Elsevier

Atherosclerosis

Volume 133, Issue 2, September 1997, Pages 183-192
Atherosclerosis

Effect of the oxidation state of LDL on the modulation of arterial vasomotor response in vitro.

https://doi.org/10.1016/S0021-9150(97)00124-XGet rights and content

Abstract

Although it is established that highly oxidized LDL modify both vasodilator and vasoconstrictor responses in normal and atherosclerotic arterial tissue, there is a paucity of data on the relationship between the degree of the oxidative modification of LDL and vasomotor response. We therefore compared the impact of native LDL (Nat-LDL), and of partially (P-oxLDL), of moderately (M-oxLDL) and of highly oxidized LDL (H-oxLDL) on the vasomotor response of isolated human internal mammary artery and of rat thoracic aorta. Copper-mediated oxidative modification for up to 24 h at 37°C was characterised by a progressive increase in the net negative electrical charge of LDL, and in the content of oxysterols; by contrast, lipid hydroperoxide and TBARS content peaked in M-oxLDL at 6 h. Neither basal vascular tone nor vasoconstriction induced by KCl (100 mmol/l) were modified significantly in arterial segments in relation to the degree of LDL oxidation. While Nat-LDL did not modify the contractile response of rat aorta to norepinephrine, increase in the degree of oxidative modification of LDL progressively and significantly shifted the norepinephrine response curve to the right (EC50 values for Nat-LDL, M-oxLDL and H-oxLDL: 1.2±0.5×10−8, 3.5±1×10−7, 1.3±0.4×10−6 mol/l respectively) with reduction in the maximal effect (74.5±12.2 and 100.1±6.2% for H-oxLDL and M-oxLDL respectively, P<0.05 versus controls). Similar findings were made in human arteries treated with H-oxLDL (P<0.05 for EC50 and maximal response versus controls). The acetylcholine-induced, endothelial-dependent relaxation of rat aortic segments was significantly and progressively impaired with increase in the degree of LDL oxidation, maximal relaxation with H-oxLDL being 3-fold less (P<0.05) than Nat-LDL at the same protein concentration (100 μg/ml). Acetylated LDL was without effect. Our data indicate that the increase in the degree of copper-mediated, oxidative modification of LDL parallels progressive reduction in the vasomotor response of the arterial wall to norepinephrine-induced contraction and to acetylcholine-induced relaxation subsequent to precontraction. Our data are consistent with the hypothesis that the major oxysterols (7-ketocholesterol, 7β-hydroxycholesterol) present in Ox-LDL underlie such effects.

Introduction

Low density lipoproteins (LDL) constitute the major vehicle for cholesterol transport in human plasma. Considerable evidence is now available to support the hypothesis that oxidative modification of LDL underlies the atherogenicity of these particles in vivo [1]. Indeed, oxidized forms of LDL and apo B-100, in addition to oxidized lipids and oxysterols, have been identified in atherosclerotic plaques [2]. The biological oxidation of LDL involves marked modification of its structural and compositional characteristics, including peroxidation of lipids leading to lipid degradation with formation of aldehydic products, hydrolysis of phosphatidylcholine to its lyso-derivative, oxysterol formation, fragmentation of apo B-100, increased net negative charge causing elevated electrophoretic mobility and increased density 3, 4, 5, 6, 7.

It is now well established that patients presenting atherosclerotic arterial disease often exhibit abnormal vasomotor phenomena such as vasospasm, blood flow reduction, thrombus formation and predisposition to hypertension, which may lead to a variety of clinical syndromes including angina, acute myocardial infarction and sudden cardiac death 8, 9. The mechanisms which underlie impaired vasomotion of the coronary arteries remain to be clarified. The vascular endothelium is intimately implicated in the modulation of vasomotor tone as a result of its capacity to release several vasoactive substances; these factors include endothelium-derived relaxing factors (EDRF) assimilated to nitric oxide (NO), and prostacyclin (PGI2), which may induce relaxation of underlying smooth muscle cells, as well as vasoactive molecules such as endothelin 1 and angiotensin II 10, 11.

Several mechanisms implicating the action of Ox-LDL have been proposed to account for the impairment of endothelial-mediated relaxation which typically occurs in arteries presenting atherosclerotic disease 11, 12. These include (1) an increased synthesis of the vasoactive factor endothelin 1 [13], a finding which could not be confirmed by Jougasaki et al. [14]; (2) an inhibition of PGI2 release by endothelial cells in the presence of Ox-LDL, lipid peroxides or hydrogen radical [15]; (3) reduced synthesis of NO by endothelium 16, 17in response to inhibition of NO synthase by Ox-LDL [18]; and (4) an inactivation of NO as a result of the production of superoxide anions [19]. In addition, evidence has been provided for the selective inhibition of vascular smooth muscle cell relaxation in rabbit and human arteries by Ox-LDL. The mechanism of such inhibition involves attenuation of the agonist-induced rise in tissue content of cyclic nucleotides [20]. Moreover, the enrichment of arterial tissue with cholesterol and lipids may influence transmembrane ionic movements, and the calcium content of smooth muscle cells, thereby leading to alteration in the regulation of vascular tone [21]. Furthermore, Ox-LDL has been shown to directly induce contraction of arterial rings pretreated with norepinephrine [22]or thromboxane mimetics 23, 24. In addition, Ox-LDL potentiates the contractile response of blood vessels to other contractile agonists 11, 22, 25. These vascular effects of Ox-LDL mimic vascular dysfunction observed during atherosclerosis and hyperlipidemia, although marked variability between individuals has been observed 26, 27, 28, 29.

Until now, studies of the effects of Ox-LDL on arterial vasomotricity have involved the use of lipoprotein preparations which have been extensively oxidized in the presence of copper ions in vitro 11, 16, 19, 20, 22, 23, 24, 30, 31, 32, 33, 34, 35. Indeed, there is a paucity of data on the relationship between the degree of LDL oxidation and the vasomotor response of the arterial wall. The purpose of the present study was therefore to investigate the effects of progressive degrees of oxidative modification of human LDL on arterial vasomotricity in both human and rat vessels, and to compare such effects to those of native LDL (Nat-LDL).

Section snippets

Blood samples

Blood (250 ml) was drawn from healthy normolipidemic human subjects by venipuncture into sterile plastic sacs containing citrate–dextrose solution at the local Blood Transfusion Center (CNTS, Rungis, France). Plasma was subsequently separated by low speed centrifugation at 500×g for 20 min at 4°C.

Isolation of LDL

The major particle subspecies of LDL was isolated from plasma in the density interval 1.024–1.050 g/ml by sequential preparative ultracentrifugation. EDTA (final concentration, 0.1%) and gentamycin

Characteristics of native and modified LDL

The degree of oxidative modification of LDL was characterized by the content of lipid peroxides and the thiobarbituric acid-reactive products of peroxidation which include MDA and other aldehydes (Table 1). Lipoperoxides and TBARS were present in partially oxidized LDL (P-oxLDL) at levels of 48–72 nmol lipid peroxides/mg LDL protein and 27–57 nmol equivalents MDA/mg LDL protein, respectively. The content of these oxidation products attained a maximum in moderately oxidized LDL (M-oxLDL) (24 h

Discussion

Our present studies demonstrate for the first time that a progressive increase in the degree of oxidation modification of LDL tends to lead to a pronounced alteration in arterial vasomotricity, with significant reduction in endothelial-dependent acetylcholine-induced relaxation and concomitant reduction in norepinephrine-induced vasoconstriction. Such observations were made not only in rat aorta but also in human mammary artery. Thus, Ox-LDL not only appears to inhibit the ability of blood

Acknowledgements

This study was partially supported by the Faculty of Medicine, University Pierre et Marie Curie (Paris VI) and by INSERM.

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