内皮细胞向间充质转化促进系统性硬皮病的内皮功能障碍和皮肤纤维化。

Endothelial-to-mesenchymal transition contributes to endothelial dysfunction and dermal fibrosis in systemic sclerosis.

机构信息

Department of Experimental and Clinical Medicine, Section of Anatomy and Histology, University of Florence, Florence, Italy.

Department of Experimental and Clinical Medicine, Section of Internal Medicine, Rheumatology Unit, Azienda Ospedaliero-Universitaria Careggi (AOUC), University of Florence, Florence, Italy.

出版信息

Ann Rheum Dis. 2017 May;76(5):924-934. doi: 10.1136/annrheumdis-2016-210229. Epub 2017 Jan 6.

DOI:10.1136/annrheumdis-2016-210229

PMID:28062404

Abstract

OBJECTIVE

Systemic sclerosis (SSc) features multiorgan fibrosis orchestrated predominantly by activated myofibroblasts. Endothelial-to-mesenchymal transition (EndoMT) is a transdifferentiation by which endothelial cells (ECs) lose their specific morphology/markers and acquire myofibroblast-like features. Here, we determined the possible contribution of EndoMT to the pathogenesis of dermal fibrosis in SSc and two mouse models.

METHODS

Skin sections were immunostained for endothelial CD31 or vascular endothelial (VE)-cadherin in combination with α-smooth muscle actin (α-SMA) myofibroblast marker. Dermal microvascular ECs (dMVECs) were prepared from SSc and healthy skin (SSc-dMVECs and H-dMVECs). H-dMVECs were treated with transforming growth factor-β1 (TGFβ1) or SSc and healthy sera. Endothelial/mesenchymal markers were assessed by real-time PCR, immunoblotting and immunofluorescence. Cell contractile phenotype was assayed by collagen gel contraction.

RESULTS

Cells in intermediate stages of EndoMT were identified in dermal vessels of either patients with SSc or bleomycin-induced and urokinase-type plasminogen activator receptor (uPAR)-deficient mouse models. At variance with H-dMVECs, SSc-dMVECs exhibited a spindle-shaped appearance, co-expression of lower levels of CD31 and VE-cadherin with myofibroblast markers (α-SMA+ stress fibres, S100A4 and type I collagen), constitutive nuclear localisation of the EndoMT driver Snail1 and an ability to effectively contract collagen gels. Treatment of H-dMVECs either with SSc sera or TGFβ1 resulted in the acquisition of a myofibroblast-like morphology and contractile phenotype and downregulation of endothelial markers in parallel with the induction of mesenchymal markers. Matrix metalloproteinase-12-dependent uPAR cleavage was implicated in the induction of EndoMT by SSc sera.

CONCLUSIONS

In SSc, EndoMT may be a crucial event linking endothelial dysfunction and development of dermal fibrosis.

摘要

目的

系统性硬化症（SSc）的特征是多器官纤维化，主要由活化的肌成纤维细胞协调。内皮细胞向间充质转化（EndoMT）是一种转分化，其中内皮细胞（ECs）失去其特定的形态/标志物，并获得成肌纤维细胞样特征。在这里，我们确定了 EndoMT 对 SSc 皮肤纤维化发病机制和两种小鼠模型的可能贡献。

方法

皮肤切片用内皮细胞 CD31 或血管内皮（VE）-钙黏蛋白与α-平滑肌肌动蛋白（α-SMA）成肌纤维细胞标志物联合免疫染色。从 SSc 和健康皮肤中制备真皮微血管内皮细胞（dMVECs）（SSc-dMVECs 和 H-dMVECs）。用转化生长因子-β1（TGFβ1）或 SSc 和健康血清处理 H-dMVECs。通过实时 PCR、免疫印迹和免疫荧光评估内皮/间充质标志物。通过胶原蛋白凝胶收缩测定细胞收缩表型。

结果

在 SSc 患者或博来霉素诱导和尿激酶型纤溶酶原激活受体（uPAR）缺陷型小鼠模型的真皮血管中均鉴定出处于 EndoMT 中间阶段的细胞。与 H-dMVECs 不同，SSc-dMVECs 表现出梭形外观，同时表达较低水平的 CD31 和 VE-钙黏蛋白与成肌纤维细胞标志物（α-SMA+应激纤维、S100A4 和 I 型胶原），EndoMT 驱动因子 Snail1 的核内定位以及有效收缩胶原蛋白凝胶的能力。用 SSc 血清或 TGFβ1 处理 H-dMVECs 会导致获得成肌纤维细胞样形态和收缩表型，并伴随着内皮标志物的下调和间充质标志物的诱导。基质金属蛋白酶-12 依赖性 uPAR 裂解被认为是 SSc 血清诱导 EndoMT 的原因。