Research on influence mechanism of G protein coupled receptor kinase interacting protein 1 on differentiation of bone marrow mesenchymal stem cells into endothelial cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

DONG Xiancheng , YIN Jian , YUN Bo , LÜ Bin ,  YIN Guoyong

Department of Orthopedics, the First Affiliated Hospital of Nanjing Medical University, Nanjing Jiangsu, 210029, P.R.China;

Corresponding author：

YIN Guoyong, Email: guoyong_yin@sina.com

Keywords：

G protein coupled receptor kinase interacting protein 1; bone marrow mesenchymal stem cells; endothelial cells; vascular endothelial growth factor receptor 3; mouse

DOI：

10.7507/1002-1892.201709090

Video：

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ObjectiveTo investigate the mechanism of G protein coupled receptor kinase interacting protein 1 (GIT1) affecting angiogenesis by comparing the differentiation of bone marrow mesenchymal stem cells (BMSCs) differentiated into endothelial cells between GIT1 wild type mice and GIT1 gene knockout mice.MethodsMale and female GIT1 heterozygous mice were paired breeding, and the genotypic identification of newborn mice were detected by PCR. The 2nd generation BMSCs isolated from GIT1 wild type mice or GIT1 gene knockout mice were divided into 4 groups, including wild type control group (group A), wild type experimental group (group A1), GIT1 knockout control group (group B), and GIT1 knockout experimental group (group B1). The cells of groups A1 and B1 were cultured with the endothelial induction medium and the cells of groups A and B with normal cluture medium. The expressions of vascular endothelial growth factor receptor 2 (VEGFR-2), VEGFR-3, and phospho-VEGFR-2 (pVEGFR-2), and pVEGFR-3 proteins were detected by Western blot. The endothelial cell markers [von Willebrand factor (vWF), platelet-endothelial cell adhesion molecule 1 (PECAM-1), and vascular endothelial cadherin (VE-Cadherin)] were detected by flow cytometry. The 2nd generation BMSCs of GIT1 wild type mice were divided into 4 groups according to the different culture media: group Ⅰ, primary cell culture medium; group Ⅱ, cell culture medium containing SAR131675 (VEGFR-3 blocker); group Ⅲ, endothelial induction medium; group Ⅳ, endothelial induction medium containing SAR131675. The endothelial cell markers (vWF, PECAM-1, and VE-Cadherin) in 4 groups were also detected by flow cytometry.ResultsWestern blot results showed that there was no obviously difference in protein expressions of VEGFR-2 and pVEGFR-2 between groups; and the expressions of VEGFR-3 and pVEGFR-3 proteins in group A1 were obviously higher than those in groups A, B, and B1. The flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group A1 than in groups A, B, and B1 (P<0.05), and in group B1 than in groups A and B (P<0.05); but no significant difference was found between groups A and B (P>0.05). In the VEGFR-3 blocked experiment, the flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group Ⅲ than in groupsⅠ, Ⅱ, and Ⅳ, and in group Ⅳ than in groups Ⅰ and Ⅱ (P<0.05); but no significant difference was found between groups Ⅰ and Ⅱ (P>0.05).ConclusionGIT1 mediates BMSCs of mice differentiation into endothelial cells via VEGFR-3, thereby affecting the angiogenesis.

Citation： DONG Xiancheng, YIN Jian, YUN Bo, LÜ Bin, YIN Guoyong. Research on influence mechanism of G protein coupled receptor kinase interacting protein 1 on differentiation of bone marrow mesenchymal stem cells into endothelial cells. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(3): 257-263. doi: 10.7507/1002-1892.201709090 Copy

1.	Schmalzigaug R, Phee H, Davidson CE, et al. Differential expression of the ARF GAP genes GIT1 and GIT2 in mouse tissues. J Histochem Cytochem, 2007, 55(10): 1039-1048.
2.	Brown MC, Cary LA, Jamieson JS, et al. Src and FAK kinases cooperate to phosphorylate paxillin kinase linker, stimulate its focal adhesion localization, and regulate cell spreading and protrusiveness. Mol Biol Cell, 2005, 16(9): 4316-4328.
3.	Zhang S, Hisatsune C, Matsu-Ura T, et al. G-protein-coupled receptor kinase-interacting proteins inhibit apoptosis by inositol 1, 4, 5-triphosphate receptor-mediated Ca²⁺ signal regulation. J Biol Chem, 2009, 284(42): 29158-29169.
4.	Yin G, Sheu TJ, Menon P, et al. Impaired angiogenesis during fracture healing in GPCR kinase 2 interacting protein-1(GIT1) knock out mice. PLoS One, 2014, 9(2): e89127.
5.	Alam A, Blanc I, Gueguen-Dorbes G, et al. SAR131675, a potent and selective VEGFR-3-TK inhibitor with antilymphangiogenic, antitumoral, and antimetastatic activities. Mol Cancer Ther, 2012, 11(8): 1637-1649.
6.	Pang J, Hoefen R, Pryhuber GS, et al.GIT1 is required for pulmonary vascular development. Circulation, 2009, 119(11): 1524-1532.
7.	Robciuc MR, Kivelä R, Williams IM, et al. VEGFB/VEGFR1-induced expansion of adipose vasculature counteracts obesity and related metabolic complications. Cell Metab, 2016, 23(4): 712-724.
8.	Pan CC, Shah N, Kumar S, et al. Angiostatic actions of capsicodendrin through selective inhibition of VEGFR2-mediated AKT signaling and disregulated autophagy. Oncotarget, 2016, 8(8): 12675-12685.
9.	Kalitin NN, Karamysheva AF. RARalpha mediates all-trans-retinoic acid-induced VEGF-C, VEGF-D, and VEGFR3 expression in lung cancer cells. Cell Biol Int, 2016, 40(4): 456-464.
10.	Saj A, Arziman Z, Stempfle D, et al. A combined ex vivo and in vivo RNAi screen for notch regulators in Drosophila reveals an extensive notch interaction network. Dev Cell, 2010, 18(5): 862-876.
11.	Heitzler P. Biodiversity and noncanonical Notch signaling. Curr Top Dev Biol, 2010, 92: 457-481.
12.	Tammela T, Zarkada G, Wallgard E, et al. Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation. Nature, 2008, 454(7204): 656-660.
13.	Kume T. Novel insights into the differential functions of Notch ligands in vascular formation. J Angiogenes Res, 2009, 1: 8.
14.	Gale NW, Dominguez MG, Noguera I, et al. Haploinsufficiency of delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development. Proc Natl Acad Sci U S A, 2004, 101(45): 15949-15954.
15.	Limbourg FP, Takeshita K, Radtke F, et al. Essential role of endothelial Notch1 in angiogenesis. Circulation, 2005, 111(14): 1826-1832.
16.	Siekmann AF, Lawson ND. Notch signalling limits angiogenic cell behaviour in developing zebrafish arteries. Nature, 2007, 445(7129): 781-784.
17.	Thurston G, Noguera-Troise I, Yancopoulos GD. The Delta paradox: DLL4 blockade leads to more tumour vessels but less tumour growth. Nat Rev Cancer, 2007, 7(5): 327-331.
18.	Hayashi H, Kume T. Foxc2 transcription factor as a regulator of angiogenesis via induction of integrin beta3 expression. Cell Adhesion Migr, 2009, 3(1): 24-26.
19.	Benedito R, Rocha SF, Woeste M, et al. Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF-VEGFR2 signaling. Nature, 2012, 484(7392): 110-114.

1. Schmalzigaug R, Phee H, Davidson CE, et al. Differential expression of the ARF GAP genes GIT1 and GIT2 in mouse tissues. J Histochem Cytochem, 2007, 55(10): 1039-1048.
2. Brown MC, Cary LA, Jamieson JS, et al. Src and FAK kinases cooperate to phosphorylate paxillin kinase linker, stimulate its focal adhesion localization, and regulate cell spreading and protrusiveness. Mol Biol Cell, 2005, 16(9): 4316-4328.
3. Zhang S, Hisatsune C, Matsu-Ura T, et al. G-protein-coupled receptor kinase-interacting proteins inhibit apoptosis by inositol 1, 4, 5-triphosphate receptor-mediated Ca²⁺ signal regulation. J Biol Chem, 2009, 284(42): 29158-29169.
4. Yin G, Sheu TJ, Menon P, et al. Impaired angiogenesis during fracture healing in GPCR kinase 2 interacting protein-1(GIT1) knock out mice. PLoS One, 2014, 9(2): e89127.
5. Alam A, Blanc I, Gueguen-Dorbes G, et al. SAR131675, a potent and selective VEGFR-3-TK inhibitor with antilymphangiogenic, antitumoral, and antimetastatic activities. Mol Cancer Ther, 2012, 11(8): 1637-1649.
6. Pang J, Hoefen R, Pryhuber GS, et al.GIT1 is required for pulmonary vascular development. Circulation, 2009, 119(11): 1524-1532.
7. Robciuc MR, Kivelä R, Williams IM, et al. VEGFB/VEGFR1-induced expansion of adipose vasculature counteracts obesity and related metabolic complications. Cell Metab, 2016, 23(4): 712-724.
8. Pan CC, Shah N, Kumar S, et al. Angiostatic actions of capsicodendrin through selective inhibition of VEGFR2-mediated AKT signaling and disregulated autophagy. Oncotarget, 2016, 8(8): 12675-12685.
9. Kalitin NN, Karamysheva AF. RARalpha mediates all-trans-retinoic acid-induced VEGF-C, VEGF-D, and VEGFR3 expression in lung cancer cells. Cell Biol Int, 2016, 40(4): 456-464.
10. Saj A, Arziman Z, Stempfle D, et al. A combined ex vivo and in vivo RNAi screen for notch regulators in Drosophila reveals an extensive notch interaction network. Dev Cell, 2010, 18(5): 862-876.
11. Heitzler P. Biodiversity and noncanonical Notch signaling. Curr Top Dev Biol, 2010, 92: 457-481.
12. Tammela T, Zarkada G, Wallgard E, et al. Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation. Nature, 2008, 454(7204): 656-660.
13. Kume T. Novel insights into the differential functions of Notch ligands in vascular formation. J Angiogenes Res, 2009, 1: 8.
14. Gale NW, Dominguez MG, Noguera I, et al. Haploinsufficiency of delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development. Proc Natl Acad Sci U S A, 2004, 101(45): 15949-15954.
15. Limbourg FP, Takeshita K, Radtke F, et al. Essential role of endothelial Notch1 in angiogenesis. Circulation, 2005, 111(14): 1826-1832.
16. Siekmann AF, Lawson ND. Notch signalling limits angiogenic cell behaviour in developing zebrafish arteries. Nature, 2007, 445(7129): 781-784.
17. Thurston G, Noguera-Troise I, Yancopoulos GD. The Delta paradox: DLL4 blockade leads to more tumour vessels but less tumour growth. Nat Rev Cancer, 2007, 7(5): 327-331.
18. Hayashi H, Kume T. Foxc2 transcription factor as a regulator of angiogenesis via induction of integrin beta3 expression. Cell Adhesion Migr, 2009, 3(1): 24-26.
19. Benedito R, Rocha SF, Woeste M, et al. Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF-VEGFR2 signaling. Nature, 2012, 484(7392): 110-114.

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Research on influence mechanism of G protein coupled receptor kinase interacting protein 1 on differentiation of bone marrow mesenchymal stem cells into endothelial cells

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