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(Stroke. 2004;35:2687.)
© 2004 American Heart Association, Inc.
Articles |
From the Department of Neurology and Neuroscience, Burke/Cornell Medical Research Institute, Weill Medical College of Cornell, White Plains, NY.
Correspondence to Dr Rajiv R. Ratan, Burke/Cornell Medical Research Institute, 785 Mamaroneck Avenue, White Plains, NY 10605. E-mail rratan{at}caregroup.harvard.edu
| Abstract |
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Key Words: acute care hypoxia ischemia stroke transcription
| Introduction |
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The above list is not exhaustive, but the heterogeneity of stroke argues that one needs a therapeutic approach that recognizes and addresses the complexity inherent in its diverse causes and outcomes. One strategy to address this complexity is combinatorial therapy, which has been used successfully in the treatment of cancer, HIV infection, and tuberculosis. Indeed, several labs have begun to apply the principles of combinatorial therapy to stroke, with promising preclinical results.2,3
An alternative to combinatorial therapy is to identify single agents that act on a multivalent, but thoughtfully coordinated, homeostatic response.4 Such a coordinated response would involve multiple gene programs (eg, survival, repair, and revascularization) that act in various cell types (neuronal, glial, and endothelial). A biological paradigm that provides insight into multivalent protective responses in the brain is ischemic preconditioning.5 Ischemic preconditioning is a fascinating biological phenomenon in which exposure of animals (and likely humans as well6) to a short, sublethal ischemic insult provides immediate and lasting resistance to a subsequent, more severe insult. The lasting resistance appears to result in part from the de novo expression of genes involved in homeostatic responses to hypoxia and ischemia. A central working hypothesis of our group and others is that pharmacological agents that activate programs of gene expression normally induced by ischemic preconditioning will be effective agents for prevention and treatment of stroke.7
| Hypoxia Homeostasis, Hypoxia Inducible Factor-1, and Ischemic Preconditioning |
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Over a decade ago, Gregg Semenza and colleagues at Johns Hopkins University Medical School used a series of transgenic mice to identify a sequence in the 3' untranslated region of the Epo gene that is required for its upregulation by hypoxia.15 Using a portion of this sequence to make a DNA affinity column combined with industrial quantities of lysates from cultured Chinese hamster ovary cells exposed to a hypoxia mimic, the Semenza group purified a heterodimeric complex of proteins that bind to DNA to regulate hypoxia gene expression.15 They designated this complex as hypoxia-inducible factor 1 (HIF-1). It is composed of a 120 kDa HIF-1
subunit and a 91 to 94 kDa HIF-1ß subunit. Established inducers of Epo expression, including iron chelators, cobalt chloride, and hypoxia, induce HIF-1. Of note, the canonical binding site for HIF-1 (5'-GCGTG-3') is found in the promoter regions of many of the genes one would choose to compensate for hypoxia-Epo, VEGF, glycolytic enzymes, heme oxygenase, glucose transporters, neuroglobin, etc, and although Epo is limited in its expression to the liver, kidney, and brain, HIF-1 appears to be ubiquitously expressed in all tissues, including brain.
The evidence that HIF-1 is activated in response to ischemic preconditioning in brain is extensive: (1) ischemic preconditioning in the brain requires new transcription and is not dependent on the activation of glutamate receptors;16 (2) a 3-hour exposure (the duration of exposure required for ischemic preconditioning) to 8% O2 induces HIF-1 in the neonatal brain;17 (3) preconditioning with pharmacological activators of HIF-1 (deferoxamine or cobalt chloride) rather than hypoxia confers significant protection in the central nervous system (CNS);17 (4) infusion of soluble Epo receptor (intracerebroventricularly) reverses the protective effect of ischemic preconditioning.18
Protein Hydroxylation, HIF-1 Stabilization, and Activation of a Central Homeostatic Response to Stroke
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and enhance HIF-1dependent transcription may be viable agents for human stroke therapy. Indeed, over the past decade the signaling paths involved in HIF-1 activation have been deciphered in great detail. Under normoxic conditions, HIF-1
is constitutively transcribed and translated. However, the stability of the protein is drastically reduced by the hydroxylation of HIF-1 at prolines 402 and 564 by HIF-1 prolyl hydroxylases. Hydroxylated HIF-1 recruits the E3-ubiquitin ligase Von Hippel Lindau (VHL) protein. VHL protein tags HIF-1 with ubiquitin groups and targets it for degradation by the proteasome.19
As expected from this model, the prolyl hydroxylases involved in regulating HIF-1
stability are oxygen-dependent.20,21 Thus, under conditions of hypoxia, these enzymes function with low efficiency, resulting in HIF-1
that is not hydroxylated. Because nonhydroxylated HIF-1
cannot interact with VHL, it becomes stabilized. Stable HIF-1
can then bind to its heterodimeric partner HIF-1ß, and together these proteins can act in the nucleus to transactivate genes involved in adaptation to hypoxic-ischemic stress.
The prolyl hydroxylases not only require oxygen, but iron and 2-oxoglutarate21,22 to hydroxylate the critical prolines on HIF-1
. Iron and 2-oxoglutarate are thus rational pharmacological targets for inhibiting the HIF-1 prolyl hydroxylases and activating HIF-1 under conditions of normoxia, or for augmenting HIF-1 activation under conditions of ischemia.23 It is not surprising that some of the best-established activators of HIF-1 are chelators of iron. Indeed, previous studies from our laboratory demonstrated that 2 distinct chelators of iron, deferoxamine mesylate and mimosine both significantly induce HIF-1 and its target genes in neurons and protect neurons from oxidative stress.24 Subsequent studies by 2 other groups demonstrated that delivery of desferoxamine mesylate to neonatal or adult animals reduces infarct volume in models of focal ischemia.17 The findings provided experimental substance to the notion that inhibitors of the prolyl hydroxylase are neuroprotective.
| Which Prolyl Hydroxylase Inhibitors Will Be Best for Stroke Therapy? |
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Another strategy to identify novel, safe prolyl hydroxylase inhibitors is to screen a library of drugs already approved for use in humans (FDA-approved drugs). Such a screen might elucidate agents that would more potently activate HIF-1 in the CNS; additionally, agents that activate HIF-1 preferentially in glia, neurons, or endothelial cells might be identified. The screen holds the obvious clinical benefit that all of the agents have passed a series of rigorous tests for safety in humans. Our laboratory has been involved in screening an FDA-approved library for novel HIF-1 activators over the past few years. A number of agents have been identified and preliminary studies are ongoing to document when, where, and how these agents can activate HIF-1 in the CNS.
| There Is Good and Bad in All of Us: HIF-1 Is No Different |
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but a host of other proteins as well. The important message for physicians who treat stroke is that pharmacological inhibition of the HIF-prolyl hydroxylases is not equivalent to HIF-1
overexpression. | HIF-1 Prolyl Hydroxylases: Promising But As Yet Unproven Therapeutics for Stroke |
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Despite the details that have yet to be elucidated, the notion of being able to pharmacologically activate a multifaceted homeostatic response that works at a cellular, local, and systemic level to alleviate the discrepancy between substrate (oxygen/glucose) supply and demand holds tremendous intuitive appeal. It is our fervent hope that such a strategy, which takes advantage of endogenous homeostatic pathways, will be more efficacious while less toxic.
| Acknowledgments |
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| Footnotes |
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Received July 12, 2004; accepted August 5, 2004.
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