, restored LC3B expression and reduced HIF1A and sFLT1 expression in H2O2-exposed HTR-8/SVneo trophoblast cells. HMP improved the mitochondrial electron chain enzyme activity, indicating that a reduction in OS alleviates mitochondrial stress and also reduces anti-angiogenic responses. In reducing placental trophoblast OS, HMP presents a potential novel therapeutic approach for the treatment of PE. Future investigation is warranted regarding the in vivo use of HMP.

A Photoactivated Ru (II) Polypyridine Complex Induced Oncotic Necrosis of A549 Cells by Activating Oxidative Phosphorylation and Inhibiting DNA Synthesis as Revealed by Quantitative Proteomics.

The ruthenium polypyridine complex [Ru(dppa)2(pytp)] (PF6)2 (termed as ZQX-1), where dppa = 4,7-diphenyl-1,10-phenanthroline and pytp = 4'-pyrene-2,2':6',2''-terpyridine, has been shown a high and selective cytotoxicity to hypoxic and cisplatin-resistant cancer cells either under irradiation with blue light or upon two-photon excitation. The IC50 values of ZQX-1 towards A549 cancer cells and HEK293 health cells are 0.16 ± 0.09 µM and >100 µM under irradiation at 420 nm, respectively. However, the mechanism of action of ZQX-1 remains unclear. In this work, using the quantitative proteomics method we identified 84 differentially expressed proteins (DEPs) with |fold-change| ≥ 1.2 in A549 cancer cells exposed to ZQX-1 under irradiation at 420 nm. Bioinformatics analysis of the DEPs revealed that photoactivated ZQX-1 generated reactive oxygen species (ROS) to activate oxidative phosphorylation signaling to overproduce ATP; it also released ROS and pyrene derivative to damage DNA and arrest A549 cells at S-phase, which synergistically led to oncotic necrosis and apoptosis of A549 cells to deplete excess ATP, evidenced by the elevated level of PRAP1 and cleaved capase-3. Moreover, the DNA damage inhibited the expression of DNA repair-related proteins, such as RBX1 and GPS1, enhancing photocytotoxicity of ZQX-1, which was reflected in the inhibition of integrin signaling and disruption of ribosome assembly. Importantly, the photoactivated ZQX-1 was shown to activate hypoxia-inducible factor 1A (HIF1A) survival signaling, implying that combining use of ZQX-1 with HIF1A signaling inhibitors may further promote the photocytotoxicity of the prodrug.

NPAS2 promotes aerobic glycolysis and tumor growth in prostate cancer through HIF-1A signaling

BackgroundProstate cancer (PCa), one of the common malignant tumors, is the second leading cause of cancer-related deaths in men. The circadian rhythm plays a critical role in disease. Circadian disturbances are often found in patients with tumors and enable to promote tumor development and accelerate its progression. Accumulating evidence suggests that the core clock gene NPAS2 (neuronal PAS domain-containing protein 2) has been implicated in tumors initiation and progression. However, there are few studies on the association between NPAS2 and prostate cancer. The purpose of this paper is to investigate the impact of NPAS2 on cell growth and glucose metabolism in prostate cancer.MethodsQuantitative real-time PCR (qRT-PCR), immunohistochemical (IHC) staining, western blot, GEO (Gene Expression Omnibus) and CCLE (Cancer Cell Line Encyclopedia) databases were used to analyze the expression of NPAS2 in human PCa tissues and various PCa cell lines. Cell proliferation was assessed using MTS, clonogenic assays, apoptotic analyses, and subcutaneous tumor formation experiments in nude mice. Glucose uptake, lactate production, cellular oxygen consumption rate and medium pH were measured to examine the effect of NPAS2 on glucose metabolism. The relation of NPAS2 and glycolytic genes was analyzed based on TCGA (The Cancer Genome Atlas) database.ResultsOur data showed that NPAS2 expression in prostate cancer patient tissue was elevated compared with that in normal prostate tissue. NPAS2 knockdown inhibited cell proliferation and promoted cell apoptosis in vitro and suppressed tumor growth in a nude mouse model in vivo. NPAS2 knockdown led to glucose uptake and lactate production diminished, oxygen consumption rate and pH elevated. NPAS2 increased HIF-1A (hypoxia-inducible factor-1A) expression, leading to enhanced glycolytic metabolism. There was a positive correlation with the expression of NPAS2 and glycolytic genes, these genes were upregulated with overexpression of NPAS2 while knockdown of NPAS2 led to a lower level.ConclusionNPAS2 is upregulated in prostate cancer and promotes cell survival by promoting glycolysis and inhibiting oxidative phosphorylation in PCa cells.

Succinate aggravates intestinal injury in mice with necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the most prevalent gastrointestinal disorder that predominantly threatens preterm newborns. Succinate is an emerging metabolic signaling molecule that was recently studied in relation to the regulation of intestinal immunity and homeostasis. We aimed to investigate the relationship between NEC and gut luminal succinate and preliminarily explored the effect of succinate on NEC pathogenesis. Fecal samples from human neonates and mouse pups were analyzed by HPLC - MS/MS and 16S rRNA gene sequencing. C57BL/6 mice were randomly divided into four groups: control, NEC, Lsuc, and Hsuc. The mortality, weight gain, and intestinal pathological changes in four mouse groups were observed. Inflammatory cytokines and markers of macrophages were identified by quantitative real-time PCR. Succinate receptor 1 (SUCNR1) localization was visualized by immunohistochemistry. The protein levels of SUCNR1 and hypoxia-inducible factor 1a (HIF-1a) were quantified by western blotting. The levels of succinate in feces from NEC patients were higher than those in feces from non-NEC patients (P <0.05). In the murine models, succinate levels in intestinal content samples were also higher in the NEC group than in the control group (P <0.05). The change in succinate level was closely related to intestinal flora composition. In samples from human neonates, relative to the control group, the NEC group showed a higher abundance of Enterobacteriaceae and a lower abundance of Lactobacillaceae and Lactobacillus (P <0.05). In the murine models, relative to the control group, increased abundance was observed for Clostridiaceae, Enterococcaceae, Clostridium_sensu_stricto_1, and Enterococcus, whereas decreased abundance was observed for Lactobacillaceae and Lactobacillus (P <0.05). Increased succinate levels prevented mice from gaining weight, damaged their intestines, and increased their mortality; upregulated the gene expression of interleukin-1β (IL-1β), IL-6, IL-18 and tumor necrosis factor (TNF); and downregulated the gene expression of IL-10 and transforming growth factor (TGF)-β. Exogenous succinic acid increased inducible nitric oxide synthase (iNOS) gene expression but decreased Arginase-1 (Arg1) gene expression; and increased the protein expression of SUCNR1 and HIF-1a. Succinate plays an important role in the development of necrotizing enterocolitis severity, and the activation of the HIF-1a signaling pathway may lead to disease progression.

Arginyltransferase1 mediates a pVHL‐independent oxygen sensing pathway in mammalian cells

The cellular response to oxygen availability is a fundamental process in all eukaryotes that regulates energy metabolism and death/survival decisions.The mechanisms for oxygen sensing likely co-evolved with the domestication of mitochondria as an adaptation to the oxygenation of the earth atmosphere. However, in mammalian cells, the only known oxygen sensing mechanism depends on the Von Hippel–Lindau tumor suppressor protein (pVHL), which is only found in metazoans but not in other species. This discrepancy points to a potential missing link in oxygen sensing pathways. In this study, we found that oxygen sensing in mammalian cells is also regulated by Arginyltransferase 1 (ATE1), which is ubiquitously conserved in eukaryotes and influences the degradation of many proteins by posttranslational arginylation via the principle of N-end rule. We report that ATE1 centrally controls the hypoxic response and glycolysis in mammalian cells by arginylating Hypoxia-inducible factor 1a (HIF1a), and subsequently regulating HIF1a signaling pathways in a transcription-independent manner. Moreover, we find that ATE1-mediated N-arginylation of HIF1a is sensitive to its oxygen-dependent hydroxylation mediated by the oxygen sensor prolyl hydroxylase (PHD). However, ATE1 promotes HIF1a degradation independently of the conventional pVHL E3 ubiquitin ligase pathway, but depending on typical N-end degradation machineries, the UBR E3 Ubiquitin ligase family. Bioinformatic analysis of human tumor data reveal that the ATE1/UBR and the pVHL pathway jointly regulate oxygen sensing in vivo with tissue specificities. Finally, a phylogenetic analysis suggests that ATE1 was derived from mitochondrial gene transfer and involved in oxygen-sensing in multiple lineages of eukaryotes. Our findings present an unexplored role of ATE1-mediated arginylation as a novel pathway for cellular oxygen sensing and adaptation in mammalian cells.

HIF1A signaling selectively supports proliferation of breast cancer in the brain

Blood-borne metastasis to the brain is a major complication of breast cancer, but cellular pathways that enable cancer cells to selectively grow in the brain microenvironment are poorly understood. We find that cultured circulating tumor cells (CTCs), derived from blood samples of women with advanced breast cancer and directly inoculated into the mouse frontal lobe, exhibit striking differences in proliferative potential in the brain. Derivative cell lines generated by serial intracranial injections acquire selectively increased proliferative competency in the brain, with reduced orthotopic tumor growth. Increased Hypoxia Inducible Factor 1A (HIF1A)-associated signaling correlates with enhanced proliferation in the brain, and shRNA-mediated suppression of HIF1A or drug inhibition of HIF-associated glycolytic pathways selectively impairs brain tumor growth while minimally impacting mammary tumor growth. In clinical specimens, brain metastases have elevated HIF1A protein expression, compared with matched primary breast tumors, and in patients with brain metastases, hypoxic signaling within CTCs predicts decreased overall survival. The selective activation of hypoxic signaling by metastatic breast cancer in the brain may have therapeutic implications.

A HIF1a-Dependent Pro-Oxidant State Disrupts Synaptic Plasticity and Impairs Spatial Memory in Response to Intermittent Hypoxia.

Sleep apnea causes cognitive deficits and is associated with several neurologic diseases. Intermittent hypoxia (IH) is recognized as a principal mediator of pathophysiology associated with sleep apnea, yet the basis by which IH contributes to impaired cognition remains poorly defined. Using a mouse model exposed to IH, this study examines how the transcription factor, hypoxia inducible factor 1a (HIF1a), contributes to disrupted synaptic physiology and spatial memory. In wild-type mice, impaired performance in the Barnes maze caused by IH coincided with a loss of NMDA receptor (NMDAr)-dependent long-term potentiation (LTP) in area CA1 and increased nuclear HIF1a within the hippocampus. IH-dependent HIF1a signaling caused a two-fold increase in expression of the reactive oxygen species (ROS) generating enzyme NADPH oxidase 4 (NOX4). These changes promoted a pro-oxidant state and the downregulation of GluN1 within the hippocampus. The IH-dependent effects were not present in either mice heterozygous for Hif1a (HIF1a+/−) or wild-type mice treated with the antioxidant manganese (III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP). Our findings indicate that HIF1a-dependent changes in redox state are central to the mechanism by which IH disrupts hippocampal synaptic plasticity and impairs spatial memory. This mechanism may enhance the vulnerability for cognitive deficit and lower the threshold for neurologic diseases associated untreated sleep apnea.

Metabolic Rewiring in Response to Biguanides Is Mediated by mROS/HIF-1a in Malignant Lymphocytes

Metabolic flexibility allows cells to adapt to various environments and limits the efficacy of metabolic drugs. Therapeutic targeting of cancer metabolism relies on defining limiting requirements and vulnerabilities in the highly dynamic metabolic network. Here, we characterize the metabolic reprogramming and identify cancer-specific metabolic vulnerabilities in response to the pharmacological inhibition of mitochondrial complex I. Our work reveals the adaptation mechanism in malignant lymphocytes providing resistance against the biguanides phenformin and metformin by transcriptionally reprogramming glucose metabolism. Metabolic adaptation to complex I inhibition is mediated by mitochondrial reactive oxygen species (mROS) serving as a mitochondrial stress signal activating hypoxia-inducible factor-1a (HIF-1a). Inhibition of the mROS/HIF-1a axis through antioxidants or direct suppression of HIF-1a selectively disrupts metabolic adaptation and survival during complex I dysfunction in malignant lymphocytes. Our results identify HIF-1a signaling as a critical factor in resistance against biguanide-induced mitochondrial dysfunction, allowing selective targeting of metabolic pathways in leukemia and lymphoma.

Duration‐dependent effects of IH‐stimulated Hif1a signaling on adult hippocampal neurogenesis

Intermittent hypoxia (IH), is a hallmark consequence of sleep apnea, a common untreated disorder associated with impaired neurological health. We have recently demonstrated that thirty days of IH impairs long term potentiation and reduced the proportion of neurons generated during adult neurogenesis. Hypoxia Inducible Factor 1a (HIF1a) is a transcription factor important to stem cell development that is regulated by the state of oxygenation. The objective of this on‐going study is to characterize how the duration of IH exposure influences the generation of adult‐born neurons and to determine the role of IH‐dependent HIF1a signaling in the generation of adult‐born neurons in the hippocampus. We hypothesize that IH has duration dependent influence over neurons generated through adult neurogenesis and involves HIF1a dependent signaling. We used a cre‐loxP transgenic strategy to birth label a discrete cohort of stem cells that experience room air for 30 days (control), 10 days of IH +20 days of recovery in room air (IH10+20RA), or 30 days of IH (IH30) in wild‐type reporter mice (Ai27) and in Ai27‐HIF1a+/flox mice. In wild‐type mice, exposure to IH10+20RA led to higher proportion of birth‐labelled neurons compared to the control (+19% ± 5%, n=6) while the proportion of neurons following IH30 decreased (−40% ± 10%, n=4). In Ai27‐HIF1a+/− mice, the proportion of birth‐labeled neurons was similar between control and IH10+20RA groups. However, following IH30 the proportion of birth‐labeled neurons was markedly decreased when compared to control in Ai27‐Hif1a+/− and to the IH30 wild‐type group. Dendritic complexity, as determined by Sholl analysis, was similar among labeled neurons in both wild‐type and Ai27‐HIF1a+/− groups suggesting that neither IH nor IH‐dependent HIF1a signaling influences dendritic complexity. These findings indicate that exposure duration is factor impacting how IH affects the generation of adult‐born neurons through a HIF1a signaling shaping how IH influences, associated with sleep apnea affects the generation of adult‐born neurons.Support or Funding InformationThis work was supported by NIH R01 NS10742101 (AJG).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reduced HIF1a Signaling Enhances Apneas During Perinatal Development

Immature neonatal breathing is characterized by the occurrence of periodic apneas that disrupt the breathing rhythm and cause intermittent hypoxia (IH). IH has been proposed as a factor that facilitates the development of respiratory control allowing for the transition from unstable to stable breathing rhythms where apneas no longer occur. In adults, IH increases hypoxia inducible factor 1a (HIF1a) signaling that remodels respiratory control and augments peripheral chemoreflexes. However, it is unclear whether HIF1a signaling contributes to the stabilization of immature neonatal breathing during the first days of perinatal life. To address this, we used whole body plethysmography to track perinatal development of breathing beginning at postnatal day zero (P0) in mice heterozygous for hypoxia inducible factor 1a (HIF1a+/−) and wildtype littermates (WT). At P0 we observed unstable breathing rhythms characterized by periodic apneas in both genotypes. However, in HIF1a+/− (n=4) apnea number was 50% greater and apnea duration was 172% longer when compared to the WT (n=3). Our ongoing experiments suggest that the switch to stable breathing rhythms occurs between postnatal days three and four. Our findings indicate that HIFa signaling is important to the of the neonatal breathing rhythm.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Duration Dependent Effects of Intermittent Hypoxia (IH) on Adult Hippocampal Neurogenesis

Patients with sleep apnea commonly exhibit cognitive decline and injury to the hippocampal formation. Rodents exposed to IH, a primary consequence of the condition, also show impaired hippocampal based learning and memory. While these impairments correlate with suppressed long‐term potentiation, synaptic plasticity is only one process that supports normal function in this brain structure. Adult neurogenesis is also important for maintaining normal hippocampal physiology. Although Hypoxia Inducible Factor 1a (HIF1a) signaling is to important cellular development and homeostatic responses to hypoxia, chronic IH‐dependent HIF1a activity can promote pathophysiology. In this ongoing study, we test the hypothesis that IH causes HIF1a dependent signaling in neural stem cells (NSCs) to disrupt the adult neurogenesis. To follow the fate of wildtype NSCs and NSCs heterozygous for HIF1a (HIF1a+/−), we used a birth labeling strategy in transgenic mice. After labeling NSCs, we exposed mice to 0 (control), 10, or 30 days of IH. Tissue was harvested for immunohistochemical study at the 33 to 35 day time point. Relative to control, 10 days of IH increased the proportion of GCs generated from wildtype NSCs (by 23% ± 1% SEM, n=3); whereas, 30 days IH suppressed this proportion (−9% ± 2% SEM, n=2). Sholl analysis showed dendritic complexity was similar across all wildtype GCs. 30 days IH exaggerated the loss of GCs generated from HIF1a+/− NSCs (by −30% ± 5%, n=3). These findings indicate that IH has duration‐dependent effects on adult neurogenesis that impact the terminal differentiation of NSCs to GCs and involves IH‐dependent HIF1a signaling in NSCs. These findings suggest that IH‐dependent HIF1a signaling has both positive and negative effects on adult neurogenesis. This mechanism could be a contributor to the negative effects on learning and memory commonly seen in sleep apnea patients.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

HIF1A is a critical downstream mediator for hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome characterized by overwhelming immune activation. A steroid and chemotherapy-based regimen remains as the first-line of therapy but it has substantial morbidity. Thus, novel, less toxic therapy for HLH is urgently needed. Although differences exist between familial HLH (FHL) and secondary HLH (sHLH), they have many common features. Using bioinformatic analysis with FHL and systemic juvenile idiopathic arthritis, which is associated with sHLH, we identified a common hypoxia-inducible factor 1A (HIF1A) signature. Furthermore, HIF1A protein levels were found to be elevated in the lymphocytic choriomeningitis virus infected Prf1−/− mouse FHL model and the CpG oligodeoxynucleotide-treated mouse sHLH model. To determine the role of HIF1A in HLH, a transgenic mouse with an inducible expression of HIF1A/ARNT proteins in hematopoietic cells was generated, which caused lethal HLH-like phenotypes: severe anemia, thrombocytopenia, splenomegaly, and multi-organ failure upon HIF1A induction. Mechanistically, these mice show type 1 polarized macrophages and dysregulated natural killler cells. The HLH-like phenotypes in this mouse model are independent of both adaptive immunity and interferon-γ, suggesting that HIF1A is downstream of immune activation in HLH. In conclusion, our data reveal that HIF1A signaling is a critical mediator for HLH and could be a novel therapeutic target for this syndrome.

MP32-08 THE EFFECT OF HYPOXIA-INDUCIBLE FACTOR-1? EXPRESSION IN BIOPSY SPECIMEN AFTER REPERFUSION ON THE EARLY RECOVERY OF GRAFT FUNCTION AFTER CADAVERIC KIDNEY TRANSPLANTATION

You have accessJournal of UrologyTransplantation & Vascular Surgery: Renal Transplantation & Vascular Surgery II1 Apr 2016MP32-08 THE EFFECT OF HYPOXIA-INDUCIBLE FACTOR-1? EXPRESSION IN BIOPSY SPECIMEN AFTER REPERFUSION ON THE EARLY RECOVERY OF GRAFT FUNCTION AFTER CADAVERIC KIDNEY TRANSPLANTATION Teruyuki Oda, Takeshi Ishimura, and Masato Fujisawa Teruyuki OdaTeruyuki Oda More articles by this author , Takeshi IshimuraTakeshi Ishimura More articles by this author , and Masato FujisawaMasato Fujisawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1303AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Ischemia/reperfusion injury during kidney transplantation delays allograft recovery. Hypoxia-inducible factor-1a (HIF-1a) is considered to be the key regulator of cell response to hypoxia and regulates several genes responsible for tissue protection. In this study, we evaluated the expression levels of major proteins mediating HIF-1a signaling by immunohistochemical staining of graft biopsy specimens at 1-h of revascularization during cadaveric kidney transplantation, and examined the correlation between these findings and recovery of allograft function. METHODS Between 1996 and 2015, 46 patients underwent cadaveric kidney transplantation at our institution. Of these, 15 patients received kidneys from heart beating donors (HBDs) and 31 from non-heart beating donors (NHBDs). Immunohistochemical staining was performed to evaluate the expression levels of HIF-1a-related proteins, including PI3K, phosphorylated (p)-Akt, p-mammalian target of rapamycin (mTOR), p-eIF4E, p-S6 ribosomal protein, and HIF-1a in graft biopsy specimens at 1 h of revascularization (1H). Based on the percentage of positive staining area in the specimen, immunohistochemical findings were scored from 0 to 3. 0-hour biopsy specimens from 10 who underwent living-donor kidney transplantation (0H) were used as control. Delayed graft function (DGF) was defined as the need for dialysis within the first week after kidney transplantation. We compared the staining score of each protein and several clinical parameters between patients with and without DGF. RESULTS Expression levels of all 6 proteins were elevated at 1 H compared with those at 0H, with significant differences for pAKT, pS6, and HIF-1a. Thirty five patients experienced DGF. We observed significant higher expression of all 6 proteins, in patients without DGF than in DGF patients. Univariate analysis identified the expression levels of p-Akt, p-S6, and HIF-1a, in addition to donor type (HBD/NHBD), cold ischemic time, and donor age as significant predictors of DGF. Of these 6 significant parameters, only the expression levels of HIF-1a and donor type were found to be independently associated with DGF on multivariate analysis. CONCLUSIONS In conclusion, up-regulation of HIF-1a in allograft biopsy specimens after reperfusion might be a predictor of early recovery of graft function in cadaveric kidney transplantation. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e430 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Teruyuki Oda More articles by this author Takeshi Ishimura More articles by this author Masato Fujisawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Oxymatrine suppresses proliferation and induces apoptosis of hemangioma cells through inhibition of HIF-1a signaling.

Oxymatrine (OMT), a natural quinolizidine alkaloid, has been known to have anti-inflammation, anti-anaphylaxis, and chemopreventive effects on various cancer cells. To clarify the underlying role and molecular mechanisms of OMT in human hemangioma (HA), in the present study, we examined the expression of hypoxia-inducible factor-1a (HIF-1a) and vascular endothelial growth factor (VEGF) in different phases of human HA. After HA derived endothelial cells (HDEC) were pretreated with different concentrations of OMT, cell proliferation, apoptosis, and cycle distribution were evaluated by MTT assay and flow cytometry analysis, respectively. The effects of OMT on expression of HIF-1a signaling were determined by real-time PCR and western blot assays. Our results showed that, the expression of HIF-1a and VEGF was significantly increased in proliferating phase HA, but decreased in involuting phase HA. Moreover, OMT in vitro inhibited proliferative activities and induced cell apoptosis and cycle arrest in G0/G1 phase in HA cells with decreased expression of HIF-1a, VEGF, Bcl-2, and CyclinD1, and increased expression of p53. Taken together, our findings suggest that, the expression of HIF-1a and VEGF is increased in proliferating phase HA, and OMT suppresses cell proliferation and induces cell apoptosis and cycle arrest in proliferative phase HA through inhibition of the HIF-1a signaling pathway, suggesting OMT may provide a novel therapeutic strategy for the treatment of HA.

AML1/ETO Cooperates with HIF1α to Promote Leukemogenesis through DNMT3a Transactivation

The t(8;21)(q22;q22) translocation, resulting in a chimeric protein AML1/ETO (A/E), is one of the most common chromosomal abnormalities in acute myeloid leukemia (AML). It has been reported that additional mutagenic “hits” are required for A/E to be a leukemic driver, but it is unclear why most A/E-positive patients don’t carry such gene mutations. We hypothesized that unconventional and more universal events might cooperate with A/E to drive leukemogenesis. Hypoxia inducible factor 1a (HIF1a) is a transcription factor mediating the cellular response to hypoxia in malignant cells. To identify the key molecule responsible for A/E-driven leukemia, we focused on HIF1a signaling, as it is selectively activated in AML stem cells in a hypoxia-independent fashion.Using qPCR analysis, we measured HIF1a mRNA levels in bone marrow mononuclear cells from 73 A/E-positive and 59 A/E-negative patients with newly diagnosed AML and 15 healthy donors. HIF1a was highly expressed in A/E-positive patients, as compared to A/E-negative patients and healthy controls. The expression of HIF1a was positively correlated with the expression of A/E, independent of KIT mutation. Patients were grouped into quartiles according to HIF1a expression levels (Q1-Q4, each quartile containing 25% of patients) and divided into high HIF1a (HIF1a high, Q4; n = 33) and low HIF1a (HIF1a low, Q1-Q3; n = 99). AE-positive patients with high HIF1a expression had a significantly shorter OS (median = 19.0 months vs. not reached, P = 0.015) and EFS (median = 9.0 months vs. 24.0 months, P = 0.001) compared to those with low HIF1a expression. Notably, HIF1a-associated inferior prognosis in A/E-positive patients was KIT mutation-independent. In patients carrying wild-type KIT [n = 53, about 73% (53/73) of the entire A/E-patients], those with high HIF1a levels had a shorter OS (median = 23.1 months vs. not reached, P = 0.008) and EFS (median = 9.4 months vs. not reached, P = 0.001) than those with low HIF1a levels. Multivariate analysis revealed that high HIF1a levels were independent prognostic indicators for both OS and EFS (P = 0.014, HR = 3.574 and P = 0.004, HR = 4.304, respectively) in AE-positive patients. Mechanistic studies revealed that A/E and HIF1a formed a positive regulatory circuit, in which A/E bound to HIF1a gene promoter and transactivated HIF1a or vice versa. Co-expression of AE and HIF1a in leukemia cells caused a higher cell proliferation rate in vitro and more serious leukemic status in mice. To gain insight into the molecular basis of A/E-HIF1a functional cooperation in leukemogenesis, we examined the mRNA levels of DNMT1, DNMT3a and DNMT3b expression in bone marrow samples from above-mentioned AML patients and healthy donors using qPCR. The result showed that DNMT3a, but not DNMT1 and DNMT3b, was significantly overexpressed in A/E-positive patients, as compared to A/E-negative patients and healthy donors. DNMT3a levels were positively correlated with both A/E and HIF1a levels. We therefore focused on evaluating the role of DNMT3a in A/E-positive AML. We found that DNMT3a expression was positively correlated with both A/E and HIF1a levels. As an individual event, A/E or HIF1a was enriched on DNMT3a gene promoter and synergistically increased DNMT3a transcription and enhanced global DNA methylation. Pharmacological or genetic interventions in the AE-HIF1a loop resulted in DNA hypomethylation, a reexpression of hypermethylated tumor suppressor p15INK4band the blockage of leukemia growth.In summary, we showed that A/E and HIF1a form a regulatory circuit and cooperatively control aggressive leukemia growth through DNMT3a transactivation and subsequent DNA hypermethylation. Thus, the HIF1a-DNMT3a nexus serves as a reliable marker, which identifies patients with a poor prognosis in an otherwise prognostically favorable AML group, and represents an innovative therapeutic target in high-risk AE-positive leukemia. DisclosuresNo relevant conflicts of interest to declare.

Abstract 18906: Congenital Right Ventricular Hypertrophy Compensation is Characterized by Sustained Angiogenesis

The right ventricle (RV) is hypertrophied at birth and in the neonatal period. The RV regresses to a normal phenotype in the first month of life correlated with a concomitant drop PVR over that time. The RV may remain hypertrophied and compensated in response to maintained afterload in patients with congenital heart disease (CHD) for decades; whereas the hypertrophied RV acquired in adulthood from pulmonary arterial hypertension (PAH) quickly transitions to failure, and remains their primary determinant of mortality in PAH. Potentially, the transition from RV compensation to failure has been attributed to ischemia from deficient capillary density from inadequate angiogenesis in RV hypertrophy. Angiogenesis is largely regulated by the transcription factor hypoxia-inducible factor 1a (HIF1a), which governs the transcription of proangiogenic signaling effectors. Two such effectors strongly promoting angiogenesis under HIF1a regulation are vascular endothelial growth factor (VEGF) and stromal derived factor 1 (SDF1), which promote endothelial cell survival/proliferation and recruitment of CXCR4+ stem cells, respectively. We hypothesized that sustained HIF1a-driven angiogenesis maintains adequate RV vascularity in hypertrophy, thus preventing the transition from RV compensation to failure in CHD patients. The free walls of neonatal rat RVs had elevated levels of HIF1a (immunoblot and nuclear localization) compared to those of adult RVs, adult LVs, and neonatal LVs. This was associated with increased VEGF and SDF1 expression (Immunoblot and ELISA), which correlated with increased angiogenesis (matrigel assay) and CXCR4+ stem cell recruitment (Boyden chamber migration assay). Neonatal RVs had elevated expression vascular density (CD31 expression) and tissue perfusion (Lectin) compared to adult RVs, further supporting proangiogenic signaling. Human compensated RV hypertrophy myocardium from CHD patients had elevated SDF1 and CXCR4 expression compared to normal adult RV and LV myocardium (immunoblot), suggesting HIF1a driven angiogenic pathways are maintained in CHD tissues. Therapies that maintain HIF1a signaling may delay the transition from compensation to failure in adults with acquired PAH.

Suppression of Hypoxia-Inducible Factor-1a Contributes to the Antiangiogenic Activity of Red Propolis Polyphenols in Human Endothelial Cells 3

Polyphenol-enriched fractions from natural sources have been proposed to interfere with angiogenesis in pathological conditions. We recently reported that red propolis polyphenols (RPP) exert antiangiogenic activity. However, molecular mechanisms of this activity remain unclear. Here, we aimed at characterizing molecular mechanisms to explain the impact of RPP on endothelial cells' (EC) physiology. We used in vitro and ex and in vivo models to test the hypothesis that RPP inhibit angiogenesis by affecting hypoxia-inducible factor-1a (HIF1a) stabilization in EC. RPP (10 mg/L) affected angiogenesis by reducing migration and sprouting of EC, attenuated the formation of new blood vessels, and decreased the differentiation of embryonic stem cells into CD31-positive cells. Moreover, RPP (10 mg/L) inhibited hypoxia- or dimethyloxallylglycine-induced mRNA and protein expression of the crucial angiogenesis promoter vascular endothelial growth factor (VEGF) in a time-dependent manner. Under hypoxic conditions, RPP at 10 mg/L, supplied for 1–4 h, decreased HIF1a protein accumulation, which in turn attenuatedVEGFgene expression. In addition, RPP reduced the HIF1a protein half-life from ~58 min to 38 min under hypoxic conditions. The reduced HIF1a protein half-life was associated with an increase in the von Hippel-Lindau (pVHL)-dependent proteasomal degradation of HIF1a. RPP (10 mg/L, 4 h) downregulated Cdc42 protein expression. This caused a corresponding increase in pVHL protein levels and a subsequent degradation of HIF1a. In summary, we have elucidated the underlying mechanism for the antiangiogenic action of RPP, which attenuates HIF1a protein accumulation and signaling.