Supplementary MaterialsSupplementary information 41598_2019_54877_MOESM1_ESM. environment and proteolytic activity in the lysosomes. Transgenic overexpression of SphK1 in SphK2-deficient mice rescued aggravation of atherosclerosis and abnormalities of autophagosomes and lysosomes in macrophages with reductions of sphingosine, suggesting at least partial overlapping actions of two SphKs. Taken together, these results indicate that SphK2 is required for autophagosome- and lysosome-mediated catabolism of intracellular lipid droplets to impede the development of atherosclerosis; therefore, SphK2 may be a novel target for treating atherosclerosis. mice and mice, both with an ApoE-deficient background and found that mice, but not mice, show aggravation of atherosclerosis Indocyanine green because of defective autophagic breakdown of LDs in macrophages. These observations indicate that SphK2 is a novel key factor essential for autophagosome- and lysosome-mediated LD catabolism and may be a target in the development of new therapies for atherosclerosis. Results Genetic disruption of aggravates atherosclerosis in mice To investigate roles of the two SphK isoforms in atherosclerosis, we fed four mice groups with different SphK genotypes, i.e. Sphk2Sphk2background, on a WD for 12 weeks, followed by determinations of the aortic plaque lesion areas. The plaque lesions MSH4 in the spread aortae, as evaluated with Oil Red O (ORO) staining, were increased by approximately 60% in mice compared with control mice than control mice (Fig.?1b). The ORO-positive cross-sectional area of the abdominal aorta in mice was also increased compared with control mice (Supplemental Fig.?S1). Plasma concentrations of total cholesterol and triglyceride, plasma lipoprotein profiles, liver histology and cardiovascular parameters were all similar between mice and control mice (Supplemental Figs.?S2, S3a,b). Both groups of mice had similar body weights after 12 weeks of WD feeding, although the basal body weight of mice at 8 weeks was slightly lower than that of control mice (Supplemental Fig.?S3c). These results suggest that SphK2 has a protective role in atherosclerotic lesion formation in the aorta without affecting a plasma lipid profile. Open in a separate window Figure 1 Aggravation of atherosclerosis in SphK2-, but not SphK1-, deficient mice. (a) En face aortic lesion areas stained with ORO from control mice that harbor the -galactosidase (LacZ) gene at the SphK2 gene locus15. Macroscopically, mouse aortae showed much more intense blue color in X-gal staining compared with or control donor mice (Fig.?2d). The ORO-stained atherosclerotic lesion area in the spread aortae was greater in mice that received BM compared with mice that received mice. In tissue lysates from the aortae of WD-fed mice, the expression of p62 (Sqstm1), a ubiquitin-binding scaffold protein which recruits ubiquitinated autophagosomal contents to the phagophore isolation membrane by binding to phagophore-bound LC33, was increased compared with control mice (Fig.?2e), suggesting impaired progress of autophagic degradation processes in the aortae of mice. Furthermore, double immunofluorescence staining of aortic sections showed enhanced accumulation of LAMP2-positive macrophages and an increase of p62 expression in LAMP2-positive plaque macrophages (yellow) in mice compared with control mice (Fig.?2f). Previous studies16C19 showed that both SphK1 and SphK2 were localized in the cytosol and cell organelles, which include lysosomes and early and late endosomes. Immunostaining of peritoneal macrophages showed that both SphK1 and SphK2 were highly co-localized in LAMP2-positive lysosomes in macrophages (Fig.?2g, arrowheads). These observations together suggest that aggravation of atherosclerosis in mice may be accompanied by defective autophagy in plaque macrophages. Sphingosine level in macrophages was elevated approximately 4-fold in mice compared with control mice (Supplemental Fig.?S5a). Those of dihydrosphingosine and ceramides (16:0, 18:0, 20:0, 22:0 and 24:0) were also significantly increased in macrophages compared with mice was higher than that in control Indocyanine green mice (Supplemental Fig.?S5b), which is in agreement with previous reports23,24. Feeding mice with a WD resulted in a Indocyanine green further increase in plasma S1P, which was also consistent with a previous report25. The gene expression of S1P receptors and sphingolipid-metabolizing enzymes except SphK2 was similar or slightly increased in macrophages compared with macrophages We determined the lipid deposition in peritoneal macrophages freshly harvested from WD-fed and mice showed greater areas of ORO-positive LDs (Fig.?3a) and contained higher total cellular cholesterol (Fig.?3b) and esterified cholesterol (Fig.?3c), compared with macrophages from macrophages only slightly increased cholesterol content (Supplemental Fig.?S6). Open Indocyanine green in a separate window Figure Indocyanine green 3 Increased cholesterol accumulation and impaired autophagy in SphK2?/? macrophages. Peritoneal macrophages were.