Name: analysis of extracellular vesicle-mediated vascular calcification using in vitro and in vivo models
Uploaded: 2023-01-27T14:50:00
Description: Watch this Scientific Journal Video about Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models at JoVE.com

这项工作得到了美国国立卫生研究院（NIH）国家心脏，肺和血液研究所（1R01HL160740和5 T32GM132054-04）和佛罗里达心脏研究基金会的资助。我们要感谢Kassandra Gomez帮助合成和成像水凝胶。

体内工作由佛罗里达国际大学机构动物护理和使用委员会（IACUC）批准和监督，并符合当前的美国国立卫生研究院（NIH）指南。对于该协议，该过程不会因小鼠的应变，体重，年龄和性别而异。正在研究的钙化类型，饮食和治疗可能会改变研究的长度和所用小鼠的重量，并且可能取决于研究中使用的小鼠的特定品系和性别。对于该协议，使用雄性和雌性C57BL / 6J小鼠，并喂食钙化饮食。小鼠?...

<ol>
	<li>Bakhshian Nik, A., Hutcheson, J. D., Aikawa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+as+mediators+of+cardiovascular+calcification.">Extracellular vesicles as mediators of cardiovascular calcification.</a> Frontiers in Cardiovascular Medicine. 4, 78 (2017).</li><li>Ho, C. Y., Shanahan, C. 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X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+vascular+calcification+in+chronic+kidney+disease.">Mechanisms of vascular calcification in chronic kidney disease.</a> Journal of the American Society of Nephrology. 19 (2), 213-216 (2008).</li><li>Mir, B., Goettsch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+as+delivery+vehicles+of+specific+cellular+cargo.">Extracellular vesicles as delivery vehicles of specific cellular cargo.</a> Cells. 9 (7), 1601 (2020).</li><li>Ruiz, J. L., Hutcheson, J. D., Aikawa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+calcification:+Current+controversies+and+novel+concepts.">Cardiovascular calcification: Current controversies and novel concepts.</a> Cardiovascular Pathology. 24 (4), 207-212 (2015).</li><li>New, S. E., Aikawa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+extracellular+vesicles+in+de+novo+mineralization:+An+additional+novel+mechanism+of+cardiovascular+calcification.">Role of extracellular vesicles in de novo mineralization: An additional novel mechanism of cardiovascular calcification.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 33 (8), 1753-1758 (2013).</li><li>Aikawa, E., Hutcheson, J. 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执行协议时，重要的是要注意获得成功结果的关键步骤。在小鼠主动脉的分离过程中，正确进行灌注至关重要。注射PBS时，必须注意不要刺穿右心室。这将导致液体直接从心室泄漏出来，无法通过肺部循环，在主动脉内留下血液。一旦灌注正确进行并开始显微解剖，必须在完成和扫描之前从主动脉中取出所有脂肪和脂肪组织。剩余的脂肪将导致主动脉在将数据标准化为总组织大小时显得更大。 </p...

钙化是心血管疾病死亡率和发病率的最重要预测指标1.钙化会由于钙和磷酸盐矿物质的堆积而改变动脉壁力学2。在动脉粥样硬化中，钙化会加剧局部压力并导致斑块破裂，这是心脏病发作的主要原因。内侧钙化（通常由慢性肾脏疾病引起）更为普遍，可导致显著的动脉僵硬、功能障碍和心脏超负荷2，3。目前，没有治疗或预防血管钙化的治疗选择。
血管平滑肌细胞 （VSMC） 采用成骨细胞样表型并释放钙化细胞外囊泡 （EV），这些囊泡对新生矿物质进行成核，从而驱动钙化4，5，6。这个过程类似于骨7中成骨细胞的生理矿化。尽管血管壁和骨基质中的矿化终点相似，但钙化EVs的起源机制在两种组织中不同8。有许多类型的模型用于研究血管钙化。在体外，细胞培养模型用专门的培养基模拟VSMC的成骨转变和随后的矿物形成。
在研究 体内 钙化时，使用的模型取决于所研究的钙化类型。高脂血症小鼠模型通常用于研究动脉粥样硬化性钙化，这在富含脂质的斑块中显得更局灶性9。相比之下，内侧钙化在整个脉管系统中更为普遍，并且通常使用慢性肾脏疾病模型进行研究，该模型采用富含腺嘌呤的饮食方案来诱导肾衰竭或手术技术切除肾脏的重要部分10，11。积极的血管钙化模型使用了高脂血症和慢性肾脏疾病模型的组合12。该协议提供了一种评估小鼠主动脉血管钙化的方法，用于内侧和动脉粥样硬化钙化，从主动脉壁中提取EV，并观察从 体外 细胞培养模型获得的EV中的矿化潜力。未来的研究可以将这些程序用于血管钙化的机制分析，并评估潜在的治疗干预措施。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>8-well chambered coverglass</td>
			<td>Thermo Scientific</td>
			<td>155409PK</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Syringe</td>
			<td>BD</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr>
			<td>20 G 1 inch Needle</td>
			<td>BD</td>
			<td>305175</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen, High Concentraion, Rat Tail</td>
			<td>Corning</td>
			<td>354249</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Worthington Biochemical</td>
			<td>LS004174</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-8254</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Dish</td>
			<td>Living Systems Instrumentation</td>
			<td>DD-90-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Pan and Wax</td>
			<td>United Scientific Supplies</td>
			<td>DSPA01-W</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cytiva</td>
			<td>SH30022.FS</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Sigma-Aldrich</td>
			<td>26675-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>LI-COR Odyssey</td>
			<td>LI-COR</td>
			<td>DLx</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Curved Scissors (24 mm Blade)&#160;</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5913</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Spring Scissors (13 mm Blade)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5677</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Spring Scissors (5 mm Blade)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5600</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Tweezers (0.10 x 0.06 mm Tip)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-4976</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima MAX-TL Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>B11229</td>
			<td></td>
		</tr>
		<tr>
			<td>OsteoSense 680EX</td>
			<td>Perkin Elmer</td>
			<td>NEV10020EX</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Protease Inhibitor</td>
			<td>Thermo Scientific</td>
			<td>A32963</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fischer Chemical</td>
			<td>P217</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA Lysis and Extraction Buffer</td>
			<td>G Biosciences</td>
			<td>786-489</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fischer Chemical</td>
			<td>BP358</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Thermo Scientific</td>
			<td>A4782602</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>S0751</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy HTX Multimode Reader</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plate, 96-well</td>
			<td>Thermo Fisher</td>
			<td>167008</td>
			<td></td>
		</tr>
		<tr>
			<td>T-Pins</td>
			<td>United Scientific Supplies</td>
			<td>TPIN02-PK/100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Hydrochloride</td>
			<td>Fischer Chemical</td>
			<td>BP153</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of extracellular vesicle-mediated vascular calcification using in vitro and in vivo models

心血管疾病是世界上导致死亡的主要原因，血管钙化是心血管事件最重要的预测指标;然而，目前没有血管钙化的治疗或治疗选择。钙化始于专门的细胞外囊泡（EV），其通过聚集钙和磷酸根离子作为成核病灶。该协议描述了获取和评估鼠主动脉钙化并分析相关提取的EV的方法。首先，对小鼠进行大体解剖以收集任何相关器官，例如肾脏、肝脏和肺。然后，分离鼠主动脉并从主动脉根部切除到股动脉。然后将两到三个主动脉汇集并在消化溶液中孵育，然后进行超速离心以分离感兴趣的 EV。接下来，通过在高磷酸盐溶液中孵育并测量 340 nm 波长处的光吸光度来确定 EV 的矿化潜力。最后，使用胶原蛋白水凝胶观察EV在 体外产生的钙化矿物形成和成熟。

提取主动脉后，使用近红外光学扫描仪成像显示主动脉的视觉表现以及血管钙化（图1）。扫描荧光图像中的像素强度值表示钙化的分布，此处使用彩色热图显示。定量方法包括识别阳性阈值并报告主动脉面积的百分比，其值大于该阈值和/或报告主动脉内像素的平均荧光强度。如图 2 和 图3所示，市售的原代人冠状动脉平滑肌细胞...

Watch this Scientific Journal Video about Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models at JoVE.com

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

本文介绍了通过分离小鼠主动脉，然后提取钙化的细胞外囊泡来观察矿化潜力来获取和评估血管钙化的方法。

使用体外和体内模型分析细胞外囊泡介导的血管钙化

Cardiovascular disease is the leading cause of death in the world, and vascular calcification is the most significant predictor of cardiovascular events; ...

该协议提供了一种可用于帮助评估的方法，并在未来帮助开发血管钙化的治疗方法，血管钙化是心血管疾病最重要的预测指标。分离EV的能力使我们能够进一步研究导致血管钙化的机制，以及血管平滑肌细胞的表型变化。该技术提供了对血管平滑肌细胞介导钙化的早期分子机制的见解。了解这一点可以研究所理解的机制的治疗选择。任何感兴趣的电动汽车都可以使用这种方法进行研究。它不仅限于血管平滑肌细胞、成骨细胞或血管钙化。对小鼠实施安乐死后，用镊子提起皮肤，并在中线切开一个切口。去除多余的皮肤或脂肪。然后去除中线外的生殖器官、膀胱和脂肪。然后将胃肠道器官移到右侧，沿着中线的肠道移动。一旦发现，抬起中线，并将胃肠道切除到胃部。通过将肾脏从中线抬起来切除肾脏。切得尽可能靠近肾脏。通过抬起肺叶并从中线切开来去除肝脏。然后通过使用10毫升注射器将冷PBS注入右心室来灌注心脏和主动脉。等待肺部膨胀，然后变白。切除肺部和任何多余的横膈膜或胸腔。要去除骨头，请打开一把宽的剪刀，并将它们放在鼠标尾巴上方的下部区域。向下切开，从皮肤上抬起脊柱，然后去除中线两侧的脂肪。一旦皮肤脱落，通过在心脏上方切割来切除脊柱和完整的器官。如果运送到立体镜，请将脊柱和完整的器官放在冰桶中的PBS烧杯中。将脊柱和器官移入解剖盘后，用冰冷的PBS填充，并用针钉住脊柱和胸腔。在解剖显微镜下，用镊子小心地提起心脏，然后开始在脊柱上方和主动脉下方切割。继续此操作，直到到达脊柱底部。从解剖盘中取出脊柱，并固定心脏和任何多余的脂肪或肌肉。从心脏正下方的区域开始，识别食道、腔静脉和主动脉。切除食道和腔静脉，以获得清晰的主动脉视野。使用显微解剖钳和剪刀，开始提起脂肪组织，并尽可能靠近主动脉切割。沿着内侧线继续，直到所有主动脉和股动脉都暴露在外。在心脏处，去除所有脂肪组织，露出主动脉弓处的三个分支。通过将显微夹层剪刀插入左心室并切割主动脉周围的肌肉，从左心室移除主动脉根部。分离并清洁主动脉后，使用近红外扫描仪对主动脉进行成像，以可视化血管钙化。使用自定义 MATLAB 脚本，量化钙示踪剂的总信号，归一化为总扫描主动脉区域。将 MATLAB 从主动脉的近红外扫描中指向 TIF 文件的位置，打开单个文件，然后从 TIF 文件中提取像素强度值。选择钙化程度最大的主动脉作为彩色映射图像上的最大值。当窗口上出现“图像中有多少个标本”命令时，输入当前图像中的主动脉数量，然后逐个选择每个主动脉。选择主动脉后，MATLAB 将创建带有主动脉总面积和钙化面积掩码的二进制图像。然后使用这些遮罩图像的值来确定总钙化面积、主动脉总面积、钙化阳性面积百分比和钙化面积的平均强度。扫描主动脉后，立即汇集两到三个主动脉以产生足够的蛋白质浓度。在 1.5 毫升消化溶液中在 37 摄氏度下孵育两到三个混合主动脉两小时。收集溶液并以1， 000g离心15分钟以除去细胞碎片。接下来，除去微泡，再次将上清液以33， 000g离心30分钟。收集并评估上清液的钙化潜力。通过离心从条件收集的培养基中除去细胞碎片后，将收集的上清液以33， 000g旋转30分钟。收集上清液并将其转移到新管中。然后将1%的300毫摩尔磷酸二氢钠加入细胞外囊泡样品中，并通过移液混合溶液。将200微升混合物溶液转移到96孔板中，并将96孔板在酶标仪中以37摄氏度孵育。将酶标仪设置为每分钟记录340纳米波长的吸光度。将300微升胶原蛋白溶液移液到八孔室玻璃的每个孔中，并在37摄氏度和5%二氧化碳下孵育72小时。孵育后，向孔中加入300微升DMEM作为对照。将先前收集的300微升细胞外囊泡培养基样品加入剩余的孔中，并孵育六天，如前所述。在第10天，将2.5微升OsteoSense和DMEM混合物移液到每个孔中，并孵育24小时。在孵育结束时，使用倒置显微镜通过盖玻片室的底部或从顶部使用长工作距离物镜对带有OsteoSense荧光滤光片的水凝胶进行成像。使用 ImageJ 中提供的颗粒分析选项评估钙化次数、钙化大小和收集的图像。然后导航到图像，选择调整，然后单击阈值以二值化图像，以便只有 OsteoSense 信号显示为白色。然后使用“分析”并单击“分析颗粒”命令以获取图像中每个钙化的信息。使用相同的阈值参数以确保分析的每个图像的一致性。解剖主动脉的近红外光学扫描仪成像显示，与对照小鼠的两个主动脉相比，患有慢性肾脏疾病的小鼠的整个主动脉具有更高的OsteoSense信号。从在促钙化培养基中培养的血管平滑肌细胞获得的条件培养基在340纳米处显示出比对照培养基更高的吸光度。与对照样品相比，促钙化样品的吸光度增加快1.5倍。来自在促钙化条件下培养的细胞的条件培养基含有矿物质沉积物。在显微解剖主动脉时，仔细切割主动脉周围的脂肪很重要。您希望分析尽可能多的主动脉，但这可能非常乏味。一旦分离出EV，就可以完成额外的测试，例如碱性磷酸酶活性测定或免疫体。这些测试将进一步显示电动汽车中存在的机制和蛋白质。

Research

JoVE Journal

Biology

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

雪旺的星形胶质细胞使用相互作用分析<em在体外检测

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

科研

生物学

In vivo and in vitro Studies of Adaptor-clathrin Interaction

In vivo and in vitro Studies of Adaptor-clathrin Interaction

适配器，网格蛋白相互作用在体内和体外研究

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

MG53介导的细胞膜使用维修的可视化在体内在体外系统

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Mouse Sperm Cryopreservation and Recovery using the IÃƒâ€šÃ‚Â·Cryo Kit

精子冷冻保存和恢复使用I ·冷冻包

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

在体外合成在人体细胞修饰蛋白表达的mRNA的感应

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

基于 X 射线 Microcomputed 层析成像的玉米血管束微米尺度分型技术研究

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

细胞外通量分析技术在视网膜组织外种中的能量代谢测定

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

In Vitro Analysis of E3 Ubiquitin Ligase Function

体外 E3泛素连接酶功能分析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...