Fujiwara Masao, Muragaki Yasuteru, Ooshima Akira
Department of Plastic and Reconstructive Surgery, Tenri Hospital, 200 Mishima, Tenri, Nara, 632-8552, Japan.
Arch Dermatol Res. 2005 Oct;297(4):161-9. doi: 10.1007/s00403-005-0596-2. Epub 2005 Oct 26.
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.
瘢痕疙瘩是伤口愈合过程中过度瘢痕形成导致的肿瘤样病变。从组织学上看,与正常真皮或正常瘢痕相比,瘢痕疙瘩的血管密度增加。然而,瘢痕疙瘩成纤维细胞的血管生成活性尚不清楚。在本研究中,我们调查了瘢痕疙瘩成纤维细胞的血管生成活性。转化生长因子-β1(TGF-β1)和血管内皮生长因子(VEGF)作为血管生成因子的成分进行了研究。用酶联免疫吸附测定(EIA)和Northern印迹分析测量条件培养基中TGF-β1和VEGF的表达。还通过添加TGF-β1和中和性抗TGF-β1抗体研究了TGF-β1在VEGF产生中的作用。进行改良的Boyden小室试验以评估血管内皮细胞的趋化活性。通过将成纤维细胞植入严重联合免疫缺陷(SCID)小鼠形成的结节的新生血管形成来评估体内血管生成活性。EIA显示,与正常成纤维细胞相比,瘢痕疙瘩成纤维细胞培养后条件培养基中TGF-β1和VEGF的浓度分别增加了2.5倍和6倍。Northern印迹分析显示,与正常成纤维细胞相比,瘢痕疙瘩成纤维细胞中TGF-β1和VEGF mRNA的表达分别上调了3.6倍和6倍。向瘢痕疙瘩成纤维细胞培养物中添加TGF-β1可使VEGF产生增加3.5倍,而正常成纤维细胞培养物中则增加6倍。中和性抗TGF-β1抗体将VEGF分泌降低至对照水平,表明TGF-β1介导了VEGF表达的上调。改良的Boyden小室试验表明,瘢痕疙瘩成纤维细胞条件培养基诱导血管内皮细胞的趋化活性比正常成纤维细胞条件培养基更强(七倍)。抗VEGF抗体将趋化性抑制至基础水平。当SCID小鼠背部植入成纤维细胞时,瘢痕疙瘩成纤维细胞形成的结节比正常成纤维细胞形成的结节大三倍。虽然在瘢痕疙瘩成纤维细胞结节中观察到丰富的新生血管形成,但在正常成纤维细胞结节中新生血管形成很少。体外和体内研究均证实,与正常成纤维细胞相比,瘢痕疙瘩成纤维细胞的血管生成活性明显更高,并且清楚地表明TGF-β1和VEGF参与其中。这些结果表明,瘢痕疙瘩中的血管生成是由内源性TGF-β1和VEGF促进的。