Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-β Signaling

Summary

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-β in MS-1 endothelial cells.

Abstract

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-β, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-β and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

Introduction

Endothelial-mesenchymal transition (EndMT) is the process by which a differentiated endothelial cell undergoes a variety of molecular changes, resulting in a fibroblast-like mesenchymal cell¹. EndMT was initially described as an endothelial cell transformation during development of the heart^2,3. In early heart development, the heart tube consists of an inner endocardium and an outer myocardium. These two layers are separated by a layer of extracellular matrix called the cardiac jelly. The embryonic endocardial cells, which acquire endothelial cell markers, transit into mesenchymal cells....

Protocol

1. Induction of EndMT

1. Maintain MS-1 cells in standard culture conditions and avoid confluency. A source of MS-1 cells is described in the Table of Materials. For MS-1 cells, use Minimum Essential Medium-α (MEM-α) with 10% fetal calf serum (FCS), 50 U/mL penicillin, and 50 μg/mL streptomycin.
2. Wash MS-1 cells on 10 cm dish with 1x phosphate buffered saline (PBS) and add 1.0 mL of trypsin to the plate. Incubate for 5 min at 37 °C.
3. Detach the cells using 9 mL of culture media. Collect cell suspension in a 15 mL tube.
4. Centrifuge the cell suspension at 300–400 x g for 5 min at room....

Results

TGF-β is a potent inducer of EndMT in various endothelial cells. After 24 h treatment with TGF-β in MS-1 cells, staining for F-actin shows reorganization of actin stress fibers (Figure 1A)²⁰. Pretreatment with a ROCK inhibitor Y-27632 inhibits the induction of actin reorganization²⁰. MS-1 endothelial cells change from a classical cobblestone-like morphology to a mesenchymal spindle-shaped morphology upon TGF-β t.......

Discussion

It has been reported that activated Ras and TGF-β treatment for 24 h induced EndMT in MS-1 cells, while TGF-β alone failed to induce EndMT in this short period²¹. Consistently, we observed that TGF-β substantially induced EndMT after longer treatment (48–72 h) in MS-1 cells²⁰. EndMT has been repeatedly observed after prolonged treatment with TGF-β (2–6 days) in various endothelial cells such as human umbilical vein endothelial cells (HUVEC), .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MS-1 cells	American Type Culture Collection	CRL-2279
MEM-alpha	Thermo Fisher Scientific	32571036
TGF-beta2	R&D	302-B2-002
4 well Lab-Tek II Chamber Slide	Thermo Fisher Scientific	154526
Y-27632	Sigma-Aldrich	Y0503
Blocking One	nacalai tesque	03953-95
phalloidin-tetramethylrhodamine B isothiocyanate	Sigma-Aldrich	P1951
TOTO-3 iodide	Thermo Fisher Scientific	T3604
VE cadherin monoclonal antibody (BV13)	Thermo Fisher Scientific	14-1441-82
alpha-SMA Cy3 monoclonal antibody (1A4)	Sigma-Aldrich	C6198
Alexa Fluor 488 goat anti-mouse IgG (H+L)	Thermo Fisher Scientific	A-11001
Cover slip	Thermo Fisher Scientific	174934
Collagen solution	Nitta gelatin Inc.	Cellmatrix I-P
Collagen dilution buffer	Nitta gelatin Inc.	Cellmatrix I-P
LNA miRNA inhibitor	EXIQON	miRCURY LNAmicroRNA Power Inhibitor (Negative Control B and target miRNA)
synthetic miRNA duplex	Qiagen	miScript miRNA Mimic
Lipofectamine RNAiMAX	Thermo Fisher Scientific	13778030
Lipofectamine 2000	Thermo Fisher Scientific	11668027