EXPRESSION AND SIGNIFICANCE OF HYPOXIA INDUCIBLE FACTOR 1α IN RAT MODEL OF HETEROTOPIC OSSIFICATION AFTER Achilles TENOTOMY_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YUDu ^1,2 ,  XIAOHaijun ² , XUEFeng ² , PANMingmang ¹ , JUJinyong ² , TANGGuo ¹

1. Third Affiliated Clinical College of Southern Medical University, Guangzhou Guangdong, 510515, P. R. China;
2. Department of Orthopedics, Fengxian Hospital Affiliated to Southern Medical University;

Corresponding author：

XIAOHaijun, Email: xiaohaijun89@126.com

Keywords：

Hypoxia inducible factor lα; Heterotopic ossification; Achilles tendon; Rat

DOI：

10.7507/1002-1892.20160224

Video：

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Objective To explore the role and significance of hypoxia inducible factor lα (HIF-lα) and hypoxia microenvironment in the pathogenesis of post-traumatic heterotopic ossification by detecting the expression of HIF-lα in rat model of heterotopic ossification after Achilles tenotomy. Methods A total of 140 male Sprague Dawley rats, aged 8-10 weeks, and weighing (210.1±10.6) g, were randomly divided into experimental group (n=70) and control group (n=70). In experimental group, the Achilles tendon was cut off and clamped to prepare post-traumatic heterotopic ossification model; in control group, only Achilles tendon was exposed. The general condition of rats was observed after operation, and at 2, 3, 4, 5, 6, 7, 8, 10, 12, and 14 days after operation, the Achilles tendon tissue was harvested from 6 rats for gross observation, histological observation, and immunohistochemical staining observation, and real-time fluorescence quantitative PCR and Western blot were used to detect the expressions of HIF-lα gene and protein at different time points in 2 groups. The X-ray films were taken and histological examination was done at 10 weeks after operation to evaluate the formation of heterotopic ossification. Results During the experiment, 1 rat died in experimental group at 3 days after operation, and the other rats survived to the end of the experiment. Gross and histological staining showed that the Achilles tendon had no obvious change, with normal tendon structure in control group at each time point. In experimental group, atrophy and necrosis of Achilles tendon stump were observed, with infiltration of inflammatory cells; and the hardness of Achilles tendon tissue gradually increased with the time; there were a large number of irregular connective tissue and cartilage cells. When compared with control group, the HIF-lα mRNA and protein expressions were significantly increased in experimental group at each time point (P < 0.05). Immunohistochemical staining showed that HIF-lα was positive in experimental group. According to the results of X-ray films and histological examination at 10 weeks after operation, heterotopic ossification was found in experimental group, but no heterotopic ossification in control group. Conclusion The expression of HIF-lα significantly increases at early stage of post-traumatic heterotopic ossification after Achilles tenotomy, suggesting that the local hypoxia microenvironment plays an important role in the pathogenesis of heterotopic ossification.

Citation： YUDu, XIAOHaijun, XUEFeng, PANMingmang, JUJinyong, TANGGuo. EXPRESSION AND SIGNIFICANCE OF HYPOXIA INDUCIBLE FACTOR 1α IN RAT MODEL OF HETEROTOPIC OSSIFICATION AFTER Achilles TENOTOMY. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(9): 1098-1103. doi: 10.7507/1002-1892.20160224 Copy

1.	Downey J, Lauzier D, Kloen P, et al. Prospective heterotopic ossification progenitors in adult human skeletal muscle. Bone, 2015, 71:164-170.
2.	Wu J, Xue F, Shi WZ, et al. Fetuin A is down-regulated in rats during heterotopic ossification after Achilles tenotomy. Biotech Histochem, 2016, 91(4):229-236.
3.	Agarwal S, Loder SJ, Brownley C, et al. BMP signaling mediated by constitutively active Activin type 1 receptor (ACVR1) results in ectopic bone formation localized to distal extremity joints. Dev Biol, 2015, 400(2):202-209.
4.	Lin L, Shen Q, Leng H, et al. Synergistic inhibition of endochondral bone formation by silencing HIF-lα and Runx2 in trauma-induced heterotopic ossification. Mol Ther, 2011, 19(8):1426-1432.
5.	张丹, 任利玲.缺氧诱导因子1α在组织工程成骨和成血管中的作用.中国修复重建外科杂志, 2016, 30(4):504-509.
6.	Zimmermann SM, Würgler-Hauri CC, Wanner GA, et al. Echinomycin in the prevention of heterotopic ossification-an experimental antibiotic agent shows promising results in a murine model. Injury, 2013, 44(4):570-575.
7.	Winkler S, Niedermair T, Füchtmeier B, et al. The impact of hypoxia on mesenchymal progenitor cells of human skeletal tissue in the pathogenesis of heterotopic ossification. Int Orthop, 2015, 39(12):2495-2501.
8.	Pfander D, Cramer T, Schipani E, et al. HIF-lalpha controls extracellular matrix synthesis by epiphyseal chondrocytes. J Cell Sci, 2003, 116(Pt 9):1819-1826.
9.	石伟哲, 肖海军, 薛锋, 等.基质金属蛋白酶9在大鼠跟腱异位骨化模型中的动态变化研究.中国修复重建外科杂志, 2014, 28(9):1133-1138.
10.	吴江, 肖海军, 薛锋, 等.选择性与非选择性环氧化酶2抑制剂预防大鼠跟腱异位骨化的实验研究.中国修复重建外科杂志, 2014, 28(3):371-376.
11.	Majmundar AJ, Wong WJ, Simon MC. Hypoxia-inducible factors and the response to hypoxic stress. Mol Cell, 2010, 40(2):294-309.
12.	Kaplan FS, Glaser DL, Hebela N, et al. Heterotopic ossification. J Am Acad Orthop Surg, 2004, 12(2):116-125.
13.	Kan L, Kessler JA. Evaluation of the cellular origins of heterotopic ossification. Orthopedics, 2014, 37 (5):329-340.
14.	Agarwal S, Loder S, Brownley C, et al. Inhibition of Hif1α prevents both trauma-induced and genetic heterotopic ossification. Proc Natl Acad Sci U S A, 2016, 113(3):E338-E347.
15.	Wang CJ, Huang KE, Sun YC, et al. VEGF modulates angiogenesis and osteogenesis in shockwave-promoted fracture healing in rabbits. J Surg Res, 2011, 171(1):114-119.
16.	Mistry IN, Smith PJ, Wilson DI, et al. Probing the epigenetic regulation of HIF-1α transcription in developing tissue. Mol BioSyst, 2015, 11(10):2780-2785.
17.	Tomanek RJ, Lotun K, Clark EB, et al. VEGF and bFGF stimulate myocardial vascularization in embryonic chick. Am J Physiol, 1998, 274(5 Pt 2):H1620-H1626.
18.	Dilling CF, Wada AM, Lazard ZW, et al. Vessel formation is induced prior to the appearance of cartilage in BMP-2-mediated heterotopic ossification. J Bone Miner Res, 2010, 25(5):1147-1156.
19.	Kaplan FS, Glaser DL, Shore EM, et al. Hematopoietic stem-cell contribution to ectopic skeletogenesis. J Bone Joint Surg (Am), 2007, 89(2):347-357.
20.	Lin L, Shen Q, Xue T, et al. Heterotopic ossification induced by Achilles tenotomy via endochondral bone formation:expression of bone and cartilage related genes. Bone, 2010, 46(2):425-431.

1. Downey J, Lauzier D, Kloen P, et al. Prospective heterotopic ossification progenitors in adult human skeletal muscle. Bone, 2015, 71:164-170.
2. Wu J, Xue F, Shi WZ, et al. Fetuin A is down-regulated in rats during heterotopic ossification after Achilles tenotomy. Biotech Histochem, 2016, 91(4):229-236.
3. Agarwal S, Loder SJ, Brownley C, et al. BMP signaling mediated by constitutively active Activin type 1 receptor (ACVR1) results in ectopic bone formation localized to distal extremity joints. Dev Biol, 2015, 400(2):202-209.
4. Lin L, Shen Q, Leng H, et al. Synergistic inhibition of endochondral bone formation by silencing HIF-lα and Runx2 in trauma-induced heterotopic ossification. Mol Ther, 2011, 19(8):1426-1432.
5. 张丹, 任利玲.缺氧诱导因子1α在组织工程成骨和成血管中的作用.中国修复重建外科杂志, 2016, 30(4):504-509.
6. Zimmermann SM, Würgler-Hauri CC, Wanner GA, et al. Echinomycin in the prevention of heterotopic ossification-an experimental antibiotic agent shows promising results in a murine model. Injury, 2013, 44(4):570-575.
7. Winkler S, Niedermair T, Füchtmeier B, et al. The impact of hypoxia on mesenchymal progenitor cells of human skeletal tissue in the pathogenesis of heterotopic ossification. Int Orthop, 2015, 39(12):2495-2501.
8. Pfander D, Cramer T, Schipani E, et al. HIF-lalpha controls extracellular matrix synthesis by epiphyseal chondrocytes. J Cell Sci, 2003, 116(Pt 9):1819-1826.
9. 石伟哲, 肖海军, 薛锋, 等.基质金属蛋白酶9在大鼠跟腱异位骨化模型中的动态变化研究.中国修复重建外科杂志, 2014, 28(9):1133-1138.
10. 吴江, 肖海军, 薛锋, 等.选择性与非选择性环氧化酶2抑制剂预防大鼠跟腱异位骨化的实验研究.中国修复重建外科杂志, 2014, 28(3):371-376.
11. Majmundar AJ, Wong WJ, Simon MC. Hypoxia-inducible factors and the response to hypoxic stress. Mol Cell, 2010, 40(2):294-309.
12. Kaplan FS, Glaser DL, Hebela N, et al. Heterotopic ossification. J Am Acad Orthop Surg, 2004, 12(2):116-125.
13. Kan L, Kessler JA. Evaluation of the cellular origins of heterotopic ossification. Orthopedics, 2014, 37 (5):329-340.
14. Agarwal S, Loder S, Brownley C, et al. Inhibition of Hif1α prevents both trauma-induced and genetic heterotopic ossification. Proc Natl Acad Sci U S A, 2016, 113(3):E338-E347.
15. Wang CJ, Huang KE, Sun YC, et al. VEGF modulates angiogenesis and osteogenesis in shockwave-promoted fracture healing in rabbits. J Surg Res, 2011, 171(1):114-119.
16. Mistry IN, Smith PJ, Wilson DI, et al. Probing the epigenetic regulation of HIF-1α transcription in developing tissue. Mol BioSyst, 2015, 11(10):2780-2785.
17. Tomanek RJ, Lotun K, Clark EB, et al. VEGF and bFGF stimulate myocardial vascularization in embryonic chick. Am J Physiol, 1998, 274(5 Pt 2):H1620-H1626.
18. Dilling CF, Wada AM, Lazard ZW, et al. Vessel formation is induced prior to the appearance of cartilage in BMP-2-mediated heterotopic ossification. J Bone Miner Res, 2010, 25(5):1147-1156.
19. Kaplan FS, Glaser DL, Shore EM, et al. Hematopoietic stem-cell contribution to ectopic skeletogenesis. J Bone Joint Surg (Am), 2007, 89(2):347-357.
20. Lin L, Shen Q, Xue T, et al. Heterotopic ossification induced by Achilles tenotomy via endochondral bone formation:expression of bone and cartilage related genes. Bone, 2010, 46(2):425-431.

Chinese Journal of Reparative and Reconstructive Surgery

EXPRESSION AND SIGNIFICANCE OF HYPOXIA INDUCIBLE FACTOR 1α IN RAT MODEL OF HETEROTOPIC OSSIFICATION AFTER Achilles TENOTOMY

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