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

Andreas Zietzer,Marko Bulic,Alina Lisann Jahnel,Georg Nickenig,Nikos Werner,Felix Jansen,Philip Düsing,Mohammed Rabiul Hosen,Peter Dörmann,Katharina Gutbrod

doi:10.1007/s00018-021-04049-5

Abstract

BackgroundPro-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 ResultsA 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.ConclusionlEVs mediate the induction of apoptosis in endothelial cells in response to hyperglycemic injury through intercellular transfer of ceramides.Graphical abstract

Highlights

Pro-apoptotic and pro-inflammatory ceramides are crucially involved in atherosclerotic plaque development
While adding 5 mM glucose did not have any effect on cell viability or the induction of apoptosis, the addition of 30 mM glucose for 72 h led to a decline of human coronary artery endothelial cells (HCAECs) viability and an induction of apoptosis (Fig. 1B + C)
We found that injury with an addition of 30 mM glucose for 72 h led to an increase of the total ceramide levels in large extracellular vesicles (lEVs)

Summary

Introduction

Pro-apoptotic and pro-inflammatory ceramides are crucially involved in atherosclerotic plaque development. Conclusion lEVs mediate the induction of apoptosis in endothelial cells in response to hyperglycemic injury through intercellular transfer of ceramides. In response to cellular injury from extracellular stressors, is mainly regulated by sphingomyelinases Of these sphingomyelinases, isoform 3 (sphingomyelin phosphodiesterase 3 = SMPD3, which codes for the protein neutral sphingomyelinase 2 = nSMase2) is the most responsive isoform with regard to important inductors of atherosclerotic plaque development, such as oxidative stress, inflammation, and apoptosis [9, 10]. Diabetes-associated hyperglycemia triggers atherosclerotic plaque development through the impairment of endothelial function and regeneration [15, 16] This again involves the induction of endothelial apoptosis through oxidative stress and the accumulation of ceramides [17,18,19,20]

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