Bu eser Ulusal kalp tarafından desteklenmiştir, akciğer ve kan Enstitüsü R01088554, T32HL007284 ve Amerikan Kalp Derneği HGUGM bursu 14PRE20420024 verir. Biz özellikle St. Jude hücresel görüntüleme paylaşılan araştırma Randall Wakefield ve Linda Horner elektron mikroskobu görüntüler için Adam Straub temsilcisi ayirt görüntüleri hakkında yardım almak için ve onun için Pooneh Bagher dahil olmak üzere çekirdek, teşekkür el yazması Protokolü değerli geribildirim.

1. kaplama hücre VCCC <ol><li>kültür büyük 225 cm 2 şişeler EC ve SMC % 70-90 birleşmesi kadar 37 ° C'de steril koşullarda. SMC daha daha fazla EC kullanılan sağlamak; birincil insan EC ve SMC, EC genellikle 3 büyük şişeler SMC 1 büyük şişesi için kullanıyorsanız.
	<ol><li>Alt geçitler Primer hücre kullanın ve geçit 10 kullanmayın. Co kültürlü olmak alan kültür ortamı seçin. Birincil insan koroner arter için EC, EBM-2 EC Bazal medya EGM2 MV büyüme faktörleri ile kullanın. İçin birincil insan koroner SMC, SmBM SMC Bazal medya SmGM-2 büyüme faktörleri ile kullanın. Önce kültürlü hücrelerle, büyüme faktörleri medyaya ekle.</li>
	</ol></li> <li>VCCC gün 1 inşaatı: sprey büyük (150 mm) Petri yemekler ve kapakları dezenfektan ile (örneğin, Cavicide) ve uzak bir kağıt havlu veya hav bırakmayan mendil ile silin. O zaman Petri yemekler % 70 etanol (alkol) ile sprey ve mahallede hava kuru koyun.</li>
	<li>(Tablo reçetesi görmek) filtre ekler tabak steril koşullarda açın ve (filtre alt tarafı) SMC tarafında ekleme fibronektin çözüm ile kat. INSERT sağlanan 6-şey plaka tutarak, damlalıklı fibronektin çözüm yan aracılığıyla filtre yarısı alt (en fazla 1 mL) kaplı emin plaka, altına kesmiş.</li>
	<li>Sonra 30 dk fibronektin çözüm, oda sıcaklığında hücre kültür Hood ile tedavinin ekler plaka dışında al, herhangi bir fazla çözüm ve ters çevir'i temiz Petri kabına yarısı altına yerleştirerek, vakum. Bir kenara koyun. Filtre Ekle membran aşırı çözüm emme olduğunda bozmamaya dikkat edin; Bu yavaşça plaka devirme ve çözüm ekleme kenarına kapalı vakumlama önlenebilir.</li>
	<li>Trypsinize 225 cm 2 şişeye SMC, 3 mL Önceden ısıtılmış 2 X tripsin-EDTA çözüm, ve bir kez hücreleri plaka kaldırdı ile 9 mL SMC medya tripsin (3:1, medya: tripsin) nötralize etmek için ekleyin. Aktarım için konik bir tüp, mix iyi ve damlalıklı 10 µL üzerine cep numarasını belirlemek için bir hemasitometre.
	<ol><li>Sayısı 5 x 5 hücre sayısı kutuları (Yani, 18), bu hücre sayısı 1 mL (180.000) almak için 10.000 çarpmak. O zaman 12 mL (toplam hacmi hücre süspansiyon, 2.16 milyon SMC) tarafından çarpın. Wells için istediğiniz sayıyı plaka için yeterli SMC olduğundan emin olun. 
		Not: Örneğin, 1 de 75.000 SMC olmalı ve böylece 18 wells 1.35 milyon toplam SMC olurdu. 1 mL başına 180.000 hücreleri tarafından gerekli 1.35 milyon Hücre Böl ve kaplama için gerekli olacak hücre süspansiyon 7.5 mL sonuçlanır. Sonra gerekli son hacim hesaplamak (18 kuyu x 750 µL 13.5 mL =). Böylece, hücre süspansiyon 7.5 mL al, ses getirmek Önceden ısıtılmış SMC medya ve mix iyi ile ilâ 13,5 mL.</li>
	</ol></li> <li>Plaka 750 µL yaklaşık her süzgeç, alt tarafında üzerine 75.000 SMC bir medya ve hücre çözüm bile ziyan değil dikkatli içeren hücre süspansiyon. Kapağı üstüne yerleştirin ve dikkatle Petri kabına ekler ile 37 ° C'de kuluçka makinesi içine gecede transfer. 
	Not: Diğer hücre tipleri için optimum hücre yoğunluğu ampirik olarak belirlenmesi gerekir.</li>
	<li>2 günde temiz 6 iyi yemekler taze, Önceden ısıtılmış SMC medya 2 mL ile doldurulması. Tabakları da kuluçka kaldırın. Ekler tek tek dışarı almak ve medya tarafından emme kaldırmak sonra yer SMC medya ile 6-iyi çanak içine.</li>
	<li>Filtre (EC yan) karşı tarafına 1 mL için en az 30 dk. %37 0.5 sığır jelatin çözeltisi ile kat ° C.</li>
	<li>EC 225 cm 2 flask(s) Önceden ısıtılmış 2 3 mL ile trypsinize X tripsin-EDTA (10 x seyreltilmiş steril Dulbecco &#39; s PBS) hücreleri plaka dışına Asansör için balonun nazik dokunarak ile çözüm. 
	Not: Şişeye geri 37 ° C'de kuluçka 1-3 dakika içine yerleştirmek için gerekli olabilir.
	<ol>Bir kez <li>hücreleri plaka kaldırdı tripsin (3:1) nötralize etmek için 9 mL EC medya ekleyin. Konik bir tüp, havuzu birlikte transfer, mix iyi ve damlalıklı 10 µL üzerine cep numarasını belirlemek için bir hemasitometre.
		<ol><li>Sayısı 5 x 5 hücre sayısı kutuları (Yani, 60), bu hücre sayısı 1 mL (600.000) almak için 10.000 çarpmak. O zaman 12 mL (toplam hacmi hücre süspansiyon, 7.2 milyon EC) tarafından çarpın. Wells için istediğiniz sayıyı plaka için yeterli EC olduğundan emin olun. 
			Not: İyi yaklaşık 360.000 EC gereklidir. Örneğin, 18 wells 6.48 milyon EC gerektirir 600.000 EC 1 mL başına gerekli 6.48 milyon Hücre Böl ve kaplama için gerekli olacak hücre süspansiyon 10.8 mL sonuçlanır. Sonra gerekli son hacim hesaplamak (18 kuyu 1 mL = 18 mL x). Böylece, hücre süspansiyon 10.8 mL al, birim kadar getirmek ile 18 mL önceden EC medya ısıttı ve yavaşça resuspend.</li>
		</ol></li></ol></li> <li>1 mL her INSERT filtre üst kısmı üzerine 360.000 EC plaka. 24 h yerine orta gün 4'te 37 ° C'de rahatsız edilmeden kuluçkaya hücreleri izin. Hasat günde 5.</li>
</ol> 2. VCCC gelen kesirler hasat <ol><li>4 ° C Oda izolasyonda gerçekleştirmek ve tüm aletleri buz üzerinde tutmak</li>
	<li>İzole bireysel ekleme sayısı göz önünde bulundurun ve lizis arabellek MEJ (için izole filtreler) etiketli bir 50 mL tüp ekleyin. O zaman etiket iki 10 mm tabağı; bir at, bir SMC için.</li>
	<li>Bağlı olarak nasıl konsantre lysate olması gerekiyor; 15 için ekler lizis arabellek hacmi, 500 µL lizis arabelleği MEJ tüpü ve Petri kabına başına 250 µL tavsiye edilir ayarlama.</li>
	<li>İş ekler bir seferde bir 6-şey plakalı. Hücre kültür hood ve buz soğuk bir odaya ışınlamaya ortamda kapalı emme 
	Not: Aşağıdaki talimatlar 1 yalıtım eklemek için geçerlidir.</li>
	<li>1 x PBS SMC yan ucun üzerine damlalıklı 10 µL.</li>
	<li>Ekleme, bir ucunu hücrelere aşağı kazımak için hücre kaldırıcı kullanmak ve onu arabellekte lizis var SMC Petri kabına için hırsızı hücrelerden transfer</li>
	Hücre lifter çanak lizis arabellekte dokundu bir kez <li>Bu kadar edilmiş tamamen sildi ve kağıt havlu üzerinde kurutulur Ekle filtresi dokunmak izin vermez. Kalan SMC hücre artıkları tamamen kaldırmak için kazıma SMC filtre en az bir iki kez yineleyin.</li>
	<li>Üzerinde ekleme ve damlalıklı 10 µL 1 x PBS, INSERT filtre üst/EC yan içine açın. 2.6 ve 2.7 hücre kazıyıcı ve EC Petri kapları ile yineleyin.</li>
	<li>Kalan hücre ve PBS Bulamaç için lizis arabelleği ile EC Petri kabına pipet ve transfer filtresiyle EC tarafındaki toplanır. Hücreleri her iki tarafında en az EC/SMC kirlenme MEJ kesir terk olarak filtre dışına kazıma çok kapsamlı olması.</li>
	<li>Neşteri plastik eklemek filtresinden kesmek için dikkatli kullanın. Kesme 70-80 %'plastik uzak bunu ve forseps tamamen kapalı plastik eklemek yapı filtresi çekmek için kullanın. Filtre ile lizis tampon MEJ etiketli 50 mL konik tüp içine koyun ve lizis arabellekte oturur ve ıslak kalır olun.</li>
	<li>Tüm ekler iþlenene kadar gruplar halinde 6, yalıtım adımları yineleyin.</li>
	<li>Farklı tedavi grupları ayrı tüpler ve yemekleri tutulmasını sağlamak.</li>
	<li>Girdap 50 mL MEJ konik tüp 15 tam güçte s veya iyi kadar karışık. En fazla protein ve sıvı tüp terk olarak tüp yan duvarlar boyunca sürükleyerek Forseps ile MEJ konik filtreleri kaldırın. Bu noktadan sonra filtreleri atın.</li>
	Bir kez tüm filtreleri MEJ konik tüp sizden çıkarmak <li>yapmak bir tüm sıvı ve proteinler tüp altına çekmek için bir santrifüj hızlı spin (10-15 s max hız ~ 16.000 x g). MEJ tüp ve EC ve SMC Petri yemekler içeriğini ayrı, daha küçük santrifüj tüpler içine aktarmak.</li>
	<li>İsteğe bağlı: MEJ/SMC/EC tüpler içeriğini solüsyon içeren temizleyicide 4 üç 10 s bakliyat için buza ayarlamak veya hafifçe el ile homojenize.</li>
	<li>Maksimum hızda (~ 16.000 x g) 15 dk santrifüj kapasitesi 4'te ° C. damlalıklı süpernatant dışarı ve lysate bicinchoninic tahlil yöntemi kullanarak protein içeriği için tahlil.</li>
</ol> 3. VCCC Tr yüz ayirt için hasat <ol><li>üzerinde gün 5 in VCCC kuluçka kuluçka makinesi ekler kaldırmak. Hücre kültür mahallede çalışma, SMC medya her INSERT filtre ve durulama 1 x PBS ile iki kez daha düşük Wells kapalı emme gerek. Tekrar EC yüzü.</li>
	<li>Ekler bozulmadan ve plaka tutarak, nazik bir shaker bir yürüyüş 4 ° C Oda %4 paraformaldehyde (PFA) 10-20 dk için ekleyin. 1 x PBS Ekle filtresiyle 5 min için her iki yıkama ve iki kere tekrar edin. 
	Dikkat: PFA toksik ve dikkatle ele alınması gerekir.</li>
	<li>Engelleme, birincil ve ikincil antikor incubations plaka, her iki taraf için filtre antikor içinde tutulur sağlanması + çözüm engelleme gerçekleştirmek (9 mL PBS, 25 µL Triton X100, 50 mg sığır serum albümin, 500 µL normal serum)</li>
	<li>Bir slaytta bir neşter ile plastik eklemek dışında geçiren filtre filtre takmak için monte edilmiş kolu ve montaj orta ile mikroskop Slayttaki yer.</li>
</ol> 4. VCCC için enine ayirt hasat <ol><li>üzerinde gün 5 in VCCC kuluçka kuluçka makinesi ekler kaldırmak. Hücre kültür başlık Ekle filtrede her iki medya kapalı emme ve her iki tarafında iki kez 1 x PBS ile filtre durulayın.</li>
	<li>Ekler koruma sağlam ve 6-şey plaka eklemek %4 PFA ve gecede bir yürüyüş 4 ° C odada nazik bir shaker üzerinde kuluçkaya. 
	Dikkat: PFA toksik ve dikkatle ele alınması gerekir.</li>
</ol> 5. VCCC enine ayirt için gömme <ol><li>sonra filtre ekler yaklaşık %4 24 h için düzeltilen PFA, transfer % 70 alkol en az 24 saat, ancak birkaç hafta kadar.</li>
	<li>(Gece) bir otomatik doku işlemcisindeki gerçekleştirmek uzun tarih filtreleri işlemek. Program aşağıdaki gibi kullanın: % 70 alkol 30 dk, % 85 alkol 30 dk, % 95 alkol 30 dk, %100 alkol 30 dk, %100 alkol 45 dk, %100 alkol 45 dk, Ksilen 30 dk, Ksilen 30 dk, Ksilen 45 min, 60 ° C için alkol 30 dk, 30 dk alkol 60 ° C , 60 ° C alkol 45 dk.</li>
	<li>İşlendikten sonra hasat filtreleri Sıvı parafin, filtre kasete tutmak içine gömme istasyona transfer.</li>
	<li>Dikkatle arasındaki sınırda tutmak için forseps kullanarak kaset, filtreyi kaldırın. Filtre, yarıya makasla yarım şeklinde iki yarı dairesel şekillendirme. Her 2 yarıya gömme istasyonu uzun gömme doldurmak için yeterli Sıvı parafin ile kalıp sadece soğuk plaka parçasındaki yatıyordu.</li>
	Bir kez <li>Sıvı parafin ile dolu çok kısa bir süre soğuk plaka gömme kalıp touch, sonra her biri için filtre (yan kısmak) kalıp katıştırma içine embed; bu kesit sırasında bir kenarına doğru filtre ortasından doğraması ki sağlar .</li>
	<li>Gömme sırasında forseps yerine parafin onları tek başına yeterince soğuk olana düşmesini içine yarım önlemek için filtre tutmak için kullanın. Tamamen katılaşmış kadar soğuk bir tabak için gömme kalıp taşımak. 
	Not: blok buz tepsiler üzerinde soğutma yardımcı olur buruşuk bölümleri önlemek için.</li>
</ol> 6. Kesit ve enine ayirt VCCC boyama <ol><li>parafin blok için kesit ve montaj slayt: 5 µm bölümleri kesmek, Bölüm 42 ° C su banyosu yerleştirin ve pozitif yüklü kullanarak bölümleri Seç slaytlar. Sonra slaytlar gecede kurumaya 42 ° C fırında tutun.</li>
	<li>Rehidrasyon adımları başlamadan önce slaytlar parafin eritmek için 60 ° c fırında ve slayt VCCC bölümlerine uygun yardım. Kısaca serin, o zaman slaytlar arasında Ksilen, % 100 iki değişiklik rehydrate alkol, % 95 alkol, bir değişim % 70 alkol ve iki değişiklikler distile su.</li>
	<li>Mikrodalga veya bölümler slaytın dışına düşebilir gibi Isıtma dahil antijen alma yöntemleri kaçının.</li>
	<li>Gerçekleştir, oda sıcaklığında, sonra gece kuluçka 4 ° C'de birincil antikor ile 1 h için standart engelleme adımları ve 1 h için ikincil antikor oda sıcaklığında yıkayarak takip ettim. Montaj montaj medya ve coverslip.</li>
</ol>

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+injury+induces+vascular+smooth+muscle+cell+proliferation+in+highly+localized+regions+of+a+direct+contact+co-culture+system.">Endothelial injury induces vascular smooth muscle cell proliferation in highly localized regions of a direct contact co-culture system.</a> Cell Biochem Biophys. 52 (1), 37-46 (2008).</li><li>Scott-Drechsel, 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=A+new+flow+co-culture+system+for+studying+mechanobiology+effects+of+pulse+flow+waves.">A new flow co-culture system for studying mechanobiology effects of pulse flow waves.</a> Cytotechnology. 64 (6), 649-666 (2012).</li><li>van Buul-Wortelboer, M. F., 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=Reconstitution+of+the+vascular+wall+in+vitro.+A+novel+model+to+study+interactions+between+endothelial+and+smooth+muscle+cells.">Reconstitution of the vascular wall in vitro. A novel model to study interactions between endothelial and smooth muscle cells.</a> Exp Cell Res. 162 (1), 151-158 (1986).</li><li>Wilhelm, I., Fazakas, C., Krizbai, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+the+blood-brain+barrier.">In vitro models of the blood-brain barrier.</a> Acta Neurobiol Exp (Wars). 71 (1), 113-128 (2011).</li><li>Heberlein, K. R., 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=A+novel+mRNA+binding+protein+complex+promotes+localized+plasminogen+activator+inhibitor-1+accumulation+at+the+myoendothelial+junction.">A novel mRNA binding protein complex promotes localized plasminogen activator inhibitor-1 accumulation at the myoendothelial junction.</a> Arterioscler Thromb Vasc Biol. 32 (5), 1271-1279 (2012).</li><li>Straub, A. C., 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=Compartmentalized+connexin+43+s-nitrosylation/denitrosylation+regulates+heterocellular+communication+in+the+vessel+wall.">Compartmentalized connexin 43 s-nitrosylation/denitrosylation regulates heterocellular communication in the vessel wall.</a> Arterioscler Thromb Vasc Biol. 31 (2), 399-407 (2011).</li><li>Cucina, 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=Vascular+endothelial+growth+factor+increases+the+migration+and+proliferation+of+smooth+muscle+cells+through+the+mediation+of+growth+factors+released+by+endothelial+cells.">Vascular endothelial growth factor increases the migration and proliferation of smooth muscle cells through the mediation of growth factors released by endothelial cells.</a> J Surg Res. 109 (1), 16-23 (2003).</li><li>Heberlein, K. R., 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=Plasminogen+activator+inhibitor-1+regulates+myoendothelial+junction+formation.">Plasminogen activator inhibitor-1 regulates myoendothelial junction formation.</a> Circ Res. 106 (6), 1092-1102 (2010).</li><li>Isakson, B. E., Best, A. K., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+protein+on+actin+bridges+between+endothelium+and+smooth+muscle+in+arterioles+demonstrates+heterogeneous+connexin+expression+and+phosphorylation.">Incidence of protein on actin bridges between endothelium and smooth muscle in arterioles demonstrates heterogeneous connexin expression and phosphorylation.</a> Am J Physiol Heart Circ Physiol. 294 (6), H2898-H2904 (2008).</li><li>Biwer, L. A., Isakson, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum-mediated+signalling+in+cellular+microdomains.">Endoplasmic reticulum-mediated signalling in cellular microdomains.</a> Acta Physiol (Oxf). 219 (1), 162-175 (2017).</li></ol>

Yazarlar ifşa gerek yok.

VCCC MEJs vitrorecapitulating açısından avantajları vardır, ama eğer VCCC-ebilmek var olmak kullanmak için belirli uygulama belirlerken tartışma noktaları vardır. Örneğin, farklı hücre türleri bu model ile kullanılmak üzere olduğunda optimize gerekir. Biz birincil insan hücreleri kullandık ama sığır aortas24veya wildtype ya da nakavt fareler hücreleri izole ve VCCC1,5,14'...

Küçük çaplı direnç arterlerde endotel (EC) ve düz kas hücreleri (SMC) bir ince iç elastik lamina (IEL) ayırır. Hücreler bu elastik matris deliklerden proje ve doğrudan sitoplazmik bağlantı yolu ile gap junction kanal1,2. Bu eşsiz yapının myoendothelial Kavşağı (MEJ) olarak adlandırılır. MEJ küçük (yaklaşık 0.5 µm vasküler yatağın bağlı olarak 0.5 µm x), hücresel projeksiyon ağırlıklı olarak endotel hücreleri oluşur fakat düz kas de3,4kaynaklı. Müfettişler bir dizi seçmeli olarak endotel ve düz kas5 arasında çift yönlü sinyal kolaylaştırmak için özellikle önemli bir konuma işleme MEJ oluşan ağlar sinyal büyük karmaşıklığı göstermiştir ,6,7,8,9,10.
Ancak, sinyal yolları MEJ, mekanik bir diseksiyon sağlam bir arter zordur. MEJ hücresel bir projeksiyon olduğundan, şu anda bir vivo içinde MEJ damar duvarından izole etmek mümkün değil. Bu nedenle, VCCC model1 geliştirilmiştir. Önemlisi, VCCC fizyolojik endotel Morfoloji11 ve apikal arasında sinyal polarizasyon çoğaltır ve hücre12MEJ bölümleri. Endotel hücre, MEJ için polarize olan alfa hemoglobin keşif kolaylaştırdı bu benzersiz model oldu. Alfa hemoglobin13ifade değil geleneksel kültürlü endotel hücreleri aksine bu. Araştırmacılar mikrovasküler endotel baktılar, özellikle niyet küçük çaplı direnç arterlerde oluşan sinyal yolları incelemek için ise VCCC, kültürlü endotel hücreleri kullanmak daha uygun olabilir.
Sağlam bir plastik eklemek küçük çaplı gözenekleri (çapı 0.4 µm) olan bir filtre içeren ortak kültür iki farklı hücre tipleri için kullanarak hücrelerin katmanlar arasında geçiş engeller. 10 µm mesafe içinde vivoönemli ölçüde daha uzun, ama hala MEJs protein Yerelleştirme ve ikinci haberci1 sinyal de dahil olmak üzere, in vivo özelliklerinin çoğunu çoğaltır hücreleri arasındaki sonuçlar , 14. Ayrıca, hücre türüne özgü bir agonist veya antagonisti belirli hücresel yuvası eklenmesi ile sinyal hedefleme için VCCC sağlar. Örneğin, EC kalsiyum şelasyon yapın için BAPTA-AM ile yükleme ve SMC phenylephrine14ile uyarıcı. Aksine diğer açıklamalar ortak kültür modelleri15,16,17,18,19,20,21, bu sağlar mRNA ve protein, filtre gözenekleri içinde farklı MEJ kesir dahil olmak üzere farklı kesirler yalıtma hakkında talimatlar. MRNA yerelleştirme veya transkripsiyon22, protein fosforilasyon12,23ve protein etkinlik12değişiklikler belirli incelenmesi için bu teknik için VCCC eklenmesini sağlar. İki hücre tipleri farklı biçimler11,18,24yılında kültür mümkündür ancak bu makalede EC filtre ve SMC üstünde tepe-in alt kaplama anlatacağız.

<table><tbody><tr><td>24 mm diameter, 0.4 &amp;micro;m Transwell Polyester membrane filter insert</td><td>Corning</td><td>3450</td><td>This is one 6 well plate of inserts</td></tr><tr><td>225 cm flask</td><td>Corning</td><td>431082</td><td></td></tr><tr><td>6 well plates (without filter inserts)</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>Primary human coronary artery endothelial cells</td><td>Lonza</td><td>CC-2585</td><td></td></tr><tr><td>Primary human coronary artery smooth muscle cells</td><td>Lonza</td><td>CC-2583</td><td></td></tr><tr><td>EBM-2 EC Basal media</td><td>Lonza</td><td>CC-3156</td><td></td></tr><tr><td>EC growth factors (EGM2 MV Single quots)</td><td>Lonza</td><td>CC-4147</td><td>Add to basal media before use</td></tr><tr><td>SmBM SMC basal media</td><td>Lonza</td><td>CC-3181</td><td></td></tr><tr><td>SMC growth factors (SmGM-2 SingleQuot)</td><td>Lonza</td><td>CC-4149</td><td>Add to basal media before use</td></tr><tr><td>0.5% Trypsin-EDTA 10X</td><td>Gibco</td><td>15400-054</td><td>Dilute to 2X with sterile dPBS</td></tr><tr><td>Hemocytometer</td><td>VWR</td><td>15170-208</td><td></td></tr><tr><td>large petri dishes for SMC Plating (150mm)</td><td>Corning</td><td>353025</td><td></td></tr><tr><td>Bovine gelatin</td><td>Sigma</td><td>G-9382</td><td></td></tr><tr><td>Human fibronectin</td><td>Corning</td><td>356008</td><td>5&amp;micro;g/ml of fibronectin and store at -20</td></tr><tr><td>10x Hanks&#039; Buffered salt solution (HBSS)</td><td>Gibco</td><td>14065-056</td><td>Dilute to 1x with sterile water</td></tr><tr><td>10x Dulbecco&#039;s Phosphate buffered saline (dPBS)</td><td>Gibco</td><td>14200-075</td><td>Dilute to 1x with sterile water</td></tr><tr><td>Disposable Curved Scalpel (30mm cutting edge) with handle</td><td>Amazon</td><td>MDS15210</td><td></td></tr><tr><td>Forceps</td><td>Fisher</td><td>17-467-201</td><td></td></tr><tr><td>Cell lifter</td><td>Corning</td><td>3008</td><td></td></tr><tr><td>Cell scraper</td><td>BD Falcon</td><td>353085</td><td></td></tr><tr><td>50 mL conical tube</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>10 mm petri dishes</td><td>Falcon</td><td>35-1008</td><td></td></tr><tr><td>Rneasy mini kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>RNA lysis reagent</td><td>Qiagen</td><td>79306</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127</td><td>Make 4% solution with dPBS</td></tr><tr><td>70% ethanol</td><td>Fisher</td><td>04-355-305</td><td></td></tr><tr><td>Cavicide disinfectant</td><td>Fisher</td><td>131000</td><td></td></tr><tr><td>RIPA protein lysis buffer</td><td>Sigma</td><td>R0278</td><td></td></tr><tr><td>Sonic dismembranator</td><td>Fisher</td><td>Model 50</td><td></td></tr><tr><td>1x PBS for harvesting EC/SMC and immunocytochemistry</td><td>Gibco</td><td>10010023</td><td>does not need to be sterile</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Embedding/Sectioning/Staining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Paraffin</td><td>Fisher</td><td>P31-500</td><td></td></tr><tr><td>Automated tissue processer</td><td>Excelsior</td><td>ES</td><td></td></tr><tr><td>Embedding station + cold plate</td><td>Leica</td><td>HistoCore Arcadia</td><td></td></tr><tr><td>Tissue Tek Accu-Edge Microtome blade</td><td>VWR</td><td>25608-961 or 25608-964</td><td></td></tr><tr><td>Microscope slides</td><td>Thermo Fisher</td><td>22-230-900</td><td></td></tr><tr><td>Coverslips</td><td>Thermo Fisher</td><td>12-548-5E</td><td></td></tr><tr><td>Prolong Gold mounting medium</td><td>Thermo Fisher</td><td>P36931</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Other Solutions&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;**Perform all steps in sterile conditions if using the solution on live cells&lt;/strong&gt;</td></tr><tr><td>Sterile dPBS</td><td></td><td></td><td>Autoclave water to sterilize. Using the sterile water make DPBS (450mL sterile water: 50mL 10X DPBS), mix and filter sterilize (0.2&amp;micro;m filter).&lt;br/&gt; Final concentration: 1X</td></tr><tr><td>sterile HBSS</td><td></td><td></td><td>Autoclave water to sterilize. Using the sterile water make HBSS (450mL sterile water: 50mL 10X HBSS), mix and filter sterilize (0.2&amp;micro;m filter).&lt;br/&gt; Final concentration: 1X</td></tr><tr><td>Fibronectin stock solution</td><td></td><td></td><td>Use 50mL of sterile water to fibronectin to make stock of 1mL aliquots, then freeze until needed&lt;br/&gt; Final concentration: 100&amp;micro;g/mL</td></tr><tr><td>Fibronectin to coat SMC side of VCCC</td><td></td><td></td><td>thaw one aliquot and add 1mL of fibronectin (100&amp;micro;g/mL) to 19mLs of 1X HBSS to reach a final concentration of 5&amp;micro;g/mL.&lt;br/&gt; Final concentration: 5&amp;micro;g/mL</td></tr><tr><td>2x Trypsin EDTA solution</td><td></td><td></td><td>Dilute the 10X 0.5% Trypsin-EDTA to 2X with 1X dPBS (40mL DPBS: 10mL 10X Trypsin) .&lt;br/&gt; Final concentration: 2X</td></tr><tr><td>Blocking/antibody solution</td><td></td><td></td><td>(9mL 1X PBS, 25mL Triton X100, 50mg bovine serum albumin, 500mL normal serum)</td></tr><tr><td>Bovine gelatin to coat EC VCCC</td><td></td><td></td><td>Mix 0.5g bovine gelatin into 100mL distilled water. Autoclave prior to first use and store at room temperature. Keep in sterile conditions.&lt;br/&gt; Final concentration: 0.50%</td></tr><tr><td>1x PBS for harvesting cells</td><td></td><td></td><td>does not need to be sterile</td></tr></tbody></table>

a cell culture model of resistance arteries

Myoendothelial kavşak (MEJ), küçük çaplı direnç arterler içinde benzersiz bir sinyal microdomain yerelleştirme spesifik proteinlerin ve vasküler sesi ve kan basıncı kontrol edebilirsiniz sinyal süreçlerin sergiler. Bu da bir projeksiyon endotel veya düz kas hücresi ve küçük nedeniyle (ortalama olarak, bir alan ~ 1 µm2), MEJ boyutu gibi yalıtım modunda çalışmaya zordur. Ancak, vitro MEJ oluşumu, endotel hücre polarizasyon ve proteinler ve süreçleri direnç damar duvarındaki sinyalizasyon disseksiyonu sağlar vasküler hücre ortak kültürü (VCCC) denilen bir hücre kültür modeli geliştirdik arterler. VCCC uygulamalar vardır ve farklı hücre türleri uygun şekilde uyarlanabilir. Model içinde vitro MEJs oluşabilir 0.4 µm gözenekli filtre karşı taraflarında yetiştirilen iki hücre tipleri oluşur. Burada VCCC yolu ile endotel, izolasyon ve kaplama hücre oluşturmak nasıl açıklamak MEJ ve daha sonra protein yalıtım veya etkinlik için kullanılabilir düz kas kesirler deneyleri. Katıştırılmış ve kesitli immünfloresan analiz için filtre sağlam hücre katmanları ile düzeltilebilir. Önemlisi, birçok buluşlar bu modelden sağlam direnç arterler, fizyolojik önemini vurgulamış kullanarak teyit edilmiştir.

VCCC için kullanılan filtre ekler var küçük, 0.4 µm delik. Şekil 1A, VCCC filtre ilave bir tr yüz görünümünü MEJ vitrooluşabilir gözenekler gösterir. Şematik filtre alt tarafında bir gün önce endotel hücreleri karşı tarafta (şekil 1B) kaplama kaplama düz kas hücreleri gösterir. Sonra hücreleri kaplama, EC, MEJ ve SMC kesirler izole üç gün sonra olabilir. Bunun bir örneği <strong ...

Watch this Scientific Journal Video about A Cell Culture Model of Resistance Arteries at JoVE.com

A Cell Culture Model of Resistance Arteries

Hücre kültür manken direnç arter yollar endotel, düz kas, veya endotel ve düz kas (myoendothelial junction) arasında sinyal diseksiyon için izin açıklanmıştır. Bu hücre kültür model kullanarak agonistler veya protein yalıtım, elektron mikroskopi veya ayirt seçici uygulama yararlı olabilir.

Hücre kültür manken direnç arter

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

a-cell-culture-model-of-resistance-arteries

Research

JoVE Journal

Medicine

7.8K Görüntüleme.  University of Virginia School of Medicine. Hücre kültür manken direnç arter yollar endotel, düz kas, veya endotel ve düz kas (myoendothelial junction) arasında sinyal diseksiyon için izin açıklanmıştır. Bu hücre kültür model kullanarak agonistler veya protein yalıtım, elektron mikroskopi veya ayirt seçici uygulama yararlı olabilir.

Video: A Cell Culture Model of Resistance Arteries

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

İzolasyon ve Kemirgen Aort eksizyonu; Kardiyovasküler Hastalığı Çalışmasında Çok Yönlü Tekniği

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

Tıp

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall. In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for quantifying i) pressure-induced changes in internal elastic lamina branching angles and adventitial collagen straightness using Fiji and ii) collagen and elastin volume densities determined using Ilastik software. Preferably all mechanical and imaging measurements are performed on live, perfused arteries, however, an alternative approach using standard video-microscopy pressure myography in combination with post-fixation imaging of re-pressurized vessels is discussed. This alternative method provides users with different options for analysis approaches. The inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed, and future development and additions to the protocol are proposed.

We describe simultaneous mechanical testing and 3D-imaging of the arterial wall of isolated, live human resistance arteries, and Fiji and Ilastik image analyses for the quantification of elastin and collagen spatial organization and volume densities. We discuss the use of these data in mathematical models of arterial wall mechanics.

Canlı, insan direnç arterler tarafından etiket içermeyen Floresans mikroskobu kolajen ve Elastin basınç bağımlı Microarchitectures değerlendirilmesi

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations ...

Biyomühendislik

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Aort Kalsifikasyon ve Enflamasyon Vasküler düz kas hücrelerinin ve Görüntüleme Kalsifikasyon

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of various important processes in the cardiovascular system. Recently, although several biological studies have been performed using such 3D models of human arteries, they have not been applied to study vascular targeting. This paper presents a new method to fabricate real-sized, reconstructed human arterial models using a 3D printing technique, line them with human endothelial cells (ECs), and study particle targeting under physiological flow. These models have the advantage of replicating the physiological size and conditions of blood vessels in the human body using low-cost components. This technique may serve as a new platform for studying and understanding drug targeting in the cardiovascular system and may improve the design of new injectable nanomedicines. Moreover, the presented approach may provide significant tools for the study of targeted delivery of different agents for cardiovascular diseases under patient-specific flow and physiological conditions.

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Here, we present a new protocol to study and map the targeted deposition of drug carriers to endothelial cells in fabricated, real-sized, three-dimensional human artery models under physiological flow. The presented method may serve as a new platform for targeting drug carriers within the vascular system.

Akış Altında Damar İlacı Hedeflemesi Üzerinde Çalışmak için In Vitro 3D Hücre Kültürlü Arteriyel Modeller

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

Sıçan izole direnç arter Video mikroskobu kullanan vasküler kontrol mekanizmalarının değerlendirilmesi

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection (TAAD). However, human TAAD sometimes cannot be characterized by animal models. There is an apparent species gap between clinical human studies and animal experiments that may hinder the discovery of therapeutic drugs. In contrast, the conventional cell culture model is unable to simulate in vivo biomechanical stimuli. To this end, microfabrication and microfluidic techniques have developed greatly in recent years, providing novel techniques for establishing organoids-on-a-chip models that replicate the biomechanical microenvironment. In this study, a human aorta smooth muscle cell organ-on-a-chip (HASMC-OOC) model was developed to simulate the pathophysiological parameters of aortic biomechanics, including the amplitude and frequency of cyclic strain experienced by human aortic smooth muscle cells (HASMCs) that play a vital role in TAAD. In this model, the morphology of HASMCs became elongated in shape, aligned perpendicularly to the strain direction, and presented a more contractile phenotype under strain conditions than under static conventional conditions. This was consistent with the cell orientation and phenotype in native human aortic walls. Additionally, using bicuspid aortic valve-related TAAD (BAV-TAAD) and tricuspid aortic valve-related TAAD (TAV-TAAD) patient-derived primary HASMCs, we established BAV-TAAD and TAV-TAAD disease models, which replicate HASMC characteristics in TAAD. The HASMC-OOC model provides a novel in vitro platform that is complementary to animal models for further exploring the pathogenesis of TAAD and discovering therapeutic targets.

Here, we developed a human aorta smooth muscle cell organ-on-a-chip model to replicate the in vivo biomechanical strain of smooth muscle cells in the human aortic wall.

Aort Duvarındaki Biyomekanik Suşu Özetlemek İçin İnsan Aort Düz Kas Hücresi Organ-On-A-Chip Modelinin Yapımı

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

Fare Direnç arterlerden mikrovasküler endotel Tüpler İzolasyonu

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Biyoloji

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Koroner Anjiyogenezin In Vitro Modeli

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

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.

İzolasyon ve Fare aort endotel hücreleri İlköğretim Kültür

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

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

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

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

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Murin Aort Kırma Yaralanması: Verimli<emIn vivo</em&gt; Düz Kas Hücresi çoğalması ve Endotel Fonksiyon Modeli

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