Fig 1.
Aldosterone exerts cytotoxic actions to cultured HUVECs.
HUVECs were either left untreated (“Ctrl”), or treated with applied concentrations of aldosterone (1–1000 nM) for indicated time point, cell survival was tested by MTT assay (A and B), and cell death was tested by LDH release assay (C and D). Data were expressed as the mean ± SD. For each assay, n = 5. Experiments in this figure were repeated four times, and similar results were obtained. * p < 0.05 vs. “Ctrl” group.
Fig 2.
Aldosterone induces caspase-3-dependent apoptotic death in HUVECs.
HUVECs were treated with applied concentrations of aldosterone (1–1000 nM) for indicate time, cell apoptosis was evidenced by Annexin V FACS assay (A, representative FACS images were shown in lower panel), histone DNA ELISA assay (B), caspase-3 activity assay (C, lower panel) and Western blot assaying of cleavead-caspase-3 (“C-Caspase-3”) (C, upper panel). HUVECs, pre-treated with the caspase-3 inhibitor z-DVED-fmk (“ZDVED”, 25 μM)/AC-DEVD-CHO (“AC-DEVD”, 25 μM), or the pan caspase inhibitor z-VAD-fmk (“ZVAD”, 25 μM) for 1 hour, were stimulated with aldosterone (100 nM), caspase-3 activity and cell apoptosis were analyzed by the caspase-3 activity assay (D) and Annexin V FACS assay (E), respectively; Cell survival was tested by the MTT assay (F). Data were expressed as the mean ± SD. For each assay, n = 5. Experiments in this figure were repeated three times, and similar results were obtained. * p < 0.05 vs. “Ctrl” group. # p < 0.05 vs. aldosterone (100 nM) only group (D-F).
Fig 3.
Aldosterone mainly induces C18 ceramide production in HUVECs.
HUVECs were treated with applied concentrations of aldosterone (1–1000 nM) for 4 hours, total cellular ceramide level was analyzed by the DAG kinase assay, and was normalized to the untreated control (“Ctrl”) group (A), individual ceramide level was detected by LS-MS assay as described (F). HUVECs, pretreated with PDMP (10 μM), S1P (10 μM) or C6 ceramide (25 μM) for 1 hour, were stimulated with aldosterone (100 nM), cellular ceramide was analyzed (B); Cell survival was tested by MTT assay (C), and cell apoptosis was tested by the caspase-3 activity assay (D) or the Histone DNA ELISA assay (E). For each assay, n = 3. Experiments in this figure were repeated three times, and similar results were obtained. * p < 0.05 vs. “Ctrl” group. ** p < 0.05 (C-E).
Fig 4.
Eplerenone blocks aldosterone-induced ceramide production and following HUVEC cytotoxicity.
HUVECs, pretreated with Eplerenone (10 μM) for 1 hour, were stimulated with aldosterone (100 nM), C18 ceramide (A, LS-MS assay), C24 ceramide (B, LS-MS assay), and total ceramide (C, DAG kinase assay) were analyzed after 4 hours; Cell viability was tested by MTT assay (D), and cell apoptosis was tested by the caspase-3 activity assay (E) and Histone DNA ELISA assay (F). For each assay, n = 3. Experiments in this figure were repeated three times, and similar results were obtained. * p < 0.05.
Fig 5.
Ceramide synthase 1 mediates aldosterone-induced C18 ceramide production and following HUVEC damages.
Expression of CerS-1 and tubulin (the equal loading) in HUVECs expressing scramble control shRNA or CerS-1 shRNA (two colonies) was shown (A), CerS-1 expression (vs. tubulin) was quantified (A). Above cells were treated with or without aldosterone (100 nM), C18 and C24 ceramide production (B and C), cell survival (D) and cell apoptosis (Caspase-3 activity, E and Histone DNA EILSA OD, F) were tested. Expression of CerS-1 and β-actin (the equal loading) in HUVECs expressing CerS-1-cDNA or the empty vector (p-Super-puro) was shown, CerS-1 expression (vs. β-actin) was quantified (G); Aldo (100 nM)-induced C18 ceramide production (H, 4 hours), cell viability reduction (I, 24 hours), and caspase-3 activity (J, 12 hours) in above cells were tested. For each assay, n = 3. Experiments in this figure were repeated three times, and similar results were obtained. * p < 0.05.