Microarray cluster analysis of irradiated growth plate zones following laser microdissection.
OBJECTIVE
Genes and pathways involved in early growth plate chondrocyte recovery after fractionated irradiation were sought as potential targets for selective radiorecovery modulation.


METHODS
Three groups of six 5-week male Sprague-Dawley rats underwent fractionated irradiation to the right tibiae over 5 days, totaling 17.5 Gy, and then were killed at 7, 11, and 16 days after the first radiotherapy fraction. The growth plates were collected from the proximal tibiae bilaterally and subsequently underwent laser microdissection to separate reserve, perichondral, proliferative, and hypertrophic zones. Differential gene expression was analyzed between irradiated right and nonirradiated left tibia using RAE230 2.0 GeneChip microarray, compared between zones and time points and subjected to functional pathway cluster analysis with real-time polymerase chain reaction to confirm selected results.


RESULTS
Each zone had a number of pathways showing enrichment after the pattern of hypothesized importance to growth plate recovery, yet few met the strictest criteria. The proliferative and hypertrophic zones showed both the greatest number of genes with a 10-fold right/left change at 7 days after initiation of irradiation and enrichment of the most functional pathways involved in bone, cartilage, matrix, or skeletal development. Six genes confirmed by real-time polymerase chain reaction to have early upregulation included insulin-like growth factor 2, procollagen type I alpha 2, matrix metallopeptidase 9, parathyroid hormone receptor 1, fibromodulin, and aggrecan 1.


CONCLUSIONS
Nine overlapping pathways in the proliferative and hypertrophic zones (skeletal development, ossification, bone remodeling, cartilage development, extracellular matrix structural constituent, proteinaceous extracellular matrix, collagen, extracellular matrix, and extracellular matrix part) may play key roles in early growth plate radiorecovery.
Damron
Timothy A
TA
SUNY Upstate Medical University, Syracuse, NY, USA. damront@upstate.edu
Zhang
Mingliang
M
Pritchard
Meredith R
MR
Middleton
Frank A
FA
Horton
Jason A
JA
Margulies
Bryan M
BM
Strauss
Judith A
JA
Farnum
Cornelia E
CE
Spadaro
Joseph A
JA
D053674:Aggrecans / Q000235:genetics / Q000378:metabolism; D000818:Animals; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D019583:Dose Fractionation, Radiation; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D000071235:Fibromodulin; D020869:Gene Expression Profiling; D006132:Growth Plate / Q000378:metabolism / Q000473:pathology / Q000528:radiation effects*; D006984:Hypertrophy / Q000235:genetics / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000235:genetics / Q000378:metabolism; D053685:Laser Therapy; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D042282:Microdissection / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000235:genetics / Q000378:metabolism; D013977:Tibia / Q000528:radiation effects; D015854:Up-Regulation
D053674:Aggrecans; D024042:Collagen Type I; D016326:Extracellular Matrix Proteins; C000604334:Fmod protein, rat; D011509:Proteoglycans; D044168:Receptor, Parathyroid Hormone, Type 1; D000071235:Fibromodulin; D007335:Insulin-Like Growth Factor II; D020780:Matrix Metalloproteinase 9