title: Mitochondrial Membrane Potential and Nuclear and Gene Expression Changes During Human Disc Cell Apoptosis: In Vitro and In Vivo Annulus Findings.
abstract: METHODS
  A study using cultured human annulus cells and human annular tissue.
  
  
  OBJECTIVE
  To further explore and define mitochondrial mechanisms related to disc cell apoptosis in vitro and in vivo.
  
  
  BACKGROUND
  Mitochondrial-dependent intrinsic signaling pathways are a well-recognized component of apoptosis (programmed cell death). Disc cell apoptosis is important because it is a major mechanism by which cell numbers decrease during disc degeneration. Our objective was to further explore and define mitochondrial mechanisms related to disc cell apoptosis.
  
  
  METHODS
  High-content screening techniques were used to study nuclear morphology and mitochondrial membrane potentials in cultured annulus cells. Gene expression in annulus tissue was studied with microarray analysis.
  
  
  RESULTS
  Cultured cells showed significantly increased nuclear size (an indicator of apoptosis) with increasing Thompson grade (P < 0.00001 by analysis of variance). A significant negative correlation for mitochondrial potential (which results from the difference in electrical potential generated by the electrochemical gradient across the inner membrane of the mitochondrion) versus Thompson grade was identified in cultured human annulus cells in control conditions (r = 0.356, P < 0.0001). When exposed to the K ionophore valinomycin at sublethal levels to induce apoptosis, a significant reduction in mitochondrial potential was identified versus nontreated cells. Gene expression patterns in more degenerated Thompson grade III, IV, and V discs versus healthier grade I and II discs showed significant upregulation of a number of genes with well-recognized apoptosis roles in mitochondrial potential decline (ITM2B, beta-2-microglobulin, and cathepsin B, DAP, GAS1, and PDCD5) and TNF-α associations (cathepsin B, RAC1, and PPT1).
  
  
  CONCLUSIONS
  Data presented here show the in vivo expression of apoptosis-related genes associated with the loss of mitochondrial membrane integrity and decreased mitochondrial membrane potential with increasing Thompson scores. These data, which mimic our novel, direct cell-based in vitro findings, stress the importance of mitochondrial changes related to apoptosis and TNF-α during human disc degeneration.
  
  
  METHODS
  N/A.
authors.affiliation: From the Department of Orthopaedic Surgery, Carolinas HealthCare System, Charlotte, NC.
authors.forename: Helen E
authors.initials: HE
authors.lastname: Gruber
authors.forename: Gretchen L
authors.initials: GL
authors.lastname: Hoelscher
authors.forename: Synthia
authors.initials: S
authors.lastname: Bethea
authors.forename: Edward N
authors.initials: EN
authors.lastname: Hanley
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D053144:Cell Nucleus Shape; D002478:Cells, Cultured; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation; D057166:High-Throughput Screening Assays; D006801:Humans; D007403:Intervertebral Disc / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D055959:Intervertebral Disc Degeneration / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology*; D020411:Oligonucleotide Array Sequence Analysis; D011188:Potassium / Q000378:metabolism; D014634:Valinomycin / Q000633:toxicity
chemical_list: D014634:Valinomycin; D011188:Potassium