Volume 14 Issue 7 - July 2, 2010 PDF
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VEGF-A/VEGFR-2 signaling leading to CREB phosphorylation is a shared pathway underlying the protective effect of preconditioning on neurons and endothelial cells.
Hsueh-Te Lee,1 Ying-Chao Chang,4 Yi-Fang Tu,2,3 and Chao-Ching Huang1,2*
1Department of Pediatrics, 2Institute of Clinical Medicine, and 3Department of Emergency Medicine, National Cheng Kung University Hospital, Tainan70428, Taiwan, and 4Department of Pediatrics, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine,Kaohsiung 83301, Taiwan
 
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Hypoxic-ischemia (HI) encephalopathy is a major cause of neonatal mortality and neurological disabilities in childhood. A new approach to studying treatment for neonatal HI encephalopathy is investigating states of tolerance. Although neurons are the cellular target of preconditioning, ischemic tolerance occurring on vascular cells might also contribute greatly to neuroprotection. Studies have suggested that preconditioning improves microvascular perfusion and protects cerebral blood vessels from ischemia, however, the signaling mechanisms by which preconditioning protects vascular endothelial cells have not been well determined. More importantly, very few studies have examined whether there is a shared signaling pathway that underlies preconditioning-induced protection in neuron and endothelial cells.

Our previous study on 7-day-old rat pups showed that ligating the carotid artery 1 h before hypoxia produces damage in the ipsilateral cerebral hemisphere, in contrast, ligating the artery 24 h before hypoxia provided complete neuroprotection. The protective effect of the 24-h artery ligation preconditioning required the activation of cAMP response element-binding protein (CREB), a transcription factor implicated in synaptic plasticity, memory and survival of nervous system. However, it remains to be determined whether this 24-h artery ligation preconditioning affects CREB activation in neurons and vascular endothelial cells. If so, then, the preconditioning-activated transducer and upstream pathway that leads to CREB activation in neurons and endothelial cells warrants investigation because the molecules that protect both vessels and neurons will be attractive therapeutic targets.

Vessels and nerves guide each other to their target during development. This neuronal and vascular cross-talk includes the use of common signals, such as vascular endothelial growth factor (VEGF)-A, to determine the fate of neurons and vascular endothelial cells. Recent insights into the role of VEGF-A in a variety of neurological disorders suggest that VEGF-A and its downstream effectors may be promising therapeutic targets in these diseases. Because VEGF-A is highly expressed in neurons and vessels throughout the early postnatal period, VEGF-A may be a major protector through its dual neuronal and vascular effects in the developing brain.

VEGF-A binds to two cell-surface tyrosine kinase receptors, VEGFR-1 and VEGFR-2, which activate several downstream pathways. The VEGF/VEGFR-2 pathway was also involved in the neuroprotection of hypoxic preconditioning in a neonatal murine model of excitotoxic brain injury. However, the exact cellular phenotypes that are involved in the protective effects of VEGF/VEGFR-2 signaling during preconditioning remain undetermined. Whether VEGF-A/VEGFR-2 signaling leading to CREB activation is the shared pathway underlying the protective effect of preconditioning in neurons and endothelial cells in the developing brain.

Here, we tested the hypothesis that VEGF-A/VEGFR-2 signaling that leads to CREB activation is the shared pathway underlying the protective effect of preconditioning in neurons and endothelial cells. VEGF-A, VEGFR-1, or VEGFR-2 was inhibited by antisense oligodeoxynucleotides (ODN) in vivo, and by a VEGF-A neutralizing antibody or VEGFR-2 inhibitor in vitro. CREB phosphorylation (pCREB), and VEGF-A and VEGFR-2 expression were increased and co-localized in vascular endothelial cells and neurons in the ipsilateral cerebral cortex 24 h post-ligation. The antisense ODN blockades of VEGF-A and VEGFR-2 decreased pCREB and reduced the protection of 24-h ligation preconditioning. Furthermore, oxygen-glucose deprivation (OGD) preconditioning upregulated VEGF-A, VEGFR-2, and pCREB levels, and protected immortalized H19-7 neuronal cells and b.End3 vascular endothelial cells against 24-h OGD cell death. Blocking VEGF-A or VEGFR-2 reduced CREB activation and the effects of OGD preconditioning in neuronal cells and endothelial cells. Transfecting a serine-133 phosphorylation mutant CREB also inhibited the protective effect of OGD preconditioning. We conclude that VEGF-A/VEGFR-2 signaling leading to CREB phosphorylation is the shared pathway underlying the preconditioning-induced protective effect in neurons and vascular endothelial cells in the developing brain.
Fig. In the 24-hour ligation group, the neurons (arrowheads) and vessels (arrows) with increased VEGFR- 2 expression also coexpressed pCREB.
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