[Show abstract][Hide abstract] ABSTRACT: Reperfusion of ischemic cardiac tissue is the standard treatment for improving clinical outcome following myocardial infarction but is inevitably associated with ischemia-reperfusion injury (IRI). Ischemic myocardial injury can be alleviated by exposing the heart to brief episodes of sublethal ischemia-reperfusion prior to the ischemic insult, a phenomenon that has been termed ischemic preconditioning (IPC). Similarly, remote IPC (RIPC) is defined as transient episodes of ischemia at a distant site before a subsequent prolonged injury of the target organ. In this setting, adaptive responses to hypoxia/ischemia in peripheral tissues include the release of soluble factors that have the potential to protect cardiomyocytes remotely. Oxygen fluctuations is a hallmark of insufficient tissue perfusion and ischemic episodes. Emerging evidence indicates that prolyl hydroxylase oxygen sensors (PHDs) and hypoxia-inducible transcription factors (HIFs) are critical regulators of IPC and RIPC. In this review, we discuss recent findings concerning the role of the PHD-HIF axis in IPC and RIPC-mediated cardioprotection and examine molecular pathways and cell types that might be involved. We also appraise the therapeutic value of targeting the PHD-HIF axis to enhance cardiac tolerance against IRI.
Frontiers in Physiology 05/2015; 6:137. DOI:10.3389/fphys.2015.00137 · 3.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inactivation of the von Hippel-Lindau (VHL) tumor suppressor drives the development of clear-cell renal cell carcinoma (ccRCC) through hypoxia-inducible factors (HIFs). Although ccRCC cells exhibit constitutive normoxic HIF signaling, the potential role of hypoxia in this setting is not fully understood. We show here that the ccRCC cell lines RCC4 and RCC10, which express mutant versions of VHL, have reduced HIF1α expression in hypoxia, whereas HIF2α expression is increased or not affected. Similar findings were observed in normoxia after abrogation of prolyl hydroxylase activity by siRNA or pharmacological inhibition, and by siRNA inhibition of mutant VHL. This reduction of HIF1α protein is due to proteasome-dependent degradation and is mediated by the E3 ubiquitin ligase SART1. HIF1α degradation favors ccRCC proliferation, in line with the previously recognized tumor suppressor capability of HIF1α. Our data indicate that mutant VHL can protect HIF1α from SART1-dependent degradation in normoxic conditions, but this protection is lost in hypoxic settings, favoring hypoxia-dependent ccRCC proliferation. This mechanism of HIF1α degradation might operate in some VHL-related clear-cell renal carcinomas in which the deletion of HIF1α locus does not occur.Oncogene advance online publication, 27 April 2015; doi:10.1038/onc.2015.113.
[Show abstract][Hide abstract] ABSTRACT: The presence of hypoxic regions in solid tumors is an adverse prognostic factor for patient outcome. Here, we show that hypoxia induces the expression of Ephrin-A3 through a novel hypoxia-inducible factor (HIF)-mediated mechanism. In response to hypoxia, the coding EFNA3 mRNA levels remained relatively stable, but HIFs drove the expression of previously unknown long noncoding (lnc) RNAs from EFNA3 locus and these lncRNA caused Ephrin-A3 protein accumulation. Ephrins are cell surface proteins that regulate diverse biological processes by modulating cellular adhesion and repulsion. Mounting evidence implicates deregulated ephrin function in multiple aspects of tumor biology. We demonstrate that sustained expression of both Ephrin-A3 and novel EFNA3 lncRNAs increased the metastatic potential of human breast cancer cells, possibly by increasing the ability of tumor cells to extravasate from the blood vessels into surrounding tissue. In agreement, we found a strong correlation between high EFNA3 expression and shorter metastasis-free survival in breast cancer patients. Taken together, our results suggest that hypoxia could contribute to metastatic spread of breast cancer via HIF-mediated induction of EFNA3 lncRNAs and subsequent Ephrin-A3 protein accumulation.Oncogene advance online publication, 14 July 2014; doi:10.1038/onc.2014.200.
[Show abstract][Hide abstract] ABSTRACT: The mammalian target of rapamycin (mTOR) pathway, which is essential for cell proliferation, is repressed in certain cell types in hypoxia. However, hypoxia-inducible factor 2α (HIF2α) can act as a proliferation-promoting factor in some biological settings. This paradoxical situation led us to study whether HIF2α has a specific effect on mTORC1 regulation. Here we show that activation of the HIF2α pathway increases mTORC1 activity by upregulating expression of the amino acid carrier SLC7A5. At the molecular level we also show that HIF2α binds to the Slc7a5 proximal promoter. Our findings identify a link between the oxygen-sensing HIF2α pathway and mTORC1 regulation, revealing the molecular basis of the tumor-promoting properties of HIF2α in von Hippel-Lindau-deficient cells. We also describe relevant physiological scenarios, including those that occur in liver and lung tissue, wherein HIF2α or low-oxygen tension drive mTORC1 activity and SLC7A5 expression.
[Show abstract][Hide abstract] ABSTRACT: The tumor microenvironment of transplanted and spontaneous mouse tumors is profoundly deprived of oxygenation as confirmed by positron emission tomographic (PET) imaging. CD8 and CD4 tumor-infiltrating T lymphocytes (TIL) of transplanted colon carcinomas, melanomas, and spontaneous breast adenocarcinomas are CD137 (4-1BB)-positive, as opposed to their counterparts in tumor-draining lymph nodes and spleen. Expression of CD137 on activated T lymphocytes is markedly enhanced by hypoxia and the prolyl-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Importantly, hypoxia does not upregulate CD137 in hypoxia-inducible factor (HIF)-1α-knockout T cells, and such HIF-1α-deficient T cells remain CD137-negative even when becoming TILs, in clear contrast to co-infiltrating and co-transferred HIF-1α-sufficient T lymphocytes. The fact that CD137 is selectively expressed on TILs was exploited to confine the effects of immunotherapy with agonist anti-CD137 monoclonal antibodies to the tumor tissue. As a result, low-dose intratumoral injections avoid liver inflammation, achieve antitumor systemic effects, and permit synergistic therapeutic effects with PD-L1/B7-H1 blockade. SIGNIFICANCE: CD137 (4-1BB) is an important molecular target to augment antitumor immunity. Hypoxia in the tumor microenvironment as sensed by the HIF-1α system increases expression of CD137 on tumor-infiltrating lymphocytes that thereby become selectively responsive to the immunotherapeutic effects of anti-CD137 agonist monoclonal antibodies as those used in ongoing clinical trials.
Cancer Discovery 06/2012; 2(7):608-23. DOI:10.1158/2159-8290.CD-11-0314 · 19.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Both malignant and stromal components in tumors are influenced by the physiologic conditions of the microenvironment. Hypoxia is a prominent feature of solid tumors as a result of defective vascularization and intense metabolic activity. The gene-expression control mechanisms that adapt tissues to hypoxia are exploited by tumors to promote angiogenesis and vasculogenesis. The functions of infiltrating immune cells (macrophages and lymphocytes) and other stromal components are also influenced by a limited O(2) supply. Hypoxia-inducible factors (HIF) are the main molecular transcriptional mediators in the hypoxia response. The degradation and activity of HIF-1α and HIF-2α are tightly controlled by the fine-tuned action of oxygen-sensing prolyl and asparaginyl hydroxylase enzymes. Recent evidence indicates that hypoxia can modulate the differentiation and function of T lymphocytes and myeloid cells, skewing their cytokine-production profiles and modifying the expression of costimulatory receptors. This conceivably includes tumor-infiltrating lymphocytes. Hypoxia not only directly affects tumor-infiltrating leukocytes but also exerts effects on tumor cells and vascular cells that indirectly cause selective chemokine-mediated recruitment of suppressive and proangiogenic T-cell subsets. This review focuses on changes induced by hypoxia in immune cells infiltrating solid malignancies. Such changes may either promote or fight cancer, and thus are important for immunotherapy.
Clinical Cancer Research 12/2011; 18(5):1207-13. DOI:10.1158/1078-0432.CCR-11-1591 · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The fine regulation of mitochondrial function has proved to be an essential metabolic adaptation to fluctuations in oxygen availability. During hypoxia, cells activate an anaerobic switch that favors glycolysis and attenuates the mitochondrial activity. This switch involves the hypoxia-inducible transcription factor-1 (HIF-1). We have identified a HIF-1 target gene, the mitochondrial NDUFA4L2 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2). Our results, obtained employing NDUFA4L2-silenced cells and NDUFA4L2 knockout murine embryonic fibroblasts, indicate that hypoxia-induced NDUFA4L2 attenuates mitochondrial oxygen consumption involving inhibition of Complex I activity, which limits the intracellular ROS production under low-oxygen conditions. Thus, reducing mitochondrial Complex I activity via NDUFA4L2 appears to be an essential element in the mitochondrial reprogramming induced by HIF-1.
[Show abstract][Hide abstract] ABSTRACT: Von Hippel Lindau (Vhl) gene inactivation results in embryonic lethality. The consequences of its inactivation in adult mice, and of the ensuing activation of the hypoxia-inducible factors (HIFs), have been explored mainly in a tissue-specific manner. This mid-gestation lethality can be also circumvented by using a floxed Vhl allele in combination with an ubiquitous tamoxifen-inducible recombinase Cre-ER(T2). Here, we characterize a widespread reduction in Vhl gene expression in Vhl(floxed)-UBC-Cre-ER(T2) adult mice after dietary tamoxifen administration, a convenient route of administration that has yet to be fully characterized for global gene inactivation. Vhl gene inactivation rapidly resulted in a marked splenomegaly and skin erythema, accompanied by renal and hepatic induction of the erythropoietin (Epo) gene, indicative of the in vivo activation of the oxygen sensing HIF pathway. We show that acute Vhl gene inactivation also induced Epo gene expression in the heart, revealing cardiac tissue to be an extra-renal source of EPO. Indeed, primary cardiomyocytes and HL-1 cardiac cells both induce Epo gene expression when exposed to low O(2) tension in a HIF-dependent manner. Thus, as well as demonstrating the potential of dietary tamoxifen administration for gene inactivation studies in UBC-Cre-ER(T2) mouse lines, this data provides evidence of a cardiac oxygen-sensing VHL/HIF/EPO pathway in adult mice.
PLoS ONE 07/2011; 6(7):e22589. DOI:10.1371/journal.pone.0022589 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hypoxia inducible factors (HIF1 and HIF2) have emerged as central regulators of the activity of myeloid cells at inflammatory sites where O(2) is frequently limited. Novel insights in the field have revealed that the expression of HIFs by myeloid cells is not exclusively induced by hypoxia but also in response to central inflammatory mediators independently of O(2) shortage. This has substantially elevated the biological significance of HIFs in the context of inflammatory diseases. As a consequence, the loss of HIF1 or HIF2 in myeloid cells specifically compro-mises some of the processes driven by myeloid cells, such as bactericidal activity and myeloid invasion, as well as inflammation-associated detrimental consequences.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to explore the possible involvement of the angiopoietin (Ang)-1, -2/Tie-2 system in the development, growth, and metastases evolution of gastroenteropancreatic-neuroendocrine tumors (GEP-NETs). We prospectively examined the serum levels of Tie-2, Ang-1, and Ang-2 by ELISA in 42 patients with proven GEP-NETs and 27 controls. We also determined the expression of the Ang/Tie-2 system in freshly isolated peripheral blood monocytes and in tumor cells from malignant primary tumors and/or liver metastases samples from GEP-NET patients by flow cytometry and/or RT-PCR. Furthermore, the function of the Ang/Tie-2 system in monocytes from controls and patients was assessed by a chemotaxis assay. GEP-NET patients showed enhanced serum levels of soluble form of Tie-2 (sTie-2), Ang-1, and Ang-2 (P<0.05 in all cases), compared to controls. sTie-2 and Ang-2 levels were significantly higher in GEP-NETs with metastases compared to those with no metastases. In addition, a significant correlation was detected between Ang-2 levels and chromogranin A or sTie-2 concentrations or 5-hydroxy-indole acetic acid excretion (r=0.71, r=0.60, and r=0.81 respectively, P<0.01 in all cases). Furthermore, we observed an enhanced expression of Ang-1, Ang-2, and Tie-2 in freshly isolated tumor cells from GEP-NET both by immunohistochemistry and by RT-PCR. Interestingly, an enhanced expression and function of Tie-2 was detected in monocytes from GEP-NET patients. Our data suggest that the Ang/Tie-2 system is involved in the growth and development of metastases of GEP-NETs, and that favors the recruitment of Tie-2(+) monocytes to the tumor site, where they can promote inflammation and angiogenesis.
Endocrine Related Cancer 12/2010; 17(4):897-908. DOI:10.1677/ERC-10-0020 · 4.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Under hypoxic conditions, mitochondria can represent a threat to the cell because of their capacity to generate toxic reactive oxygen species (ROS). However, cardiomyocytes are equipped with an oxygen-sensing pathway that involves prolyl hydroxylase oxygen sensors and hypoxia-inducible factors (HIFs), which induces a tightly regulated programme to keep ischaemic mitochondrial activity under control. The aim of this review is to provide an update on the pathways leading to mitochondrial reprogramming, which occurs in the myocardium during ischaemia, with particular emphasis on those induced by HIF activation. We start by studying the mechanisms of mitochondrial damage during ischaemia and upon reperfusion, highlighting the importance of the formation of the mitochondrial permeability transition pore during reperfusion and its consequences for cardiomyocyte survival. Next, we analyse hypoxia-induced metabolic reprogramming through HIF and its important consequences for mitochondrial bioenergetics, as well as the phenomenon known as the hibernating myocardium. Subsequently, we examine the mechanisms underlying ischaemic preconditioning, focusing, in particular, on those that involve the HIF pathway, such as adenosine signalling, sub-lethal ROS generation, and nitric oxide production. Finally, the role of the mitochondrial uncoupling proteins in ischaemia tolerance is discussed.
Cardiovascular Research 11/2010; 88(2):219-28. DOI:10.1093/cvr/cvq256 · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factor (VEGF) regulates angiogenesis, but also has important, yet poorly characterized roles in neuronal wiring. Using several genetic and in vitro approaches, we discovered a novel role for VEGF in the control of cerebellar granule cell (GC) migration from the external granule cell layer (EGL) toward the Purkinje cell layer (PCL). GCs express the VEGF receptor Flk1, and are chemoattracted by VEGF, whose levels are higher in the PCL than EGL. Lowering VEGF levels in mice in vivo or ectopic VEGF expression in the EGL ex vivo perturbs GC migration. Using GC-specific Flk1 knock-out mice, we provide for the first time in vivo evidence for a direct chemoattractive effect of VEGF on neurons via Flk1 signaling. Finally, using knock-in mice expressing single VEGF isoforms, we show that pericellular deposition of matrix-bound VEGF isoforms around PC dendrites is necessary for proper GC migration in vivo. These findings identify a previously unknown role for VEGF in neuronal migration.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 11/2010; 30(45):15052-66. DOI:10.1523/JNEUROSCI.0477-10.2010 · 6.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Macrophages play a relevant role in innate and adaptive immunity depending on the balance of the stimuli received. From an analytical and functional point of view, macrophage stimulation can be segregated into three main modes, as follows: innate, classic, and alternative pathways. These differential activations result in the expression of specific sets of genes involved in the release of pro- or anti-inflammatory stimuli. In the present work, we have analyzed whether specific metabolic patterns depend on the signaling pathway activated. A [1,2-(13)C(2)]glucose tracer-based metabolomics approach has been used to characterize the metabolic flux distributions in macrophages stimulated through the classic, innate, and alternative pathways. Using this methodology combined with mass isotopomer distribution analysis of the new formed metabolites, the data show that activated macrophages are essentially glycolytic cells, and a clear cutoff between the classic/innate activation and the alternative pathway exists. Interestingly, macrophage activation through LPS/IFN-gamma or TLR-2, -3, -4, and -9 results in similar flux distribution patterns regardless of the pathway activated. However, stimulation through the alternative pathway has minor metabolic effects. The molecular basis of the differences between these two types of behavior involves a switch in the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) from the liver type-PFK2 to the more active ubiquitous PFK2 isoenzyme, which responds to Hif-1alpha activation and increases fructose-2,6-bisphosphate concentration and the glycolytic flux. However, using macrophages targeted for Hif-1alpha, the switch of PFK2 isoenzymes still occurs in LPS/IFN-gamma-activated macrophages, suggesting that this pathway regulates ubiquitous PFK2 expression through Hif-1alpha-independent mechanisms.
The Journal of Immunology 07/2010; 185(1):605-14. DOI:10.4049/jimmunol.0901698 · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Liver ischemia/reperfusion (I/R) injury is a frequent cause of organ dysfunction. Loss of the oxygen sensor prolyl hydroxylase domain enzyme 1 (PHD1) causes tolerance of skeletal muscle to hypoxia. We assessed whether loss or short-term silencing of PHD1 could likewise induce hypoxia tolerance in hepatocytes and protect them against hepatic I/R damage.
Hepatic ischemia was induced in mice by clamping of the portal vessels of the left lateral liver lobe; 90 minutes later livers were reperfused for 8 hours for I/R experiments. Hepatocyte damage following ischemia or I/R was investigated in PHD1-deficient (PHD1(-/-)) and wild-type mice or following short hairpin RNA-mediated short-term inhibition of PHD1 in vivo.
PHD1(-/-) livers were largely protected against acute ischemia or I/R injury. Among mice subjected to hepatic I/R followed by surgical resection of all nonischemic liver lobes, more than half of wild-type mice succumbed, whereas all PHD1(-/-) mice survived. Also, short-term inhibition of PHD1 through RNA interference-mediated silencing provided protection against I/R. Knockdown of PHD1 also induced hypoxia tolerance of hepatocytes in vitro. Mechanistically, loss of PHD1 decreased production of oxidative stress, which likely relates to a decrease in oxygen consumption as a result of a reprogramming of hepatocellular metabolism.
Loss of PHD1 provided tolerance of hepatocytes to acute hypoxia and protected them against I/R-damage. Short-term inhibition of PHD1 is a novel therapeutic approach to reducing or preventing I/R-induced liver injury.
[Show abstract][Hide abstract] ABSTRACT: Hypoxia-inducible factor-1alpha (HIF-1alpha) protein is degraded under normoxia by its association to von Hippel-Lindau protein (pVHL) and further proteasomal digestion. However, human renal cells HK-2 treated with 15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) accumulate HIF-1alpha in normoxic conditions. Thus, we aimed to investigate the mechanism involved in this accumulation. We found that 15d-PGJ(2) induced an over-accumulation of HIF-1alpha in RCC4 cells, which lack pVHL and in HK-2 cells treated with inhibitors of the pVHL-proteasome pathway. These results indicated that pVHL-proteasome-independent mechanisms are involved, and therefore we aimed to ascertain them. We have identified a new lysosomal-dependent mechanism of HIF-1alpha degradation as a target for 15d-PGJ(2) based on: (1) HIF-1alpha colocalized with the specific lysosomal marker Lamp-2a, (2) 15d-PGJ(2) inhibited the activity of cathepsin B, a lysosomal protease, and (3) inhibition of lysosomal activity did not result in over-accumulation of HIF-1alpha in 15d-PGJ(2)-treated cells. Therefore, expression of HIF-1alpha is also modulated by lysosomal degradation.
Cellular and Molecular Life Sciences CMLS 06/2009; 66(13):2167-80. DOI:10.1007/s00018-009-0039-x · 5.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Low oxygen tension areas are found in inflamed or diseased tissues where hypoxic cells induce survival pathways by regulating the hypoxia-inducible transcription factor (HIF). Macrophages are essential regulators of inflammation and, therefore, we have analyzed their response to hypoxia. Murine peritoneal elicited macrophages cultured under hypoxia produced higher levels of IFN-gamma and IL-12 mRNA and protein than those cultured under normoxia. A similar IFN-gamma increment was obtained with in vivo models using macrophages from mice exposed to atmospheric hypoxia. Our studies showed that IFN-gamma induction was mediated through HIF-1alpha binding to its promoter on a new functional hypoxia response element. The requirement of HIF-alpha in the IFN-gamma induction was confirmed in RAW264.7 cells, where HIF-1alpha was knocked down, as well as in resident HIF-1alpha null macrophages. Moreover, Ag presentation capacity was enhanced in hypoxia through the up-regulation of costimulatory and Ag-presenting receptor expression. Hypoxic macrophages generated productive immune synapses with CD8 T cells that were more efficient for activation of TCR/CD3epsilon, CD3zeta and linker for activation of T cell phosphorylation, and T cell cytokine production. In addition, hypoxic macrophages bound opsonized particles with a higher efficiency, increasing their phagocytic uptake, through the up-regulated expression of phagocytic receptors. These hypoxia-increased immune responses were markedly reduced in HIF-1alpha- and in IFN-gamma-silenced macrophages, indicating a link between HIF-1alpha and IFN-gamma in the functional responses of macrophages to hypoxia. Our data underscore an important role of hypoxia in the activation of macrophage cytokine production, Ag-presenting activity, and phagocytic activity due to an HIF-1alpha-mediated increase in IFN-gamma levels.
The Journal of Immunology 04/2009; 182(5):3155-64. DOI:10.4049/jimmunol.0801710 · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A key function of blood vessels, to supply oxygen, is impaired in tumors because of abnormalities in their endothelial lining. PHD proteins serve as oxygen sensors and may regulate oxygen delivery. We therefore studied the role of endothelial PHD2 in vessel shaping by implanting tumors in PHD2(+/-) mice. Haplodeficiency of PHD2 did not affect tumor vessel density or lumen size, but normalized the endothelial lining and vessel maturation. This resulted in improved tumor perfusion and oxygenation and inhibited tumor cell invasion, intravasation, and metastasis. Haplodeficiency of PHD2 redirected the specification of endothelial tip cells to a more quiescent cell type, lacking filopodia and arrayed in a phalanx formation. This transition relied on HIF-driven upregulation of (soluble) VEGFR-1 and VE-cadherin. Thus, decreased activity of an oxygen sensor in hypoxic conditions prompts endothelial cells to readjust their shape and phenotype to restore oxygen supply. Inhibition of PHD2 may offer alternative therapeutic opportunities for anticancer therapy.
[Show abstract][Hide abstract] ABSTRACT: Aerobic organisms developed mechanisms to protect themselves against a shortage of oxygen (O(2)). Recent studies reveal that O(2) sensors, belonging to the novel class of 2-oxoglutarate dependent iron(ii)-dioxygenases, have more important roles in metabolism than anticipated. Here, we provide a "metabolo-centric" overview of the role of the PHD/FIH members of this family in metabolism, in particular on how they regulate O(2) supply and consumption, energy compensation and conservation, O(2) conformance and hypoxia tolerance, redox and pH homeostasis, and other vital metabolic processes with implications in health and disease. These insights may offer novel opportunities for the treatment of ischemic diseases.