Published online before print October 11, 2007, doi: 10.1161/ATVBAHA.107.148551
© 2007 American Heart Association, Inc.
Vascular Biology |
A Novel Class of Prolyl Hydroxylase Inhibitors Induces Angiogenesis and Exerts Organ Protection Against Ischemia
From the Division of Nephrology and Endocrinology (M.N.), University of Tokyo School of Medicine, Japan; the Institute of Medical Sciences (Y.I., S.T., T.M.), Divisions of Nephrology, Hypertension, and Metabolism and of Neurology, Tokai University School of Medicine, Kanagawa, Japan; the Center for Tsukuba Advanced Research Alliance and Institute of Basic Medical Sciences (T.Y., O.O.), University of Tsukuba, Japan; the Center for Tsukuba Advanced Research Alliance (Y.F.-K.), University of Tsukuba, Japan; the Center for Tsukuba Advanced Research Alliance and JST-ERATO Environmental Response Project (M.Y.), University of Tsukuba, and the Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan; the Service de Nephrologie (C.v.Y.d.S.), Universite Catholique de Louvain, Brussels, Belgium; and the Basic Medical Science and Molecular Medicine (N.H.), Tokai University School of Medicine, Kanagawa, Japan.
Correspondence to Toshio Miyata, MD, PhD, Institute of Medical Sciences and Division of Nephrology, Hypertension and Metabolism, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan. E-mail t-miyata{at}is.icc.u-tokai.ac.jp
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Objective— Hypoxia inducible factor (HIF) plays a pivotal role in the adaptation to ischemic conditions. Its activity is modulated by an oxygen-dependent hydroxylation of proline residues by prolyl hydroxylases (PHD).
Methods and Results— We discovered 2 unique compounds (TM6008 and TM6089), which inhibited PHD and stabilized HIF activity in vitro. Our docking simulation studies based on the 3-dimensional structure of human PHD2 disclosed that they preferentially bind to the active site of PHD. Whereas PHD inhibitors previously reported inhibit PHD activity via iron chelation, TM6089 does not share an iron chelating motif and is devoid of iron chelating activity. In vitro Matrigel assays and in vivo sponge assays demonstrated enhancement of angiogenesis by local administration of TM6008 and TM6089. Their oral administration stimulated HIF activity in various organs of transgenic rats expressing a hypoxia-responsive reporter vector. No acute toxicity was observed up to 2 weeks after a single oral dose of 2000 mg/kg for TM6008. Oral administration of TM6008 protected neurons in a model of cerebrovascular disease. The protection was associated with amelioration of apoptosis but independent of enhanced angiogenesis.
Conclusions— The present study uncovered beneficial effects of novel PHD inhibitors preferentially binding to the active site of PHD.
We discovered 2 compounds (TM6008 and TM6089) that inhibited PHD and stabilized HIF activity. Local administration of TM6008 and TM6089 enhanced angiogenesis, and their oral administration stimulated HIF activity in transgenic rats expressing a hypoxia-responsive reporter vector. Oral administration of TM6008 protected neurons in a model of ischemic cerebrovascular disease.
Key Words: hypoxia • hypoxia inducible factor • structure based drug design • stroke • ischemia
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Oxygen supply declines under ischemic conditions in many human vascular diseases including ischemic heart disease, chronic kidney failure, and stroke. The resulting hypoxia causes functional impairment of cells as well as structural tissue damage and triggers a broad spectrum of cellular defenses such as angiogenesis, erythropoiesis, glycolysis, and antioxidative enzymes.
Hypoxia-inducible factor (HIF), a heterodimeric nuclear factor, is a crucial intermediate in these defensive mechanisms.1–3 Under normoxic conditions, HIF is constitutively transcribed and translated. Its stability is drastically reduced by the oxygen-dependent enzymatic hydroxylation of proline residues by prolyl hydroxylases (PHD).4–9 Hydroxylated HIF recruits the E3-ubiquitin ligase, von Hippel Lindau protein (pVHL)10,11 which, in turn, tags HIF with ubiquitin groups and targets it for degradation by the proteasome.12,13 Under hypoxic conditions, HIF is not hydroxylated but binds to its heterodimeric partner HIF-1β. The resulting protein complex transactivates in the nucleus a host of genes involved in the adaptation to hypoxic stress.14
Activation of HIF may prove therapeutic for vascular disorders. Most treatments for ischemic and hypoxic disorders are currently focused on symptomatic relief and correction of etiologic factors. Drugs dissolving thrombi are also used to restore blood flow in the acute phase. As yet no compound enhancing organ resistance to hypoxia is clinically available. HIF activates a "master gene" switch that results in a broad and coordinated downstream reaction, protecting tissues against the consequences of hypoxia. The availability of less cumbersome non-toxic small molecular activators of HIF should prove very useful for therapeutic intervention.15,16
To obtain such novel compounds and to understand a molecular mechanism of PHD inhibition, we performed docking simulation based on the 3-dimensional structure of human PHD2. We further documented the in vitro and in vivo effectiveness of the novel PHD inhibitors we identified.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Please see the supplemental data section at http://atvb.ahajournals. org for detailed Methods.
Docking Simulations
The X-ray crystal structure of human PHD2 was obtained from the Protein Data Bank17 (PDB code: 2HBT). Throughout the present study, the software system MOE (Molecular Operating Environment, version 2005.06) and the MMFF94s force field18 were used. Binding sites were characterized using the alpha site finder function19 in MOE. The docking of small molecules and the target sites was performed by the program Ph4Dock.20
PHD Activity
PHD activity was determined as described by Kaule et al.21 In brief, mitochondrial fraction of IRPTC homogenates was reacted with the tested compounds and ODD peptide of HIF-1
. ODD-dependent hydroxylase activity was assessed by counting the radioactivity of [1-14C]-succinate converted from [5-14C]-2-OG by PHD.
Transition Metal Chelation
The chelating activity of the tested compounds for transition metal ions was measured by the method of Price et al22 with some modifications.
Capillary Network Formation
Capillary network formation was examined by Matrigel assays (BD Biosciences) as described previously.23
Sponge Assays
Sponge angiogenesis assays were performed as described previously.24
Hypoxia-Sensing Transgenic Rat
Stimulation of the HIF-HRE system by systemic administration of TM6008 or TM6089 was evaluated using the hypoxia-sensing transgenic rat strain.24 Expression of the hypoxia-responsive luciferase gene was estimated by semiquantitative RT-PCR as described previously.25
Cerebral Ischemic Injury Model
Transient global ischemia of Mongolian gerbils was achieved by bilateral carotid occlusion.26 Animals were then randomly divided into 3 experimental groups: Groups 1 (TM6008) and 2 (vehicle) animals underwent transient global ischemia. Group 3 animals were sham-operated and served as controls.
We also measured cortical microperfusion by laser-Doppler flowmetry in gerbil forebrain ischemia treated with TM6008 or vehicle.
Statistics
Differences among groups were assessed by Kruskal-Wallis test or ANOVA. The statistical significance was determined by 2-tailed Mann–Whitney U test or Student t test. Data are expressed as means±SD. Values are considered significant at P<0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Identification of Novel HIF-Stimulating Compounds
Thirty-seven compounds that have structural similarities to FG-0041, a previously reported PHD inhibitor supposedly acting through iron chelation,27 were selected from a chemical database. The chemical structures of these compounds are shown in supplemental Figure I. Their HIF-stimulating activity was tested by an in vitro screening assay which used cells expressing luciferase controlled by hypoxia responsive element (HRE) (supplemental Figure II). Cobalt, a well known chemical mimicker of hypoxia by stabilizing HIF-
subunit,28 was used as a positive control. Two derivatives, TM6008 and TM6089, exhibited strong HIF-stimulating activities (Figure 1). TM6008 is 6-amino-1, 3-di methyl-5-(2-pyridin-2-yl-quinoline-4-carbonyl)-1H-pyrimidine-2, 4- dione, and TM6089 is 6-amino-1,3-di-methyl-5-[2-(pyridin-2- ylsulfanyl)-acetyl]-1H-pyrimidine-2,4-dione.
|
PHD Inhibition
The inhibitory effect of our compounds on the oxygen-dependent hydroxylation of HIF- subunit by PHD was evaluated. All tested compounds inhibited PHD activity in a dose-dependent manner (Figure 2). TM6008 was the most effective, exceeding cobalt chloride.
|
In Vitro Transition Metal Chelation of PHD Inhibitors
Previously reported PHD inhibitors, such as 3,4-DHB,29 S956711,29 and FG-0041,27 share an iron chelating motif. Although chemical structures of TM6008 and TM6089 differ significantly from previous PHD inhibitors, TM6008 also share this motif. By contrast, TM6089 lacks this motif.
We therefore evaluated their abilities to chelate transition metals in vitro by copper-catalyzed oxidation of ascorbic acid. 3,4-DHB, S965711, and TM6008 chelated transition metal (copper) and inhibited the autoxidation of ascorbic acid in a dose-dependent manner (IC 50 values were 330, 31.4, and 0.57 µmol/L, respectively). By contrast, TM6089 did not chelate transition metal even at the concentration of 100 µmol/L.
Binding Mode to Human PHD
PHD produces trans-4-hydroxyproline from 2-OG and L-proline (Pro) in the presence of Fe(II). The crystal structure of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxia response in normal cells, has been recently reported.30 Based on the 3-dimensional structure of this PHD, we undertook docking simulations between our 2 PHD inhibitors and human PHD2. The docking modes of TM6008 and TM6089 are shown in Figure 3. TM6008 binds to the active site of PHD2 by chelating 2 nitrogen atoms with the iron atom. By contrast, TM6089 binds to the active site by nonchelating mechanism. The sulfur and 1 carbonyl oxygen atom of TM6089 point to the iron atom. The disposition of these 3 atoms, however, is unfavorable to form coordinate bonds. The binding mode of TM6089 demonstrates that TM6089 is a unique inhibitor without iron chelating affinity.
|
Toxicity and Pharmacokinetics
TM6008 and TM6089 did not exhibit cytotoxicity at the tested concentrations (up to 100 µmol/L). No acute toxicity was observed in mice up to 2 weeks after a single oral dose of 2000 mg/kg for TM6008, whereas the 50% lethal dose of TM6089 was 500 mg/kg. Pharmacokinetics studies in rats given an oral dose of 50 mg/kg of each compound disclosed plasma Tmax, Cmax, and T1/2 values of 3.5 hour, 0.9 µg/mL and 1.5 hour for TM6008, and 1.0 hour, 0.5 µg/mL, and 0.6 hour for TM6089.
Demonstration of the In Vivo Effectiveness
As VEGF is regulated by the HIF-HRE system, we examined whether TM6008 and TM6089 stimulate angiogenesis.
Firstly, we examined whether local injection of our compounds stimulates angiogenesis in vivo. For this purpose, we introduced small sponges under the skin of mice and measured their hemoglobin contents and vessel numbers after 10 days to estimate the stimulation of the HIF-HRE system. Injection of TM6008 increased angiogenesis as demonstrated by an increase of the hemoglobin content, and by an increased vessel number on immunohistochemical evaluation of the sponges. TM6089 also enhanced angiogenesis in the sponge assays (Figure 4A through 4C).
|
To investigate whether systemic administration of TM6008 and TM6089 stimulates in vivo the HIF-HRE system in various organs, we used the hypoxia-sensing transgenic rats. In the kidney expression of the reporter gene was not detected under basal conditions (amplification of 40 cycles), but expression of the reporter gene was obviously induced after a single oral dose 100 mg/kg of TM6008 and TM6089 (detected at 31.0±0.85 cycles and 31.0±2.05 cycles, respectively). In the liver, expression of the reporter gene, which was undetectable under basal conditions, was also induced after TM6008 administration (detected at 32.3±0.35 cycles), whereas TM6089 was ineffective. In the heart, the reporter gene was detected under basal conditions and both TM6008 and TM6089 increased its expression (1.37±1.00 and 6.69±5.45 fold increase, respectively). No attempt was made to evaluate the expression of the transgene in the brain because the pharmacokinetics studies showed that neither of the tested compounds crossed the blood-brain barrier.
Next, we evaluated capillary network formation by Matrigel assays. When endothelial cells were seeded onto Matrigel at subconfluent density, they developed tube-like structures at 9 hours. Quantification of capillary network formation by measuring the tube length revealed promotion of capillary network formation by TM6008, confirming the results of the sponge assays (Figure 4D).
Prevention of Neuronal Cell Death Induced by Hypoxia
PHD inhibitors might protect cells against hypoxic damage. To test this hypothesis we used the delayed neuronal death model in gerbil. Nontoxic TM6008 (100 mg/kg/d) was given orally for 7 days in gerbils after a 5-minute transient global cerebral ischemia. The pathological outcome of neuronal cells was examined after 7 day administration of TM6008 in CA1 hippocampus with light microscopy.
In contrast with nonischemic gerbils (Figure 5A), gerbils subjected to ischemia and given vehicle alone (Figure 5B) exhibited in most pyramidal neurons ischemic cell damage, characterized by shrunken, darkly stained cytoplasm, and pyknotic nuclei with accumulation of glial cells. In the TM6008-treated animals, only a few neurons showed ischemic changes (Figure 5C). The number of viable neurons in the CA1 hippocampus, was higher in the TM6008-treated animals than in the vehicle-treated gerbils (166±73 versus 61±55, P<0.05). The number of viable neurons in the CA1 hippocampus of the TM6008-treated animals was not statistically different from that observed in the nonischemia control group (227±50). Further, treatment with TM6008 decreased the number of apoptotic cells (Figure 5D). The final plasma concentration of TM6008 in these experiments was 7.8±2.9 µg/mL. Thus, TM6008 clearly protected against hypoxia-induced apoptotic neuronal death.
|
Next, we examined whether the protective effect of TM6008 against delayed neuronal death was attributable to enhanced angiogenesis. There was no statistically significant difference of the number of VEGF-positive cells between TM6008- and vehicle-treated groups (10.17±5.02 versus 9.12±1.55, respectively). Further, there was no significant difference of the value of cortical microperfusion at 7 days after occlusion between TM6008- and vehicle-treated groups (25.0±9.3 versus 29.4±8.7, respectively).
To clarify a neuroprotective mechanism of TM6008 in global ischemia models, we immunohistochemically stained with EPO, GLUT-1, and GLUT-3. The number of GLUT-3–positive cells in the CA1 hippocampus was significantly higher in TM6008 treated than in the vehicle-treated gerbils (26.9±7.5 versus 15.3±8.6, P<0.05). However, there was no statistically significant difference in EPO or GLUT-1–positive cells in the CA1 hippocampus between TM6008- and vehicle-treated groups (2.9±2.9 versus 3.3±1.7; and 5.5±2.1 versus 6.4±1.8, respectively).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We identified novel molecules able to inhibit PHD activity and stabilize HIF. Our docking simulation studies based on the 3 dimensional structure of PHD2 have disclosed the molecular events required to inhibit PHD and therefore stabilize HIF. The target of these PHD inhibitors is the PHD active site.
Most of the PHD inhibitors reported so far, eg, 3,4-DHB, S956711 and FG-0041, are believed to inhibit the enzyme by iron chelating mechanism.27,29 Iron chelating compounds could have nonspecific binding affinity to the iron containing proteins or iron ions and may not be desirable from the therapeutic point of view because iron is an essential cofactor for a host of important cellular functions, including oxidative phosphorylation and arachidonic acid signaling. To our surprise, the docking simulations demonstrated that TM6089 could preferentially bind to the active site of PHD2 without chelating to the iron atom. Indeed, TM6089 is devoid of iron chelating activity in vitro. Thus, iron chelation is not a necessary intermediate of PHD inhibition. According to our knowledge, TM6089 is the first unique PHD inhibitor which stimulates HIF activity without iron chelation.
The in vivo relevance of our novel PHD inhibitors was first demonstrated by the sponge assay in mice. Previous reports have shown that the hemoglobin contents of the sponge implants and the surrounding granuloma tissue correlated with the degree of angiogenesis.31 Accordingly, both 3,4-DHB and S956711 were shown to raise the number of vessels in the sponge. In this study, we demonstrated not only an augmented number of vessels by immunohistochemistry but also an increased hemoglobin content in the sponge after local administration of TM6008 and TM6089.
Of great interest, these effects of TM6008 and TM6089 are not restricted locally but extend to several organs. To reach this conclusion, we used a hypoxia-sensing transgenic rat expressing a hypoxia-responsive reporter vector using a HRE of the 5' VEGF untranslated region.25 These transgenic rats have the unique asset to allow a sensitive and specific evaluation of HIF stimulation. As a consequence of systemic administration of TM6008 and TM6089 to these rats, expression of the reporter gene was considerably upregulated in the kidney, liver, and heart.
To extend these findings, we used less toxic TM6008 and obtained therapeutically relevant results in studies using gerbils. In gerbils, transient brain ischemia followed by reperfusion results in neuronal death in selectively vulnerable brain regions such as the hippocampal CA1 sector and caudate-putamen. The discovery that, in this model, TM6008 rescued neurons from apoptotic cell death in the CA1 hippocampus is noteworthy. Whereas TM6008 did not cross the blood-brain barrier, TM6008 protected the brain in a model of global cerebral ischemia. This is likely attributable to an increase in permeability of the blood-brain barrier, as previous reports showed that ischemic injury in this model destroys the blood-brain barrier and allows passage of compounds which do not penetrate the barrier under normal conditions.32
Mechanisms of neuroprotection by TM6008 can theoretically be multifactorial because HIF regulates a wide range of protective genes such as those involved in erythropoiesis (EPO, transferring, and hepcidin), angiogensis (VEGF), antioxidative stress (HO-1), glycolysis (Glut-1, Glut-3, and aldolase A), and so on. Angiogenic effects of TM6008 shown by the Matrigel assays and sponge assays stimulated us to study whether enhanced angiogenesis played a role in neuronal protection in our model. However, we could not find enhanced angiogenesis in the brain of gerbils treated with TM6008 by counting VEGF-positive vessels or measuring blood flow by laser Doppler flowmetry. Therefore, it is unlikely that the protective effect of TM6008 was related to angiogenesis in the gerbil forebrain ischemia model. This may be explained by different concentrations of TM6008 among the assays. Although we could not measure the local concentrations of TM6008 in the damaged brain, it is likely that the concentration of the gerbil forebrain treated with TM6008 p.o. is lower than those obtained by local administration such as sponge assays and Matrigel assay.
We next focused on effects of TM6008 on neuronal apoptosis. Our terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays demonstrated that TM6008 decreased the number of apoptotic cells in the brain, and other potential neuroprotective mechanisms by TM6008 include antiapoptotic effects mediated by other HIF-regulated genes such as EPO,33 VEGF,34 and glucose transporters.35 EPO is a pleiotropic cytokine36 and induces neuroprotection via the antiapoptotic signaling cascades like Bcl-XL through direct binding to the Bcl-X promoter.37 Antiapoptotic effects of VEGF contribute to reduction of ischemic brain damage in addition to its angiogenic effects.38 The glucose transporter GLUT-1 is also positively regulated through HIF-1, and the microinfusion of virus vectors bearing the GLUT-1 isoform into the brain tissue reduced seizure-induced39 and ischemic neuronal damage in vivo.40 However, our immunohistochemical analysis could not demonstrate upregulation of these genes. In contrast, we observed upregulation of Glut-3. Glut-3 is also regulated by HIF,41 and recent studies suggested a critical role of Glut-3 in protecting against a decline in brain glucose uptake under ischemic conditions.42
These results fit with the observations collected during various therapeutic strategies related to HIF target genes. For instance, cobalt chloride has been used as a conventional HIF stabilizer. It is generally believed to replace the iron present in PHD, but recent studies demonstrated that cobalt also depletes intracellular ascorbate,28 a substrate of PHD. Cobalt is effective in a variety of hypoxia-related disorders including cerebrovascular disease.23,43,44 In addition to PHD, there are other factors regulating the HIF stability/activity. Factor-inhibiting-HIF (FIH) hydroxylates regulates HIF activation via controlling CBP/p300 recruitment. The phosphoinositide 3-kinase (PI3K)/Akt pathway and the protein kinase C signaling have also been implicated in the regulation of HIF-. Whether these pathways can be a good target for therapeutic approaches is a future subject to be pursed.
The protective effect of TM6008 against ischemia-induced cerebral lesions suggested its potential usefulness in other ischemic disorders such as cardiac or kidney diseases. It should not be forgotten that HIF stimulation acts as a general switch for several proteins such as VEGF, erythropoietin, etc. Although these proteins are protective under hypoxic conditions, recent demonstration that both erythropoietin and VEGF accelerates diabetic retinopathy independently45 should call for caution. Their administration during several months warrants long-term experimental studies before concluding to its safety. On the other hand, the short-term use of PHD inhibitors for acute hypoxic damage will probably prove safe.
|
Acknowledgments |
|---|
Sources of Funding
This study was supported by grants from the Program for Promotion of Fundamental Studies in Health Sciences of the Pharmaceuticals and Medical Devices Agency (PMDA) and from the Japan Society for the Promotion of Science for Scientific Research.
Disclosures
None.
|
Footnotes |
|---|
Original received May 24, 2007; final version accepted September 26, 2007.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Marx J. How cells endure low oxygen. Science. 2004; 303: 1454–1456.
2. Semenza GL, Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol. 1992; 12: 5447–5454.
3. Wang GL, Semenza GL. General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia. Proc Natl Acad Sci U S A. 1993; 90: 4304–4308.
4. Epstein AC, Gleadle JM, McNeill LA, Hewitson KS, O’Rourke J, Mole DR, Mukherji M, Metzen E, Wilson MI, Dhanda A, Tian YM, Masson N, Hamilton DL, Jaakkola P, Barstead R, Hodgkin J, Maxwell PH, Pugh CW, Schofield CJ, Ratcliffe PJ. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell. 2001; 107: 43–54.[CrossRef][Medline] [Order article via Infotrieve]
5. Schofield CJ, Ratcliffe PJ. Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol. 2004; 5: 343–354.[CrossRef][Medline] [Order article via Infotrieve]
6. Semenza GL. Hydroxylation of HIF-1: oxygen sensing at the molecular level. Physiology (Bethesda). 2004; 19: 176–182.[CrossRef][Medline] [Order article via Infotrieve]
7. Masson N, Ratcliffe PJ. HIF prolyl and asparaginyl hydroxylases in the biological response to intracellular O(2) levels. J Cell Sci. 2003; 116: 3041–3049.
8. Bruick RK, McKnight SL. A conserved family of prolyl-4-hydroxylases that modify HIF. Science. 2001; 294: 1337–1340.
9. Semenza GL. HIF-1, O(2), and the 3 PHDs: how animal cells signal hypoxia to the nucleus. Cell. 2001; 107: 1–3.[CrossRef][Medline] [Order article via Infotrieve]
10. Hon WC, Wilson MI, Harlos K, Claridge TDW, Schofield CJ, Pugh CW, Maxwell PH, Ratcliffe PJ, Stuart DI, Jones EY. Structural basis for the recognition of hydroxyproline in HIF-1 alpha by pVHL. Nature. 2002; 417: 975–978.[CrossRef][Medline] [Order article via Infotrieve]
11. Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, Salic A, Asara JM, Lane WS, Kaelin WG. HIFa targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science. 2001; 292: 464–468.
12. Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME, Wykoff CC, Pugh CW, Maher ER, Ratcliffe RJ. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature. 1999; 399: 271–275.[CrossRef][Medline] [Order article via Infotrieve]
13. Ohh M, Park CW, Ivan M, Hoffman MA, Kim TY, Huang LE, Pavletich N, Chau V, Kaelin WG. Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein. Nat Cell Biol. 2000; 2: 423–427.[CrossRef][Medline] [Order article via Infotrieve]
14. Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med. 2003; 9: 677–684.[CrossRef][Medline] [Order article via Infotrieve]
15. Giaccia A, Siim BG, Johnson RS. HIF-1 as a target for drug development. Nat Rev Drug Discov. 2003; 2: 803–811.[CrossRef][Medline] [Order article via Infotrieve]
16. Hewitson KS, Schofield CJ. The HIF pathway as a therapeutic target. Drug Discov Today. 2004; 9: 704–711.[CrossRef][Medline] [Order article via Infotrieve]
17. Bernstein FC, Koetzle TF, Williams GJ, Meyer EF Jr, Brice MD, Rodgers JR, Kennard O, Shimanouchi T, Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977; 112: 535–542.[Medline] [Order article via Infotrieve]
18. Halgren TA. Merck molecular force field. I. Basis, form, scope, parameterization, and performance of MMFF94. J Comp Chem. 1996; 17: 490–519.[CrossRef]
19. Edelsbrunner H, Facello M, Fu R, Liang J. Measuring proteins and voids in proteins. Proceedings of the 28th Hawaii International Conference on Systems Science. 1995; 256–264.
20. Goto J, Kataoka R, Hirayama N. Ph4Dock-Pharmacophore-based protein-ligand docking. J Med Chem. 2004; 47: 6804–6811.[CrossRef][Medline] [Order article via Infotrieve]
21. Kaule G, Gunzler V. Assay for 2-oxoglutarate decarboxylating enzymes based on the determination of [1-14C] succinate: Application to prolyl 4-hydroxylase. Anal Biochem. 1990; 184: 291–297.[CrossRef][Medline] [Order article via Infotrieve]
22. Price DL, Rhett PM, Thorpe SR, Baynes JW. Chelating activity of advanced glycation end-product (AGE) inhibitors. J Biol Chem. 2001; 276: 48967–48972.
23. Tanaka T, Kojima I, Ohse T, Ingelfinger JR, Adler S, Fujita T, Nangaku M. Cobalt promotes angiogenesis via hypoxia-inducible factors and protects ischemic tubulointerstitium in the remnant kidney. Lab Invest. 2005; 85: 1292–1307.[CrossRef][Medline] [Order article via Infotrieve]
24. Muramatsu M, Katada J, Hayashi I, Majima M. Chymase as a proangiogenic factor. A possible involvement of chymase-angiotensin-dependent pathway in the hamster sponge angiogenesis model. J Biol Chem. 2000; 275: 5545–5552.
25. Tanaka T, Miyata T, Inagi R, Fujita T, Nangaku M. Hypoxia in renal disease with proteinuria and/or glomerular hypertension. Am J Pathol. 2004; 165: 1979–1992.
26. Kirino T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 1982; 239: 57–69.[CrossRef][Medline] [Order article via Infotrieve]
27. Ivan M, Haberberger T, Gervasi DC, Michelson KS, Gunzler V, Kondo K, Yang H, Sorokina I, Conaway RC, Conaway JW, Kaelin WG Jr. Biochemical purification and pharmacological inhibition of a mammalian prolyl hydroxylase acting on hypoxia-inducible factor. Proc Natl Acad Sci U S A. 2002; 99: 13459–13464.
28. Salnikow K, Donald SP, Bruick RK, Zhitkovich A, Phang JM, Kasprzak KS. Depletion of intracellular ascorbate by the carcinogenic metals nickel and cobalt results in the induction of hypoxic stress. J Biol Chem. 2004; 279: 40337–40344.
29. Warnecke C, Griethe W, Weidemann A, Jurgensen JS, Willam C, Bachmann S, Ivashchenko Y, Wagner I, Frei U, Wiesener M, Eckardt KU. Activation of the hypoxia-inducible factor-pathway and stimulation of angiogenesis by application of prolyl hydroxylase inhibitors. FASEB J. 2003; 17: 1186–1188.
30. McDonough MA, Li V, Flashman E, Chowdhury R, Mohr C, Lienard BM, Zondlo J, Oldham NJ, Clifton IJ, Lewis J, McNeill LA, Kurzeja RJ, Hewitson KS, Yang E, Jordan S, Syed RS, Schofield CJ. Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2). Proc Natl Acad Sci U S A. 2006; 103: 9814–9819.
31. Majima M, Isono M, Ikeda Y, Hayashi I, Hatanaka K, Harada Y, Katsumata O, Yamashina S, Katori M, Yamamoto S. Significant roles of inducible cyclooxygenase (COX)-2 in angiogenesis in rat sponge implants. Jpn J Pharmacol. 1997; 75: 105–114.[Medline] [Order article via Infotrieve]
32. Picozzi P, Todd NV, Crockard HA. Regional blood-brain barrier permeability changes after restoration of blood flow in postischemic gerbil brains: a quantitative study. J Cereb Blood Flow Metab. 1985; 5: 10–16.[Medline] [Order article via Infotrieve]
33. Zaman K, Ryu H, Hall D, O’Donovan K, Lin KI, Miller MP, Marquis JC, Baraban JM, Semenza GL, Ratan RR. Protection from oxidative stress– induced apoptosis in cortical neuronal cultures by iron chelators is associated with enhanced dna binding of hypoxia-inducible factor-1 and atf-1/creb and increased expression of glycolytic enzymes, p21waf1/cip1, and erythropoietin. J Neurosci. 1999; 15: 9821–9830.
34. Hayashi T, Abe K, Itoyama Y. Reduction of ischemic damage by application of vascular endothelial growth factor in rat brain after transient ischemia. J Cereb Blood Flow Metab. 1998; 18: 887–895.[CrossRef][Medline] [Order article via Infotrieve]
35. Vannucci SJ, Clark RR, Koehler-Stec E, Li K, Smith CB, Davies P, Maher F, Simpson IA. Glucose transporter expression in brain: Relationship to cerebral glucose utilization. Dev Neurosci. 1998; 20: 369–379.[CrossRef][Medline] [Order article via Infotrieve]
36. Liu J, Narasimhan P, Yu F, Chan PH. Neuroprotection by hypoxic preconditioning involves oxidative stress-mediated expression of hypoxia-inducible factor and erythropoietin. Stroke. 2005; 36: 1264–1269.
37. Wen TC, Sadamoto Y, Tanaka J, Zhu PX, Nakata K, Ma YJ, Hata R, Sakanaka M. Erythropoietin protects neurons against chemical hypoxia and cerebral ischemic injury by up-regulating Bcl-xL expression. J Neurosci Res. 2002; 67: 795–803.[CrossRef][Medline] [Order article via Infotrieve]
38. Sun FY, Guo X. Molecular and cellular mechanisms of neuroprotection by vascular endothelial growth factor. J Neurosci Res. 2005; 79: 180–4.[CrossRef][Medline] [Order article via Infotrieve]
39. McLaughlin J, Roozendaal B, Dumas T, Gupta A, Ajilore O, Hsieh J, Ho D, Lawrence M, McGaugh JL, Sapolsky R. Sparing of neuronal function postseizure with gene therapy. Proc Natl Acad Sci. 2000; 97: 12804–12809.
40. Lawrence MS, Sun GH, Kunis DM, Saydam TC, Dash R, Ho DY, Sapolsky RM, Steinberg GK. Overexpression of the glucose transporter gene with a herpes simplex viral vector protects striatal neurons against stroke. J Cereb Blood Flow Metab. 1996; 16: 181–185.[CrossRef][Medline] [Order article via Infotrieve]
41. O’Rourke JF, Pugh CW, Bartlett SM, Ratcliffe PJ. Identification of hypoxically inducible mRNAs in HeLa cells using differential-display PCR. Role of hypoxia-inducible factor-1. Eur J Biochem. 1996; 241: 403–410.[Medline] [Order article via Infotrieve]
42. Zovein A, Flowers-Ziegler J, Thamotharan S, Shin D, Sankar R, Nguyen K, Gambhir S, Devaskar SU. Postnatal hypoxic-ischemic brain injury alters mechanisms mediating neuronal glucose transport. Am J Physiol Regul Integr Comp Physiol. 2004; 286: R273–R282.
43. Bergeron M, Gidday JM, Yu AY, Semenza GL, Ferriero DM, Sharp FR. Role of hypoxia-inducible factor-1 in hypoxia-induced ischemic tolerance in neonatal rat brain. Ann Neurol. 2000; 48: 285–296.[CrossRef][Medline] [Order article via Infotrieve]
44. Matsumoto M, Makino Y, Tanaka T, Tanaka H, Ishizaka N, Noiri E, Fujita T, Nangaku M. Induction of renoprotective gene expression by cobalt ameliorates ischemic injury of the kidney in rats. J Am Soc Nephrol. 2003; 14: 1825–1832.
45. Watanabe D, Suzuma K, Matsui S, Kurimoto M, Kiryu J, Kita M, Suzuma I, Ohashi H, Ojima T, Murakami T, Kobayashi T, Masuda S, Nagao M, Yoshimura N, Takagi H. Erythropoietin as a retinal angiogenic factor in proliferative diabetic retinopathy. N Engl J Med. 2005; 353: 782–792.
This article has been cited by other articles:
 |
|
 |
|
  K. Takeda, T. Ichiki, E. Narabayashi, K. Inanaga, R. Miyazaki, T. Hashimoto, H. Matsuura, J. Ikeda, T. Miyata, and K. Sunagawa Inhibition of Prolyl Hydroxylase Domain-Containing Protein Suppressed Lipopolysaccharide-Induced TNF-{alpha} Expression Arterioscler Thromb Vasc Biol, December 1, 2009; 29(12): 2132 - 2137. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Schultz, V. Murthy, J. B. Tatro, and D. Beasley Prolyl hydroxylase 2 deficiency limits proliferation of vascular smooth muscle cells by hypoxia-inducible factor-1{alpha}-dependent mechanisms Am J Physiol Lung Cell Mol Physiol, June 1, 2009; 296(6): L921 - L927. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Miyata and C. van Ypersele de Strihou Translation of basic science into clinical medicine: novel targets for diabetic nephropathy Nephrol. Dial. Transplant., May 1, 2009; 24(5): 1373 - 1377. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||