Oxidation of chylomicron remnants and vascular dysfunction

Atherosclerosis Supplements 9 (2008) 57–61

Oxidation of chylomicron remnants and vascular dysfunction Kathleen M. Botham ∗ Department of Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London NW1 0TU, UK Received 11 January 2008; received in revised form 26 February 2008; accepted 13 May 2008

Abstract Recent evidence suggests that chylomicron remnants (CMRs), the lipoproteins which carry dietary lipids in the blood, may play a direct role in the initiation of atherosclerosis by influencing vascular function. Unlike low-density lipoprotein (LDL), CMR do not require prior oxidation to bring about potentially pro-atherogenic effects on vascular endothelial cell function and macrophage foam cell formation. However, CMR carry oxidized lipids from the diet and may also become oxidized in the body, thus it is important to establish how the oxidative state of the particles may modulate these effects. Pharmacological studies have demonstrated that oxidation of CMR significantly enhances their inhibitory effects on endothelium-dependent vascular relaxation and their potentiation of vasoconstriction in rat and pig arteries. In striking contrast to the effects of LDL oxidation, however, the induction of macrophage foam cell formation has been found to be inversely related to the oxidative state of CMR. Thus, oxidation of CMR has potentially pro-atherogenic effects on endothelial function, but appears to protect against foam cell generation. These findings indicate that the oxidative state of CMR may cause important changes in the atherogenicity of the particles. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Oxidized chylomicron remnants; Endothelium-dependent vascular dysfunction; Macrophage foam cell formation

1. Introduction Atherosclerotic lesion development begins with vascular endothelial dysfunction and the accumulation of lipid engorged macrophage foam cells in the artery wall causing the formation of fatty streaks, the first visible lesions in blood vessels [1,2]. Low-density lipoprotein (LDL) is known to play a major role in these initiating events in atherogenesis [3], but there is now a great deal of evidence to indicate that chylomicron remnants (CMRs), the lipoproteins which carry lipids from the diet from the gut to the liver, also have potentially atherogenic effects on these processes [4]. Although LDL is strongly implicated in atherosclerosis development, extensive studies have established that oxidation of the particles, a process which can occur in the artery wall by the action of cell-associated lipoxygenase and/or myeloperoxidase, is required for many of its effects [3]. In contrast, CMR have been demonstrated to cause vascular endothelial dysfunction and induce macrophage foam cell formation without prior oxidation. However, like LDL, CMR ∗

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are taken up and retained by the artery wall [5] and thus they may be oxidized in a similar way. In addition, it has been shown that oxidized lipids from the diet, including peroxidized fatty acids and oxysterols formed when fats are cooked at high temperatures, are absorbed and transported in CMR [6]. Thus, oxidized CMR (oxCMR), are likely to be present in the circulation and in the blood vessel wall. In addition, the CMR may be protected from oxidation by lipophilic antioxidants, such as plant cartenoids or vitamins present in the diet. It is clearly important for the understanding of the atherogenicity of CMR, therefore, to establish how their oxidation influences their effects on the vasculature. This article will review current knowledge concerning the effects of the oxidative state of CMR on their interactions with endothelial cells and macrophages, and how this may influence the events initiating atherosclerosis.

2. Oxidized chylomicron remnants and endothelial function Maintaining the balance between vasodilation and vasoconstriction is a major function of the endothelium [2].

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K.M. Botham / Atherosclerosis Supplements 9 (2008) 57–61

Table 1 The effects of CMR on endothelial function in rat and pig arteries Experiment

CMR

oxCMR

Control

+CMR

Control

+oxCMR

Perfused rat aorta rings Contraction to PE Relaxation to CCh Relaxation to SNAP

0.32 ± 0.05(6) 88.7 ± 2.6(6) 99.8 ± 0.6 (6)

0.37 ± 0.04(6) 80.2 ± 5.7(6) 100.9 ± 0.9(6)

0.34 ± 0.55(6) 91.6 ± 2.4(6) 101.3 ± 2.0(6)

0.51 ± 0.04(6)* 71.5 ± 7.2(6)* 101.2 ± 1.8(6)

Rat aorta rings Contraction to PE Relaxation to CCh Relaxation to SNAP

0.36 ± 0.04(6) 85.2 ± 5.7(6) 103.4 ± 0.8(6)

0.42 ± 0.05(6) 62.5 ± 7.7(6)* 103.5 ± 0.9(6)

0.44 ± 0.55(6) ND ND

0.59 ± 0.07(6)* ND ND

Pig coronary artery rings Relaxation to BK Relaxation to 5-HT Relaxation to SNAP

108.6 ± 4.1(5) 7.9 ± 1.2(6) ND

110.2 ± 7.9(5) 2.4 ± 0.6(6)* ND

91.3 ± 4.2(5) ND 91.5 ± 3.8(5)

79.4 ± 6.7(5)* ND 99.7 ± 2.9(5)

Ring segments from rat aortas (rat aorta rings) or pig coronary arteries (pig coronary artery rings) were incubated with CMR or oxCMR (16–20 ␮M cholesterol, 45 min) and concentration contraction or relaxation responses to phenylephrine (PE), bradykinin (BK), 5-hydroxytryptamine (5-HT), carbachol (CCh) or the nitric oxide donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) were determined. In perfusion experiments (perfused rat aorta rings), ring segments were taken from rat aortas after perfusion with CMR or oxCMR (0.48 ␮mol cholesterol, 2 h) and concentration contraction or relaxation responses to PE, CCh or SNAP were measured in the absence of CMR in the organ baths. Data shown are the mean ± S.E.M. from the number of experiments shown in parentheses, and are expressed as maximum tension (g/ml) (contraction) or % maximum relaxation after contraction with 3 ␮M PE (rat aorta) or 10–30 nM U44069 (pig coronary artery); ND: not determined. * P < 0.05 vs. corresponding control.

These processes are known to be modulated in the development of atherosclerosis, and oxidized LDL (oxLDL) is believed to play a part in the observed effects [7]. However, since dietary fats have been shown to influence endothelial function [8], we and others have evaluated the effects of CMR. In organ bath experiments in which rat aorta rings were suspended between parallel wires and the wall tension continuously monitored, we found that agonist-induced vasodilation is inhibited by CMR (Table 1), and that this change is abolished by the nitric oxide (NO) donor S-nitrosoN-acetyl-d,l-penicillamine (SNAP) [9] indicating that it is mediated by endothelium-derived NO production, and Doi et al. [10] have reported similar results with remnant lipoproteins from hyperlipidemic subjects using rabbit aortas. Our studies also demonstrated this effect following perfusion of the rat aorta ex vivo with CMR even when the lipid particles were absent from the organ bath solution (Table 1) [11]. Oxidation of CMR significantly enhanced their effects on endothelium-dependent vasorelaxation and also potentiated the vasoconstrictor response to phenylephrine (Table 1) [12]. Furthermore, studies by Doi et al. [13] have indicated that antioxidants abolished the inhibition of vascular relaxation caused by human remnant lipoproteins in rabbit aorta rings. In addition, we have found that oxCMR are taken up more readily than CMR by the artery wall during perfusion of the rat aorta, and this difference is enhanced in vessels from hypercholesterolemic rats [14]. Further studies with pig coronary arteries using model CMR-like particles (CRLPs) containing porcine apolipoprotein (apo) E demonstrated that, in the pig vessels, CRLPs have comparable effects to CMR in the rat aorta. Endothelium relaxation to 5-hydroxytryptamine was inhibited, but signifi-

cant inhibition of relaxation to bradykinin required oxidation of the particles [15] (Table 1). This latter change was shown to be mediated by NO, while endothelium-dependent hyperpolarising factor (EDHF) is not involved. The endothelium of porcine coronary arteries produces NO continuously, even in the resting unstimulated state, and evidence was found to suggest that oxCMR inhibit both agonist stimulated and tonic activity of the NO pathway, and are effective almost immediately [16]. Thus, both the entry into the artery wall of CMR and the impairment of endothelium-dependent vascular relaxation they cause is markedly enhanced by oxidation of the particles, indicating that modulation of the oxidative state of CMR may be important for their effects on endothelial function. The cellular mechanisms by which CMR influence endothelial function have been investigated in our laboratory and others using human umbilical vein endothelial cells (HUVEC). CRLPs containing human apoE and post-prandial triglyceride-rich lipoproteins (TRLs) (a lipoprotein fraction enriched in CMR but which also contains chylomicrons and very low density lipoprotein) were found to increase the production of the vasodilator prostaglandin I2 as well as that of the vasoconstrictor thromboxane, via the induction of cyclo-oxgenase-2 (COX-2) activity [17,18]. Experiments using CRLPs and TRLs from hypertriglyceridemic subjects have shown that these particles increase the expression of the antioxidant enzyme heme oxygenase-1 (HO-1) and have also provided evidence to suggest that these effects may be mediated by the nuclear factor-␬B and mitogen-activated protein kinase cell signalling pathways [18–20]. Few studies have addressed the role of the oxidative state of CMR in these cellular processes; however, our preliminary work has

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indicated that the induction of COX-2 and HO-1 [18] is significantly enhanced when CRLPs are oxidized (Dalla Riva, J., Botham K.M., Wheeler-Jones, C.P.D., unpublished results). These findings suggest that CMR influence endothelial cell function by causing a change in the balance between the production of vasodilator and vasoconstrictor mediators which results in a pro-inflammatory response, but the part, if any, played by the oxidative state of the particles requires further investigation.

3. Oxidized chylomicron remnants and macrophage foam cell formation Foam cells are formed when macrophages which have invaded the artery wall take up lipoproteins from the sub-endothelial space and store the lipid intracellularly, eventually becoming so engorged that they take on a foamy appearance. Oxidized LDL (oxLDL) is known to play a major role in foam cell formation [3], but studies in our laboratory and others have shown that CMR or CRLPs containing human apoE also induce extensive lipid accumulation in human monocyte-derived macrophages (HMDMs) and in human and murine macrophage cell lines [4,21–23]. In the presence of similar levels of lipoprotein cholesterol, CRLPs caused

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HMDM or macrophages derived from the human monocyte cell line THP-1 to accumulate comparable amounts of cholesterol and higher levels of triacylglycerol (TG) as compared to oxLDL [23]. In order to test whether increased oxidation of CMR is associated with increased uptake of the particles by macrophages and greater intracellular lipid accumulation, as occurs with LDL, we used model CRLPs in three different oxidative states; untreated; after oxidation with CuSO4 (oxCRLPs); or protected from oxidation by incorporation of the antioxidant lipophilic drug, probucol (pCRLPs) or the tomato pigment, lycopene, together with THP-1 macrophages. Contrary to expectations, the results clearly showed that oxidation of CMR, in striking contrast to LDL, inhibits their uptake and induction of lipid accumulation by macrophages, while protection from oxidation enhances the process (Fig. 1) [24–26], and a similar trend towards lower TG accumulation after oxidation of physiological CMR from rats was found with J774 macrophages, although in this case a much shorter incubation period (8 h) was used [27]. We also found that efflux of cholesterol from the macrophages after loading with CRLPs was limited and comparable to that reported for oxLDL, regardless of the oxidative state of the particles, possibly because lipid from CMR, like that from oxLDL, is sequestered inside lysosomes after uptake [28].

Fig. 1. The effects of the oxidative state of CRLPs on their uptake and induction of lipid accumulation in macrophages. THP-1 macrophages were incubated with CRLPs, oxCRLPs or CRLPs containing probucol (pCRLPs) (30 ␮g cholesterol/ml) (A) Confocal microscopy images after 16 h incubation with DiI-labelled CRLPs. (B) Total lipid accumulation after 48 h (n = 4). *P < 0.01 vs. CRLPs.

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Fig. 2. DiI-labelled CRLPs, oxCRLPs or pCRLPs (30 ␮g cholesterol/ml) were incubated with THP-1 macrophages (2 h) in the presence/absence of LDL and/or lactoferrin and cell-associated fluorescence was evaluated by FACS (n = 3). *P < 0.05, **P < 0.01 vs. corresponding no additions.

The uptake of LDL by macrophages is mediated by the LDL receptor (LDLr), which is down-regulated as cellular cholesterol concentrations rise, while oxLDL is taken up by scavenger receptors, including scavenger receptor-A (SR-A) and CD36, in an unregulated manner, hence oxLDL causes foam cell formation while native LDL does not [3]. CMR have been shown to be taken up and degraded by macrophages in numerous studies with human and animal primary macrophages and cell lines. Uptake appears to be mainly via apoE-dependent pathways mediated by the LDLr and the LDLr-related protein (LRP), although CD36 and phagocytosis may also play minor roles [4,26]. The expression of mRNA for the LDLr was found to be decreased by CRLPs as well as by oxLDL in studies in our laboratory [29], thus, as might be expected, the delivery cholesterol to macrophages by CMR also down-regulates the expression of this receptor. Using specific inhibitors of the pathways believed to be involved in macrophage uptake of CMR, we investigated the effects of the oxidative state of CRLPs on their mechanism of uptake by THP-1 macrophages using CRLPs, oxCRLPs and pCRLPs. Our findings indicated that neither oxidation nor protection from oxidation of the particles changes the main pathways of internalisation of CRLPs into the cells, with uptake occurring mainly via the LRP and the LDLr (Fig. 2) and CD36 and phagocytosis have only a minor role, regardless of the oxidative state of the lipoprotein particles [26]. The differential rates of uptake of CRLPs of different oxidative states may be due to changes in their interaction with the LDLr and the LRP caused by differences in the conformation of the apoE in the particles and/or in the number of apoE molecules able to bind to the receptors [26]. It is clear from these studies that oxidative modification of CMR as compared to LDL has profoundly different effects on macrophage foam cell formation. In sharp contrast to the effects of oxidation of LDL, oxidation of CMR inhibits their uptake by macrophages and reduces lipid accumulation in the cells. This difference is likely to be due to the different receptor mechanisms involved, since oxidation of LDL causes a

shift to uptake via the unregulated scavenger receptors, while oxidation of CMR does not. These surprising findings provide a possible explanation for the puzzling finding that probucol, which in general has provided strong support for the benefits of antioxidants in atherosclerosis, increases lesion development in apoE- or LDLr-deficient mice, since delayed clearance of CMR in such animals leads to their accumulation in the blood [30]. The failure of large scale clinical trials to show any cardiovascular benefit of dietary supplements of lipophilic antioxidants such as vitamin E and ␤-carotene [31], may be due to a number of factors, including subject characteristics, background medication and anti-oxidant genotypes, but the promotion of foam cell formation by CMR containing antioxidants may also be a contributory factor, since the potentially detrimental effects of CMR carrying the antioxidant may counteract the beneficial effects of their subsequent incorporation into LDL.

4. Summary and conclusions It has been established in numerous studies that CMR influence vascular function by causing endothelial dysfunction and promoting macrophage foam cell formation, and the molecular mechanisms mediating these effects are beginning to be elucidated [4,18–20]. However, although it is known that oxidation of LDL markedly alters its effects on the vascular cells and that oxCMR are likely to occur in the body, the influence of the oxidative state of CMR on events in the vasculature has not been studied extensively. Experiments in our laboratory have provided evidence to suggest that oxidation of CMR enhances their inhibition of endothelium-dependent vascular relaxation via down-regulation of NO production (Table 1), and effects of oxidation on the cellular mechanisms regulating endothelial function have also been demonstrated. A striking and surprising finding from our work is that oxidation of CMR inhibits their uptake and induction of foam formation by macrophages, while protection from oxidation strongly enhances this effect (Fig. 1). This sharp contrast to the effects of LDL oxidation is likely to be due to the absence of a shift to uptake by scavenger receptors after oxidation of the particles, as uptake of CMR was shown to occur mainly via the LDLr and the LRP regardless of their oxidative state. Although most of the studies on the effects of oxidation were done with model CRLPs, we have found that the effects of these particles are generally similar to those of physiological CMR [4,9,11,15,22,23]. These findings indicate that the oxidative state of CMR may have an important impact on the atherogenic properties of the particles, highlighting the need for further study in this area.

Conflict of interest statement There are no conflicts of interest arising from this manuscript.

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