Figure 1.
Chemical structure of kahweol.
Figure 2.
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.
Figure 3.
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.
Figure 4.
Kahweol inhibits endothelial cell proliferation.
Survival curves of proliferative (squares) and non-proliferative (circles) HUVEC endothelial cells treated with kahweol. Concentrations are represented in logarithmic scale. Depicted data are means of values of three independent experiments (each one with quadruplicate samples). Standard deviation values (in all the cases lower than 20% of mean values) are not represented for the sake of clarity.
Figure 5.
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.
Figure 6.
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.
Figure 7.
Kahweol inhibits HUVEC endothelial cell invasion in a dose-dependent manner.
Invading controls and 25 and 75 µM kahweol-treated HUVEC cell count values are represented by using diamonds, squares and triangles, respectively. As negative controls, the number of untreated invading HUVEC to wells not containing chemoattractant was determined (crosses). Data are given as number of invading cells and they are means of two different assays (each one carried out in triplicate).
Figure 8.
Kahweol inhibits HUVEC MMP-2 and uPA in a dose dependent manner.
A) Gelatin zymography of MMP-2 in conditioned media of HUVEC after treatment with different kahweol concentrations. B) In situ determination of kahweol effects on HT-1080 gelatinases, as determined by gelatin zymography with the presence of kahweol in the incubation substrate buffer. C) Plasminogen zymography of HUVEC uPA after treatment with different kahweol concentrations.
Figure 9.
Kahweol inhibits HUVEC endothelial cell COX-2 expression and MCP-1 secretion in a dose-dependent manner.
A) Typical results of a Western blot assay using anti-COX-2 antibodies. B) Quantification of the normalized relative inhibitory effect. Data are given as percentage, taking the normalized levels of COX-2 in control cells as 100%, and they are means±S.D. of three different assays. C) Quantification of the amount of MCP-1 secreted by HUVEC after a 24 h treatment in the presence of different concentrations of kahweol. Data are given as femtograms of secreted MCP-1 per cell, 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.