AIM： To explore the interaction between macrophages and chemokines [monocyte chemoattractant protein 1（MCP-1/CCL2） and fractalkine/CX3CL1] and the effects of their interaction on neovascularization.METHODS： Human ...AIM： To explore the interaction between macrophages and chemokines [monocyte chemoattractant protein 1（MCP-1/CCL2） and fractalkine/CX3CL1] and the effects of their interaction on neovascularization.METHODS： Human peripheral blood mononuclear cells, donated by healthy volunteers, were separated and cultured in RPMI-1640 medium containing 10% fetal bovine serum, then induced into macrophages by stimulation with 30 μg/L granulocyte macrophage-colony stimulating factor（GM-CSF）. The expression of CCR2 and/or CX3CR1 in the macrophages was examined using flow cytometry. Macrophages were then stimulated with recombinant human CCL2（rh-CCL2） or recombinant human CX3CL1（rh-CX3CL1）. The expression of angiogenesis-related genes, including VEGF-A, THBS-1 and ADAMTS-1 were examined using real-time quantitative polymerase chain reaction（PCR）. Supernatants from stimulated macrophages were used in an assay of human retinal endothelial cell（HREC） proliferation. Finally, stimulated macrophages were cocultured with HREC in a migration assay.RESULTS： The expression rate of CCR2 in macrophages stimulated by GM-CSF was 42%±1.9%. The expression rate of CX3CR1 was 71%±3.3%. Compared with vehicle-treated groups, gene expression of VEGF-A in the macrophages was greater in 150 mg/L CCL2-treated groups（P〈0.05）, while expression of THBS-1 and ADAMTS-1 was significantly lower（P〈0.05）. By contrast, compared with vehicle-treated groups, expression of VEGF-A in 150 mg/L CX3CL1-treated groups was significantly lower（P〈0.05）, while expression of THBS-1 and ADAMTS-1 was greater（P〈0.05）. Supernatants from CCL2 treated macrophages promoted proliferation of HREC（P〈0.05）, while supernatants from CX3CL1-treated macrophages inhibited the proliferation of HREC（P〈0.05）. HREC migration increased when co-cultured with CCL2-treated macrophages, but decreased with CX3CL1-treated macrophages（P〈0.05）.CONCLUSION： CCL2 and CX3CL1 exert different effects in regulation of macrophage in expression of angiogenesisrelated factors, including VEGF-A, THBS-1 and ADAMTS-1. Our findings suggest that CCL2 and CX3CL1 may be candidate proteins for further exploration of novel targets for treatment of ocular neovascularization.展开更多
基金Supported by the National Natural Science Foundation of China(No.30972712No.31600736+5 种基金No.81671641)Jiangsu Province’s Key Provincial Talents Program(No.RC2011104)the Natural Science Foundation of Jiangsu Province of China(No.BK20151208)Jiangsu Provincial Medical Youth Talent(No.QNRC2016718)the Soochow Scholar Project of Soochow University(No.R5122001)Jiangsu Provincial Medical Innovation Team(No.CXTDA2017039)
文摘AIM： To explore the interaction between macrophages and chemokines [monocyte chemoattractant protein 1（MCP-1/CCL2） and fractalkine/CX3CL1] and the effects of their interaction on neovascularization.METHODS： Human peripheral blood mononuclear cells, donated by healthy volunteers, were separated and cultured in RPMI-1640 medium containing 10% fetal bovine serum, then induced into macrophages by stimulation with 30 μg/L granulocyte macrophage-colony stimulating factor（GM-CSF）. The expression of CCR2 and/or CX3CR1 in the macrophages was examined using flow cytometry. Macrophages were then stimulated with recombinant human CCL2（rh-CCL2） or recombinant human CX3CL1（rh-CX3CL1）. The expression of angiogenesis-related genes, including VEGF-A, THBS-1 and ADAMTS-1 were examined using real-time quantitative polymerase chain reaction（PCR）. Supernatants from stimulated macrophages were used in an assay of human retinal endothelial cell（HREC） proliferation. Finally, stimulated macrophages were cocultured with HREC in a migration assay.RESULTS： The expression rate of CCR2 in macrophages stimulated by GM-CSF was 42%±1.9%. The expression rate of CX3CR1 was 71%±3.3%. Compared with vehicle-treated groups, gene expression of VEGF-A in the macrophages was greater in 150 mg/L CCL2-treated groups（P〈0.05）, while expression of THBS-1 and ADAMTS-1 was significantly lower（P〈0.05）. By contrast, compared with vehicle-treated groups, expression of VEGF-A in 150 mg/L CX3CL1-treated groups was significantly lower（P〈0.05）, while expression of THBS-1 and ADAMTS-1 was greater（P〈0.05）. Supernatants from CCL2 treated macrophages promoted proliferation of HREC（P〈0.05）, while supernatants from CX3CL1-treated macrophages inhibited the proliferation of HREC（P〈0.05）. HREC migration increased when co-cultured with CCL2-treated macrophages, but decreased with CX3CL1-treated macrophages（P〈0.05）.CONCLUSION： CCL2 and CX3CL1 exert different effects in regulation of macrophage in expression of angiogenesisrelated factors, including VEGF-A, THBS-1 and ADAMTS-1. Our findings suggest that CCL2 and CX3CL1 may be candidate proteins for further exploration of novel targets for treatment of ocular neovascularization.
