Senescent vs. non-senescent cells in the human annulus in vivo: cell harvest with laser capture microdissection and gene expression studies with microarray analysis.
BACKGROUND
Senescent cells are well-recognized in the aging/degenerating human disc. Senescent cells are viable, cannot divide, remain metabolically active and accumulate within the disc over time. Molecular analysis of senescent cells in tissue offers a special challenge since there are no cell surface markers for senescence which would let one use fluorescence-activated cell sorting as a method for separating out senescent cells.


METHODS
We employed a novel laser capture microdissection (LCM) design to selectively harvest senescent and non-senescent annulus cells in paraffin-embedded tissue, and compared their gene expression with microarray analysis. LCM was used to separately harvest senescent and non-senescent cells from 11 human annulus specimens.


RESULTS
Microarray analysis revealed significant differences in expression levels in senescent cells vs non-senescent cells: 292 genes were upregulated, and 321 downregulated. Genes with established relationships to senescence were found to be significantly upregulated in senescent cells vs. non-senescent cells: p38 (MPAK14), RB-Associated KRAB zinc finger, Discoidin, CUB and LCCL domain, growth arrest and DNA-damage inducible beta, p28ING5, sphingosine-1-phosphate receptor 2 and somatostatin receptor 3; cyclin-dependent kinase 8 showed significant downregulation in senescent cells. Nitric oxidase synthase 1, and heat shock 70 kDa protein 6, both of which were significantly down-regulated in senescent cells, also showed significant changes. Additional genes related to cytokines, cell proliferation, and other processes were also identified.


CONCLUSIONS
Our LCM-microarray analyses identified a set of genes associated with senescence which were significantly upregulated in senescent vs non-senescent cells in the human annulus. These genes include p38 MAP kinase, discoidin, inhibitor of growth family member 5, and growth arrest and DNA-damage-inducible beta. Other genes, including genes associated with cell proliferation, extracellular matrix formation, cell signaling and other cell functions also showed significant modulation in senescent vs non-senescent cells. The aging/degenerating disc undergoes a well-recognized loss of cells; understanding senescent cells is important since their presence further reduces the disc's ability to generate new cells to replace those lost to necrosis or apoptosis.
Gruber
Helen E
HE
Department of Orthopaedic Surgery, Carolinas Medical Center, Charlotte, NC, USA. helen.gruber@carolinashealthcare.org
Hoelscher
Gretchen L
GL
Ingram
Jane A
JA
Zinchenko
Natalia
N
Hanley
Edward N
EN
D000328:Adult; D016922:Cellular Senescence / Q000235:genetics*; D005260:Female; D020869:Gene Expression Profiling; D006651:Histocytochemistry; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology; D007834:Lasers; D008297:Male; D042282:Microdissection; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis