We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.
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.
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 °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'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.
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
2. Matrix Gel Preparation
3. Mix Matrix Gel with Cells
4. Mouse Preparation
5. Matrix Gel Injection
6. Matrix Gel Plug Isolation: 14 Days after Injection
7. Paraffin Process the Matrix Gel Plug
8. Stain Sections with Hematoxylin and Eosin Stain (H&E)13
9. Stain Other Slides for Human Capillaries and Pericytes
10. Quantify Capillary Density and Structure14
Representative H&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.
Figure 1. Tissue Orientation when Embedded in a Tissue Cassette. After the matrix gel plug (yellow) is isolated, it is in between the mouse'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. Please click here to view a larger version of this figure.
Figure 2. Representative H & 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 (α smooth muscle actin (αSMA)) are labeled in red (bottom right), and nuclei are stained blue with DAPI stain. Scale is 25 µm. This figure has been modified from a previous publication8. Please click here to view a larger version of this figure.
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 μl 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.
The authors have nothing to disclose.
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.
PBS (Phosphate buffered saline) | Corning | 21-031-CV | |
0.25% Trypsin/0.53mM EDTA | Corning | 25-053-CI | |
Endothelial Cell Media (ECM) kit | Sciencell | 1001 | includes media, EC growth supplement, and penicillin/streptomycin, each supplied at the appropriate volume for easy mixing |
Pericyte Media (PM) kit | Sciencell | 1201 | includes media, Pericyte growth supplement, and penicillin/streptomycin, each supplied at the appropriate volume for easy mixing |
primary human pulmonary microvascular endothelial cells | Pulmonary Hypertension Breakthrough Initiative | this cell type is also available commercially. Cells used at passage 1-4 | |
primary human pulmonary pericytes | Pulmonary Hypertension Breakthrough Initiative | this cell type is not available commercially, but brain paricytes are. Cells used at passage 1-4 | |
bFGF (basic Fibroblast Growth Factor) | Peprotech | 100-18B | stock solution is 50ug/ml in 0.1% BSA in PBS, aliquots at 50 uL and stored at -20 degrees |
Matrigel Basement Membrane Matrix | BD | 356237 | |
28G 1cc Insluin Syringe | BD | 329410 | |
SCID (Severe Combined Immune Deficiency) mice | The Jackson Laboratory | 5557 | NOD.SCID IL2R gamma knockout strain is the best strain; 4-6 weeks of age |
1.5mL microcentrifuge tubes, sterile | Thermo Fisher Scientific | 05-408-129 | |
15 mL screw top tubes, sterile | BD Biosciences | 352096 | |
PAP pen | Life Technologies | 8899 | |
hemocytometer | ThermoFisher Scientific | 02-671-6 | |
Nair hair removal cream | Walmart | ||
anti-human CD31 primary antibody | LifeSpan Biosciences | LS-B4737 | working solution is 1:50 |
anti-human Smooth Muscle actin CY3 primary fluorescent antibody | Sigma | C6198 | working solution is 1:300 |
goat anti-rabbit secondary antibody; 488/green | ThermoFisher Scientific | A-11008 | working solution is 1:250 |
Prolong Gold DAPI solution | Cell Signaling | 8961S | |
microscope slides | VWR | 48300-047 | |
no. 1.5 cover slips | Thermo Fisher Scientific | 12-544-D | |
citrate buffer 10x | Millipore | 21545 | |
extra fine surgical scissors | Fine Science Tools | 14084-08 | |
Formalin (paraformaldehyde) | Thermo Fisher Scientific | 245-685 | |
Tissue cassettes | Simport | M492-12 | |
goat serum | Dako | X0907 |