这项工作得到了国家心脏, 肺和血液研究所的资助 R01088554, T32HL007284 和美国心脏协会 predoctoral 奖学金14PRE20420024。我们特别感谢 St. 裘德细胞成像共享的研究核心, 包括兰德尔和霍纳的电子显微镜图像, 亚当 Straub 为协助与代表性的免疫荧光图像, 和 Pooneh 巴盖尔·阿萨迪为她对原稿协议有价值的反馈。

1. 用于 VCCC 的电镀单元 <ol> <li> 区域性大 225 cm 2 瓶 EC 和 SMC 在不育条件下在37和 #176; C, 直到70-90% 汇合。确保使用的 EC 比 SMC 多;如果使用初级人类 ec 和 smc, 通常3大烧瓶 ec 到1大烧瓶 smc. 
	<ol> <li> 使用较低通道的主单元格, 并且不使用过去的段落10。选择基于单元格的区域性媒体共。对于原发性人冠状动脉 ec, 使用 EBM-2 ec 基底介质与 EGM2 MV 生长因子。对于原发性人冠状动脉 smc, 使用 SmBM smc 基底介质与 SmGM-2 生长因子。在使用培养细胞之前, 将生长因子添加到介质中. </li>
	</ol> </li> <li> VCCC 1 天的构造: 用消毒剂 (如 、Cavicide) 喷洒大 (150 mm) 培养皿和盖子, 然后用纸巾或无绒抹布擦拭。然后用70% 乙醇 (乙醇) 喷雾培养皿, 将它们放在引擎盖上晾干. </li>
	<li> 在无菌条件下打开过滤器插入板 (参见 材料表 ), 并将插入物的 SMC 侧 (过滤器的底部) 涂上纤维连接蛋白溶液。保持插入在提供的6井板, 吸管纤维连接蛋白解决方案通过侧缝到底部的板, 确保底部一半的过滤器覆盖 (不超过1毫升). </li>
	<li> 经过30分钟的处理, 纤维连接蛋白溶液在室温下在细胞培养罩, 采取插入出的板, 真空任何过剩的解决方案, 并倒置, 放置到底部一半的清洁培育皿。预留。在吸痰过剩溶液时, 一定不要干扰过滤器插入膜;这可以避免通过轻轻地倾斜板, 并清除解决方案边缘的插入. </li>
	<li> 处理 225 cm 2 烧瓶与3毫升的 5ml 2X 胰蛋白酶-EDTA 溶液, 一旦细胞解除了板块, 添加9毫升 smc 介质, 以中和胰蛋白酶 (3:1, 媒体: 胰蛋白酶)。转移到锥形管, 混合好, 和吸管10和 #181; L 到例上以确定单元格号. 
	<ol> <li> 计算 5 x 5 框中的单元格数 ( 即 , 18), 将其乘以1万以获取 1 mL (18万) 中的单元格数。然后乘以12毫升 (细胞悬浮总体积, 216万 SMC)。确保有足够的 SMC, 以满足所需的油井数量. 
		注: 例如, 1 井应该有 7.5万 smc, 因此18口井将有135万总 smc。除以每1毫升18万细胞所需的135万细胞, 这导致7.5 毫升的细胞悬浮, 将需要电镀。然后计算所需的最终体积 (18 井 x 750 和 #181; L = 13.5 毫升)。因此, 采取7.5 毫升的细胞悬浮, 使体积高达13.5 毫升与 5ml SMC 介质, 并混合良好. </li>
	</ol> </li> <li> 板750和 #181; L 单元悬浮物包含大约 7.5万 SMC 到每个过滤器的底部, 注意不要泄漏任何介质和电池解决方案。将盖子放在顶部, 并在37和 #176 中小心地将培养皿与插入器一起转移到恒温箱中. 
	注: 其他细胞类型的最佳细胞密度将需要根据经验确定. </li>
	<li> 在2天, 用2毫升新鲜的 5ml SMC 介质填充干净的6个菜肴。从孵化器中取出盘子。一次取出一个插入物, 然后通过抽吸将介质取出, 然后放入 SMC 介质中的6井盘中. </li>
	<li> 将1毫升的0.5% 牛凝胶溶液涂在过滤器 (EC 侧) 的另一侧, 在37和 #176 上至少30分钟; C. </li>
	<li> 处理 225 cm 2 烧瓶, 3 毫升的 5ml 2X 胰蛋白酶-EDTA (10x 稀释在无菌 Dulbecco 和 #39; s PBS) 溶液中, 轻轻敲击烧瓶, 以解除板上的细胞. 
	注: 可能需要将烧瓶放回37和 #176 的恒温箱中; C 为1-3 分钟。
	<ol> <li> 一旦细胞从板上脱落, 加入9毫升 EC 培养基来中和胰蛋白酶 (3:1)。转移到锥形管, 池一起, 混合好, 和吸管10和 #181; L 到例上以确定单元格数。
		<ol> <li> 计算 5 x 5 框中的单元格数 ( 即 , 60), 将其乘以1万以获取 1 mL (60万) 中的单元格数。然后乘以12毫升 (细胞悬浮总体积, 720万 EC)。确保有足够的 EC, 以满足所需的油井数量. 
			注: 每个井需要大约 36万 EC。例如, 18 口井需要 648万 EC。除以每1毫升 60万 EC 所需的648万细胞, 这结果在10.8 毫升的细胞悬浮, 将需要电镀。然后计算所需的最终体积 (18 口井 x 1 毫升 = 18 毫升)。因此, 采取10.8 毫升的细胞悬浮, 使体积高达18毫升与 5ml EC 介质, 并轻轻重. </li>
		</ol> </li> </ol> </li> <li> 板 1 mL 36万 EC 到每个插入过滤器的顶部。让细胞在37和 #176 不受干扰地孵育; C 为 24 h. 4 天更换培养基。5天收获. </li>
</ol> 2。从 VCCC <ol> 中获取分数 <li> 在4和 #176 中执行隔离, 并将所有仪器放在冰上. </li>
	<li> 请记住单独插入的数量, 并将裂解缓冲器添加到标记为梅杰的50毫升管中 (用于隔离过滤器)。然后贴上两个 10 mm 的盘子;一个用于 EC, 一个用于 SMC. </li>
	<li> 根据裂解物的浓度需要调整裂解缓冲液的体积; 对于15插入, 500 和 #181; 梅杰管的裂解缓冲液和250和 #181; 推荐使用每培养皿. </li>
	<li> 一次使用一个 6-好的插入板。吸入细胞培养罩中的培养基, 然后将其输送到冰上的冷室. 
	注意: 以下说明用于1插入隔离. </li>
	<li> 吸管10和 #181; L 1x PBS 到插入的 SMC 一侧. </li>
	<li> 使用细胞提升器将细胞刮到插入物的一端, 并将电池从升降器转移到具有裂解缓冲液的 SMC 培养皿中. </li>
	<li> 当电池升降器触到盘中的裂解缓冲液后, 不要让它接触到插入式滤光片, 直到它被完全擦干并在纸巾上晾干。重复对 smc 过滤器的刮擦至少一到两次, 以完全去除其余的 smc 细胞碎片. </li>
	<li> 将 "插入" 和 "吸管 10" #181; 将 1x PBS 转到插入过滤器的上部/EC 一侧。重复步骤2.6 和2.7 与细胞刮刀和 EC 培养皿. </li>
	<li> 用吸管从过滤器的 ec 一侧收集剩余的电池和 PBS 浆料, 并将其转移到 ec 培养皿中进行裂解缓冲。要非常彻底地刮除过滤器两侧的细胞, 以使梅杰分数最小的 EC/SMC 污染. </li>
	<li> 小心使用手术刀从塑料插入物中切出过滤器。做到这一点, 削减70-80% 它远离塑料和使用镊子拉过滤器完全关闭塑料插入结构。将过滤器放在50毫升锥形管中, 用裂解缓冲液标记梅杰, 确保它位于裂解缓冲液中, 并保持湿润. </li>
	<li> 按6组重复执行隔离步骤, 直到处理完所有插入操作. </li>
	<li> 确保不同的治疗组保持在单独的管和菜中. </li>
	<li> 涡旋50毫升梅杰锥形管在全强度十五年代或直到混合好。将过滤器从梅杰的锥形带钳上取下, 将它们沿管子的侧壁拖动, 以使试管中的蛋白质和液体达到最大值。在此点之后, 丢弃筛选器. </li>
	<li> 一旦所有的过滤器已经从梅杰锥管中删除, 做一个快速旋转 (10-15 s 在最大速度〜 1.6万 x g) 在离心机, 把所有的液体和蛋白质的底部的管。将梅杰管和 EC 和 SMC 培养皿的内容转移到分离的小型离心管中. </li>
	<li> 可选: 几种梅杰/SMC/EC 管的内容在设置4三十年代脉冲在冰或质轻轻地用手. </li>
	<li> 离心机在最大转速 (〜 1.6万 x g) 15 分钟在4和 #176; c. 用二辛可宁测定法吸管出上清液和测定蛋白质含量的裂解物. </li>
</ol> 3。在 VCCC 孵化的5天, 为 En 免疫荧光 <ol> <li> 收割 VCCC, 从孵化箱中取出插入物。在细胞培养罩中工作, 从插入式过滤器的下井中抽离 SMC 介质, 用 1x PBS 冲洗两次。在 EC 方面重复. </li>
	<li> 保持插入完好, 并在板中, 添加4% 甲醛 (粉煤灰) 10-20 分钟在一个步入式的4和 #176; C 室在一个温和的振动筛。将插入过滤器的两侧用 1x PBS 清洗5分钟, 重复两次. 
	注意: 粉煤灰是有毒的, 必须小心处理. </li>
	<li> 在板中执行阻断、主、二级抗体孵化, 确保过滤器的两侧都保存在抗体 + 阻断液中 (9 毫升 PBS、25和 #181; l X100、50毫克牛血清白蛋白、500和 #181; l 正常血清) </li>
	<li> 要在幻灯片上安装滤镜, 请将过滤器从塑料插入物中取出, 并将手术刀安装在手柄上, 并放置在带有安装介质的显微镜幻灯片上. </li>
</ol> 4。采集 VCCC 的横向免疫荧光 <ol> <li> 在 VCCC 孵化的5天, 从孵化箱中取出插入物。吸出介质从两边的插入过滤器在细胞培养罩和冲洗每一侧的过滤器两次与 1x PBS. </li>
	<li> 保持插入完好, 并在6井板, 增加4% 粉煤灰和孵化过夜在一个步入式的4和 #176; C 室在一个温和的振动筛. 
	注意: 粉煤灰是有毒的, 必须小心处理. </li>
</ol> 5。嵌入 VCCC 为横向免疫荧光 <ol> <li> 在过滤器插入物被固定了大约 24 h 在4% 煤灰之后, 转移到70% 乙醇为至少 24 h, 但至多几星期. </li>
	<li> 在自动组织处理器上长时间 (通宵) 运行过滤器。使用程序如下: 70% 乙醇30分钟, 85% 乙醇30分, 95% 乙醇30分, 100% 乙醇30分, 100% 乙醇45分, 100% 乙醇45分, 二甲苯30分, 二甲苯30分, 二甲苯45分, 60 和 #176; c 石蜡 30 min, 60 和 #176; c 石蜡 30 min, 60, #176; C 石蜡 45 min. </li>
	<li> 经过处理后, 将采集到的过滤器转移到液体石蜡中, 将过滤器保存在卡带中. </li>
	<li> 从卡带上取下过滤器, 小心地用镊子把它放在边缘。用剪刀把过滤器切成两半, 形成半圆状半。放置在埋置站的冷板部分的2一半, 只要足够长的时间, 以填补嵌入模具与液体石蜡. </li>
	<li> 一旦充满液体石蜡, 很简单地将嵌入模具接触到冷板, 然后将每半部分的过滤器嵌入模具 (切边); 这确保在剖切过程中, 一个将从过滤器的中心向边缘切割.</li>
	<li> 在嵌入过程中, 使用镊子将过滤器放置到位, 以防止两个部分相互掉落, 直到石蜡冷却到足以让它们单独站立为止。将嵌入模具移至冷板, 直到完全凝固. 
	注: 冰盘上的冷却块有助于防止皱纹的部分. </li>
</ol> 6。VCCC 横向免疫荧光的切片和染色 <ol> <li> 用于石蜡切片和滑动安装: 切割5和 #181; m 节, 将剖面放置到42和 #176 中; C 水浴, 并拿起的部分使用正电荷幻灯片.然后在42和 #176 中一夜之间保持幻灯片; C 烘箱晾干. </li>
	<li> 在开始补液步骤之前, 在60和 #176 烘烤幻灯片; C 将石蜡融化, 并帮助将 VCCC 部分粘附到幻灯片上。冷却简单, 然后水化的幻灯片通过两个变化的二甲苯, 100% 乙醇, 95% 乙醇, 一个变化的70% 乙醇, 和两个变化的蒸馏水. </li>
	<li> 避免抗原检索方法, 涉及微波或加热的部分可能脱落的幻灯片. </li>
	<li> 在室温下执行1小时的标准阻塞, 然后在4和 #176 中使用主抗体进行过夜孵育, 其次是室温下的洗涤步骤和1小时的二次抗体。安装介质和片. </li>
</ol>

<ol>
	<li>Isakson, B. E., 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=Heterocellular+contact+at+the+myoendothelial+junction+influences+gap+junction+organization.">Heterocellular contact at the myoendothelial junction influences gap junction organization.</a> Circ Res. 97 (1), 44-51 (2005).</li><li>Little, T. L., Xia, J., 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=Dye+tracers+define+differential+endothelial+and+smooth+muscle+coupling+patterns+within+the+arteriolar+wall.">Dye tracers define differential endothelial and smooth muscle coupling patterns within the arteriolar wall.</a> Circ Res. 76 (3), 498-504 (1995).</li><li>Heberlein, K. R., Straub, A. C., 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=The+myoendothelial+junction:+breaking+through+the+matrix?.">The myoendothelial junction: breaking through the matrix?.</a> Microcirculation. 16 (4), 307-322 (2009).</li><li>Straub, A. C., Zeigler, A. C., 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=The+myoendothelial+junction:+connections+that+deliver+the+message.">The myoendothelial junction: connections that deliver the message.</a> Physiology (Bethesda). 29 (4), 242-249 (2014).</li><li>Isakson, B. E., Ramos, S. I., 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=Ca2++and+inositol+1,4,5-trisphosphate-mediated+signaling+across+the+myoendothelial+junction.">Ca2+ and inositol 1,4,5-trisphosphate-mediated signaling across the myoendothelial junction.</a> Circ Res. 100 (2), 246-254 (2007).</li><li>Dora, K. A., Doyle, M. P., 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=Elevation+of+intracellular+calcium+in+smooth+muscle+causes+endothelial+cell+generation+of+NO+in+arterioles.">Elevation of intracellular calcium in smooth muscle causes endothelial cell generation of NO in arterioles.</a> Proc Natl Acad Sci U S A. 94 (12), 6529-6534 (1997).</li><li>Sonkusare, S. K., 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=Elementary+Ca2++signals+through+endothelial+TRPV4+channels+regulate+vascular+function.">Elementary Ca2+ signals through endothelial TRPV4 channels regulate vascular function.</a> Science. 336 (6081), 597-601 (2012).</li><li>Ledoux, 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=Functional+architecture+of+inositol+1,4,5-trisphosphate+signaling+in+restricted+spaces+of+myoendothelial+projections.">Functional architecture of inositol 1,4,5-trisphosphate signaling in restricted spaces of myoendothelial projections.</a> Proc Natl Acad Sci U S A. 105 (28), 9627-9632 (2008).</li><li>Boerman, E. M., Everhart, J. E., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+age+decreases+local+calcium+signaling+in+endothelium+of+mouse+mesenteric+arteries+in+vivo.">Advanced age decreases local calcium signaling in endothelium of mouse mesenteric arteries in vivo.</a> Am J Physiol Heart Circ Physiol. 310 (9), H1091-H1096 (2016).</li><li>Toussaint, F., Charbel, C., Blanchette, A., Ledoux, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMKII+regulates+intracellular+Ca(2)(+)+dynamics+in+native+endothelial+cells.">CaMKII regulates intracellular Ca(2)(+) dynamics in native endothelial cells.</a> Cell Calcium. 58 (3), 275-285 (2015).</li><li>Truskey, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Cell+Vascular+Smooth+Muscle+Cell+Co-Culture+Assay+For+High+Throughput+Screening+Assays+For+Discovery+of+Anti-Angiogenesis+Agents+and+Other+Therapeutic+Molecules.">Endothelial Cell Vascular Smooth Muscle Cell Co-Culture Assay For High Throughput Screening Assays For Discovery of Anti-Angiogenesis Agents and Other Therapeutic Molecules.</a> Int J High Throughput Screen. 2010 (1), 171-181 (2010).</li><li>Biwer, L. 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=Two+functionally+distinct+pools+of+eNOS+in+endothelium+are+facilitated+by+myoendothelial+junction+lipid+composition.">Two functionally distinct pools of eNOS in endothelium are facilitated by myoendothelial junction lipid composition.</a> Biochim Biophys Acta. 1861 (7), 671-679 (2016).</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=Endothelial+cell+expression+of+haemoglobin+alpha+regulates+nitric+oxide+signalling.">Endothelial cell expression of haemoglobin alpha regulates nitric oxide signalling.</a> Nature. 491 (7424), 473-477 (2012).</li><li>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=Localized+expression+of+an+Ins(1,4,5)P3+receptor+at+the+myoendothelial+junction+selectively+regulates+heterocellular+Ca2++communication.">Localized expression of an Ins(1,4,5)P3 receptor at the myoendothelial junction selectively regulates heterocellular Ca2+ communication.</a> J Cell Sci. 121 (Pt 21), 3664-3673 (2008).</li><li>Axel, D. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cell-rich+and+lipid-rich+plaques+in+a+transfilter+coculture+system+with+human+vascular+cells.">Induction of cell-rich and lipid-rich plaques in a transfilter coculture system with human vascular cells.</a> J Vasc Res. 33 (4), 327-339 (1996).</li><li>Hastings, N. E., Simmers, M. B., McDonald, O. G., Wamhoff, B. R., Blackman, 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=Atherosclerosis-prone+hemodynamics+differentially+regulates+endothelial+and+smooth+muscle+cell+phenotypes+and+promotes+pro-inflammatory+priming.">Atherosclerosis-prone hemodynamics differentially regulates endothelial and smooth muscle cell phenotypes and promotes pro-inflammatory priming.</a> Am J Physiol Cell Physiol. 293 (6), C1824-C1833 (2007).</li><li>Herzog, D. P., Dohle, E., Bischoff, I., Kirkpatrick, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+communication+in+a+coculture+system+consisting+of+outgrowth+endothelial+cells+and+primary+osteoblasts.">Cell communication in a coculture system consisting of outgrowth endothelial cells and primary osteoblasts.</a> Biomed Res Int. , 320123 (2014).</li><li>Jacot, J. G., Wong, J. 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>

作者没有什么可透露的。

VCCC 在综述 MEJs 的体外方面有许多优点, 但是在确定是否可以将 VCCC 用于特定的应用程序时, 还有一些讨论点。例如, 如果要与此模型一起使用不同的单元格类型, 则需要对其进行优化。我们已经使用了主要的人类细胞, 但它可以隔离的细胞从牛脉24, 或野生或淘汰赛小鼠, 并使用它们在 VCCC1,5,14。
<p class...

在小直径阻力动脉, 薄内弹性板 (IEL) 分离内皮 (EC) 和平滑肌细胞 (SMC)。细胞可以通过这个弹性矩阵的孔, 并通过缝隙连接通道的直接细胞质连接1,2。这种独特的结构称为 myoendothelial 结 (梅杰)。梅杰是一个小 (大约0.5 µm x 0.5 µm, 取决于血管床), 细胞投射主要组成的内皮细胞, 但可能起源于平滑肌以及3,4。许多调查人员已经证明了在梅杰上有选择地发生的信号网络的巨大复杂性, 使它成为促进内皮和平滑肌之间双向信号传递的一个特别重要的位置5 ,6,7,8,9,10。
然而, 在梅杰的信号通路的机械解剖是很难在一个完整的动脉。由于梅杰是细胞投射, 目前无法将体内的梅杰从血管壁隔离出来。因此, 开发了 VCCC 模型1 。重要的是, VCCC 复制的生理内皮形态学11和信号的极化之间的顶端和梅杰部分的细胞12。正是这种独特的模型, 促进了发现α血红蛋白是在内皮细胞, 极化到梅杰。这与常规培养的内皮细胞不表达α血红蛋白13形成对比。对于对微血管内皮有兴趣的研究人员来说, 使用 VCCC 中培养的内皮细胞可能更合适, 特别是如果目的是解剖在小直径阻力动脉中发生的信号通路。
使用一个坚固的塑料插入物, 含有小孔直径的过滤器 (直径0.4 µm), 以培养两种不同的细胞类型, 防止细胞在层间迁移。它导致细胞之间的10µm 距离, 这是明显长于在体内, 但仍然复制许多的体内特征的 MEJs, 包括蛋白质本地化和第二信使信令1,14. 此外, VCCC 允许通过在特定的蜂窝室中加入一个激动剂或拮抗器来锁定特定的细胞类型的信号。例如, 加载 EC 与 BAPTA, 以螯合钙, 并刺激 SMC 与肾上腺素14。与其他描述共培养模式的15,16,17,18,19,20,21, 这提供关于分离 mRNA 和蛋白质的不同分数的说明, 包括过滤毛孔内的不同的梅杰分数。这项技术添加到 VCCC 允许具体调查的变化 mRNA 本地化或转录22, 蛋白质磷酸化12,23, 和蛋白质活动12。本文将描述 EC 在过滤器和 SMC 底部的电镀, 虽然可以在不同的构象中培养两个单元类型11,18,24。

<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 结 (梅杰), 一个独特的信号 microdomain 在小口径阻力动脉, 展品定位的特定的蛋白质和信号的过程, 可以控制血管的音调和血压。由于它是一个投射从内皮或平滑肌细胞, 并且由于它的小尺寸 (平均, 面积为 ~ 1 µm2), 梅杰很难孤立地研究。然而, 我们已经开发了一种细胞培养模型, 称为血管细胞共育 (VCCC), 允许体外梅杰形成, 内皮细胞极化, 并解剖的信号蛋白和过程中血管壁的阻力动脉.VCCC 有多种应用, 可以适应不同的细胞类型。该模型由两个细胞类型生长在一个过滤器的相反两侧的0.4 µm 孔, 其中体外MEJs 可以形成。在这里, 我们描述如何创建 VCCC 通过电镀细胞和分离的内皮, 梅杰, 和平滑的肌肉分数, 然后可以用于蛋白质分离或活动化验。具有完整单元格层的过滤器可以固定、嵌入和分段进行荧光分析。重要的是, 这个模型的许多发现都被证实使用了完整的阻力动脉, 凸显了它的生理相关性。

用于 VCCC 的过滤器插入物有小, 0.4 µm 孔。图 1A, VCCC 滤镜嵌入的en 面视图显示了梅杰可以在体外形成的毛孔。该示意图描绘的是光滑的肌肉细胞镀上的过滤器的下部前一天内皮细胞被镀在对面 (图 1B)。一旦细胞被镀, EC, 梅杰, 和 SMC 分数可以隔离三天后。其中的一个示例显示在图 2A中, 其中纤?...

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

A Cell Culture Model of Resistance Arteries

一个细胞培养模型的阻力动脉描述, 允许解剖的信号通路的内皮, 平滑肌, 或之间的内皮和平滑肌 (myoendothelial 交界处)。选择性应用激动剂或蛋白质隔离, 电子显微镜, 或免疫荧光可以利用这种细胞培养模型。

阻力动脉的细胞培养模型

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 次观看.  University of Virginia School of Medicine. 一个细胞培养模型的阻力动脉描述, 允许解剖的信号通路的内皮, 平滑肌, 或之间的内皮和平滑肌 (myoendothelial 交界处)。选择性应用激动剂或蛋白质隔离, 电子显微镜, 或免疫荧光可以利用这种细胞培养模型。

视频: 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.

隔离和小鼠主动脉切除;心血管疾病研究的一个多功能的技术

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

科研

医学

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.

用无标签荧光显微镜评价活体、人体阻力动脉中胶原蛋白和弹性蛋白的压力依赖性 Microarchitectures

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

生物工程

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.

主动脉钙化和炎症血管平滑肌细胞和成像的钙化

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.

研究流动下血管药物靶向的体外3D细胞培养动脉模型

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.

利用大鼠离体阻力动脉的视频显微镜对血管控制机制的评价

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.

构建人主动脉平滑肌细胞器官芯片模型，用于概括主动脉壁中的生物力学应变

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.

从鼠标阻力动脉分离微血管内皮管

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

生物学

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.

冠状血管生成体模型

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.

隔离和鼠标主动脉内皮细胞原代培养

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.

小鼠主动脉夹层损伤:高效<em&gt;体内</em&gt;平滑肌细胞增殖和内皮功能模型

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