EFFECT OF CALCITONIN GENE-RELATED PEPTIDE ON PROLIFERATION AND MIGRATION OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

TUO Yonghua ¹ , GUO Xiaolei ¹ , ZHANG Xinxin ² , WANG Zhao ¹ , ZHOU Jian ¹ , ZHANG Yongtao ¹ , XIA Liheng ¹ , WEN Jun ¹ , JIN Dan. ¹

1Department of Trauma and Orthopaedics, Nanfang Hospital, Southern Medical University, Guangzhou Guangdong, 510515, P.R.China;;
2Department of Orthopaedics, Cancer Hospital and Institute, Chinese Academy of Medical Sciences.;

Corresponding author：

Keywords：

Calcitonin gene-related peptide; Angiogenesis; Cell migration; Cell prol iferation; Bone tissue engineering

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

Objective Tissue engineered bone implanted with sensory nerve can effectively promote angiogenesis and repair of bone defects. To investigate the effects of calcitonin gene-related peptide (CGRP) on proliferation and migration of human umbilical vein endothelial cells (HUVECs) for further revealing the mechanism of tissue engineered bone implanted with sensory nerve promoting angiogenesis. Methods HUVECs were collected from human umbilical core, and identified through von Willebrand factor (vWF) and CD31 immunofluorescence. The HUVECs were treated with CGRP and were ivided into 6 groups according to CGRP concentration: group A (0 mol/L), group B (1 × 10—12 mol/L), group C (1 × 10—11 mol/L), group D (1 × 10—10 mol/L), group E (1 × 10—9 mol/L), and group F (1 × 10—8 mol/L). The expression of the CGRP1 receptor (CGRP1R) was observed in HUVECs by cell immunofluorescence. The growth rate of HUVECs was detected through AlarmarBlue at 1, 2, 3, 4, and 5 days. Transwell chamber was used to detect the abil ity of cell migration. ELISA assay was used to detect the vascular endothel ial growth factor (VEGF) secretion and the protein expression of focal adhesion kinase (FAK) was examined using Western blot. Results HUVECs were identified through morphology, vWF and CD31 immunofluorescence. HUVECs expressed CGRP1R. CGRP could stimulate HUVECs prol iferation in a time- and concentration-dependent manners; the cell growth rates of groups B-F were significantly higher than that of group A at all time (P lt; 0.05); group F had highest cell growth rate. The number of cell migration of group B-F was significantly higher than that of group A (P lt; 0.05), which increased more than 3 times. Groups B-F had higher amount of VEGF than group A (P lt; 0.05), and groups C and D had highest amount of VEGF. FAK expression of groups B-F was significantly increased at 3, 7, and 10 days after CGRP treatment when compared with group A (P lt; 0.05). Conclusion CGRP may enhance the proliferation and migration of HUVECs by increasing the secretion of VEGF and expression of FAK.

Citation： TUO Yonghua,GUO Xiaolei,ZHANG Xinxin,WANG Zhao,ZHOU Jian,ZHANG Yongtao,XIA Liheng,WEN Jun,JIN Dan.. EFFECT OF CALCITONIN GENE-RELATED PEPTIDE ON PROLIFERATION AND MIGRATION OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(4): 495-500. doi: Copy

1.	Deleu J, Trueta J. Vascularisation of bone grafts in the anterior chamber of the eye. J Bone Joint Surg (Br), 1965, 47: 319-329.
2.	Brandi ML, Collin-Osdoby P. Vascular biology and the skeleton. J Bone Miner Res, 2006, 21(2): 183-192.
3.	Towler DA. The osteogenic-angiogenic interface: novel insights into the biology of bone formation and fracture repair. Curr Osteoporos Rep, 2008, 6(2): 67-71.
4.	Hauge EM, Qvesel D, Eriksen EF, et al. Cancellous bone remodeling occurs in specialized compartments lined by cells expressing osteoblastic markers. J Bone Miner Res, 2001, 16(9): 1575-1582.
5.	Hill EL, Elde R. Distribution of CGRP-, VIP-, D beta H-, SP-, and NPY-immunoreactive nerves in the periosteum of the rat. Cell Tissue Res, 1991, 264(3): 469-480.
6.	Hukkanen M, Konttinen YT, Santavirta S, et al. Rapid proliferation of calcitonin gene-related Peptide immunoreactive nerves during healing of rat tibial fracture suggests neural involvement in bone growth and remodelling. Neuroscience, 1993, 54(4): 969-979.
7.	Aoki M, Tamai K, Saotome K. Substance P- and calcitonin gene-related peptide-immunofluorescent nerves in the repair of experimental bone defects. Int Orthop, 1994, 18(5): 317-324.
8.	张元平, 崔继秀, 裴国献, 等. 神经化组织工程骨构建的初步观察. 中华创伤骨科杂志, 2005, 7(1): 60-65.
9.	覃俊君, 王簕, 陈思圆, 等. 血管束、感觉神经束植入组织工程骨降钙素基因相关肽和受体的时空分布. 中华创伤骨科杂志, 2009, 11(8): 742-746.
10.	刘勇, 裴国献, 江汕, 等. 组织工程骨神经化构建的组织学研究. 中国矫形外科杂志, 2009, 17(16): 1246-1249.
11.	Lamalice L, Boeuf F, Huot J. Endothelial cell migration during angiogenesis. Circ Res, 2007, 100(6): 782-794.
12.	Li J, Kreicbergs A, Bergstrom J, et al. Site-specific CGRP innervation coincides with bone formation during fracture healing and modeling: A study in rat angulated tibia. J Orthop Res, 2007, 25(9): 1204-1212.
13.	Maayan C, Bar-On E, Foldes AJ, et al. Bone mineral density and metabolism in familial dysautonomia. Osteoporos Int, 2002, 13(5): 429-433.
14.	Akopian A, Demulder A, Ouriaghli F, et al. Effects of CGRP on human osteoclast-like cell formation: A possible connection with the bone loss in neurological disorders? Peptides, 2000, 21(4): 559-564.
15.	Bjurholm A, Kreicbergs A, Schultzberg M, et al. Neuroendocrine regulation of cyclic AMP formation in osteoblastic cell lines (UMR-106-01, ROS 17/2.8, MC3T3-E1, and Saos-2) and primary bone cells. J Bone Miner Res, 1992, 7(9): 1011-1019.
16.	Kawase T, Howard GA, Roos BA, et al. Diverse actions of calcitonin gene-related peptide on intracellular free Ca2+ concentrations in UMR 106 osteoblastic cells. Bone, 1995, 16(4 Suppl): 379S-384S.
17.	Bernard GW, Shih C. The osteogenic stimulating effect of neuroactive calcitonin gene-related peptide. Peptides, 1990, 11(4): 625-632.
18.	Shih C, Bernard GW. Calcitonin gene related peptide enhances bone colony development in vitro. Clin Orthop Relat Res, 1997, (334): 335-344.
19.	Collin-Osdoby P. Role of vascular endothelial cells in bone biology. J Cell Biochem, 1994, 55(3): 304-309.
20.	Schmid J, Wallkamm B, Hammerle CH, et al. The significance of angiogenesis in guided bone regeneration. A case report of a rabbit experiment. Clin Oral Implants Res, 1997, 8(3): 244-248.
21.	Liping Wang, Xiaoyou Shi, Rong Zhao, et al. Calcitonin-gene-related peptide stimulates stromal cell osteogenic differentiation and inhibits RANKL induced NF-kB activation, osteocalstogenesis and bone resorption. Bone, 2009, 11(29): 1369-1379.
22.	Lamalice L, Le Boeuf F, Huot J. Endothelial cell migration during angiogenesis. Circ Res, 2007, 100(6): 782-794.
23.	Detmar M. Molecular regulation of angiogenesis in the skin. J Inves Dermatol, 1996, 106(2): 207-208.
24.	Hamadi A, Bouali M, Dontenwill M, et al. Regulation of focal adhesion dynamics and disassembly by phosphorylation of FAK at tyrosine 397. J Cell Sci, 2005, 118(Pt 19): 4415-4425.
25.	Hamid R, Rotshteyn Y, Radadi L, et al. Comparison of alamar blue and MTT asssys for high through-put screening. Toxicol In Vitro, 2004, 18(5): 703-710.
26.	Sieg DJ, Hauck CR, Ilic D, et al. FAK integrates growth factor and integrin signals to promote cell migration. Nat Cell Biol, 2000, 2(5): 249-256.
27.	Wang HB, Dembo M, Hanks SK, et al. Focal adhesion kinase is involved in mechanosensing during fibroblast migration. Proc Natl Acad Sci U S A, 2001, 98(20): 11295-11300.
28.	Lee OH, Lee DJ, Kim YM, et al. Sphingosine 1-phosphate stimulates tyrosine phosphorylation of focal adhesion kinase and chemotactic motility of endothelial cells via the G (i) protein-linked phospholipase C pathway. Biochem Biophys Res Commun, 2000, 268(1): 47-53.
29.	Qin L, Zhang M. Maspin regulates endothelial cell adhesion and migration through an integrin Signaling pathway. J Biol Chem, 2010, 285(42): 32360-32369.
30.	Cary LA, Chang JF, Guan JL. Stimulation of cell migration by over-expression of focal adhesion kinase and its association with Src and Fyn. J Cell Sci, 1996, 109(Pt 7): 1787-1794.
31.	Kornberg LJ, Shaw LC, Spoerri PE, et al. Focal adhesion kinase overexpression induces enhanced pathological retinal angiogenesis. Invest Ophthalmol Vis Sci, 2004, 45(12): 4463-4469.32 Nakamura J, Shigematsu S, Yamauchi K, et al. Biphasic function of focal adhesion kinase in endothelial tube formation induced by fibril-forming collagens. Biochem Biophys Res Commun, 2008, 374(4): 699-703.

1. Deleu J, Trueta J. Vascularisation of bone grafts in the anterior chamber of the eye. J Bone Joint Surg (Br), 1965, 47: 319-329.
2. Brandi ML, Collin-Osdoby P. Vascular biology and the skeleton. J Bone Miner Res, 2006, 21(2): 183-192.
3. Towler DA. The osteogenic-angiogenic interface: novel insights into the biology of bone formation and fracture repair. Curr Osteoporos Rep, 2008, 6(2): 67-71.
4. Hauge EM, Qvesel D, Eriksen EF, et al. Cancellous bone remodeling occurs in specialized compartments lined by cells expressing osteoblastic markers. J Bone Miner Res, 2001, 16(9): 1575-1582.
5. Hill EL, Elde R. Distribution of CGRP-, VIP-, D beta H-, SP-, and NPY-immunoreactive nerves in the periosteum of the rat. Cell Tissue Res, 1991, 264(3): 469-480.
6. Hukkanen M, Konttinen YT, Santavirta S, et al. Rapid proliferation of calcitonin gene-related Peptide immunoreactive nerves during healing of rat tibial fracture suggests neural involvement in bone growth and remodelling. Neuroscience, 1993, 54(4): 969-979.
7. Aoki M, Tamai K, Saotome K. Substance P- and calcitonin gene-related peptide-immunofluorescent nerves in the repair of experimental bone defects. Int Orthop, 1994, 18(5): 317-324.
8. 张元平, 崔继秀, 裴国献, 等. 神经化组织工程骨构建的初步观察. 中华创伤骨科杂志, 2005, 7(1): 60-65.
9. 覃俊君, 王簕, 陈思圆, 等. 血管束、感觉神经束植入组织工程骨降钙素基因相关肽和受体的时空分布. 中华创伤骨科杂志, 2009, 11(8): 742-746.
10. 刘勇, 裴国献, 江汕, 等. 组织工程骨神经化构建的组织学研究. 中国矫形外科杂志, 2009, 17(16): 1246-1249.
11. Lamalice L, Boeuf F, Huot J. Endothelial cell migration during angiogenesis. Circ Res, 2007, 100(6): 782-794.
12. Li J, Kreicbergs A, Bergstrom J, et al. Site-specific CGRP innervation coincides with bone formation during fracture healing and modeling: A study in rat angulated tibia. J Orthop Res, 2007, 25(9): 1204-1212.
13. Maayan C, Bar-On E, Foldes AJ, et al. Bone mineral density and metabolism in familial dysautonomia. Osteoporos Int, 2002, 13(5): 429-433.
14. Akopian A, Demulder A, Ouriaghli F, et al. Effects of CGRP on human osteoclast-like cell formation: A possible connection with the bone loss in neurological disorders? Peptides, 2000, 21(4): 559-564.
15. Bjurholm A, Kreicbergs A, Schultzberg M, et al. Neuroendocrine regulation of cyclic AMP formation in osteoblastic cell lines (UMR-106-01, ROS 17/2.8, MC3T3-E1, and Saos-2) and primary bone cells. J Bone Miner Res, 1992, 7(9): 1011-1019.
16. Kawase T, Howard GA, Roos BA, et al. Diverse actions of calcitonin gene-related peptide on intracellular free Ca2+ concentrations in UMR 106 osteoblastic cells. Bone, 1995, 16(4 Suppl): 379S-384S.
17. Bernard GW, Shih C. The osteogenic stimulating effect of neuroactive calcitonin gene-related peptide. Peptides, 1990, 11(4): 625-632.
18. Shih C, Bernard GW. Calcitonin gene related peptide enhances bone colony development in vitro. Clin Orthop Relat Res, 1997, (334): 335-344.
19. Collin-Osdoby P. Role of vascular endothelial cells in bone biology. J Cell Biochem, 1994, 55(3): 304-309.
20. Schmid J, Wallkamm B, Hammerle CH, et al. The significance of angiogenesis in guided bone regeneration. A case report of a rabbit experiment. Clin Oral Implants Res, 1997, 8(3): 244-248.
21. Liping Wang, Xiaoyou Shi, Rong Zhao, et al. Calcitonin-gene-related peptide stimulates stromal cell osteogenic differentiation and inhibits RANKL induced NF-kB activation, osteocalstogenesis and bone resorption. Bone, 2009, 11(29): 1369-1379.
22. Lamalice L, Le Boeuf F, Huot J. Endothelial cell migration during angiogenesis. Circ Res, 2007, 100(6): 782-794.
23. Detmar M. Molecular regulation of angiogenesis in the skin. J Inves Dermatol, 1996, 106(2): 207-208.
24. Hamadi A, Bouali M, Dontenwill M, et al. Regulation of focal adhesion dynamics and disassembly by phosphorylation of FAK at tyrosine 397. J Cell Sci, 2005, 118(Pt 19): 4415-4425.
25. Hamid R, Rotshteyn Y, Radadi L, et al. Comparison of alamar blue and MTT asssys for high through-put screening. Toxicol In Vitro, 2004, 18(5): 703-710.
26. Sieg DJ, Hauck CR, Ilic D, et al. FAK integrates growth factor and integrin signals to promote cell migration. Nat Cell Biol, 2000, 2(5): 249-256.
27. Wang HB, Dembo M, Hanks SK, et al. Focal adhesion kinase is involved in mechanosensing during fibroblast migration. Proc Natl Acad Sci U S A, 2001, 98(20): 11295-11300.
28. Lee OH, Lee DJ, Kim YM, et al. Sphingosine 1-phosphate stimulates tyrosine phosphorylation of focal adhesion kinase and chemotactic motility of endothelial cells via the G (i) protein-linked phospholipase C pathway. Biochem Biophys Res Commun, 2000, 268(1): 47-53.
29. Qin L, Zhang M. Maspin regulates endothelial cell adhesion and migration through an integrin Signaling pathway. J Biol Chem, 2010, 285(42): 32360-32369.
30. Cary LA, Chang JF, Guan JL. Stimulation of cell migration by over-expression of focal adhesion kinase and its association with Src and Fyn. J Cell Sci, 1996, 109(Pt 7): 1787-1794.
31. Kornberg LJ, Shaw LC, Spoerri PE, et al. Focal adhesion kinase overexpression induces enhanced pathological retinal angiogenesis. Invest Ophthalmol Vis Sci, 2004, 45(12): 4463-4469.32 Nakamura J, Shigematsu S, Yamauchi K, et al. Biphasic function of focal adhesion kinase in endothelial tube formation induced by fibril-forming collagens. Biochem Biophys Res Commun, 2008, 374(4): 699-703.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF CALCITONIN GENE-RELATED PEPTIDE ON PROLIFERATION AND MIGRATION OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS

Abstract Full text Figures/Tables Video References Cited by

Format

Content