RESUMO
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.
RESUMO
The chorioallantoic membrane (CAM) is a highly vascularized avian extraembryonic membrane widely used as an in vivo model to study angiogenesis and its inhibition in response to tissues, cells, or soluble factors. In recent years, the use of CAM has become an integral part of the biocompatibility testing process for developing biomaterials intended for regenerative strategies and tissue engineering applications. In this study, we used the chicken ex ovo CAM assay to investigate the angiogenic potential of innovative acellular biopolymer polyhydroxybutyrate/chitosan (PHB/CHIT) scaffold, which is intended for the treatment of hard tissue defects, depending on treatment with pro- and anti-angiogenic substances. On embryonic day (ED) 7, the experimental biomaterials were placed on the CAM alone or soaked in vascular endothelial growth factor (VEGF-A), saline solution (PHY), or tyrosine kinase inhibitor (SU5402). After 72 h, the formation of vessels was analyzed in the surrounding area of the scaffold and inside the pores of the implants, using markers of embryonic endothelium (WGA, SNA), myofibroblasts (α-SMA), and macrophages (KUL-01). The morphological and histochemical analysis showed strong angiogenic potential of untreated scaffolds without additional effect of the angiogenic factor, VEGF-A. The lowest angiogenic potential was observed in scaffolds soaked with SU5402. Gene expression of pro-angiogenic growth factors, i.e., VEGF-A, ANG-2, and VE-CAD, was upregulated in untreated scaffolds after 72 h, indicating a pro-angiogenic environment. We concluded that the PHB/CHIT has a strong endogenous angiogenic potential and could be promising biomaterial for the treatment of hard tissue defects.
RESUMO
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).
