Hypoxia-inducible Factor Response Research Articles

Novel bioassays based on 3D-printed device for sensing of hypoxia and p53 pathway in 3D cell models.

Cell-based assays are widely exploited for drug screening and biosensing, providing useful information about bioactivity of target analytes and complex biological samples. It is well recognized that 3D cell models are required to achieve highly valuable information, also from the perspective of replacing animal models. However, bioassays relying on 3D cell models are generally highly demanding in terms of facilities, equipment, and skilled personnel requirements. To reduce cost, increase sustainability, and provide a flexible 3D cell-based platform for bioassays, we here report a novel approach based on a 3D-printed microtissue device. To assess the suitability of this strategy for reporter gene technology, we selected to monitor two molecular pathways which were of interest in several applications, hypoxia signaling and the p53 pathway. The investigation of such pathways is highly relevant in fields spanning from drug screening to bioactivity monitoring for industrial by-product valorization. Microtissues of human hepatocarcinoma (HepG2) and human embryonic kidney (Hek293T) cell lines were obtained with a low-cost and sustainable chip platform and bioassays were developed to monitor the hypoxia-inducible factors (HIFs) and the p53 tumor suppressor pathway. HepG2 and Hek293T 3D cell models were genetically engineered to express the Luc2P from Photinus pyralis firefly either under the regulation of p53 or HIF response elements. The bioassays allowed quantitative assessment of hypoxia and tumoral activity with 1,10-phenanthroline for HIF and with doxorubicin for p53 pathway activation, respectively, showing good potential for applications of this sustainable and low-cost 3D-printed microfluidic platform for bioactivity analyses, drug screening, and precision medicine.

Deubiquitinating enzyme mutagenesis screens identify a USP43-dependent HIF-1 transcriptional response

The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1α isoform, and while USP43 does not alter HIF-1α stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia and phosphorylation dependent manner to increase the nuclear pool of HIF-1. Together, our results highlight the multifunctionality of DUBs, illustrating that they can provide important signalling functions alongside their catalytic roles.

Expression of prolyl hydroxylase domains, the upstream regulators of HIF, in the brain of the anoxia-tolerant crucian carp during anoxia-reoxygenation.

The hypoxia-inducible factor (HIF) is considered key in the transcriptional response to low oxygen. Yet, the role of HIF in the absence of oxygen (anoxia) and in preparation for reoxygenation remains unclear. Recent studies suggest that mounting a HIF response may be counterproductive for anoxia survival. We here studied one of the champions of anoxia survival, the crucian carp (Carassius carassius), and hypothesized that expression of prolyl hydroxylase domains (PHDs; the upstream regulators of HIF) are upregulated to circumvent an energy-costly activation of HIF in anoxia and to prepare for reoxygenation. We measured whole brain mRNA and protein levels of the three isoforms PHD1, PHD2, and PHD3, coded for by multiple paralogs of the genes egln2, egln1, and egln3, using quantitative PCR and Western blotting in the brain of crucian carps exposed to 5 days normoxia or anoxia, and 5 days anoxia followed by 3 or 24 h of reoxygenation. The mRNA levels of most egln paralogs were increased in anoxia and upon reoxygenation, with egln3 showing the largest increase in mRNA level (up to 17-fold) and highest relative mRNA abundance (up to 75% of expressed egln). The protein level of all PHDs was maintained in anoxia and increased upon reoxygenation. We then explored PHD distribution in different brain regions and found PHD immunoreactivity to be associated with axonal branches and showing region-specific changes during anoxia-reoxygenation. Our results support an overall upregulation of egln under prolonged anoxia and PHDs upon reoxygenation in crucian carp, likely aimed at suppressing HIF responses, although regional differences are apparent in such a complex organ as the brain.NEW & NOTEWORTHY We report a profound upregulation of most egln paralog mRNA levels in anoxia and upon reoxygenation, with egln3ii showing the largest, a 17-fold increase, and highest relative mRNA abundance. The relative abundance of prolyl hydroxylase domain (PHD) proteins was maintained during anoxia and increased at reoxygenation. PHD immunoreactivity was localized to axonal branches with region-specific changes during anoxia-reoxygenation. These dynamic and regional changes in crucian carp, champion of anoxia tolerance, are most likely adaptive and call for further mechanistic studies.

Contribution of HIF-P4H isoenzyme inhibition to metabolism indicates major beneficial effects being conveyed by HIF-P4H-2 antagonism

Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases (HIF-P4Hs 1–3) are druggable targets in renal anemia, where pan-HIF-P4H inhibitors induce an erythropoietic response. Preclinical data suggest that HIF-P4Hs could also be therapeutic targets for treating metabolic dysfunction, although the contributions of HIF-P4H isoenzymes in various tissues to the metabolic phenotype are inadequately understood. Here, we used mouse lines that were gene-deficient for HIF-P4Hs 1 to 3 and two preclinical pan-HIF-P4H inhibitors to study the contributions of these isoenzymes to the anthropometric and metabolic outcome and HIF response. We show both inhibitors induced a HIF response in wildtype white adipose tissue (WAT), liver, and skeletal muscle and alleviated metabolic dysfunction during a 6-week treatment period, but they did not alter healthy metabolism. Our data indicate that HIF-P4H-1 contributed especially to skeletal muscle and WAT metabolism and that its loss lowered body weight and serum cholesterol levels upon aging. In addition, we found HIF-P4H-3 had effects on the liver and WAT and its loss increased body weight, adiposity, liver weight and triglyceride levels, WAT inflammation, and cholesterol levels and resulted in hyperglycemia and insulin resistance, especially during aging. Finally, we demonstrate HIF-P4H-2 affected all tissues studied; its inhibition lowered body and liver weight and serum cholesterol levels and improved glucose tolerance. We found very few HIF target metabolic mRNAs were regulated by the inhibition of three isoenzymes, thus suggesting a potential for selective therapeutic tractability. Altogether, these data provide specifications for the future development of HIF-P4H inhibitors for the treatment of metabolic diseases.

The effect of HIF on metabolism and immunity.

Cellular hypoxia occurs when the demand for sufficient molecular oxygen needed to produce the levels of ATP required to perform physiological functions exceeds the vascular supply, thereby leading to a state of oxygen depletion with the associated risk of bioenergetic crisis. To protect against the threat of hypoxia, eukaryotic cells have evolved the capacity to elicit oxygen-sensitive adaptive transcriptional responses driven primarily (although not exclusively) by the hypoxia-inducible factor (HIF) pathway. In addition to the canonical regulation of HIF by oxygen-dependent hydroxylases, multiple other input signals, including gasotransmitters, non-coding RNAs, histone modifiers and post-translational modifications, modulate the nature of the HIF response in discreet cell types and contexts. Activation of HIF induces various effector pathways that mitigate the effects of hypoxia, including metabolic reprogramming and the production of erythropoietin. Drugs that target the HIF pathway to induce erythropoietin production are now approved for the treatment of chronic kidney disease-related anaemia. However, HIF-dependent changes in cell metabolism also have profound implications for functional responses in innate and adaptive immune cells, and thereby heavily influence immunity and the inflammatory response. Preclinical studies indicate a potential use of HIF therapeutics to treat inflammatory diseases, such as inflammatory bowel disease. Understanding the links between HIF, cellular metabolism and immunity is key to unlocking the full therapeutic potential of drugs that target the HIF pathway.

Abstract 3766: Correlating locus-specific changes in histone trimethylation and gene expression in hypoxia

Abstract Low oxygen availability (hypoxia) is a common feature of many solid tumors and is associated with poor prognosis and resistance to therapy. The main regulator of cellular responses to hypoxia is the transcription factor HIF (hypoxia-inducible factor). In addition, several histone lysine demethylases (KDMs) are 2-OG dependent dioxygenases and require oxygen for their activity. When oxygen availability is limited, the activity of these KDMs is inhibited leading to global histone hypermethylation, potentially altering gene expression. However, some KDMs are also known to be direct transcriptional targets of HIF, increasing their expression in hypoxia and counteracting this histone hypermethylation. Furthermore, HIF can facilitate recruitment of histone methyltransferase activity to chromatin in cis, leading to local increases in methylation status. Histone methylation can be associated with both gene activation and gene repression, and it remains unclear how the balance of these widespread changes relates to locus-specific regulation of gene expression. Furthermore, previous attempts to analyze regulation of histone methylation by ChIP-seq in hypoxia have not employed normalization methods that take into account global changes in histone modification. We have performed ChIP-seq analysis of histone trimethylation (H3K4me3, H3K9me3, H3K27me3 and H3K36me3) in PC3 prostate cancer cells incubated in normoxia or (16hrs, 1%) hypoxia. We have then examined the effects of the HIF-transcriptional pathway on these marks using the same cell line following deletion of HIF-1b to block the HIF response. We employ an integrated spike-in with drosophila chromatin to provide a normalization control. We then relate our findings to changes in gene expression using RNA-seq analysis, again normalized to a spike-in control. We observe global increases in all 4 histone trimethylations across the genome in hypoxia. In particular, we observe hypoxic increases in histone trimethylation at all gene loci. However, while basal levels of histone trimethylation correlated with basal transcript levels as previously reported, hypoxic increases in promoter-associated histone H3K4me3 trimethylation bore no relation to either the direction or magnitude of gene regulation in hypoxia. Although, more subtle increases were observed across the gene body at gene loci induced by hypoxia. These findings question the currently accepted model that changes in H3K4me3 instruct changes in transcription in hypoxia and suggest that the overall effect of histone trimethylation is a more subtle balance of competing effects. Citation Format: Jessica D. Kindrick, Peter J. Ratcliffe, WIlliam Doug Figg, David R. Mole. Correlating locus-specific changes in histone trimethylation and gene expression in hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3766.

Hypoxia-Inducible Factors Regulate Osteoclasts in Health and Disease.

Hypoxia-inducible factors (HIFs) have become key transcriptional regulators of metabolism, angiogenesis, erythropoiesis, proliferation, inflammation and metastases. HIFs are tightly regulated by the tissue microenvironment. Under the influence of the hypoxic milieu, HIF proteins allow the tissue to adapt its response. This is especially critical for bone, as it constitutes a highly hypoxic environment. As such, bone structure and turnover are strongly influenced by the modulation of oxygen availability and HIFs. Both, bone forming osteoblasts and bone resorbing osteoclasts are targeted by HIFs and modulators of oxygen tension. Experimental and clinical data have delineated the importance of HIF responses in different osteoclast-mediated pathologies. This review will focus on the influence of HIF expression on the regulation of osteoclasts in homeostasis as well as during inflammatory and malignant bone diseases.

HIF-1-induced mitochondrial ribosome protein L52: a mechanism for breast cancer cellular adaptation and metastatic initiation in response to hypoxia.

Background: Hypoxia is a hallmark of the physical microenvironment of solid tumors. As a key factor that regulates tumor development and progression, hypoxia can reprogram the expression of multiple genes, whose biological function and molecular mechanism in cancer remain largely unclear. The mitochondrial ribosome protein family consists of nuclear-encoded mitochondrial proteins that are responsible for protein synthesis in the mitochondria.Methods: A high-throughput RNA sequencing assay was carried out to identify differentially expressed mRNAs between breast cancer tissues and adjacent normal tissues as well as breast tumors with metastasis and those without metastasis. Our clinical samples and TCGA database were analyzed to observe the clinical value of mitochondrial ribosome protein L52 (MRPL52) in human breast cancer. Potent hypoxia response elements in the promoter region of MRPL52 were identified and validated by chromatin immunoprecipitation and luciferase reporter assays. Functional experiments were performed using breast cancer cell lines with MRPL52 ectopic expression and knockdown cultured in a 20% or 1% O2 environment.Results: MRPL52 expression was upregulated in human breast cancer and was significantly associated with aggressive clinicopathological characteristics and a higher metastatic risk of breast cancer patients. We found that the overexpression of MRPL52 in breast cancer is induced by hypoxia-inducible factor-1 in response to hypoxic exposure. The role of MRPL52 in suppressing apoptosis and promoting migration and invasion of hypoxic breast cancer cells was demonstrated by our experimental evidence. Mechanistically, MRPL52 promoted PTEN-induced putative kinase 1 /Parkin-dependent mitophagy to remove oxidatively damaged mitochondria and prevent uncontrolled reactive oxygen species (ROS) generation, thus repressing activation of the mitochondrial apoptotic cascade. Additionally, MRPL52 augmented epithelial-mesenchymal transition, migration and invasion of hypoxic breast cancer cells by activating the ROS-Notch1-Snail signaling pathway. Benefited from this bidirectional regulatory mechanism, MRPL52 is responsible for maintaining ROS levels in a window that can induce tumorigenic signal transduction without causing cytotoxicity in hypoxic breast cancer cells.Conclusions: This work elucidates the molecular mechanism by which MRPL52 mediates hypoxia-induced apoptotic resistance and metastatic initiation of breast cancer, and provides new insights into the interplay between cancer and the tumor microenvironment.

The role of hypoxia‐inducible factors in age‐related macular degeneration

AbstractPurposePathophysiological mechanisms of age‐related macular degeneration (AMD) have been attributed mainly to local tissue ischemia or the presence of low‐grade inflammatory reaction. A fundamental difference between both types of AMD is the increased vascular endothelial growth factor (VEGF) level in neovascular AMD (nAMD), a result of underlying tissue hypoxia and upregulation of hypoxia‐inducible factor (HIF) in the retinal pigment epithelium (RPE), leading to choroidal neovascularization (CNV). Here, we will present a summary of our studies in the benefits of mitigating HIF responses in models of nAMD.MethodsWe have addressed the hypoxia response in RPE cells in vitro (retinal cells cultures) and in vivo (laser‐induced CNV model), using tissue ischemia molecular modulators (UPARANT), HIF‐specific inhibitors (Echinomycin) and HIF molecular negative regulators (prolyl hydroxylase domain 2) as a negative‐modulators of HIF.ResultsWe have demonstrated that expression of HIF is upregulated in RPE cells in vivo during progression of CNV, and was associated with increased VEGF levels. Collectively, our data has demonstrated a role for HIF in nAMD, and that negative‐regulation of the hypoxia pathway in RPE cells abnegates the increase in VEGF, together with other HIF‐mediated factors, significantly reducing CNV associated with nAMD.ConclusionsDue to the protracted nature of AMD, the multifactorial mechanisms involved, and current therapeutic limitations there is a great need for long‐term therapies that can mitigate multiple angiogenic and inflammatory factors known to be associated with nAMD, allowing for sustainable treatments. We have established a molecular role for HIFs in nAMD progression, and have identified molecules capable of regulating hypoxia processes that have pronounced translational therapeutic potential.ReferenceH André et. al. IOVS 2015; M Cammalleri et al. IOVS 2016; A Takei et al. Sci Rep 2017; F. Plastino et al. EVER2019 abstract.

The insights into molecular pathways of hypoxia-inducible factor in the brain.

The objectives of this present work were to review recent developments on the role of hypoxia-inducible factor (HIF) in the survival of cells under normoxic versus hypoxic and inflammatory brain conditions. The dual nature of HIF effects appears well established, based on the accumulated evidence of HIF playing both the role of adaptive factor and mediator of cell demise. Cellular HIF responses depend on pathophysiological conditions, developmental phase, comorbidities, and administered medications. In addition, HIF-1α and HIF-2α actions may vary in the same tissues. The multiple roles of HIF in stem cells are emerging. HIF not only regulates expression of target genes and thereby influences resultant protein levels but also contributes to epigenetic changes that may reciprocally provide feedback regulations loops. These HIF-dependent alterations in neurological diseases and its responses to treatments in vivo need to be examined alongside with a functional status of subjects involved in such studies. The knowledge of HIF pathways might be helpful in devising HIF-mimetics and modulating drugs, acting on the molecular level to improve clinical outcomes, as exemplified here by clinical and experimental data of selected brain diseases, occasionally corroborated by the data from disorders of other organs. Because of complex role of HIF in brain injuries, prospective therapeutic interventions need to differentially target HIF responses depending on their roles in the molecular mechanisms of neurologic diseases.

CLIC4/Arf6 Pathway.

Increased expression of CLIC4 (chloride intracellular channel 4) is a feature of endothelial dysfunction in pulmonary arterial hypertension, but its role in disease pathology is not fully understood. To identify CLIC4 effectors and evaluate strategies targeting CLIC4 signaling in pulmonary hypertension. Proteomic analysis of CLIC4-interacting proteins in human pulmonary artery endothelial cells identified regulators of endosomal trafficking, including Arf6 (ADP ribosylation factor 6) GTPase activating proteins and clathrin, while CLIC4 overexpression affected protein regulators of vesicular trafficking, lysosomal function, and inflammation. CLIC4 reduced BMPRII (bone morphogenetic protein receptor II) expression and signaling as a result of Arf6-mediated reduction in gyrating clathrin and increased lysosomal targeting of the receptor. BMPRII expression was restored by Arf6 siRNA, Arf inhibitor Sec7 inhibitor H3 (SecinH3), and inhibitors of clathrin-mediated endocytosis but was unaffected by chloride channel inhibitor, indanyloxyacetic acid 94 or Arf1 siRNA. The effects of CLIC4 on NF-κB (nuclear factor-kappa B), HIF (hypoxia-inducible factor), and angiogenic response were prevented by Arf6 siRNA and SecinH3. Sugen/hypoxia mice and monocrotaline rats showed elevated expression of CLIC4, activation of Arf6 and NF-κB, and reduced expression of BMPRII in the lung. These changes were established early during disease development. Lung endothelium-targeted delivery of CLIC4 siRNA or treatment with SecinH3 attenuated the disease, reduced CLIC4/Arf activation, and restored BMPRII expression in the lung. Endothelial colony-forming cells from idiopathic pulmonary hypertensive patients showed upregulation of CLIC4 expression and Arf6 activity, suggesting potential importance of this pathway in the human condition. Arf6 is a novel effector of CLIC4 and a new therapeutic target in pulmonary hypertension.

Determination of hypoxia-inducible factor-1 by using a ratiometric colorimetric test based on click-mediated growth of gold nanoparticles.

The authors describe a significantly improved colorimetric nanoprobe for the determination of transcription factors (TFs). It is making use of click-mediated growth of gold nanoparticles (AuNPs) to amplify the signal-to-noise ratio. Hypoxia-inducible factor-1 (HIF-1) is an important TF that acts as a mediator of cell response to hypoxia. So, the detection of HIF-1 was chosen as the model analyte. Specifically, target HIF-1 is designed to bind to the hypoxia response element within DNA duplex. The click chemistry between the DNA duplex and alkynyl-functionalized AuNPs (AF-AuNPs) is then inhibited because of significant steric hindrance. As a result, the AF-AuNPs grow into larger-sized highly-aggregated irregular nanostructures, which in turn enable colorimetric determination. The ratio of absorbances at 620 and 560nm increases in the 0.5 to 10nM HIF-1 concentration range, and the detection limit is 0.27nM. This is better by a factor of 100 than that of aggregation-based colorimetric assays. The nanoprobe is selective and can be used in complex samples. Conceivably, it may also be extended to the determination of other TFs by simply changing the used DNA duplex. Graphical abstract Schematic of a nanoprobe for detecting hypoxia-inducible factor-1 (HIF-1). Three concepts are involved: the binding of HIF-1 and hypoxia response element, the Cu+-catalyzed click chemistry between P1/P2 duplex and alkynyl-functionalized AuNPs (AF-AuNPs), and the AuNPs growth with hydroxylamine and HAuCl4.

Cellular adaptive response and regulation of HIF after low dose gamma-radiation exposure

Purpose: Cellular damage due to low dose of γ-radiation (≤0.1 Gy) is generally extrapolated from observing the effects at higher doses. These estimations are not accurate. This has led to uncertainties while assessing the radiation risk factors at low doses. Although there are reports on the radiation induced adaptive response, the mechanism of action is not fully elucidated, leading to the uncertainties. One of the outcomes of low dose radiation exposure is believed to be an adaptive response. The mechanism of adaptive response is not fully understood. Therefore, the study was undertaken to understand the role of hypoxia inducible factor (HIF) on radiation induced adaptive response.Materials and methods: Human breast cancer cell line MCF-7 cells pre-exposed to low dose γ-radiation (0.1 Gy; priming dose) were exposed to 2 Gy (challenging dose) 8 h after the priming dose and studied for the adaptive response. Cell death was measured by 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay and apoptosis was measured by fluorescence-activated cell sorting analysis. DNA damage was measured by alkaline comet assay. HIF transcription activity was assayed using transiently transfected plasmid having HIF consensus sequence and luciferase as the reporter gene.Results: Cells when exposed to 0.1 Gy priming dose 8 h prior to the higher dose (2 Gy; challenging dose) results in lower amount of radiation induced damages compared to the cells exposed to 2 Gy alone. Cobalt chloride treatment in place of priming dose also results in the protection to cells when exposed to challenging dose. There was up-regulation of HIF activity when cells were exposed to priming dose, indicating the role of HIF in radiation induced response.Conclusion: Results indicate the γ-radiation induced adaptive response. One of the mechanism proposed is up-regulation of HIF after low dose exposure, which protects the cells from damages when they are exposed to challenging dose of 2 Gy radiation.

Aryl Hydrocarbon Receptor Nuclear Translocator in Vascular Smooth Muscle Cells Is Required for Optimal Peripheral Perfusion Recovery.

BackgroundLimb ischemia resulting from peripheral vascular disease is a common cause of morbidity. Vessel occlusion limits blood flow, creating a hypoxic environment that damages distal tissue, requiring therapeutic revascularization. Hypoxia‐inducible factors (HIFs) are key transcriptional regulators of hypoxic vascular responses, including angiogenesis and arteriogenesis. Despite vascular smooth muscle cells’ (VSMCs’) importance in vessel integrity, little is known about their functional responses to hypoxia in peripheral vascular disease. This study investigated the role of VSMC HIF in mediating peripheral ischemic responses.Methods and ResultsWe used Arnt SMKO mice with smooth muscle–specific deletion of aryl hydrocarbon receptor nuclear translocator (ARNT, HIF‐1β), required for HIF transcriptional activity, in a femoral artery ligation model of peripheral vascular disease. Arnt SMKO mice exhibit impaired perfusion recovery despite normal collateral vessel dilation and angiogenic capillary responses. Decreased blood flow manifests in extensive tissue damage and hypoxia in ligated limbs of Arnt SMKO mice. Furthermore, loss of aryl hydrocarbon receptor nuclear translocator changes the proliferation, migration, and transcriptional profile of cultured VSMCs. Arnt SMKO mice display disrupted VSMC morphologic features and wrapping around arterioles and increased vascular permeability linked to decreased local blood flow.ConclusionsOur data demonstrate that traditional vascular remodeling responses are insufficient to provide robust peripheral tissue reperfusion in Arnt SMKO mice. In all, this study highlights HIF responses to hypoxia in arteriole VSMCs critical for the phenotypic and functional stability of vessels that aid in the recovery of blood flow in ischemic peripheral tissues.

High-intensity interval training in hypoxia does not affect muscle HIF responses to acute hypoxia in humans.

The myocellular response to hypoxia is primarily regulated by hypoxia-inducible factors (HIFs). HIFs thus conceivably are implicated in muscular adaptation to altitude training. Therefore, we investigated the effect of hypoxic versus normoxic training during a period of prolonged hypoxia ('living high') on muscle HIF activation during acute ischaemia. Ten young male volunteers lived in normobaric hypoxia for 5 weeks (5 days per week, ~ 15.5h per day, FiO2: 16.4-14.0%). One leg was trained in hypoxia (TRHYP, 12.3% FiO2) whilst the other leg was trained in normoxia (TRNOR, 20.9% FiO2). Training sessions (3 per week) consisted of intermittent unilateral knee extensions at 20-25% of the 1-repetition maximum. Before and after the intervention, a 10-min arterial occlusion and reperfusion of the leg was performed. Muscle oxygenation status was continuously measured by near-infrared spectroscopy. Biopsies were taken from m. vastus lateralis before and at the end of the occlusion. Irrespective of training, occlusion elevated the fraction of HIF-1α expressing myonuclei from ~ 54 to ~ 64% (P < 0.05). However, neither muscle HIF-1α or HIF-2α protein abundance, nor the expression of HIF-1α or downstream targets selected increased in any experimental condition. Training in both TRNOR and TRHYP raised muscular oxygen extraction rate upon occlusion by ~ 30%, whilst muscle hyperperfusion immediately following the occlusion increased by ~ 25% in either group (P < 0.05). Ten minutes of arterial occlusion increased HIF-1α-expressing myonuclei. However, neither normoxic nor hypoxic training during 'living high' altered muscle HIF translocation, stabilisation, or transcription in response to acute hypoxia induced by arterial occlusion.

Hypoxia favors myosin heavy chain beta gene expression in an Hif-1alpha-dependent manner

The potentiation of the naturally limited regenerative capacity of the heart is dependent on an understanding of the mechanisms that are activated in response to pathological conditions such as hypoxia. Under these conditions, the expression of genes suggested to support cardiomyocyte survival and heart adaptation is triggered. Particularly important are changes in the expression of myosin heavy chain (MHC) isoforms. We propose here that alterations in the expression profiles of MHC genes are induced in response to hypoxia and are primarily mediated by hypoxia inducible factor (HIF). In in vitro models of mouse embryonic stem cell-derived cardiomyocytes, we showed that hypoxia (1% O2) or the pharmacological stabilization of HIFs significantly increased MHCbeta (Myh7) gene expression. The key role of HIF-1alpha is supported by the absence of these effects in HIF-1alpha-deficient cells, even in the presence of HIF-2alpha. Interestingly, ChIP analysis did not confirm the direct interaction of HIF-1alpha with putative HIF response elements predicted in the MHCalpha and beta encoding DNA region. Further analyses showed the significant effect of the mTOR signaling inhibitor rapamycin in inducing Myh7 expression and a hypoxia-triggered reduction in the levels of antisense RNA transcripts associated with the Myh7 gene locus. Overall, the recognized and important role of HIF in the regulation of heart regenerative processes could be highly significant for the development of novel therapeutic interventions in heart failure.

CHCHD4 Regulates Intracellular Oxygenation and Perinuclear Distribution of Mitochondria.

Hypoxia is a characteristic of the tumor microenvironment and is known to contribute to tumor progression and treatment resistance. Hypoxia-inducible factor (HIF) dimeric transcription factors control the cellular response to reduced oxygenation by regulating the expression of genes involved in metabolic adaptation, cell motility, and survival. Alterations in mitochondrial metabolism are not only a downstream consequence of HIF-signaling but mitochondria reciprocally regulate HIF signaling through multiple means, including oxygen consumption, metabolic intermediates, and reactive oxygen species generation. CHCHD4 is a redox-sensitive mitochondrial protein, which we previously identified and showed to be a novel regulator of HIF and hypoxia responses in tumors. Elevated expression of CHCHD4 in human tumors correlates with the hypoxia gene signature, disease progression, and poor patient survival. Here, we show that either long-term (72 h) exposure to hypoxia (1% O2) or elevated expression of CHCHD4 in tumor cells in normoxia leads to perinuclear accumulation of mitochondria, which is dependent on the expression of HIF-1α. Furthermore, we show that CHCHD4 is required for perinuclear localization of mitochondria and HIF activation in response to long-term hypoxia. Mutation of the functionally important highly conserved cysteines within the Cys-Pro-Cys motif of CHCHD4 or inhibition of complex IV activity (by sodium azide) redistributes mitochondria from the perinuclear region toward the periphery of the cell and blocks HIF activation. Finally, we show that CHCHD4-mediated perinuclear localization of mitochondria is associated with increased intracellular hypoxia within the perinuclear region and constitutive basal HIF activation in normoxia. Our study demonstrates that the intracellular distribution of the mitochondrial network is an important feature of the cellular response to hypoxia, contributing to hypoxic signaling via HIF activation and regulated by way of the cross talk between CHCHD4 and HIF-1α.

Oxygen sensors as therapeutic targets in kidney disease

Hypoxia is a common clinical problem that has profound effects on renal homeostasis. Prolyl-4-hydroxylases PHD1, 2 and 3 function as oxygen sensors and control the activity of hypoxia-inducible factor (HIF), an oxygen-sensitive transcription factor that regulates a multitude of hypoxia responses, which help cells and tissues to adapt to low oxygen environments. This review provides an overview of the molecular mechanisms that govern these hypoxia responses and discusses clinical experience with compounds that inhibit prolyl-4-hydroxylases to harness HIF responses for therapy in nephrology.

Acidic pH Is a Metabolic Switch for 2-Hydroxyglutarate Generation and Signaling

2-Hydroxyglutarate (2-HG) is an important epigenetic regulator, with potential roles in cancer and stem cell biology. The d-(R)-enantiomer (d-2-HG) is an oncometabolite generated from α-ketoglutarate (α-KG) by mutant isocitrate dehydrogenase, whereas l-(S)-2-HG is generated by lactate dehydrogenase and malate dehydrogenase in response to hypoxia. Because acidic pH is a common feature of hypoxia, as well as tumor and stem cell microenvironments, we hypothesized that pH may regulate cellular 2-HG levels. Herein we report that cytosolic acidification under normoxia moderately elevated 2-HG in cells, and boosting endogenous substrate α-KG levels further stimulated this elevation. Studies with isolated lactate dehydrogenase-1 and malate dehydrogenase-2 revealed that generation of 2-HG by both enzymes was stimulated severalfold at acidic pH, relative to normal physiologic pH. In addition, acidic pH was found to inhibit the activity of the mitochondrial l-2-HG removal enzyme l-2-HG dehydrogenase and to stimulate the reverse reaction of isocitrate dehydrogenase (carboxylation of α-KG to isocitrate). Furthermore, because acidic pH is known to stabilize hypoxia-inducible factor (HIF) and 2-HG is a known inhibitor of HIF prolyl hydroxylases, we hypothesized that 2-HG may be required for acid-induced HIF stabilization. Accordingly, cells stably overexpressing l-2-HG dehydrogenase exhibited a blunted HIF response to acid. Together, these results suggest that acidosis is an important and previously overlooked regulator of 2-HG accumulation and other oncometabolic events, with implications for HIF signaling.

Abstract PR03: Hypoxia preconditioning affects metastatic seeding and proliferation through vascular and immune function, in a process regulated by lung endothelial HIFa

Abstract Metastases are the main cause of mortality in solid cancers. Current evidence shows that tumor cells are present in circulation from early stages of malignancy, and this initial dissemination implies that early diagnosis alone is not a consistent or reliable predictor of outcome. A deeper understanding of factors affecting extravasation and secondary proliferation is crucial to develop strategies to prevent and/or treat metastatic disease. Our research and that of others have shown that tumor dissemination rely on properties of the tumor cells, but also on the individual cell types in the microenvironments, as well as on the physiological context in the host organ. Tumor progression is, consequently, a function of the individual cell types in the microenvironments of the primary tumor and metastatic sites. Activation of hypoxia-inducible factors (HIFs) in most solid tumor types is directly correlated with poor prognosis. However, we have shown that when in host organ cells, and specifically endothelial cells, the role of HIF is isoform-specific: mice lacking endothelial HIF-1α develop less lung metastases, whereas those lacking endothelial HIF-2α have more. This is a very intriguing phenomenon that revealed a decisive role for the endothelium in allowing or preventing metastatic success, shown to be, in part, dependent on an imbalanced production of nitric oxide (NO), with clear NO-driven effects on vessel function. Upon exposure of mice to low atmospheric oxygen levels, we found that there is a time-dependent activation of HIF-1α and HIF-2α in lung tissue, which results in differential gene expression and inflammatory response/inflammatory cell recruitment, affecting both extravasation and secondary proliferation of circulating tumor cells. In this study, mice were hypoxia-conditioned prior to intravenous injection of cancer cells, to investigate the effect of hypoxia-driven vascular changes on pulmonary metastases. Mice exposed to either short or prolonged hypoxia show increased number of lung monocytes when compared to normoxic controls. However, acute hypoxia (24h), which correlates with higher HIF-1α levels, results in increased numbers of lung tumors, whereas chronic hypoxia exposure (10d, higher HIF-2α activation) results in fewer and less proliferative tumors. Using a variety of approaches, including cytokine and transcriptional screens, imaging and cell-specific HIF-deletion models, both in vivo and ex-vivo, we are investigating which parameters affecting metastatic disease are changed during acute and prolonged hypoxia, resulting in differential metastatic incidence. We found that both endothelial permeability and endothelial-derived signaling is affected differently upon acute or chronic hypoxia stimulus, with also different impact on extravasation and immune cell recruitment and activation. We aim to identify what molecular mechanism(s) underlying hypoxia, HIF and vascular endothelium responses condition the establishment of secondary tumors. Citation Format: Cristina Branco, Helene Rundqvist, Renato Colaco, Randall S. Johnson. Hypoxia preconditioning affects metastatic seeding and proliferation through vascular and immune function, in a process regulated by lung endothelial HIFa. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr PR03.