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Anti-vascular endothelial growth factor acts on retinal microglia/macrophage activation in a rat model of ocular inflammation.

Couturier A, Bousquet E, Zhao M, Naud MC, Klein C, Jonet L, Tadayoni R, de Kozak Y, Behar-Cohen F - Mol. Vis. (2014)

Bottom Line: To dissociate the effect of anti-VEGF on microglia and macrophages subsequent to its antiangiogenic effect, we chose a model of acute intraocular inflammation.Neutralizing rat anti-VEGF antibodies significantly decreased ocular VEGF levels but did not decrease the endotoxin-induced uveitis (EIU) clinical score or the number of infiltrating cells and cytokines in ocular media (interleukin [IL]-1β, IL-6, tumor necrosis factor [TNF]-α, and monocyte chemoattractant protein [MCP]-1).Eyes treated with anti-VEGF showed a significantly decreased number of activated microglia and macrophages in the retina and the choroid and decreased iNOS-positive microglia.

View Article: PubMed Central - PubMed

Affiliation: Inserm, U1138, Team 17, Physiopathology of ocular diseases : Threrapeutic innovations, Université René Descartes Sorbonne Paris Cité, Centre de Recherche des Cordeliers, Paris, France.

ABSTRACT

Purpose: To evaluate whether anti-vascular endothelial growth factor (VEGF) neutralizing antibodies injected in the vitreous of rat eyes influence retinal microglia and macrophage activation. To dissociate the effect of anti-VEGF on microglia and macrophages subsequent to its antiangiogenic effect, we chose a model of acute intraocular inflammation.

Methods: Lewis rats were challenged with systemic lipopolysaccharide (LPS) injection and concomitantly received 5 µl of rat anti-VEGF-neutralizing antibody (1.5 mg/ml) in the vitreous. Rat immunoglobulin G (IgG) isotype was used as the control. The effect of anti-VEGF was evaluated at 24 and 48 h clinically (uveitis scores), biologically (cytokine multiplex analysis in ocular media), and histologically (inflammatory cell counts on eye sections). Microglia and macrophages were immunodetected with ionized calcium-binding adaptor molecule 1 (IBA1) staining and counted based on their differential shapes (round amoeboid or ramified dendritiform) on sections and flatmounted retinas using confocal imaging and automatic quantification. Activation of microglia was also evaluated with inducible nitric oxide synthase (iNOS) and IBA1 coimmunostaining. Coimmunolocalization of VEGF receptor 1 and 2 (VEGF-R1 and R2) with IBA1 was performed on eye sections with or without anti-VEGF treatment.

Results: Neutralizing rat anti-VEGF antibodies significantly decreased ocular VEGF levels but did not decrease the endotoxin-induced uveitis (EIU) clinical score or the number of infiltrating cells and cytokines in ocular media (interleukin [IL]-1β, IL-6, tumor necrosis factor [TNF]-α, and monocyte chemoattractant protein [MCP]-1). Eyes treated with anti-VEGF showed a significantly decreased number of activated microglia and macrophages in the retina and the choroid and decreased iNOS-positive microglia. IBA1-positive cells expressed VEGF-R1 and R2 in the inflamed retina.

Conclusions: Microglia and macrophages expressed VEGF receptors, and intravitreous anti-VEGF influenced the microglia and macrophage activation state. Taking into account that anti-VEGF drugs are repeatedly injected in the vitreous of patients with retinal diseases, part of their effects could result from unsuspected modulation of the microglia activation state. This should be further studied in other ocular pathogenic conditions and human pathology.

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Related in: MedlinePlus

Ionized calcium-binding adaptor molecule 1 immunostaining on flatmounted retinas. A–B: Example of flatmounted retinas from rat eyes with endotoxin-induced uveitis (EIU) injected with vehicle (A) or with antivascular endothelial growth factor (VEGF) antibodies (B); Bar = 50 µm. Arrowhead show round reactive ionized calcium-binding adaptor molecule 1 (IBA1)-positive cells. C–D: Extraction of cell contours used for automatic labeling of IBA1 staining. E-F: Quantification of round and total IBA-1 positive cells on flat-mounted retina from EIU control and anti-VEGF treated eyes. Bar = 50 µm.
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f3: Ionized calcium-binding adaptor molecule 1 immunostaining on flatmounted retinas. A–B: Example of flatmounted retinas from rat eyes with endotoxin-induced uveitis (EIU) injected with vehicle (A) or with antivascular endothelial growth factor (VEGF) antibodies (B); Bar = 50 µm. Arrowhead show round reactive ionized calcium-binding adaptor molecule 1 (IBA1)-positive cells. C–D: Extraction of cell contours used for automatic labeling of IBA1 staining. E-F: Quantification of round and total IBA-1 positive cells on flat-mounted retina from EIU control and anti-VEGF treated eyes. Bar = 50 µm.

Mentions: On the flatmounted retinas, numerous round amoeboid microglia were observed in the vehicle-injected EIU rats (Figure 3A–C, arrows), while more ramified cells were observed after anti-VEGF treatment (Figure 3B-D). To count the total number of microglia in the inner neuroretina and the relative number of round and ramified IBA1-positive cells, the surface of the fluorescently stained IBA1-positive cells was analyzed automatically after the different microglia shapes were captured on the flatmounted retina as illustrated in Figure 3C,D. This automated quantification showed that although no significant difference was observed in the total surface of IBA1 staining between the anti-VEGF and vehicle-treated eyes (n = 10 retinas per group, p>0.05), the area of fluorescence resulting from round IBA1 immunolabeled cells was significantly decreased after anti-VEGF treatment compared to the vehicle-treated eyes (n = 10 retinas per group, p<0.05; Figure 3E,F). NOS2 is expressed in activated macrophages and microglia and is a marker of the activation state [13,38]. As shown in Figure 4, double-stained IBA1/NOS2 positive round cells were identified in the retinas of eyes with EIU treated with vehicle (Figure 4A–C), but none were found in the anti-VEGF-treated eyes (Figure 4D-F). NOS2 expression was also decreased at the mRNA levels in the anti-VEGF-treated retinas compared to the vehicle-treated retinas (Figure 4G). TNF-α expression by microglia is also a marker of its activation, but TNF-α expression was not significantly reduced in the whole neuroretina of eyes treated with anti-VEGF compared to controls even if a trend was observed (data not shown).


Anti-vascular endothelial growth factor acts on retinal microglia/macrophage activation in a rat model of ocular inflammation.

Couturier A, Bousquet E, Zhao M, Naud MC, Klein C, Jonet L, Tadayoni R, de Kozak Y, Behar-Cohen F - Mol. Vis. (2014)

Ionized calcium-binding adaptor molecule 1 immunostaining on flatmounted retinas. A–B: Example of flatmounted retinas from rat eyes with endotoxin-induced uveitis (EIU) injected with vehicle (A) or with antivascular endothelial growth factor (VEGF) antibodies (B); Bar = 50 µm. Arrowhead show round reactive ionized calcium-binding adaptor molecule 1 (IBA1)-positive cells. C–D: Extraction of cell contours used for automatic labeling of IBA1 staining. E-F: Quantification of round and total IBA-1 positive cells on flat-mounted retina from EIU control and anti-VEGF treated eyes. Bar = 50 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4067232&req=5

f3: Ionized calcium-binding adaptor molecule 1 immunostaining on flatmounted retinas. A–B: Example of flatmounted retinas from rat eyes with endotoxin-induced uveitis (EIU) injected with vehicle (A) or with antivascular endothelial growth factor (VEGF) antibodies (B); Bar = 50 µm. Arrowhead show round reactive ionized calcium-binding adaptor molecule 1 (IBA1)-positive cells. C–D: Extraction of cell contours used for automatic labeling of IBA1 staining. E-F: Quantification of round and total IBA-1 positive cells on flat-mounted retina from EIU control and anti-VEGF treated eyes. Bar = 50 µm.
Mentions: On the flatmounted retinas, numerous round amoeboid microglia were observed in the vehicle-injected EIU rats (Figure 3A–C, arrows), while more ramified cells were observed after anti-VEGF treatment (Figure 3B-D). To count the total number of microglia in the inner neuroretina and the relative number of round and ramified IBA1-positive cells, the surface of the fluorescently stained IBA1-positive cells was analyzed automatically after the different microglia shapes were captured on the flatmounted retina as illustrated in Figure 3C,D. This automated quantification showed that although no significant difference was observed in the total surface of IBA1 staining between the anti-VEGF and vehicle-treated eyes (n = 10 retinas per group, p>0.05), the area of fluorescence resulting from round IBA1 immunolabeled cells was significantly decreased after anti-VEGF treatment compared to the vehicle-treated eyes (n = 10 retinas per group, p<0.05; Figure 3E,F). NOS2 is expressed in activated macrophages and microglia and is a marker of the activation state [13,38]. As shown in Figure 4, double-stained IBA1/NOS2 positive round cells were identified in the retinas of eyes with EIU treated with vehicle (Figure 4A–C), but none were found in the anti-VEGF-treated eyes (Figure 4D-F). NOS2 expression was also decreased at the mRNA levels in the anti-VEGF-treated retinas compared to the vehicle-treated retinas (Figure 4G). TNF-α expression by microglia is also a marker of its activation, but TNF-α expression was not significantly reduced in the whole neuroretina of eyes treated with anti-VEGF compared to controls even if a trend was observed (data not shown).

Bottom Line: To dissociate the effect of anti-VEGF on microglia and macrophages subsequent to its antiangiogenic effect, we chose a model of acute intraocular inflammation.Neutralizing rat anti-VEGF antibodies significantly decreased ocular VEGF levels but did not decrease the endotoxin-induced uveitis (EIU) clinical score or the number of infiltrating cells and cytokines in ocular media (interleukin [IL]-1β, IL-6, tumor necrosis factor [TNF]-α, and monocyte chemoattractant protein [MCP]-1).Eyes treated with anti-VEGF showed a significantly decreased number of activated microglia and macrophages in the retina and the choroid and decreased iNOS-positive microglia.

View Article: PubMed Central - PubMed

Affiliation: Inserm, U1138, Team 17, Physiopathology of ocular diseases : Threrapeutic innovations, Université René Descartes Sorbonne Paris Cité, Centre de Recherche des Cordeliers, Paris, France.

ABSTRACT

Purpose: To evaluate whether anti-vascular endothelial growth factor (VEGF) neutralizing antibodies injected in the vitreous of rat eyes influence retinal microglia and macrophage activation. To dissociate the effect of anti-VEGF on microglia and macrophages subsequent to its antiangiogenic effect, we chose a model of acute intraocular inflammation.

Methods: Lewis rats were challenged with systemic lipopolysaccharide (LPS) injection and concomitantly received 5 µl of rat anti-VEGF-neutralizing antibody (1.5 mg/ml) in the vitreous. Rat immunoglobulin G (IgG) isotype was used as the control. The effect of anti-VEGF was evaluated at 24 and 48 h clinically (uveitis scores), biologically (cytokine multiplex analysis in ocular media), and histologically (inflammatory cell counts on eye sections). Microglia and macrophages were immunodetected with ionized calcium-binding adaptor molecule 1 (IBA1) staining and counted based on their differential shapes (round amoeboid or ramified dendritiform) on sections and flatmounted retinas using confocal imaging and automatic quantification. Activation of microglia was also evaluated with inducible nitric oxide synthase (iNOS) and IBA1 coimmunostaining. Coimmunolocalization of VEGF receptor 1 and 2 (VEGF-R1 and R2) with IBA1 was performed on eye sections with or without anti-VEGF treatment.

Results: Neutralizing rat anti-VEGF antibodies significantly decreased ocular VEGF levels but did not decrease the endotoxin-induced uveitis (EIU) clinical score or the number of infiltrating cells and cytokines in ocular media (interleukin [IL]-1β, IL-6, tumor necrosis factor [TNF]-α, and monocyte chemoattractant protein [MCP]-1). Eyes treated with anti-VEGF showed a significantly decreased number of activated microglia and macrophages in the retina and the choroid and decreased iNOS-positive microglia. IBA1-positive cells expressed VEGF-R1 and R2 in the inflamed retina.

Conclusions: Microglia and macrophages expressed VEGF receptors, and intravitreous anti-VEGF influenced the microglia and macrophage activation state. Taking into account that anti-VEGF drugs are repeatedly injected in the vitreous of patients with retinal diseases, part of their effects could result from unsuspected modulation of the microglia activation state. This should be further studied in other ocular pathogenic conditions and human pathology.

Show MeSH
Related in: MedlinePlus