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Src regulates angiogenic factors and vascular permeability after focal cerebral ischemia-reperfusion - PubMed

  • ️Wed Jan 01 2014

Src regulates angiogenic factors and vascular permeability after focal cerebral ischemia-reperfusion

L Zan et al. Neuroscience. 2014.

Abstract

Developing new strategies to treat cerebral ischemic-reperfusion injury will require a better understanding of the mechanisms that underlie vascular permeability. In this study we examined the temporal expression of Src and angiogenic factors in rat brain after focal cerebral ischemia and reperfusion and analyzed the relationships among those factors. We also investigated the effect of Src inhibitor PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) in ischemic reperfusion. Rats were subjected to middle cerebral artery occlusion for 90 min followed by reperfusion with or without PP1 treatment. Src mRNA increased at 3h after reperfusion and then gradually declined. Phosphorylation of Src at Y418 displayed a biphasic increase. Phosphorylation increased as early as 3h and peaked at 6h; after decreasing, it peaked again at 3-7 days. Increases in Src mRNA and phosphorylation correlated positively with levels of vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2), and negatively with levels of angiopoietin-1 (Ang-1) and zonula occludens-1 (ZO-1). Changes in the expression of these factors correlated with the progress of vascular permeability, especially early after reperfusion. Hence, dynamic temporal changes in Src Y418 phosphorylation may modulate vascular permeability after cerebral ischemia and reperfusion. PP1 effectively decreased Src Y418 phosphorylation and the expression of VEGF and Ang-2 and increased the expression of Ang-1 and ZO-1. It also reduced cerebral infarct size and neurologic dysfunction. Therefore, Src may represent a new therapeutic target for reducing tissue damage caused by increased vascular permeability.

Keywords: PP1; Src; angiogenic factors; cerebral ischemia; vascular permeability; zonula occludens-1.

Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

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Figures

Fig. 1
Fig. 1

Changes in Src mRNA and Y418 phosphorylation after ischemia and reperfusion. (A) Top: RT-PCR of Src mRNA at the indicated time points after reperfusion. Bottom: bar graph showing mRNA expression relative to that of β-actin. (B) Top: Western blot of Src Y418 phosphorylation at the indicated time points after reperfusion. Bottom: bar graph showing protein expression relative to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are representative of at least three independent experiments. (C) Immunohistochemical staining for Src (Y418) at the indicated time points after ischemic reperfusion. Scale bar = 20 μm. (D) Bar graph shows quantification analysis of pSrc-positive cells per 40x field at the indicated time points after ischemic reperfusion. Data in A (n = 6 rats/group), B (n = 6 rats/group), and D (n = 7 rats/group) are presented as means ± S.D. and were analyzed by one-way ANOVA followed by the Bonferroni post hoc test. *p < 0.05 compared with the sham group.

Fig. 2
Fig. 2

Changes in expression of VEGF, Ang-1, Ang-2, and Evans blue (EB) extravasation after ischemia and reperfusion. (A) Top: RT-PCR of VEGF, Ang-1, and Ang-2 mRNA at the indicated time points after reperfusion. Bottom: bar graph showing mRNA expression relative to that of β-actin. (B) Top: Western blot of VEGF, Ang-1, and Ang-2 at the indicated time points after reperfusion. Bottom: bar graph showing protein expression relative to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (C) Immunohistochemical staining shows VEGF and Ang-2 immunoreactivity in neuron-like and glial-like cells in the peri-infarct region. Ang-1 immunostaining was weak at 1 day after ischemic reperfusion. Scale bar = 20 μm. (D) Quantification analysis of VEGF-, Ang-2-, and Ang-1-positive cells per 40x field at the indicated time points after ischemic reperfusion. (E) EB extravasation increased at 3 h and peaked at 1 day after reperfusion before returning to baseline at 14 days. Data in A (n = 6 rats/group), B (n = 6 rats/group), D (n = 7 rats/group), and E (n = 7 rats/group) are presented as means ± S.D. and were analyzed by one-way ANOVA followed by the Bonferroni post hoc test. *p < 0.05 compared with the sham group.

Fig. 3
Fig. 3

Changes in ZO-1 expression after ischemia and reperfusion. (A) RT-PCR of ZO-1 mRNA at the indicated time points after reperfusion. β-actin was used as a loading control. (B) Western blot of ZO-1 protein after reperfusion. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (C) ZO-1 immunoreactivity was observed in vascular-like structures, especially in the adjacent vessels. ZO-1 was strongly expressed in the brains of sham-operated rats, but it was weakly expressed at 12 h and 1 day after ischemic reperfusion. Scale bar = 50 μm. (D) Quantification analysis of ZO-1-positive microvessels per 40x field at the indicated time points after ischemic reperfusion. Data in A, B, and D are presented as means ± S.D. and were analyzed by one-way ANOVA followed by the Bonferroni post hoc test. n = 7 rats per group; *p < 0.05 compared with the sham group.

Fig. 4
Fig. 4

Changes in expression of Src, angiogenic factors, and ZO-1 in PP1-treated rats after ischemia and reperfusion. (A) Left: RT-PCR of Src, VEGF, Ang-1, Ang-2, and ZO-1 mRNA in DMSO-, PP3-, and PP1-treated rats 1 day after reperfusion. Right: bar graph showing mRNA expression relative to that of β-actin. (B) Left: Western blot of Src (Y418), VEGF, Ang-1, Ang-2, and ZO-1 in DMSO-, PP3-, and PP1-treated rats 12 h after reperfusion. Right: bar graph showing protein expression relative to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (C) Bar graph shows quantification analysis of the immunopositive cells for pSrc, VEGF, Ang-2, Ang-1, and of the immunopositive microvessels for ZO-1. Immunohistochemical staining revealed a decrease in immunoreactivity of Src (Y418), VEGF, and Ang-2, and an increase in immunoreactivity of Ang-1 and ZO-1 in PP1-treated rats, compared with that in the DMSO- and PP3-treated rats after ischemic reperfusion. Data in A, B, and C are presented as means ± S.D. and were analyzed by one-way ANOVA followed by the Bonferroni post hoc test. n = 7 rats per group; *p < 0.05 compared with the sham group, #p < 0.05 compared with DMSO-and PP3-treated groups. (D) Representative immunohistochemical staining for pSrc, VEGF, Ang-2, Ang-1, and ZO-1 in the sham group, DMSO-, PP3-, and PP1-treated groups at 1 day after ischemic reperfusion. Scale bar = 20 μm.

Fig. 5
Fig. 5

Effect of Src inhibition with PP1 on Evans blue (EB) extravasation, brain water content, infarct volume, and neurologic score after ischemia and reperfusion. (A) Extravasation of EB was significantly reduced in the PP1-treated rats 1 day after reperfusion compared with that in DMSO- and PP3-treated rats. (B) PP1 treatment also significantly reduced post-ischemic water content. (C) Representative images of TTC-stained brain sections from control (left and middle panels) and PP1-treated rats. (D) The infarct volume was significantly decreased in the PP1-treated group compared to that in control groups. Data in A, B, and D are presented as means ± S.D. and were analyzed by one-way ANOVA followed by the Bonferroni post hoc test. (E) PP1 pretreatment decreased the neurologic score 1 day after ischemia and reperfusion. Data were analyzed with the non-parametric Kruskal-Wallis analysis of ranks and are represented as box-and-whisker plots. The upper and lower limits of the boxes indicate the 75th and 25th percentiles (interquartile range), respectively, and the horizontal line in each box represents the median. n = 7 rats per group; *p < 0.05 compared with the sham group, #p < 0.05 compared with DMSO- and PP3-treated groups.

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