Modified forms of low density lipoprotein and atherosclerosis

Atherosclerosis, 98 (1993) 1-9 0 1993 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved. 0021-9150/93/$06.00

Printed and Published in Ireland

ATHERO 04945

Review Article and Viewpoint

Modified forms of low density lipoprotein and atherosclerosis Michael

Aviram

Lipid Research Laboratory, Rambam Medical Center, Rappaport Family Institute for Research in the Medical Sciences, Technion Faculty of Medicine, Haifa (Israel)

Summary

Modified forms of low density lipoprotein (LDL) are associated with increased atherogenicity. Modified LDL, in comparison with native LDL, demonstrates enhanced cellular uptake by macrophages, foam cell formation and also causes the secretion of cytokines and growth factors from arterial wall cells. Nonenzymatic modifications of LDL (proteoglycans, glycosylation, immune complexes) and enzymatic modifications (lipases, oxygenases) were shown to affect the physicochemical (size, charge) as well as the biological (cellular uptake, secretion) properties of the lipoprotein, Of special interest is the oxidative modification of LDL which was demonstrated to occur in vivo. The mechanism of this process involves cellular lipid peroxidation and requires the binding of LDL to its receptor on macrophages. Some of the modifications can render the LDL more susceptible to other types of modifications (lipid modifications, aggregation, oxidation). As atherosclerosis is a multifactorial disease and since lipases and oxygenases exist in cells of the arterial wall, several forms of modified LDL may exist in vivo. These modifications can occur either in parallel or along different stages of atherogenesis. Inhibition of such LDL modifications may arrest the development of the atherosclerotic lesion.

Key words: Modified LDL; Oxidized LDL; Macrophages; Phospholipases; Lipases; Atherosclerosis

Enzymatic and non-enzymatic modifications of LDL

Correspondence

to: Michael Aviram D.Sc., Lipid Research

Laboratory, Rambam Medical Center, Rappaport Family Institute for Research in the Medical Sciences, Technion Faculty of Medicine, Haifa, Israel.

During the last decade, it has been demonstrated that low density lipoprotein (LDL), the major cholesterol carrier in human plasma, can be modified by enzymatic as well as by nonenzymatic processes [ 1,2] and the atherogenicity of

2

have resulted from the action of neuraminidase on LDL) was isolated from the plasma of patients with coronary heart disease and was shown to induce foam cell formation in vitro [13]. Glycosylation of the LDL apolipoprotein B-100 may also play an important role in cellular lipid accumulation 1141. Although various forms of modified LDL have

LDL could be attributed to these modifications t31. The modifications of LDL could occur in vivo following interactions(s) of the lipoprotein with cells of the arterial wall [4-61, blood cells [7-101, plasma constituents such as immune complexes Ill] and also components of the arterial wall matrix [12]. A sialic acid-poor LDL (that may

CARBOHYDRATES ------ APOLIPOPROTEIN B-100

PHOSPHOLIPIDS

CHOLESTERYL

LINOLEATE PL- OOH CE-OOH

TRIGLYCERIDES CH2 O&OR,

I CI+O*

/ COR2

@

I

---F

C%0+COR3

Cholestenone

7-ketocholesterol

TG-OOH /

5,~ epoxycholesterol

Fig. I, Enzymatic modifications of LDL lipid constituents. CEase, cholesterol esterase; LPL, lipoprotein lipase; HL, hepatic lipase; PLase, phospholipase; LPO, lipoxygenase; CO, cholesterol oxidase.

3

been described, the in vivo relevance of most of these modified forms of LDL is not clear yet. The most studied form of modified LDL, the acetyl LDL (AC-LDL), cannot be formed under in vivo conditions [ 1.51. Thus, studies were designed to identify other forms of modified LDL that may occur in vivo. Cells of the arterial wall were shown to contain lipoprotein lipase (LPL), cholesterol esterase (CEase), phospholipase (PLase) AZ, PLase C, PLase D and also lipoxygenases (LPO). Enzymatic modifications of the lipid moieties of LDL might take place in the arterial wall [16-231. Figure 1 demonstrates various enzymatic LDL-lipid modifications and points to the site of action of each enzyme. LDL cholesteryl ester (CE), phospholipids and triglycerides (TG) are subjected to hydrolysis of their fatty acids and the formation

TABLE

of new products. Hydrolysis of the LDL-CE enriches the lipoprotein with unesterified cholesterol whereas TG hydrolysis results in the formation of diglycerides, monoglycerides and glycerol. PLase A2 produce lysolecithin whereas PLase C action on the LDL phospholipids results in the formation of inositol phosphate and diacylglycerol. PLase D release the alcohol polar group of the phospholipids and produce phosphatidic acid on the surface of the lipoprotein. The action of lipoxygenases can produce hydroperoxides from the fatty acids of the LDL CE, PL and TG moieties following lipolysis of these moieties and the release of free fatty acids. The cholesterol moiety of LDL on its surface as well as on its core CE is also subjected to oxidative modifications. Enzymatic action of cholesterol oxidase (CO) on the LDL unesterified cholesterol was recently

I

PROPERTIES

OF ENZYMATICALLY

MODIFIED

The modified LPL/HL Size Negative charge CE content UC content TG content PL content FFA content Vitamin E content Heparin binding TNBS reactivity Apo B-100 fragmentation Susceptiblity to oxidation Imrnunoreactivity with mAb BlB6 Degradation in tibroblasts Degradation in macrophages

N N N N N + N N + N N

LDL

enzyme CEase

PLase A,

PLase C

PLase D

N + N N N

+ + N N N

+ N N N

N N N N.D. N

_ _

N

+ N.D. N N.D. N

N

N N N N N N

N

N

+

+

+

N.D.

+

N

+

+

+

+

+

+

N + N N + N

+ + +

+

N

co N N N _

LPL, lipoprotein lipase; HL, hepatic lipase; CEase, cholesterol esterase; PLase, phospholipase, CO, cholesterol oxidase; +, increased; -, decreased; N, normal (like native LDL); N.D., not determined; CE, cholesteryl ester; UC, unesterilied cholesterol; TG, triglycerides; PL, phospholipids; FFA, free fatty acids; TNBS, trinitrobenzensulfonic acid; mAb BlB6, monoclonal antibody towards the LDL receptor binding domain on the LDL apo B-100.

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shown to result in the formation of atherogenic cholestenone-rich LDL [24]. A list of the properties of the enzymatically modified LDLs is given in Table 1. Non-enzymatic modifications of LDL include its binding to immune complexes [ll], proteoglycans [12] and carbohydrates [25], and also chemical derivatization of the lipoprotein [ 151. The physicochemical changes in the modified forms of LDL involve alterations in the LDL lipids composition, as well as in the lipoprotein conformation, configuration and sometimes also particle aggregation. LDL isolated from atherosclerotic lesions was shown to have negative charge, fragmentation of its apo-B-100 and reduced content of its polyunsaturated fatty acids, and it was shown to cholesterol-load macrophages [26]. Macrophage involvement in the atherosclerotic process consists of damaging potential (proteases, oxygen radicals), scavenging functions (chemotaxis, uptake of modified LDL) and secretory functions (growth factor, cytokines). Endothelial cells and smooth muscle cells also play very important roles in atherogenesis. Moditications of LDL by cells of the arterial wall including macrophages, endothelial cells (EC) and smooth muscle cells (SMC), involve both enzymatic and non-enzymatic actions [4-61.

MACROPHAGE

Oxidation of LDL The contribution of the lipid peroxidation to the pathogenesis of atherosclerosis is based on: (1) the finding of oxidized LDL and lipid peroxides in areas of the atherosclerotic plaque [27,28]; (2) the increased susceptibility of LDL from atherosclerotic patients to undergo lipid peroxidation; (3) the anti atherogenicity of antioxidant therapy. The early lesion in human atherosclerosis is composed of monocyte-derived macrophages which accumulate cholesterol which is originated mostly from plasma LDL [ 151.This native LDL has to undergo some modification in order to contribute to macrophage cholesterol accumulation in vitro [l-3]. Whereas macrophages predominate in the early lesion, in the advanced atherosclerotic plaque, arterial smooth muscle cells play a major role by their proliferation, migration and cholesterol accumulation. A role for oxidation in LDL modification was shown in early studies on the cytotoxicity of LDL to vascular SMC [29] and on endothelial cell modification of the lipoprotein [30,31]. Furthermore, evidence was shown of the inhibition of LDL oxidation and for the regression of atherosclerosis by the antioxidant effect of probucol [32,33]. Arterial wall cells, including macrophages, EC

- MEDIATED LDL OXIDATION

(1)

Macrophage Fig. 2. Requirement for LDL binding to its cellular receptor (LDL-R) for macrophage-mediated binding. (2) Cell-mediated oxidation of LDL. (3) Production of oxidized LDL. (4) Macrophage receptor (S-R).

LDL lipid peroxidation. (1) LDL uptake of Ox-LDL via the scavenger

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and SMC can mediate the lipid peroxidation of LDL [4-61. The mechanism of cell-mediated oxidation of LDL, however, is not yet clear. Macrophages take up oxidized LDL (Ox-LDL) via specific scavenger receptors [34-361 at enhanced rate, and this interaction can lead to cellular cholesterol accumulation and foam cell formation. Recently, we have found [37] that a specific binding of LDL to its receptor on macrophages is required in order to permit subsequent oxidation of the lipoprotein (Fig. 2). The oxidized LDL can then be taken up by macrophages to form foam cells. Receptor-dependent, cell-mediated oxidation of LDL may, however, be under low oxidative stress, a protective mechanism from the accumulation of extracellular oxidized LDL. This mechanism may suggest an additional role for the LDL receptor in promoting the rapid removal of plasma LDL. This is achieved by LDL conversion to OxLDL, which unlike native LDL is rapidly taken up via the macrophage scavenger receptor. The cellular uptake of the lipoprotein cholesterol will then block the synthesis of LDL receptors and thus a further formation of atherogenic Ox-LDL will be prevented. Ox-LDL also affect the properties of various other systems including the contractility of the arterial wall and the secretion of chemotactic and growth factors [38]. Oxidized LDL was shown to enhance platelet activation [39], and macrophage uptake of Ox-LDL was demonstrated to be enhanced by platelet secretory products [40-451. Blood coagulation and the thrombotic-tibrinolytic systems are also perturbed by Ox-LDL [46,47]. Under pathological conditions such as atherosclerosis [48], hypercholesterolemia [49], diabetes [50] and hypertension [51], the presence of abnormal LDL, with increased susceptibility to undergo lipid peroxidation was demonstrated. Hypolipidemic drug therapy was shown to be associated with reduced propensity of LDL to undergo such oxidation [52,53]. Similarly, both the synthetic and the natural isomers of pcarotene reinduced LDL oxidation [54]. On the other hand, LDL lipid modifications or its association with proteoglycans were shown to render the lipoprotein more susceptible to oxidative modification [12,22,24]. Oxidized LDL, in turn was shown to have increased tendency to aggregate [55]. Although evidence accumulates to suggest

the importance of the peroxidation of both LDL fatty acids and cholesterol in the pathogenesis of atherosclerosis, the other modifications of LDL are most likely to occur as well, either in parallel or in addition to lipoprotein oxidation. Some of the lipid peroxidation of LDL may occur in plasma but most of these changes probably take place in the injured arterial wall, where protective and antioxidant mechanisms are perturbed [56]. LDL oxidation involves the peroxidation of the polyunsaturated fatty acids on the surface phospholipids and in the core cholesteryl ester moieties of the lipoprotein. In addition to the LDL fatty acids, the cholesterol on the surface (unesteritied cholesterol) and in the core of the LDL molecule (cholesteryl ester) can be also oxidized. Selective oxidation of the fatty acids on different lipid moieties of LDL (phospholipids or cholesteryl ester) as well as selevtive cholesterol oxidation on the surface or the core of LDL may be operative under different conditions. Figure 3 summarizes the lipid peroxidation of LDL fatty acids and cholesterol moieties. Factors which affect the susceptibility of LDL to undergo lipid peroxidation [1,3,56] include the composition and location of its polyunsaturated fatty acids (linoleic acid is located mainly in the core CE of LDL whereas arachidonic acid is mainly on the LDL surface phospholipids) and its antioxidants content vitamin E, P-carotene, ubiquinol). Extrinsic factors such as the extracellular content of copper ions, vitamin E concentration, the arterial matrix and also the cellular oxidative systems (oxygenases, superoxides), also have important roles in the lipid peroxidation of LDL. Cellular cholesterol accumulation functions

and arterial wall

The role of modified lipoproteins in atherosclerosis was attributed mainly to their ability to cause macrophage cholesterol accumulation [l-3]. However, the ability of the macrophages to take up LDL cholesterol may serve at first as a protective mechanism against the accumulation of atherogenic modified lipoproteins in the plasma [l]. Only when the macrophages are massively

LDL OXIDATION

-FA PL

CHOL

, TG , CE

18:2

-

30%

, 6% , 64%

20:4

-

68%

, 7% , 25%

UC , CE

PUFA

J(

OX -

A

FA

oX-LDL \

OX-CHOL

7 -KETO FA -

HYDROPEROXIDES

ALDEHYDES

(MDA,HNE)

5,6

-

-

CHOLESTEROL

EPOXY-CHOLESTEROL

CHOLESTENON 7 -

OH-CHOLESTEROL

25-OH-CHOLESTEROL 3,5,6

-

CHOLESTEN

TRIOL

Fig. 3. Oxidation of the LDL fatty acids (FA) and cholesterol (Chol) moieties. The polyunsaturated fatty acids (PUFA) such as arachidonate (C20:4) and linoleate (C-18:2) on the lipoprotein phospholipids, triglycerides and cholesteyl ester (PL, TG, CE) can be oxidized and produce hydroperoxides and aldehydes. The cholesterol on both LDL surface (unesterified cholesterol) and in its core CE, can be also oxidize to several cholesterol oxide derivatives.

loaded with cholesterol, they may exert atherogenie properties [ 1,3]. This latter phase may involve the release of cellular cytokines, growth factors, proteases, elastase, collagenase and oxygen radicals, which contribute to the development of the atherosclerotic plaque [3,38]. The ability of cells to maintain their appropriate cholesterol requirements is dependent on the balance between cholesterol influx and efflux. Thus, in foam cell formation, the effect of modified lipoproteins on cellular cholesterol eMux may be as important as their role in macrophage cholesterol influx. In the advanced atherosclerotic lesion, cholesterol accumulates not only in macrophages, but also in arterial smooth muscle cell (SMC) and thus contributes to the formation of the complicated

fibrous plaque. Cellular cholesterol accumulation in the arterial wall affects the properties of cells [1,3,51. It is important thus to assess the biological effects of modified lipoproteins not only on cellular cholesterol accumulation, but also on other events which occur during atherogenesis, i.e. endothelial cell injury, chemotaxis, smooth muscle cell proliferation and the secretion of cytokines and growth factors by the arterial wall cells. While the lipid peroxidation of LDL is under intensive investigation [1,3], little is known about the peroxidation of the lipid constituents of cells in the arterial wall. Under certain conditions, the lipid components of these cells are prone to oxidative stress [5,57].

7

macrophage activation During atherogenesis, results in the production of oxygen reactive species [58], and SMC are also capable of producing free radicals and potent oxidants [5]. These oxidative stresses can contribute to the oxidation of the lipid constituents of the cells themselves. Stimulation of cellular enzymatic systems which are associated with lipid peroxidation, such as the NADPH oxidase, xanthine oxidase and lipoxygenase, may also contribute to cellular lipid peroxidation. The cellular uptake of oxidized LDL by macrophages or by SMC can possibly lead to a subsequent peroxidation of cellular lipids. Conclusion The complicated processes that occur during atherogenesis seem to involve the participation of more than one form of modified lipoprotein and include lipid-modified LDL, aggregated LDL and oxidized LDL. Different types of oxidative modifications of LDL may occur by the different cells of the arterial wall [58]. Furthermore, some LDL modifications can render the lipoprotein more susceptible to another type of modification. It is suggested to analyze the peroxidation products of fatty acid on the LDL phospholipids and on its cholesteryl ester as well as the various cholesterol oxide derivatives which are formed as a result of cholesterol oxidation on the surface (unesterified cholesterol) or in the core of the lipoprotein (cholesteryl ester). At early stages of atherosclerosis, macrophage-mediated oxidation of LDL is probably dominant, whereas at later stages, LDL oxidation by SMC may become more important. Thus, the mechanism and regulation of cell-mediated oxidation of LDL should be studied by using macrophages as well as SMC. Recently, it was shown that macrophage activation enhances the ability of the cells to take up LDL and even more so when these cells were loaded with cholesterol [59]. The effect of cellular cholesterol content and of cell activation on cell-mediated oxidation of LDL should be analyzed. The requirement for a specific cellular oxidative enzymatic system(s) is not clear yet. Understanding of the involvement of such enzymes in LDL oxidation may enable us to use some intervention means in order to control cell-mediated oxidation of LDL. It is important to study the effects of the modified forms of LDL not only on macrophages but also

on SMC cellular cholesterol metabolism, on cell proliferation and on their secretory properties. The possibility of macrophages being mediators of the effects of modified LDL on SMC functions should be also studied. Modified lipoproteins affect multiple functions of all major cells of the arterial wall. Studies of modified forms of LDL thus should concentrate not only on their effect on cellular cholesterol metabolism but also on other cellular functions. These functions include scavenging functions, damaging potential and secretory properties, all of which considerably affect the behaviour of cells in the arterial wall and the progression of the atherosclerotic process. Not only the lipid constituents of the LDL can undergo lipid peroxidation, but the cellular lipids in macrophages and in SMC can be also prone to lipid peroxidation under certain pathological conditions. Such oxidative stress may have an important effect on various cellular functions and studies should be designed to address this important issue. References Steinberg, D., Parthasarathy, S., Carew, T.E., Khoo, J.C. and Witztum, J.L., Beyond cholesterol: modification of low density lipoprotein that increase its atherogenicity, N. Engl. J. Med., 320 (1989) 915. Aviram, M., Effect of lipoproteins and platelets on macrophage cholesterol metabolism. In: Harris, J.R. (Ed.), Blood Cell Biochemistry, Vol. 2, Megakaryocytes, Platelets, Macrophages and Eosinophils, Plenum Publishing Co., N.Y., Chapter 7, 1991, pp. 179-208. Witztum, J.L. and Steinberg, D., Role of oxidized low density lipoprotein in atherogenesis, J. Clin. Invest., 88 (1991) 1785. Van Hinsberg, V.W.M., Marielle, S., Lewis, H. and Kempen, H., Role of endothelial cells and their products in the modification of LDL, Biochim. Biophys. Acta, 878 (1986) 49. Heinecke, J.W., Baker, L., Rosen, H. and Chait, A., Superoxide-mediated modification of low density hpoprotein by arterial smooth muscle cells, J. Clin. Invest., 77 (1986) 757. Leake, D.S. and Rankin, S., The oxidative modification of LDL by macrophages, Biochem. J., 270 (1990) 741. Aviram, M., Platelet-modified low-density lipoprotein: studies in normals and in patient with homozygous familial hypercholesterolemia, Clin. Biochem., 29 (1987) 91. Aviram, M., Fuhrman, B., Keidar, S.. Maor, I., Rosenblat, M., Dankner, G. and Brook, J.G., Plateletmodified low density lipoprotein induces macrophage cholesterol accumulation and platelet activation, J. Clin.

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