Syed Farhatullah, Thomas Alexis N, Singh Subir, Kolluru Venkatesh, Emeigh Hart Susan G, Bayat Ardeshir
Plastic and Reconstructive Surgery Research, School of Translational Medicine, Manchester Interdisciplinary Biocentre, University of Manchester, Manchester, United Kingdom.
PLoS One. 2012;7(2):e31430. doi: 10.1371/journal.pone.0031430. Epub 2012 Feb 24.
Dupuytren's disease (DD) is a benign, fibroproliferative disease of the palmar fascia, with excessive extracellular matrix (ECM) deposition and over-production of cytokines and growth factors, resulting in digital fixed flexion contractures limiting hand function and patient quality of life. Surgical fasciectomy is the gold standard treatment but is invasive and has associated morbidity without limiting disease recurrence. Injectable Collagenase Clostridium histolyticum (CCH)--Xiaflex®--is a novel, nonsurgical option with clinically proven in vivo reduction of DD contractures but with limited in vitro data demonstrating its cellular and molecular effects. The aim of this study was to delineate the effects of CCH on primary fibroblasts isolated from DD and non-DD anatomical sites (using RTCA, LDH, WST-1, FACS, qRT-PCR, ELISA and In-Cell Quantitative Western Blotting) to compare the efficacy of varying concentrations of Xiaflex® against a reagent grade Collagenase, Collagenase A. Results demonstrated that DD nodule and cord fibroblasts had greater proliferation than those from fat and skin. Xiaflex® exposure resulted in dose- and time-dependent inhibition of cellular spreading, attachment and proliferation, with cellular recovery after enzyme removal. Unlike Collagenase A, Xiaflex® did not cause apoptosis. Collagen expression patterns were significantly (p<0.05) different in DD fibroblasts across anatomical sites - the highest levels of collagen I and III were detected in DD nodule, with DD cord and fat fibroblasts demonstrating a smaller increase in both collagen expression relative to DD skin. Xiaflex® significantly (p<0.05) down-regulated ECM components, cytokines and growth factors in a dose-dependent manner. An in vitro scratch wound assay model demonstrated that, at low concentrations, Xiaflex® enabled a faster fibroblast reparatory migration into the wound, whereas, at high concentrations, this process was significantly (p<0.05) inhibited. This is the first report elucidating potential mechanisms of action of Xiaflex® on Dupuytren fibroblasts, offering a greater insight and a better understanding of its effect in DD.
掌腱膜挛缩症(DD)是一种手掌筋膜的良性纤维增生性疾病,伴有细胞外基质(ECM)过度沉积以及细胞因子和生长因子过度产生,导致手指固定性屈曲挛缩,限制手部功能和患者生活质量。手术筋膜切除术是金标准治疗方法,但具有侵入性且伴有相关发病率,同时无法限制疾病复发。注射用溶组织梭菌胶原酶(CCH)——Xiaflex®——是一种新型非手术选择,临床已证实其在体内可减轻DD挛缩,但体外数据有限,无法证明其细胞和分子效应。本研究的目的是利用实时无标记细胞分析技术(RTCA)、乳酸脱氢酶(LDH)、水溶性四氮唑盐-1(WST-1)、荧光激活细胞分选术(FACS)、定量逆转录聚合酶链反应(qRT-PCR)、酶联免疫吸附测定(ELISA)和细胞内定量蛋白质免疫印迹法,阐明CCH对从DD和非DD解剖部位分离的原代成纤维细胞的影响,以比较不同浓度的Xiaflex®与试剂级胶原酶A的疗效。结果表明,DD结节和条索的成纤维细胞比脂肪和皮肤来源的成纤维细胞增殖能力更强。暴露于Xiaflex®会导致细胞铺展、黏附和增殖受到剂量和时间依赖性抑制,去除酶后细胞可恢复。与胶原酶A不同,Xiaflex®不会导致细胞凋亡。DD成纤维细胞在不同解剖部位的胶原蛋白表达模式存在显著差异(p<0.05)——在DD结节中检测到最高水平的I型和III型胶原蛋白,DD条索和脂肪成纤维细胞中这两种胶原蛋白的表达相对于DD皮肤的增加幅度较小。Xiaflex®以剂量依赖性方式显著下调(p<0.05)ECM成分、细胞因子和生长因子。体外划痕伤口试验模型表明,在低浓度下,Xiaflex®能使成纤维细胞更快地向伤口迁移进行修复,而在高浓度下,这一过程受到显著抑制(p<0.05)。这是第一份阐明Xiaflex®对掌腱膜挛缩症成纤维细胞潜在作用机制的报告,为深入了解其在DD中的作用提供了更多见解。