Name: modified in vivo matrix gel plug assay for angiogenesis studies
Uploaded: 2023-06-30T14:15:00.000+00:00
Duration: 9 min 3 s
Description: Watch this Scientific Journal Video about Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies at JoVE.com

This work was funded by Natural Science Foundation of Zhejiang Province (LY22H020005), and National Natural Science Foundation of China (81873466).

All procedures involving animal subjects were approved by the Institutional Animal Care and Use Committee (IACUC) of Wenzhou Medical University (XMSQ2021-0057, July 19th, 2021). All reagents and consumables are listed in the Table of Materials.
1. Culture medium preparation
<ol>
	<li>10x M199 culture medium: Dissolve M199 powder to 10x concentration with 90 mL of deionized water and add 10 mL of fetal bovine serum (FBS), then pass through a 0.22 &#181;m filter. Store the medium at 4 &#176;C for up to 2 months.</li>
	<li>Complete endothelial culture medium: Add 50 mL of FBS, 5 mL of penicillin/streptomycin, and 5 mL of endothelial cell growth supplement to 460 mL of endothelial cell medium (ECM). Store the medium at 4 &#176;C for up to 1 month.</li>
</ol>
2. Vascular cell preparation
<ol>
	<li>Culture 1 x 105 vascular cells (primary cultured endothelial progenitor cells14, endothelial cells, or endothelial cell lines) with 8 mL of complete endothelial culture medium in 100 mm tissue culture dish at 37 &#176;C and 5% CO2 to 70% confluency.</li>
	<li>Remove the culture medium and rinse the dish 2x with 1x phosphate buffered saline (PBS) to remove unattached cells and debris. Remove PBS and add 3 mL of 0.25% trypsin containing 2.21 mM EDTA, and incubate at 37 &#176;C for 1 min.</li>
	<li>Neutralize the trypsin with 7 mL of complete endothelial culture medium, and gently rinse cells off the culture dish. Confirm cell detachment under a light microscope (40x magnification).</li>
	<li>Collect cell suspension in a 15 mL tube and centrifuge at 400 x g for 10 min. Remove supernatant and resuspend cells with 5 mL of complete endothelial culture medium.</li>
	<li>Count cells using a hemocytometer and move suspension containing 2 x 106 cells to a sterile 1.5 mL tube. Each plug contains 1.5 x 106 cells, additional 25% cells are used in case of waste. Each group includes at least 3 plugs. 
	NOTE: After trials it was found that 1.5 x 106 cells in 300 &#181;L of matrix gel led to the proper development of angiogenesis and was therefore chosen for experimentation.</li>
	<li>Centrifuge cell suspension at 400 x g for 5 min to pellet cells, and then remove supernatant.</li>
</ol>
3. Matrix gel preparation
<ol>
	<li>Pre-cool sterile 1.5 mL tubes containing cell pellet in a 4 &#176;C water bath. Pre-cool 30G 1 mL insulin syringes in a 4 &#176;C refrigerator.</li>
	<li>Completely thaw matrix gel in a 4 &#176;C water bath . Do not mix or vortex. Pre-cool 10x M199 and test reagent/drug-of-interest in a 4 &#176;C water bath.</li>
	<li>Mix pre-cooled matrix gel with 10x M199 containing 10% FBS and the reagent to be tested at a volume ratio of 8.8:1:0.2 to get a matrix gel and M199 mixture containing 1% FBS and the reagent.</li>
	<li>Resuspend cells with 400 &#181;L of the matrix gel mixture, mix gently to avoid forming bubbles. Keep the tube on ice until mice are prepared for injection. Keep the matrix gel mixture on ice all the time to avoid coagulation. 
	&#8203;NOTE: Here, 300 &#181;L of matrix gel mixture containing 1.5 x 106 cells was used to form gel plug. Prepare 25% extra gel according to 25% additional cells in step 2.</li>
</ol>
4. Mouse preparation
<ol>
	<li>Anesthetize 6-8 week old male Nu/Nu mouse (18-25 g) in the chamber of the animal anesthesia device with isoflurane (3% isoflurane in 100% oxygen at a flowrate of 1 L/min). After successful anaesthetization, confirmed by the absence of righting reflex and toe pinch reflex, move the mouse out of the chamber, move the mouse out of the chamber and put an anesthesia mask on the mouse and change the concentration of isoflurane to 1.5% in 100% oxygen at a flowrate of 1 L/min. 
	NOTE: Provide thermal support to the animal using a heating pad throughout the procedure.</li>
	<li>Use vet ointment on eyes to prevent dryness. Tape the limbs of the mice on the operation board in the prone position.</li>
</ol>
5. Matrix gel mixture injection
<ol>
	<li>Load 300 &#181;L of the matrix gel mixture into a 1 mL insulin syringe with a 30G needle. Avoid bubble formation. Quickly load the matrix gel (within 2 min) to avoid solidification during this time. Place the insulin syringe loaded with matrix gel on ice immediately.</li>
	<li>Clean the skin on the back of mice using 75% alcohol pads. Subcutaneously inject 300 &#181;L of matrix gel mixture into one side of the back of mice.</li>
	<li>Gently remove the needle from the injection site to prevent leakage of the matrix gel mixture. Check for a small hump at the injection site (Figure 1).</li>
	<li>Place the mouse on the heating pad for 2 min to let the matrix gel mixture coagulate and form plug.</li>
	<li>Repeat steps 5.2. to 5.4. to create plug on the other side of the back of the mouse. Mark the edges of the humps using a marker pen.</li>
	<li>Observe the mouse until it has regained sufficient consciousness to maintain sternal recumbency. Put the mouse in a separate cage until it is fully recovered. House the mice in SPF-class experimental animal laboratory at 22 &#177; 2 &#176;C with a 12 h light/dark cycle for 7 days.</li>
</ol>
6. Dextran-FITC injection through the tail vein
<ol>
	<li>After 7 days of matrix gel injection, resuspend 0.5 mg of dextran-FITC with 500 &#181;L of double distilled water (ddH2O) and then violently vortex to obtain the dextran-FITC solution at the final concentration of 1 &#181;g/&#181;L. 
	NOTE: Use dextran-FITC with molecular weight of ~150 kDa for angiogenesis assay, and use dextran-FITC with molecular weight of ~4 kDa for vascular permeability assay.</li>
	<li>Load 50 &#181;L of dextran-FITC solution into the 29G syringes.</li>
	<li>Fix the mouse on the tail vein injection instrument. Clean the tail with 75% alcohol cotton ball and gently inject 50 &#181;L of dextran-FITC through tail vein.</li>
	<li>Compress the injection site with cotton swab for 1 min to stanch bleeding, and then put the mice back in the cage for 30 min.</li>
	<li>Repeat steps 6.3. and 6.4. until all mice have received dextran-FITC injection.</li>
</ol>
7. Matrix gel plug collection
<ol>
	<li>Intraperitoneally inject 1% pentobarbital sodium in PBS (200 mg/kg body weight) and euthanize the mice with an IACUC approved procedure.</li>
	<li>Cut the skin along the marked border of the plug using surgical scissors and remove the skin above the matrix gel plug.</li>
	<li>Collect the plug and rinse in a small beaker with 1x PBS to wash away excess blood (Figure 2A). The color of plug can roughly delineate the degree of blood abundance and the content of neo-vessels (Figure 3A).</li>
</ol>
8. Embedding matrix gel plug and section preparation
<ol>
	<li>Cover the button of tissue embedding cassette with about 0.5 mL of tissue embedding gel, then immediately place matrix gel plug on the tissue embedding gel in the desired orientation as shown in Figure 2B. Then, embed the plug with additional tissue embedding gel.</li>
	<li>Put the cassettes into a -80 &#176;C freezer for 12 h to solidify.</li>
	<li>For thick section preparation, take out the solidified plug block and cut 12 &#181;m thick sections using the freezing microtome according to the instruction, and mount onto microscope slides.</li>
	<li>Slice 5-10 sections from each plug block.</li>
</ol>
9. Quantification of angiogenesis (Figure 3)
<ol>
	<li>Acquire fluorescence images from 5 independent fields of the section with an inverted fluorescence microscope at 488 nm wavelength under 10x magnification.</li>
	<li>Open the image file with Image J software (https://imagej.en.softonic.com/) and click the Angiogenesis Analyze button. Then, click Analyze HUVEC Phase Contrast button, and switch to Stat Results Table to gather information. Measure all 5 acquired fluorescence images for each section.</li>
	<li>Analyze the total length of neo-vessels from different groups to statistically evaluate the difference in angiogenesis between these groups. 
	&#8203;NOTE: Total master segments length indicates the total length of neo-vessels. The reagent that increases the length of neo-vessels is termed pro-angiogenic, while the reagent that decreases the length of neo-vessels is anti-angiogenic.</li>
</ol>
10. Quantification of vascular permeability (Figure 4)
<ol>
	<li>Acquire fluorescence images from 5 independent fields of the section with an inverted fluorescence microscope at 488 nm wavelength under 20x magnification.</li>
	<li>Open the image file with Image J software. Click the Freehand Selection button and circle the leakage area, then click on the Analyze menu and select the Measure option to get the area information of the leakage area. Use the ratio of leakage area and the area of image to indicate vascular permeability.</li>
	<li>Analyze the ratio of leakage area and area of the image from different groups to statistically evaluate the difference in vascular permeability between these groups.</li>
</ol>

Simplifying Angiogenesis Studies with a Modified Matrix Gel Plug Assay

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The authors declare no conflict of interest.

We present a reliable and convenient method for the quantitative evaluation of angiogenesis in vivo without staining. In this protocol, vascular cells were mixed with matrix gel in the presence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of Nu/Nu mice to form gel plug (Figure 1). After 7 days of gel plug formation, dextran-FITC was intravenously injected and circulated for 30 min. The gel plug was collected and embedded with tissue embedding gel, and 12 &#181;m sections were sliced for photography (Figure 2). Dextran-FITC can indicate functional vessels without staining, and the length of neo-vessels can be used to quantitatively evaluate the pro-angiogenic or anti-angiogenic function of reagents. In the example presented in this protocol, palmitate treatment reduced length of neo-vessels, while FGF1 increased length of neo-vessels (Figure 3), which indicated that palmitate is anti-angiogenic, while FGF1 is pro-angiogenic. Besides that, this protocol can also be used in the evaluation of vascular permeability (Figure 4).
Caution should be exercised on the selection of the recipient mice, handling and injecting the matrix gel, collecting gel plug, and detecting the length of the vessels. Immunodeficient mice aged 6-8 weeks are recommended, although C57BL/6 mice are also workable. Considering the variability in the plug, 3-5 plugs in each group is recommended. The number of vascular cells should be even between different groups. Matrix gel should be carefully handled according to the instructions and should not be used if solidified or containing particulate matter or numerous bubbles. In the&#160;matrix gel preparation step, the final concentration of matrix gel should not be less than 80%, and vortex is not allowed as bubbles may be formed. It should be noted that the shape of gel plug may influence the reproducibility of the results, so heating pad was used to accelerate the solidification of matrix gel plug. A 37 &#176;C animal incubator can also be used. Besides that, during gel plug embedding and section preparation, plugs and sections should be protected against bright light, and photos should be taken under a fluorescence microscope as soon as possible.
Among the various in vivo angiogenesis assays, the matrix gel plug assay is one of the most widely used methods because it is versatile, less costly, and easy to perform11. However, the staining process in ordinary matrix gel plug assay is error prone. The plug is fragile and with high water content. The dehydration and fixation steps included in the staining process may distort the gel plug, and further influence the evaluation of the length of the vessels. Besides that, the scattered vascular cells in the plug may be stained and recognized as functional vessels in ordinary matrix gel plug assay. One of the most important advantages of this modified matrix gel plug assay is that the functional vessels can be visualized without staining. FITC-labeled dextran was used to indicate vessels, and only functional vessels that have blood flow can be indicated, which contributes to the convenience and reliability of this assay.
Besides the length of neo-vessels, the permeability also affects the function of neo-vessels20. Fluorescent-labeled dextran with low molecular weight (~4,400 Da) can pass through the gap in vessels and can be used to indicate the permeability of neo-vessels. If needed, researchers can evaluate both angiogenesis and vascular permeability in the same gel plug by using both fluorescent-labeled dextran with low molecular weight and that with high molecular weight21.
Angiogenesis can be affected by the in vivo environment22, such as diabetes13,14,19, etc. This in vivo environment is hard to reproduce in these in vitro models. However, in this modified protocol, the in vivo environment can be easily reproduced by choosing proper recipient mice. Besides that, the interaction between different vascular cells also affects the process of angiogenesis. Different kinds of vascular cells, including endothelial cells, smooth muscle cells and pericytes, can be added to form neo-vessels in the plug in this protocol to investigate the contribution of different vascular cells in angiogenesis.
However, there are also some limitations for this method. The formation of neo-vessels can be affected by the shape of gel plug. Moreover, the neo-vessels formed in the gel plug is not even. There are more functional vessels at the edge of gel plug than that at the center (Figure 2A). Thus, the results may be affected by the proficiency of performer.

Angiogenesis, the process of formation of neo-vessels from pre-existing vessels, plays a critical role in many physiological and pathological processes, such as embryonic development, wound healing, atherosclerosis, tumor development, etc.1,2,3,4,5. This dynamic process involves several steps, including the degradation of the matrix, vascular cell proliferation, migration and self-organization to form tubular structures and the stabilization of the neo-vessels6. Promoting angiogenesis has been demonstrated to be critical in the treatment of myocardial infarction, stroke and other kinds of ischemic diseases7 while inhibiting angiogenesis has been considered a promising strategy in the treatment of cancers8 and rheumatoid diseases9. Angiogenesis has been considered an organizing principle for drug discovery10. Thus, the construction of a reliable and convenient method to assess the extent of angiogenesis is critical for mechanical research or drug discovery in angiogenesis-dependent diseases.
Several in vitro and in vivo models have been developed to evaluate angiogenesis11. Among these, two-dimensional (2-D) models, like matrix gel tube formation assay12, cannot form functional tubular structures. The animal models, such as the hind limb ischemia model13,14, can reproduce the angiogenesis process but are complex and require a laser speckle blood flow imaging system. 3D models of vascular morphogenesis, like matrix gel plug assay, provide a simple platform that can mimic the process of angiogenesis in vivo15, but the detection of angiogenesis requires immunohistochemistry or immunofluorescence staining16,17,18, which are variable and poorly visualized.
Here, we describe a protocol for a modified matrix gel plug assay where vascular cells were mixed with matrix gel and subcutaneously injected into the back of mice to form a plug. In the plug, vascular cells need to degrade the matrix, proliferate, migrate, and self-organize to finally form functional vessels with blood flow in the internal environment. Thereafter, fluorescent-labeled dextran is injected via the tail vein, to flow through the plug, and the label is visualized to indicate neo-vessels. The content of angiogenesis can be quantitatively evaluated by the length of the vessels. This method can form functional vessels that cannot be produced in 2-D angiogenesis models12, and does not need complex stain process as in ordinary matrix gel plug assay11. It also does not require expensive specific instruments like laser speckle blood flow imaging system in hind limb ischemia model13,14,19. This method is versatile, low-cost, quantifiable, and easy to perform, and can be used to determine the pro- or anti-angiogenic capability of drugs or be used in mechanical research involved in angiogenesis.

<table><tbody><tr><td>Adhesion Microscope Slides</td><td>CITOTEST</td><td>188105</td><td></td></tr><tr><td>Anesthesia System</td><td>RWD</td><td>R640-S1</td><td></td></tr><tr><td>Cell Counter</td><td>Invitrogen</td><td>AMQAX1000</td><td></td></tr><tr><td>Cell Culture Dish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>Cryoslicer</td><td>Thermo Fisher</td><td>CryoStar NX50</td><td></td></tr><tr><td>Dextrans-FITC-150kDa</td><td>WEIHUA BIO</td><td>WH007N07</td><td></td></tr><tr><td>Dextrans-FITC-4kDa</td><td>WEIHUA BIO</td><td>WH007N0705</td><td></td></tr><tr><td>Embedding Cassettes</td><td>CITOTEST</td><td>80203-0007</td><td></td></tr><tr><td>Endothelial Cell Medium</td><td>ScienCell</td><td>35809</td><td></td></tr><tr><td>Endothelial Growth Supplements</td><td>ScienCell</td><td>1025</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10100147C</td><td></td></tr><tr><td>Fibroblast Growth Factor 1</td><td>AtaGenix</td><td>9043p-082318-A01</td><td>FGF1</td></tr><tr><td>Fluorescence Microscope</td><td>Nikon</td><td>ECLIPSE Ni</td><td></td></tr><tr><td>Heating Pad</td><td>Boruida</td><td>30-50-30</td><td></td></tr><tr><td>Insulin Syringe</td><td>BD</td><td>300841</td><td></td></tr><tr><td>Isoflurane</td><td>RWD</td><td>R510-22-10</td><td></td></tr><tr><td>Laboratory&amp;nbsp;Balance</td><td>Sartorius</td><td>BSA124S-CW</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>356234</td><td>Matrix gel</td></tr><tr><td>Medium 199 powder</td><td>Gibco</td><td>31100-035</td><td></td></tr><tr><td>Microtubes</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>Optimal Cutting Temperature (OCT) Compound</td><td>SUKURA</td><td>4583</td><td>Tissue embedding gel</td></tr><tr><td>Palmitate Acid</td><td>KunChuang</td><td>KC001</td><td></td></tr><tr><td>Penicillin-Streptomycin Liquid</td><td>Solarbio</td><td>P1400</td><td></td></tr><tr><td>Phosphate Buffer Saline</td><td>Solarbio</td><td>P1022</td><td></td></tr><tr><td>Surgical Instruments</td><td>RWD</td><td>RWD</td><td></td></tr><tr><td>Tail Vein Injection Instrument</td><td>KEW BASIS</td><td>KW-XXY</td><td></td></tr><tr><td>Trypsin-EDTA Solution</td><td>Solarbio</td><td>T1320</td><td></td></tr><tr><td>Ultra-Low Temperature Freezer</td><td>eppendorf</td><td>U410</td><td></td></tr><tr><td>Vascular Endothelial Growth Factor</td><td>CHAMOT</td><td>CM058-5HP</td><td>VEGF</td></tr></tbody></table>

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.

Figure 1 is the flowchart depicting how to prepare the mixture of matrix gel, vascular cells, culture medium and reagent. The mixture was then subcutaneously injected into the back of Nu/Nu mice and heated using a heating pad to accelerate its coagulation to finally form gel plug.
Figure 2A is the flowchart to indicate vessels with fluorescent labeled dextran. Fluorescent labeled dextran was injected via the tail vein and circle for 30 min, so it can enter the functional vessel in the gel plug. Thereafter, the gel plugs were collected, embedded using tissue embedding gel (the orientation of matrix gel when embedded was indicated in Figure 2B). A 12 &#181;m of thick section was sliced from the plugs (5 slices from each side) and fluorescent pictures were taken for quantitative analysis of angiogenesis and/or vascular permeability.
Figure 3 is the influence of anti-angiogenic reagent palmitate and pro-angiogenic reagent fibroblast growth factor 1 (FGF1) on angiogenesis in the gel plug. Figure 3A is the appearance of gel plugs with vehicle, palmitate or FGF1. Blood can enter the functional neo-vessel in the gel plug, which makes the plug red to varying degrees. Figure 3B is the fluorescent image of matrix gel plugs, in which the functional vessels were visualized by dextran-FITC with high molecular weight. Figure 3C is the quantitative result of vessel length of different group. Palmitate can significantly decrease the length of neo-vessels, while FGF1 treatment can obviously increase the length.
Figure 4 compares the vascular permeability in gel plugs with or without vascular endothelial growth factor (VEGF) treatment. Figure 4A is the fluorescent image of matrix gel plugs, in which the leakage area is visualized by dextran-FITC with low molecular weight and indicated by arrows. Figure 4B is the quantitative result of leakage area. The increased leakage area in VEGF treatment group revealed that VEGF can increase vascular permeability.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65567/65567fig01.jpg" /> 
Figure 1. Flowchart showing formation of a gel plug in mice. Vascular cells cultured in monolayer culture are dissociated, pelleted, resuspended with endothelial cell medium (ECM), and counted (I-IV). After pelleting and resuspension in matrix gel mixture (containing 8.8 volume matrix gel, 1 volume 10x M199 supplemented with 10% FBS and 0.2 volume reagent), 300 &#181;L of gel mixture was subcutaneously injected into the back of the mice (V-VII) to form plugs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65567/65567fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65567/65567fig02.jpg" /> 
Figure 2. Flowchart showing dextran-FITC injection and quantification of angiogenesis. (A)&#160;Dextran-FITC was injected via the tail vein (I). After 30 min, gel plug was acquired, embedded and sections were sliced using freezing microtome (II, III). Thereafter, fluorescence images were taken using fluorescence microscope (IV) and angiogenesis was quantitatively analyzed using Image J software (V). (B) The diagram of matrix gel plug orientation when embedded in tissue embedding cassette. Abbreviations: OCT = optimum cutting temperature compound. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65567/65567fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65567/65567fig03.jpg" /> 
Figure 3. Evaluating angiogenesis using modified matrix gel plug assay. (A) Appearance of representative matrix gel plugs. (B)&#160;The fluorescent picture of functional vessels in matrix gel plugs indicated by Dextran-FITC. (C)&#160;The quantitative analysis of the length of functional vessels in matrix gel plugs using one-way ANOVA. *p&#60;0.05 versus vehicle; ***p&#60;0.001 versus vehicle. The scale bar is 100 &#956;m. Abbreviations: FGF1 = Fibroblast growth factor 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65567/65567fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65567/65567fig04.jpg" /> 
Figure 4. Evaluating vascular permeability using modified matrix gel plug assay. (A) The representative fluorescence image of vessels in gel plug, the arrows indicate leakage site. (B) The quantitative analysis of leakage area to evaluate vascular permeability using Student&#39;s t-test. ***p&#60;0.001. The scale bar is 100 &#956;m. Abbreviations: VEGF = vascular endothelial growth factor. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65567/65567fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies at JoVE.com

Author Spotlight: Investigating Angiogenesis and Vessel Permeability Through a Modified Matrix Gel Plug Assay

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.

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 ...

modified-in-vivo-matrix-gel-plug-assay-for-angiogenesis-studies

Nanyang Technological University

Research

JoVE Journal

Biology

834 Views.  Pingyang Affiliated Hospital of Wenzhou Medical University. Ons onderzoek richt zich op angiogenese, het proces van vorming van nieuwe vaten uit reeds bestaande vaten. Het speelt een cruciale rol in veel fysiologische en pathologische processen, zoals embryonale ontwikkeling of genezing van atherosclerose, tumorontwikkeling, enz. De constructie van een betrouwbaar model om de mate van angiogenese te evalueren is van cruciaal belang voor myocardonderzoek of de ontdekking van geneesmiddelen voor angiogenese-afhankelijke ziekten.De meeste bestaand...

Video: Auteur in de schijnwerpers: onderzoek naar angiogenese en vatpermeabiliteit door middel van een gemodificeerde matrix-gelplugtest

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

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.

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

Neuroscience

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Bioengineering

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.

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 ...

Medicine

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner.

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

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;

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 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.

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

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

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