HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis
June 24th, 2019
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.
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
View the full transcript and gain access to thousands of scientific videos
Overview
This study evaluates the effects of the water extract of Ruta graveolens on vessel network formation using a tube formation assay on a gelled basement matrix.
Key Study Components
Area of Science
- Neuroscience
- Vascular biology
- Botanical extracts
Background
- Ruta graveolens is known for its medicinal properties.
- Understanding vessel formation is crucial for various biological processes.
- Tube formation assays are standard methods to assess angiogenesis.
- The role of plant extracts in vascular biology is an emerging field of study.
Purpose of Study
- To investigate the impact of Ruta graveolens on angiogenesis.
- To utilize a tube formation assay for assessing vascular network development.
- To contribute to the understanding of natural compounds in promoting vascular health.
Methods Used
- Water extraction of Ruta graveolens.
- Tube formation assay conducted on a gelled basement matrix.
- Assessment of vessel network formation.
- Analysis of results to determine the efficacy of the extract.
Main Results
- The water extract of Ruta graveolens promoted vessel network formation.
- Results indicated significant differences compared to control conditions.
- Findings support the potential use of Ruta graveolens in vascular therapies.
- Further studies are suggested to explore underlying mechanisms.
Conclusions
- Ruta graveolens has a positive effect on angiogenesis.
- The study highlights the potential of botanical extracts in vascular research.
- Future research could expand on the therapeutic applications of these findings.
Frequently Asked Questions
Executive Industry Relevance
The tube formation assay provides a quantitative in vitro model to assess how natural compounds modulate angiogenesis, a critical process in oncology, wound healing, and vascular disease. By linking compound exposure to endothelial network formation and pathway-specific dependencies (e.g., MEK/ERK), the assay supports mechanistic de-risking in early discovery. This enables R&D teams to prioritize bioactive natural products with defined mechanisms of action before investing in lead optimization.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates therapeutic hypotheses by testing whether natural products inhibit or promote endothelial tube formation.
- Operational Value: Clarifies pathway involvement (e.g., MEK/ERK dependence) to de-risk target engagement.
- Predictive Value: Supports portfolio triage by identifying compounds with measurable effects on angiogenic potential.
Screening & Assay Development
- Assay Readiness: Enables standardized evaluation of compound effects on HUVEC network formation using a gelled basement matrix.
- Quantitative Output: Generates measurable tube-like structures that reflect angiogenic capacity.
- Reproducibility: Requires optimized cell seeding density to ensure consistent tube formation across replicates.
Translational & Preclinical Research
- Disease Relevance: Models angiogenesis in contexts such as tumor vascularization and ischemic tissue repair.
- Translational Continuity: Bridges in vitro findings to preclinical validation by establishing mechanistic links (e.g., MEK/ERK modulation).
- Risk-Adjusted Decisions: Informs go/no-go criteria based on whether compound effects are pathway-dependent and reproducible.
Pipeline & Workflow Integration
The assay fits within the discovery continuum from target validation to lead identification, particularly for natural product screening programs focused on vascular modulation.
- Discovery Biology: Tests whether compounds alter endothelial cell behavior in a biologically relevant angiogenesis model.
- Screening: Delivers reproducible, quantitative readouts for compound library evaluation.
- Analytics: Provides imaging-based metrics of tube formation that can be correlated with pathway activation states.
- Translational Research: Supports mechanistic follow-up by linking phenotypic outcomes to signaling cascades like MEK/ERK.
- Enterprise Reuse: Functions as a modular assay platform adaptable to various compound classes and endothelial cell types.
Operational & Enterprise Impact
- Scientific Value: Mechanistic de-risking through pathway-specific validation (e.g., MEK/ERK dependence).
- Operational Value: Standardized protocol with defined cell density and matrix requirements.
- Strategic Value: Enables data-driven prioritization of natural products with defined MoA.
- Portfolio Impact: Reduces false positives by requiring both phenotypic effect and pathway validation.
Implementation Considerations
- Cell culture expertise to maintain HUVEC viability and passage consistency (passages 2–5).
- Access to basement matrix (e.g., Matrigel) and optimized seeding density determined via pilot experiments.
- Standardized imaging and analysis tools for quantifying tube network formation.
- Ability to modulate signaling pathways (e.g., via transfection) to confirm mechanism of action.
- Natural product preparation workflow (extraction, lyophilization, aliquoting) for reproducible dosing.
