Name: molecular analysis of endothelial-mesenchymal transition induced by transforming growth factor-&#946; signaling
Uploaded: 2018-08-03T13:30:00
Description: Watch this Scientific Journal Video about Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-ÃŽÂ² Signaling at JoVE.com

Ringraziamo Zea Borok e Kohei Miyazono per suggerimenti in preparazione del manoscritto. H.I.S e M.H. sono supportati da Uehara Memorial Foundation Research Fellowship e H.I.S è supportato dal Osamu Hayaishi Memorial Scholarship per studiare all'estero. Questo lavoro è stato supportato da una sovvenzione da Takeda Science Foundation (A.S.).

1. induzione di EndMT
<ol><li>Mantenere MS-1 cellule in condizioni di coltura standard ed evitare confluency. Una fonte di cellule MS-1 è descritta nella Tabella materiali. Per cellule MS-1, è possibile utilizzare Medium-α minimo essenziale (MEM-α) con 10% siero fetale di vitello (FCS), 50 U/mL di penicillina e 50 μg/mL di streptomicina.</li>
	<li>Cellule di lavare MS-1 su 10 cm piatto con 1x tamponato fosfato salino (PBS) e aggiungono 1,0 mL di tripsina alla piastra. Incubare per 5 min a 37 ° C....

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	<li>Sanchez-Duffhues, G., Garcia de Vinuesa, A., Ten Dijke, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial-to-mesenchymal+transition+in+cardiovascular+diseases:+Developmental+signaling+pathways+gone+awry.">Endothelial-to-mesenchymal transition in cardiovascular diseases: Developmental signaling pathways gone awry.</a> Developmental Dynamics. , (2017).</li><li>Markwald, R. R., Fitzharris, T. P., Smith, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+analysis+of+endocardial+cytodifferentiation.">Structural analysis of endocardial cytodifferentiation.</a> Developmental Biology. 42 (1), 160-180 (1975).</li><li>Eisenberg, L. M., Markwald, R. 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C., Ten Dijke, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta-Induced+Endothelial-Mesenchymal+Transition+in+Fibrotic+Diseases.">TGF-beta-Induced Endothelial-Mesenchymal Transition in Fibrotic Diseases.</a> International Journal of Molecular Sciences. 18 (10), (2017).</li><li>Mihira, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta-induced+mesenchymal+transition+of+MS-1+endothelial+cells+requires+Smad-dependent+cooperative+activation+of+Rho+signals+and+MRTF-A.">TGF-beta-induced mesenchymal transition of MS-1 endothelial cells requires Smad-dependent cooperative activation of Rho signals and MRTF-A.</a> Journal of Biochemistry. 151 (2), 145-156 (2012).</li><li>Hashimoto, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial-mesenchymal+transition+in+bleomycin-induced+pulmonary+fibrosis.">Endothelial-mesenchymal transition in bleomycin-induced pulmonary fibrosis.</a> American Journal of Respiratory Cell and Molecular Biology. 43 (2), 161-172 (2010).</li><li>Suzuki, H. I., Miyazono, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+microRNA+biogenesis:+crosstalk+between+p53+network+and+microRNA+processing+pathway.">Dynamics of microRNA biogenesis: crosstalk between p53 network and microRNA processing pathway.</a> Journal of Molecular Medicine (Berl). 88 (11), 1085-1094 (2010).</li><li>Suzuki, H. I., Miyazono, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+complexity+of+microRNA+generation+cascades.">Emerging complexity of microRNA generation cascades.</a> Journal of Biochemistry. 149 (1), 15-25 (2011).</li><li>Nicoloso, M. S., Spizzo, R., Shimizu, M., Rossi, S., Calin, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs--the+micro+steering+wheel+of+tumour+metastases.">MicroRNAs--the micro steering wheel of tumour metastases.</a> Nature Reviews Cancer. 9 (4), 293-302 (2009).</li><li>Lagendijk, A. K., Goumans, M. J., Burkhard, S. B., Bakkers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-23+restricts+cardiac+valve+formation+by+inhibiting+Has2+and+extracellular+hyaluronic+acid+production.">MicroRNA-23 restricts cardiac valve formation by inhibiting Has2 and extracellular hyaluronic acid production.</a> Circulation Research. 109 (6), 649-657 (2011).</li><li>Katsura, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-31+is+a+positive+modulator+of+endothelial-mesenchymal+transition+and+associated+secretory+phenotype+induced+by+TGF-beta.">MicroRNA-31 is a positive modulator of endothelial-mesenchymal transition and associated secretory phenotype induced by TGF-beta.</a> Genes Cells. 21 (1), 99-116 (2016).</li><li>Suzuki, H. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+TGF-beta-mediated+endothelial-mesenchymal+transition+by+microRNA-27.">Regulation of TGF-beta-mediated endothelial-mesenchymal transition by microRNA-27.</a> Journal of Biochemistry. 161 (5), 417-420 (2017).</li><li>Camenisch, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+distinct+TGFbeta+ligand+requirements+during+mouse+and+avian+endocardial+cushion+morphogenesis.">Temporal and distinct TGFbeta ligand requirements during mouse and avian endocardial cushion morphogenesis.</a> Developmental Biology. 248 (1), 170-181 (2002).</li><li>Krenning, G., Moonen, J. R., van Luyn, M. J., Harmsen, M. 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È stato segnalato che attivato trattamento Ras e TGF-β per 24 h ha indotto la EndMT in cellule di MS-1, mentre TGF-β da solo non è riuscito a indurre EndMT in questo breve periodo21. Coerentemente, abbiamo osservato che TGF-β sostanzialmente indotto EndMT dopo trattamento più lungo (48-72 h) in MS-1 cellule20. EndMT è stato più volte osservato dopo il trattamento prolungato con TGF-β (2 – 6 giorni) in varie cellule endoteliali come cordone ombelicale umano cellul...

Transizione endothelial-mesenchimale (EndMT) è il processo mediante il quale una cellula endoteliale differenziata subisce una varietà di cambiamenti molecolari, risultante in un fibroblasto-come delle cellule mesenchimali1. EndMT è stato inizialmente descritto come una trasformazione delle cellule endoteliali durante lo sviluppo del cuore2,3. Nello sviluppo iniziale del cuore, il tubo del cuore è costituito da un endocardio interno e un esterno miocardio. Questi due strati sono separati da uno strato di matrice extracellulare chiamato la gelatina cardiaca. Le cellule embrionali endocardial, che acquisiscono gli indicatori delle cellule endoteliali, transitano in cellule mesenchimali, invadono la gelatina cardiaca sottostante e promuovano la formazione dei cuscini cardiaci, fornendo le basi per le valvole atrioventricolari e setto e le valvole semilunari. Inoltre, EndMT è stata suggerita per essere fonti di periciti e cellule muscolari lisce vascolari in altri sistemi vascolare embrionale tra cui vasi coronarici, aorta addominale e dell'arteria polmonare4,5,6. Inoltre, EndMT è implicato nella fisiologico angiogenici spuntano7.
Raccogliendo la prova ha suggerito che EndMT è anche coinvolto in molteplici malattie cardiovascolari e altre malattie1,8. EndMT-collegato condizioni includono calcificazione vascolare, aterosclerosi, ipertensione arteriosa polmonare, malformazione cavernosa, fibrosi dell'organo, che ritocca dell'innesto della vena, disfunzione dell'allotrapianto in trapianto di rene e cancro8, 9,10,11,12,13,14,15,16,17, 18. un recente rapporto descritto che diversi marcatori molecolari di EndMT possono essere uno strumento per la stima di diagnosi e prognosi della disfunzione renale dell'innesto nel trapianto di rene17. Modulazione delle vie di segnalazione cellulare EndMT-correlati sono stati indicati per migliorare le condizioni di malattia diversi tra cui fibrosi cardiaca e vena innesto rimodellamento in animale modelli8,15. Pertanto, la comprensione dei meccanismi sottostanti EndMT è importante sviluppare strategie diagnostiche e terapeutiche EndMT di targeting.
EndMT è caratterizzata da perdita di giunzioni della cellula-cellula, aumento potenziale migratorio, downregulation dei geni endoteliali specifici quali VE-caderina e il upregulation dei geni mesenchimali compreso actina α-liscia del muscolo (α-SMA). Inoltre, EndMT e transizione epiteliale-mesenchimale (EMT), un processo simile che converte le cellule epiteliali in cellule mesenchimali, sono associati con alterata produzione di vari componenti della matrice extracellulare, che possono contribuire allo sviluppo di tessuti fibrosi8,19.
Recentemente, parecchi studi in vitro di EndMT hanno delucidato dettagli dei meccanismi molecolari di EndMT15,20. EndMT è indotta da varie vie di segnalazione compreso (TGF) del fattore di crescita trasformante-β, Wnt e tacca1. Fra loro, TGF-β svolge ruoli fondamentali nell'induzione di EMT e di EndMT. In EndMT, prolungata esposizione ai risultati di TGF-β in EndMT in varie cellule endoteliali, mentre breve esposizione sembra essere insufficiente21. Abbiamo descritto qui un semplice protocollo per induzione di EndMT, in cui miglio SVEN 1 (MS-1) cellule endoteliali microvascolari del pancreas del mouse subiscono EndMT in vitro dopo l'esposizione prolungata a TGF-β20. In questo modello, più a valle analisi possono essere eseguite per studiare le caratteristiche di marchio di garanzia di EndMT, tra cui i cambiamenti morfologici, downregulation dei marcatori endoteliali, upregulation di mesenchymal marcatori e geni infiammatori, del citoscheletro riorganizzazioni e contrazione del gel di collagene.
I microRNA (Mirna) sono ~ 22 nt piccoli RNA regolatori che dirigono posttranscriptional repressione di vari mRNA target22,23. Attraverso il riconoscimento di destinazione di sequenza-mediata del seme, Mirna sopprimono centinaia di geni bersaglio e modulano varie funzioni cellulari quali la motilità, proliferazione e differenziazione cellulare. Questo è anche il caso di regolamento di EMT ed EndMT, e diversi miRNA sono stati segnalati come regolatori di EMT ed EndMT24,25. Il modello EndMT presentato in questa recensione sono facilmente abbinabili con procedure di modulazione di miRNA per testare i ruoli di Mirna in EndMT. L'esame attuale riassume le nostre procedure sperimentali per studiare EndMT TGF-β-indotta in cellule MS-1 e include anche il confronto delle condizioni di induzione di EndMT di TGF-β in altre cellule endoteliali.

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	<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

Plasticità fenotipica delle cellule endoteliali è alla base dello sviluppo del sistema cardiovascolare, malattie cardiovascolari e varie circostanze connesse con la fibrosi dell'organo. In queste condizioni, differenziate cellule endoteliali acquisiranno fenotipi mesenchymal-like. Questo processo è chiamato transizione endoteliali mesenchimali (EndMT) ed è caratterizzato da downregulation dei marcatori endoteliali, upregulation di indicatori mesenchymal e cambiamenti morfologici. EndMT è indotta da diverse vie di segnalazione, compreso (TGF) del fattore di crescita trasformante-β, Wnt e tacca e regolata da meccanismi molecolari simili a quelli di transizione epiteliale-mesenchimale (EMT) importante per gastrulazione, fibrosi del tessuto, e metastasi del cancro. Comprensione dei meccanismi di EndMT è importante sviluppare approcci diagnostici e terapeutici EndMT di targeting. Induzione robusto del EndMT in vitro è utile per caratterizzare comune firme di espressione di gene, identificare meccanismi molecolari trattabili e dello schermo per modulatori di EndMT. Qui, descriviamo un metodo in vitro per l'induzione di EndMT. Le cellule endoteliali microvascolari del pancreas di mouse MS-1 subiscono EndMT dopo una prolungata esposizione a TGF-β e mostrano upregulation dei marcatori mesenchimali ed i cambiamenti morfologici così come induzione di più infiammatoria chemochine e citochine. Sono inclusi anche i metodi per l'analisi dei microRNA (miRNA) modulazione. Questi metodi forniscono una piattaforma per studiare i meccanismi alla base di EndMT e il contributo di Mirna a EndMT.

TGF-β è un potente induttore di EndMT in varie cellule endoteliali. Dopo il trattamento 24 ore con TGF-β in cellule MS-1, macchiatura per F-actina Mostra riorganizzazione dell'actina stress fibre (Figura 1A)20. Il pretrattamento con un inibitore ROCK Y-27632 inibisce l'induzione di actina riorganizzazione20. MS-1 cellule endoteliali modificare da una classica morfologia di ciottoli-come a una morfologia fusif...

Watch this Scientific Journal Video about Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-ÃŽÂ² Signaling at JoVE.com

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â² Signaling

Un protocollo per l'induzione in vitro di transizione endoteliali mesenchimali (EndMT), che è utile per investigare le vie di segnalazione cellulare coinvolti nella EndMT, è descritto. In questo modello sperimentale, EndMT è indotta dal trattamento con TGF-β in cellule endoteliali MS-1.

Analisi molecolare di transizione endoteliali mesenchimali indotta dalla trasformazione di segnalazione di fattore di crescita-β

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

Research

JoVE Journal

Biology

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

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Identificazione dei fenotipi inibizione della crescita indotta da Espressione di effettori batterica di tipo III nel lievito

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Ricerca

Biologia

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Video Bioinformatica Analisi delle embrionali umane crescita Colony cellule staminali

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

Volatili Blueberry erbivori indotta e intra-impianto di segnalazione

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2.  These HIPVs are mainly regulated by the defensive ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

L&#39;analisi in vitro dei complessi CFTR PDZ-dipendenti macromolecolari di segnalazione

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

La stimolazione meccanica indotta calcio Wave Propagation in monostrati cellulari: l&#39;esempio di bovini cellule endoteliali corneali

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

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

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Metabolic Etichettatura e con membrana frazionamento per proteomica comparativa Analisi del<em&gt; Arabidopsis thaliana</em&gt; Culture sospensione cellulare

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

Rilevamento delle modifiche droga indotte recettore post-internalizzazione Tratta di Analisi Colocalizational

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Fecale Analisi glucocorticoidi: non invasivo di monitoraggio surrenale in equidi

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Identificazione di segnale intracellulare eventi indotta in cellule vitali per interazione con le cellule vicine sottoposti apoptotica delle cellule morte

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...