Activation of neutral sphingomyelinase 2 through hyperglycemia contributes to endothelial apoptosis via vesicle-bound intercellular transfer of ceramides

Abstract

Background Pro-apoptotic and pro-inflammatory ceramides are crucially involved in atherosclerotic plaque development. Local cellular ceramide accumulation mediates endothelial apoptosis, especially in type 2 diabetes mellitus, which is a major cardiovascular risk factor. In recent years, large extracellular vesicles (lEVs) have been identified as an important means of intercellular communication and as regulators of cardiovascular health and disease. A potential role for lEVs as vehicles for ceramide transfer and inductors of diabetes-associated endothelial apoptosis has never been investigated. Methods and Results A mass-spectrometric analysis of human coronary artery endothelial cells (HCAECs) and their lEVs revealed C16 ceramide (d18:1–16:0) to be the most abundant ceramide in lEVs and to be significantly increased in lEVs after hyperglycemic injury to HCAECs. The increased packaging of ceramide into lEVs after hyperglycemic injury was shown to be dependent on neutral sphingomyelinase 2 (nSMase2), which was upregulated in glucose-treated HCAECs. lEVs from hyperglycemic HCAECs induced apoptosis in the recipient HCAECs compared to native lEVs from untreated HCAECs. Similarly, lEVs from hyperglycemic mice after streptozotocin injection induced higher rates of apoptosis in murine endothelial cells compared to lEVs from normoglycemic mice. To generate lEVs with high levels of C16 ceramide, ceramide was applied exogenously and shown to be effectively packaged into the lEVs, which then induced apoptosis in lEV-recipient HCAECs via activation of caspase 3. Intercellular transfer of ceramide through lEVs was confirmed by use of a fluorescently labeled ceramide analogue. Treatment of HCAECs with a pharmacological inhibitor of nSMases (GW4869) or siRNA-mediated downregulation of nSMase2 abrogated the glucose-mediated effect on apoptosis in lEV-recipient cells. In contrast, for small EVs (sEVs), hyperglycemic injury or GW4869 treatment had no effect on apoptosis induction in sEV-recipient cells. Conclusion lEVs mediate the induction of apoptosis in endothelial cells in response to hyperglycemic injury through intercellular transfer of ceramides. Graphical abstract

Ceramide Metabolism in Cardiovascular Disease: A Network With High Therapeutic Potential

“…33,34 In the context of extracellular vesicles, our group showed that hyperglycemia-induced activation of NSM-2 promotes endothelial apoptosis through the intercellular transfer of ceramides via extracellular vesicles. 35…”

“…In this context, hyperglycemia has been associated with endothelial ceramide accumulation. 35,54 While in the early years of ceramide research, the induction of cellular apoptosis was thought to be the leading cause of ceramide-associated cellular injury, 55 this has been challenged, as increases in cellular ceramide levels have been shown to occur only at later stages in apoptosis. 56,57 In macrophages, endothelial cells, hepatocytes, and cancerous cell lines, such as MCF7 (Michigan Cancer Foundation-7) breast cancer cells, NSM-2 and related ceramide was found to mediate cellular effects of TNF (tumor necrosis factor)-α receptor.…”

Contribution of specific ceramides to obesity-associated metabolic diseases

“…Furthermore, ceramide accumulation through increased de novo ceramide production may dictate endothelial cell fate and injury [ 197 ]. Endothelial cell apoptosis in response to hyperglycemia has been related to the intercellular transfer of high concentrations of C 16:0 ceramides in large extracellular vesicles derived from nSMase2-dependent sphingomyelin hydrolysis, thereby causing endothelial dysfunction in obesity and diabetes [ 310 ]. By correlating C 16:0 ceramide levels in thoracic adipose tissue and circulation with the deregulation of the vascular redox state and inflammation in human atherosclerotic patients, together with complementary experiments on human tissue ex vivo and primary cultured cells in vitro, it has been suggested that adipose-tissue-derived C 16:0 ceramides increase the risk of cardiovascular death by acting on endothelial cells to reduce vasodilation, induce inflammation, and promote oxidative stress via eNOS uncoupling [ 299 ].…”

Ceramide Acyl Chain Length and Its Relevance to Intracellular Lipid Regulation

“…Similarly, C16-ceramides drive apoptosis in human coronary artery endothelial cells (HCAECs). Under hyperglycemic conditions, Zietzer and colleagues showed that C16-ceramides dominated HCAEC-derived large extracellular vesicles (lEVs), the latter of which triggered HCAEC apoptosis [ 62 ]. Inhibition of neutral sphingomyelinase 2 (nSMase2), significantly reduced C16-ceramides (as well as other less dominant ceramide species) in lEVs, which resulted in the abrogation of hyperglycemic-induced apoptosis in HCAECs [ 62 ].…”

“…As mentioned earlier, the acyl length of ceramides is linked to its toxicity, with C16- and C18-ceramides being notorious in the development of various diseases [ 29 , 32 , 41 , 62 ]. Saddoughi et al reported that I2PP2A preferentially binds to C18-ceramides over C14-, C16-, C20-, C22-, and C24-ceramides [ 75 ], suggesting that C18-ceramides are a relatively more potent inhibitor of Akt compared to other ceramides.…”