EFFECT OF MACROPHAGE MIGRATION INHIBITORY FACTOR ON VASCULAR REPAIR OF STEROID-INDUCED AVASCULAR NECROSIS OF FEMORAL HEAD IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

MAJun , ZHANGChen , ZHANGEryang , BANWenrui , LÜLeifeng , DANGXiaoqian ,  WANGKunzheng

The First Department of Orthopaedics, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an Shaanxi, 710004, P. R. China;

Corresponding author：

WANGKunzheng, Email: wangkunzheng1955@163.com

Keywords：

Steroid-induced avascular necrosis of femoral head; Macrophage migration inhibitory factor; Vascular repair; In vitro experiment

DOI：

10.7507/1002-1892.20160202

Video：

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Objective To interpret the mechanisms of vascular repair disorders in steroid-induced avascular necrosis of the femoral head (SANFH) via detection of the changes of proliferation, migration, and macrophage migration inhibitory factor (MIF)/vascular endothelial growth factor (VEGF) expressions of endothelial cells (ECs) under hypoxia/glucocorticoid. Methods According to culture conditions, human umbilical vein ECs (HUVECs) at passage 3 were divided into group A (normal), group B (1.0×10^-6 mol/L dexamethasone), group C (hypoxia), and group D (hypoxia+1.0×10^-6 mol/L dexamethasone). The cell activity was detected by AlamarBlue; the number of viable cells was detected in live/dead cell staining; the cell morphology was observed after cytoskeleton staining; cell migration ability was compared by scratch test; and the levels of MIF and VEGF expressions were detected by ELISA. Results At 24 hours after culture, the cell activity and the number of living cells in group C were significantly higher than those in the other 3 groups, showing significant difference between groups (P < 0.05), and group D had the worst cell activity and least living cells. Cytoskeleton staining showed that cells had normal morphology in groups A and B; cells had rich cytoskeleton and secretion granules in group C; cytoskeleton form disorder and nucleus pyknosis were observed in group D. Scratch test showed that the cell migration ability of group C was strongest while cell migration ability of group D was weakest. Accumulated concentration of MIF and VEGF in 4 groups significantly increased with time extending. Accumulated concentration of MIF in group C were significantly higher than that in other 3 groups at each time point (P < 0.05). Within 24 hours after intervention, stage concentration of MIF during 1-8 hours was significantly lower than that during 0-1 hour and 8-24 hours in every group (P < 0.05). Stage concentration of MIF in group C was significantly higher than other groups during 0-1 hour and 8-24 hours (P < 0.05). Within 2 hours after intervention, stage concentration of MIF in 4 groups during 0.5-1 hour was significantly higher than that during other stages (P < 0.05). Accumulated concentration of VEGF in group C was significantly higher than that in other groups at 8 and 24 hours (P < 0.05). The stage concentration of VEGF in groups C and D during 8-24 hours was significantly higher than that during 0-1 hour and 1-8 hours (P < 0.05). There was no significant difference in the stage concentration of VEGF within and among group A, B, C, and D at every stage within 2 hours after intervention (P > 0.05). Conclusion In hypoxia environment, the proliferation and migration of ECs is enhanced, and the secretion of VEGF and MIF is increased. High concentration of dexamethasone will suppress the process above, which induces vascular repair disorders and aggravating SANFH.

Citation： MAJun, ZHANGChen, ZHANGEryang, BANWenrui, LÜLeifeng, DANGXiaoqian, WANGKunzheng. EFFECT OF MACROPHAGE MIGRATION INHIBITORY FACTOR ON VASCULAR REPAIR OF STEROID-INDUCED AVASCULAR NECROSIS OF FEMORAL HEAD IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(8): 998-1005. doi: 10.7507/1002-1892.20160202 Copy

1.	Koo K, Mont MA, Jones LC, et al. Epidemiology of osteonecrosis in the USA//Osteonecrosis. Berlin:Springer, 2014:39-45.
2.	Kang JS, Moon KH, Kwon DG, et al. The natural history of asymptomatic osteonecrosis of the femoral head. Int Orthop, 2013, 37(3):379-384.
3.	Seamon J, Keller T, Saleh J, et al. The pathogenesis of nontraumatic osteonecrosis. Arthritis, 2012, 2012:601763.
4.	Zhao FC, Li ZR, Guo KJ. Clinical analysis of osteonecrosis of the femoral head induced by steroids. Orthop Surg, 2012, 4(1):28-34.
5.	Weinstein RS. Glucocorticoid-induced osteoporosis and osteonecrosis. Endocrinol Metab Clin North Am, 2012, 41(3):595-611.
6.	Vijay V, Tyagi V, Mehtani A. Comment on Zeng et al.:Vascularised greater trochanter bone graft, combined free iliac flap and impaction bone grafting for osteonecrosis of the femoral head. Int Orthop, 2013, 37(7):1421.
7.	Lee YJ, Lee JS, Kang EH, et al. Vascular endothelial growth factor polymorphisms in patients with steroid-induced femoral head osteonecrosis. J Orthop Res, 2012, 30(1):21-27.
8.	Zhang C, Ma J, Li M, et al. 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.
9.	Haddad FS, Glynn A, Javad Parvizi MD. The pathology and management of osteonecrosis of the hip//The young adult hip in sport. London:Springer, 2014:63-72.
10.	Xu X, Pang J, Chen Y, et al. Macrophage migration inhibitory factor (MIF) deficiency exacerbates aging-induced cardiac remodeling and dysfunction despite improved inflammation:role of autophagy regulation. Sci Rep, 2016, 6:22488.
11.	Bruchfeld A, Wendt M, Miller EJ. Macrophage migration inhibitory factor in clinical kidney disease. Front Immunol, 2016, 7:8.
12.	Zapatero MC, Pérez P, Vázquez MJ, et al. Discovery of novel inhibitors of the tautomerase activity of macrophage migration inhibitory factor (MIF). J Biomol Screen, 2016, 21(5):446-458.
13.	Roger T, Schneider A, Weier M, et al. High expression levels of macrophage migration inhibitory factor sustain the innate immune responses of neonates. Proc Natl Acad Sci U S A, 2016, 113(8): E997-E1005.
14.	Huang WS, Duan LP, Huang B, et al. Macrophage migration inhibitory factor (MIF) family in arthropods:Cloning and expression analysis of two MIF and one D-dopachrome tautomerase (DDT) homologues in mud crabs, Scylla paramamosain. Fish Shellfish Immunol, 2016, 50:142-149.
15.	Wen F, Zheng J, Yu J, et al. Macrophage migration inhibitory factor in the regulation of myoblast proliferation and differentiation. Biosci Biotechnol Biochem, 2016, 80(7):1313-1320.
16.	Wang D, Luo L, Chen W, et al. Significance of the vascular endothelial growth factor and the macrophage migration inhibitory factor in the progression of hepatocellular carcinoma. Oncol Rep, 2014, 31(3):1199-1204.
17.	Chen D, Xia M, Hayford C, et al. Expression of human tissue factor pathway inhibitor on vascular smooth muscle cells inhibits secretion of macrophage migration inhibitory factor and attenuates atherosclerosis in ApoE-/-mice. Circulation, 2015, 131(15):1350-1360.
18.	Giannice R, Erreni M, Allavena P, et al. Chemokines mRNA expression in relation to the Macrophage Migration Inhibitory Factor (MIF) mRNA and Vascular Endothelial Growth Factor (VEGF) mRNA expression in the microenvironment of endometrial cancer tissue and normal endometrium:a pilot study. Cytokine, 2013, 64(2):509-515.
19.	Asare Y, Schmitt M, Bernhagen J. The vascular biology of macrophage migration inhibitory factor (MIF). Expression and effects in inflammation, atherogenesis and angiogenesis. Thromb Haemost, 2013, 109(3):391-398.
20.	Zis O, Zhang S, Dorovini-Zis K, et al. Hypoxia signaling regulates macrophage migration inhibitory factor (MIF) expression in stroke. Mol Neurobiol, 2015, 51(1):155-167.
21.	Choi M, Lee M, Rhim T. Dexamethasone-conjugated polyethylenimine/MIF siRNA complex regulation of particulate matter-induced airway inflammation. Biomaterials, 2013, 34(30): 7453-7461.
22.	Gagala J, Buraczynska M, Mazurkiewicz T, et al. Prevalence of genetic risk factors related with thrombophilia and hypofibrinolysis in patients with osteonecrosis of the femoral head in Poland. BMC Musculoskelet Disord, 2013, 14:264.
23.	Chen SB, Hu H, Gao YS, et al. Prevalence of clinical anxiety, clinical depression and associated risk factors in chinese young and middleaged patients with osteonecrosis of the femoral head. PLoS One, 2015, 10(3):e0120234.
24.	van der Jagt D, Mokete L, Pietrzak J, et al. Osteonecrosis of the femoral head:evaluation and treatment. J Am Acad Orthop Surg, 2015, 23(2):69-70.
25.	Harvey TW, Engel JE, Chade AR. Vascular endothelial growth factor and podocyte protection in chronic hypoxia:effects of endothelin-A receptor antagonism. Am J Nephrol, 2016, 43(2):74-84.
26.	Janaszak-Jasiecka A, Bartoszewska S, Kochan K, et al. miR-429 regulates the transition between Hypoxia-Inducible Factor (HIF) 1A and HIF3A expression in human endothelial cells. Sci Rep, 2016, 6: 22775.
27.	Geenen IL, Molin DG, van den Akker NM, et al. Endothelial cells (ECs) for vascular tissue engineering:venous ECs are less thrombogenic than arterial ECs. J Tissue Eng Regen Med, 2015, 9(5): 564-576.
28.	Ferrelli F, Pastore D, Capuani B, et al. Serum glucocorticoid inducible kinase (SGK)-1 protects endothelial cells against oxidative stress and apoptosis induced by hyperglycaemia. Acta Diabetol, 2015, 52(1):55-64.
29.	Xia W, Hou M. Macrophage migration inhibitory factor induces autophagy to resist hypoxia/serum deprivation-induced apoptosis via the AMP-activated protein kinase/mammalian target of rapamycin signaling pathway. Mol Med Rep, 2016, 13(3):2619-2626.
30.	Calan M, Kume T, Yilmaz O, et al. A possible link between luteinizing hormone and macrophage migration inhibitory factor levels in polycystic ovary syndrome. Endocr Res, 2016.[Epub ahead of print].

1. Koo K, Mont MA, Jones LC, et al. Epidemiology of osteonecrosis in the USA//Osteonecrosis. Berlin:Springer, 2014:39-45.
2. Kang JS, Moon KH, Kwon DG, et al. The natural history of asymptomatic osteonecrosis of the femoral head. Int Orthop, 2013, 37(3):379-384.
3. Seamon J, Keller T, Saleh J, et al. The pathogenesis of nontraumatic osteonecrosis. Arthritis, 2012, 2012:601763.
4. Zhao FC, Li ZR, Guo KJ. Clinical analysis of osteonecrosis of the femoral head induced by steroids. Orthop Surg, 2012, 4(1):28-34.
5. Weinstein RS. Glucocorticoid-induced osteoporosis and osteonecrosis. Endocrinol Metab Clin North Am, 2012, 41(3):595-611.
6. Vijay V, Tyagi V, Mehtani A. Comment on Zeng et al.:Vascularised greater trochanter bone graft, combined free iliac flap and impaction bone grafting for osteonecrosis of the femoral head. Int Orthop, 2013, 37(7):1421.
7. Lee YJ, Lee JS, Kang EH, et al. Vascular endothelial growth factor polymorphisms in patients with steroid-induced femoral head osteonecrosis. J Orthop Res, 2012, 30(1):21-27.
8. Zhang C, Ma J, Li M, et al. 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.
9. Haddad FS, Glynn A, Javad Parvizi MD. The pathology and management of osteonecrosis of the hip//The young adult hip in sport. London:Springer, 2014:63-72.
10. Xu X, Pang J, Chen Y, et al. Macrophage migration inhibitory factor (MIF) deficiency exacerbates aging-induced cardiac remodeling and dysfunction despite improved inflammation:role of autophagy regulation. Sci Rep, 2016, 6:22488.
11. Bruchfeld A, Wendt M, Miller EJ. Macrophage migration inhibitory factor in clinical kidney disease. Front Immunol, 2016, 7:8.
12. Zapatero MC, Pérez P, Vázquez MJ, et al. Discovery of novel inhibitors of the tautomerase activity of macrophage migration inhibitory factor (MIF). J Biomol Screen, 2016, 21(5):446-458.
13. Roger T, Schneider A, Weier M, et al. High expression levels of macrophage migration inhibitory factor sustain the innate immune responses of neonates. Proc Natl Acad Sci U S A, 2016, 113(8): E997-E1005.
14. Huang WS, Duan LP, Huang B, et al. Macrophage migration inhibitory factor (MIF) family in arthropods:Cloning and expression analysis of two MIF and one D-dopachrome tautomerase (DDT) homologues in mud crabs, Scylla paramamosain. Fish Shellfish Immunol, 2016, 50:142-149.
15. Wen F, Zheng J, Yu J, et al. Macrophage migration inhibitory factor in the regulation of myoblast proliferation and differentiation. Biosci Biotechnol Biochem, 2016, 80(7):1313-1320.
16. Wang D, Luo L, Chen W, et al. Significance of the vascular endothelial growth factor and the macrophage migration inhibitory factor in the progression of hepatocellular carcinoma. Oncol Rep, 2014, 31(3):1199-1204.
17. Chen D, Xia M, Hayford C, et al. Expression of human tissue factor pathway inhibitor on vascular smooth muscle cells inhibits secretion of macrophage migration inhibitory factor and attenuates atherosclerosis in ApoE-/-mice. Circulation, 2015, 131(15):1350-1360.
18. Giannice R, Erreni M, Allavena P, et al. Chemokines mRNA expression in relation to the Macrophage Migration Inhibitory Factor (MIF) mRNA and Vascular Endothelial Growth Factor (VEGF) mRNA expression in the microenvironment of endometrial cancer tissue and normal endometrium:a pilot study. Cytokine, 2013, 64(2):509-515.
19. Asare Y, Schmitt M, Bernhagen J. The vascular biology of macrophage migration inhibitory factor (MIF). Expression and effects in inflammation, atherogenesis and angiogenesis. Thromb Haemost, 2013, 109(3):391-398.
20. Zis O, Zhang S, Dorovini-Zis K, et al. Hypoxia signaling regulates macrophage migration inhibitory factor (MIF) expression in stroke. Mol Neurobiol, 2015, 51(1):155-167.
21. Choi M, Lee M, Rhim T. Dexamethasone-conjugated polyethylenimine/MIF siRNA complex regulation of particulate matter-induced airway inflammation. Biomaterials, 2013, 34(30): 7453-7461.
22. Gagala J, Buraczynska M, Mazurkiewicz T, et al. Prevalence of genetic risk factors related with thrombophilia and hypofibrinolysis in patients with osteonecrosis of the femoral head in Poland. BMC Musculoskelet Disord, 2013, 14:264.
23. Chen SB, Hu H, Gao YS, et al. Prevalence of clinical anxiety, clinical depression and associated risk factors in chinese young and middleaged patients with osteonecrosis of the femoral head. PLoS One, 2015, 10(3):e0120234.
24. van der Jagt D, Mokete L, Pietrzak J, et al. Osteonecrosis of the femoral head:evaluation and treatment. J Am Acad Orthop Surg, 2015, 23(2):69-70.
25. Harvey TW, Engel JE, Chade AR. Vascular endothelial growth factor and podocyte protection in chronic hypoxia:effects of endothelin-A receptor antagonism. Am J Nephrol, 2016, 43(2):74-84.
26. Janaszak-Jasiecka A, Bartoszewska S, Kochan K, et al. miR-429 regulates the transition between Hypoxia-Inducible Factor (HIF) 1A and HIF3A expression in human endothelial cells. Sci Rep, 2016, 6: 22775.
27. Geenen IL, Molin DG, van den Akker NM, et al. Endothelial cells (ECs) for vascular tissue engineering:venous ECs are less thrombogenic than arterial ECs. J Tissue Eng Regen Med, 2015, 9(5): 564-576.
28. Ferrelli F, Pastore D, Capuani B, et al. Serum glucocorticoid inducible kinase (SGK)-1 protects endothelial cells against oxidative stress and apoptosis induced by hyperglycaemia. Acta Diabetol, 2015, 52(1):55-64.
29. Xia W, Hou M. Macrophage migration inhibitory factor induces autophagy to resist hypoxia/serum deprivation-induced apoptosis via the AMP-activated protein kinase/mammalian target of rapamycin signaling pathway. Mol Med Rep, 2016, 13(3):2619-2626.
30. Calan M, Kume T, Yilmaz O, et al. A possible link between luteinizing hormone and macrophage migration inhibitory factor levels in polycystic ovary syndrome. Endocr Res, 2016.[Epub ahead of print].

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EFFECT OF MACROPHAGE MIGRATION INHIBITORY FACTOR ON VASCULAR REPAIR OF STEROID-INDUCED AVASCULAR NECROSIS OF FEMORAL HEAD IN VITRO

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