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.

Ethics Statement: Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee at Stanford University School of Medicine.
NOTE: Animals are under anesthetization with 3% vaporizer isoflurane and 3% supply of O2 gas. Use of vet ointment on eyes may help to prevent dryness while under anesthesia.
1. Cell Preparation
<ol>
	<li>Grow human endothelial and pericyte cell cultures in 100 mm plates with 10 ml of the appropriate media. Replace 10 ml of fresh medium every 2 - 3 days until cells reach 80% confluency.
	<ol>
		<li>Culture endothelial cells (ECs) in complete endothelial cell media (ECM) supplemented with company supplied 5% fetal bovine serum (FBS), 10% penicillin/streptomycin and 10% endothelial cell growth supplement.</li>
		<li>Culture pericytes in complete pericyte media (PM) supplemented with company supplied 2% FBS, 10% penicillin/streptomycin and 10% pericyte growth supplement.</li>
	</ol>
	</li>
	<li>Prepare cell mixtures
	<ol>
		<li>Calculate the required number of cell for the experimental and control groups. 
		NOTE: Each plug in the experimental group contains one million (1 x 106) human ECs and two hundred thousand (2 x 105) pericytes. For the control group, each plug contains 1.2 million human ECs only. The negative control group has matrix gel only. Each group should include at least four plugs, which would require two mice as plugs can be injected into each side of a mouse&#39;s lower back. Three plugs would serve as triplicate for the experiment and the fourth could be used in case one fails. Prepare 25% extra cells to match the extra volume of gel.</li>
		<li>Collect and count cells from culture plates.
		<ol>
			<li>To detach cells, remove media and wash once with room temperature 1x phosphate buffered saline (PBS). Remove PBS, add 1 ml 0.25% Trypsin/2.21 mM EDTA, and incubate for 1 min. Wash cells off the plate by adding 2 ml medium and collect in a 15 ml tube.</li>
			<li>Count cell using a hemocytometer according to the manufacturer&#39;s instructions.</li>
			<li>Centrifuge the appropriate number of cells into a pellet at 400 x g for 5 min. For the experimental group, centrifuge both ECs and pericytes down together into one pellet.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Matrix Gel Preparation
<ol>
	<li>Thaw out matrix gel completely at 4 &#176;C for 4 hr or overnight to obtain a homogenous solution. No mixing is required.</li>
	<li>Aliquot matrix gel in 1.5 ml tubes and store at 4 &#176;C.</li>
	<li>Pre-chill sterile 1.5 ml tubes and 28 G 1 cc insulin syringes at 4 &#176;C. Caution: Keep matrix gel on ice at all times.</li>
	<li>Mix cold matrix gel with bFGF at a volume ratio of 1:100 (final concentration of bFGF= 0.5 &#956;g/ml; bFGF stock solution= 50 &#956;g/ml) After mixing matrix gel solution by pipetting up and down several times, incubate on ice for 30 - 60 min before mixing with the cells in step 3.1. 
	NOTE: Thaw the stock bFGF before mixing. Calculate the number of plugs needed for injection. For example, each plug requires 200 &#956;l matrix gel. Therefore, prepare 25% extra gel.</li>
</ol>
3. Mix Matrix Gel with Cells
<ol>
	<li>For each group, resuspend the cell pellet (containing appropriate cell number) with the calculated volume of matrix gel containing bFGF. Mix gently to avoid foaming and leave in the 15 ml tube on ice until mice are prepared for injection.</li>
</ol>
4. Mouse Preparation
<ol>
	<li>Anesthetize 1&#160;SCID mice for 5 min in 3% Isoflurane plus 3% O2 in an induction chamber. Focus on one experimental group, or 2 mice, at a time.</li>
	<li>Remove the&#160;mouse from the induction chamber, place on a heating pad ventral side down and quickly attach a snout nozzle to the mouse&#39;s face via a non-rebreathing system to supply anesthesia throughout the injection procedure. Switch the gas flow from the induction chamber to the non-rebreathing system and lower the gas pressure to 1.5% Isoflurane and 1.5% O2.</li>
	<li>Remove hair from both lateral hind regions (around 1 cm diameter) with a shaver or hair removal cream.</li>
	<li>Wipe the skin area with a 70% alcohol pad.</li>
	<li>Return the mouse to the induction chamber and repeat steps 4.2 - 4.4 with the second mouse.</li>
</ol>
5. Matrix Gel Injection
<ol>
	<li>Prepare one mouse for injection by attaching to the non-rebreathing system and placing on the heating pad ventral side down.</li>
	<li>Load the matrix gel into a pre-chilled 28 G 1 cc insulin syringe one mouse&#160;at a time.
	<ol>
		<li>Load the complete 500 &#956;l&#160;volume of matrix gel for two&#160;plugs (200 &#956;l per plug plus 25% extra) into the syringe.</li>
		<li>Make sure to inject the matrix gel into the mice within 3 min of loading the cells into the syringe to prevent the cells from settling to the side of the syringe and to prevent matrix gel from solidifying inside of the syringe.</li>
	</ol>
	</li>
	<li>Lift the back skin to locate subcutaneous space, then inject 200 &#956;l of the matrix gel slowly and evenly into the subcutaneous space of the back posterior of the rib cage.</li>
	<li>Remove the injection needle slowly, being careful to prevent matrix gel from leaking out of the injection site. A bump will form at the site of injection. Wipe injection site with an alcohol pad.</li>
	<li>Repeat injection steps 5.2 - 5.5 on the other side of mouse&#39;s back, then leave the mouse on its back for 1 min to allow gelation of the matrix gel on the heat pad.</li>
	<li>Outline both bumps using a permanent marker to identify the bump after 14 days. After some hair grows back, it will be easier to find the bump.</li>
</ol>
6. Matrix Gel Plug Isolation: 14 Days after Injection
<ol>
	<li>Euthanize the mice humanely by exposing the animals to &#62;5 min of carbon dioxide inhalation followed by cervical dislocation to assure death.</li>
	<li>Remove the matrix gel plug from the mouse&#39;s back.
	<ol>
		<li>Gently remove regrown hair around injection site where the plug is located by repeating step 4.3. The marker line indicates the size of original bump under the skin.</li>
		<li>Excise the plug along with the surrounding skin and muscle layers attached on the top and bottom sides of the plug, respectively.
		<ol>
			<li>Using fine surgical scissors, make an incision on the marked border of the plug that is perpendicular to the skin and cut through to the muscle underneath the plug. Then, carefully cut around the periphery of the plug along the marked border.</li>
			<li>Remove the plug, keeping it sandwiched between the skin and muscle layers. Immediately rinse in a small beaker with 1x PBS to wash away excess blood. The plug is now ready to be fixed (next step, 6.3).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Fix the plug in 4% paraformaldehyde.
	<ol>
		<li>Place the entire plug, including the skin and muscle layers, in a 50 ml tube with 25 ml of 4% paraformaldehyde for 24 hr at room temperature without rocking. The next day, transfer the plug into 25 ml of 70% ethanol for another 24 hr before paraffin embedding. 
		NOTE: Handle paraformaldehyde with caution. Wear gloves.</li>
	</ol>
	</li>
</ol>
7. Paraffin Process the Matrix Gel Plug
<ol>
	<li>Prepare the plug for paraffin embedding.
	<ol>
		<li>Orient the plug as shown in Figure 1a; place the plug in a tissue cassette so that the side of the plug is facing up and both the skin and muscle layers are visible across the surface of the tissue block that will be cut first during sectioning.</li>
	</ol>
	</li>
	<li>Paraffin embed the plug according to standard paraffin embedding protocols13.</li>
	<li>Cut 8 - 10 &#956;m thick sections off of the tissue block and mount onto microscope slides according to standard paraffin sectioning protocols13.</li>
	<li>Store the slides in a cool dry place at room temperature until ready for staining.</li>
</ol>
8. Stain Sections with Hematoxylin and Eosin Stain (H&#38;E)13
<ol>
	<li>De-paraffinize the tissue sections by passing the slides through 100% xylene twice, 100% ethanol, 95% ethanol, 70% ethanol and lastly 1x PBS, each for 5 min at room temperature. Leave slides in 1x PBS until the next step.</li>
	<li>Pipette 100 &#956;l H&#38;E solution onto the section and incubate for 1 - 5 min, or until there is clear contrast between blue nuclei and pink cytoplasm in a cell as viewed under an upright light microscope at 10X magnification. Be careful not to let any H&#38;E solution touch the objective.</li>
	<li>Wash H&#38;E solution off of the slide by dunking the slide in a large beaker of tap water, then place the slide in a rack in the beaker and flush with running water for 2 min.</li>
	<li>Move to step 9.8 to mount the slides.</li>
</ol>
9. Stain Other Slides for Human Capillaries and Pericytes
<ol>
	<li>De-paraffinize the tissue sections by passing the slides through 100% xylene twice, 100% ethanol, 95% ethanol, 70% ethanol and lastly 1x PBS, each for 5 min at room temperature. Leave slides in 1x PBS until the next step.</li>
	<li>Boil the sections in antigen retrieval solution.
	<ol>
		<li>In a 2 L beaker, prepare boiling antigen retrieval solution using 1x citrate buffer (pH 7.0) at 95 &#176;C on a heating block.</li>
		<li>Once boiling, place the slides in a metal rack and submerge in the boiling antigen retrieval solution for 20 min.</li>
		<li>Carefully remove the beaker from the heating block with the slides still submerged and allow the solution to slowly return to room temperature for about 30 min.</li>
		<li>Place the slides in 1x PBS until next step.</li>
	</ol>
	</li>
	<li>Block the sections in blocking buffer (1x PBS, 5% Goat Serum, 0.3% Triton X-100).
	<ol>
		<li>Draw a hydrophobic circle around the edges of each section with a PAP pen.</li>
		<li>Place the slides in a humidity tray with distilled water at the bottom of the tray.</li>
		<li>Pipette 100 &#956;l blocking buffer onto the sections making sure all of the tissue is covered by fluid (may require more than 100 &#956;l).</li>
		<li>Incubate sections with blocking buffer for 1 hr.</li>
	</ol>
	</li>
	<li>Incubate sections with primary antibody.
	<ol>
		<li>Prepare one solution with two primary antibodies to probe each section for CD31 (ECs) and smooth muscle actin (pericytes) diluted in dilution buffer (1x PBS, 1%BSA, 0.3% Triton X-100). 
		NOTE: Here, anti-CD31 concentration is 1:50 and anti-smooth muscle actin concentration is 1:300.</li>
		<li>Incubate the sections overnight at 4 &#176;C with 100 &#956;l primary antibodies in a humidity tray containing distilled water.</li>
	</ol>
	</li>
	<li>Wash the slides three times in 1x PBST (1x PBS + 0.1% Tween20) for 5 min each wash.</li>
	<li>Incubate sections with secondary antibody.
	<ol>
		<li>Prepare a solution with a fluorophore conjugated goat anti-rabbit secondary antibody to recognize the anti-CD31 rabbit antibody diluted in dilution buffer.
		<ol>
			<li>NOTE: The smooth muscle actin primary antibody is already conjugated with a CY3 fluorophore, so no secondary antibody staining is needed. Goat-anti rabbit secondary antibody concentration is 1:250.</li>
		</ol>
		</li>
		<li>Incubate the sections for 1 hr with 100 &#956;l secondary antibody in the humidity tray containing distilled water.</li>
	</ol>
	</li>
	<li>Wash the slides three times in 1x PBST for 5 min each wash.</li>
	<li>Mount the slides.
	<ol>
		<li>Pipette 2 - 3 drops (~ 40 - 60 &#956;l) of antifade mounting solution with DAPI nuclear staining onto each section.</li>
		<li>Gently place a no. 1.5 cover slip over the sections and mounting solution, allowing the fluid to spread out over the sections and to the edges of the cover slip. Gently use a finger to press out air bubbles that may be located over the section.</li>
		<li>Let the slides dry for at least 1 hr before handling.</li>
	</ol>
	</li>
</ol>
10. Quantify Capillary Density and Structure14
<ol>
	<li>Count the number of capillaries in H&#38;E stained sections, expressed as #/mm2.
	<ol>
		<li>Acquire photos of four different fields on an upright light microscope under 40X magnification. 
		NOTE: Capillaries are identified as tubes filled with red blood cells.</li>
		<li>Alternatively, instead of manually counting capillaries, use image analysis software (e.g., Wimasis Image Analysis) to quantify various angiogenesis parameters such as vessel number, average vessel length and diameter, and branch points.</li>
	</ol>
	</li>
	<li>Analyze capillary structure in immunofluorescent stained sections.
	<ol>
		<li>Acquire photos of four different fields on an inverted fluorescent microscope with FITC and TxRed filter cube.
		<ol>
			<li>Use a FITC cube to image ECs by excitation at 488 nm wavelength and use a TxRed cube to image pericytes by excitation at 594 nm wavelength.
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			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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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. 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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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No competing financial interests enclosed.

The matrix gel plug assay has proven to be a convenient and powerful method to evaluate gene regulation in angiogenesis, angiogenic and antiangiogenic compounds in vivo, and to supplement in vitro tests. Here, we describe in detail a novel matrix gel plug assay of human angiogenesis that investigates the interaction between endothelial cells and pericytes during vessel formation.
There are a few novel and critical steps in this protocol. Cells in low passage (passage 1 - 4) are preferable because young cells will be more viable during the 14-day experimental setting. The number of endothelial cells used is minimum one million and the ratio of EC to pericyte is 5:1; however, increased pericyte cell number will enhance its coverage on capillaries. For the experimental group of endothelial cells alone, the total number of cells injected should be the sum of endothelial cells and pericytes, which is 1.2 million, therefore, the total cell number per plug is consistent. It is important to calculate the appropriate amounts of cell numbers and matrix gel volume. The basic formula is 1.2 million cells in 200 &#956;l of matrix gel. In addition, before injection, keep pipet tips, syringes, matrix gel, and cell pellets on ice at all times. Since matrix gel is viscous and in order to avoid air bubbles forming during cell suspension, cut about 1 cm off of the tip of 1 ml pipet tips to allow for better flow. Do not mix matrix gel with cells until mice are ready to be injected. Each plug injection should take less than one minute. One mouse can carry two plugs, one on each side of its back. When extracting the plugs make sure to keep the plug sandwiched between the muscle and skin layers, then the matrix gel plug will be well collected after isolation.
The drawbacks of the matrix gel plug assay are that it is time consuming, costly, and involves tedious and delicate steps such as injection and plug isolation. If these steps were to fail, the results would be ruined and the process would have to be repeated again with new mice and materials.
However, the data is reproducible and provides flexibility in terms of experimental design. For example, growth factors or inhibitors could be administered to the plug at different stages of vascular development, which can be used for drug validation. Furthermore, incorporation of other cell types, such as growth-arrested cells, transfected cell lines or tumor cell lines, into the matrix gel plug is another possibility for manipulating the endothelial cell phenotype during angiogenesis. Our protocol utilizes this flexibility and introduces human-derived pericytes along with ECs to allow formation of fully functional, pericyte covered, hybrid vessels in vivo. Our protocol also describes how to analyze the histological sections from these plugs for critical angiogenesis endpoints, including tube number and tube length, with these two cell types present.

Angiogenesis is the process by which new blood vessels are formed from a pre-existing vascular network1 and is the focus of ongoing research across many areas ranging from normal development to disease. This dynamic process involves the proliferation and migration of endothelial cells (ECs) and recruitment of pericytes to construct a vascular tube that is directed toward the site that needs oxygen and nutrient delivery2. To study this process requires an equally dynamic assay, most importantly one that can recapitulate the three-dimensional nature of tube formation. In vitro 3D matrix assays have been developed to address this need and have worked well to allow researchers to define the discrete steps in space and time in which angiogenesis takes place3,4,5,6. However, these in vitro 3D matrix models are limited to studying non-perfused vessels and therefore lack critical components pertinent to the angiogenesis process (e.g., circulating growth and inhibitory factors, unnatural tension/forces across the vascular bed) and fail to simulate the complex environment present in live tissue. To address this limitation, several in vivo angiogenesis assays have been developed7, including the matrix gel plug assay which will be the focus of our report8,9.
The matrix gel plug assay is a well-established in vivo angiogenesis assay that appeals to researchers as it provides a robust platform to test the roles of different cells and substances in angiogenesis. Matrix gel is a commercially available basement membrane solution that is secreted by the Engelbreth-Holm-Swarm (EHS) mouse sarcoma cell line that solidifies into a gel-like material at 37 &#176;C. The matrix gel can be mixed with cells and/or substances, such as growth factors, and injected subcutaneously into the mouse. The host ECs will invade the plug over 14 days, form a vascular network, and become perfused with the host&#39;s blood. To date, matrix gel plug assays have focused exclusively on the study of endothelial cell behavior during angiogenesis, however, to the best of our knowledge no effort has yet been made to determine whether this assay can be used to co-culture endothelial cells and pericytes to study how these two cell types interact during angiogenesis. Specifically, understanding the relationship between ECs and pericytes is valuable for studying diseases where blood vessel loss is pathologic, including microvascular ischemia and peripheral vascular disease10,11,12.
Here, we describe a protocol that introduces human-derived pericytes to the matrix gel mixture along with human ECs and fibroblast growth factor (bFGF). This mixture can then be injected subcutaneously in the dorsum of SCID mice to allow formation of fully functional, pericyte coated, hybrid vessels. Our protocol describes how to prepare matrix gel plugs containing human ECs either with or without human pericytes, placement into SCID mice and how to analyze the histological sections for critical angiogenesis endpoints.

<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.

Representative H&#38;E and immunofluorescent staining of matrix gel plug sections are shown in Figure 2. Sections from EC only plugs display some vessels that are mostly not perfused with blood (Figure 2 top left, black arrows) whereas plugs containing both ECs and pericytes display several perfused vessels with larger diameters and complete pericyte coverage, as evidenced by positive SMA staining immediately adjacent to CD31-positive ECs. These results suggest that pericytes play a substantial role in nascent blood vessel formation and that this dynamic process can be recapitulated and easily analyzed in an in vivo model.
<img alt="Figure 1" src="/files/ftp_upload/54617/54617fig1.jpg" /> 
Figure 1. Tissue Orientation when Embedded in a Tissue Cassette. After the matrix gel plug (yellow) is isolated, it is in between the mouse&#39;s muscle (purple) and skin (green) layers. The plug is placed in the tissue cassette as shown so that all three layers can be seen across the surface that will be cut first during sectioning. A) View from the top of the cassette, B) View from the side of the cassette. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54617/54617fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54617/54617fig2.jpg" /> 
Figure 2. Representative H &#38; E (top row) and Immunofluorescence (bottom row) of Matrix Gel. Images show the appearance of ECs alone (left images) and ECs plus healthy pericytes (Pc) (right images). Human and murine ECs are labeled in green (bottom images) and pericytes (&#945; smooth muscle actin (&#945;SMA)) are labeled in red (bottom right), and nuclei are stained blue with DAPI stain. Scale is 25 &#181;m. This figure has been modified from a previous publication8. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54617/54617fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biology

9.6K Views.  Stanford University.  We presenteren een protocol voor humane endotheliale pericyte-interacties in muizen te bestuderen onder toepassing van een variatie van de matrix gel plug angiogenese assay. 

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.

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.

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.

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.

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.

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.

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.

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;

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.

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

Simplifying Angiogenesis Studies with a Modified Matrix Gel Plug Assay

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.

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.

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

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

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