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, invade the underlying cardiac jelly, and promote formation of the cardiac cushions, providing the foundation for the atrioventricular valves and septum and the semilunar valves. Furthermore, EndMT has been suggested to be sources of pericytes and vascular smooth muscle cells in other embryonic vascular systems including coronary vessels, abdominal aorta, and pulmonary artery^4,5,6. In addition, EndMT is implicated in physiological angiogenic sprouting⁷.

Accumulating evidence has suggested that EndMT is also involved in multiple cardiovascular diseases and other diseases^1,8. EndMT-associated conditions include vascular calcification, atherosclerosis, pulmonary arterial hypertension, cavernous malformation, organ fibrosis, vein graft remodeling, allograft dysfunction in kidney transplantation, and cancer^{8,9,10,11,12,13,14,15,16,17,18}. A recent report described that several molecular EndMT markers can be a tool for diagnosis and prognosis prediction of renal graft dysfunction in kidney transplantation¹⁷. Modulation of EndMT-related cellular signaling pathways have been shown to ameliorate several disease conditions including cardiac fibrosis and vein graft remodeling in animal models^8,15. Therefore, understanding the mechanisms underlying EndMT is important to develop diagnostic and therapeutic strategies targeting EndMT.

EndMT is characterized by loss of cell-cell junctions, increase in migratory potential, downregulation of endothelial-specific genes such as VE-cadherin, and upregulation of mesenchymal genes including α-smooth muscle actin (α-SMA). In addition, EndMT and epithelial-mesenchymal transition (EMT), a similar process that converts epithelial cells to mesenchymal cells, are associated with altered production of various extracellular matrix components, which may contribute to the development of tissue fibrosis^8,19.

Recently, several in vitro studies of EndMT have elucidated details of molecular mechanisms of EndMT^15,20. EndMT is induced by various signaling pathways including transforming growth factor (TGF)-β, Wnt, and Notch¹. Among them, TGF-β plays pivotal roles in the induction of both EMT and EndMT. In EndMT, prolonged exposure to TGF-β results in EndMT in various endothelial cells, while short exposure appears to be insufficient²¹. We here described a straightforward protocol for EndMT induction, in which MILE SVEN 1 (MS-1) mouse pancreatic microvascular endothelial cells undergo EndMT in vitro after prolonged exposure to TGF-β²⁰. In this model, multiple downstream analyses can be performed to investigate hallmark features of EndMT, including morphological changes, downregulation of endothelial markers, upregulation of mesenchymal markers and inflammatory genes, cytoskeletal rearrangements, and collagen gel contraction.

MicroRNAs (miRNAs) are ~22 nt small regulatory RNAs that direct posttranscriptional repression of various mRNA targets^22,23. Through seed sequence-mediated target recognition, miRNAs suppress hundreds of target genes and modulate diverse cellular functions such as cell differentiation, proliferation, and motility. This is also the case for regulation of EMT and EndMT, and several miRNAs have been reported as regulators of EMT and EndMT^24,25. The EndMT model presented in this review can be easily combined with miRNA modulation procedures to test the roles of miRNAs in EndMT. The present review summarizes our experimental procedures to investigate TGF-β-induced EndMT in MS-1 cells and also includes comparison of conditions of EndMT induction by TGF-β in other endothelial 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 temperature.
5. Carefully remove the supernatant and suspend the cell pellet in pre-warmed culture media.
6. Count viable cells using Trypan Blue solution and a standard hemocytometer or an automatic cell counter.
7. Plate MS-1 cells onto uncoated standard culture plates at 0.5 x 10³ cells per cm² for long term culture. For example, plate 5.0 x 10³ cells per well in a 6 well culture plate. Use the same culture media including the same concentration of FCS. In MS-1 cells, TGF-β induces EndMT in long term culture (at least 48–72 h) with the use of FCS.
8. Stimulate endothelial cells with TGF-β2 (a final concentration of 1 ng/mL) after 24 h.
9. Culture cells in a humidified incubator (5% CO₂, 37 °C). Monitor cells daily when focusing on morphological changes.
10. Replace the media with fresh pre-warmed culture media containing TGF-β2 after 48 h of treatment with TGF-β in long term culture.
11. Proceed to the downstream analyses. Typically, actin reorganization is observed after 24 h of treatment, and downregulation of endothelial markers and upregulation of mesenchymal markers are observed after 48–72 h of treatment with TGF-β.

2. Immunocytochemical Analysis

1. Maintain MS-1 cells in standard culture conditions.
2. Plate MS-1 cells onto 4 well cell culture chamber slides at 1.0 x 10³ cells per well (1.7 cm²). Slides are pre-coated with gelatin. Use 0.5–1.0 mL of culture media per well.
3. Stimulate endothelial cells with TGF-β2 (a final concentration of 1 ng/mL) after 24 h. When analyzing the involvement of ROCK in EndMT, add a ROCK inhibitor Y-27632 (10 μM) 1 h prior to TGF-β treatment. When assessing the effect of TGF-β signaling inhibition, TGF-β receptor kinase inhibitors can be used.
4. Culture cells in a humidified incubator (5% CO₂, 37 °C). Actin reorganization is observed in MS-1 cells after 24 h of treatment with TGF-β. Other changes become obvious after 48–72 h of treatment. For 72 h culture, replace the media with fresh media containing TGF-β after 48 h of treatment with TGF-β.
5. F-actin staining
   1. After 24 h of TGF-β treatment, remove media, fix cells with 4% paraformaldehyde in 1x PBS for 20 min at room temperature, and rinse cells with 1x PBS.
   2. Incubate with 0.2% Triton X-100 for 5 min at room temperature.
   3. Rinse cells three times with 1x PBS.
   4. Incubate with phalloidin-tetramethylrhodamine B isothiocyanate from Amanita phalloides diluted 150-fold with blocking buffer for 1 h at room temperature.
   5. Rinse cells three times with 1x PBS.
   6. Stain the nuclei with cyanine nucleic acid binding dyes for 5 min at room temperature.
   7. Rinse slides and mount coverslips face down on a slide using slide mounting media.
   8. Observe with a confocal microscope. Use a 543 nm laser and an emission filter of 560–615 nm for detection of fluorescence of phalloidin. Use a 633 nm laser and an emission filter longer than 650 nm for nuclear staining. Upon treatment with TGF-β, formation of thick stress fibers would be observed.
6. VE-cadherin and α-SMA staining
   1. After 72 h of TGF-β treatment, remove media, fix cells with cold 50% methanol and 50% acetone (0.5–1.0 mL per well) for 5 min.
   2. Rinse cells three times with 1x PBS (0.5–1.0 mL per well).
   3. Incubate with primary antibodies in blocking buffer overnight at 4 °C in the dark according to the manufacturer's recommended concentration. Use VE-cadherin monoclonal antibody (BV13, 1:100 dilution) and Cy3-conjugated α-SMA monoclonal antibody (1A4, 1:200 dilution)²⁰.
   4. Rinse cells three times with 1x PBS.
   5. Incubate cells with a green dye-conjugated secondary antibody to VE-cadherin antibody in blocking buffer for 1 h at room temperature in the dark according to the manufacturer's recommended concentration.
   6. Rinse cells three times with 1x PBS.
   7. Stain the nuclei with cyanine nucleic acid binding dyes for 5 min at room temperature.
   8. Rinse slides and mount coverslips face down on a slide using slide mounting media.
   9. Observe with a confocal microscope. Use a 543 nm laser and an emission filter of 560–615 nm for detection of α-SMA (Cy3). Use a 488 nm laser and an emission filter of 505–550 nm for detection of VE-cadherin. Typically, upon treatment with TGF-β treatment, decrease in VE-cadherin signals and increase in α-SMA signals would be observed.

3. Three-dimensional Collagen Gel Contraction Assay

1. Perform culture of MS-1 cells with/without TGF-β for 72 h prior to collagen gel contraction assay.
2. Prepare the type I collagen gel on ice. Mix cold collagen solution, 10x concentrated MEM medium, and collagen dilution buffer containing 0.05 N NaOH, 2.2% NaHCO₃, and 200 mM HEPES pH 7.4 at an 8:1:1 ratio.
3. Mix 200 mL of control or TGF-β-treated endothelial cell suspensions (1.0 x 10⁶ cells/200 μL) and 800 μl of the collagen gel solution. For TGF-β treated samples, add TGF-β2 (1 ng/mL) to the cell suspension.
4. Add 1.0 mL of the mixture to each well of 12-well culture plates and allow to solidify in the incubator at 37 °C for 30–60 min.
5. After gelatinization, overlay 1.0 mL of MEM-α containing 10% FCS, 50 U/mL penicillin, and 50 μg/mL streptomycin to float the gel. For TGF-β treated samples, add TGF-β2 (1 ng/mL) to the media.
   NOTE: For TGF-β treated samples, add TGF-β to both the gel and culture media.
6. Incubate the floating gels at 37 °C for 48 h.
7. Scan the images of gels from the bottom of plates using a scanner. Alternatively, record the images of gels using a digital camera at a fixed distance above the gels.
8. Quantify the gel surface area based on pixel number using ImageJ. Select the gel using a freehand selection or related selection tools, or select the gel using "Image | Adjust | Color Threshold" tool, and then determine the area of gel using "Analyze | Measure" command.

4. Inhibition of miRNA Activities by Locked Nucleic Acid-based miRNA Inhibitors

1. Maintain MS-1 cells in standard culture conditions.
2. Transfect Locked Nucleic Acid (LNA) miRNA inhibitors, synthetic miRNA duplexes or siRNAs (20 or 50 nM) using lipofection reagents according to the manufacturer's instruction. Details and sources of LNA miRNA inhibitors, synthetic miRNA duplexes, and siRNAs were described in our previous reports^26,27. In MS-1 cells, standard transfection procedures based on the manufacturer's instruction work well for introduction of LNA miRNA inhibitors, synthetic miRNA duplexes or siRNAs.
3. After 16–48 h, stimulate endothelial cells with TGF-β2 (a final concentration of 1 ng/mL).
4. Proceed to various downstream analyses including RNA expression analysis (quantitative RT-PCR and RNA-sequencing (RNA-seq) analyses), immunocytochemical analysis, and reporter assay. Downstream analyses have been described in our previous reports^20,26,27.

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