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

Vi takker Zea Borok og Kohei Miyazono for forslag i utarbeidelsen av manuskriptet. H.I.S. og M.H. støttes av forskningsstipend for Uehara Memorial Foundation, og H.I.S. er støttet av Osamu Hayaishi Memorial stipend for studier i utlandet. Dette arbeidet ble støttet av et stipend fra Takeda Science Foundation (AS).

1. induksjon av EndMT
<ol><li>Opprettholde MS-1 celler i standard oppdrettsforholdene og unngå confluency. En kilde til MS-1 celler er beskrevet i Tabellen for materiale. For MS-1 celler, kan du bruke Minimum nødvendig Medium-α (MEM-α) med 10% fosterets kalv serum (FCS), 50 U/mL penicillin og 50 μg/mL streptomycin.</li>
	<li>Vask MS-1 celler på 10 cm rett med 1 x fosfat bufret saltvann (PBS) og tilsett 1,0 mL av trypsin til platen. Inkuber i 5 min på 37 ° C.</li>
	<li>Koble celler med 9 mL kult...

<ol>
	<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. 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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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Det har blitt rapportert at aktivert Ras og TGF-β behandling for 24 h indusert EndMT i MS-1 celler, mens TGF-β alene mislyktes å indusere EndMT i denne korte perioden21. Konsekvent, observerte vi at TGF-β vesentlig indusert EndMT etter lengre behandling (48-72 h) i MS-1 celler20. EndMT er observert flere ganger etter langvarig behandling med TGF-β (2 – 6 dager) i forskjellige endotelceller som menneskelige umbilical vene endotelceller (HUVEC), menneskelige kutan mikr...

Endothelial-mesenchymal overgang (EndMT) er prosessen der en differensiert endothelial celle gjennomgår ulike molekylær endringer, som resulterer i en fibroblast-lignende mesenchymal celle1. EndMT ble først beskrevet som en endothelial celle transformasjon under utviklingen av hjertet2,3. I tidlig heart utvikling består hjertet røret av en indre endocardium og en ytre myokard. Disse to lagene er atskilt av et ekstracellulær matrix kalles cardiac gelé. Embryonale endocardial celler som skaffer endothelial celle markører, transitt i mesenchymal celler, invadere underliggende cardiac gelé og fremme dannelsen av cardiac puter, som er grunnlaget for atrioventrikulær ventiler og septum og semilunar ventiler. Videre har EndMT blitt foreslått for å være kilder til pericytes og vaskulær glatte muskelcellene i andre embryonale vaskulære systemer inkludert koronar fartøy, abdominal aorta og lungearterien4,5,6. I tillegg er EndMT innblandet i fysiologiske angiogenic spirende7.
Samler bevis har foreslått at EndMT er også involvert i flere karsykdommer og andre sykdommer1,8. EndMT-assosiert tilstandene omfatter vaskulær forkalkninger, atherosclerosis, pulmonal arteriell hypertensjon, kavernøse misdannelse, orgel fibrose, vene pode remodeling, allograft dysfunksjon i nyre transplantasjon, og kreft8, 9,10,11,12,13,14,15,16,17, 18. en fersk rapport beskrevet at flere molekylære EndMT markører kan være et verktøy for diagnose og prognose prediksjon av nyre pode dysfunksjon i nyre transplantasjon17. Modulering av EndMT-relaterte mobilnettet signalveier har vist seg å forbedre flere sykdom betingelser inkludert cardiac fibrose og vene pode remodeling i dyr modeller8,15. Forstå mekanismene er underliggende EndMT derfor viktig å utvikle diagnostiske og terapeutiske strategier målretting EndMT.
EndMT er preget av tap av celle-celle veikryss, økning i vandrende potensial, downregulation av endothelial-spesifikke gener som VE-cadherin og oppregulering av mesenchymal gener inkludert α-glatt muskel utgangen (α-SMA). I tillegg er EndMT og epithelial-mesenchymal overgang (EMT), en tilsvarende prosess som konverterer epitelceller mesenchymal celler, forbundet med meditativ produksjon av ulike ekstracellulær matrix komponenter som kan bidra til utvikling vev fibrose8,19.
Flere i vitro studier av EndMT har nylig belyst detaljer om molekylære mekanismer EndMT15,20. EndMT er indusert av ulike signalveier inkludert omforming vekst faktor (TGF)-β, Wnt, og hakk1. Blant dem spiller TGF-β viktige roller i induksjon av både EMT og EndMT. I EndMT, langvarig eksponering for TGF-β resultater i EndMT i forskjellige endotelceller, mens kort eksponering synes å være utilstrekkelige21. Vi her har beskrevet en enkel protokoll for EndMT induksjon, i hvilken MILE SVEN 1 (MS-1) musen bukspyttkjertelen mikrovaskulær endotelceller gjennomgå EndMT i vitro etter langvarig eksponering for TGF-β20. I denne modellen, kan flere nedstrøms analyser utføres for å undersøke hallmark funksjoner i EndMT, inkludert morfologiske endringer, downregulation av endothelial markører, oppregulering av mesenchymal markører og provoserende gener, cellen cytoskjelett rearrangements og kollagen gel sammentrekning.
MicroRNAs (miRNAs) er ~ 22 nt liten regulatoriske RNAs som direkte posttranscriptional undertrykkelse av ulike mRNA mål22,23. Gjennom frø sekvens-mediert målet anerkjennelse, miRNAs undertrykke hundrevis av målet gener og modulere mangfoldig cellular funksjonene som celledifferensiering, spredning og motilitet. Dette er også tilfelle for regulering av EMT og EndMT, og flere miRNAs har blitt rapportert som regulatorer av EMT og EndMT24,25. EndMT modellen presentert i denne gjennomgangen kan lett kombineres med miRNA modulering prosedyrer å teste rollene som miRNAs i EndMT. Stede gjennomgangen oppsummerer våre eksperimentelle prosedyrer for å undersøke TGF-β-indusert EndMT i MS-1 celler og inneholder sammenligning av betingelser for EndMT induksjon av TGF-β i andre endotelceller.

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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

Fenotypiske plastisitet av endotelceller ligger under kardiovaskulære systemutvikling, kardiovaskulære sykdommer og ulike forhold knyttet til orgel fibrose. I disse forholdene erverve differensiert endotelceller mesenchymal-lignende fenotyper. Denne prosessen kalles endothelial-mesenchymal overgang (EndMT) og er preget av downregulation av endothelial markører, oppregulering av mesenchymal markører og morfologiske endringer. EndMT er indusert av flere signalveier, inkludert omforming vekst faktor (TGF)-β, Wnt og hakk, og regulert av molekylære mekanismer ligner på de av epitelial-mesenchymal overgang (EMT) viktig for gastrulation, vev fibrosis, og kreft metastasering. Forstå mekanismene av EndMT er viktig å utvikle diagnostiske og terapeutiske metoder rettet mot EndMT. Robust induksjon av EndMT i vitro er nyttig å karakterisere felles genuttrykk signaturer, identifisere druggable molekylære mekanismer og skjerm for modulatorer av EndMT. Her beskriver vi en i vitro metode for induksjon av EndMT. MS-1 musen bukspyttkjertelen mikrovaskulær endotelceller gjennomgå EndMT etter langvarig eksponering for TGF-β og Vis oppregulering av mesenchymal markører og morfologiske endringer og induksjon av flere inflammatorisk chemokines og cytokiner. Metoder for analyse av microRNA (miRNA) modulering er også inkludert. Disse metodene gir en plattform for å undersøke mekanismene bak EndMT og bidrag av miRNAs til EndMT.

TGF-β er en potent induser av EndMT i forskjellige endotelceller. Etter 24 timer behandling med TGF-β i MS-1 celler viser flekker for F-utgangen omorganisering av utgangen stress fiber (figur 1A)20. Forbehandling med en ROCK hemmer Y-27632 hemmer induksjon av utgangen omorganisering20. MS-1 endotelceller endres fra en klassisk brosteinsbelagte-lignende morfologi til en mesenchymal spindel-formet morfologi på ...

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

En protokoll for i vitro induksjon av endothelial-mesenchymal overgang (EndMT), som er nyttig for å undersøke mobilnettet signalveier involvert i EndMT, er beskrevet. I denne eksperimentelle modellen er EndMT indusert av behandling med TGF-β i MS-1 endotelceller.

Molekylær analyse av Endothelial-mesenchymal overgang av omforming vekst faktor-β signalisering

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

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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

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 ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

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

Herbivore-indusert Blueberry flyktige og Intra-plante signaleringsprotokoll

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

In Vitro Analyse av PDZ-avhengige CFTR makromolekylær signalveier Komplekser

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

Mekanisk stimulering-indusert Kalsium wave forplantning i cellemonolagene: The Eksempel på storfe Hornhinnen endotelceller

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

Induksjon og analyse av Epiteliale til Mesenchymale Transition

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

Metabolsk Merking og Membran Fraksjone for komparativ proteomikk analyser av<em&gt; Arabidopsis thaliana</em&gt; Suspension cellekulturer

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

Sporing Drug-induced Endringer i Receptor Post-intern Trafficking etter Colocalizational Analysis

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

Fecal Glukokortikoid analyse: Ikke-invasiv Adrenal Monitoring i Equids

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

Identifikasjon av intracellulære signal Hendelser indusert i levende celler ved Interaksjon med nabocellene gjennomgår celledød ved apoptose

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