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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2423-2429

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2423-2429.)
© 1997 American Heart Association, Inc.

Articles

Local Application of LDL Promotes Intimal Thickening in the Collared Carotid Artery of the Rabbit

Katelijne E. Matthys; Cor E. Van Hove; Mark M. Kockx; Luc J. Andries; Nancy Van Osselaer; Arnold G. Herman; ; Hidde Bult

From the Divisions of Pharmacology (K.E.M., C.E. Van H., N. Van O., A.G.H., H.B.) and Physiology (L.J.A.), University of Antwerp, and the Department of Pathology, General Hospital Middelheim, (M.M.K.), Antwerp, Belgium.

Correspondence to Katelijne E. Matthys, Division of Pharmacology, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium. E-mail matthysk{at}uia.ua.ac.be


*    Abstract
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Abstract Oxidized LDL (oxLDL) has been implicated in atherogenesis on the basis of in vitro studies and is present in atherosclerotic lesions. The aim of this study was to investigate the effects of LDL and oxLDL on intimal thickening in vivo. Intimal thickening was evoked by the placement of silicone collars around the carotid arteries of rabbits for 2 weeks. The collars were connected to osmotic minipumps containing LDL (7 µg h-1, n=16 arteries), oxLDL (Cu2+ oxidized, 7 µg h-1, n=16), or phosphate-buffered saline (5 µL h-1, n=16). Segments proximal to the collars served as controls. Collar placement without lipoprotein application resulted in the appearance of {alpha}-SMC actin–immunoreactive cells in the intima, thereby increasing the intimal thickness from 5±1 to 26±5 µm. The perivascular infusion of LDL or oxLDL within the collar significantly enhanced the development of the intima ninefold and sevenfold, respectively. The large intimas resulting from lipoprotein exposure were infiltrated by macrophages and T lymphocytes, and the intimal collagen area was increased from 5±2% in the discrete collar-induced intima to {approx}20% in the lipoprotein-evoked lesions. In conclusion, the local vascular application of LDL, oxidized in vitro or possibly in vivo, elicited an inflammatory-fibroproliferative response characteristic of arteriosclerotic lesions, thereby demonstrating an active role for this class of lipoproteins in the disease process.


Key Words: LDL • intima • atherosclerosis • collagen • leukocyte


*    Introduction
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Atherosclerotic lesions develop in the intima of conduit arteries. In humans, intimal thickenings are already present at birth in some arteries at orifices and branching points,1 and these thickenings increase with time.2 This spontaneously developing intima contains SMCs, connective tissue, and isolated macrophages and is considered to be an adaptation to mechanical wall stress. Although not pathological at this stage, the thickened intima marks locations where atherosclerosis tends to develop later in life under the influence of atherogenic stimuli, eg, hypercholesterolemia.

The long-standing and continuously refined "response-to-injury" hypothesis3 considers atherosclerotic lesions as the result of an excessive inflammatory-fibroproliferative response to various forms of insults to the endothelium and smooth muscle. Furthermore, the presence of T lymphocytes in atherosclerotic lesions at all stages of development points to an important immunological component in atherogenesis.4 In hypercholesterolemia-induced atherosclerosis, the major "noxious" agent is assumed to be oxLDL.5 6

There is a large body of literature describing multiple actions of oxLDL in vitro, which may contribute to its atherogenicity5 6 : eg, chemoattraction of monocytes7 and T lymphocytes,8 enhanced uptake by macrophages,9 and induction of SMC proliferation.10 11 12 However, no clear evidence has been presented for a direct causal role of oxLDL in atherogenesis in vivo. The aim of this study was to introduce LDL and oxLDL in an in vivo model of intimal thickening. In this model, intimal SMC accumulation was induced in the rabbit common carotid artery by the positioning of a silicone collar around the vessel.13 14 The delivery of lipoprotein to the artery by means of an implanted osmotic minipump connected to the collar significantly enhanced collar-induced intimal thickening.


*    Methods
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Experimental Model of Intimal Thickening
Male New Zealand White rabbits (2.5 to 3.5 kg, 24 animals, n=48 carotid arteries) were anesthetized with sodium pentobarbital (30 mg/kg body weight IV), and the common carotid arteries were exposed surgically. After removal of the fibroadipose periadventitia, nonocclusive, flexible, silicone collars (inlet/outlet diameter 1.8 mm; Silicone MED-4211, Nusil Technology) were placed around the carotid arteries. 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 PBS (5 µL h-1, composition in mmol/L: NaCl 154, Na2HPO4 8, NaH2PO4 2, EDTA 0.2; n=16 arteries) or the lipoprotein solution (LDL or oxLDL, 7 µg h-1; n=16 arteries each) continuously and locally to the carotid arteries for 14 days.

Because the LDL was of human origin, another series of experiments (12 animals, n=24 carotid arteries) was conducted using human and rabbit albumin (essentially fatty acid free, Sigma; 7 µg h-1, n=8 arteries each and PBS, n=8 arteries) to evaluate the contribution of antigenic stimulation in the process of intimal thickening. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication No 85-23; revised, 1985).

Preparation of oxLDL and Lipid Peroxidation Assay
Human LDL (5 mg/mL in 0.15 mol/L NaCl, 0.01% EDTA, pH 7.4, purchased from Sigma) was Cu2+ oxidized, and determination of TBARS, expressed as µmol/L MDA equivalents (a measure of lipid peroxidation), was performed as previously described.15 The concentration of TBARS in the LDL solution before oxidation was below the detection limit of the assay (<1 µmol/L). After oxidation, 6 µmol/L was measured, which corresponded to 30 nmol MDA equivalents per milligram protein. Like oxLDL, the native LDL used in the experiments was subjected to 24 hours of cold dialysis against EDTA-containing PBS, conditions that do not allow oxidative modification of LDL.16 Finally, the lipoprotein preparations were sterile filtered, the protein concentration was measured (BCA assay, Pierce) and adjusted to 1.4 mg/mL, and polymyxin B (2 µg/mL) was added to prevent the effect of possible endotoxin contamination.

Histological Evaluation of Artery Segments
After 14 days the rabbits were again anesthetized (sodium pentobarbital 30 mg/kg body weight IV); 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 (2 mm) cut from the collar-wrapped vessel segments and from the proximal segments outside the collar (controls) were either formalin fixed and paraffin embedded or snap-frozen in liquid N2 for light microscopy and immunohistochemistry.

Staining with hematoxylin/eosin and Sirius red/hematoxylin (collagen stain) was performed. Under polarized light, the cross-striated pattern of fibrillar collagen reflects the light, which enabled us to distinguish interstitial crossbanded collagen from nonfibrillar collagen.17 Immunohistochemical detection of SMCs ({alpha}-SMC actin, 1A4, Sigma), macrophages18 (CD68, EBM/11, Dako), T lymphocytes19 (CD43, L11/135, Serotec), and ECs (CD31, JC/70A, Dako) was done using specific monoclonal antibodies visualized by the indirect peroxidase antibody conjugate technique. Staining for CD68 was done on frozen preparations.

Quantification of Intimal Thickening and Components
Intimal thickness (measured at 20 random sites covering the whole ring and averaged per artery) and the percentage of the intimal area occupied by collagen were measured on one transverse section at 400x magnification using a computer-assisted color image analysis system (PC-image Colour, Foster Findlay Associates). Segmentation of the collagen area was done by interactive (de visu) selection of the appropriate red-green-blue threshold levels corresponding to the red areas under study. The presence of the endothelium was assessed by measuring the length of the lumen perimeter covered by CD31-positive cells and expressing it as a percent of the total lumen perimeter. Intimal leukocytes were quantified by measuring the CD68- and CD43-immunoreactive areas and expressing them as percent of the total intimal area. We also measured the thickness of the media as a rough parameter of its integrity.

Statistical Analysis
All data are expressed as the mean±SEM; n refers to the number of arteries. Differences between the collared segment receiving PBS (PBS-collar), oxLDL (oxLDL-collar), LDL (LDL-collar), human or rabbit albumin (albumin-collar), and the control segments (proximal) were evaluated by ANOVA followed by the Student-Newman-Keuls test with a significance level of .05. If the variances were unequal (Levene test for homogeneity of variances, P<.05), logarithmically transformed values were analyzed. If the distribution of the variable was not normal (Kolmogorov-Smirnov test, P<.05), the nonparametric Kruskal-Wallis test was used.


*    Results
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Native LDL and oxLDL Promote Collar-Induced Intimal Thickening
Control vessel segments proximal to the collars retained their normal appearance. They did not develop intimal thickening (Fig 1Down) and were covered by "flat" ECs, which formed a virtually complete lining (Table 1Down). The positioning of a collar resulted in a discrete intimal thickening (Fig 1Down and Fig 2ADown), reaching 26±5 µm, which was mostly circular. Again, a continuous layer of anti-CD31–staining cells lined the interior of the artery (Table 1Down), thereby confirming the presence of an intact endothelium. The ECs sometimes appeared more cuboidal than in the control segments.



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  		Figure 1. Intimal thickening in the rabbit carotid artery after 14 days of collaring with local infusion of PBS (n=16 arteries), LDL (n=16), or oxLDL (n=16). Segments proximal to the collars did not develop intimal thickening and served as controls. *Collar different from control; #lipoprotein-collar different from PBS-collar; +LDL-collar different from oxLDL-collar; ANOVA, P<.05.


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  		Table 1. Length of Intima Covered by ECs (%) and Medial Thickness



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  		Figure 2. Intimal thickenings in rabbit carotid arteries induced by the positioning of a perivascular collar connected to an osmotic mini pump. A, Discrete intimal thickening was induced even when the pump contained only PBS. Sirius red/hematoxylin stain; scale bar=60 µm; arrowhead points to internal elastic membrane. B, A much larger intima was induced when the pump contained oxLDL. Pronounced collagen deposition occurred in the deeper layers of the intimal lesion. Sirius red/hematoxylin stain; scale bar=60 µm; arrowhead points to internal elastic membrane. C, Immunohistochemical stain for {alpha}-SMC actin of the intimal lesion evoked by collaring combined with oxLDL infusion. The expression of {alpha}-SMC actin appeared to be less in the deeper collagen-rich areas. Scale bar=30 µm; arrowhead points to internal elastic membrane.

Delivery of LDL or oxLDL to the vessel segment within the collar significantly increased the development of the intima ninefold and sevenfold, respectively (Fig 1Up and Fig 2BUp and 2CUp). This corresponded to mean intimal thicknesses of 246±23 and 170±14 µm, the LDL-evoked intima being larger than the oxLDL-induced lesion. The thickening was not eccentric but again involved the whole inner surface of the vessel. ECs, often displaying a cuboidal shape, completely lined the intima in the oxLDL segments, whereas some cell loss was observed in the LDL segments (Table 1Up). Only in the LDL-treated segments was thinning of the media observed (Table 1Up).

The application of human and rabbit albumin increased the intimal thickness about twofold over the effect obtained by collaring alone (46±6 and 62±8 µm, respectively, versus 25±3 µm in the collared segment; n=8 arteries in each group), but this effect was significantly less than the effect of lipoprotein exposure (ANOVA, P<.05).

Cellular Composition of the Intima
The intima induced by collaring alone was mainly composed of {alpha}-SMC–actin immunoreactive cells, as previously described.13 Very rarely, some intimal or adhering monocytes or T lymphocytes could be detected (Fig 3ADown and 3CDown and Table 2Down). Mononuclear cells were never present in the proximal control segments. The large intimas evoked by application of both types of lipoprotein also contained {alpha}-SMC actin–immunoreactive cells (Fig 2CUp). However, expression of this SMC-specific protein sometimes appeared inhomogeneous and was rather low in the largest intimas. Furthermore, great numbers of macrophages and T lymphocytes adhered to and infiltrated the intima and, to a lesser extent, the media (Fig 3BDown and 3DDown and Table 2Down). Fat-red staining did not reveal the presence of foam cells or lipid deposits in the intima or media.



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  		Figure 3. Intimal leukocyte infiltration in the collared rabbit carotid artery in the absence (A and C) or presence (B and D) of oxLDL. A and B, Immunohistochemical stain for CD68, demonstrating monocytes/macrophages. Without oxLDL (A), rare immunoreactive cells are present adjacent to the internal elastic membrane. In the presence of oxLDL (B), many immunoreactive cells are observed in the thickened intima. C and D, Immunohistochemical stain for CD43, demonstrating T lymphocytes. In the absence of oxLDL (C), few immunoreactive lymphocytes are found adhering to or infiltrating the small, collar-induced intima. The large, oxLDL-enhanced intimas (D) contained many immunoreactive cells. Scale bars=40 µm; arrowheads point to internal elastic membrane.


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  		Table 2. Intimal Macrophages and T Lymphocytes

The application of human albumin also induced significant leukocyte infiltration. In these segments, more macrophages were observed than in those vessels treated with rabbit albumin, although the latter also showed some infiltration (Table 2Up). The presence of T lymphocytes was highly variable in both cases, ranging from almost complete absence to massive infiltration in the segments exposed to human albumin.

Intimal Collagen Deposition
Collaring alone resulted in diffuse collagen deposition surrounding the intimal SMCs. Collagen composed 5±2% of the intimal area. Intimal collagen accumulation was enhanced to 22±5% or 19±2%, respectively, after the artery segment had received either native LDL or oxLDL. In the lipoprotein-treated segments, the collagen appeared more tightly packed and was most abundant in the deeper layers of the intima (Fig 2AUp and 2BUp).

By polarized light microscopy, most of the collagen in the large, lipoprotein-induced intimas showed enhanced birefringence, as opposed to the discrete collagen deposition induced by collaring alone, thus identifying the former type of collagen as fibrillar.


*    Discussion
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 *Discussion
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Collaring of the rabbit carotid artery evokes a small intima, mainly consisting of SMCs. The mechanism presumably involves discrete injury of the medial smooth muscle layer leading to cell activation, combined with hindrance of transmural flow, thereby resulting in high concentrations of growth factors and cytokines throughout the vessel wall.14 The SMCs indeed displayed an activated synthetic phenotype, as previously demonstrated by transmission electron microscopy.13 In this respect, the collar-induced intima bears a resemblance to the normal human intima2 and intimal areas adjacent to atheromata.20 The endothelium was intact, as demonstrated by CD31 staining, but the more cuboidal aspect of some cells and the previous findings of a prominent, rough endoplasmic reticulum and intense staining for von Willebrand factor21 reflect cell activation. Furthermore, we demonstrated the deposition of fibrinogen in the media 6 hours after collaring, which strongly suggests increased permeability of the endothelium.22 As the process of intimal growth reaches a plateau after 2 weeks,13 an additional stimulus is apparently required to sustain the growth of the intimal lesions.

Oxidatively modified LDL has received a great deal of attention in recent years for its suspected causal role in atherogenesis.5 6 OxLDL23 and immunocompetent activated T cells4 that recognize oxLDL24 are present in atherosclerotic lesions. A multitude of in vitro actions of oxLDL that could be involved in atherogenesis has been described. In vitro, oxLDL is chemotactic for monocytes7 and T lymphocytes.8 Minimally oxidized LDL stimulates ECs and SMCs to secrete monocyte chemotactic peptide-1 (MCP-1)25 and growth factors involved in the differentiation and proliferation of monocytes.26 Furthermore, oxLDL may promote mononuclear leukocyte adhesion to the endothelium, as it induces the expression of P-selectin on ECs27 and enhances the cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-1) in vitro.28 The oxidative stress–sensitive nuclear transcription factor {kappa}B (NF-{kappa}B) may be a crucial intermediate in the inflammatory activation of the endothelium.29 In addition, oxLDL stimulates DNA synthesis10 11 12 and enhances collagen production30 in vascular SMC cultures.

In contrast to the impressive amount of in vitro data, reports dealing with the effect of oxLDL on the arterial wall in vivo are scant. Oxidized lipids in the diet accelerate the development of fatty streaks in cholesterol-fed rabbits.31 Using intravital microscopy, Lehr et al32 33 34 demonstrated the increased rolling and sticking of fluorescently labeled leukocytes to the endothelium of arterioles and postcapillary venules after injection of oxLDL. The group of Berliner et al,35 who introduced the concept of minimally oxidized LDL, reported that injection of this modified lipoprotein in mice induced monocyte binding at susceptible sites in the aorta and stimulated the release of macrophage colony stimulating factor (M-CSF) and expression of mRNA for JE (the mouse homologue of MCP-1) in the liver.36 Although these short-term experiments confirm that oxLDL is biologically active in vivo, a role for oxLDL in atherosclerotic lesion formation remains to be determined. Therefore, it was of interest to conduct experiments with longer exposures to oxLDL. Because intravenously administered oxLDL is rapidly cleared by the liver,37 we applied the lipoproteins locally. The same human lipoprotein preparation, native or Cu2+ oxidized in vitro, was used in all experiments. The choice of human instead of rabbit lipoprotein was based on the known antigenic polymorphism of rabbit LDL, which would have led to unpredictable immunological activation in the different animals.38 With human lipoprotein, this confounding factor is avoided because all of the rabbits, at least theoretically, experience similar antigenic stimulation.

Intimal thickening in the collared rabbit carotid artery was greatly enhanced by prolonged in situ application of oxLDL and even more of LDL. A slight increase of intimal thickening was also observed with human albumin, but this was far below the effects of the lipoproteins. Furthermore, rabbit albumin had the same small effect as human albumin. Hence, antigenic stimulation does not appear to play a major role in the stimulation of intimal growth. Many intimal cells in the lipoprotein-evoked intimal lesions were recognized by an antibody against {alpha}-SMC actin, although local loss of this SMC-specific protein occurred, especially in the largest intimas. This phenomenon has been described as part of the phenotypic modulation of SMCs in intimal atherosclerotic lesions and proliferating cell cultures.39 Large stretches of ECs had a cuboidal aspect, probably related to cell activation as discussed above. Therefore, LDL and oxLDL appear to reinforce the collar-induced activation of medial SMCs and ECs and hence, the whole process of intimal thickening. A systemic effect of the lipoproteins due to possible leakage into the circulation does not seem to be involved, since the intima in the contralateral artery, which was collared but not treated with lipoproteins, was not larger than in a previous series of experiments. Although native LDL may have proatherogenic effects of its own, eg, stimulation of SMC proliferation,40 we suspect that in situ oxidative modification of LDL has occurred, as it did in the rabbits immunized with LDL.41 ECs and SMCs,42 neutrophils,43 monocytes,43 lymphocytes,44 and fibroblasts45 are all capable of oxidizing LDL and are present in the collared vascular segment.13 Furthermore, the slight desquamation of the endothelium, the thinning of the media, and the more pronounced effect of LDL suggest that more vascular injury and activation occurred than with oxLDL, which could be caused by active oxidative processes.

A difficulty with this type of in vivo experiment is estimation of the concentration of active test compound that builds up locally at the carotid artery. Our assumptions are the following: On the basis of the dimensions of the carotid artery and collar, a "tissue-free" space corresponding to 100 µL is available around the artery within the collar. The osmotic minipump delivers 5 µL/h to this space. Hence, 1.4 mg/mL of lipoprotein in the osmotic minipump would give a concentration of 70 µg/mL around the artery after 1 hour. However, the disappearance of the product by breakdown, diffusion in all directions, and drainage via lymph vessels must also be taken into account but is even harder, if not impossible, to estimate. In the literature, LDL and oxLDL are used in concentrations ranging from 10 to 500 µg/mL, eg, in cell culture or organ bath experiments. The LDL concentration at branching points of the abdominal aorta in rabbits fed cholesterol for 16 days is 50 µg LDL cholesterol per gram of tissue.46 Based on a cholesterol-to-protein ratio of 2.5:1,47 this corresponds to 20 µg protein per gram of tissue, or 1 µg/50 mg. Fifty milligrams is the approximate weight of the segment of carotid artery that was exposed to lipoprotein in the collar. In our experiments, 7 µg was delivered to that segment every hour. Also, 290 µg apolipoprotein B per 100 mg dry defatted tissue is present in normal arteries of 40-year-old humans.48 Considering the above and the fact that LDL accumulation in the vessel wall may exceed its plasma concentration due to interactions with the extracellular matrix,49 we believe that the lipoprotein concentration used in the present experiments is relevant to the in vivo situation in hypercholesterolemia.

OxLDL may stimulate SMC proliferation and/or migration directly10 11 12 or indirectly through the attraction of leukocytes. The simultaneous infiltration into the intima of both monocytes and T lymphocytes in this model and in the cholesterol-fed rabbit19 suggests that they are responding to a common stimulus, which may be oxLDL. Inhibition of mononuclear leukocyte infiltration by monoclonal antibodies significantly reduced intimal thickening in the electrically stimulated rabbit carotid artery,50 thus identifying leukocytes as promoters of intimal growth. Macrophages indeed produce many growth factors for SMCs51 and have been shown to stimulate mitogenesis in cocultures.52 T lymphocytes have also been shown to stimulate SMC proliferation in vitro53 but appeared to inhibit lesion formation in vivo,54 55 possibly by the production of interferon-{gamma}.56 57 The net effect probably depends on the interplay between growth-promoting and -inhibiting activities and may even be perfectly balanced, as suggested by the finding that human albumin did not induce more intimal thickening than did rabbit albumin, despite significantly more leukocyte infiltration. It could also mean that leukocytes are not the main source of growth factors acting on SMCs to stimulate proliferation and collagen production and points to direct effects of the lipoproteins on medial SMCs. The absence of foam cells may relate to the fate of the administered lipoproteins. After 14 days, a loose fatty sheet was present around the vessel segment within the collar. This suggests that lipoprotein-derived lipids accumulate to a certain degree in situ and could explain the absence of significant lipid deposits in the media and intima.

The thick, lipoprotein-promoted intimas often showed less staining for {alpha}-SMC actin in the deeper layers of the intima. This correlated with large collagen deposits in those regions. Such pronounced collagen production was never seen in the absence of lipoprotein infusion. In the collar-induced intimas, only collagen type IV, which is found exclusively in the basal lamina, was detected.13 By polarized light microscopy, the large collagen areas in the lipoprotein-enhanced intimas were identified as deposits of interstitial collagen. Therefore, the lipoprotein-induced lesions more closely resemble human atherosclerotic lesions that contain increased amounts of type IV and fibrillar collagen (types I and III and others)58 and lesions in the cholesterol-fed rabbit that show collagen deposition in the deeper layers of the intima.59 60 The mechanisms of the enhanced biosynthetic activity of SMCs could be manyfold. In vitro, oxLDL has been demonstrated to stimulate collagen synthesis directly30 and also indirectly through stimulation of SMC proliferation and the associated phenotypic change from the contractile to the synthetic state.61 Stimulation of collagen synthesis by leukocyte-derived cytokines could also be involved and is supported by the joint occurrence of collagen mRNA–containing SMCs and macrophages in human atherosclerotic vessels,62 although this was not confirmed by another similar study.63 Both macrophages51 and T lymphocytes64 produce transforming growth factor-ß, the most potent stimulus for collagen production in SMCs.65 However, in human plaques, type I procollagen mRNA was negatively associated with the spatial presence of T lymphocytes, possibly due to the production of the inhibitory cytokine interferon-{gamma}.63

In conclusion, local vascular application of LDL, oxidized in vitro or possibly in vivo, elicited an inflammatory and fibroproliferative response characteristic of arteriosclerotic lesions, thereby demonstrating an active role for this class of lipoproteins in the disease process.


*    Selected Abbreviations and Acronyms
 
EC = endothelial cell
MDA = malondialdehyde
oxLDL = oxidized LDL
SMC = smooth muscle cell
TBARS = thiobarbituric acid–reactive substances


*    Acknowledgments
 
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. The authors wish to thank Rita Van Den Bossche, Hermine Fret, and Ludo Zonnekeyn for technical assistance 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.

Received September 9, 1996; accepted May 14, 1997.


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 *References
 

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