Upregulation of transforming growth factor-beta1 and vascular endothelial growth factor in cultured keloid fibroblasts: relevance to angiogenic activity

Arch Dermatol Res. 2005 Oct;297(4):161-9. doi: 10.1007/s00403-005-0596-2. Epub 2005 Oct 26.

Abstract

Keloids are tumor-like lesions that result from excessive scar formation during healing of wounds. Histologically, keloids show an increased blood vessel density compared with normal dermis or normal scars. However, the angiogenic activity of keloid fibroblasts remains unknown. In this study, we investigated angiogenic activity of keloid fibroblasts. Transforming growth factor-beta1 (TGF-beta1) and vascular endothelial growth factor (VEGF) were investigated as elements of the angiogenic factors. Expressions of TGF-beta1 and VEGF in conditioned medium were measured with enzyme-linked immunosorbent assay (EIA) and Northern blot analysis. Participation of TGF-beta1 in the production of VEGF was also investigated with addition of TGF-beta1 and a neutralizing anti-TGF-beta1 antibody. A modified Boyden chamber assay was performed to assess the chemotactic activity of vascular endothelial cells. Angiogenic activity in vivo was evaluated by neovascularization of nodules formed by implantation of fibroblasts into severe combined immunodeficiency (SCID) mice. EIA showed that the concentrations of TGF-beta1 and VEGF in conditioned medium were increased 2.5- and 6-fold, respectively, after the culture of keloid fibroblasts compared with normal fibroblasts. Northern blot analysis revealed that the expression of TGF-beta1 and VEGF mRNA was upregulated 3.6- and 6-fold, respectively, in keloid fibroblasts compared with normal fibroblasts. Addition of TGF-beta1 to keloid fibroblast cultures increased VEGF production by 3.5-fold, while there was a 6-fold in culture of normal fibroblasts. A neutralizing anti-TGF-beta1 antibody reduced VEGF secretion to control levels, suggesting that TGF-beta1 mediated the upregulation of VEGF expression. A modified Boyden chamber assay demonstrated that the chemotactic activity of vascular endothelial cells was more strongly (sevenfold) induced by keloid fibroblast-conditioned medium than by normal fibroblast-conditioned medium. Anti-VEGF antibody inhibited chemotaxis to basal levels. When SCID mice underwent implantation of fibroblasts into the back, the nodules formed by keloid fibroblasts were three times larger than those formed by normal fibroblasts. Although abundant neovascularization was observed in keloid fibroblast nodules, neovascularization was scarce in normal fibroblast nodules. Both in vitro and in vivo studies confirmed the significantly higher angiogenic activity of keloid fibroblasts compared with normal fibroblasts, and TGF-beta1 and VEGF were clearly shown to be involved. These results suggest that angiogenesis in keloids is promoted by endogenous TGF-beta1 and VEGF.

MeSH terms

  • Adolescent
  • Adult
  • Animals
  • Antibodies / immunology
  • Blotting, Northern
  • Cells, Cultured
  • Endothelium, Vascular / chemistry
  • Endothelium, Vascular / pathology
  • Endothelium, Vascular / physiopathology
  • Enzyme-Linked Immunosorbent Assay
  • Female
  • Fibroblasts / chemistry
  • Fibroblasts / metabolism*
  • Fibroblasts / pathology
  • Humans
  • Keloid / genetics
  • Keloid / immunology
  • Keloid / pathology
  • Keloid / physiopathology*
  • Male
  • Mice
  • Mice, SCID
  • Neovascularization, Pathologic*
  • RNA, Messenger / analysis
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Transforming Growth Factor beta / genetics*
  • Transforming Growth Factor beta / immunology
  • Transforming Growth Factor beta / metabolism
  • Up-Regulation*
  • Vascular Endothelial Growth Factor A / genetics*
  • Vascular Endothelial Growth Factor A / metabolism

Substances

  • Antibodies
  • RNA, Messenger
  • Transforming Growth Factor beta
  • Vascular Endothelial Growth Factor A