Rabbit annulus fibrosus cell apoptosis induced by mechanical overload via a mitochondrial apoptotic pathway

Abstract

In order to investigate the apoptotic pathway of rabbit annulus fibrosus (AF) cells induced by mechanical overload, an experimental air-pressure model was established in this study to pressurize the rabbit AF cells in vitro. Cells were randomly divided into five groups in which the cells were exposed to a continuous pressure of 1.1 MPa for different lengths of time (0, 5, 12, 24 and 36 h). The cell proliferation and apoptosis were detected by cell counting kit-8 (CCK-8) assay and flow cytometry; the alterations in mitochondrial membrane potential were measured by fluorescence microscopy and fluorescence spectrophotometer; the activities of caspase-8 and 9 were determined by spectrophotometry. The results showed that after the cells were subjected to the pressure for 24 or 36 h, the cell proliferation was inhibited; the ratio of cell apoptosis was increased; the mitochondrial membrane potential was decreased; the activity of caspase-9 was enhanced; no activity changes were observed in caspase-8. The results suggested that treatment with a pressure of 1.1 MPa for more than 24 h can lead to the proliferation inhibition and the apoptosis of rabbit AF cells in vitro, and the mitochondrial-dependent pathway is implicated in the pressure-induced AF cell apoptosis.

Keywords

pressure / annulus fibrosus / apoptosis / mitochondrial membrane potential / caspase

References

[1] KasraM., MerrymanW.D., LovelessK.N., et al.. Frequency response of pig intervertebral disc cells subjected to dynamic hydrostatic pressure. J Orthop Res, 2006, 24(10): 1967-1973

[2] AliR., LeM.C.L., RichardsonS.M., et al.. Connective tissue growth factor expression in human intervertebral disc: implications for angiogenesis in intervertebral disc degeneration. Biotech Histochem, 2008, 83(5): 239-245

[3] HoogendoornR., DoulabiB.Z., HuangC.L., et al.. Molecular changes in the degenerated goat intervertebral disc. Spine (Phila Pa 1976), 2008, 33(16): 1714-1721

[4] LeM.C.L., FreemontA.J., HoylandJ.A.. Accelerated cellular senescence in degenerate intervertebral discs: a possible role in the pathogenesis of intervertebral disc degeneration. Arthritis Res Ther, 2007, 9(3): R45

[5] JungerS., Gantenbein-RitterB., LezuoP., et al.. Effect of limited nutrition on in situ intervertebral disc cells under simulated-physiological loading. Spine (Phila Pa 1976), 2009, 34(12): 1264-1271

[6] RajasekaranS., VenkatadassK., NareshB.J., et al.. 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

[7] TschoekeS.K., HellmuthM., HostmannA., et al.. Apoptosis of human intervertebral discs after trauma compares to degenerated discs involving both receptor-mediated and mitochondrial-dependent pathways. J Orthop Res, 2008, 26(7): 999-1006

[8] WeiA., BrisbyH., ChungS.A., et al.. Bone morphogenetic protein-7 protects human intervertebral disc cells in vitro from apoptosis. Spine J, 2008, 8(3): 466-474

[9] ZhaoC.Q., JiangL.S., DaiL.Y.. Programmed cell death in intervertebral disc degeneration. Apoptosis, 2006, 11(12): 2079-2088

[10] JonesP., GardnerL., MenageJ., et al.. Intervertebral disc cells as competent phagocytes in vitro: implications for cell death in disc degeneration. Arthritis Res Ther, 2008, 10(4): R86

[11] LotzJ.C., ChinJ.R.. Intervertebral disc cell death is dependent on the magnitude and duration of spinal loading. Spine (Phila Pa 1976), 2000, 25(12): 1477-1483

[12] WuertzK., UrbanJ.P., KlasenJ., et al.. Influence of extracellular osmolarity and mechanical stimulation on gene expression of intervertebral disc cells. J Orthop Res, 2007, 25(11): 1513-1522

[13] LeeC.R., IatridisJ.C., PovedaL., et al.. In vitro organ culture of the bovine intervertebral disc: effects of vertebral endplate and potential for mechanobiology studies. Spine (Phila Pa 1976), 2006, 31(5): 515-522

[14] KasraM., GoelV., MartinJ., et al.. Effect of dynamic hydrostatic pressure on rabbit intervertebral disc cells. J Orthop Res, 2003, 21(4): 597-603

[15] YuW.R., LiuT., FehlingsT.K., et al.. Involvement of mitochondrial signaling pathways in the mechanism of Fas-mediated apoptosis after spinal cord injury. Eur J Neurosci, 2009, 29(1): 114-131

[16] RannouF., LeeT.S., ZhouR.H., et al.. Intervertebral disc degeneration: the role of the mitochondrial pathway in annulus fibrosus cell apoptosis induced by overload. Am J Pathol, 2004, 164(3): 915-924

[17] ParkJ.B., ParkI.C., ParkS.J., et al.. Anti-apoptotic effects of caspase inhibitors on rat intervertebral disc cells. J Bone Joint Surg Am, 2006, 88(4): 771-779

[18] OzakiT., IizukaK., SuzukiM., et al.. Threshold-dependent DNA synthesis by pure pressure in human aortic smooth muscle cells: Gialpha-dependent and -independent pathways. Biochem Biophys Res Commun, 1999, 256(1): 212-217

[19] HuttonW.C., ElmerW.A., BodenS.D., et al.. The effect of hydrostatic pressure on intervertebral disc metabolism. Spine (Phila Pa 1976), 1999, 24(15): 1507-1515

[20] KroeberM., UnglaubF., GuehringT., et al.. Effects of controlled dynamic disc distraction on degenerated intervertebral discs: an in vivo study on the rabbit lumbar spine model. Spine (Phila Pa 1976), 2005, 30(2): 181-187

[21] UnglaubF., GuehringT., OmlorG., et al.. Controlled distraction as a therapeutic option in moderate degeneration of the intervertebral disc — an in vivo study in the rabbit-spine model. Z Orthop Ihre Grenzgeb, 2006, 144(1): 68-73

[22] LiuG.Z., IshiharaH., OsadaR., et al.. Nitric oxide mediates the change of proteoglycan synthesis in the human lumbar intervertebral disc in response to hydrostatic pressure. Spine (Phila Pa 1976), 2001, 26(2): 134-141

[23] Yu JS, Qiu GX, Yang BL, et al. The difference and its significance of actin in inverterbral disc cells under cyclic hydrostatic pressure in vitro. Chin J Orthopaedics, 2005, (09):52–56

[24] ArigaK., YonenobuK., NakaseT., et al.. Mechanical stress-induced apoptosis of endplate chondrocytes in organ-cultured mouse intervertebral discs: an ex vivo study. Spine (Phila Pa 1976), 2003, 28(14): 1528-1533

[25] WilkeH.J., NeefP., CaimiM., et al.. New in vivo measurements of pressures in the intervertebral disc in daily life. Spine (Phila Pa 1976), 1999, 24(8): 755-762

[26] HishikawaK., OemarB.S., NakakiT.. Static pressure regulates connective tissue growth factor expression in human mesangial cells. J Biol Chem, 2001, 276(20): 16797-16803

[27] LeM.C.L., FrainJ., FotheringhamA.P., et al.. Human cells derived from degenerate intervertebral discs respond differently to those derived from non-degenerate intervertebral discs following application of dynamic hydrostatic pressure. Biorheology, 2008, 45(5): 563-575

[28] ScoltockA.B., CidlowskiJ.A.. Activation of intrinsic and extrinsic pathways in apoptotic signaling during UV-C-induced death of Jurkat cells: the role of caspase inhibition. Exp Cell Res, 2004, 297(1): 212-223

[29] SchulerM., Bossy-WetzelE., GoldsteinJ.C., et al.. p53 induces apoptosis by caspase activation through mitochondrial cytochrome C release. J Biol Chem, 2000, 275(10): 7337-7342

[30] MazumderS., PlescaD., AlmasanA.. Caspase-3 activation is a critical determinant of genotoxic stress-induced apoptosis. Methods Mol Biol, 2008, 414: 13-21

[31] RudnerJ., Lepple-WienhuesA., BudachW., et al.. Wild-type, mitochondrial and ER-restricted Bcl-2 inhibit DNA damage-induced apoptosis but do not affect death receptor-induced apoptosis. J Cell Sci, 2001, 114(Pt23): 4161-4172

[32] AndrewsH.E., NicholsP.P., BatesD., et al.. Mitochondrial dysfunction plays a key role in progressive axonal loss in multiple sclerosis. Med Hypotheses, 2005, 64(4): 669-677

[33] vanR.B.J., VerhoevenA.J., KuijpersT.W.. Mitochondria in neutrophil apoptosis. Int J Hematol, 2006, 84(3): 199-204

[34] EevaJ., NuutinenU., RopponenA., et al.. Feedback regulation of mitochondria by caspase-9 in the B cell receptor-mediated apoptosis. Scand J Immunol, 2009, 70(6): 574-583