Natural products include a diverse set of compounds of drug discovery that are currently being actively used to target tumor angiogenesis. In the present study, we evaluated the anti-angiogenic activities of secondary metabolite usnic acid isolated from Usena antarctica. We investigated the in vitro effects on proliferation, migration, and tube formation of VEGF- and bFGF-stimulated HUVECs. Ex ovo anti-angiogenic activity was evaluated using the CAM assay. Our findings demonstrated that usnic acid in the concentration of 33.57 µM inhibited VEGF (25 ng/mL) and bFGF (30 ng/mL)-induced HUVECs proliferation, migration, and tube formation. The ex ovo CAM model was used to confirm the results obtained from in vitro studies. VEGF- and bFGF-induced vessel formation was inhibited by usnic acid after 72 h in over 2-fold higher concentrations compared to in vitro. Subsequently, histological sections of affected chorioallantoic membranes were stained with hematoxylin–eosin and alcian blue to determine the number and diameter of vessels as well as the thickness of the individual CAM layers (ectoderm, mesoderm, endoderm). Usnic acid was able to suppress the formation of VEGF- and bFGF-induced vessels with a diameter of less than 100 μm, which was demonstrated by the reduction of mesoderm thickness as well.

Dimethyl sulfoxide (DMSO) is widely used as a solvent for small hydrophobic drug molecules. However, the safe volume allowing to avoid its embryotoxic effect has been poorly studied. In this study, we documented the effects of dimethyl sulfoxide (DMSO) in the developing chicken embryo at morphological, histological, and molecular levels. We focused on the developing chicken liver as the main organ involved in the process of detoxification. In our study, 100% DMSO was administered subgerminally onto the eggshell membrane (membrana papyracea) at various volumes (5, 10, 15, 20, 25, 30, 35, and 50 µL) on 4th embryonic day (ED). We focused on histopathological alterations of the liver structure, and noticed the overall impact of DMSO on developing chicken embryos (embryotoxicity, malformation). At the molecular level, we studied cytochrome P450 complex (CYP) isoform’s activities in relation to changes of CYP1A5, CYP3A37, and CYP3A80 gene expression. Total embryotoxicity after application of different doses of DMSO on ED 4, and the embryo lethality increased with increasing DMSO amounts. Overall mortality after DMSO administration ranged below 33%. Mortality was increased with higher amounts of DMSO, mainly from 20 µL. The highest mortality was observed for the highest dose of DMSO over 35 µL. The results also showed a decrease in body weight with increased application volumes of DMSO. At the histological level, we observed mainly the presence of lipid droplets and dilated bile canaliculi and sinusoids in samples over the administration of 25 µL of DMSO. While these findings were not statistically significant, DMSO treatment caused a significant different up-regulation of mRNA expression in all studied genes. For CYP1A5, CYP3A37, and CYP3A80 DMSO volumes needed were 15 µL, 10 µL, and 20 µL, respectively. A significant down-regulation of all studied CYP isoform was detected after application of a DMSO dose of 5 µL. Regarding the morphological results, we can assume that the highest safe dose of DMSO without affecting chicken embryo development and its liver is up to 10 µL. This conclusion is corroborated with the presence of number of malformations and body weight reduction, which correlates with histological findings. Moreover, the gene expression results showed that even the lowest administered DMSO volume could affect hepatocytes at the molecular level causing down-regulation of cytochrome P450 complex (CYP1A5, CYP3A37, CYP3A80).

In recent years, the chorioallantoic membrane (CAM) has emerged as a crucial component of biocompatibility testing for biomaterials designed for regenerative strategies and tissue engineering applications. This study explores angiogenic potential of an innovative acellular and porous biopolymer scaffold, based on polyhydroxybutyrate and chitosan (PHB/CHIT), using the ex ovo quail CAM assay as an alternative to the conventional chick CAM test. On embryonic day 6 (ED6), we placed the tested biomaterials on the CAM alone or soaked them with various substances, including vascular endothelial growth factor (VEGF-A), saline, or the endogenous angiogenesis inhibitor Angiostatin. After 72 h (ED9), we analyzed blood vessels formation, a sign of ongoing angiogenesis, in the vicinity of the scaffold and within its pores. We employed marker for cell proliferation (PHH3), embryonic endothelium (WGA, SNA), myofibroblasts (α-SMA), and endothelial cells (QH1) for morphological and histochemical analysis. Our findings demonstrated the robust angiogenic potential of the untreated scaffold without additional influence from the angiogenic factor VEGF-A. Furthermore, gene expression analysis revealed an upregulation of pro-angiogenic growth factors, including VEGF-A, ANG-2, and VE-Cadherin after 5 days of implantation, indicative of a pro-angiogenic microenvironment. These results underscore the inherent angiogenic potential of the PHB/CHIT composite. Additionally, monitoring of CAM microvilli growing to the scaffold provides a methodology for investigating the biocompatibility of materials using the ex ovo quail CAM assay as a suitable alternative model compared to the chicken CAM platform. This approach offers a rapid screening method for biomaterials in the field of tissue repair/regeneration and engineering.

The chorioallantoic membrane (CAM) is a highly vascularized avian extraembryonic membrane, widely used as an in vivo model for angiogenesis, with predominant application in tumor research. CAM assays are performed under in ovo or ex ovo conditions; however, a clear rationale for model selection is lacking, and comparative morphological data remain limited. In this study, we compared chicken embryo CAM development under both incubation conditions. Morphological analyses revealed pronounced incubation-dependent differences in CAM maturation, with the mesoderm showing the most dynamic alterations. Ex ovo CAMs exhibited early mesodermal thickening, and peaks in angiogenesis; indicative of accelerated but less coordinated tissue and vascular development. In ovo CAMs showed a gradual and well-coordinated increase in mesodermal thickness and vascularization, peaking at later developmental stages. Differences were also observed in epithelial organization: ex ovo CAMs exhibited disrupted ectodermal architecture, whereas in ovo CAMs maintained stable epithelial stratification. Functional assessment using GFP-labelled human fibrosarcoma cells (HT1080) demonstrated significantly higher embryo survival and larger tumor growth in the in ovo model, while ex ovo conditions were associated with increased mortality and reduced tumor size. These findings demonstrate that incubation conditions significantly influence CAM morphology and functional performance, highlighting the importance of careful model selection in angiogenesis research.

Lead sulfide nanoparticles (PbS NPs) are promising materials for biomedical imaging. However, their safety during embryonic development remains inadequately explored. This study investigated for the first time the developmental toxicity, hepatic effects, angiogenic activity, and irritation potential of PbS NPs using the Chick Embryotoxicity Screening Test (CHEST) and the chorioallantoic membrane (CAM) assay. Fertilized chicken eggs were exposed to various concentrations of PbS NPs (6%, 10%, 12%, 20%, 25%, 50%, and 100%). Developmental toxicity was assessed by mortality rate, body and selected organ weights, malformations, liver histological alterations, and RT-qPCR of antioxidant-related genes. Vascular evaluation was quantitatively assessed using the CAM assay with the following irritation potential analysis. Embryonic exposure to PbS NPs resulted in concentration-dependent developmental effects, with embryo mortality reaching 56% at the highest concentration and the presence of developmental malformations in surviving embryos. No histological alterations were observed in the liver; however, higher concentrations altered α-smooth muscle actin and cell proliferation activity and modulated antioxidant gene expression. In the CAM assay, the highest concentrations of PbS NPs significantly reduced total vessel area, branching, and total vessel length while inducing only slight irritation, characterized by vascular dilatation. These findings demonstrate concentration-dependent developmental and vascular responses following direct embryonic exposure to PbS NPs under the experimental conditions. Only the highest concentrations caused significant morphological, angiogenic, and molecular alterations after short-time PbS NP exposure. This study provides a comprehensive safety evaluation of PbS NPs during chicken embryonic development and supports their potential for further investigation in biomedical imaging. Further, it indicates that the combination of CHEST and CAM assays as complementary models can be used for preclinical developmental nanotoxicity assessment.