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 under forberedelse af manuskript. H.I.S. og M.H. understøttes af Uehara Memorial Foundation Research Fellowship, og H.I.S. er understøttet af Osamu Hayaishi Mindelegat for Study Abroad. Dette arbejde blev støttet af en bevilling fra Takeda Science Foundation (A.S.).

1. induktion af EndMT
<ol><li>Vedligeholde MS-1 celler i standard dyrkningsbetingelserne og undgå confluency. En kilde til MS-1 celler er beskrevet i Tabel af materialer. For MS-1 celler, skal du bruge Minimum afgørende Medium-α (MEM-α) med 10% føtalt kalveserum (FCS), 50 U/mL penicillin og 50 μg/mL streptomycin.</li>
	<li>Vaske MS-1 celler på 10 cm parabol med 1 x fosfatbufferet saltopløsning (PBS) og tilsættes 1,0 mL trypsin til pladen. Inkuber i 5 min. ved 37 ° C.</li>
	<li>Frigør celler ...

<ol>
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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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Det er blevet rapporteret, at aktiveret Ras og TGF-β behandling for 24 h induceret EndMT i MS-1 celler, mens TGF-β alene undladt at fremkalde EndMT i denne korte periode21. Konsekvent, konstaterede vi, at TGF-β væsentligt induceret EndMT efter længere behandling (48-72 h) i MS-1 celler20. EndMT er gentagne gange blevet observeret efter langvarig behandling med TGF-β (2-6 dage) i forskellige endotelceller såsom menneskelige umbilical vene endotelceller (HUVEC), mennes...

Endotel-mesenchymale overgang (EndMT) er den proces, hvorved en differentieret endotel celle gennemgår en række forskellige molekylære ændringer, hvilket resulterer i en fibroblast-lignende mesenchymale celler1. EndMT blev oprindeligt beskrevet som en endotel celle transformation under udviklingen af hjertet2,3. I tidlig udvikling af hjertet består hjerte rør af en indre endokardiet og en ydre myokardiet. Disse to lag er adskilt af et lag af ekstracellulære matrix kaldes den kardiale jelly. De embryonale endokardial celler, som erhverver endotel celle markører, transit til mesenchymale celler, invadere de underliggende hjerte gelé og fremme dannelsen af den kardiale puder, giver grundlaget for atrieventrikelklapperne og septum og semilunar ventiler. Desuden er EndMT blevet foreslået for at være kilder til pericytes og vaskulære glatte muskelceller i andre embryonale vaskulære systemer, herunder koronar fartøjer, abdominal aorta og lungepulsåren4,5,6. Derudover er EndMT impliceret i fysiologiske angiogene spiring7.
Akkumulere beviser har foreslået, at EndMT også er involveret i flere hjerte-kar-sygdomme og andre sygdomme1,8. EndMT-associerede betingelser omfatter vaskulære forkalkning, åreforkalkning, pulmonal arteriel hypertension, bundløs misdannelser, orgel fibrose, vene graft remodeling, allograft dysfunktion i nyre transplantation og kræft8, 9,10,11,12,13,14,15,16,17, 18. en nylig rapport beskrev, at flere molekylære EndMT markører kan være et redskab til diagnose og prognose forudsigelse af renal graft dysfunktion i nyre transplantation17. Graduering af EndMT-relaterede cellulær signalering veje har vist sig at forbedre adskillige sygdomstilstande, herunder hjerte fibrose og vene graft remodeling i dyre modeller8,15. Derfor er forstå mekanismerne underliggende EndMT vigtigt at udvikle diagnostiske og terapeutiske strategier rettet mod EndMT.
EndMT er karakteriseret ved tab af celle-celle vejkryds, forøgelse af vandrende potentiale, downregulation i endothelial-specifikke gener som VE-cadherin, og opregulering af mesenchymale gener herunder α-glat muskel aktin (α-SMA). Desuden er EndMT og epitel-mesenchymale overgang (EMT), en lignende proces, der konverterer epitelceller mesenchymale celler, forbundet med ændret produktion af forskellige ekstracellulære matrix komponenter, som kan bidrage til udviklingen af væv fibrose8,19.
For nylig, flere in vitro- undersøgelser af EndMT har belyst detaljer molekylære mekanismer i EndMT15,20. EndMT er foranlediget af forskellige signaling veje herunder omdanne vækst faktor (TGF)-β, Wnt, og hak1. Blandt dem spiller TGF-β afgørende roller i induktion af både EMT og EndMT. Lang tids udsættelse for TGF-β resultater i EndMT i forskellige endotelceller, i EndMT, mens korte eksponering ser ud til at være utilstrækkelig21. Vi har her beskrevet en enkel protokol for EndMT induktion, i hvilke MILE SVEN 1 (MS-1) mus i bugspytkirtlen mikrovaskulære endotelceller gennemgå EndMT i vitro efter langvarig udsættelse for TGF-β20. I denne model, kan flere downstream analyser udføres for at undersøge hallmark egenskaber i EndMT, herunder morfologiske ændringer, downregulation i endothelial markører, opregulering af mesenchymale markører og inflammatoriske gener, cytoskeletal rearrangementer, og kollagen gel sammentrækning.
MicroRNA (miRNAs) er ~ 22 nt små lovgivningsmæssige RNA'er, der direkte posttranskriptionelle undertrykkelse af forskellige mRNA mål22,23. Gennem seed sekvens-medieret mål anerkendelse, miRNAs undertrykke hundredvis af target gener og modulere forskellige cellulære funktioner såsom Celledifferentiering, spredning og motilitet. Dette er også tilfældet for regulering af EMT og EndMT, og flere miRNAs er blevet rapporteret som regulatorer af EMT og EndMT24,25. Den EndMT model præsenteret i denne gennemgang kan let kombineres med miRNA graduering procedurer til test af miRNAs rolle i EndMT. Denne undersøgelse opsummerer vores eksperimentelle procedurer for at undersøge TGF-β-induceret EndMT i MS-1 celler og omfatter også sammenligning af EndMT induktion af TGF-β forhold 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

Fænotypisk plasticitet i endothelial celler ligger til grund for systemudvikling af hjerte-kar-, hjerte-kar-sygdomme og forskellige betingelser forbundet med orgel fibrose. I disse betingelser erhverve differentieret endotelceller mesenchymale-lignende fænotyper. Denne proces kaldes endotel-mesenchymale overgang (EndMT) og er kendetegnet ved downregulation i endothelial markører, opregulering af mesenchymale markører og morfologiske ændringer. EndMT er foranlediget af flere signaling veje, herunder omdanne vækst faktor (TGF)-β, Wnt og hak, og reguleret af molekylære mekanismer ligner dem af epitel-mesenchymale overgang (EMT) vigtig for gastrulation, væv fibrose, og kræft metastaser. Forstå mekanismerne i EndMT er vigtigt at udvikle diagnostiske og terapeutiske strategier rettet mod EndMT. Robust induktion af EndMT i vitro er nyttige til at karakterisere fælles gene expression signaturer, identificere druggable molekylære mekanismer og skærmen for modulatorer af EndMT. Her, beskriver vi en in vitro- metode til induktion af EndMT. MS-1 mus i bugspytkirtlen mikrovaskulære endotelceller underkastes EndMT efter langvarig udsættelse for TGF-β og vise opregulering af mesenchymale markører og morfologiske ændringer samt induktion af flere inflammatoriske kemokiner og cytokiner. Metoder til analyse af mikroRNA (miRNA) graduering er også inkluderet. Disse metoder giver en platform til at undersøge mekanismerne bag EndMT og bidrag af miRNAs til EndMT.

TGF-β er en potent inducer af EndMT i forskellige endotelceller. Efter 24 timer behandling med TGF-β i MS-1 celler viser farvning for F-actin reorganisering af actin stress fibre (figur 1A)20. Forbehandling med en ROCK hæmmer Y-27632 hæmmer induktion af actin reorganisering20. MS-1 endotelceller Skift fra en klassisk brostensbelagte-lignende morfologi til en mesenchymale spindel-formet morfologi på TGF-β b...

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 protokol til in vitro- induktion i endothelial-mesenchymale overgang (EndMT), som er nyttige for efterforskningen cellulær signalering veje involveret i EndMT, er beskrevet. I denne eksperimentelle model, er EndMT foranlediget af behandling med TGF-β i endothelial celler, MS-1.

Molekylær analyse af endotel-mesenchymale overgang induceret af Transforming Growth Factor-β signalering

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

Planteæder-induceret Blueberry Flygtige stoffer og intra-anlæg Signaling

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 af PDZ-afhængige CFTR Makromolekylære signalering 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 Stimulation-induceret Calcium bølgeudbredelse i cellemonolag: eksempel med Bovin hornhindeendothelceller

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

Induktion og analyse af Epithelial 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

Metabolisk mærkning og membranfraktionering for Komparativ proteomiske analyse af<em&gt; Arabidopsis thaliana</em&gt; Suspension Cell Cultures

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 lægemiddelinduceret Ændringer i Receptor Post-internalisering Trafficking af 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

Fækal Glukokortikoid Analyse: Non-invasive Adrenal Overvågning i dyr fra hestefamilien

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

Identifikation af intracellulær signalering begivenheder induceret i levedygtige celler ved interaktion med naboceller Gennemgår apoptotisk celledød

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