Usnic Acid Isolated from Usnea antarctica (Du Rietz) Reduced In Vitro Angiogenesis in VEGF- and bFGF-Stimulated HUVECs and Ex Ovo in Quail Chorioallantoic Membrane (CAM) Assay

Abstract

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.

Keywords: CAM; HUVECs; VEGF; angiogenesis; bFGF; usnic acid.

Figure 1

Chemical structure of usnic acid.

Figure 2

VEGF and bFGF promote HUVECs proliferation. Cells were treated with different concentrations (10–40 ng/mL) of VEGF (A) or bFGF (B) for 48 h, and cell viability was detected by MTT assay. Each treatment was performed in triplicate and repeated three times.

Figure 3

Inhibitory effect of usnic acid (UA) on cell viability. HUVECs cells were treated with vehicle (DMSO) and various concentrations of UA (10, 50, 100 μM) in the absence (A) or presence (B) of VEGF (25 ng/mL) and (C) bFGF (30 ng/mL) for 48 h. Values are means ± SD from three independent experiments (* p < 0.05 versus control; # p < 0.05; ## p < 0.01 versus VEGF alone; $$ p < 0.01; $$$ p < 0.001 versus bFGF alone).

Figure 4

Influence of IC₅₀ of usnic acid (UA) alone or co-incubated with VEGF (25 ng/mL) or bFGF (30 ng/mL) on the migration of HUVECs. Representative images of HUVECs scratch wound assay are shown for the 0 h and 24 h time points (100× magnification, scale bar 1000 μm).

Figure 5

Effects of usnic acid (UA, 33.57 μM) on tube formation. (A) Representative micrographs of the human umbilical vein endothelial cells (HUVECs) tube formation showing network formation after 24 h treatment (100× magnification, scale bar 1000 μm). Graphs show automatic parameter detection by ImageJ software. Tube segments are colored in yellow, green, and dark blue; the master junction in pink; and nodes surrounded by junction in red surrounded by blue. Quantification of the number of master segments (B), total segment length (C), number of master junctions (D), and number of nodes (E). Data are expressed as mean ± SD of three independent experiments (* p < 0.05; ** p < 0.01 versus control; ### p < 0.001 versus VEGF alone; $ p < 0.05; $$ p < 0.01 versus bFGF alone).

Figure 6

Fibrin gel bead assay to evaluate the anti-angiogenic potential of usnic acid (UA). (A) Representative images of HUVEC-coated beads embedded in a fibrin gel in the absence or presence of VEGF (25 ng/mL) or bFGF (30 ng/mL) treated with IC₅₀ of UA photographed at day 7 (100× magnification; scale bar 200 μm). Graphs representing sprout area (px) (B) and number of sprouts (C). In all graphs, values are given as average ± SEM (n = 3 per condition). * p < 0.05 compared to control; ## p < 0.01 compared to VEGF alone; $$ p < 0.01 compared to bFGF alone.

Figure 7

The effects of usnic acid (UA) alone on capillary formation ex ovo in the quail vascular system. (A) Representative images of CAMs at 0 h and 72 h after incubation with UA (75 μg/mL, 100 μg/mL). The results are summarized in the graphs as vessel density (B), total vessel network length (C), and total branch (D). Each group contained 10 CAMs, and the experiment was repeated three times.

Figure 8

Usnic acid (UA) reduced capillary formation ex ovo in the VEGF-stimulated quail vascular system. (A) Representative images of CAMs at 0 h and 72 h after incubation with UA (75 μM, 100 μM) in combination with VEGF (25 ng/mL). The results are summarized in the graphs as vessel density (B), total vessel network length (C), and total branch (D). Each group contained 10 CAMs, and the experiment was repeated three times. Error bars represent ± SD (# p < 0.05; ## p < 0.01 versus VEGF alone).

Figure 9

Usnic acid (UA) reduced capillary formation ex ovo in the bFGF-stimulated quail vascular system. (A) After incubation for 72 h, CAMs were photographed with a digital camera. Each group contained 10 CAMs, and the experiment was repeated three times. The results are summarized in the graphs as vessel density (B), total vessel network length (C), and total branch (D). Error bars represent ± SD ($ < 0.05 versus bFGF alone).

Figure 10

H–E/alcian blue staining of chorioallantoic membranes showing the chorionic epithelium (ectoderm, Ec), the intermediate vascularized mesenchyme (mesoderm, M) with vessels (asterisks), and the deep allantoic epithelium (endoderm, En). Evaluation of CAM tissue response to 0.9% NaCl (A), VEGF 25 ng/mL (B), bFGF 30 ng/mL (C), UA 75 μM (D), UA 75 μM + VEGF (E), UA 75 μM + bFGF (F), UA 100 μM (G), UA 100 μM + VEGF (H), and UA 100 μM + bFGF (I) on ED9. Original magnification 40×, scale bar 50 μm. Graphs represent number of vessels <50 μm (J), <100 μm (K), >100 μm (L) and thickness of ectoderm (M), mesoderm (N), and endoderm (O). Error bars (* p < 0.05, ** p < 0.01 versus control; # p < 0.05, ## p < 0.01 versus VEGF alone; $ p < 0.05, $$ p < 0.01 versus bFGF alone).