Assays to Examine Endothelial Cell Migration, Tube Formation, and Gene Expression Profiles

Abstract

Common methods for studying angiogenesis in vitro include the tube formation assay, the migration assay, and the study of the endothelial genome. The formation of capillary-like tubes in vitro on basement membrane matrix mimics many steps of the angiogenesis process in vivo and is used widely as a screening test for angiogenic or antiangiogenic factors. Other assays related to the study of angiogenesis include the cell migration assay, the study of gene expression changes during the process of angiogenesis, and the study of endothelial-derived microparticles. Protocols for these procedures will be described here.

1 Introduction

The study of a complicated biological process such as angiogenesis requires the use of appropriate in vitro models to facilitate an understanding of the signaling pathways in angiogenesis. About 30 years ago, Montesano and Orci reported that bovine microvascular endothelial cells formed tubelike structures in a 3D collagen-I gel and that this process may serve as an in vitro angiogenesis model [1]. This model was expanded by George E. Davis to represent vasculogenesis or angiogenesis [2]; however, the assay required a period of several days, and was limited by the fact that not all the cells formed tubes, making it difficult to perform any type of quantification. In 1988 Kubota et al. showed that the use of a reconstituted basement membrane matrix enabled endothelial cells to attach and align rapidly and to form capillary-like tubes within hours, representing many of the steps of angiogenesis in vivo. These capillary-like tubes had characteristics of endothelial cells in vivo, including the ability to form tight cell-cell contacts and to take up acetylated low-density lipoprotein [3]. The basement membrane matrix tube formation assay has been shown to be an easy, rapid, and reliable assay and is widely used to study the signaling pathways and mechanisms of angiogenesis, along with migration assays and proliferation assays [4].

Additional important assays for the study of endothelial cells include the analyses of the endothelial genome as well as the study of endothelial vesicles. A large amount of vesicles are found to originate from eukaryotic cells and are present in almost all body fluids [5]. These vesicles are called exosomes, microparticles, or microvesicles, according to their origins and characteristics [6]. These vesicular structures are pivotal in cellular communication both under physiological and pathological conditions [7 , 8]. For instance, extracellular vesicles derived from platelets are reported to promote angiogenesis [9 , 10]. Likewise, endothelial-derived microparticles containing proteins or mRNA of the matrix metalloproteinase family were also reported to contribute to angiogenesis [11 , 12].

In this chapter we describe the Matrigel tube formation assay, the scratch migration assay [13], the transcriptome analysis of brain microvascular endothelial cells purified by PECAM-1 antibody [14], and the preparation of extracellular vesicles, including microparticles and microvesicles.

2 Materials

2.1 Growth Factor Reduced (GFR) Matrigel Matrix (BD Bioscience, San Jose, CA)

Matrigel is the most commonly used commercial basement membrane. A solubilized matrix extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, it contains laminin, collagen IV, entactin, and heparin sulfate proteoglycan (perlecan). GFR BD Matrigel matrix contains reduced concentrations of growth factors compared to standard Matrigel matrix, resulting in a lower basal level of tube formation, which is advantageous for testing factors that increase angiogenesis.

2.2 Rat Brain Endothelial Cells

1. The rat brain microvascular endothelial cell line, RBE4.

2. Endothelial growth medium (EGM), as endothelial basal medium (EBM-2) with supplements (catalogue # CC-4147, Lonza, Cambridge, MA), which consist of 5 mL fetal bovine serum, hEGF, hydrocortisone, GA-1000 (gentamicin, amphotericin-B), VEGF, hFGF-B, R3-IGF-1, and ascorbic acid. The working medium is EBM-2 with 100 units/mL penicillin-streptomycin without serum and any supplements.

3. RBE4 cells were plated on collagen-I-coated flasks or plates.

2.3 Cell Detachment Solution

0.05 % trypsin-EDTA solution, stored at 4 °C.

2.4 Fixation Solution

4 % paraformaldehyde (PFA).

2.5 Animals for Brain Endothelial Cell Preparation

CD57 mice (6–8 weeks old) were used. For each preparation pool the cortexes from 3 mice.

2.6 Reagents for Endothelial Cell Purification

1. HBSS without calcium/magnesium, referred to as HBSS in this chapter.

2. Collagenase-A dissolved in H₂O and stored as 200 mg/mL stock in −20 °C. The working solution is 2 mg/mL diluted in HBSS with calcium/magnesium and warmed to 37 °C for use.

3. Rat antibody anti-mouse PECAM-1 (clone MEC13.3, Pharmingen, Franklin Lakes, NJ).

4. Dynabeads M-450 with sheep anti-rat IgG (Dynal Biotech).

5. Magnetic separator (Dynal Biotech).

2.7 Reagents for Total RNA Preparation and Measurement

1. 14.3 M β-mercaptoethanol (β-ME).

2. Ethanol (70, 80, and 96–100 %), molecular reagent grade.

3. RNeasy Plus Micro Kit (Qiagen).

4. RNA 6000 Pico kits including reagents and chips (Agilent Technologies, Santa Clara, CA).

5. Ovation Pico WTA system (NuGEN, San Carlos, CA).

2.8 Exosome-Free Fetal Bovine Serum (FBS)

The FBS used for conditioned culture medium to prepare extracellular vesicles is prepared by ultracentrifugation at 100,000 × g for 16 h prior to use, to remove bovine exosomes.

3 Methods

3.1 Matrigel Tube Formation

1. Thaw the Matrigel on ice or in cold room completely and slowly (see Notes 1 , 2). Keep it on ice until use.

2. Keep the multiwell plate on ice. Use precooled tips to add 100 μL soluble matrix into wells of 48-well plate, making sure that it covers the well evenly, with no bubbles in the gel mixture.

3. Incubate the plates with Matrigel at 37 °C for 30–60 min. This process causes the matrix to polymerize into insoluble gels. The Matrigel is now ready for use.

4. Detach RBE4 cells with detachment solution when they reach 80–90 % confluence. Collect and suspend the cells in EBM-2 with 0.5 % FBS.

5. Count the cells with a hemocytometer, diluting the cells in EBM-2 with 0.5 % FBS to 2.5 × 10⁴ per cells/mL (see Note 3).

6. Seed the diluted RBE4 cells on to the surface of Matrigel-coated wells at a density of 5 × 10³ cells/well (see Note 4).

7. Incubate the plate in the CO₂ incubator for 6–18 h, with temperature at 37 °C (see Note 5).

8. Examine the cells using a phase-contrast microscope, and take 2–3 images for each well using the light microscope (see Note 6).

9. Count the number of complete rings in each well. The number of rings is a measure of the capability to form tubes and a reflection of the angiogenic ability (see Note 7).

3.2 Scratch Migration Assay

1. Use RBE4 cells when confluent, wash twice with PBS, then add 3 mL cell detachment solution, and incubate at 37 °C for 5 min.

2. Add fresh 3 mL EGM, gently pipette to resuspend the cells, and centrifuge the suspension to get a cell pellet.

3. Resuspend the cell pellet with EGM, and plate 2 × 10⁵ cells per well in a 6-well plate pre-coated with collagen-I or 5 × 10⁴ cells per well in a 24-well plate.

4. Return the plates to the CO₂ incubator at 37 °C. Change to fresh EGM every 2–3 days until the cells are almost confluent (see Note 8).

5. Change to working medium, and incubate overnight for serum starvation (see Note 9).

6. Scrape the cell monolayer in a straight line with a p200 or p1000 pipette tip (see Note 10). Remove the debris by washing twice with warm working media, and then replace with fresh working medium.

7. To enable imaging the area with scratch injury, mark the location of the scratch with reference points on the outer bottom of the culture plate using a razor blade or an ultrafine tip marker.

8. Return the cells back to the CO₂ incubator for 8–24 h (see Note 11).

9. After the incubation, wash the cells twice with PBS gently to remove detached or dead cells, and fix cells with 4 % PFA for 10 min at 4 °C (see Note 12).

10. Place the culture plate under a phase-contrast microscope, locate the scratch injury site using the reference point, and acquire images from at least 4 random areas (see Note 13).

11. Quantify the number of migrated cells by counting cells that cross into the scratched area from their reference points (see Note 14).

3.3 The Transcriptome of Mouse Brain Microvascular Endothelial Cells (See Note 15)

3.3.1 Preparation of Anti-Mouse PECAM-1 Dynabeads

1. Take 12 μL of bead slurry; mix well in a 15 mL tube with 0.1 % BSA in PBS (see Note 16).

2. Place the tube in a magnetic separator for 1–2 min, then remove the supernatant.

3. Repeat washing as above for 3 times.

4. Resuspend the beads in 100 μL of 0.1 % BSA in PBS, then add 1 mL HBSS.

5. Add the rat anti-mouse PECAM-1 into the beads solution, at the ratio of 1 μg antibody for every 40 μL of beads. Incubate overnight on a rotator at 4 °C (or 2 h at RT) for antibody linkage.

6. Wash the beads/antibody mix with 2 mL 0.1 % BSA in PBS, for 3 times.

7. Resuspend the beads in original volume of 12 μL 0.1 % BSA to maintain beads at 4 × 10⁸ beads/mL. Store the beads at 4 °C and they are ready for the purification. Use within 1–2 weeks.

3.3.2 Tissue Dissection and Dissociation

1. Sacrifice the mice by intracardiac perfusion with PBS, and wash the body with a generous amount of 70 % ethanol.

2. Harvest the brains in HBSS on ice; dissect the brains to remove the cerebral cortex.

3. Roll the cortical tissue on 3MM filter paper (autoclaved) to remove the big vessels.

4. Transfer the tissue to a new dish with a small amount of HBSS and finely mince the tissue with scissors.

5. Transfer the minced tissue to a 50 mL tube with warm collagenase-A solution (see Note 17).

6. Incubate the tissue in collagenase solution at 37 °C with gentle shaking for 20 min.

7. Using 14 gauge catheters attached to a syringe, triturate the enzyme suspension until there are no obvious big pieces.

8. Filter the enzyme suspension through a 70 μm cell strainer into a new 50 mL tube.

9. Centrifuge the cell suspension at 4 °C, 400 × g for 8 min.

10. Resuspend the pellet with 10 mL HBSS, centrifuge again for washing.

3.3.3 Purification of Endothelial Cells

1. Resuspend the pellet with 4 mL HBSS, and transfer into a new 15 mL tube.

2. Add anti-PECAM-1-coated Dynabeads, and mix by carefully rotating the tube up and down.

3. Incubate at room temperature for 10 min, or at 4 °C for 30 min, with gentle rotation for binding of endothelial cells to anti-PECAM-coated Dynabeads.

4. Mount the tube on a magnetic separator and wait for 1–2 min; then remove the supernatant (see Note 18).

5. Remove the tube from the magnetic separator and resuspend the Dynabeads with 4 mL HBSS by vigorous trituration (see Note 19).

6. Repeat steps 4, 5 for 3 times, then remove the supernatant.

7. After the last wash, directly add 400 μL Buffer RLT plus (with 1 % β-ME) for RNA preparation or freeze the Dynabead-bound cells in −80 °C for later preparation.

3.3.4 Preparation of RNA from Purified Endothelial Cells

Following the protocol suggested by the manufacturer:

1. Transfer the lysate of endothelial cells with Dynabeads to a 1.5 mL Eppendorf tube.

2. Homogenize the cell lysate with a 20 gauge needle attached to syringe, passing the cell lysate through the needle several times.

3. Transfer the lysate to the gDNA Eliminator spin column and centrifuge at 10,000 × g for 30 s.

4. Add 400 μL 70 % ethanol to the flow-through and mix well by gently pipetting.

5. Transfer the mixture to an RNeasy MinElute spin column in collection tube, and centrifuge at 10,000 × g for 30 s. Discard the flow-through.

6. Wash the RNeasy MinElute spin column with 700 μL Buffer RW1 and 500 μL Buffer RPE, respectively, by centrifuge at 10,000 × g for 30 s.

7. Wash again with 500 μL 80 % ethanol at ≥8,000 g for 2 min. And completely dry the column by centrifuge in a new collection tube at maximum speed for 5 min.

8. Transfer the column to a new collection tube; elute the RNA sample with 30 μL H₂O. Aliquot the RNA samples on ice and store at −80 °C.

3.3.5 Quantification and Qualification of RNA Samples for Microarray Service

1. Check the quantity of RNA samples. Apply 1 μL of RNA onto the NanoDrop, for quantification of RNA concentration, and the ratio of A₂₆₀/A₂₈₀ and A₂₃₀/A₂₈₀.

2. Check the integrity of the RNA samples with Agilent 2100 Bioanalyzer. Apply 1 μL of RNA (diluted to concentrations less than 1 ng/mL) to Eukaryotic RNA Pico chip following the protocol of manufacturer. The respective ribosomal RNAs should appear as sharp peaks. The apparent ratio of 28S rRNA to 18S rRNA should be approximately 2. The RIN (RNA integrity number) score should be larger than 7.0.

3. Use 500 pg–50 ng of RNA in a volume less than 5 μL for amplification to get cDNA with the NuGEN Ovation Pico WTA system, then fragmentation and labeling with Encore Biotin Module. The individual samples are hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 microarray.

3.4 Preparation of Extracellular Vesicles

1. Use cells at 70 % confluence in several 15 cm culture dishes (see Note 20).

2. Discard culture media and wash thoroughly with pre-warmed PBS three times (see Note 21).

3. Add culture media with exosome-free FBS, and incubate 24–48 h to collect conditioned media with extracellular vesicles (see Note 22).

4. Collect the conditioned medium and centrifuge at 300 × g , 4 °C for 10 min, and followed by 2,000 × g , 4 °C for 20 min (see Note 23).

5. (a) To get microparticles, further centrifuge the supernatant at 20,000 × g , 4 °C for 20 min, resulting in a pellet containing the microparticles (see Note 24). (b) To get microvesicles, filter the supernatant with a 0.2 μm filter, and then ultracentrifuge at 100,000 × g (Rotor 70Ti) for 2 h, which results in a pellet containing the microvesicles (see Note 25).

6. Wash the pellets with filtered PBS, then resuspended in filtered PBS, and store at −80 °C (see Note 26).