Blood vessels function as conduits for the delivery of O2 and nutrients. Hypoxia-inducible factor 1 (HIF-1) mediates adaptive transcriptional responses to hypoxia/ischemia that include expression of angiogenic cytokines/growth factors by hypoxic cells and expression of cognate receptors for these ligands by vascular cells and their progenitors. Impairment of HIF-1-dependent responses to hypoxia is a major factor contributing to the impaired vascular responses to ischemia that are associated with aging and diabetes.
Breast cancers contain regions of intratumoral hypoxia in which reduced O2 availability activates the hypoxia-inducible factors HIF-1 and HIF-2, which increase the transcription of genes encoding proteins that are required for many important steps in cancer progression. Recently, HIFs have been shown to play critical roles in the metastasis of breast cancer to the lungs through the transcriptional activation of genes encoding angiopoietin-like 4 and L1 cell adhesion molecule, which promote the extravasation of circulating cancer cells from the lung vasculature, and the lysyl oxidase family members LOX, LOXL2, and LOXL4, which promote invasion and metastatic niche formation. Digoxin, a drug that inhibits HIF-1 activity, blocks primary tumor growth, vascularization, invasion, and metastasis in ex vivo and in vivo assays.
acriflavine; angiopoietin-like 4; collagen crosslinking; digoxin; extravasation; HIF-1; lysyl oxidase; metastatic niche formation
Oxygen homeostasis represents an essential organizing principle of metazoan evolution and biology. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of transcriptional responses to changes in O2 concentration. HIF-1 is a heterodimer of HIF-1α and HIF-1β subunits. O2-dependent degradation of the HIF-1α subunit is mediated by prolyl hydroxylase, von Hippel-Lindau protein (VHL)/Elongin-C E3 ubiquitin ligase, and the proteasome. O2-independent degradation of HIF-1α is regulated by the competition of RACK1 and HSP90 for binding to HIF-1α. RACK1 binding results in the recruitment of the Elongin-C E3 ubiquitin ligase, leading to VHL-independent ubiquitination and degradation of HIF-1α. In this report, we show that calcineurin inhibits the ubiquitination and proteasomal degradation of HIF-1α. Calcineurin is a serine/threonine phosphatase that is activated by calcium and calmodulin. The phosphatase activity of calcineurin is required for its regulation of HIF-1α. RACK1 binds to the catalytic domain of calcineurin and is required for HIF-1α degradation induced by the calcineurin inhibitor cyclosporine A. Elongin-C and HIF-1α each bind to RACK1 and dimerization of RACK1 is required to recruit Elongin-C to HIF-1α. Phosphorylation of RACK1 promotes its dimerization and dephosphorylation by calcineurin inhibits dimerization. Serine 146 within the dimerization domain is phosphorylated and mutation of serine 146 impairs RACK1 dimerization and HIF-1α degradation. These results indicate that intracellular calcium levels can regulate HIF-1α expression by modulating calcineurin activity and RACK1 dimerization.
The outflow tract myocardium and other regions corresponding to the location of the major coronary vessels of the developing chicken heart, display a high level of hypoxia as assessed by the hypoxia indicator EF5. The EF5 positive tissues were also specifically positive for nuclear-localized hypoxia inducible factor-1 alpha (HIF-1α), the oxygen-sensitive component of the hypoxia inducible factor-1 (HIF-1) heterodimer. This led to our hypothesis that there is a “template” of hypoxic tissue that determines the stereotyped pattern of the major coronary vessels. In this study we disturbed this template by altering ambient oxygen levels (hypoxia 15%; hyperoxia 75-40%) during the early phases of avian coronary vessel development, in order to alter tissue hypoxia, HIF-1α protein expression and its downstream target genes without high mortality. We also altered HIF-1α gene expression in the embryonic outflow tract cardiomyocytes by injecting an adenovirus containing a constitutively active form of HIF-1α (AdCA5). We assayed for coronary anomalies using anti-alpha-smooth muscle actin immunohistology. When incubated under abnormal oxygen levels or injected with a low titer of the AdCA5, coronary arteries displayed deviations from their normal proximal connections to the aorta. These deviations were similar to known clinical anomalies of coronary arteries. These findings indicated that developing coronary vessels may be subject to a level of regulation that is dependent on differential oxygen levels within cardiac tissues and subsequent HIF-1 regulation of gene expression.
Chronic intermittent hypoxia (IH) associated with sleep-disordered breathing is an important cause of hypertension, which results from carotid body-mediated activation of the sympathetic nervous system. IH triggers increased levels of reactive oxygen species (ROS) in the carotid body, which induce increased synthesis and stability of hypoxia-inducible factor 1α (HIF-1α) and calpain-dependent degradation of HIF-2α. HIF-1 activates transcription of the Nox2 gene, encoding NADPH oxidase 2, which generates superoxide. Loss of HIF-2 activity leads to decreased transcription of the Sod2 gene, encoding manganese superoxide dismutase, which converts superoxide to hydrogen peroxide. Thus, IH disrupts the balance between HIF-1-dependent pro-oxidant and HIF-2-dependent anti-oxidant activities, and this loss of redox homeostasis underlies the pathogenesis of autonomic morbidities associated with IH.
Cardiorespiratory homeostasis; Obstructive sleep apnea; Oxidative stress; Oxygen homeostasis
Tumors contain well-oxygenated (aerobic) and poorly oxygenated (hypoxic) regions, which were thought to utilize glucose for oxidative and glycolytic metabolism, respectively. In this issue of the JCI, Sonveaux et al. show that human cancer cells cultured under hypoxic conditions convert glucose to lactate and extrude it, whereas aerobic cancer cells take up lactate via monocarboxylate transporter 1 (MCT1) and utilize it for oxidative phosphorylation (see the related article beginning on page 3930). When MCT1 is inhibited, aerobic cancer cells take up glucose rather than lactate, and hypoxic cancer cells die due to glucose deprivation. Treatment of tumor-bearing mice with an inhibitor of MCT1 retarded tumor growth. MCT1 expression was detected exclusively in nonhypoxic regions of human cancer biopsy samples, and in combination, these data suggest that MCT1 inhibition holds potential as a novel cancer therapy.
Microvascular ischemia and infections are associated with the development of chronic rejection following lung transplantation. The von Hippel–Lindau protein (VHL) controls protein levels of hypoxia-inducible factors (HIFs), regulates vascular repair, and improves tissue perfusion. Here, we studied the role of VHL in microvascular repair by orthotopically transplanting tracheas into mice with VHL haplodeficiency in Tie2 lineage cells. We showed that VHL haplodeficiency prolonged airway microvascular perfusion and promoted tissue blood flow through the production of the angiogenic factors, SDF-1 and angiopoietin 1. VHL-haplodeficient pulmonary endothelial cells exhibited increased angiogenic activity, resistance to serum deprivation-induced cell death, and enhanced microvascular repair. By contrast, in recipient mice with HIF-1α deficiency in Tie2 lineage cells, microvascular repair was significantly diminished and suggested that recipient-derived HIF-1α normally participates in the repair of alloimmune-mediated microvascular damage. To evaluate the translational impact of our findings, we compared VHL-haplodeficient mice with wild-type controls using a model of Aspergillus airway infection. In 83 % of the VHL-haplodeficient recipients, Aspergillus fumigatus was noninvasive in contrast to 75 % of wild-type mice in which the mold was deeply invasive. Our study demonstrated that stabilization of HIF-1α in angiogenic cells, through Tie2 cell VHL haplodeficiency, promoted airway microvascular regeneration and vascular normalization and thereby minimized tissue ischemia and hypoxia. By also mitigating the virulence of A. fumigatus, a common pathogen and itself a risk factor for the development of lung transplant rejection, the selective enhancement of HIF-1α expression has the prospect of offering several novel therapeutic effects to transplant recipients.
Microvascular loss and prolonged ischemia occurs with acute rejection.Von Hippel-Lindau (VHL) protein controls hypoxia inducible factors (HIFs).In tracheal allografts, VHL haplodeficient Tie2 cells promote neovascularization.Reduced transplant ischemia limits Aspergillus invasion.
Electronic supplementary material
The online version of this article (doi:10.1007/s00109-013-1063-8) contains supplementary material, which is available to authorized users.
HIF-1α; Aspergillus fumigatus; VHL; Orthotopic tracheal transplant; Obliterative bronchiolitis; Chronic lung transplant rejection; Microvascular perfusion
An important physiological response to changes in local or systemic oxygenation is the modulation of vascular tone, which is mediated in part by changes in the activities of the 3 NO synthase (NOS) isoforms. In arterial smooth muscle cells, acute hypoxia induces increased vascular tone, which is attenuated if hypoxia persists. In this issue of the JCI, Ward et al. demonstrate that changes in O2 concentration have effects on neuronal NOS enzymatic activity and gene expression that contribute to vascular homeostasis under conditions of acute and chronic hypoxia.
Hypoxia-inducible factors (HIFs) mediate adaptive physiological responses to hypoxia. In human cancers that are accessible for O2 electrode measurements, intratumoral hypoxia is common and is associated with increased risk of mortality. HIF activity in regions of intratumoral hypoxia mediates angiogenesis, epithelial-mesenchymal transition, stem cell maintenance, invasion, metastasis, and resistance to radiation therapy and chemotherapy. A growing number of drugs have been identified that inhibit HIF activity by a variety of molecular mechanisms. Because many of these drugs are already FDA-approved for other indications, clinical trials can (and should) be initiated to test the hypothesis that incorporation of HIF inhibitors into current standard-of-care therapy will increase the survival of cancer patients.
Oxygen homeostasis represents an organizing principle for understanding metazoan evolution, development, physiology, and pathobiology. The hypoxia-inducible factors (HIFs) are transcriptional activators that function as master regulators of oxygen homeostasis in all metazoan species. Rapid progress is being made in elucidating homeostatic roles of HIFs in many physiological systems, determining pathological consequences of HIF dysregulation in chronic diseases, and investigating potential targeting of HIFs for therapeutic purposes.
Hypoxia-inducible factor 1 (HIF-1) is a heterodimer composed of HIF-1α and HIF-1β subunits. HIF-1 is known to promote tissue vascularization by activating the transcription of genes encoding angiogenic factors, which bind to receptors on endothelial cells (ECs) and bone marrow-derived angiogenic cells (BMDACs). In this study, we analysed whether HIF-1 activity in the responding ECs and BMDACs is also required for cutaneous vascularization during burn wound healing.
Methods and results
We generated mice with floxed alleles at the Hif1a or Arnt locus encoding HIF-1α and HIF-1β, respectively. Expression of Cre recombinase was driven by the Tie2 gene promoter, which is expressed in ECs and bone marrow cells. Tie2Cre+ and Tie2Cre− mice were subjected to burn wounds of reproducible diameter and depth. Deficiency of HIF-1α or HIF-1β in Tie2-lineage cells resulted in delayed wound closure, reduced vascularization, decreased cutaneous blood flow, impaired BMDAC mobilization, and decreased BMDAC homing to burn wounds.
HIF-1 activity in Tie2-lineage cells is required for the mobilization and homing of BMDACs to cutaneous burn wounds and for the vascularization of burn wound tissue.
Hypoxia; Wound healing; Conditional knockout; Angiogenesis
Metastasis involves critical interactions between cancer and stromal cells. Intratumoral hypoxia promotes metastasis through activation of hypoxia-inducible factors (HIFs). We demonstrate that HIFs mediate paracrine signaling between breast cancer cells (BCCs) and mesenchymal stem cells (MSCs) to promote metastasis. In a mouse orthotopic implantation model, MSCs were recruited to primary breast tumors and promoted BCC metastasis to LNs and lungs in a HIF-dependent manner. Coculture of MSCs with BCCs augmented HIF activity in BCCs. Additionally, coculture induced expression of the chemokine CXCL10 in MSCs and the cognate receptor CXCR3 in BCCs, which was augmented by hypoxia. CXCR3 expression was blocked in cocultures treated with neutralizing antibody against CXCL10. Conversely, CXCL10 expression was blocked in MSCs cocultured with BCCs that did not express CXCR3 or HIFs. MSC coculture did not enhance the metastasis of HIF-deficient BCCs. BCCs and MSCs expressed placental growth factor (PGF) and its cognate receptor VEGFR1, respectively, in a HIF-dependent manner, and CXCL10 expression by MSCs was dependent on PGF expression by BCCs. PGF promoted metastasis of BCCs and also facilitated homing of MSCs to tumors. Thus, HIFs mediate complex and bidirectional paracrine signaling between BCCs and MSCs that stimulates breast cancer metastasis.
T cell differentiation into distinct functional effector and inhibitory subsets is regulated in part by the cytokine environment present at the time of antigen recognition. Here, we show that hypoxia-inducible factor 1 (HIF-1), a key metabolic sensor, regulates the balance between T regulatory (Treg) and TH17 differentiation. HIF-1α enhances TH17 development through direct transcriptional activation of RORvt, and via tertiary complex formation with RORvt and p300 recruitment to the IL17 promoter, thereby regulating TH17 signature genes. Concurrently, HIF-1α attenuates Treg development by binding Foxp3 and targeting it for proteasomal degradation. Importantly this regulation occurs under both normoxic and hypoxic conditions. Mice with HIF-1α deficient T cells are resistant to induction of TH17-dependent experimental autoimmune encephalitis associated with diminished TH17 and increased Treg cells. These findings highlight the importance of metabolic cues in T cell fate determination and suggest that metabolic modulation could ameliorate certain T cell-based immune pathologies.
Recurrent apneas are characterized by transient repetitive cessations of breathing (two breaths duration or longer) resulting in periodic decreases in arterial blood PO2 or chronic intermittent hypoxia (IH). Patients with recurrent apneas and experimental animals exposed to chronic IH exhibit cardio-respiratory morbidities. The purpose of this article is to highlight the current information on the transcriptional mechanisms associated with chronic IH. Studies on rodents and cell cultures have shown that IH activates a variety of transcription factors including the hypoxia-inducible factor-1 (HIF-1), c-fos (immediate early gene), nuclear factor of activated T-Cells (NFAT), and nuclear factor kB (NF-kB). The signaling pathways associated with transcriptional activation associated with IH differ from continuous hypoxia (CH). Compared to same duration and intensity of CH, IH is more potent in activating HIF-1 and c-fos and also results in long-lasting accumulation of HIF-1α and c-fos mRNA, a phenomenon that was not seen with CH. IH-evoked transcriptional activation by HIF-1, c-fos as well as the resulting activator protein-1 (AP-1) requires reactive oxygen species (ROS)-mediated signaling and involves complex feed-forward interactions between HIF-1 and ROS. Chronic IH evoked cardio-respiratory responses are absent in Hif-1a+/− mice, and hypertension elicited by chronic IH is absent in mice lacking NFAT3c. These studies indicate that cardio-respiratory responses to chronic IH depend on complex interactions between various transcription factors resulting in alterations in several down stream genes and their protein products.
Hypoxia-inducible factor-1; NFAT; Activator protein-1; Nuclear factor kB; intermittent hypoxia; reactive oxygen species; NADPH oxidase
Hypoxia-inducible factor 1 (HIF-1) mediates adaptive responses to reduced oxygen availability by regulating gene expression. A critical cell-autonomous adaptive response to chronic hypoxia controlled by HIF-1 is reduced mitochondrial mass and/or metabolism. Exposure of HIF-1-deficient fibroblasts to chronic hypoxia results in cell death due to excessive levels of reactive oxygen species (ROS). HIF-1 reduces ROS production under hypoxic conditions by multiple mechanisms including: a subunit switch in cytochrome c oxidase from the COX4-1 to COX4-2 regulatory subunit that increases the efficiency of complex IV; induction of pyruvate dehydrogenase kinase 1, which shunts pyruvate away from the mitochondria; induction of BNIP3, which triggers mitochondrial selective autophagy; and induction of microRNA-210, which blocks assembly of Fe/S clusters that are required for oxidative phosphorylation. HIF-1 is also required for ischemic preconditioning and this effect may be due in part to its induction of CD73, the enzyme that produces adenosine. HIF-1-dependent regulation of mitochondrial metabolism may also contribute to the protective effects of ischemic preconditioning.
MCM proteins are components of a DNA helicase that plays an essential role in DNA replication and cell proliferation. However, MCM proteins are present in excess relative to origins of replication, suggesting they may serve other functions. Decreased proliferation is a fundamental physiological response to hypoxia in many cell types and hypoxia-inducible factor 1 (HIF-1) has been implicated in this process. Here, we demonstrate that multiple MCM proteins bind directly to the HIF-1α subunit and synergistically inhibit HIF-1 transcriptional activity via distinct O2-dependent mechanisms. MCM3 inhibits transactivation domain function whereas MCM7 enhances HIF-1α ubiquitination and proteasomal degradation. HIF-1 activity decreases when quiescent cells re-enter the cell cycle and this effect is MCM dependent. Exposure to hypoxia leads to MCM2–7 downregulation in diverse cell types. These studies reveal a function of MCM proteins apart from their DNA helicase activity and establish a direct link between HIF-1 and the cell cycle machinery.
The pyruvate kinase isoforms PKM1 and PKM2 are alternatively spliced products of the PKM2 gene. PKM2, but not PKM1, alters glucose metabolism in cancer cells and contributes to tumorigenesis by mechanisms that are not explained by its known biochemical activity. We show that PKM2 gene transcription is activated by hypoxia-inducible factor 1 (HIF-1). PKM2 interacts directly with the HIF-1α subunit and promotes transactivation of HIF-1 target genes by enhancing HIF-1 binding and p300 recruitment to hypoxia response elements, whereas PKM1 fails to regulate HIF-1 activity. Interaction of PKM2 with prolyl hydroxylase 3 (PHD3) enhances PKM2 binding to HIF-1α and PKM2 coactivator function. Mass spectrometry and anti-hydroxyproline antibody assays demonstrate PKM2 hydroxylation on proline-403/408. PHD3 knockdown inhibits PKM2 coactivator function, reduces glucose uptake and lactate production, and increases O2 consumption in cancer cells. Thus, PKM2 participates in a positive feedback loop that promotes HIF-1 transactivation and reprograms glucose metabolism in cancer cells.
Oxidative stress contributes to tissue injury in conditions ranging from cardiovascular disease to stroke, spinal cord injury, neurodegeneration, and perhaps even aging. Yet the efficacy of antioxidants in human disease has been mixed at best. We need a better understanding of the mechanisms by which established antioxidants combat oxidative stress.
Iron chelators are well-established inhibitors of oxidative death in both neural and non-neural tissues, but their precise mechanism of action remains elusive. The prevailing but not completely substantiated view is that iron chelators prevent oxidative injury by suppressing Fenton Chemistry and the formation of highly reactive hydroxyl radicals. Here we show that iron chelation protects, rather unexpectedly, by inhibiting the hypoxia-inducible factor prolyl 4-hydroxylase isoform 1 (PHD1), an iron and 2-oxoglutarate-dependent dioxygenase. PHD1 and its isoforms 2 and 3 are best known for stabilizing transcriptional regulators involved in hypoxic adaptation, such as HIF-1α and CREB. Yet we find that global HIF–PHD inhibition protects neurons even when HIF-1α and CREB are directly suppressed. Moreover, two global HIF-PHD inhibitors continued to be neuroprotective even in the presence of diminished HIF-2α levels, which itself increases neuronal susceptibility to oxidative stress. Finally, RNA interference to PHD1 but not isoforms PHD2 or PHD3 prevents oxidative death, independent of HIF activation. Together, these studies suggest that iron chelators can prevent normoxic oxidative neuronal death through selective inhibition of PHD1 but independent of HIF-1α and CREB; and that HIF-2α not HIF-1α regulates susceptibility to normoxic oxidative neuronal death.
Hypoxia; neuroprotection; CREB; transcription factor; Hypoxia-ischemia; RNA interference
Cancer cells feature altered glucose metabolism that allows their rapid growth. They consume large amounts of glucose to produce lactate, even in the presence of ample oxygen, which is known as the Warburg effect. Pyruvate kinase M2 (PKM2) contributes to the Warburg effect by previously unknown mechanisms. Hypoxia-inducible factor 1 (HIF-1) mediates PKM2 gene transcription and metabolic reprogramming in cancer cells. The recent discovery of novel physical and functional interactions between PKM2 and HIF-1 in cancer cells has provided insight into molecular mechanisms underlying the Warburg effect.
HIF-1; hypoxia; metabolism; glycolysis; Warburg effect
Chronic rejection, manifested as small airway fibrosis (obliterative bronchiolitis [OB]), is the main obstacle to long-term survival in lung transplantation. Recent studies demonstrate that the airways involved in a lung transplant are relatively hypoxic at baseline and that OB pathogenesis may be linked to ischemia induced by a transient loss of airway microvasculature. Here, we show that HIF-1α mediates airway microvascular repair in a model of orthotopic tracheal transplantation. Grafts with a conditional knockout of Hif1a demonstrated diminished recruitment of recipient-derived Tie2+ angiogenic cells to the allograft, impaired repair of damaged microvasculature, accelerated loss of microvascular perfusion, and hastened denudation of epithelial cells. In contrast, graft HIF-1α overexpression induced via an adenoviral vector prolonged airway microvascular perfusion, preserved epithelial integrity, extended the time window for the graft to be rescued from chronic rejection, and attenuated airway fibrotic remodeling. HIF-1α overexpression induced the expression of proangiogenic factors such as Sdf1, Plgf, and Vegf, and promoted the recruitment of vasoreparative Tie2+ cells. This study demonstrates that a therapy that enhances vascular integrity during acute rejection may promote graft health and prevent chronic rejection.
The vascular system delivers oxygen and nutrients to every cell in the vertebrate organism. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of hypoxic/ischaemic vascular responses, driving transcriptional activation of hundreds of genes involved in vascular reactivity, angiogenesis, arteriogenesis, and the mobilization and homing of bone marrow-derived angiogenic cells. This review will focus on the pivotal role of HIF-1 in vascular homeostasis, the involvement of HIF-1 in vascular diseases, and recent advances in targeting HIF-1 for therapy in preclinical models.
Angiogenesis; Cell therapy; Gene therapy; HIF-1; Hypoxia; Vascularization
Hypoxia-inducible factor 1 (HIF-1) plays a key role in the reprogramming of cancer metabolism by activating transcription of genes encoding glucose transporters and glycolytic enzymes, which take up glucose and convert it to lactate; pyruvate dehydrogenase kinase 1, which shunts pyruvate away from the mitochondria; and BNIP3, which triggers selective mitochondrial autophagy. The shift from oxidative to glycolytic metabolism allows maintenance of redox homeostasis and cell survival under conditions of prolonged hypoxia. Many metabolic abnormalities in cancer cells increase HIF-1 activity. As a result, a feed-forward mechanism can be activated that drives HIF-1 activation and may promote tumor progression.