In vitro and ex vivo retina angiogenesis assays (original) (raw)

A novel ex vivo murine retina angiogenesis (EMRA) assay

Experimental Eye Research, 2013

Keywords: angiogenesis bevacizumab endothelium mouse ranibizumab retina VEGF a b s t r a c t Pathological retinal angiogenesis results from the imbalance of pro-angiogenic and anti-angiogenic factors. In particular, vascular endothelial growth factor (VEGF) plays a pivotal role in retinal neovascularization and various therapeutic VEGF blockers have evolved over time. Nevertheless, new retinal angiogenesis models are crucial for investigating anti-angiogenic therapies and bringing them to patients. Here, we developed a novel ex vivo murine retina angiogenesis (EMRA) assay in which endothelial sprouts originate from mature and quiescent retinal vessels. In this model, retina fragments from adult mice are embedded in a three-dimensional fibrin gel in the presence of human recombinant VEGF. Starting from the 3rde4th day of incubation, endothelial cell sprouts invading the fibrin gel can be observed under an inverted microscope and measured at different time points thereafter. The effect of VEGF is dose-dependent, maximal stimulation being observed at day 7 for retina fragments stimulated with 25e75 ng/ml of the growth factor. To assess whether the EMRA assay is suitable for testing the activity of anti-angiogenic compounds, retina fragments were incubated with VEGF in the presence of the neutralizing anti-VEGF antibodies bevacizumab and ranibizumab. The results demonstrate that both antibodies inhibit VEGF activity in a dose-dependent manner. In conclusion, the EMRA assay represents a new ex vivo model of retinal neovascularization suitable for the rapid screening of novel anti-angiogenic therapeutics.

A Critical Analysis of the AvailableIn VitroandEx VivoMethods to Study Retinal Angiogenesis

Journal of Ophthalmology, 2017

Angiogenesis is a biological process with a central role in retinal diseases. The choice of the ideal method to study angiogenesis, particularly in the retina, remains a problem. Angiogenesis can be assessed throughin vitroandin vivostudies. In spite of inherent limitations,in vitrostudies are faster, easier to perform and quantify, and typically less expensive and allow the study of isolated angiogenesis steps. We performed a systematic review of PubMed searching for original articles that appliedin vitroor ex vivo angiogenic retinal assays until May 2017, presenting the available assays and discussing their applicability, advantages, and disadvantages. Most of the studies evaluated migration, proliferation, and tube formation of endothelial cells in response to inhibitory or stimulatory compounds. Other aspects of angiogenesis were studied by assessing cell permeability, adhesion, or apoptosis, as well as by implementing organotypic models of the retina. Emphasis is placed on how ...

Rapid Analysis of Angiogenesis Drugs in a Live Fluorescent Zebrafish Assay

Arteriosclerosis, Thrombosis, and Vascular Biology, 2003

Solid tumors require an adequate supply of blood vessels to survive, grow, and metastasize. 1-3 New blood vessels that nourish growing tumors form by angiogenesis. Drugs shown to have antiangiogenic activity are currently in clinical cancer trials. 4 To date, anti-angiogenic drugs have had mixed success in clinical application. Many new compounds may need to be tested to identify drugs capable of treating a wide range of tumors. The ideal assay for screening new compounds should involve blood vessels growing in their natural environment, such as a whole living organism, yet be amenable to rapid analysis. No current assays provide such a unique combination. We describe here an assay using the zebrafish (Danio rerio) that provides the relevance of an in vivo environment as well as the potential for high throughput drug screening. The zebrafish has become a well accepted model for studies of vertebrate developmental biology. The vascular system has been well described and shown to be highly conserved in the zebrafish. 5,6 Many zebrafish blood vessels form by angiogenic sprouting and appear to require the same proteins that are necessary for blood vessel growth in mammals. In addition, anti-angiogenic compounds, such as PTK787/ZK222584 and SU5416, have been shown to affect the formation of zebrafish blood vessels. 7,8 Current methods of visualizing blood vessels in the zebrafish include whole mount in situ hybridization, 9,10 detection of endogenous alkaline phosphatase activity, 8 and microangiography. 11 The first two methods are time consuming and involve fixation of embryos and larvae prior to analysis. Microangiography is also labor intensive and only useful for visualization of patent blood vessels in a complete circulatory system. Transgenic zebrafish with fluorescent blood vessels 12,13 represent a less labor-intensive way of visualizing blood vessels in the zebrafish. We have generated a transgenic line with fluorescent blood vessels by driving expression of a green reef coral fluorescent protein (G-RCFP 14) with a promoter for the vascular endothelial growth factor receptor 2 gene (VEGFR2, also referred to as Flk-1 or KDR). VEGFR2 is one of several receptors for VEGF family members in humans and is expressed specifically in blood vessels. 15 Several zebrafish VEGFR2 cDNAs have been cloned and their expression pattern described. 7,9,10,16 We used a 6.5-KB genomic fragment 5Ј to the VEGFR2 initiation codon to drive G-RCFP expression specifically in zebrafish blood vessels. Hereafter, the stable transgenic line generated is referred to as TG(VEGFR2:G-RCFP), using standard zebrafish nomenclature. The expression pattern of G-RCFP in TG(VEGFR2:G-RCFP) embryos mirrored that of VEGFR2 in situ hybridization (not shown). Please see Figure I, available at http:// atvb.ahajournals.org, for detailed fluorescent expression in TG(VEGFR2:G-RCFP) embryos. The bright, consistent fluorescence of angiogenic blood vessels in TG(VEGFR2:G-RCFP) zebrafish embryos suggested that they could provide an ideal tool for testing angiogenesis drugs. Jain et al 17 described several factors that are important for the design of an optimal angiogenesis assay, including ease of experimentation, cost-effectiveness, rapidity, reproducibility, and ability to quantify vessel formation. Pairs of adult zebrafish produce thousands of eggs routinely and are relatively inexpensive to maintain, thus fulfilling the first two criteria. To test the utility of the TG(VEGFR2:G-RCFP) line for angiogenesis drug screening, we subjected embryos to compounds known to have anti-angiogenic activity, including SU5416 18,19 and SU6668, 20 two indolinone-based small molecules demonstrated to inhibit VEGF-induced vascular endothelial cell proliferation in vitro. Furthermore, SU5416 and SU6668 have been reported to inhibit the formation of metastases and microvessel formation and increase apoptosis of both tumor cells and tumor endothelial cells in mouse xenograft models. 21 SU5416 is believed to specifically target VEGF receptors, 18,22 while SU6668 inhibits the basic fibroblast growth

Review of the Ocular Angiogenesis Animal Models

Seminars in Ophthalmology, 2009

Increasing interest in developing reliable and reproducible models to study angiogenesis has emerged due to recent advances in the treatment of eye disease with pathologic angiogenesis. This review provides a summary of the principal ocular animal models for angiogenesis. Models of anterior segment neovascularization include the corneal micropocket assay, used to study the influence of specific molecules/proteins in angiogenesis, and corneal chemical and suture induced injury, which mimic more closely the complex nature of the human disease. Angiogenesis models of the posterior segment include the well-known laser-induced injury of the choroid/Bruch's membrane, as well as the oxygen induced retinopathy and models of injections of pro-angiogenic/inflammatory molecules. In addition, knockout or knock-in transgenic mice provide powerful tools in studying the role of specific proteins in angiogenesis.

Zebrafish angiogenesis: a new model for drug screening

Angiogenesis, 1999

Angiogenesis is necessary for tumor growth, making inhibition of vessel formation an excellent target for cancer therapy. Current assays for angiogenesis, however, are too complex to be practical for drug screening. Here, we demonstrate that the zebrafish is a viable whole animal model for screening small molecules that affect blood vessel formation. Blood vessel patterning is highly characteristic in the developing zebrafish embryo and the subintestinal vessels (SIVs) can be stained and visualized microscopically as a primary screen for compounds that affect angiogenesis. Small molecules added directly to the fish culture media diffuse into the embryo and induce observable, dose-dependent effects. To evaluate the zebrafish as a model, we used two angiogenesis inhibitors, SU5416 and TNP470, both of which have been tested in mammalian systems. Both compounds caused a reduction in vessel formation when introduced to zebrafish embryos prior to the onset of angiogenesis. Short duration ...

In vivo models of angiogenesis

Journal of Cellular and Molecular Medicine, 2006

The process of building new blood vessels (angiogenesis) and controlling the propagation of blood vessels (anti-angiogenesis) are fundamental to human health, as they play key roles in wound healing and tissue growth. More than 500 million people may stand to benefit from anti-or pro-angiogenic treatments in the coming decades*. The use of animal models to assay angiogenesis is crucial to the search for therapeutic agents that inhibit angiogenesis in the clinical setting. Examples of persons that would * Correspondence to: Klas NORRBY, MD, PhD benefit from these therapies are cancer patients, as cancer growth and spread is angiogenesis-dependent, and patients with aberrant angiogenesis in the eye, which may lead to blindness or defective sight. Recently, anti-angiogenesis therapies have been introduced successfully in the clinic, representing a turning point in tumor therapy and the treatment of macular degeneration and heralding a new era for the treatment of several commonly occurring angiogenesis-related diseases. On the other hand, pro-angiogenic therapies that promote compensatory angiogenesis in hypoxic tissues, such as those subjected to ischemia in myocardial or cerebral hypoxia due to occluding lesions in the coronary or cerebral arteries, respectively, and in cases of poor wound healing, are also being developed. In this review, the current major and newly introduced preclinical angiogenesis assays are described and discussed in terms of their specific advantages and disadvantages from the biological, technical, economical and ethical perspectives. These assays include the corneal micropocket, chick chorioallantoic membrane, rodent mesentery, subcutaneous (s.c.) sponge/matrix/alginate microbead, s.c. Matrigel plug, s.c. disc, and s.c. directed in vivo angiogenesis assays, as well as, the zebrafish system and several additional assays. A note on quantitative techniques for assessing angiogenesis in patients is also included. The currently utilized preclinical assays are not equivalent in terms of efficacy or relevance to human disease. Some of these assays have significance for screening, while others are used primarily in studies of dosage-effects, molecular structure activities, and the combined effects of two or more agents on angiogenesis. When invited to write this review, I was asked to describe in some detail the rodent mesenteric-window angiogenesis assay, which has not received extensive coverage in previous reviews.

Selective Inhibition of Retinal Angiogenesis by Targeting PI3 Kinase

2009

Ocular neovascularisation is a pathological hallmark of some forms of debilitating blindness including diabetic retinopathy, age related macular degeneration and retinopathy of prematurity. Current therapies for delaying unwanted ocular angiogenesis include laser surgery or molecular inhibition of the pro-angiogenic factor VEGF. However, targeting of angiogenic pathways other than, or in combination to VEGF, may lead to more effective and safer inhibitors of intraocular angiogenesis. In a small chemical screen using zebrafish, we identify LY294002 as an effective and selective inhibitor of both developmental and ectopic hyaloid angiogenesis in the eye. LY294002, a PI3 kinase inhibitor, exerts its anti-angiogenic effect in a dose-dependent manner, without perturbing existing vessels. Significantly, LY294002 delivered by intraocular injection, significantly inhibits ocular angiogenesis without systemic side-effects and without diminishing visual function. Thus, targeting of PI3 kinase pathways has the potential to effectively and safely treat neovascularisation in eye disease.

The Mouse Retina as an Angiogenesis Model

Investigative Ophthalmology & Visual Science, 2010

The mouse retina has been used extensively over the past decades to study both physiologic and pathologic angiogenesis. Over time, various mouse retina models have evolved into well-characterized and robust tools for in vivo angiogenesis research. This article is a review of the angiogenic development of the mouse retina and a discussion of some of the most widely used vascular disease models. From the multitude of studies performed in the mouse retina, a selection of representative works is discussed in more detail regarding their role in advancing the understanding of both the ocular and general mechanisms of angiogenesis. (Invest Ophthalmol Vis Sci.