Fig 2: Effect of ET-1 on expression levels of AR and testosterone secretion. (A) Lysed PC3 cells were analyzed by western blotting and membranes were incubated with antibodies against AR and β-actin. (B) Protein expression levels of AR in the presence or absence of BQ123. (C) Protein expression levels of AR in the presence or absence of BQ788. (D) Protein expression levels of AR in the presence or absence of BOSE. Quantification was normalized to β-actin and control PC3 cells. The cells were stimulated with 100 nM ET-1 and previously incubated with BQ123 or BQ788 for 96 h. (E) Testosterone secretion in presence or absence of BQ123. (F) Testosterone secretion in presence or absence of BQ788. Data are presented as the mean ± SD (n=3 independent experiments). *P<0.05, **P<0.01 (Kruskal Wallis test/Dunn's multiple comparisons test). Quantification was normalized to control PC3 cells. The values 180 and 40 correspond to mass units (kDa). ET-1, endothelin-1; BQ123, 2-[(3R,6R,9S,12R,15S)-6-(1H-indol-3-ylmethyl)-9-(2-methylpropyl)-2,5,8,11,14-pentaoxo-12-propan-2-yl-1,4,7,10,13-pentazabicyclo[13.3.0]octadecan-3-yl] acetic acid; BQ788, 2,6-Dimethylpiperidinecarbonyl-γ-Methyl-Leu-Nin-(Methoxycarbonyl)-D-Trp-D-Nle,-N-[N-[N-[(2,6-Dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl]-1-(methoxycarbonyl)-D-tryptophyl]-D-norleucine sodium salt; BOSE, bosentan; AR, androgen receptor.

Fig 3: Putative mechanisms involving ET-1 system whereby MPs released under VEGFi treatment causes endothelial cell dysfunction. VEGF/VEGFR inhibition causes an increase in ECMPs release which suggests an endothelial injury. In addition to carry ET-1, MPs induce endothelial cells damage by increasing ET-1 production, inflammation and ROS production through ETA and ETB activation but also by down-regulating eNOS/NO signalling. The dysregulation of endothelial cell function induced by MPs may induce vascular tone alterations and endothelial dysfunction, and may explain, at least partially, molecular mechanisms underlying VEGFi-associated hypertension.

Fig 4: Post-treatment MPs increase pre-pro-ET-1 gene expression in HAEC. (A) Graph represents the expression of ET-1 assessed by immunoblotting in plasma isolated MPs from patients pre-treatment and post-treatment with VEGFi (n = 17). The image shows six samples from one patient and is representative of the 17 patients, pre-treatment and post-treatment. (B) Pre-pro-ET-1 mRNA levels assessed by qPCR in HAEC exposed to total pre-treatment (grey bar) and post-treatment (black bar) MPs for 8 h and non-stimulated (NS, white bar) HAEC (n = 6–7). (C) ET-1 levels measured by ELISA in media from HAEC stimulated with vehicle or vatalanib (VEGFi) for 8 h (n = 6–7). Results represent the mean ± SEM. Data were analysed using t-test or one-way ANOVA followed by a post hoc Tukey test when appropriate. *P < 0.01 vs. NS, †P < 0.05 vs. pre-treatment. S, subject.

Fig 5: MPs increase ROS production in HAEC, which is attenuated by ET-1 receptors antagonism. NADPH-stimulated O2− production was assessed by lucigenin assay in (A) HAEC exposed to total pre-treatment and post-treatment MPs for 5, 15, 30, and 60 min (n = 5–6) and (B) HAEC exposed to post-treatment MPs for 5, 30, and 60 min in presence or absence of BQ123 (ETA antagonist, 1 µmol/L) or BQ788 (ETB antagonist, 1 µmol/L) (n = 5–9). Results represent the mean ± SEM. Data were analysed using one-way ANOVA followed by a post hoc Tukey test. *P < 0.05, **P < 0.01, ***P < 0.001 vs. NS; φP < 0.05 vs. post-treatment.