HIF Genes Research Articles

Pilot Study on the Effect of Patient Condition and Clinical Parameters on Hypoxia-Induced Factor Expression: HIF1A, EPAS1 and HIF3A in Human Colostrum Cells

Hypoxia-inducible factor 1 (HIF-1) may play a role in mammary gland development, milk production and secretion in mammals. Due to the limited number of scientific reports on the expression of HIF genes in colostrum cells, it was decided to examine the expression of HIF1A, HIF3A and EPAS1 in the these cells, collected from 35 patients who voluntarily agreed to provide their biological material for research, were informed about the purpose of the study and signed a consent to participate in it. The expression of HIF genes was assessed using qPCR. Additionally, the influence of clinical parameters (method of delivery, occurrence of stillbirths in previous pregnancies, BMI level before pregnancy and at the moment of delivery, presence of hypertension during pregnancy, presence of Escherichia coli in vaginal culture, iron supplement and heparin intake during pregnancy) on the gene expression was assessed, revealing statistically significant correlations. The expression of HIF1A was 3.5-fold higher in the case of patients with the presence of E. coli in vaginal culture (p = 0.041) and 2.5 times higher (p = 0.031) in samples from women who used heparin during pregnancy. Approximately 1.7-fold higher expression of the EPAS1 was observed in women who did not supplement iron during pregnancy (p = 0.046). To our knowledge, these are the first studies showing the relationship between HIF expression in cells from breast milk and the method of delivery and health condition of women giving birth. The assessment of HIF expression requires deeper examination in a larger study group, and the results of further studies will allow to determine whether HIF can become biomarkers in pregnancy pathology states.

Effects of Environmental Hypoxia on Serum Hematological and Biochemical Parameters, Hypoxia-Inducible Factor (hif) Gene Expression and HIF Pathway in Hybrid Sturgeon (Acipenser schrenckii ♂ × Acipenser baerii ♀).

Hypoxia is a globally pressing environmental problem in aquatic ecosystems. In the present study, a comprehensive analysis was performed to evaluate the effects of hypoxia on physiological responses (hematology, cortisol, biochemistry, hif gene expression and the HIF pathway) of hybrid sturgeons (Acipenser schrenckii ♂ × Acipenser baerii ♀). A total of 180 hybrid sturgeon adults were exposed to dissolved oxygen (DO) levels of 7.00 ± 0.2 mg/L (control, N), 3.5 ± 0.2 mg/L (moderate hypoxia, MH) or 1.00 ± 0.1 mg/L (severe hypoxia, SH) and were sampled at 1 h, 6 h and 24 h after hypoxia. The results showed that the red blood cell (RBC) counts and the hemoglobin (HGB) concentration were significantly increased 6 h and 24 h after hypoxia in the SH group. The serum cortisol concentrations gradually increased with the decrease in the DO levels. Moreover, several serum biochemical parameters (AST, AKP, HBDB, LDH, GLU, TP and T-Bil) were significantly altered at 24 h in the SH group. The HIFs are transcription activators that function as master regulators in hypoxia. In this study, a complete set of six hif genes were identified and characterized in hybrid sturgeon for the first time. After hypoxia, five out of six sturgeon hif genes were significantly differentially expressed in gills, especially hif-1α and hif-3α, with more than 20-fold changes, suggesting their important roles in adaptation to hypoxia in hybrid sturgeon. A meta-analysis indicated that the HIF pathway, a major pathway for adaptation to hypoxic environments, was activated in the liver of the hybrid sturgeon 24 h after the hypoxia challenge. Our study demonstrated that hypoxia, particularly severe hypoxia (1.00 ± 0.1 mg/L), could cause considerable stress for the hybrid sturgeon. These results shed light on their adaptive mechanisms and potential biomarkers for hypoxia tolerance, aiding in aquaculture and conservation efforts.

The putative role of ferroptosis in gastric cancer: a review.

Ferroptosis is a unique cell death modality triggered by iron-dependent lipid peroxidation, with cysteine metabolism and glutathione-dependent antioxidant defence responses as the primary triggering mechanisms. Ferroptosis is an independent tumour suppression mechanism and has been implicated in various disorders. In tumourigenesis, ferroptosis plays a dual role in promoting and inhibiting tumours. P53, NFE2L2, BAP1, HIF, and other tumour suppressor genes regulate ferroptosis, releasing damage-associated molecular patterns or lipid metabolites to influence cellular immune responses. Ferroptosis is also involved in tumour suppression and metabolism. The combination of amino acid, lipid, and iron metabolism is involved in the initiation and execution of ferroptosis, and metabolic regulatory mechanisms also play roles in malignancies. Most investigations into ferroptosis in gastric cancer are concentrated on predictive models, not the underlying processes. This review investigates the underlying mechanisms of ferroptosis, tumour suppressor genes, and the tumour microenvironment.

Effect of secondary attack by Aeromonas hydrophila on the expression level of hif genes in common carp (Cyprinus carpio).

Hypoxia-inducible factors (hifs) are involved in infectious diseases inflammatory reactions, and immune regulation. Common carp, a representative allotetraploid species that has undergone genome-wide replication events, has important research value. In this study, common carp were infected twice with Aeromonas hydrophila. Liver tissues of common carp were collected at 4h, 12h, 24h, 48h, 3days, 7days post-first infection and 4h, 12h, 24h post-second infection. The mRNA levels of hif genes were determined at different time points. The hif2a-2, hif3a-2, hif3b-1 and hif3b-2 expression levels in the infected group were upregulated when compared with those in the control group, whereas the expression levels of other genes were downregulated after the second infection. This indicates that the effect of A.hydrophila infection on gene expression pattern is dependent on the host, pathogen, infected tissue and gene. Pressure analysis of the hif gene family revealed that the non-synonymous substitution to synonymous substitution ratio of 12 hif genes was <1, which indicated that they were in a state of purification and selection. Combined with the differences between copy genes, the polyclonal antibodies against Hif1b-1 and Hif1b-2 were successfully prepared in this study. Western blot analysis showed that the protein expression of Hif1b-1 and Hif1b-2 reached to the highest level 48h after the first infection. After the second A.hydrophila infection, the protein expression levels of Hif1b-1 and Hif1b-2 reached the highest levels at 4 and 48h, respectively. This may indicate that the Hif1b-1 and Hif1b-2 genes in common carp play an important role in the immune mechanism at the protein level. The findings of this study will lay the foundation for future studies on the immune regulatory function of common carp hif genes, which may aid in devising novel therapeutic strategies for common carp diseases, such as A.hydrophila infection.

Interleukin 13 (IL-13) alters hypoxia-associated genes and upregulates CD73.

Interleukin 13 (IL-13) is a pleiotropic cytokine that has been shown to be important in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) and other type 2 inflammation-related diseases. Increased IL-13 expression can elicit several pro-inflammatory effects, including eosinophilia, and pathology such as increased mucus secretion. Polypogenesis in chronic rhinosinusitis (CRS) can be caused by hypoxia, which can also lead to hyperpermeability of airway epithelium and epithelium-to-mesenchymal translation through the upregulation of hypoxia-associated genes, such as HIF1. Whether T-helper 2 (Th2) inflammatory cytokines, such as IL-13, can also induce sinonasal epithelial hypoxia-associated genes is currently unknown. Human air-liquid interface (ALI) sinonasal epithelial cell cultures treated with recombinant IL-13 were analyzed by real-time polymerase chain reaction (PCR) and flow cytometry to determine the effect on epithelial cells. Whole tissue from CRSwNP subjects showed increased HIF1A gene expression. Treatment of fully differentiated human ALI cultures with IL-13 resulted in a concurrent increase in HIF1A and ARNT messenger RNA (mRNA) expression. However, the level of EPAS1 expression was significantly reduced. IL-13 also had a dose-dependent response on the expression of HIF genes and the time course experiment showed peak expression of HIF1A and ARNT at 5 to 7 days poststimulation. Remarkably, CD73 surface expression also peaked at day 5 poststimulation. Our data suggests that IL-13 can induce hypoxia signaling pathway genes leading to surface expression of CD73, which has an anti-inflammatory effect.

Physiological and genomic evidence that selection on the transcription factor Epas1 has altered cardiovascular function in high-altitude deer mice.

Evolutionary adaptation to extreme environments often requires coordinated changes in multiple intersecting physiological pathways, but how such multi-trait adaptation occurs remains unresolved. Transcription factors, which regulate the expression of many genes and can simultaneously alter multiple phenotypes, may be common targets of selection if the benefits of induced changes outweigh the costs of negative pleiotropic effects. We combined complimentary population genetic analyses and physiological experiments in North American deer mice (Peromyscus maniculatus) to examine links between genetic variation in transcription factors that coordinate physiological responses to hypoxia (hypoxia-inducible factors, HIFs) and multiple physiological traits that potentially contribute to high-altitude adaptation. First, we sequenced the exomes of 100 mice sampled from different elevations and discovered that several SNPs in the gene Epas1, which encodes the oxygen sensitive subunit of HIF-2α, exhibited extreme allele frequency differences between highland and lowland populations. Broader geographic sampling confirmed that Epas1 genotype varied predictably with altitude throughout the western US. We then discovered that Epas1 genotype influences heart rate in hypoxia, and the transcriptomic responses to hypoxia (including HIF targets and genes involved in catecholamine signaling) in the heart and adrenal gland. Finally, we used a demographically-informed selection scan to show that Epas1 variants have experienced a history of spatially varying selection, suggesting that differences in cardiovascular function and gene regulation contribute to high-altitude adaptation. Our results suggest a mechanism by which Epas1 may aid long-term survival of high-altitude deer mice and provide general insights into the role that highly pleiotropic transcription factors may play in the process of environmental adaptation.

Multicenter prospective study of angiogenesis polymorphism validation in HCC patients treated with sorafenib. An INNOVATE study protocol.

Although sorafenib is the upfront standard of care for advanced hepatocellular carcinoma (HCC), molecular predictors of efficacy have not been identified yet. In the ALICE-1 study, rs2010963 of VEGF-A and VEGF-C proved to be independent predictive factors for progression-free survival (PFS) and overall survival (OS) in multivariate analysis. The ALICE-1 study results were confirmed in the ALICE-2 study, in which VEGF and VEGFR SNPs were analyzed. In the ePHAS study we analyzed the SNPs of eNOS. In univariate analysis, patients homozygous for an eNOS haplotype (HT1: T-4b at eNOS-786/eNOS VNTR) had significantly shorter median PFS and OS than those with other haplotypes. These data were confirmed in the validation set. This nonpharmacological, interventional, prospective multicenter study aims to determine whether eNOS, HIF-1, VEGF, Ang2 and VEGFR polymorphisms play a role in predicting the objective response rate, PFS, and OS of advanced HCC patients treated with sorafenib. The study will involve 160 advanced HCC patients with Child-Pugh class A disease. The primary aim is to validate the prognostic or predictive roles of eNOS, Ang2, HIF-1, VEGF and VEGFR polymorphisms in relation to the clinical outcome (PFS) of HCC patients treated with sorafenib. Overall, our data may suggest that polymorphism analysis of the VEGF, VEGFR-2, HIF and eNOS genes can identify HCC patients who are more likely to benefit from sorafenib.

The effect of zinc deficiency and diet restriction on gene expression of hypoxia‐inducible factors in rat's kidney

We reported that plasma erythropoietin (EPO) concentration, kidney EPO concentration and kidney EPO mRNA level were changed by moderate zinc deficiency or diet restriction. It is not established how moderate zinc deficiency or diet restriction induces these alterations. We investigated whether moderate zinc deficiency or diet restriction affects renal hypoxia‐inducible factor genes expression in rats.Thirty 3‐week‐old male Sprague‐Dawley rats were assigned into 3 dietary groups (n=10 each) and fed on dietary regimens for 4 weeks: control group (AIN‐93G; Control), moderate zinc‐deficient group (4.5 mg Zn/kg) and pair‐fed group (AIN‐93G; diet restriction). Kidney was separated into cortex and medulla for RNA isolation. Quantitative real time‐PCR using TaqMan proves was performed for mRNA measurement of hypoxia‐inducible factor (Hif) genes (Hif‐1a, Hif‐1an and Hif‐3a). Statistical analysis was performed by ANOVA and PLSD with separate variance approach (when normality was satisfied) or the Kruskal‐Wallis and Conover‐Iman tests (when normality was not satisfied). P value less than 0.05 was considered significant. Kidney medulla mRNA expressions per organ weight of Hif‐1a and Hif‐1an were significantly decreased by zinc deficiency or diet restriction compared with Control. Kidney medulla Hif‐3a and cortex Hif‐1a, Hif‐1an and Hif‐3a mRNA expressions per organ weight were not significantly different among all groups.These results suggest that kidney hypoxia‐inducible factor gene expressions are decreased by zinc deficiency and diet restriction.Support or Funding InformationThis work was supported by KAKENHI grant‐in‐aid for Young Scientists (B) (23700928) and (15K16216).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Next Generation Sequencing Is a Useful Tool for the Diagnosis of Congenital/Idiopathic Erythrocytoses

Introduction: Erythrocytoses are characterized by an elevated red cell mass. The most widely studied disease is Polycythemia Vera (PV), a myeloproliferative neoplasm due to the acquired JAK2-V617F mutation. However, other types of erythrocytoses exist and are of major importance. They can be either inherited (Congenital Erythrocytosis-CE) or diagnosed in adult patients with no family history (Idiopathic Erythrocytosis-IE). CE/IE are not associated with myeloproliferation but they can be associated with severe thrombo-embolic or haemorrhagic events, pulmonary arterial hypertension and, rarely, tumours. The 8 genes identified so far as causing CE lie at the crossroads of major biological pathways (metabolism, inflammation, oncogenesis) and are implicated in multiple diseases. These genes are involved (i) in the regulation of the hypoxia pathway, PHD2 (also called EGLN1), HIF-2A (EPAS1), VHL, (ii) in proliferation and differentiation of erythroid progenitors (EPOR), or (iii) in mature cell function, haemoglobins (HBB, HBA1, HBA2) or bisphosphoglyceratemutase (BPGM). However, in 80% of cases the cause remains unknown meaning that no proper diagnosis can be made, no prognosis or advice can be provided to CE/IE patients and their families, and no curative treatment exists.Method: We created and developed a national network in France to (i) identify, (ii) collect and (iii) analyze the genomic abnormalities in patients suspected of CE/IE. The selection of patients was performed using a clinical and biological data sheet including mandatory further tests in order to exclude patients with PV or obvious secondary erythrocytosis related to lung, cardiac or renal disorder. Next generation sequencing (NGS) has been used to analyse the presence of mutations in 17 genes (VHL, PHD1, PHD2 and PHD3, HIF1A, HIF2A, HIF3A, FH, BPGM, and 8 other candidate genes). SureDesign software (Agilent, Santa Clara, CA) was used to design the custom HaloPlex capture assay. For sequence capture, HaloPlex Target Enrichment System Kit (AgilentR), for Illumina sequencing was used, according to the manufacturer's instructions.Results: To date, samples from 103 patients have been recorded, among whom 46 have been tested using NGS approach. Variants in 10 (21%) patients [9 males and 1 female ; median age 50 y. (12-71)] with unknown significance have been detected, including 4 in PHD genes, 5 in HIF genes, and 1 in JAK2 gene.In patients with variants, a familial history of erythrocytosis was noted in 2. No independent thrombotic complication was reported in the 10 patients. The proportion of variants detected (21%) was close to the classical rate of genomic abnormalities usually observed in CE/IE. In 2 patients (one with a PHD2 and one with a JAK2 variants), the erythropoietin was low, whereas for the others, the erythropoietin was normal. Of note, the median age of the patients was surprisingly high, suggesting that the diagnostic was not previously performed due to the absence of available tests. Indeed, the diagnostic approaches using NGS techniques led to a considerable time gain and facilitated the identification of certain molecular abnormalities associated with CE/IEConclusion: NGS is a useful tool to explore mutations in CE/IE, but identifies genetic variants in only 20% of patients with such disorder. In vitro, in cellulo and in vivo (including zebrafish models) functional studies are currently performed to validate the clinical relevance of these variants. Further exams including whole exome sequencing are planned to achieve a right diagnosis in the 80% remaining CE/IE patients without identified genomic abnormalities. DisclosuresNo relevant conflicts of interest to declare.

Expression of HIF1a and HIF2a in bone metastatic clear cell renal cell cancer and use as prognostic markers.

e15523 Background: Bone metastasis is a frequent complication of ccRCC and predictors of this type of progression are scanty. The aim of this study was to analyse HIF and HIF regulated gene expressions in bone metastatic ccRCC. Methods: A pre-target therapy era cohort of 125 ccRCC patients with &lt;100 month follow-up period have been collected with paraffin embedded primary tumors. 65 patients developed bone metastasis where 45 metastases were available for analysis. RNA was isolated from FFPE tissues and gene expression of HIF1A, HIF2A as well as HIF-regulated genes GLUT1, LDH5, GAPDH, CAIX, VEGF, VEGFR2 and EPOR were measured by RT-PCR using B2M as control. Protein product of these genes was measured on TMA arrays using standardized immunohistochemistry and an image analysis program (Mirax Viewer, 3DHistech). Results: Expression of HIF1A and HIF2A was significantly increased in bone metastatic primary ccRCC tumor tissues both at mRNA and protein levels. Significant upregulation of 3 out of 7 HIF-regulated genes (VEGFR2, EPOR and LDH5) in the primary tumor tissues of bone metastatic ccRCC was proved at mRNA levels and confirmed at protein level for VEGFR2 as well. In bone metastatic tissues a significant overexpression of HIF2A and VEGF mRNA was also detected compared to the primary site. Prognostic power of the protein expression in primary tumor was found for HIF1A (p= .001), HIF2A (p = .041), and VEGFR2 (p = .001) (confirmed at mRNA levels as well) for overall survival at time of surgical removal of primary renal tumor using Kaplan-Meier method and log-rank statistics. To further test the power of HIF1A and HIF2A as prognostic factors we have performed multivariate analysis considering available clinicopathological variables (gender, Fuhrman grade, stage) and found that HIF2A protein level outperformed all other factors in the model. Conclusions: These data indicate that HIF genes are overexpressed in bone metastatic ccRCC among which HIF2A expression at mRNA and protein levels can be used as a strong independent prognostic marker.

A novel series of G-quadruplex ligands with selectivity for HIF-expressing osteosarcoma and renal cancer cell lines

A series of naphthalene derivatives with disubstituted triazole side-arms have been assembled by click chemistry. Lead compounds show a high level of selectivity for renal, osteo- and Ewing’s sarcomas that express the HIF-1α transcription factor. They also interact selectively with the quadruplex DNAs located in the promoter of the HIF genes and it is suggested that the mechanism of action involves inhibition of transcription by drug-mediated quadruplex stabilization in these regions.

Highlights of This Issue

In this study, Trujillo and coworkers show that the field of histologically normal tissue up to 1 centimeter from human breast tumors contains a population of epithelial cellswith telomerase levels similar to the patient-matched tumor. This population has a mechanism for limitless replicative capacity and exists within tissue that has previously beenshown to exhibit genomic instability and a microenvironment that supports tumor initiation. Because adequate surgical margins are often no more than a few millimeters from the tumor margin, in many cases, this tissue is not removed during breast-conserving surgery and may have implications in local recurrence rates.The transmembrane serine protease hepsin is a highly upregulated gene in prostate cancer that promotes tumor progression and metastasis in preclinical models. Based on its substrate profile, Ganesan and colleagues investigated whether hepsin activates the precursor form of the plasminogen-like growth factor macrophage-stimulating protein (MSP). They show that both soluble and cell surface–expressed hepsin efficiently convert the inactive zymogen-like MSP precursor into a functionally competent heterodimeric form that induces phosphorylation of its cognate receptor RON and downstream signaling proteins promoting epithelial cell motility. These findings suggest that hepsin regulates the MSP/RON signaling pathway in cancer, immune disorders, and tissue homeostasis.FIP200 is a component of the ULK1/Atg13/FIP200 complex essential for autophagy induction in mammalian cells, which has been previously implicated in breast cancer. It is not clear, however, how autophagy function of FIP200 may contribute to its role in breast or other cancers. Here, Bae and Guan present data suggesting a novel function for FIP200 in the regulation of DNA damage response and cell survival through its activity in autophagy.These results suggest the possibility of FIP200 or other autophagy proteins as a potential treatment to enhance the efficiency of cancer therapy using DNA damage–inducing agents.To adapt to unfavorable growth conditions such as hypoxia, mTORC1, a critical regulator of cell growth, is inactivated. mTORC1 inhibition by hypoxia is mediated by REDD1, a HIF target gene. As a consequence of VHL inactivation, HIF and hypoxia-inducible genes are constitutively upregulated in clear-cell renal cell carcinoma (ccRCC), but whether this leads to REDD1 upregulation is unclear and mTORC1 is commonly activated in ccRCC. Kucejovaand colleagues show that REDD1 is consistently upregulated in ccRCC and that, whereas in some tumors REDD1 is engaged in mTORC1 inhibition, in others, strategies have evolved to uncouple mTORC1 from REDD1. Specifically, Kucejova and colleagues found somatic mutations in REDD1 and TSC1, which is required for mTORC1 inhibition by REDD1.

Characterization of genetic and phenotypic diversity of invasive nontypeable Haemophilus influenzae.

The ability of unencapsulated (nontypeable) Haemophilus influenzae (NTHi) to cause systemic disease in healthy children has been recognized only in the past decade. To determine the extent of similarity among invasive nontypeable isolates, we compared strain R2866 with 16 additional NTHi isolates from blood and spinal fluid, 17 nasopharyngeal or throat isolates from healthy children, and 19 isolates from middle ear aspirates. The strains were evaluated for the presence of several genetic loci that affect bacterial surface structures and for biochemical reactions that are known to differ among H. influenzae strains. Eight strains, including four blood isolates, shared several properties with R2866: they were biotype V (indole and ornithine decarboxylase positive, urease negative), contained sequence from the adhesin gene hia, and lacked a genetic island flanked by the infA and ksgA genes. Multilocus sequence typing showed that most biotype V isolates belonged to the same phylogenetic cluster as strain R2866. When present, the infA-ksgA island contains lipopolysaccharide biosynthetic genes, either lic2B and lic2C or homologs of the losA and losB genes described for Haemophilus ducreyi. The island was found in most nasopharyngeal and otitis isolates but was absent from 40% of invasive isolates. Overall, the 33 hmw-negative isolates were much more likely than hmw-containing isolates to have tryptophanase, ornithine decarboxylase, or lysine decarboxylase activity or to contain the hif genes. We conclude (i) that invasive isolates are genetically and phenotypically diverse and (ii) that certain genetic loci of NTHi are frequently found in association among NTHi strains.

Adhesin Expression in Matched Nasopharyngeal and Middle Ear Isolates of Nontypable Haemophilus influenzae from Children with Acute Otitis Media † 866

The HMW1 and HMW2 proteins, Hia, and hemagglutinating pili are important adherence factors in nontypable H. influenzae. To gain insight into the relative importance of these adhesins in nasopharyngeal colonization and localized respiratory tract disease, we assessed their expression in simultaneous nasopharyngeal and middle ear isolates of nontypable H. influenzae from 17 children with acute otitis media. In all patients, including 11 with bilateral disease, the matched isolates were isogenic based on total protein profiles and genomic fingerprints. Of the nasopharyngeal isolates, 14 expressed only HMW1/HMW2-like proteins, one expressed only Hia, one expressed only pili, and one expressed both Hia and pili. Further analysis revealed concordance between nasopharyngeal isolates and the matched middle ear isolates for expression of HMW1/HMW2-like proteins and Hia. In contrast, in the two children with piliated organisms colonizing the nasopharynx, the corresponding middle ear isolates were nonpiliated and could not be enriched for piliation. Nevertheless, Southern analysis revealed that these two middle ear isolates contained all five hif genes required for pilus biogenesis and had no evidence of major genetic rearrangement. In summary, the vast majority of isolates of nontypable H. influenzae associated with acute otitis media express HMW1/HMW2-like proteins, with expression present in both the nasopharynx and the middle ear. A minority of isolates express Hia, again both in the nasopharynx and the middle ear. A smaller fraction of isolates express pili, which are irreversibly inactivated upon transit from the nasopharynx to the middle ear. We speculate that the HMW adhesins and Hia are important at multiple steps in the pathogenesis of otitis media, while pili contribute to early colonization and then become dispensible.