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The sphingolipid de novo synthesis pathway is considered a promising target for pharmacological intervention in atherosclerosis. However, its potential is hampered by the fact that the substance’s atherogenic mechanism is not completely understood. To unravel the complex mechanisms, we utilized the sphingomyelin synthase 2 (Sms2) gene knockout approach to test our hypothesis that selectively decreasing plasma lipoprotein SM, can play an important role in preventing atherosclerosis.
We prepared Sms2 and Apoe double knockout (KO) mice. They showed a significant decrease in plasma lipoprotein SM levels (35%, P<0.01) and a significant increase in ceramide and dihydroceramide levels (87.5 and 27%, P<0.01, respectively), but no significant changes in other tested sphingolipids, cholesterol, and triglyceride. Non-HDL lipoproteins from the double KO mice showed a reduction of SM but not cholesterol and displayed a less tendency toward aortic sphingomyelinase-mediated lipoprotein aggregation in vitro and retention in aortas in vivo, compared to controls. More important, at age 19 weeks, Sms2 KO/Apoe KO mice showed a significant reduction in atherosclerotic lesions of the aortic arch and root (52%, P<0.01), compared to controls. We also found that the Sms2 KO/Apoe KO brachiocephalic artery (BCA) contained significantly less SM, ceramide, free cholesterol, and cholesteryl ester (35, 32, 58, and 60%, P<0.01, respectively), than that of Apoe KO BCA.
Decreasing plasma SM levels through decreasing SMS2 activity could become a promising treatment for atherosclerosis.
Sphingomyelin (SM), which is the second most abundant phospholipid in mammalian plasma, appears in all major lipoproteins. Up to 18% of total plasma phospholipid exists as SM,1 with the ratio of phosphatidylcholine (PC)/SM varying widely among lipoprotein subclasses.2 Atherogenic lipoproteins such as VLDL and LDL are SM-enriched.1, 3 The SM content of atherosclerotic lesions is higher than that of normal arterial tissue.4
Williams and Tabas have suggested that subendothelial retention and aggregation of atherogenic lipoproteins play a very important role in atherogenesis.5–6 SM-rich LDL retained in atherosclerotic lesions is acted on by an arterial wall sphingomyelinase that appears to promote aggregation and retention, initiating the early phase of atherosclerosis development.7 We have found that plasma SM levels in Apoe KO mice are four-fold higher than those in WT mice,8 and this may partially explain the increased atherosclerosis found in these animals.9 Our laboratory and others have also discovered that chemical inhibition of sphingolipid biosynthesis significantly decreases plasma SM levels, thus lessening atherosclerotic lesions in Apoe KO mice.10–11
We have evidence that human plasma SM levels are an independent risk factor for coronary heart disease,12–13 and that these levels are prognostic in patients with acute coronary syndrome.13 All these data suggest that plasma SM plays a critical role in the development of atherosclerosis. However, in mouse studies we and others have found that after inhibiting sphingolipid de novo synthesis, all other tested sphingolipids, including SM, ceramide, sphingosine, sphingosine-1-phosphate, and glycosphingolipids, are significantly decreased.10–11 Consequently, we could not rule out the effect of sphingolipids other than SM on mouse atherogenicity.
The biochemical synthesis of SM occurs through the action of serine palmitoyltransferase (SPT), 3-ketosphinganine reductase, ceramide synthase, dihydroceramide desaturase, and sphingomyelin synthase (SMS).14 SMS is the last enzyme for SM biosynthesis, and it utilizes ceramide and PC as substrates to produce SM and diacylglycerol. Therefore, its activity should directly influence SM levels in cells and in the circulation. The liver and small intestine are the major contributors of plasma SM. The liver assembles lipids (SM, PC, cholesterol, and triglyceride) and apolipoproteins, secreting the end products, VLDL and HDL, into the circulation.15 Following hydrolysis of dietary SM in the lumen of the small intestine,16 the backbone sphingoid bases and fatty acids, which are taken up by the enterocytes, and can be used to resynthesize SM. This SM can participate in chylomicron assembly, and then be secreted.1
Two Sms genes, Sms1 and Sms2, have been cloned and characterized for their cellular localizations.17 Both are expressed in the liver and small intestine.17 SMS1 is found in the trans-golgi apparatus, while SMS2 is predominantly located in the plasma membranes.17 We as well as other investigators have shown that SMS1 and SMS2 expression positively correlates with levels of cellular SM, as well as SM in membrane lipid rafts.18–20 We have reported that macrophage-specific Sms2 deficiency decreases atherosclerosis in a mouse model.21 We also found that Sms2 deficiency decreases, while Sms2 overexpression increases, plasma SM levels.22 In this study, we prepared Sms2 KO/Apoe KO mice and evaluated the impact of total Sms2 deficiency on atherosclerosis. Our hypothesis is that decreasing plasma SM, but not other sphingolipids, could play an important role in preventing the development of atherosclerosis.
Sms2 KO mice originally from a 129 mouse genetic background were backcrossed with C57BL/6 animals for five generations. To prepare double KO mice, we crossed Sms2 KO animals with Apoe KO mice. The resulting double heterozygous animals were crossed to prepare double homozygous Sms2/Apoe DKO mice and Apoe KO littermate controls. The pups were weaned at 21 days and fed standard mouse chow until they were 8 weeks old. They were then utilized for studies of aortic retention of non-HDL lipoproteins in vivo. The reason to choose such young animals for the retention studies is that differences in pre-existing lesion size could contribute to differences in the retention of atherogenic lipoproteins.23 The second set of Sms2/Apoe DKO and Apoe KO mice were fed standard mouse chow until they were 19 weeks old. At this point the Apoe KO mice had developed substantial numbers of atherosclerotic lesions, and these animals were utilized for lesion size determination. All animal procedures were approved by the SUNY Downstate Medical Center Animal Care and Use Committee.
We measured SM, PC, and ceramide levels in plasma by LC/MS/MS, as previously described.24
We assessed lipoprotein aggregation as previously described,8 with some modifications. Since Apoe KO mice have no normal LDL and VLDL,9 we used non-HDL to define the lipoproteins which are not HDL. Briefly, non-HDL lipoproteins (d<1.063, 40 μg cholesterol from Sms2/Apoe DKO and Apoe KO mice were incubated with wildtype (WT) mouse aorta homogenate (95 μg total protein) as a source of mammalian sphingomyelinase in 0.1 M Tris-HCl buffer, pH 7.0, at 37°C for 4 hours. The turbidity of samples was assessed by measuring the OD at 430 nm.
Mouse (Sms2/Apoe DKO and Apoe KO) aorta homogenate (95μg total protein) was incubated with 1 μg of NBD-SM in 0.1 M Tris-HCl buffer, pH 7.0, at 37°C for 4 hours. The lipids were extracted and separated by thin layer chromatography, and the product, NBD-ceramide, was measured.
Non-HDL lipoprotein subendothelial retention was determined according to a previously published procedure.23 Briefly, non-HDL lipoproteins (d<1.063) were isolated by ultracentrifugation of Apoe KO mouse plasma. The lipoproteins were labeled with Alexa Fluor 647 and purified according to the manufacturer’s protocol. Alexa Fluor labels protein and does not label SM or PC, which would exchange off onto endogenous lipoproteins. Approximately 500 μg of labeled lipoproteins were injected into the femoral vein of each mouse. After 18 hours, the animals were anesthetized and the hearts perfusion-fixed in situ with 4% paraformaldehyde in PBS. The aortic roots were collected, frozen, and then cut serially at 10 μm intervals from the aortic sinus. Images were obtained with a confocal microscope using 638-nm excitation and an LP660 emission filter. The total intimal fluorescent area was quantified by taking the average of six sections spaced 30μm apart. Every image was captured with the same parameters of the microscope. The mean fluorescent areas were quantified using Image–Pro Plus version 4.5 software (Media Cybernetics Inc.).
The aorta was dissected and the arch photographed. Aortic lesion en face assay was performed, as previously described.21 For morphometric lesion analysis, sections were stained with Harris’ hematoxylin and eosin. Total intimal lesion area was quantified by taking the average of six sections spaced 30μm apart, beginning at the base of the aortic root. Images were viewed and captured with a Nikon Labophot 2 microscope equipped with a SPOT RT3 color video camera attached to a computerized imaging system with Image–Pro Plus version 4.5 software.
Each experiment was conducted at least three times. Data are typically expressed as mean ± SD. Data between two groups were analyzed by the unpaired, two-tailed Student’s t test, and among multiple groups by ANOVA followed by the Student-Newman-Keuls (SNK) test. A P value of less than 0.05 was considered significant.
We measured plasma SM, total cholesterol, total phospholipids, and triglyceride using enzymatic assays in Sms2/Apoe DKO mice, finding that there was a significant decrease in SM levels (30%, P<0.01), but not in other lipids (Table 1), as compared with Apoe KO animals. We then utilized LC/MS/MS to measure plasma sphingolipid levels. As indicated in Table 2, Sms2/Apoe DKO mice showed a significant decrease in plasma SM levels (35%, P<0.01) as well as SM/PC ratio (46%, P<0.01), which confirmed the previous results. They also demonstrated a significant increase in plasma ceramide levels (36%, P<0.01) (Table 2), mainly in ceramide 24:0, 24:1, and 22:0 (Supplement Table I) and dihydroceramide (27%, P<0.05) (Supplement Table I). However, neither sphingosine nor sphingosine-1-phosphate showed any significant changes (Supplement Table I). The distribution of lipids was determined by FPLC of pooled plasma samples. Non-HDL-SM was decreased in Sms2/Apoe DKO mice (Fig. 1A), but no changes were observed in non-HDL-cholesterol levels (Fig. 1B), compared with Apoe KO animals. HDL-SM and HDL-Cholesterol levels did not show differences between the two groups of mice.
We felt it important to determine whether SM reduction in non-HDL lipoproteins from Sms2/Apoe DKO mice would contribute to a lowering of atherogenicity in these particles. As previously noted, there is evidence to suggest that hydrolysis of lipoprotein SM by an arterial wall sphingomyelinase may lead to lipoprotein aggregation and retention.7–8 We therefore reasoned that the decrease of non-HDL lipoprotein with SM might decrease susceptibility to aggregation induced by aortic sphingomyelinase. This might occur through decreasing substrate availability to the enzyme.8, 26 As shown in Figure 2A, non-HDL particles from Sms2/Apoe DKO mice were indeed less significantly aggregated after treatment with aortic sphingomyelinase, compared with controls (P<0.01). We also measured sphingomyelinase activity in 8 week-old Sms2/Apoe DKO and Apoe KO mouse aortas, finding no significant differences (Fig. 2B).
We next sought to investigate non-HDL lipoprotein retention in vivo. We used a heterologous approach8 (injecting Apoe KO non-HDL lipoproteins into Sms2/Apoe DKO and Apoe KO mice) to observe particle aortic retention. We knew that after injecting fluorescently labeled Apoe KO non-HDL lipoproteins into Sms2/Apoe DKO mice, the exogenous particles can be immediately incorporated into the endogenous non-HDL lipoprotein pool which is so called “SM-poor non-HDL” lipoprotein pool in the circulation.8 We found that 8-week-old Sms2/Apoe DKO and Apoe KO mice demonstrated either no atherosclerotic lesions, or very small ones (Fig. 2C). However, the double KO mice had significantly fewer fluorescent areas in the aortas than Apoe KO animals (Fig. 2D), indicating that SM-poor non-HDL lipoproteins had a lower tendency to be retained in the aortas, compared with that of SM-rich particles.
For further evaluation of the impact of total Sms2 deficiency on atherogenesis, we dissected mouse aortas and photographed them. We also measured proximal and whole aortic lesion areas. At age 19 weeks, we found that all mice (18/18) had lesions in the aortic arch. However, the Sms2/Apoe DKO animals had noticeably less lesion areas than the Apoe KO mice (Fig. 3A).
We likewise found that the Sms2/Apoe DKO animals had 52% reduction in lesion area (Figs. 3B and C), compared to Apoe KO ones. This difference was statistically significant (P<0.02). We then isolated the brachiocephalic arteries (BCA) from both mice, and extracted lipid from them. Using LC/MS/MS, we found that the double KO mice had significantly lower free cholesterol and cholesteryl ester levels in BCA than the Apoe KO mice (by 58 and 60%, P<0.01 and P<0.001, respectively) (Table 3). More important, we also found that SM and ceramide levels in BCA were significantly decreased (by 35 and 32%, P<0.01) in Sms2/Apoe DKO BCA, compared to Apoe KO mice (Table 3). However, BCA dihydroxylceramide sphingosine, and sphingosine-1-phosphate levels showed no significant changes (Supplement Table II). Likewise, BCA PC levels were not statistically distinguishable between the two groups of mice (Table 3).
In this study, we have demonstrated that disruption of the Sms2 gene in an Apoe-deficient background caused: 1) a significant decrease of plasma SM and increase of ceramide levels; 2) no significant changes of plasma total cholesterol and triglyceride levels; 3) a significant reduction of non-HDL lipoprotein aggregation in vitro catalyzed by aortic sphingomyelinase; 4) a significant reduction of non-HDL lipoprotein retention in the aortas in vivo; 5) a significant reduction of atherosclerotic lesions in the aortic arch and root; and 6) a significant reduction of SM, ceramide, free cholesterol, and cholesteryl ester in the brachiocephalic arteries (BCA), the most susceptible region for atherosclerosis development. To our knowledge, our study is the first direct study testing the beneficial effect of plasma SM reduction, in terms of anti-atherogenesis. Moreover, we are the first to measure all the important sphingolipids in BCA from an atherogenic mouse model.
SM, an amphathic phospholipid located in the surface monolayer of all classes of plasma lipoproteins (LDL/VLDL, 70–75%; HDL, 25–30%),1 has significant effects on lipoprotein metabolism. However, there is even now no clear answer to one of the fundamental questions: what factors determine the levels of SM in the circulation? In our previous study22 and this study, we found that Sms2 is one of the factors that influence plasma SM levels. We also found that SM-deficient non-HDL particles from double KO mice have less potential for being aggregated after arterial sphingomyelinase treatment, compared with controls (Fig. 2A), indicating less atherogenic properties in these particles. The non-HDL lipoprotein aggregation results confirmed previous observations that non-HDL lipoproteins from Apoe KO mice,26 or adenovirus-mediated Sms2 overexpressed mice27 or liver-specific Sms2 transgenic mice22 have a stronger potential for aggregation after a mammalian sphingomyelinase treatment. More important, in this study we utilized aorta homogenate, instead of macrophage culture medium, as a source of sphingomyelinase, indicating that aortic enzyme has the ability to aggregate atherogenic lipoproteins in vitro.
The most striking result springing from this study is confirmation of non-HDL particle in vivo retention and atherosclerosis development. Sms2/Apoe DKO and Apoe KO mice at the age of 8 weeks had the same levels of aorta sphingomyelinase activity (Fig. 2B), and demonstrated either no atherosclerotic lesions, or very small ones (Fig. 2C). However, the double KO mice had significantly less fluorescence-labeled non-HDL lipoprotein retention in the aortic wall than the single KO mice (Fig. 2C and D). As a consequence, at the age of 19 weeks the double KO mice developed significantly smaller atherosclerotic lesions than the Apoe KO animals (Fig. 3). It is known that non-HDL lipoprotein subendothelial retention is an early step in atherogenesis28. It is believed that SM-rich non-HDL lipoproteins retained in atherosclerotic lesions are hydrolyzed by an arterial wall sphingomyelinase that promotes aggregation by converting SM to ceramide.5, 29 Tabas’s group provided convincing evidence that Apoe KO mice lacking sphingomyelinase have decreased development of early atherosclerotic lesions.23 In this study, we investigate this retention/aggregation event in another angle: reducing SM content of non-HDL lipoproteins through SMS deficient approach, thus leading to less non-HDL lipoprotein retention/aggregation in aorta, and preventing the development of atherosclerosis.
Lipid analysis of the brachiocephalic arteries (BCA) indicates that plasma SM reduction can be reflected by BCA lipid level reduction. It can be argued that Sms2 deficiency is the reason for SM reduction in BCA from the double KO mice. However, it is well-known that lipoprotein retention makes a contribution to the SM in the aorta. 7, 23, 30 Plasma SM, but not cholesterol levels, were significantly decreased in Sms2/Apoe DKO mice (Table 1). Because of that, we believe that the significant reduction of non-HDL lipoprotein retention in aorta (Figs. 2C and D) is directly related to the significant reduction of SM, cholesterol, and cholesteryl ester levels in the aorta (Table 3). Moreover, we found that aortic ceramide levels were significantly decreased (Supplement Table II) in the double KO mice. This suggests that the reduction of lipoprotein retention causes the less SM retained in aorta, thus leading to lower sphingomyelinase-mediated ceramide production, which can overbalance the Sms2 deficiency-mediated ceramide accumulation in the mouse aorta.
In our previous two studies,21, 31 we reported that macrophage-specific Sms2 deficiency significantly decreases SM in plasma membrane lipid rafts, increases cholesterol efflux, and decreases inflammatory responses, thus decreasing atherosclerosis. Since Sms2/Apoe DKO mice used in this study are general Sms2 deficient mice, the macrophage-mediated anti-inflammation and anti-atherosclerosis properties may also play a role in the reduction of atherosclerosis observed in this study. We found Apoe KO/Sms2 KO macrophages show significantly less sensitivity to lysenin-mediated cytolysis than Apoe KO cells (P<0.01), confirming the critical and physiological role of SMS2 in regulating SM levels in cell membrane microdomains (lipid rafts) (Supplemental Fig. I). We also found that Sms2 deficiency attenuates macrophage NFκB, p-38, and p44/42 activation in Apoe deficiency background (Supplemental Fig. II) and IL-6 and TNFα secretion (Supplemental Fig. III). However, there are three fundamental differences between the previous studies and this study:1) macrophage-Sms2 KO/Ldlr KO mice had same plasma SM levels as Ldlr KO mice,21 while Sms2 KO/Apoe KO mice had lower plasma SM levels than their controls (Table 1 and and2);2); 2) western type diet was used to induce atherosclerosis in macrophage-Sms2 KO/Ldlr KO mice and their controls,21 while chow diet was used in this study; and 3) no mammalian sphingomyelinase-mediated atherogenic lipoprotein aggregation was observed in macrophage-Sms2 KO/Ldlr KO mice and their controls (Liu and Jiang, unpublished observation), while such aggregation was observed in both Sms2 KO/Apoe KO and Apoe KO mice, and the former had significantly less tendency than that of latter (Fig. 2). It is quite possible that both plasma SM and cell membrane SM levels play additive or synergistic roles in the development of atherosclerosis.
Roles have been proposed for ceramide in atherogenesis. Ceramide has been shown to induce apoptosis of certain cells lining the vascular wall, a process implicated in plaque erosion and thrombosis.32 Ceramide mediates an inflammatory response initiated by cytokines or oxidized LDL, a response that upregulates adhesion molecule expression and induces adhesion and migration of monocytes, both important events in the initiation and progression of atherogenesis.33 Plasma ceramide may contribute to maladaptive inflammation in patients with coronary heart disease.34 It has been reported that plasma ceramide levels in Apoe KO mice are higher than those in WT mice.35 Plasma ceramides may possibly correlate with an increase in LDL oxidation, becoming a risk factor for atherosclerosis.35 In general, ceramide is a proatherogenic factor. However, in this study we found that plasma ceramide levels in Sms2/Apoe DKO mice are increased, so that ceramide level changes could not be a reason for the reduction of atherosclerosis in the double KO mice. To further address this issue we measured and compared plasma ceramide and SM levels in three sets of mice with atherosclerosis: 1) Apoe KO mice with or without myriocin10 (Sphingolipid de novo synthesis inhibitor); 2) Ldlr KO mice with or without sphingolipid-rich-diet36 (The experimental diet was formulated by supplementing the control diet with 1% sphingolipids at the expense of sucro; 3 Apoe KO and Apoe KO/Sms2 KO mice (this study). As shown in Supplemental Table III, we found that SM but not ceramide levels are positively related to the development of atherosclerosis. Decreasing SM decreases atherosclerosis and increasing SM increases atherosclerosis. Ceramide level seems not always correlate with atherogenic consequences in the studies. These results support the notion that SM levels dominate the proatherogenic consequences.
Sms2 deficiency may have an impact on other sphingolipid levels, including sphingosine and sphingosine-1-phosphate, which have antiatherogenic properties.37–38 However, we did not observe significant changes of these two important sphingolipids in the plasma and the BCA (Table 2 and 4), suggesting that Sms2 deficiency-mediated antiatherogenic properties might not relate to both sphingolipids.
In conclusion, SMS2 contributed physiologically to de novo SM biosynthesis and plasma SM levels. SMS2 deficiency caused lower atherogenic lipoprotein retention and reduced atherosclerosis in Apoe KO mice. Thus, SMS2 should be considered a potential therapeutic target for the treatment of atherosclerosis.
This work was supported by National Institutes of Health grant HL093419 to X.C.J. The authors wish to thank Mrs. Tom Beyer, Rob Christe and Michael Kalbfleisch for technical support.