Inverse relationship between circulating sphingosine-1-phosphate and precursor species and coronary artery calcification score in type 2 diabetes

Abstract

Background: Sphingosine 1-phosphate (S1P) is a key mediator of lipid signaling with strong immunomodulatory and anti-inflammatory effects. Circulating S1P levels including S1P in high-density lipoproteins (HDL) were demonstrated to be inversely associated with cardiovascular diseases (CVD). However, no studies are available regarding a potential implication of S1P on the risk of CVD in type 2 diabetes (T2D). The objective of this study is to determine if the increased CVD risk in T2D may involve an alteration of circulating S1P species as well as their precursors.

Methods: A total of 168 and 31 patients with T2D (154 men and 45 women) with available Coronary artery calcification (CAC) score from the DIACART and CERABIAB cohorts, respectively, were included in the study. Quantification of S1P species and their precursors was carried out by LC-MS/MS in plasma and isolated HDL. CAC score was modeled as a binary variable (0/1 below or equal/above 100) using CAC < 100 for reference. S1P species or precursors were modeled as binary variables dichotomized at the median (0/1: below or equal/above the median). The relationships between S1P species and CAC score modeled as a binary variable (below or equal/above 100) was evaluated by linear regression analyses. In vitro experiments were conducted to evaluate the contribution of HDL-S1P content on anti-inflammatory properties of HDL particles.

Results: Multivariate analysis revealed that plasma S1P levels, especially d18:1-S1P, and sphingosine in HDL were inversely associated with the high risk of CVD (CAC > 100) in patients with T2D. Clustering of HDL according to their concentration in S1P species and their precursors revealed that S1P-impoverished HDL is a major feature of patients with a CAC > 100. In vitro analysis of monocyte adhesion and inflammation in human umbilical vein endothelial cells as well as inflammatory phenotype of human macrophages demonstrated that low HDL-S1P exhibited impaired anti-inflammatory properties in comparison to high HDL-S1P.

Conclusion: This study unraveled that circulating S1P and their precursors are biomarkers of coronary atherosclerosis in T2D, which may underlie the lower abundance of S1P and anti-inflammatory activities of HDL. Trial registration ClinicalTrials.gov number, NCT02431234.

Keywords: Atherosclerosis; Cardiovascular diseases; High-density lipoprotein; Sphingosine-1-phosphate; Type 2 diabetes.

Fig. 1

Biosynthesis pathways of sphingosine-1-phosphate species. Major biosynthesis pathways of Sphingosine-1-Phosphate species (d16:1-S1P, d18:0-S1P, d18:1-S1P and d18:2-S1P) and their precursors (dihydrosphingosine and sphingosine) (in red). Dihydrosphingosine (Sphinganine); S1P, Sphingosine-1-Phosphate; SphK, sphingosine kinase; SPT, Serine palmitoyl transferase

Fig. 2

Correlation between sphingosine-1-phosphate and precursors species with cholesterol in high-density lipoproteins. Correlation between HDL S1P species, d18:2-S1P panel A, d18:1-S1P panel B, d16:1-S1P panel C, d18:0-S1P (panel F) or precursor, sphingosine panel D, dihydrosphingosine panel E and plasma HDL-cholesterol levels in patients with type 2 diabetes (n = 199). r indicates pearson correlation coefficient, dotted line indicate 95% confidence interval

Fig. 3

Circulating sphingosine-1-phosphate and precursor species and risk to develop a high coronary artery calcification score. Relationship between plasma levels of S1P species panel A and precursors panel B with coronary artery calcification score, CAC > 100. Relationship between plasma levels of HDL-S1P species panel C and precursors panel D with CAC > 100. Adjusted model included independent variables associated with coronary calcium score including age, sex, LDL-cholesterol, CKD, status with regard to use of statins, dual antiplatelet therapy, angiotensin converting enzyme inhibitors/angiotensin II receptor blocker and beta blockers. OR, odd ratio; CI, confidence interval. n = 199 T2D patients

Fig. 4

Clustering of high-density lipoproteins according to the amount of sphingosine-1-phosphate and precursor species. Hierarchical cluster of type 2 diabetic patients based on HDL-S1P species and precursors levels. Three clusters were identified and referred to C1 (n = 26, red), C2 (n = 70, green) and C3 (n = 72, blue) panel A. PCA score panel B. Box plot of HDL-d18:1-S1P levels according to clusters panel C. Variable importance in projection (VIP) score plot panel D. ***p < 0.0005 Cluster 3 vs Cluster 1

Fig. 5

Clinical and biochemical features of type 2 diabetes patients with high-density lipoproteins impoverished in sphingosine-1-phosphate species. Variations of major biological and biochemical parameters among patients with type 2 diabetes belonging to cluster 3 relative to those belonging to cluster 1 panel A. HDL-S1P levels expressed relative to HDL-cholesterol levels, total S1P panel B, d16:1-S1P (panel C), d18:1-S1P panel D and d18:2-S1P panel E. Cluster 1 (C1, n = 26, red), Cluster 2 (C2, n = 70, green) and Cluster 3, (C3, n = 72, blue). *p < 0.05; **p < 0.005; ***p < 0.0005 Cluster 3 vs Cluster 1

Fig. 6

Anti-inflammatory activities of high-density lipoproteins according to the amount of sphingosine-1-phosphate and precursor species. Anti-inflammatory properties of HDL particles isolated patients with type 2 diabetes belonging to cluster 3 (the lowest HDL-S1P; blue bars) relative to those belonging to cluster 1 (the highest HDL-S1P; red bars) evaluated in A human umbilical endothelial cells treated or not with TNFα at both cell surface protein (flow cytometry) and mRNA levels (RT-QPCR) and in C human THP-1 macrophages treated or not with LPS. B Calcein-labelled monocyte-recruitment by endothelial cells was evaluated by flow cytometry and expressed in percentage of FITC-positive cells. Data are presented in means ± SEM. *p < 0.05; **p < 0.005; ***p < 0.0005