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Cardiovascular Research 2000 47(1):173-182; doi:10.1016/S0008-6363(00)00090-0
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Copyright © 2000, European Society of Cardiology

Local application of advanced glycation end products and intimal hyperplasia in the rabbit collared carotid artery

Herta M Crauwels*, Arnold G Herman and Hidde Bult

Division of Pharmacology, University of Antwerp (UIA), Wilrijk, Belgium

* Corresponding author. Tel.: +32-3-820-2737; fax: +32-3-820-2567 crauhert{at}uia.ua.ac.be

Received 3 January 2000; accepted 23 March 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Accumulation of advanced glycation end products (AGEs) in the vessel wall has been implicated in atherogenesis. The aim of our study was to examine the effects of local application of glycated bovine serum albumin (AGE-BSA) on collar-induced intimal hyperplasia in a diabetes-free setting. Methods: Intimal thickening was induced by placing a collar around the carotid artery of rabbits. Via a catheter attached to an osmotic minipump, control BSA or AGE-BSA was administered locally to the vessel wall in a dose of 1.5 or 15 µg h–1 during 14 days. Vessels receiving phosphate buffered saline (PBS, 5 µl h–1) were used as controls. Results: Infusion of AGE-BSA 15 µg h–1 significantly enhanced intimal thickening as compared to control BSA or PBS. Positive remodelling, measured as an increase in the area comprised by the external elastic lamina and preservation of lumen size, was only significant after treatment with the higher dose of AGE-BSA. In all other groups, intimal thickening was accompanied by a decrease of the lumen without outward displacement. Infusion of control BSA or AGE-BSA changed the cell composition of the neointima, with a significant enhancement in the number of T-lymphocytes and macrophages and a reduction in the percentage of intimal area occupied by smooth muscle cells. These effects were however similar for control BSA as well as AGE-BSA. Conclusions: It is concluded that infusion of control BSA or AGE-BSA may aggravate collar-induced intimal thickening by evoking an inflammatory response. This supports the concept that inflammation contributes to atherogenesis. Further, the significant enhancement in intimal hyperplasia by AGE-BSA suggests that glycated proteins provide an additional stimulus for the development of atherosclerotic lesions.

KEYWORDS Experimental; Vasculature; Organism; Pathophysiology; Atherosclerosis; Inflammation; Rabbit; Remodelling; Diabetes


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Despite progress in the early detection and treatment of the disease, diabetes is still a well-known risk factor for accelerated macrovascular disease. Compared to healthy subjects, diabetics develop atherosclerosis that is more severe and occurs earlier in life. Correction of the typical risk factors such as dyslipidemia, hypertension and obesity does not change this, which indicates that hyperglycemia on its own might be responsible for this phenomenon.

Long-term exposure of proteins to glucose leads to the formation of irreversible advanced glycation end products (AGEs), which accumulate in the vessel wall and other tissues [1,2]. Over the past years, increasing evidence has been gathered about the putative role of AGEs in atherogenesis [3,4], especially in regard to enhanced atherosclerosis in diabetic patients. Many in vitro studies suggest that AGEs have biological properties that might promote atherogenesis [5–10]. Accumulation of AGEs in the vessel wall can lead to cross-linking with extravasated lipoproteins, which could enhance cholesterol accumulation, an important step in the development of atherosclerotic lesions. Furthermore, AGEs could stimulate atherosclerosis by their interaction with nitric oxide [11]. However, in vivo data linking AGEs to the development of atherosclerosis is mostly indirect [12,13]. In a limited number of studies, exogenous AGEs have been administered systemically to healthy rats [14,15], rabbits [14,16] and mice [17], and only few of them focused on vascular disease [14,16].

In the present study, the atherogenic properties of AGEs were evaluated in an in vivo model of intimal thickening in the rabbit carotid artery, independently of other risk factors such as hypercholesterolemia or diabetes. In contrast to previous studies, AGEs were administered locally to the vessel wall as opposed to systemically, and unglycated proteins were used as control. Infusion of glycated bovine serum albumin (AGE-BSA) significantly enhanced neointima formation in this model.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Glycation of BSA
BSA (for cell culture applications, endotoxin and fatty acid free, sterile, Boehringer Mannheim) was incubated (50 mg ml–1, PBS) with 1 M glucose for 7 weeks in the presence of antibiotics (penicillin 100 IE ml–1, gentamycin 40 µg ml–1), protease inhibitor (aprotinin 2 µg ml–1) and a chelator of heavy metals (EDTA 0.5 mM). This results in the formation of brown pigments with specific fluorescence. Control BSA was incubated under the same conditions, but in the absence of glucose. Samples were taken for fluorescence measurements (emission 440 nm, excitation 370 nm) at regular times at a fixed protein concentration (1 mg ml–1). Incubation was stopped after 7 weeks. At that time fluorescence levels had reached a plateau (Fig. 1), with values being 36.0 arbitrary units for AGE-BSA versus 1.8 arbitrary units for control BSA. One arbitrary unit was defined as the fluorescence of BSA (1 mg ml–1) before incubation. Regression analysis did not point to a significant rise in fluorescence in the control samples (without glucose). The fluorescence of AGE-BSA on the other hand, increased 20-fold in comparison to non-glycated BSA. Subsequently, the control BSA and AGE-BSA preparations were dialysed (4°C) extensively against PBS. All preparations were filtered aseptically and stored in aliquots at –80°C. Protein concentrations were determined using the BCA method (Bicinchoninic acid, Pierce), and expressed as mg protein ml–1.


Figure 1
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  		Fig. 1 Time-dependent increase in fluorescence during incubation of BSA with or without 1 M glucose. One arbitrary unit was defined as the fluorescence of BSA (1 mg ml–1) before incubation.

 
2.2 Cytotoxicity
To evaluate cytotoxicity, the viability of murine J774A.1 macrophages after 24 h exposure to various concentrations of control BSA or AGE-BSA was assessed by measuring the uptake of the supravital dye neutral red by viable cells as previously described [18]. For the in vivo experiments, products were used in concentrations that were not toxic in the in vitro assay.

2.3 Experimental model of intimal thickening
Male New Zealand White rabbits (2.5–3.0 kg) were anaesthetised with sodium pentobarbital (Nembutal®, Sanofi, 30 mg kg–1, i.v.) and both carotid arteries were exposed surgically. A non-occlusive silicone (MED-4211, C.C.M.P) collar (inlet/outlet diameter 1.8 mm; length 19.7 mm) was placed around each artery and closed with silicone glue. Collars were attached to an osmotic minipump (Alzet, 2 ML2, Charles River France), placed subdermally in the thoracic region to deliver products continuously and locally to the vessel wall (Fig. 2). In a pilot experiment non-glycated, non-incubated BSA was administered in doses of 15 (n=5) and 150 µg h–1 (n=6) for 14 days. AGE-BSA was administered in doses of 1.5 (n=8) and 15 µg h–1 (n=5) during 14 days, while the contralateral arteries were treated with the same dose of control BSA, i.e. BSA incubated without glucose. Vessels with a collar unattached to a minipump (n=5) and vessels receiving PBS (5 µl h–1; n=8) were used as controls. Polymixin B (PMB, 20 IE ml–1) was added to all administered solutions to capture possible contaminating endotoxins. All the animals were kept on a normal chow diet and were given water ad libitum throughout the whole study. The studies were reviewed 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.


Figure 2
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  		Fig. 2 Perivascular collar placement in the rabbit carotid artery. PBS, control BSA or AGE-BSA were continuously (14 days) administered to the vessel wall via an osmotic minipump.

 
2.4 Histological evaluation of artery segments
After 14 days, rabbits were again anaesthetised (sodium pentobarbital, 30 mg kg–1, i.v.); collared arteries were dissected, clamped, removed and placed in a cold, aerated (95% O2–5% CO2) Krebs–Ringer solution. Animals were killed with an overdose of sodium pentobarbital (60 mg kg–1, i.v.). The vessels were carefully cleaned of loose connective tissue and collar. Rings (2 mm) of the collared segment (collar) and of the part of the vessel proximal to the collar (sham) were either formaldehyde (4%)-fixed and paraffin-embedded, or snap-frozen in liquid nitrogen for light microscopy and immunohistochemistry. Transverse sections (5 µm) were cut and staining with haematoxylin/eosin and Verhoeff–Van Giesson (elastin) was performed. Immunohistochemical detection of smooth muscle cells ({alpha}-SMC-actin, Sigma), macrophages (RAM11, Dako), T-lymphocytes (CD43: L11/135, Serotec), monocytes and neutrophils (CD14: MY-14, Coulter Counter), endothelial cells (CD31: JC/70A, Dako) and VCAM-1 (Rb1/9, gift of Dr. M. Cybulsky) was done using specific monoclonal antibodies. The specificity of these antibodies for rabbit tissue and the selectivity for the cells mentioned above was confirmed by flow cytometry cell sorting analysis. Sections were developed with the ABC IgG-method (Vectastain Kit) or by the indirect peroxidase antibody conjugate technique. The chromogen used was 3-amino-9-ethyl-carbazole (AEC).

2.5 Quantification of intimal thickening and remodelling
Intima (magnification 400x) and media (magnification 200x) thickness were measured at 20 and 12 sites, respectively, covering the whole section and averaged per segment (one section per vessel segment). To study vascular remodelling, the perimeters of the external elastic lamina (EEL) and the lumen were measured in collared segments and sham segments. These values were used to calculate the total vessel area (i.e. the area comprised by the EEL) and the lumen area using the formula perimeter2 (4{pi})–1, to normalise for artefacts due to fixation [19,20].

2.6 Cell composition of the neointima
A semi-quantitative score was used to evaluate the presence of different cell types in intima and media, using the following scale: 0: immunoreactive cells absent, 1: 1–10 cells, 2: 11–30 cells, 3: >30 cells present per cross section (one section per vessel segment). To validate the scoring system, the immunoreactivity for {alpha}-SMC-actin and T-lymphocytes (CD43) was also measured using a computer-assisted colour image analysis system (PC-image Colour, Foster Findlay Associates). The immunoreactive area was measured at four sites covering the whole section, expressed as percentage of the intimal area at those sites, and averaged per segment. The presence of the endothelium was assessed by checking immunoreactivity for CD31.

2.7 Statistical analysis
All results are expressed as mean±S.E.M; n represents the number of arteries. For the statistical analysis, the SPSS for Windows package was used. A 5% level of significance was selected. Evaluation was done using analysis of variance (one-way ANOVA) followed by Bonferroni, or the non-parametric Mann–Whitney U-test when there was no homogeneity of variances. Where appropriate, Student's t-test for paired measurements was used.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effect of BSA and glycated BSA on intimal thickness
It has been shown by Minick et al. [21] that repeated injection of BSA in healthy rabbits induced lesion formation. Therefore, we first performed a dose-finding study to evaluate the effect of BSA alone, and to assess the optimal concentration to be used in an in vivo system. PMB (20 IE ml–1) was added to the BSA-solutions to capture possible contaminating endotoxins. To exclude effects of PMB itself, one group without PMB was included in the study. Four different treatments were applied, randomly assigned among rabbits: collar only, non-incubated BSA 15 µg h–1 with PMB, non-incubated BSA 150 µg h–1 without PMB, and non-incubated BSA 150 µg h–1 with PMB. After 14 days, carotid arteries were removed and evaluated histologically and immunohistochemically. The results of one rabbit in the last group (BSA 150 µg h–1 with PMB) were discarded because of the formation of an occlusive thrombus. Collar placement caused local intimal thickening as compared to sham operated segments (data not shown). Local administration of non-incubated BSA caused a dose-dependent increase (one-way ANOVA, P<0.01) in neointima formation as compared to a collar only (Fig. 3). Addition of PMB did not cause significantly different results. Therefore, in subsequent experiments, PMB (20 IE ml–1) was included in all solutions.


Figure 3
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  		Fig. 3 Dose-dependent increase in collar-induced intimal thickness after administration of non-incubated BSA. Co-infusion of polymixin B (PMB, 20 IE ml–1) did not influence the response to BSA 150 µg h–1. * Different from collar (P<0.05); ** different from collar and BSA 15 µg h–1 (P<0.01) (one-way ANOVA). Data are expressed as mean±S.E.M.

 
In the final experiment AGE-BSA was administered to the vessel wall in doses of 15 µg h–1 (n=5), 1.5 µg h–1 (n=8) and 0 µg h–1 (PBS 5 µl h–1, n=8). The contralateral carotid arteries received the same concentrations of control BSA (incubated in the absence of glucose). Infusion of PBS (5 µl h–1) did not influence neointima formation as compared to vessels with a collar only (results compared to the dose-finding study). Administration of the lower dose (1.5 µg h–1) of control BSA or AGE-BSA did not increase intimal thickening as compared to PBS infusion (Fig. 4). Also, infusion of 15 µg h–1 of control BSA did not significantly enhance the neointima formation. Administration of AGE-BSA 15 µg h–1 however, caused a significant enhancement (one-way ANOVA, P<0.05) in intimal hyperplasia as compared to all other groups (Fig. 4).


Figure 4
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  		Fig. 4 Administration of AGE-BSA 15 µg h–1 significantly increased collar-induced intimal thickness as compared to all other groups (# one-way ANOVA, P<0.01). Data are expressed as mean±S.E.M.

 
3.2 Effect of BSA and glycated BSA on media thickness
The media thickness of collared segments was not different from the media thickness of sham-operated segments proximal to the collars (paired t-test, P>0.05). There was also no difference in media thickness between the different treatment groups (one-way ANOVA, P>0.05), although there was a tendency to a decrease in media thickness with higher doses of BSA or AGE-BSA (Table 1). This tendency is however consistent with the positive remodelling observed in collared segments treated with the higher dose of either protein. Results obtained in the dose-finding study with non-incubated BSA were similar (data not shown). The integrity of the media was preserved in all segments, as there was never a significant loss in {alpha}-SMC-actin immunoreactivity in the media (results not shown).


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  		Table 1 Placement of a collar or administration of control BSA or AGE-BSA did not influence media thicknessa

 
3.3 Vascular remodelling
The area within the EEL and the luminal area were compared between collared and sham-operated segments proximal to the collar (paired samples t-test), and among treatment groups (one-way ANOVA). Luminal area was reduced in collared segments as compared to sham-operated segments, when vessels were treated with PBS or proteins (control BSA or AGE-BSA) in the lower dose (Fig. 5A). In vessels treated with the higher dose of proteins, luminal area was preserved, in spite of the increased intimal thickness in vessels treated with AGE-BSA 15 µg h–1 (Fig. 4). The preservation of the lumen size was due to an increase (paired t-test, P<0.01) in the area comprised by the EEL after infusion of AGE-BSA 15 µg h–1 (Fig. 5B), as compared to sham-operated segments. For BSA 15 µg h–1, there was a tendency for an increase in vessel area, but this was not significant. Comparison of different treatment groups (one-way ANOVA) showed expansion of segments treated with the higher dose of BSA or AGE-BSA as compared to all other groups, while sham-operated segments did not differ among treatment groups. Similar results were obtained in the dose-finding study where positive remodelling was statistically significant after infusion of non-incubated BSA at a dose of 150 µg h–1 (data not shown).


Figure 5
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  		Fig. 5 Vascular remodelling. A. Decreased luminal area in collared vessels treated with PBS or the low dose (1.5 µg h–1) of proteins, but not with the high dose (15 µg h–1) of BSA or AGE-BSA. B. Infusion of BSA or AGE-BSA at 15 µg h–1 resulted in an increased area within the EEL. Data are expressed as mean±S.E.M. * Significantly different from sham-operated segment (paired t-test, P<0.05) # significantly different from PBS and 1.5 µg h–1 (one-way ANOVA, P<0.05).

 
3.4 Cellular composition of neointima
Besides an increase in intimal thickness, protein infusion also changed the cell composition of the neointima (Fig. 6). A collar alone, or infusion of PBS induces an intima consisting mainly of smooth muscle cells with very few leukocytes. In the protein-treated vessels, there was a significant inflammatory component in the media and the neointima. The leukocytes in the inner media and intima were mainly T-lymphocytes and macrophages, as assessed by immunoreactivity for CD43 and RAM11, respectively. CD14-immunoreactive cells, which include neutrophils and monocytes, were less common. Mann–Whitney U-tests showed a significant increase in the median score of the numbers of lymphocytes (CD43; P<0.01) and macrophages (RAM11; P<0.05) in the intima after infusion of control BSA and AGE-BSA, as compared to vessels treated with PBS (Table 2). The number of CD14-reactive cells was not significantly different among groups. Although AGE-BSA induced larger neointimas, the relative amount of leukocytes was not enhanced as compared to control BSA. The validity of the scoring system was confirmed by the strong positive correlation between the immunoreactive area for CD43 (µm2), measured by means of image analysis and the score of the CD43-immunoreactivity in the intima (rs=0.87, P<0.01, results not shown). When the immunoreactive area of CD43 was expressed as percentage of the intimal area, to correct for the difference in size of the intima, the relative CD43-immunoreactivity in the intima remained different among treatment groups (Mann–Whitney U, Table 3).


Figure 6
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  		Fig. 6 Intimal thickening and lymphocyte infiltration in collared rabbit carotid arteries receiving PBS 5 µl h–1 (A,B), control BSA 15 µg h–1 (C,D) or AGE-BSA 15 µg h–1 (E,F). A,C,E: haematoxylin/eosin stain; B,D,F: immunohistochemical staining for CD43. Scale bar 50 µm, arrow indicates IEL, arrowheads indicate immunoreactive cells.

 

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  		Table 2 Effect of control BSA and AGE-BSA on infiltration of neutrophils and monocytes (CD14), T-lymphocytes (CD43) or macrophages (RAM11) in the intimaa

 

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  		Table 3 Effect of BSA and AGE-BSA on proportion of intima occupied by {alpha}-SMC-actin and CD43-immunoreactive cellsa

 
The endothelium was intact, as checked by CD31-immunoreactivity, but the endothelial cells had a cuboidal shape, pointing to an activated state. VCAM-1 was rarely expressed in PBS-treated segments, but was clearly upregulated in most vessels treated with control BSA and AGE-BSA (data not shown).

The area comprised by {alpha}-SMC-actin-immunoreactive cells was measured using the image analysis system, and expressed as percentage of total intimal area (Table 3). Although the absolute area of {alpha}-SMC-immunoreactive cells augmented after administration of control BSA or AGE-BSA (results not shown), the percentage of the intimal area containing {alpha}-SMC-actin was significantly decreased in a dose-dependent way, as compared to PBS infusion. This effect was similar for control BSA and AGE-BSA.

Finally, there were significant correlations (Spearman test) between intimal thickness and the scores of the leukocyte numbers in the intima. The correlation was weak for CD14 (rs=0.34, P<0.05), and stronger for CD43 (rs=0.52, P<0.01; Fig. 7) or RAM11 (rs=0.53, P<0.01). Even after correction for intimal thickness, there was still a positive correlation between CD43-immunoreactivity and intima (rs=0.50, P<0.01).


Figure 7
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  		Fig. 7 Correlation between intimal thickness and the score for CD43-immunoreactivity in the intima.

 
In the dose-finding study with non-incubated BSA, similar results concerning the cellular composition of the neointima were obtained (data not shown). Protein infusion induced a dose-dependent infiltration of leukocytes in intima and media, which correlated well with intimal thickness, and a decrease in the percentage of intimal area occupied by smooth muscle cells.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Advanced glycosylation, resulting from the spontaneous covalent reaction of circulating glucose with protein amino groups, leads to the accumulation of AGEs in the vessel wall and other tissues during the normal ageing process. AGEs are believed to be involved in the removal of senescent proteins [22] via interaction with a receptor for AGEs (RAGE), expressed by macrophages, endothelial cells and smooth muscle cells [23]. However, immunohistochemical data have also linked AGE-accumulation to the development of experimental [24] and clinical atherosclerosis, particularly in diabetic patients, where AGE-formation is more excessive [12,13]. In vitro data show increased vascular permeability [5,14], induction of the expression of several cytokines and adhesion molecules [10,22], and impairment of endothelium-dependent relaxation [25].

The aim of the present in vivo study was to investigate whether local infusion of AGEs could enhance intimal hyperplasia independently of diabetes or hypercholesterolemia, in a collar model in the rabbit. In contrast to previous studies [14,16], AGEs were administered locally to the vessel wall as opposed to systemically, and unglycated proteins were used as controls.

Placement of a non-occlusive collar around the carotid artery induces neointima formation, which is not seen in sham-operated vessels [26]. The endothelium remains present, but the cuboidal shape of the endothelial cells and the increased synthesis and deposition of von Willebrand factor, point to an activated state. The process probably starts with an activation of cells by discrete media damage followed by obstruction of transmural flow resulting in high concentrations of cytokines or growth factors in the vicinity of the vessel wall [27]. However, without an additional stimulus, the neointima formed remains discrete, and thickening is complete after 14 days [26].

Previously, we reported that perivascular infusion of ‘fresh’ rabbit serum albumin (RSA, 7 µg h–1) only modestly increased intimal thickening with infiltration of a small amount of T-lymphocytes and macrophages [28]. In contrast, upon incubation in the absence or the presence of glucose, RSA unexpectedly created severe media damage, as demonstrated by smooth muscle cell loss and deposition of calcium complexes in the vessel wall (results not shown). Incubated RSA and AGE-RSA were also toxic to murine macrophages in vitro, and FPLC showed an abundant presence of low molecular weight impurities. Therefore, in further experiments bovine serum albumin (BSA) especially purified for cell culture applications was used, as this proved to result in control BSA and AGE-BSA preparations with less low molecular weight contaminants and no cytotoxicity in the concentrations used in our study.

We first examined the effect of local administration of native, unglycated BSA to the vessel wall, and found that it dose-dependently increased intimal hyperplasia. Therefore, doses of 15 µg h–1 or less were selected for future studies. Related phenomena have been described in the rabbit by Minick et al. [21], who showed that systemic administration of heterologous serum protein by repeated intravenous injection of large amounts of BSA (10 mg kg–1) resulted in proliferative fibromuscular lesions, resembling diffuse intimal thickening in humans. This was attributed to the deposition of immune complexes, triggering inflammatory reactions. When combined with a cholesterol-rich diet, fatty-proliferative lesions developed at these sites predisposed by the immunological injury [29]. In contrast to the study of Minick et al. [21], BSA was administered locally to the vessel wall, resulting in a local build-up of protein at a site of the vessel prone to lesion formation by collar placement. The presence of T-lymphocytes in the neointima points to an inflammatory reaction, which was however local and not systemic. The absence of intimal thickening in segments proximal to the collar or in the contralateral artery, indicates that the increased intimal hyperplasia probably results from a direct interaction of the protein with the vessel wall rather than via circulating immune complexes. Also, even in assuming that all products administered via the osmotic minipumps end up in the circulation, the systemic BSA-load of 0.13 mg kg–1 day–1 at dose 15 µg h–1 was significantly less than the dose (10 mg kg–1) administered by Minick et al., and therefore the immune system would be triggered only modestly in comparison.

Previously, Vlassara et al. [16] showed a modest intimal thickening in the aorta of normal rabbits after long-term systemic administration of glycated rabbit serum albumin (AGE-RSA, 16 mg kg–1 day–1). From that study, it is not clear whether the effects were AGE-related, since a control group receiving unglycated RSA was not included, and the report by Minick et al. [21] and the present study show that control albumin (RSA as well as BSA) may exert pro-atherogenic effects.

In the collar model, infusion of AGE-BSA 15 µg h–1 enhanced neointima formation as compared to control BSA, whereas a lower dose (1.5 µg h–1) did not raise intimal thickening. Control BSA and AGE-BSA both caused a local inflammatory response, presumably due to immune stimulation by these heterologous proteins. However, this immune response appeared to be qualitatively and quantitatively similar for both proteins (see below), pointing to an additional mechanism that accounts for the increased effects of AGE-BSA. In spite of the increased intimal thickness, the size of the lumen was preserved at 15 µg h–1 AGE-BSA due to a compensatory expansion of the vessel. This positive remodelling [30], as shown by a rise in the area comprised by the EEL, was only significant in vessels treated with the higher dose of AGE-BSA. In the other groups, intimal hyperplasia was not accompanied by outward displacement of the vessel, resulting in a reduction of the luminal area.

A possible explanation for the rather modest stimulation of intimal thickening by AGE-BSA could be that the prolonged incubation of BSA with glucose resulted in a mixture of advanced glycosylation end products, without early and intermediate glycosylation products. Few studies have compared the effects of early and advanced glycosylation products. Angulo et al. [31] showed impaired endothelium-dependent relaxation of the isolated rat aorta upon incubation with glycosylated human hemoglobin. Since AGEs could not be detected in that product, these results were attributed to early glycosylation products. On the other hand, Bucala et al. [32] suggested that nitric oxide is quenched in vitro by advanced glycosylation end products only, though not all AGEs exhibited this quenching activity. It could therefore be that the effects of AGEs are highly dependent on the composition of the mixture of various products that is formed. As in vitro this could vary substantially according to the experimental conditions, this might lead to variable results with exogenous AGE-administration.

Infusion of AGE-BSA or control BSA changed the cellular composition of the intima as compared to a collar only or solvent infusion. The number of leukocytes, predominantly T-lymphocytes and macrophages, in intima and media was significantly enhanced, pointing towards an inflammatory reaction in protein-treated vessels. This observation was however similar for control BSA as well as AGE-BSA. The endothelial cells covering the neointima had a cuboidal shape and therefore appear to be in an activated state. Also, the expression of VCAM-1 points to an increased interaction of the endothelium with circulating monocytes and lymphocytes. Exposure of EC to AGEs generates reactive oxygen intermediates, resulting in oxidant stress and the activation of NF-{kappa}B [6]. This has many implications given the broad spectrum of genes with recognition sites for NF-{kappa}B, which upon activation could promote lesion formation [33]. The process of intimal thickening starts by migration of smooth muscle cells from the media and subsequent proliferation. AGEs are able to induce the chemotactic migration of smooth muscle cells in vitro, probably by the production of TGF-β [9]. Monocytes express PDGF in response to AGEs, which is mitogenic and chemotactic for vascular SMC and is believed to play a central role in intimal proliferation [8], and also macrophages are known to produce mitogenic factors for smooth muscle cells [34,35]. Indeed, the absolute amount of smooth muscle cells was raised by control BSA and AGE-BSA, but the relative intimal area covered by smooth muscle cells decreased in protein-treated vessels. Therefore, the increase in intimal thickness was not only due to a larger number of smooth muscle cells. It could however be that this method underestimated the number of smooth muscle cells in the intima, since IFN-{gamma} released from T-lymphocytes at sites of vascular inflammation has been shown to suppress {alpha}-actin expression in smooth muscle cells [36]. T-lymphocytes have been shown to stimulate smooth muscle cell proliferation in vitro [37], but in vivo they appear to inhibit lesion formation, possibly by the production of IFN-{gamma} [38]. These, both stimulating and inhibitory, effects could possibly explain the observation that an abundance of T-lymphocytes in the intima and media did not always promote intimal hyperplasia.

In summary, perivascular administration of control BSA caused a local inflammatory response as indicated by the accumulation of T-lymphocytes and macrophages in the vessel wall. The continuous inflammatory stimulus resulted in an aggravation of collar-induced intimal thickening. This is in agreement with the ‘response-to-injury’ hypothesis, suggesting that atherosclerosis is the result of an excessive inflammatory–fibroproliferative response to various forms of insults to the endothelium and smooth muscle [39]. The presence of T-lymphocytes in atherosclerotic lesions at all stages of development, points to an immunological component in atherogenesis [36].

Finally, AGE-BSA enhanced intimal thickening, suggesting that glycated proteins provide an additional stimulus for intimal hyperplasia. However, the observations with control BSA emphasise the necessity to include proper controls in future studies with exogenously administered AGEs.

Time for primary review 24 days.


   Acknowledgements
 
This work was supported by grant No. 7.0019.96 of the Fund for Scientific Research (FWO, Levenslijn), Flanders. The authors which to thank Dr. M Cybulsky (Toronto, Canada) for the kind gift of the VCAM-1 antibody. We also thank Francois Jordaens, Rita Van den Bossche, Ludo Zonnekeyn and Hermine Fret for technical assistance, and Liliane Van Den Eynde for secretarial assistance. The results have been presented in part at the Winter Meeting of the British Pharmacological Society in Brighton, January 1999.


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

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