ET-1 induces CLL activation and proliferation through ET_A receptor.

<p>Purified CLL cells, pretreated or not with 0.1 µM BQ-123, were stimulated with 100 nM ET-1 for 4 hours (n = 7) in panel A, or with an agonist specific for toll-like receptor 9 (TLR9) (CpG oligonucleotides) and IL-2 with/without ET-1 addition for 5 days (n = 15) in panel B, or with endothelial cell contact (HC) for 4 days (n = 11) in panel C. Histograms depict the formazan release by metabolically active CLL cells as fold change compared to unstimulated control (untreated CLL cells cultured alone). Data are presented as mean±SEM of 6 independent experiments. Of note, the CLL activation is significantly increased upon ET-1 stimulation and inhibited by blocking ET_AR signaling in all conditions. (D) After 1 h of serum-starvation, CLL cells (n = 8) were stimulated or not with ET-1 overnight. PI analysis was performed to define cell cycle phases. Histograms represent the percentage of CLL cells in S-phase after treatment compared to unstimulated control. (E) CFSE-labeled CLL cells were cultured for 4 days alone in complete medium (control) or on endothelial layers. Where indicated, CLL cells were incubated for 20 min with 0.1 µM BQ-123 before co-culture. The proliferative measure was inspected for 4 days, gating the CD19+ live CLL cells. The histograms represent cumulative data at 96 hours of 5 independent experiments by using 9 CLL patients. Data are shown as mean values ± SEM of the percentage of dividing CLL cells. In panel F, the percentages of divided CLL cells at 48 h, 72 h and 96 h measured by CFSE dilution in a representative CLL sample are shown. HUVEC cells stimulate CLL cells to divide. The addition of BQ-123 counteracts the EC-mediated proliferative stimuli. (*p<0.05, **p<0.01, ***p<0.001).