Radiation attenuates physiological angiogenesis by differential expression of VEGF, Ang-1, tie-2 and Ang-2 in rat brain

Abstract

The etiology of radiation-induced cerebrovascular rarefaction remains unknown. In the present study, we examined the effect of whole-brain irradiation on endothelial cell (EC) proliferation/apoptosis and expression of various angiogenic factors in rat brain. F344 × BN rats received either whole-brain irradiation (a single dose of 10 Gy γ rays) or sham irradiation and were maintained for 4, 8 and 24 h after irradiation. Double immunofluorescence staining was employed to visualize EC proliferation/apoptosis in brain. The mRNA and protein expression levels of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), endothelial-specific receptor tyrosine kinase (Tie-2), and Ang-2 in brain were determined by real-time RT-PCR and immunofluorescence staining. A significant reduction in CD31-immunoreactive cells was detected in irradiated rat brains compared with sham-irradiated controls. Whole-brain irradiation significantly suppressed EC proliferation and increased EC apoptosis. In addition, a significant decrease in mRNA and protein expression of VEGF, Ang-1 and Tie-2 was observed in irradiated rat brains. In contrast, whole-brain irradiation significantly upregulated Ang-2 expression in rat brains. The present study provides novel evidence that whole-brain irradiation differentially affects mRNA and protein expression of VEGF, Ang-1, Tie-2 and Ang-2. These changes are closely associated with decreased EC proliferation and increased EC apoptosis in brain.

FIG. 1

Effect of whole-brain irradiation on endothelial cell proliferation in rat brain. Representative images show double immunofluorescence staining for CD31 in green and Ki67 in red (panels A–L). Whole-brain irradiation resulted in a marked decrease in the fluorescence intensity of CD31 labeling of endothelial cells (panels A, D, G and J). Ki67 labeling of proliferating cells was dramatically reduced in irradiated rats (panels B, E, H and K). CD31⁺/Ki67⁺ proliferating endothelial cells (white arrows) were more abundant in sham-irradiated controls compared with irradiated rats (panels C, F, I and L). Quantitative analysis indicated that radiation significantly reduced proliferating endothelial cells in rat brains compared with sham-irradiated controls (panels M and N). Data shown are means ± SEM for each group (n = 4). *Statistically significant difference from control (P < 0.05). Panels A–C: Sham irradiation (Control); panels D–F: 4 h postirradiation; panels G–I: 8 h postirradiation; panels J–L: 24 h postirradiation. Panels A, D, G and J: green staining of CD31-positive immunoreactivity; panels B, E, H and K: red staining of Ki67-positive immunoreactivity; panels C, F, I and L: merged images of the green and red fluorescence staining. Original magnification of the images in panels A–L is 100×.

FIG. 2

Effect of whole-brain irradiation on endothelial cell apoptosis in rat brain. Representative images show double immunofluorescence staining for CD31 in green and cleaved caspase-3 in red (panels A–L). Whole-brain irradiation resulted in a marked decrease in the fluorescence intensity of CD31 labeling of endothelial cells (panels A, D, G and J). Cleaved caspase-3 labeling of apoptotic cells was dramatically increased in irradiated rats (panels B, E, H and K). CD31⁺/cleaved caspase-3⁺ apoptotic endothelial cells (white arrows) were more abundant in irradiated rats than in sham-irradiated controls (panels C, F, I and L). Quantitative analysis indicated that radiation significantly increased endothelial cell apoptosis in rat brains compared with sham-irradiated controls (panels M and N). Data shown are means ± SEM for each group (n = 4). *Statistically significant difference from control (P < 0.05). Panels A–C: sham irradiation (Control); panels D–F: 4 h postirradiation; panels G–I: 8 h postirradiation; panels J–L: 24 h postirradiation. Panels A, D, G and J: green staining of CD31-positive immunoreactivity; panels B, E, H and K: red staining of cleaved caspase-3-positive immunoreactivity; panels C, F, I and L: merged images of the green and red fluorescence staining. Original magnification of the images in panels A–L is 100×.

FIG. 3

Effect of whole-brain irradiation on mRNA expression of VEGF, Ang-1, Tie-2 and Ang-2 in rat brain. Compared with sham-irradiated controls, radiation significantly downregulated mRNA expression levels of VEGF (panel A), Ang-1 (panel B) and Tie-2 (panel C) in rat brains. In contrast, mRNA expression levels of Ang-2 were upregulated by radiation (panel D). Data shown are means ± SEM for each group (n = 4). *Statistically significant difference from control (P < 0.05).

FIG. 4

Effect of whole-brain irradiation on VEGF protein expression in rat brain. Quantitative analysis indicated that radiation significantly reduced protein expression levels of VEGF in rat brains compared with sham-irradiated controls. Data shown are means ± SEM for each group (n = 4). *Statistically significant difference from control (P < 0.05).

FIG. 5

Effect of whole-brain irradiation on Ang-1 protein expression in rat brain. Quantitative analysis indicated that radiation significantly reduced protein expression levels of Ang-1 in rat brains compared with sham-irradiated controls. Data shown are means ± SEM for each group (n = 4). *Statistically significant difference from control (P < 0.05).

FIG. 6

Effect of whole-brain irradiation on Tie-2 protein expression in rat brain. Quantitative analysis indicated that radiation significantly reduced protein expression levels of Tie-2 in rat brains compared with sham-irradiated controls. Data shown are means ± SEM for each group (n = 4). *Statistically significant difference from control (P < 0.05).

FIG. 7

Effect of whole-brain irradiation on Ang-2 protein expression in rat brain. Quantitative analysis indicated that radiation significantly increased protein expression levels of Ang-2 in rat brains compared with sham-irradiated controls. Data shown are means ± SEM for each group (n = 4). *Statistically significant difference from control (P < 0.05).