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.

We have recently shown that the blood cell-derived secretome of remote ischaemic (RIC)-conditioned individuals provides an external source of neuroprotection. In this study, we identified the bioactive compounds from the total proteins released by those cells. Our main strategy was to separate protein–protein complexes while maintaining their native structure and testing their bioactive properties. Subsequently, we identified up- and downregulated bioactive proteins. We uncovered two bioactive fractions composed of 18 proteins. Most of the protein peaks were unchanged; however, RIC mediated a decrease in two peaks (comprising seven proteins) and an increase in one peak (identified as haptoglobin). When focussing on the biological activity of these proteins, we found positive impacts on the regulation of cellular metabolic processes and an increase in biological processes related to the acute phase response and inflammation in the RIC-treated samples. Although we have identified the 18 proteins that exert the greatest cytoprotection, additional studies are needed to elucidate their particular function and detailed mechanisms of action.