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Upregulation of transforming growth factor-β1 and vascular endothelial growth factor in cultured keloid fibroblasts: relevance to angiogenic activity

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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-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) were investigated as elements of the angiogenic factors. Expressions of TGF-β1 and VEGF in conditioned medium were measured with enzyme-linked immunosorbent assay (EIA) and Northern blot analysis. Participation of TGF-β1 in the production of VEGF was also investigated with addition of TGF-β1 and a neutralizing anti-TGF-β1 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-β1 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-β1 and VEGF mRNA was upregulated 3.6- and 6-fold, respectively, in keloid fibroblasts compared with normal fibroblasts. Addition of TGF-β1 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-β1 antibody reduced VEGF secretion to control levels, suggesting that TGF-β1 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-β1 and VEGF were clearly shown to be involved. These results suggest that angiogenesis in keloids is promoted by endogenous TGF-β1 and VEGF.

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References

  1. Appleton I, Brown NJ, Willoughby DA (1996) Apoptosis, necrosis, and proliferation: possible implications in the etiology of keloids. Am J Pathol 149:1441–1447

    PubMed  CAS  Google Scholar 

  2. Ashcroft GS, Dodsworth J, van Boxtel E, Tarnuzzer RW, Horan MA, Schultz GS, Ferguson MW (1997) Estrogen accelerates cutaneous wound healing associated with an increase in TGF-β1 levels. Nat Med 3:1209–1215

    Article  PubMed  CAS  Google Scholar 

  3. Beer TW, Baldwin HC, Goddard JR, Gallagher PJ, Wright DH (1998) Angiogenesis in pathological and surgical scars. Hum Pathol 29:1273–1278

    Article  PubMed  CAS  Google Scholar 

  4. Berse B, Hunt JA, Diegel RJ, Morganelli P, Yeo K, Brown F, Fava RA (1999) Hypoxia augments cytokine (transforming growth factor-beta (TGF-β) and IL-1)-induced vascular endothelial growth factor secretion by human synovial fibroblasts. Clin Exp Immunol 115:176–182

    Article  PubMed  CAS  Google Scholar 

  5. Bettinger DA, Yager DR, Diegelmann RF, Cohen IK (1996) The effect of TGF-β on keloid fibroblast proliferation and collagen synthesis. Plast Reconstr Surg 98:827–833

    Article  PubMed  CAS  Google Scholar 

  6. Brogi E, Wu T, Namiki A, Isner JM (1994) Indirect angiogenic cytokines upregulate VEGF and bFGF gene expression in vascular smooth muscle cells, whereas hypoxia upregulates VEGF expression only. Circulation 90:649–652

    PubMed  CAS  Google Scholar 

  7. Chua CC, Hamdy RC, Chua BH (2000) Mechanism of transforming growth factor-β1-induced expression of vascular endothelial growth factor in murine osteoblastic MC3T3-E1 cells. Biochim Biophys Acta 1497:69–76

    Article  PubMed  CAS  Google Scholar 

  8. Ehrlich HP, Desmouliere A, Diegelmann RF, Cohen IK, Compton CC, Garner WL, Kapanci Y, Gabbiani G (1994) Morphological and immunochemical differences between keloid and hypertrophic scar. Am J Pathol 145:105–113

    PubMed  CAS  Google Scholar 

  9. English RS, Shenefelt PD (1999) Keloids and hypertrophic scars. Dermatol Surg 25:631–638

    Article  PubMed  CAS  Google Scholar 

  10. Estrem SA, Domayer M, Bardach J, Cram AE (1987) Implantation of human keloid into athymic mice. Laryngoscope 97:1214–1218

    Article  PubMed  CAS  Google Scholar 

  11. Fajardo LF, Prionas SD, Kwan HH, Kowalski J, Allison AC (1996) Transforming growth factor β1 induces angiogenesis in vivo with a threshold pattern. Lab Invest 74:600–608

    PubMed  CAS  Google Scholar 

  12. Ferrara N, Gerber HP, LeCouter J (2003) The biology of VEGF and its receptors. Nat Med 9:669–676

    Article  PubMed  CAS  Google Scholar 

  13. Gajdusek CM, Luo Z, Mayberg MR (1993) Basic fibroblast growth factor and transforming growth factor beta-1: synergistic mediators of angiogenesis in vitro. J Cell Physiol 157:133–144

    Article  PubMed  CAS  Google Scholar 

  14. Gira AK, Brown LF, Washington CV, Cohen C, Arbiser JL (2004) Keloids demonstrate high-level epidermal expression of vascular endothelial growth factor. J Am Acad Dermatol 50:850–853

    Article  PubMed  Google Scholar 

  15. Goumans MJ, Valdimarsdottir G, Itoh S, Rosendahl A, Sideras P, ten Dijke P (2002) Balancing the activation state of the endothelium via two distinct TGF-β type I receptors. EMBO J 21:1743–1753

    Article  PubMed  CAS  Google Scholar 

  16. Hicklin DJ, Ellis LM (2005) Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol 23:1011–1027

    Article  PubMed  CAS  Google Scholar 

  17. Hoeben A, Landuyt B, Highley MS, Wildiers H, Van Oosterom AT, De Bruijn EA (2004) Vascular endothelial growth factor and angiogenesis. Pharmacol Rev 56:549–580

    Article  PubMed  CAS  Google Scholar 

  18. Jain RK (2003) Molecular regulation of vessel maturation. Nat Med 9:685–693

    Article  PubMed  CAS  Google Scholar 

  19. Kischer CW, Thies AC, Chvapil M (1982) Perivascular myofibroblasts and microvascular occlusion in hypertrophic scars and keloids. Hum Pathol 13:819–824

    Article  PubMed  CAS  Google Scholar 

  20. Kischer CW, Pindur J, Shetlar MR, Shetlar CL (1989) Implants of hypertrophic scars and keloids into the nude (athymic) mouse: viability and morphology. J Trauma 29:672–677

    PubMed  CAS  Google Scholar 

  21. Kischer CW (1992) The microvessels in hypertrophic scars, keloids and related lesions: a review. J Submicrosc Cytol Pathol 24:281–296

    PubMed  CAS  Google Scholar 

  22. Le AD, Zhang Q, Wu Y, Messadi DV, Akhondzadeh A, Nguyen AL, Aghaloo TL, Kelly AP, Bertolami CN (2004) Elevated vascular endothelial growth factor in keloids: relevance to tissue fibrosis. Cells Tissues Organs 176:87–94

    Article  PubMed  CAS  Google Scholar 

  23. Lee TY, Chin GS, Kim WJ, Chau D, Gittes GK, Longaker MT (1999) Expression of transforming growth factor beta 1, 2, and 3 proteins in keloids. Ann Plast Surg 43:179–184

    Article  PubMed  CAS  Google Scholar 

  24. Miyata M, Biro S, Kaieda H, Eto H, Orihara K, Kihara T, Obata H, Matsushita N, Matsuyama T, Tei C (2001) Apolipoprotein J/clusterin is induced in vascular smooth muscle cells after vascular injury. Circulation 104:1407–1412

    Article  PubMed  CAS  Google Scholar 

  25. Niessen FB, Spauwen PH, Schalkwijk J, Kon M (1999) On the nature of hypertrophic scars and keloids: a review. Plast Reconstr Surg 104:1435–1458

    Article  PubMed  CAS  Google Scholar 

  26. Pepper MS, Vassalli JD, Orci L, Montesano R (1993) Biphasic effect of transforming growth factor-β1 on in vitro angiogenesis. Exp Cell Res 204:356–363

    Article  PubMed  CAS  Google Scholar 

  27. Peltonen J, Hsiao LL, Jaakkola S, Sollberg S, Aumailley M, Timpl R, Chu ML, Uitto J (1991) Activation of collagen gene expression in keloids: co-localization of type I and VI collagen and transforming growth factor-β1 mRNA. J Invest Dermatol 97:240–248

    Article  PubMed  CAS  Google Scholar 

  28. Pertovaara L, Kaipainen A, Mustonen T, Orpana A, Ferrara N, Saksela O, Alitalo K (1994) Vascular endothelial growth factor is induced in response to transforming growth factor-β in fibroblastic and epithelial cells. J Biol Chem 269:6271–6274

    PubMed  CAS  Google Scholar 

  29. Polo M, Kim YJ, Kucukcelebi A, Hayward PG, Ko F, Robson MC (1998) An in vivo model of human proliferative scar. J Surg Res 74:187–195

    Article  PubMed  CAS  Google Scholar 

  30. Rahban SR, Garner WL (2003) Fibroproliferative scars. Clin Plast Surg 30:77–89

    Article  PubMed  Google Scholar 

  31. Renner U, Lohrer P, Schaaf L, Feirer M, Schmitt K, Onofri C, Arzt E, Stalla GK (2002) Transforming growth factor-β stimulates vascular endothelial growth factor production by folliculostellate pituitary cells. Endocrinology 143:3759–3765

    Article  PubMed  CAS  Google Scholar 

  32. Roberts AB, Sporn MB, Assoian RK, Smith JM, Roche NS, Wakefield LM, Heine UI, Liotta LA, Falanga V, Kehrl JH, Fauci AS (1986) Transforming growth factor type β: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci USA 83:4167–4171

    Article  PubMed  CAS  Google Scholar 

  33. Saadeh PB, Mehrara BJ, Steinbrech DS, Dudziak ME, Greenwald JA, Luchs JS, Spector JA, Ueno H, Gittes GK, Longaker MT (1999) Transforming growth factor-β1 modulates the expression of vascular endothelial growth factor by osteoblasts. Am J Physiol 277:C628–C637

    PubMed  CAS  Google Scholar 

  34. Schierle HP, Scholz D, Lemperle G (1997) Elevated levels of testosterone receptors in keloid tissue: an experimental investigation. Plast Reconstr Surg 100:390–395

    Article  PubMed  CAS  Google Scholar 

  35. Steinbrech DS, Mehrara BJ, Chau D, Rowe NM, Chin G, Lee T, Saadeh PB, Gittes GK, Longaker MT (1999) Hypoxia upregulates VEGF production in keloid fibroblasts. Ann Plast Surg 42:514–519

    Article  PubMed  CAS  Google Scholar 

  36. Tuan TL, Nichter LS (1998) The molecular basis of keloid and hypertrophic scar formation. Mol Med Today 4:19–24

    Article  PubMed  CAS  Google Scholar 

  37. Vladutiu AO (1993) The severe combined immunodeficient (SCID) mouse as a model for the study of autoimmune diseases. Clin Exp Immunol 93:1–8

    Article  PubMed  CAS  Google Scholar 

  38. Wahid S, Blades MC, De Lord D, Brown I, Blake G, Yanni G, Haskard DO, Panayi GS, Pitzalis C (2000) Tumour necrosis factor-alpha (TNF-α) enhances lymphocyte migration into rheumatoid synovial tissue transplanted into severe combined immunodeficient (SCID) mice. Clin Exp Immunol 122:133–142

    Article  PubMed  CAS  Google Scholar 

  39. Wang X, Smith P, Pu LL, Kim YJ, Ko F, Robson MC (1999) Exogenous transforming growth factor β2 modulates collagen I and collagen III synthesis in proliferative scar xenografts in nude rats. J Surg Res 87:194–200

    Article  PubMed  CAS  Google Scholar 

  40. Wu Y, Zhang Q, Ann DK, Akhondzadeh A, Duong HS, Messadi DV, Le AD (2003) Increased vascular endothelial growth factor may account for an elevated level of plasminogen activator inhibitor-1 via activating ERK1/2 in keloid fibroblasts. Am J Physiol Cell Physiol 286:905–912

    Article  Google Scholar 

  41. Xia W, Phan TT, Lim IJ, Longaker MT, Yang GP (2004) Complex epithelial-mesenchymal interactions modulate transforming growth factor-β expression in keloid-derived cells. Wound Repair Regen 12:546–556

    Article  PubMed  Google Scholar 

  42. Yoshimoto H, Ishihara H, Ohtsuru A, Akino K, Murakami R, Kuroda H, Namba H, Ito M, Fujii T, Yamashita S (1999) Overexpression of insulin-like growth factor-1 (IGF-I) receptor and the invasiveness of cultured keloid fibroblasts. Am J Pathol 154:883–889

    PubMed  CAS  Google Scholar 

  43. Zhang Q, Wu Y, Ann DK, Messadi DV, Tuan TL, Kelly AP, Bertolami CN, Le AD (2003) Mechanisms of hypoxic regulation of plasminogen activator inhibitor-1 gene expression in keloid fibroblasts. J Invest Dermatol 121:1005–1012

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Plastic and Reconstructive Surgery, Tenri Hospital, 200 Mishima, Tenri, Nara, 632-8552, Japan

    Masao Fujiwara

  2. Department of Pathology, Wakayama Medical University, 811-1 Kimiidera, Wakayama, 641-0012, Japan

    Masao Fujiwara, Yasuteru Muragaki & Akira Ooshima

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  1. Masao Fujiwara
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  2. Yasuteru Muragaki
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Correspondence to Masao Fujiwara.

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Fujiwara, M., Muragaki, Y. & Ooshima, A. Upregulation of transforming growth factor-β1 and vascular endothelial growth factor in cultured keloid fibroblasts: relevance to angiogenic activity. Arch Dermatol Res 297, 161–169 (2005). https://doi.org/10.1007/s00403-005-0596-2

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