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

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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.
3. 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 °C for 60 min.....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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
1,000 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 μm 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