Neuroprotection by Hypoxic Preconditioning Requires Sequential Activation of Vascular Endothelial Growth Factor Receptor and Akt

Abstract

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.

Arsenic trioxide prevents radiation-enhanced tumor invasiveness and inhibits matrix metalloproteinase-9 through downregulation of nuclear factor κB

“…Consequently, pharmaceutical inhibitors of MMPs or a v b 3 -integrin expression have successfully prevented radiation-induced tumor cell invasion (Qian et al, 2002;Wick et al, 2002). Based on the presented data, radiation significantly induced MMP-9 expression in human cervical cancer cells and also in LLC cells.…”

Targeting Astrocytes for Stroke Therapy

“…A neutralizing antibody against VEGF prevented the protection mediated by hypoxic preconditioning observed in vitro. 110 In rats, hypoxic preconditioning was mirrored by an increase in VEGF levels in astrocytes. 111 It should be noted, however, that astrocytes are not the sole source of VEGF in the brain.…”

The neuroprotective mechanism of brain ischemic preconditioning

“…They also demonstrated that the preconditioning treatment inhibited the augmentation of neuronal apoptosis in the penumbral region to prevent the spread of infarction, and that the preconditioning-induced neuroprotection was due to the persistent activation of Akt in the penumbra [37] . Wick et al demonstrated that neuroprotection by hypoxic preconditioning required sequential activation of vascular endothelial growth factor (VEGF) receptor and Akt [38] . Neurons incubated at 5% O 2 for 9 h showed increased levels of the VEGF, VEGF receptor-2 (VEGFR-2), phosphorylated Akt/PKB, and ERK1.…”