Ceramides and risk of major adverse cardiovascular events: A meta-analysis of longitudinal studies

Journal Pre-proof Ceramides And Risk Of Major Adverse Cardiovascular Events: A Meta-Analysis Of Longitudinal Studies Alessandro Mantovani, MD, Clementina Dugo, MD PII:

S1933-2874(20)30005-2

DOI:

https://doi.org/10.1016/j.jacl.2020.01.005

Reference:

JACL 1540

To appear in:

Journal of Clinical Lipidology

Received Date: 13 September 2019 Revised Date:

9 January 2020

Accepted Date: 12 January 2020

Please cite this article as: Mantovani A, Dugo C, Ceramides And Risk Of Major Adverse Cardiovascular Events: A Meta-Analysis Of Longitudinal Studies, Journal of Clinical Lipidology (2020), doi: https:// doi.org/10.1016/j.jacl.2020.01.005. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 National Lipid Association. All rights reserved.

CERAMIDES AND RISK OF MAJOR ADVERSE CARDIOVASCULAR EVENTS: A META-ANALYSIS OF LONGITUDINAL STUDIES

Alessandro Mantovani, MD1, Clementina Dugo, MD2 1

Section of Endocrinology, Diabetes and Metabolism, University and Azienda Ospedaliera Universitaria Integrata of Verona, Verona, Italy

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Division of Cardiology, ‘‘IRCCS Sacro Cuore - Don Calabria’’ Hospital, Negrar (VR), Italy

Word count: abstract 233; text 2,756 (excluding title page and references); table: n. 1; figure: n. 1; Supplementary Tables n. 4; Supplementary Figures n. 4; n. 30 references. Running title: ceramides and CVD

Address of correspondence: Dr. Alessandro Mantovani, MD Section of Endocrinology, Diabetes and Metabolism University and Azienda Ospedaliera Universitaria Integrata Piazzale Stefani, 1 37126 Verona Tel. 045/8123110 Fax: 045/8027314 E-mail: [email protected]

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ABSTRACT Background – Recent cohort studies evaluated the association between some previously identified high-risk ceramides [Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/22:0), Cer(d18:1/24:0) and Cer(d18:1/24:1)] and risk of major adverse cardiovascular events in adult population. Objective - The objective of this meta-analysis was to investigate the magnitude of such associations. Methods – We searched publication databases using appropriate keywords to identify cohort studies (published up to July 30, 2019), in which association between previously identified highrisk ceramides and major adverse cardiovascular events was reported. Data from eligible studies were extracted and meta-analysis was performed using random-effects modeling. Results – Seven cohort studies with aggregate data on 29,818 individuals (2,736 new cases of cardiovascular events over a median follow-up of 6 years) were included. Higher plasma levels of Cer(d18:1/16:0) (random effects hazard ratio [HR] per standard deviation 1.21, 95% confidence interval [CI] 1.11-1.32, I2=88%), Cer(d18:1/18:0) (HR 1.19, 95% CI 1.10-1.27, I2=68%), and Cer(d18:1/24:1) (HR 1.17, 95% CI 1.08-1.27, I2=83%) were associated with major adverse cardiovascular events. Conversely, no association with plasma levels of Cer(d18:1/22:0) (HR 1.14 95% CI 0.88-1.47, I2=88%) and Cer(d18:1/24:0) (HR 0.97, 95% CI 0.89-1.05, I2=73%) was found. Subgroup analyses did not substantially modify the findings. Conclusions – Higher plasma levels of Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/24:1) were associated with major adverse cardiovascular events, whereas plasma levels of Cer(d18:1/22:0) and Cer (d18:1/24:0) were not. Additional research is required to elucidate the different role of ceramides on pathways involved in cardiovascular disease.

Keywords: ceramides; cardiovascular disease; CVD; major adverse cardiovascular events; cardiovascular events.

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INTRODUCTION Ceramides are a class of sphingolipids consisting of sphingosine or a related base linked to a fatty acid by an amide bond (1,2). Compared to glycerophospholipids, sphingolipids (such as ceramides, gangliosides and sphingomyelins) are low in quantities, as they are present in the human body at levels of <20% of their glycerolipid counterparts (1,2). Specifically, ceramides are found in small amounts [i.e., <1 mole percent (mol%)] among cell membranes, but their concentration can rise by 10-fold in specific physiological or pathological conditions (3,4). Importantly, along with their structural functions in cellular membranes, ceramides also play a role as second messengers in intra- and inter-cellular signaling pathways (1-4). In this context, they can activate transcription mediators involved in the development of atherosclerotic processes (1-4).

Over the last decade, accumulating evidence, mostly derived from in vitro experiments and in vivo animal studies, has supported the role of distinct ceramides as potential mediators or biomarkers of several disease mechanisms, including cardiovascular disease. In this regard, recently, some large cohort studies have identified specific plasma ceramides [namely Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/24:1)] as significant predictors of cardiovascular morbidity and mortality in the general population and in patients with established coronary artery disease (CAD) or acute coronary syndrome, regardless of traditional cardiovascular risk factors and other confounders (5-14). In addition, higher plasma levels of such ceramides have been also significantly associated with higher angiographic severity of coronary stenosis in patients with CAD (15,16), as well as with lower post-stress myocardial wall perfusion in patients with established/suspected CAD, who underwent myocardial perfusion scintigraphy (17,18)

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However, to date, the magnitude of the association between specific plasma ceramides and the risk of major adverse cardiovascular events is not established. Thus, we herein report the results of a meta-analysis of large cohort studies investigating the association between previously identified high-risk plasma ceramide molecules [namely Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/22:0), Cer(d18:1/24:0) and Cer(d18:1/24:1)] and the risk of major adverse cardiovascular events. We believed that the clarification of the effect of various ceramides on the risk of cardiovascular events may have important clinical implications for the prevention and management of coronary artery disease.

MATERIALS AND METHODS Registration of protocol The protocol of this meta-analysis was registered in advance in PROSPERO database (International Prospective Register of Systematic Reviews) with the following number: CRD42019143301.

Data Sources and Searches Studies were included if they were (prospective or retrospective) cohort studies investigating the association between the previously identified high-risk plasma ceramides [i.e., Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/22:0), Cer(d18:1/24:0) and Cer(d18:1/24:1)] and the risk of major adverse cardiovascular events among adults with or without prior history of coronary artery disease. No restrictions in terms of race or ethnicity were adopted. Exclusion criteria were: (i) abstracts, case reports, reviews, editorials, practice guidelines and cross-sectional studies; (ii) studies conducted in pediatric populations; (iii) studies with a follow-up duration <1 year; (iv) studies where not reported any hazard ratio (HR) and 95% confidence interval (CI) for major

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adverse cardiovascular events; (v) studies evaluating the relationship between total plasma ceramide levels and major adverse cardiovascular events.

Data Extraction and Quality Assessment Seven cohort studies were identified by systematically searching PubMed, Scopus and Web of Science from January 1, 2000 to July 15, 2019 (date last searched) using the free text terms “ceramides” (OR “ceramide” OR “ceramide molecules”) AND “cardiovascular disease” OR “incident cardiovascular disease” OR “fatal and non-fatal cardiovascular events” OR “major adverse cardiovascular events”. Non-English-language papers were excluded. Two investigators (AM and CD) independently examined titles and abstracts and obtained full texts of potentially relevant papers. Working independently, we read the papers and determined whether they met inclusion criteria. Discrepancies were resolved by discussion. For all eligible studies, we extracted information regarding study design, study size, source of data, population characteristics, duration of follow-up, outcome of interest, and confounding factors.

Two authors (AM and CD) independently assessed the risk of bias. Seeing that all the eligible studies had a cohort design, the Newcastle-Ottawa Scale (NOS) was used to judge study quality, as recommended by the Cochrane Collaboration.

Data Synthesis and Analysis The full adjusted HRs (expressed per 1-standard deviation of circulating levels of ceramides) of all eligible cohort studies were pooled, and an overall estimate of effect size was calculated using a random-effects model. Publication bias was assessed using funnel plot, the Begg’s test and the Egger's regression test (19-21). Given the expected heterogeneity of the eligible studies, in order

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to explore the possible sources of heterogeneity among studies and to assess the robustness of the associations, we also conducted sensitivity/subgroup analyses by quality of studies (based on the NOS scale), study population, and study country. Furthermore, we assessed for possibly excessive influence of individual studies using a meta-analysis influence test that eliminated each of the included studies at a time. Finally, we also conducted multivariate meta-regression analyses to assess the association of age, body mass index and LDL-cholesterol levels with the risk of major adverse cardiovascular events carried out by specific ceramides [i.e., Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0) and Cer(d18:1/24:1)].

All statistical tests were two sided and used a significance level of p<0.05. We used STATA® 14.2 (Stata, College Station, TX) for all statistical analyses. Specifically, metan command was used for random effects meta-analyses.

RESULTS As reported in Supplementary Figure 1, after literature research and study selection, we included seven cohort studies (5-11) for a total of 29,818 adult individuals (mean age 60.9 years, mean body mass index 27.3 kg/m2, mean LDL-cholesterol 3.3 mmol/l, median triglycerides 1.5 mmol/L and 64.9% men) with 2,736 cases of major adverse cardiovascular events over a median follow-up of 6.0 years (interquartile range: 4.0-6.0 years) (Table 1). The cohort studies (12-14) excluded at the eligibility step of PRISMA diagram are reported in the Supplementary Table 1. The distribution of eligible cohort studies by estimate of the association between previously identified high-risk plasma ceramide molecules and the risk major of adverse cardiovascular outcome is plotted in Figure 1. Six eligible cohort studies (6-11) provided data for major adverse cardiac events, while one (5) provided data only for cardiovascular death. Overall, we found that higher plasma levels of

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Cer(d18:1/16:0) (random effects hazard ratio [HR] per standard deviation 1.21, 95% confidence interval [CI] 1.11-1.32, I2=88%), Cer(d18:1/18:0) (HR 1.19, 95% CI 1.10-1.27, I2=68%), and Cer(d18:1/24:1) (HR 1.17, 95% CI 1.08-1.27, I2=83%) were associated with major adverse cardiovascular events. Conversely, no association with plasma levels of Cer(d18:1/22:0) (HR 1.14 95% CI 0.88-1.47, I2=88%) and Cer(d18:1/24:0) (HR 0.97, 95% CI 0.89-1.05, I2=73%) was found. Similar findings were also observed after stratification the eligible studies by outcome (i.e., major adverse cardiovascular events vs. cardiovascular death) (Figure 1). Information on the association between plasma levels of Cer(d18:1/20:0) and risk of major adverse cardiovascular events was available only for an eligible study and was not statistically significant (8).

In order to investigate potential sources of heterogeneity across eligible studies, we performed sensitivity analyses, as reported in Supplementary Table 2. Restricting the analysis to “highquality” cohort studies (NOS >8 stars), we observed that the overall estimates were substantially consistent with the pooled primary analyses and, importantly, we also noted a relative reduction of heterogeneity. When the comparison was stratified by study population, the association between plasma levels of Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/24:1) and major adverse cardiovascular events was maintained only among individuals at high cardiovascular risk. When the comparison was stratified by study countries, only the association between Cer(d18:1/24:1) levels and risk of major adverse cardiovascular events remained across all countries.

As shown in Supplementary Figure 2, the Egger's regression tests (but not the Begg’s tests) showed statistically significant asymmetry for the funnel plots of Cer(d18:1/16:0) and Cer(d18:1/24:1), thus suggesting a potential publication bias. Subsequently, we used the non-

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parametric trim and fill analysis (22) to adjust for funnel plot asymmetry. With regard to funnel plot asymmetry of Cer(d18:1/16:0), the results showed no trimming performed and data unchanged. With regard to funnel plot asymmetry of Cer(d18:1/24:1), the non-parametric trim and fill analysis did not confirm the existence of publication bias (Supplementary Table 3 and Supplementary Figure 3). In addition, eliminating each of the eligible studies from the analysis had no significant effect on the risk of major adverse cardiovascular events (Supplementary Figure 4).

Lastly, we also conducted specific multivariate meta-regression analyses (Supplementary Table 4) showing the lack of any significant association of age, body mass index and LDL-cholesterol levels with the risk of major adverse cardiovascular events carried out by specific ceramides [i.e., Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0) and Cer(d18:1/24:1)] across eligible studies. Similar findings were also observed after additional adjustment for use of statins (when available across eligible studies) (data not shown).

DISCUSSION Our meta-analysis provides evidence for a significant association between specific ceramides and risk of incident major adverse cardiovascular events. Specifically, this meta-analysis includes seven unique, cohort studies (5-11) with aggregate data on nearly 30,000 adults and approximately 3,000 new cases of major adverse cardiovascular events over a median follow-up of 6 years. We observed that higher plasma levels of Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/24:1) were significantly associated with major adverse cardiovascular events, whereas plasma levels of Cer(d18:1/22:0) and Cer (d18:1/24:0) were not. In our meta-analysis, most studies (5,7,8,9,11) have investigated such associations in patients with established or suspected coronary artery

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disease, whereas only two studies (6,10) have examined the associations in apparently healthy individuals. As reported in Supplementary Table 3, from our meta-analysis we excluded the study of Alshehry et al. (12) and the study of Anroedh et al. (13), as the hazard ratios were not given per standard deviation of plasma ceramide levels. Specifically, in the study of Alshehry et al. (12) only plasma levels of Cer(d18:1/24:1) were associated with an increased risk of fatal and non-fatal cardiovascular events, whereas in the study of Anroedh et al. (13) only plasma levels of Cer(d18:1/16:0) were associated with adverse cardiovascular outcome. However, it is important to highlight that these findings are, at least in part, consistent with the eligible studies included in our meta-analysis. We also excluded the study of Lemaitre et al. (14), as they provided hazard ratios only for incident heart failure and not for major adverse cardiovascular events (Supplementary Table 3). In addition, it is important to underline that we have not included studies evaluating the relationship between total plasma ceramides and risk of cardiovascular events, as we believe that such association (if it exists) does not provide additional (useful) information on this topic. Indeed, examining the association between total ceramides and the risk of cardiovascular events, it is not possible to differentiate the specific role of different ceramides (e.g., those with long or very long acyl-chain lengths). In addition, one may also speculate that the relationship between total plasma ceramides and risk of cardiovascular events may be partly dampened by ceramides that do not show a strong relationship with cardiovascular disease.

Differences between findings of eligible studies may be due to various factors, such as study population, study countries, differences in definition of major adverse cardiovascular events and also differences in mass spectrometry method for quantification of ceramide species. For instance, limiting the analysis to eligible studies with NOS>8 (6,10,11), that are studies with elevated standard spectrometry method for quantification of lipid species, we observed a reduction of

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heterogeneity and, again, that plasma levels of Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/24:1) were associated with major adverse cardiovascular events.

The results of our meta-analysis may be of clinical relevance, as they further reinforce the existence of an association between specific ceramides and major adverse cardiovascular events in patients with established/suspected coronary artery disease, as well as in apparently healthy individuals. In particular, our meta-analysis sheds light that such association may be particularly strong for plasma ceramides with long acyl-chain lengths and for those with unsaturated fat compounds, even after adjustment for traditional lipids and other established cardiovascular risk factors. In this context, recent studies also reported that plasma levels of specific ceramides [mostly Cer(d18:1/16:0), Cer(d18:1/18:0) and Cer(d18:1/24:1)] were independently associated with the presence of inducible myocardial ischemia in patients with established or suspected coronary artery disease referred for clinically indicated myocardial perfusion scintigraphy (17,18). In addition, in the ATHEROREMO-IVUS study Cheng et al. showed that plasma levels of specific ceramides [mostly Cer(d18:1/16:0)] were significantly associated with a vulnerable coronary plaque morphology in approximately 600 patients with stable CAD or suspected acute coronary syndrome (16). In another prospective study of nearly 500 patients who underwent elective coronary angiography, Meeusen et al. observed that plasma levels of Cer(d18:1/16:0), Cer(d18:1/18:0) and Cer(d18:1/24:1) were independently associated with an increased risk of adverse cardiovascular outcomes over a mean follow-up of 4 years (9). In that study, however, the authors did not find significant associations between the aforementioned ceramides and the angiographic severity of coronary-artery stenosis (at baseline) (9). Interestingly, de Carvalho et al. showed myocardial up-regulation of three ceramide-producing enzymes (namely ceramide synthase 6, serine palmitoyl transferase-2, neutral sphingomyelinase) in a rodent model of acute

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myocardial infarction after ligation of left anterior descending artery (8). Recently, in a crosssectional study of 167 consecutive patients with established or suspected CAD, who underwent urgent or elective coronary angiography, Mantovani et al. documented that higher levels of specific plasma ceramides were independently associated with a higher severity of coronaryartery stenosis in the left anterior descending artery (15). However, given the design of available studies, it is important to highlight that we are not able to establish whether circulating ceramides are effectors or merely markers of cardiovascular events. To further complicate the context, an alternative hypothesis may be that ceramides are simply biomarker for increased circulating fatty acids (23).

In the last years, interestingly, it has been also reported that some specific interventions may modify the plasma levels of specific ceramides (7,24,25). For instance, in the post-hoc analysis of the Prevention with Mediterranean Diet (PREDIMED) trial, Wang et al. showed that Mediterranean dietary intervention might mitigate the possible adverse effect of higher plasma ceramide levels on cardiovascular events (7). In a single-centre, randomized, double-blind, placebo-controlled trial involving 37 patients with metabolic syndrome assigned to pioglitazone or placebo, Warshauer et al. documented that pioglitazone may induce a decrease in plasma ceramide levels, probably due to some changes in insulin resistance and adiponectin levels (24).

At present, the putative pathophysiological mechanisms underpinning the association between plasma ceramides and risk of major adverse cardiovascular events are not completely understood. However, accumulating experimental data indicate that different ceramide species may be implicated in several atherosclerotic processes, including increased uptake of lipoproteins, accumulation of cholesterol within macrophages, platelet activation, regulation of nitric oxide

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synthesis, production of reactive oxygen species and pro-inflammatory cytokines (1-4). In addition, some studies have also suggested that ceramides with long acyl-chain lengths [e.g., Cer(d18:1/16:0) and Cer(d18:1/18:0)] may induce insulin resistance and/or hepatic steatosis (1,2,23,26-29). In this regard, it was demonstrated that these ceramides interfere with insulin signaling at the level of AKT (a serine/threonine-specific protein kinase that plays a key role in this pathway) (2). There is also evidence that ceramide included in various lipoproteins (e.g., LDL) may exert adverse effects on cells (30). However, this aspect is not specifically considered in the eligible studies of this meta-analysis.

Our meta-analysis has some important limitations (strictly associated to the characteristics of the eligible studies). First, the observational design of the eligible studies does not allow establishing a causal relationship between plasma levels of ceramides and major adverse cardiovascular events. Second, although we used a random-effects model, the interpretation of the results may require some caution, because of the (expected) high heterogeneity observed in the overall analyses. We believe that the high heterogeneity mainly reflects a mix of different patients, coming from various parts of the world, with different cardiovascular profile risk, in which the plasma ceramides have been measured with different methods. Third, the varying degree of confounder adjustment across the eligible studies may interfere with a systematic assessment of the effect of specific ceramides on the outcome of interest.

Despite these limitations, our study has also strengths. This meta-analysis provides a comprehensive assessment of the prognostic impact of specific ceramides on the long-term risk of major adverse cardiovascular events. These results were obtained by analyzing approximately

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3,000 new cases of incident fatal and non-fatal cardiovascular events among approximately 30,000 individuals over a median follow-up of 6 years.

In conclusion, our meta-analysis is the first to document that higher plasma levels of Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/24:1) were significantly associated with increased risk of major adverse cardiovascular events. Conversely, plasma levels of Cer(d18:1/22:0) and Cer (d18:1/24:0) were not. Further research is required to elucidate the different effects of plasma ceramides with various acyl-chain lengths and also with different saturated/unsaturated compounds on signaling pathways involved in cardiovascular disease.

Declarations of interest: none. Financial support: none.

Author Contributions Study concept and design: AM; acquisition of data: AM, CD; statistical analysis of data: AM; analysis and interpretation of data: AM, CD; drafting of the manuscript: AM; critical revision of the manuscript for important intellectual content: CD.

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21. Egger M, Smith GD, Phillips AN. Meta-analysis: principles and procedures. BMJ 1997; 315: 1533–7. 22. Duval S, Tweedie R. A nonparametric “trim and fill” method of accounting for publication bias in meta-analysis. Journal of the American Statistical Association 2000; 95: 89-98. 23. Bandet CL, Tan-Chen S, Bourron O, Le Stunff H, Hajduch E. Sphingolipid Metabolism: New Insight into Ceramide-Induced Lipotoxicity in Muscle Cells. Int J Mol Sci. 2019; 20(3). 24. Warshauer JT, Lopez X, Gordillo R, Hicks J, Holland WL, Anuwe E, Blankfard MB, Scherer PE, Lingvay I. Effect of pioglitazone on plasma ceramides in adults with metabolic syndrome. Diabetes Metab Res Rev 2015; 31: 734-44. 25. Mathews AT, Famodu OA, Olfert MD, Murray PJ, Cuff CF, Downes MT, Haughey NJ, Colby SE, Chantler PD, Olfert IM, McFadden JW. Efficacy of nutritional interventions to lower circulating ceramides in young adults: FRUVEDomic pilot study. Physiol Rep. 2017; 5(13). 26. Turpin SM, Nicholls HT, Willmes DM, Mourier A, Brodesser S, Wunderlich CM, Mauer J, Xu E, Hammerschmidt P, Brönneke HS, Trifunovic A, LoSasso G, Wunderlich FT, Kornfeld JW, Blüher M, Krönke M, Brüning JC. Obesity-induced CerS6-dependent C16:0 ceramide production promotes weight gain and glucose intolerance. Cell Metab. 2014; 20: 678-86. 27. Raichur S, Wang ST, Chan PW, Li Y, Ching J, Chaurasia B, Dogra S, Öhman MK, Takeda K, Sugii S, Pewzner-Jung Y, Futerman AH, Summers SA. CerS2 haploinsufficiency inhibits βoxidation and confers susceptibility to diet-induced steatohepatitis and insulin resistance. Cell Metab 2014; 20: 687-95. 28. Hla T, Kolesnick R. C16:0-ceramide signals insulin resistance. Cell Metab 2014; 20: 703-705. 29. Gosejacob D, Jäger PS, Vom Dorp K, Frejno M, Carstensen AC, Köhnke M, Degen J, Dörmann P, Hoch M. Ceramide Synthase 5 Is Essential to Maintain C16:0-Ceramide Pools

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Table 1. Principal cohort studies examining the association between ceramides and the risk of incident fatal and non-fatal cardiovascular events (ordered by publication year). Author, Reference

Characteristics of study

Laaksonen et al. European Heart Journal 2016; 37, 1967-1976 [5]

Prospective cohort study: 1,580 adults (age 62 years; males 59%, 2 BMI 25 kg/m , LDL-cholesterol 2.8 mmol/L, triglycerides 1.4 mmol/L, statin use 62.6%) referred to elective coronary angiography for suspected stable angina pectoris recruited at the Haukeland University Hospital in Bergen (BECAC study) who were followed-up for 4.6 years and 1,637 patients (age 63 years; males 78%, BMI 2 26 kg/m , LDL 2.6 mmol/L, triglycerides 1 mmol/L, statin use 27.2%) with a primary diagnosis of ACS referred for invasive management at four Swiss university hospitals (SPUM-ACS study) who were followed-up for 1 year Population-based cohort study: 8,101 apparently healthy patients (age 48 years; males 2 47%, BMI 26 kg/m , LDLcholesterol 3.3 mmol/L, triglycerides 1.3 mmol/L) from FINRISK 2002. Follow-up: 13 years Cohort study: 980 participants (age 68 years; males 45%, BMI 2 30 kg/m , LDL-cholesterol 3.4

Havulinna et al. Arterioscler Thromb Vasc Biol 2016; 36: 24242430 [6]

Wang et al. Circulation 2017; 135: 2028-2040

Methods for ceramide measurements 5500 QTRAP (SCIEX, Framingham, MA) mass spectrometer equipped with an Eksigent 100-XL UHPLC system

Primary Endpoints

Adjustments

Main Results

NOS

Cardiovascular death

Total cholesterol, triglycerides, HDLcholesterol, LDLcholesterol, age, gender, smoking status, previous acute myocardial infarction, diabetes, hypertension, prior stroke

Among the previously identified high-risk ceramides, only plasma levels of Cer(d18:1/16:0) and Cer(d18:1/24:1) were associated with an increased risk of cardiovascular death in all both cohorts

8

Targeted liquid chromatography– tandem mass spectrometry assay

Major adverse cardiac and cerebrovascular events

Total cholesterol, HDL-cholesterol, blood pressure, diabetes mellitus, and smoking

Among the previously identified high-risk ceramides, plasma levels of Cer(d18:1/16:0), Cer(d18:1/18:0) and Cer(d18:1/24:1) were significantly higher in patients with adverse cardiovascular outcome when compared to asymptomatic subjects

9

Liquid chromatography tandem mass

MACEs (i.e., nonfatal acute myocardial

Age, sex, body mass index, family history of premature

Among the previously identified high-risk ceramides, the last quartiles of plasma levels of Cer(d18:1/16:0),

7

19

[7]

de Carvalho et al. JACC Basic Transl Sci 2018; 3: 163175 [8]

mmol/L, triglycerides 1.6 mmol/L) from the PREDIMED trial including 230 incident cases of CVD and 787 randomly selected participants at baseline. Follow-up: 4.5 years Longitudinal study: 327 discovery cohort patients (age 57 years; males 90%, BMI 26 kg/m2, LDL-cholesterol 3.1 mmol/L, triglycerides 1.2 mmol/L) and 119 validation cohort patients (age 66 years; 2 males 72%, BMI 29 kg/m , LDLcholesterol 3.2 mmol/L). Follow-up: 1 year

spectrometry technique

infarction, nonfatal stroke, or cardiovascular death)

Hydrophilic interaction LC (HILIC) MS/MS

Major adverse cardiac and cerebrovascular events

MACEs (i.e., Combined primary end point of myocardial infarction, percutaneous intervention, coronary artery bypass, stroke, or death) MACEs (i.e., fatal and non-fatal cardiovascular events)

Meeusen et al. Arterioscler Thromb Vasc Biol. 2018; 38: 1933-1939 [9]

Longitudinal study: 495 participants (age 60 years; males 62%, BMI 28 kg/m2, LDLcholesterol 3.1 mmol/L, triglycerides 1.7 mmol/L, statin use 28.5%) before non-urgent coronary angiography. Follow-up: 4 years

API 5000 MS/MS (AB Sciex, Framingham, MA)

Peterson et al. J Am Heart Assoc. 2018;7: e007931. [10]

Community-based study: 2,642 FHS (Framingham Heart Study) participants (age 66 years; males 46%, BMI 28 kg/m2, LDLcholesterol 2.7 mmol/L, triglycerides 1.3 mmol/L, statin use 42.7%) and 3,134 SHIP (Study of Health in Pomerania) participants (age 54 years; males 48%, BMI 28 kg/m2, LDLcholesterol 5.5 mmol/L,

Liquid chromatography/mass spectrometry assay

coronary heart disease, smoking status, histories of hypertension, dyslipidemia, and type 2 diabetes mellitus Global Registry of Acute Coronary Events Risk score

Cer(d18:1/22:0), Cer(d18:1/24:0), and Cer(d18:1/24:1) were associated with adverse cardiovascular outcome

Among the previously identified high-risk ceramides, plasma levels of Cer(d18:1/16:0), Cer(d18:1/22:0) and Cer(d18:1/24:1) were associated with adverse cardiovascular outcome

6

Age, sex, body mass index, hypertension, smoking, LDLcholesterol, HDLcholesterol, triglycerides, serum glucose, family history of coronary artery disease

Among the previously identified high-risk ceramides, plasma levels of Cer(d18:1/16:0), Cer(d18:1/18:0) and Cer(d18:1/24:1) were associated with adverse cardiovascular outcome

6

Age, sex, body mass index, hypertension, diabetes mellitus, smoking status, antihypertensive medications, the ratio of total/HDL cholesterol, triglycerides, and lipid-lowering medication

Among the previously identified high-risk ceramides, only plasma levels of Cer(d18:1/24:1) were associated with adverse cardiovascular outcome

9

20

Hilvo et al. European Heart Journal 2019, in press [11]

triglycerides 1.8 mmol/L, statin use 14.6%) were followed for 6 and 8 years respectively Longitudinal study: Three large cohort studies: 3,789 patients (age 62 years; males 72%, LDLcholesterol 2.9 mmol/L, triglycerides 1.5 mmol/L, statin use 72.6%) from WECAC (The Western Norway Coronary Angiography Cohort); 5,991 patients (age 65 years; males 83%, LDL-cholesterol 3.9 mmol/L, triglycerides 1.6 mmol/L, statin use 49.9%) from LIPID (Long-Term Intervention with Pravastatin in Ischaemic Disease) trial; and 1,023 patients (age 62 years; males 84%, LDLcholesterol 3 mmol/L, triglycerides 1.6 mmol/L, statin use 75.6%) from KAROLA (Langzeiterfolge der KARdiOLogischen Anschlussheilbehandlung) Follow-up: 6 years

High-throughput LCMS/MS assay

MACEs (i.e., composite endpoint, which included CV death, MI, and stroke)

Age, sex, statin treatment (WECAC, KAROLA), diabetes mellitus, hypertension, current smoking, previous MI, previous stroke, stratified by vitamin B intervention (WECAC) and treatment group (LIPID)

Among the previously identified high-risk ceramides, plasma levels of Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/24:1) were associated with adverse cardiovascular outcome in WEDAC and LIPID cohorts

9

Abbreviations: BMI, body mass index; Cer, ceramides; MACEs, major adverse cardiovascular events.

21

Supplementary Table 1. Cohort study excluded at the eligibility step of PRISMA diagram. Author, Reference Alshehry et al. Circulation 2016; 134: 1637-1650 [12]

Anroedh et al. J. Lipid Res. 2018; 59: 1729–1737 [13]

Lemaitre et al. Circ Heart Fail. 2019; 12: e005708 [14]

Characteristics of study

Primary Endpoints

Reasons for exclusion

Prospective study: 3,154 T2DM patients randomly selected from 7,376 participants of ADVANCE trial (Action in Diabetes and Vascular Disease: Preterax and DiamicronMR Controlled Evaluation). Follow-up: 5 years Cohort study: 581 patients underwent diagnostic coronary angiography or percutaneous coronary intervention for stable angina pectoris (SAP) or acute coronary syndrome (ACS). Follow-up 4.7 years Prospective cohort study: 4,249 study participants from Cardiovascular Health Study followed up for a median period of 9.4 years

MACEs (i.e., fatal and non-fatal cardiovascular events)

HRs were expressed in terms of unit increase in Ln-transformed variable and not for 1-standard deviation

MACEs (i.e., all-cause mortality, nonfatal ACS, or unplanned coronary revascularization)

HRs were normalized to the interquartile range and not for 1-standard deviation

Incident heart failure

No data for major adverse cardiovascular events

Abbreviations: MACEs, major adverse cardiovascular events; T2DM, type 2 diabetes.

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Supplementary Table 2. Subgroup analyses – Relationship between previously identified high risk ceramide molecules and risk of fatal and nonfatal cardiovascular events in longitudinal studies stratified by Newcastle-Ottawa Scale category, study population, and study country.

Subanalyses

Cer(d18:1/16:0)

Cer(d18:1/18:0)

Cer(d18:1/22:0)

Cer(d18:1/24:0)

Cer(d18:1/24:1)

NOS >8

Random-effects HR 1.13 (95% CI 1.07-1.20) 2 I = 43.8% Number of studies: 3

Random-effects HR 1.18 (95% CI 1.09-1.21) 2 I =0% Number of studies: 2

NA

Random-effects HR 0.86 (95% CI 0.69-1.07) 2 I = 82.4% Number of studies: 2

Random-effects HR 1.13 (95% CI 1.08-1.18) 2 I = 0% Number of studies: 5

NOS ≤8

Random-effects HR 1.47 (95% CI 1.11.-1.95) 2 I = 92.4% Number of studies: 4

Random-effects HR 1.30 (95% CI 1.03-1.64) 2 I = 81.0% Number of studies: 6

Random-effects HR 1.14 (95% CI 0.89-1.47) 2 I =88.2% Number of studies: 2

Random-effects HR 1.02 (95% CI 0.92-1.13) 2 I =63.1% Number of studies: 4

Random-effects HR 1.31 (95% CI 1.06-1.61) 2 I =86.3% Number of studies: 2

Population with high CVD risk

Random-effects HR 1.29 (95% CI 1.16-1.45) 2 I = 90.8% Number of studies: 5

Random-effects HR 1.19 (95% CI 1.09-1.29) 2 I =70.5% Number of studies: 8

Random-effects HR 1.14 (95% CI 0.89-1.47) 2 I =88.2% Number of studies: 2

Random-effects HR 1.02 (95% CI 0.92-1.13) 2 I = 63.1% Number of studies: 4

Random-effects HR 1.18 (95% CI 1.08-1.29) 2 I = 84.6% Number of studies: 5

Population with lowmoderate CVD risk

Random-effects HR 1.05 (95% CI 0.93-1.19) 2 I = 53.1% Number of studies: 3

Random-effects HR 1.21 (95% CI 1.10-1.32) 2 I = 53.1% Number of studies: 1

NA

Random-effects HR 0.86 (95% CI 0.69-1.07) 2 I = 82.4% Number of studies: 3

Random-effects HR 1.14 (95% CI 1.04-1.26) 2 I = NA Number of studies: 1

Asia

Random-effects HR 1.03 (95% CI 1.01-1.05) 2 I = NA Number of studies: 1

Random-effects HR 1.05 (95% CI 0.99-1.12) 2 I = NA Number of studies: 1

Random-effects HR 1.01 (95% CI 0.99-1.03) 2 I = NA Number of studies: 1

Random-effects HR 1.00 (95% CI 0.99-1.01) 2 I = NA Number of studies: 1

Random-effects HR 1.015 (95% CI 1.01-1.02) 2 I = NA Number of studies: 1

Europe

Random-effects HR 1.24 (95% CI 1.13-1.37) 2 I = 77.7% Number of studies: 5

Random-effects HR 1.20 (95% CI 1.13-1.27) 2 I = 34.6% Number of studies: 3

Random-effects HR 1.32 (95% CI 1.10-1.58) 2 I = NA Number of studies: 1

Random-effects HR 0.99 (95% CI 0.84-1.17) 2 I = 78.1% Number of studies: 4

Random-effects HR 1.17 (95% CI 1.10-1.25) 2 I = 38.8% Number of studies: 8

United States

Random-effects HR 1.17 (95% CI 0.71-1.92) 2 I = 85.5% Number of studies: 2

Random-effects HR 1.42 (95% CI 1.11-1.82) 2 I = 85.5% Number of studies: 1

NA

Random-effects HR 0.84 (95% CI 0.64-1.12) 2 I = 73.6% Number of studies: 2

Random-effects HR 1.43 (95% CI 1.08-1.89) 2 I = NA Number of studies: 1

Abbreviations: CI, confidence interval; HR, hazard ratio; NA, not applicable.

23

Supplementary Table 3. Non-parametric trim and fill analysis for potential publication bias regarding overall estimates of Cer(d18:1/24:1).

Panel A - Cer(d18:1/24:1) Interaction 1 2 3 4

Estimate 0.160 0.121 0.109 0.092

Tn 29 37 40 40

# to trim 2 3 4 4

diff 45 16 6 0

Trimming estimator: Linear. Meta-analysis type: Random-effects model.

Method Fixed Random

Pooled estimate 1.017 1.099

95% lower CI 1.012 1.023

95% upper CI 1.022 1.180

Z value

P value

6.795 2.591

<0.0001 0.010

N of studies 13 13

Test for heterogeneity: Q= 60.579 on 12 degrees of freedom (p<0.001). Moment-based estimate of between studies variance =0.009.

24

Supplementary Table 4. Multivariate regression analyses for the risk of major adverse cardiovascular events carried out by specific ceramides [i.e., Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0) and Cer(d18:1/24:1)].

Coefficient

95% Confidence

P values

interval Hazard Ratio by Cer(d18:1/16:0) (Log Scale) (n=6 studies) Age (years)

0.020

-0.48 to 0.09

0.464

BMI (Kg/m2)

-0.09

-0.36 to 0.18

0.408

LDL-cholesterol

-0.02

-0.47 to 0.42

0.886

(mmol/L) Hazard Ratio by Cer(d18:1/18:0) (Log Scale) (n=3 studies) Age (years)

-0.039

-0.17 to 0.09

0.165

BMI (Kg/m2)

0.13

-0.69 to 0.97

0.289

LDL-cholesterol

-1.36

-5.67 to2.96

0.156

(mmol/L) Hazard Ratio by Cer(d18:1/24:0) (Log Scale) (n=6 studies) Age (years)

-0.006

-0.06 to 0.04

0.769

BMI (Kg/m2)

0.051

-0.16 to 0.26

0.533

LDL-cholesterol

-0.07

-0.39 to 0.26

0.603

(mmol/L) Hazard Ratio by Cer(d18:1/24:1) (Log Scale) (n=5 studies) Age (years)

-0.011

-0.10 to 0.08

0.656

BMI (Kg/m2)

0.111

-0.36 to 0.58

0.416

LDL-cholesterol

-0895

-3.32 to 1.53

0.254 25

(mmol/L)

26

FIGURE LEGENDS

Figure 1. Forest plot and pooled estimates of the effect of previously identified high-risk ceramides [namely Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/22:0), Cer(d18:1/24:0) and Cer(d18:1/24:1); Panels from A to E, respectively) on the risk of adverse cardiovascular outcome in seven eligible cohort studies stratified by outcome of interest. Note: Information on the association between plasma levels of Cer(d18:1/20:0) and risk of major adverse cardiovascular events was available only for an eligible study (8).

Supplementary Figure 1. The PRISMA flow diagram of the meta-analysis.

Supplementary Figure 2. Funnel plot of standard error by log-hazard ratio of Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0) and Cer(d18:1/24:1) (Panels from A to D, respectively) for the risk of major adverse cardiovascular events.

Supplementary Figure 3. Funnel plots of standard error by log-hazard ratio of Cer(d18:1/24:1) for the risk of major adverse cardiovascular events. The new hypothetical studies assessed by trim and fill analysis are indicated as square around the data symbol. Note: Regarding the funnel plot asymmetry of Cer(d18:1/16:0), the results showed no trimming performed and data unchanged.

Supplementary Figure 4. Influence of each individual study on the overall meta-analysis estimate for each ceramide [i.e., Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0) and Cer(d18:1/24:1); Panels from A to D respectively]. The graph shows the results of an influence analysis in which the meta-analysis is re-estimated omitting each study in turn.

27

A) Cer(d18:1/16:0)

B) Cer(d18:1/18:0)

D) Cer(d18:1/24:0)

E) Cer(d18:1/24:1)

C) Cer(d18:1/22:0)

HIGHLIGHTS • The magnitude of the association between ceramides and CVD remains uncertain. • Cer16, Cer18, and Cer24:1 were associated with CVD. • No association with Cer22 and Cer24:0 was observed.