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

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

Antioxidants Supplementation Reduces Ceramide Synthesis Improving the Cardiac Insulin Transduction Pathway in a Rodent Model of Obesity

Obesity-related disruption in lipid metabolism contributes to cardiovascular dysfunction. Despite numerous studies on lipid metabolism in the left ventricle, there is no data describing the influence of n-acetylcysteine (NAC) and α-lipoic acid (ALA), as glutathione precursors, on sphingolipid metabolism, and insulin resistance (IR) occurrence. The aim of our experiment was to evaluate the influence of chronic antioxidants administration on myocardial sphingolipid state and intracellular insulin signaling as a potential therapeutic strategy for obesity-related cardiovascular IR. The experiment was conducted on male Wistar rats fed a standard rodent chow or a high-fat diet with intragastric administration of NAC or ALA for eight weeks. Cardiac and plasma sphingolipid species were assessed by high-performance liquid chromatography (HPLC). The proteins expressed from sphingolipid and insulin signaling pathways were determined by Western blot. Antioxidant supplementation markedly reduced ceramide accumulation by lowering the expression of selected proteins from the sphingolipid pathway and simultaneously increased the myocardial sphingosine-1-phosphate level. Moreover, NAC and ALA augmented the expression of GLUT4 and the phosphorylation state of Akt (Ser473) and GSK3β (Ser9), which improved the intracellular insulin transduction pathway. Based on our results, we may postulate that NAC and ALA have a beneficial influence on the cardiac ceramidose under IR conditions.

Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction

Aberrant ceramide build-up in preeclampsia, a serious disorder of pregnancy, causes exuberant autophagy-mediated trophoblast cell death. The significance of ceramide accumulation for lysosomal biogenesis in preeclampsia is unknown. Here we report that lysosome formation is markedly increased in trophoblast cells of early-onset preeclamptic placentae, in particular in syncytiotrophoblasts. This is accompanied by augmented levels of transcription factor EB (TFEB). In vitro and in vivo experiments demonstrate that ceramide increases TFEB expression and nuclear translocation and induces lysosomal formation and exocytosis. Further, we show that TFEB directly regulates the expression of lysosomal sphingomyelin phosphodiesterase (L-SMPD1) that degrades sphingomyelin to ceramide. In early-onset preeclampsia, ceramide-induced lysosomal exocytosis carries L-SMPD1 to the apical membrane of the syncytial epithelium, resulting in ceramide accumulation in lipid rafts and release of active L-SMPD1 via ceramide-enriched exosomes into the maternal circulation. The SMPD1-containing exosomes promote endothelial activation and impair endothelial tubule formation in vitro. Both exosome-induced processes are attenuated by SMPD1 inhibitors. These findings suggest that ceramide-induced lysosomal biogenesis and exocytosis in preeclamptic placentae contributes to maternal endothelial dysfunction, characteristic of this pathology.

Cardiomyocyte Krüppel-like Factor 5 Promotes De Novo Ceramide Biosynthesis and Contributes to Eccentric Remodeling in Ischemic Cardiomyopathy

Background: We previously showed that cardiomyocyte Krüppel-like factor (KLF)-5 regulates cardiac fatty acid oxidation. As heart failure has been associated with altered fatty acid oxidation, we investigated the role of cardiomyocyte KLF5 in lipid metabolism and pathophysiology of ischemic heart failure. Methods: Using rtPCR and Western Blot, we investigated the KLF5 expression changes in a myocardial infarction (MI) mouse model and heart tissue from patients with ischemic heart failure. Using 2D-echocardiography, we evaluated the effect of KLF5 inhibition after MI using pharmacological KLF5 inhibitor ML264 and mice with cardiomyocyte specific KLF5 deletion (αMHC-KLF5 -/- ). We identified the involvement of KLF5 in regulating lipid metabolism and ceramide accumulation after MI using liquid-chromatography-tandem-mass-spectrometry, and Western Blot and rtPCR analysis of ceramide-metabolism-related genes. We lastly evaluated the effect of cardiomyocyte-specific KLF5 overexpression (αMHC-rtTA-KLF5) on cardiac function and ceramide metabolism, and rescued the phenotype using myriocin to inhibit ceramide biosynthesis. Results: KLF5 mRNA and protein levels were higher in human ischemic heart failure samples and in rodent models 24h, 2- and 4-weeks post-permanent left coronary artery ligation. αMHC-KLF5 -/- mice and mice treated with ML264 had higher ejection fraction and lower ventricular volume and heart weight after MI. Lipidomic analysis showed that αMHC-KLF5 -/- mice with MI had lower myocardial ceramide levels compared with littermate control mice with MI although basal ceramide content of αMHC-KLF5 -/- mice was not different from control mice. KLF5 ablation suppressed the expression of serine-palmitoyl-transferase-long-chain-base-subunit (SPTLC)1 and SPTLC2, which regulate de novo ceramide biosynthesis. We confirmed our previous findings that myocardial SPTLC1 and SPTLC2 levels are increased in heart failure patients. Consistently, αMHC-rtTA-KLF5 mice showed increased SPTLC1 and SPTLC2 expression, higher myocardial ceramide levels, and systolic dysfunction beginning 2-weeks after KLF5 induction. Treatment of αMHC-rtTA-KLF5 mice with myriocin that inhibits SPT, suppressed myocardial ceramide levels and alleviated systolic dysfunction. Conclusions: KLF5 is induced during the development of ischemic heart failure in humans and mice and stimulates ceramide biosynthesis. Genetic or pharmacological inhibition of KLF5 in mice with MI prevents ceramide accumulation, alleviates eccentric remodeling, and increases ejection fraction. Thus, KLF5 emerges as a novel therapeutic target for the treatment of ischemic heart failure.

KLF5 Is Induced by FOXO1 and Causes Oxidative Stress and Diabetic Cardiomyopathy

Rationale: Diabetic cardiomyopathy (DbCM) is a major complication in type-1 diabetes (T1D), accompanied by altered cardiac energetics, impaired mitochondrial function and oxidative stress. Previous studies indicate that T1D is associated with increased cardiac expression of Krüppel-like factor-5 (KLF5) and Peroxisome Proliferator Activated Receptor (PPAR)α that regulate cardiac lipid metabolism. Objective: In this study, we investigated the involvement of KLF5 in DbCM and its transcriptional regulation. Methods and Results: KLF5 mRNA levels were assessed in isolated cardiomyocytes from cardiovascular patients with diabetes and was higher compared with non-diabetic individuals. Analyses in human cells and diabetic mice with cardiomyocyte-specific FOXO1 deletion showed that FOXO1 bound directly on the KLF5 promoter and increased KLF5 expression. Diabetic mice with cardiomyocyte-specific FOXO1 deletion had lower cardiac KLF5 expression and were protected from DbCM. Genetic, pharmacologic gain and loss of KLF5 function approaches and AAV-mediated Klf5 delivery in mice showed that KLF5 induces DbCM. Accordingly, the protective effect of cardiomyocyte FOXO1 ablation in DbCM was abolished when KLF5 expression was rescued. Similarly, constitutive cardiomyocyte-specific KLF5 overexpression caused cardiac dysfunction. KLF5 caused oxidative stress via direct binding on NADPH oxidase (NOX)4 promoter and induction of NOX4 expression. This was accompanied by accumulation of cardiac ceramides. Pharmacologic or genetic KLF5 inhibition alleviated superoxide formation, prevented ceramide accumulation and improved cardiac function in diabetic mice. Conclusions: Diabetes-mediated activation of cardiomyocyte FOXO1 increases KLF5 expression, which stimulates NOX4 expression, ceramide accumulation and causes DbCM.

TNFa is Involved in the Development of Disuse Muscle Atrophy Through the Acid Sphingomyelinase/ Ceramide Up-Regulation and Pro-Oxidant Signaling

Background: There is paucity of data indicating the role of cytokines including TNFa in the development of disuse muscle atrophy, despite the growing interest to this problem emerging in the recent years. The aim of the present study was to test the hypothesis that TNFa/ TNFR1 may be involved in the development of disuse muscle atrophy caused by unloading through aSMase/ ceramide/ ROS mechanism. Methods: The experiments were performed on male Wistar rats (180 – 230g) subjected to 4 or 14 days of hindlimb suspension (HS) and treated with clomipramine (HS+Clom) or vehicle. The following parameters were studied: TNFR1, aSMase, nSMase and Nox2 proteins and ceramide in detergent-resistant membrane (DRM) fraction isolated from soleus muscle homogenates, pro-oxidant/anti-oxidant and pro-apoptotic/anti-apoptotic activities, immune fluorescence intensity and distribution of ceramide, Nox2, Nox4 and caveolin-3 on longitudinal and transverse muscle sections. The relative muscle mass, cross-sectional area (CSA) and Feret’s diameter (FD) of muscle fibers were used to confirm muscle atrophy. Statistical analysis was performed using one-way ANOVA followed by the Bonferroni post hoc test, or Cruskall-Wallis and Mann-Whitney U test. Results: Disuse caused an increase in membrane TNFR1, aSMase, ceramide abundance in DRM, up-regulation of pro-oxidant and pro-apoptotic capacities (increased Nox2, Nox4, TBA-active products, Bax/Bcl-2 ratio, elevated activity of caspase-3/7 and -6). The most of alterations were maximal on the 4 th day of unloading. The inhibitor of aSMase clomipramine attenuated ceramide accumulation, decreased pro-oxidant and pro-apoptotic activities and diminished muscle atrophy induced by unloading. It has been shown that in suspended for 14 days rats the loss in relative muscle mass, CSA and FD averaged -35%, -65% and -49%, respectively, whereas in clomipramine treated rats it was -25%, -45% and -25%, in comparison with the control values. Clomipramine also mitigated the inhibition of the mTORC1/p70S6 kinase inhibition caused by 14-day HS. Conclusions: The obtained results indicate the involvement of aSMase/ ceramide pathway in the development of disuse muscle atrophy. This effect may be triggered by TNFR1 and realized through enhanced prooxidant NADPH oxidase activity and pro-apoptotic signaling.

Sphingolipid Profiling Reveals Different Extent of Ceramide Accumulation in Bovine Retroperitoneal and Subcutaneous Adipose Tissues

Sphingolipids are bioactive lipids that can modulate insulin sensitivity, cellular differentiation, and apoptosis in a tissue-specific manner. However, their comparative profiles in bovine retroperitoneal (RPAT) and subcutaneous adipose tissue (SCAT) are currently unknown. We aimed to characterize the sphingolipid profiles using a targeted lipidomics approach and to assess whether potentially related sphingolipid pathways are different between SCAT and RPAT. Holstein bulls (n = 6) were slaughtered, and SCAT and RPAT samples were collected for sphingolipid profiling. A total of 70 sphingolipid species were detected and quantified by UPLC-MS/MS in multiple reaction monitoring (MRM) mode, including ceramide (Cer), dihydroceramide (DHCer), sphingomyelin (SM), dihydrosphingomyelin (DHSM), ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), galactosylceramide (GalCer), glucosylceramide (GluCer), lactosylceramide (LacCer), sphinganine (DHSph), and sphingosine (Sph). Our results showed that sphingolipids of the de novo synthesis pathway, such as DHSph, DHCer, and Cer, were more concentrated in RPAT than in SCAT. Sphingolipids of the salvage pathway and the sphingomyelinase pathway, such as Sph, S1P, C1P, glycosphingolipid, and SM, were more concentrated in SCAT. Our results indicate that RPAT had a greater extent of ceramide accumulation, thereby increasing the concentration of further sphingolipid intermediates in the de novo synthesis pathway. This distinctive sphingolipid distribution pattern in RPAT and SCAT can potentially explain the tissue-specific activity in insulin sensitivity, proinflammation, and oxidative stress in RPAT and SCAT.