Effect of natural hirudin on angiogenesis of human microvascular endothelial cells
10.7507/1002-1892.201806055
- Author:
Guanyu LIN
1
Author Information
1. Department of Plastic and Aesthetic Surgery, First Affiliated Hospital of Guangxi Medical University
- Publication Type:Journal Article
- Keywords:
angiogenesis;
Hirudin;
microvascular endothelial cells;
Notch signaling pathway;
three-dimensional culture
- From:
Chinese Journal of Reparative and Reconstructive Surgery
2018;32(12):1586-1591
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To explore the effect of natural hirudin on proliferation of human microvascular endothelial cells (HMVECs) and its preliminary mechanism of promoting angiogenesis. Methods: Three-dimensional culture models of HMVECs were established in vitro and observed by inverted phase contrast microscopy after 24 hours of culturing. Then, the three-dimensional culture models of HMVECs were treated with different concentrations (1, 4, and 7 ATU/mL) of the natural hirudin, respectively, and Dulbecco's modified Eagle's medium containing 10% fetal bovine serum as control. The cell proliferations of 4 groups were detected by cell counting kit 8 (CCK-8) method at 24, 48, and 72 hours; the angiogenesis of 4 groups were observed by tube formation assay at 24 hours; the expressions of vascular endothelial growth factor (VEGF) and Notch1 of HMVECs in 4 groups were observed by immunofluorescence staining at 24 hours. Results: The observation of cells in three-dimensional culture models showed that HMVECs attached to Matrigel well, and the cells formed tube structure completely after 24 hours. The results of CCK-8 test showed that the absorbance ( A) value of 1 and 4 ATU/mL groups were higher than that of control group at each time point ( P<0.05), and A value of 4 ATU/mL group was the highest. The A value of 7 ATU/mL group was significantly lower than those of 1 and 4 ATU/mL groups and control group ( P<0.05). The tube formation assay showed that the tube structure was more in 1 and 4 ATU/mL groups than in 7 ATU/mL group and control group, and in 4 ATU/mL group than in 1 ATU/mL group, showing significant differences ( P<0.05). There was no significant difference between 7 ATU/mL group and control group ( P>0.05). The results of immunofluorescence staining showed that compared with control group, the Notch1 expression was higher in 1 and 4 ATU/mL groups and lower in 7 ATU/mL group; and there was significant difference between 4 and 7 ATU/mL groups and control group ( P<0.05). The VEGF expression was higher in 1, 4, and 7 ATU/mL groups than in control group, in 4 ATU/mL group than in 1 and 7 ATU/mL groups, showing significant differences ( P<0.05). Conclusion: Natural hirudin can promote angiogenesis at low and medium concentrations, but suppress angiogenesis at high concentrations. Its mechanism may be related to the VEGF-Notch signal pathway.
