A subscription to JoVE is required to view this content. Sign in or start your free trial.
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 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.
1. RGWE Preparation
2. Cell Culture
3. Transfection
4. Tube Formation Assay
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...
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...
The authors have nothing to disclose.
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.
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 |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved