Muir Amanda B, Dods Kara, Henry Steven J, Benitez Alain J, Lee Dale, Whelan Kelly A, DeMarshall Maureen, Hammer Daniel A, Falk Gary, Wells Rebecca G, Spergel Jonathan, Nakagawa Hiroshi, Wang Mei-Lun
*Division of Gastroenterology, Hepatology, and Nutrition, The Children's Hospital of Philadelphia, PA †Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania ‡Department of Bioengineering, University of Pennsylvania §University of Pennsylvania School of Engineering and Applied Science, Philadelphia ||Division of Gastroenterology, Seattle Children's Hospital ¶Gastroenterology Division, Department of Medicine #Abramson Cancer Center, University of Pennsylvania, Philadelphia **Division of Allergy and Immunology, The Children's Hospital of Philadelphia, Philadelphia, PA.
J Pediatr Gastroenterol Nutr. 2016 Aug;63(2):200-9. doi: 10.1097/MPG.0000000000001100.
Eosinophilic esophagitis (EoE) is an immune-mediated allergic disease characterized by progressive esophageal dysmotility and fibrotic stricture associated with chronic esophageal fibroblast activation. It remains unknown how esophageal fibroblasts respond to EoE-relevant matrix stiffness or inflammatory cytokines.
Immunofluorescence was used to evaluate α-smooth muscle actin (α-SMA) expression in endoscopic esophageal biopsies. Primary esophageal fibroblasts from adult and pediatric patients with or without EoE were exposed to transforming growth factor (TGF)β to determine gene expression, collagen-matrix contractility, and cytoskeletal organization. The influence of matrix stiffness upon fibroblast behavior was assessed on the engineered surface of polyacrylamide gels with varying stiffness. Fibroblast traction forces were measured using microfabricated-post-array-detectors.
EoE esophageal fibroblasts had enhanced α-SMA expression. TGFβ not only stimulated enhanced fibroblast-specific gene expression but also promoted fibroblast-mediated collagen-matrix contraction, despite disease state or age of patients as the origin of cells. Unlike conventional monolayer cell, culture conditions using plastic surface (1 GPa) that activates fibroblasts constitutively, our engineered platforms recapitulating physiologically relevant stiffness (1-20 kPa) revealed that matrix stiffness defines the extent of α-SMA expression, intracellular collagen fibril organization, SMAD3 phosphorylation, and fibroblast traction force.
Matrix stiffness may critically influence TGFβ-mediated gene expression and functions of esophageal fibroblasts ex vivo independent of age and disease conditions. These findings provide a novel insight into the pathogenesis of fibrostenotic disease in EoE.
嗜酸性粒细胞性食管炎（EoE）是一种免疫介导的过敏性疾病，其特征为进行性食管动力障碍和与慢性食管成纤维细胞活化相关的纤维化狭窄。目前尚不清楚食管成纤维细胞如何对与EoE相关的基质硬度或炎性细胞因子作出反应。
采用免疫荧光法评估内镜下食管活检组织中α平滑肌肌动蛋白（α-SMA）的表达。将成年和儿科EoE患者及非EoE患者的原代食管成纤维细胞暴露于转化生长因子（TGF）β，以确定基因表达、胶原基质收缩性和细胞骨架组织。在具有不同硬度的聚丙烯酰胺凝胶工程表面上评估基质硬度对成纤维细胞行为的影响。使用微制造的柱阵列检测器测量成纤维细胞的牵引力。
EoE食管成纤维细胞的α-SMA表达增强。TGFβ不仅刺激成纤维细胞特异性基因表达增强，还促进成纤维细胞介导的胶原基质收缩，无论细胞来源患者的疾病状态或年龄如何。与使用塑料表面（1 GPa）持续激活成纤维细胞的传统单层细胞培养条件不同，我们模拟生理相关硬度（1-20 kPa）的工程平台显示，基质硬度决定了α-SMA表达程度、细胞内胶原纤维组织、SMAD3磷酸化及成纤维细胞牵引力。
基质硬度可能在体外对TGFβ介导的食管成纤维细胞基因表达和功能产生关键影响，且与年龄和疾病状况无关。这些发现为EoE纤维化狭窄疾病的发病机制提供了新见解。
