Effect of natural hirudin on angiogenesis of human microvascular endothelial cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIN Guanyu ¹ , LIN Bojie ¹ , ZHU Jiangying ¹ , PAN Xinyuan ² ,  YIN Guoqian ¹

1. Department of Plastic and Aesthetic Surgery, the First Affiliated Hospital of Guangxi Medical University, Nanning Guangxi, 530021, P.R.China;
2. Department of Burn and Plastic Surgery, Minzu Hospital of Zhuang Autonomous Region, Nanning Guangxi, 530001, P.R.China;

Corresponding author：

YIN Guoqian, Email: yingq61@163.com

Keywords：

Hirudin; microvascular endothelial cells; three-dimensional culture; Notch signaling pathway; angiogenesis

DOI：

10.7507/1002-1892.201806055

Video：

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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), andA 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.

Citation： LIN Guanyu, LIN Bojie, ZHU Jiangying, PAN Xinyuan, YIN Guoqian. Effect of natural hirudin on angiogenesis of human microvascular endothelial cells. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(12): 1586-1591. doi: 10.7507/1002-1892.201806055 Copy

1.	张和韡, 王丽萍. 水蛭素的研究进展. 中国中西医结合肾病杂志, 2013, 14(1): 76-78.
2.	毛全高. 水蛭素剂型研究进展. 安徽医药, 2015, 19(12): 2250-2254.
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8.	Lv S, Cheng G, Zhou Y, et al. Thymosin beta4 induces angiogenesis through Notch signaling in endothelial cells. Mol Cell Biochem, 2013, 381(1-2): 283-290.
9.	Sainson RC, Harris AL. Regulation of angiogenesis by homotypic and heterotypic notch signalling in endothelial cells and pericytes: from basic research to potential therapies. Angiogenesis, 2008, 11(1): 41-51.
10.	Wen Z, Shen Y, Berry G, et al. The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway. Sci Transl Med, 2017, 9(399). doi: 10.1126/scitranslmed.aal3322.
11.	Peng L, Pan X, Yin G. Natural hirudin increases rat flap viability by anti-inflammation via PARs/p38/NF-κB pathway. BioMed Research International, 2015, 2015(12): 1-7.
12.	Fichter AM, Ritschl LM, Robitzky LK, et al. Impact of different antithrombotics on the microcirculation and viability of perforator-based ischaemic skin flaps in a small animal model. Scientific Reports, 2016, 6(1). doi: 10.1038/srep35833.
13.	Donayre CE, Ouriel K, Rhee RY, et al. Future alternatives to heparin: low-molecular-weight heparin and hirudin. J Vasc Surg, 1992, 15(4): 675-682.
14.	Huang B, Chen T, Bau D, et al. EGFR is a pivotal regulator of thrombin-mediated inflammation in primary human nucleus pulposus culture. Scientific Reports, 2017, 7(1): 1-15.
15.	Turpin B, Miller W, Rosenfeldt L, et al. Thrombin drives tumorigenesis in colitis-associated colon cancer. Cancer Res, 2014, 74(11): 3020-3030.
16.	Xu Z, Zhu L, Yao M, et al. PTEN plays an important role in thrombin-mediated lung cancer cell functions. Biomed Res Int, 2015, 2015: 459170.
17.	任荣梅, 白怀. 血管生成三维培养模型的研究进展. 医学综述, 2015, 21(16): 2881-2884.
18.	滕伟, 郭志坤. 细胞三维培养的研究进展. 医学综述, 2008, 14(12): 1767-1770.
19.	赵燕娜, 许健, 邓同乐. 三维细胞培养技术在再生医学研究中的应用. 科技通报, 2011, 27(4): 531-535.
20.	Ganesan MK, Finsterwalder R, Leb H, et al. Three-dimensional coculture model to analyze the cross talk between endothelial and smooth muscle cells. Tissue Eng Part C: Methods, 2017, 23(1): 38-49.
21.	Sahara M, Hansson EM, Wernet O, et al. Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells. Cell Res, 2014, 24(7): 820-841.

1. 张和韡, 王丽萍. 水蛭素的研究进展. 中国中西医结合肾病杂志, 2013, 14(1): 76-78.
2. 毛全高. 水蛭素剂型研究进展. 安徽医药, 2015, 19(12): 2250-2254.
3. 郭应信, 殷国前, 李佳荃, 等. 天然、重组水蛭素对随意皮瓣淤血模型血管内皮细胞生长因子的影响. 中国组织工程研究与临床康复, 2011, 15(7): 1210-1214.
4. 郭应信, 殷国前, 李佳荃. 天然与重组水蛭素对大鼠随意皮瓣淤血模型超氧化物歧化酶、丙二醛、内皮素变化的影响. 中国组织工程研究与临床康复, 2011, 15(24): 4453-4456.
5. Pan XY, Peng L, Han ZQ, et al. Hirudin promotes angiogenesis by modulating the cross-talk between p38 MAPK and ERK in rat ischemic skin flap tissue. Tissue Cell, 2015, 47(3): 301-310.
6. Pitulescu ME, Schmidt I, Giaimo BD, et al. Dll4 and Notch signalling couples sprouting angiogenesis and artery formation. Nature Cell Biology, 2017, 19(8): 915-927.
7. Kovall RA, Gebelein B, Sprinzak D, et al. The canonical notch signaling pathway: structural and biochemical insights into shape, sugar, and force. Dev Cell, 2017, 41(3): 228-241.
8. Lv S, Cheng G, Zhou Y, et al. Thymosin beta4 induces angiogenesis through Notch signaling in endothelial cells. Mol Cell Biochem, 2013, 381(1-2): 283-290.
9. Sainson RC, Harris AL. Regulation of angiogenesis by homotypic and heterotypic notch signalling in endothelial cells and pericytes: from basic research to potential therapies. Angiogenesis, 2008, 11(1): 41-51.
10. Wen Z, Shen Y, Berry G, et al. The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway. Sci Transl Med, 2017, 9(399). doi: 10.1126/scitranslmed.aal3322.
11. Peng L, Pan X, Yin G. Natural hirudin increases rat flap viability by anti-inflammation via PARs/p38/NF-κB pathway. BioMed Research International, 2015, 2015(12): 1-7.
12. Fichter AM, Ritschl LM, Robitzky LK, et al. Impact of different antithrombotics on the microcirculation and viability of perforator-based ischaemic skin flaps in a small animal model. Scientific Reports, 2016, 6(1). doi: 10.1038/srep35833.
13. Donayre CE, Ouriel K, Rhee RY, et al. Future alternatives to heparin: low-molecular-weight heparin and hirudin. J Vasc Surg, 1992, 15(4): 675-682.
14. Huang B, Chen T, Bau D, et al. EGFR is a pivotal regulator of thrombin-mediated inflammation in primary human nucleus pulposus culture. Scientific Reports, 2017, 7(1): 1-15.
15. Turpin B, Miller W, Rosenfeldt L, et al. Thrombin drives tumorigenesis in colitis-associated colon cancer. Cancer Res, 2014, 74(11): 3020-3030.
16. Xu Z, Zhu L, Yao M, et al. PTEN plays an important role in thrombin-mediated lung cancer cell functions. Biomed Res Int, 2015, 2015: 459170.
17. 任荣梅, 白怀. 血管生成三维培养模型的研究进展. 医学综述, 2015, 21(16): 2881-2884.
18. 滕伟, 郭志坤. 细胞三维培养的研究进展. 医学综述, 2008, 14(12): 1767-1770.
19. 赵燕娜, 许健, 邓同乐. 三维细胞培养技术在再生医学研究中的应用. 科技通报, 2011, 27(4): 531-535.
20. Ganesan MK, Finsterwalder R, Leb H, et al. Three-dimensional coculture model to analyze the cross talk between endothelial and smooth muscle cells. Tissue Eng Part C: Methods, 2017, 23(1): 38-49.
21. Sahara M, Hansson EM, Wernet O, et al. Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells. Cell Res, 2014, 24(7): 820-841.

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