Name: molecular analysis of endothelial-mesenchymal transition induced by transforming growth factor-&#946; signaling
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We thank Zea Borok and Kohei Miyazono for suggestions in preparation of manuscript. H.I.S. and M.H. are supported by the Uehara Memorial Foundation Research Fellowship, and H.I.S. is supported by the Osamu Hayaishi Memorial Scholarship for Study Abroad. This work was supported by a grant from Takeda Science Foundation (A.S.).

1. Induction of EndMT
<ol>
	<li>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-&#945; (MEM-&#945;) with 10% fetal calf serum (FCS), 50 U/mL penicillin, and 50 &#956;g/mL streptomycin.</li>
	<li>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 &#176;C.</li>
	<li>Detach the cells using 9 ...

<ol>
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It has been reported that activated Ras and TGF-&#946; treatment for 24 h induced EndMT in MS-1 cells, while TGF-&#946; alone failed to induce EndMT in this short period21. Consistently, we observed that TGF-&#946; substantially induced EndMT after longer treatment (48&#8211;72 h) in MS-1 cells20. EndMT has been repeatedly observed after prolonged treatment with TGF-&#946; (2&#8211;6 days) in various endothelial cells such as human umbilical vein endothelial cells (HUVEC), ...

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 heart2,3. 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 artery4,5,6. In addition, EndMT is implicated in physiological angiogenic sprouting7.
Accumulating evidence has suggested that EndMT is also involved in multiple cardiovascular diseases and other diseases1,8. EndMT-associated conditions include vascular calcification, atherosclerosis, pulmonary arterial hypertension, cavernous malformation, organ fibrosis, vein graft remodeling, allograft dysfunction in kidney transplantation, and cancer8,9,10,11,12,13,14,15,16,17,18. 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 models8,15. 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 &#945;-smooth muscle actin (&#945;-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 fibrosis8,19.
Recently, several in vitro studies of EndMT have elucidated details of molecular mechanisms of EndMT15,20. EndMT is induced by various signaling pathways including transforming growth factor (TGF)-&#946;, Wnt, and Notch1. Among them, TGF-&#946; plays pivotal roles in the induction of both EMT and EndMT. In EndMT, prolonged exposure to TGF-&#946; 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-&#946;20. 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 targets22,23. 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 EndMT24,25. 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-&#946;-induced EndMT in MS-1 cells and also includes comparison of conditions of EndMT induction by TGF-&#946; in other endothelial cells.

<table border="0" cellpadding="0" cellspacing="0" style="width:1435px;" width="1434">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:361px;">MS-1 cells</td>
			<td style="width:289px;">American Type Culture Collection</td>
			<td style="width:593px;">CRL-2279</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MEM-alpha</td>
			<td style="width:289px;">Thermo Fisher Scientific</td>
			<td style="width:593px;">32571036</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">TGF-beta2</td>
			<td>R&#38;D</td>
			<td style="width:593px;">302-B2-002</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">4 well Lab-Tek II Chamber Slide</td>
			<td style="width:289px;">Thermo Fisher Scientific</td>
			<td style="width:593px;">154526</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Y-27632&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:593px;">Y0503</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Blocking One</td>
			<td style="width:289px;">nacalai tesque</td>
			<td style="width:593px;">03953-95</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">phalloidin-tetramethylrhodamine B isothiocyanate</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">TOTO-3 iodide</td>
			<td style="width:289px;">Thermo Fisher Scientific</td>
			<td style="width:593px;">T3604</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">VE cadherin monoclonal antibody (BV13)</td>
			<td style="width:289px;">Thermo Fisher Scientific</td>
			<td>14-1441-82</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">alpha-SMA Cy3 monoclonal antibody (1A4)</td>
			<td>Sigma-Aldrich</td>
			<td>C6198</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Alexa Fluor 488 goat anti-mouse IgG (H+L)</td>
			<td style="width:289px;">Thermo Fisher Scientific</td>
			<td style="width:593px;">A-11001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Cover slip</td>
			<td style="width:289px;">Thermo Fisher Scientific</td>
			<td>174934</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Collagen solution</td>
			<td style="width:289px;">Nitta gelatin Inc.</td>
			<td>Cellmatrix I-P</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:361px;">Collagen dilution buffer</td>
			<td style="width:289px;">Nitta gelatin Inc.</td>
			<td>Cellmatrix I-P</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">LNA miRNA inhibitor</td>
			<td>EXIQON&#160;</td>
			<td colspan="2">miRCURY LNAmicroRNA Power Inhibitor (Negative Control B and target miRNA)</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">synthetic miRNA duplex</td>
			<td>Qiagen&#160;</td>
			<td>miScript miRNA Mimic</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Lipofectamine RNAiMAX</td>
			<td style="width:289px;">Thermo Fisher Scientific</td>
			<td style="width:593px;">13778030</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Lipofectamine 2000</td>
			<td style="width:289px;">Thermo Fisher Scientific</td>
			<td style="width:593px;">11668027</td>
			<td></td>
		</tr>
	</tbody>
</table>

molecular analysis of endothelial-mesenchymal transition induced by transforming growth factor-&#946; signaling

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)-&#946;, 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-&#946; 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.

TGF-&#946; is a potent inducer of EndMT in various endothelial cells. After 24 h treatment with TGF-&#946; 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 up...

Watch this Scientific Journal Video about Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-Î² Signaling at JoVE.com

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

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-&#946; in MS-1 endothelial cells.

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...

This method can help answer key questions in vascular and cancer biology fields, such as mechanisms of endothelial plasticity in various pathological conditions, including cancer. The main advantage of this technique is that robust induction of in vitro EndMT is accomplished by treatment with TGF-in MS-1 endothelial cells. First grow the MS-1 cells in standard culture conditions and avoid the culture from being confluent.Next, on a 10 cm dish, wash the MS-1 cells with 1x phosphate-buffered saline. Then add 1 mL of trypsin to the plate and incubate the plate at 37 degrees Celsius for five minutes. Then add 9 mL of culture media to the trypsinized cells for detachment.Then collect the trypsinized cell suspension in a 15 mL tube. Centrifuge the cell suspension at 300 to 400 x G for five minutes at room temperature. Next, use an automatic cell counter to count the number of viable cells.Then plate the MS-1 cells on uncoated standard culture plates for long-term culture. After 24 hours, stimulate the endothelial cells with a final concentration of 1 ng per milliliter of TGF-2. Then incubate the culture plate at 37 degrees Celsius with 5%carbon dioxide.After 48 hours of treatment, replace the medium with fresh, pre-warmed culture medium containing TGF-2 For the downstream analysis, plate the MS-1 cells on an 8-well culture chamber slide pre-coated with gelatin. After 24 hours, add 10 M of ROCK inhibitor to the cell culture. After an hour, stimulate the endothelial cells with a final concentration of 1 ng per milliliter of TGF-2.Then incubate the cells at 37 degrees Celsius. For a 72 hours long culture, replace the old medium with fresh TGF-2-containing medium after 48 hours. After one whole day of TGF-treatment, remove the medium.Then fix the cells with 4%paraformaldehyde and phosphate-buffered saline for 20 minutes at room temperature. After the fixing period is over, rinse the cells with phosphate-buffered saline. Then incubate the cells with 0.2%Triton X-100 for five minutes at room temperature.After five minutes, again rinse the cells with 1x phosphate-buffered saline three times. After three washes, incubate the cells with diluted fluorescent phallotoxin containing blocking buffer for an hour at room temperature. Post incubation, wash the cells with 1x phosphate-buffered saline, thrice.Then stain the cell nuclei using cyanine nucleic acid binding dye for five minutes at room temperature. After five minutes, rinse the slide, and mount a cover slip on the slide using slide mounting media. Observe the cells under a confocal microscope.The cells show reorganization of the thick stress fibers upon TGF-2 treatment. This indicates the potency of TGF-2 in inducing EndMT in endothelial cells. After 72 hours of TGF-2 treatment, discard the medium and fix the cells with 0.5 to 1 ml of 50%cold methanol and 50%acetone for five minutes.Then rinse the cells with 1x phosphate-buffered saline, thrice. After the three washes are complete, incubate the cells with primary antibodies in blocking buffer overnight at 4 degrees Celsius, in the dark. The following day, again wash the cells with 1x phosphate-buffered saline, thrice.Then incubate the cells with green dye conjugated secondary antibody to VE-cadherin in blocking buffer, for one hour in the dark. Again wash the cells with 1x phosphate-buffered saline, thrice. Then stain the cell nuclei using cyanine nucleic acid binding dye for five minutes at room temperature.After five minutes, rinse the slide, and mount a cover slip on the slide using slide mounting medium. Then observe the cells under a confocal microscope. The cells show decreased expression of VE-cadherin, and increased smooth muscle actin expression upon TGF-2 treatment for 48 to 72 hours.Before the collagen gel contraction assay, culture the MS-1 cells in the presence and absence of TGF-2 for 72 hours. Then prepare the type 1 collagen gel on ice. Then mix 800 l of the collagen gel solution with 200 mL of the control, or TGF-2-treated endothelial cells.Next, add 1 mL of this mixture to each well of a 12-well culture plate. Let the gel solidify at 37 degrees Celsius for 30 to 60 minutes. After the solidification of the gel, add 1 mL of MEM Alpha, containing 10%fetal calf serum, 50 units per milliliter of penicillin, and 50 g per milliliter of streptomycin, to float the gel.Then detach the gel from the wall of the well, and incubate the floating gel at 37 degrees Celsius for 48 hours. After 48 hours of incubation, scan the images of the gel from the bottom of the plate using a scanner. Distinct morphological changes are visible, transforming the cells from the cobblestone to the mesenchymal spindle shaped architecture due to exposure to TGF-2 treatment for 72 hours.Interestingly, TGF beta 2 treatment for 48 to 72 hours shows an increase in both the the mRNA and the protein expression levels of smooth muscle actin in the MS-1 cells. The gel contraction assay also shows that upon TGF-2 treatment, the collagen type 1 gel contracts significantly. Interestingly, deprivation of TGF-2 decreases the smooth muscle actin to basal level and the cells resume the cobblestone morphology, indicating that EndMT is a dynamic process.In fact, inhibiting microRNA 31 by LNA microRNA inhibitor using the MS-1 cells, attenuates the induction of mesenchymal markers. However, the inhibitor does not affect the induction of TGF-target genes such as Fibronectin 1, PAI-1, and Smad 7. Following this procedure as a method like gene expression, knockdown, or microarray modulation, can be performed in order to answer additional questions like specific gene and microRNA function in EndMT.

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