Local injection of angiopoietin 2 promotes angiogenesis in tissue engineered bone and repair of bone defect with autophagy induction <i>in vivo</i> _Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YIN Jian , WANG Bin , ZHU Chao , SUN Chao ,  LIU Xinhui

Department of Orthopedics, the Affiliated Jiangning Hospital, Nanjing Medical University, Nanjing Jiangsu, 211100, P.R.China;

Corresponding author：

LIU Xinhui, Email: professorlxh2008@163.com

Keywords：

Angiopoietin 2; autophagy; angiogenesis; tissue engineered bone; bone defect; rabbit

DOI：

10.7507/1002-1892.201804105

Video：

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ObjectiveTo investigate the mechanism of early vascularization of the tissue engineered bone in the treatment of rabbit radial bone defect by local injection of angiopoietin 2 (Ang-2).MethodsForty-eight New Zealand white rabbits were established unilateral 1.5 cm long radius defect models. After implantation of hydroxyapatite/collagen scaffolds in bone defects, the rabbits were randomly divided into 2 groups: control group (group A) and Ang-2 group (group B) were daily injected with 1 mL normal saline and 1 mL saline-soluble 400 ng/mL Ang-2 at the bone defect within 2 weeks after operation, respectively. Western blot was used to detect the expressions of autophagy related protein [microtubule associated protein 1 light chain 3 (LC3), Beclin-1], angiogenesis related protein [vascular endothelial growth factor (VEGF)], and autophagy degradable substrate protein (SQSTMl/p62) in callus. X-ray films examination and Lane-Sandhu X-ray scoring were performed to evaluate the bone defect repair at 4, 8, and 12 weeks after operation. The rabbits were sacrificed at 12 weeks after operation for gross observation, and the angiogenesis of bone defect area was observed by HE staining.ResultsWestern blot assay showed that the relative expressions of LC3-Ⅱ/LC3-Ⅰ, Beclin-1, and VEGF in group B were significantly higher than those in group A, and the relative expression of SQSTMl/p62 was significantly lower than that in group A (P<0.05). Radiographic and gross observation of specimens showed that only a few callus were formed in group A, the bone defect was not repaired; more callus were formed and complete repair of bone defect was observed in group B. The Lane-Sandhu scores in group B were significantly higher than those in group A at 4, 8, and 12 weeks after operation (P<0.05). HE staining showed that the Harvard tubes in group B were well arranged and the number of new vessels was significantly higher than that in group A (t=–11.879, P=0.000).ConclusionLocal injection of appropriate concentration of Ang-2 may promote early vascularization and bone defect repair of tissue engineered bone in rabbits by enhancing autophagy.

Citation： YIN Jian, WANG Bin, ZHU Chao, SUN Chao, LIU Xinhui. Local injection of angiopoietin 2 promotes angiogenesis in tissue engineered bone and repair of bone defect with autophagy induction in vivo . Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(9): 1150-1156. doi: 10.7507/1002-1892.201804105 Copy

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2.	Schumann P, Lindhorst D, von See C, et al. Accelerating the early angiogenesis of tissue engineering constructs in vivo by the use of stem cells cultured in matrigel. J Biomed Mater Res A, 2014, 102(6): 1652-1662.
3.	Lane JM, Sandhu HS. Current approaches to experimental bone grafting. Orthop Clin North Am, 1987, 18(2): 213-225.
4.	Nandi SK, Kundu B, Ghosh SK, et al. Efficacy of nano-hydroxyapatite prepared by an aqueous solution combustion technique in healing bone defects of goat. J Vet Sci, 2008, 9(2): 183-191.
5.	Yuan J, Cui L, Zhang WJ, et al. Repair of canine mandibular bone defects with bone marrow stromal cells and porous beta-tricalcium phosphate. Biomaterials, 2007, 28(6): 1005-1013.
6.	Nishida J, Shimamura T. Methods of reconstruction for bone defect after tumor excision: a review of alternatives. Med Sci Monit, 2008, 14(8): R107-R113.
7.	Bernstein P, Bornhäuser M, Günther KP, et al. Bone tissue engineering in clinical application: assessment of the current situation. Orthopade, 2009, 38(11): 1029-1037.
8.	Wang MO, Vorwald CE, Dreher ML, et al. Evaluating 3D-printed biomaterials as scaffolds for vascularized bone tissue engineering. Adv Mater, 2015, 27(1): 138-144.
9.	Koupaei N, Karkhaneh A, Daliri Joupari M. Preparation and characterization of (PCL-crosslinked-PEG)/hydroxyapatite as bone tissue engineering scaffolds. J Biomed Mater Res A, 2015, 103(12): 3919-3926.
10.	Yu H, Zeng X, Deng C, et al. Exogenous VEGF introduced by bioceramic composite materials promotes the restoration of bone defect in rabbits. Biomed Pharmacother, 2018, 98: 325-332.
11.	Liu Z, Yuan X, Liu M, et al. Antimicrobial peptide combined with BMP2-modified mesenchymal stem cells promotes calvarial repair in an osteolytic model. Mol Ther, 2018, 26(1): 199-207.
12.	Kobayashi N, Hashimoto Y, Otaka A, et al. Porous alpha-tricalcium phosphate with immobilized basic fibroblast growth factor enhances bone regeneration in a canine mandibular bone defect model. Materials (Basel), 2016, 9(10): pii: E853.
13.	Silva de Oliveira JC, Okamoto R, Sonoda CK, et al. Evaluation of the osteoinductive effect of PDGF-BB associated with different carriers in bone regeneration in bone surgical defects in rats. Implant Dent, 2017, 26(4): 559-566.
14.	Gao Y, Li C, Wang H, et al. Acceleration of bone-defect repair by using A-W MGC loaded with BMP2 and triple point-mutant HIF1α-expressing BMSCs. J Orthop Surg Res, 2015, 10: 83.
15.	Liu X, Zhang G, Hou C, et al. Vascularized bone tissue formation induced by fiber-reinforced scaffolds cultured with osteoblasts and endothelial cells. Biomed Res Int, 2013, 2013: 854917.
16.	赵萍萍, 刘陶文. Ang/Tie2 系统与肿瘤血管生成的关系. 医学综述, 2011, 17(21): 3250-3252.
17.	Augustin HG, Koh GY, Thurston G, et al. Control of vascular morphogenesis and homeostasis through the angiopoietin-Tie system. Nat Rev Mol Cell Biol, 2009, 10(3): 165-177.
18.	Huang JJ, Shi YQ, Li RL, et al. Angiogenesis effect of therapeutic ultrasound on HUVECs through activation of the PI3K-Akt-eNOS signal pathway. Am J Transl Res, 2015, 7(6): 1106-1115.
19.	殷建, 殷照阳, 杨海源, 等. 自噬现象及其在脊髓缺血再灌注损伤中作用的研究进展. 中国脊柱脊髓杂志, 2015, 25(6): 557-562.
20.	Jiang F. Autophagy in vascular endothelial cells. Clin Exp Pharmacol Physiol, 2016, 43(11): 1021-1028.
21.	Goyal A, Gubbiotti MA, Chery DR, et al. Endorepellin-evoked autophagy contributes to angiostasis. J Biol Chem, 2016, 291(37): 19245-19256.
22.	Li R, Du J, Chang Y. Role of autophagy in hypoxia-induced angiogenesis of RF/6A cells in vitro. Curr Eye Res, 2016, 41(12): 1566-1570.
23.	Kumar S, Guru SK, Pathania AS, et al. Autophagy triggered by magnolol derivative negatively regulates angiogenesis. Cell Death Dis, 2013, 4: e889.
24.	Fiedler U, Scharpfenecker M, Koidl S, et al. The Tie-2 ligand angiopoietin-2 is stored in and rapidly released upon stimulation from endothelial cell Weibel-Palade bodies. Blood, 2004, 103(11): 4150-4156.
25.	Gill KA, Brindle NP. Angiopoietin-2 stimulates migration of endothelial progenitors and their interaction with endothelium. Biochem Biophys Res Commun, 2005, 336(2): 392-396.
26.	Helfrich I, Edler L, Sucker A, et al. Angiopoietin-2 levels are associated with disease progression in metastatic malignant melanoma. Clin Cancer Res, 2009, 15(4): 1384-1392.
27.	Sfiligoi C, de Luca A, Cascone I, et al. Angiopoietin-2 expression in breast cancer correlates with lymph node invasion and short survival. Int J Cancer, 2003, 103(4): 466-474.
28.	Goede V, Coutelle O, Neuneier J, et al. Identification of serum angiopoietin-2 as a biomarker for clinical outcome of colorectal cancer patients treated with bevacizumab-containing therapy. Br J Cancer, 2010, 103(9): 1407-1414.
29.	Wang X, Bullock AJ, Zhang L, et al. The role of angiopoietins as potential therapeutic targets in renal cell carcinoma. Transl Oncol, 2014, 7(2): 188-195.
30.	Scholz A, Harter PN, Cremer S, et al. Endothelial cell-derived angiopoietin-2 is a therapeutic target in treatment-naive and bevacizumab-resistant glioblastoma. EMBO Mol Med, 2016, 8(1): 39-57.
31.	Falcón BL, Hashizume H, Koumoutsakos P, et al. Contrasting actions of selective inhibitors of angiopoietin-1 and angiopoietin-2 on the normalization of tumor blood vessels. Am J Pathol, 2009, 175(5): 2159-2170.
32.	Coxon A, Bready J, Min H, et al. Context-dependent role of angiopoietin-1 inhibition in the suppression of angiogenesis and tumor growth: implications for AMG 386, an angiopoietin-1/2-neutralizing peptibody. Mol Cancer Ther, 2010, 9(10): 2641-2651.
33.	Theelen TL, Lappalainen JP, Sluimer JC, et al. Angiopoietin-2 blocking antibodies reduce early atherosclerotic plaque development in mice. Atherosclerosis, 2015, 241(2): 297-304.
34.	He J, Bao Q, Zhang Y, et al. Yes-associated protein promotes angiogenesis via signal transducer and activator of transcription 3 in endothelial cells. Circ Res, 2018, 122(4): 591-605.
35.	Williams JA, Thomas AM, Li G, et al. Tissue specific induction of p62/Sqstm1 by farnesoid X receptor. PLoS One, 2012, 7(8): e43961.
36.	Yin ZY, Yin J, Huo YF, et al. Rapamycin facilitates fracture healing through inducing cell autophagy and suppressing cell apoptosis in bone tissues. Eur Rev Med Pharmacol Sci, 2017, 21(21): 4989-4998.
37.	Li X, Wei J, Aifantis KE, et al. Current investigations into magnetic nanoparticles for biomedical applications. J Biomed Mater Res A, 2016, 104(5): 1285-1296.
38.	Li X, Gao H, Uo M, et al. Effect of carbon nanotubes on cellular functions in vitro. J Biomed Mater Res A, 2009, 91(1): 132-139.

1. Seale NM, Varghese S. Biomaterials for pluripotent stem cell engineering: From fate determination to vascularization. J Mater Chem B, 2016, 4(20): 3454-3463.
2. Schumann P, Lindhorst D, von See C, et al. Accelerating the early angiogenesis of tissue engineering constructs in vivo by the use of stem cells cultured in matrigel. J Biomed Mater Res A, 2014, 102(6): 1652-1662.
3. Lane JM, Sandhu HS. Current approaches to experimental bone grafting. Orthop Clin North Am, 1987, 18(2): 213-225.
4. Nandi SK, Kundu B, Ghosh SK, et al. Efficacy of nano-hydroxyapatite prepared by an aqueous solution combustion technique in healing bone defects of goat. J Vet Sci, 2008, 9(2): 183-191.
5. Yuan J, Cui L, Zhang WJ, et al. Repair of canine mandibular bone defects with bone marrow stromal cells and porous beta-tricalcium phosphate. Biomaterials, 2007, 28(6): 1005-1013.
6. Nishida J, Shimamura T. Methods of reconstruction for bone defect after tumor excision: a review of alternatives. Med Sci Monit, 2008, 14(8): R107-R113.
7. Bernstein P, Bornhäuser M, Günther KP, et al. Bone tissue engineering in clinical application: assessment of the current situation. Orthopade, 2009, 38(11): 1029-1037.
8. Wang MO, Vorwald CE, Dreher ML, et al. Evaluating 3D-printed biomaterials as scaffolds for vascularized bone tissue engineering. Adv Mater, 2015, 27(1): 138-144.
9. Koupaei N, Karkhaneh A, Daliri Joupari M. Preparation and characterization of (PCL-crosslinked-PEG)/hydroxyapatite as bone tissue engineering scaffolds. J Biomed Mater Res A, 2015, 103(12): 3919-3926.
10. Yu H, Zeng X, Deng C, et al. Exogenous VEGF introduced by bioceramic composite materials promotes the restoration of bone defect in rabbits. Biomed Pharmacother, 2018, 98: 325-332.
11. Liu Z, Yuan X, Liu M, et al. Antimicrobial peptide combined with BMP2-modified mesenchymal stem cells promotes calvarial repair in an osteolytic model. Mol Ther, 2018, 26(1): 199-207.
12. Kobayashi N, Hashimoto Y, Otaka A, et al. Porous alpha-tricalcium phosphate with immobilized basic fibroblast growth factor enhances bone regeneration in a canine mandibular bone defect model. Materials (Basel), 2016, 9(10): pii: E853.
13. Silva de Oliveira JC, Okamoto R, Sonoda CK, et al. Evaluation of the osteoinductive effect of PDGF-BB associated with different carriers in bone regeneration in bone surgical defects in rats. Implant Dent, 2017, 26(4): 559-566.
14. Gao Y, Li C, Wang H, et al. Acceleration of bone-defect repair by using A-W MGC loaded with BMP2 and triple point-mutant HIF1α-expressing BMSCs. J Orthop Surg Res, 2015, 10: 83.
15. Liu X, Zhang G, Hou C, et al. Vascularized bone tissue formation induced by fiber-reinforced scaffolds cultured with osteoblasts and endothelial cells. Biomed Res Int, 2013, 2013: 854917.
16. 赵萍萍, 刘陶文. Ang/Tie2 系统与肿瘤血管生成的关系. 医学综述, 2011, 17(21): 3250-3252.
17. Augustin HG, Koh GY, Thurston G, et al. Control of vascular morphogenesis and homeostasis through the angiopoietin-Tie system. Nat Rev Mol Cell Biol, 2009, 10(3): 165-177.
18. Huang JJ, Shi YQ, Li RL, et al. Angiogenesis effect of therapeutic ultrasound on HUVECs through activation of the PI3K-Akt-eNOS signal pathway. Am J Transl Res, 2015, 7(6): 1106-1115.
19. 殷建, 殷照阳, 杨海源, 等. 自噬现象及其在脊髓缺血再灌注损伤中作用的研究进展. 中国脊柱脊髓杂志, 2015, 25(6): 557-562.
20. Jiang F. Autophagy in vascular endothelial cells. Clin Exp Pharmacol Physiol, 2016, 43(11): 1021-1028.
21. Goyal A, Gubbiotti MA, Chery DR, et al. Endorepellin-evoked autophagy contributes to angiostasis. J Biol Chem, 2016, 291(37): 19245-19256.
22. Li R, Du J, Chang Y. Role of autophagy in hypoxia-induced angiogenesis of RF/6A cells in vitro. Curr Eye Res, 2016, 41(12): 1566-1570.
23. Kumar S, Guru SK, Pathania AS, et al. Autophagy triggered by magnolol derivative negatively regulates angiogenesis. Cell Death Dis, 2013, 4: e889.
24. Fiedler U, Scharpfenecker M, Koidl S, et al. The Tie-2 ligand angiopoietin-2 is stored in and rapidly released upon stimulation from endothelial cell Weibel-Palade bodies. Blood, 2004, 103(11): 4150-4156.
25. Gill KA, Brindle NP. Angiopoietin-2 stimulates migration of endothelial progenitors and their interaction with endothelium. Biochem Biophys Res Commun, 2005, 336(2): 392-396.
26. Helfrich I, Edler L, Sucker A, et al. Angiopoietin-2 levels are associated with disease progression in metastatic malignant melanoma. Clin Cancer Res, 2009, 15(4): 1384-1392.
27. Sfiligoi C, de Luca A, Cascone I, et al. Angiopoietin-2 expression in breast cancer correlates with lymph node invasion and short survival. Int J Cancer, 2003, 103(4): 466-474.
28. Goede V, Coutelle O, Neuneier J, et al. Identification of serum angiopoietin-2 as a biomarker for clinical outcome of colorectal cancer patients treated with bevacizumab-containing therapy. Br J Cancer, 2010, 103(9): 1407-1414.
29. Wang X, Bullock AJ, Zhang L, et al. The role of angiopoietins as potential therapeutic targets in renal cell carcinoma. Transl Oncol, 2014, 7(2): 188-195.
30. Scholz A, Harter PN, Cremer S, et al. Endothelial cell-derived angiopoietin-2 is a therapeutic target in treatment-naive and bevacizumab-resistant glioblastoma. EMBO Mol Med, 2016, 8(1): 39-57.
31. Falcón BL, Hashizume H, Koumoutsakos P, et al. Contrasting actions of selective inhibitors of angiopoietin-1 and angiopoietin-2 on the normalization of tumor blood vessels. Am J Pathol, 2009, 175(5): 2159-2170.
32. Coxon A, Bready J, Min H, et al. Context-dependent role of angiopoietin-1 inhibition in the suppression of angiogenesis and tumor growth: implications for AMG 386, an angiopoietin-1/2-neutralizing peptibody. Mol Cancer Ther, 2010, 9(10): 2641-2651.
33. Theelen TL, Lappalainen JP, Sluimer JC, et al. Angiopoietin-2 blocking antibodies reduce early atherosclerotic plaque development in mice. Atherosclerosis, 2015, 241(2): 297-304.
34. He J, Bao Q, Zhang Y, et al. Yes-associated protein promotes angiogenesis via signal transducer and activator of transcription 3 in endothelial cells. Circ Res, 2018, 122(4): 591-605.
35. Williams JA, Thomas AM, Li G, et al. Tissue specific induction of p62/Sqstm1 by farnesoid X receptor. PLoS One, 2012, 7(8): e43961.
36. Yin ZY, Yin J, Huo YF, et al. Rapamycin facilitates fracture healing through inducing cell autophagy and suppressing cell apoptosis in bone tissues. Eur Rev Med Pharmacol Sci, 2017, 21(21): 4989-4998.
37. Li X, Wei J, Aifantis KE, et al. Current investigations into magnetic nanoparticles for biomedical applications. J Biomed Mater Res A, 2016, 104(5): 1285-1296.
38. Li X, Gao H, Uo M, et al. Effect of carbon nanotubes on cellular functions in vitro. J Biomed Mater Res A, 2009, 91(1): 132-139.

Chinese Journal of Reparative and Reconstructive Surgery

Local injection of angiopoietin 2 promotes angiogenesis in tissue engineered bone and repair of bone defect with autophagy induction in vivo

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