October 2017
Volume 58, Issue 12
Open Access
Retinal Cell Biology  |   October 2017
Inhibition of Placenta Growth Factor Reduces Subretinal Mononuclear Phagocyte Accumulation in Choroidal Neovascularization
Author Notes
  • 1Department of Ophthalmology, Charité Universitätsmedizin Berlin, Berlin, Germany 
  • Correspondence: Antonia M. Joussen, Department of Ophthalmology, Charité Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany; antonia.joussen@charite.de
  • Nadine Reichhart, Department of Ophthalmology, Charité Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany; nadine.reichhart@charite.de
Investigative Ophthalmology & Visual Science October 2017, Vol.58, 4997-5006. doi:10.1167/iovs.16-21283
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Sergio Crespo-Garcia, Caitlin Corkhill, Christophe Roubeix, Anja-Maria Davids, Norbert Kociok, Olaf Strauss, Antonia M. Joussen, Nadine Reichhart; Inhibition of Placenta Growth Factor Reduces Subretinal Mononuclear Phagocyte Accumulation in Choroidal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2017;58(12):4997-5006. doi: 10.1167/iovs.16-21283.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: The cellular immune response driven by mononuclear phagocytes (MPs) is crucial for choroidal neovascularization (CNV) progression. Case reports show that a switch from pure anti-vascular endothelial growth factor-A (VEGF-A) intravitreal treatment to aflibercept, a drug with combined anti-VEGF-A and anti-placenta growth factor (PlGF) activity, can be beneficial for patients who do not respond to anti-VEGF-A alone. Since MPs harbor VEGFR1, we hypothesize that the interplay of P1GF/vascular endothelial growth factor receptor 1 (VEGFR1) in immune cells plays a pivotal role for CNV.

Methods: CNV was induced with laser, and immune cells and neovascularization were analyzed in vivo and ex vivo. Immunohistochemistry was employed for protein detection. Differential expression of angiogenic factors and macrophage polarization markers were assessed by quantitative PCR (qPCR). One day after laser, intravitreal injection of aflibercept or anti-PlGF was performed.

Results: In the early inflammatory phase after laser, Plgf but not Vegfa was significantly upregulated. VEGF-A upregulation is limited to the scar, whereas PlGF shows a wider distribution. M1 (proinflammatory) macrophage markers were upregulated in the early phase of CNV. However, M2 (proangiogenic) markers showed more inconsistent dynamics. We demonstrated that both aflibercept and anti-PlGF treatments decrease the overall amount of activated subretinal MPs, and especially of those expressing PlGF. These data correlated with a reduction in leakage associated to CNV. Aflibercept showed a stronger reduction in both parameters.

Conclusions: The results hint at an interplay between PlGF/VEGFR1 and MPs that is important in the early phase of CNV. A combined inhibition of VEGF-A and PlGF is superior to a specific anti-PlGF treatment in terms of subretinal MP recruitment.

Wet age-related macular degeneration (AMD) has a high economic and epidemiologic impact in the Western hemisphere, being one of the major causes for vision loss among elderly people. Current AMD treatments are based on local vascular endothelial growth factor A (VEGF-A) blockade.1 Bevacizumab, a recombinant humanized monoclonal antibody, binds all VEGF-A isoforms. Ranibizumab is a humanized antigen-binding fragment with higher affinity for VEGF-A. Aflibercept, in contrast, is a fusion protein consisting of ligand-binding elements vascular endothelial growth factor receptor (VEGFR)-1 and VEGFR2, and works as a decoy receptor binding not only VEGF-A, but also VEGF-B and placenta growth factor (PlGF).2,3 There are no remarkable differences detectable among these agents regarding their clinical efficacy in randomized clinical trial settings.1 Nevertheless, there are case reports about patients who do not respond well to certain anti-VEGF-A therapies or develop tolerance during the treatment course. In some cases, patients benefit from switching from a pure anti-VEGF-A therapy (ranibizumab, bevacizumab) to aflibercept, a therapy that combines anti-VEGF-A with anti-PlGF activity.414 This raises the question whether PlGF plays a specific role in the course of neovascular AMD. 
PlGF has been extensively characterized with regard to its role in pathologic neovascularization, in the setting of tumor growth and wound healing in particular.15,16 Literature concerning its role in retinal pathology is sparser, although it has been reported that mice lacking PlGF show less neovascularization after laser impact.17 Another group demonstrated similar results after pharmacologic blockade of PlGF.18 
Whereas VEGF-A activates both VEGFR1 and VEGFR2, PlGF is a specific agonist for VEGFR1. VEGFR1 blockade led to reduction in subretinal neovascularization experimentally.19 VEGFR1 is expressed by endothelial cells,20 retinal pigment epithelium (RPE),21 and mononuclear phagocytes (MPs) (e.g., microglia, macrophages).20 VEGFR1/PlGF interaction has been described as chemoattractive in regard to macrophage recruitment,22,23 and it is important to remark that MPs play a crucial, although not fully understood, role in the development of choroidal neovascularization (CNV). Extensive study of different signaling pathways, such as CCL-2 and CCR-2, has proven that macrophage recruitment has an impact on CNV progression during experimental neovascular AMD. Deficiency and polymorphisms of CX3CR1, for instance, lead to altered recruitment of subretinal MPs and thus contribute to AMD progression by hampering the physiological phagocytic activity for tissue maintenance and inducing higher cytokine production.2429 Interestingly, when VEGFR1 was blocked, the infiltration of MPs was substantially reduced.30 Thus PlGF-dependent VEGFR1 signaling might be a relevant therapeutic target in retinal inflammation. 
In this study we used laser-induced CNV to examine the influence of PlGF on MP recruitment, based on the assertion that PlGF, as a ligand of VEGFR1, is crucial for neovascularization in AMD. Furthermore, we investigated whether VEGFR1/PlGF activation determines the activation of distinct subsets of MPs involved in subretinal inflammation and neovascularization. Specific inhibition of PlGF was less effective in reducing both MP recruitment and leakage associated with CNV than aflibercept, pointing toward a synergistic role of PlGF and VEGF-A via VEGFR1. 
Materials and Methods
Animal Model
All animal experiments were designed and conducted according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the governmental authorities (LaGeSo, G0103/14, Berlin). 
B6N.Cg-Tg (Csf1r-EGFP)1Hume/J mice are referred to as MacGreen.31 Transgenic MacGreen mice have an enhanced green fluorescent protein (EGFP) reporter under the Csf1r promoter. Thus all myeloid-derived macrophages are GFP positive. C57BL/6J mice (Charles River, Erkrath, Germany) were used as controls. All animals undergoing laser-induced CNV were 10 to 15 weeks old, weighed over 20 g, and tested negative for RD8 mutation.32 
Laser-Induced Choroidal Neovascularization
After application of 2.5% phenylephrine-hydrochloride and 0.5% Tropicamid (Charité Apotheke, Berlin, Germany) for pupil dilatation, animals were anesthetized with intraperitoneal injection of a mix of 1% ketamine hydrochloride (Actavis, Munich, Germany) and 0.1% xylazine (Rompun; Bayer Vital GmbH, Leverkusen, Germany) at a dosage of 0.1 mL/10 mg body weight. A glass coverslip was lubricated with Corneregel (Bausch & Lomb GmbH, Berlin, Germany) and used as a lens before performing four or five well-distributed laser spots surrounding the optic nerve head with an argon laser (120 mW, 100 ms, 50 μm). Only animals that showed the appearance of a heat-induced bubble, indicative for the rupture of the Bruch's membrane, were included in the study. Retinal bleeding after laser was an exclusion criterion. 
Intravitreal Injection
Animals were anesthetized as described above. Intravitreal injection was performed 24 hours after laser treatment. Animals were randomly assigned to receive aflibercept (n = 10; 10 mg/mL; Bayer, Leverkusen, Germany) or rabbit polyclonal antibody against PlGF (n = 12; 5 mg/mL, Ref. no. ab4592; Abcam, Cambridge, UK) dissolved in phosphate-buffered saline (PBS, pH = 7.1). Blocking effect of the employed antibody was evaluated using ELISA (Supplementary Fig. S1). Vehicle controls (n = 5) received PBS only. Fc portion effect was assessed injecting IgG (n = 12; 40 mg/mL; Merck Millipore, Darmstadt, Germany) in another control group. Each eye received 2 μL treatment solution intravitreally, delivered with a 34-gauge needle (World Precision Instruments, Sarasota, FL, USA) attached to a glass syringe (10 μL; Hamilton Bonaduz, Bonaduz, Switzerland). Subsequent fundus evaluation demonstrated no adverse effects from intravitreal injection. Retinal bleeding after injection was an exclusion criterion. 
Scanning Laser Ophthalmoscopy
In vivo imaging using a scanning laser ophthalmoscope (SLO) was performed as described previously by our group.33 Animals with dilated pupils were anesthetized and placed onto a customized platform. Corneas were moisturized with Hylo-Vision (NaCl 0.1%; Omnivision, Puchheim, Germany) to optimize imaging. Spectralis HRA-OCT (Heidelberg Engineering, Heidelberg, Germany) was settled with a c-curve of 3.5 and incorporated a lens of 50°. 
Eyes were examined with the FA mode (diode laser, wavelength of 488 nm) at different time points (before laser and day [D]4, D7, and D14 after laser) distinguishing between inner and outer retina by adjusting the knob at the setup.33 Fundus angiography (FAG) was carried out 5 minutes after subcutaneous injection of fluorescein (5 mg/kg, Fluorescein 10%; Alcon, Freiburg, Germany) and served to evaluate vasculature and leakage. Images were analyzed using Heidelberg Eye Explorer 1.7.0.0 software. Quantification of leakage in FAG images was performed in a masked fashion using ImageJ (National Institutes of Health, Bethesda, MD, USA). 
Analysis of Retinal Whole Mounts
Eyes were enucleated at time points before laser and D4, D7, or D14 after laser, and fixed for 13 minutes in paraformaldehyde 4% at room temperature (RT). A peripheral incision along the limbus line permitted the removal of the cornea and the lens. The posterior eyecup was subjected to four radial cuts allowing its flattening. The neural retina was separated from the sclera by sectioning the optic nerve. Both retinal and scleral samples were permeabilized overnight with tris-buffered saline supplemented with 5% Triton X-100. After repeated washes, samples were blocked using normal goat serum and incubated with the desirable primary antibody overnight at 4°C (Supplementary Table S1). Species-appropriate secondary antibodies were applied for 1 hour at RT. Tissue was mounted onto glass slides and subjected to imaging with a LSM 510 confocal microscope (Zeiss, Jena, Germany). Subretinal mononuclear phagocytes associated to laser scar were counted manually in a masked fashion. CNV volume was detected using IsolectinB4 (IB4), a vascular endothelial marker, and was analyzed by sets of images (z-stack mode) and quantified using ImageJ. 
Immunohistochemistry on Paraffin Sections
Eyes were enucleated, fixed in Davidson fixative, and embedded in paraffin. Sections of 5-μm thickness were deparaffinized and rehydrated. Different unmasking treatments were applied before blocking the samples with BSA 5% (Supplementary Table S1). Primary antibodies (listed in Supplementary Table S1; all antibodies were validated by the supplier) were incubated overnight, followed by multiple washes and incubation with species-appropriate secondary antibodies. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (3 μM; Invitrogen, Carlsbad, CA, USA). Isotype controls were evaluated as shown in Supplementary Figure S2. Imaging was performed using an Axio Imager M2 fluorescence microscope (Zeiss) with ZEN lite 2012 software (Zeiss). 
RNA Isolation and Quantitative RT-PCR (qPCR)
Enucleated eyes, or cell lysates for the in vitro studies, were preserved at −80°C. For RNA isolation, retinas were dissected and placed in lysis buffer. RNA extraction was performed using RNeasy Mini Kit (Qiagen, Hilden, Germany). QuantiTect Reverse Transcription Kit (Qiagen) was used for cDNA synthesis. 
For quantification, samples were run on a Rotor Gene Q (Qiagen) using QuantiNova SYBR Green RT-PCR Kit (Qiagen) for Il4r, Cd206, and Arg1 and QuantiTect SYBR Green PCR Kit (Qiagen) for all other markers tested. All reactions were prepared in triplicate. Gene expression levels were normalized to Gapdh expression. Target gene expression was analyzed using the comparative CT (cycle threshold) method.34 Primer sequences are listed in Supplementary Table S2
Statistical Analyses
Results are presented as mean ± SEM. N numbers represent the number of animals unless indicated differently, and all experiments were repeated at least three times. Experimental data were analyzed using analysis of variance (ANOVA), and P values of different treatments were evaluated using Dunnett's test. When comparing only two groups, statistical analysis was performed using unpaired t-test. Values with statistical significance are indicated as * when P < 0.05, ** when P < 0.01, and *** when P < 0.001. 
Results
Placenta Growth Factor Under Control and Laser CNV Conditions
Vascular endothelial growth factor A was weakly expressed in vessels of the adult nonlasered retina (Fig. 1A), but was present in the RPE. In laser-treated eyes, marked VEGF-A expression was present at the lesion site in the subretinal space (Fig. 1B). Immunostaining of sagittal sections further confirmed VEGF-A expression in the scar (Fig. 1C). 
Figure 1
 
PlGF in the control and CNV retina. (A) Weak VEGF-A (red) expression in a retinal flat mount of a nonlasered animal. Vessels are marked with IB4 (green). Scale bar: 100 μm. (B) Costaining against IB4 (green) and VEGF-A (red) in RPE flat mounts at D0, D4, and D14 after laser. Scale bar: 100 μm. (C) Staining of sagittal sections of the retina against VEGF-A (red). Nuclei were counterstained with DAPI. Scale bar: 50 μm. (D) PlGF (red) expression found in vessels (IB4, green) of a flat mount of a nonlasered animal. Scale bar: 100 μm. (E) Costaining of RPE flat mounts against IB4 (green) and PlGF (red) at D0, D4, and D14 after laser. Scale bar: 100 μm. (F) Staining of sagittal sections of the retina against PlGF (red). Magnification of the scar (inset). Nuclei were counterstained with DAPI. Scale bar: 50 μm. (GJ) Fold change in expression of different genes involved in angiogenesis, normalized to Gapdh and compared to control. N = 6–12.
Figure 1
 
PlGF in the control and CNV retina. (A) Weak VEGF-A (red) expression in a retinal flat mount of a nonlasered animal. Vessels are marked with IB4 (green). Scale bar: 100 μm. (B) Costaining against IB4 (green) and VEGF-A (red) in RPE flat mounts at D0, D4, and D14 after laser. Scale bar: 100 μm. (C) Staining of sagittal sections of the retina against VEGF-A (red). Nuclei were counterstained with DAPI. Scale bar: 50 μm. (D) PlGF (red) expression found in vessels (IB4, green) of a flat mount of a nonlasered animal. Scale bar: 100 μm. (E) Costaining of RPE flat mounts against IB4 (green) and PlGF (red) at D0, D4, and D14 after laser. Scale bar: 100 μm. (F) Staining of sagittal sections of the retina against PlGF (red). Magnification of the scar (inset). Nuclei were counterstained with DAPI. Scale bar: 50 μm. (GJ) Fold change in expression of different genes involved in angiogenesis, normalized to Gapdh and compared to control. N = 6–12.
Placenta growth factor, on the other hand, was expressed in vessels of nonlasered adult healthy retina (Fig. 1D). After laser impact, we found no difference in the expression of PlGF in vessels. PlGF expression was detectable in the subretinal space at the laser scar site at time points corresponding with macrophage recruitment and new vessel growth, analogous to VEGF-A expression (Fig. 1E). Interestingly, PlGF expression in sagittal sections was not limited to the lesion, but was also detectable in peripheral areas corresponding with vascular plexus, far from the laser scar, predominantly in the outer plexiform layer (OPL) (Fig. 1F). 
Expression levels of Plgf and Vegfa together with their corresponding receptors were analyzed across the time course of CNV formation (Figs. 1G–J). No significant upregulation in Vegf expression was observed in the whole retina lysates (Fig. 1G). A significant increase in Plgf expression was observed 1 day after laser (D1) and returned to basal levels by D4 (Fig. 1H). Flt1 showed a similar pattern to sFlt1, with significant upregulation at D10 (Figs. 1I, 1J). Vegfb levels were unaltered after laser (Supplementary Fig. S3). 
Mononuclear Phagocytes in CNV
IBA-1 is a marker for MPs ex vivo (Supplementary Fig. S4A). In the untreated retina, MPs presented a ramified phenotype, while during the early inflammatory phase after laser, they switched to a more amoeboid shape defined as the activated phenotype (Fig. 2A). Furthermore, a subset of MPs that was exclusively detected in the subretinal space of lasered retinas expressed PlGF. This subset of PlGF-positive amoeboid MPs was found not only in the scar site but also in peripheral regions of the subretinal space, attached to the RPE (Fig. 2B). 
Figure 2
 
Mononuclear phagocyte dynamics in CNV. (A) Costaining of retinal flat mounts against Iba-1 (red) to visualize mononuclear phagocytes and IB4 (green) for vessels before laser and at D4 and D7 after laser in MacGreen mouse. Amoeboid-like cells are marked by arrows. (B) MacGreen mouse was lasered and retinas were observed 14 days afterward. Costaining of retinal flat mounts against PlGF (red) and GFP (green). Certain subsets of mononuclear phagocytes in the subretinal space (GFP-positive) also express PlGF. Scale bar represents 50 μm. (CI) Bar charts representing fold change in expression of different genes involved in inflammation, normalized to Gapdh and compared to control, sorted by their polarization into M1. N = 5–12.
Figure 2
 
Mononuclear phagocyte dynamics in CNV. (A) Costaining of retinal flat mounts against Iba-1 (red) to visualize mononuclear phagocytes and IB4 (green) for vessels before laser and at D4 and D7 after laser in MacGreen mouse. Amoeboid-like cells are marked by arrows. (B) MacGreen mouse was lasered and retinas were observed 14 days afterward. Costaining of retinal flat mounts against PlGF (red) and GFP (green). Certain subsets of mononuclear phagocytes in the subretinal space (GFP-positive) also express PlGF. Scale bar represents 50 μm. (CI) Bar charts representing fold change in expression of different genes involved in inflammation, normalized to Gapdh and compared to control, sorted by their polarization into M1. N = 5–12.
Expression of MP markers pointing to M1 polarization (Cd68, Cd86, Il1b, Tnfa) was significantly upregulated in the initial inflammatory phase after laser (D1–D4; Figs. 2C–F). On the other hand, M2 markers (Il4r, Cd206) showed a decrease in expression, being significant at D14 (Figs. 2G, 2H). Arg1, another M2 marker, was significantly upregulated at D4, but decreased at later time points (Fig. 2I). 
Effect of Anti-PlGF and Aflibercept on CNV
Differential gene expression was evaluated comparing the different treatments (PBS, aflibercept, or anti-PlGF antibody) to IgG control. Expression of Vegfa, Plgf, and its receptor, Flt1, was reduced significantly after anti-PlGF antibody or aflibercept injection (Figs. 3A–C). Soluble Vegfr1 (sFlt1) was unaltered among the different treatment groups (Fig. 3D). 
Figure 3
 
Angiogenic effect of anti-PlGF in CNV. (AD) Fold change in expression of different genes involved in angiogenesis at D7 after laser, normalized to Gapdh and compared to IgG and PBS controls. Statistical significance was tested comparing to IgG controls. αPlGF stands for anti-PlGF. N = 6–9. (EG) SLO fundus angiography (FAG) sequential images at distinct time points before and after laser treatment and subsequent intravitreal injection (D4, D7, and D14) of IgG (E) PBS (F), anti-PlGF (G), and aflibercept (AFL) (H), respectively, showing differences in leakage area. (I) Bar chart represents the quantification of the leakage area at D14 after laser in the different treated groups (IgG = 17 scars, PBS = 12 scars, anti-PlGF = 35 scars, AFL = 29 scars). (J) Bar chart represents the quantification of the CNV volume at D14 after laser in the different treated groups (IgG = 5 scars, PBS = 5 scars, anti-PlGF = 11 scars, AFL = 17 scars).
Figure 3
 
Angiogenic effect of anti-PlGF in CNV. (AD) Fold change in expression of different genes involved in angiogenesis at D7 after laser, normalized to Gapdh and compared to IgG and PBS controls. Statistical significance was tested comparing to IgG controls. αPlGF stands for anti-PlGF. N = 6–9. (EG) SLO fundus angiography (FAG) sequential images at distinct time points before and after laser treatment and subsequent intravitreal injection (D4, D7, and D14) of IgG (E) PBS (F), anti-PlGF (G), and aflibercept (AFL) (H), respectively, showing differences in leakage area. (I) Bar chart represents the quantification of the leakage area at D14 after laser in the different treated groups (IgG = 17 scars, PBS = 12 scars, anti-PlGF = 35 scars, AFL = 29 scars). (J) Bar chart represents the quantification of the CNV volume at D14 after laser in the different treated groups (IgG = 5 scars, PBS = 5 scars, anti-PlGF = 11 scars, AFL = 17 scars).
Leakage was evaluated in vivo among the different treatment groups at D7 after laser by FAG (Figs. 3E–H). Animals that underwent aflibercept treatment had a significantly smaller leakage area compared to both controls. Eyes injected with anti-PlGF antibody showed a smaller leakage area than the control groups, but larger than the ones treated with aflibercept. However, no significant differences were found between anti-PlGF antibody and controls (IgG and PBS) or aflibercept, respectively. (Fig. 3I). 
CNV volume was verified in sclera flat mounts stained against IB4 (Fig. 3J). Eyes injected with either aflibercept or anti-PlGF antibody showed a significantly reduced CNV volume with respect to IgG-injected controls. When compared to PBS controls, only aflibercept-injected animals showed a significant decrease of the CNV volume. 
PlGF-Positive Immune Cells
MPs in transgenic MacGreen mice were analyzed in vivo by means of their EGFP fluorescence. Immunohistochemical analysis proved that GFP is coexpressed in IBA-1– and CD11B-positive cells (Supplementary Figs. S4A, S4B). Fundus autofluorescence (FA) images revealed no remarkable differences, at different time points after laser, between the anti-PlGF antibody and aflibercept groups except for the disappearance of the autofluorescence scar in eyes treated with aflibercept (Figs. 4A–D). The IgG- and PBS-injected control groups, however, had more GFP-positive cells in general throughout the time course. 
Figure 4
 
Inflammatory effect of anti-PlGF in CNV. (AC) SLO fundus autofluorescence (FA) sequential images at selected time points before and after laser treatment and subsequent intravitreal injection (D4, D7, and D14) of IgG (A) PBS (B), anti-PlGF (C), and aflibercept (D), showing differences in mononuclear phagocyte behavior after laser. (EH) Costaining against GFP (green) and PlGF (red) in retina flat mounts. Representative images of subsets of subretinal macrophages in the scar site for IgG (E), PBS (F), anti-PlGF (G), and aflibercept (H). Scale bar represents 50 μm. (I) Quantification of double GFP- and PlGF-positive subretinal macrophages ex vivo comparing the different treatment groups: IgG = 18 scars; PBS = 5 scars; anti-PlGF = 13 scars; aflibercept (AFL) = 10 scars. Amoe refers to amoeboid cells whereas Ram stands for ramified ones.
Figure 4
 
Inflammatory effect of anti-PlGF in CNV. (AC) SLO fundus autofluorescence (FA) sequential images at selected time points before and after laser treatment and subsequent intravitreal injection (D4, D7, and D14) of IgG (A) PBS (B), anti-PlGF (C), and aflibercept (D), showing differences in mononuclear phagocyte behavior after laser. (EH) Costaining against GFP (green) and PlGF (red) in retina flat mounts. Representative images of subsets of subretinal macrophages in the scar site for IgG (E), PBS (F), anti-PlGF (G), and aflibercept (H). Scale bar represents 50 μm. (I) Quantification of double GFP- and PlGF-positive subretinal macrophages ex vivo comparing the different treatment groups: IgG = 18 scars; PBS = 5 scars; anti-PlGF = 13 scars; aflibercept (AFL) = 10 scars. Amoe refers to amoeboid cells whereas Ram stands for ramified ones.
Ex vivo quantitative analysis of the subretinal MPs related to the scar site at D14 after laser demonstrated significant differences between the groups in terms of cell numbers and activation status (Figs. 4E–H). Control groups showed the highest total of activated PlGF-positive myeloid cells. Anti-PlGF treatment significantly reduced these numbers. Aflibercept animals displayed the least amount of PlGF-positive immune cells (Fig. 4I). The analysis of the total amount of GFP-positive immune cells showed similar results to the double GFP- and PlGF-positive subset. Furthermore, GFP-positive ramified cells were likely not expressing PlGF (Supplementary Fig. S5). 
We demonstrated in vitro that monocytes isolated from bone marrow express Vegfa, Plgf, and Flt1. After application of lipopolysaccharide (LPS) to mimic inflammation, Vegfa and Plgf expression were upregulated whereas Flt1 was significantly downregulated (Supplementary Methods, Supplementary Fig. S6). 
Regarding M1–M2 polarization of the MPs, gene expression of most of the markers revealed no significant differences among the treated retina groups at D7 after laser (Figs. 5A–G). Regarding M1 polarization, downregulation was detected for Cd68 expression in the aflibercept group (Fig. 5A) and Il1b expression in the anti-PlGF group (Fig. 5C), whereas Tnfa expression was reduced among all groups (with significance only in the PBS group) when compared to IgG injection (Fig. 5D). Only Arg1 expression, a classical indicator for M2, was significantly reduced in both the anti-PlGF– and the aflibercept-treated groups (Fig. 5G). 
Figure 5
 
Mononuclear phagocyte polarization after anti-PlGF treatment. (AG) Bar charts represent fold change in expression, normalized to Gapdh and compared to IgG controls, of different genes involved in M1–M2 polarization. α-PlGF stands for anti-PlGF. N = 5–12.
Figure 5
 
Mononuclear phagocyte polarization after anti-PlGF treatment. (AG) Bar charts represent fold change in expression, normalized to Gapdh and compared to IgG controls, of different genes involved in M1–M2 polarization. α-PlGF stands for anti-PlGF. N = 5–12.
Discussion
This study aimed to improve the understanding of the role and importance of PlGF in neovascular AMD, under the hypothesis that PlGF coordinates MP recruitment. While VEGF-A expression is restricted to the CNV area, PlGF is expressed more generally throughout the retina. Analysis of inflammation associated with CNV revealed gene expression differences for markers of immune cell polarization during CNV. Furthermore, a subpopulation of MPs was PlGF positive and was present only after CNV. Intravitreal injection of anti-PlGF reduced significantly the number of PlGF-positive MPs in the subretinal space, although not as efficiently as aflibercept. The same occurred with leakage area associated with CNV. These data suggest an interplay between VEGFR1/PlGF and MPs that might be crucial for CNV progression. 
VEGF-A expression in the retina has been extensively studied for RPE cells,3538 Müller cells,39 and macrophages.36,40 Literature concerning PlGF is sparser. However, recent works exploring the role of PlGF in the retina emphasize its importance in neovascular AMD. Rakic and colleagues,17 in accordance with our findings, identified PlGF in the CNV lesion and found an upregulation of Plgf in the microdissected scar during the early phase after laser. Their histologic analysis of retinal sagittal sections showed a nonsignificant reduction in lesion size when compared to Plgf/ or wild type (WT) treated systemically with an inhibitor of VEGFR1.17 Concerning the neovascular area, another group found a dosage-dependent effect of systemic PlGF inhibition using a monoclonal antibody. Furthermore, inhibiting the VEGFR1 signaling cascade by using Flt1-TK/ mice also reduced CNV area, a similar effect to that seen with Plgf/ mice or anti-PlGF therapy in WT mice.18 A third study analyzed systemic inhibition of VEGFR1 and VEGFR2 by means of leakage area and gene expression. Plgf expression was upregulated in the early phase after laser, whereas Vegfa levels remained unchanged throughout the experimental time course. These results are in accordance with our data, although they showed a more sustained increase in Plgf expression in general.19 
The scope of the studies mentioned above, however, was solely the angiogenic effect of PlGF. Variation in VEGF-A and PlGF mRNA levels and vascular leakage after CNV, compared to our results, are due to differences in experimental setting: The employed treatment in our study was local (intravitreal injection) instead of systemic (intraperitoneal injection), and a single injection was performed instead of repeated interventions. Furthermore, the laser model used in our work is milder than that in other studies (e.g., reduced laser power and diameter,41,42 fewer laser impacts per eye, analysis of the entire retina versus a scar microdissection17). Laser parameters seem to be critical in terms of VEGF-A upregulation. For instance, compared to us, Rohrer et al.41 reported VEGF-A upregulation of 60-fold 3 days after laser using double spot size. The work of Xu et al.42 showed a 2-fold VEGF-A increase 2 days after laser using higher laser power and bigger spot size. Furthermore, VEGF-A levels were highly fluctuating during the 14-day time course. Thus the selection of our laser parameters was based on a previous work in which we analyzed the immune response associated with CNV in vivo and ex vivo,33 and they were designed to better resemble the inflammatory dynamics of AMD, rather than nonspecific inflammation associated with more severely damaged tissue. 
For a long time, macrophage-derived VEGF-A was thought to be an important factor for inflammation-driven angiogenesis in CNV.2628 Interestingly, using several myeloid-specific VEGF-A knockout models, a recent report proved that myeloid-derived VEGF-A production does not contribute significantly to CNV and vascular leakage.43 Based on this controversy, myeloid-derived PlGF might be a better candidate to take into account for this role.4446 Mononuclear phagocytes in the retina represent a very heterogeneous and dynamic cell type. Different stimuli polarize such cells, altering the gene expression of surface markers and cytokines.47 Differences in the expression profile are used to classify MPs into the so-called M1 or M2 polarized groups, although this classification has been controversially discussed for years since there is no clear evidence of functional implications. Several lines of evidence have highlighted that M1 macrophages are associated more with residential MPs whereas M2 are recruited (e.g., bone marrow-derived).48 There is a consensus that M2, the proangiogenic type of macrophages, support neovascularization in laser-induced CNV.44,49 Among other factors, it has been shown that PlGF upregulation promotes polarization toward M2.45 PlGF-positive subsets of immune cells detected in the subretinal space might be involved in polarization processes since they are observed only after laser. It is important to note that the dynamics of M1–M2 polarization in the early inflammatory phase of CNV have not yet been extensively described. A recent work by Yang and colleagues49 has partly characterized these dynamics by means of qPCR, using a more intensive laser CNV model than ours. M1 macrophages were associated more with the early phase of CNV whereas M2 were, in general, predominant in the later phase. Our findings regarding M1 are very similar, although we found differences in M2 marker dynamics, most likely due to the usage of the much milder laser model (4 laser impacts versus 10 impacts). The lower levels of M2 markers in our scenario might be explained by lower levels of recruited immune cells. 
The blocking capability of the employed anti-PlGF antibody has not been validated functionally, and this has to be taken as a limitation of the study. After treatment with anti-PlGF or aflibercept, gene expression levels of Plgf and Flt1 were reduced compared to controls, and only Vegfa for the case of aflibercept. The treatment effect was validated in terms of leakage inhibition in vivo and CNV volume ex vivo. Injection of anti-PlGF alone was less effective than aflibercept. The lack of significance in some gene expression changes may be explained by a dilution effect of the whole retina and by limiting the readout to a single time point. The exploration of MPs in vivo after treatment did not show apparent differences in activation among the groups, although in anti-PlGF– and aflibercept-treated animals, the autofluorescent scar was decreased and, in many cases, vanished at D14 whereas it persisted in controls. A quantitative analysis of MPs in vivo was handicapped by the extension of autofluorescence signal associated with the laser scar, which prevented detailed visualization of the number of cells in situ. Thus, an analysis of PlGF-positive subsets associated with the scar was feasible only ex vivo. Both treatments significantly decreased the number of these cells at D14 after laser, and aflibercept was more effective than anti-PlGF alone. The impact of the treatment, regarding macrophage polarization markers, gave inconclusive results. Nevertheless, there are interesting hints such as a significant decrease in Il1b and Cd68, both M1 markers, and Arg1, a relevant marker for determining proangiogenic polarization of MPs.49 
Since we observed a superior effect, regarding both the reduction of leakage area as well as the number and activation of MPs, when treating with aflibercept compared to pure anti-PlGF, we hypothesize an interaction of VEGF-A and PlGF via VEGFR1 contributing to CNV progression. This hypothesis is also supported by data from Huo and colleagues,44 reporting an increase in the inhibitory effect of anti-VEGF therapy on vessel density after laser burn when combined with anti-PlGF. A protein interplay between VEGF-A and PlGF would partly explain the reported results of “clinical switching,” when pure anti-VEGF-A therapy is substituted for by aflibercept. Thus, it is appealing to think that the specific anti-PlGF effect might be due to certain preconditions in some patients, making them more susceptible to this therapy. 
In summary, in the experimental setting of laser-induced CNV using our current readout, an anti-PlGF therapy is not sufficient to explain the switch responder cases. Nevertheless, this work presents relevant results indicating that certain subsets of MPs involved in CNV might be connected to PlGF signaling and thus susceptible to specific blockade. 
Acknowledgments
The authors thank Gabriele Fels and Karin Oberländer (Charité, Germany) for their excellent technical support. MacGreen mice were provided by Helmut Kettenmann (Max Dellbrück Center, Germany). Thanks go to Rita Rosenthal (Charité, Germany) for providing access to the confocal microscope. Aflibercept employed in the study was provided by Bayer. 
Supported by a research grant by Bayer and a Marie Curie grant from the European Commission in the framework of the REVAMMAD ITN (Initial Training Research network) project number 316990. CR is supported by an Einstein Foundation Berlin grant. 
Disclosure: S. Crespo-Garcia, None; C. Corkhill, None; C. Roubeix, None; A.-M. Davids, None; N. Kociok, None; O. Strauss, Bayer (C); A.M. Joussen, Bayer (C); N. Reichhart, Bayer (C, F, R) 
References
Ba J, Peng RS, Xu D, et al. Intravitreal anti-VEGF injections for treating wet age-related macular degeneration: a systematic review and meta-analysis. Drug Des Devel Ther. 2015; 9: 5397–5405.
Deissler HL, Lang GK, Lang GE. Capacity of aflibercept to counteract VEGF-stimulated abnormal behavior of retinal microvascular endothelial cells. Exp Eye Res. 2014; 122: 20–31.
Papadopoulos N, Martin J, Ruan Q, et al. Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab. Angiogenesis. 2012; 15: 171–185.
Cho H, Shah CP, Weber M, Heier JS. Aflibercept for exudative AMD with persistent fluid on ranibizumab and/or bevacizumab. Br J Ophthalmol. 2013; 97: 1032–1035.
Fassnacht-Riederle H, Becker M, Graf N, Michels S. Effect of aflibercept in insufficient responders to prior anti-VEGF therapy in neovascular AMD. Graefes Arch Clin Exp Ophthalmol. 2014; 252: 1705–1709.
Fujii A, Imai H, Kanai M, Azumi A. Effect of intravitreal aflibercept injection for age-related macular degeneration with a retinal pigment epithelial tear refractory to intravitreal ranibizumab injection. Clin Ophthalmol. 2014; 8: 1199–1202.
Grewal DS, Gill MK, Sarezky D, Lyon AT, Mirza RG. Visual and anatomical outcomes following intravitreal aflibercept in eyes with recalcitrant neovascular age-related macular degeneration: 12-month results. Eye (Lond). 2014; 28: 895–899.
Ho VY, Yeh S, Olsen TW, et al. Short-term outcomes of aflibercept for neovascular age-related macular degeneration in eyes previously treated with other vascular endothelial growth factor inhibitors. Am J Ophthalmol. 2013; 156: 23–28, e22.
Kumar N, Marsiglia M, Mrejen S, et al. Visual and anatomical outcomes of intravitreal aflibercept in eyes with persistent subfoveal fluid despite previous treatments with ranibizumab in patients with neovascular age-related macular degeneration. Retina. 2013; 33: 1605–1612.
Messenger WB, Campbell JP, Faridi A, et al. Injection frequency and anatomic outcomes 1 year following conversion to aflibercept in patients with neovascular age-related macular degeneration. Br J Ophthalmol. 2014; 98: 1205–1207.
Michalewski J, Nawrocki J, Trebinska M, Michalewska Z. Switch to a single dose of aflibercept in bevacizumab nonresponders with AMD. Can J Ophthalmol. 2014; 49: 431–435.
Patel KH, Chow CC, Rathod R, et al. Rapid response of retinal pigment epithelial detachments to intravitreal aflibercept in neovascular age-related macular degeneration refractory to bevacizumab and ranibizumab. Eye (Lond). 2013; 27: 663–667, quiz 668.
Wykoff CC, Brown DM, Maldonado ME, Croft DE. Aflibercept treatment for patients with exudative age-related macular degeneration who were incomplete responders to multiple ranibizumab injections (TURF trial). Br J Ophthalmol. 2014; 98: 951–955.
Yonekawa Y, Andreoli C, Miller JB, et al. Conversion to aflibercept for chronic refractory or recurrent neovascular age-related macular degeneration. Am J Ophthalmol. 2013; 156: 29–35.e2.
Fischer C, Mazzone M, Jonckx B, Carmeliet P. FLT1 and its ligands VEGFB and PlGF: drug targets for anti-angiogenic therapy? Nat Rev Cancer. 2008; 8: 942–956.
Kim KJ, Cho CS, Kim WU. Role of placenta growth factor in cancer and inflammation. Exp Mol Med. 2012; 44: 10–19.
Rakic JM, Lambert V, Devy L, et al. Placental growth factor, a member of the VEGF family, contributes to the development of choroidal neovascularization. Invest Ophthalmol Vis Sci. 2003; 44: 3186–3193.
Van de Veire S, Stalmans I, Heindryckx F, et al. Further pharmacological and genetic evidence for the efficacy of PlGF inhibition in cancer and eye disease. Cell. 2010; 141: 178–190.
Huang H, Shen J, Vinores SA. Blockade of VEGFR1 and 2 suppresses pathological angiogenesis and vascular leakage in the eye. PLoS One. 2011; 6: e21411.
Ribatti D. The discovery of the placental growth factor and its role in angiogenesis: a historical review. Angiogenesis. 2008; 11: 215–221.
Saint-Geniez M, Maldonado AE, D'Amore PA. VEGF expression and receptor activation in the choroid during development and in the adult. Invest Ophthalmol Vis Sci. 2006; 47: 3135–3142.
Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signalling - in control of vascular function. Nat Rev Mol Cell Biol. 2006; 7: 359–371.
Incio J, Tam J, Rahbari NN, et al. PlGF/VEGFR-1 signaling promotes macrophage polarization and accelerated tumor progression in obesity. Clin Cancer Res. 2016; 22: 2993–3004.
Sennlaub F, Auvynet C, Calippe B, et al. CCR2(+) monocytes infiltrate atrophic lesions in age-related macular disease and mediate photoreceptor degeneration in experimental subretinal inflammation in Cx3cr1 deficient mice. EMBO Mol Med. 2013; 5: 1775–1793.
Ambati J, Anand A, Fernandez S, et al. An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice. Nat Med. 2003; 9: 1390–1397.
Liu J, Copland DA, Horie S, et al. Myeloid cells expressing VEGF and arginase-1 following uptake of damaged retinal pigment epithelium suggests potential mechanism that drives the onset of choroidal angiogenesis in mice. PLoS One. 2013; 8: e72935.
Krause TA, Alex AF, Engel DR, Kurts C, Eter N. VEGF-production by CCR2-dependent macrophages contributes to laser-induced choroidal neovascularization. PLoS One. 2014; 9: e94313.
Itaya M, Sakurai E, Nozaki M, et al. Upregulation of VEGF in murine retina via monocyte recruitment after retinal scatter laser photocoagulation. Invest Ophthalmol Vis Sci. 2007; 48: 5677–5683.
Xie P, Kamei M, Suzuki M, et al. Suppression and regression of choroidal neovascularization in mice by a novel CCR2 antagonist, INCB3344. PLoS One. 2011; 6: e28933.
Huang H, Parlier R, Shen JK, Lutty GA, Vinores SA. VEGF receptor blockade markedly reduces retinal microglia/macrophage infiltration into laser-induced CNV. PLoS One. 2013; 8: e71808.
Sasmono RT, Oceandy D, Pollard JW, et al. A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse. Blood. 2003; 101: 1155–1163.
Mattapallil MJ, Wawrousek EF, Chan CC, et al. The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes. Invest Ophthalmol Vis Sci. 2012; 53: 2921–2927.
Crespo-Garcia S, Reichhart N, Hernandez-Matas C, et al. In vivo analysis of the time and spatial activation pattern of microglia in the retina following laser-induced choroidal neovascularization. Exp Eye Res. 2015; 139: 13–21.
Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008; 3: 1101–1108.
Blaauwgeers HG, Holtkamp GM, Rutten H, et al. Polarized vascular endothelial growth factor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaris. Evidence for a trophic paracrine relation. Am J Pathol. 1999; 155: 421–428.
Grossniklaus HE, Ling JX, Wallace TM, et al. Macrophage and retinal pigment epithelium expression of angiogenic cytokines in choroidal neovascularization. Mol Vis. 2002; 8: 119–126.
Saint-Geniez M, Kurihara T, Sekiyama E, Maldonado AE, D'Amore PA. An essential role for RPE-derived soluble VEGF in the maintenance of the choriocapillaris. Proc Natl Acad Sci U S A. 2009; 106: 18751–18756.
Aiello LP, Northrup JM, Keyt BA, Takagi H, Iwamoto MA. Hypoxic regulation of vascular endothelial growth factor in retinal cells. Arch Ophthalmol. 1995; 113: 1538–1544.
Pierce EA, Avery RL, Foley ED, Aiello LP, Smith LE. Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization. Proc Natl Acad Sci U S A. 1995; 92: 905–909.
Xiong M, Elson G, Legarda D, Leibovich SJ. Production of vascular endothelial growth factor by murine macrophages: regulation by hypoxia, lactate, and the inducible nitric oxide synthase pathway. Am J Pathol. 1998; 153: 587–598.
Rohrer B, Long Q, Coughlin B, et al. A targeted inhibitor of the alternative complement pathway reduces angiogenesis in a mouse model of age-related macular degeneration. Invest Ophthalmol Vis Sci. 2009; 50: 3056–3064.
Xu J, Zhu D, Sonoda S, et al. Over-expression of BMP4 inhibits experimental choroidal neovascularization by modulating VEGF and MMP-9. Angiogenesis. 2012; 15: 213–227.
Liyanage SE, Fantin A, Villacampa P, et al. Myeloid-derived vascular endothelial growth factor and hypoxia-inducible factor are dispensable for ocular neovascularization--brief report. Arterioscler Thromb Vasc Biol. 2016; 36: 19–24.
Huo X, Li Y, Jiang Y, et al. Inhibition of ocular neovascularization by co-inhibition of VEGF-A and PLGF. Cell Physiol Biochem. 2015; 35: 1787–1796.
Jetten N, Verbruggen S, Gijbels MJ, Post MJ, De Winther MP, Donners MM. Anti-inflammatory M2, but not pro-inflammatory M1 macrophages promote angiogenesis in vivo. Angiogenesis. 2014; 17: 109–118.
Shen J, Xie B, Dong A, Swaim M, Hackett SF, Campochiaro PA. In vivo immunostaining demonstrates macrophages associate with growing and regressing vessels. Invest Ophthalmol Vis Sci. 2007; 48: 4335–4341.
Chambers SE, O'Neill CL, O'Doherty TM, Medina RJ, Stitt AW. The role of immune-related myeloid cells in angiogenesis. Immunobiology. 2013; 218: 1370–1375.
Jin N, Gao L, Fan X, Xu H. Friend or foe? Resident microglia vs bone marrow-derived microglia and their roles in the retinal degeneration. Mol Neurobiol. 2017; 54: 4094–4112.
Yang Y, Liu F, Tang M, et al. Macrophage polarization in experimental and clinical choroidal neovascularization. Sci Rep. 2016; 6: 30933.
Figure 1
 
PlGF in the control and CNV retina. (A) Weak VEGF-A (red) expression in a retinal flat mount of a nonlasered animal. Vessels are marked with IB4 (green). Scale bar: 100 μm. (B) Costaining against IB4 (green) and VEGF-A (red) in RPE flat mounts at D0, D4, and D14 after laser. Scale bar: 100 μm. (C) Staining of sagittal sections of the retina against VEGF-A (red). Nuclei were counterstained with DAPI. Scale bar: 50 μm. (D) PlGF (red) expression found in vessels (IB4, green) of a flat mount of a nonlasered animal. Scale bar: 100 μm. (E) Costaining of RPE flat mounts against IB4 (green) and PlGF (red) at D0, D4, and D14 after laser. Scale bar: 100 μm. (F) Staining of sagittal sections of the retina against PlGF (red). Magnification of the scar (inset). Nuclei were counterstained with DAPI. Scale bar: 50 μm. (GJ) Fold change in expression of different genes involved in angiogenesis, normalized to Gapdh and compared to control. N = 6–12.
Figure 1
 
PlGF in the control and CNV retina. (A) Weak VEGF-A (red) expression in a retinal flat mount of a nonlasered animal. Vessels are marked with IB4 (green). Scale bar: 100 μm. (B) Costaining against IB4 (green) and VEGF-A (red) in RPE flat mounts at D0, D4, and D14 after laser. Scale bar: 100 μm. (C) Staining of sagittal sections of the retina against VEGF-A (red). Nuclei were counterstained with DAPI. Scale bar: 50 μm. (D) PlGF (red) expression found in vessels (IB4, green) of a flat mount of a nonlasered animal. Scale bar: 100 μm. (E) Costaining of RPE flat mounts against IB4 (green) and PlGF (red) at D0, D4, and D14 after laser. Scale bar: 100 μm. (F) Staining of sagittal sections of the retina against PlGF (red). Magnification of the scar (inset). Nuclei were counterstained with DAPI. Scale bar: 50 μm. (GJ) Fold change in expression of different genes involved in angiogenesis, normalized to Gapdh and compared to control. N = 6–12.
Figure 2
 
Mononuclear phagocyte dynamics in CNV. (A) Costaining of retinal flat mounts against Iba-1 (red) to visualize mononuclear phagocytes and IB4 (green) for vessels before laser and at D4 and D7 after laser in MacGreen mouse. Amoeboid-like cells are marked by arrows. (B) MacGreen mouse was lasered and retinas were observed 14 days afterward. Costaining of retinal flat mounts against PlGF (red) and GFP (green). Certain subsets of mononuclear phagocytes in the subretinal space (GFP-positive) also express PlGF. Scale bar represents 50 μm. (CI) Bar charts representing fold change in expression of different genes involved in inflammation, normalized to Gapdh and compared to control, sorted by their polarization into M1. N = 5–12.
Figure 2
 
Mononuclear phagocyte dynamics in CNV. (A) Costaining of retinal flat mounts against Iba-1 (red) to visualize mononuclear phagocytes and IB4 (green) for vessels before laser and at D4 and D7 after laser in MacGreen mouse. Amoeboid-like cells are marked by arrows. (B) MacGreen mouse was lasered and retinas were observed 14 days afterward. Costaining of retinal flat mounts against PlGF (red) and GFP (green). Certain subsets of mononuclear phagocytes in the subretinal space (GFP-positive) also express PlGF. Scale bar represents 50 μm. (CI) Bar charts representing fold change in expression of different genes involved in inflammation, normalized to Gapdh and compared to control, sorted by their polarization into M1. N = 5–12.
Figure 3
 
Angiogenic effect of anti-PlGF in CNV. (AD) Fold change in expression of different genes involved in angiogenesis at D7 after laser, normalized to Gapdh and compared to IgG and PBS controls. Statistical significance was tested comparing to IgG controls. αPlGF stands for anti-PlGF. N = 6–9. (EG) SLO fundus angiography (FAG) sequential images at distinct time points before and after laser treatment and subsequent intravitreal injection (D4, D7, and D14) of IgG (E) PBS (F), anti-PlGF (G), and aflibercept (AFL) (H), respectively, showing differences in leakage area. (I) Bar chart represents the quantification of the leakage area at D14 after laser in the different treated groups (IgG = 17 scars, PBS = 12 scars, anti-PlGF = 35 scars, AFL = 29 scars). (J) Bar chart represents the quantification of the CNV volume at D14 after laser in the different treated groups (IgG = 5 scars, PBS = 5 scars, anti-PlGF = 11 scars, AFL = 17 scars).
Figure 3
 
Angiogenic effect of anti-PlGF in CNV. (AD) Fold change in expression of different genes involved in angiogenesis at D7 after laser, normalized to Gapdh and compared to IgG and PBS controls. Statistical significance was tested comparing to IgG controls. αPlGF stands for anti-PlGF. N = 6–9. (EG) SLO fundus angiography (FAG) sequential images at distinct time points before and after laser treatment and subsequent intravitreal injection (D4, D7, and D14) of IgG (E) PBS (F), anti-PlGF (G), and aflibercept (AFL) (H), respectively, showing differences in leakage area. (I) Bar chart represents the quantification of the leakage area at D14 after laser in the different treated groups (IgG = 17 scars, PBS = 12 scars, anti-PlGF = 35 scars, AFL = 29 scars). (J) Bar chart represents the quantification of the CNV volume at D14 after laser in the different treated groups (IgG = 5 scars, PBS = 5 scars, anti-PlGF = 11 scars, AFL = 17 scars).
Figure 4
 
Inflammatory effect of anti-PlGF in CNV. (AC) SLO fundus autofluorescence (FA) sequential images at selected time points before and after laser treatment and subsequent intravitreal injection (D4, D7, and D14) of IgG (A) PBS (B), anti-PlGF (C), and aflibercept (D), showing differences in mononuclear phagocyte behavior after laser. (EH) Costaining against GFP (green) and PlGF (red) in retina flat mounts. Representative images of subsets of subretinal macrophages in the scar site for IgG (E), PBS (F), anti-PlGF (G), and aflibercept (H). Scale bar represents 50 μm. (I) Quantification of double GFP- and PlGF-positive subretinal macrophages ex vivo comparing the different treatment groups: IgG = 18 scars; PBS = 5 scars; anti-PlGF = 13 scars; aflibercept (AFL) = 10 scars. Amoe refers to amoeboid cells whereas Ram stands for ramified ones.
Figure 4
 
Inflammatory effect of anti-PlGF in CNV. (AC) SLO fundus autofluorescence (FA) sequential images at selected time points before and after laser treatment and subsequent intravitreal injection (D4, D7, and D14) of IgG (A) PBS (B), anti-PlGF (C), and aflibercept (D), showing differences in mononuclear phagocyte behavior after laser. (EH) Costaining against GFP (green) and PlGF (red) in retina flat mounts. Representative images of subsets of subretinal macrophages in the scar site for IgG (E), PBS (F), anti-PlGF (G), and aflibercept (H). Scale bar represents 50 μm. (I) Quantification of double GFP- and PlGF-positive subretinal macrophages ex vivo comparing the different treatment groups: IgG = 18 scars; PBS = 5 scars; anti-PlGF = 13 scars; aflibercept (AFL) = 10 scars. Amoe refers to amoeboid cells whereas Ram stands for ramified ones.
Figure 5
 
Mononuclear phagocyte polarization after anti-PlGF treatment. (AG) Bar charts represent fold change in expression, normalized to Gapdh and compared to IgG controls, of different genes involved in M1–M2 polarization. α-PlGF stands for anti-PlGF. N = 5–12.
Figure 5
 
Mononuclear phagocyte polarization after anti-PlGF treatment. (AG) Bar charts represent fold change in expression, normalized to Gapdh and compared to IgG controls, of different genes involved in M1–M2 polarization. α-PlGF stands for anti-PlGF. N = 5–12.
Supplement 1
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×