Abstract:

Objective To detect the mRNA and protein levels of VEGFA and VEGFR2 in mouse liver tissue and liver cells of mouse infected with Echinococcus multilocularis(Em), in order to explore the roles of vascular endothelial growth factor A (VEGFA) and endothelial growth factor receptor 2 (VEGFR2) in tissue angiogenesis. Methods Twenty female C57BL/6 mice were randomly divided into two groups, the experimental group and the control group, 10 animals each. Each mouse in the experimental group was directly injected Em metacestodes into the liver, while the control group mice were injected with PBS in the same way. Sera were separated from the whole blood of mice on days 0, 16, 37, 58, 81, and 105 after infection. The mice were euthanized on day 120 to observe the growing status of metacestode and collect. Metacestode tissue, liver tissue around metacestode, and normal liver tissue of control group mice for examining the angiogenesis in metacestode tissue by HE staining. The expression and distribution of VEGFA and VEGFR2 in these tissue samples were detected by immunohistochemistry. The liver cells and protoscoleces were sampled for in vitro incubation in three groups in 12 Petri dishes each group, comprising the group of liver cells-protoscoleces co-incubation, liver cells and protoscoleces. The culture supernatant, liver cells and protoscoleces in each group were collected on incubation day 1, 2 and 3 to assay the levels of VEGFA, VEGFR2 mRNA and protein in normal liver tissue, metacestode tissue, liver tissues around metacestode within 4 mm, and liver cells after co-incubation with protoscoleces using qRT-PCR and Western blotting. ELISA was performed to detect the content of VEGFA in peripheral blood, as well as the protein levels of VEGFA in normal liver tissue, metacestode tissue, liver tissue around metacestode, as well as in the culture supernatant of the co-incubation group, the liver cell group and the protoscolex group. Results qRT-PCR showed that the relative transcription levels of VEGFA mRNA in healthy liver tissues, liver tissues around metacestode, and metacestode tissue were 1.033 ± 0.102, 1.222 ± 0.501, and 0.276 ± 0.092, respectively. The relative transcription level of VEGFA in metacestode tissue was lower than those in the normal liver tissue and the liver tissue around metacestode (P < 0.05); while the transcription of VEGFR2 mRNA was 1.042 ± 0.071, 1.836 ± 0.062, and 0.226 ± 0.077, respectively. In comparision, the relative transcription level of VEGFR2 in the liver tissue around metacestode was higher than that in normal liver tissue (P < 0.01), whereas the transcription in metacestode tissue was lower than those in the normal liver tissue and the liver tissue around metacestode (P < 0.01). Western blotting showed that the proteins of GAPDH, VEGFA and VEGFR2 were not detected in the protein extracts of metacestode tissue, while the relative expression of VEGFA protein in the liver tissue around metacestode and normal liver tissues were 0.920 ± 0.096 and 0.816 ± 0.129, and those of VEGFR2 protein in the two groups were 1.439 ± 0.160 and 0.515 ± 0.022, respectively (P < 0.05). ELISA indicated that the content of VEGFA in the normal liver tissue, liver tissue around metacestode, and metacestode tissue was (6.581 ± 0.722), (6.363 ± 0.638), and (0.670 ± 0.105) pg, respectively. The immunohistochemical assay for VEGFA by scoring showed the scores of normal liver tissue, liver tissue around metacetode, and metacestode tissue was 3.552 ± 0.683, 3.355 ± 0.807, and 9.450 ± 1.292, respectively, revealing the expression of VEGFA in metacestode tissue being higher than those in the other two liver tissue samples (P < 0.05); while the assay for VEGFR2 scored 0.361 ± 0.547, 4.093 ± 1.042, 5.275 ± 1.003, respectively, with the expressionlevel in metacestode tissue being the highest among the three tissue samples (P < 0.01). ELISA results presented that the VEGFA level in peripheral blood on days 0, 16, 37, 58, 81, and 105 of the experimental group was (73.233 ± 7.651), (156.925 ± 5.111), (176.571 ± 40.343), (204.212 ± 9.601), (201.335 ± 24.161) and (185.745 ± 37.902) pg/ml, respectively, which were significantly higher than those of the control group of (70.355 ± 24.751), (56.144 ± 14.736), (81.094 ± 13.753), (76.172 ± 5.689), (64.393 ± 19.060) and (70.871 ± 23.966) pg/ml, respectively(P < 0.05). qRT-PCR results showed that the relative transcription levels of VEGFA and VEGFR2 mRNA on day 2 of the co-incubation group were 9.380 ± 1.165 and 2.764 ± 0.871, respectively, indicating significantly higher than those of the liver cell group of 1.028 ± 0.252 and 1.062 ± 0.201, respectively(P < 0.05). Western blotting showed that the relative expression levels of VEGFA and VEGFR2 protein on day 2 in the co-incubation group were 1.500 ± 0.148 and 1.540 ± 0.079, respectively, while those in the liver cell group were 1.322 ± 0.050 and 0.303 ± 0.003, and the expression levels in the metacestode group were higher than those in the liver vell group (P < 0.01). ELISA demonstrated that the content of VEGFA in the culture supernatant liver cell group on the incubation day 1, 2, and 3 was (24.923 ± 1.427), (151.760 ± 4.282), and (223.033 ± 10.061) pg/ml, respectively, all significantly lower than those in the co-incubation group (P < 0.01), which were (95.218 ± 4.932), (240.295 ± 15.121) and (366.148 ± 4.822) pg/ml, respectively. Conclusion E. multilocularis infection induces host cells to continuously express VEGFA, which spreads to the surrounding tissue by secretion enters the blood circulation, and further inducing the peripheral cells to highly express VEGFR2 targeting to the infection site, promoting angiogenesis in metacestode tissue.

Key words: Alveolar echinococcosis, Vascular endothelial growth factor, Vascular endothelial growth factor receptor 2, Angiogenesis