Temporal profile of Src, SSeCKS, and angiogenic factors after focal cerebral ischemia: correlations with angiogenesis and cerebral edema

Abstract

A better understanding of the underlying mechanisms of angiogenesis and vascular permeability is necessary for the development of therapeutic strategies for ischemic injury. The purpose of this study was to examine the spatial and temporal expression of Src and Src-suppressed C kinase substrate (SSeCKS) in brain after middle cerebral artery occlusion (MCAO) and elucidate the relationships among Src, SSeCKS, and the key angiogenic factors present after stroke. Rats were subjected to either MCAO or sham operation. Reverse transcriptase-polymerase chain reaction and Western blotting results revealed that Src gradually increased starting as early as 2 h after MCAO and remained high for 1 day. In contrast, SSeCKS decreased after MCAO. Src expression correlated positively with that of vascular endothelial growth factor and angiopoietin-2, and negatively with that of SSeCKS, angiopoietin-1, and zonula occludens-1. However, SSeCKS had the reverse correlations. Changes in the expression of these factors correlated with the progress of angiogenesis and cerebral edema. Dynamic temporal changes in Src and SSeCKS expression may modulate angiogenesis and cerebral edema formation after focal cerebral ischemia.

Fig. 1

Changes in Src and SSeCKS expression at the mRNA and protein levels after MCAO. (A) (Top) RT-PCR of Src and SSeCKS mRNA at the indicated time points after MCAO; (Bottom) bar graph showing mRNA expression relative to that of β-actin. (B) (Top) Western blot of Src and SSeCKS at the indicated time points after MCAO; (Bottom) bar graph showing protein expression relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are representative of at least three independent experiments and presented as means ± S.D.; n = 6 per group; ^#P < 0.05, *P < 0.01 compared with the sham group. (C) Immunohistochemical staining for Src and SSeCKS at the indicated time points after MCAO. Scale bar = 20 μm.

Fig. 2

Changes in expression of VEGF, Ang-1, and Ang-2 after MCAO. (A) (Top) RT-PCR of VEGF, Ang-1, and Ang-2 mRNA at the indicated time points after MCAO; (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 MCAO; (Bottom) bar graph showing protein expression relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are presented as means ± S.D.; n = 6/group; ^#P < 0.05, *P < 0.01. (C) Immunohistochemical staining revealed VEGF and Ang-2 immunoreactivity in neuron-like and glial-like cells in the ischemic boundary area after MCAO. Ang-1 immunostaining was especially weak at 1 day after ischemia. Scale bar = 20 μm.

Fig. 3

Neovascularization in rat brain after MCAO. (A) Brain sections were immunostained for CD31 to identify capillaries. Scale bar = 50 μm. (B) The bar graph shows microvessel counts at different time points after MCAO. Microvessels were counted from 10 fields of the ischemic penumbra area under a microscope at 400× magnification. Data are means ± S.D.; n = 7 per group; *P < 0.01 compared with the sham group.

Fig. 4

Effect of MCAO on brain water content and ZO-1 expression. (A) Brain water content was calculated as the weight difference between wet and dry samples. Data are means ± S.D.; n = 7 per group; *P < 0.01 compared with sham group. (B) Northern blot of ZO-1 mRNA at the indicated time points after MCAO. β-actin was used as a loading control. (C) Western blot of ZO-1 protein after MCAO. GAPDH was used as a loading control. Data are presented as means ± S.D.; n = 6 per group, ^#P < 0.05, *P < 0.01 compared with the sham group. (D) ZO-1 immunoreactivity was observed around the vessels, especially at sites of endothelial cell–cell contact in control brain. ZO-1 was weakly expressed at 1 day after MCAO, and strongly expressed at 7 days. Scale bar = 50 μm.