Name: huvec tube-formation assay to evaluate the impact of natural products on angiogenesis
Uploaded: 2019-06-24T13:00:00.000+00:00
Duration: 6 min 12 s
Description: Watch this Scientific Journal Video about HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis at JoVE.com

This work has been funded by Fondi di Ateneo to Luca Colucci-D&#39;Amato and VALERE Program funds to Maria Teresa Gentile and AIRC fund IG18999 to Maurizio Bifulco.

1. RGWE Preparation
<ol>
	<li>Collect R. graveolens leaves from plants during the spring/summer months under the supervision of a botanist. 
	NOTE: In this case, leaves were collected at the Experimental Section of Medicinal Plants in the Botanical Garden of Naples, Italy5. The plant is spontaneous, perennial, and is present in the Mediterranean regions (Figure 1).</li>
	<li>Weigh 250 g of leaves and finely chop them with scissors.</li>
	<li>Put the leaves in a conical flask and add 1 L of distilled water to 250 g of chopped leaves and bring that to a boil at 110 &#176;C for 60 min. Cover the flask with aluminum foil to avoid excessive evaporation.</li>
	<li>Use 3 mm-funnel filter paper to separate the boiled leaves from the liquid phase that represents the water extract.</li>
	<li>Filter the liquid phase through a 0.22 &#181;m filter, collecting it in a beaker. Cover the beaker with parafilm and freeze the extract at -80 &#176;C overnight.</li>
	<li>Make 10 - 15 holes with a needle in the parafilm and lyophilize the extract in a lyophilizer. Note that it can take a couple of days to obtain the powder.</li>
	<li>Weigh the powder obtained, divide it into aliquots, and store it at 4 &#176;C until necessary. It is possible to store it for a year.</li>
	<li>When necessary for the experiments, prepare a stock solution, diluting the powder with distilled water to a standard concentration of 50 mg/mL. Separate it into small-volume (e.g., 1 mL) aliquots. This preparation can be stored at -20 &#176;C until used but cannot be stored at 4 &#176;C for more than a couple of days because of the oxidations of the molecules. Avoid freeze and thaw.</li>
</ol>
2. Cell Culture
<ol>
	<li>Cultivate HUVECs in endothelial cell growth medium: endothelial basal medium supplemented with 1 &#956;g/mL hydrocortisone and 1 ng/mL epidermal growth factor, 10% FBS, and penicillin/streptomycin at 37 &#176;C in an atmosphere of 5% CO2.</li>
	<li>When 80% confluent, split the cells at the ratio of 1:3 every passage. Use cells from two to five passages, that do not show apparent differences in response to growth factors. After the sixth passage, cells become senescent and do not form tubes on the gelled basement matrix.</li>
</ol>
3. Transfection
<ol>
	<li>When the HUVECs are 70% confluent, transfect them with the desired gene.</li>
	<li>For a 100 mm plate, use 1 &#181;g of empty pCDNA3 vector as control and 1 &#181;g of pCDNA3 vector containing the gene of interest (constitutively active MEK [caMEK], in this case).</li>
	<li>Complex 1 &#181;g of DNA with 3 &#181;L of lipofectamine 2,000, diluted with reduced serum medium (see Table of Materials) to the final volume of 200 &#181;L. Remove the medium from the cells and replace it with 1 mL of fresh complete medium; then, add the transfecting solution to the cells. Incubate the cells in the incubator at 37 &#176;C, with 5% CO2. It is possible to also use other transfection agents different from lipofectamine 2,000, following the manufacturer&#8217;s instructions.</li>
	<li>Add 7 mL of complete fresh medium 3 h after the transfection and, then, further incubate the cells at 37 &#176;C for 24 - 48 h before proceeding with the next assays. The day after the transfection, replace the medium with fresh complete medium.</li>
</ol>
4. Tube Formation Assay
<ol>
	<li>Cool a 96-well plate and pipette tips at 4 &#176;C for 1 h before preparing the gelled basement.</li>
	<li>At the moment of the basement preparation, put the plate on ice (0 &#176;C) and add 50 &#956;L of cold matrix in each well while avoiding the formation of bubbles. Note that the matrix-containing vial should be kept on ice during the procedure since the matrix becomes solid at room temperature.</li>
	<li>Put the plate in the incubator at 37 &#176;C with 5% CO2 for at least 30 min to allow the basement to polymerize.</li>
	<li>In the meantime, harvest the cells. Wash the HUVECs with 1 mL of a 0.25% trypsin/0.53 mM EDTA solution; then, add 2 mL of the trypsin-EDTA solution and incubate at 37 &#176;C for 5 min. Observe the cells under an inverted microscope to verify that the cell layer is completely dispersed. Then, collect the medium in which the cells are suspended and harvest the detached cells by centrifugation at 264 x g for 3 min. Resuspend them in 1 mL of phosphate-buffered saline (PBS).</li>
	<li>To count the cells, add 0.2 mL of the cell suspension to 0.5 mL of PBS and 0.3 mL of 0.4% trypan blue solution. After 5 min at room temperature, count the cells in a B&#252;rker chamber.</li>
	<li>Resuspend the cells in complete medium at the concentration of 2 x 104 cells/50 &#181;L. Seed them onto the gelled basement at the concentration of 2 x 104 cells per well and let them settle on it.</li>
	<li>Pay attention to the number of the cells. Too many or too few cells may not form tubes in the right way on the gelled basement.</li>
	<li>Prepare increasing doses of RGWE (0.01, 0.1, and 1 mg/mL), diluting the stock solution in culture medium or rutin (12, 120, and 300 &#181;g/mL), all in the final volume of 50 &#181;L/well, and add them to 50 &#956;L/well of cell suspension. The final volume in each well will be 100 &#956;L. Prepare four to six wells for each experimental condition. As a control, dilute distilled water (vehicle) in the culture medium at a ratio of 1:50.</li>
	<li>Incubate the cells in the incubator at 37 &#176;C and 5% CO2 for a time ranging from 6 to 24 h.</li>
	<li>Observe the cells within a time range spanning from 6 to 24 h after seeding, using a phase-contrast microscope at a magnification of 10X. HUVECs are able to form tube-like structures within 6 h but it is possible to observe better results after 12 - 18 h. Photograph the tube-like structures on each well and image an average from three to five random fields in each well.</li>
	<li>After the image acquisition, the cells can be detached from the basement matrix and counted to evaluate the effect of the treatment on the number of cells. For each well, remove the medium, add dispase at a concentration of 24 U/mL, and put the plate at 37 &#176;C for 1 h. Then, from each well, collect the medium in which the cells are suspended and harvest the detached cells by centrifugation at 264 x g for 3 min. Resuspend them in 0.2 mL of PBS. To count the cells, add 0.2 mL of cell suspension to 0.5 mL of PBS and 0.3 mL of 0.4% trypan blue solution. After 5 min at room temperature, count the cells in the B&#252;rker chamber.</li>
	<li>To quantify the results of the tube formation assay, it is possible to count the number of branch points in which at least three tubes join. Using the appropriate image software, count for each field in each micrograph the number of branch points and calculate the mean &#177; the standard error (SE) for each experimental condition. Use a two-tailed t-test to evaluate statistical significance.</li>
</ol>

<ol>
	<li>Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+life,+disease+and+medicine.">Angiogenesis in life, disease and medicine.</a> Nature. 438 (7070), 932-936 (2005).</li><li>Carmeliet, P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+and+clinical+applications+of+angiogenesis.">Molecular mechanisms and clinical applications of angiogenesis.</a> Nature. 473 (7347), 298-307 (2011).</li><li>Ferrara, N., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+as+a+therapeutic+target.">Angiogenesis as a therapeutic target.</a> Nature. 438 (7070), 967-974 (2005).</li><li>Ravishankar, D., Rajora, A. K., Greco, F., Osborn, H. M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavonoids+as+prospective+compounds+for+anti-cancer+therapy.">Flavonoids as prospective compounds for anti-cancer therapy.</a> The International Journal of Biochemistry &amp; Cell Biology. 45, 2821-2831 (2013).</li><li>Gentile, M. T., 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=Ruta+graveolens+water+extract+inhibits+cell-cell+network+formation+in+human+umbilical+endothelial+cells+via+MEK-ERK1/2+pathway.">Ruta graveolens water extract inhibits cell-cell network formation in human umbilical endothelial cells via MEK-ERK1/2 pathway.</a> Experimental Cell Research. 364 (1), 50-58 (2018).</li><li>Butler, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+products+to+drugs:+natural+product-derived+compounds+in+clinical+trials.">Natural products to drugs: natural product-derived compounds in clinical trials.</a> Natural Product Reports. 25, 475-516 (2008).</li><li>Sulaiman, R. S., Basavarajappa, H. D., Corson, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+product+inhibitors+of+ocular+angiogenesis.">Natural product inhibitors of ocular angiogenesis.</a> Experimental Eye Research. 129, 161-171 (2014).</li><li>Risau, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+angiogenesis.">Mechanisms of angiogenesis.</a> Nature. 386 (6626), 671-674 (1997).</li><li>Trung, N. X. <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+models+for+angiogenesis.">In vitro models for angiogenesis.</a> Journal of Science &amp; Development. 13 (4), 850-858 (2015).</li><li>Ucuzian, A. A., Greisler, H. P. <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+Models+of+Angiogenesis.">In vitro Models of Angiogenesis.</a> World Journal of Surgery. 31, 654-663 (2007).</li><li>Simons, M., 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=American+Heart+Association+Council+on+Basic+Cardiovascular+Sciences+and+Council+on+Cardiovascular+Surgery+and+Anaesthesia.+State-of-the-Art+Methods+for+Evaluation+of+Angiogenesis+and+Tissue+Vascularisation:+A+Scientific+Statement+From+the+American+Heart+Association.">American Heart Association Council on Basic Cardiovascular Sciences and Council on Cardiovascular Surgery and Anaesthesia. State-of-the-Art Methods for Evaluation of Angiogenesis and Tissue Vascularisation: A Scientific Statement From the American Heart Association.</a> Circulation Research. 116 (11), e99-e132 (2015).</li><li>Arnaoutova, I., Kleinman, H. K. <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+angiogenesis:+endothelial+cell+tube+formation+on+gelled+basement+membrane+extract.">In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract.</a> Nature Protocols. 5 (4), 628-635 (2010).</li><li>DeCicco-Skinner, K. L., 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=Endothelial+cell+tube+formation+assay+for+the+in+vitro+study+of+angiogenesis.">Endothelial cell tube formation assay for the in vitro study of angiogenesis.</a> Journal of Visualized Experiments. (91), e51312 (2014).</li></ol>

The authors have nothing to disclose.

Natural compounds are characterized by a high chemical diversity and biochemical specificity and represent a source of potentially therapeutic molecules. Here, we show how to obtain water extract from the plant R. graveolens and&#160;propose the tube formation assay as an easy-to-perform, reliable, and quantitative method useful to investigate RGWE&#39;s effects on angiogenesis. It is important to boil the R. graveolens leaves for 1 h to be sure to obtain the complete water extract. Boiling for less than 1 h did not allow for the extraction of all the molecules, and the extract could not exert the expected biological effect.
Tube formation assay represents an in vitro test to study the molecular mechanisms underlying the several steps that lead to the formation of new blood vessels. This assay allows researchers to identify compounds able to modulate angiogenesis, as well as the proteins and signaling cascades involved. Moreover, this assay allows researchers to test substances that can influence, at the same time, endothelial cell proliferation, adhesion, migration, and protease activity, all important mechanisms in blood vessel formation. Using only this kind of test, we show that RGWE, but not its major component rutin, is able to reduce the ability of HUVECs to form tube-like structures without affecting cell viability and that this effect depends on MEK-ERK pathway activation. However, it is important to perform the test with the right number of cells. In fact, too few or too many cells could not allow the right formation of the tubes. For this reason, it is advisable to perform a preliminary test to find the correct number of cells. Moreover, the number of cell passages is as important as the cell number since, to obtain the correct tube formation assay, cells have to be passaged twice to five times. After passage 6, senescence mechanisms occur that can impair the right tube formation.
The tube formation assay is a very reproducible assay, and it sheds light on the physiology of endothelial cells, even if it is not the gold standard for the three-dimensional study of tube formation8,9,10,11. Three-dimensional collagen and fibrin models have been demonstrated to be better for investigations of vascular tubulogenesis, sprouting, and endothelial cell-pericyte interaction. However, compared to the tube formation assay on gelled basement matrix, these tests are more time- and money-consuming11, suggesting that the first could be a good test to obtain preliminary results that can be the basis for more focused in vivo studies. Finally, the tube formation assay can be carried out in 24 h, since nontransformed HUVECs are able to form tube-like structures within 6 h12,13.

Angiogenesis is a physiological process that leads to the formation of new blood vessels from preexisting ones and occurs during embryogenesis and organ growth. In adulthood, angiogenesis is activated only in the cycling ovary, in the placenta during pregnancy, and during wound healing and repair. Angiogenesis depends on the ability of endothelial cells to proliferate, differentiate, and migrate to form an intact vascular network1. However, in several disorders, such as inflammatory, metabolic, and rheumatic diseases, angiogenic processes are altered and angiogenesis becomes excessive. Moreover, uncontrolled angiogenic processes also stimulate tumor progression and metastasis1. For these reasons, in the last decade, research studies are focused on the development of new therapeutic strategies aimed at the inhibition of excessive angiogenesis in cancer, ocular, joint, or skin disorders2,3.
Vascular endothelial growth factor (VEGF) represents the main target of current antiangiogenic therapies4, and several anti-VEGF monoclonal antibodies have been developed and synthesized to prevent excessive angiogenesis. However, these synthetic drugs show severe side effects and have an unfavorable cost-to-benefit ratio5,6. Therefore, it is imperative to find new therapeutic strategies to limit excessive angiogenesis with minimal side effects to complement and combine with currently used drugs. These new drugs can be found among natural products that are characterized by a high chemical diversity and biochemical specificity.
In this article, we propose a simple method to evaluate the impact of the RGWE on the ability of HUVECs to form tubules on a gelled basement matrix in vitro5. Indeed, RGWE is a mixture of secondary metabolites such as flavonoids and polyphenols among which rutin is the major component5. Many of them have been already tested as anti-inflammatory and vasoprotective agents7,8,9,10,11. Moreover, we have recently demonstrated that RGWE, but not rutin, is able to inhibit the HUVEC ability to form tubules on a gelled basement matrix and that this phenomenon is mediated by the MEK-ERK pathway, indicating RGWE as a potential therapeutic tool able to prevent excessive new blood vessel formation5.

<table><tbody><tr><td>HUVEC cells</td><td>Clontech</td><td>C2519A</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>10270106</td><td></td></tr><tr><td>EBM-2 basal medium</td><td>Clontech</td><td>cc3156</td><td></td></tr><tr><td>Single quot kit- supplemets and growth factors</td><td>clontech</td><td>cc4147</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>354234</td><td></td></tr><tr><td>96-well plates</td><td>Thermo Scientific</td><td>167008</td><td></td></tr><tr><td>15 mL conical tubes</td><td>Sarstedt</td><td>62,554,502</td><td></td></tr><tr><td>10 mL disposable serological pipette</td><td>Sarstedt</td><td>861,254,001</td><td></td></tr><tr><td>5 mL disposable serological pipette</td><td>Sarstedt</td><td>861,253,001</td><td></td></tr><tr><td>1,000 &amp;mu;L pipette</td><td>Gilson</td><td>Pipetman classic</td><td></td></tr><tr><td>100 &amp;mu;L pipette</td><td>Gilson</td><td>Pipetman classic</td><td></td></tr><tr><td>20 &amp;mu;L pipette</td><td>Gilson</td><td>Pipetman classic</td><td></td></tr><tr><td>p1000 pipette tips</td><td>Sarstedt</td><td></td><td></td></tr><tr><td>p20-200 pipette tips</td><td>Sarstedt</td><td>70,760,502</td><td></td></tr><tr><td>Burker chamber</td><td>Fortuna</td><td></td><td></td></tr><tr><td>Trypan blu stain</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>mill-ex 0.22 &amp;mu;m filters</td><td>Millipore</td><td>SLGS033SS</td><td></td></tr><tr><td>Lyophilizer</td><td>VirTis-SP Scientific</td><td></td><td></td></tr><tr><td>Incubator</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;</td><td>AirCos</td><td></td><td></td></tr><tr><td>Pen-Strep</td><td>Gibco</td><td>15070-063</td><td></td></tr><tr><td>100 mm dish</td><td>Sarstedt</td><td>833,902</td><td></td></tr><tr><td>pcDNA3</td><td>Invitrogen</td><td>v79020</td><td></td></tr><tr><td>Lipofectamine-2000</td><td>Invitrogen</td><td>11668027</td><td></td></tr><tr><td>Opti-MEM</td><td>Gibco</td><td>31985070</td><td>Reduced serum medium</td></tr><tr><td>Rutin</td><td>Sigma-Aldrich</td><td>R5143-50G</td><td></td></tr><tr><td>Axiovert 25 microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>AmScope MD500 camera</td><td>AmScope</td><td></td><td></td></tr><tr><td>Dispase</td><td>Thermo Scientific</td><td>D4818</td><td></td></tr><tr><td>Lab heater</td><td>Falc</td><td></td><td></td></tr><tr><td>ParaFilm</td><td>American National Can</td><td></td><td></td></tr></tbody></table>

huvec tube-formation assay to evaluate the impact of natural products on angiogenesis

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the formation of new blood vessels and involve several signal transduction pathways. Here we show that the tube formation assay is a simple in vitro method to evaluate the impact of natural products on angiogenesis and to investigate the molecular mechanisms involved. In particular, in the presence of the water extract of Ruta graveolens (RGWE), endothelial cells are no longer able to form a cell-cell network and that the RGWE effects on human umbilical vein endothelial cell (HUVEC) tube formation is abolished by the constitutive activation of MEK.

To evaluate the influence of RGWE on angiogenesis, we carried out a tube formation assay on a gelled basement matrix. When cultivated on it, HUVECs form tube-like structures that originate from cells that appear elongated and that connect each other to form a cell-cell network (Figure 2). In Figure 3, we show that the number of branches in HUVECs treated with RGWE was significantly lower as compared to the control conditions. Notably, in the presence of 0.1 mg/mL and 1 mg/mL RGWE, the number of the branches are lower by 40% and 60%, respectively, compared to the control condition. Since rutin has been indicated as the major component of RGWE5, we analyzed its effect on HUVEC tube-formation assay. As shown in Figure 4, rutin alone is not able to affect the HUVECs&#39; ability to form a cell-cell network. Then, we used the tube formation assay to investigate the molecular mechanisms underlying the RGWE-induced inhibition of tube formation. The MEK/ERK intracellular signaling pathway exerts a pivotal role in angiogenic processes. HUVECs transfected by caMEK were treated with RGWE and cultivated on gelled basement matrix. As shown in Figure 5, in caMEK-transfected endothelial cells, RGWE no longer inhibits tube formation, while mock-transfected cells, used as control, still form a cell-cell network on the gelled basement matrix and are responsive to RGWE, thus indicating that the RGWE&#39;s effect in angiogenesis is mediated by the MEK-ERK pathway.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58591/58591fig1.jpg" /> 
Figure 1:&#160;Ruta graveolens.&#160;A Ruta graveolens shrub. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58591/58591fig1large.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/58591/58591fig2.jpg" /> 
Figure 2: HUVECs form tube-like structures on a gelled basement matrix. Representative microscopic photographs of HUVECs cultured in polystyrene dish (left) and on gelled basement matrix (right). Scale bar = 10 &#181;m.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58591/58591fig3.jpg" /> 
Figure 3: RGWE inhibits tube formation in HUVECs. (A) High-power microscopic photographs of HUVECs cultured on a gelled basement matrix treated with RGWE (0, 0.01, 0.1, and 1.0 mg/mL). The scale bar is 10 &#181;m. (B) The percentage of the HUVEC branch point (dark gray) in the presence of RGWE (0.01, 0.1, and 1.0 mg/mL) compared to untreated cells (0 mg/mL) and the trypan blue exclusion test (light gray) on HUVECs treated (0.01, 0.1, and 1 mg/mL) or not (0 mg/mL) with RGWE for 24 h. *p &#60; 0.01 vs. the control condition (0 mg/mL). The results are expressed as the mean &#177; the SE of three independent experiments. The statistical significance was obtained by a two-tailed t-test.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58591/58591fig4.jpg" /> 
Figure 4: Rutin does not affect HUVEC tube formation. (A) High-power microscopic photographs of HUVECs seeded on a gelled basement matrix and treated (12, 120, and 300 &#181;g/mL) or not (0 &#181;g/mL) with rutin for 24 h. The scale bar is 10 &#181;m. (B) The percentage of HUVEC branch point (dark gray) in the presence of increasing doses of rutin (12, 120, and 300 &#181;/mL) compared to control conditions (0 &#181;g/mL) and the trypan blue exclusion test (light gray) on HUVECs treated (12, 120, and 300 &#181;/mL) or not (0 &#181;g/mL) with rutin for 24 h. The results are expressed as the mean &#177; the SE of three independent experiments. Statistical significance was obtained by a two-tailed t-test.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58591/58591fig5.jpg" /> 
Figure 5: MEK pathway mediates RGWE effects on HUVEC tube formation. (A) High-power microscopic photographs of HUVECs transfected with empty vector (mock transfection) or with caMEK, seeded on the gelled basement matrix and treated with increasing doses of RGWE (0, 0.01, 0.1, and 1 mg/mL). The scale bar is 10 &#181;m. (B) The percentage of the number of branch points in HUVECs mock-transfected (dark gray) and in HUVECs transfected with caMEK (light gray) and treated with increasing doses of RGWE (0.01, 0.1, and 1 mg/mL) compared to control conditions (0 mg/mL). *p &#60; 0.01 vs. the control condition; &#167;p &#60; 0.05 vs. cells transfected with the empty vector and treated with the same amount of RGWE. The results are expressed as the mean &#177; the SE of three independent experiments. The statistical significance was obtained by a two-tailed t-test.

Watch this Scientific Journal Video about HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis at JoVE.com

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Here, we evaluate the effects of the water extract of Ruta graveolens on vessel network formation by using a tube formation assay on a gelled basement matrix.

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...

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16.5K Views.  University of Campania "Luigi Vanvitelli". Here, we evaluate the effects of the water extract of Ruta graveolens on vessel network formation by using a tube formation assay on a gelled basement matrix.

Video: HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

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, 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 often do not resemble vessels in vivo. Here we demonstrate an optimized 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. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

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

Biology

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 ...

Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is unregulated proliferation and cell division. Inhibition of proliferation by halting cell cycle progression has been shown to be a promising approach for cancer treatment, including LC. 
miRNA therapeutics have emerged as important post-transcriptional gene regulators and are increasingly being studied for use in cancer treatment. In recent work, we utilized two miRNAs, miR-143 and miR-506, to regulate cell cycle progression. A549 non-small cell lung cancer (NSCLC) cells were transfected, gene expression alterations were analyzed, and apoptotic activity due to the treatment was finally analyzed. Downregulation of cyclin-dependent kinases (CDKs) were detected (i.e., CDK1, CDK4 and CDK6), and cell cycle halted at the G1/S and G2/M phase transitions. Pathway analysis indicated potential antiangiogenic activity of the treatment, which endows the approach with multifaceted activity. Here, described are the methodologies used to identify miRNA activity regarding cell cycle inhibition, induction of apoptosis, and effects of treatment on endothelial cells by inhibition of angiogenesis. It is hoped that the methods presented here will support future research on miRNA therapeutics and corresponding activity and that the representative data will guide other researchers during experimental analyses.

miRNA therapeutics have significant potential in regulating cancer progression. Demonstrated here are analytical approaches used for identification of the activity of a combinatorial miRNA treatment in halting cell cycle and angiogenesis.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is ...

Cancer Research

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

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 ...

Analyzing Angiogenesis: In Vitro Assay for Tube Formation on BM Matrix

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of mesenchymal to endothelial-like transition. Traditionally, this process is evaluated by an in vitro tube-formation assay, where the cells organize themselves into tube-like structures when seeded over a polymerized basement membrane preparation. Although several structural features can be measured in photomicrographs of the structures, to assess the real commitment of EVT to the endothelial-type phenotype, biochemical analysis of cell extracts is required. Scraping the cells from the culture dish to obtain RNA and/or protein extracts is not an alternative since the tube-like structures are severely contaminated by the bulk of proteins from the polymerized basement membrane. Thus, a strategy to separate the cells from the basement membrane proteins prior to the preparation of cell extracts is needed. Here, a simple, fast, and cost-effective method to recover the tube-like structures from the in vitro tube-formation assay and the subsequent analysis by biochemical techniques is presented. Tube-like structures formed by HTR8/SVneo cells, an EVT cell line, were liberated from the polymerized basement membrane by a short incubation with PBS supplemented with ethylenediaminetetraacetic acid (EDTA). After serial washes, a ready-to-use pellet of purified tube-like structures can be obtained. This pellet can be subsequently processed to obtain RNA and protein extracts. qPCR analysis evidenced the induced expression of VE-cadherin and alphav-integrin, two endothelial cell markers, in EVT-derived tube-like structures compared to control cells, which was consistent with the induction of the endothelial cell marker, CD31, evaluated by immunofluorescence. Western blot analysis of the tube-like structures' protein extracts revealed the overexpression of RECK in transfected HTR8/SVneo cells. Thus, this simple method allows to obtain cell extracts from the in vitro tube-formation assay for the subsequent analysis of RNAs and protein expression.

The present protocol allows the recovery of tube-like structures formed in the classical in vitro angiogenesis-tube formation assay where cells, such as trophoblast cells, organize in capillary-like structures over a layer of extracellular basement membrane matrix. The recovered structures are used for RNA and protein isolation for various biochemical analyses.

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of ...

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 ...

Study of Angiogenesis Using Scratch Wound Migration and Spheroid Sprouting Assays

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

Author Spotlight: Investigating Angiogenesis Through Challenges and Innovations in Assay Development

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Summary

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Chapters in this video

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

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

Rat Mesentery Angiogenesis Assay

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

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

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay