In this study, we have demonstrated for the first time that disruption of the SMS2 gene caused: 1) a 60% reduction of SMS activity in the liver, although SMS1 mRNA levels were not influenced; 2) a significant decrease of plasma SM and increase of ceramide levels; 3) a significant reduction of liver and hepatocyte plasma membrane SM levels; 4) a significant increase in plasma apoE levels, but not in those of apoA-I or apoB; and 5) a significant induction of cholesterol efflux from macrophages toward the plasma. Moreover, we have shown that overexpression of SMS2 in the liver had the opposite effect from a deficiency of it.
SM, an amphathic phospholipid located in the surface monolayer of all classes of plasma lipoproteins (LDL/VLDL, 70-75%; HDL, 25–30%),29
has significant effects on lipoprotein metabolism. But there is even now no clear answer to the one fundamental question: what factors determine the levels of SM in the circulation? In this study, we have partially answered that question: SMS2 is one of those factors, and it influences tissue and also plasma SM levels.
We found that human plasma SM level is an independent risk factor for coronary heart disease.12, 13
We believe that SM on LDL retained in atherosclerotic lesions is hydrolyzed by an arterial wall sphingomyelinase, which promotes aggregation by converting SM to ceramide.4, 6
There are two ways of preventing this atherogenic event, the first being to reduce sphingomyelinase levels. Indeed, it has recently been reported that apoE KO mice lacking sphingomyelinase have decreased development of early atherosclerotic lesions and, more important, decreased retention of atherogenic lipoproteins, compared with apoE KO matched for similar lipoprotein levels.30
The second way of preventing atherogenicity is by reducing SM levels in the atherogenic lipoproteins, through inhibition of the SM biosynthesis pathway in the lipoprotein-producing tissues, such as the liver and small intestine. We have demonstrated that SMS2 deficiency causes lower plasma SM levels, while liver-specific SMS2 overexpression causes higher ones, compared with controls. We also found that SM-enriched non-HDL particles from SMS2LTg mice have a stronger potential for aggregation after mammalian sphingomyelinase treatment, compared with controls (), indicating a proatherogenic property in these particles. The non-HDL aggregation study also confirmed our previous study, that SM-enriched non-HDL particles from adenovirus-mediated SMS2 overexpressed mice have a stronger potential for aggregation after sphingomyelinase treatment.31
It is interesting that there were no effects on cholesterol levels in our animals. It is known that cholesterol levels in membranes paralleled SM levels. It is expected that decreasing SM levels would influence cholesterol levels, but this phenominon was not observed in this study. Previously, we also found that inhibition of SM de novo
synthesis does not influence plasma cholesterol levels9
and that decreasing membrane SM levels does not always accompany with decreasing cholesterol levels.11
There are other mechanisms other than cholesterol-binding govern SM levels on the membrane and in the circulation.
The relationship between SMS2 activity and apoE in the plasma is unexpected. In this study, we found that SMS2 deficiency increases plasma apoE (Supplement Figs. VIA and B
), while SMS2 overexpression in the liver decreases it (Supplement Figs. VIC and D
). It is very likely that an SM increase or decrease alters the structure of the cellular membranes. Lipid rafts and caveolae are a subset of membrane microdomains that are enriched with SM, cholesterol, and glycosphingolipids.32
The changes of SM levels in such microdomains might thereby influence the conformation of nascent apoE associated with these structures. It has been reported that apoE expression leads to increases in the secretion of SM, which is colocalized with apoE-enriched lipoproteins, suggesting the importance of the SM-containing membranes.33
The SM content of the Golgi is in rapid equilibrium with SM in the plasma membranes, and SM-enriched microdomains have been described in the Golgi membranes.34
Indeed, we found that SMS2 KO cells have lower SM levels in the plasma membranes, while SMS2LTg hepatocytes have higher ones (), suggesting that SMS2 activity might be related to liver apoE secretion. Another possibility is that changes in levels of ceramide (a substrate of SMS) may also have an impact on plasma apoE, since ceramide is a known second messenger,35
and one of the components of large lipid rafts.36
Increased ceramide in the SMS2 KO liver () might be related to increased apoE secretion, while decreased ceramide in SMS2LTg liver () might be related to the opposite. However, this possibility may be quite illusory. It has been reported that inhibiting the degradation of cellular ceramide, or supplementing exogenous ceramide to the macrophages, decreases rather than increases apoE secretion from the macrophages.33
Of course, the mechanism of apoE secretion from the macrophages may be different from that of the hepatocytes. The details of this mechanism deserve further investigation.
Plasma from SMS2 deficient mice significantly enhanced cholesterol efflux from the macrophages, while that from SMS2LTg animals significantly reduced it, compared with their respective controls (). This observation could have important implications for the vessel wall homeostatic response to atherogenic insult. There are two possibilities that might explain this phenomenon. ApoE is a mediator of cholesterol efflux28
which plays an important role in reverse cholesterol transport.37
Therefore, changes in plasma ApoE may account for the differences in cholesterol efflux. Alternatively, changes in plasma SM may be the explanation. SM-depletion in tissue culture medium can prevent apoA-1 mediated cholesterol efflux from CHO cells, while SM supplement promote the efflux from these cells.38
Although we observed opposite in macrophage cholesterol efflux (), one thing seems to be clear that SM levels in microenvironment can influence cholesterol efflux and different cells have different outcomes.
SMS2 deficiency also has impact on other sphingolipid levels, including three important second messengers, ceramide, sphingosine, and sphingosine-1-phosphate (). The metabolism of these lipids is tightly linked. While we are considering the effect of SM on atherosclerosis, we should not ignore the potential impact of these bioactive lipids. Indeed, pharmacological inhibition of SM de novo
synthesis, not only decrease SM levels, but also decrease ceramide, sphingosine, and sphingosine-1-phosphate.9
These changes might contribute to the reduction of atherosclerosis observed in apoE KO mice. 9
In summary, SMS2 deficiency has antiatherogenic properties, while SMS2 liver overexpression has very much the opposite effect. We therefore believe that regulation of liver SMS2 activity could become a promising treatment for atherosclerosis.