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Cardiovascular Research 2003 59(1):189-199; doi:10.1016/S0008-6363(03)00353-5
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Copyright © 2003, European Society of Cardiology

Plaque-associated endothelial dysfunction in apolipoprotein E-deficient mice on a regular diet. Effect of human apolipoprotein AI

Herta M. Crauwelsa,*, Cor E. Van Hovea, Paul Holvoetb, Arnold G. Hermana and Hidde Bulta

aDivision of Pharmacology (T2), University of Antwerp (UIA), Universiteitsplein 1, B-2610 Wilrijk, Belgium
bCentre for Experimental Surgery and Anaesthesiology, University of Leuven (KUL), Leuven, Belgium

herta.crauwels{at}ua.ac.be

* Corresponding author. Tel.: +32-3-820-2737; fax: +32-3-820-2567.

Received 24 September 2002; accepted 17 March 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Apolipoprotein E-deficient mice (apoE–/–) on a regular diet become hypercholesterolemic and develop atherosclerosis, but endothelium-dependent relaxation remains undisturbed for up to 6 months. We investigated whether vasomotor dysfunction develops in aged apoE–/–, whether the defect was systemic (hypercholesterolemia-dependent) or focal (plaque-related), and the effect of human apolipoprotein AI transgenesis (apoAI/E–/–). Methods: Arteries of apoE–/– (n = 5), apoAI/E–/– (n = 6) and C57Bl/6J (WT, n = 4) mice (18 months) were systematically dissected for isometric tension recording and subsequent morphometry. Results: Acetylcholine (ACh)-induced relaxation was impaired (P<0.01) in atherosclerotic segments of apoE–/– (26±14%) as compared to WT mice (93±2%). Similar reduced (P<0.01) responses to adenosine 5'-triphosphate (apoE–/– 38±14, WT 94±3%) and the calcium ionophore A23187 [GenBank] (apoE–/– 19±6%, WT 97±2%) pointed to a post-receptor defect. Indeed, responses to exogenous nitric oxide were impaired in atherosclerotic segments as well (apoE–/– 71±7%, WT 92±1%, P<0.05). Furthermore, relaxations inversely correlated with plaque size (ACh rs=–0.74, P<0.01). In adjacent plaque-free segments however, responses to ACh (apoE–/– 92±3%, WT 97±1%) and all other agents were preserved, despite the prolonged hypercholesterolemia. ApoAI improved vasomotor responses in atherosclerotic segments. However, negative correlations between maximal relaxation and plaque area remained in apoAI/E–/– mice (ACh rs=–0.67, P<0.01). Indeed, covariate analysis of variance did not point to direct protection of vasomotor function by apoAI when the smaller lesions were taken into account. Conclusions: Endothelial dysfunction in apoE–/– mice is not affected by hypercholesterolemia alone, but is strictly associated with plaque formation. Human apoAI transgenesis—known to raise HDL—attenuated atherogenesis, thereby indirectly improving relaxation responses in apoE–/– mice.

KEYWORDS Atherosclerosis; Nitric oxide; Endothelial function; Vasodilation; Histopathology; Hypercholesterolemia; apoAI; HDL


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Apolipoprotein E-deficient (apoE–/–) mice develop hypercholesterolemia, mainly characterised by increased β-VLDL [1]. Atherosclerosis spontaneously progresses in these animals without the need for cholesterol supplementation. Plaques systematically develop in the aortic root and gradually along the aorta, while other arteries remain largely unaffected [2]. The microscopic appearance and cellular composition of the plaques is remarkably similar to those in humans [3]. Impaired endothelium-dependent relaxation is an early hallmark of atherosclerosis in rabbits [4], mice [5] and humans [6,7]. In humans, endothelial dysfunction is a systemic process, that can occur in the absence of atherosclerosis, merely by the presence of risk factors such as hypercholesterolemia [8,9]. In previous studies, vasomotor dysfunction was never documented in apoE–/– mice, unless atherogenesis was accelerated by combination with LDL receptor-deficiency [5] or a Western type diet [10–13]. Also, plaque size was never documented or taken into account. Plaques in apoE–/– mice fed a high-fat diet are different in composition and more lipid-rich [2]. Furthermore, high plasma lipid levels are associated with increased oxidative stress [14], compromising vascular reactivity [15,16].

Epidemiological studies have identified both high density lipoprotein (HDL) and its major structural protein, apolipoprotein AI (apoAI) as independent predictors of reduced coronary heart disease, suggesting an antiatherogenic role for HDL [17]. Furthermore, elevated HDL ameliorates abnormal vasoreactivity in human coronary arteries with or without atherosclerosis [18,19] and apoAI was the strongest predictor of maximum acetylcholine-relaxation in subcutaneous arteries from hypercholesterolemic patients [20]. In vitro studies showed that apoAI [21] and HDL [22] reversed the impaired endothelium-dependent relaxation induced by oxidized LDL in porcine coronary arteries and in rabbit aorta, respectively. Deckert et al. [13] showed a partial improvement by apoAI of the modest shift in the acetylcholine-relaxation in apoE–/– mice induced by a high fat, cholate-containing diet. However, they did not document atherosclerotic lesions, and it is not clear whether the improvement was due to the retarded atherogenesis reported for apoAI [23,24].

Though apoE–/– mice develop atherosclerotic plaques, endothelium-dependent relaxation remains undisturbed up to 6 months on a normal diet [5,11,13]. Therefore, we investigated whether endothelial function deteriorates in aged apoE–/– mice, whether it was a local (plaque-related) or a systemic (hypercholesterolemia-related) event, and whether it was improved by apoAI. To this end, atherosclerosis-prone and atherosclerosis-resistant segments of the aorta and the carotid artery of wild type (C57Bl/6J), apoE–/– and apoAI-overexpressing apoE–/– mice were exposed to three endothelium-dependent and two endothelium-independent relaxing agents. Unlike previous reports, the localisation and size of the plaques was taken into account, and a regular diet was used to avoid additional effects of excessive oxidative stress.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals
C57Bl/6J (WT, n = 4), apoE–/– (n = 5) and apoAI/E–/– (n = 6) mice were generated at CEHA (University of Leuven) as described [25]. Mice were given tap water ad libitum, fed a regular rodent diet (Pavan Services, Belgium) without cholesterol (<0.06%) or fat supplementation, and studied at the age of 18±0.2 months. The studies were approved by the Ethical Committee of the University, and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

2.2 Isolation and mounting of blood vessels
After anaesthesia (sodium pentobarbital, 75 mg kg–1, i.p.) blood was collected (vena cava) for determination of total cholesterol (Sigma kit 401-25). Carotid arteries and the aorta were carefully removed and cleaned of adherent tissue. The aorta was systematically sectioned (Fig. 1): the first mm of the aorta after the heart is defined the aortic root; the thoracic aorta was divided in 5x2 mm segments starting 3 mm from the origin of the left subclavian artery (where the azygos vein crosses over the aorta) down to the diaphragm. Every segment was first used in the functional study with subsequent morphometric evaluation. Segments of the aorta (2 mm) and the aortic root (1 mm) were mounted between two parallel tungsten wire hooks in 10-ml organ baths. Tension was measured isometrically with a Statham UC2 force transducer (Gould) connected to a data acquisition system (Moise 3, EMKA Technologies). Carotid artery segments (2 mm) were mounted in a wire (40 µm) myograph (Danish Myotechnology) for isometric tension recording. All vessels were immersed in Krebs–Ringer solution (37°C; 95% O2/5% CO2; pH 7.4) containing (mM): NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, CaEDTA 0.025, glucose 11.1 and piroxicam 0.1. Segments of the aorta and the aortic root were gradually stretched until a stable loading tension of 20 and 10 mN, respectively, was attained, which had been determined in pilot experiments to bring the segments to their optimal length–tension relationship. Carotid arteries were set to their normalised diameter according to the method of Mulvany and Halpern [26].


Figure 1
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  		Fig. 1 Segmental differences in plaque formation and protection by human apoAI (n = 6) in the thoracic aorta of apoE–/– mice (n = 5, A). The scheme (B) shows the location and code of the different aorta segments. The strong regional variation in plaque development is further illustrated by cross sections (elastin staining) of an atherosclerotic segment (C, TA1) and the neighbouring plaque-free segment (C, TA2) within the same animal. Data represent mean±S.E.M. Mann–Whitney U, *P<0.05, **P<0.01 versus apoE–/–; scale bar represents 250 µm.

 
2.3 Vasomotor studies
Cumulative concentration–response curves were made for phenylephrine (3 nM–30 µM) and the negative logarithm of the concentration (pD2) resulting in 50% of the maximal response (Emax) was assessed for each vessel segment. For relaxation studies, vessels were precontracted with 35 mM KCl or with the pD2-concentration of phenylephrine. Endothelium-dependent relaxation was assessed by cumulative concentration–response curves for acetylcholine (ACh, 3 nM–10 µM), adenosine 5'-triphosphate (ATP, 30 nM–100 µM) or calcium ionophore (A23187 [GenBank] , 10 nM–1 µM). Endothelium-independent relaxation was evaluated by cumulative concentration–response curves for exogenous nitric oxide (NO, acidified NaNO2, 100 nM–100 µM) and papaverine 100 µM. For each concentration of relaxing agent, the minimum force was expressed as percentage relaxation, with the initial contraction being zero relaxation. The contribution of endogenous NO-pathways was assessed by inhibition of the endothelial NO synthase (eNOS) with a combination of N{omega}-nitro-L-arginine (L-NA, 300 µM) and N{omega}-nitro-L-arginine methyl ester (L-NAME, 300 µM), or inhibition of the inducible isoform (iNOS) with N-(3-(aminomethyl)benzyl)acetamidine (1400W, 10 µM) [27]. Minimally contracted vessels (low phenylephrine concentration) were incubated with NOS-inhibitors for at least 15 min. Afterwards, the phenylephrine concentration was gradually titrated to adjust the contraction to the same level as in the previous relaxation curve in the absence of the NOS-inhibitors.

2.4 Histological quantification of plaque size and composition
After the functional study, each individual segment was placed in formaldehyde (4%, 24 h), embedded in optimal cutting temperature compound (OCT) and kept at –80°C for histological analysis. Transverse sections (6 µm) were stained for elastin (Verhoeff–Van Giesson), collagen (Trichrome–Masson) and lipid (oil red O). Immunohistochemical detection of smooth muscle cells ({alpha}-SMC-actin, Clone 1A4, FITC-labelled, Sigma), macrophages (Mac-3, M3/84, Pharmingen) and iNOS (SA-200, Biomol) was done using specific monoclonal antibodies. Sections were developed with the ABC IgG-method (Vectastain Kit) [28]. The chromogen used was 3-amino-9-ethyl-carbazole.

From each segment, three sections—taken at intervals of 150 µm (aortic root) or 250 µm (all other vessels)—were analysed. The cross-sectional area of the intima (total area between the lumen and the internal elastic lamina) was quantified on elastin-stained sections by means of point counting using a grid (0.07x0.07 mm) on the microscope eye piece [29]. Areas of oil red O, {alpha}-SMC-actin, macrophages and calcium-deposits were measured using a computer-assisted colour image analysis system (PC-image Colour, Foster Findlay Associates) and expressed as percentage of the intimal area. The average of the three sections per segment was calculated and used for further analysis.

2.5 Statistical analysis
Results are expressed as mean±S.E.M.; n represents the number of mice. Non-parametric tests were used to analyse the presence or absence of plaques (Chi-square) and the areas of the intima, macrophages and SMCs (Mann–Whitney U). Since Kolmogorov–Smirnov analysis indicated that Emax and pD2 values of all agonists were normally distributed, these variables were evaluated using univariate factorial analysis of variance (ANOVA; SPSS release 10.5, SPSS) followed by the Student–Newman–Keuls procedure. To evaluate lesion impact, data from all the individual segments of the aorta (aortic root, TA1->5) were combined and Spearman correlation coefficients (rs) were determined. Furthermore, the cross-sectional intimal area was included as covariate in the factorial analysis of variance (ANCOVA) of vasomotor responses. A 5% level of significance was selected.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Plaque formation
Serum cholesterol was raised in apoE–/– mice (457±39 mg dl–1 versus 62±8 in WT), and was not influenced by apoAI (478±46 mg dl–1). Vessels of WT mice were free of atherosclerosis. In apoE–/– mice, plaques were predominantly found in the aortic root and in the proximal and distal aorta segments (TA1 and TA5, Fig. 1). The central thoracic aorta (TA2, TA3 and TA4) was usually minimally affected. Furthermore, macroscopically visible lesions were observed in the aortic arch and its side branches, and around the carotid bifurcation. In apoAI/E–/– mice the plaque area of the aortic root, TA1 and TA5 was smaller as compared to apoE–/– mice (Fig. 1A). This was due on one hand to a reduced frequency of plaques (16/36 segments of apoAI/E–/– and 23/30 of apoE–/– mice, Chi-square, P<0.01). In addition—if plaques were present—they were also smaller in the aortic root (apoAI/E–/–: cross sectional area of intima: 0.11±0.04 mm2, 4/6 segments; apoE–/–: 0.27±0.01 mm2, 5/5 segments; P<0.01). A similar tendency was seen in the aorta (0.06±0.02 mm2, 12/30 segments versus 0.11±0.02 mm2, 18/25 segments).

3.2 Histological evaluation of atherosclerotic lesions
Plaques in the aorta of apoE–/– mice varied both in size and composition, ranging from small foam cell deposits to advanced plaques (Fig. 2A) associated with calcium deposition. A large part of the plaques was occupied by collagen (Fig. 2B). Lipid was identified as oil red O-positive material (Fig. 2A). All plaques contained macrophages (Fig. 2E) with associated iNOS expression (Fig. 2F). Cells expressing {alpha}-SMC-actin were less frequent (Fig. 2C), although some plaques were covered with a fibrous cap (Fig. 2D). Larger lesions usually showed acellular zones with cholesterol clefts and often deep medial involvement with fragmentation of the internal elastic lamina. In those cases, macrophages—associated with iNOS expression—were identified in the media. Calcium deposits were almost exclusively observed in larger plaques in the aortic root.


Figure 2
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  		Fig. 2 Cross-sections of the aorta of apoE–/– mice stained for lipid (A, oil red O), collagen (B, Trichrome–Masson), smooth muscle cells (C, D, {alpha}-SMC-actin), macrophages (E, Mac-3) and iNOS (F). Arrowheads indicate lipid droplets (A), smooth muscle cells (C, D), macrophages (E) and iNOS (F); arrows indicate calcium deposition (A) and elastic fragmentation (E, F); scale bar represents 50 µm.

 
Although plaques were smaller in apoAI/E–/– mice, the relative macrophage content—mostly associated with iNOS expression—was unaffected (apoAI/E–/– 7.8±2.3%, apoE–/– 8.7±1.2%), while the SMC content was doubled (apoAI/E–/– 3.4±1.2%; apoE–/– 1.5±0.4%). Furthermore, oil red O staining (4.2±1.6 versus 7.3±1.8%) and calcium deposition (5.5±3.7 versus 7.2±2.9%) tended to be less in apoAI/E–/– mice.

3.3 Endothelium-dependent relaxation
In apoE–/– mice, the relaxation evoked by ACh was significantly impaired as compared to WT mice in the aortic root (Fig. 3A) and the atherosclerosis-prone aorta segments TA1 (Fig. 3B) and TA5 (pD2 6.99±0.22; Emax 58±7% versus 7.54±0.09; 96±2%; P<0.05). In atherosclerosis-resistant aorta segments (TA2, Fig. 3C) and the carotid artery (Fig. 3D) however, the ACh-relaxation was unaltered. ApoAI strongly improved the ACh-relaxation in atherosclerosis-prone segments, without influencing responses in atherosclerosis-resistant segments (Fig. 3). Identical results were obtained in the aortic root when constricted with 35 mM KCl—a condition which inhibits hyperpolarisation pathways—though maximal responses were smaller as compared to phenylephrine-constricted vessels (apoE–/–: 13±7% versus 35±7% in WT, P<0.01 and versus 34±6% in apoAI/E–/–, P<0.01). A combination of L-NA and L-NAME 300 µM, used to fully inhibit endothelial NO-release, completely abolished (<5%) the relaxation in all arteries of WT, apoE–/– and apoAI/E–/– mice. The selective iNOS inhibitor 1400W was without effect in any strain (not shown).


Figure 3
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  		Fig. 3 Decreased endothelium-dependent relaxation to ACh in phenylephrine-constricted rings of the aortic root (A) and atherosclerosis-prone (TA1, B), but not in atherosclerosis-resistant aorta segments (TA2, C) or the carotid artery (D) of apoE–/– mice (n = 5). In severely atherosclerotic segments of apoE–/– mice, relaxation was almost absent, in which case a pD2-value could not be calculated. Human apoAI (n = 6) is protective of this vascular dysfunction. Data represent mean±S.E.M. ANOVA; Emax: **P<0.01, pD2: *P<0.05 versus WT (n = 4).

 
While relaxation responses did not show relationships to cholesterol levels (Spearman, P>0.05), statistically significant negative correlations existed between intimal area and Emax (Fig. 4; apoE–/–: rs=–0.74, apoAI/E–/–: rs=–0.67; P<0.01) and pD2 (apoE–/–: rs=–0.62, apoAI/E–/–: rs=–0.58; P<0.01) for ACh in both apoE–/– strains, indicating that the relaxation was only impaired in case of plaque formation. Even when plaque-free segments were removed from the analysis, the negative correlations remained significant (apoE–/–: rs=–0.67, apoAI/E–/–: rs=–0.87; P<0.01). Furthermore, a factorial ANOVA with strain and segment as factors indicated a significant difference in the Emax for ACh between apoE–/– and apoAI/E–/– mice (F = 6.72, P = 0.013) and between segments (F = 11.83, P<0.001). However, inclusion of intimal area as covariate in the analysis revealed that the beneficial effect of apoAI was entirely determined by the reduced atherosclerosis (ANCOVA, effect of lesion size: F = 51.24, P<0.001; effect of strain: F = 1.59, P = 0.214; effect of segment: F = 9.21, P<0.001).


Figure 4
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  		Fig. 4 Scatterplot of all individual aorta segments (aortic root, TA1->5) showing negative correlations (Spearman, P<0.01) between cross-sectional intimal area and maximal ACh-relaxation (Emax) in apoE–/– mice (rs=–0.74), which is similar in apoAI/E–/– mice (rs=–0.67), despite the vascular protection.

 
Relaxations to ATP and A23187 [GenBank] were also impaired in atherosclerotic segments (TA5), but not in largely intact vessels (TA4; Fig. 5). The latter even displayed a paradoxical and unexplained increase in sensitivity to ATP. Again significant (P<0.01) negative correlations between plaque size and Emax were apparent for both apoE–/– strains (ATP, apoE–/– rs=–0.80; apoAI/E–/– rs=–0.73 and A23187 [GenBank] , apoE–/– rs=–0.70; apoAI/E–/– rs=–0.75).


Figure 5
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  		Fig. 5 Relaxation to ATP (A, B) and A23187 (C, D) is impaired in atherosclerotic segments (TA5; A, C) of apoE–/– mice (n = 5), but not in largely intact vessels (TA4; B, D). Human apoAI (n = 6) protects from vascular dysfunction in apoE–/– mice. Data represent mean±S.E.M. ANOVA, *P<0.05, **P<0.01 versus WT (n = 4).

 
Finally, there were no statistically significant relationships (Spearman, P>0.05) between the absolute or relative area of macrophages or SMCs and relaxation parameters for ACh, ATP, and A23187 [GenBank] in either strain.

3.4 Endothelium-independent relaxation
Both the sensitivity to exogenous NO and the maximal relaxation were significantly reduced in atherosclerotic aorta segments (TA1, Fig. 6A; pD2 4.46±0.14; Emax 54±8% versus 5.35±0.10; 92±1%), but not in intact aorta segments (TA2, Fig. 6B; pD2 5.20±0.10; Emax 89±2% versus 5.35±0.06; 91±1%) or the carotid artery (pD2 4.46±0.07; Emax 78±6% versus 4.60±0.15; 75±11%) of apoE–/– mice as compared to WT. In apoAI/E–/– mice, the NO-induced relaxation was unaltered in all vessel types (Fig. 6A–E). Nevertheless, intimal area was inversely correlated to Emax (apoE–/–: rs=–0.83, apoAI/E–/–: rs=–0.74; P<0.01) and pD2 (apoE–/–: rs=–0.80, apoAI/E–/–: rs=–0.76; P<0.01) for exogenous NO in both apoE–/– strains. Also, even though in a factorial analysis with strain and segment as factors, Emax for NO differed between apoE–/– and apoAI/E–/– mice (F = 12.51, P = 0.001) and between segments (F = 13.63, P<0.001), inclusion of intimal area as covariate revealed that the beneficial effect of apoAI was entirely determined by the retarded atherogenesis (ANCOVA, effect of lesion size: F = 6.45, P = 0.016; effect of strain: F = 1.18, P = 0.284; effect of segment: F = 14.03, P<0.001). L-NA/L-NAME did not alter the response to exogenous NO in any strain or vessel (not shown).


Figure 6
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  		Fig. 6 Strong impairment in the relaxation to exogenous NO in atherosclerotic aorta segments (TA1, A) of apoE–/– mice (n = 5), but not in atherosclerosis-resistant segments (TA2, B). The relaxation to NO in TA1 is improved by human apoAI (B). Data represent mean±S.E.M. ANOVA, **P<0.01 versus WT (n = 4) and apoAI/E–/– (n = 6).

 
The relaxation to papaverine 100 µM was complete in all segments, even in case of severe atherosclerosis (not shown).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Although apoE–/– mice are a widely used model in lipid and atherosclerosis research, endothelial dysfunction has never been demonstrated when these animals are fed a regular diet for up to 6 months [5,11,13]. Here we report that in aged apoE–/– mice on a regular diet, endothelial dysfunction (1) develops in atherosclerotic segments, (2) is significantly correlated to lesion size, (3) does not occur in adjacent plaque-free segments, despite the prolonged hypercholesterolemia, and (4) is improved by overexpression of apoAI.

In apoE–/– mice, plaques were predominantly found in the aortic root, the aortic arch, and the proximal and distal segments of the thoracic aorta, as previously reported [1–3]. Despite the prolonged (18 months) hypercholesterolemia, the central thoracic aorta was not or minimally affected. Because of this consistent regional diversity, endothelial function could be investigated both in the presence and absence of atherosclerotic lesions within the same animal. Relaxation induced by endothelium-dependent agonists (ACh, ATP and A23187 [GenBank] ) and exogenous NO was significantly impaired in the aortic root and atherosclerotic aorta segments of aged apoE–/– mice on a regular diet. Furthermore, significant correlations existed between these defects and plaque size, but not plaque composition. However, despite exposure to the same prolonged hypercholesterolemia, the response to all agents remained normal in the adjacent plaque-free segments. There even was an unexplained improvement in the response to ATP, possibly due to changes in the sensitivity towards purinergic stimulation or in the signal transduction. The undisturbed (endothelium-independent) relaxation to papaverine, even in the most severe atherosclerotic segments, showed that the dilator capacity of the arteries was not affected by anatomical changes induced by plaque development. Collectively, these findings indicate that endothelium-dependent relaxation is not affected by hypercholesterolemia alone. As opposed to the human situation [8], endothelial dysfunction in apoE–/– mice is not a systemic process, but a local phenomenon, associated with lesion formation. These results also emphasise the importance for proper documentation of plaque dimensions in murine vasomotor studies.

An important finding is that human apoAI protects vascular function in apoE–/– mice by improving both endothelium-dependent relaxation and responses to exogenous NO. Transgenic expression of human apoAI in apoE–/– mice increases plasma HDL [24,30], without affecting total plasma cholesterol. Plaque formation was significantly retarded in apoAI-overexpressing mice—especially in atherosclerosis-prone regions—in agreement with previous observations [23,24]. Furthermore, the reduced calcium and lipid deposition suggested a less advanced plaque type in apoAI-overexpressing mice. Also, the decreased absolute number of macrophages, and tendency to increased SMC content, as described in apoE–/– mice fed a Western type diet [31,32], suggests remodelling of the plaques towards a more stable phenotype. However, when taking into account the reduced plaque area in apoAI/E–/– mice, the relative amount of macrophages was identical and the increase of SMCs did not reach significance, despite a marked tendency.

A direct protective effect of HDL-apoAI on vasomotor function was suggested in apoE–/– mice fed a high-fat diet [13], based on the fact that total plasma cholesterol was not affected. Plaque size was however not documented. Given the strong correlation between plaque size and endothelial function in apoE–/– mice, the beneficial effect of apoAI on vasomotor function could be secondary to the reduced plaque size and frequency. A direct protective effect of apoAI/HDL would implicate that for a given plaque size, the relaxation would be less impaired in apoAI/E–/– as compared to apoE–/– mice. To test this hypothesis, plaque size and composition were taken into account in the analysis of vasomotor responses, using an ANCOVA. This confirmed the significant correlation between endothelial dysfunction and plaque size for both apoE–/– and apoAI/E–/– mice, but showed no evidence for direct protective effects of apoAI-overexpression on any vasomotor response when the smaller lesions in apoAI/E–/– mice were taken into account. Furthermore, cellular composition of the plaques was not determinant for the relaxation, as there were no relationships with the absolute or relative amount of macrophages or SMCs in either strain. The present results therefore indicate that the protective role for HDL-apoAI on vascular function is secondary to the attenuated plaque formation, and stress again the necessity to document plaque size.

The ACh-relaxation in large elastic arteries, i.e. the aorta and the carotid artery of C57Bl/6J mice is solely mediated by NO acting via cGMP-dependent pathways [33]. Inhibition of eNOS also completely abolished the relaxation in all segments of both apoE–/– strains, indicating that a defect in the NO-mediated pathway is responsible for the endothelial dysfunction in apoE–/– mice. Selective defects of the muscarinic receptors, as proposed for the rabbit aorta and human coronary arteries [6,34], are unlikely in apoE–/– mice since atherosclerosis impaired the response to ATP and the receptor-independent relaxing agent A23187 [GenBank] as well. Together with the defective response to exogenous NO, this pointed to a post-receptor defect. A shortage of L-arginine or tetrahydrobiopterin was suggested in apoE/LDL receptor double knockouts [35], and inactivation of eNOS by enhanced binding to caveolin in hypercholesterolemic conditions was described in vitro [36]. Although we cannot exclude these possibilities, the impaired response to exogenous NO suggests that other—or at least additional—mechanisms, such as accelerated NO degradation, play an important role. Indeed, enhanced superoxide production has been described in apoE–/– mice on a Western type diet [11,12] and superoxide accelerates NO degradation [16]. Apart from xanthine oxidase, cytochrome P450, NADH/NADPH oxidase and cyclooxygenase, uncoupling of NO synthases when the substrate L-arginine or the cofactor tetrahydrobiopterin is limited, could result in superoxide generation [10,16]. Though we here showed iNOS upregulation in plaque macrophages, iNOS was probably not responsible for superoxide production, since the selective inhibitor 1400W did not improve the ACh-relaxation. Further, the lack of effect of L-NA/L-NAME on the relaxation to exogenous NO, indicates that eNOS is an unlikely source of superoxide anions in apoE–/– mice.

It remains intriguing that murine arteries—in contrast to the human situation—are protected from endothelial dysfunction in the absence of plaques, despite severe hypercholesterolemia. The mechanism involved is as yet unclear. The preserved relaxation in atherosclerosis-resistant segments was not due to compensatory upregulation of EDHF-like pathways, as seen in other disease models [37]. Murine vessels are able to produce large amounts of NO [33], which exerts anti-atherogenic effects [38,39]. Upregulation of eNOS-expression as reported for apoE–/– mice [10], or cellular defence mechanisms such as extracellular SOD as described in arteries of apoE–/– and LDLr–/– mice, could account for the protection from chronic and systemic oxidative stress. Alternatively, the SMCs could become more sensitive to NO when the endogenous bioavailability becomes compromised, as shown for eNOS-deficient mice [40].

In conclusion, this is the first documentation of impaired endothelium-dependent relaxation in apoE–/– mice on a regular diet. The defect involves the endogenous NOS pathway, is not receptor-dependent, and also occurs with exogenous NO. The dysfunction however only developed in segments with overt atherosclerosis, and is strongly correlated to lesion size. The preserved endothelium-dependent relaxation in intact vessel segments cannot be accounted for by compensatory upregulation of EDHF-like pathways. These results therefore indicate that endothelial dysfunction in mice is a focal and not a systemic, hypercholesterolemia-dependent defect. Murine arteries are able to maintain endothelium-dependent relaxation as long as plaques do not develop. This was further substantiated by our finding that the beneficial effects of transgenesis of human apoAI in the protection against the impaired responses to ACh, A23187 [GenBank] , ATP and exogenous NO proved to be secondary to the strong retardation of atherogenesis.

Time for primary review 24 days.


   Acknowledgements
 
The financial support by the Fund for Scientific Research-Flanders (F.W.O. grants G.0079.98 and G.0263.01) and IUAP P5/02 is greatly appreciated. The authors wish to thank Ludo Zonnekeyn and Rita Van den Bossche for technical, and Liliane Van Den Eynde for secretarial assistance.


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

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