Blockade of integrins αvβ3/αvβ5 increases ceramide in adherent endothelial cells, independent of cell detachment. (A) HBMECs (10⁶ cells/10-cm plate) were allowed to spread on plates coated with vitronectin and blocked with 1% BSA. RGDfV (▪), RADfV (▨; 25 μg/mL), or vehicle control (□, DMSO) was added 2 hours after plating. Cells were labeled with [³H]palmitic acid at the time of addition of peptides. Following overnight incubation, cells were collected, lipids extracted, and [³H]ceramide was assessed by TLC as described in “Materials and methods.” Results are depicted as percent [³H]ceramide of total [³H]lipids extracted. Bars are means of 27 (vehicle), 31 (RADfV), or 33 (RGDfV) repeat experiments, each performed in triplicate. P < .001 between vehicle and RGDfV and between RADfV and RGDfV, but P = .085 between vehicle and the control peptide, RADfV. (B) Experiment was performed as in panel A; cells were collected at different time points; P = .004 by one-way ANOVA; n = 3. (C) Cells were labeled and treated, and lipids were extracted and resolved on TLC as in panel A. The TLC plate was exposed to film for 7 days. (D-E) HBMECs (10⁶ cells/10-cm dish) were allowed to adhere for 2 hours to plates coated with BSA-blocked vitronectin (D-E); PLL that was blocked with 1% BSA (D); or PLL that was left unblocked (E). Cells were labeled with [³H]palmitic acid and treated with RGDfV (G; ▪) or RADfV (A; ▨; 25 μg/mL), or vehicle control (-; □). Ceramide was determined as in panel A. In panel D, P values on VN were vehicle versus RGDfV (P = .003) and RADfV versus RGDfV (P = .014). In vehicle control–treated cells on VN versus on PLL/BSA, P = .001, and RADfV on VN versus vehicle-control cells on PLL/BSA had P = .010. However, P value was not significant (P > .17 to .37) for all comparisons between RGDfV-treated cells on VN and any of the treatment conditions on PLL/BSA. In panel E, P values were as follows: VN versus PLL (vehicle controls, □), P = .291; vehicle versus RGDfV on VN, P = .001; RADfV versus RGDfV on VN, P = .001; and vehicle versus RGDfV on PLL, P = .021. P value for RGDfV-treated cells on VN versus PLL was not significant (P = .087). (F) HBMECs (10⁶ per 10-cm plate) were incubated for 24 hours with C₁₆-ceramide that was prepared as described in “Materials and methods.” They were then harvested, fixed, permeabilized, stained with propidium iodide, and analyzed by flow cytometry for the sub G₀/G₁ fraction. P < .001 by one-way ANOVA; n = 3. (G) Lysates from 0.3 × 10⁶ control or ceramide-treated HBMECs incubated as in panel E were resolved by SDS-PAGE. PARP cleavage was assessed by Western blotting. Tubulin blot was used as loading control. Fold cleavage compared with control cells was calculated from the digitized images and corrected for tubulin.