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Cardiovascular Research 1998 37(1):239-246; doi:10.1016/S0008-6363(97)00202-2
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Copyright © 1998, European Society of Cardiology

Exposure to oxidized low-density lipoprotein in vivo enhances intimal thickening and selectively impairs endothelium-dependent dilation in the rabbit

Katelijne E Matthysa,*, Cor E Van Hovea, Mark M Kockxc, Luc J Andriesb, Nancy Van Osselaera, Arnold G Hermana and Hidde Bulta

aDivision of Pharmacology (UIA), University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium
bDivision of Physiology (RUCA), University of Antwerp, Antwerp, Belgium
cDepartment of Pathology, General Hospital Middelheim, Antwerp, Belgium

* Corresponding author. Tel.: +32 3 8202636; Fax: +32 3 8202567; E-mail: matthysk@uia.ua.ac.be

Received 14 April 1997; accepted 1 August 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Based on in vitro studies, oxidized low-density lipoprotein (oxLDL) has been implicated in atherogenesis and the associated deficiency in endothelium-dependent relaxation. The aim of this study was to investigate the effects of in vivo exposure to oxLDL on intimal thickening and relaxing behaviour. Methods: Intimal thickening was evoked by the placement of silicone collars around the carotid arteries of the rabbit for 3 or 14 days. OxLDL (Cu2+-oxidized, 7 µg/h) or the vehicle phosphate-buffered saline (PBS) was infused in the collars via subdermally implanted osmotic minipumps. Results: The collared vessels receiving PBS developed discrete intimal thickening after 14 days (intima/media (I/M) ratio 11±2%). OxLDL infusion resulted in intimal thickening after 3 days and significantly enhanced the intimal thickness by 14 days (I/M ratio 98±16%). Collaring alone for 3 or 14 days and 3 days exposure to oxLDL did not impair the endothelium-dependent relaxations to acetylcholine or calcium ionophore, nor to the NO donors glyceryl trinitrate (GTN) and S-nitroso-N-acetylpenicillamine (SNAP). However, the sensitivity to acetylcholine was decreased after exposure to oxLDL for 14 days (–logEC50 oxLDL 6.95±0.11 vs. 7.52±0.11 collar alone) and the maximal relaxation to the endothelium-dependent agonist was reduced by 50%, this in the presence of a virtually intact endothelium. Complete relaxation was still obtained with the nitric oxide donors. Conclusion: Our results show for the first time that local vascular exposure to oxLDL in vivo promotes intimal thickening and inhibits endothelium-dependent dilation, thereby supporting an active role for oxLDL in the morphological and functional changes observed in atherosclerotic blood vessels.

KEYWORDS Atherosclerosis; Low-density lipoprotein; Nitric oxide; Intima; Rabbit; Vasodilation; Endothelium


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
In humans, intimal thickenings are already present at birth in some arteries at orifices and branching points [1]and increase with time [2]. This spontaneously developing intima consists of smooth muscle cells, connective tissue and isolated macrophages, and is considered an adaptation to mechanical wall stress. Intimal thickenings are locations where fatty streaks, the first manifestations of atherosclerosis, tend to develop later in life under the influence of atherogenic stimuli, e.g. hypercholesterolaemia. According to the ‘response-to-injury’ hypothesis, atherosclerotic plaque formation is an excessive inflammatory-fibroproliferative reaction to various forms of insults to the vessel wall [3].

Oxidized low-density lipoprotein (oxLDL) has been implicated in atherogenesis based on in vitro studies and is present in atherosclerotic lesions [4]. Oxidatively modified LDL may promote lesion formation by stimulating the endothelial expression of mononuclear cell adhesion molecules, chemotactic factors and growth factors. In addition, direct chemotactic attraction of mononuclear cells and stimulation of smooth muscle cell proliferation have been described [5].

Atherosclerotic blood vessels display hyperreactivity to certain contractile agonists, e.g. serotonin, and impaired endothelium-dependent dilation, both of which may contribute to ischemia-related clinical symptoms [6]. Endothelium-mediated relaxation of blood vessels largely depends on the release of endothelium-derived relaxing factor (EDRF) [7], identified as nitric oxide (NO) [8], which relaxes the underlying smooth muscle by stimulating the formation of cyclic GMP.

The altered vascular reactivity of atherosclerotic vessels may be caused by oxLDL, as suggested by experiments with isolated blood vessels. In vitro, oxLDL elicits vasoconstriction, enhances agonist-stimulated contractions and inhibits endothelium-dependent relaxations [9, 10].

The aim of this study was to investigate the vascular reactivity of arterial segments exposed to oxLDL in vivo. To that purpose, a previously described rabbit model of intimal thickening, based on the positioning of a silicone collar around the carotid artery [11], was adapted for the perivascular infusion of oxLDL in vivo.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Experimental model of intimal thickening
Male New Zealand white rabbits (2.5–3.5 kg, n = 14 animals) were anaesthetized with sodium pentobarbital (30 mg/kg i.v.) and both common carotid arteries were exposed surgically. Non-occlusive, biologically inert, flexible silicone collars (inlet/outlet diameter 1.8 mm, interior volume 134 mm3, Silicone MED-4211, Nusil Technology, CA, USA) were placed around the carotid arteries and closed with silicone glue [11]. The interior of each collar was connected to an osmotic minipump (Alzet, Charles River France) placed subdermally in the thoracic region. The pumps delivered the vehicle phosphate-buffered saline (PBS, composition in mM: NaCl 154, Na2HPO4 8, NaH2PO4 2, EDTA 0.2, pH 7.4) or the test compound oxLDL (7 µg/h) continuously and locally to the carotid arteries for 3 (n = 6) or 14 (n = 8) days. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

2.2 Preparation of oxidized LDL and lipid peroxidation assay
LDL (human, 5 mg/ml) was purchased from Sigma (St. Louis, MO, USA) and dialysed for 24 h against PBS to remove EDTA. Oxidation of LDL (200 µg/ml in PBS) was performed under sterile conditions by the addition of CuCl2 (6.4 µM) during 16 h at 37°C, and stopped by adding EDTA (200 µM) and keeping on ice for 1 h. Before and after oxidation, a sample was taken for the determination of thiobarbituric acid reactive substances as a measure of lipid peroxidation (TBARS, expressed as µM malondialdehyde (MDA) equivalents, calculated from a standard curve with tetramethoxypropane which decomposes to MDA under the assay conditions). To that purpose, a sample volume of 0.5 ml was mixed with 2 ml 0.25 M HCl containing 3.75% thiobarbituric acid and 15% trichloroacetic acid, and boiled for 15 min. After centrifugation at 1500xg for 15 min, absorbance was read at 532 nm. The concentration of TBARS in the LDL solution before oxidation was below de detection limit of the assay (<1 µM). After oxidation, 6 µM was measured which corresponded to 30 nmol MDA equivalents/mg protein. After the oxidation of LDL with Cu2+, the oxLDL solution was dialysed for 24 h against PBS containing 200 µM EDTA to remove the Cu2+ ions, and concentrated by keeping the dialysis bag in Aquacide I powder (Calbiochem, La Jolla, CA, USA) for 8 h. Finally, the oxLDL preparation was sterile filtered, the protein concentration was measured (BCA assay, Pierce, Rockford, IL, USA), adjusted to 1.4 mg/ml and polymyxin B 2 µg/ml was added to prevent the effect of possible endotoxin contamination. This endotoxin-binding antibiotic was also added to the PBS control solution. Compared with previous series of experiments, polymyxin B did not influence the intimal thickness in collared carotid arteries.

2.3 Histological evaluation of artery segments
After 3 or 14 days, the rabbits were again anaesthetized, the collared carotid arteries were clamped, removed and placed in an aerated (95% O2/5% CO2) physiological salt solution. The animals were killed by an overdose of pentobarbital. The arteries were prepared free of surrounding tissue and carefully released from the collars. Rings of 2 mm were cut of the carotid segment proximal to each collar and of the collar-wrapped segment. The rings were formalin-fixed, paraffin-embedded and stained with haematoxylin–eosin, Sirius red-haematoxylin (collagen) and a monoclonal antibody against {alpha}-smooth muscle cell (SMC) actin (1A4, Sigma), which was visualized by the indirect peroxidase antibody conjugate technique.

Intimal thickness (measured at 20 random sites covering the whole ring and averaged per artery) was measured on one transverse section at 400x magnification using a computer-assisted colour image analysis system (PC-image Colour, Foster Findlay Associates, Newcastle-upon-Tyne, UK). Values are given in µm and as intima to media (I/M) ratio (%). The presence of the endothelium was assessed by measuring the length of the lumen perimeter covered by CD31-positive cells and expressing it as percentage of the total lumen perimeter. We also measured the total cross-sectional area of the media as a rough parameter of its integrity.

2.4 Organ bath studies of vascular reactivity
The vessel segments within the collar and the proximal segments easily provided for two 2-mm rings, which enabled organ bath studies in parallel with the histological evaluation. Proximal and collar rings were suspended in organ chambers filled with 25 ml Krebs–Ringer solution (NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, CaEDTA 0.025, glucose 11.1 mM) maintained at 37°C and continuously gassed with 95% O2/5% CO2. Indomethacin (10 µM, Merck Sharp and Dohme, Brussels, Belgium) was always added to the bath solution to prevent possible interference due to the release of vasoactive prostanoids. Tension was measured isometrically with a Statham UC2 force transducer (Gould, Cleveland, OH, USA) connected to a data acquisition system (Moise 3, EMKA Technologies, Paris, France). The preparations were gradually stretched to a tension of 6 g, which had been determined in preliminary experiments to bring the segments to their optimal length-tension relationship. The segments were then allowed to equilibrate for 45 min. Subsequently, the rings were contracted with a depolarizing potassium solution (50 mM). Cumulative concentration–response curves were made for serotonin (10–9–10–5 M, Janssen Chimica, Geel, Belgium) and phenylephrine ({alpha}1-agonist, 3x10–8–3x10–5 M, Sigma). Additionally, cumulative concentration–response curves were constructed for acetylcholine (10–9–10–5 M, Sigma) and calcium ionophore (A23187 [GenBank] , 10–9–3x10–7 M, Sigma), after half-maximal contraction with phenylephrine. Furthermore, the effect of the NO donors glyceryl trinitrate (GTN, 3x10–9–10–5 M, Merck, Darmstadt, Germany) and S-nitroso-N-acetylpenicillamine (SNAP, 3x10–9–10–5 M, only in the 14-day experiments, gift from Schwarz Pharma, Monheim, Germany) were tested. Between concentration–response curves, segments were allowed to equilibrate for 30 min, during which the bath solution was exchanged three times.

2.5 Statistical analysis
All data are expressed as the mean±standard error of the mean (s.e.m.); n refers to the number of animals. As the data on intimal thickness displayed a large variation, non-parametric statistics were used, with a significance level of 1%. The collared segments were compared with their respective proximal control segments (same vessel) by the Wilcoxon signed-ranks test for two related samples. The comparison between the collared segments receiving PBS (PBS-collar) and the collared segments receiving oxLDL (oxLDL-collar) was done by the Mann–Whitney U-test for two independent samples (different rabbits). The raw data (gram contraction) of the vascular reactivity experiments were fitted to a logistic function to estimate pD2 values [12]. The pD2 is the negative logarithm of the molar concentration (EC50) producing half of the maximal response (Emax). The pD2 reflects the sensitivity to an agonist and is independent of the Emax. Both parameters showed normal distributions (Kolmogorov–Smirnov test) and homogeneity of variances (Levene's test) in the different experimental groups. Differences between the PBS-collar group, the oxLDL-collar group and the proximal control groups were evaluated by analysis of variance (ANOVA) followed by the Student–Newman–Keuls test with a significance level of 5%.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Collar-induced intimal thickening is enhanced by oxidized LDL
Control vessel segments proximal to the collars retained their normal appearance. They did not develop intimal thickening (Fig. 1) and were covered by a continuous layer of flat endothelial cells (Table 1) resting directly on the internal elastic lamina. At 14 days, the I/M ratios were 1.2±0.1% and 1.4±0.3% for the control segments of the PBS-collar and the oxLDL-collar, respectively.


Figure 1
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  		Fig. 1 Collar-induced intimal thickening is enhanced by oxLDL application. Rabbit carotid arteries were collared for 3 (n = 6) or 14 days (n = 8) and treated with PBS (open bars) or oxLDL (filled bars). Results shown are means±s.e.m. Intimal thickness is not increased in the control segments proximal to the collars (lower graph). *Collar different from control, #oxLDL-collar different from PBS-collar, non-parametric statistics as described in Section 2.5, P<0.01.

 

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  		Table 1 Length of intima covered by endothelial cells (%) and medial area after 14 days

 
The positioning of a collar resulted in discrete intimal thickening only after 14 days (Fig. 1, Fig. 2A). By that time, the median value (n = 8) was 26 µm, with a range of 5 to 65 µm. The intima was circular and mainly composed of {alpha}-SMC actin-immunoreactive cells, as previously described [11]. The I/M ratio increased from 2±0.4% at 3 days to 11±2% after 14 days. Again, a continuous layer of anti-CD31-staining cells lined the interior of the artery (Table 1), thereby confirming the presence of an intact endothelium.


Figure 2
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  		Fig. 2 Intimal thickenings in collared carotid arteries without or with oxLDL infusion. (A) Discrete intimal thickening is induced if the pump contains only PBS. (B) A much larger intima is induced if the pump contains oxidized LDL. Pronounced collagen deposition (dark grey bands) occurs in the deeper layers of the intimal lesion. Sirius red/haematoxylin; scale bar=50 µm. Small arrows point to the internal elastic membrane separating the intima from the media; large arrows point to the external elastic membrane separating the media from the adventitia.

 
Delivery of oxLDL to the vessel segment within the collar significantly enhanced intimal thickening at 3 and 14 days, compared to the effect of collaring alone (Fig. 1). Accumulation of intimal {alpha}-SMC actin immunoreactive cells was observed after 3 days and significantly enhanced the I/M ratio to 5±0.4%. Two weeks of oxLDL application resulted in a large intima (Fig. 2B) and an I/M ratio of 98±16%, a 9-fold increase compared to the effect of collaring alone. The median intimal thickness in these oxLDL-treated collared segments was 189 µm, with a range of 78 to 242 µm. The thickening was not eccentric but again involved the whole inner surface of the vessel. As in the PBS-exposed discrete intima, many intimal cells of the large oxLDL-induced intima were recognized by an antibody against {alpha}-SMC actin, identifying them as smooth muscle cells (not shown). Endothelial cells lined the intima completely (Table 1). Pronounced collagen deposition was observed, especially in the deeper layers of the intima (Fig. 2B).

The cross-sectional area of the media was not affected by the collar or oxLDL at any time (Table 1), nor did visible loss of {alpha}-SMC actin reactivity occur.

3.2 Vasoconstrictor responses
The contraction of the rabbit carotid artery to a maximally effective concentration of a depolarizing K+ solution (50 mM) was reduced after 3 days of collaring, without further decline after 14 days (Table 2). Application of oxLDL did not enhance the loss of contractile force at 3 days, but did so after 14 days. As the response to K+ reflects the inherent contractile capacity of the vessel rings, the concentration–response curves to the receptor-dependent agonists serotonin and phenylephrine were expressed as percentages of the reaction to 50 mM K+ (Fig. 3).


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  		Table 2 Contractile responses to K+, serotonin and phenylephrine

 

Figure 3
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  		Fig. 3 Vasoconstrictor responses to serotonin and phenylephrine. Rabbit carotid arteries were collared for 3 (n = 6) or 14 days (n = 8) and treated with PBS (filled circles) or oxLDL (filled squares). Contractile responses to serotonin and phenylephrine are expressed as percentages of the contraction evoked by 50 mM K+. The control rings proximal of the PBS-collar (open circles) and the oxLDL-collar (not shown) reacted similarly. #Left shift compared to control, *enhanced maximal response compared to control, +oxLDL-collar different from PBS-collar, ANOVA, P<0.05.

 
Collaring for 3 days, without oxLDL treatment, significantly increased the sensitivity (Table 2) and the Emax (as % of K+-induced contraction) to serotonin (Fig. 3). The latter was even more increased if the vessel had been exposed to oxLDL. At 14 days, the hypercontractility to serotonin was still present in the oxLDL-treated vessels, whereas the reaction of the PBS-collared segments had almost returned to normal (Fig. 3).

In addition to serotonin, PBS-collared rings also showed an increased sensitivity (Table 2) and Emax to phenylephrine (Fig. 3). The enhancing effect on the Emax was present only at the 3-day time point and was further enhanced by oxLDL (Fig. 3).

3.3 Endothelium-dependent relaxations to acetylcholine and calcium ionophore
Relaxations were studied after the segments had been preconstricted half-maximally with phenylephrine. As collared segments developed less force in response to contractile agonists, the initial phenylephrine contractions differed among the groups studied. We previously demonstrated that different levels of phenylephrine-induced contractions do not influence the characteristics of the relaxation response to acetylcholine [12]. To further evaluate the possible influence of the height of the initial contraction on the sensitivity to endothelium-dependent relaxation, we also compared the initial contractile forces and the pD2 values for all the individual acetylcholine concentration–response curves. As there was no significant correlation between both parameters (not shown), direct comparison of the pD2 values obtained in the different groups under study is allowed.

Three days of collaring in the absence or presence of oxLDL did not influence the sensitivity to acetylcholine or calcium ionophore (Table 3), and complete relaxation was reached for both agonists.


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  		Table 3 Endothelium-dependent relaxations to acetylcholine and calcium ionophore and endothelium-independent relaxations to the nitrovasodilators glyceryl trinitrate (GTN) and S-nitroso-N-acetylpenicillamine (SNAP)

 
At 14 days, PBS-collared segments still had a normal sensitivity and Emax to acetylcholine and calcium ionophore (Table 3, Fig. 4), in spite of the presence of a discrete intima at that time. OxLDL exposure for 14 days significantly decreased the relaxation to both endothelium-dependent agonists: the relaxation curves were shifted to the right and the response to the highest dose of acetylcholine reached only 50% of the initial contraction (Table 3, Fig. 4).


Figure 4
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  		Fig. 4 Relaxing responses to endothelium-dependent and -independent agonists. Rabbit carotid arteries were collared for 14 days (n = 8) and treated with PBS (filled circles) or oxLDL (filled squares). Concentration–relaxation curves were obtained after half-maximal constriction of the segments with phenylephrine and are expressed as percentage residual contraction. The initial contraction was 4.3±0.4 g in the control segments and 2.7±0.3 and 1.5±0.4 g in the PBS- and oxLDL-collared segments, respectively. The control rings proximal of the PBS-collar (open circles) and the oxLDL-collar (not shown) reacted similarly. #Right shift compared to control, *decreased maximal response compared to control, +left shift compared to control, ANOVA, P<0.05.

 
3.4 Endothelium-independent relaxations to the NO donors glyceryl trinitrate and S-nitroso-N-acetylpenicillamine
At 3 days, both collared groups showed a normal sensitivity (Table 3) and complete relaxation to GTN.

At 14 days, the GTN concentration–response curve had shifted to the right in the oxLDL-exposed vessels (Table 3, Fig. 4), but not in the PBS-collared rings. Complete relaxation was attained in all segments. Because of the decreased sensitivity of oxLDL-treated rings to GTN, we also used the NO donor SNAP. In contrast to nitroglycerin, this nitrosothiol does not need enzymatic biotransformation in order to release NO. The relaxation to SNAP in the oxLDL-exposed segments was comparable to the proximal control segments. In the contralateral PBS-treated rings, the sensitivity to SNAP was slightly increased.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Atherosclerotic lesion formation is associated with alterations in the vascular reactivity, e.g. inhibition of endothelium-dependent, EDRF/NO-mediated relaxations and hyperreactivity to contractile agonists, e.g. serotonin [9]. The raised serotonin levels in the plasma of patients with ischaemic heart disease, resulting from the increased release of hyperactive platelets [13, 14], further compromise the normal, dilated state of the blood vessels.

Discrete intimal thickening can be induced experimentally in the rabbit carotid artery by the positioning of a collar around the vessel [11, 15]. This model of intimal thickening not only displays histological but also functional features of a pre-atherosclerotic stage. Indeed, collaring induces supersensitivity to serotonin [16], a feature that also precedes the appearance of fatty streaks in hypercholesterolaemia-based rabbit models of atherosclerosis [17].

Intimal thickening in the rabbit carotid artery ceases after about 2 weeks collaring [11]. Apparently, the formation of atherosclerotic lesions requires an additional stimulus, which could be provided by oxidatively modified LDL [4, 5]. Intimal thickening was indeed enhanced by prolonged in situ application of oxLDL. The effect was already significant after 3 days and became very pronounced after 14 days. Smooth muscle cells, collagen and leukocytes [18]were observed in the hyperplastic intima. The integrity of the endothelium was preserved, as demonstrated by CD31 staining. This was not the case if native LDL was used (not shown). Infusion of native LDL also caused thinning of the media and complete loss of contractile activity, in addition to intimal thickening. We suspect that in situ oxidative modification of LDL has occurred, as it did in rabbits immunized with LDL [19]. The oxidation of LDL in the vicinity of the media may have initiated lipid peroxidation in the smooth muscle cell membranes, leading to extensive vascular injury. Therefore, these segments could not be investigated in the organ bath.

Enhanced contractile responses to serotonin have been described in several animal models of atherosclerosis [17, 20–24]and in atherosclerotic human arteries [25]. They are sometimes associated with hyperreactivity to {alpha}-adrenergic stimulation [24, 26]. As collaring induced an aspecific loss of the contractile capacity of the vessels, we expressed the responses to the receptor-dependent agonists serotonin and phenylephrine as percentages of the response to 50 mM K+. Collaring increased the sensitivity of the carotid artery to the contractile effects of serotonin and phenylephrine. This was not further enhanced by oxLDL. However, the maximal contractile responses to phenylephrine, and especially to serotonin, appeared to be increased by collaring, and this was reinforced by the application of oxLDL. Hence, the modulation of the contractile behaviour induced by collaring and oxLDL parallels the changes observed in other in vivo models of atherosclerosis.

According to some authors, the malfunctioning of the NO signaling pathway is the major cause of the hyperreactivity to contractile agonists [6, 27]. As collaring alone did not impair the relaxations to the endothelium-dependent agonists, this mechanism is probably not involved in the collar-induced hypercontractility. However, increased contractility resulting from a decrease in the basal release of EDRF can not be excluded and could explain the increased sensitivity to SNAP-induced relaxation [28]. On the other hand, a specific supersensitivity of the 5-HT2 receptors [21, 29]or 5-HT1-like receptors [30]on the smooth muscle cells themselves may be involved. The exact mechanism underlying the development of hypercontractility to serotonin in the collared carotid artery of the rabbit remains to be investigated. The persistently high maximal contraction to serotonin in the vessels exposed to oxLDL for 14 days coincides with and could partly result from the deficiency of the endogenous NO pathway. The latter was not due to endothelial cell loss, since CD31 staining showed a continuous layer of endothelial cells. A defective EDRF/NO signaling pathway is a well-described feature in atherosclerotic blood vessels of experimental animals and humans [6, 9, 27]. The mechanisms underlying the reduced availability of bioactive EDRF are likely multifactorial [31], but a major factor appears to be the increased degradation by reactive oxygen species generated in the vessel wall, a process in which oxLDL may be involved [32]. Hence, also the modulation of the relaxing responses by oxLDL in vivo appears similar to the changes observed in atherosclerosis.

Collaring alone did not impair the relaxations to the endothelium-dependent agonists acetylcholine and calcium ionophore, nor to the NO donors GTN and SNAP. In the oxLDL-treated segments, however, the relaxation to GTN but not to SNAP became impaired. The normal reaction to exogenous NO supplied by SNAP suggests that the smooth muscle is still responsive to NO, and supports the idea that impaired production and/or increased oxidative breakdown of endogenous NO underlies the loss of endothelium-dependent relaxation. The reduced responsiveness to GTN could be due to increased oxidant stress in the vascular wall leading to oxygen radical-mediated inactivation of the GTN-transforming enzyme or depletion of cellular thiols. Enhanced superoxide anion production has indeed been demonstrated to be involved in nitrate tolerance [33].

In summary, we demonstrated for the first time in vivo that chronic oxLDL application to the vessel wall induces similar morphological and functional changes as described in atherosclerotic blood vessels, thereby supporting an active role for oxLDL in the disease process.

Time for primary review 31 days.


   Acknowledgements
 
The authors wish to thank Rita Van Den Bossche, Hermine Fret and Ludo Zonnekeyn for technical and Liliane Van Den Eynde for secretarial assistance. The results have been partly presented at the Annual Meeting of the European Vascular Biology Association in Göteborg, Sweden, June 1996. The work was supported by the Belgian Programme on Interuniversity Poles of Attraction Initiated by the Belgian State, Prime Minister's Office, Science Policy Programming and by the Belgian Fund for Medical Research, Grants no. G.3009.93 and 3.0068.94.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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