Selective Reduction in the Sphingomyelin Content of Atherogenic Lipoproteins Inhibits Their Retention in Murine Aortas and the Subsequent Development of Atherosclerosis
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.
- Sms2 knockout mice
- Sms2 and Apoe double kncockout mice
- plasma sphingomyelin
- plasma cholesterol
- non-HDL lipoprotein aggregation and retention
- 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 Tabas5,6 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 Researchers10,11 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 disease12,13 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, researchers10,11 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. 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 Researchers18–20 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.
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 μg of cholesterol from Sms2/Apoe DKO and Apoe KO mice) were incubated with wild-type mouse aorta homogenate (95 μg 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 μg of total protein) was incubated with 1 μg 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 μg 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-μm 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 μm 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 μm 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.
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.
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 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 http://atvb.ahajournals.org) 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.
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 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 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 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 were not statistically distinguishable between the 2 groups of mice (Table 3).
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 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 study22 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 mice22 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 fluorescence-labeled 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 al23 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 SMS-deficient 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 sphingomyelinase-mediated 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 macrophage-mediated 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 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 Figure I). We also found that Sms2 deficiency attenuates macrophage nuclear factor κB, p-38, and p44/42 activation in an Apoe deficiency background (supplemental Figure II) and interleukin 6 and tumor necrosis factor α 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 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 myriocin10 (a sphingolipid de novo synthesis inhibitor); (2) Ldlr KO mice with or without a sphingolipid-rich diet36 (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.
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 HL093419 from the National Institutes of Health (Dr Jiang).
Drs Fan and Shi made equal contributions.
Received on: March 14, 2010; final version accepted on: August 16, 2010.
Nilsson A, Duan RD. Absorption and lipoprotein transport of sphingomyelin. J Lipid Res. 2006; 47: 154–171.
Zilversmit DB, Mc CE, Jordan PH, Henly WS, Ackerman RF. The synthesis of phospholipids in human atheromatous lesions. Circulation. 1961; 23: 370–375.
Williams KJ, Tabas I. The response-to-retention hypothesis of early atherogenesis. Arterioscler Thromb Vasc Biol. 1995; 15: 551–561.
Schissel SL, Tweedie-Hardman J, Rapp JH, Graham G, Williams KJ, Tabas I. Rabbit aorta and human atherosclerotic lesions hydrolyze the sphingomyelin of retained low-density lipoprotein: proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins. J Clin Invest. 1996; 98: 1455–1464.
Jeong T, Schissel SL, Tabas I, Pownall HJ, Tall AR, Jiang X. Increased sphingomyelin content of plasma lipoproteins in apolipoprotein e knockout mice reflects combined production and catabolic defects and enhances reactivity with mammalian sphingomyelinase. J Clin Invest. 1998; 101: 905–912.
Park TS, Panek RL, Mueller SB, Hanselman JC, Rosebury WS, Robertson AW, Kindt EK, Homan R, Karathanasis SK, Rekhter MD. Inhibition of sphingomyelin synthesis reduces atherogenesis in apolipoprotein e-knockout mice. Circulation. 2004; 110: 3465–3471.
Hojjati MR, Li Z, Zhou H, Tang S, Huan C, Ooi E, Lu S, Jiang XC. Effect of myriocin on plasma sphingolipid metabolism and atherosclerosis in apoe-deficient mice. J Biol Chem. 2005; 280: 10284–10289.
Jiang XC, Paultre F, Pearson TA, Reed RG, Francis CK, Lin M, Berglund L, Tall AR. Plasma sphingomyelin level as a risk factor for coronary artery disease. Arterioscler Thromb Vasc Biol. 2000; 20: 2614–2618.
Schlitt A, Blankenberg S, Yan D, von Gizycki H, Buerke M, Werdan K, Bickel C, Lackner KJ, Meyer J, Rupprecht HJ, Jiang XC. Further evaluation of plasma sphingomyelin levels as a risk factor for coronary artery disease. Nutr Metab. 2006; 3: 5.
Hamilton RL, Moorehouse A, Havel RJ. Isolation and properties of nascent lipoproteins from highly purified rat hepatocytic golgi fractions. J Lipid Res. 1991; 32: 529–543.
Huitema K, van den Dikkenberg J, Brouwers JF, Holthuis JC. Identification of a family of animal sphingomyelin synthases. EMBO J. 2004; 23: 33–44.
Miyaji M, Jin ZX, Yamaoka S, Amakawa R, Fukuhara S, Sato SB, Kobayashi T, Domae N, Mimori T, Bloom ET, Okazaki T, Umehara H. Role of membrane sphingomyelin and ceramide in platform formation for fas-mediated apoptosis. J Exp Med. 2005; 202: 249–259.
Van der Luit AH, Budde M, Zerp S, Caan W, Klarenbeek JB, Verheij M, Van Blitterswijk WJ. Resistance to alkyl-lysophospholipid-induced apoptosis due to downregulated sphingomyelin synthase 1 expression with consequent sphingomyelin- and cholesterol-deficiency in lipid rafts. Biochem J. 2007; 401: 541–549.
Liu J, Huan C, Chakraborty M, Zhang H, Lu D, Kuo MS, Cao G, Jiang XC. Macrophage sphingomyelin synthase 2 deficiency decreases atherosclerosis in mice. Circ Res. 2009; 105: 295–303.
Liu J, Zhang H, Li Z, Hailemariam TK, Chakraborty M, Qiu D, Bui HH, Peake DA, Kuo MS, Wadgaonkar R, Cao G, Jiang XC. Sphingomyelin synthase 2 is one of the determinants for plasma and liver sphingomyelin levels in mice. Arterioscler Thromb Vasc Biol. 2009.
Devlin CM, Leventhal AR, Kuriakose G, Schuchman EH, Williams KJ, Tabas I. Acid sphingomyelinase promotes lipoprotein retention within early atheromata and accelerates lesion progression. Arterioscler Thromb Vasc Biol. 2008; 28: 1723–1730.
Li Z, Li Y, Chakraborty M, Fan Y, Bui HH, Peake DA, Kuo MS, Xiao X, Cao G, Jiang XC. Liver-specific deficiency of serine palmitoyltransferase subunit 2 decreases plasma sphingomyelin and increases apolipoprotein e levels. J Biol Chem. 2009; 284: 27010–27019.
Kuo MS, Kalbfleisch JM, Rutherford P, Gifford-Moore D, Huang XD, Christie R, Hui K, Gould K, Rekhter M. Chemical analysis of atherosclerotic plaque cholesterol combined with histology of the same tissue. J Lipid Res. 2008; 49: 1353–1363.
Schissel SL, Jiang X, Tweedie-Hardman J, Jeong T, Camejo EH, Najib J, Rapp JH, Williams KJ, Tabas I. Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral ph: implications for atherosclerotic lesion development. J Biol Chem. 1998; 273: 2738–2746.
Dong J, Liu J, Lou B, Li Z, Ye X, Wu M, Jiang XC. Adenovirus-mediated overexpression of sphingomyelin synthases 1 and 2 increases the atherogenic potential in mice. J Lipid Res. 2006; 47: 1307–1314.
Schissel SL, Keesler GA, Schuchman EH, Williams KJ, Tabas I. The cellular trafficking and zinc dependence of secretory and lysosomal sphingomyelinase, two products of the acid sphingomyelinase gene. J Biol Chem. 1998; 273: 18250–18259.
Nievelstein PF, Fogelman AM, Mottino G, Frank JS. Lipid accumulation in rabbit aortic intima 2 hours after bolus infusion of low density lipoprotein: a deep-etch and immunolocalization study of ultrarapidly frozen tissue. Arterioscler Thromb. 1991; 11: 1795–1805.
Hailemariam TK, Huan C, Liu J, Li Z, Roman C, Kalbfeisch M, Bui HH, Peake DA, Kuo MS, Cao G, Wadgaonkar R, Jiang XC. Sphingomyelin synthase 2 deficiency attenuates NFκB activation. Arterioscler Thromb Vasc Biol. 2008; 28: 1519–1526.
Mallat Z, Tedgui A. Current perspective on the role of apoptosis in atherothrombotic disease. Circ Res. 2001; 88: 998–1003.
Chatterjee S. Sphingolipids in atherosclerosis and vascular biology. Arterioscler Thromb Vasc Biol. 1998; 18: 1523–1533.
de Mello VD, Lankinen M, Schwab U, Kolehmainen M, Lehto S, Seppanen-Laakso T, Oresic M, Pulkkinen L, Uusitupa M, Erkkila AT. Link between plasma ceramides, inflammation and insulin resistance: association with serum il-6 concentration in patients with coronary heart disease. Diabetologia. 2009; 52: 2612–2615.
Kimura T, Sato K, Malchinkhuu E, Tomura H, Tamama K, Kuwabara A, Murakami M, Okajima F. High-density lipoprotein stimulates endothelial cell migration and survival through sphingosine 1-phosphate and its receptors. Arterioscler Thromb Vasc Biol. 2003; 23: 1283–1288.