Macrophage-specific promoter SP146-C1 enhances vascular endothelial growth factor C expression in atherosclerotic mice
Li Sijin1, Feng Xiaoteng1, Wang Yiru1, Qin Hewei2, Liu Ping1
1Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China; 2Henan Province Hospital of TCM, Zhengzhou 450002, Henan Province, China
Abstract: BACKGROUND: The expression efficiency of recombinant adeno-associated virus serotype 9 (rAAV9) carrying the macrophage-specific promoter synthetic promoter 146-C1 (SP146-C1) and the exogenous gene vascular endothelial growth factor C (VEGFC) in atherosclerosis is uncertain.
OBJECTIVE: To investigate the expression efficiency of rAAV9-SP146-C1-VEGFC in atherosclerotic mice and its effect on lymphangiogenesis.
METHODS: Thirty ApoE-/- mice were fed high-fat diet for 12 weeks to establish atherosclerosis models and were randomly divided into six groups, five in each group: 7-, 14-, 21-, 28-, and 35-day transfection groups and control group. The mice in the transfection groups were transfected with 5.0×1011 vg rAAV9-SP146-C1-VEGFC by caudal vein injection. In the control group, the mice were injected with the same amount of control virus rAAV9-SP146-C1-Scramble. Animals in the first five groups were killed under anesthesia at 7, 14, 21, 28 and 35 days after transfection, respectively, and those in the control group were killed under anesthesia at 7 days. Serum, femur, tibia, heart and aorta tissue samples were collected and retained in each group. The femur and tibia of mice in each group were used to extract bone marrow-derived macrophages. The gene expression of vascular endothelial growth factor C (VEGFC), vascular endothelial growth factor receptor 3 (VEGFR3), Podoplanin and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) in bone marrow-derived macrophages and the aorta were detected by RT-qPCR. VEGFC protein expression levels in bone marrow-derived macrophages and the aorta were detected by western blot, serum level of VEGFC was detected by ELISA, and VEGFC expression in the aortic sinus and LYVE-1 expression around the aorta and in the myocardium was detected by immunofluorescence.
RESULTS AND CONCLUSION: The serum level of VEGFC, the mRNA expression of VEGFC, VEGFR3, Podoplanin, and LYVE-1 in bone marrow-derived macrophages and the aorta, the protein expression of VEGFC in bone marrow-derived macrophages, and the fluorescence intensity of VEGFC in aortic sinus plaques were significantly increased in the 7-day transfection group compared with the control group (P < 0.05, P < 0.01). Serum VEGFC level of mice transfected with rAAV9-SP146-C1-VEGFC gradually increased with time and began to decrease at 28 days. mRNA levels of VEGFC, VEGFR3, Podoplanin and LYVE-1 in mouse aorta and bone marrow-derived macrophages, VEGFC protein level in bone marrow-derived macrophages, VEGFC fluorescence intensity in aortic sinus plaques, LYVE-1 fluorescence intensity around the aortic sinus and in the myocardium gradually increased with time (P < 0.05). In addition, the mRNA level of LYVE-1 in the aorta and the fluorescence intensity of LYVE-1 around the aortic sinus and in the myocardium were the highest at 28 days (P < 0.05), and gradually decreased (P < 0.05). The expression of the other indicators reached the peak at 21 days. To conclude, rAAV9-SP146-C1-VEGFC could effectively transfect bone marrow-derived macrophages and promote lymphatic hyperplasia in atherosclerotic mice.
Key words: atherosclerosis, macrophage, vascular endothelial growth factor C, macrophage specific promoter, aorta