Microvascular Experimentation in the Chick Chorioallantoic Membrane as a Model for Screening Angiogenic Agents including from Gene-Modified Cells

The chick chorioallantoic membrane (CAM) assay model of angiogenesis has been highlighted as a relatively quick, low cost and effective model for the study of pro-angiogenic and anti-angiogenic factors. The chick CAM is a highly vascularised extraembryonic membrane which functions for gas exchange, nutrient exchange and waste removal for the growing chick embryo. It is beneficial as it can function as a treatment screening tool, which bridges the gap between cell based in vitro studies and in vivo animal experimentation. In this review, we explore the benefits and drawbacks of the CAM assay to study microcirculation, by the investigation of each distinct stage of the CAM assay procedure, including cultivation techniques, treatment applications and methods of determining an angiogenic response using this assay. We detail the angiogenic effect of treatments, including drugs, metabolites, genes and cells used in conjunction with the CAM assay, while also highlighting the testing of genetically modified cells. We also present a detailed exploration of the advantages and limitations of different CAM analysis techniques, including visual assessment, histological and molecular analysis along with vascular casting methods and live blood flow observations.

The Releasate of Avascular Cartilage Demonstrates Inherent Pro-Angiogenic Properties In Vitro and In Vivo

Objective Cartilage is avascular and numerous studies have identified the presence of single anti- and pro-angiogenic factors in cartilage. To better understand the maintenance hyaline cartilage, we assessed the angiogenic potential of complete cartilage releasate with functional assays in vitro and in vivo. Design We evaluated the gene expression profile of angiogenesis-related factors in healthy adult human articular cartilage with a transcriptome-wide analysis generated by next-generation RNAseq. The effect on angiogenesis of the releasate of cartilage tissue was assessed with a chick chorioallantoic membrane (CAM) assay as well as human umbilical vein endothelial cell (HUVEC) migration and proliferation assays using conditioned media generated from tissue-engineered cartilage derived from human articular and nasal septum chondrocytes as well as explants from bovine articular cartilage and human nasal septum. Experiments were done with triplicate samples of cartilage from 3 different donors. Results RNAseq data of 3 healthy human articular cartilage donors revealed that the majority of known angiogenesis-related factors expressed in healthy adult articular cartilage are pro-angiogenic. The releasate from generated cartilage as well as from tissue explants, demonstrated at least a 3.1-fold increase in HUVEC proliferation and migration indicating a pro-angiogenic effect of cartilage. Finally, the CAM assay demonstrated that cartilage explants can indeed attract vessels; however, their ingrowth was not observed. Conclusion Using multiple approaches, we show that cartilage releasate has an inherent pro-angiogenic capacity. It remains vessel free due to anti-invasive properties associated with the tissue itself.

Extending the Applicability of In Ovo and Ex Ovo Chicken Chorioallantoic Membrane Assays to Study Cytostatic Activity in Neuroblastoma Cells

PurposeThe chick chorioallantoic membrane (CAM) assay can provide an alternative versatile, cost-effective, and ethically less controversial in vivo model for reliable screening of drugs. In the presented work, we demonstrate that CAM assay (in ovo and ex ovo) can be simply employed to delineate the effects of cisplatin (CDDP) and ellipticine (Elli) on neuroblastoma (Nbl) cells in terms of their growth and metastatic potential.MethodsThe Nbl UKF-NB-4 cell line was established from recurrent bone marrow metastases of high-risk Nbl (stage IV, MYCN amplification, 7q21 gain). Ex ovo and in ovo CAM assays were optimized to evaluate the antimetastatic activity of CDDP and Elli. Immunohistochemistry, qRT-PCR, and DNA isolation were performed.ResultsEx ovo CAM assay was employed to study whether CDDP and Elli exhibit any inhibitory effects on growth of Nbl xenograft in ex ovo CAM assay. Under the optimal conditions, Elli and CDDP exhibited significant inhibition of the size of the primary tumor. To study the efficiency of CDDP and Elli to inhibit primary Nbl tumor growth, intravasation, and extravasation in the organs, we adapted the in ovo CAM assay protocol. In in ovo CAM assay, both studied compounds (CDDP and Elli) exhibited significant (p < 0.001) inhibitory activity against extravasation to all investigated organs including distal CAM.ConclusionsTaken together, CAM assay could be a helpful and highly efficient in vivo approach for high-throughput screening of libraries of compounds with expected anticancer activities.

Neuroprotection in early stages of Alzheimer’s disease is promoted by transthyretin angiogenic properties

Background While still controversial, it has been demonstrated that vascular defects can precede the onset of other AD hallmarks features, making it an important therapeutic target. Given that the protein transthyretin (TTR) has been established as neuroprotective in AD, here we investigated the influence of TTR in the vasculature. Methods We evaluated the thickness of the basement membrane and the length of brain microvessels, by immunohistochemistry, in AβPPswe/PS1A246E (AD) transgenic mice and non-transgenic mice (NT) bearing one (TTR+/−) or two (TTR+/+) copies of the TTR gene. The angiogenic potential of TTR was evaluated in vitro using the tube formation assay, and in vivo using the chick chorioallantoic membrane (CAM) assay. Results AD transgenic mice with TTR genetic reduction, AD/TTR+/−, exhibited a thicker BM in brain microvessels and decreased vessel length than animals with normal TTR levels, AD/TTR+/+. Further in vivo investigation, using the CAM assay, revealed that TTR is a pro-angiogenic molecule, and the neovessels formed are functional. Also, TTR increased the expression of key angiogenic molecules such as proteins interleukins 6 and 8, angiopoietin 2, and vascular endothelial growth factor, by endothelial cells, in vitro, under tube formation conditions. We showed that while TTR reduction also leads to a thicker BM in NT mice, this effect is more pronounced in AD mice than in NT animals, strengthening the idea that TTR is a neuroprotective protein. We also studied the effect of TTR tetrameric stabilization on BM thickness, showing that AD mice treated with the TTR tetrameric stabilizer iododiflunisal (IDIF) displayed a significant reduction of BM thickness and increased vessel length, when compared to non-treated littermates. Conclusion Our in vivo results demonstrate the involvement of TTR in angiogenesis, particularly as a modulator of vascular alterations occurring in AD. Since TTR is decreased early in AD, its tetrameric stabilization can represent a therapeutic avenue for the early treatment of AD through the maintenance of the vascular structure.

Neonauclea formicaria (Rubiaceae) Leaf Extract Inhibits Vascularization in the Chorioallantoic Membrane of Duck Embryos

Plants are reservoirs of bioactive compounds with the potential for pharmaceutical use. In this study, the secondary metabolites of Neonauclea formicaria leaf crude ethanolic extract were determined using phytochemical screening. The plant's leaf extract was then used to test its angiogenesis activity using the chorioallantoic membrane (CAM) assay. Four concentrations of the extract were prepared—0.1 mg/L, 1.0 mg/L, 10.0 mg/L, and 100.0 mg/L and were topically applied on the CAM. Phytochemical screening revealed that N. formicaria leaves contain heavy amounts of flavonoids and tannins, while alkaloids, saponins, and steroids were present in trace amounts. The crude ethanolic extract was anti-angiogenic, as indicated by the significant decrease of vascular density at higher concentrations (P<0.05). The 100 mg/L extract concentration showed the highest vascular inhibition (50.93%) among the other concentrations, suggesting its angiopreventive potential (P<0.05). Further investigation on the embryo's gross morphometry revealed no significant effects in the weight, crown-rump length, head-beak length, forelimb length, and hind limb length. Also, these indices were not associated with the angiogenesis activity on the CAM. Further studies exploring the specific metabolites of the different plant parts of N. formicaria and the plant's angiopreventive potential are recommended.

Determination of Ocular Irritancy Potential of Ophthalmic products using HET-CAM Method

Objective: The research is about the stability of ophthalmic products as they start to produce irritancy effects during their shelf life. The aim of the present study is to assess the stability of certain ophthalmic products by the HET-CAM (Hen’s Egg Test-Chorioallatonic membrane) Method. Method: Incubated Eggs were utilized for performing the HET-CAM assay according to the protocol prescribed by ICCVAM (The Inter agency Coordinating Committee on the Validation of Alternative Methods). Results: Calculation of various irritancy factors like hemorrhage time, vascular lysis time and coagulation time and to calculate HET-CAM score. Statistical interpretation of the data obtained by one-way ANOVA. Conclusion: Results have shown that the few ophthalmic products are slight and moderate irritants. None of the products are strong irritants. However further studies to be conducted to confirm the potential and safety of the products.