In Vitro and in Vivo Models for Studying Angiogenesis

Vijay Avin BR, Molecular Biomedicine Laboratory, Sahyadri Sceince College, Shimoga, Karnataka, India

Growth of new blood vesselsAdvanced cancers can secrete

angiogenic factors (VEGF, bFGF etc).

Basic research to elucidate molecular mechanisms of angiogenesis:Identify and characterize regulatory pathways that mediate various steps of angiogenesis such as endothelial cell migration, invasion, and tubulogenesis.

To develop treatments for cancer and other diseases associated with angiogenesis: Identification of compounds that inhibit or stimulate key steps in the angiogenesis process.

Angiogenesis Research

Methods to Study Angiogenesis

In vitro ModelsIn Vivo Models

Important Considerations forDeveloping

Angiogenesis Studies Choose appropriate endothelial cell source.

Establish acceptable dynamic range to measure stimulation and/or inhibition of angiogenesis.

Incorporate appropriate extracellular matrix (ECM) protein(s) to facilitate cell functionality and assay outcome.

Endothelial Cells are the most important tool for

in vitro studies of

Angiogenesis……

Tumor cell survival is dependent on the health and proliferation of endothelial cells in surrounding blood

vessels………

Sources of Endothelial CellsLarge vessel– aortic (e.g., HAEC)– umbilical vein (e.g. HUVEC)– pulmonary artery

Microvascular (e.g., HMVEC)– brain– lung– dermis (e.g., HDMEC)– myocardium

Human Umbilical Vein Endothelial Cells HUVEC

Most commonly used EC type for angiogenesis studies

ECM provides a physiological substrate that supports key cellular functions:

• Structural organization of cells and tissue.

• Cell attachment, survival, and proliferation.

• Induction and maintenance of cell differentiation.

• Can influence signal transduction and regulation of gene expression.

Examples: gelatin, fibronectin, vitronectin, laminin, collagen & Matrigel

Extracellular Matrix

In vitro models for AngiogenesisCord Formation Assay

Tube Formation Assay

Cell Migration Assay

Cell Proliferation Assay

Gelatin Zymography

Cord Formation Assay

Endothelial cells are incubated in growth factor containing matrigel

Then they were trypsinized and resuspended in the same medium and dispersed onto the matrigel.

Cord formation in each well is monitored and photographed using an inverted microscope.

Control

+ Inhibitor

The endothelial cells are isolated and cultured in medium in gelatin coated flasks.

After gelation at 37C for 30 min the gels are overlaid with basal medium supplemented with test substances at desired concentrations.

Gels are examined and the tube length is determined for each well followed by determination of each group by using software.

Tube Formation Assay

Cell Migration Assay

Polycarbonate + 12uM

Cell Proliferation AssayControl

+ Inhibitor

The proliferation studies are based on cell counting, thymidine incorporation (or) Immunohistochemical staining for proliferation (or) cell death.

Endothelial cells are isolated and cultured in medium at 37C in a humidified atmosphere containing 5 % CO2. Cell proliferation is determined using a 5-bromo-2'-deoxyuridine (BrdU) colorimetric assay kit.

Gelatin Zymography

MMP MMP +Inhibitor

This assay can also be called as Matrix Metalloproteinase (MMP) assay.

Gelatin is used as a substrate and is incorporated into poly acryl amide gels.

Samples are mixed with buffer loaded onto the gel and electrophoreses.

After electrophoreses the gels are incubated in activity buffer analyzed by densitography

Invasion assay

Rabbit or rat corneal assaySponge implantation modelsMatrigel plugsWound healing assay

In Vivo Models

Sponge Implantation Method

This model is used for the evaluation of angiogenesis and anti- angiogenic agents.

The mechanism involved in this is stimulation of inflammation by foreign substance leads to the

angiogenesis.

Sponge implant modelsSterile absorbable gel foam is used as a sponge .Incision is made at midline of the anaesthetized animal and gel piece is inserted in to subcutaneously.At 14th day the animals are sacrificed and gel foams are harvested and quantification is done for angiogenesis activity.

Matrigel plug AssayUsed for the evaluation of both angiogenic and anti-angiogenic agents.

The mostly used animal model is mice.

The mechanism involved in this model is injection of foreign substances in to the animal leads to the stimulation of the inflammatory cells including macrophages and neutrophils that leads to the stimulation of angiogenesis.

Matrigel is a gelatinous material derived from mouse tumor cells

Matrigel plugs

Mice injected with VEGF supplemented Matrigel were left untreated or were treated with the angiogenesis inhibitor which fully

suppresses angiogenesis, as visible macroscopically and microscopically.

Corneal Angiogenesis AssayA pocket is created in the cornea where the compound of interest is inserted.

To induce an angiogenic response, a variety of materials including sponges, ethylene vinyl copolymer, or Hydron, containing an angiogenic substance (i.e. FGF-2 of VEGF) are implanted in "pockets"

The vascular response can then be monitored by direct observation using either a microscope or can be quantified by computer image analysis after perfusion of the cornea.

Wound Healing Assay

Two circular holes of approximately 5 mm in diameter are punched with a tissue puncher through the dorsal skin of an anesthetized mouse.

Wound size, scar formation and re-epithelization of the wounds should be recorded daily by photography and by measuring the wound area with calipers.

Treatment can consist of pro-or anti-angiogenic compounds, and their effects on angiogenesis is determined post mortem after the regenerated tissue has been excised, fixed and stained. Transgenic or knock-out mice can be used, when available, to study the specific effects of particular genes.

Inflammation, new tissue formation and remodeling

Hind Limb Ischemia Model

This method is mostly used for the evaluation of angiogenesis substances.

The mechanism involved in this model is haemodynamic changes leads to the formation of new blood vessels i.e. while large vessels with low flow tend to augmentation of blood flow which leads to the stimulation of vascular sprouting and maintain the potency of the newly formed collateral vessels thereby providing blood flow to theischemic tissue

the animals are anaesthetized and incision is making in the skin overlying the middle portion of the hind limb. Then the proximal end of the femoral artery is ligating and distal portion of saphenous artery is ligating and artery and their side branches were dissected free. The femoral artery and attached side branched are excised and overlying skin is then closed.

IN- OVA ASSAY

Pilot method for most of the angiogenesis evaluation studies.

Fertilized chicken eggs on the second day of incubation is and incubated at 37C at constant humidity.

On day 3 small hole is drill at narrow end and the albumin is withdrawn.

At the 7th day of incubation a small square window is open in the shell and test substances are implanted on the top of the membrane.

The window was sealed and reincubated. Eggs are incubated up to appropriate incubation time and angiogenesis is quantified

Xenograft model On day third window made

and removing 2-3 drops of albumin

Resealed and kept for incubation

On day 10 plastic rings along with cells were placed and kept for incubation

On day 17th the eggs were opened and observed for the formation of solid tumor

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