Ringraziamo Ph.D. Hsia Ru-ching, presso l&#39;Electron Microscopy Core Facility della University of Maryland School of Medicine per il suo supporto tecnico nel trattamento dei campioni di scansione microscopica elettronica.

Dichiarazione di etica: I protocolli per la manipolazione degli animali sono stati approvati dal Comitato istituzionale per la cura degli animali (IACUC) dell&#39;Università del Maryland (protocollo numero 0416009) e condotto secondo gli standard internazionali AAALAC. 1. Procedura chirurgica <ol><li> Tecnica anestetica <ol><li> Sterilizzare tutti gli strumenti utilizzati per la chirurgia di sopravvivenza con sterilizzazione a vapore a 121 ° C per 30 min. </li><li> Indurre l&#39;anestesia attraverso un serbatoio di induzione con 100% O 2 e 2,5% isoflurano consegnato tramite vaporizzatore di precisione. Post-induzione, interrompere l&#39;isoflurano e sciacquare la camera con O 2 . Mantenere l&#39;anestesia con 1,5-2% di isoflurano tramite maschera e 1 L / min O 2 per inalazione. </li><li> Attaccare sia la camera di induzione e la maschera facciale ad un detergente per carbone per l&#39;assorbimento del gas di scarico per proteggere il personale. Garantire un adeguato livello anestetico da dimostrareChe non vi è risposta a stimoli nocivi (pizzicotto del piede). </li><li> Creare un campo operativo costituito da un vassoio chirurgico con rilievo isotermico per fornire un supporto termico durante l&#39;intervento chirurgico. Un ulteriore pad isotermico fornirà un supporto termico agli animali nella loro gabbia di recupero. </li></ol></li><li> Preparazione degli animali <ol><li> Eseguire la seguente inchiesta sui maschi C57BL / 6 maschi di 10-12 settimane. </li><li> Rimuovere i peli sulla superficie addominale ventrale dell&#39;animale dallo sterno fino alla regione inguinale con un agente depilatore o un tagliatore elettrico con una lama di 40. <ol><li> In caso di agente depilatorio, applicare questo composto alla zona chirurgica per 2-3 minuti e poi rimuoverlo con cotone. Utilizziamo un composto commercialmente disponibile di idrossido di calcio e idrossido di sodio. </li></ol></li><li> Preparare l&#39;area sopra la spalla con alcol al 70% e iniettare sottocutanea carprofen (5 mg / kg) con un ago da 25 gauge o più piccolo. QuestoIl trattamento fornirà analgesia postoperatoria per l&#39;animale. </li><li> Trasferisci l&#39;animale al campo chirurgico e posiziona nella ricostruzione dorsale. </li><li> Preparare il sito chirurgico sciacquando 8-12% di providone-iodio con un applicatore di cotone pulito o garza di cotone. Quindi sciacquare la pelle due volte con alcool al 70%. </li><li> Mettere il lubrificante oculare in entrambi gli occhi per ridurre l&#39;incidenza della desquamazione corneale. Coprire il sito chirurgico con un drappo sterile. </li></ol></li><li> Tecnica operativa <ol><li> Effettuare un&#39;incisione mediana di laparotomia addominale di circa 2-2,5 cm di lunghezza con un bisturi che inizia immediatamente caudale il processo xiphoid e si estende verso il bacino. </li><li> Mobilizzare l&#39;intestino e il duodeno e riflettere lateralmente a destra. Roll up una striscia di cotone sterile confezionato e imbevuto di salina sterile per l&#39;iniezione per consentire l&#39;imballaggio della viscera per migliorare l&#39;esposizione. </li><li> Con il piccolo intestino mobilitato al lato destro dellaL&#39;addome esporre il retroperitoneo ed esporre l&#39;aorta addominale dalla vena renale sinistra alla biforcazione aortica ( Figura 1 ). </li><li> Con un applicatore sterile a cotone, consegnare 30 frantumazioni consecutive, ogni cinque secondi in durata. </li><li> Rimuovere l&#39;imballaggio e consentire al viscera di tornare alla propria posizione nativa. </li><li> Chiudere la fascia con una sutura monofilamento assorbibile 4-0 (polidioxanone). La pelle è chiusa con una sutura monofilamento non in assorbimento 6-0 (nylon). </li></ol></li><li> Recupero e cura post-procedura <ol><li> Dopo la procedura, posizionare l&#39;animale in una gabbia di recupero con biancheria pulita su un tampone isotermico per continuare il supporto termico finché l&#39;animale non è in grado di ambulare normalmente. Non lasciare l&#39;animale incustodito fino a dimostrare la capacità di mantenere la ricostruzione sternale. </li><li> Monitorare l&#39;animale ogni ora per i primi 4 h dopo l&#39;intervento chirurgico. Una volta che l&#39;animale sta ambulando normalmente ritornare i<html> T alla camera di affiliazione assegnata. L&#39;animale non sarà ospitato in compagnia di un altro animale fino a quando non sarà completamente recuperato. </li><li> Monitorare l&#39;animale due volte al giorno per i primi 72 h dopo l&#39;intervento chirurgico e almeno 3 volte la settimana dopo. Il monitoraggio include la pesatura dell&#39;animale tre volte alla settimana. </li><li> Amministrare carpofen (5 mg / kg) sottocutanea due volte al giorno per i primi 72 h dopo l&#39;intervento chirurgico. </li></ol></li></ol> 2. Approvvigionamento di tessuti <ol><li> Metodo di eutanasia <ol><li> Eutanizzare gli animali in momenti prestabiliti. </li><li> Indurre l&#39;anestesia attraverso un serbatoio di induzione con 100% O 2 e 2,5% isoflurano consegnato tramite vaporizzatore di precisione. <ol><li> Per studiare l&#39;integrità dell&#39;endotelio, somministrare tintura blu all&#39;animale. NOTA: Evans Blue è un colorante azoto caricato negativamente con un&#39;elevata affinità di legame per l&#39;albumina e può solo macchiare i vasi sanguigni in assenza di un endotilo intattoS = &quot;xref&quot;&gt; 6. </li><li> Per gli studi di integrità endoteliale, al momento dell&#39;eutanasia, perfusione degli animali con 5 mL di 0.3% Evans Blue colorante seguita da 5 mL di PBS a pressione fisiologica per 5 minuti. L&#39;animale viene mantenuto in un piano chirurgico di anestesia durante la procedura di perfusione. </li></ol></li><li> Dopo aver raggiunto un piano profondo anestetico, aprire il petto con una sternotomia. Fare una lacerazione nell&#39;atrio destro per consentire il drenaggio del sangue dall&#39;animale e accedere al ventricolo sinistro è accessibile con un ago da 21 G e iniettare la soluzione salina fosfatizzata (PBS) finché l&#39;effluente dal atrio destro non è chiaro. </li><li> Dopo la perfusione con PBS, entrare nell&#39;addome attraverso un&#39;incisione midline. </li><li> Ancora una volta, mobilitare l&#39;intestino sottile al lato destro dell&#39;addome che esprima l&#39;aorta infrarenale, seziona acutamente l&#39;aorta dai tessuti adiacenti e lo accetta dalla vena renale sinistra alla biforcazione aortica. </li><li> Conservare l&#39;aorta esaurita in un paraformald del 4%Ehyde soluzione fino al processo di trasformazione del tessuto. <ol><li> Per gli studi di integrità endoteliale, aprire l&#39;aorta longitudinalmente e fissarlo a un foglio di cera esponendo l&#39;intera superficie della lumina 6 . Eseguire una valutazione qualitativa dell&#39;integrità endoteliale per il grado di colorazione con il colorante Evans Blue 6 . </li><li> Per altri esami istologici, tagliare l&#39;aorta trasversalmente e inserirlo in composti ottimali di taglio (OCT) come dettato dal metodo istologico da utilizzare 7 . </li></ol></li></ol></li></ol>

<ol>
	<li>Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+angiogenesis+and+arteriogenesis.">Mechanisms of angiogenesis and arteriogenesis.</a> Nat Med. 6 (4), 389-395 (2000).</li><li>Carmeliet, P., Moons, L., Collen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+angiogenesis,+arterial+stenosis,+atherosclerosis+and+hemostasis.">Mouse models of angiogenesis, arterial stenosis, atherosclerosis and hemostasis.</a> Cardiovasc Res. 39 (1), 8-33 (1998).</li><li>Baker, A. B., 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=Heparanase+Alters+Arterial+Structure,+Mechanics,+and+Repair+Following+Endovascular+Stenting+in+Mice.">Heparanase Alters Arterial Structure, Mechanics, and Repair Following Endovascular Stenting in Mice.</a> Circ Res. 104 (3), 380-387 (2009).</li><li>Petrov, L., Laurila, H., Hayry, P., Vamvakopoulos, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+aortic+angioplasty+for+genomic+studies+of+neointimal+hyperplasia.">A mouse model of aortic angioplasty for genomic studies of neointimal hyperplasia.</a> J Vasc Res. 42 (4), 292-300 (2005).</li><li>Li, J., 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=Vascular+smooth+muscle+cells+of+recipient+origin+mediate+intimal+expansion+after+aortic+allotransplantation+in+mice.">Vascular smooth muscle cells of recipient origin mediate intimal expansion after aortic allotransplantation in mice.</a> Am J Path. 158 (6), 1943-1947 (2001).</li><li>Radu, M., Chernoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+assay+to+test+blood+vessel+permeability.">An in vivo assay to test blood vessel permeability.</a> J Vis Exp. (73), e50062 (2013).</li><li>Turbett, G. R., Sellner, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+optimal+cutting+temperature+compound+can+inhibit+amplification+by+polymerase+chain+reaction.">The use of optimal cutting temperature compound can inhibit amplification by polymerase chain reaction.</a> Diagn Mol Pathol. 6 (5), 298-303 (1997).</li><li>Puchtler, H., Waldrop, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+mechanism+of+Verhoeff's+elastica+stain:+a+convenient+stain+for+myelin+sheaths.">On the mechanism of Verhoeff's elastica stain: a convenient stain for myelin sheaths.</a> Histochem. 62 (3), 233-247 (1979).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proc Natl Acad Sci U S A. 105 (7), 2415-2420 (2008).</li><li>Nelson, P. R., Yamamura, S., Mureebe, L., Itoh, H., Kent, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smooth+muscle+cell+migration+and+proliferation+are+mediated+by+distinct+phases+of+activation+of+the+intracellular+messenger+mitogen-activated+protein+kinase.">Smooth muscle cell migration and proliferation are mediated by distinct phases of activation of the intracellular messenger mitogen-activated protein kinase.</a> J Vasc Surg. 27 (1), 117-125 (1998).</li><li>Rzucidlo, E. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+disruption+and+matrix+metalloproteinase-9+expression+during+reperfusion+injury:+mechanical+versus+embolic+focal+ischemia+in+spontaneously+hypertensive+rats.">Blood-brain barrier disruption and matrix metalloproteinase-9 expression during reperfusion injury: mechanical versus embolic focal ischemia in spontaneously hypertensive rats.</a> Stroke. 33 (11), 2711-2717 (2002).</li><li>Yu, 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=MARCKS+Signaling+Differentially+Regulates+Vascular+Smooth+Muscle+and+Endothelial+Cell+Proliferation+through+a+KIS-,+p27kip1-+Dependent+Mechanism.">MARCKS Signaling Differentially Regulates Vascular Smooth Muscle and Endothelial Cell Proliferation through a KIS-, p27kip1- Dependent Mechanism.</a> PLoS One. 10 (11), e0141397 (2015).</li><li>Banai, S., 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=Rabbit+ear+model+of+injury-induced+arterial+smooth-muscle+cell-proliferation+-+kinetics,+reproducibility,+and+implications.">Rabbit ear model of injury-induced arterial smooth-muscle cell-proliferation - kinetics, reproducibility, and implications.</a> Circ Res. 69 (3), 748-756 (1991).</li></ol>

Questo lavoro è stato finanziato dal Dipartimento per gli Affari dei Veterani per lo sviluppo della carriera (1IK2BX001553-01) (TSM) e la cure vascolari EJ Wylie Scholarship (TSM).

Abbiamo caratterizzato gli effetti di un modello di lesioni aortiche murine che provoca l&#39;iperplasia mediale e la disfunzione della barriera endoteliale. Il distacco parziale CE lungo l&#39;aorta intima ha accompagnato la perdita di contatto cellulare e miglioramento delle protuberanze delle cellule. Corrispondentemente, la funzione di barriera endoteliale è stata significativamente compromessa, che ha stimolato i percorsi di segnalazione sensibili al mitogeno, portando alla proliferazione di VSMC e ad ispessimento...

La restenosi Seguendo procedure cardiovascolari (chirurgia bypass, angioplastica o stenting) è un problema significativo riducendo la durata di queste procedure. Tutte le procedure di rivascolarizzazione sono afflitte da restenosi. Le strategie presenti per prevenire la restenosi (stent che eluiscono farmaci e palloni rivestiti di droga) inibiscono sia la cellula muscolare liscia vascolare (VSMC) che la proliferazione delle cellule endoteliali (EC). Di conseguenza, questi interventi prevengono la restenosi mediata dal VSMC, ma impediscono anche la rigenerazione dell&#39;endotelio. Senza un endotelio intatto, i pazienti devono essere su potenti agenti antiagglomeranti per ridurre il rischio di trombosi in situ a rischio di complicazioni emorragiche. Una terapia ideale inibirà la proliferazione del VSMC, promuovendo la rigenerazione dell&#39;endotelio. Pertanto, vi è la necessità di studiare simultaneamente la proliferazione VSMC e la funzione di barriera endoteliale i n vivo . Attualmente, ci sono severModelli di topo restenosi 1 . Questi modelli includono lesioni carotide e lesioni del filo femorale 2 . I modelli aortici includono il posizionamento dello stent 3 , la lesione a palloncino 4 e l&#39;allograft aortico 5 . Tutti i modelli presenti sono limitati. La legatura carotide genera una lesione neointimale mediata dal flusso e non ha lesioni endoteliali. Inoltre, le arterie carotide e femorali hanno molte pieghe meno strati cellulari rispetto ai vasi umani, limitando il loro valore traduzionale. L&#39;aorta del topo, che è di circa 1,3 mm di diametro, è l&#39;unica vaso che si avvicina ad un&#39;arteria umana (coronaria) clinicamente rilevante (3). Nonostante il potenziale di traslazione dei modelli murali aortici della malattia, i modelli correnti hanno limitazioni. Questi modelli richiedono abilità microchirurgiche avanzate e attrezzature specializzate come palloncini e stents per angioplastica. Qui, noi presentiamoUna nuova tecnica riproducibile per indurre contemporaneamente la proliferazione del VSMC e disturbare la funzione di barriera endoteliale.

<table border="0" cellpadding="0" cellspacing="0" width="920">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Ocular lubricant</td>
			<td style="width:160px;">Dechra</td>
			<td style="width:163px;">17033-211-38</td>
			<td style="width:247px;">Pharmaceutical agents</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Isoflurane</td>
			<td>VetOne</td>
			<td>502017</td>
			<td>Pharmaceutical agents</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carprofen</td>
			<td>Zoetis</td>
			<td>26357</td>
			<td>Pharmaceutical agents</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Precision vaporizer</td>
			<td>Summit Medical</td>
			<td>10675</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Charcoal scavenger</td>
			<td>Bickford Inc.</td>
			<td>80120</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Isothermal pad</td>
			<td>Harvard Apparatus</td>
			<td>50-7053-R</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Sterile cotton-tipped applicator</td>
			<td>Fisher Scientific</td>
			<td>23-400-124</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">4-0 absorbable monofilament suture&#160;</td>
			<td>Ethicon, Inc</td>
			<td>J310</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">5-0 non-absorbable monofilament suture</td>
			<td>Ethicon,Inc</td>
			<td>1666</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">21-gauge x 1 inch needle</td>
			<td>BD Biosciences</td>
			<td>305165</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">25-gauge x 1 inch&#160; needle</td>
			<td>BD Biosciences</td>
			<td>305125</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dry sterilizer</td>
			<td>Cellpoint&#160;</td>
			<td>7770</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-09</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adson forceps</td>
			<td>Fine Science Tools</td>
			<td>11006-12</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Dumont #5 fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Vannas Spring Scissors 3mm cutting edge</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Needle driver</td>
			<td>Fine Science Tools</td>
			<td>91201-13</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel handle #4</td>
			<td>Fine Science Tools</td>
			<td>10004-13</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel blades #10</td>
			<td>Fine Science Tools</td>
			<td>10010-00</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS&#160;</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td>Reagents for tissue processing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Evans Blue</td>
			<td>Sigma-Aldrich</td>
			<td>E2129</td>
			<td>Reagents for tissue processing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Reagents for tissue processing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Modeling wax</td>
			<td>Bego</td>
			<td>40001</td>
			<td>Reagents for tissue processing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OCT compound</td>
			<td>Tissue-Tek Sakura</td>
			<td>4583</td>
			<td>Reagents for tissue processing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mayer&#39;s hematoxylin solution</td>
			<td>Sigma-Aldrich</td>
			<td>MHS16</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eosin Y solution alcoholic&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>HT110316</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Elastin stain kit</td>
			<td>Sigma-Aldrich</td>
			<td style="width:163px;">HT25A</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Click-it Edu Alexa-488 Imaging Kit</td>
			<td>Invitrogen</td>
			<td>C10337</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Anti-Erk1/2 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4695</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Anti-phospho-Erk1/2 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4370</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Anti-p27kip1 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>3698</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Trichloroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T9159</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
	</tbody>
</table>

murine aortic crush injury: an efficient in vivo model of smooth muscle cell proliferation and endothelial function

La ricostruzione arteriosa, sia in caso di angioplastica che di intervento di bypass, comporta traumi iatrogeni che provocano disturbi endoteliali e proliferazione delle cellule muscolari lisce vascolari (VSMC). I modelli murini comuni studiano piccole vasi come le arterie carotide e femorali. Qui descriviamo un sistema in vivo in cui sia la proliferazione VSMC che la funzione di barriera endoteliale possono essere valutate contemporaneamente in un grande recipiente. Abbiamo studiato la risposta aortica infrarenale alla lesione nei topi C57BL / 6. L&#39;aorta è stato ferito dalla vena renale sinistra alla biforcazione aortica da 30 crush transmural di durata di 5 secondi con un applicatore a cotone. I cambiamenti morfologici sono stati valutati con l&#39;istologia convenzionale. Lo spessore della parete aorta è stato misurato dalla superficie luminare all&#39;adventitia. L&#39;integrazione di EdU e la colorazione di contatori con DAPI e l&#39;alpha-actina è stata utilizzata per dimostrare la proliferazione del VSMC. L&#39;attivazione di ERK1 / 2, un moderatore noto della formazione di iperplasia intima, è stato scoraggiatoEstratti da Western Blot. L&#39;effetto dell&#39;infiammazione è stato determinato da immunohistochemistry per le cellule B, le cellule T e macrofagi . Le sezioni di endotelio sono state visualizzate con la microscopia elettronica a scansione (SEM). La funzione di barriera endoteliale è stata determinata con la colorazione Evans Blue. La lesione transmurale ha provocato l&#39;ispessimento della parete aortica. Questa lesione ha causato la proliferazione del VSMC, soprattutto 3 giorni dopo la lesione e l&#39;attivazione precoce di ERK1 / 2 e la diminuzione della espressione p27 kip1. La lesione non ha determinato l&#39;aumento delle cellule B, delle cellule T o dell&#39;infiltrazione dei macrofagi nella parete del vaso. La lesione ha causato denudazione parziale della cellula endoteliale e la perdita del contatto cellulare. La lesione ha causato una significativa perdita di funzione barriera endoteliale, che è tornata alla base dopo sette giorni. Il modello di lesioni aortiche blasfonde trasversali murine fornisce un sistema efficace per studiare contemporaneamente sia la proliferazione VSMC che la funzione di barriera endoteliale in un grande recipiente.

Le sezioni trasversali delle aorta incorporate in OCT sono state sezionate e macchiate con ematoxilina e eosina e poi contate con la macchia Verhoeff-Van Gieson (VVG) per identificare la lamina elastica interna ed esterna 7 . L&#39;ispessimento della parete aortica indotta da lesioni a crisi è aumentato rispetto alle aorte degli animali trattati con una procedura sham (laparotomia e piccola mobilitazione intestinale). Lo spessore delle pareti, valutato dalla dist...

Watch this Scientific Journal Video about Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function at JoVE.com

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

La restenosi dopo procedure cardiovascolari (chirurgia bypass, angioplastica o stenting) è un problema significativo che riduce la durata di queste procedure. Una terapia ideale potrebbe inibire la proliferazione delle cellule muscolari lisce (VSMC), promuovendo la rigenerazione dell&#39;endotelio. Descriviamo un modello per la valutazione simultanea della proliferazione VSMC e della funzione endoteliale in vivo.

Lesione aortica di crusca a murina: un efficace<em&gt; In Vivo</em&gt; Modello di proliferazione cellulare liscia e funzione endoteliale

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

murine-aortic-crush-injury-an-efficient-vivo-model-smooth-muscle-cell

Research

JoVE Journal

Biology

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

8.3K Views.  Baltimore Veterans Affairs Medical Center.  La restenosi dopo procedure cardiovascolari (chirurgia bypass, angioplastica o stenting) è un problema significativo che riduce la durata di queste procedure. Una terapia ideale potrebbe inibire la proliferazione delle cellule muscolari lisce (VSMC), promuovendo la rigenerazione dell'endotelio. Descriviamo un modello per la valutazione simultanea della proliferazione VSMC e della funzione endoteliale in vivo. 

Video: Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

Focale Ca 2 + Rilevamento transitoria nel muscolo liscio

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Ricerca

Biologia

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

Esplorare arteriosa Muscolo liscio KV7 Funzione canale del potassio con morsetto Elettrofisiologia Patch e Myography pressione

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

Valutazione della funzione muscolare del muscolo estensore lungo delle dita<em&gt; Ex vivo</em&gt; E Muscolo tibiale anteriore<em&gt; In situ</em&gt; In Mouse

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

Misura di Smooth funzione muscolare nei isolato carta igienica-applicazioni Farmacologia Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

A due fotoni Imaging di Intracellulare Ca<sup&gt; 2+</sup&gt; Manipolazione e l&#39;ossido nitrico Produzione in endoteliale e della muscolatura liscia cellule di un isolato Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Isolamento e colture primarie di cellule di topo aortica endoteliali

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

L&#39;isolamento di Murine coronarica vascolari cellule muscolari lisce

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease processes. For these reasons, measuring proliferation is a common tool used to assess cell function. Cell proliferation can be measured simply by counting; however, this is an indirect means of measuring proliferation. One common means of directly detecting cells preparing to divide is by incorporation of labeled nucleoside analogs. These include the radioactive nucleoside analog 3H-thymidine plus non-radioactive nucleoside analogs such as 5-bromo-2&#8217; deoxyuridine (BrdU) and 5-ethynyl-2&#8242;-deoxyuridine (EdU). Incorporation of EdU is detected by click chemistry, which has several advantages when compared to BrdU. In this report, we provide a protocol for measuring proliferation by the incorporation of EdU. This protocol includes options for various readouts, along with the advantages and disadvantages of each. We also discuss places where the protocol can be optimized or altered to meet the specific needs of the experiment planned. Finally, we touch on the ways that this basic protocol can be modified for measuring other cell metabolites.

Proliferation is a critical part of cellular function, and a common readout used to assess potential toxicity of new drugs. Measuring proliferation is, therefore, a frequently used assay in cell biology. Here we present a simple, versatile method of measuring proliferation that can be used in adherent and non-adherent cells.

Misurazione della proliferazione delle cellule muscolari lustiche vascolari utilizzando la chimica dei clic

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease ...

In vivo Measurement of Knee Extensor Muscle Function in Mice

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

Misurazione in vivo della funzione muscolare estensore del ginocchio nei topi

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the ...

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

This paper describes an explant culture-based method for the isolation and culturing of primary, patient-specific human aortic smooth muscle cells and dermal fibroblasts. Furthermore, a novel method is presented for measuring cell contraction and subsequent analysis, which can be used to study patient-specific differences in these cells.

Isolamento delle cellule muscolari lisce aortiche primarie specifiche del paziente e misurazioni semiquantitative di contrazione in tempo reale in vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in ...

Lesione aortica di crusca a murina: un efficace

Riepilogo

Esplora Altri Video

Capitoli in questo video

Focale Ca^{2 +} Rilevamento transitoria nel muscolo liscio

Esplorare arteriosa Muscolo liscio KV7 Funzione canale del potassio con morsetto Elettrofisiologia Patch e Myography pressione

Valutazione della funzione muscolare del muscolo estensore lungo delle dita

Misura di Smooth funzione muscolare nei isolato carta igienica-applicazioni Farmacologia Research

A due fotoni Imaging di Intracellulare Ca

Isolamento e colture primarie di cellule di topo aortica endoteliali

L'isolamento di Murine coronarica vascolari cellule muscolari lisce

Misurazione della proliferazione delle cellule muscolari lustiche vascolari utilizzando la chimica dei clic

Misurazione in vivo della funzione muscolare estensore del ginocchio nei topi

Isolamento delle cellule muscolari lisce aortiche primarie specifiche del paziente e misurazioni semiquantitative di contrazione in tempo reale in vitro