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Endogenous VEGF is required for visual function: evidence for a survival role on müller cells and photoreceptors.

Saint-Geniez M, Maharaj AS, Walshe TE, Tucker BA, Sekiyama E, Kurihara T, Darland DC, Young MJ, D'Amore PA - PLoS ONE (2008)

Bottom Line: After 14 days of VEGF neutralization, there was no effect on the inner and outer retina vasculature, but a significant increase in apoptosis of cells in the inner and outer nuclear layers.By four weeks, the increase in neural cell death was associated with reduced thickness of the inner and outer nuclear layers and a decline in retinal function as measured by electroretinograms. siRNA-based suppression of VEGF expression in a Müller cell line in vitro supports the existence of an autocrine role for VEGF in Müller cell survival.These results indicate an important role for endogenous VEGF in the maintenance and function of adult retina neuronal cells and indicate that anti-VEGF therapies should be administered with caution.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Background: Vascular endothelial growth factor (VEGF) is well known for its role in normal and pathologic neovascularization. However, a growing body of evidence indicates that VEGF also acts on non-vascular cells, both developmentally as well as in the adult. In light of the widespread use of systemic and intraocular anti-VEGF therapies for the treatment of angiogenesis associated with tumor growth and wet macular degeneration, systematic investigation of the role of VEGF in the adult retina is critical.

Methods and findings: Using immunohistochemistry and Lac-Z reporter mouse lines, we report that VEGF is produced by various cells in the adult mouse retina and that VEGFR2, the primary signaling receptor, is also widely expressed, with strong expression by Müller cells and photoreceptors. Systemic neutralization of VEGF was accomplished in mice by adenoviral expression of sFlt1. After 14 days of VEGF neutralization, there was no effect on the inner and outer retina vasculature, but a significant increase in apoptosis of cells in the inner and outer nuclear layers. By four weeks, the increase in neural cell death was associated with reduced thickness of the inner and outer nuclear layers and a decline in retinal function as measured by electroretinograms. siRNA-based suppression of VEGF expression in a Müller cell line in vitro supports the existence of an autocrine role for VEGF in Müller cell survival. Similarly, the addition of exogenous VEGF to freshly isolated photoreceptor cells and outer-nuclear-layer explants demonstrated VEGF to be highly neuroprotective.

Conclusions: These results indicate an important role for endogenous VEGF in the maintenance and function of adult retina neuronal cells and indicate that anti-VEGF therapies should be administered with caution.

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Related in: MedlinePlus

VEGF neutralization does not affect retinal vascular perfusion or permeability.Mice infected with Ad- or Ad-sFlt1 for 14 days were perfused with h.m.w fluorescein dextran (green), then eyes were dissected and (A) flat mounted or (B) sectioned and immunostained for collagen IV, and visualized with a Cy3-conjugated secondary antibody (red). (A) Flat mounts of retinas from mice expressing sFlt1 perfused with fluorescein dextran shows no gross abnormalities or perfusion defects, and no changes in capillary density in the inner retina compared to the control mice expressing Ad- (n = 6 mice/condition). (B) Immunofluorescent localization of collagen IV in sections of fluorescein-perfused retinas revealed nearly complete overlay of FITC-dextran and collagen IV of the inner retinal vessels in both Ad- and Ad-sFlt1 (arrows, third column). (C) Quantification of fluorescein- and collagen IV-positive vessels of the innermost retinal vasculature in Ad-sFlt1 (n = 5) compared to Ad- (n = 4) showed no reduction in the number of perfused vessels, indicating the absence of vascular damage. (D) Fluorescein angiography of experimental mice seven days after infection showing no opacity in the vitreous 4-5 min after injection in either group, indicating no change in retinal vascular permeability (n = 6 mice/condition). Scale bar is 1 mm in A and 100 µm in B.
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pone-0003554-g002: VEGF neutralization does not affect retinal vascular perfusion or permeability.Mice infected with Ad- or Ad-sFlt1 for 14 days were perfused with h.m.w fluorescein dextran (green), then eyes were dissected and (A) flat mounted or (B) sectioned and immunostained for collagen IV, and visualized with a Cy3-conjugated secondary antibody (red). (A) Flat mounts of retinas from mice expressing sFlt1 perfused with fluorescein dextran shows no gross abnormalities or perfusion defects, and no changes in capillary density in the inner retina compared to the control mice expressing Ad- (n = 6 mice/condition). (B) Immunofluorescent localization of collagen IV in sections of fluorescein-perfused retinas revealed nearly complete overlay of FITC-dextran and collagen IV of the inner retinal vessels in both Ad- and Ad-sFlt1 (arrows, third column). (C) Quantification of fluorescein- and collagen IV-positive vessels of the innermost retinal vasculature in Ad-sFlt1 (n = 5) compared to Ad- (n = 4) showed no reduction in the number of perfused vessels, indicating the absence of vascular damage. (D) Fluorescein angiography of experimental mice seven days after infection showing no opacity in the vitreous 4-5 min after injection in either group, indicating no change in retinal vascular permeability (n = 6 mice/condition). Scale bar is 1 mm in A and 100 µm in B.

Mentions: Conceived and designed the experiments: MSG. Performed the experiments: MSG. Analyzed the data: MSG. Wrote the paper: MSG. Oversaw all aspects of the work, collaborated on study design, interpretation of results and manuscript preparation: PAD. Responsible for all aspects of the study: MSG. Contributed materially to all figures except Figure 7 and Supplemental Figures 4 and 5: MSG. Analyzed all the data, assembled the figures and wrote the first draft of the manuscript: MSG. Developed and characterized the sFlt model and assisted in characterization of the vascular phenotype (Figures 2–5): ASRM. Collaborated with mouse work and participated in the vascular analysis (Figures 2–5): TEW. Developed the method for isolation of photoreceptor sheets and is responsible for Figure 7 and Supplemental Figures 4 and 5: BAT. Carried out the analysis of retinal function via electroretinograms (Figure 5): ES. Participated in immunohistochemistry, tissue culture and RT-PCR: TK. Conducted studies on VEGF expression in the inner retina and contributed to Figure 1: DCD. Oversaw the development and characterization of photoreceptor sheets: MJY. Contributed to Figure 7 and Supplemental Figures 4 and 5: MJY.


Endogenous VEGF is required for visual function: evidence for a survival role on müller cells and photoreceptors.

Saint-Geniez M, Maharaj AS, Walshe TE, Tucker BA, Sekiyama E, Kurihara T, Darland DC, Young MJ, D'Amore PA - PLoS ONE (2008)

VEGF neutralization does not affect retinal vascular perfusion or permeability.Mice infected with Ad- or Ad-sFlt1 for 14 days were perfused with h.m.w fluorescein dextran (green), then eyes were dissected and (A) flat mounted or (B) sectioned and immunostained for collagen IV, and visualized with a Cy3-conjugated secondary antibody (red). (A) Flat mounts of retinas from mice expressing sFlt1 perfused with fluorescein dextran shows no gross abnormalities or perfusion defects, and no changes in capillary density in the inner retina compared to the control mice expressing Ad- (n = 6 mice/condition). (B) Immunofluorescent localization of collagen IV in sections of fluorescein-perfused retinas revealed nearly complete overlay of FITC-dextran and collagen IV of the inner retinal vessels in both Ad- and Ad-sFlt1 (arrows, third column). (C) Quantification of fluorescein- and collagen IV-positive vessels of the innermost retinal vasculature in Ad-sFlt1 (n = 5) compared to Ad- (n = 4) showed no reduction in the number of perfused vessels, indicating the absence of vascular damage. (D) Fluorescein angiography of experimental mice seven days after infection showing no opacity in the vitreous 4-5 min after injection in either group, indicating no change in retinal vascular permeability (n = 6 mice/condition). Scale bar is 1 mm in A and 100 µm in B.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2571983&req=5

pone-0003554-g002: VEGF neutralization does not affect retinal vascular perfusion or permeability.Mice infected with Ad- or Ad-sFlt1 for 14 days were perfused with h.m.w fluorescein dextran (green), then eyes were dissected and (A) flat mounted or (B) sectioned and immunostained for collagen IV, and visualized with a Cy3-conjugated secondary antibody (red). (A) Flat mounts of retinas from mice expressing sFlt1 perfused with fluorescein dextran shows no gross abnormalities or perfusion defects, and no changes in capillary density in the inner retina compared to the control mice expressing Ad- (n = 6 mice/condition). (B) Immunofluorescent localization of collagen IV in sections of fluorescein-perfused retinas revealed nearly complete overlay of FITC-dextran and collagen IV of the inner retinal vessels in both Ad- and Ad-sFlt1 (arrows, third column). (C) Quantification of fluorescein- and collagen IV-positive vessels of the innermost retinal vasculature in Ad-sFlt1 (n = 5) compared to Ad- (n = 4) showed no reduction in the number of perfused vessels, indicating the absence of vascular damage. (D) Fluorescein angiography of experimental mice seven days after infection showing no opacity in the vitreous 4-5 min after injection in either group, indicating no change in retinal vascular permeability (n = 6 mice/condition). Scale bar is 1 mm in A and 100 µm in B.
Mentions: Conceived and designed the experiments: MSG. Performed the experiments: MSG. Analyzed the data: MSG. Wrote the paper: MSG. Oversaw all aspects of the work, collaborated on study design, interpretation of results and manuscript preparation: PAD. Responsible for all aspects of the study: MSG. Contributed materially to all figures except Figure 7 and Supplemental Figures 4 and 5: MSG. Analyzed all the data, assembled the figures and wrote the first draft of the manuscript: MSG. Developed and characterized the sFlt model and assisted in characterization of the vascular phenotype (Figures 2–5): ASRM. Collaborated with mouse work and participated in the vascular analysis (Figures 2–5): TEW. Developed the method for isolation of photoreceptor sheets and is responsible for Figure 7 and Supplemental Figures 4 and 5: BAT. Carried out the analysis of retinal function via electroretinograms (Figure 5): ES. Participated in immunohistochemistry, tissue culture and RT-PCR: TK. Conducted studies on VEGF expression in the inner retina and contributed to Figure 1: DCD. Oversaw the development and characterization of photoreceptor sheets: MJY. Contributed to Figure 7 and Supplemental Figures 4 and 5: MJY.

Bottom Line: After 14 days of VEGF neutralization, there was no effect on the inner and outer retina vasculature, but a significant increase in apoptosis of cells in the inner and outer nuclear layers.By four weeks, the increase in neural cell death was associated with reduced thickness of the inner and outer nuclear layers and a decline in retinal function as measured by electroretinograms. siRNA-based suppression of VEGF expression in a Müller cell line in vitro supports the existence of an autocrine role for VEGF in Müller cell survival.These results indicate an important role for endogenous VEGF in the maintenance and function of adult retina neuronal cells and indicate that anti-VEGF therapies should be administered with caution.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Background: Vascular endothelial growth factor (VEGF) is well known for its role in normal and pathologic neovascularization. However, a growing body of evidence indicates that VEGF also acts on non-vascular cells, both developmentally as well as in the adult. In light of the widespread use of systemic and intraocular anti-VEGF therapies for the treatment of angiogenesis associated with tumor growth and wet macular degeneration, systematic investigation of the role of VEGF in the adult retina is critical.

Methods and findings: Using immunohistochemistry and Lac-Z reporter mouse lines, we report that VEGF is produced by various cells in the adult mouse retina and that VEGFR2, the primary signaling receptor, is also widely expressed, with strong expression by Müller cells and photoreceptors. Systemic neutralization of VEGF was accomplished in mice by adenoviral expression of sFlt1. After 14 days of VEGF neutralization, there was no effect on the inner and outer retina vasculature, but a significant increase in apoptosis of cells in the inner and outer nuclear layers. By four weeks, the increase in neural cell death was associated with reduced thickness of the inner and outer nuclear layers and a decline in retinal function as measured by electroretinograms. siRNA-based suppression of VEGF expression in a Müller cell line in vitro supports the existence of an autocrine role for VEGF in Müller cell survival. Similarly, the addition of exogenous VEGF to freshly isolated photoreceptor cells and outer-nuclear-layer explants demonstrated VEGF to be highly neuroprotective.

Conclusions: These results indicate an important role for endogenous VEGF in the maintenance and function of adult retina neuronal cells and indicate that anti-VEGF therapies should be administered with caution.

Show MeSH
Related in: MedlinePlus