HIF-1α Response Research Articles

Gas Tunnel Engineering of Prolyl Hydroxylase Reprograms Hypoxia Signaling in Cells.

Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O2) concentrations. Here, we have reprogrammed cellular hypoxia (low O2) signaling via gas tunnel engineering of prolyl hydroxylase 2 (PHD2), a non-heme iron dependent O2 sensor. Using computational modeling and protein engineering techniques, we identify a gas tunnel and critical residues therein that limit the flow of O2 to PHD2's catalytic core. We show that systematic modification of these residues can open the constriction topology of PHD2's gas tunnel. Using kinetic stopped-flow measurements with NO as a surrogate diatomic gas, we demonstrate up to 3.5-fold enhancement in its association rate to the iron center of tunnel-engineered mutants. Our most effectively designed mutant displays 9-fold enhanced catalytic efficiency (kcat/KM=830±40 M-1 s-1) in hydroxylating a peptide mimic of hypoxia inducible transcription factor HIF-1α, as compared to WT PHD2 (kcat/KM=90±9 M-1 s-1). Furthermore, transfection of plasmids that express designed PHD2 mutants in HEK-293T mammalian cells reveal significant reduction of HIF-1α and downstream hypoxia response transcripts under hypoxic conditions of 1 % O2. Overall, these studies highlight activation of PHD2 as a new pathway to reprogram hypoxia responses and HIF signaling in cells.

Hypoxia-inducible Factor 1α Contributes to Matrix Metalloproteinases 2/9 and Inflammatory Responses in Periodontitis.

Periodontitis is a prevalent condition characterized by inflammation and tissue destruction within the periodontium, with hypoxia emerging as a contributing factor to its pathogenesis. Hypoxia-inducible factor 1α (HIF-1α) has a crucial role in orchestrating adaptive responses to hypoxic microenvironments and has been implicated in various inflammatory-related diseases. Understanding the interplay between HIF-1α, matrix metalloproteinases (MMPs), and inflammatory responses in periodontitis could provide insights into its molecular mechanisms. We investigated the relationship between HIF-1α, MMP2, and MMP9 in gingival crevicular fluid (GCF) and periodontal ligament stem cells (PDLSCs) from periodontitis patients. The expression levels of HIF-1α, MMP2, MMP9, and inflammatory factors (IL-6, IL-1β, TNF-α) were assessed using enzyme-linked immunosorbent assay (ELISA) and real-time PCR (RT-PCR). Additionally, osteogenic differentiation of PDLSCs was identified by alkaline phosphatase activity. Significantly elevated levels of HIF-1α, MMP2, and MMP9 were observed in GCF of periodontitis patients compared to controls. Positive correlations were found between HIF-1α and MMP2/MMP9, as well as with IL-6, IL-1β, and TNF-α. Modulation of HIF-1α expression in PDLSCs revealed its involvement in MMP2/9 secretion and inflammatory responses, with inhibition of HIF-1α mitigating these effects. Furthermore, HIF-1α inhibition alleviated the reduction in osteogenic differentiation induced by inflammatory stimuli. Our findings elucidate the regulatory role of HIF-1α in MMP expression, inflammatory responses, and osteogenic differentiation in periodontitis. In conclusion, targeting HIF-1α signaling pathways may offer therapeutic opportunities for managing periodontitis and promoting periodontal tissue regeneration.

Novel mechanisms of intestinal flora regulation in high-altitude hypoxia

BackgroundThis study investigates the molecular mechanisms behind firmicutes-mediated macrophage (Mψ) polarization and glycolytic metabolic reprogramming through HIF-1α in response to intrinsic mucosal barrier injury induced by high-altitude hypoxia. MethodsEstablishing a hypoxia mouse model of high altitude, we utilized single-cell transcriptome sequencing to identify key cell types involved in regulating intestinal mucosal barrier damage caused by high-altitude hypoxia. Through proteomic analysis of colonic tissue Mψ and metabolomic analysis of Mψ metabolites, we determined crucial proteins and metabolic pathways influencing intestinal mucosal barrier damage induced by high-altitude hypoxia. Mechanistic validation was conducted using RAW264.7 Mψ in vitro by assessing cell viability with CCK-8 assay following treatment with different metabolites. The hypoxia mouse model was further validated in vivo by transplanting gut microbiota of Firmicutes. Histological examinations through H&E staining assessed colonic cell morphology and structure, while the FITC-dextran assay evaluated intestinal tissue permeability. Hypoxia probe signal intensity in mouse colonic tissue was assessed via metronidazole staining. Various experimental techniques, including flow cytometry, immunofluorescence, ELISA, Western blot, and RT-qPCR, were employed to study the impact of HIF-1α/glycolysis pathway and different gut microbiota metabolites on Mψ polarization. ResultsBioinformatics analysis revealed that single-cell transcriptomics identified Mψ as a key cell type, with their polarization pattern playing a crucial role in the intestinal mucosal barrier damage induced by high-altitude hypoxia. Proteomics combined with metabolomics analysis indicated that HIF-1α and the glycolytic pathway are pivotal proteins and signaling pathways in the intestinal mucosal barrier damage caused by high-altitude hypoxia. In vitro cell experiments demonstrated that activation of the glycolytic pathway by HIF-1α led to a significant upregulation of mRNA levels of IL-1β, IL-6, and TNFα while downregulating mRNA levels of IL-10 and TGFβ, thereby promoting M1 Mψ activation and inhibiting M2 Mψ polarization. Further mechanistic validation experiments revealed that the metabolite butyric acid from Firmicutes bacteria significantly downregulated the protein expression of HIF-1α, GCK, PFK, PKM, and LDH, thus inhibiting the HIF-1α/glycolytic pathway that suppresses M1 Mψ and activates M2 Mψ, consequently alleviating the hypoxic symptoms in RAW264.7 cells. Subsequent animal experiments confirmed that Firmicutes bacteria inhibited the HIF-1α/glycolytic pathway to modulate Mψ polarization, thereby mitigating intestinal mucosal barrier damage in high-altitude hypoxic mice. ConclusionThe study reveals that firmicutes, through the inhibition of the HIF-1α/glycolysis pathway, mitigate Mψ polarization, thereby alleviating intrinsic mucosal barrier injury in high-altitude hypoxia.

Hypoxia decreases mitochondrial ROS production in cells

We re-examined the reported increase in mitochondrial ROS production during acute hypoxia in cells. Using the Amplex Ultrared/horseradish peroxidase assay we found a decrease, not increase, in hydrogen peroxide release from HEK293 cells under acute hypoxia, at times ranging from 1 min to 3 h. The rates of superoxide/hydrogen peroxide production from each of the three major sites (site IQ in complex I and site IIIQo in complex III in mitochondria, and NADH oxidases (NOX) in the cytosol) were decreased to the same extent by acute hypoxia, with no change in the cells’ ability to degrade added hydrogen peroxide. A similar decrease in ROS production under acute hypoxia was found using the diacetyldichlorofluorescein assay. Using a HIF1α reporter cell line we confirmed earlier observations that suppression of superoxide production by site IIIQo decreases HIF1α expression, and found similar effects of suppressing site IQ or NOX. We conclude that increased mitochondrial ROS do not drive the response of HIF1α to acute hypoxia, but suggest that cytosolic H2O2 derived from site IQ, site IIIQo and NOX in cells is necessary to permit HIF1α stabilization by other signals.

The Relationship Between Expression of HIF-1α and Clinical Response in Cervical Cancer Patients with Neoadjuvant Chemotherapy at Dr. Soetomo Surabaya Hospital

This investigation aims to analyze the relationship between expression of Hypoxia Inducible factors-1 αlfa (HIF-1α) to assess the response to neoadjuvant chemotherapy in cervical cancer patients. This research is a Quast Experimental Non-Randomized Before and After with no control design with a sample size of 30 stage IIIB cervical cancer patients by inclusion and exclusion criteria, with pretest and posttest treatment, namely biopsy for immunohistochemical examination and abdominal ultrasound to evaluate lesion diameter cervix after three cycles of chemotherapy. This study showed a decrease in HIF-1α expression before and after chemotherapy (p=0,003) and a decrease in the diameter of cervical lesions before and after chemotherapy (p=0,000). Correlation between decreased HIF-1α expression and decreased diameter of cervical lesions (p=<0,05). Response to neoadjuvant chemotherapy: 25 patients (83,3 %) showed a negative response and 5 patients (16,7 %) showed a positive response. Strong HIF-1α expression before chemotherapy gave a negative response 23 and 25 subjects with a negative response after neoadjuvant chemotherapy. This study found a relationship between high HIF-α expression before chemotherapy with a negative chemotherapy response and low HIF-1α expression after neoadjuvant chemotherapy with a positive chemotherapy response in cervical cancer patients at Dr Soetomo Hospital, Surabaya

Abstract 5413: In vitro modeling of the migration ability of lung adenocarcinoma cell lines under hypoxic conditions

Abstract Hypoxia is a common condition in the tumor microenvironment that contributes to tumor cell migration, invasion, and metastasis, partly through inducing epithelial-mesenchymal transition (EMT). One of the key proteins in this process is hypoxia-inducible factor 1 (HIF-1), which regulates the expression of various genes as transcription factor and is vital for the cellular response in tissue hypoxia. Several studies have investigated the causation between hypoxic conditions and increased in vitro tumor cell motility and the role of potential regulatory signaling pathways. Understanding the precise molecular mechanisms underlying hypoxia-induced responses is crucial for better targeting tumor progression. However, establishing sound and predictive models is urgently needed to achieve this.In this study, we investigated the migration ability of four non-small cell lung cancer cell lines carrying different mutations of the growth factor pathway (H1975: EGFR T790M, PF901: BRAF V600E, PF139: KRAS G12C mutant, and H838: triple WT) under normoxic, hypoxic, and hypoxia-mimicking CoCl2-treated conditions. The motility of the cells was investigated by 48 h video microscopy (single cell random migration) and 72 h scratch assay. We analyzed the expression of crucial genes and proteins, including HIF-1α, as well as molecules associated with epithelial-mesenchymal transition (vimentin, n-cadherin, and e-cadherin), proliferation (PCNA), and apoptosis (PARP), using Western blot and qPCR techniques. Our work showed no change in cellular motility between any of the cell lines following treatment with CoCl2 compared to normoxic conditions. However, PF139 and PF901 cell lines showed a significant decrease in single-cell motility upon hypoxia. Interestingly, immunoblot analysis revealed stabilization of HIF-1α in response to both CoCl2 and hypoxia in the meantime. Additionally, we observed cell line-dependent changes in EMT marker expression upon CoCl2 treatment and hypoxia. Our work complements previous results published by our group and others on the tumor-promoting role of hypoxia, demonstrating that short-term exposure to hypoxia may act as an acute stressor for tumor cell lines. Our work suggests that the tumor progression-promoting effects are tissue-specific and should be investigated using long-term hypoxia models. Acknowledgments: This work was supported by the Hungarian Thematic Excellence Program (TKP2021-EGA-44) and the National Laboratories Program - National Laboratory for Tumor Biology (NLP-17). Sára Eszter Surguta is a grantee of the ITM-NKFIA funded Cooperative Doctoral Program (1018567). Citation Format: Sára Eszter Surguta, Marcell Baranyi, Laura Svajda, Ivan Ranđelović, Mihály Cserepes, József Tóvári. In vitro modeling of the migration ability of lung adenocarcinoma cell lines under hypoxic conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5413.

The autocrine action of salidroside on osteoclast during osteoclastogenesis via hypoxia-inducible factor-1α pathway.

The objective of this study was to investigate the potential of salidroside (SAL) (a major active compound in Rhodiola rosea L.) in regulating osteoclast differentiation and function by modulating the HIF-1α pathway and its downstream target genes. The expression of HIF-1α and its downstream target genes was examined at both mRNA and protein levels in osteoclasts treated with SAL. Immunofluorescence analysis was performed to assess the nuclear translocation and transcriptional activity of HIF-1α in response to SAL. MTT, flow cytometry, qPCR, TRAP staining and bone resorption assays were used to evaluate the potential effect of salidroside on osteoclasts. SAL enhanced the expression of HIF-1α and its downstream target genes in osteoclasts. Immunofluorescence analysis confirmed the facilitation of HIF-1α nuclear translocation and transcriptional activity by SAL. In addition, SAL enhanced osteoclast viability, differentiation and bone resorption activity in an autocrine manner through HIF-1α/VEGF, IL-6 and ANGPTL4 pathways. SAL promotes osteoclast proliferation, differentiation and bone resorption through HIF-1α/VEGF, IL-6 and ANGPTL4 pathways.

Oxidative Stress Accompanies HIF1-Dependent Impairment of Glucose Metabolism in the Hippocampus of Adult Rats That Survived Prenatal Severe Hypoxia

Introduction: Many socially significant diseases are associated with prenatal developmental disorders. Previously, we showed the pathological role of hypoxia-inducible factor-1 (HIF1) in post-hypoxic reoxygenation. This study aimed to investigate the effect of prenatal severe hypoxia (PSH) on HIF1α protein expression as well as on HIF1-dependent activity of the pentose phosphate pathway (PPP) and anaerobic glycolysis in the hippocampus (HPC) of offspring that reached adulthood. Methods: PSH was induced during the critical period of fetal hippocampal formation on gestation days 14–16 in a hypobaric chamber (180 Torr, 5% oxygen, 3 h). Subsequent studies were conducted on both the HPC of adult control and PSH rats under normal conditions, as well as in response to severe hypoxia (SH) or psycho-emotional stress (“learned helplessness” [LH] model). We evaluated HIF1α protein levels using both immunohistochemistry and Western blotting techniques. The amount of glucose-6-phosphate dehydrogenase (G6PD) was also determined by Western blotting. Colorimetric enzymatic assays were employed to analyze enzymatic activity of lactate dehydrogenase (LDH), the concentration of lactate, NADPH, reduced glutathione (GSHred), and malonic dialdehyde (MDA). Results: We showed that PSH caused a stable increase in the content of HIF1α protein in the HPC, which was accompanied by an increase in the efficiency of anaerobic glycolysis. This was confirmed by increased LDH activity and lactate concentration. At the same time, the amounts of G6PD, NADPH, and GSHred decreased in the HPC of PSH rats, whereas the concentration of MDA, an oxidative stress marker, exceeded the control values. In a series of experiments using the LH or SH stress, it was shown that in the HPC of control rats, there was an increase in the amount of HIF1α in response to stress, which was also accompanied by more efficient anaerobic glycolysis and decrease of PPP-dependent NADPH production, similar to the intact PSH rats. In PSH rats, emotional stress resulted in higher HIF1α levels without affecting glycolysis or PPP. Conclusion: Therefore, the increased content and activity of the transcription factor HIF1α in the HPC of adult rats exposed to prenatal hypoxia leads to an imbalance between glycolysis and PPP, which is accompanied by oxidative stress.

SUMOylation indirectly suppresses activity of the HIF-1α pathway in intestinal epithelial cells

The hypoxia-inducible factor (HIF) is a master regulator of the cellular transcriptional response to hypoxia. While the oxygen-sensitive regulation of HIF-1α subunit stability via the ubiquitin-proteasome pathway has been well described, less is known about how other oxygen-independent post-translational modifications impact the HIF pathway. SUMOylation, the attachment of SUMO (small ubiquitin-like modifier) proteins to a target protein, regulates the HIF pathway, although the impact of SUMO on HIF activity remains controversial. Here, we examined the effects of SUMOylation on the expression pattern of HIF-1α in response to pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG) in intestinal epithelial cells. We evaluated the effects of SUMO-1, SUMO-2, and SUMO-3 overexpression and inhibition of SUMOylation using a novel selective inhibitor of the SUMO pathway, TAK-981, on the sensitivity of HIF-1α in Caco-2 intestinal epithelial cells. Our findings demonstrate that treatment with TAK-981 decreases global SUMO-1 and SUMO-2/3 modification and enhances HIF-1α protein levels, whereas SUMO-1 and SUMO-2/3 overexpression results in decreased HIF-1α protein levels in response to DMOG. Reporter assay analysis demonstrates reduced HIF-1α transcriptional activity in cells overexpressing SUMO-1 and SUMO-2/3, whereas pretreatment with TAK-981 increased HIF-1α transcriptional activity in response to DMOG. In addition, HIF-1α nuclear accumulation was decreased in cells overexpressing SUMO-1. Importantly, we showed that HIF-1α is not directly SUMOylated, but that SUMOylation affects HIF-1α stability and activity indirectly. Taken together, our results indicate that SUMOylation indirectly suppresses HIF-1α protein stability, transcriptional activity, and nuclear accumulation in intestinal epithelial cells.

MYC overrides HIF-1α to regulate proliferating primary cell metabolism in hypoxia

Hypoxia requires metabolic adaptations to sustain energetically demanding cellular activities. While the metabolic consequences of hypoxia have been studied extensively in cancer cell models, comparatively little is known about how primary cell metabolism responds to hypoxia. Thus, we developed metabolic flux models for human lung fibroblast and pulmonary artery smooth muscle cells proliferating in hypoxia. Unexpectedly, we found that hypoxia decreased glycolysis despite activation of hypoxia-inducible factor 1α (HIF-1α) and increased glycolytic enzyme expression. While HIF-1α activation in normoxia by prolyl hydroxylase (PHD) inhibition did increase glycolysis, hypoxia blocked this effect. Multi-omic profiling revealed distinct molecular responses to hypoxia and PHD inhibition, and suggested a critical role for MYC in modulating HIF-1α responses to hypoxia. Consistent with this hypothesis, MYC knockdown in hypoxia increased glycolysis and MYC over-expression in normoxia decreased glycolysis stimulated by PHD inhibition. These data suggest that MYC signaling in hypoxia uncouples an increase in HIF-dependent glycolytic gene transcription from glycolytic flux.

Targeting 2-Oxoglutarate-Dependent Dioxygenases Promotes Metabolic Reprogramming That Protects against Lethal SARS-CoV-2 Infection in the K18-hACE2 Transgenic Mouse Model.

Dysregulation of host metabolism is a feature of lethal SARS-CoV-2 infection. Perturbations in α-ketoglutarate levels can elicit metabolic reprogramming through 2-oxoglutarate-dependent dioxygenases (2-ODDGs), leading to stabilization of the transcription factor HIF-1α. HIF1-α activation has been reported to promote antiviral mechanisms against SARS-CoV-2 through direct regulation of ACE2 expression (a receptor required for viral entry). However, given the numerous pathways HIF-1α serves to regulate it is possible that there are other undefined metabolic mechanisms contributing to the pathogenesis of SARS-CoV-2 independent of ACE2 downregulation. In this study, we used invitro and invivo models in which HIF-1α modulation of ACE2 expression was negated, allowing for isolated characterization of the host metabolic response within SARS-CoV-2 disease pathogenesis. We demonstrated that SARS-CoV-2 infection limited stabilization of HIF-1α and associated mitochondrial metabolic reprogramming by maintaining activity of the 2-ODDG prolyl hydroxylases. Inhibition of 2-ODDGs with dimethyloxalylglycine promoted HIF-1α stabilization following SARS-CoV-2 infection, and significantly increased survival among SARS-CoV-2-infected mice compared with vehicle controls. However, unlike previous reports, the mechanism by which activation of HIF-1α responses contributed to survival was not through impairment of viral replication. Rather, dimethyloxalylglycine treatment facilitated direct effects on host metabolism including increased glycolysis and resolution of dysregulated pools of metabolites, which correlated with reduced morbidity. Taken together, these data identify (to our knowledge) a novel function of α-ketoglutarate-sensing platforms, including those responsible for HIF-1α stabilization, in the resolution of SARS-CoV-2 infection and support targeting these metabolic nodes as a viable therapeutic strategy to limit disease severity during infection.

Role of hypoxia inducible factor HIF-1α in heart valves.

The 2016 Albert Lasker Basic Medical Research Award and subsequently the 2019 Nobel Prize in Physiology or Medicine were awarded to William Kaelin, Jr., Sir Peter Ratcliffe, and Gregg Semenza for their work on how cells sense and adapt to hypoxic conditions. Their work showed that the changes in gene expression, cell metabolism, and tissue remodelling that occur in response to low oxygen concentrations are orchestrated by the transcription factor, hypoxia inducible factor-1α (HIF-1α). While the effects mediated by HIF-1α have been widely studied, its role in heart valves has only recently been investigated. These studies have shown that HIF-1α expression is evident in mechanisms that regulate the structure and function of heart valves. These include embryonic development, the regulation of the extracellular matrix, angiogenesis and the initiation of the calcification process. This review provides a background on the role and function of HIF-1α in response to hypoxia and a discussion of the available evidence of its involvement in the regulation of heart valves in health and disease.

Prolonged intermittent hypoxia causes mitochondrial dysfunction due to O-GlcNacylation of DRP1 in models of COPD (chronic obstructive pulmonary disease)

Background: Sarcopenia, or skeletal muscle loss, is common complication in COPD and contributes to adverse clinical outcomes including mortality. As the disease progresses, a significant proportion of COPD patients develop nocturnal hypoxemia which causes tissue-level hypoxia in the skeletal muscle which we have termed prolonged intermittent hypoxia (PIH). We have demonstrated sarcopenia, mitochondrial dysfunction, and dysregulated hypoxia-inducible factor 1-α (HIF1-α) responses both in vitro and in vivo in response to PIH. We also noted multiple transcriptional targets of HIF1-α were upregulated in the glycolytic pathway. We hypothesized that PIH upregulates the Hexosamine Biosynthetic (HB) pathway, a branch of glycolysis responsible for post-translational glycosylation of proteins, to cause O-GlcNAcylation of DRP1 leading to increased mitochondrial fission, fragmented mitochondrial networks, mitochondrial dysfunction, and sarcopenia. Methods: Our in vitro model of PIH exposed differentiated, murine C2C12 myotubes to 16h normoxia/8h hypoxia [1% oxygen] for 3 days, while our in vivo model exposed C57BL/6 mice to 12h normoxia/12h hypoxia [10% oxygen] for 21 days. Protein extraction and immunoblots were performed to probe for enzymes in the HB, mTORC1 pathways, O-GlcNAcylation, and puromycin incorporation. Imaging of mitochondrial structure was determined by staining with MitoTracker orange and electron microscopy. Immunoprecipitation and detection of Drp1 O-GlcNAcylation was performed by incubating protein lysates with Wheat Germ Agglutinin coated Agarose beads. To determine responses to inhibition of O-GlcNAcylation, we performed CRISPR-Cas9 RNP deletion of GFAT, the first enzyme in the HB pathway. Results: We found significant increase in O-GlcNAcylated proteins in our PIH models . This was accompanied by increased expression of HIF1-α, mitochondrial dysfunction, and upregulated enzymes in the HB pathway. O-GlcNAcylated modification of DRP1 was significantly upregulated due to PIH in vitro. GFAT KO myotubes demonstrated significant reductions in global O-GlcNAcylation of proteins and O-GlcNAcylated Drp1 with PIH. Interestingly, protein synthesis was rescued in GFAT KO myotubes as determined by puromycin incorporation, restoration of mTORC1 signaling, and reversal of a sarcopenic phenotype. Conclusion: Our findings demonstrate a potential mechanism for mitochondrial dysfunction and sarcopenia due to PIH which may be translationally relevant for patients with COPD. Future studies focused on O-GlcNAcylated modification of DRP1 and the functional consequences are needed to identify novel therapeutic targets to reverse sarcopenia in COPD. Supported in part by: NIH RO1 GM119174; RO1 DK113196; P50 AA024333; RO1 AA021890; 3U01AA026976 - 03S1; UO1 AA 026976; R56HL141744;UO1 DK061732; 5U01DK062470-17S2; R21 AR 071046; Howard and Helen Trevey Endowment; (SD); K12 HL141952 (AA) and the American College of Gastroenterology Clinical Research Award and NIH KO8 AAAA028794 (NW). 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.

An optimized reporter of the transcription factor hypoxia-inducible factor 1α reveals complex HIF-1α activation dynamics in single cells

Immune cells adopt a variety of metabolic states to support their many biological functions, which include fighting pathogens, removing tissue debris, and tissue remodeling. One of the key mediators of these metabolic changes is the transcription factor hypoxia-inducible factor 1α (HIF-1α). Single-cell dynamics have been shown to be an important determinant of cell behavior; however, despite the importance of HIF-1α, little is known about its single-cell dynamics or their effect on metabolism. To address this knowledge gap, here we optimized a HIF-1α fluorescent reporter and applied it to study single-cell dynamics. First, we showed that single cells are likely able to differentiate multiple levels of prolyl hydroxylase inhibition, a marker of metabolic change, via HIF-1α activity. We then applied a physiological stimulus known to trigger metabolic change, interferon-γ, and observed heterogeneous, oscillatory HIF-1α responses in single cells. Finally, we input these dynamics into a mathematical model of HIF-1α-regulated metabolism and discovered a profound difference between cells exhibiting high versus low HIF-1α activation. Specifically, we found cells with high HIF-1α activation are able to meaningfully reduce flux through the tricarboxylic acid cycle and show a notable increase in the NAD+/NADH ratio compared with cells displaying low HIF-1α activation. Altogether, this work demonstrates an optimized reporter for studying HIF-1α in single cells and reveals previously unknown principles of HIF-1α activation.

Epigenetic Differences Arise in Endothelial Cells Responding to Cobalt–Chromium

Cobalt-chromium (Co-Cr)-based alloys are emerging with important characteristics for use in dentistry, but the knowledge of epigenetic mechanisms in endothelial cells has barely been achieved. In order to address this issue, we have prepared a previously Co-Cr-enriched medium to further treat endothelial cells (HUVEC) for up to 72 h. Our data show there is important involvement with epigenetic machinery. Based on the data, it is believed that methylation balance in response to Co-Cr is finely modulated by DNMTs (DNA methyltransferases) and TETs (Tet methylcytosine dioxygenases), especially DNMT3B and both TET1 and TET2. Additionally, histone compaction HDAC6 (histone deacetylase 6) seems to develop a significant effect in endothelial cells. The requirement of SIRT1 seems to have a crucial role in this scenario. SIRT1 is associated with a capacity to modulate the expression of HIF-1α in response to hypoxia microenvironments, thus presenting a protective effect. As mentioned previously, cobalt is able to prevent HIF1A degradation and maintain hypoxia-related signaling in eukaryotic cells. Together, our results show, for the first time, a descriptive study reporting the relevance of epigenetic machinery in endothelial cells responding to cobalt-chromium, and it opens new perspectives to better understand their repercussions as prerequisites for driving cell adhesion, cell cycle progression, and angiogenesis surrounding this Co-Cr-based implantable device.

HIF-1α Expression Increases Preoperative Concurrent Chemoradiotherapy Resistance in Hyperglycemic Rectal Cancer.

Simple SummaryPreoperative Concurrent Chemoradiotherapy (CCRT) is one of the standard treatments for patients with locally advanced rectal cancer. However, the efficacy of CCRT in hyperglycemic rectal cancer patients does not seem to be as expected. Therefore, we used clinical specimens, ectopic hyperglycemia animal models, and rectal cancer cells in a high-glucose environment to confirm how the high-glucose environment causes radiation resistance. The results confirmed that the high-glycemic environment could induce the overexpression of HIF-1α to cause CCRT tolerance in rectal cancer and suggest that combining HIF-1α inhibitors could reverse radioresistance in a high glucose environment. In future, HIF-1α inhibitors might be used as sensitizers to improve the efficacy of hyperglycemic rectal cancer patients receiving CCRT.Purpose: Preoperative concurrent chemoradiotherapy (CCRT) is the standard treatment for locally advanced rectal cancer patients. However, the poor therapeutic efficacy of CCRT was found in rectal cancer patients with hyperglycemia. This study investigated how hyperglycemia affects radiochemotherapy resistance in rectal cancer. Methods and Materials: We analyzed the correlation between prognosis indexes with hypoxia-inducible factor-1 alpha (HIF-1α) in rectal cancer patients with preoperative CCRT. In vitro, we investigated the effect of different concentrated glucose of environments on the radiation tolerance of rectal cancers. Further, we analyzed the combined HIF-1α inhibitor with radiation therapy in hyperglycemic rectal cancers. Results: The prognosis indexes of euglycemic or hyperglycemic rectal cancer patients after receiving CCRT treatment were investigated. The hyperglycemic rectal cancer patients (n = 13, glycosylated hemoglobin, HbA1c > 6.5%) had poorer prognosis indexes. In addition, a positive correlation was observed between HIF-1α expression and HbA1c levels (p = 0.046). Therefore, it is very important to clarify the relationship between HIF-1α and poor response in patients with hyperglycemia receiving pre-operative CCRT. Under a high glucose environment, rectal cancer cells express higher levels of glucose transport 1 (GLUT1), O-GlcNAc transferase (OGT), and HIF-1α, suggesting that the high glucose environment might stimulate HIF-1α expression through the GLUT1-OGT-HIF-1α pathway promoting tolerance to Fluorouracil (5-FU) and radiation. In the hyperglycemic rectal cancer animal model, rectal cancer cells confirmed that radiation exposure reduces apoptosis by overexpressing HIF-1α. Combining HIF-1α inhibitors was able to reverse radioresistance in a high glucose environment. Lower HIF-1α levels increased DNA damage in tumors leading to apoptosis. Conclusions: The findings here show that hyperglycemia induces the expression of GLUT1, OGT, and HIF-1α to cause CCRT tolerance in rectal cancer and suggest that combining HIF-1α inhibitors could reverse radioresistance in a high glucose environment. HIF-1α inhibitors may be useful for development as CCRT sensitizers in patients with hyperglycemic rectal cancer.

Modulating the Siah2-PHD3-HIF1α axis and/or autophagy potentially retard colon cancer proliferation possibly, due to the damping of colon cancer stem cells

BackgroundHypoxic microenvironment of colon cancer is associated with HIF-1α upregulation. HIF-1α response elements are responsible for autophagy induction that promotes tumor proliferation. Moreover, HIF-1α induces tumor cell proliferation via maintaining cancer stem cells (CSCs) survival. Siah2 is E3 ubiquitin ligase that indirectly stabilizes HIF-1α. We hypothesized that dual inhibition of Siah2 as well as autophagy could be a promising approach that may inhibit CSCs growth. Aim of the workThis study investigated the possible effect of vitamin K3 as a Siah2 inhibitor and hydroxychloroquine as an autophagy inhibitor in colon cancer management. The effect (if any) of these agents on CSCs growth will be also manipulated. MethodsColon cancer was induced by dimethylhydrazine. MDA and GSH were selected as oxidative stress markers, Expression of HIF-1α, Caspase-3, VEGF, MMP-9, EpCAM, SCF, and CA19.9 were assayed using immunoassay. The Western blot technique was used to assess LC3Ⅰ, CD44, and CD133 whereas RT-PCR was used to investigate PHD3 and CD44 in colon tissues. Additionally, Ki-67 and Siah2 were detected immunohistochemically. Resultsvitamin K3 and hydroxychloroquine either alone or in combination downregulated the expression of Siah2 and HIF-1α through upregulating PHD3 in colon tissues. This combination significantly downregulated MDA, Ki-67, VEGF, and MMP-9 expression and upregulated the expression of GSH and caspase-3. LC3Ⅰ was also upregulated. Interestingly, these therapeutic options were correlated with down-regulation of the cancer stem cell marker such as CD44 and EpCAM. ConclusionOur results suggested that suppression of both Siah2-PHD3-HIF-1α axis and autophagy retard colon cancer proliferation and dampened CSCs

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

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

Hypoxia-induced circWSB1 promotes breast cancer progression through destabilizing p53 by interacting with USP10

BackgroundHypoxia has long been considered as a hallmark of solid tumors and is closely associated with tumor progression. Circular RNAs (circRNAs) have been identified as a critical modulator in various cancers. However, the connections between hypoxia and circRNAs are largely unknown.MethodsHere, we investigated the expression profile of circRNAs in breast cancer (BC) MCF-7 cells under hypoxia and normoxia using microarray. We identified a novel hypoxia-responsive circRNA named circWSB1, whose expression pattern, potential diagnostic value and prognostic significance were assessed by qRT-PCR and in situ hybridization. Loss- and gain-of-function investigations in vivo and in vitro were performed to determine the biological functions of circWSB1. Mechanistically, chromatin immunoprecipitation and dual luciferase reporter assays were carried out to analyze the biogenesis of circWSB1. Furthermore, biotin-labeled RNA pull-down, mass spectrometry, RNA immunoprecipitation, fluorescent in situ hybridization, RNA electrophoretic mobility shift, deletion-mapping, co-immunoprecipitation assays and rescue experiments were applied to investigate the interaction between circWSB1 and Ubiquitin-specific peptidase 10 (USP10) as well as the relationship between USP10 and p53.ResultsWe found that the expression of circWSB1 was significantly upregulated in BC tissues and correlated with poor clinical outcomes, which might serve as an independent prognostic factor for BC patients. Ectopic expression of circWSB1 promoted the proliferation of BC cell in vitro and in vivo. Mechanistically, circWSB1 was transcriptionally upregulated by HIF1α in response to hypoxia and could competitively bind to deubiquitinase USP10 to prevent the access of p53 to USP10 in BC cells, leading to degradation of p53 and tumor progression of BC.ConclusionsTaken together, our findings disclose a novel mechanism that hypoxia-inducible circWSB1 could interact with USP10 to attenuate USP10 mediated p53 stabilization and promote the progression of BC, providing an alternative prognostic biomarker and therapeutic target for BC.

Neurogenic Hypertension Mediated Mitochondrial Abnormality Leads to Cardiomyopathy: Contribution of UPRmt and Norepinephrine-miR- 18a-5p-HIF-1α Axis.

Aims: Hypertension increases the risk of heart disease. Hallmark features of hypertensive heart disease is sympathoexcitation and cardiac mitochondrial abnormality. However, the molecular mechanisms for specifically neurally mediated mitochondrial abnormality and subsequent cardiac dysfunction are unclear. We hypothesized that enhanced sympatho-excitation to the heart elicits cardiac miR-18a-5p/HIF-1α and mitochondrial unfolded protein response (UPRmt) signaling that lead to mitochondrial abnormalities and consequent pathological cardiac remodeling.Methods and Results: Using a model of neurogenic hypertension (NG-HTN), induced by intracerebroventricular (ICV) infusion of Ang II (NG-HTN; 20 ng/min, 14 days, 0.5 μl/h, or Saline; Control, 0.9%) through osmotic mini-pumps in Sprague-Dawley rats (250–300 g), we attempted to identify a link between sympathoexcitation (norepinephrine; NE), miRNA and HIF-1α signaling and UPRmt to produce mitochondrial abnormalities resulting in cardiomyopathy. Cardiac remodeling, mitochondrial abnormality, and miRNA/HIF-1α signaling were assessed using histology, immunocytochemistry, electron microscopy, Western blotting or RT-qPCR. NG-HTN demonstrated increased sympatho-excitation with concomitant reduction in UPRmt, miRNA-18a-5p and increased level of HIF-1α in the heart. Our in silico analysis indicated that miR-18a-5p targets HIF-1α. Direct effects of NE on miRNA/HIF-1α signaling and mitochondrial abnormality examined using H9c2 rat cardiomyocytes showed NE reduces miR-18a-5p but increases HIF-1α. Electron microscopy revealed cardiac mitochondrial abnormality in NG-HTN, linked with hypertrophic cardiomyopathy and fibrosis. Mitochondrial unfolded protein response was decreased in NG-HTN indicating mitochondrial proteinopathy and proteotoxic stress, associated with increased mito-ROS and decreased mitochondrial membrane potential (ΔΨm), and oxidative phosphorylation. Further, there was reduced cardiac mitochondrial biogenesis and fusion, but increased mitochondrial fission, coupled with mitochondrial impaired TIM-TOM transport and UPRmt. Direct effects of NE on H9c2 rat cardiomyocytes also showed cardiomyocyte hypertrophy, increased mitochondrial ROS generation, and UPRmt corroborating the in vivo data.Conclusion: In conclusion, enhanced sympatho-excitation suppress miR-18a-5p/HIF-1α signaling and increased mitochondrial stress proteotoxicity, decreased UPRmt leading to decreased mitochondrial dynamics/OXPHOS/ΔΨm and ROS generation. Taken together, these results suggest that ROS induced mitochondrial transition pore opening activates pro-hypertrophy/fibrosis/inflammatory factors that induce pathological cardiac hypertrophy and fibrosis commonly observed in NG-HTN.