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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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VEGF increases annexin A2 expression through VEGFR2. A: RF/6A cells were pretreated with 10 µM or 100 µM SU10944 for 30 min and then treated with 25 ng/ml of VEGF for 1 h. Annexin A2 expression was detected by real-time PCR and western blot analysis. B: RF/6A cells were pretreated with 2 µg/ml goat IgG or neutralizing anti–VEGFR2 antibody for 1 h and then stimulated with 25 ng/ml of VEGF for 1 h. Total RNA was isolated, and quantitative real-time PCR was performed. C: RF/6A cells were pretreated with anti-VEGF antibody or VEGFR2 for 1 h, and then treated with 25 ng/ml VEGF for1 h. Annexin A2 expression was detected with real-time PCR. Double asterisks (**) represent p<0.01 compared with VEGF-treated and VEGFR2-treated samples.
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f4: VEGF increases annexin A2 expression through VEGFR2. A: RF/6A cells were pretreated with 10 µM or 100 µM SU10944 for 30 min and then treated with 25 ng/ml of VEGF for 1 h. Annexin A2 expression was detected by real-time PCR and western blot analysis. B: RF/6A cells were pretreated with 2 µg/ml goat IgG or neutralizing anti–VEGFR2 antibody for 1 h and then stimulated with 25 ng/ml of VEGF for 1 h. Total RNA was isolated, and quantitative real-time PCR was performed. C: RF/6A cells were pretreated with anti-VEGF antibody or VEGFR2 for 1 h, and then treated with 25 ng/ml VEGF for1 h. Annexin A2 expression was detected with real-time PCR. Double asterisks (**) represent p<0.01 compared with VEGF-treated and VEGFR2-treated samples.

Mentions: The VEGF-induced expression levels of the mRNA and protein of annexin A2 respectively were suppressed by 87% and 75% in RF/6A cells after pretreatment with SU10944 (Figure 4A). VEGF-induced annexin A2 expression after pretreatment of RF/6A cells with 2 µg/ml neutralizing antibody against monkey VEGFR2 was suppressed by 69% (Figure 4B). The results indicate that VEGFR2 plays a major role in VEGF-induced annexin A2 expression, although we cannot exclude a role for VEGFR1. Pretreatment of RF/6A cells with 2 µg/ml antibody against monkey VEGF almost entirely inhibited ischemia-induced annexin A2 expression (Figure 4C). The expression levels of annexin A2 rose markedly in the simultaneous presence of VEGF and VEGFR2 compared with VEGF or VEGFR2 singly (p<0.001).


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)

VEGF increases annexin A2 expression through VEGFR2. A: RF/6A cells were pretreated with 10 µM or 100 µM SU10944 for 30 min and then treated with 25 ng/ml of VEGF for 1 h. Annexin A2 expression was detected by real-time PCR and western blot analysis. B: RF/6A cells were pretreated with 2 µg/ml goat IgG or neutralizing anti–VEGFR2 antibody for 1 h and then stimulated with 25 ng/ml of VEGF for 1 h. Total RNA was isolated, and quantitative real-time PCR was performed. C: RF/6A cells were pretreated with anti-VEGF antibody or VEGFR2 for 1 h, and then treated with 25 ng/ml VEGF for1 h. Annexin A2 expression was detected with real-time PCR. Double asterisks (**) represent p<0.01 compared with VEGF-treated and VEGFR2-treated samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: VEGF increases annexin A2 expression through VEGFR2. A: RF/6A cells were pretreated with 10 µM or 100 µM SU10944 for 30 min and then treated with 25 ng/ml of VEGF for 1 h. Annexin A2 expression was detected by real-time PCR and western blot analysis. B: RF/6A cells were pretreated with 2 µg/ml goat IgG or neutralizing anti–VEGFR2 antibody for 1 h and then stimulated with 25 ng/ml of VEGF for 1 h. Total RNA was isolated, and quantitative real-time PCR was performed. C: RF/6A cells were pretreated with anti-VEGF antibody or VEGFR2 for 1 h, and then treated with 25 ng/ml VEGF for1 h. Annexin A2 expression was detected with real-time PCR. Double asterisks (**) represent p<0.01 compared with VEGF-treated and VEGFR2-treated samples.
Mentions: The VEGF-induced expression levels of the mRNA and protein of annexin A2 respectively were suppressed by 87% and 75% in RF/6A cells after pretreatment with SU10944 (Figure 4A). VEGF-induced annexin A2 expression after pretreatment of RF/6A cells with 2 µg/ml neutralizing antibody against monkey VEGFR2 was suppressed by 69% (Figure 4B). The results indicate that VEGFR2 plays a major role in VEGF-induced annexin A2 expression, although we cannot exclude a role for VEGFR1. Pretreatment of RF/6A cells with 2 µg/ml antibody against monkey VEGF almost entirely inhibited ischemia-induced annexin A2 expression (Figure 4C). The expression levels of annexin A2 rose markedly in the simultaneous presence of VEGF and VEGFR2 compared with VEGF or VEGFR2 singly (p<0.001).

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