Chorioallantoic Membrane Vasculature Research Articles

Short communication: Characterizing arterial and venous blood gases over the gas exchange surface, the chorioallantoic membrane, of embryonic American alligators (Alligator mississippiensis) at two points of development

Assessments of arterial and venous blood gases are required to understand the function of respiratory organs in animals at different stages of development. We measured blood gases in the arteries entering and veins leaving the chorioallantoic membrane (CAM) in embryonic alligators (Alligator mississippiensis). The CAM accounts for virtually all gas exchange in these animals, and we hypothesized that the CAM vasculature would be larger in eggs incubated in hypoxia (10% O2 for 50% or 70% of incubation), which would be reflected in a lower partial pressure of CO2 (PCO2). Contrary to this hypothesis, our measurements revealed no effects of hypoxic incubation on PCO2, and seemingly no increase in vascularization of the CAM in response to incubation in 10% O2. PCO2 was lower on the venous side, but only significantly different from arterial blood at 70% of incubation. The calculated blood flow to the CAM increased with development and was lower in both groups of alligators that had been incubated in hypoxia. Future studies should include measurements of blood parameters taken from embryos held in conditions that mirror incubation O2 levels, in combination with direct measurements of CAM artery blood flow.

Differential Angiogenic Induction Impacts Nasal Polyp Tissue Growth.

In chronic rhinosinusitis with nasal polyps, inflammatory edema drives tissue remodeling favoring anomalous growth of the nasal mucosa, but a proangiogenic contribution of nasal polyp in support of tissue growth is still controversial. The chorioallantoic membrane of chicken embryo model was employed to address the potentiality of nasal tissue fragments to modulate angiogenesis. Fifty-seven fertilized eggs were implanted with polyp or healthy nasal mucosa tissue or were kept as non-implanted controls. The embryos' size, length, and development stage, and chorioallantoic membrane vasculature morphology were evaluated after 48h. Quantitative computer vision techniques applied to digital chorioallantoic membrane images automatically calculated the branching index as the ratio between the areas of the convex polygon surrounding the vascular tree and the vessels' area. Ethics approval and consent to participate: the study was approved by the Human Research Ethics Committee of the Federal University of São Paulo (CAAE number: 80763117.1.0000.5505) and by the Animal Research Ethics Committee of University of São Paulo (nº CEUA 602-2019). Mucosal, but not polyp tissue implants, hampered embryo development and induced underdeveloped chorioallantoic membranes with anastomosed, interrupted, and regressive vessels. Vessels' areas and branching indexes were higher among the chorioallantoic membranes with polyp implants and controls than among those with healthy mucosa implants. Nasal polyp presents differential angiogenic induction that impacts tissue growth.

Exploration of Chick Embryo and Chorioallantoic Membrane on Imaging Navigated Platforms for Anticancer Pharmaceutical Evaluations.

Conforming to the current replace-reduce-refine 3Rs' guidelines in animal experiments, a series of explorative efforts have been made to set up operable biomedical imaging-guided platforms for qualitative and quantitative evaluations on pharmacological effects of tumor vascular-disrupting agents (VDAs), based on the chick embryos (CEs) with its chorioallantoic membrane (CAM), in this overview. The techniques and platforms have been hierarchically elaborated, from macroscopic to microscopic and from overall to specific aspects. A protocol of LED lamplight associated with a new deep-learning algorithm was consolidated to screen out weak CEs by using the CAM vasculature imaging. 3D magnetic resonance imaging (MRI) and laser speckle contrast imaging (LSCI) to monitor the evolution of CE and vascular changes in CAM are introduced. A LSCI-CAM platform for studying the effects of VDAs on normal and cancerous vasculature of CAM and possible molecular mechanisms has been demonstrated. Finally, practical challenges and future perspectives are highlighted. The aim of this article is to help peers in biomedical research to familiarize with the CAM platform and to optimize imaging protocols for the evaluation of vasoactive pharmaceuticals, especially anticancer vascular targeted therapy.

Bridging the In Vitro to In Vivo gap: Using the Chick Embryo Model to Accelerate Nanoparticle Validation and Qualification for In Vivo studies.

We postulate that nanoparticles (NPs) for use in therapeutic applications have largely not realized their clinical potential due to an overall inability to use in vitro results to predict NP performance in vivo. The avian embryo and associated chorioallantoic membrane (CAM) has emerged as an in vivo preclinical model that bridges the gap between in vitro and in vivo, enabling rapid screening of NP behavior under physiologically relevant conditions and providing a rapid, accessible, economical, and more ethical means of qualifying nanoparticles for in vivo use. The CAM is highly vascularized and mimics the diverging/converging vasculature of the liver, spleen, and lungs that serve as nanoparticle traps. Intravital imaging of fluorescently labeled NPs injected into the CAM vasculature enables immediate assessment and quantification of nano-bio interactions at the individual NP scale in any tissue of interest that is perfused with a microvasculature. In this review, we highlight how utilization of the avian embryo and its CAM as a preclinical model can be used to understand NP stability in blood and tissues, extravasation, biocompatibility, and NP distribution over time, thereby serving to identify a subset of NPs with the requisite stability and performance to introduce into rodent models and enabling the development of structure-property relationships and NP optimization without the sacrifice of large populations of mice or other rodents. We then review how the chicken embryo and CAM model systems have been used to accelerate the development of NP delivery and imaging agents by allowing direct visualization of targeted (active) and nontargeted (passive) NP binding, internalization, and cargo delivery to individual cells (of relevance for the treatment of leukemia and metastatic cancer) and cellular ensembles (e.g., cancer xenografts of interest for treatment or imaging of cancer tumors). We conclude by showcasing emerging techniques for the utilization of the CAM in future nano-bio studies.

Development and characterization of a chick embryo chorioallantoic membrane (CAM) based platform for evaluation of vasoactive medications.

Among various anti-cancer therapies, tumor vascular disrupting agents (VDAs) play a crucial role, for which their off-targeting effects on normal vessels need also to be investigated. The purpose of this study was to set up an in-ovo platform that combines a laser speckle contrast imaging (LSCI) modality with chick embryo chorioallantoic membrane (CAM) to real-time monitor vascular diameters and perfusion without and with intravascular injection. Two eggshell windows for both observation or measurement and injection were opened. Dynamic blood perfusion images and corresponding statistic graphs were acquired by using a LSCI unit on CAMs from embryo date (ED) 9 to ED15. A dedicated fine needle catheter was made for slow intravascular administration over 30min with simultaneous LSCI acquisition. To verify the connectivity between CAM vessels and the embryonic circulations in the egg, contrast-enhanced 3D micro computed tomography (μCT), 2D angiography and histology were executed. This platform was successfully established to acquire, quantify and demonstrate vascular and hemodynamic information from the CAM. Chick embryos even with air cell opened remained alive from ED9 to ED15. Through collecting LSCI derived CAM vascular diameter and perfusion parameters, ED12 was determined as the best time window for vasoactive drug studies. A reverse correlation between CAM vessel diameter and blood perfusion rate was found (p<0.002). Intravascular infusion and simultaneous LSCI acquisition for 30min in ovo proved feasible. Contrast-enhanced angiography and histomorphology could characterize the connectivity between CAM vasculature and embryonic circulation. This LSCI-CAM platform was proved effective for investigating the in-ovo hemodynamics, which paves the road for further preclinical research on vasoactive medications including VDAs.

Utilisation of Chick Embryo Chorioallantoic Membrane as a Model Platform for Imaging-Navigated Biomedical Research.

The fertilised chick egg and particularly its chorioallantoic membrane (CAM) have drawn continuing interest in biomedicine and bioengineering fields, especially for research on vascular study, cancer, drug screening and development, cell factors, stem cells, etc. This literature review systemically introduces the CAM’s structural evolution, functions, vascular features and the circulation system, and cell regulatory factors. It also presents the major and updated applications of the CAM in assays for pharmacokinetics and biodistribution, drug efficacy and toxicology testing/screening in preclinical pharmacological research. The time course of CAM applications for different assays and their advantages and limitations are summarised. Among these applications, two aspects are emphasised: (1) potential utility of the CAM for preclinical studies on vascular-disrupting agents (VDAs), promising for anti-cancer vascular-targeted therapy, and (2) modern imaging technologies, including modalities and their applications for real-time visualisation, monitoring and evaluation of the changes in CAM vasculature as well as the interactions occurring after introducing the tested medical, pharmaceutical and biological agents into the system. The aim of this article is to help those working in the biomedical field to familiarise themselves with the chick embryo CAM as an alternative platform and to utilise it to design and optimise experimental settings for their specific research topics.

In vivo evidence of angiogenesis inhibition by β2-glycoprotein I subfractions in the chorioallantoic membrane of chicken embryos.

The vascular network expansion and functioning are important factors affecting normal intra-uterine fetal development. This study addressed the previously reported antiangiogenic potential of beta-2-glycoprotein I (β2GPI) in vivo in the chick embryo model of angiogenesis. The effects of two naturally occurring β2GPI forms on the development of the chorioallantoic membrane (CAM) vessels and the chicken embryo were investigated. β2GPI monomers and dimers were obtained by fractioned purification and characterized using SDS-PAGE, immunoblot, and ELISA. The egg exposure was performed by injection of small volumes of 2.5 µg/mL solutions of the β2GPI subfractions. Angiogenesis was evaluated through quantitative measurements of vascular architecture parameters in the captured CAM images, using computational analysis of texture contrasts and computer vision techniques. Quantitative information was assigned to the CAM vasculature modifications. In vivo, the β2GPI dimer completely halted the formation of CAM vessels and led to embryo death after 48 h of exposure. The β2GPI monomer allowed the embryo to develop up to the 10th day, despite early changes of CAM vessels. The impaired normal vessel growth proceeded as a self-limited effect. The β2GPI monomer-exposed eggs showed reduced vascularization on the 6th day of incubation, but embryos were viable on the 10th day of incubation, with ingurgitated CAM vessels implying sequelae of the angiogenesis inhibition. Both subfractions impaired CAM vasculature development. The β2GPI dimer proved to be largely more harmful than the β2GPI monomer. β2GPI modification by cleavage or dimerization may play a role in angiogenesis control in vivo.

Angiogenesis in the Avian Embryo Chorioallantoic Membrane: A Perspective on Research Trends and a Case Study on Toxicant Vascular Effects.

The chorioallantoic membrane (CAM) of the avian embryo is an intrinsically interesting gas exchange and osmoregulation organ. Beyond study by comparative biologists, however, the CAM vascular bed has been the focus of translational studies by cardiovascular life scientists interested in the CAM as a model for probing angiogenesis, heart development, and physiological functions. In this perspective article, we consider areas of cardiovascular research that have benefited from studies of the CAM, including the themes of investigation of the CAM’s hemodynamic influence on heart and central vessel development, use of the CAM as a model vascular bed for studying angiogenesis, and the CAM as an assay tool. A case study on CAM vascularization effects of very low doses of crude oil as a toxicant is also presented that embraces some of these themes, showing the induction of subtle changes in the pattern of the CAM vasculature growth that are not readily observed by standard vascular assessment methodologies. We conclude by raising several questions in the area of CAM research, including the following: (1) Do changes in patterns of CAM growth, as opposed to absolute CAM growth, have biological significance?; (2) How does the relative amount of CAM vascularization compared to the embryo per se change during development?; and (3) Is the CAM actually representative of the mammalian systemic vascular beds that it is presumed to model?

Gap junctions regulate vessel diameter in chick chorioallantoic membrane vasculature by both tone-dependent and structural mechanisms.

In this study, we examined the impact of gap junction blockade on chick chorioallantoic membrane microvessels. Expression of Cx37, Cx40/42, and Cx43 in chick chorioallantoic membrane tissue was studied by PCR, Western blot, and confocal immunofluorescence microscopy. Vessel diameter changes occurring under gap junction blockade with carbenoxolone (175µmol/L), palmitoleic acid (100µmol/L), 43 GAP27 (1mmol/L) were analyzed by intravital microscopy. To analyze vascular tone, chick chorioallantoic membrane vessels were exposed to a vasodilator cocktail consisting of acetylcholine (10μmol/L), adenosine (100μmol/L), papaverine (200μmol/L), and sodium nitroprusside (10μmol/L). In chick chorioallantoic membrane lysates, Western blot analysis revealed the expression of Cx40 and Cx43. Immunofluorescence in intact chick chorioallantoic membrane vasculature showed only Cx43, limited to arterial vessel walls. Upon gap junction blockade (3hours) arterial and venous diameters decreased to 0.50±0.03 and 0.36±0.06 (carbenoxolone), 0.72±0.08 and 0.63±0.15 (palmitoleic acid) and 0.77±0.004 and 0.58±0.05 (GAP27), relative to initial values. Initially, diameter decrease was dominated by increasing vascular tone. After 6hours, however, vessel tone was reduced, suggesting structural network remodeling. Our findings suggest a major role for connexins in mediating acute and chronic diameter changes in developing vascular networks.

Vascular alteration in relation to fosfomycine: In silico and in vivo investigations using a chick embryo model

Fosfomycin residues are found in the egg following administration in the layer hen. In this regard, some aspects of embryo-toxicity of fosfomycin have been documented previously. The exact mechanism by which fosfomycin causes embryo-toxicity is not clearly understood. We hypothesis that fosfomycin may alter vasculature as well as normal expression of genes, which are associated with vascular development. Therefore, the present study aimed to address these issues through in silico and in vivo investigations. At first, embryo-toxicity and anti-angiogenic effects of fosfomycin were tested using computerized programs. After that, fertile chicken eggs were treated with fosfomycin and chorioallantoic membrane vasculature was assessed through morphometric, molecular and histopathological assays. The results showed that fosfomycin not only interacted with VEGF-A protein and promoter, but also altered embryonic vasculature and decreased expression level of VEGF-A. Reticulin staining of treated group was also confirmed decreased vasculature. The minor groove of DNA was the preferential binding site for fosfomycin with its selective binding to GC-rich sequences. We suggested that the affinity of fosfomycin for VEGF-A protein and promoter as well as alteration of the angiogenic signaling pathway may cause vascular damage during embryonic growth. Hence, veterinarians should be aware of such effects and limit the use of this drug during the developmental stages of the embryo, particularly in breeder farms. Considering the anti-angiogenic activity and sequence selectivity of fosfomycin, a major advantage that seems to be very promising is the fact that it is possible to achieve a sequence-selective binding drug for cancer.

Ethyl acetate extract of Citrullus colocynthis (Linn.) Schrad. fruit suppresses angiogenesis

Context: Anti-angiogenesis is a targeted therapy that uses drugs or other substances to stop tumors from making new blood vessels. Citrullus colocynthis is a medicinal plant that has anticancer property mediated by apoptosis. Aims: To evaluate the anti-angiogenic potential of C. colocynthis fruits extract through chorioallantoic membrane (CAM) assay. Methods: For the CAM assay, heparin (an anti-angiogenic agent) was used as a positive control, and quercetin (Q) as the marker compound in the extracts to demonstrate the inhibition of angiogenesis. The reduction in the number of blood vessels in each area and in each image was used as a measure to determine the anti-angiogenic activity of the extract. Results: From the results of phytochemical screening, the polyphenol and flavonoid contents of the ethyl acetate extract were 0.6708 mg equivalent of gallic acid per gram of the dry extract and 7.65 mg equivalent of Q per gram of the dry extract, respectively. From the results of CAM assay, Q caused marked reduction in the formation of new blood vessels. Similarly, ethyl acetate extract of C. colocynthis fruits (50 µg/mL) showed 53.52% inhibition of blood vessel formation in the CAM vasculature, whereas 25 µg/mL of Q exhibited 64.78% inhibition. The analysis with Bonferroni’s post-hoc test showed statistically significant difference at p&lt;0.0001, with respect to the positive control, heparin. The EC50 values obtained by non-linear regression analysis using GraphPad Prism software 8.1.2 were 14.74 µg/mL (Q) and 44.13 µg/mL (ethyl acetate fruit extract of C. colocynthis). Conclusions: Ethyl acetate extract of C. colocynthis fruits demonstrated potential angiogenesis suppression activity.

Nanoparticles characterization using the CAM assay.

The current chapter highlights the use of chorioallantoic membrane (CAM) of fertilized chicken egg for the characterization of nanoparticles applied in cancer nanomedicine. The CAM assay represents a promising alternative to mouse models in term of costs, ease of use, rapidity and ethical issues in particular for the screening of nanoformulations. Hence, the features of nanoparticles including blood retention, biocompatibility, active targeting or tumor accumulation, angiogenic activity, drug delivery and tumor elimination might be simply evaluated via the CAM model. In particular, in this model, embryo organs and morphology, CAM vasculature and blood cells, transplanted tumors on CAM were typically monitored and used for the evaluation of the nanomaterials. With the above advantages, the CAM assay, as highly valuable in vivo model, could be used regularly in pharmaceutical industries.

An Improved In Vivo Methodology to Visualise Tumour Induced Changes in Vasculature Using the Chick Chorionic Allantoic Membrane Assay.

Decreasing the vascularity of a tumour has proven to be an effective strategy to suppress tumour growth and metastasis. Anti-angiogenic therapies have revolutionized the treatment of advanced-stage cancers, however there is still demand for further improvement. This necessitates new experimental models that will allow researchers to reliably study aspects of angiogenesis. The aim of this study was to demonstrate an in vivo technique in which the highly vascular and accessible chorioallantoic membrane (CAM) of the chick embryo is used to study tumour-induced changes in the macro and microvessels. Two cancer cell lines (human melanoma (C8161) and human prostate cancer (PC3)) were selected as model cells. Human dermal fibroblasts were used as a control. One million cells were labelled with green fluorescent protein and implanted on the CAM of the chick embryo at embryonic development day (EDD) 7 and angiogenesis was evaluated at EDDs 10, 12 and 14. A fluorescently-tagged lectin (lens culinaris agglutinin (LCA)) was injected intravenously into the chick embryo to label endothelial cells. The LCA is known to label the luminal surface of endothelial cells, or dextrans, in the CAM vasculature. Macrovessels were imaged by a hand-held digital microscope and images were processed for quantification. Microvessels were evaluated by confocal microscopy. Tumour invasion was assessed by histological and optical sectioning. Tumour cells (C8161 and PC3) produced quantifiable increases in the total area covered by blood vessels, compared to fibroblasts when assessed by digital microscopy. Tumour invasion could be demonstrated by both histological and optical sectioning. The most significant changes in tumour vasculature observed were in the microvascular structures adjacent to the tumour cells, which showed an increase in the endothelial cell coverage. Additionally, tumour intravasation and tumour thrombus formation could be detected in the areas adjacent to tumour cells. The fragility of tumour blood vessels could be demonstrated when tumour cells seeded on a synthetic scaffold were grown on CAM. We report on a modification to a well-studied CAM in vivo assay, which can be effectively used to study tumour induced changes in macro and microvasculature.

Thermal treatments prior to and during the beginning of incubation affects development of the broiler embryo and yolk sac membranes, and live performance and carcass characteristics

This study evaluated temperature during preincubation and embryonic day 0 (E0) E0 to E5 of incubation on broiler embryo development and subsequent live performance. Freshly laid eggs from a single 41-wk-old Ross 708 broiler breeder flock produced on a single day were weighed individually for weight matching purposes, stored overnight, and assigned to 4 treatment combinations of 2 preincubation temperatures (23.9 or 29.4°C)× 2 E0 to E5 temperatures (38.1 or 37.5°C). The 29.4°C preincubation temperature decreased (P ≤ 0.05) yolk sac membrane (YSM) vasculature at E6 and E7, and increased (P ≤ 0.05) embryo weight and length but decreased (P ≤ 0.05) yolk sac weight (YSW) at E15. No subsequent main effects were observed. The 38.1°C incubation temperature increased YSM vasculature at E7, chorioallantoic membrane (CAM) vasculature at E8 and E10, and egg weight loss, embryo weight, and embryo length at E15 and chick length at E21 in the presence of reduced BW and YSW (P ≤ 0.05). This was followed by greater male BW at 35 d, as well as improved FCR in females 0 to 14 d and in males 15 to 35 d (P ≤ 0.05). Pectoralis major and minor yields were increased (P ≤ 0.05) at 50 d of age in males and females, respectively. There were no interactions observed with regards to broiler live performance and carcass yield, which probably negated the importance of the interactions observed for preincubation temperature by E0 to E5 incubation temperature that affected YSM vasculature, CAM vasculature area, egg weight loss, embryo weight, yolk sac weight, and chick length.

Abstract B02: Ultrasound evaluation of anti-angiogenic therapy on patient-derived renal cell carcinoma xenograft tumors in the chicken embryo model

Abstract Introduction: The avian embryo patient-derived xenograft (PDX) model's use as a platform for pre-clinical drug testing is a promising application. However, conventional methods of monitoring, such as tumor-take rates, light microscopy, and histology, either do not provide sufficient detail for in-depth therapy evaluation or are reserved for end-point analysis. Ultrasound monitoring permits non-destructive longitudinal evaluation of tumor growth, progression, and angiogenesis in the chorioallantoic membrane (CAM) xenograft ex ovo model. High-frequency ultrasound was selected for its low cost, lack of ionizing radiation, and high spatial and temporal resolutions. Methods: A subset of tumor initiating cells (RCC243) was isolated from a patient-derived parental renal carcinoma cell line (RCC22). Cells were grown to confluence, pelleted, and combined with an equal volume of Matrigel. On the ninth day of embryonic development (EDD-9), the CAM surface of 8 animals was pierced, and 10µL of the cell-Matrigel mix was deposited into the opening. Half of the embryos were treated every two days with 10µL of TAK-441, a Hedgehog inhibitor with hypothesized anti-angiogenic effects. Three-dimensional anatomical (B-mode) and contrast-enhanced images were acquired using a Vevo 2100 ultrasound system (VisualSonics Inc.) equipped with a 20 MHz linear array transducer. On EDD-18, tumor volumes were assessed using the B-mode images. The CAM vasculature was then cannulated with a glass capillary needle, and a 50µL solution of Vevo MicroMarker (VisualSonics Inc.) microbubble contrast agent (2 x 109 microbubbles/mL) was injected in a pulsatile manner. Tumor blood perfusion (blood volume, velocity, and flow) was then assessed using destruction-reperfusion contrast ultrasound. Image analysis was performed within manually segmented tumor volumes. Results: Hedgehog inhibition of RCC243 tumors via TAK-441 therapy produced a significant decrease in mean tumor volume (vehicle: 187.68 ± 69.55 mm3 vs. treatment: 78.94 ± 52.35 mm3; p &amp;lt; 0.05) and blood flow (vehicle: 645.7 ± 261.5 mm3/min vs. treatment: 190.8 ± 133.4 mm3/min; p &amp;lt; 0.05). There was a non-significant trend of reduced blood volume (vehicle: 97.0 ± 64.7 mm3 vs. treatment: 33.7 ± 23.8 mm3; p = 0.12), and flow velocity (vehicle: 6.34 ± 1.07 mm/s vs. treatment: 5.52 ± 1.18 mm/s; p = 0.34) in the treated tumors. Conclusions: This proof of principal study shows that tumors implanted in an ex ovo chick CAM model can successfully be imaged using high-frequency ultrasound and quantitative measures of tumor volume, blood volume, velocity and flow can be obtained. Responses to anti-angiogenic therapy can be successfully quantified. This approach to therapy evaluation is inexpensive and permits observation of tumor-induced angiogenesis over a 9 day timeframe. Citation Format: Matthew R. Lowerison, Chantalle J. Willie, Siddika Pardhan, Nicholas E. Power, Ann F. Chambers, Hon S. Leong, James C. Lacefield. Ultrasound evaluation of anti-angiogenic therapy on patient-derived renal cell carcinoma xenograft tumors in the chicken embryo model. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B02.

Blood flow distribution following adrenergic blockade and nitric oxide production inhibition in embryonic American alligators, Alligator mississippiensis (879.4)

Assessment of cardiovascular function in developing egg‐laying amniotes has been primarily limited to measurements made via catheters in the chorioallantoic membrane (CAM) and potentially invasive femoral and major outflow tract flow probes. Here we assess the capacity of embryonic alligators, Alligator mississippiensis, to regulate blood flow distribution using a minimally invasive microsphere distribution technique at 70 and 90% of development, in eggs incubated in normoxia or 10% hypoxia. Measurements were made in normoxia, acute 10% hypoxia, and acute 10% hypoxia following nitric oxide inhibition or alpha‐receptor blockade. We hypothesized that the embryonic response is concomitant with a redistribution of blood flow to the brain, heart, and CAM vasculature. Embryonic alligators respond to hypoxia with an initial bradycardia followed by a hypertensive tachycardia, however chronic hypoxic incubation blunts this response. Our data suggest that cardiac output is reduced during acute hypoxia in normoxic‐incubated embryos while it is maintained the hypoxic group. In addition, tonic nitric oxide dilation is present at 70% of incubation however inhibition of this tone does not modify total cardiac output at 90% of incubation. Our data suggest that hypoxic development may modify the patterns of blood flow redistribution in embryonic alligators and the mechanisms used to achieve this.Grant Funding Source: NSF CAREER IBN IOS‐0845741 to DAC

Photodynamic Effect of Hypericin after Topical Application in the Ex Ovo Quail Chorioallantoic Membrane Model

Photosensitizing properties of hypericin are well known, and the chicken chorioallantoic membrane has previously been used to test photodynamic effects of hypericin and other substances. In our study the photodynamic effect of hypericin in the ex ovo quail chorioallantoic membrane model was evaluated. Steady-state and time-resolved fluorescence spectroscopy of hypericin solution in PEG-400 and its mixture in PBS was performed to assess and characterize the process of aggregation and disaggregation of hypericin during the drug formulation preparation. A therapeutical formulation (2 µg/g of embryo weight) was topically applied on CAM into the silicone ring. Hypericin was excited by diode laser with wavelength 405 nm, fluence rate 140 mW/cm2, and fluence 16.8 J/cm2. Hypericin in 100% PEG-400 exhibits typical fluorescence spectra with a maximum of about 600 nm, while hypericin 10% PEG-400 formulation exhibits almost no fluorescence. Time resolved spectra analysis showed fluorescence decay of hypericin in 100% PEG-400 solution with a mean lifetime of 5.1 ns and in 10% PEG 4.1 ns. Damage of quail chorioallantoic membrane vasculature after photodynamic therapy ranged from hemorrhage and vanishing of capillary vessels to thrombosis, lysis, and hemorrhage of larger vessels.The presented findings suggest that quail embryos can be used as a suitable model to test the effect of hypericin and other photodynamic compounds.

Gene Transfer to Chicks Using Lentiviral Vectors Administered via the Embryonic Chorioallantoic Membrane

The lack of affordable techniques for gene transfer in birds has inhibited the advancement of molecular studies in avian species. Here we demonstrate a new approach for introducing genes into chicken somatic tissues by administration of a lentiviral vector, derived from the feline immunodeficiency virus (FIV), into the chorioallantoic membrane (CAM) of chick embryos on embryonic day 11. The FIV-derived vectors carried yellow fluorescent protein (YFP) or recombinant alpha-melanocyte-stimulating hormone (α-MSH) genes, driven by the cytomegalovirus (CMV) promoter. Transgene expression, detected in chicks 2 days after hatch by quantitative real-time PCR, was mostly observed in the liver and spleen. Lower expression levels were also detected in the brain, kidney, heart and breast muscle. Immunofluorescence and flow cytometry analyses confirmed transgene expression in chick tissues at the protein level, demonstrating a transduction efficiency of ∼0.46% of liver cells. Integration of the viral vector into the chicken genome was demonstrated using genomic repetitive (CR1)-PCR amplification. Viability and stability of the transduced cells was confirmed using terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay, immunostaining with anti-proliferating cell nuclear antigen (anti-PCNA), and detection of transgene expression 51 days post transduction. Our approach led to only 9% drop in hatching efficiency compared to non-injected embryos, and all of the hatched chicks expressed the transgenes. We suggest that the transduction efficiency of FIV vectors combined with the accessibility of the CAM vasculature as a delivery route comprise a new powerful and practical approach for gene delivery into somatic tissues of chickens. Most relevant is the efficient transduction of the liver, which specializes in the production and secretion of proteins, thereby providing an optimal target for prolonged study of secreted hormones and peptides.

Neuropilin-1 regulates platelet-derived growth factor receptor signalling in mesenchymal stem cells

Using human MSCs (mesenchymal stem cells) lacking VEGF (vascular endothelial growth factor) receptors, we show that the pro-angiogenic receptor neuropilin-1 associates with phosphorylated PDGFRs [PDGF (platelet-derived growth factor) receptors], thereby regulating cell signalling, migration, proliferation and network assembly. Neuropilin-1 co-immunoprecipitated and co-localized with phosphorylated PDGFRs in the presence of growth factors. Neuropilin-1 knockdown blocked PDGF-AA-induced PDGFRα phosphorylation and migration, reduced PDGF-BB-induced PDGFRβ activation and migration, blocked VEGF-A activation of both PDGFRs, and attenuated proliferation. Neuropilin-1 prominently co-localized with both PDGFRs within MSC networks assembled in Matrigel™ and in the chorioallantoic membrane vasculature microenvironment, and its knockdown grossly disrupted network assembly and decreased PDGFR signalling. Thus neuropilin-1 regulates MSCs by forming ligand-specific receptor complexes that direct PDGFR signalling, especially the PDGFRα homodimer. This receptor cross-talk may control the mobilization of MSCs in neovascularization and tissue remodelling.

Processing of fluorescence angiograms for the quantification of vascular effects induced by anti-angiogenic agents in the CAM model

The chicken embryo chorioallantoic membrane (CAM) is widely used as an in vivo model to study the vascular effects of angiogenesis modulating agents. The main goal of the present study was to develop and validate a quantitative method to characterize time-dependent changes mainly in the capillary network of the CAM. To that end, the CAM capillaries were monitored in ovo, between days 7 and 13 of embryo development, using an epi-fluorescence microscope equipped with a sensitive camera following the intravenous injection of a fluorescent agent. We present a method by which the fluorescence angiograms of the CAM vasculature were recorded and analyzed by a multistep mathematical procedure to obtain a skeleton representation of the vessels and capillaries. From this skeleton descriptors were extracted, including the number of branching points/mm 2, the mean area of the vessel network meshes, and the mean of the 3rd quartile of the mesh area histogram. A qualitative visual assessment of the vasculature based on the number and the size of the avascular zones was obtained for comparison. To illustrate this approach, the activity of a neutralizing anti-vascular endothelial growth factor antibody, Avastin ® (Bevacizumab), was then quantified. Blood vessel growth inhibition and changes in the architecture of the capillary plexus after topical application of this drug on the CAM surface were monitored using the three descriptors mentioned above.