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...

<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 tha...

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>1000 &#956;L pipette</td>
			<td>Gilson</td>
			<td>Pipetman classic</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;L pipette</td>
			<td>Gilson</td>
			<td>Pipetman classic</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;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 um 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>CO2</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. Not...

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 ...

Tube-formation Assay is an in vitro test to study the molecular mechanisms underlying several steps that lead to the formation of new blood vessels.This assay allows to identify compounds and signaling cascades associated to angiogenesis.With one test, we're able to show that a natural compound like RGWE reduces the ability of endothelial cells to form tube-like structure, do not affect cell viability, and that this effect depends on the activation of the MEK/ERK pathway.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 tubes.For this reason, it is advisable to perform a preliminary test to find out the correct number of the cells.Luca Olga Pastorino, PhD student in my lab, will show the procedures.First, collect Ruta graveolens leaves during the spring and summer months.Put 250 grams of chopped leaves in a conical flask, and add one liter of distilled water, boil, and filter, as described in the manuscript.The next day, use a lyophilizer to lyophilize the leaf extract.After obtaining the powder, weigh it, and divide it into aliquots.Culture the HUVECs in endothelial cell growth medium, according to the text protocol.When the cells reach 80%confluency, passage the cells at a ratio of one to three.Use the cells obtained between passage two and passage five.Once the passaged HUVECs reach 70%confluency, transvect them with one microgram of the vector containing the gene of interest.Combine one microgram of DNA with three microliters of diluted Lipofectamine 2000.Then, remove the medium from the HUVECs, and replace it with one milliliter of fresh complete medium.Add the transvecting solution to the cells, and incubate the cells at 37 degrees Celsius, and five percent carbon dioxide.Three hours after transvection, add seven milliliters of fresh complete medium, and incubate the cells for an additional 24 to 48 hours.Immediately before basement preparation, put a pre-chilled, 96-well plate on ice, and add 50-microliters of cold matrix to each well.While the plate incubates, wash the HUVECs with one milliliter of PBS/EDTA solution.Then, add one-milliliter of Trypsin-EDTA solution to the cells, and incubate for five minutes at 37 degrees Celsius.Collect the medium containing the suspended cells, and harvest the cells via centrifugation.Then, resuspend the cells in one milliliter of PBS.Transfer 0.2-milliliters of the cell suspension to 0.5-milliliters of PBS with trypan blue solution.After holding the suspension at room temperature for five minutes, count the cells in a B

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16.0K Views.  University of Campania "Luigi Vanvitelli". Tube-formation Assay is an in vitro test to study the molecular mechanisms underlying several steps that lead to the formation of new blood vessels.This assay allows to identify compounds and signaling cascades associated to angiogenesis.With one test, we're able to show that a natural compound like RGWE reduces the ability of endothelial cells to form tube-like structure, do not affect cell viability, and that this effect depends on the activation of the MEK/ERK pathway.It is important to pe...