Kahweol inhibits in vivo angiogenesis and does not induce endothelial cell-specific apoptosis in the quail CAM assay.

A) CAM assay. Dotted circles identify the position of the methyl cellulose discs after incubation, carried out as described in Materials and methods. In controls, methyl cellulose discs were prepared with the vehicle (DMSO). In treatments, methyl cellulose discs contained 50 nmol of kahweol. B) Detection of apoptosis in the quail CAM assay. Arrows point to apoptotic cells. C) Angiogenesis assay in the zebrafish model. The arrow points to the caudal region with narrower and disrupted intersegmental vessels in kahweol treated zebrafish embryos.

Kahweol inhibits endothelial cell sprouting from aortic rings in a dose-dependent manner.

Aortic ring assay was performed as described in Materials and methods. A) Negative of photographs (x20) of aortic rings (lateral view) after 10 days of incubation in a 3D collagen gel overlayed with complete medium in the presence of 20 mg/mL VEGF, 0.05% DMSO (the vehicle taken as a control), or kahweol at 1, 5 and 25 μM (K1, K5, K25, respectively). Experiments were repeated at least three times. B) Microvessel time course for the different treatments mentioned in A. Data are given as microvessel total count at different incubation times (spanning from 3 to to 10 days), and they are means±S.D. of three different experiments. C) Microvessel total count after 10 days of incubation. Data are given as microvessel total count, and they are means±S.D. of three different experiments. *Statistically significant (p<0.01) as compared to control values, according to a two-tailed Student′s t-test.

Kahweol inhibits tubule formation of endothelial cells on Matrigel in a dose-dependent manner.

Data are representative of, at least, three independent experiments.(C-) Negative controls, HUVEC on Matrigel with no treatment. (C +) Positive controls, HUVEC on Matrigel treated with 50 μM suramin.

Kahweol inhibits endothelial cell migration.

Photographs were taken on untreated (control) and 75 μM kahweol-treated HUVEC cells at 0, 8 and 24 h after “wounding”. Data are representative of, at least, three independent experiments. At the right, the counting of HUVEC migration into the “wounded” area at 8 and 24 h after “wounding” is depicted. Data are given as percentages of re-occupied “wounded” area and they are means±S.D. of three different experiments. White bars are control values and grey bars correspond to treatments. *Statistically significant (p<0.01) as compared to control values, according to a two-tailed Student′s t-test.