阻力动脉的细胞培养模型
1Department of Molecular Physiology and Biophysics,University of Virginia School of Medicine, 2Robert M. Berne Cardiovascular Research Center,University of Virginia School of Medicine, 3Department of Hematology,St. Jude Children’s Research Hospital, 4Research Histology Core,University of Virginia School of Medicine
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. 用于 VCCC 的电镀单元 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. 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 …
Representative Results
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?…
Discussion
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…Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 24mm diameter, 0.4µm Transwell Polyester membrane filter insert | Corning | 3450 | This is one 6 well plate of inserts |
| 225cm2 flask | Corning | 431082 | |
| 6 well plates (without filter inserts) | Corning | 3506 | |
| Primary human coronary artery endothelial cells | Lonza | CC-2585 | |
| Primary human coronary artery smooth muscle cells | Lonza | CC-2583 | |
| EBM-2 EC Basal media | Lonza | CC-3156 | |
| EC growth factors (EGM2 MV Single quots) | Lonza | CC-4147 | Add to basal media before use |
| SmBM SMC basal media | Lonza | CC-3181 | |
| SMC growth factors (SmGM-2 SingleQuot) | Lonza | CC-4149 | Add to basal media before use |
| 0.5% Trypsin-EDTA 10X | Gibco | 15400-054 | Dilute to 2X with sterile dPBS |
| Hemocytometer | VWR | 15170-208 | |
| large petri dishes for SMC Plating (150mm) | Corning | 353025 | |
| Bovine gelatin | Sigma | G-9382 | |
| Human fibronectin | Corning | 356008 | 5µg/ml of fibronectin and store at -20 |
| 10X Hanks' Buffered salt solution (HBSS) | Gibco | 14065-056 | Dilute to 1x with sterile water |
| 10X Dulbecco's Phosphate buffered saline (dPBS) | Gibco | 14200-075 | Dilute to 1x with sterile water |
| Disposable Curved Scalpel (30mm cutting edge) with handle | Amazon | MDS15210 | |
| Forceps | Fisher | 17-467-201 | |
| Cell lifter | Corning | 3008 | |
| Cell scraper | BD Falcon | 353085 | |
| 50mL conical tube | Corning | 352070 | |
| 10mm petri dishes | Falcon | 35-1008 | |
| Rneasy mini kit | Qiagen | 74104 | |
| RNA lysis reagent | Qiagen | 79306 | |
| Paraformaldehyde | Sigma | 158127 | Make 4% solution with dPBS |
| 70% ethanol | Fisher | 04-355-305 | |
| Cavicide disinfectant | Fisher | 131000 | |
| RIPA protein lysis buffer | Sigma | R0278 | |
| Sonic dismembranator | Fisher | Model 50 | |
| 1X PBS for harvesting EC/SMC and immunocytochemistry | Gibco | 10010023 | does not need to be sterile |
| Name | Company | Catalog Number | Comments |
| Embedding/Sectioning/Staining | |||
| Paraffin | Fisher | P31-500 | |
| Automated tissue processer | Excelsior | ES | |
| Embedding station + cold plate | Leica | HistoCore Arcadia | |
| Tissue Tek Accu-Edge Microtome blade | VWR | 25608-961 or 25608-964 | |
| Microscope slides | Thermo Fisher | 22-230-900 | |
| Coverslips | Thermo Fisher | 12-548-5E | |
| Prolong Gold mounting medium | Thermo Fisher | P36931 | |
| Name | Company | Catalog Number | Comments |
| Other Solutions | **Perform all steps in sterile conditions if using the solution on live cells | ||
| Sterile dPBS | Autoclave water to sterilize. Using the sterile water make DPBS (450mL sterile water: 50mL 10X DPBS), mix and filter sterilize (0.2µm filter). Final concentration: 1X |
||
| sterile HBSS | Autoclave water to sterilize. Using the sterile water make HBSS (450mL sterile water: 50mL 10X HBSS), mix and filter sterilize (0.2µm filter). Final concentration: 1X |
||
| Fibronectin stock solution | Use 50mL of sterile water to fibronectin to make stock of 1mL aliquots, then freeze until needed Final concentration: 100µg/mL |
||
| Fibronectin to coat SMC side of VCCC | thaw one aliquot and add 1mL of fibronectin (100µg/mL) to 19mLs of 1X HBSS to reach a final concentration of 5µg/mL. Final concentration: 5µg/mL |
||
| 2x Trypsin EDTA solution | Dilute the 10X 0.5% Trypsin-EDTA to 2X with 1X dPBS (40mL DPBS: 10mL 10X Trypsin) . Final concentration: 2X |
||
| Blocking/antibody solution | (9mL 1X PBS, 25mL Triton X100, 50mg bovine serum albumin, 500mL normal serum) | ||
| Bovine gelatin to coat EC VCCC | Mix 0.5g bovine gelatin into 100mL distilled water. Autoclave prior to first use and store at room temperature. Keep in sterile conditions. Final concentration: 0.50% |
||
| 1X PBS for harvesting cells | does not need to be sterile |
References
- Isakson, B. E., Duling, B. R. Heterocellular contact at the myoendothelial junction influences gap junction organization. Circ Res. 97 (1), 44-51 (2005).
- Little, T. L., Xia, J., Duling, B. R. Dye tracers define differential endothelial and smooth muscle coupling patterns within the arteriolar wall. Circ Res. 76 (3), 498-504 (1995).
- Heberlein, K. R., Straub, A. C., Isakson, B. E. The myoendothelial junction: breaking through the matrix?. Microcirculation. 16 (4), 307-322 (2009).
- Straub, A. C., Zeigler, A. C., Isakson, B. E. The myoendothelial junction: connections that deliver the message. Physiology (Bethesda). 29 (4), 242-249 (2014).
- Isakson, B. E., Ramos, S. I., Duling, B. R. Ca2+ and inositol 1,4,5-trisphosphate-mediated signaling across the myoendothelial junction. Circ Res. 100 (2), 246-254 (2007).
- Dora, K. A., Doyle, M. P., Duling, B. R. Elevation of intracellular calcium in smooth muscle causes endothelial cell generation of NO in arterioles. Proc Natl Acad Sci U S A. 94 (12), 6529-6534 (1997).
- Sonkusare, S. K., et al. Elementary Ca2+ signals through endothelial TRPV4 channels regulate vascular function. Science. 336 (6081), 597-601 (2012).
- Ledoux, J., et al. Functional architecture of inositol 1,4,5-trisphosphate signaling in restricted spaces of myoendothelial projections. Proc Natl Acad Sci U S A. 105 (28), 9627-9632 (2008).
- Boerman, E. M., Everhart, J. E., Segal, S. S. Advanced age decreases local calcium signaling in endothelium of mouse mesenteric arteries in vivo. Am J Physiol Heart Circ Physiol. 310 (9), H1091-H1096 (2016).
- Toussaint, F., Charbel, C., Blanchette, A., Ledoux, J. CaMKII regulates intracellular Ca(2)(+) dynamics in native endothelial cells. Cell Calcium. 58 (3), 275-285 (2015).
- Truskey, G. A. Endothelial Cell Vascular Smooth Muscle Cell Co-Culture Assay For High Throughput Screening Assays For Discovery of Anti-Angiogenesis Agents and Other Therapeutic Molecules. Int J High Throughput Screen. 2010 (1), 171-181 (2010).
- Biwer, L. A., et al. Two functionally distinct pools of eNOS in endothelium are facilitated by myoendothelial junction lipid composition. Biochim Biophys Acta. 1861 (7), 671-679 (2016).
- Straub, A. C., et al. Endothelial cell expression of haemoglobin alpha regulates nitric oxide signalling. Nature. 491 (7424), 473-477 (2012).
- Isakson, B. E. Localized expression of an Ins(1,4,5)P3 receptor at the myoendothelial junction selectively regulates heterocellular Ca2+ communication. J Cell Sci. 121 (Pt 21), 3664-3673 (2008).
- Axel, D. I., et al. Induction of cell-rich and lipid-rich plaques in a transfilter coculture system with human vascular cells. J Vasc Res. 33 (4), 327-339 (1996).
- Hastings, N. E., Simmers, M. B., McDonald, O. G., Wamhoff, B. R., Blackman, B. R. Atherosclerosis-prone hemodynamics differentially regulates endothelial and smooth muscle cell phenotypes and promotes pro-inflammatory priming. Am J Physiol Cell Physiol. 293 (6), C1824-C1833 (2007).
- Herzog, D. P., Dohle, E., Bischoff, I., Kirkpatrick, C. J. Cell communication in a coculture system consisting of outgrowth endothelial cells and primary osteoblasts. Biomed Res Int. , 320123 (2014).
- Jacot, J. G., Wong, J. Y. Endothelial injury induces vascular smooth muscle cell proliferation in highly localized regions of a direct contact co-culture system. Cell Biochem Biophys. 52 (1), 37-46 (2008).
- Scott-Drechsel, D., et al. A new flow co-culture system for studying mechanobiology effects of pulse flow waves. Cytotechnology. 64 (6), 649-666 (2012).
- van Buul-Wortelboer, M. F., et al. Reconstitution of the vascular wall in vitro. A novel model to study interactions between endothelial and smooth muscle cells. Exp Cell Res. 162 (1), 151-158 (1986).
- Wilhelm, I., Fazakas, C., Krizbai, I. A. In vitro models of the blood-brain barrier. Acta Neurobiol Exp (Wars). 71 (1), 113-128 (2011).
- Heberlein, K. R., et al. A novel mRNA binding protein complex promotes localized plasminogen activator inhibitor-1 accumulation at the myoendothelial junction. Arterioscler Thromb Vasc Biol. 32 (5), 1271-1279 (2012).
- Straub, A. C., et al. Compartmentalized connexin 43 s-nitrosylation/denitrosylation regulates heterocellular communication in the vessel wall. Arterioscler Thromb Vasc Biol. 31 (2), 399-407 (2011).
- Cucina, A., et al. Vascular endothelial growth factor increases the migration and proliferation of smooth muscle cells through the mediation of growth factors released by endothelial cells. J Surg Res. 109 (1), 16-23 (2003).
- Heberlein, K. R., et al. Plasminogen activator inhibitor-1 regulates myoendothelial junction formation. Circ Res. 106 (6), 1092-1102 (2010).
- Isakson, B. E., Best, A. K., Duling, B. R. Incidence of protein on actin bridges between endothelium and smooth muscle in arterioles demonstrates heterogeneous connexin expression and phosphorylation. Am J Physiol Heart Circ Physiol. 294 (6), H2898-H2904 (2008).
- Biwer, L. A., Isakson, B. E. Endoplasmic reticulum-mediated signalling in cellular microdomains. Acta Physiol (Oxf). 219 (1), 162-175 (2017).
Cite This Article
Biwer, L. A., Lechauve, C., Vanhoose, S., Weiss, M. J., Isakson, B. E. A Cell Culture Model of Resistance Arteries. J. Vis. Exp. (127), e55992, doi:10.3791/55992 (2017).