Inhibitors of acid sphingomyelinase decrease endothelial apoptosis induced by αvβ3/αvβ5-integrin inhibition. (A) HBMECs (106 cells/10-cm plate) were seeded on plates coated with vitronectin and blocked with 1% BSA or on plates coated with PLL/BSA (▧). They were then incubated with RGDfV (▪) or vehicle control (□ or ▧). Cells were collected 24 hours later and apoptosis was assessed by PI staining. Desipramine (5-20 μM) was added where indicated starting 2 hours prior to the addition of RGDfV or prior to plating on PLL/BSA. P = .001 for increasing doses of desipramine on vitronectin with RGDfV, and P < .001 for desipramine on PLL/BSA, by one-way ANOVA (n = 3). (B) Cells were plated on vitronectin/BSA and treated and analyzed as in panel A, except that the inhibitor was imipramine. P < .001 by one-way ANOVA in respect to increasing doses of imipramine; n = 3. (C) Cells were plated on vitronectin, treated with RGDfV as indicated, and analyzed as in panels A-B, except that the inhibitor was SR33557. (D-E) Cells were plated on vitronectin and treated with RGDfV or vehicle as in panels A-C. Imipramine (Imip; 5-20 μM) or SR33557 (SR; 1-10 μM) was added where indicated, starting 2 hours prior to RGDfV or vehicle. Staurosporine (50 nM) was used as a positive control. Cells were collected after 24 hours, cell lysates were resolved on 10% SDS-PAGE, and apoptosis was detected by PARP cleavage. Tubulin was used as loading control.
