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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

Decreased retina thickness and loss of visual function in sFlt1 expressing mice.(A) Semi-thin epon sections from retinas of experimental mice expressing sFlt1 for 28 days showed substantial thinning of the INL (black double arrowheads line) and the ONL (white double arrowheads line) (n = 3 for Ad- and n = 4 for Ad-sFlt1). (B–D) TEM of retinas from sFlt1 expressing mice at 28 days. (B–C) Micrographs taken in the INL region revealed apoptotic Müller cells (MC) and amacrine cells (A). Both cells displayed condensation of the chromatin, membrane swelling and rupture mitochondria (arrows). (D) In the ONL, a high percentage of photoreceptors appeared apoptotic (white asterisk). Cell death left numerous empty spaces containing membranous debris (arrowhead). Processes from the Müller cells that normally fill the intercellular space appeared shrunken (MP). (E) Scotopic ERG recordings of experimental mice 28 days post-infection using a flash intensity of +10 dB revealed a marked reduction of both a- and b-wave amplitudes in sFlt1-expressing mice (n = 6 for Ad- and n = 10 for Ad-sFlt1). GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer, OPL: outer plexiform layer, Ch: choroid. Scale bars are 100 µm in A and 5 µm in B to D.
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pone-0003554-g005: Decreased retina thickness and loss of visual function in sFlt1 expressing mice.(A) Semi-thin epon sections from retinas of experimental mice expressing sFlt1 for 28 days showed substantial thinning of the INL (black double arrowheads line) and the ONL (white double arrowheads line) (n = 3 for Ad- and n = 4 for Ad-sFlt1). (B–D) TEM of retinas from sFlt1 expressing mice at 28 days. (B–C) Micrographs taken in the INL region revealed apoptotic Müller cells (MC) and amacrine cells (A). Both cells displayed condensation of the chromatin, membrane swelling and rupture mitochondria (arrows). (D) In the ONL, a high percentage of photoreceptors appeared apoptotic (white asterisk). Cell death left numerous empty spaces containing membranous debris (arrowhead). Processes from the Müller cells that normally fill the intercellular space appeared shrunken (MP). (E) Scotopic ERG recordings of experimental mice 28 days post-infection using a flash intensity of +10 dB revealed a marked reduction of both a- and b-wave amplitudes in sFlt1-expressing mice (n = 6 for Ad- and n = 10 for Ad-sFlt1). GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer, OPL: outer plexiform layer, Ch: choroid. Scale bars are 100 µm in A and 5 µm in B to D.

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)

Decreased retina thickness and loss of visual function in sFlt1 expressing mice.(A) Semi-thin epon sections from retinas of experimental mice expressing sFlt1 for 28 days showed substantial thinning of the INL (black double arrowheads line) and the ONL (white double arrowheads line) (n = 3 for Ad- and n = 4 for Ad-sFlt1). (B–D) TEM of retinas from sFlt1 expressing mice at 28 days. (B–C) Micrographs taken in the INL region revealed apoptotic Müller cells (MC) and amacrine cells (A). Both cells displayed condensation of the chromatin, membrane swelling and rupture mitochondria (arrows). (D) In the ONL, a high percentage of photoreceptors appeared apoptotic (white asterisk). Cell death left numerous empty spaces containing membranous debris (arrowhead). Processes from the Müller cells that normally fill the intercellular space appeared shrunken (MP). (E) Scotopic ERG recordings of experimental mice 28 days post-infection using a flash intensity of +10 dB revealed a marked reduction of both a- and b-wave amplitudes in sFlt1-expressing mice (n = 6 for Ad- and n = 10 for Ad-sFlt1). GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer, OPL: outer plexiform layer, Ch: choroid. Scale bars are 100 µm in A and 5 µm in B to D.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2571983&req=5

pone-0003554-g005: Decreased retina thickness and loss of visual function in sFlt1 expressing mice.(A) Semi-thin epon sections from retinas of experimental mice expressing sFlt1 for 28 days showed substantial thinning of the INL (black double arrowheads line) and the ONL (white double arrowheads line) (n = 3 for Ad- and n = 4 for Ad-sFlt1). (B–D) TEM of retinas from sFlt1 expressing mice at 28 days. (B–C) Micrographs taken in the INL region revealed apoptotic Müller cells (MC) and amacrine cells (A). Both cells displayed condensation of the chromatin, membrane swelling and rupture mitochondria (arrows). (D) In the ONL, a high percentage of photoreceptors appeared apoptotic (white asterisk). Cell death left numerous empty spaces containing membranous debris (arrowhead). Processes from the Müller cells that normally fill the intercellular space appeared shrunken (MP). (E) Scotopic ERG recordings of experimental mice 28 days post-infection using a flash intensity of +10 dB revealed a marked reduction of both a- and b-wave amplitudes in sFlt1-expressing mice (n = 6 for Ad- and n = 10 for Ad-sFlt1). GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer, OPL: outer plexiform layer, Ch: choroid. Scale bars are 100 µm in A and 5 µm in B to D.
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