Dong LI, Qi Chun SONG, Yan ZHAO, Da Peng DUAN

The effect of endostatin on angiogenesis and osteogenesis of steroid-induced osteonecrosis of the femoral head in a rabbit model

Objective: This study aimed to investigate whether endostatin, a crucial anti-angiogenic factor, plays a negative role in angiogenesis and osteogenesis and aggravates the progression of osteonecrosis of the femoral head induced by steroid use in a rabbit model. Methods: 66 New Zealand white rabbits were randomly divided into four groups: glucocorticoid model (GC) group (GC group, n = 18), glucocorticoid model and endostatin group (GC&ES group, n = 18), ES group (ES group, n = 18), and blank control group (CON group, n = 12). In the GC group, 10 μg/ kg lipopolysaccharide (LPS) was intravenously injected into the ear margin, and 24h after LPS injection, 20 mg/kg GC methylprednisolone (MPS) was injected into the gluteus muscle three times, each time at an interval of 24h. The animals of the GC&ES group were given as same treatment as the GC group, except for the addition of ES. MPS was not used in the ES group and CON group. ES group was only given ES, while the CON group was only given the same amount of normal saline. All animals successfully established models of femoral head necrosis, and then the difference among the Immunohistochemistry, Quantitative polymerase chain reaction (qPCR) analysis, Enzyme-linked immunosorbent assay, Biomechanical test, etracyclline-calcein double labeling, and Van Gieson staining indices were compared among the four groups. Results: The combination of MPS and LPS was successful in establishing the femoral head necrosis model in New Zealand white rabbits. The incidence of osteonecrosis after MPS and LPS intervention was 70% (7/10), while that plus ES was 100% (10/10). At the same time, after MPS and LPS intervention, while the empty bone lacuna rate of the femoral head was significantly increased, the number of osteoblasts was decreased. Also, the expressions of CD31 positive cells, Runx2, Osterix, COL1A1, and VEGF mRNA in the femoral head were decreased, and the levels of osteogenesis-related protein b-ALP, OCN, and angiogenic factor VEGF in the femoral head were decreased. The percentage of the trabecular bone area (%Tb.Ar), trabecular thickness (Tb.Th), trabecular number (Tb.N), labeled perimeter percent (%L.Pm), mineral apposition rate (MAR), and bone formation rate (BFR/BS) in the femoral head after MPs and LPS intervention detected by tetracycline calcein double labeling and Van Gieson staining decreased significantly, except trabecular separation (Tb.Sp) increased significantly. The compressive strength (CS), elastic modulus (EM), and strain energy (SE) of the femoral head examed by biomechanical measurement decreased significantly. All the above changes were more obvious after adding ES intervention. ES mRNA in the femoral head was undifferentiated and increased in the GC, ES, and GC&ES group compared with group CON.

PDF

___

1. Torgashin AN, Rodionova SS, Shumsky AA, et al. Treatment of aseptic necrosis of the femoral head. Clinical guidelines. Clinical guidelines. Naučnopraktičeskaâ revmatologiâ. 2021;58(6):637-645. [CrossRef]
2. Mont MA, Cherian JJ, Sierra RJ, Jones LC, Lieberman JR. Nontraumatic osteonecrosis of the femoral head: where do we stand today? A ten-year update. J Bone Joint Surg Am. 2015;97(19):1604-1627. [CrossRef]
3. Mont MA, Salem HS, Piuzzi NS, Goodman SB, Jones LC. Nontraumatic osteonecrosis of the femoral head: where do we stand today?: A 5-year update. J Bone Joint Surg Am. 2020;102(12):1084-1099. [CrossRef]
4. Mutijima E, De Maertelaer V, Deprez M, Malaise M, Hauzeur JP. The apoptosis of osteoblasts and osteocytes in femoral head osteonecrosis: its specificity and its distribution. Clin Rheumatol. 2014;33(12):1791-1795. [CrossRef]
5. Tian L, Wen Q, Dang X, You W, Fan L, Wang K. Immune response associated with toll-like receptor 4 signaling pathway leads to steroid-induced femoral head osteonecrosis. BMC Musculoskelet Disord. 2014;15:18. [CrossRef]
6. Assouline-Dayan Y, Chang C, Greenspan A, Shoenfeld Y, Gershwin ME. Pathogenesis and natural history of osteonecrosis. Semin Arthritis Rheum. 2002;32(2):94-124. [CrossRef]
7. Aaron RK. Concepts of the pathogenesis of osteonecrosis. Tech Orthop. 2001;16(1):101-104. [CrossRef]
8. Jones LC, Hungerford DS. The pathogenesis of osteonecrosis. Instr Course Lect. 2007;56(56):179-196.
9. Panin MA, Zagorodniy NV, Karchebnyi NN, Sadkov IA, Zakirova AR. Modern view on pathogenesis of non traumatic osteonecrosis. N N Priorov J Traumatol Orthop. 2017;2:69-75.
10. Huard J. Stem cells, blood vessels, and angiogenesis as major determinants for musculoskeletal tissue repair. J Orthop Res. 2019;37(6):1212-1220. [CrossRef]
11. Yamashiro DJ, Cohn SL. Angiogenesis. Pediatr Oncol. 2005.
12. Feige JJ, Pagès G, Soncin F. Molecular Mechanisms of Angiogenesis; 2014. [CrossRef]
13. Wicki A, Christofori G. The Angiogenic Switch in Tumorigenesis; Tumor Angiogenesis. 2008;1(4):67-88.
14. Sinkovics J Horák A. Angiogenesis, antiangiogenesis [Angiogenesis, anti-angiogenesis]. Orv Hetil; 1998;139(20):1269.
15. Hellsten Y, Hoier B, Gliemann L. What turns off the angiogenic switch in skeletal muscle? Exp Physiol. 2015;100(7):772-773. [CrossRef]
16. Printers C, Carmeliet p, jain r. Angiogenesis in health and disease. Nat Med. 2003;9(6):653-660.
17. Veikkola T, Alitalo K. VEGFs, receptors and angiogenesis. Semin Cancer Biol. 1999;9(3):211-220. [CrossRef]
18. Wang Y, Xia CJ, Wang BJ, Ma XW, Zhao DW. The association between vegf -634c/g polymorphisms and osteonecrosis of femoral head: A meta-analysis. Int J Clin Exp Med. 2015;8(6):9313-9319.
19. Hang D, Wang Q, Guo C, Chen Z, Yan Z. Treatment of osteonecrosis of the femoral head with vegf165 transgenic bone marrow mesenchymal stem cells in mongrel dogs. Cells Tissues Organs. 2012;195(6):495-506. [CrossRef]
20. Yamaguchi N. An analysis of the functional mechanisms of endostatin - the anti-angiogenic activity of endostatin is mediated by its multiple binding ability. Connect Tissue. 2004;36(3):171-178.
21. Abdollahi A, Hlatky L, Huber PE. Endostatin: the logic of antiangiogenic therapy. Drug Resist Updat. 2005;8(1-2):59-74. [CrossRef]
22. Ahluwalia A, Jones MK, Deng X, Sandor Z, Szabo S, Tarnawski AS. An imbalance between vegf and endostatin underlies impaired angiogenesis in gastric mucosa of aging rats. Am J Physiol Gastrointest Liver Physiol. 2013;305(4):G32 5-G332. [CrossRef]
23. Asai K, Kanazawa H, Otani K, Shiraishi S, Hirata K, Yoshikawa J. Imbalance between vascular endothelial growth factor and endostatin levels in induced sputum from asthmatic subjects. J Allergy Clin Immunol. 2002;110(4):571-575. [CrossRef]
24. Nagashima M, Asano G, Yoshino S. Imbalance in production between vascular endothelial growth factor and endostatin in patients with rheumatoid arthritis. J Rheumatol. 2000;27(10):2339-2342.
25. Takeshita S, Kawamura Y, Takabayashi H, Yoshida N, Nonoyama S. Imbalance in the production between vascular endothelial growth factor and endostatin in Kawasaki disease. Clin Exp Immunol. 2005;139(3):575-579. [CrossRef]
26. Zhang C, Ma J, Li M, Li XH, Dang XQ, Wang KZ. Repair effect of coexpression of the hVEGF and hBMP genes via an adeno-associated virus vector in a rabbit model of early steroid-induced avascular necrosis of the femoral head. Transl Res. 2015;166(3):269-280. [CrossRef]
27. Hankenson KD, Dishowitz M, Gray C, Schenker M. Angiogenesis in bone regeneration. Injury. 2011;42(6):556-561. [CrossRef]
28. Grosso A, Burger MG, Lunger A, Schaefer DJ, Banfi A, Di Maggio N. It takes two to tango: coupling of angiogenesis and osteogenesis for bone regeneration. Front Bioeng Biotechnol. 2017;5:68. [CrossRef]
29. Yamaguchi N, Anand-Apte B, Lee M, et al. Endostatin inhibits vegf-induced endothelial cell migration and tumor growth independently of zinc binding. EMBO J. 1999;18(16):4414-4423. [CrossRef]
30. Holstein JH, Karabin-Kehl B, Scheuer C, et al. Endostatin inhibits callus remodeling during fracture healing in mice. J Orthop Res. 2013;31(10):1579-1584. [CrossRef]
31. Lazaridou M, Gkalitsiou V, Maloutas D, Volitakis A. Bevacizumab and osteonecrosis of the jaw: report of a case and review of the literature. Hellen Arch Oral Maxillofac Surg. 2014;15(2)77-86.
32. Wan YY, Tian GY, Guo HS, et al. Endostatin, an angiogenesis inhibitor, ameliorates bleomycin-induced pulmonary fibrosis in rats. Respir Res. 2013;14(1):56. [CrossRef].
33. Yamamoto T, Irisa T, Sugioka Y, et al. Effects of pulse methylprednisolone on bone and marrow tissues: corticosteroid-induced osteonecrosis in rabbits. Arthritis Rheum. 1997;40(11):2055-2064. [CrossRef].
34. Qin L, Zhang G, Sheng H, Yeung K, Leung K. Multiple bioimaging modalities in evaluation of an experimental osteonecrosis induced by combination of lipopolysaccharide and methylprednisolone. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi Zhongguo Xiufu Chong Jian Waike Zazhi Chin J Reparat Reconstr Surg. 2008;22(3):258-264.
35. Folkman J. Antiangiogenesis in cancer therapy--endostatin and its mechanisms of action. Exp Cell Res. 2006;312(5):594-607. [CrossRef]
36. Sodha NR, Clements RT, Boodhwani M, Xu SH, Sellke FW. Endostatin and angiostatin are increased in diabetic patients with coronary artery disease and associated with impaired coronary collateral formation. AJP Heart Circ Physiol. 2008;296(2):H428-H434.
37. Panchal VR, Rehman J, Nguyen AT, et al. Reduced pericardial levels of endostatin correlate with collateral development in patients with ischemic heart disease. J Am Coll Cardiol. 2004;43(8):1383-1387. [CrossRef]
38. Marneros AG, She H, Zambarakji H, Hashizume H, Olsen BR. Endogenous endostatin inhibits choroidal neovascularization. FASEB J. 2008;21(14): 3809-3818.
39. Bai YJ, Huang LZ, Zhou AY, Zhao M, Yu WZ, Li XX. Antiangiogenesis effects of endostatin in retinal neovascularization. J Ocul Pharmacol Ther. 2013;29(7): 619-626. [CrossRef]
40. Gammal SE, Moennig B, Hoffmann K, Altmeyer P. Three-Dimensional Reconstruction of Terminal Blood Spaces in the Proximal Tibia Metaphysis of the Growing Rat — a Model to Study Normal Angiogenesis. Springer Berlin Heidelberg; 1995.
41. Sipola A, Ilvesaro J, Birr E, et al. Endostatin inhibits endochondral ossification. J Gene Med. 2007;9(12):1057-1064. [CrossRef]
42. Lau KW, Kapur S, Kesavan C, Baylink DJ. Up-regulation of the wnt, estrogen receptor, insulin-like growth factor-i, and bone morphogenetic protein pathways in C57BL/6J osteoblasts as opposed to c3h/hej osteoblasts in part contributes to the differential anabolic response to fluid shear. J Biol Chem. 2006;281(14):9576-9588. [CrossRef]