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Retina  |   July 2015
M2 Macrophages Enhance Pathological Neovascularization in the Mouse Model of Oxygen-Induced Retinopathy
Author Affiliations & Notes
  • Yedi Zhou
    Department of Ophthalmology Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
  • Shigeo Yoshida
    Department of Ophthalmology Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
  • Shintaro Nakao
    Department of Ophthalmology Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
  • Takeru Yoshimura
    Department of Ophthalmology Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
  • Yoshiyuki Kobayashi
    Department of Ophthalmology Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
  • Takahito Nakama
    Department of Ophthalmology Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
  • Yuki Kubo
    Department of Ophthalmology Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
  • Kohta Miyawaki
    Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
  • Muneo Yamaguchi
    Department of Ophthalmology Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
  • Keijiro Ishikawa
    Department of Ophthalmology Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
  • Yuji Oshima
    Department of Ophthalmology Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
  • Koichi Akashi
    Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
  • Tatsuro Ishibashi
    Department of Ophthalmology Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
  • Correspondence: Shigeo Yoshida, Department of Ophthalmology, Kyushu University Graduate School of Medical Sciences, Fukuoka, 812-8582, Japan; yosida@eye.med.kyushu-u.ac.jp
Investigative Ophthalmology & Visual Science July 2015, Vol.56, 4767-4777. doi:10.1167/iovs.14-16012
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      Yedi Zhou, Shigeo Yoshida, Shintaro Nakao, Takeru Yoshimura, Yoshiyuki Kobayashi, Takahito Nakama, Yuki Kubo, Kohta Miyawaki, Muneo Yamaguchi, Keijiro Ishikawa, Yuji Oshima, Koichi Akashi, Tatsuro Ishibashi; M2 Macrophages Enhance Pathological Neovascularization in the Mouse Model of Oxygen-Induced Retinopathy. Invest. Ophthalmol. Vis. Sci. 2015;56(8):4767-4777. doi: 10.1167/iovs.14-16012.

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

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Abstract

Purpose: To investigate the roles played by M2 macrophages in a mouse model of oxygen-induced retinopathy (OIR).

Methods: Oxygen-induced retinopathy was induced in C57BL/6J mice by exposing postnatal day seven (P7) pups to 75% oxygen and then returning them to room air at P12. Real-time RT-PCR and immunofluorescence staining were used to assess the levels and distributions of different macrophage markers. Bone marrow–derived M1 and M2 macrophages and mannosylated clodronate liposomes (MCLs) were injected into the vitreous on P12 to examine the effects at P17. M2 macrophages were cocultured with human retinal endothelial cells (HRECs) to examine their effects on proliferation and tube formation.

Results: The results showed that the M2 macrophages, rather than M1 phenotype, were highly expressed in OIR mice. The number of M2 macrophages had increased significantly at P17, and the increase was closely associated with the presence of neovascular tufts in the OIR retinas. Selective depletion of M2 macrophages suppressed the pathological neovascularization and promoted physiological revascularization. In contrast, intravitreal injection of bone marrow–derived M2 macrophages or the culture supernatants promoted pathological neovascularization and inhibited physiological revascularization. In an in vitro coculture system, M2-polarized macrophages significantly promoted proliferation and tube formation of HRECs.

Conclusions: These results indicated that M2 macrophages, rather than M1, play an important role in promoting retinal pathological neovascularization probably by producing secreted factors. Thus, targeting M2 macrophages could be a potential therapeutic option for inhibiting retinal pathological neovascularization.

Intraocular neovascularization is a major cause of reduced vision in patients with diseases such as proliferative diabetic retinopathy (PDR), retinal vein occlusion, retinopathy of prematurity (ROP), and age-related macular degeneration (AMD).1 The mouse model of oxygen-induced retinopathy (OIR) is widely used to investigate the mechanism(s) causing retinal neovascularization.2 In this model, there are two types of retinal neovascularization: one is the pathological neovascularization with a sprouting of abnormal vessels from the surface of the retina into the vitreous; and the other is a physiological revascularization of avascular areas with functional intraretinal vasculature. 
We recently determined the global gene expression profile of hypoxic retinas obtained from a mouse model of OIR using gene microarray analyses.3 The results showed that one of the main functional set of gene expression was related to post-ischemic inflammation. Subsequent multiplex ELISA determined that the protein levels of several chemokines in hypoxic retinas were markedly altered. The levels of chemokines that attract granulocyte-macrophage lineages, viz, chemokine (C-X-C motif) ligand (CXCL) 1, chemokine (C-C motif) ligand (CCL) 2, CCL3, CCL4, and CXCL12 were enhanced,3 but other chemokines that are related mainly to eosinophils, mast cells, and lymphocytes were not altered. These findings indicated that the macrophages recruited into the ischemic retina may play a role in the post-ischemic inflammation and possibly the subsequent retinal neovascularization. This is reasonable because macrophages carry out a wide variety of biologic functions including neovascularization.49 
Macrophages are important angiogenic effector cells that produce a number of growth stimulators, inhibitors and proteolytic enzymes that are capable of modulating new vessel formation.10 The infiltration of macrophages also occurs in patients with several intraocular angiogenic diseases such as PDR and AMD.1115 
In the mouse model of OIR, the number of macrophages and microglia is increased in response to the ischemic condition, and some of these cells are closely associated with the neovascular tufts on the inner surface of the retina.16 In addition, an intravitreal injection of clodronate liposomes, a macrophage depletion drug, significantly reduced both the avascular areas and neovascular tufts.17 We have also shown that CCL2, CCL3, and CCL4, are involved in retinal neovascularization by recruiting macrophages in the mouse model of OIR.3,18 These findings indicated that macrophages probably play important roles in intraocular neovascularization. 
It has been recently reported that macrophages are made up of at least two phenotypes, M1 and M2.19,20 The classically activated M1 or proinflammatory macrophages are thought to play a critical role in destroying foreign organisms and tumor cells. Alternatively, activated M2 or immunosuppressive macrophages are believed to be important in debris scavenging, wound healing, and angiogenesis. They may also play some essential roles in chronic infections, tumorigenesis, and tumor metastasis.9,2123 Nevertheless, the roles played by these two phenotypes of macrophages during intraocular neovascularization has not been fully determined. 
Thus, the purpose of this study was to investigate the roles played by M1 and M2 macrophages in retinal neovascularization using a mouse model of OIR. We shall show that the M2 macrophages play an essential role in promoting retinal pathological neovascularization. The possible mechanism(s) by which M2 macrophages predominate in OIR retinas is discussed. 
Materials and Methods
Mouse Model of OIR
C57BL/6J mice (Kyudo Company, Tosu, Saga, Japan) were treated according to the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The experimental procedures were approved by the Institutional Animal Care and Use Committee of Kyushu University (Fukuoka, Japan). 
Oxygen-induced retinopathy was induced in the mice as described in detail.2,24,25 Briefly, pups were exposed to 75% oxygen on postnatal day P7 for 5 days and then returned to room air on P12. Control mice were kept in room air during the entire postnatal period. Mice were killed by cervical dislocation, and the eyes were enucleated. 
Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from whole retinas at the selected time points. The MagDEA RNA kit (Precision System Science, Pleasanton, CA, USA) was used according to the protocol included with the kit.3 The RNA concentrations were quantified and cDNA were synthesized with a First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany). Quantitative RT-PCR was performed and analyzed using Taqman gene expression assays (Applied Biosystems, Foster City, CA, USA) and a LightCycler 96 Real-time PCR System (Roche). Twenty microliters of the PCR mixture containing 1 μL of primer was loaded into each LightCycler well. The reference numbers for the assays used were: Mm99999915_g1 (glyceraldehyde 3-phosphate dehydrogenase, GAPDH), Mm00802529_m1 (F4/80), Mm00485148_m1 (CD206), Mm00711660_m1 (CD80), Mm00440502_m1 (NOS2), Mm00474091_m1 (CD163), Mm00443260_g1(TNF-α), Mm00446190_m1 (IL-6), and Mm00433287_m1 (bFGF). GAPDH was used as the endogenous control. An initial step of 10 minutes at 95°C was used to activate the HotStart DNA polymerase, and then a two-step cycling program including 45 cycles of 95°C for 20 seconds and 60°C for 40 seconds was used for the Taqman assays. The LightCycler 96 Real-time PCR System software was used to detect the probe, calculate the threshold cycles (Ct values), and additional analyses. 
Histology and Immunofluorescent Staining
Eyes were enucleated and fixed in 4% paraformaldehyde for 1 hour. The corneas and muscles were removed, and the eyecups were placed in 4% paraformaldehyde for another 1 hour. Then, the entire retina was dissected from the eyecup, and after rinsing and blocking, the retinas were incubated with the primary antibodies or conjugated antibodies overnight at 4°C. After rinsing with phosphate buffered saline with Tween (PBST), the retinas were flat-mounted on cover slides with an aqueous mounting medium (Thermo Scientific, Fremont, CA, USA). 
In other retinas, 20-μm sections were cut with a cryostat (Leica CM 1800, Bannockburn, IL, USA). After washing and blocking, the flat-mounted retinas or cryostat sections were incubated with the primary antibodies overnight at 4°C. The secondary antibodies were added for 1 hour at room temperature, and the nuclei were counterstained with DAPI (Hoechst 33342; Molecular Probes, Eugene, OR, USA). 
The cryostat sections and flat-mounts were incubated with the following antibodies: Alexa Fluor 647 anti-mouse CD206 (1:100 dilution; Biolegend, San Diego, CA, USA), anti-mouse CD80 (B7-1) FITC (1:200 dilution; eBioscience, San Diego, CA, USA), FITC anti-mouse F4/80 antibody (1:200 dilution; Biolegend), fluorescein-labeled isolectin B4 (1:150 dilution; Vector Laboratories, Burlingame, CA, USA), dyLight 594 labeled isolectin B4 (1:150 dilution; Vector Laboratories), and CD31 antibody (1:20 dilution; R&D System, Minneapolis, MN, USA). The sizes of the avascular areas and neovascular tufts areas were quantified after staining with fluorescein-labeled isolectin B4 according to a described protocol.2,25 The retinas were photographed under a fluorescent microscope (BZ-9000; KEYENCE, Osaka, Japan) and merged to obtain whole-mount retinal images using image-joint software BZ-Analyzer (KEYENCE).3,26,27 Hematoxylin and eosin (H&E) staining was performed in thick paraffin sections as described.26,28 
Selective Depletion of M2 Macrophages in Retinas
Mannosylated clodronate liposomes (MCLs) selectively deplete M2 macrophages and microglia.2931 Mannosylated clodronate liposomes and control liposomes containing only PBS (PBS-Liposomes; Encapsular Nano Sciences, Brentwood, TN, USA) were injected intravitreally (0.5 μL/eye) with a selective depletion of M2 macrophages at P12 of OIR mice.17,30 Only one injection was given to each eye, and the eyes were enucleated at P17. 
Quantification of CD206-Positive Cells
Immunofluorescent staining of flat-mounted retinas was performed as described. To quantify the number of CD206-positive cells in the flat-mounted retinas, eight randomly selected areas (500 × 500 μm) were studied. Four or eight retinas were analyzed for each group. 
Preparation of M1 and M2 Macrophages From Mouse Bone Marrow
Bone marrow–derived monocytes were collected from the femoral shafts of adult C57BL/6J or enhanced enhanced green fluorescent protein (EGFP) male mice,32 and they were incubated in 10 mL of RPMI 1640 supplemented with GlutaMAX (Gibco, Invitrogen, Carlsbad, CA, USA) containing 10% FBS.33 Then, 50 ng/mL of macrophage colony-stimulating factor (M-CSF), a secreted cytokine, was added to the solution (R&D System). Five days later, the cells were stimulated with 1 μg/mL LPS and 20 ng/mL IFN-γ (R&D System) for 24 hours to induce differentiation into the M1 phenotype. Other cells were stimulated with 20 ng/mL of IL-4 (R&D System), IL-10 (R&D System), or IL-13 (R&D System) for 24 hours, to induce a polarization of the cells into M2 phenotypes.19 The expressions of macrophage markers were determined by real-time RT-PCR (Supplementary Figs. S3A, S3B). 
Intravitreal Treatment of M1 and M2 Macrophages
Bone marrow–derived M1 or M2 macrophages were collected after polarization, and 1×105 cells in 0.5 μL PBS/eye was injected intravitreally into P12 pups with a 33-G needle on a Hamilton syringe (Hamilton, Reno, NV, USA) just after they were returned to room air. Eyes were enucleated at P17. 
Intravitreal Treatment of M2 Macrophage Culture Supernatants
M2 macrophage culture supernatants and uncultured medium 24 hours after the polarization were injected intravitreally (0.5 μL/eye) into P12 pups using a 33-G needle on a Hamilton syringe. Eyes were enucleated at P17. 
Human Retinal Endothelial Cells in Culture
Human retinal endothelial cells (HRECs; Cell Systems Corporation, Kirkland, WA, USA) were cultured on collagen-coated dishes (Iwaki, Tokyo, Japan) and maintained in 6% CO2 at 37°C in CS-C complete medium (Cell Systems Corporation). Cells at passages 7 through 8 were used for the studies. 
MTT Assays
Human retinal endothelial cells (3 × 104 cells/500 μL) were seeded into 24-wells plates, cultured with C-SC medium (10% FBS), and allowed to attach. The medium was removed 24 hours later, and the cells were starved in C-SC (1% FBS) medium for 24 hours. The medium was replaced by RPMI1640 with 1 × GlutaMAX (Gibco, Invitrogen) in 10% FBS, and the mixture was added to the lower chamber of a Transwell migration assay system with 0.4-μm pores (Corning, NY, USA). Then 5 × 104/100 μL of M2 macrophages were added to the upper chamber, and the cells in the upper and lower chambers were cocultured for 24 hours. The cells were washed gently with PBS twice, and fresh medium (500 μL) was added to each well together with MTT (5 mg/mL, 60 μL; Sigma-Aldrich Corp., St. Louis, MO, USA). After 5 hours of incubation, the supernatants were decanted, and the formazan precipitates were solubilized by the addition of 500 uL of 100% DMSO (Sigma-Aldrich Corp.) and placed on a shaker for 10 minutes. Then 150 uL of the DMSO solution was transferred to 96-format plate wells. The absorbance at 490 nm was measured with an Immuno Mini NJ-2300 plate reader (System Instruments, Tokyo, Japan). 
Tube Formation Assay
The tube formation assay of HRECs was performed as described in detail.3436 In brief, growth factor–reduced Matrigel matrix (BD Biosciences, Franklin Lakes, NJ, USA) was allowed to polymerize for 30 minutes at 37°C. After gel formation, serum starved HRECs were suspended in serum and growth factor-free RPMI1640 with 1 × GlutaMAX (Gibco), and finally seeded at a density of 8 × 104 cells/500 μL. The HRECs were cultured at 37°C for 1 hour, and 5 × 104/100 μL of M2 macrophages in RPMI1640 with 1 × GlutaMAX (Gibco) were added to the upper chamber of the Trasnswell migration assay system (Corning) with 0.4-μm pore size. Cultured supernatant without M2 macrophages were used as the control group. Three independent wells were examined for each group. After culturing for 24 hours, four to five images were photographed randomly with a phase contrast microscope (Olympus CK2; Olympus, Tokyo, Japan) coupled to a digital camera (Olympus DP21). The images were analyzed by Angiogenesis Analyzer for ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). 
Statistical Analyses
All results are presented as the means ± SEMs. The significance of differences between each group was determined by Dunnett's tests or Student's t-tests. Differences were considered statistically significant at P less than 0.05. Statistical analyses were performed using the software of JMP 9.0.2 (SAS Institute, Cary, NC, USA). 
Results
Induction of M2 Macrophage Markers in OIR Retinas
To determine whether M1 and M2 macrophages were involved in the development of retinal neovascularization in OIR mice, real-time RT-PCR was performed and the expression of the mRNAs of CD206, CD163, CD80, NOS2, and F4/80 in the retinas of OIR mice were compared with that of the control pups kept in room air. CD206 and CD163 were used as M2 macrophage markers,37,38 CD80 and NOS2 (also known as iNOS) were used as M1 macrophage markers,39,40 and F4/80 was used as a pan macrophage marker.16,41 The expressions of the mRNA of both CD206 and CD163 were significantly upregulated in the OIR retinas compare with the room air controls. The increase was first detected at P12, reached a peak at P21, and then was decreased at P25. The expression of the mRNA of CD206 was significantly increased from P14 to P25 (P < 0.05 at P21 and P25; P < 0.01 at P14 and P17; Fig. 1A). The expression of the mRNA of CD163 was significantly increased at P17 and P21 (P < 0.05, Fig. 1B), the expression of the mRNA of CD80 was significantly increased at P17 and P25 (P < 0.05, Fig. 1C), and that of NOS2 at P17 and P21 (P < 0.05, Fig. 1D). However, the increase degree of CD80 and NOS2 were less than that of CD206 and CD163. 
Figure 1
 
Expressions of the mRNAs of macrophage markers determined by real-time RT-PCR in retinas of OIR mice. (A) Expression of the mRNA of CD206 was significantly increased from P14 to P25. (B) Expression of the mRNA of CD163 was significantly increased at both P17 and P21. (C) Expression of the mRNA of CD80 was significantly increased at P17 and P25. (D) Expression of the mRNA of NOS2 was significantly increased at P17 and P21, but decreased at P12 and P25. (E) Expression of the mRNA of F4/80 was significantly increased at all the time points in OIR retinas. **P < 0.01, *P < 0.05 compared to the room air controls at the same or earlier time points. Retinas of mice raised in room air served as controls. Error bars represent the means ± SEMs (n = 4/group).
Figure 1
 
Expressions of the mRNAs of macrophage markers determined by real-time RT-PCR in retinas of OIR mice. (A) Expression of the mRNA of CD206 was significantly increased from P14 to P25. (B) Expression of the mRNA of CD163 was significantly increased at both P17 and P21. (C) Expression of the mRNA of CD80 was significantly increased at P17 and P25. (D) Expression of the mRNA of NOS2 was significantly increased at P17 and P21, but decreased at P12 and P25. (E) Expression of the mRNA of F4/80 was significantly increased at all the time points in OIR retinas. **P < 0.01, *P < 0.05 compared to the room air controls at the same or earlier time points. Retinas of mice raised in room air served as controls. Error bars represent the means ± SEMs (n = 4/group).
The expression of F4/80 was also increased at P17 and peaked at P21, which was similar to that of CD206 and CD163 (P < 0.05 at P12, P14 and P21; P < 0.01 at P17 and P25; Fig. 1E). 
Predominance of M2 Macrophages Over M1 in OIR Retinas
We next coimmunostained flat-mounted retinas with antibodies against CD80-CD206 and CD206-F4/80 at P17. A large number of CD206-positive cells and F4/80-positive cells were detected in the retinas, and their distribution overlapped (Fig. 2A). In contrast, much fewer CD80-positive cells were found, and their locations did not overlap the areas of the CD206-positive cells (Fig. 2B). These findings suggest that M2 macrophages play a more important role than M1 macrophages in retinal neovascularization. 
Figure 2
 
Immunohistologic staining for macrophages in OIR mice retinas at P17. (A) CD206 (red) costained with F4/80 (green) in the flat-mounted OIR retina at P17. A large number of CD206-positive cells presented on the retina, and the location of the F4/80-positive cells overlapped the location of the CD206-positive cells. Scale bar: 100 μm. (B) CD206 (red) costained with CD80 (green) in the flat-mounted OIR retina at P17. A few CD80-positive cells were found, and they did not overlap the locations of the CD206-positive cells. Scale bar: 100 μm. (C) CD206 (red) costained with isolectin B4 (green) in the flat-mounted OIR retinas at P17 when the pathological neovascularization is maximal. The distribution of CD206-positive cells overlapped that of the isolectin B4 cells. Scale bar: 100 μm. (D) CD206 (red) costained with isolectin B4 (green) in the flat-mounted retinas of room air control mice at P17. Scale bar: 100 μm. (E) CD206 (red) and CD31 (green) in cryosection of OIR eyes, and H&E staining of paraffin section of OIR eyes at P17. The distribution of CD206-positive cells overlapped that of CD31-positive cells, and the cells were distributed in the inner layer of the retina. Scale bar: 100 μm.
Figure 2
 
Immunohistologic staining for macrophages in OIR mice retinas at P17. (A) CD206 (red) costained with F4/80 (green) in the flat-mounted OIR retina at P17. A large number of CD206-positive cells presented on the retina, and the location of the F4/80-positive cells overlapped the location of the CD206-positive cells. Scale bar: 100 μm. (B) CD206 (red) costained with CD80 (green) in the flat-mounted OIR retina at P17. A few CD80-positive cells were found, and they did not overlap the locations of the CD206-positive cells. Scale bar: 100 μm. (C) CD206 (red) costained with isolectin B4 (green) in the flat-mounted OIR retinas at P17 when the pathological neovascularization is maximal. The distribution of CD206-positive cells overlapped that of the isolectin B4 cells. Scale bar: 100 μm. (D) CD206 (red) costained with isolectin B4 (green) in the flat-mounted retinas of room air control mice at P17. Scale bar: 100 μm. (E) CD206 (red) and CD31 (green) in cryosection of OIR eyes, and H&E staining of paraffin section of OIR eyes at P17. The distribution of CD206-positive cells overlapped that of CD31-positive cells, and the cells were distributed in the inner layer of the retina. Scale bar: 100 μm.
M2 Macrophages Associated With Neovascular Tufts in OIR Retinas
To determine the relationship between M2 macrophages and neovascularization, flat-mounted retinas from the OIR mice and control mice were immunostained with CD206 and isolectin B4. In addition, the cryostat sections of eyes from OIR mice were immunostained with CD206 and CD31. The results showed that the CD206-positive cells were concentrated in and around the neovascular tufts that extended into the vitreous of the OIR retinas (Figs. 2C, 2E). Fewer CD206-positive cells were found in the control retinas (Fig. 2D). Overall, the cryosection revealed that the CD206-positive macrophages were mainly located in the inner layers of the retina during the neovascularization process (Fig. 2E). 
Increase in Number of CD206-Positive Cells in OIR Retinas
To determine the changes in the number of M2 macrophages in the OIR retina, the numbers of CD206-positive cells were counted at different times after the hyperoxygen exposure in OIR and normoxic exposure in control retinas. We found that CD206-positive cells were not significantly altered at P12 and P14. However, a significant increase was detected at P17 (181 ± 7.54/mm2, P < 0.001; Figs. 3A, 3C) and at P21 (156 ± 15.11/mm2, P < 0.001) in the OIR groups over the room air control retinas (Figs. 3B, 3C). 
Figure 3
 
Number of CD206-positive cells increases during the pathological progression of OIR. (A) Immunofluorescent double staining of CD206 (red) and isolectin B4 (green) in flat-mounted OIR retina at P17. A large number of CD206-positive cells were present in the retina. Scale bar: 500 μm. (B) Immunofluorescent double staining of CD206 (red) and isolectin B4 (green) in flat-mounted control retina at P17. Fewer CD206-positive cells were present at the same time points. Scale bar: 500 μm. (C) Number of CD206-positive cells in the retinas counted at each time point for the OIR and control retinas. The retinas from mice treated by hyperoxygen had significantly larger numbers of CD206-positive cells at P17 and P21 compare with the normoxic controls. **P < 0.001 at the same time points. Error bars represent the means ± SEMs (n = 4/group).
Figure 3
 
Number of CD206-positive cells increases during the pathological progression of OIR. (A) Immunofluorescent double staining of CD206 (red) and isolectin B4 (green) in flat-mounted OIR retina at P17. A large number of CD206-positive cells were present in the retina. Scale bar: 500 μm. (B) Immunofluorescent double staining of CD206 (red) and isolectin B4 (green) in flat-mounted control retina at P17. Fewer CD206-positive cells were present at the same time points. Scale bar: 500 μm. (C) Number of CD206-positive cells in the retinas counted at each time point for the OIR and control retinas. The retinas from mice treated by hyperoxygen had significantly larger numbers of CD206-positive cells at P17 and P21 compare with the normoxic controls. **P < 0.001 at the same time points. Error bars represent the means ± SEMs (n = 4/group).
M2 Macrophage Depletion Suppresses Pathological Neovascularization and Promote Physiological Revascularization in OIR Retinas
To examine the role played by the M2 macrophages in OIR retinas, we examined the effects of a selective inhibition of M2 macrophages by MCLs on retinal neovascularization. We first validated that MCLs depleted M2 macrophages. In the PBS liposomes–injected control group, a large number of CD206-positive cells were found in the retinas (101.5 ± 7.00/mm2, Supplementary Figs. S1A, S1C), however in the MCLs-injected group, the number of CD206-positive cells was significantly reduced (6.63 ± 1.35/mm2, P < 0.001, Supplementary Figs. S1B, S1C). 
Real-time RT-PCR showed that the expression of CD206 was significantly reduced after MCLs injection (68.52% decrease, P < 0.001, Supplementary Fig. S1D). However, the injection of MCLs did not affect the level of expressions of both CD80 (P > 0.05, Supplementary Fig. S1E) and NOS2 (P > 0.05, Supplementary Fig. S1F). 
We then tested the effects of MCLs on physiological revascularization and pathological neovascularization in the OIR model. We measured the sizes of the avascular areas representing physiological revascularization and the areas of neovascular tufts representing pathological neovascularization at P17 in OIR flat-mounted retinas given MCLs or PBS-liposome as controls. 
The sizes of the areas of neovascular tufts and the avascular areas were significantly decreased after MCLs injection compare with the control group (35.8% and 18.8% decrease, P < 0.01 and P < 0.05, respectively; n = 23, Figs. 4A–F). These findings indicated that the pathological neovascularization was suppressed and physiological revascularization was enhanced in the retinas after depleting the M2 macrophages. 
Figure 4
 
M2 macrophage depletion suppresses pathological neovascularization and promotes physiological revascularization in OIR retinas. The retinas were stained by isolectin B4, and flat-mounted retinas were examined by fluorescence microscopy. Representative images of flat-mounted OIR retinas that have been intravitreal injected with PBS-liposome (A, C) and MCLs (B, D). The red color represents areas of neovascular tufts and white represents avascular areas. In the retinas with MCLs injection, the size of the avascular areas (E) and neovascular tufts (F) were both significantly smaller than that of the controls. **P < 0.01, *P < 0.05. Error bars represent the means ± SEMs (n = 23/group). Scale bars: 500 μm.
Figure 4
 
M2 macrophage depletion suppresses pathological neovascularization and promotes physiological revascularization in OIR retinas. The retinas were stained by isolectin B4, and flat-mounted retinas were examined by fluorescence microscopy. Representative images of flat-mounted OIR retinas that have been intravitreal injected with PBS-liposome (A, C) and MCLs (B, D). The red color represents areas of neovascular tufts and white represents avascular areas. In the retinas with MCLs injection, the size of the avascular areas (E) and neovascular tufts (F) were both significantly smaller than that of the controls. **P < 0.01, *P < 0.05. Error bars represent the means ± SEMs (n = 23/group). Scale bars: 500 μm.
Bone Marrow–Derived M2 Macrophages Promote Pathological Neovascularization and Reduce Physiological Revascularization in OIR Retinas
To further confirm the role of M1 and M2 macrophages in retinal neovascularization, bone marrow–derived M1 and M2 macrophages from C57BL/6J mice or control PBS were injected into the vitreous of OIR mice at P12, and retinas were analyzed at P17. Both the pathological neovascular tufts and avascular areas were increased in the M2 macrophage–injected group compared with the control group (53.5% and 21.2% increase respectively, P < 0.01 and P < 0.05, n = 16, Figs. 5A–H). In contrast, M1 macrophage injection suppressed both the pathological neovascular tufts and avascular areas compared with the control group (41.3% and 46.9% decrease respectively, P < 0.01, n = 16, Figs. 5A–H). 
Figure 5
 
M2 macrophages promote pathological neovascularization and reduce physiological revascularization in OIR retinas. Representative images of flat-mounted OIR retinas that had an intravitreal injection of PBS (A, D), bone marrow–derived M1 (B, E), and M2 macrophages (C, F). In the retinas with M1 macrophages injection, both the avascular areas (G) and neovascular tufts (H) were significantly suppressed compare to the PBS control. On the other hand, in the retinas with M2 macrophages injection, the size of the avascular areas (G) and neovascular tufts areas (H) were significantly increased. **P < 0.01, *P < 0.05. Error bars represent the means ± SEMs (n = 16/group). Scale bars: 500 μm.
Figure 5
 
M2 macrophages promote pathological neovascularization and reduce physiological revascularization in OIR retinas. Representative images of flat-mounted OIR retinas that had an intravitreal injection of PBS (A, D), bone marrow–derived M1 (B, E), and M2 macrophages (C, F). In the retinas with M1 macrophages injection, both the avascular areas (G) and neovascular tufts (H) were significantly suppressed compare to the PBS control. On the other hand, in the retinas with M2 macrophages injection, the size of the avascular areas (G) and neovascular tufts areas (H) were significantly increased. **P < 0.01, *P < 0.05. Error bars represent the means ± SEMs (n = 16/group). Scale bars: 500 μm.
To determine whether the injected M2 macrophages survive and how long they survive in the retina, bone marrow–derived M2 macrophages from EGFP mice were injected intravitreally in OIR mice at P12. EGFP-positive cells were detected around the neovascular tufts (arrowheads) at P17 (Supplementary Figs. S2A, S2B). These findings indicate that there was an infiltration of exogenous M2 macrophages into the retina and were still surviving. A large number of EGFP-positive cells was detected at P14 and P17, but only a small number could be recognized at P21 and P25 (Supplementary Fig. S2C). Although bone marrow–derived M1 macrophages from EGFP mice were injected intravitreally, they did not infiltrate around the neovascular tufts (data not shown). 
To determine whether the M2 macrophage directly promoted neovascularization or indirectly promoted neovascularization by producing secreted factors, the supernatants of M2 macrophages in culture and uncultured medium were also injected intravitreally. The results showed that cultured supernatants increased the size of both the avascular areas and neovascular tufts significantly, but the increases were smaller than injecting M2 macrophages–containing medium (19.1% and 32.2% increase respectively, P < 0.05, n = 12, Figs. 6A–F). 
Figure 6
 
M2 macrophages cultured supernatants enhance physiological revascularization and pathological neovascularization in OIR retinas. Representative images of flat-mounted OIR retinas from eyes that had an intravitreal injection of cultured supernatant (A, C) or uncultured medium (B, D). After injecting cultured supernatant, the size of the avascular areas (E) and neovascular tufts areas (F) were significantly increased compare with the group injected with uncultured medium. *P < 0.05. Error bars represent the means ± SEMs (n = 12/group). Scale bars: 500 μm.
Figure 6
 
M2 macrophages cultured supernatants enhance physiological revascularization and pathological neovascularization in OIR retinas. Representative images of flat-mounted OIR retinas from eyes that had an intravitreal injection of cultured supernatant (A, C) or uncultured medium (B, D). After injecting cultured supernatant, the size of the avascular areas (E) and neovascular tufts areas (F) were significantly increased compare with the group injected with uncultured medium. *P < 0.05. Error bars represent the means ± SEMs (n = 12/group). Scale bars: 500 μm.
Figure 7
 
M2 macrophages promote proliferation and tube formation of HRECs in vitro. The optical density (490 nm) of the HRECs which cocultured with M1 or M2 macrophages was significantly higher after 24 hours compare with those cultured alone without M2 macrophages (A). **P < 0.01, *P < 0.05. Error bars represent the means ± SDs (n = 9). Tube formation assay photographs were taken after cultured alone (B), coculture with M1 (C) or M2 macrophages (D). Quantitative assessment of the tube formation was done by measuring the lengths of the tubes in each group. Coculturing with M1 macrophages did not affect the tube formation significantly, whereas the group cocultured with M2 macrophages had a significant increase in tube formation compare with that cultured alone (E). **P < 0.01. Error bars represent the means ± SEMs (n = 12/group).
Figure 7
 
M2 macrophages promote proliferation and tube formation of HRECs in vitro. The optical density (490 nm) of the HRECs which cocultured with M1 or M2 macrophages was significantly higher after 24 hours compare with those cultured alone without M2 macrophages (A). **P < 0.01, *P < 0.05. Error bars represent the means ± SDs (n = 9). Tube formation assay photographs were taken after cultured alone (B), coculture with M1 (C) or M2 macrophages (D). Quantitative assessment of the tube formation was done by measuring the lengths of the tubes in each group. Coculturing with M1 macrophages did not affect the tube formation significantly, whereas the group cocultured with M2 macrophages had a significant increase in tube formation compare with that cultured alone (E). **P < 0.01. Error bars represent the means ± SEMs (n = 12/group).
To determine which cytokines play significant roles in the retinal neovascularization, real-time RT-PCR was performed using mouse bone marrow–derived M0, M1, and M2 macrophages (Supplementary Fig. S3). The results showed that the inflammatory cytokines, TNF-α and IL-6, were increased more in M1 macrophages compare with monocytes and M2 macrophages. In contrast, bFGF was expressed at much higher levels in M2 than M1 macrophages and monocytes. 
Bone Marrow–Derived M2 Macrophages Enhance Proliferation and Tube Formation of Cultured HRECs
To further investigate the influence of M2 macrophages on retinal neovascularization, HRECs were cocultured with M2 macrophages. The results of MTT assay showed that after 24 hours, the proliferation of HRECs was enhanced by coculturing with both M1 and M2 macrophages (P < 0.05 for M1, P < 0.01 for M2, n = 9, Fig. 7A). 
To examine the effect of M2 macrophages on tube formation, we also cocultured M2 macrophages with HRECs in a tube formation assay with the Transwell system. The total length of the tubes in the group that was cocultured with M1 macrophages was slightly increased, but the increase was not statistically significant (P = 0.062, n = 12, Figs. 7B, 7C, 7E). Coculturing with M2 macrophages significantly increased tube formation after 24 hours compared with those without M2 macrophages (P < 0.01, n = 12, Figs. 7B, 7D, 7E). These findings indicated that M2 macrophages promoted a greater increase in the length of the tubes of HRECs in vitro. 
Discussion
Our results showed that the majority of F4/80-positive cells stained positive for CD206, and the number of M2 macrophages was significantly higher than M1 macrophages in the retinas of OIR mice (Figs. 115523). In addition, a selective depletion of M2 macrophages by MCLs promoted a physiological revascularization and suppressed pathological neovascularization (Fig. 4). In contrast, intravitreally injected M2 macrophages in OIR mice survived and infiltrated into the retinas (Supplementary Fig. S2), increased the pathological neovascular tufts, and reduced physiological revascularization (Fig. 5). Finally, M2 macrophages enhanced the degree of proliferation and the number of tube formation of HRECs in vitro (Fig. 7). These results indicated that M2 macrophages may be more important for the progression of pathological neovascularization than physiological revascularization. Our results are consistent with those of earlier studies that showed that M2 macrophages are associated with the pathogenesis of angiogenesis in tumors.42,43 
The CD206-positive M2 macrophages were concentrated in and around the neovascular tufts that extended into the vitreous at P17 where the pathological neovascularization is maximum (Figs. 2C, 2E). On the other hand, there were fewer M2 macrophages detected in the retina of normoxic-exposed mice (Fig. 2D). These results indicated that the function of M2 macrophages may be primarily related to the progression of pathological neovascularization rather than physiological revascularization. Because a large number of retinal endothelial cells greatly increased during this pathological process, the increase in the neovascular tufts may in turn lead to a reduction of physiological revascularization as was found. 
Bone marrow–derived M2 macrophages enhanced pathological neovascularization and reduced physiological revascularization, and bone marrow–derived M1 macrophages had an opposite effects in OIR mice (Fig. 5). However, both M1 and M2 macrophages enhanced the proliferation and tube formation of HRECs in vitro with a more significant increase by M2 phenotype (Fig. 7). Therefore, the exogenous addition of M1 macrophages might primarily play more important roles in enhancing physiological revascularization than pathological neovascularization. 
In addition, the increase of pathological neovascularization may play a significant role in supplying oxygen to the hypoxic inner retina, and the physiological revascularization may be further inhibited by the reduction of retinal hypoxia. These findings agree with the results of our recent study that showed an over-expression of M2 macrophages in epiretinal fibrovascular membranes (FVMs) of patients with PDR, and much fewer M1 macrophages were present.44 
It is known that M2 macrophages can be polarized by M-CSF with IL-4, IL-10, and/or IL-13.19 Macrophage colony-stimulating factor is constitutively expressed at all times in OIR mice, while GM-CSF is not present.16 In addition, M-CSF is reported to be required for pathological neovascularization but not for physiological revascularization in OIR mice.45 The predominance of M-CSF over GM-CSF indicates that ischemic retinas may be a M2 macrophage-dominant microenvironment in OIR mice. We recently demonstrated that the concentrations of M-CSF and IL-13, but not GM-CSF or IL-4, were significantly higher in the vitreous of patients with PDR than in control patients. Moreover, the concentration of IL-13 was significantly correlated with the presence of FVMs indicating that IL-13 is closely associated with FVM formation in eyes with PDR.46 Therefore, an increase of M2 macrophages in OIR mice may be due to, at least in part, local M2 polarization after being recruited into the eyes. 
There is a new concept that M2 macrophages can be divided into three groups: M2a, M2b, and M2c, and that each can be polarized by a different chemokine system.19 Although there has not been a study that demonstrated an upregulation of IL-4, IL-13, or IL-10 in OIR retinas, only IL-13 is upregulated in the vitreous of patients with PDR according to our study.46 Therefore, it is likely that M2a is a functional subtype. If M2a is the predominant subtype in OIR mice, bFGF may be the factor that induces neovascularization.43,47,48 Consistent with this, injecting cultured supernatants of M2 macrophages tended to enhance pathological neovascularization although the effect was not as strong as injecting a medium containing M2 cells (Fig. 6). This would indicate that M2 macrophages can exert their functions by producing some type of secreted factors. Moreover, in vitro analysis demonstrated that bFGF was expressed at a much higher level in M2 macrophages than M1 (Supplementary Fig. S3). Further studies are needed to determine which one of three subtypes of M2 macrophages is actually involved in the ischemia-induced retinal neovascularization. 
Similar experiments on OIR mice have been reported by Marchetti,49 who used human umbilical cord blood–derived cells that differentiated in vitro into M2 macrophages. They reported that injecting M2 macrophages reduced the pathological neovascularization and promoted physiological revascularization, which was contrary to our results. However, we believe that the discrepancy comes from different experimental designs; in their studies, human cells were injected into mouse eyes, which may lead to more inflammatory reactions due to the difference of species. Moreover, they only injected at P7 and checked the retinas at P17. During the 5 days in hyperoxygen, the environment may influence the functions or even polarize the macrophages into another phenotype. In our studies, mouse bone marrow–derived M2 macrophages were injected into vitreous at P12 immediately after the mice were returned to the room air at which time the macrophages may start to affect the pathological neovascularization. 
In summary, M2 macrophages, rather than M1 macrophages, play important roles in promoting pathological retinal neovascularization in the mouse model of OIR. Thus, cell therapies targeting M2 macrophages could be a potential therapeutic option for inhibiting retinal pathological neovascularization. 
Acknowledgments
The authors thank Masayo Eto, Kinuko Sasada, and Hiroko Miura for their excellent technical assistance. Yedi Zhou thanks the China Scholarship Council (CSC) to support his studies. 
Supported by Japan Society for the Promotion of Science (JSPS) KAKENHI (Tokyo, Japan) Grant Numbers 24249083, 26293374, and 26670757, and the Takeda Science Foundation (Osaka, Japan). 
Disclosure: Y. Zhou, None; S. Yoshida, None; S. Nakao, None; T. Yoshimura, None; Y. Kobayashi, None; T. Nakama, None; Y. Kubo, None; K. Miyawaki, None; M. Yamaguchi, None; K. Ishikawa, None; Y. Oshima, None; K. Akashi, None; T. Ishibashi, None 
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Figure 1
 
Expressions of the mRNAs of macrophage markers determined by real-time RT-PCR in retinas of OIR mice. (A) Expression of the mRNA of CD206 was significantly increased from P14 to P25. (B) Expression of the mRNA of CD163 was significantly increased at both P17 and P21. (C) Expression of the mRNA of CD80 was significantly increased at P17 and P25. (D) Expression of the mRNA of NOS2 was significantly increased at P17 and P21, but decreased at P12 and P25. (E) Expression of the mRNA of F4/80 was significantly increased at all the time points in OIR retinas. **P < 0.01, *P < 0.05 compared to the room air controls at the same or earlier time points. Retinas of mice raised in room air served as controls. Error bars represent the means ± SEMs (n = 4/group).
Figure 1
 
Expressions of the mRNAs of macrophage markers determined by real-time RT-PCR in retinas of OIR mice. (A) Expression of the mRNA of CD206 was significantly increased from P14 to P25. (B) Expression of the mRNA of CD163 was significantly increased at both P17 and P21. (C) Expression of the mRNA of CD80 was significantly increased at P17 and P25. (D) Expression of the mRNA of NOS2 was significantly increased at P17 and P21, but decreased at P12 and P25. (E) Expression of the mRNA of F4/80 was significantly increased at all the time points in OIR retinas. **P < 0.01, *P < 0.05 compared to the room air controls at the same or earlier time points. Retinas of mice raised in room air served as controls. Error bars represent the means ± SEMs (n = 4/group).
Figure 2
 
Immunohistologic staining for macrophages in OIR mice retinas at P17. (A) CD206 (red) costained with F4/80 (green) in the flat-mounted OIR retina at P17. A large number of CD206-positive cells presented on the retina, and the location of the F4/80-positive cells overlapped the location of the CD206-positive cells. Scale bar: 100 μm. (B) CD206 (red) costained with CD80 (green) in the flat-mounted OIR retina at P17. A few CD80-positive cells were found, and they did not overlap the locations of the CD206-positive cells. Scale bar: 100 μm. (C) CD206 (red) costained with isolectin B4 (green) in the flat-mounted OIR retinas at P17 when the pathological neovascularization is maximal. The distribution of CD206-positive cells overlapped that of the isolectin B4 cells. Scale bar: 100 μm. (D) CD206 (red) costained with isolectin B4 (green) in the flat-mounted retinas of room air control mice at P17. Scale bar: 100 μm. (E) CD206 (red) and CD31 (green) in cryosection of OIR eyes, and H&E staining of paraffin section of OIR eyes at P17. The distribution of CD206-positive cells overlapped that of CD31-positive cells, and the cells were distributed in the inner layer of the retina. Scale bar: 100 μm.
Figure 2
 
Immunohistologic staining for macrophages in OIR mice retinas at P17. (A) CD206 (red) costained with F4/80 (green) in the flat-mounted OIR retina at P17. A large number of CD206-positive cells presented on the retina, and the location of the F4/80-positive cells overlapped the location of the CD206-positive cells. Scale bar: 100 μm. (B) CD206 (red) costained with CD80 (green) in the flat-mounted OIR retina at P17. A few CD80-positive cells were found, and they did not overlap the locations of the CD206-positive cells. Scale bar: 100 μm. (C) CD206 (red) costained with isolectin B4 (green) in the flat-mounted OIR retinas at P17 when the pathological neovascularization is maximal. The distribution of CD206-positive cells overlapped that of the isolectin B4 cells. Scale bar: 100 μm. (D) CD206 (red) costained with isolectin B4 (green) in the flat-mounted retinas of room air control mice at P17. Scale bar: 100 μm. (E) CD206 (red) and CD31 (green) in cryosection of OIR eyes, and H&E staining of paraffin section of OIR eyes at P17. The distribution of CD206-positive cells overlapped that of CD31-positive cells, and the cells were distributed in the inner layer of the retina. Scale bar: 100 μm.
Figure 3
 
Number of CD206-positive cells increases during the pathological progression of OIR. (A) Immunofluorescent double staining of CD206 (red) and isolectin B4 (green) in flat-mounted OIR retina at P17. A large number of CD206-positive cells were present in the retina. Scale bar: 500 μm. (B) Immunofluorescent double staining of CD206 (red) and isolectin B4 (green) in flat-mounted control retina at P17. Fewer CD206-positive cells were present at the same time points. Scale bar: 500 μm. (C) Number of CD206-positive cells in the retinas counted at each time point for the OIR and control retinas. The retinas from mice treated by hyperoxygen had significantly larger numbers of CD206-positive cells at P17 and P21 compare with the normoxic controls. **P < 0.001 at the same time points. Error bars represent the means ± SEMs (n = 4/group).
Figure 3
 
Number of CD206-positive cells increases during the pathological progression of OIR. (A) Immunofluorescent double staining of CD206 (red) and isolectin B4 (green) in flat-mounted OIR retina at P17. A large number of CD206-positive cells were present in the retina. Scale bar: 500 μm. (B) Immunofluorescent double staining of CD206 (red) and isolectin B4 (green) in flat-mounted control retina at P17. Fewer CD206-positive cells were present at the same time points. Scale bar: 500 μm. (C) Number of CD206-positive cells in the retinas counted at each time point for the OIR and control retinas. The retinas from mice treated by hyperoxygen had significantly larger numbers of CD206-positive cells at P17 and P21 compare with the normoxic controls. **P < 0.001 at the same time points. Error bars represent the means ± SEMs (n = 4/group).
Figure 4
 
M2 macrophage depletion suppresses pathological neovascularization and promotes physiological revascularization in OIR retinas. The retinas were stained by isolectin B4, and flat-mounted retinas were examined by fluorescence microscopy. Representative images of flat-mounted OIR retinas that have been intravitreal injected with PBS-liposome (A, C) and MCLs (B, D). The red color represents areas of neovascular tufts and white represents avascular areas. In the retinas with MCLs injection, the size of the avascular areas (E) and neovascular tufts (F) were both significantly smaller than that of the controls. **P < 0.01, *P < 0.05. Error bars represent the means ± SEMs (n = 23/group). Scale bars: 500 μm.
Figure 4
 
M2 macrophage depletion suppresses pathological neovascularization and promotes physiological revascularization in OIR retinas. The retinas were stained by isolectin B4, and flat-mounted retinas were examined by fluorescence microscopy. Representative images of flat-mounted OIR retinas that have been intravitreal injected with PBS-liposome (A, C) and MCLs (B, D). The red color represents areas of neovascular tufts and white represents avascular areas. In the retinas with MCLs injection, the size of the avascular areas (E) and neovascular tufts (F) were both significantly smaller than that of the controls. **P < 0.01, *P < 0.05. Error bars represent the means ± SEMs (n = 23/group). Scale bars: 500 μm.
Figure 5
 
M2 macrophages promote pathological neovascularization and reduce physiological revascularization in OIR retinas. Representative images of flat-mounted OIR retinas that had an intravitreal injection of PBS (A, D), bone marrow–derived M1 (B, E), and M2 macrophages (C, F). In the retinas with M1 macrophages injection, both the avascular areas (G) and neovascular tufts (H) were significantly suppressed compare to the PBS control. On the other hand, in the retinas with M2 macrophages injection, the size of the avascular areas (G) and neovascular tufts areas (H) were significantly increased. **P < 0.01, *P < 0.05. Error bars represent the means ± SEMs (n = 16/group). Scale bars: 500 μm.
Figure 5
 
M2 macrophages promote pathological neovascularization and reduce physiological revascularization in OIR retinas. Representative images of flat-mounted OIR retinas that had an intravitreal injection of PBS (A, D), bone marrow–derived M1 (B, E), and M2 macrophages (C, F). In the retinas with M1 macrophages injection, both the avascular areas (G) and neovascular tufts (H) were significantly suppressed compare to the PBS control. On the other hand, in the retinas with M2 macrophages injection, the size of the avascular areas (G) and neovascular tufts areas (H) were significantly increased. **P < 0.01, *P < 0.05. Error bars represent the means ± SEMs (n = 16/group). Scale bars: 500 μm.
Figure 6
 
M2 macrophages cultured supernatants enhance physiological revascularization and pathological neovascularization in OIR retinas. Representative images of flat-mounted OIR retinas from eyes that had an intravitreal injection of cultured supernatant (A, C) or uncultured medium (B, D). After injecting cultured supernatant, the size of the avascular areas (E) and neovascular tufts areas (F) were significantly increased compare with the group injected with uncultured medium. *P < 0.05. Error bars represent the means ± SEMs (n = 12/group). Scale bars: 500 μm.
Figure 6
 
M2 macrophages cultured supernatants enhance physiological revascularization and pathological neovascularization in OIR retinas. Representative images of flat-mounted OIR retinas from eyes that had an intravitreal injection of cultured supernatant (A, C) or uncultured medium (B, D). After injecting cultured supernatant, the size of the avascular areas (E) and neovascular tufts areas (F) were significantly increased compare with the group injected with uncultured medium. *P < 0.05. Error bars represent the means ± SEMs (n = 12/group). Scale bars: 500 μm.
Figure 7
 
M2 macrophages promote proliferation and tube formation of HRECs in vitro. The optical density (490 nm) of the HRECs which cocultured with M1 or M2 macrophages was significantly higher after 24 hours compare with those cultured alone without M2 macrophages (A). **P < 0.01, *P < 0.05. Error bars represent the means ± SDs (n = 9). Tube formation assay photographs were taken after cultured alone (B), coculture with M1 (C) or M2 macrophages (D). Quantitative assessment of the tube formation was done by measuring the lengths of the tubes in each group. Coculturing with M1 macrophages did not affect the tube formation significantly, whereas the group cocultured with M2 macrophages had a significant increase in tube formation compare with that cultured alone (E). **P < 0.01. Error bars represent the means ± SEMs (n = 12/group).
Figure 7
 
M2 macrophages promote proliferation and tube formation of HRECs in vitro. The optical density (490 nm) of the HRECs which cocultured with M1 or M2 macrophages was significantly higher after 24 hours compare with those cultured alone without M2 macrophages (A). **P < 0.01, *P < 0.05. Error bars represent the means ± SDs (n = 9). Tube formation assay photographs were taken after cultured alone (B), coculture with M1 (C) or M2 macrophages (D). Quantitative assessment of the tube formation was done by measuring the lengths of the tubes in each group. Coculturing with M1 macrophages did not affect the tube formation significantly, whereas the group cocultured with M2 macrophages had a significant increase in tube formation compare with that cultured alone (E). **P < 0.01. Error bars represent the means ± SEMs (n = 12/group).
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