APOE-knockout in rabbits causes loss of cells in nucleus pulposus and enhances the levels of inflammatory catabolic cytokines damaging the intervertebral disc matrix

Autoři: Anja Beierfuß aff001;  Monika Hunjadi aff002;  Andreas Ritsch aff002;  Christian Kremser aff003;  Claudius Thomé aff004;  Demissew Shenegelegn Mern aff004
Působiště autorů: Laboratory Animal Facility, Medical University of Innsbruck, Innsbruck, Austria aff001;  Department of Internal Medicine I, Medical University of Innsbruck, Innsbruck, Austria aff002;  Department of Radiology, Medical University of Innsbruck, Innsbruck, Austria aff003;  Department of Neurosurgery, Medical University of Innsbruck, Innsbruck, Austria aff004
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225527


Rabbits with naturally high levels of cholesterol ester transfer protein (CETP), unlike rodents, have become an interesting animal model for the study of lipid metabolism and atherosclerosis, as they have similarities to humans in lipid metabolism, cardiovascular physiology and susceptibility to develop atherosclerosis. Rodents, such as mice, are not prone to atherosclerosis as they lack the mass and activity of CETP, as a key player in lipoprotein metabolism. Recently, APOE-knockout in rabbits has been shown to promote atherosclerosis and associated premature IVD degeneration that mimic the symptoms of atherosclerosis and structural changes of IVDs in humans. Here we examined whether APOE-knockout promoted IVD degeneration in rabbits is associated with imbalanced inflammatory catabolic activities, as the underlying problem of biological deterioration that mimic the symptoms of advanced IVD degeneration in humans. We analysed in lumbar nucleus pulposus (NP) of APOE-knockout rabbits the cell viabilities and the intracellular levels of inflammatory, catabolic, anti-catabolic and anabolic proteins derogating IVD matrix. Grades of IVD degeneration were evaluated by magnetic resonance imaging. NP cells were isolated from homozygous APOE-knockout and wild-type New Zealand White rabbits of similar age. Three-dimensional cell culture with low-glucose was completed in alginate hydrogel. Cell proliferation and intracellular levels of target proteins were examined by MTT and ELISA assays. Alike human NP cells of different disc degeneration grades, NP cells of APOE-knockout and wild-type rabbits showed significantly different in vivo cell population densities (p<0.0001) and similar in vitro proliferation rates. Furthermore, they showed differences in overexpression of selective inflammatory and catabolic proteins (p<0.0001) similar to those found in human NP cells of different disc degeneration grades, such as IL-1β, TNF-α, ADAMTS-4, ADAMTS-5 and MMP-3. This study showed that premature IVD degeneration in APOE-knockout rabbits was promoted by the accumulation of selective inflammatory catabolic factors that enhanced imbalances between catabolic and anabolic factors mimicking the symptoms of advanced IVD degeneration in humans. Thus, APOE-knockout rabbits could be used as a promising model for therapeutic approaches of degenerative disc disorders.

Klíčová slova:

Atherosclerosis – Collagens – Cytokines – Inflammation – Magnetic resonance imaging – Nutrients – Rabbits – Cell signaling structures


1. Makarand VR, Irving MS. Role of Cytokines in Intervertebral Disc Degeneration: Pain and Disc-content. Nat Rev Rheumatol. 2014; 10(1):44–56. doi: 10.1038/nrrheum.2013.160 24166242

2. De Geer CM. Cytokine Involvement in Biological Inflammation Related to Degenerative Disorders of the Intervertebral Disk: A Narrative Review. J Chiropr Med. 2018; 17(1):54–62. doi: 10.1016/j.jcm.2017.09.003 29628809

3. Johnson ZI, Schoepflin ZR, Choi H, Shapiro IM, Risbud MV. Disc in flames: Roles of TNF-α and IL-1β in intervertebral disc degeneration. Eur Cell Mater. 2015; 30:104–16. 26388614

4. Roberts S, Evans H, Trivedi J, Menage J. Histology and pathology of the human intervertebral disc. J Bone Joint Surg Am. 2006; 88 Suppl 2:10–4.

5. Walker BF. The prevalence of low back pain: a systematic review of the literature from 1966 to 1998. J Spinal Disord. 2000; 13:205–217. doi: 10.1097/00002517-200006000-00003 10872758

6. Martin BI, Deyo RA, Mirza SK, Turner JA, Comstock BA, Hollingworth W, et al. Expenditures and health status among adults with back and neck problems. JAMA. 2008; 299:656–664. doi: 10.1001/jama.299.6.656 18270354

7. Stewart WF, Ricci JA, Chee E, Morganstein D, Lipton R. Lost productive time and cost due to common pain conditions in the US workforce. JAMA. 2003; 290:2443–2454. doi: 10.1001/jama.290.18.2443 14612481

8. Battié MC, Videman T, Kaprio J, Gibbons LE, Gill K, Manninen H, et al. The Twin Spine Study: contributions to a changing view of disc degeneration. Spine J. 2009; 9:47–59. doi: 10.1016/j.spinee.2008.11.011 19111259

9. Adams MA, Freeman BJ, Morrison HP, Nelson IW, Dolan P. Mechanical initiation of intervertebral disc degeneration. Spine 2000; 25:1625–1636. doi: 10.1097/00007632-200007010-00005 10870137

10. Wang D, Nasto LA, Roughley P, Leme AS, Houghton AM, Usas A, et al. Spine degeneration in a murine model of chronic human tobacco smokers. Osteoarthritis Cartilage 2012; 20:896–905. doi: 10.1016/j.joca.2012.04.010 22531458

11. Cheung KM, Karppinen J, Chan D, Ho DW, Song YQ, Sham P, et al. Prevalence and pattern of lumber magnetic resonance changes in a population study of one thousand fourty-three individuals. Spine 2009; 34:934–940. doi: 10.1097/BRS.0b013e3181a01b3f 19532001

12. Kanayama M, Togawa D, Takahashi C, Terai T, Hashimoto T. Cross-sectional magnetic resonance imaging study of lumbar disc degeneration in 200 healthy individuals. J Neurosurgery Spine 2009; 11:501–507.

13. Buckwalter JA. Aging and degeneration of the human intervertebral disc. Spine 1995; 20(11):1307–1314. doi: 10.1097/00007632-199506000-00022 7660243

14. Kalb S, Martirosyan NL, Kalani MYS, Broc GG, Theodore N. Genetics of the Degenerated Intervertebral Disc. World Neurosurg. 2012; 77(3–4):491–501. doi: 10.1016/j.wneu.2011.07.014 22120330

15. Mern DS, Beierfuß A, Thomé C, Hegewald AA. Enhancing human nucleus pulposus cells for biological treatment approaches of degenerative intervertebral disc diseases: a systematic review. J Tissue Eng Regen Med. 2014; 8(12):925–36. doi: 10.1002/term.1583 22927290

16. Urban JP, Smith S, Fairbank JC. Nutrition of the intervertebral disc. Spine 2004; 29:2700–2709. doi: 10.1097/01.brs.0000146499.97948.52 15564919

17. Rajasekaran S, Babu JN, Arun R, Armstrong BR, Shetty AP, Murugan S. ISSLS prize winner: A study of diffusion in human lumbar discs: a serial magnetic resonance imaging study documenting the influence of the endplate on diffusion in normal and degenerate discs. Spine 2004; 29:2654–67. doi: 10.1097/01.brs.0000148014.15210.64 15564914

18. Grunhagen T, Shirazi-Adl A, Fairbank JC, Urban JP. Intervertebral disk nutrition: a review of factors influencing concentrations of nutrients and metabolites. Orthop Clin North Am. 2011; 42(4):465–477. doi: 10.1016/j.ocl.2011.07.010 21944584

19. Rajasekaran S, Venkatadass K, Naresh Babu J, Ganesh K, Shetty AP. Pharmacological enhancement of disc diffusion and differentiation of healthy, ageing and degenerated discs: Results from in-vivo serial post-contrast MRI studies in 365 human lumbar discs. Eur Spine J. 2008; 17(5):626–643. doi: 10.1007/s00586-008-0645-6 18357472

20. Leino-Arjas P, Kaila-Kangas L, Solovieva S, Riihimäki H, Kirjonen J, Reunanen A. Serum lipids and low back pain: an association? A follow-up study of a working population sample. Spine 2006; 31(9):1032–1037. doi: 10.1097/01.brs.0000214889.31505.08 16641781

21. Leino-Arjas P, Kauppila L, Kaila-Kangas L, Shiri R, Heistaro S, Heliövaara M. Serum lipids in relation to sciatica among Finns. Atherosclerosis. 2008;197(1):43–9. doi: 10.1016/j.atherosclerosis.2007.07.035 17825307

22. Crock HV, Yoshizawa H. The blood supply of the lumbar vertebral column. Clin Orthop Relat Res. 1976;(115):6–21. 1253499

23. Chiras J, Morvan G, Merland JJ. The angiographic appearances of the normal intercostal and lumbar arteries. Analysis and the anatomic correlation of the lateral branches. J Neuroradiol. 1979; 6(3):169–196. 553951

24. Kauppila LI. Blood supply of the lower thoracic and lumbosacral regions. Postmortem aortography in 38 young adults. Acta Radiol. 1994;35(6):541–544. 7946674

25. Kauppila LI, Tallroth K. Postmortem angiographic findings for arteries supplying the lumbar spine: their relationship to low-back symptoms. J Spinal Disord. 1993;6(2):124–129. 8504223

26. Zhdanov VS, Sternby NH, Vikhert AM, Galakhov IE. Development of atherosclerosis over a 25 year period: an epidemiological autopsy study in males of 11 towns. Int J Cardiol. 1999;68(1):95–106. doi: 10.1016/s0167-5273(98)00339-8 10077406

27. Zhao CQ, Wang LM, Jiang LS, Dai LY. The cell biology of intervertebral disc aging and degeneration. Ageing Res Rev 2007; 6: 247–261. doi: 10.1016/j.arr.2007.08.001 17870673

28. Mahley RW. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 1988; 240(4852):622–630. doi: 10.1126/science.3283935 3283935

29. Mabuchi H, Itoh H, Takeda M, Kajinami K, Wakasugi T, Koizumi J, et al. A young type III hyperlipoproteinemic patient associated with apolipoprotein E deficiency. Metabolism. 1989; 38(2):115–9. doi: 10.1016/0026-0495(89)90249-7 2492364

30. Bellosta S, Mahley RW, Sanan DA, Murata J, Newland DL, Taylor JM, et al. Macrophage-specific expression of human apolipoprotein E reduces atherosclerosis in hypercholesterolemic apolipoprotein E-null mice. J Clin Invest 1995; 96(5):2170–179. doi: 10.1172/JCI118271 7593602

31. Mahley RW, Ji ZS. Remnant lipoprotein metabolism: key pathways involving cell-surface heparan sulfate proteoglycans and apolipoprotein E. J Lipid Res. 1999; 40(1):1–16. 9869645

32. Ji D, Zhao G, Songstad A, Cui X, Weinstein EJ. Efficient creation of an APOE knockout rabbit. Transgenic Res. 2015; 24(2):227–235. doi: 10.1007/s11248-014-9834-8 25216764

33. Niimi M, Yang D, Kitajima S, Ning B, Wang C, Li S, et al. ApoE knockout rabbits: A novel model for the study of human hyperlipidemia. Atherosclerosis. 2016;245:187–193. doi: 10.1016/j.atherosclerosis.2015.12.002 26724529

34. Beierfuß A, Dietrich H, Kremser C, Hunjadi M, Ritsch A, Rülicke T, et al. Knockout of Apolipoprotein E in rabbit promotes premature intervertebral disc degeneration: A new in vivo model for therapeutic approaches of spinal disc disorders. PLoS One 2017; 12(11):e0187564. doi: 10.1371/journal.pone.0187564 29099857

35. Mern DS, Fontana J, Beierfuß A, Thomé C, Hegewald AA. A combinatorial relative mass value evaluation of endogenous bioactive proteins in three-dimensional cultured nucleus pulposus cells of herniated intervertebral discs: identification of potential target proteins for gene therapeutic approaches. PLoS One 2013; 8(11):e81467. doi: 10.1371/journal.pone.0081467 24278441

36. Mern DS, Beierfuβ A, Fontana J, Thomé C, Hegewald AA. Imbalanced protein expression patterns of anabolic, catabolic, anti-catabolic and inflammatory cytokines in degenerative cervical disc cells: new indications for gene therapeutic treatments of cervical disc diseases. PLoS One 2014; 9(5):e96870. doi: 10.1371/journal.pone.0096870 24804684

37. Mähler M, Berard M, Feinstein R, Gallagher A, Illgen-Wicke B, Pritchett-Coring K, et al. FELASA recommendations for the health monitoring of mouse, rat, hamster, guinea pig and rabbit colonies in breeding and experimental units. Laboratory Animals. 2014;48(3):178–192. doi: 10.1177/0023677213516312 24496575

38. Pfirrmann CW, Metzdorf A, Zanetti M, Hodler J, Boos N. Magnetic resonance classification of lumbar intervertebral disc degeneration. Spine. 2001;26(17):1873–1878. doi: 10.1097/00007632-200109010-00011 11568697

39. Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics. 1977;33(1):159–174. 843571

40. Liebscher T, Haefeli M, Wuertz K, Nerlich AG, Boos N. Age-related variation in cell density of human lumbar intervertebral discs. Spine 2011;36:153–159. doi: 10.1097/BRS.0b013e3181cd588c 20671592

41. Bibby SR, Urban JP. Effect of nutrient deprivation on the viability of intervertebral disc cells. Eur Spine J. 2004; 13(8):695–701. doi: 10.1007/s00586-003-0616-x 15048560

42. Soukane DM, Shirazi-Adl A, Urban JP. Computation of coupled diffusion of oxygen, glucose and lactic acid in an intervertebral disc. J Biomech. 2007; 40(12):2645–2654. doi: 10.1016/j.jbiomech.2007.01.003 17336990

43. Boos N, Weissbach S, Rohrbach H, Weiler C, Spratt KF, Nerlich AG. Classification of age-related changes in lumbar intervertebral discs: 2002 Volvo Award in basic science. Spine 2002; 27(23):2631–2644. doi: 10.1097/00007632-200212010-00002 12461389

44. Solberg LA, Strong JP. Risk factors and atherosclerotic lesions. A review of autopsy studies. Arteriosclerosis. 1983;3(3):187–198. doi: 10.1161/01.atv.3.3.187 6342587

45. Le Maitre CL, Hoyland JA, Freemont AJ. Catabolic cytokine expression in degenerate and herniated human intervertebral discs: IL-1beta and TNF-alpha expression profile. Arthritis Res Ther. 2007; 9(4):R77. doi: 10.1186/ar2275 17688691

46. Wang J, Markova D, Anderson DG, Zheng Z, Shapiro IM, Risbud MV. TNF-α and IL-1β promote a disintegrin-like and metalloprotease with thrombospondin type I motif-5-mediated aggrecan degradation through syndecan-4 in intervertebral disc. J Biol Chem. 2011; 286:39738–39749. doi: 10.1074/jbc.M111.264549 21949132

47. Malfait AM, Liu RQ, Ijiri K, Komiya S, Tortorella MD. Inhibition of ADAM-TS4 and ADAM-TS5 prevents aggrecan degradation in osteoarthritic cartilage. J Biol Chem. 2002; 277:22201–22208. doi: 10.1074/jbc.M200431200 11956193

48. Wang ZH, Yang ZQ, He XJ, Kamal BE, Xing Z. Lentivirus mediated knockdown of aggrecanase-1 and -2 promotes chondrocyte-engineered cartilage formation in vitro. Biotechnol Bioeng. 2010; 107(4):730–736. doi: 10.1002/bit.22862 20632367

49. Mern DS, Tschugg A, Hartmann S, Thomé C. Self-complementary adeno-associated virus serotype 6 mediated knockdown of ADAMTS4 induces long-term and effective enhancement of aggrecan in degenerative human nucleus pulposus cells: A new therapeutic approach for intervertebral disc disorders. PLoS One. 2017; 12(2):e0172181. doi: 10.1371/journal.pone.0172181 28207788

50. Vo NV, Hartman RA, Yurube T, Jacobs LJ, Sowa GA, Kang JD. Expression and regulation of metalloproteinases and their inhibitors in intervertebral disc aging and degeneration. Spine J. 2013; 13(3): 331–341. doi: 10.1016/j.spinee.2012.02.027 23369495

51. Mern DS, Thomé C. Identification and characterization of human nucleus pulposus cell specific serotypes of adeno-associated virus for gene therapeutic approaches of intervertebral disc disorders. BMC Musculoskelet Disord. 2015; 16: 341. doi: 10.1186/s12891-015-0799-4 26552484

52. Mern DS, Tschugg A, Hartmann S, Thomé C. Self-complementary adeno-associated virus serotype 6 mediated knockdown of ADAMTS4 induces long-term and effective enhancement of aggrecan in degenerative human nucleus pulposus cells: A new therapeutic approach for intervertebral disc disorders. PLoS One. 2017; 12(2), e0172181. doi: 10.1371/journal.pone.0172181 28207788

Článek vyšel v časopise


2019 Číslo 11