Fantofarone

Neutrophils rapidly undergo spontaneous apoptosis following their release from the bone marrow. Although central to leukocyte homeostasis, the mechanisms that regulate neutrophil apoptosis remain poorly understood. We show here that apoptosis of cultured neutrophils is preceded by a substantial increase in the intracellular levels of 16 and 24 carbon atom (C16- and C24)-ceramides, which are lipid second messengers of apoptosis and stress signaling. Treatment of neutrophils with fumonisin B2, a selective inhibitor of the de novo pathway of ceramide synthesis, prevented accumulation of C16- and C24-ceramides. Moreover, fumonisin B2 significantly reduced caspase-3, -8, and -9 activation and apoptosis in these cells. Conversely, 3-O-methylsphingomyelin and fantofarone, which are specific inhibitors of neutral and acid sphingomyelinases, respectively, neither inhibited C16- and C24-ceramide production nor decreased the apoptosis rate in neutrophils, indicating that in these cells, ceramides are not generated from membrane sphingomyelin. Further experiments showed that increasing endogenous C16- and C24-ceramide levels by using DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol and (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, two inhibitors of ceramide metabolism, enhances caspase-3, -8, and -9 activity and increases neutrophil apoptosis. Similarly, apoptosis was induced rapidly when synthetic C16- and/or C24-ceramides were added to neutrophil cultures. Finally, GM-CSF, a cytokine that delays neutrophil apoptosis, abrogated C16- and C24-ceramide accumulation totally in cultured neutrophils, whereas Fas ligation accelerated apoptosis in these cells without affecting de novo ceramide production. We conclude that de novo generation of C16- and C24-ceramides contributes to spontaneous neutrophil apoptosis via caspase activation and that GM-CSF exerts its antiapoptotic effects on neutrophils, at least partly through inhibition of ceramide accumulation.

Matrix ligation of integrins αvβ3/αvβ5 is critical for endothelial survival and angiogenesis. We have previously shown that ceramide, a proapoptotic lipid second messenger, increases during endothelial anoikis (detachment-induced apoptosis). We now show that RGDfV, an integrin αvβ3/αvβ5 cyclic function-blocking peptide, increased ceramide and decreased sphingomyelin in human brain microvascular endothelial cells (HBMECs) plated on vitronectin, suggesting that sphingomyelin hydrolysis contributes to RGDfV-induced ceramide increase. Desipramine and imipramine, inhibitors of acid sphingomyelinase (ASMase), suppressed RGDfV-induced ceramide increase. Importantly, desipramine, imipramine, and a third ASMase inhibitor, SR33557, but not inhibitors of neutral sphingomyelinase, suppressed RGDfV-induced apoptosis, suggesting that ASMase was required for integrin-mediated apoptosis. Myriocin, an inhibitor of de novo ceramide synthesis, had no effect on RGDfV-induced HBMEC apoptosis. Interestingly, ASMase inhibitors also suppressed the RGDfV-induced loss of spreading on vitronectin. RGDfV induced a similar increase in ceramide and apoptosis in HBMECs on poly-l-lysine or vitronectin, although cells detached only from vitronectin, indicating that cell detachment was not required for RGDfV-induced apoptosis. Our results suggest involvement of ASMase and ceramide in endothelial apoptosis induced by inhibition of integrins αvβ3/αvβ5, and propose a novel molecular mechanism for the antiangiogenic effect of RGDfV.