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

Empagliflozin maintains capillarization and improves cardiac function in a murine model of left ventricular pressure overload

Patients with type 2 diabetes treated with Sodium glucose transporter 2 (SGLT2) inhibitors show reduced mortality and hospitalization for heart failure (HF). SGLT2 inhibitors are considered to activate multiple cardioprotective pathways; however, underlying mechanisms are not fully described. This study aimed to elucidate the underlying mechanisms of the beneficial effects of SGLT2 inhibitors on the failing heart. We generated a left ventricular (LV) pressure overload model in C57BL/6NCrSlc mice by transverse aortic constriction (TAC) and examined the effects of empagliflozin (EMPA) in this model. We conducted metabolome and transcriptome analyses and histological and physiological examinations. EMPA administration ameliorated pressure overload-induced systolic dysfunction. Metabolomic studies showed that EMPA increased citrulline levels in cardiac tissue and reduced levels of arginine, indicating enhanced metabolism from arginine to citrulline and nitric oxide (NO). Transcriptome suggested possible involvement of the insulin/AKT pathway that could activate NO production through phosphorylation of endothelial NO synthase (eNOS). Histological examination of the mice showed capillary rarefaction and endothelial apoptosis after TAC, both of which were significantly improved by EMPA treatment. This improvement was associated with enhanced expression phospho-eNOS and NO production in cardiac endothelial cells. NOS inhibition attenuated these cardioprotective effects of EMPA. The in vitro studies showed that catecholamine-induced endothelial apoptosis was inhibited by NO, arginine, or AKT activator. EMPA activates the AKT/eNOS/NO pathway, which helps to suppress endothelial apoptosis, maintain capillarization and improve systolic dysfunction during LV pressure overload.

Hepatocyte Growth Factor Protects Endothelial Barrier Against Oxidative Stress and Mitochondria-dependent Apoptosis

Background: Pulmonary microvascular endothelial cells(PMVECs) were incomplex and endothelial barrier was destroyed in the pathogenesis progress of acute lung injury(ALI)/acute respiratory distress syndrome(ARDS). Previous studies have demonstrated that hepatocyte growth factor (HGF) could decrease endothelial apoptosis. Nevertheless, the mechanism by which HGF-suppressed oxidative stress contributes to endothelial mitochondria-dependent apoptosis is poorly understood. Methods: In our current study, we introduced lipopolysaccharide(LPS)-induced PMEVCs with HGF treatment. To investigate the effects of mTOR/STAT-3 pathway in endothelial oxidative stress and mitochondria-dependent apoptosis, mammalian TOR (mTOR) inhibitor rapamycin and signal transducer and activator of transcription 3 (STAT-3) inhibitor S3I-201 were respectively used to inhibit mTOR/STAT-3 signaling. Moreover, lentivirus vector-mediated HGF, mTORC1(raptor) and mTORC2(rictor) gene knockdown modification were introduced to evaluate mTORC1 and mTORC2 pathway. Calcium measurement, ROS production, mitochondrial membrane potential and protein complex I expression, cell proliferation, apoptosis and endothelial junction protein were detected to evaluate HGF effects.Results: Our study demonstrated that HGF protected endothelium via the suppression of ROS production and intracellular calcium uptake, which leading to increased mitochondrial membrane potential (JC-1 and mitochondria tracker green detection)and specific proteins(complex I), decreased endothelial apoptosis specific protein(Caspase-3), raised anti-apoptosis mRNA level(Bcl-2 and Bcl-xL), and increased endothelial junction proteins (VE-cadherin and occludin). Reversely, mTOR inhibitor rapamycin and STAT-3 inhibitor S3I-201 could raise oxidative stress and mitochondria-dependent apoptosis even with HGF treatment in LPS-induced endothelial cells. Similarly, mTORC1 as well as mTORC2, have the same protective effects in mitochondria damage and apoptosis. Conclusion: In all, these reveal that mTOR/STAT-3 signaling mediate the HGF suppression effects to oxidative level, mitochondria-dependent apoptosis and endothelial junction protein in LPS-stimulated PMVECs, which contributing to the endothelial survival and barrier integrity.

Resveratrol attenuates cigarette smoke induced endothelial apoptosis by activating Notch1 signaling mediated autophagy

Background Increasing evidence shows that endothelial apoptosis contributes to cigarette smoke (CS)-induced disease progression, such as chronic obstructive pulmonary disease (COPD). Our previous studies have validated Notch1 as an anti-apoptotic signaling in CS-induced endothelial apoptosis. Resveratrol (RESV) is a naturally occurring polyphenol that exhibits an anti-apoptotic activity in endothelial cells that exposed to many kinds of destructive stimulus. However, the effects of resveratrol on Notch1 signaling in CS-induced endothelial apoptosis have not yet been fully elucidated. Therefore, the aim of this study was to examine whether RESV can protect endothelial cells from CS-induced apoptosis via regulating Notch1 signaling. Methods Human umbilical vein endothelial cells (HUVECs) were pretreated with RESV for 2 h, followed by cotreatment with 2.5%CSE for 24 h to explore the role of RESV in CSE induced endothelial apoptosis. 3-methyladenine (3-MA) or rapamycin was used to alter autophagic levels. Lentivirus Notch1 intracellular domain (LV-N1ICD), γ-secretase inhibitor (DAPT) and Notch1 siRNA were used to change Notch1 expression. The expression of Notch1, autophagic and apoptotic markers were examined by Western blot and the apoptosis rate was detected by Flow cytometry analysis. Results Our results showed that activating autophagy reduced CSE-induced endothelial apoptosis, while blocking autophagy promoted cell apoptosis in HUVECs. RESV pretreatment attenuated the CSE-induced endothelial apoptosis and activated Notch1 signaling. RESV pretreatment also increased LC3b-II and Beclin1 production, decreased p62 and mTOR expression. 3-MA treatment inhibited autophagy and aggravated CSE induced apoptosis, while rapamycin promoted autophagy, led to a decrease in cell apoptosis. LV-N1ICD transfection upregulated autophagy and reduced apoptosis. However, this protective effect was abolished by 3-MA treatment. In cells treated with DAPT or Notch1 siRNA, autophagy was decreased, while apoptosis was increased. RESV partly rescued the DAPT or Notch1 siRNA induced apoptosis by activating Notch1 signaling. Conclusion In HUVECs, RESV attenuates CSE induced endothelial apoptosis by inducing autophagy in a Notch1-dependent manner.