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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 signaling in the adult retina.(A) Sections of eyes from adult VEGF-lacZ mice stained for LacZ using x-gal (blue) revealed VEGF expression in the GCL (arrowheads) and INL layer (asterisks). (B) Flat-mounted retinas from adult VEGF-lacZ mice stained for lacZ (blue) and NG2 (brown) demonstrated VEGF expression by NG2-positive cells associated with microvessels (arrowhead) and by some astrocytes (arrow) in the GCL. (C) Sections of eyes from adult VEGFR2-lacZ mice stained for lacZ revealed expression of VEGFR2 in the GCL, in the INL (asterisks), and in the photoreceptors where a strong lacZ staining was observed in the inner segments (arrows). (D) Immunohistochemistry for VEGFR2 in sections of adult retina revealed expression in vascular cells (arrowhead), in Müller cells processes, and in the IS of the photoreceptors (arrows). (E) Immunohistochemistry for VEGFR1 revealed a spotty expression in the IPL and OPL. VEGFR1 was also detected in the photoreceptor IS. (F) Staining of Müller cells using CRALBP revealed an expression pattern similar to VEGFR2 (compare with D). (G) Expression of VEGF and its receptors, VEGFR1 and VEGFR2, by Müller cells was confirmed by RT-PCR of RNA from adult rat retina and isolated rat Müller cells. (H) Expression of VEGFR1 and VEGFR2 in photoreceptors (PR) isolated from adult mouse retinas. (I) Immunoprecipitation (IP) of VEGFR2 from pooled adult mouse retinas, followed by immunoblotting for phosphorylated tyrosine revealed VEGFR2 expression (bottom panel) and activation (top panel) in the adult retina. As a control, lysates of porcine aortic endothelial (PAE) cells overexpressing VEGFR2 either untreated or stimulated with VEGF were immunoprecipitated and immunoblotted as described above. GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, IS: inner segment, OS: outer segment. Scale bar is 100 µm.
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pone-0003554-g001: VEGF signaling in the adult retina.(A) Sections of eyes from adult VEGF-lacZ mice stained for LacZ using x-gal (blue) revealed VEGF expression in the GCL (arrowheads) and INL layer (asterisks). (B) Flat-mounted retinas from adult VEGF-lacZ mice stained for lacZ (blue) and NG2 (brown) demonstrated VEGF expression by NG2-positive cells associated with microvessels (arrowhead) and by some astrocytes (arrow) in the GCL. (C) Sections of eyes from adult VEGFR2-lacZ mice stained for lacZ revealed expression of VEGFR2 in the GCL, in the INL (asterisks), and in the photoreceptors where a strong lacZ staining was observed in the inner segments (arrows). (D) Immunohistochemistry for VEGFR2 in sections of adult retina revealed expression in vascular cells (arrowhead), in Müller cells processes, and in the IS of the photoreceptors (arrows). (E) Immunohistochemistry for VEGFR1 revealed a spotty expression in the IPL and OPL. VEGFR1 was also detected in the photoreceptor IS. (F) Staining of Müller cells using CRALBP revealed an expression pattern similar to VEGFR2 (compare with D). (G) Expression of VEGF and its receptors, VEGFR1 and VEGFR2, by Müller cells was confirmed by RT-PCR of RNA from adult rat retina and isolated rat Müller cells. (H) Expression of VEGFR1 and VEGFR2 in photoreceptors (PR) isolated from adult mouse retinas. (I) Immunoprecipitation (IP) of VEGFR2 from pooled adult mouse retinas, followed by immunoblotting for phosphorylated tyrosine revealed VEGFR2 expression (bottom panel) and activation (top panel) in the adult retina. As a control, lysates of porcine aortic endothelial (PAE) cells overexpressing VEGFR2 either untreated or stimulated with VEGF were immunoprecipitated and immunoblotted as described above. GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, IS: inner segment, OS: outer segment. Scale bar is 100 µm.

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 signaling in the adult retina.(A) Sections of eyes from adult VEGF-lacZ mice stained for LacZ using x-gal (blue) revealed VEGF expression in the GCL (arrowheads) and INL layer (asterisks). (B) Flat-mounted retinas from adult VEGF-lacZ mice stained for lacZ (blue) and NG2 (brown) demonstrated VEGF expression by NG2-positive cells associated with microvessels (arrowhead) and by some astrocytes (arrow) in the GCL. (C) Sections of eyes from adult VEGFR2-lacZ mice stained for lacZ revealed expression of VEGFR2 in the GCL, in the INL (asterisks), and in the photoreceptors where a strong lacZ staining was observed in the inner segments (arrows). (D) Immunohistochemistry for VEGFR2 in sections of adult retina revealed expression in vascular cells (arrowhead), in Müller cells processes, and in the IS of the photoreceptors (arrows). (E) Immunohistochemistry for VEGFR1 revealed a spotty expression in the IPL and OPL. VEGFR1 was also detected in the photoreceptor IS. (F) Staining of Müller cells using CRALBP revealed an expression pattern similar to VEGFR2 (compare with D). (G) Expression of VEGF and its receptors, VEGFR1 and VEGFR2, by Müller cells was confirmed by RT-PCR of RNA from adult rat retina and isolated rat Müller cells. (H) Expression of VEGFR1 and VEGFR2 in photoreceptors (PR) isolated from adult mouse retinas. (I) Immunoprecipitation (IP) of VEGFR2 from pooled adult mouse retinas, followed by immunoblotting for phosphorylated tyrosine revealed VEGFR2 expression (bottom panel) and activation (top panel) in the adult retina. As a control, lysates of porcine aortic endothelial (PAE) cells overexpressing VEGFR2 either untreated or stimulated with VEGF were immunoprecipitated and immunoblotted as described above. GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, IS: inner segment, OS: outer segment. Scale bar is 100 µm.
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Related In: Results  -  Collection

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pone-0003554-g001: VEGF signaling in the adult retina.(A) Sections of eyes from adult VEGF-lacZ mice stained for LacZ using x-gal (blue) revealed VEGF expression in the GCL (arrowheads) and INL layer (asterisks). (B) Flat-mounted retinas from adult VEGF-lacZ mice stained for lacZ (blue) and NG2 (brown) demonstrated VEGF expression by NG2-positive cells associated with microvessels (arrowhead) and by some astrocytes (arrow) in the GCL. (C) Sections of eyes from adult VEGFR2-lacZ mice stained for lacZ revealed expression of VEGFR2 in the GCL, in the INL (asterisks), and in the photoreceptors where a strong lacZ staining was observed in the inner segments (arrows). (D) Immunohistochemistry for VEGFR2 in sections of adult retina revealed expression in vascular cells (arrowhead), in Müller cells processes, and in the IS of the photoreceptors (arrows). (E) Immunohistochemistry for VEGFR1 revealed a spotty expression in the IPL and OPL. VEGFR1 was also detected in the photoreceptor IS. (F) Staining of Müller cells using CRALBP revealed an expression pattern similar to VEGFR2 (compare with D). (G) Expression of VEGF and its receptors, VEGFR1 and VEGFR2, by Müller cells was confirmed by RT-PCR of RNA from adult rat retina and isolated rat Müller cells. (H) Expression of VEGFR1 and VEGFR2 in photoreceptors (PR) isolated from adult mouse retinas. (I) Immunoprecipitation (IP) of VEGFR2 from pooled adult mouse retinas, followed by immunoblotting for phosphorylated tyrosine revealed VEGFR2 expression (bottom panel) and activation (top panel) in the adult retina. As a control, lysates of porcine aortic endothelial (PAE) cells overexpressing VEGFR2 either untreated or stimulated with VEGF were immunoprecipitated and immunoblotted as described above. GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, IS: inner segment, OS: outer segment. Scale bar is 100 µm.
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