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Developmental Biology

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

Published: June 24th, 2019

DOI:

10.3791/58591

1Laboratory of Cellular and Molecular Neuropathology, Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania "Luigi Vanvitelli", 2Department of Molecular Medicine and Biotechnologies, University of Naples "Federico II" Naples

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 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.

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 ....

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1. RGWE Preparation

  1. 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).
  2. Weigh 250 g of leaves and finely chop them with scissors.

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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.......

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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 propose the tube formation assay as an easy-to-perform, reliable, and quantitative method useful to investigate RGWE'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.......

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

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Name Company Catalog Number Comments
HUVEC cells Clontech C2519A
FBS Invitrogen 10270106
EBM-2 basal medium Clontech cc3156
Single quot kit- supplemets and growth factors clontech cc4147
Matrigel Corning 354234
96-well plates Thermo Scientific 167008
15 mL conical tubes Sarstedt 62,554,502
10 mL disposable serological pipette Sarstedt 861,254,001
5 mL disposable serological pipette Sarstedt 861,253,001
1000 μL pipette Gilson Pipetman classic
100 μL pipette Gilson Pipetman classic
20 μL pipette Gilson Pipetman classic
p1000 pipette tips Sarstedt
p20-200 pipette tips Sarstedt 70,760,502
Burker chamber Fortuna
Trypan blu stain Gibco 15250-061
DPBS Gibco 14190-094
mill-ex 0.22 um filters Millipore SLGS033SS
Lyophilizer VirTis-SP Scientific
Incubator Thermo Scientific
CO2 AirCos
Pen-Strep Gibco 15070-063
100 mm dish Sarstedt 833,902
pcDNA3 Invitrogen v79020
Lipofectamine-2000 Invitrogen 11668027
Opti-MEM Gibco 31985070 Reduced serum medium
Rutin Sigma-Aldrich R5143-50G
Axiovert 25 microscope Zeiss
AmScope MD500 camera AmScope
Dispase Thermo Scientific D4818
Lab heater Falc
ParaFilm American National Can

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