The present study was conducted to investigate cellular and molecular features of chronic graft-versus-host disease fibroblasts (GVHD-Fbs) and to assess the effectiveness of nilotinib as a fibrosis modulator. Growth kinetics, phenotype, and differentiation of cultured skin biopsy-derived GVHD-Fbs were compared with normal fibroblasts from both a dermal cell line (n-Fbs) and healthy individuals undergoing cosmetic surgery (n-skin-Fbs). Collagen genes (COL1α1/COL1α2) and p-SMAD2 expression were assessed by real-time PCR and immunofluorescence. The in vivo effects of nilotinib on chronic GVHD (cGVHD)-affected skin were investigated by immunohistochemistry; the relationship to TGF-β plasma levels was assessed. Although the morphology, phenotype, and differentiation of cultured GVHD-Fbs were comparable to normal fibroblasts, growth was slower and senescence was reached earlier. The expression of COL1α1 and COL1α2 mRNAs was respectively 4 and 1.6 times higher in cGVHD-Fbs (P = .02); the addition of TGF-β increased n-Fbs, but not GVHD-Fbs, collagen gene expression. Compared with the baseline, the addition of 1 μM nilotinib induced 86.5% and 49% reduction in COL1α1 and COL1α2 expression in cultured GVHD-Fbs, respectively (P< .01). In vivo immunohistochemistry analysis of skin biopsy specimens from patients with cGVHD showed strong baseline staining for COL1α1 and COL1α2, which decreased sharply after 180 days of nilotinib; immunofluorescence revealed TGF-β inhibition and p-Smad2 reduction at the intracellular level. Of note, nilotinib treatment was associated with normalization of TGF-β levels both in culture supernatants and in plasma. In general, the data show that cGVHD fibroblasts promote fibrosis through abnormal collagen production induced by hyperactive TGF-β signaling. TGF-β inhibition at the intracellular and systemic level represents an essential antifibrotic mechanism of nilotinib in a clinical setting.

中文翻译：

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尼罗替尼治疗受慢性移植物抗宿主病影响的患者通过下调 TGF-β 和 p-SMAD 通路来减少胶原蛋白的产生和皮肤纤维化。

本研究旨在研究慢性移植物抗宿主病成纤维细胞 (GVHD-Fbs) 的细胞和分子特征，并评估尼罗替尼作为纤维化调节剂的有效性。将培养的皮肤活检衍生的 GVHD-Fbs 的生长动力学、表型和分化与来自真皮细胞系 (n-Fbs) 和接受整容手术的健康个体 (n-skin-Fbs) 的正常成纤维细胞进行比较。通过实时 PCR 和免疫荧光评估胶原基因 (COL1α1/COL1α2) 和 p-SMAD2 表达。通过免疫组织化学研究了尼罗替尼对受慢性 GVHD (cGVHD) 影响的皮肤的体内作用；评估了与 TGF-β 血浆水平的关系。尽管培养的 GVHD-Fbs 的形态、表型和分化与正常成纤维细胞相当，生长较慢，衰老较早。在 cGVHD-Fbs 中 COL1α1 和 COL1α2 mRNA 的表达分别高出 4 倍和 1.6 倍（P = .02）；添加 TGF-β 增加了 n-Fbs，但不增加 GVHD-Fbs，胶原基因的表达。与基线相比，添加 1 μM 尼罗替尼分别诱导培养的 GVHD-Fbs 中 COL1α1 和 COL1α2 表达降低 86.5% 和 49%（P<.01）。对 cGVHD 患者皮肤活检标本的体内免疫组织化学分析显示，COL1α1 和 COL1α2 的基线染色较强，在使用尼罗替尼 180 天后急剧下降；免疫荧光显示细胞内水平的 TGF-β 抑制和 p-Smad2 减少。值得注意的是，尼罗替尼治疗与培养上清液和血浆中 TGF-β 水平的正常化有关。一般来说，数据显示，cGVHD 成纤维细胞通过过度活跃的 TGF-β 信号传导诱导的异常胶原生成促进纤维化。细胞内和全身水平的 TGF-β 抑制代表了尼罗替尼在临床环境中的基本抗纤维化机制。