Name: in vivo study of human endothelial-pericyte interaction using the matrix gel plug assay in mouse
Uploaded: 2016-12-19T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse Video at JoVE.com

Dr. K. Yuan was supported by an American Heart Association Scientist Development Grant (15SDG25710448) and the Pulmonary Hypertension Association Proof of Concept Award (SPO121940). Dr. V. de Jesus Perez was supported by a career development award from the Robert Wood Johnson Foundation, an NIH K08 HL105884-01 award, a Pulmonary Hypertension Association Award, a Biomedical Research Award from the American Lung Association and a Translational Research and Applied Medicine award from Stanford University.

倫理文：動物を対象とする手順は、スタンフォード大学医学部での施設内動物管理使用委員会によって承認されています。 注：動物は、3％の気化イソフルランおよびO 2ガスの3％の供給と麻酔下にあります。目に獣医軟膏の使用は、麻酔下ながら乾燥を防ぐのに役立つかもしれません。 1.細胞の調製<ol><li>適切なメディアの10ミリリットルと100ミリメートルプレートにヒ?...

<ol>
	<li>Risau, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+angiogenesis.">Mechanisms of angiogenesis.</a> Nature. 386 (6626), 671-674 (1997).</li><li>Ribatti, D., Nico, B., Crivellato, 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+role+of+pericytes+in+angiogenesis.">The role of pericytes in angiogenesis.</a> Int J Dev Biol. 55 (3), 261-268 (2011).</li><li>Koh, W., Stratman, A. N., Sacharidou, A., Davis, G. E. <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+three+dimensional+collagen+matrix+models+of+endothelial+lumen+formation+during+vasculogenesis+and+angiogenesis.">In vitro three dimensional collagen matrix models of endothelial lumen formation during vasculogenesis and angiogenesis.</a> Methods Enzymol. 443, 83-101 (2008).</li><li>Liu, Y., Senger, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-specific+activation+of+Src+and+Rho+initiates+capillary+morphogenesis+of+endothelial+cells.">Matrix-specific activation of Src and Rho initiates capillary morphogenesis of endothelial cells.</a> FASEB J. 18 (3), 457-468 (2004).</li><li>Grant, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+endothelial+cells+with+a+laminin+A+chain+peptide+(SIKVAV)+in+vitro+and+induction+of+angiogenic+behavior+in+vivo.">Interaction of endothelial cells with a laminin A chain peptide (SIKVAV) in vitro and induction of angiogenic behavior in vivo.</a> J Cell Physiol. 153 (3), 614-625 (1992).</li><li>Madri, J. A., Pratt, B. M., Tucker, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+modulation+of+endothelial+cells+by+transforming+growth+factor-beta+depends+upon+the+composition+and+organization+of+the+extracellular+matrix.">Phenotypic modulation of endothelial cells by transforming growth factor-beta depends upon the composition and organization of the extracellular matrix.</a> J Cell Biol. 106 (4), 1375-1384 (1988).</li><li>Brooks, P. C., Montgomery, A. M., Cheresh, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+10-day-old+chick+embryo+model+for+studying+angiogenesis.">Use of the 10-day-old chick embryo model for studying angiogenesis.</a> Methods Mol Biol. 129, 257-269 (1999).</li><li>Yuan, 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=Activation+of+the+Wnt/planar+cell+polarity+pathway+is+required+for+pericyte+recruitment+during+pulmonary+angiogenesis.">Activation of the Wnt/planar cell polarity pathway is required for pericyte recruitment during pulmonary angiogenesis.</a> Am J Pathol. 185 (1), 69-84 (2015).</li><li>Johns, A., Freay, A. D., Fraser, W., Korach, K. S., Rubanyi, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+estrogen+receptor+gene+prevents+17+beta+estradiol-induced+angiogenesis+in+transgenic+mice.">Disruption of estrogen receptor gene prevents 17 beta estradiol-induced angiogenesis in transgenic mice.</a> Endocrinology. 137 (10), 4511-4513 (1996).</li><li>Chen, C. W., 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=Human+pericytes+for+ischemic+heart+repair.">Human pericytes for ischemic heart repair.</a> Stem Cells. 31 (2), 305-316 (2013).</li><li>Richards, O. C., Raines, S. M., Attie, A. 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B., Bocker, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunohistochemistry: Basics and Methods. , 1-153 (2010).</li><li>Tata, D. A., Anderson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+investigation+of+capillary+structure.">A new method for the investigation of capillary structure.</a> J Neurosci Methods. 113 (2), 199-206 (2002).</li></ol>

マトリックスゲルプラグアッセイは、血管形成、in vivoでの血管新生および抗血管新生化合物における遺伝子調節を評価するため、およびインビトロ試験を補完するために便利で強力な方法であることが証明されています。ここでは、詳細には血管形成中の内皮細胞と周皮細胞との間の相互作用を調べ、ヒト血管新生の新規マトリックスゲルプラグアッセイを記述する。 <p clas...

血管新生は、新しい血管が既存の血管ネットワーク1から形成されるプロセスであり、正常な発達、疾患の範囲の多くの地域を横切って進行中の研究の焦点です。この動的なプロセスは、酸素や栄養素デリバリー2を必要とするサイトに向けられている血管の管を構築するために、内皮細胞（EC）および周皮細胞の動員の増殖および移動を伴います。このプロセスを研究するためには、管形成の3次元性を再現することができます1、最も重要なのは、同じように動的分析を必要とします。 in vitroでの3Dマトリックスアッセイは、この必要性に対処するために開発されており、研究者は、血管新生が生じ3、4、5、6をとる空間および時間における個別のステップを定義することができるように十分に取り組んできました。しかし、これらのインビトロ 3Dマトリックスモデルは、非灌流血管研究に限られていますdは、したがって、血管新生プロセスに関連する重要なコンポーネントが不足している（ 例えば、血管床を横切って成長と阻害因子、不自然なテンション/力を循環させる）と、生きている組織に存在する複雑な環境をシミュレートすることができません。この制限に対処するために、いくつかのin vivoでの血管新生アッセイは、当社の報告書8、9の焦点になるマトリックスゲルプラグアッセイを含む、7を開発されてきました。 マトリックスゲルプラグアッセイは、血管新生に異なる細胞や物質の役割をテストするための堅牢なプラットフォームを提供するので、研究者にアピールする十分に確立されたin vivoでの血管新生アッセイです。マトリックスゲルは、37℃でゲル状物質に固化エンゲル-Holm-Swarm（EHS）マウス肉腫細胞株によって分泌される市販の基底膜溶液です。マトリックスゲルは、細胞および/またはそのような成長因子、および麟蹄などの物質と混合することができますマウスに皮下CTED。ホストのECは、14日間にわたってプラグに侵入血管網を形成し、宿主の血液で灌流になります。現在までに、マトリックスゲルプラグアッセイこのアッセイは、に共培養内皮細胞および周皮細胞を使用することができるかどうかを決定するために努力がまだ行われていない我々の知る限り、しかし、血管形成中の内皮細胞の挙動の研究にのみ焦点を当てていますこれら2つの細胞型は、血管新生の間にどのように相互作用するかを研究します。具体的には、ECおよび周皮細胞との間の関係を理解することは、血管の損失は微小血管虚血および末梢血管疾患10、11、12を含む、病理学的である疾患を研究するための貴重なものです。 ここでは、ヒトECおよび線維芽細胞成長因子（bFGF）と共にマトリックスゲル混合物にヒト由来周皮細胞を導入するプロトコルを記載しています。次いで、この混合物を皮下I注射することができますnはSCIDマウスの背は、完全に機能的な、周皮細胞被覆され、ハイブリッド血管の形成を可能にします。私たちのプロトコルが持つまたはSCIDマウスとどのように重要な血管新生エンドポイントの組織切片を分析するために人間の周皮細胞、配置があってもなくても、人間のECを含むマトリックスゲルプラグを準備する方法について説明します。

<table border="0" cellpadding="0" cellspacing="0" width="806">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">PBS (Phosphate buffered saline)</td>
			<td style="width:115px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">0.25% Trypsin/0.53mM EDTA</td>
			<td style="width:115px;">Corning</td>
			<td style="width:119px;">25-053-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Endothelial Cell Media (ECM) kit</td>
			<td style="width:115px;">Sciencell</td>
			<td style="width:119px;">1001</td>
			<td>includes media, EC growth supplement, and penicillin/streptomycin, each supplied at the appropriate volume for easy mixing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Pericyte Media (PM) kit</td>
			<td style="width:115px;">Sciencell</td>
			<td style="width:119px;">1201</td>
			<td>includes media, Pericyte growth supplement, and penicillin/streptomycin, each supplied at the appropriate volume for easy mixing</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:405px;">primary human pulmonary microvascular endothelial cells</td>
			<td style="width:115px;">Pulmonary Hypertension Breakthrough Initiative</td>
			<td style="width:119px;"></td>
			<td>this cell type is also available commercially. Cells used at passage 1-4</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:405px;">primary human pulmonary pericytes</td>
			<td style="width:115px;">Pulmonary Hypertension Breakthrough Initiative</td>
			<td style="width:119px;"></td>
			<td>this cell type is not available commercially, but brain paricytes are.&#160; Cells used at passage 1-4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">bFGF (basic Fibroblast Growth Factor)</td>
			<td style="width:115px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
			<td>stock solution is 50ug/ml in 0.1% BSA in PBS, aliquots at 50 uL and stored at -20 degrees</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Matrigel Basement Membrane Matrix</td>
			<td style="width:115px;">BD</td>
			<td style="width:119px;">356237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">28G 1cc Insluin Syringe</td>
			<td style="width:115px;">BD</td>
			<td style="width:119px;">329410</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">SCID (Severe Combined Immune Deficiency) mice</td>
			<td style="width:115px;">The Jackson Laboratory</td>
			<td>5557</td>
			<td>NOD.SCID IL2R gamma knockout strain is the best strain; 4-6 weeks of age</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">1.5mL microcentrifuge tubes, sterile</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">15 mL screw top tubes, sterile</td>
			<td style="width:115px;">BD Biosciences</td>
			<td style="width:119px;">352096</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">PAP pen</td>
			<td style="width:115px;">Life Technologies</td>
			<td style="width:119px;">8899</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">hemocytometer</td>
			<td style="width:115px;">ThermoFisher Scientific</td>
			<td style="width:119px;">02-671-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Nair hair removal cream</td>
			<td style="width:115px;">Walmart</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">anti-human CD31 primary antibody</td>
			<td style="width:115px;">LifeSpan Biosciences</td>
			<td>LS-B4737</td>
			<td>working solution is 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-human Smooth Muscle actin CY3 primary fluorescent antibody</td>
			<td style="width:115px;">Sigma</td>
			<td>C6198</td>
			<td>working solution is 1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">goat anti-rabbit secondary antibody; 488/green</td>
			<td style="width:115px;">ThermoFisher Scientific</td>
			<td style="width:119px;">A-11008</td>
			<td>working solution is 1:250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Prolong Gold DAPI solution</td>
			<td style="width:115px;">Cell Signaling</td>
			<td style="width:119px;">8961S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">microscope slides</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">48300-047</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">no. 1.5 cover slips</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12-544-D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">citrate buffer 10x</td>
			<td style="width:115px;">Millipore</td>
			<td style="width:119px;">21545</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">extra fine surgical scissors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td style="width:119px;">14084-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">Formalin (paraformaldehyde)</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">245-685</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Tissue cassettes</td>
			<td style="width:115px;">Simport</td>
			<td>M492-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">goat serum</td>
			<td style="width:115px;">Dako</td>
			<td style="width:119px;">X0907</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo study of human endothelial-pericyte interaction using the matrix gel plug assay in mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

マトリックスゲルプラグ部は、図に示されているのECおよび周皮細胞の両方を含むプラグが、いくつかの灌流を表示するのに対し、代表的H＆Eおよび免疫蛍光染色は、ECからの2セクションは唯一のプラグは、主に血液で灌流されていないいくつかの船（左図2トップ、黒矢印）を表示しますCD31陽性のECのすぐ隣に正のSMA染色により?...

Watch this Scientific Journal Video about In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse Video at JoVE.com

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse Video (Video) | JoVE

私たちは、マトリックスゲルプラグ血管新生アッセイの変動を用いて、マウスにおけるヒト内皮 - 周皮細胞の相互作用を研究するためのプロトコルを提示します。

マウスにおけるマトリックスゲルプラグアッセイを用いたヒト内皮-周皮細胞の相互作用のin vivo研究で

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

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Biology

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

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Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism Video (Video) | JoVE

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells Video (Video) | JoVE

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products Video (Video) | JoVE

ギャップ結合細胞間コミュニケーション：毒物および毒素の有害効果を評価するための機能バイオマーカー、および天然物の健康上の利点

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...