Name: in vivo study of human endothelial-pericyte interaction using the matrix gel plug assay in mouse
Uploaded: 2016-12-19T14:00:00.000+00:00
Duration: 8 min 16 s
Description: Watch this Scientific Journal Video about In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse 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.

<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. D. <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+blood+vessels,+endothelial+cells,+and+vascular+pericytes+in+insulin+secretion+and+peripheral+insulin+action.">The role of blood vessels, endothelial cells, and vascular pericytes in insulin secretion and peripheral insulin action.</a> Endocr Rev. 31 (3), 343-363 (2010).</li><li>Ricard, N., 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=Increased+pericyte+coverage+mediated+by+endothelial-derived+fibroblast+growth+factor-2+and+interleukin-6+is+a+source+of+smooth+muscle-like+cells+in+pulmonary+hypertension.">Increased pericyte coverage mediated by endothelial-derived fibroblast growth factor-2 and interleukin-6 is a source of smooth muscle-like cells in pulmonary hypertension.</a> Circulation. 129 (15), 1586-1597 (2014).</li><li>Buchwalow, I. 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>

No competing financial interests enclosed.

את assay תקע ג&#39;ל מטריקס הוכיחה להיות שיטה נוחה וחזקה כדי להעריך ויסות הגנים ב אנגיוגנזה, תרכובות angiogenic ו antiangiogenic in vivo, וכדי להשלים בדיקות במבחנה. כאן אנו מתארים בפרוטרוט assay תקע ג&#39;ל מטריקס הרומן של אנגיוגנזה אדם שחוקר את האינטראקציה בין תאי האנדותל pericytes במה...

אנגיוגנזה הוא התהליך שבו כלי דם חדשים נוצרים מרשת וסקולרית קיים 1 והוא במוקד מחקר המתמשך על פני תחומים רבים, החל התפתחות תקינה למחלות. תהליך דינאמי זה כרוך ההתפשטות וההגירה של תאי האנדותל (EC) וגיוס pericytes לבנות צינור כלי דם כי היא מכוונת כלפי האתר שצריך העברת חמצן וחומרי הזנה 2. כדי ללמוד את התהליך הזה דורש assay דינמית לא פחות, והכי חשוב אחד שיכול לשחזר את הטבע תלת ממדי של היווצרות צינור. במבחנת מבחני מטריקס 3D פותחו כדי לתת מענה לצורך הזה עבדו היטב על מנת לאפשר לחוקרים לקבוע את הצעדים הבדידים במרחב ובזמן שבו אנגיוגנזה מתקיים 3, 4, 5, 6. עם זאת, מודלים מטריקס במבחנה 3D אלה מוגבלים ללימוד כלי הלא perfusedד ולכן הם חסרים רכיבים קריטיים רלוונטיים לתהליך אנגיוגנזה (למשל, במחזור צמיחה וגורם מעכבים, מתח טבעי / כוחות לרוחב מיטת כלי הדם) ולא מצליח לדמות את הסביבה המורכבת נוכח רקמת חיים. כדי לטפל מגבלה זו, כמה in vivo מבחני אנגיוגנזה פותחו 7, כולל assay תקע מטריקס ג&#39;ל אשר יהיה המוקד של הדו&quot;ח שלנו 8, 9. את assay תקע ג&#39;ל מטריקס הוא assay אנגיוגנזה ומבוסס in vivo שמושך חוקר כפי שהוא מספק פלטפורמה יציבה על מנת לבחון את התפקידים של תאים וחומרים שונים אנגיוגנזה. ג&#39;ל מטריקס הוא פתרון קרום במרתף זמין מסחרי כי מופרש על ידי Engelbreth-הולם-הנחיל (EHS) שורת תאי סרקומה עכבר מבצרת לחומר דמוי ג&#39;ל על 37 מעלות צלזיוס. הג&#39;ל מטריקס ניתן לערבב עם תאים ו / או חומרים, כגון גורמי גדילה, ו Injected מתחת לעור לתוך העכבר. מארח ECS יפלוש התקע למעלה מ -14 ימים, יוצר רשת כלי דם, ולהפוך להיות perfused עם של דם המארח. נכון להיום, מבחני תקע ג&#39;ל מטריקס התמקדו אך ורק על חקר התנהגות תא האנדותל במהלך אנגיוגנזה, עם זאת, למיטב ידיעתנו לא נעשה מאמץ עדיין לקבוע אם assay זה יכול לשמש כדי תאי האנדותל שיתוף תרבות pericytes ללמוד כיצד סוגי תאים אלה שני אינטראקציה במהלך אנגיוגנזה. באופן ספציפי, הבנת הקשר בין ECS ו pericytes היא בעל ערך לחקר מחלות שבו אובדן כלי דם הוא פתולוגי, כולל איסכמיה כלי דם ומחלות היקפיות וסקולרית 10, 11, 12. כאן אנו מתארים פרוטוקול מציג pericytes נגזרות האנושיות לתערובת ג&#39;ל מטריקס יחד עם ECS האדם גורם גדילה פיברובלסטים (bFGF). תערובת זו לאחר מכן ניתן מוזרק מתחת לעור in את dorsum של עכברי SCID כדי לאפשר היווצרות של מצופית מתפקד במלואה, pericyte, כלי היברידי. הפרוטוקול שלנו מתאר כיצד להכין אטמי ג&#39;ל מטריקס המכילים ECS אדם עם או בלי pericytes האנוש, שם לעכברי SCID וכיצד לנתח את הסעיפים היסטולוגית עבור נקודות קצה אנגיוגנזה קריטיות.

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

נציג צביעת H &amp; E ו- immunofluorescent סעיפי תקע ג&#39;ל מטריקס מוצגת באיור 2. חלקים מן EC רק תקעים להציג כמה כלים כי הם בעיקר לא perfused עם דם (איור 2 צד ימין למעלה, חיצים שחורים) ואילו תקעים המכילים גם ECS ו pericytes להציג כמה perfused כלי בקטרים ​​גדולים וכ?...

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

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

אנו מציגים פרוטוקול ללמוד אינטראקציות אנדותל-pericyte אדם העכבר באמצעות וריאציה של assay אנגיוגנזה תקע ג&#39;ל מטריקס.

In vivo לחקר האדם האנדותל-pericyte אינטראקציה באמצעות assay Plug מטריקס ג&#39;ל עכבר

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

in-vivo-study-human-endothelial-pericyte-interaction-using-matrix-gel

Research

JoVE Journal

Biology

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

9.6K Views.  Stanford University.  אנו מציגים פרוטוקול ללמוד אינטראקציות אנדותל-pericyte אדם העכבר באמצעות וריאציה של assay אנגיוגנזה תקע ג'ל מטריקס. 

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

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

<em&gt; In vivo</em&gt; Assay לבדיקת חדירות כלי דם

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

Medicine

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

הדמיה בזמן אמת של אינטראקציות heterotypic טסיות דם-נויטרופילים על האנדותל הופעל במהלך דלקת של כלי דם והיווצרות קרישים בעכברים חיים

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Immunology and Infection

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro. However, a limitation of this assay is a lack of endothelial &#8211; mural cell interactions, which are key to the molecular and phenotypic regulation of endothelial cell function in vivo. The protocol given here presents a methodology for the incorporation of mural cells into the bead angiogenesis assay and demonstrates a tight association of endothelial cells and pericytes during sprouting in vitro. The protocol also details a methodology for effective silencing of target genes using siRNA in endothelial cells for mechanistic studies. Altogether, this protocol provides an in vitro assay that more appropriately models the diverse cell types involved in sprouting angiogenesis, and provides a more physiologically-relevant platform for therapeutic assessment and novel discovery of mechanisms of angiogenesis regulation.

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

שילוב Pericytes חרוז תא אנדותל הלבלוב Assay

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including ...

Developmental Biology

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

קרנית Micropocket Assay: דגם של אנגיוגנזה בעין העכבר

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Neuroscience

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

Assay גיבוש האנדותל תא Tube אסטרטגי: השוואת מטריקס ו- Growth Factor מופחתת מטריקס

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

האדם Bioengineering רשתות כלי הדם של עכברים Immunodeficient

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

ב מבחני מבחנה כדי להעריך דם מוח מחסום כמו רשת כלי שיטפון ושיבוש

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired factors are introduced into cold-liquid ECM-mimic gel which, after subcutaneous injection, solidifies to form an environment mimicking the cancer milieu. This matrix permits the penetration of host cells, such as endothelial cells, and therefore, the formation of vasculature.
Herein we propose a new modified matrigel plug assay, which can be exploited to illustrate the angiogenic potential of a pool of factors secreted by cancer cells, as opposed to a specific factor (e.g., bFGF and VEGF) or agent. The plug containing ECM-mimic gel is utilized to introduce the host (i.e., mouse) with a pool of factors secreted to the C.M. of fast-growing tumor-generating glioblastoma cells. We have previously described an extensive comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant-derived clone, in this system model, showing induced angiogenesis in the U-87 MG parental cells. The C.M. is prepared by filtering collected media from confluent tissue culture plates of either cell line following 48 hr incubation. Hence, it contains only factors secreted by the cells, without the cells themselves. Described here is the combination of two imaging modalities, microbubbles contrast-enhanced ultrasound imaging and intravital fibered-confocal endomicroscopy, for an accurate, real-time characterization of the extent, morphology and functionality of newly-formed blood vessels within the plugs.

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

פונקציונליות ניטור ומורפולוגיה של כלי דם גויס על ידי גורמים מופרשים על ידי תאים מניבי גידול בצמיחה מהיר

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

במבחנה בדיקת הנבטה תלת מימדית של אנגיוגנזה באמצעות תאי גזע עובריים של עכבר לצורך מידול מחלות כלי דם ובדיקות סמים

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

פישוט מחקרי אנגיוגנזה עם בדיקת תקע ג'ל מטריצה שונה

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized equipment, or are hard to perform for subsequent quantitative analysis. Here we present a modified matrix gel plug assay to evaluate angiogenesis in vivo. In this protocol, vascular cells were mixed with matrix gel in the presence or absence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of recipient mice. After 7 days, phosphate buffer saline containing dextran-FITC is injected via the tail vein and circulated in vessels for 30 min. Matrix gel plugs are collected and embedded with tissue embedding gel, then 12 &#181;m sections are cut for fluorescence detection without staining. In this assay, dextran-FITC with high molecular weight (~150,000 Da) can be used to indicate functional vessels for detecting their length, while dextran-FITC with low molecular weight (~4,400 Da) can be used to indicate the permeability of neo-vessels. In conclusion, this protocol can provide a reliable and convenient method for the quantitative study of angiogenesis in vivo.

מחבר זרקור: חקירת אנגיוגנזה וחדירות כלי דם באמצעות בדיקת תקע ג'ל מטריצה שונה

The method presented here can evaluate the effect of reagents on angiogenesis or vascular permeability in vivo without staining. The method uses dextran-FITC injection via the tail vein to visualize neo-vessels or vascular leakage.

שונה בבדיקת תקע ג'ל מטריצת Vivo למחקרי אנגיוגנזה

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized ...

In vivo לחקר האדם האנדותל-pericyte אינטראקציה באמצעות assay Plug מטריקס ג'ל עכבר

Summary

Explore More Videos

Chapters in this video