VEGF-modified Macrophages Repair Vascular Endothelial Injury In Atherosclerotic Mice

Objective:This study aimed to investigate whether the vascular endothelial growth factor（VEGF）modified mouse macrophages can be used as a carrier of VEGF to deliver VEGF to the injured artery,promote the repair of damaged endothelium and inhibit intimal hyperplasia in the wire-induced carotid artery injury model in early atherosclerotic mice,and to explore its related mechanisms.Methods:1.Cell experiments:Subculture the untransfected group RAW264.7 cells,stable transfected cell line VEGF-RAW264.7(simultaneously transfected with human VEGF₁₆₅ and green fluorescent protein ZsGreen1 gene)and transfected control group ZsGreen1-RAW264.7（only transfected with ZsGreen1）which previously constructed and identified in our laboratory.After 48 h of cell culture,the supernatant of cell culture was collected and the production of nitric oxide（NO）was determined by Griess method.The expression of chemokine receptor type 2（CCR2）and chemokine receptor type 5（CCR5）proteins in each group was detected by Western Blot,so as to preliminarily explore the mechanism of difference in the amount of recruited cells in the endothelium.2.Animal experiments:Wire-induced carotid artery injury model was established in ApoE^-/-mice.These mice were randomly divided into following groups and each mouse received injection into tail vein immediately after induction of artery injury:VEGF-RAW264.7 group,ZsGreen1-RAW264.7 group and PBS group（containing no cell）.Three days after injury,the expression of VEGF protein in the left common carotid artery was detected by Western Blot.One week after injury,the location and number of cells expressing green fluorescent protein in the whole aorta and the left and right common carotid arteries of the mice were detected by using in vivo image system.Three weeks after the injury,the left common carotid arteries of some animals in each group were fabricated frozen sections,and the reendothelialization of the damaged vascular endothelium was demonstrated by endothelial marker CD31immunofluorescence staining.The localization of vWF（another endothelial marker）and Zs Green1（spontaneous green fluorescent protein）double positive cells in the vascular wall was observed under the laser confocal microscope to find out whether the injected VEGF-RAW264.7 cells directly formed new endothelial cells on the surface of damaged vascular lumen.The left common carotid arteries of the remaining animals in each group were made into paraffin sections.After H&E staining,neointimal area and neointima-to-media area ratio were calculated by image analysis to evaluate the degree of intimal hyperplasia.Results:1.Cell experiments:In the supernatant after 48 hours of cell culture,the NO production of VEGF-RAW264.7 group was about 5 times higher than that of RAW264.7 group and ZsGreen1-RAW264.7 group（P<0.01）,and there was no significant difference between the last two groups.Western Blot showed that the expression of CCR2 protein was higher in each group without significant difference.However,CCR5 weakly expressed in RAW264.7 and ZsGreen1-RAW264.7 cells,and significantly increased in VEGF-RAW264.7 cells（P<0.01）,suggesting that the chemotaxis ability of VEGF-RAW264.7 cells to the damaged site remained unchanged,while the ability to retain in the tissue was significantly increased.2.Animal experiments:In vivo image system showed both VEGF-RAW264.7and ZsGreen1-RAW264.7 cells injected intravenously would spontaneously recruit to the injured endothelium of the left common carotid artery,but not to the uninjured arteries.Moreover,compared with ZsGreen1-RAW264.7 group,VEGF-RAW264.7cells could be recruited to the damaged intima more（P<0.01）.Western Blot showed that the expression of VEGF protein in the left common carotid artery of the VEGF-RAW264.7 cell treatment group was 5 times higher than that of the other two groups（P<0.01）.After 3 weeks of injury,immunofluorescence staining showed that the reendothelialization rate of the damaged endothelium in VEGF-RAW264.7 group was 90.06%±6.27%,significantly higher than that in ZsGreen1-RAW264.7 group（61.70%±9.48%）and PBS group（60.57%±9.21%）.In addition,the laser confocal microscopy co-localization detected that VEGF-RAW264.7 cells directly incorporate into newly formed endothelium.H&E staining showed that the neointimal area and neointima/media ratio in VEGF-RAW264.7 group were dramatically reduced compared with those in ZsGreen1-RAW264.7 group or PBS control group（P<0.01）.Conclusion:VEGF-modified macrophage therapy could accelerate the reendothelialization of injured arteries and inhibit intimal hyperplasia,thus preventing the development of atherosclerosis.The mechanism may be through targeted delivery of VEGF to injured sites,the increase of local NO concentration and directly incorporating into newly formed endothelium.