Neuroprotection by hypoxic preconditioning requires sequential activation of vascular endothelial growth factor receptor and Akt.
Hypoxic preconditioning provides protection against ischemic brain lesions in animal models of cerebral ischemia-hypoxia. To analyze the underlying molecular mechanisms, we developed an in vitro model of hypoxic neuroprotection in cerebellar granule neurons (CGN) by reducing the oxygen tension to 1-5% for 1-24 hr. Exposure to 5% O2 for 9 hr resulted in reduction of cell death after potassium deprivation, treatment with 100 microm glutamate, or 500 microm 3-nitroproprioninc acid (3-NP) by 46, 22, and 55%, respectively. Shorter (1 or 3 hr) or longer (>12 hr) intervals or pretreatment with lower oxygen tension failed to rescue CGN from death. In contrast, toxicity of four different chemotherapeutic drugs [1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, cisplatine, topotecane, and vincristine] was unaffected by hypoxic preconditioning. The induction of protective effects was dependent on new protein synthesis. Protein levels of B-cell lymphoma protein-2 (BCL-2), BCL-x(L/S), heat shock protein 70/90, and BCL-2-associated death protein remained unaltered. CGN incubated at 5% O2 for 9 hr showed increased levels of the vascular endothelial growth factor (VEGF), the VEGF receptor-2 (VEGFR-2), phosphorylated Akt/protein kinase B (PKB), and extracellular signal-regulated kinase 1 (ERK1). Incubation with a neutralizing anti-VEGF antibody, a monoclonal antibody to VEGFR-2, wortmannin, or antisense-Akt/PKB, but not treatment with U0126, an ERK-inhibitor, reverted the resistance acquired by hypoxic preconditioning. Inhibition of VEGFR-2 blocked the activation of Akt/PKB. Finally, pretreatment with recombinant VEGF resulted in a hypoxia-resistant phenotype in the absence of hypoxic preconditioning. Our data are indicating a sequential requirement for VEGF/VEGFR-2 activation and Akt/PKB phosphorylation for neuronal survival mediated by hypoxic preconditioning and propose VEGF as a hypoxia-induced neurotrophic factor.
Wick
Antje
A
Laboratory of Neurodegeneration, Department of Neurology, University of Tübingen, 72076 Tübingen, Germany.
Wick
Wolfgang
W
Waltenberger
Johannes
J
Weller
Michael
M
Dichgans
Johannes
J
Schulz
Jörg B
JB
D000818:Animals; D016923:Cell Death; D015687:Cell Hypoxia / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D016228:Endothelial Growth Factors / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008222:Lymphokines / Q000378:metabolism / Q000494:pharmacology; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D009574:Nitro Compounds; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011188:Potassium / Q000378:metabolism; D011422:Propionates / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000378:metabolism*; D040262:Receptors, Vascular Endothelial Growth Factor; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D004791:Enzyme Inhibitors; D008222:Lymphokines; D009574:Nitro Compounds; D016376:Oligonucleotides, Antisense; D011422:Propionates; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D017978:Receptors, Growth Factor; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C015392:3-nitropropionic acid; D011188:Potassium; D010100:Oxygen