| Background and Purpose Vascular endothelial growth factor (VEGF), initially discovered as an angiogenic and vascular permeability factor, has been recognized to participate in neuroprotection, neurogenesis, anti-inflammatory process, neurite outgrowth and ischemic brain remodeling. Therefore, VEGF was a potential pharmaceutical target for treating ischemic stroke. However, accumulating evidence from both basic and preclinical trials demonstrated high risk-to-benefit ratio of VEGF application for ischemic stroke. A very possible reason of the high risk-to-benefit ratio might be its severe side effects such as brain edema and hemorrhagic transformation which could be induced by blood-brain barrier (BBB) leakage. BBB, a vital element of neurovascular unit, plays an important role in the maintenance of the central nervous system homeostasis. When BBB is disrupted by ischemic stroke, many serum proteins that are deleterious to neurons enter the brain and further worsen ischemic injury. Preventing BBB disruption and brain edema associated with VEGF administration may contribute to better treatment effect and lower risk-to-benefit ratio. In order to abolish its detrimental effects on BBB and brain edema after ischemic stroke, the underlying mechanisms have to be completely clarified. Due to the progress of microarray, it was possible to describe the full view of gene changes in distinguished circumstances. Here, the present study was designed to investigate what genes VEGF would influence after cerebral ischemia and also the underlying signal pathways.MethodsMice were randomized to sham group in which underwent sham suture middle cerebral artery occlusion (MCAO) surgery or the other group in which they received 90 min MCAO, and the successful models were randomized into 2 groups, MCAO group, mice received 1μl phosphate buffered saline (PBS,0.01M, pH 7.4) intracerebroventricularly in the right lateral ventricle 3 h after reperfusion; VEGF group, in which 1μl rhVEGF-A165 (10μg/ml) was injected into the right lateral ventricle 3 h after reperfusion. To investigate whether intracerebraventricularly delivered VEGF disrupt the BBB or improve stroke outcomes after brain ischemia, Evans Blue leakage, IgG leakage, brain water content, infarct volumes and neurological deficits were measured in 3 groups (sham group, MCAO group, and VEGF group). To clarify which genes are influenced by VEGF administration after stroke, Agilent SurePrint G3 Mouse Gene Expression 8×60K Microarray which including 39,430 protein-coding transcripts was performed. Quantile normalization and subsequent data processing were performed using the GeneSpring GX software package (version 12.0, Agilent Technologies). Differentially expressed mRNAs with statistical significance between 2 groups were determined by two-tailed Student’s t test P< 0.05 and fold change> 2. Orosomucoid (Orm) 1 protein expression was monitored via western blotting, immunohistochemistry and immunofluorescence. Moreover, to confirm that NF-κB could bind the ORM1 promoter, antibody super shifts were carried out via binding reactions including recombinant mouse NF-κB/p65 protein and biotin-labeled 0RM1 promoter oligonucleotide. Lastly, to investigate whether NF-κB pathway is inhibited by VEGF administration, electrophoretic mobility shift assay was used to measure the DNA binding ability of NF-κB, and mRNA and protein levels of key points in NF-κB pathway were detected via qRT-PCR and western blotting.ResultsThere were significant differences in leakage of Evans blue dye, IgG and brain water content of the ischemic hemisphere among 3 groups. However, there was no obvious difference in the infarct volume, accelerated rotarod test or mNSS between MCAO group and VEGF group. After transient focal cerebral ischemia,3,381 mRNAs changed significantly compared with sham group (2,340 up-regulated by 2-fold and 1,041 down-regulated by 0.5-fold). Compared to MCAO group,38 mRNAs changed significantly in VEGF group (19 up-regulated by 2-fold and 19 down-regulated by 0.5-fold). Fifteen of these 38 mRNAs were also altered after MCAO in comparison to sham group. Gene Ontology function annotations of the 15 genes include cell differentiation, regulation of immune system process, acute phase response, complement activation, phagocytosis, protein homooligomerization, protein deubiquitination, glucuronosyltransferase activity, generation of precursor metabolites and energy, hormone-mediated signalling pathway, genetic imprinting, melanocortin receptor binding, MAP kinase kinase kinase activity, et al. As the most sharply changed gene among these 15 genes, Orml was increased in MCAO group in comparison to sham group and decreased in VEGF group when compared to MCAO group as detected by western blotting, immunohistochemistry and immunofluorescence. Bioinformatics analysis showed there were 2 NF-κB binding sites in ORM1 promoter, and antibody super-shifted electrophoretic mobility shift assay (EMSA) verified that NF-κB could bind ORM1 promoter. The DNA-binding activity of NF-κB/p65 was increased after MCAO and decreased after VEGF delivery. IKKa, IKKβ, phosphor-IKKa, IκBα, phosphor-IκBα, p65, phosphor-p65 were all increased in MCAO group when compared to sham group and decreased in VEGF group in comparison to MCAO group.ConclusionsVEGF aggravated blood-brain-barrier disruption and brain edema after focal cerebral ischemia probably by decreasing Orml through inhibition of NF-κB pathway. |
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