Department of Internal Medicine II, University Hospital Bonn, University of Bonn, Venusberg-Campus 1, 53127, Bonn, Germany.
Institute of Molecular Physiology and Biotechnology of Plants, University of Bonn, Karlrobert-Kreiten-Str. 13, 53115, Bonn, Germany.
Cell Mol Life Sci. 2021 Dec 24;79(1):48. doi: 10.1007/s00018-021-04049-5.
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.
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.
lEVs mediate the induction of apoptosis in endothelial cells in response to hyperglycemic injury through intercellular transfer of ceramides.
促凋亡和促炎神经酰胺在动脉粥样硬化斑块的发展中起着至关重要的作用。局部细胞神经酰胺积累介导内皮细胞凋亡，尤其是在 2 型糖尿病中，2 型糖尿病是心血管疾病的主要危险因素。近年来，大型细胞外囊泡（lEVs）已被确定为细胞间通讯的重要手段，并作为心血管健康和疾病的调节剂。lEVs 是否可以作为神经酰胺转移的载体以及诱导与糖尿病相关的内皮细胞凋亡的诱导物，这一潜在作用尚未得到研究。
通过对人冠状动脉内皮细胞（HCAEC）及其 lEVs 的质谱分析，发现 C16 神经酰胺（d18：1-16：0）是 lEVs 中含量最丰富的神经酰胺，并且在 HCAEC 发生高血糖损伤后，lEVs 中的神经酰胺含量显著增加。高血糖诱导 HCAEC 中神经酰胺包装到 lEVs 中增加依赖于中性鞘氨醇酶 2（nSMase2），高血糖处理的 HCAEC 中 nSMase2 上调。与未处理的 HCAEC 的天然 lEV 相比，来自高血糖 HCAEC 的 lEV 可诱导接受细胞发生凋亡。同样，链脲佐菌素注射后高血糖小鼠的 lEV 比正常血糖小鼠的 lEV 诱导更高的内皮细胞凋亡率。为了产生具有高浓度 C16 神经酰胺的 lEV，应用外源性神经酰胺并证明其可有效地包装到 lEV 中，然后通过激活 caspase 3 诱导 lEV 接受的 HCAEC 凋亡。通过使用荧光标记的神经酰胺类似物证实了通过 lEV 进行神经酰胺的细胞间转移。用 nSMase 的药理学抑制剂（GW4869）处理 HCAEC 或用 siRNA 下调 nSMase2 可阻断葡萄糖对 lEV 接受细胞凋亡的影响。相反，对于小细胞外囊泡（sEVs），高血糖损伤或 GW4869 处理对 sEV 接受细胞的凋亡诱导没有影响。
lEV 通过神经酰胺的细胞间转移介导内皮细胞对高血糖损伤的凋亡诱导。
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