Gene expression profiles from hypertrophic scar fibroblasts before and after IL-6 stimulation.
The structural rearrangement of collagen fibres in hypertrophic scar causes abnormal contracture, low tensile strength, and raised scars, which cause functional impairment and disfigurement. It is hypothesized that changes in the genes of cytokines, extracellular matrix proteins, and proteins regulating programmed cell death are related to hypertrophic scar formation. To test this hypothesis, fibroblasts were cultured from hypertrophic scars and their response to interleukin-6 (IL-6) stimulation was studied by defining their gene expression profiles. Affymetrix gene chip analysis was used to identify up- or down-regulation in the 12 625 genes present in the affymetrix array. RT-PCR and ELISA assays were used to validate microarray expression profiles further. Comparison of gene profiles showed an increase of 12 genes in hypertrophic scar fibroblasts compared with normal skin fibroblasts, while the expression of 14 genes decreased. Thirty-three genes were affected by IL-6 treatment in the hypertrophic scar fibroblasts, while 57 genes were affected in normal skin fibroblasts. Messenger RNA to beta-actin ratios for matrix metalloproteinase-1 (MMP-1) and MMP-3 were increased with IL-6 in normal skin fibroblasts from 2.43 +/- 0.06 to 5.50 +/- 0.45 and from 0.75 +/- 0.09 to 1.98 +/- 0.01, respectively. No change in these matrix metalloproteinases could be shown with IL-6 stimulation in hypertrophic scar fibroblasts. Secreted protein levels of pro-MMP-1 and MMP-3 were elevated in the supernatants from normal skin fibroblasts from 2.00 +/- 0.09 and 1.72 +/- 0.10 ng/ml to 4.60 +/- 0.12 and 3.41 +/- 0.20 ng/ml, respectively, after treatment with IL-6 (p < 0.05). No changes were observed in hypertrophic scar fibroblasts treated with IL-6. Values are means +/- SEM. The absence of any up-regulation of MMP-1 and MMP-3 in hypertrophic scar fibroblasts, in response to IL-6, suggests that suppression of matrix metalloproteinases may play a role in the excessive accumulation of collagen formed in hypertrophic scars. While the pathogenesis of abnormal hypertrophic scars remains poorly understood, the use of gene expression arrays may prove helpful in identifying the mechanisms responsible for this type of abnormal scar formation and in formulating an effective therapeutic protocol.
Dasu
Mohan R K
MR
Shriners Hospitals for Children, and Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77550, USA.
Hawkins
Hal K
HK
Barrow
Robert E
RE
Xue
Hui
H
Herndon
David N
DN
D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D017439:Cicatrix, Hypertrophic / Q000378:metabolism* / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology*; D008297:Male; D020781:Matrix Metalloproteinase 1 / Q000096:biosynthesis / Q000235:genetics; D019278:Matrix Metalloproteinase 3 / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000166:cytology / Q000378:metabolism
D015850:Interleukin-6; D019278:Matrix Metalloproteinase 3; D020781:Matrix Metalloproteinase 1