Name: molecular analysis of endothelial-mesenchymal transition induced by transforming growth factor-β signaling
Uploaded: 2018-08-03T13:30:00.000+00:00
Duration: 7 min 49 s
Description: Watch this Scientific Journal Video about Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-Î² Signaling at JoVE.com

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 mL of culture media. Collect cell suspension in a 15 mL tube.</li>
	<li>Centrifuge the cell suspension at 300&#8211;400 x g for 5 min at room temperature.</li>
	<li>Carefully remove the supernatant and suspend the cell pellet in pre-warmed culture media.</li>
	<li>Count viable cells using Trypan Blue solution and a standard hemocytometer or an automatic cell counter.</li>
	<li>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-&#946; induces EndMT in long term culture (at least 48&#8211;72 h) with the use of FCS.</li>
	<li>Stimulate endothelial cells with TGF-&#946;2 (a final concentration of 1 ng/mL) after 24 h.</li>
	<li>Culture cells in a humidified incubator (5% CO2, 37 &#176;C). Monitor cells daily when focusing on morphological changes.</li>
	<li>Replace the media with fresh pre-warmed culture media containing TGF-&#946;2 after 48 h of treatment with TGF-&#946; in long term culture.</li>
	<li>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&#8211;72 h of treatment with TGF-&#946;.</li>
</ol>
2. Immunocytochemical Analysis
<ol>
	<li>Maintain MS-1 cells in standard culture conditions.</li>
	<li>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&#8211;1.0 mL of culture media per well.</li>
	<li>Stimulate endothelial cells with TGF-&#946;2 (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 &#956;M) 1 h prior to TGF-&#946; treatment. When assessing the effect of TGF-&#946; signaling inhibition, TGF-&#946; receptor kinase inhibitors can be used.</li>
	<li>Culture cells in a humidified incubator (5% CO2, 37 &#176;C). Actin reorganization is observed in MS-1 cells after 24 h of treatment with TGF-&#946;. Other changes become obvious after 48&#8211;72 h of treatment. For 72 h culture, replace the media with fresh media containing TGF-&#946; after 48 h of treatment with TGF-&#946;.</li>
	<li>F-actin staining
	<ol>
		<li>After 24 h of TGF-&#946; treatment, remove media, fix cells with 4% paraformaldehyde in 1x PBS for 20 min at room temperature, and rinse cells with 1x PBS.</li>
		<li>Incubate with 0.2% Triton X-100 for 5 min at room temperature.</li>
		<li>Rinse cells three times with 1x PBS.</li>
		<li>Incubate with phalloidin-tetramethylrhodamine B isothiocyanate from Amanita phalloides diluted 150-fold with blocking buffer for 1 h at room temperature.</li>
		<li>Rinse cells three times with 1x PBS.</li>
		<li>Stain the nuclei with cyanine nucleic acid binding dyes for 5 min at room temperature.</li>
		<li>Rinse slides and mount coverslips face down on a slide using slide mounting media.</li>
		<li>Observe with a confocal microscope. Use a 543 nm laser and an emission filter of 560&#8211;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-&#946;, formation of thick stress fibers would be observed.</li>
	</ol>
	</li>
	<li>VE-cadherin and &#945;-SMA staining
	<ol>
		<li>After 72 h of TGF-&#946; treatment, remove media, fix cells with cold 50% methanol and 50% acetone (0.5&#8211;1.0 mL per well) for 5 min.</li>
		<li>Rinse cells three times with 1x PBS (0.5&#8211;1.0 mL per well).</li>
		<li>Incubate with primary antibodies in blocking buffer overnight at 4 &#176;C in the dark according to the manufacturer&#39;s recommended concentration. Use VE-cadherin monoclonal antibody (BV13, 1:100 dilution) and Cy3-conjugated &#945;-SMA monoclonal antibody (1A4, 1:200 dilution)20.</li>
		<li>Rinse cells three times with 1x PBS.</li>
		<li>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&#39;s recommended concentration.</li>
		<li>Rinse cells three times with 1x PBS.</li>
		<li>Stain the nuclei with cyanine nucleic acid binding dyes for 5 min at room temperature.</li>
		<li>Rinse slides and mount coverslips face down on a slide using slide mounting media.</li>
		<li>Observe with a confocal microscope. Use a 543 nm laser and an emission filter of 560&#8211;615 nm for detection of &#945;-SMA (Cy3). Use a 488 nm laser and an emission filter of 505&#8211;550 nm for detection of VE-cadherin. Typically, upon treatment with TGF-&#946; treatment, decrease in VE-cadherin signals and increase in &#945;-SMA signals would be observed.</li>
	</ol>
	</li>
</ol>
3. Three-dimensional Collagen Gel Contraction Assay
<ol>
	<li>Perform culture of MS-1 cells with/without TGF-&#946; for 72 h prior to collagen gel contraction assay.</li>
	<li>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.</li>
	<li>Mix 200 mL of control or TGF-&#946;-treated endothelial cell suspensions (1.0 x 106 cells/200 &#956;L) and 800 &#956;l of the collagen gel solution. For TGF-&#946; treated samples, add TGF-&#946;2 (1 ng/mL) to the cell suspension.</li>
	<li>Add 1.0 mL of the mixture to each well of 12-well culture plates and allow to solidify in the incubator at 37 &#176;C for 30&#8211;60 min.</li>
	<li>After gelatinization, overlay 1.0 mL of MEM-&#945; containing 10% FCS, 50 U/mL penicillin, and 50 &#956;g/mL streptomycin to float the gel. For TGF-&#946; treated samples, add TGF-&#946;2 (1 ng/mL) to the media. 
	NOTE: For TGF-&#946; treated samples, add TGF-&#946; to both the gel and culture media.</li>
	<li>Incubate the floating gels at 37 &#176;C for 48 h.</li>
	<li>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.</li>
	<li>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 &#34;Image | Adjust | Color Threshold&#34; tool, and then determine the area of gel using &#34;Analyze | Measure&#34; command.</li>
</ol>
4. Inhibition of miRNA Activities by Locked Nucleic Acid-based miRNA Inhibitors
<ol>
	<li>Maintain MS-1 cells in standard culture conditions.</li>
	<li>Transfect Locked Nucleic Acid (LNA) miRNA inhibitors, synthetic miRNA duplexes or siRNAs (20 or 50 nM) using lipofection reagents according to the manufacturer&#39;s instruction. Details and sources of LNA miRNA inhibitors, synthetic miRNA duplexes, and siRNAs were described in our previous reports26,27. In MS-1 cells, standard transfection procedures based on the manufacturer&#39;s instruction work well for introduction of LNA miRNA inhibitors, synthetic miRNA duplexes or siRNAs.</li>
	<li>After 16&#8211;48 h, stimulate endothelial cells with TGF-&#946;2 (a final concentration of 1 ng/mL).</li>
	<li>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 reports20,26,27.</li>
</ol>

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The authors have nothing to disclose.

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), human cutaneous microvascular endothelial cells (HCMEC), human coronary endothelial cells (HCEC), mouse lung endothelial cells (MLEC), rat brain endothelial cells (BEC), bovine aortic endothelial cells (BAEC), immortalized bovine retinal microvascular endothelial cells (iBREC), and ovine aortic valve endothelial cells8,18,29,30,31,32,33,34. In Table 1, we summarized the conditions of mesenchymal marker induction by TGF-&#946; in various endothelial cells8,18,20,21,29,30,31,32,33,34. Comparison of these reports suggests that each endothelial cell line has different thresholds for EndMT. Differential sensitivity to TGF-&#946; or other mechanisms may underlie diverse EndMT-related pathological conditions in different organs. This should be considered together with the results of in vivo studies.
The details of in vitro EndMT induction protocol need to be tailored in different cell lines: e.g., concentration of TGF-&#946;, concentration of serum, addition or removal of other growth factors, and culture period. For example, HUVECs often poorly respond to TGF-&#946; and EndMT would be induced by combination with other factors such as PDGF-BB and inflammatory stimuli and/or modulation of serum concentration and other medium components in a longer culture setting29,35. In addition, overconfluency would mitigate the responses to TGF-&#946;.
EndMT is a dynamic process and an opposite process called mesenchymal-endothelial transition (MEndoT) has been recently reported36. While induction of mesenchymal markers by TGF-&#946; alone is reversible in MS-1 cells (Figure 2), it has been reported that downregulation of endothelial markers in MS-1 cells with activated Ras persisted after withdrawal of TGF-&#946;21. Irreversible EndMT has been also reported in iBREC and ovine aortic valve endothelial cells33,34. In addition, a previous report showed that the EndMT process persisted after withdrawal of TGF-&#946; due to a sustained activation of Ras-GTP and methylation of the RASAL1 promoter in human coronary endothelial cells37. Thus, comparison of these models may be also useful to understand the regulatory mechanisms of endothelial cell plasticity.
Responsiveness of endothelial cells to EndMT induction appears to be substantially heterogeneous38. Xiao et al. reported that tumor-specific endothelial cells from a mammary tumor model show distinct forms of EndMT in response to TGF-&#946; stimulation38. TGF-&#946;-driven EndMT is also modulated by other signaling pathways including FGF-2, BMP-7, and IL-1&#946;35,37,38. We also found that TNF-&#945; enhances TGF-&#946;-induced EndMT and EndMT-SP in MS-1 cells26. Furthermore, it is well known that EndMT is induced by other signaling pathways including Wnt, Notch, and hypoxia1. Thus, combination with other stimuli in various endothelial cells may be important to understand heterogeneity of EndMT responsiveness.
The EndMT protocol presented here can be easily modified to investigate the regulators of EndMT. In fact, we have characterized various regulators of EndMT in MS-1 cells20,26,27. A guanine nucleotide exchange factor Arhgef5 and myocardin-related transcription factor-A (MRTF-A) are induced by Smad signals, and both contribute to &#945;-SMA upregulation in MS-1 cells20. Thus, TGF-&#946; signaling activates both Rho signals and MRTF-A to induce EndMT. In addition, we also found that constitutively active miR-31 and TGF-&#946;-inducible miR-27b positively regulate EndMT induction26,27. In MS-1 cells, constitutively active miR-31 is also necessary for TGF-&#946;-induced EndMT-SP26. Overall, these in vitro EndMT induction procedures would be useful for understanding the biology of EndMT, identifying EndMT-associated gene signatures, and developing strategies to modulate this process.

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><tbody><tr><td>MS-1 cells</td><td>American Type Culture Collection</td><td>CRL-2279</td><td></td></tr><tr><td>MEM-alpha</td><td>Thermo Fisher Scientific</td><td>32571036</td><td></td></tr><tr><td>TGF-beta2</td><td>R&amp;amp;D</td><td>302-B2-002</td><td></td></tr><tr><td>4 well Lab-Tek II Chamber Slide</td><td>Thermo Fisher Scientific</td><td>154526</td><td></td></tr><tr><td>Y-27632&amp;nbsp;</td><td>Sigma-Aldrich</td><td>Y0503</td><td></td></tr><tr><td>Blocking One</td><td>nacalai tesque</td><td>03953-95</td><td></td></tr><tr><td>phalloidin-tetramethylrhodamine B isothiocyanate</td><td>Sigma-Aldrich</td><td>P1951</td><td></td></tr><tr><td>TOTO-3 iodide</td><td>Thermo Fisher Scientific</td><td>T3604</td><td></td></tr><tr><td>VE cadherin monoclonal antibody (BV13)</td><td>Thermo Fisher Scientific</td><td>14-1441-82</td><td></td></tr><tr><td>alpha-SMA Cy3 monoclonal antibody (1A4)</td><td>Sigma-Aldrich</td><td>C6198</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgG (H+L)</td><td>Thermo Fisher Scientific</td><td>A-11001</td><td></td></tr><tr><td>Cover slip</td><td>Thermo Fisher Scientific</td><td>174934</td><td></td></tr><tr><td>Collagen solution</td><td>Nitta gelatin Inc.</td><td>Cellmatrix I-P</td><td></td></tr><tr><td>Collagen dilution buffer</td><td>Nitta gelatin Inc.</td><td>Cellmatrix I-P</td><td></td></tr><tr><td>LNA miRNA inhibitor</td><td>EXIQON&amp;nbsp;</td><td>miRCURY LNAmicroRNA Power Inhibitor (Negative Control B and target miRNA)</td><td></td></tr><tr><td>synthetic miRNA duplex</td><td>Qiagen&amp;nbsp;</td><td>miScript miRNA Mimic</td><td></td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Thermo Fisher Scientific</td><td>13778030</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Thermo Fisher Scientific</td><td>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 upon TGF-&#946; treatment (Figure 1B). TGF-&#946;-treated cells lose cell-cell contact. After treatment with TGF-&#946; for 48&#8211;72 h, immunocytochemical analyses demonstrate decreased expression of VE-cadherin and increased expression of &#945;-SMA (Figure 1C). TGF-&#946; drastically induces the expression of &#945;-SMA at both mRNA and protein levels in MS-1 cells after 48&#8211;72 h of treatment (Figures 1D and 1E). Mammalian TGF-&#946; includes TGF-&#946;1, 2, and 3 isoforms. A previous study using mouse cultured endocardial cushion explants has shown that TGF-&#946;2 but not TGF-&#946;3 is obligatory for transformation of endocardial cushion cells28. On the other hand, we previously compared the effects of three isoforms on &#945;-SMA expression and confirmed that all isoforms similarly induced &#945;-SMA expression in MS-1 cells20. Collagen gel contraction assay shows enhanced contraction of the collagen type I gel by MS-1 cells following TGF-&#946; treatment (Figure 1F). This effect suggests the acquisition of mesenchymal cell capacity to remodel extracellular matrix. These features are hallmark traits of EndMT20. In our previous report20, treatment with a ROCK inhibitor Y-27632 strongly suppressed induction of mesenchymal markers &#945;-SMA and SM22&#945; without concomitant suppression of induction of other TGF-&#946; target genes such as Fibronectin 1 and MMP-2.
EndMT is a highly dynamic process. In MS-1 cells, the effects of TGF-&#946; on &#945;-SMA expression and morphology are reversible; after TGF-&#946; deprivation, &#945;-SMA expression decreases to basal levels (Figure 2A), and cells recover a cobblestone-like morphology (Figure 2B). In addition, induction of EndMT and EMT is associated with dynamic changes in secretory capacity of multiple chemokines and cytokines. Multiple inflammatory chemokines and cytokines including CCL17, CX3CL1, CXCL16, IL-6, and Angptl2 are induced by TGF-&#946; in MS-1 cells, which we previously designated EndMT-associated secretory phenotype (EndMT-SP)26. This experimental model is useful to investigate the putative regulators of EndMT and EndMT-SP. We have previously studied the roles of several miRNAs in EndMT by enhancing or inhibiting miRNA activities26,27. In MS-1 cells, miRNA activities can be robustly modulated by miRNA mimic oligonucleotides or LNA-based miRNA inhibitors, and multiple downstream assays including RNA-seq analysis can be easily performed (Figure 3). Based on integrative transcriptome analysis, we have previously linked constitutively active miR-31 to EndMT regulation in MS-1 cells26. RNA-seq analysis showed that inhibition of miR-31 by LNA miRNA inhibitor results in attenuation of induction of mesenchymal markers (Figure 3A) and EndMT-SP genes (Figure 3C), while it does not affect induction of conventional TGF-&#946; target genes such as Fibronectin 1, PAI-1, and Smad7 (Figure 3B) and downregulation of endothelial markers (Figure 3D)26. Both TGF-&#946; and miR-31 downregulate Stk40, a negative regulator of NF-&#954;B pathway, which acts as a potential regulator of EndMT-SP. Although TGF-&#946; does not increase the expression of miR-31, TGF-&#946; induces alternative polyadenylation-mediated exclusion of internal poly(A) sequence in Stk40 3&#39;UTR, thereby enhancing targeting of Stk40 by constitutive active miR-31 and finally suppressing Stk4026. In addition, we previously examined the roles of TGF-&#946;-inducible miR-27b in EndMT27. Inhibition of miR-27 suppressed upregulation of mesenchymal markers (Figure 3A) while the effects on conventional TGF-&#946; target genes, EndMT-SP genes, and endothelial markers were marginal (Figures 3B, 3C, and 3D)27.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57577/57577fig1.jpg" /> 
Figure 1: Induction of EndMT in MS-1 cells by TGF-&#946;. (A) Actin reorganization in MS-1 cells after 24 h of TGF-&#946;2 treatment (1 ng/mL). (B) Morphological changes in MS-1 cells after 72 h of TGF-&#946;2 treatment (1 ng/mL). (C) Immunocytochemical analyses for VE-cadherin and &#945;-SMA in MS-1 cells after 72 h of TGF-&#946;2 treatment (1 ng/mL). (D, E) Expression changes in &#945;-SMA, determined by standard quantitative RT-PCR analysis (D) and Western blot analysis (E). (F) Collagen gel contraction assay. The ratio of the surface area after 72 h culture with/without TGF-&#946;2 treatment (1 ng/mL) to the original surface are displayed. Scale bars = 20 &#956;m (A and C), and 200 &#956;m (B). Figures are modified from Mihira et al.20. Error bars represent standard deviations. <a href="/files/ftp_upload/57577/57577fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57577/57577fig2.jpg" /> 
Figure 2: Reversible effects of TGF-&#946; in MS-1 cells. (A) &#945;-SMA mRNA expression and (B) cell morphology after withdrawal of TGF-&#946;2 in MS-1 cells. Scale bars = 200 &#956;m. Error bars represent standard deviations. <a href="/files/ftp_upload/57577/57577fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57577/57577fig3v2.jpg" /> 
Figure 3: RNA-seq analysis in MS-1 cells. After introduction of LNA miRNA inhibitors and treatment with TGF-&#946;2 (72 h), RNA-seq analysis was performed26,27. NC: negative control. Expression changes of representative genes are shown (A, mesenchymal markers; B, conventional TGF-&#946; target genes; C, EndMT-SP genes; and D, endothelial markers). FPKM (fragments per kilobase of transcript per million mapped reads) values were normalized with the values of a control sample (LNA-NC without TGF-&#946;2 treatment). Error bars represent standard deviations.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell type</td>
			<td>Condition of mesenchymal marker induction</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>human umbilical vein endothelial cells (HUVEC)</td>
			<td>Induction by mesenchymal differentiation medium (TGF-&#946; 5 ng/mL, PDGF-BB 25 ng/mL), 5&#8211;21 days.</td>
			<td>Krenning et al.</td>
		</tr>
		<tr>
			<td>human cutaneous microvascular endothelial cells (HCMEC)</td>
			<td>Induction by TGF-&#946;2 (10 ng/mL), without FBS, 2 days.</td>
			<td>Medici et al.</td>
		</tr>
		<tr>
			<td>human coronary endothelial cells (HCEC)</td>
			<td>Induction by TGF-&#946;1 (10 ng/mL), 6 days. Inhibition by BMP-7.</td>
			<td>Zeisberg et al.</td>
		</tr>
		<tr>
			<td>mouse lung endothelial cells (MLEC)</td>
			<td>Induction by TGF-&#946;1 (10 ng/mL), reduce to 2% FBS, without endothelial mitogen, 2 days.</td>
			<td>Zeisberg et al.</td>
		</tr>
		<tr>
			<td>rat brain endothelial cells (BEC)</td>
			<td>Induction by TGF-&#946;1 (10 ng/mL), 2 days.</td>
			<td>Krizbai et al.</td>
		</tr>
		<tr>
			<td>bovine aortic endothelial cells (BAEC)</td>
			<td>Induction by TGF-&#946;1 (1 ng/mL), 5 days.</td>
			<td>Arciniegas et al.</td>
		</tr>
		<tr>
			<td>immortalized bovine retinal microvascular endothelial cells (iBREC)</td>
			<td>Induction by TGF-&#946;2 (10 ng/mL), 3&#8211;6 days.</td>
			<td>Deissler et al.</td>
		</tr>
		<tr>
			<td>ovine aortic valve endothelial cells</td>
			<td>Induction by TGF-&#946;1 or 3 (1 ng/mL), 6 days.</td>
			<td>Paranya et al.</td>
		</tr>
		<tr>
			<td>MS-1 mouse pancreatic microvascular endothelial cells</td>
			<td>Induction by TGF-&#946; (10 ng/mL), activated Ras expression, 1 day.</td>
			<td>Hashimoto et al.</td>
		</tr>
		<tr>
			<td>MS-1 mouse pancreatic microvascular endothelial cells</td>
			<td>Induction by TGF-&#946;2 (1 ng/mL), 2&#8211;3 days.</td>
			<td>Mihira et al.&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Induction of EndMT by TGF-&#946; in various endothelial cells. Culture conditions of EndMT induction by TGF-&#946; are summarized. Changes in culture conditions including serum concentration and addition or removal of other growth factors are also noted.

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 ...

molecular-analysis-endothelial-mesenchymal-transition-induced

Nanyang Technological University

Research

JoVE Journal

Biology

11.2K Views.  Massachusetts Institute of Technology. Een protocol voor in vitro inductie van endotheel-mesenchymale overgang (EndMT), die nuttig is voor het onderzoeken van cellulaire signaalroutes die betrokken zijn bij EndMT, wordt beschreven. In dit experimenteel model wordt EndMT veroorzaakt door behandeling met TGF-β in MS-1 endotheliale cellen.

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

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Developmental Biology

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic plasticity drives invasion, dissemination and metastasis. Indeed, while most of the studies of phenotypic plasticity have been in the context of epithelial-derived carcinomas, it turns out sarcomas, which are mesenchymal in origin, also exhibit phenotypic plasticity, with a subset of sarcomas undergoing a phenomenon that resembles a mesenchymal-epithelial transition (MET). Here, we developed a method comprising the miR-200 family and grainyhead-like 2 (GRHL2) to mimic this MET-like phenomenon observed in sarcoma patient samples.We sequentially express GRHL2 and the miR-200 family using cell transduction and transfection, respectively, to better understand the molecular underpinnings of these phenotypic transitions in sarcoma cells. Sarcoma cells expressing miR-200s and GRHL2 demonstrated enhanced epithelial characteristics in cell morphology and alteration of epithelial and mesenchymal biomarkers. Future studies using these methods can be used to better understand the phenotypic consequences of MET-like processes on sarcoma cells, such as migration, invasion, metastatic propensity, and therapy resistance.

We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments.

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic ...

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...

Cancer Research

Visualization and Quantification of TGF&#946;/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and homeostasis of the vasculature system Therefore, deregulation in this signaling pathway results in severe vascular pathologies, such as pulmonary artery hypertension, hereditary hemorrhagic telangiectasia, and atherosclerosis. Endothelial cells (ECs), as the innermost layer of blood vessels, are constantly exposed to fluid shear stress (SS). Abnormal patterns of fluid SS have been shown to enhance TGF&#946;/BMP signaling, which, together with other stimuli, induce atherogenesis. In relation to this, atheroprone, low laminar SS was found to enhance TGF&#946;/BMP signaling while atheroprotective, high laminar SS, diminishes this signaling. To efficiently analyze the activation of these pathways, we designed a workflow to investigate the formation of transcription factor complexes under low laminar SS and high laminar SS conditions using a commercially available pneumatic pump system and proximity ligation assay (PLA).
Active TGF&#946;/BMP-signaling requires the formation of trimeric SMAD complexes consisting of two regulatory SMADs (R-SMAD); SMAD2/3 and SMAD1/5/8 for TGF&#946; and BMP signaling, respectively) with a common mediator SMAD (co-SMAD; SMAD4). Using PLA targeting different subunits of the trimeric SMAD-complex, i.e., either R-SMAD/co-SMAD or R-SMAD/R-SMAD, the formation of active SMAD transcription factor complexes can be measured quantitatively and spatially using fluorescence microscopy.
The usage of flow slides with 6 small parallel channels, that can be connected in series, allows for the investigation of the transcription factor complex formation and inclusion of necessary controls. 
The workflow explained here can be easily adapted for studies targeting the proximity of SMADs to other transcription factors or to transcription factor complexes other than SMADs, in different fluid SS conditions. The workflow presented here shows a quick and effective way to study the fluid SS induced TGF&#946;/BMP signaling in ECs, both quantitatively and spatially.

Here, we establish a protocol to simultaneously visualize and analyze multiple SMAD complexes using proximity ligation assay (PLA) in endothelial cells exposed to pathological and physiological fluid shear stress conditions.

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and ...

Biochemistry

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Medicine

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...

Bioengineering

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Analyzing Angiogenesis: In Vitro Assay for Tube Formation on BM Matrix

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of mesenchymal to endothelial-like transition. Traditionally, this process is evaluated by an in vitro tube-formation assay, where the cells organize themselves into tube-like structures when seeded over a polymerized basement membrane preparation. Although several structural features can be measured in photomicrographs of the structures, to assess the real commitment of EVT to the endothelial-type phenotype, biochemical analysis of cell extracts is required. Scraping the cells from the culture dish to obtain RNA and/or protein extracts is not an alternative since the tube-like structures are severely contaminated by the bulk of proteins from the polymerized basement membrane. Thus, a strategy to separate the cells from the basement membrane proteins prior to the preparation of cell extracts is needed. Here, a simple, fast, and cost-effective method to recover the tube-like structures from the in vitro tube-formation assay and the subsequent analysis by biochemical techniques is presented. Tube-like structures formed by HTR8/SVneo cells, an EVT cell line, were liberated from the polymerized basement membrane by a short incubation with PBS supplemented with ethylenediaminetetraacetic acid (EDTA). After serial washes, a ready-to-use pellet of purified tube-like structures can be obtained. This pellet can be subsequently processed to obtain RNA and protein extracts. qPCR analysis evidenced the induced expression of VE-cadherin and alphav-integrin, two endothelial cell markers, in EVT-derived tube-like structures compared to control cells, which was consistent with the induction of the endothelial cell marker, CD31, evaluated by immunofluorescence. Western blot analysis of the tube-like structures' protein extracts revealed the overexpression of RECK in transfected HTR8/SVneo cells. Thus, this simple method allows to obtain cell extracts from the in vitro tube-formation assay for the subsequent analysis of RNAs and protein expression.

The present protocol allows the recovery of tube-like structures formed in the classical in vitro angiogenesis-tube formation assay where cells, such as trophoblast cells, organize in capillary-like structures over a layer of extracellular basement membrane matrix. The recovered structures are used for RNA and protein isolation for various biochemical analyses.

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

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

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