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Vascular endothelial growth factor upregulates expression of annexin A2 in vitro and in a mouse model of ischemic retinopathy.

Zhao S, Huang L, Wu J, Zhang Y, Pan D, Liu X - Mol. Vis. (2009)

Bottom Line: Annexin A2 expression and the effect of vascular endothelial growth factor (VEGF) on annexin A2 ex pression in retinal neovascularization were assayed by real-time PCR, western blot analysis.In RF/6A cells, increased expression of annexin A2 was inhibited by the VEGF receptor 2 (VEGFR2) inhibitor SU10944, and by anti-VEGFR2 neutralizing antibody, and was increased by VEGF.Therefore, annexin A2 is an angiogenesis activator and may be a potential target for the development of effective therapeutic strategies for the treatment of retinal neovascularization.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Changhai Hospital, Second Military Medical University, Shanghai, Peoples Republic of China. zhaosh2001@sina.com

ABSTRACT

Purpose: Annexin A2 has been shown to play a role in many neovascularization diseases. We investigated the effect of vascular endothelial growth factor (VEGF) on annexin A2 expression and related intracellular signaling mechanisms in a mouse model of ischemia-induced retinal neovascularization.

Methods: Annexin A2 expression and the effect of vascular endothelial growth factor (VEGF) on annexin A2 ex pression in retinal neovascularization were assayed by real-time PCR, western blot analysis. The effect of Annexin A2 on retinal neovascularization were assayed by siRNA interference and overexpression of Annexin A2, fluorescence imaging, and immunofluorescence histochemistry in a mouse model of ischemia-induced retinal neovascularization.

Results: Expression of annexin A2 mRNA and protein were increased in the mouse model of ischemia-induced retinal neovascularization and in RF/6A cells treated with VEGF. In RF/6A cells, increased expression of annexin A2 was inhibited by the VEGF receptor 2 (VEGFR2) inhibitor SU10944, and by anti-VEGFR2 neutralizing antibody, and was increased by VEGF. Mice with ischemic retinopathy showed increased expression of annexin A2 in vascular endothelial cells, and enhanced retinal neovascularization after intraocular injection of an adenoviral vector containing an annexin A2 expression cassette. Conversely, annexin A2 knockdown suppressed retinal neovascularization in these mice.

Conclusions: These findings suggest that annexin A2 might induce retinal neovascularization through a VEGF-VEGFR2 pathway in ischemia-induced retina neovascularization. Therefore, annexin A2 is an angiogenesis activator and may be a potential target for the development of effective therapeutic strategies for the treatment of retinal neovascularization.

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Annexin A2 expression levels increased in a mouse model of hypoxia-induced retinopathy and in vitro. Relative mRNA levels of Annexin A2 were normalized by GAPDH mRNA levels A: Levels of annexin A2 and VEGF mRNA were measured by quantitative real-time PCR in mouse retinas immediately after hypoxia treatment (P13) and 12 h after return to room air (P14). Double asterisks (**) indicate a p<0.01 compared with controls. Six mice were used for each time point. B: The figures showed western blot results for annexin A2 protein in retinas from the mouse model of hypoxia-induced retinopathy (days P12–P16). C: The figures showed the effects of cytokines factors on the expression of annexin A2 mRNA. RF/6A cells were treated respectively with VEGF, IL1-β, TNF-α, FGF2, or PIGF at 25 ng/ml for 2 h. CN was Annexin A2 expression levels in RF/6A cells untreated with the cytokines. Total RNA was isolated, and quantitative real-time PCR was used to detect the expression of annexin A2 mRNA. Results are representative of three independent experiments, each performed with duplicate samples. Asterisk (*) indicates a p<0.05 compared with untreated control.
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f1: Annexin A2 expression levels increased in a mouse model of hypoxia-induced retinopathy and in vitro. Relative mRNA levels of Annexin A2 were normalized by GAPDH mRNA levels A: Levels of annexin A2 and VEGF mRNA were measured by quantitative real-time PCR in mouse retinas immediately after hypoxia treatment (P13) and 12 h after return to room air (P14). Double asterisks (**) indicate a p<0.01 compared with controls. Six mice were used for each time point. B: The figures showed western blot results for annexin A2 protein in retinas from the mouse model of hypoxia-induced retinopathy (days P12–P16). C: The figures showed the effects of cytokines factors on the expression of annexin A2 mRNA. RF/6A cells were treated respectively with VEGF, IL1-β, TNF-α, FGF2, or PIGF at 25 ng/ml for 2 h. CN was Annexin A2 expression levels in RF/6A cells untreated with the cytokines. Total RNA was isolated, and quantitative real-time PCR was used to detect the expression of annexin A2 mRNA. Results are representative of three independent experiments, each performed with duplicate samples. Asterisk (*) indicates a p<0.05 compared with untreated control.

Mentions: Annexin A2 mRNA expression was investigated in the mouse model of hypoxia-induced retinopathy. As shown in Figure 1A, VEGF mRNA levels were significantly higher in P14 mice than in P13 mice (p<0.01). Annexin A2 mRNA levels were also significantly increased. mRNA levels in P14 mice were approximately four times the levels in P13 mice. Western blot analysis demonstrated that annexin A2 protein levels were elevated in retinas from P14 to P16 compared with P13 (Figure 1B). Several cytokines associated with angiogenesis were detected. Annexin A2 mRNA expression in RF/6A cells was dramatically upregulated by VEGF and modestly upregulated by TNF-α and IL-1β (Figure 1C). FGF2 and PIGF did not significantly affect the expression of annexin A2 mRNA.


Vascular endothelial growth factor upregulates expression of annexin A2 in vitro and in a mouse model of ischemic retinopathy.

Zhao S, Huang L, Wu J, Zhang Y, Pan D, Liu X - Mol. Vis. (2009)

Annexin A2 expression levels increased in a mouse model of hypoxia-induced retinopathy and in vitro. Relative mRNA levels of Annexin A2 were normalized by GAPDH mRNA levels A: Levels of annexin A2 and VEGF mRNA were measured by quantitative real-time PCR in mouse retinas immediately after hypoxia treatment (P13) and 12 h after return to room air (P14). Double asterisks (**) indicate a p<0.01 compared with controls. Six mice were used for each time point. B: The figures showed western blot results for annexin A2 protein in retinas from the mouse model of hypoxia-induced retinopathy (days P12–P16). C: The figures showed the effects of cytokines factors on the expression of annexin A2 mRNA. RF/6A cells were treated respectively with VEGF, IL1-β, TNF-α, FGF2, or PIGF at 25 ng/ml for 2 h. CN was Annexin A2 expression levels in RF/6A cells untreated with the cytokines. Total RNA was isolated, and quantitative real-time PCR was used to detect the expression of annexin A2 mRNA. Results are representative of three independent experiments, each performed with duplicate samples. Asterisk (*) indicates a p<0.05 compared with untreated control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2697492&req=5

f1: Annexin A2 expression levels increased in a mouse model of hypoxia-induced retinopathy and in vitro. Relative mRNA levels of Annexin A2 were normalized by GAPDH mRNA levels A: Levels of annexin A2 and VEGF mRNA were measured by quantitative real-time PCR in mouse retinas immediately after hypoxia treatment (P13) and 12 h after return to room air (P14). Double asterisks (**) indicate a p<0.01 compared with controls. Six mice were used for each time point. B: The figures showed western blot results for annexin A2 protein in retinas from the mouse model of hypoxia-induced retinopathy (days P12–P16). C: The figures showed the effects of cytokines factors on the expression of annexin A2 mRNA. RF/6A cells were treated respectively with VEGF, IL1-β, TNF-α, FGF2, or PIGF at 25 ng/ml for 2 h. CN was Annexin A2 expression levels in RF/6A cells untreated with the cytokines. Total RNA was isolated, and quantitative real-time PCR was used to detect the expression of annexin A2 mRNA. Results are representative of three independent experiments, each performed with duplicate samples. Asterisk (*) indicates a p<0.05 compared with untreated control.
Mentions: Annexin A2 mRNA expression was investigated in the mouse model of hypoxia-induced retinopathy. As shown in Figure 1A, VEGF mRNA levels were significantly higher in P14 mice than in P13 mice (p<0.01). Annexin A2 mRNA levels were also significantly increased. mRNA levels in P14 mice were approximately four times the levels in P13 mice. Western blot analysis demonstrated that annexin A2 protein levels were elevated in retinas from P14 to P16 compared with P13 (Figure 1B). Several cytokines associated with angiogenesis were detected. Annexin A2 mRNA expression in RF/6A cells was dramatically upregulated by VEGF and modestly upregulated by TNF-α and IL-1β (Figure 1C). FGF2 and PIGF did not significantly affect the expression of annexin A2 mRNA.

Bottom Line: Annexin A2 expression and the effect of vascular endothelial growth factor (VEGF) on annexin A2 ex pression in retinal neovascularization were assayed by real-time PCR, western blot analysis.In RF/6A cells, increased expression of annexin A2 was inhibited by the VEGF receptor 2 (VEGFR2) inhibitor SU10944, and by anti-VEGFR2 neutralizing antibody, and was increased by VEGF.Therefore, annexin A2 is an angiogenesis activator and may be a potential target for the development of effective therapeutic strategies for the treatment of retinal neovascularization.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Changhai Hospital, Second Military Medical University, Shanghai, Peoples Republic of China. zhaosh2001@sina.com

ABSTRACT

Purpose: Annexin A2 has been shown to play a role in many neovascularization diseases. We investigated the effect of vascular endothelial growth factor (VEGF) on annexin A2 expression and related intracellular signaling mechanisms in a mouse model of ischemia-induced retinal neovascularization.

Methods: Annexin A2 expression and the effect of vascular endothelial growth factor (VEGF) on annexin A2 ex pression in retinal neovascularization were assayed by real-time PCR, western blot analysis. The effect of Annexin A2 on retinal neovascularization were assayed by siRNA interference and overexpression of Annexin A2, fluorescence imaging, and immunofluorescence histochemistry in a mouse model of ischemia-induced retinal neovascularization.

Results: Expression of annexin A2 mRNA and protein were increased in the mouse model of ischemia-induced retinal neovascularization and in RF/6A cells treated with VEGF. In RF/6A cells, increased expression of annexin A2 was inhibited by the VEGF receptor 2 (VEGFR2) inhibitor SU10944, and by anti-VEGFR2 neutralizing antibody, and was increased by VEGF. Mice with ischemic retinopathy showed increased expression of annexin A2 in vascular endothelial cells, and enhanced retinal neovascularization after intraocular injection of an adenoviral vector containing an annexin A2 expression cassette. Conversely, annexin A2 knockdown suppressed retinal neovascularization in these mice.

Conclusions: These findings suggest that annexin A2 might induce retinal neovascularization through a VEGF-VEGFR2 pathway in ischemia-induced retina neovascularization. Therefore, annexin A2 is an angiogenesis activator and may be a potential target for the development of effective therapeutic strategies for the treatment of retinal neovascularization.

Show MeSH
Related in: MedlinePlus