Abstract
Purpose: :
Pathological conditions in neovascular age-related macular degeneration (nvAMD) cause the migration of microglia/macrophage to choroidal neovascularization (CNV), which in turn exacerbates the pathogenesis and progression of nvAMD. The mechanisms regulating the recruitment process are not completely understood. This study is focused on the cytokine and chemokine signaling molecules that regulate microglia/macrophage recruitment to laser-induced CNV, an experimental model of nvAMD.
Methods: :
CNV was generated in mice by laser injury. The CNV lesions were isolated by laser capture micodissection (LCM) and RNAs were isolated from the LCM-isolated CNV and the surrounding tissues at 3, 7, and 14 days after lasering. The RT2 Profiler PCR Arrays were used to examine the expression profile of cytokine, chemokines and receptors. RT-PCR, real-time PCR and immunofluorescence (IF) staining were used to determine gene and protein expression. Neutralizing antibodies specific for various target molecules, such as MF1 (VEGFR1) and DC101 (VEGFR2), were used to block their respective signaling activity. A panel of antibodies to microglia/macrophage: Iba1, CD45, and Mac-1 (CD11b) were used to confirm the identity of microglia/macrophage in the retinal pigment epithelium (RPE)/choroidal flat mounts or retinal cross sections. Quantification of digitized images was accomplished with ImageJ.
Results: :
With the LCM-isolated CNV, mRNA of VEGFR1 and its two ligands, PlGF and VEGF-B, were detected in 3-, 7-, and 14-day CNV lesions. VEGFR2 was not detected at 3-day, but was at 7- and 14-day CNV. IF staining detected protein expression of placental growth factor (PlGF) and VEGF at the 3- and 7-day CNV. Further gene expression profiling demonstrated that 9 out of 84 key chemokines, cytokines and receptors represented in the array were expressed in the 3-day CNV; they were Abcf1, Bcl6, CxCR5, Ccr1, Il1r2, Itgam, Mif, Spp1, Tgfb1. At 3-day after laser, systemically-administrated MF1 but not DC101 significantly inhibited recruitment of CD45 (+) and Mac-1(+) cells to CNV. At 14-day CNV, microglia/macrophage expressed both VEGFR1 and VEGFR2 and both MF1 and DC101 antibodies systemically-administrated markedly inhibited recruitment of CD45 (+) and Mac-1(+) cells to CNV.
Conclusions: :
VEGFR1 and VEGFR2 can regulate CNV by controlling the recruitment of microglia/macrophage independently and synergistically, depending on the stages: VEGFR1 plays a dominant role at the 3-day CNV, whereas both VEGF receptors play pivotal roles at 14-day CNV with the enhanced efficacy with their combination. The microglia/macrophage populations recruited to CNV may interact with the other cell types within CNV, such as RPE, affecting the pathogenesis of CNV.
Keywords: choroid: neovascularization • cytokines/chemokines • microglia