Fat-Secreted Ceramides Regulate Vascular Redox State and Influence Outcomes in Patients With Cardiovascular Disease

Background Obesity is associated with increased cardiovascular risk; however, the potential role of dysregulations in the adipose tissue (AT) metabolome is unknown. Objectives The aim of this study was to explore the role of dysregulation in the AT metabolome on vascular redox signaling and cardiovascular outcomes. Methods A screen was conducted for metabolites differentially secreted by thoracic AT (ThAT) and subcutaneous AT in obese patients with atherosclerosis (n = 48), and these metabolites were then linked with dysregulated vascular redox signaling in 633 patients undergoing coronary bypass surgery. The underlying mechanisms were explored in human aortic endothelial cells, and their clinical value was tested against hard clinical endpoints. Results Because ThAT volume was associated significantly with arterial oxidative stress, there were significant differences in sphingolipid secretion between ThAT and subcutaneous AT, with C16:0-ceramide and derivatives being the most abundant species released within adipocyte-derived extracellular vesicles. High ThAT sphingolipid secretion was significantly associated with reduced endothelial nitric oxide bioavailability and increased superoxide generated in human vessels. Circulating C16:0-ceramide correlated positively with ThAT ceramides, dysregulated vascular redox signaling, and increased systemic inflammation in 633 patients with atherosclerosis. Exogenous C16:0-ceramide directly increased superoxide via tetrahydrobiopterin-mediated endothelial nitric oxide synthase uncoupling and dysregulated protein phosphatase 2 in human aortic endothelial cells. High plasma C16:0-ceramide and its glycosylated derivative were independently related with increased risk for cardiac mortality (adjusted hazard ratios: 1.394; 95% confidence interval: 1.030 to 1.886; p = 0.031 for C16:0-ceramide and 1.595; 95% confidence interval: 1.042 to 2.442; p = 0.032 for C16:0-glycosylceramide per 1 SD). In a randomized controlled clinical trial, 1-year treatment of obese patients with the glucagon-like peptide-1 analog liraglutide suppressed plasma C16:0-ceramide and C16:0-glycosylceramide changes compared with control subjects. Conclusions These results demonstrate for the first time in humans that AT-derived ceramides are modifiable regulators of vascular redox state in obesity, with a direct impact on cardiac mortality in advanced atherosclerosis. (The Interaction Between Appetite Hormones; NCT02094183)

In this study, we applied a metabolomics approach to interrogate differences in the secretome profiles of biologically distinct AT depots collected from patients with atherosclerosis. We revealed AT-secreted metabolites that are dysregulated in obesity, exert endocrine effects on the human vascular wall, and regulate vascular redox state.  Table 1.

METHODS
Baseline characteristics of study participants are detailed in Supplemental Table 2.    COHORT-WIDE GENE EXPRESSION ANALYSES. Total ribonucleic acid was extracted from whole blood, reverse-transcribed, and quantified as described (1) (Supplemental Methods).

EXTRACTION AND DEFINITION OF ENDPOINTS.
Collection and classification of mortality data are described in Supplemental Methods.
STATISTICAL ANALYSES. The differential and enrichment analyses of metabolic data presented in this study were performed using MetaboAnalyst    Figure 2). We then organized a nested study (study 2) in which we cultured 48 paired biopsies of ScAT and ThAT from 31 obese and 17 propensity-matched nonobese patients for 4 h and used the conditioned media for secretome studies (study 2) (Supplemental Table 2). We performed hypothesis-free metabolomics profiling of the AT secretome to identify differentially secreted metabolites related to the AT depot type and the obesity To identify dysregulated metabolic pathways in the AT secretome in patients with obesity, we identified metabolites that were differentially secreted from ThAT and ScAT of the obese versus lean participants from study 2 (matched for age, sex, and risk factors) (Supplemental Figure 4). SPL metabolism scored the highest pathway impact in obesity ( Figure 2D). Obesity was also related with higher secretion of ceramides from ThAT ( Figures 2E and 2F).  Adipocyte EVs showed typical morphology by transmission electron microscopy (F). The abundance of each SPL species in EVs released from adipocytes is presented in G to L. Bars in A to D indicate mean AE SEM; bars in G to L indicate median AE interquartile range; *p < 0.05 (nominal) and **p < 0.01 (false discovery rate adjusted) for mature adipocytes versus pre-adipocytes (calculated using Wilcoxon signed rank tests). Abbreviations as in Figures 1 and 2.    Table 5). The SPLs detected in the plasma of these patients and their circulating concentrations are  shown in Supplemental Figure 10 and Supplemental were the most abundant ceramides in plasma, yet specific Cer16:0 metabolites were still detected in high concentrations, although they were no longer the dominant species, as was the case for the AT.
We then explored the associations between AT and circulating ceramides and key mechanisms of vascular disease pathogenesis ( Figure  6A,  Figure 7C) and primary HAECs ( Figure 7D) significantly increased O 2 .À production.
This was confirmed using dihydroethidium staining of these cells ( Figure 7E).
Next, to determine the mechanism by which  There was no association between plasma Cer16:0 and endothelium-independent vasorelaxations of human vessels to SNP (G; n ¼ 123). P values were calculated using the Kruskal-Wallis test (B-D) or 2-way analysis of variance with dose Â group interaction terms (F,G). BMI ¼ body mass index; HDL ¼ high-density lipoprotein; HOMA-IR ¼ homeostasis model assessment of insulin resistance; LDL ¼ low-density lipoprotein; other abbreviations as in Figures 1, 2, and 5.  by suppressing the phosphorylation of its inhibitory site PP2A Tyr307 ( Figure 7M). We then used LB100, a potent inhibitor of PP2A activity, which prevented Cer16:0-induced reductions in p-eNOS Ser1177 :eNOS ( Figure 7N).  Table 6). The  Table 7). The tested metabolites were also found to be associated with cardiovascular death (Supplemental Tables 8 and 9), adverse cardiac phenotypes, and endpoints (Supplemental Figure 17, Supplemental Table 10).
There was no association between these metabolites and noncardiac mortality in the same cohort ( Figure 8I).  Although there was no effect of Cer16:0 on AKT phosphorylation at site Ser473 (L), it induced significant reduction of protein phosphatase 2 (PP2A) phosphorylation at its inhibitory site Tyr307 (M). The Cer16:0-induced reductions in p-eNOS Ser1177 /eNOS ratio was reversed by 10 mM LB100, a PP2A inhibitor (N; n ¼ 6). *p < 0.05 versus control (DMSO <1%); all presented as median (interquartile range). Abbreviations as in Figures 1, 2, 5, and 6.   Ceramides have been recognized as key signaling molecules in different cell types regulating vital cellular processes. The origin of circulating ceramides in humans is poorly understood, although the liver and AT might be key sources (9,10). We now demonstrate that SPL is the most prominent dysregulated pathway in obese ThAT compared with ScAT.
Cer16:0 is the most abundant ceramide generated in ThAT, and it is secreted largely in extracellular vesicles released from the adipocytes (Supplemental Figures 19 and 20). The secreted Cer16:0 levels from ThAT (but not ScAT) are correlated with plasma Cer16:0 levels, implying that ThAT may contribute to circulating Cer16:0 levels, although this association is suggestive and not conclusive.
Ceramides and their derivatives have been linked with cardiovascular disease (11,12). In particular, plasma Cer16:0 has been associated with increased risk for major adverse cardiovascular events, including heart failure (13,14) and myocardial infarction (11,15), in the general population. However, it is unclear whether AT-secreted ceramides play a role in the vascular complications of obesity. We now demonstrate that ThAT volume is associated with increased arterial oxidative stress and that this depot secretes more ceramides than ScAT, particularly Cer16:0. ThAT-derived ceramide levels were also related with vascular redox state.
Increased vascular oxidative stress and reduced endothelial nitric oxide bioavailability are key features of atherogenesis (16). Nitric oxide is produced by eNOS, an enzyme that becomes a source of O 2 .À when its cofactor tetrahydrobiopterin is oxidized (6).
It is unclear whether AT-derived ceramides play a role in the regulation of these fundamental mechanisms of atherogenesis. We now reveal using ex vivo human tissue and in vitro primary cultured cells that increased levels of Cer16:0 in the patients who were randomized to a low-calorie diet or liraglutide treatment (1.2 mg/day) for 52 weeks, following 8 weeks (baseline) of lowcalorie diet-induced weight loss. Box plots represent median (interquartile range). Abbreviations as in Figure 8.
Akawi et al. This effect was observed with long-chain ceramides in the initial association screening, but not with medium-chain ceramides such as C12:0-ceramide.
We also show that Cer16:0 induces eNOS uncoupling via tetrahydrobiopterin oxidation, while it affects eNOS activity via PP2A. These findings suggest that Cer16:0, the most abundant ceramide in ThAT secretome, has direct effects on the mechanisms of atherogenesis.
Although our findings suggest causal links between ceramides and vascular dysfunction, existing data on the prognostic role of circulating ceramides in humans remain unclear, with some studies reporting positive (11,12) and others negative (17)  Promising therapeutic approaches specifically targeting ceramides biosynthesis have been reported (18,19), but it is unknown whether ceramides could be modified by therapeutic interventions that are known to reprogram AT and to reduce cardiovascular disease risk, such as GLP-1 analogs (20). We explored the ef-

CONCLUSIONS
We demonstrate the ThAT secretes ceramides, and particularly Cer16:0, which has an endocrine effect on the vascular wall, inducing vascular redox state. We also show that high plasma levels of Cer16:0 have striking prognostic value for cardiac mortality, and may be a drugable target of GLP1-analogues.