Yifan Fan, Fujun Shi, Jing Liu, Jibin Dong, Hai H. Bui, David A. Peake, Ming-Shang Kuo, Guoqing Cao, Xian-Cheng Jiang

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1 Selective Reduction in the Sphingomyelin Content of Atherogenic Lipoproteins Inhibits Their Retention in Murine Aortas and the Subsequent Development of Atherosclerosis Yifan Fan, Fujun Shi, Jing Liu, Jibin Dong, Hai H. Bui, David A. Peake, Ming-Shang Kuo, Guoqing Cao, Xian-Cheng Jiang Downloaded from by guest on July 9, 2018 Objective We used the sphingomyelin (SM) synthase 2 (Sms2) gene knockout (KO) approach to test our hypothesis that selectively decreasing plasma lipoprotein SM can play an important role in preventing atherosclerosis. Methods and Results The sphingolipid de novo synthesis pathway is considered a promising target for pharmacological intervention in atherosclerosis. However, its potential is hampered because the substance s atherogenic mechanism is not completely understood. We prepared Sms2 and apolipoprotein E (Apoe) double-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%, respectively; P 0.01) but no significant changes in other tested sphingolipids, cholesterol, and triglyceride. Non high-density lipoproteins from the double-ko mice showed a reduction of SM, but not cholesterol, and displayed less tendency toward aortic sphingomyelinase-mediated lipoprotein aggregation in vitro and retention in aortas in vivo when compared with controls. More important, at the age of 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 with controls. The Sms2 KO/Apoe KO brachiocephalic artery contained significantly less SM, ceramide, free cholesterol, and cholesteryl ester (35%, 32%, 58%, and 60%, respectively; P 0.01) than that of the Apoe KO brachiocephalic artery. Conclusion Decreasing plasma SM levels through decreasing SMS2 activity could become a promising treatment for atherosclerosis. (Arterioscler Thromb Vasc Biol. 2010;30: ) Key Words: Sms2 knockout mice Sms2 and Apoe double kncockout mice plasma sphingomyelin plasma cholesterol non-hdl lipoprotein aggregation and retention atherosclerosis lipids in brachiocephalic artery 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 very-low-density lipoprotein (VLDL) and LDL, are SM enriched. 1,3 The SM content of atherosclerotic lesions is higher than that of healthy arterial tissue. 4 Williams and Tabas 5,6 suggested that subendothelial retention and aggregation of atherogenic lipoproteins play an important role in atherogenesis. 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 Plasma SM levels in apolipoprotein E (Apoe) knockout (KO) mice are 4-fold higher than those in wild-type mice, 8 and this may partially explain the increased atherosclerosis found in these animals. 9 Researchers 10,11 have also discovered that chemical inhibition of sphingolipid biosynthesis significantly decreases plasma SM levels, thus lessening atherosclerotic lesions in Apoe KO mice. 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, researchers 10,11 have found that after inhibiting sphingolipid de novo synthesis, all other tested sphin- Received on: March 14, 2010; final version accepted on: August 16, From the Department of Cardiology (Y.F.), Renmin Hospotal, Wuhan University, Wuhan, China; the Department of Cell Biology (Y.F., F.S., J.L., and X.-C.J.), State University of New York Downstate Medical Center, Brooklyn, NY; Department of Medicine, Southern Medical University (F.S.), Guangzhou, China; Department of Biochemistry, School of Pharmacy (J.D.), Fudan University, Shanghai, China; and Lilly Research Laboratories (H.H.B., D.A.P., M.-S.K., and G.C.), Eli Lilly & Company, Indianapolis, Ind. Drs Fan and Shi made equal contributions. Correspondence to Xian-Cheng Jiang, PhD, State University of New York Downstate Medical Center, 450 Clarkson Ave, Box 5, Brooklyn, NY XJiang@downstate.edu 2010 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at DOI: /ATVBAHA

2 Fan et al Atherogenic Lipoprotein Retention and Atherosclerosis 2115 Downloaded from by guest on July 9, 2018 golipids, including SM, ceramide, sphingosine, sphingosine- 1-phosphate, and glycosphingolipids, are significantly decreased. Consequently, we could not exclude the effect of sphingolipids other than SM on mouse atherogenicity. The biochemical synthesis of SM occurs through the action of serine palmitoyltransferase, 3-ketosphinganine reductase, ceramide synthase, dihydroceramide desaturase, and SM synthase (SMS). 14 SMS is the last enzyme for SM biosynthesis, and it uses 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 high-density lipoprotein (HDL), into circulation. 15 After hydrolysis of dietary SM in the lumen of the small intestine, 16 the backbone sphingoid bases and fatty acids, which are absorbed by the enterocytes, can be used to resynthesize SM. This SM can participate in chylomicron assembly and can 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, whereas SMS2 is predominantly located in the plasma membranes. 17 Researchers have shown that Sms1 and Sms2 expression positively correlates with levels of cellular SM and with SM in membrane lipid rafts. Macrophage-specific Sms2 deficiency decreases atherosclerosis in a mouse model. 21 Also, Sms2 deficiency decreases, whereas Sms2 overexpression increases, plasma SM levels. 22 In the present study, we prepared Sms2 KO/Apoe KO mice and evaluated the impact of total Sms2 deficiency on atherosclerosis. Our hypothesis was that decreasing plasma SM, but not other sphingolipids, could play an important role in preventing the development of atherosclerosis. Methods Sms2/Apoe Double-KO Mouse Preparation Sms2 KO mice, originally from a 129-mouse genetic background, were backcrossed with C57BL/6 animals for 5 generations. To prepare double-ko (DKO) 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 age 8 weeks. They were then used for studies of aortic retention of non-hdl lipoproteins in vivo. The reason for choosing such young animals for the retention studies is that differences in preexisting lesion size could contribute to differences in the retention of atherogenic lipoproteins. 23 The second set of Sms2/Apoe DKO and Apoe KO mice was fed standard mouse chow until the mice were age 19 weeks. At this point, the Apoe KO mice had developed many atherosclerotic lesions, and these animals were used for lesion size determination. All animal procedures were approved by the State University of New York Downstate Medical Center Animal Care and Use Committee. We measured SM, PC, and ceramide levels in plasma by liquid chromatography (LC)/mass spectroscopy (MS)/MS, as previously described. 24 Atherogenic Lipoprotein Aggregation Assay We assessed lipoprotein aggregation, as previously described, 8 with some modifications. Because Apoe KO mice have no normal LDL and VLDL, 9 we used non-hdl to define the lipoproteins that are not HDL. Briefly, non-hdl lipoproteins (d 1.063; 40 g of cholesterol from Sms2/Apoe DKO and Apoe KO mice) were incubated with wild-type mouse aorta homogenate (95 g of total protein) as a source of mammalian sphingomyelinase in 0.1-mol/L Tris-HCl buffer, ph 7.0, at 37 C for 4 hours. The turbidity of samples was assessed by measuring the optical density at 430 nm. Aorta Sphingomyelinase Activity Assay Mouse (Sms2/Apoe DKO and Apoe KO) aorta homogenate (95 gof total protein) was incubated with 1 g of Nitrobenzoxadiazol-SM in 0.1-mol/L 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. Total cholesterol, cholesteryl ester, SM, PC, and ceramide levels in the brachiocephalic artery (BCA) were measured according to a previously published method. 21,25 Quantification of Subendothelial Lipoprotein Retention 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 fluorescent dye (Alexa Fluor 647) and purified according to the manufacturer s protocol. Alexa Fluor labels protein and does not label SM or PC. Approximately 500 g of labeled lipoproteins was injected into the femoral vein of each mouse. After 18 hours, the animals were anesthetized and the hearts were perfusion fixed in situ with 4% paraformaldehyde in PBS. The aortic roots were collected, frozen, and then cut serially at 10- m intervals from the aortic sinus. Images were obtained with a confocal microscope using 638-nm excitation and an emission filter (model LP660). The total intimal fluorescent area was quantified by taking the average of 6 sections spaced 30 m 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. Mouse Atherosclerotic Lesion Measurement The aorta was dissected, and the arch was photographed. An aortic lesion en face assay was performed as previously described. 21 For morphometric lesion analysis, sections were stained with Harris hematoxylin-eosin. The total intimal lesion area was quantified by taking the average of 6 sections spaced 30 m apart, beginning at the base of the aortic root. Images were viewed and captured with a microscope (Nikon Labophot 2), equipped with a color video camera (SPOT RT3) attached to a computerized imaging system with Image Pro Plus version 4.5. Statistical Analysis Each experiment was conducted at least 3 times. Data are typically expressed as mean SD. Data between 2 groups were analyzed by the unpaired 2-tailed Student t test; and data among multiple groups were analyzed by ANOVA, followed by the Student-Newman-Keuls test. P 0.05 was considered significant. Results Plasma Lipid Analysis 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), compared with Apoe KO animals. We then used 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) and in the SM/PC ratio (46%, P 0.01), which confirmed the previous results. They

3 2116 Arterioscler Thromb Vasc Biol November 2010 Table 1. Mouse Plasma Lipid Measurements Level, mg/dl* Total Total Mice SM Phospholipids Cholesterol Triglyceride Apoe KO Sms2/Apoe KO *Values are expressed as mean SD (n 6). Lipids were measured by enzymatic assays. P Downloaded from by guest on July 9, 2018 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 (supplemental Table I; available online at and dihydroceramide (27%, P 0.05) (supplemental Table I). However, neither sphingosine nor sphingosine-1-phosphate showed any significant changes (supplemental Table I). The distribution of lipids was determined by fast protein liquid chromatography (FPLC) of pooled plasma samples. Non HDL-SM was decreased in Sms2/Apoe DKO mice (Figure 1A), but no changes were observed in non HDL-cholesterol levels (Figure 1B), compared with Apoe KO animals. HDL-SM and HDL-cholesterol levels did not show differences between the 2 groups of mice. Table 2. Mouse Plasma SM, PC, and Ceramide Measurements Level, mmol/ml* Mice SM PC Ceramide SM/PC Ratio Apoe KO Sms2 KO/Apoe KO *Values are expressed as mean SD (n 6). Lipids were measured by liquid chromatography/ms/ms. P Figure 1. Plasma lipoprotein analysis by FPLC in mice. A 300- L aliquot of pooled plasma (from 7 animals) was loaded into a column (Sepharose 6B) and eluted with 50-mmol/L Tris and 0.15-mol/L NaCl (ph 7.5). An aliquot of each fraction was used for the determination of cholesterol and SM. A, SM distribution in Sms2/Apoe DKO and Apoe KO mice. B, Cholesterol distribution in Sms2/Apoe DKO and Apoe KO mice. Non-HDL Particle Aggregation and Retention We believed it was 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 Therefore, we 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 (Figure 2B). Next, we sought to investigate non-hdl lipoprotein retention in vivo. We used a heterologous approach 8 (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 the 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 small ones (Figure 2C). However, the DKO mice had significantly fewer fluorescent areas in the aortas than Apoe KO animals (Figure 2D), indicating that SM-poor non-hdl lipoproteins had a lower tendency to be retained in the aortas compared with SM-rich particles. Evaluation of Atherosclerosis in Sms2/Apoe DKO Mice 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 the age of 19 weeks, we found that all mice (18/18) had lesions in the aortic arch. However, the Sms2/ Apoe DKO animals had noticeably fewer lesion areas than the Apoe KO mice (Figure 3A). Likewise, we found that the Sms2/Apoe DKO animals had a 52% reduction in lesion area (Figure 3B and C) compared with the Apoe KO animals. This difference was statistically significant (P 0.02). We then isolated the BCAs from both mice and extracted lipid from them. By using LC/MS/MS, we

4 Fan et al Atherogenic Lipoprotein Retention and Atherosclerosis 2117 Downloaded from by guest on July 9, 2018 found that the DKO mice had significantly lower free cholesterol and cholesteryl ester levels in the BCA than the Apoe KO mice (by 58% [P 0.01] and 60% [P 0.01], respectively) (Table 3). More important, we also found that SM and ceramide levels in the BCA were significantly decreased (by 35% and 32%, respectively; P 0.01) in Sms2/Apoe DKO BCA compared with Apoe KO mice (Table 3). However, BCA dihydroxyl ceramide sphingosine and sphingosine-1-phosphate levels showed no significant changes (supplemental Table II). Likewise, BCA PC levels Figure 2. Non-HDL lipoproteins from Sms2/Apoe DKO mice exhibit reduced susceptibility to aggregation in vitro and aortic retention in vivo compared with Apoe KO controls. A, In vitro aggregation of atherogenic lipoproteins induced by aorta-derived sphingomyelinase (SMase). The non-hdl particles from Sms2/Apoe DKO and Apoe KO mice were isolated, and their aggregation was assessed as previously described in Aorta Sphingomyelinase Activity Assay. OD indicates optical density. B, Aorta SMase activity assay. C, In vivo retention of atherogenic lipoproteins in mouse aorta. Light and fluorescent fields were shown on the right and left, respectively. D, Quantitative display of a fluorescent area in the aortic root. Values are expressed as mean SD (n 6). *P were not statistically distinguishable between the 2 groups of mice (Table 3). Discussion In this study, we demonstrated that disruption of the Sms2 gene in an Apoe-deficient background caused: (1) a significant decrease of plasma SM and an 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 Figure 3. Sms2/Apoe DKO mice demonstrated significantly decreased atherosclerotic lesion size compared with Apoe KO controls. A, Mice were euthanized, and the aortas were dissected and photographed. This set of pictures is representative of 3 sets from 18 animals. Atherosclerotic lesions in the aortic arch are indicated by arrows. B, Hematoxylineosin staining for the proximal aortic root. Lesions are indicated by arrows. C, Quantitative display of the root assay. Values are expressed as mean SD. *P 0.02.

5 2118 Arterioscler Thromb Vasc Biol November 2010 Table 3. SM, Ceramide, PC, FC, and CE Levels in Mouse BCA Level, mmol/whole BCA* Mice SM PC Ceramide FC CE Apoe KO Sms2 KO/Apoe KO CE indicates cholesteryl ester; FC, free cholesterol. *Values are expressed as mean SD (n 6). Lipids were measured by liquid chromatography/ms/ms. P Downloaded from by guest on July 9, 2018 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 BCAs, 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 antiatherogenesis. Moreover, we are the first to measure all the important sphingolipids in the BCA from an atherogenic mouse model. SM, an amphathic phospholipid located in the surface monolayer of all classes of plasma lipoproteins (LDL or VLDL, 70% to 75%; HDL, 25% to 30%), 1 has significant effects on lipoprotein metabolism. However, there is no clear answer to one of the fundamental questions: What factors determine the levels of SM in the circulation? In a previous study 22 and in the present study, we found that Sms2 is one of the factors that influences plasma SM levels. We also found that SM-deficient non-hdl particles from DKO mice have less potential for being aggregated after arterial sphingomyelinase treatment, compared with controls (Figure 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 adenovirus-mediated Sms2 overexpressed mice, 27 or liver-specific Sms2 transgenic mice 22 have a stronger potential for aggregation after mammalian sphingomyelinase treatment. More important, in this study, we used aorta homogenate, instead of macrophage culture medium, as a source of sphingomyelinase, indicating that the 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 (Figure 2B) and demonstrated either no atherosclerotic lesions or small lesions (Figure 2C). However, the DKO mice had significantly less fluorescencelabeled non-hdl lipoprotein retention in the aortic wall than the single KO mice (Figure 2C and D). Consequently, at the age of 19 weeks, the DKO mice developed significantly smaller atherosclerotic lesions than the Apoe KO mice (Figure 3). Non-HDL lipoprotein subendothelial retention is an early step in atherogenesis. 28 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 Devlin et al 23 provided convincing evidence that Apoe KO mice lacking sphingomyelinase have decreased development of early atherosclerotic lesions. In this study, we investigate this retention/aggregation event using another angle: reducing the SM content of non-hdl lipoproteins through an SMSdeficient approach, thus leading to less non-hdl lipoprotein retention/aggregation in the aorta and preventing the development of atherosclerosis. Lipid analysis of the BCAs indicates that plasma SM reduction can be reflected by BCA lipid-level reduction. Sms2 deficiency may be the reason for SM reduction in BCAs from the DKO 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). Thus, we believe that the significant reduction of non-hdl lipoprotein retention in the aorta (Figure 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 (supplemental Table II) in the DKO mice. This suggests that the reduction of lipoprotein retention causes the less SM retained in the aorta, thus leading to lower sphingomyelinasemediated ceramide production, which can overbalance the Sms2 deficiency mediated ceramide accumulation in the mouse aorta. In our previous studies, 21,31 it was reported that macrophage-specific Sms2 deficiency significantly decreases SM in plasma membrane lipid rafts, increases cholesterol efflux, and decreases inflammatory responses, thus decreasing atherosclerosis. Because the Sms2/Apoe DKO mice used in this study are general Sms2-deficient mice, the macrophagemediated antiinflammation and antiatherosclerosis properties may also play a role in the reduction of atherosclerosis observed in this study. We found that Apoe KO/Sms2 KO macrophages show significantly less sensitivity to lyseninmediated 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 Figure I). We also found that Sms2 deficiency attenuates macrophage nuclear factor B, p-38, and p44/42 activation in an Apoe deficiency background (supplemental Figure II) and interleukin 6 and tumor necrosis factor secretion (supplemental Figure III). However, there are 3 fundamental differences between the previous studies and the present study: (1) macrophage-sms2 KO/LDL receptor (Ldlr) KO mice had the same plasma SM levels as Ldlr KO mice, 21 whereas Sms2 KO/Apoe KO mice had lower plasma SM levels than their controls (Tables 1 and 2); (2) a Western-type

6 Fan et al Atherogenic Lipoprotein Retention and Atherosclerosis 2119 Downloaded from by guest on July 9, 2018 diet was used to induce atherosclerosis in macrophage-sms2 KO/Ldlr KO mice and their controls, 21 whereas a 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 (J.L. and X.-C.J., unpublished data, 2009), whereas such aggregation was observed in both Sms2 KO/ Apoe KO and Apoe KO mice (the former had significantly less tendency than the latter) (Figure 2). It is 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 induces 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 Plasma ceramide levels in Apoe KO mice are higher than those in wild-type 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 DKO mice. To further address this issue, we measured and compared plasma ceramide and SM levels in 3 sets of mice with atherosclerosis: (1) Apoe KO mice with or without myriocin 10 (a sphingolipid de novo synthesis inhibitor); (2) Ldlr KO mice with or without a sphingolipid-rich diet 36 (the experimental diet was formulated by supplementing the control diet with 1% sphingolipids at the expense of sucrose); and (3) Apoe KO and Apoe KO/Sms2 KO mice (present 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. The ceramide level seems to 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 sphingolipids, including sphingosine and sphingosine-1-phosphate, that have antiatherogenic properties. 37,38 However, we did not observe significant changes in these 2 important sphingolipids in the plasma and the BCA (supplemental Tables I and II), 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. Acknowledgments We thank Mrs. Tom Beyer, MS, Rob Christe, MS, and Michael Kalbfleisch, MS, for technical support. Sources of Funding This study was supported by grant HL from the National Institutes of Health (Dr Jiang). None. Disclosures References 1. Nilsson A, Duan RD. 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8 Downloaded from by guest on July 9, 2018 Selective Reduction in the Sphingomyelin Content of Atherogenic Lipoproteins Inhibits Their Retention in Murine Aortas and the Subsequent Development of Atherosclerosis Yifan Fan, Fujun Shi, Jing Liu, Jibin Dong, Hai H. Bui, David A. Peake, Ming-Shang Kuo, Guoqing Cao and Xian-Cheng Jiang Arterioscler Thromb Vasc Biol. 2010;30: ; originally published online September 2, 2010; doi: /ATVBAHA Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX Copyright 2010 American Heart Association, Inc. All rights reserved. Print ISSN: Online ISSN: The online version of this article, along with updated information and services, is located on the World Wide Web at: Data Supplement (unedited) at: Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Arteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online at:

9 Supplement Material Supplemental Table I. Mouse plasma sphingolipid measurement (LC/MS/MS) Mice Cer16 Cer22 Cer22:1 Cer24 Cer24:1 DHCer Sph S1P (nmole/ml) Apoe KO Sms2 KO/ Apoe KO * * * * * Value: mean+sd; n=6. Lipids were measured by LC/MS/MS. *P<0.02. SM, Sphingomyelin; PC, Phosphatidylcholine; DHCer, dihydroceramide; Sph, sphingosine; S1P, sphingosine-1-phosphate. 1

10 Supplemental Table II. brachiocephalicartery sphingolipid measurement (LC/MS/MS) Mice Cer16 Cer22 Cer22:1 Cer24 Cer24:1 DHCer Sph S1P (nmole/whole BCA) Apoe KO Sms2 KO/ Apoe KO * * * Value: mean+sd; n=6. Lipids were measured by LC/MS/MS. *P<0.02. SM, Sphingomyelin; PC, Phosphatidylcholine; DHCer, Dihydroxylceramide; Sph, sphingosine; S1P, sphingosine-1-phosphate. 2

11 Supplemental Table III. Ceramide and SM measurement in mice Ceramide SM Atherosclerosis (nmole/ml) (nmole/ml) Apoe KO(control) Apoe KO(myriocin) 20+3* * (10) Ldlr KO(control) Ldlr KO(SL-rich diet) * (36) Apoe KO Apoe KO/Sms2 KO 37+6* * (This study) Value: mean + SD, n=5; *P<

12 Supplemental figure legends: Supplemental Figure I: Sms2 deficiency decreases SM levels on macrophage plasma membrane SM-rich microdomains. Lysenin mediated cell lysis assay. Suppkemental Figure II: Sms2 deficiency attenuates macrophage NFkB, P38, and p-44/42 activations. Western blot of cytoplasmic IκBα, P38, phosphrylated p38 (p-p38), P44/42, phosphrylated P44/42 (p-p44/42), JUK, phosphrylated JUK (p-juk). Suppkemental Figure III: Sms2 deficient macrophage secrete less IL-6 and TNFa after LPS treatment. BMDM, bone marrow derived macrophages. Culture medium ELISA analysis. *P<

13 Supplemental Figure I. Fan et al. 150 Mφ Mort tality (% of Apo oe KO) * 0 Apoe KO Apoe/Sms2 KO

14 Supplemental Figure II. Fan et al. Apoe KO Sms2 KO/Apoe KO +LPS (min) IκBα p- P38 total- P38 p-p44/p42 total- P44/42 p-jnk total-jnk β-actini

15 Supplemental Figure III. Fan et al. A 2000 BMDMs (4h+LPS) B 5000 BMDMs (4h+LPS) Secre eted IL-6 (pg/ ml) Apoe KO * Sms2 KO/Apoe KO Secrete ed TNF-α (pg /ml) Apoe KO * Sms2 KO/Apoe KO