The pivotal role of lipoprotein lipase in atherosclerosis

Time for primary review 33 days. Atherosclerosis, the underlying cause of heart attacks, stroke and peripheral vascular disease, is responsible for over 50% of all deaths in developed countries. The disease can generally be viewed as a form of chronic inflammation that is induced and perturbed by lipid accumulation and involves a number of components, including the damaged endothelium, monocytes/macrophages, T cells, smooth muscle cells and a regulatory network of growth factors and cytokines (see Refs. 1,2 for reviews). The process is believed to be triggered by damage to the arterial endothelial cells leading to dramatic changes in their properties and increased expression of both chemokines and adhesion molecules 1,2. This causes an infiltration of both T lymphocytes and monocytes to the site of damage. The monocytes then differentiate into macrophages, internalise lipoproteins, and transform into lipid-loaded foam cells to form the fatty streak seen in early lesions 1,2. This transformation of macrophages into foam cells represents a critical initial event in the pathogenesis of atherosclerosis. It is, therefore, not surprising that a major focus in cardiovascular research has been devoted to understanding the molecular basis of foam cell formation and has resulted in the identification of a key, but complex, role of the enzyme lipoprotein lipase (LPL; EC 3.1.1.34) in the process. LPL is a central enzyme in overall lipid metabolism and transport, being responsible for catalysing the hydrolysis of triglycerides transported in the bloodstream by chylomicrons and VLDL, thereby providing non-esterified fatty acids and 2-monoacylglycerols for tissue utilization 3,4. Due to the large size of its substrates, the physiological site of LPL action is at the luminal surface of blood vessels, to which the enzyme is attached via highly charged heparan sulphate proteoglycans (HSPG) 3,4. Mature LPL is secreted to the vascular endothelium from the parenchymal cells of the adipose and muscle tissues, its major sites of synthesis, and a variety of other tissues which have also been implicated as lesser, but significant, sources of the enzyme 3,4. Of these alternative sources, studies on LPL expressed by cell types found in the locus of the vascular wall, particularly by monocyte-derived macrophages, have identified additional, pathophysiological actions of the enzyme that promote foam cell formation and, ultimately, atherosclerosis. In direct contrast to this, LPL produced by adipose tissue and muscle has a protective effect with respect to atherosclerosis. As a result of these observations, LPL has been identified as a potential therapeutic target and continues to be the subject of extensive scientific investigation. In this comprehensive and current review, the most recent evidence implicating LPL in both its pro- and anti-atherogenic roles will be discussed. In addition, data regarding the regulation of the enzyme by factors involved in atherosclerosis will be covered. The importance of LPL in the atherosclerotic process was first proposed in 1973 by Donald Zilversmit 5. He postulated that the action of LPL on circulating VLDL and chylomicrons would lead to high, localized concentrations of cholesterol-rich remnants, which would subsequently be taken up into the arterial wall and propagate lesion formation. Since this hypothesis was formulated, it has been shown to be true but, as this article will discuss, is far more complex than originally anticipated. Of the cell types present in the atherosclerotic lesion, LPL is known to be expressed by both monocyte-derived macrophages and smooth muscle cells 3,4. The differentiation of monocytes to macrophages is accompanied by a substantial increase in LPL mRNA, protein and activity levels 6 and has also recently been shown to lead to an increase in the level of apolipoprotein C-II 7, a known activator of LPL 3. These macrophages transform into foam cells through the accumulation of lipids 1,2. Detailed immunocytochemical and in situ hybridization experiments have shown that it is the macrophage-derived foam cells, and not smooth muscle cells, that are the primary source of LPL in the lesion 8. In addition, the expression of LPL mRNA, protein and enzymatic activity have all been found to increase in response to damage sustained by the arterial wall 9,10. Vascular injury has long been known to be a precursor of clinical conditions associated with atherosclerosis and is, indeed, how the well-established ‘response to injury’ hypothesis for atherogenesis was derived 1,2. The possibility that variations in macrophage LPL synthesis and secretion might constitute a hereditary component of atherosclerosis was investigated by Renier and colleagues 11 who evaluated LPL levels in macrophages from inbred mouse strains differing in their susceptibility to diet-induced atherosclerosis. It was observed that macrophages isolated from mice susceptible to atherosclerosis showed a 2- to 3-fold higher basal LPL mass, activity and mRNA levels than those from mice resistant to it, thereby inferring a contributive role for LPL in the progression of the disease. More recently, Semenkovitch et al. 12 have shown that although feeding an atherogenic diet to heterozygous LPL deficient mice results in profound dyslipidaemia, due to an increase in non-HDL lipoproteins, no differences were seen in atherosclerotic lesion area when compared to normal mice fed the same diet. It therefore appears that the decreased presence of LPL in the vascular wall might have conferred a protective effect against the highly atherogenic lipoprotein profile 12. The LPL mRNA, mass and activity levels have also been found to be substantially higher in monocyte-derived macrophages from diabetic patients when compared to those from normal subjects, and such an increase can be conferred to normal macrophages by culturing them in serum obtained from diabetic patients 13. Given that atherosclerosis is a major complication of diabetes, this observation provides further evidence for an association of LPL with atherogenesis. The most compelling data concerning the relationship between macrophage LPL and atherosclerosis has come from several recent transplantation studies in which the expression of LPL in macrophages was modulated 14–16. Such transplantation approaches were necessary because homozygous LPL deficient mice die soon after birth due to the accumulation of chylomicrons, a particular problem in the lungs where they prevent the contact of erythrocytes with the endothelium 17. Babaev et al. 14 initially transplanted irradiated female mice with foetal liver cells, the predominant site of hematopoiesis in mammalian embryogenesis, from mice with LPL−/−, LPL+/− and LPL+/+ genotypes whereas Van Eck et al. 15 carried out bone marrow transplantation from LPL−/− and LPL+/+ mice. In both cases, the mice were fed an identical atherogenic diet for several weeks and their atherosclerotic lesion areas were measured. Similar reductions, of around 50%, were seen in both studies in mice that are deficient for macrophage LPL, thereby indicating that this source of enzyme directly leads to atherosclerosis in the setting of a pro-atherogenic diet. It is also interesting to note that Van Eck and co-workers 15 observed a 2-fold reduction in apolipoprotein E (apoE) levels in macrophage LPL−/− mice. Macrophage-derived apoE has been shown to have a preventative effect upon atherosclerosis by promoting cholesterol efflux from the cells 18. That macrophage LPL knockout still has such a significantly protective effect upon atherosclerosis in the pro-atherogenic environment induced by reduction of apoE expression provides further indication of the central role played by the enzyme in the promotion of atherosclerosis. Both of the studies identified above 15,16 were carried out in C57BL/6 mice, a strain that has previously been shown to have limitations as a model for human atherosclerosis 19. When fed a pro-atherogenic diet, these mice develop only relatively mild fatty streak lesions, which are exclusively located in the proximal aorta 19. However, C57BL/6 mice that are homozygous negative for LDL receptor (LDLR−/−) display an enhanced susceptibility to diet-induced atherosclerosis throughout the length of the aorta 20. In addition, the extent of atherosclerosis in these mice can be modulated by altering the duration of the high fat diet used to generate the disease process, thus providing the framework for studies at different stages of lesion formation 20. Using this improved system, Babaev and co-workers used their same experimental approach, as previously described 14, to create macrophage LPL chimeras 16. They found that LDLR−/− mice reconstituted with LPL−/− macrophages developed significantly less atherosclerosis when challenged with an atherosclerotic diet for either 8 weeks (early stage atherosclerosis) or 19 weeks (later stage atherosclerosis) compared to those mice with LPL+/+ or LPL+/− macrophages, as determined by en face analysis of pinned-out distal aortae. However, when cross-sectional analysis of the proximal aorta, the region most susceptible to atherosclerosis, was carried out (as used in their earlier study) 14, similar results to those obtained using the en face procedure were seen at 8 weeks but no differences between genotypes were observed at 19 weeks, a time point when the lesions are extremely complex in this region. It was therefore concluded that although macrophage LPL expression undoubtedly promotes foam cell formation and atherosclerosis in vivo, its impact is limited to the macrophage-rich early lesions as opposed to more complex, advanced lesions. More recently, Clee et al. 21 have carried out studies to distinguish between the effects of plasma LPL (i.e. enzyme bound to the endothelial cells and derived predominantly from the adipose tissue and muscle) and/or vessel wall LPL (i.e. enzyme derived mainly from macrophages) on atherosclerosis susceptibility in apoE-deficient mice. They showed that decreased LPL expression from both sources (LPL+/− E−/−) was associated with a reduction in atherosclerotic lesion area compared to the LPL+/+ E−/− mice despite a pro-atherogenic lipid profile. Thus, the loss of the macrophage-derived LPL protein in the vessel wall had a dominant effect on limiting atherogenesis because a reduction was seen even in the presence of an atherogenic lipid profile caused by low plasma LPL activity. Overall, therefore, the use of the modern transgenic technologies, detailed above, have given greater credence to several previous in vitro studies (reviewed in Refs. 22,23), and firmly indicate that, within the setting of an atherogenic diet, macrophage LPL expression promotes foam cell formation. However, the involvement of the enzyme in the latter stages of the disease, characterised by complex lesions, needs to be resolved. In addition to the analysis of the relationship between elevated LPL expression and atherosclerosis, the precise mechanisms through which the enzyme causes macrophage lipid uptake and foam cell formation has also been the subject of intense research in the last few years. The findings from these studies form the focus of the next section. LPL bound to the luminal surface of the vascular endothelium is responsible for mediating the lipolysis of circulating VLDL and chylomicron particles, thereby leading to both a decrease in their size and enrichment in their cholesteryl ester content 24. Studies in vitro have shown that such remnants are readily taken up by macrophages 25. In addition to this, the free fatty acids produced by the action of LPL can be re-esterified by macrophages 26. The net outcome of these processes is the accumulation of cholesteryl esters within macrophages and, as a consequence, their transformation into foam cells 25,26. LPL-mediated hydrolysis of VLDL also leads to the production of LDL, arguably the major contributor to the development of atherosclerotic lesions 1,2. Frequently, these LDL molecules are oxidized in the intimal space by free radicals which, along with other modifications, increases their rate of uptake into macrophages through the scavenger receptors, and thereby promotes further foam cell formation 1,2. In vitro studies carried out in the last few years have also indicated the possibility that the enzymatic action of LPL may affect other cell types present in the atherosclerotic lesion, namely endothelial and smooth muscle cells. For example, Hennig and co-workers 27 have demonstrated that selected fatty acids and LPL-derived remnants of lipoproteins isolated from hypertriglyceridaemic subjects can activate vascular endothelial cells and disrupt endothelial integrity. As mentioned earlier, vascular injury is thought to be a principal cause of the initial recruitment of macrophages to the arterial wall and is, therefore, a probable initiating event in atherosclerosis. In addition, LPL enzyme activity has also been shown to lead to the proliferation of vascular smooth muscle cells 28. Such cells are known to secrete that promote the formation of a of the stages of atherogenesis of the above how the actions of LPL affect the progression of atherosclerosis. of LPL is its to as a pro-atherogenic of its a that is The of LPL to uptake of lipoproteins of its properties was initially proposed in the observation that LPL molecules associated with chylomicrons after hydrolysis and, therefore, might in their uptake in this hypothesis was not when and colleagues showed that LPL was a for the LDL protein studies have not only identified other for LPL, but have also demonstrated that the enzyme with lipoproteins and promotes their to LDL VLDL and apoE receptor (see Refs. for reviews). The net result of all the between lipoproteins and LPL or cell surface is the and accumulation of lipoproteins in the arterial and their uptake by cells. are potential by which LPL may uptake of The first is receptor uptake of lipoproteins bound to the cell surface via LPL LPL may directly as a for and, the complex may be by a of along with the uptake of potential of LPL, would promote the transformation of macrophages into foam cells. other of the involvement of LPL, in a in the atherosclerotic process have also been investigated in the last few years and are LPL has been found to substantially the uptake and of LDL, in endothelial cells and macrophages of LDL is a of its apolipoprotein by and is, therefore, more in diabetic patients Given that diabetic patients have macrophages that higher levels of LPL and that they are more to LDL, it can be proposed that higher levels of this atherogenic lipoprotein may in their than those from normal subjects, and may therefore the increased of atherosclerosis in diabetic In addition to mediating the of lipoproteins to on cell LPL can also the same with vascular proteoglycans In atherosclerotic lesions, predominant proteoglycans are present in areas in smooth muscle cells, and which is in the to the sites of macrophage infiltration et al. have recently demonstrated that the of both oxidized and LDL to and is by the presence of from in the have been found to LDL this environment using and co-workers have shown that LPL increases the of both and oxidized Due to the that are large of in the arterial and that proteoglycans substantially the of the atherosclerotic process, is potential for LPL to a accumulation of LDL and oxidized LDL on these These particularly when the recent of and is They have shown that macrophage uptake of oxidized LDL bound to macrophage-derived is upon the presence of these studies indicate that LPL is not only responsible for the accumulation of atherogenic lipoproteins, but is a in their uptake by macrophages as is known to be present in atherosclerotic lesions and has the to the of and oxidized LDL to the and VLDL to the et al. have shown that in mouse macrophages, and LPL can increase the and uptake of and oxidized LDL, and the of cholesterol derived from for this the of to increase the of and oxidized LDL to LPL and to the of LPL It is that with and for the of LPL It therefore that when is present in the cell surface be and, as a consequence, be less lipoprotein accumulation in an in in has shown that the of oxidized LDL This hypothesis was further recently using a for clinical use in the of atherosclerosis. and co-workers found that, as the LPL into In addition, their in vitro showed that LPL bound to produced by both endothelial cells and the monocyte-derived The of LDL to LPL and its uptake was also by this thereby indicating that of the action of LPL a potential for an anti-atherogenic of the evidence above how LPL as a molecular due to its to to lipoproteins and via The proposed of LPL has been by tissue studies (see Refs. above and and has also been in further on the involves cells than lipoproteins as LPL has been shown to as a adhesion protein by a between the arterial and surface As mentioned earlier, a with respect to LPL expression and atherosclerosis. As as being responsible for atherogenic in the arterial wall, it has also been shown to a anti-atherogenic as by a variety of experimental For decreased adipose tissue LPL activity has been implicated to be at in for the that is associated with and diabetes, which then a major to the atherosclerosis in these The of LPL expression in these clinical conditions is believed to be by cytokines that are present at high such as and In addition, studies on patients in the have identified a between LPL activity and of Of the patients in the LPL activity of However, this was significantly in the LPL activity a recent clinical in has also shown levels of serum LPL mass in patients with atherosclerosis compared to those in several homozygous patients have been found to develop relatively advanced atherosclerosis a number of heterozygous LPL have been identified that are associated with a pro-atherogenic lipid profile and an increased for cardiovascular disease (see Refs. for recent reviews). This the and that at of up to and are characterised by a reduction in LPL enzymatic activity the which LPL activity by the of the factors and has been found to be associated patients with mild in plasma levels and heart disease a which is by increased LPL activity and has a of is associated with an anti-atherogenic lipid profile and a of heart disease It is well-established that lipolysis of lipoproteins in muscle and adipose tissue generally the circulating lipoprotein profile in a Thus, in contrast to macrophage LPL, the enzyme derived from the parenchymal cells of adipose tissue and muscle an Clee et al. 21 have recently shown that transgenic mice with increased plasma LPL but with no changes in the expression of the enzyme in macrophages, and thus the vessel wall, have atherogenesis. of LPL is highly in the atherosclerotic lipoprotein of both apoE-deficient and LDL mice and mice against diet-induced Of particular importance are the levels of which as a for it from areas of production to the liver for into the cholesterol In addition, has been shown to prevent the of LDL by characterised by high levels are associated with decreased atherosclerosis plasma LPL activity has also been directly to levels in with a heterozygous LPL associated with levels of the which increases tissue LPL against atherosclerosis via of levels of the have recently been in several large clinical and found to cause an reduction in This was also accompanied by a reduction in both the and have been shown to LPL activity in adipose tissue and heart as as plasma levels of apolipoprotein an LPL Similar effects upon LPL have also been by a of the which have in atherosclerosis The increased hydrolysis of triglycerides in such may result in the of lipids and to and thereby their The regulation of macrophage LPL by factors that have been implicated in atherosclerosis is to a major to the and the progression of the disease. It is therefore not surprising that this has been the subject of intense research in several including the regulation of LPL expression in the muscle and the adipose tissue has been investigated in the of the anti-atherogenic potential of LPL produced by these tissues respect to macrophage LPL, an has been found in several between the role of the in atherogenesis and its action on LPL For example, of free fatty acids growth and macrophage all increase macrophage LPL expression The pro-atherogenic role of all these factors is For example, peripheral factors in diabetes, including and free fatty are known to to the high of atherosclerosis in diabetic patients represents an for atherosclerosis are also present at high levels in the atherosclerotic lesion and have been implicated in the of LDL and the production of several other pro-atherogenic factors has also been found to be present in the lesion and mice the are less susceptible to atherosclerosis In with the complex of atherosclerosis, a more for several other For macrophage LPL expression is by a of and oxidized LDL and that are known to be produced LDL including and As oxidized LDL, lipids and cytokines are to be the precise of their LPL anti-atherogenic action However, as atherosclerosis is initially a protective it is that these factors may the transformation of macrophages into foam cells the early stages of vascular damage their actions are by changes in the and of LPL in the of the progression of the disease. The expression of LPL in the muscle and the adipose tissue is by and a of including growth factors and lipid a in several between the atherogenic potential of the and its action on LPL For example, the and the which display and the expression of the LPL through The evidence in this review a pro-atherogenic role of macrophage-derived LPL whereas the enzyme expressed by the muscle and the adipose tissue in an anti-atherogenic by the circulating lipoprotein profile. therapeutic approaches therefore to decrease macrophage LPL expression its action in adipose tissue and represents a potential to such and it is that more research on this is carried It is, interesting to note that studies on the of LPL expression have identified several of For example, directly LPL expression in macrophages but not In addition, LPL mRNA levels in the liver but not the adipose tissue whereas have no effect in the liver but on the adipose tissue Such is to to other because such have atherosclerosis whereas of LPL expression by these would be to increase the through their action on the enzyme expressed by of diabetic with has been shown to LPL activity in and but not in adipose tissue More recently, mice that have been fed high cholesterol or liver have been found to a increase in LPL expression in the liver and macrophages but not in other tissues adipose and muscle) It is therefore that through more research on the regulation of LPL particularly the and the factors that are involved in the process, be developed that LPL expressed by adipose and recent studies have also identified additional, roles for For example, as LPL has been found to the proliferation of smooth muscle cells a atherogenic In addition, LPL the expression of the in macrophages via a that is through cell surface proteoglycans and protein LPL also the secretion of apoE in macrophages and with in the of macrophage expression Given the importance of apoE and in it is that more research is carried out in to the of such a role of LPL and the mechanisms of In the findings from research carried out in the last few years have not only the role of LPL in atherogenesis but the potential of the enzyme expressed by macrophages and adipose in the disease. Thus, the enzyme expressed by the adipose tissue and muscle is generally as anti-atherogenic whereas that produced by macrophages is to be In addition, an has been found on the action of several factors on LPL expression and their role in atherosclerosis and growth studies to a detailed understanding of the regulation of LPL expression and develop that be used to its action in a The in the was by research from the