In addition to controlling a switch to glycolytic metabolism and induction of erythropoiesis and angiogenesis, hypoxia promotes the undifferentiated cell state in various stem and precursor cell populations. Here, we show that the latter process requires Notch signaling. Hypoxia blocks neuronal and myogenic differentiation in a Notch-dependent manner. Hypoxia activates Notch-responsive promoters and increases expression of Notch direct downstream genes. The Notch intracellular domain interacts with HIF-1alpha, a global regulator of oxygen homeostasis, and HIF-1alpha is recruited to Notch-responsive promoters upon Notch activation under hypoxic conditions. Taken together, these data provide molecular insights into how reduced oxygen levels control the cellular differentiation status and demonstrate a role for Notch in this process.

In normoxic cells the hypoxia-inducible factor-1a (HIF-1a) is rapidly degraded by the ubiquitin-proteasome pathway, and activation of HIF-1a to a functional form requires protein stabilization. Here we show that the product of the von Hippel-Lindau (VHL) tumor suppressor gene mediated ubiquitylation and proteasomal degradation of HIF-1a under normoxic conditions via interaction with the core of the oxygen-dependent degradation domain of HIF-1a. The region of VHL mediating interaction with HIF-1a overlapped with a putative macromolecular binding site observed within the crystal structure of VHL. This motif of VHL also represents a mutational hotspot in tumors, and one of these mutations impaired interaction with HIF-1a and subsequent degradation. Interestingly, the VHL binding site within HIF-1a overlapped with one of the minimal transactivation domains. Protection of HIF-1a against degradation by VHL was a multistep mechanism, including hypoxiainduced nuclear translocation of HIF-1a and an intranuclear hypoxia-dependent signal. VHL was not released from HIF-1a during this process. Finally, stabilization of HIF-1a protein levels per se did not totally bypass the need of the hypoxic signal for generating the transactivation response.

Relative hypoxia is essential in wound healing since it normally plays a pivotal role in regulation of all the critical processes involved in tissue repair. Hypoxia-inducible factor (HIF) 1␣ is the critical transcription factor that regulates adaptive responses to hypoxia. HIF-1␣ stability and function is regulated by oxygen-dependent soluble hydroxylases targeting critical proline and asparaginyl residues. Here we show that hyperglycemia complexly affects both HIF-1␣ stability and activation, resulting in suppression of expression of HIF-1 target genes essential for wound healing both in vitro and in vivo. However, by blocking HIF-1␣ hydroxylation through chemical inhibition, it is possible to reverse this negative effect of hyperglycemia and to improve the wound healing process (i.e., granulation, vascularization, epidermal regeneration, and recruitment of endothelial precursors). Local adenovirus-mediated transfer of two stable HIF constructs demonstrated that stabilization of HIF-1␣ is necessary and sufficient for promoting wound healing in a diabetic environment. Our findings outline the necessity to develop specific hydroxylase inhibitors as therapeutic agents for chronic diabetes wounds. In conclusion, we demonstrate that impaired regulation of HIF-1␣ is essential for the development of diabetic wounds, and we provide evidence that stabilization of HIF-1␣ is critical to reverse the pathological process.angiogenesis ͉ chronic complications ͉ hypoxia ͉ hyperglycemia ͉ chronic ulcers

Hyperglycemia and hypoxia are suggested to play essential pathophysiological roles in the complications of diabetes, which may result from a defective response of the tissues to low oxygen tension. In this study, we show that in primary dermal fibroblasts and endothelial cells, hyperglycemia interferes with the function of hypoxiainducible factor-1 (HIF-1), a transcription factor that is essential for adaptive responses of the cell to hypoxia. Experiments using proteasomal and prolyl hydroxylases inhibitors indicate that hyperglycemia inhibits hypoxiainduced stabilization of HIF-1␣ protein levels against degradation and suggest that mechanisms in addition to proline hydroxylation may be involved. This effect of hyperglycemia was dose dependent and correlates with a lower transcription activation potency of HIF-1␣, as assessed by transient hypoxia-inducible reporter gene assay. Regulation of HIF-1␣ function by hyperglycemia could be mimicked by mannitol, suggesting hyperosmolarity as one critical parameter. The interference of hyperglycemia with hypoxia-dependent stabilization of HIF-1␣ protein levels was confirmed in vivo, where only very low levels of HIF-1␣ protein could be detected in diabetic wounds, as compared with chronic venous ulcers. In conclusion, our data demonstrate that hyperglycemia impairs hypoxia-dependent protection of HIF-1␣ against proteasomal degradation and suggest a mechanism by which diabetes interferes with cellular responses to hypoxia. Diabetes 53:3226 -3232, 2004 C hronic complications of diabetes are a major health problem, and it has become a priority to characterize further their pathophysiological mechanisms to develop novel, rational therapeutic strategies. Even though prolonged exposure of the tissues to hyperglycemia seems to be the primary causative factor, some other factors may play a role as far as intensive blood glucose control reduces chronic complications but does not prevent them altogether (1,2). It has become increasingly evident that hypoxia plays an important role in all diabetes complications (3). In addition to deficient blood supply as a consequence of micro-and macrovascular disease, it has been postulated that hyperglycemia induces a pseudohypoxia state. This theory is based on the finding that high glucose concentrations induce a high NADH ϩ /NAD ϩ ratio in cells even when the oxygen tension is normal (4).Adaptive responses of cells to hypoxia are mediated by the hypoxia-inducible factor-1 (HIF-1), which is a heterodimeric transcription factor composed of two subunits, HIF-1␣ and aryl hydrocarbon receptor nuclear translocator (ARNT), both constitutively expressed in mammalian cells. Regulation of HIF-1 activity is critically dependent on the degradation of the HIF-1␣ subunit in normoxia. The molecular basis of its degradation is the O 2 -dependent hydroxylation of at least one of the two proline residues (5,6) in the oxygen-dependent degradation domain of HIF-1␣ by specific prolyl 4-hydroxylases (PHDs) (7-9). In this form, HIF-1 ␣ binds to the von Hippel-Lind...

gene regulation ͉ hydroxylation ͉ signal transduction R educed oxygen levels (hypoxia) lead to a set of cellular adaptations, including increased angiogenesis and erythropoiesis and a switch to glycolytic metabolism. The cellular machinery that senses hypoxia is composed of several proteins. A critical component is the transcription factor hypoxiainducible factor 1␣ (HIF-1␣) (1). The level and activity of HIF-1␣ are controlled by oxygen-dependent prolyl (PHD) and asparaginyl factor-inhibiting HIF-1␣ (FIH-1)] hydroxylases. PHDs hydroxylate two proline residues in the degradation domain of HIF-1␣ in normoxia, which makes HIF-1␣ a substrate for the von Hippel-Lindau E3 ubiquitin ligase and proteasomal degradation. After stabilization in hypoxia, HIF-1␣ interacts with aryl hydrocarbon receptor nuclear translocator (ARNT) to bind to hypoxia response elements (HREs)

The role of tryptophan-kynurenine metabolism in psychiatric disease is well established, but remains less explored in peripheral tissues. Exercise training activates kynurenine biotransformation in skeletal muscle, which protects from neuroinflammation and leads to peripheral kynurenic acid accumulation. Here we show that kynurenic acid increases energy utilization by activating G protein-coupled receptor Gpr35, which stimulates lipid metabolism, thermogenic, and anti-inflammatory gene expression in adipose tissue. This suppresses weight gain in animals fed a high-fat diet and improves glucose tolerance. Kynurenic acid and Gpr35 enhance Pgc-1α1 expression and cellular respiration, and increase the levels of Rgs14 in adipocytes, which leads to enhanced beta-adrenergic receptor signaling. Conversely, genetic deletion of Gpr35 causes progressive weight gain and glucose intolerance, and sensitizes to the effects of high-fat diets. Finally, exercise-induced adipose tissue browning is compromised in Gpr35 knockout animals. This work uncovers kynurenine metabolism as a pathway with therapeutic potential to control energy homeostasis.

Apelin and its G-protein-coupled receptor APJ have various beneficial effects on cardiac function and blood pressure. The mechanisms that regulate apelin gene expression are not known. Because apelin gene expression has been shown to increase in cardiac ischemia, we investigated if apelin (Apln) gene expression was sensitive to hypoxia. Here we show that hypoxia increases the apelin expression in rat myocardium and in cultured cardiomyocytes. Pharmacological activation of hypoxia inducible factor by desferrioxamine (DFO) or expression of a constitutively active form of HIF-1alpha increased apelin expression in cardiomyocyte cultures. The induction of apelin by hypoxia was abolished on transient expression of the HIF inhibitory PAS protein in cardiomyocytes. Increased apelin expression induced by hypoxia or DFO was accompanied by the processing of the cellular storage form proapelin into smaller apelin peptides and increased secretion of these biologically active forms of apelin. In a rat in vivo model, acute myocardial infarction (24 h) led to a transient increase in ventricular apelin mRNA levels. Our results indicate that apelin gene is regulated by hypoxia in cardiac myocytes via the HIF pathway, suggesting a role for apelin as a potential marker for acute cardiac hypoxia with a possible compensatory role in myocardial tissue suffering from oxygen deprivation.

Therapeutic angiogenesis provides a potential alternative for the treatment of cardiovascular ischemic diseases. Vascular endothelial growth factor (VEGF) is an important component of the angiogenic response to ischemia. Here we used adeno-associated virus (AAV) gene delivery to skeletal muscle to examine the effects of VEGF vs. a stabilized form of hypoxia-inducible factor-1alpha (HIF-1alpha). The recombinant AAVs were injected into mouse tibialis anterior muscle, and their effects were analyzed by immunohistochemistry and functional assays. These analyses showed that stabilized HIF-1alpha markedly increase capillary sprouting and proliferation, whereas VEGF164 or VEGF120 induced only proliferation of endothelial cells without formation of proper capillary structures. The Evans Blue permeability assay indicated that, unlike VEGF, HIF-1alpha overexpression did not increase vascular leakiness in the transduced muscle. Doppler ultrasound imaging showed that vascular perfusion in the HIF-1alpha treated muscles was significantly enhanced when compared to the controls and not further improved by co-expression of the arteriogenic growth factors angiopoietin-1 or platelet-derived growth factor-B. Our results show that AAV-mediated transduction of a stabilized form of HIF-1alpha can circumvent the problems associated with overexpression of individual angiogenic growth factors. HIF-1alpha should thus offer a potent alternative for pro-angiogenic gene therapy.