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The role of macrophages (Mφ) in choroidal neovascularization (CNV) is still controversial due to the heterogeneity of Mφ in different microenvironments.Notch signaling is involves in a variety of ocular neovascularization including CNV.However, whether or how Notch signaling regulates the polarity convention and function of Mφ during CNV is below understood.
This study aimed to investigate the alteration of Mφ polarization and the regulation mediated by Notch signaling in CNV.
In an in vivo experiment, 58 adult male C57B6 mice were grouped to post-photocoagulative 3 day group and 7 day group randomly based on the sampling time.CNV was induced by photocoagulation of retinas by 532 nm frequency doubling laser in 23 mice in each group.The CNV area, Mφ infiltrated area, secretion of vascular endothelial growth factor (VEGF) and tumor necrosis factor-α(TNF-α) were detected by choroidal flat mounts and immunofluorescence staining.The polarization of Mφ, the secretion of VEGF and TNF-α and the expression of activated Notch intracellular domain (NICD) were detected by immunofluorescence in frozen sections.The CNV was induced in the remained 6 mice in each group, with 14 laser spots in the right eyes and the left eyes as controls.Quantity real-time PCR (qRT-PCR) was performed to detect the polarization of Mφ and the expression of angiogenesis related factors.In an in vitro experiment, bone marrow derived macrophage (BMDM) was isolated and induced with granulocyte-macrophage colony stimulating factor (GM-CSF) to differentiate into Mφ.The γ-secretion inhibitor (GSI) was added to BMDM culture environment with the polarization stimulus.The cells and the culture supernatant were collected to detect the molecule markers of M1 and M2 polarization by qRT-PCR and ELISA 24 hours later.
The CNV area was significantly increased and the Mφ infiltrated area was decreased in the post-photocoagulative 7 day group compared with the 3 day group (t=8.138, 5.272, both at P=0.000). Mφ distributed in both the peripheral and central of CNV in the 3 day group and accumulated in the central of CNV in 7 day group.qRT-PCR assay showed that the relative expressions of arginase 1 (Arg1) mRNA, mannose receptor (MR) mRNA and interleukin 6 (IL-6) mRNA in the CNV were significantly upregulated in post-photocoagulative 3 days and downregulated in 7 days (all at P<0.05). However, no significant shift was found in the expression of iNOS mRNA at the same duration.Immunofluorescence results displayed that there were more F4/80+Arg1+ cells (M2-liked Mφ) than F4/80+iNOS+ cells (M1-liked Mφ) in 3 days and 7 days (t=7.348, 4.568, both at P<0.01), and the mount of F4/80+Arg1+ cells was significantly reduced in 7 days compared with in 3 days (t=5.562, P<0.001). The choroidal flat mounts, qRT-PCR and immunofluorescence results demonstrated that VEGF had a visible expression in CNV in 3 days after photocoagulation, and it was dramatically upregulated in 7 days (t=5.776, P<0.01), but the secreted VEGF level by Mφ was higher in the third day than that in the seventh day (t=5.143, P<0.001). Similarly, the secretion level of TNF-α by Mφ was reclined in the seventh day compared with the third day (t=4.519, P<0.01). In addition, qRT-PCR and immunofluorescence results indicated that the expression of downstream molecule of Notch signaling in Mφ was not found in 3 days, but these molecules were expressed and activated in 7 days after photocoagulation (P<0.05). In vitro, the BMDMs converted to M2 after blockage of Notch signaling, and the decrease of M1 markers and increase of M2 markers were seen (all at P<0.05).
Mφ polarization phenotypes alter upon the development of CNV.The activation of Notch signaling pathway participates in the modulation of Mφ polarization and functional diversity.