Hypoxia-inducible Factor 1a Levels Research Articles

Luoshi Neiyi Prescription inhibits estradiol synthesis and inflammation in endometriosis through the HIF1A/EZH2/SF-1 pathway

Ethnopharmacological relevanceEndometriosis (EMS) is a common gynecological disease that causes dysmenorrhea, chronic pelvic pain and infertility. Luoshi Neiyi Prescription (LSNYP), a traditional Chinese medicine (TCM) formula, is used to relieve EMS in the clinic. AimsThis study aimed to examine the active components of LSNYP and the possible mechanism involved in its treatment of EMS. Materials and methodsUltrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) was used to identify the chemical components of LSNYP. Human primary ectopic endometrial stromal cells (ecESCs) and eutopic endometrial stromal cells (euESCs) were isolated, and the expression levels of hypoxia inducible factor 1A (HIF1A), enhancer of zeste homolog 2 (EZH2) and steroidogenic factor 1 (SF-1) were detected by immunofluorescence and qPCR. Cobalt chloride (CoCl2) was utilized to construct an in vitro hypoxic environment, and lentiviruses were engineered to downregulate HIF1A and EZH2 and upregulate EZH2. Subsequently, the expression levels of HIF1A, EZH2, and SF-1 were measured using qPCR or western blotting. The binding of EZH2 to the SF-1 locus in ESCs was examined via ChIP. Furthermore, the effects of LSNYP on the HIF1A/EZH2/SF-1 pathway were evaluated both in vitro and in vivo. ResultsA total of 185 components were identified in LSNYP. The protein and gene expression levels of HIF1A and SF-1 were increased, whereas those of EZH2 were decreased in ecESCs. After treating euESCs with 50 μmol L−1 CoCl2 for 24 h, cell viability and estradiol (E2) production were enhanced. Hypoxia decreased EZH2 protein expression, while si-HIF1A increased it. SF-1 was increased when EZH2 was downregulated in normal and hypoxic environments, whereas the overexpression of EZH2 led to a decrease in SF-1 expression. ChIP revealed that hypoxia reduced EZH2 binding to the SF-1 locus in euESCs. In vitro, LSNYP-containing serum decreased E2 and prostaglandin E2 (PGE2) production, inhibited cell proliferation and invasion, and reduced the expression of HIF1A, SF-1, steroidogenic acute regulatory protein (StAR), and aromatase cytochrome P450 (P450arom). In vivo, LSNYP suppressed inflammation and adhesion and inhibited the HIF1A/EZH2/SF-1 pathway in endometriotic tissues. ConclusionsLSNYP may exert pharmacological effects on EMS by inhibiting E2 synthesis and inflammation through regulation of the HIF1A/EZH2/SF-1 pathway. These results suggest that LSNYP may be a promising candidate for the treatment of EMS.

The effects of Gualou Qumai Wan combined with Sunitinib on VEGFA, HIF1A, and renal function in advanced clear cell renal cell carcinoma

[Objective:] To study the efficacy of Gualou Qumai Wan (GQW) combined with Sunitinib in the treatment of advanced clear cell renal cell carcinoma (ccRCC) and its effects on vascular endothelial growth factor A (VEGFA), hypoxia inducible factor 1A (HIF1A), and renal function. [Method:] A retrospective analysis was conducted on 20 patients with advanced ccRCC treated with GQW combined with Sunitinib at Shouguang Hospital of Traditional Chinese Medicine from January 2021 to January 2023. Analyze the patient's quality of life, serum VEGFA and HIF1A levels, renal function indicators, T lymphocyte subgroup levels, traditional Chinese medicine (TCM) symptom scores, and clinical efficacy. [Result:] The total effective rate of clinical efficacy in patients after treatment was 70%; The serum levels of VEGFA and HIF1A in patients were lower than before treatment, and the difference was statistically significant (P<0.05); The levels of serum creatinine (Scr), blood urea nitrogen (BUN), and urinary albumin (ALB) in patients were lower than before treatment, and the differences were statistically significant (P<0.05). Before and after treatment, there was no statistically significant difference in the levels of T lymphocyte subgroup among patients (P>0.05). After treatment, the TCM symptom score and EORTC QoL-C30 score of the patients all decreased compared to before treatment, and the difference was statistically significant (P<0.01). [Conclusion:] The combination of GQW and Sunitinib has a significant therapeutic effect on advanced ccRCC, and can effectively improve the levels of VEGFA, HIF1A, and renal function.

Investigation of the effect of CA IX enzyme inhibition on the EZH2 gene and histone 3 modifications

Abstract Objectives Colon cancer is the most common gastrointestinal cancer worldwide with high morbidity and mortality rates. The main purpose of our study is to elucidate the interaction mechanism of the H+ ion concentration effect in the CO2/HCO3 − buffer system of tumor-associated carbonic anhydrase IX (CA IX) enzyme inhibition in the HT-29 colon cancer cell line on cell epigenetic modifications. Methods Cell culture was performed using the human colon cancer cell line HT-29. CA IX enzyme inhibitor Acetazolamide (AZA) was administered. The results of the cell viability test and inhibition were evaluated. Extracellular pH measurements were performed. Total histone protein isolation was performed and Histone H3 modifications were analyzed by ELISA method. After RNA isolation, complementary DNA (cDNA) synthesis was carried out. RT-PCR was performed to determine the gene expression levels of hypoxia-inducible factor 1A (HIF1A), enhancer of zeste homolog 2 (EZH2) and CA IX. Results CA IX enzyme inhibition in the HT-29 cell line decreased the expression of CA IX (p<0.05) and HIF1A (p<0.01) genes and increased the expression of the EZH2 (p<0.05). There was a significant decrease in the expression of CA IX (p<0.05) and HIF1A genes as a result of inhibition with AZA performed under hypoxic conditions. It was observed that CA IX enzyme inhibition increases the expression of the EZH2 gene by more than 3 times (p<0.01). As a result of AZA inhibition, methylation levels were observed to increase in normoxic conditions, while methylation levels were observed to decrease in hypoxic conditions. Conclusions Observing the changes in the H3 modifications and changes in the expression of CA IX, HIF1A and EZH2 genes in this study supports that CA IX enzyme inhibition plays an active role in epigenetic modifications.

Analysis of Hypoxia-Inducible Factor-1a and Intercellular Adhesive Molecule-1 on Clinical Findings in Rhegmatogenous Retinal Detachment Patients

The aim of the study is to examine the role of hypoxia-inducible factor-1a (HIF-1a) and intercellular adhesive molecule (ICAM-1) in clinical findings found in patients with rhegmatogenous retinal detachment (RRD). The clinical findings are; the duration of retinal detachment, retinal quadrants involvement, and the severity of proliferative vitreoretinopathy (PVR). A cross sectional study was conducted, with 34 consecutive vitreous sample were obtained from dr. Cipto Mangunkusumo National General Hospital (RSCM), from October 2015 to October 2016. The samples were taken from RRD patient who received vitrectomy in the hospital. There were no significant difference between the ICAM-1 and HIF-1a levels to the clinical findings that is measured; duration of retinal detachment (p>0.05), extent of retinal detachment area (p>0.05) and severity of PVR (p>0.05). However, there is a significant mean difference of ICAM-1 level on the duration of retinal detachment, where < 1 week group has higher mean 49.71, (SD 49.54) than the other group, as well as in the retinal quadrants groups (2nd quadrant 19.42 (SD 2.49) and also in severity of PVR groups (PVR B 35.42, SD 30.13). The results from HIF-1a level on each sub group were insignificant. The level of ICAM-1 and HIF-1a cannot be used as predictor of the clinical findings found in the patients. ICAM-1 levels tend to be high on patients whose diagnosed with retinal detachment early, with smaller area of detachment (2nd quadrant) and lower PVR levels (PVR B). The level of HIF-1a shows a consistent pattern with minimal difference in between the sub groups.

Endothelial cells derived from elephant seals blunt angiogenic transcriptional signaling during hypoxia exposure

Northern elephant seals ( Mirounga angustirostris) conduct repeated deep dives, during which arterial blood oxygen becomes depleted. Interestingly, elephant seals require little surface recovery time from these dives, and extreme hypoxemia occurs regardless of dive duration or purpose. Recent in vitro and in silica work indicates that tight regulation of the canonical hypoxia response may protect diving seals against the inflammatory injuries typically associated with repeated hypoxia exposure. In vivo assessments of these phenomena, however, remain restricted due to both technical constraints and animals' inaccessibility while diving at sea. We developed a proliferative endothelial cell culture system from expelled placental arteries to assess the real-time molecular response to hypoxia exposure in northern elephant seals. Hypoxia inducible factor 1a (HIF-1a) protein localized to the nucleus within 1 h of exposure to 1% O2 in both species[KA1]. Hypoxia increased HIF-1a protein levels to a maximum of ~70-fold over (normoxic) baseline levels in both seal and human cells (p=0.005 for both species), though the response dynamics differed between species. HIF-1a abundance in seal cells was biphasic, with an initial rapid peak occurring within 15 min of hypoxia onset. In contrast, HIF-1a levels did not peak in human cells until 1 h in hypoxia and abundance declined continuously following this peak. Interestingly, RNAseq analysis of HIF-1a target gene expression suggested that seal cells blunt angiogenic signaling and vascular remodeling during hypoxia exposure despite sustained HIF-1a protein stabilization. Seal cell expression of STC2, NHE1/SLC9A1, and PFKL lagged behind expression of these genes in human cells during hypoxia exposure, and seal cell expression of ANGPT2 and MMP14 remained stable during long term hypoxia exposure, while expression of these genes was variable or high in human cells. Cis-regulatory analyses suggested a role for altered metabolism (rather than altered oxygen delivery) in seal cells during hypoxia via FOXK1, NFKB1, JUN and ZBTB7A. In contrast, the transcriptional signature in human cells suggests altered expression of oxygen delivery pathways via homeobox A13 (HOXA13), FOS like 1 (FOSL1) and SRF, all of which regulate angiogenesis, vascular remodeling and vasodilation. Together, these data suggest that seal endothelial cells respond to hypoxia by managing metabolic factors rather than calling for increased oxygen delivery despite rapid and sustained HIF-1a stabilization. NSF GRFP and UC Berkeley startup This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Diagnostic potential of differentially regulated microRNAs among endometriosis, endometrioid ovarian cancer, and endometrial cancer.

There is an increased risk of developing endometrioid ovarian and endometrial cancer in patients with endometriosis and there are no definitive diagnostic biomarkers available for these three associated diseases. Therefore, we evaluated the diagnostic potential of differentially expressed microRNAs (miRNAs) from the tissue samples of endometriosis, endometrioid ovarian cancer, and endometrial cancer to establish them as biomarkers for these diseases. Ten samples of each, i.e., endometriosis, endometrioid ovarian cancer, endometrial cancer and control healthy endometrium were enrolled after obtaining ethical clearance. Differential expression of miR-16, miR-20a, miR-99b, miR-125a, miR-143, and miR-145 and some of their target genes, i.e., vascular endothelial growth factor (VEGF), hypoxia inducible factor 1A (HIF1A), cyclooxygenase 2 (COX2), and tumor necrosis factor (TNF) were quantified using quantitative reverse transcription polymerase chain reaction. Receiver operating characteristic (ROC) curve analysis was performed to predict the diagnostic potential. miR-16 and miR-20a were significantly downregulated, whereas miR-99b, miR-125a, and miR-143 were significantly upregulated in all three diseased samples. miR-145 was significantly upregulated in endometriosis and endometrioid ovarian cancer but significantly downregulated in endometrial cancer. mRNA levels of VEGF, HIF1A, COX2, and TNF were significantly increased in all three diseased samples as compared to control samples. ROC curve analysis revealed that for endometriosis, miR-99b, and miR-125a were giving highest area under curve (AUC) (0.950 and 0.733, respectively), for endometrioid carcinoma of ovary miR-143 was giving highest AUC (0.933) and for endometrioid endometrial cancer miR-16 (AUC = 0.815), miR-99b (AUC = 0.920), and miR-145 (AUC = 0.985) were found to be best predictors. These findings suggest that these miRNAs can act as good predictors and discriminators of these three diseases and might serve as potential biomarkers for them.

Multiple roles for peptidylglycine α-amidating monooxygenase in the response to hypoxia.

The biosynthesis of many of the peptides involved in homeostatic control requires peptidylglycine α-amidating monooxygenase (PAM), an ancient, highly conserved copper- and ascorbate-dependent enzyme. Using the production of amidated chromogranin A to monitor PAM function in tumor cells, physiologically relevant levels of hypoxia were shown to inhibit this monooxygenase. The ability of primary pituitary cells exposed to hypoxic conditions for 4 h to produce amidated chromogranin A was similarly inhibited. The affinity of the purified monooxygenase for oxygen (Km = 99 ± 19 μM) was consistent with this result. The ability of PAM to alter secretory pathway behavior under normoxic conditions required its monooxygenase activity. Under normoxic conditions, hypoxia-inducible factor 1a levels in dense cultures of corticotrope tumor cells expressing high levels of PAM exceeded those in control cells; expression of inactive monooxygenase did not have this effect. The effects of hypoxia on levels of two PAM-regulated genes (activating transcription factor 3 [Atf3] and FK506 binding protein 2[Fkbp2]) differed in cells expressing high versus low levels of PAM. Putative hypoxia response elements occur in both human and mouse PAM, and hPAM has consistently been identified as one of the genes upregulated in response to hypoxia. Expression of PAM is also known to alter gene expression. A quarter of the genes consistently upregulated in response to hypoxia were downregulated following increased expression of PAM. Taken together, our data suggest roles for PAM and amidated peptide secretion in the coordination of tissue-specific responses to hypoxia.

Methamphetamine‐induced retinal hyperpermeability, hypoxia, and vascularization

PurposeMethamphetamine (METH), a highly addictive stimulant of neurotransmitters induces central retinal artery occlusion with retinal atrophy and neovascularization. We previously have identified METH‐induced loss of endothelial surface molecules including platelet and endothelial cell adhesion molecule‐1 (PECAM‐1) and glycocalyx in mice. However, the pathophysiologic pathway of METH‐induced retinal vascular dysfunction has been insufficiently investigated. The purpose of this study is to investigate the METH‐induced alteration of the retinal vascular circulation.Materials and methodsC57BL/6J male and female mice were administrated progressively increasing doses of METH (0 to 6 mg/kg) by repetitive intraperitoneal injections (4 times per day) for 4 weeks. Norepinephrine levels in plasma were measured by ELISA. Cross‐sections of the retina were stained with hematoxylin and eosin. Levels of hypoxia in the retina were determined by immunostaining of pimonidazole adducts with Hypoxyprobe. Retinal blood vessels were visualized with fluorescent plasma tracers injected via femoral vein injection for live imaging, and immunostained with griffonia simplicifolia lectin 1 (GSL‐1) for retinal flatmounts. Expression of various proteins was determined by immunoblot.ResultsThe level of pimonidazole adduct was increased in METH‐treated retinas, compared with saline‐treated retinas, indicating METH‐induced retinal hypoxia. Plasma norepinephrine levels, which can promote hypoxia‐inducible factor 1a (HIF‐1a) at the transcriptional level, were elevated by METH, compared with saline treatment at 1 week (2.2±0.9 vs 7.8±1.6 ng/mL, P<0.01), 2 weeks (0.8±0.3 vs 5.5±1.8 ng/mL, P<0.05), and 4 weeks (0.9±0.2 vs 2.0±0.4 ng/mL, P<0.05). Retinal protein expression levels of HIF‐1a and angiogenic proteins, such as vascular endothelial growth factor a (VEGFa), erythroblast transformation‐specific (ETS)‐related gene (ERG), and Protein C‐ets‐1 (ETS‐1) were increased in the METH‐treated retinas. Counts of retinal arterioles and capillaries were higher in METH‐treated mice than in saline‐treated mice. Moreover, protein concentration in the vitreous humor and immunoreactivity of immunoglobulin in retina, which are measures of retinal permeability, were increased by METH treatment.ConclusionOur present data suggest that METH is associated with retinal neovascularization and hyperpermeability with increases in hypoxia, plasma norepinephrine, and HIF‐1a activation.Support or Funding InformationThis work was supported by funding from the National Institute of Health (NIH) EY025632.

Exosomal microRNA-210 is a potentially non-invasive biomarker for the diagnosis and prognosis of glioma.

MicroRNAs (miRs) transferred by exosomes can function as non-invasive potential biomarkers for the diagnosis and prognosis in various types of cancer. The present study examined the diagnostic and prognostic value of serum exosomal-(exo-)miR-210 levels in association with hypoxic conditions in patients with glioma. Serum levels of exo-miR-210 were determined by quantitative PCR in samples obtained from patients with glioma. Patients were divided into low-and high-expression exo-miR-210 groups according to the median expression value. Statistical analyses were conducted to examine the potential value of exo-miR-210 in predicting the diagnosis and prognosis of patients with glioma. A significant increase in serum exo-miR-210 levels was observed in patients with glioma compared with healthy controls. Additionally, the expression levels of exo-miR-210 were increased with ascending pathological grades. Furthermore, expression levels of miR-210 in serum exosomes from patients with glioblastoma were markedly decreased following surgery and upregulated once more at the recurrences of primary tumors, indicating that exo-miR-210 could reflect alterations in malignant glioma loads. In addition, Kaplan-Meier analysis was performed to analyze overall survival (OS) time. Patients with malignant glioma with high exo-miR-210 expression exhibited a poorer OS compared with patients with low expression. Importantly, univariate and multivariate Cox regression analysis revealed that the expression levels of exo-miR-210 in glioma serum samples were independently associated with OS. Finally, increased serum exo-miR-210 expression was positively associated with high levels of hypoxia-inducible factor 1a and reflected hypoxia in patients with glioma. In conclusion, serum levels of exo-miR-210 may serve as a diagnostic, prognostic and hypoxic biomarker to reflect glioma status and hypoxic signatures.

A Strong Correlation of the HIF1a mRNA Expression Level with the Total Expression of JAK2, As Well As a Decrease in Their Level Is Observed in Leukocytes of Patients with MPN and Lymphoproliferative Cancer

Background. The hypoxia inducible factor 1a (HIF1a) is an important transcription factor regulating gene expression for an adaptive response to low oxygen level in human cells. Earlier, we observed a decrease of HIF1a and CALR mRNA, but not MPL expression in whole blood samples of patients with Ph-negative myeloproliferative neoplasm (MPN) compared with healthy volunteers (Gorbenko A., et al., Haematologica, 2017; Gorbenko A., et al., Blood, 2016). Recent transcriptome studies in granulocytes and CD34+ blood cells from MPN patients confirmed a decrease of mRNA CALR, but also revealed an increase of the relative mRNA level of HIF1a and JAK2 (Cokic VP, et al., PLoS ONE, 2015). These changes in the regulation of number genes expression may depend on the mutation JAK2V617F as was also shown (Berkofsky-Fessler W, et al., Clin Cancer Res. 2010). But some studies demonstrated transcriptomes data of the isolated blood cells from patients with lymphoproliferative disease did not find significant changes in the expression of these genes (Liao W, et al., BMC Cancer, 2015). Aims. Investigate HIF1a, CALR and JAK2 mRNA expression in patients with lymphoproliferative and myeloproliferative cancer. Methods. 10 healthy volunteers (average age 42 years, range 20-63 years, 80% of men) and 80 (average age 54 years, range 30-83 years, 46% of man), patients with MPN, also 36 (average age 62 years, range 28-79 years, 78% of man) patients with lymphoproliferative cancer after signing an informed consent were included in this study. In our study, we investigated the expression level of mRNA genes in whole blood samples obtained in test tubes with an RNA stabilizer (LTD "Formula of gene", Russia) in order to exclude the factors of preanalytical cell hypoxia. Quantitative real-time PCR was performed to determine the levels of HIF1a, CALR and JAK2 mRNA transcripts using TaqMan probes on the CFX96 (Bio-Rad). The results were calculated by ΔCt method in the software package of "R". The threshold cycles (Ct) genes and housekeeping genes (TBP, GUS, ABL) was determined using Cy0 method. The results were normalized by these reference genes. Mann-Whitney U-test was used to evaluate significance of differences between the groups, the degree of correlation (r) was assessed using Spearman test. Results. We observed that HIF1a and JAK2 mRNA expression was significantly lower in whole blood samples of all patients with MPN and lymphoproliferative cancer compared with a group of healthy volunteers (p<0,001) (Figure). We discovered a strong correlation between JAK2 and HIF1a expression in all myeloproliferative and lymphoproliferative neoplasms (r=0.83 and r=0.93 respectively, p<0,001) (Figure). It should be noted that the correlation in the blood samples of patients with MPN was observed only when the total expression of wild and mutated transcripts JAK2 (JAK2+JAK2 V617F) was assessed. No correlation was found between the level of mRNA expression and the cellular number of granulocytes or lymphocytes. The expression level of CALR mRNA also decreases in the blood cells of MPN and lymphoproliferative cancer patients (p<0.05), but we did not observe its correlation with HIF1a or JAK2 mRNA. Conclusion. We assume that the studied gene expression changes reflect the regulated metabolic processes in the cancer stem cells. Probably, the activation of the associated signaling pathways HIF1a and JAK2/STAT in the white blood cells of patients with chronic blood cancer leads to the adoptive enhancement of autophagy, causing a chronic course of the disease. The assume that the opposite shifts of HIF1a and JAK2 in the microarray research (Cokic VP, et al., PLoS ONE, 2015) can be associated with the procedure of blood cells isolation and the absence of a group of healthy people as a control. Reduced expression of CALR mRNA in patient blood cells requires further investigation. Disclosures No relevant conflicts of interest to declare.

Levels of Endocan, Angiopoietin-2, and Hypoxia-Inducible Factor-1a in Patients with Autosomal Dominant Polycystic Kidney Disease and Different Levels of Renal Function

Background: Endothelial dysfunction leading to unbalanced vasoconstriction and ischemia of renal parenchyma is increasingly proposed as an alternative pathway of renal damage in autosomal dominant polycystic kidney disease (ADPKD). However, human studies investigating the evolution of such phenomena are limited. This study investigated the levels of emerging biomarkers of endothelial function, angiogenesis and hypoxia, in ADPKD patients with different renal function. Methods: The study population consisted of three groups: 26 ADPKD patients with impaired renal function (Group A; estimated glomerular filtration rate [eGFR] 45–70 mL/min/1.73 m²), 26 ADPKD patients with preserved renal function (Group B; eGFR >70 mL/min/1.73 m²), and 26 age- and sex-matched controls with no history of renal disease. Circulating levels of endocan (endothelial cell-specific molecule-1) angiopoietin-2, and hypoxia-inducible factor-1a (HIF-1a) were determined by enzyme-linked immunosorbent assay techniques. Results: Patients in Group A had significantly higher levels of endocan (7.17 ± 0.43 ng/mL), angiopoietin-2 (5,595.43 ± 3,390), and HIF-1a (163.68 ± 37.84 pg/mL) compared to patients in Group B (6.86 ± 0.59 ng/mL, p = 0.017, 3,854.41 ± 3,014.30, p = 0.018, 136.84 ± 42.10 pg/mL, p = 0.019 respectively) or controls (4.83 ± 0.69 ng/mL, 1,069 ± 427.88 pg/mL, 70.20 ± 17.49 pg/mL, p < 0.001 for all comparisons). Of note, patients in Group B had also higher levels of all markers compared to controls (p < 0.001) despite having similar renal function. In correlation analyses within ADPKD patients, we noted strong correlations of all studied markers with asymmetric dimethylarginine (ADMA; endocan r = 0.908, p < 0.001, angiopoietin-2 r = 0.983, p < 0.001 and HIF-1a r = 0.998, p < 0.001), and only weak or modest correlations with eGFR. Conclusions: This study suggests that endothelial dysfunction causing microcirculatory changes, linked to angiogenesis and hypoxia, may come early in the course of ADPKD and could be a key regulator of renal injury progression.

An EGLN1 mutation may regulate hypoxic response in cyanotic congenital heart disease through the PHD2/HIF-1A pathway

Cyanotic congenital heart disease (CCHD), a term describing the most severe congenital heart diseases are characterized by the anatomic malformation of a right to left shunt. Although the incidence of CCHD are far less than the that of congenital heart diseases (CHD), patients with CCHD always present severe clinical features such as hypoxia, dyspnea, and heart failure. Chronic hypoxia induces hypoxemia that significantly contributes to poor prognosis in CCHD. Current studies have demonstrated that the prolyl-4-hydroxylase2 (PHD2, encoded by EGLN1)/hypoxia-inducible factor-1A (HIF-1A) pathway is a key regulator of hypoxic response. Thus, we aim to assess the associations of single polymorphisms (SNPs) of the EGLN1 gene and hypoxic response in CCHD. A missense variant of EGLN1 c.380G>C (rs1209790) was found in 46 patients (46/126), with lower hypoxia incidence and higher rate of collateral vessel formation, compared with the wild type (P < 0.05). In vitro experiments, during hypoxia, EGLN1 mutation reduced EGLN1 expression compared with the wild type, with higher HIF-1A, VEGF and EPO expression levels in the mutant. No difference in HK1 expression was observed between the mutant and wild type. CCHD patients with c.380G>C showed improved response to hypoxia compared with the wild-type counterparts. The EGLN1 c.380G>C mutation improves hypoxic response through the PHD2/HIF-1A pathway, which may provide a molecular mechanism for hypoxic response in CCHD. The effects of the EGLN1 c.380G>C mutation on CCHD prognosis deserve further investigation.

El suero de seno coronario tras un infarto de miocardio induce angiogénesis y reparación de la obstrucción microvascular. Implicación del factor inducible por hipoxia-1A

Introduction and objectivesMicrovascular obstruction (MVO) exerts deleterious effects following acute myocardial infarction (AMI). We investigated coronary angiogenesis induced by coronary serum and the role of hypoxia-inducible factor-1A (HIF-1A) in MVO repair. MethodsMyocardial infarction was induced in swine by transitory 90-minute coronary occlusion. The pigs were divided into a control group and 4 AMI groups: no reperfusion, 1minute, 1 week and 1 month after reperfusion. Microvascular obstruction and microvessel density were quantified. The proangiogenic effect of coronary serum drawn from coronary sinus on endothelial cells was evaluated using an in vitro tubulogenesis assay. Circulating and myocardial HIF-1A levels and the effect of in vitro blockade of HIF-1A was assessed. ResultsCompared with control myocardium, microvessel density decreased at 90-minute ischemia, and MVO first occurred at 1minute after reperfusion. Both peaked at 1 week and almost completely resolved at 1 month. Coronary serum exerted a neoangiogenic effect on coronary endothelial cells in vitro, peaking at ischemia and 1minute postreperfusion (32 ± 4 and 41 ± 9 tubes vs control: 3 ± 3 tubes; P < .01). Hypoxia-inducible factor-1A increased in serum during ischemia (5-minute ischemia: 273 ± 52 pg/mL vs control: 148 ± 48 pg/mL; P < .01) being present on microvessels of all AMI groups (no reperfusion: 67% ± 5% vs control: 15% ± 17%; P < .01). In vitro blockade of HIF-1A reduced the angiogenic response induced by serum. ConclusionsCoronary serum represents a potent neoangiogenic stimulus even before reperfusion; HIF-1A might be crucial. Coronary neoangiogenesis induced by coronary serum can contribute to understanding the pathophysiology of AMI.Full English text available from:www.revespcardiol.org/en

The impact of antiandrogen flutamide on the hypoxia inducible factor 1a and vascular endothelial growth factor A gene and protein expression in the pig placenta during late pregnancy.

In contrast to estradiol action, little is known about androgen signaling in placental development. The purpose of this study was to evaluate the impact of diminished androgen action on hypoxia inducible factor 1a (HIF-1a) and vascular endothelial growth factor A (VEGFA) protein expression as well as their mRNAs in the structures of fetal and maternal parts of porcine placenta during late pregnancy. Pregnant pigs were injected daily with antiandrogen flutamide, at a dose of 50 mg/kg body weight at different stages of pregnancy: between gestational days 83-89 (90 dpc) and 101-107 (108 dpc). Control groups (90 dpc or 108 dpc) were treated with vehicle (corn oil). One day after the last injection animals were sacrificed and tissues were collected. Tissue samples were frozen for mRNA isolation or fixed for immu-nohistochemistry (IHC). The expression of HIF-1a and VEGFA were investigated by real-time PCR and IHC. Flutamide treatment caused changes in both HIF-1a and VEGFA mRNA levels only in the placentas of the 90 dpc group. Relative optical density analysis showed decreased HIF-1a and increased VEGFA protein expression in the placentas obtained from flutamide-treated 108 dpc group while no differences were observed in the 90 dpc group. Experimentally induced androgen deficiency in pigs deregulates the expression of some genes important for placental blood circulation. We suggest that androgens are involved in the control of expression of HIF-1a and VEGFA in porcine placenta during late pregnancy.

Coronary Serum Obtained After Myocardial Infarction Induces Angiogenesis and Microvascular Obstruction Repair. Role of Hypoxia-inducible Factor-1A

Introduction and objectivesMicrovascular obstruction (MVO) exerts deleterious effects following acute myocardial infarction (AMI). We investigated coronary angiogenesis induced by coronary serum and the role of hypoxia-inducible factor-1A (HIF-1A) in MVO repair. MethodsMyocardial infarction was induced in swine by transitory 90-minute coronary occlusion. The pigs were divided into a control group and 4 AMI groups: no reperfusion, 1minute, 1 week and 1 month after reperfusion. Microvascular obstruction and microvessel density were quantified. The proangiogenic effect of coronary serum drawn from coronary sinus on endothelial cells was evaluated using an in vitro tubulogenesis assay. Circulating and myocardial HIF-1A levels and the effect of in vitro blockade of HIF-1A was assessed. ResultsCompared with control myocardium, microvessel density decreased at 90-minute ischemia, and MVO first occurred at 1minute after reperfusion. Both peaked at 1 week and almost completely resolved at 1 month. Coronary serum exerted a neoangiogenic effect on coronary endothelial cells in vitro, peaking at ischemia and 1minute postreperfusion (32 ± 4 and 41 ± 9 tubes vs control: 3 ± 3 tubes; P < .01). Hypoxia-inducible factor-1A increased in serum during ischemia (5-minute ischemia: 273 ± 52 pg/mL vs control: 148 ± 48 pg/mL; P < .01) being present on microvessels of all AMI groups (no reperfusion: 67% ± 5% vs control: 15% ± 17%; P < .01). In vitro blockade of HIF-1A reduced the angiogenic response induced by serum. ConclusionsCoronary serum represents a potent neoangiogenic stimulus even before reperfusion; HIF-1A might be crucial. Coronary neoangiogenesis induced by coronary serum can contribute to understanding the pathophysiology of AMI.

Tumor-associated immune factors are associated with recurrence and metastasis in non-small cell lung cancer

Dynamic interaction between tumor cells and the microenvironment is critical for tumorigenesis, and cancer immunosurveillance plays an important role in the tumor evolution. In some tumors (such as esophageal cancer, pancreatic cancer and colorectal cancer), studies have shown that the number of tumor-infiltrating lymphocytes (TILs) has a significant relationship with the prognosis, but there is little research on the prognosis of TILs and non-small cell lung cancer (NSCLC) has been performed. Therefore, it is necessary to discover the relationship between the TILs and cytokines with NSCLC prognosis and metastasis in patients. Tumor samples were carefully examined for tissue preservation and complete follow-up. A total of 107 tumor samples from NSCLC patients with radical surgical resection were enrolled for the analysis. All samples were subjected to immunohistochemistry for detection of CD3, CD4, CD8, CD28, forkhead box protein P3 (Foxp3), cytotoxic T lymphocyte-associated protein-4, cyclooxygenase2 (COX-2), transforming growth factor β 1, interleukin-2 (IL-2), interleukin-6, interleukin-10, interleukin-12 receptor and hypoxia inducible factor 1a (HIF-1a). The number, function and location of the targets were analyzed to determine their correlation with disease-free survival (DFS) and overall survival (OS). Immunhistochemical results from 107 samples indicated that the FoxP3+ regulatory TIL (HR=1.336, P=0.031), IL-2 (HR=0.595, P=0.007) and HIF-1a (HR=1.510, P=0.002) levels in tumor cells closely correlated with DFS in a COX analysis model. FoxP3+ regulatory TILs (HR=1.566, P=0.002) significantly correlated with OS and tumor node metastasis staging. The patients were divided into two groups due to the coexpression pattern of the IL-2, FoxP3+ and HIF-1a. The high-risk group had an overall worse survival than those at low risk. We confirmed that Foxp3 expression in lymphocyte and IL-2 expression in tumor cells were associated with recurrence or transfer. Furthermore, we also observed that HIF-1a expression significantly correlated with DFS and OS.

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 ...

Hypoxia-inducible factor-1a contributes to dendritic overgrowth in tuberous sclerosis

Expression of hypoxia-inducible factor 1a (HIF1a) is increased under several pathological conditions such as hyperactive mechanistic target of rapamycin complex 1 (mTORC1) in tuberous sclerosis complex (TSC). Hyperactive mTORC1 and the resulting increased dendritic complexity of neurons are shared molecular and cellular alterations in several neurological disorders associated with cognitive disabilities. Despite some evidence that HIF1a contributes to dendritic overgrowth in vitro, it remains unknown whether increased HIF1a in TSC neurons could contribute to their increased dendritic complexity. To address this use in vivo, we generated TSC neurons by deleting Tsc1 in newborn olfactory bulb (OB) neurons of conditional Tsc1 transgenic mice using neonatal electroporation. In addition to their increased dendritic complexity, Tsc1null neurons have been reported to display increased Hif1a mRNA level and HIF1a transcriptional activity. We found that Tsc1null-dependent dendritic overgrowth was prevented by knocking down HIF1a or expressing a dominant negative HIF1a. In addition, overexpressing HIF1a in wild-type developing neurons resulted in increased dendritic complexity in vivo. These data highlight that an increase in HIF1a levels contributes to abnormal dendritic patterning in developing neurons under normal conditions and hyperactive mTORC1 conditions as in TSC.

MicroRNA-18a Decreases Choroidal Endothelial Cell Proliferation and Migration by Inhibiting HIF1A Expression.

BackgroundThe aim of this study was to investigate the effect of hypoxia on the level of microRNA-18a (miR-18a) and hypoxia-inducible factor 1A (HIF1A) expressed by choroidal endothelial cells through cytological analysis.Material/MethodsAfter culturing choroidal endothelial cells (CECs) under normoxia or hypoxia, microRNAs, expressed in different ways, were screened by using GeneChip microRNA array, and the results were confirmed by real-time PCR. The bioinformatics approach was used to screen target genes of target miRNA. In addition, CECs were transfected with target miRNA mimic or inhibitor and then expression levels of targeted miRNA and genes were observed.ResultsThe GeneChip microRNA Array detected 14 miRNAs that were expressed differently. Among these miRNAs, 12 miRNAs were identified as being upregulated and 2 miRNAs as being down-regulated. MiR-18a was most significantly down-regulated. Bioinformatics analysis showed that HIF1A was the target gene of miR-18a. In CECs transfected with miR-18a mimic, there was a remarkable decrease in gene expression level and protein level of HIF1A, and thereby, significant reduction in proliferation and migration of CECs. In CECs transfected with miR-18a inhibitor, there was a significant increase in gene and protein expression levels of HIF1A, and enhanced proliferation and migration of CECs.ConclusionsWe conclude that miR-18a affects the function of CECs by inhibiting the expression of HIF1A.

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.