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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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Inhibition of the autocrine VEGF signaling in Müller cells leads to increased cell death.VEGF expression by MIO-M1 Müller cells was inhibited by transfection of siRNA into sub-confluent cells. (A–B) Seventy-two hr following transfection of the control and VEGF siRNA, VEGF mRNA levels were determined by qPCR (A) and VEGF protein secretion was quantified by ELISA (B). siVEGF transfection led to a 75% inhibition of VEGF mRNA and more than 90% reduction of VEGF protein (n = 3). (C) Inhibition of VEGF was associated with an increase in TUNEL-positive cells after three days of culture in serum-free conditions (n = 4). (D) Quantification of cell death by FACS analysis demonstrated a doubling of the number of annexin-V positive apoptotic cells in siVEGF transfected cells compared to siControl (n = 3). (E) Increased apoptosis was accompanied by a significant up-regulation of the pro-apoptotic gene, bax, and by the increase of the Bax/Bcl-2 ratio (F). Scale bar is 100 µm.
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pone-0003554-g006: Inhibition of the autocrine VEGF signaling in Müller cells leads to increased cell death.VEGF expression by MIO-M1 Müller cells was inhibited by transfection of siRNA into sub-confluent cells. (A–B) Seventy-two hr following transfection of the control and VEGF siRNA, VEGF mRNA levels were determined by qPCR (A) and VEGF protein secretion was quantified by ELISA (B). siVEGF transfection led to a 75% inhibition of VEGF mRNA and more than 90% reduction of VEGF protein (n = 3). (C) Inhibition of VEGF was associated with an increase in TUNEL-positive cells after three days of culture in serum-free conditions (n = 4). (D) Quantification of cell death by FACS analysis demonstrated a doubling of the number of annexin-V positive apoptotic cells in siVEGF transfected cells compared to siControl (n = 3). (E) Increased apoptosis was accompanied by a significant up-regulation of the pro-apoptotic gene, bax, and by the increase of the Bax/Bcl-2 ratio (F). 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)

Inhibition of the autocrine VEGF signaling in Müller cells leads to increased cell death.VEGF expression by MIO-M1 Müller cells was inhibited by transfection of siRNA into sub-confluent cells. (A–B) Seventy-two hr following transfection of the control and VEGF siRNA, VEGF mRNA levels were determined by qPCR (A) and VEGF protein secretion was quantified by ELISA (B). siVEGF transfection led to a 75% inhibition of VEGF mRNA and more than 90% reduction of VEGF protein (n = 3). (C) Inhibition of VEGF was associated with an increase in TUNEL-positive cells after three days of culture in serum-free conditions (n = 4). (D) Quantification of cell death by FACS analysis demonstrated a doubling of the number of annexin-V positive apoptotic cells in siVEGF transfected cells compared to siControl (n = 3). (E) Increased apoptosis was accompanied by a significant up-regulation of the pro-apoptotic gene, bax, and by the increase of the Bax/Bcl-2 ratio (F). Scale bar is 100 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2571983&req=5

pone-0003554-g006: Inhibition of the autocrine VEGF signaling in Müller cells leads to increased cell death.VEGF expression by MIO-M1 Müller cells was inhibited by transfection of siRNA into sub-confluent cells. (A–B) Seventy-two hr following transfection of the control and VEGF siRNA, VEGF mRNA levels were determined by qPCR (A) and VEGF protein secretion was quantified by ELISA (B). siVEGF transfection led to a 75% inhibition of VEGF mRNA and more than 90% reduction of VEGF protein (n = 3). (C) Inhibition of VEGF was associated with an increase in TUNEL-positive cells after three days of culture in serum-free conditions (n = 4). (D) Quantification of cell death by FACS analysis demonstrated a doubling of the number of annexin-V positive apoptotic cells in siVEGF transfected cells compared to siControl (n = 3). (E) Increased apoptosis was accompanied by a significant up-regulation of the pro-apoptotic gene, bax, and by the increase of the Bax/Bcl-2 ratio (F). 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