title: Mitochondrial gene expression in the human annulus: in vivo data from annulus cells and selectively harvested senescent annulus cells.
abstract: BACKGROUND
  Mitochondrial dysfunction is recognized during cell senescence and apoptosis, two important components of human disc aging/degeneration. We hypothesize that mitochondrial dysfunction is present in the degenerating and senescent annulus cells. The objective of the present study was to analyze gene expression profiles related to mitochondrial function in vivo.
  
  
  OBJECTIVE
  This study had two objectives in the analysis of gene expression patterns related to mitochondria in the human annulus: First, to assess human annulus cells in a genome-wide microarray analysis approach to evaluate mitochondrial gene expression in annulus tissue from degenerated compared with healthier discs. Second, to use laser capture microdissection (LCM) to selectively isolate senescent versus nonsenescent annulus cells to evaluate their mitochondrial gene expression patterns.
  
  
  METHODS
  Following approval by our Human Subjects Institutional Review Board, annulus cells from 20 human lumbar discs were analyzed for gene groups related to mitochondrial function; a subset was also analyzed, which focused on senescent versus nonsenescent annulus cells in a study of annulus cells from 10 lumbar discs.
  
  
  METHODS
  Human annulus tissue was used in molecular studies following institutional review board approval.
  
  
  METHODS
  Gene expression levels identified with microarray analyses were statistically evaluated using GeneSifter Web-based software (VizX Labs, Seattle, WA, USA).
  
  
  METHODS
  Human annulus specimens were assessed for gene expression related to mitochondrial function. Approaches used whole annulus tissue and senescent or nonsenescent annulus cells selectively harvested using LCM. Microarray data were analyzed using gene ontology searches and GeneSifter Web-based software.
  
  
  RESULTS
  Analysis of annulus cells compared mitochondrial gene expression patterns in annulus cells from more degenerated discs with patterns in annulus cells derived from healthier discs. Important findings included significant upregulation of p53 and several proapoptotic genes (including apoptosis-inducing factor, mitochondrion-associated 1, BCL2-like 11 [an apoptosis facilitator]; caspase 7 apoptosis-related cysteine peptidase; proteasome 26S subunit nonadenosine triphosphatase 10, programmed cell death 6, and reticulon 3). Methionine sulfoxide reductase (Msr), a repair enzyme that reduces methionine sulfoxide residues in proteins damaged by oxidation, was also significantly upregulated (2.02-fold increase). The gene "membrane-associated ring finger (C3HC4) 5" was significantly upregulated and relevant because it is believed to play a role in preventing cell senescence acting to regulate mitochondrial quality control. Nitric oxide synthase 3 (endothelial nitric oxide synthase [eNOS]) showed a 5.9-fold downregulation in more degenerated versus healthier annulus cells. In LCM-harvested senescent cells, Msr was significantly downregulated in senescent versus nonsenescent cells, a finding previously recognized in other types of senescent cells.
  
  
  CONCLUSIONS
  Novel data showed that significant gene expression patterns are present in the human annulus related to mitochondrial dysfunction; changes were identified in important genes involving apoptosis, eNOS and Msr expressions, and solute carrier genes. Because current research efforts are focusing on bioactive compounds for mitochondria, we suggest that future biologic cell-based therapies for annulus degeneration should also consider mitochondrial-focused therapies.
authors.affiliation: Department of Orthopaedic Surgery, Orthopaedic Research Biology, Cannon Building, Carolinas Medical Center, P.O. Box 32861, Charlotte, NC 28232, USA. helen.gruber@carolinashealthcare.org
authors.forename: Helen E
authors.initials: HE
authors.lastname: Gruber
authors.forename: John A
authors.initials: JA
authors.lastname: Watts
authors.forename: Gretchen L
authors.initials: GL
authors.lastname: Hoelscher
authors.forename: Synthia F
authors.initials: SF
authors.lastname: Bethea
authors.forename: Jane A
authors.initials: JA
authors.lastname: Ingram
authors.forename: Natalia S
authors.initials: NS
authors.lastname: Zinchenko
authors.forename: Edward N
authors.initials: EN
authors.lastname: Hanley
mesh_terms: D000328:Adult; D000368:Aged; D017209:Apoptosis / Q000235:genetics; D016922:Cellular Senescence / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D007403:Intervertebral Disc; D055959:Intervertebral Disc Degeneration / Q000235:genetics*; D061346:Laser Capture Microdissection; D008297:Male; D008875:Middle Aged; D008928:Mitochondria / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D055815:Young Adult