In vitro and ex vivo retina angiogenesis assays

Abstract

Pathological angiogenesis of the retina is a key component of irreversible causes of blindness, as observed in proliferative diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity. Seminal studies in the early 1980 s about the angiogenic activity exerted by mammalian retinal tissue extracts on the chick embryo chorioallantoic membrane and the later discovery of vascular endothelial growth factor (VEGF) accumulation in eyes of patients with diabetic retinopathy paved the way for the development of anti-angiogenic VEGF blockers for the treatment of retinal neovascularization. Since then, numerous preclinical and clinical studies about diabetic retinopathy and other retinal disorders have opened new lines of angiogenesis inquiry, indicating that limitations to anti-VEGF therapies may exist. Moreover, the production of growth factors other than VEGF may affect the response to anti-VEGF approaches. Thus, experimental models of retinal angiogenesis remain crucial for investigating novel anti-angiogenic therapies and bringing them to patients. To this aim, in vitro and ex vivo angiogenesis assays may be suitable for a rapid screening of potential anti-angiogenic molecules before in vivo validation of the putative lead compounds. This review focuses on the different in vitro and ex vivo angiogenesis assays that have been developed over the years based on the isolation of endothelial cells from the retina of various animal species and ex vivo cultures of neonatal and adult retina explants. Also, recent observations have shown that eye neovascularization in zebrafish (Danio rerio) embryos, an in vivo animal platform experimentally analogous to in vitro/ex vivo models, may represent a novel target for the identification of angiogenesis inhibitors. When compared to in vivo assays, in vitro and ex vivo models of retina neovascularization, including zebrafish embryo, may represent cost-effective and rapid tools for the screening of novel anti-angiogenic therapeutics.

Consensus guidelines for the use and interpretation of angiogenesis assays

“…Unfortunately, without the support of a 3D matrix, retinal cells cannot survive for long periods, thus making studies of later stages of angiogenesis impossible. To overcome this, Rezzola et al have improved the assay by embedding the retinas in different matrices after dissection [84]. In this approach, retinas can be crosscut into four equal pieces and left in serum-free media overnight.…”

Therapeutic Potential of Anti-Angiogenic Multitarget N,O-Sulfated E. Coli K5 Polysaccharide in Diabetic Retinopathy

“…Fibrin-embedded retina fragments (32,33) were treated with 75 ng/mL VEGF and increasing K5-N,OS(H) concentrations. At day 7, vessel sprouts were counted.…”

New artery of knowledge: 3D models of angiogenesis

“…This system allows to study sprouting from a native tissue, and to test the efficacy of pro-and anti-angiogenic molecules in a physiological environment Lack of flow. High results variability depending on background and age of the animal or donor (79,80,81) Retinal explant assay Studying microvessel formation and its remodelling (82,83) In vivo In-ovo chorioallantoic membrane assay Assessing the vascularisation of allografts or xenografts. Testing the effect of pro-angiogenic and anti-angiogenic treatments EC sprouting compared to more-degradable ones (53,54).…”