敲低 CD146 通过 Wnt/β-连环蛋白通路促进血管内皮细胞向间充质细胞转化。

Knockdown of CD146 promotes endothelial-to-mesenchymal transition via Wnt/β-catenin pathway.

机构信息

Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China.

Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing, China.

出版信息

PLoS One. 2022 Aug 24;17(8):e0273542. doi: 10.1371/journal.pone.0273542. eCollection 2022.

DOI:10.1371/journal.pone.0273542

PMID:36001597

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9401105/

Abstract

PURPOSE

Cardiac fibrosis is characterized by the excessive deposition of extracellular matrix (ECM) proteins and leads to the maladaptive changes in myocardium. Endothelial cells (ECs) undergoing mesenchymal transition contributes to the occurrence and development of cardiac fibrosis. CD146 is an adhesion molecule highly expressed in ECs. The present study was performed to explore the role of CD146 in modulating endothelial to mesenchymal transition (EndMT).

METHODS

C57BL/6 mice were subjected to subcutaneous implantation of osmotic minipump infused with angiotensin II (Ang Ⅱ). Adenovirus carrying CD146 short hairpin RNA (shRNA) or CD146 encoding sequence were infected into cultured human umbilical vein endothelial cells (HUVECs) followed by stimulation with Ang II or transforming growth factor-β1 (TGF-β1). Differentially expressed genes were revealed by RNA-sequencing (RNA-Seq) analysis. Gene expression was measured by quantitative real-time PCR, and protein expression and distribution were determined by Western blot and immunofluorescence staining, respectively.

RESULTS

CD146 was predominantly expressed by ECs in normal mouse hearts. CD146 was upregulated in ECs but not fibroblasts and myocytes in hearts of Ang II-infused mice and in HUVECs stimulated with Ang Ⅱ. RNA-Seq analysis revealed the differentially expressed genes related to EndMT and Wnt/β-catenin signaling pathway. CD146 knockdown and overexpression facilitated and attenuated, respectively, EndMT induced by Ang II or TGF-β1. CD146 knockdown upregulated Wnt pathway-related genes including Wnt4, LEF1, HNF4A, FOXA1, SOX6, and CCND3, and increased the protein level and nuclear translocation of β-catenin.

CONCLUSIONS

Knockdown of CD146 exerts promotional effects on EndMT via activating Wnt/β-catenin pathway and the upregulation of CD146 might play a protective role against EndMT and cardiac fibrosis.

摘要

目的

心肌纤维化的特征是细胞外基质（ECM）蛋白的过度沉积，导致心肌发生适应性变化。内皮细胞（ECs）发生间质转化有助于心脏纤维化的发生和发展。CD146 是一种在 ECs 中高度表达的黏附分子。本研究旨在探讨 CD146 在调节内皮细胞向间质转化（EndMT）中的作用。

方法

将载有 CD146 短发夹 RNA（shRNA）或 CD146 编码序列的腺病毒感染培养的人脐静脉内皮细胞（HUVECs），然后用血管紧张素 II（Ang Ⅱ）或转化生长因子-β1（TGF-β1）刺激。通过 RNA 测序（RNA-Seq）分析揭示差异表达基因。通过定量实时 PCR 测量基因表达，通过 Western blot 和免疫荧光染色分别测定蛋白表达和分布。

结果

正常小鼠心脏中的 ECs 主要表达 CD146。在 Ang Ⅱ输注小鼠的心脏和 Ang Ⅱ 刺激的 HUVECs 中，ECs 中 CD146 上调，但成纤维细胞和心肌细胞中未上调。RNA-Seq 分析显示与 EndMT 和 Wnt/β-catenin 信号通路相关的差异表达基因。CD146 敲低和过表达分别促进和减弱 Ang Ⅱ或 TGF-β1 诱导的 EndMT。CD146 敲低上调了 Wnt 通路相关基因，包括 Wnt4、LEF1、HNF4A、FOXA1、SOX6 和 CCND3，并增加了 β-catenin 的蛋白水平和核转位。