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Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:2395-2404

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:2395-2404.)
© 1999 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Cholesterol-Induced Changes of Type VIII Collagen Expression and Distribution in Carotid Arteries of Rabbit

Gabriele Plenz; A. Dorszewski; W. Völker; Y. S. Ko; N. J. Severs; G. Breithardt; H. Robenek

From the Institute of Arteriosclerosis Research, University of Münster, Department of Cell Biology and Ultrastructure Research (G.P., W.V., H.R.); the Department of Coronary Artery Disease, Section of Molecular Cardiology (A.D., G.B.); and the Hospital of the University of Münster, Department of Cardiology and Angiology (A.D., G.B.), Münster, Germany; and the National Heart and Lung Institute, Imperial College School of Medicine (Y.S.K., N.J.S.), London, England.

Correspondence to Gabriele Plenz, PhD, Institute of Arteriosclerosis Research at the University of Münster, Department of Cell Biology and Ultrastructure Research, Domagkstraße 3, D-48149 Münster, Germany. E-mail plenz{at}uni-muenster.de


*    Abstract
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Abstract—Lipoproteins play a major role in cardiovascular disease and atherosclerosis. In the vascular wall, they strongly influence the organization of extracellular matrix. The present study set out to investigate the changes in the extracellular matrix of the vessel wall induced by atherogenic diet, focusing on type VIII collagen, a vascular collagen that has not previously been investigated in detail. The influence of cholesterol diet on the expression, distribution, and deposition of type VIII collagen was examined in carotid arteries of New Zealand White rabbits. Carotid arteries of rabbits receiving diet supplemented with 1% cholesterol for 6 weeks and those on the same regimen followed by normal chow for 1 day, 10 days, 5 weeks, and 12 weeks were studied and compared with controls not exposed to the cholesterol diet. Carotid arteries of normocholesterolemic rabbits contained type VIII collagen–expressing cells in all layers, with focal accumulations of expressing cells in the subendothelial areas, the outer medial zone, and the adventitia. In response to cholesterol diet, type VIII collagen synthesis was reduced in media and adventitia and the distribution patterns changed. Expressing cells were found predominantly in the endothelium, and type VIII collagen accumulated in the intimal space. Immunogold labeling for electron microscopy revealed that type VIII collagen in the intima is associated with microfibrils extending from the internal elastic lamina. Withdrawal of cholesterol resulted in reestablishment of the normal distribution pattern. Northern and Western blot analyses supported the immunoconfocal and in situ hybridization data, demonstrating decreased type VIII collagen expression in response to cholesterol diet and progressive recovery to normal levels with time after withdrawal of cholesterol. Our study demonstrates that type VIII collagen is modulated in the presence of cholesterol. The data indicate that type VIII collagen is specifically remodeled during early experimental atherosclerosis, implying a role for this extracellular matrix component in neointimal growth.


Key Words: extracellular matrix • remodeling • animal model • atherosclerosis • smooth muscle cells


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One of the key events in the atherogenic cascade is the focal influx and accumulation of lipoproteins at arterial sites that are prone to develop atherosclerotic lesions. LDL cholesterol, and probably other lipoproteins, traverse the endothelium and undergo oxidative modification.1 2 Oxidized LDL stimulates various proatherogenic events, such as the recruitment of macrophages into arteries, the promotion of cell growth, and the formation of thrombi.3 4 5 As monocyte recruitment, macrophage differentiation, and lipoprotein influx continue, the lesion grows and develops into the fatty streak. Subsequent foam cell necrosis due to the influence of oxidatively modified lipoproteins6 7 and increased extracellular matrix (ECM) synthesis and secretion by intimal smooth muscle cells (SMCs) lead to the established atherosclerotic lesion referred to as the fibrous plaque.8 9 10

Binding of lipoproteins to the ECM of the arterial intima is considered to be an initial event in the development of atherosclerotic lesions.11 Several components of the ECM, in particular collagens, elastin, and proteoglycans, influence the uptake, accumulation, and retention of lipoproteins in the vessel wall.12 13 14 15 16 17 In the heart valve intima model18 and in the vessel wall itself,19 lipoproteins associate with collagen fibers and elastic tissues. The association of lipoproteins with arterial proteoglycans is an important factor in lipoprotein internalization, retention, and modification.15 20 21 It has been speculated that the association of lipoproteins with collagen may be a key step in lipid aggregation in the intima.18 Conversely, the most active de novo synthesis of interstitial collagen has been reported in regions of atherosclerotic lesions characterized by lipid deposition.22 In vitro studies have demonstrated that oxidized LDL stimulates the synthesis of collagen by vascular SMCs23 24 and the expression of type IV collagen by mesangial cells.25 Thus, these data suggest mutual influences between lipid and collagen that may affect lipid transport mechanisms and deposition as well as collagen synthesis.

The present study was undertaken to investigate the effects of changes in the ECM of the vessel wall induced by atherogenic diet on the expression, distribution, and deposition of type VIII collagen, a vascular collagen that has not previously been investigated in detail. Type VIII collagen is known to contribute to the maintenance of tissue integrity,26 but its location in the vascular wall and its precise role in atherogenic events is still unknown. In the present study, we therefore investigated the temporal and spatial distribution patterns of type VIII collagen in carotid arteries of normocholesterolemic and hypercholesterolemic rabbits at the mRNA and protein levels by in situ hybridization and immunolabeling, in combination with Northern and Western blot analyses.


*    Methods
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Experimental Groups
Thirty male New Zealand White rabbits (3.8±0.4 kg) were used. The animals were divided into the following treatment groups: (1) rabbits receiving regular chow for 6 weeks (n=5), (2) rabbits fed a 1% cholesterol diet for 6 weeks (n=5), and (3) rabbits receiving a 1% cholesterol diet for 6 weeks followed by normal chow for a range of periods (n=20). In this last group, animals were killed at 1 day, 10 days, 5 weeks, and 12 weeks (n=5 each) after cessation of the cholesterol diet. The rabbits were housed according to the German Animal Welfare Act specifications.

Tissue Preparation
The rabbits were euthanized under general anesthesia with ketamine (4 mg/kg body wt) and xylazine (3 mg/kg body wt). Before exsanguination by incision of the pulmonary artery after sternotomy, each animal received an injection of ethomidate (2 mg/kg body wt IV; Hypnomidate, Janssen). For the histological studies, the carotid arteries were carefully removed, placed in cryoprotective medium on cork disks, and snap-frozen in liquid nitrogen. For in vitro RNA and protein analysis, tissue from the same animals was directly frozen in liquid nitrogen.

Probes and Labeling Procedure
For in situ and in vitro RNA analyses, the recombinant cDNA clones pBSIIa1Col8, complementary to human procollagen {alpha}1(VIII) mRNA, and cG3PDH (Clontech), complementary to human G3PDH mRNA, were used. The in vitro transcription was performed according to the manufacturer's protocol with digoxigenin-labeled UTP (Boehringer Mannheim).

Northern Blot Analysis
Total RNA (0.114±0.0421 µg/mg tissue) was isolated according to Chirgwin et al.27 For Northern blot analysis, 2.0 µg total RNA was fractionated by electrophoresis under denaturing conditions on a 1.1% agarose/formaldehyde gel.28 Hybridization was performed as previously described29 at 72°C with either {alpha}1(VIII) procollagen or G3PDH antisense riboprobes (50 ng/mL). Detection was done by use of a modified detection protocol (Boehringer Mannheim) and the chemiluminogenic substrate CSPD (Tropix/Serva).

Western Blot Analysis
Twenty-five milligrams of tissue was ground to powder under liquid nitrogen, then lysed in 250 µL of sample buffer (20% SDS, 0.1 mol/L Tris pH 6.8, 10 mmol/L EDTA) and homogenized by sonication. After protein estimation by Bio-Rad DC protein assay, 2.5% 2-mercaptoethanol was added to the samples. For Western blot analyses, 15 µg was fractionated by electrophoresis under denaturing conditions on a 7.5% polyacrylamide gel30 and transferred onto Immobilon-P transfer membranes (Millipore). For detection, the membranes were blocked with TBSM (10 mmol/L Tris pH 7.6, 0.15 mol/L NaCl, 5% fat-free milk, 0.1% Tween) and incubated with the anti–type VIII collagen antibody (1:150 in TBSM). After thorough washing, the membranes were incubated with sheep anti-mouse immunoglobulin conjugated with alkaline phosphatase (Boehringer Mannheim). The alkaline phosphatase staining procedure was performed as recommended in the dark with nitro blue tetrazolium salt (67.5 mg/mL; Biomol) and 5-bromo-4-chloro-3-indolyl phosphate (X-PO4; 35 mg/mL; Biomol) as substrates. Molecular weights were estimated by reference to standard proteins (Bio-Rad). To evaluate the relative expression, the membranes were scanned (Molecular Dynamics). The absorbance units (optical density) were evaluated by use of Image Quant (Molecular Dynamics) and Microsoft Excel software. For statistical analyses, Student's t test for independent groups was used.

In Situ Hybridization
In situ hybridization was performed on cryostat sections (5 µm) by methods modified from those previously described.29 Hybridization was done overnight at 52°C with 0.3 µg digoxigenin-labeled antisense or sense riboprobe/mL hybridization solution. To evaluate potential background from the hybridization and detection procedure, slides were incubated with hybridization solution only. In general, background was not observed.

For detection of the in situ hybridization signal, a modified anti-digoxigenin alkaline phosphatase protocol (Boehringer Mannheim) was used.29 As a control to the detection procedure, the antibody was omitted. The alkaline phosphatase staining procedure was performed in the dark overnight with nitro blue tetrazolium (67.5 mg/mL) and X-PO4 (35 mg/mL) as substrates. Sections were counterstained with methylene green and mounted with Kaiser's glycerine gelatin. Background was not observed in sections hybridized with the sense probe and in hybridization solution only. Neither the antibody nor the staining procedure gave background.

Immunohistochemistry
Cell types were identified as follows: (1) for SMCs, mouse anti-human {alpha}/{gamma}-actin (HHF35; Loxo) and (2) for macrophages, mouse anti-rabbit RAM11 (MG33; Dako) were used. Type VIII collagen was localized by use of a mouse monoclonal antibody against bovine type VIII collagen (C8; Medac). The secondary antibody/detection system was donkey anti-mouse immunoglobulin conjugated to Cy3 (Chemicon).

The immunohistochemical staining procedure was performed with modifications as described.29 Different conditions were found to be optimal for each of the antibodies, as follows: HFF35 (dilution 1:200) and type VIII collagen antibody (dilution 1:200), both at room temperature for 2 hours, and RAM11 (dilution 1:500) at room temperature for 1 hour. Sections were then treated for 1 hour at room temperature with the secondary antibody (Cy3; dilution 1:500). After a washing in PBS, the slides were mounted with Fluoromount mounting medium (BDH). Negative controls included substitution of the primary antibody by mouse immunoglobulins and omission of the primary antibody. The number of the RAM11 antigen–presenting macrophages was evaluated microscopically by calculating the number of positive cells in whole sections (10 sections per animal). Data are expressed as the mean±SD. For statistical analyses, Student's t test for independent groups was used. Values of P<0.05 were considered significant.

Confocal Laser Scanning Microscopy and Correlated Histology
Immunolabeled sections were examined by confocal laser scanning microscopy with a Leica TCS 4D, equipped with an argon/krypton laser and fitted with the appropriate filter blocks for detection of Cy3 and FITC fluorescence. The FITC channel was used for viewing autofluorescent vascular structure. The images were taken with simultaneous dual-channel scanning and transformed into projection views with sets of 5 consecutive single optical sections taken at 1-µm intervals.

Sections adjacent to those used for immunolabeling, stained with hematoxylin and eosin, were examined with standard bright-field optics for comparative histological examination.

Immunoelectron Microscopy
Arterial tissues from carotid arteries of rabbits were dissected and collected in cold buffered 2% formaldehyde solution. Subsequently, they were sectioned with the aid of an oscillating tissue slicer (FTB Vibracut). Sections {approx}50 µm thick were collected and washed in PBS for preembedding immunogold labeling. Immunogold labeling of type VIII collagen in the immersed tissue slices was done in PBS in the presence of 2% BSA and 0.001% sodium azide with the anti–type VIII collagen monoclonal antibody (C8; dilution 1:40; Medac) for {approx}8 hours. The slices were washed with PBS and BSA and then treated for 8 hours with a secondary goat anti-mouse IgG antibody conjugated to 6-nm colloidal gold (70 µg protein/mL diluted 1:5) at room temperature. The antibody (M 8642; Sigma) was conjugated to colloidal gold as previously described.31 Briefly, 6-nm colloidal gold particles were prepared by reduction of chloroauric solution.32 The gold suspension was concentrated {approx}60-fold by centrifugation at 13 000g. For coupling, 0.2 mL of this gold suspension was diluted 1:10 with double-distilled water. Coupling with the antibody was carried out according to standard procedures at pH 9.0, approximately the isoelectric point of immunoglobulins. In a titration series, small amounts of protein were added to the gold suspension. Stability and saturation of the protein gold complex were checked in 20-µL aliquots mixed 1:1 with 20% sodium chloride. Unstable complexes were indicated by precipitation and by change of red to bluish colors of the solution. The final red, stable immunogold complex was further stabilized with HEPES buffer (50 mmol/L, pH 7.2) and then by the addition of 2% BSA and 0.001 sodium azide. After immunolabeling, washing, and chemical refixation of the tissue slices with 2.5% glutaraldehyde, the specimens were conventionally stained with 2% osmium tetroxide. They were washed and dehydrated in a graded series of ethanol, embedded in epoxy resin for ultrathin sectioning, and photographed in a transmission electron microscope (Philips EM 201).


*    Results
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Cellular Composition of the Carotid Arteries
In carotid arteries of rabbits fed normal chow, SMCs were strongly and homogeneously immunoreactive with the HHF35 antibody, indicating SMCs of the contractile phenotype (Figure 1ADown). By contrast, in rabbits maintained for 6 weeks on a cholesterol diet, HHF35 staining had become heterogeneously distributed, with the strongest signals in the luminal aspect of the media (Figure 1BDown). This pattern was maintained in the 1-day and 10-day cholesterol cessation groups. At 5 weeks after withdrawal of cholesterol, however, HHF35 staining appeared patchy, and after 12 weeks, the staining was homogeneous and comparable to that observed in the carotid arteries of untreated rabbits (Figure 1CDown).



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  		Figure 1. Sequence of confocal images illustrating the occurrence {alpha}/{gamma}-actin in medial SMCs. A through C, Changes in {alpha}/{gamma}-actin expression as demonstrated by single labeling. A, In carotid arteries of normocholesterolemic rabbits, medial SMCs strongly and homogeneously stained for {alpha}/{gamma}-actin. B, After a 1% cholesterol–supplemented diet, medial SMCs were immunoreactive with the HHF35 antibody, with strongest signal in the luminal part of the media. C, Twelve weeks after withdrawal of cholesterol, the signal was homogeneous and comparable to that observed in the control arteries. Magnification x400. Arrowheads indicate the internal and external elastic laminae; a, adventitia; and lu, lumen.

In carotid arteries of normocholesterolemic rabbits, RAM11-positive cells (macrophages) were not present in the media but were observed sporadically in the adventitia. After cholesterol diet and in the 1-day group, increased numbers of macrophages (9.9±3.2 and 10.5±3.4, P<0.005 versus normal chow) were located in the adventitia (Figure 2ADown). Concomitantly with time after withdrawal of cholesterol, the number of RAM11-positive macrophages decreased (Figure 2BDown and 2CDown). The relative abundance and distribution of macrophages are summarized in the TableDown.



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  		Figure 2. Immunoconfocal visualization of RAM11-positive macrophages in rabbit carotid arteries in response to cholesterol feeding. A, After cholesterol diet, RAM11-positive macrophages accumulated in the adventitia (a, arrows). Concomitantly with time after withdrawal of cholesterol, the number of macrophages decreased. B, Ten days after withdrawal of cholesterol, the number of macrophages was markedly decreased. C, In the 12-week postwithdrawal group, macrophages were found sporadically. Magnification: A, x150; B and C, x400. Arrowheads indicate internal and external elastic laminae; m, media; and lu, lumen.


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  		Table 1. Occurrence of RAM11-Positive Macrophages

Distribution Pattern of {alpha}1(VIII) Procollagen mRNA and Type VIII Collagen
In carotid arteries of rabbits fed normal chow, in situ hybridization with the procollagen {alpha}1(VIII) antisense riboprobe revealed positive signals in the endothelium, the media, and the adventitia, although the relative intensity of the signal showed significant spatial variation. Focal accumulations of expressing cells were observed in the luminal and adventitial aspects of the media (Figure 3ADown). After feeding with the 1% cholesterol-supplemented diet for 6 weeks, the expression of type VIII collagen mRNA was decreased in all layers of the vessel wall. Expressing cells were located mainly in the endothelium. The medial SMCs were only sparsely labeled (Figure 3BDown). After withdrawal of cholesterol, the normal distribution patterns were reestablished. One day after withdrawal, the expression patterns of type VIII collagen mRNA resembled those of carotid arteries immediately after cholesterol feeding. Changes were observed as early as 10 days after cholesterol withdrawal (Figure 3CDown). Increased numbers of type VIII collagen mRNA–expressing SMCs were observed throughout the entire media (Figure 3CDown). With prolonged time after cholesterol withdrawal, the number of type VIII collagen mRNA–expressing cells increased (Figure 3DDown). By 5 weeks after withdrawal, a patchy distribution of type VIII collagen mRNA was observed in all layers of the carotid arteries. The distribution patterns after 12 weeks in part resembled those shown for carotid arteries of normocholesterolemic rabbits. However, the number of type VIII collagen mRNA–expressing SMCs was increased, particularly in the adventitial part of the media and also in the adventitia (Figure 3DDown). Hybridization with the procollagen {alpha}1(VIII) sense riboprobe generally produced no background.



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  		Figure 3. In situ expression of {alpha}1(VIII) procollagen mRNA in response to 1% cholesterol–supplemented diet and cholesterol deprivation. In situ hybridization with {alpha}1(VIII) procollagen antisense riboprobes. For control, sense riboprobes and hybridization solution only were used (not shown). A, In normal carotid arteries, cells expressing type VIII collagen mRNA were localized in the endothelium, media, and adventitia. B, In carotid arteries of cholesterol-treated rabbits, type VIII collagen mRNA–expressing cells were found mainly in the endothelium and the subendothelial region. After withdrawal of cholesterol, the normal distribution pattern was reestablished. C, In the 10-day postwithdrawal group, increased numbers of type VIII collagen mRNA–expressing cells occurred in the luminal and adventitial part of the media (m) and adventitia (a). D, The distribution patterns after 12 weeks almost resembled those demonstrated for carotid arteries of normocholesterolemic rabbits. Bright field. Magnification x200. Arrowheads indicate internal and external elastic laminae; lu, lumen.

To correlate the procollagen {alpha}1(VIII) mRNA with the type VIII collagen protein expression patterns, sections were immunostained with the mouse anti–type VIII collagen antibody. In carotid arteries of normocholesterolemic rabbits, type VIII collagen was located in some endothelial cells (ECs), in the media (in particular in the luminal and adventitial aspects), and in the adventitia (Figure 4ADown). After cholesterol diet, faint labeling occurred in the media, whereas the adventitia was devoid of label. However, type VIII collagen was deposited in the intimal space. And, as indicated by the strong and homogeneous staining, type VIII collagen was concentrated along the luminal aspect of the internal elastic lamina (Figure 4BDown). One day after withdrawal of cholesterol, the distribution patterns of type VIII collagen resembled those observed directly after cholesterol diet. The first changes in the distribution patterns of immunolabeling were observed in the 10-day group, concomitant with the change in the transcription patterns described above, when deposits of type VIII collagen were again found in the luminal aspect of the media and the adventitia (Figure 4CDown). After 5 weeks, type VIII collagen immunostaining was distributed in patchy fashion over the entire media, with the intimal space staining strongly for type VIII collagen, as in the 1-day and 10-day postwithdrawal groups. By 12 weeks after withdrawal (Figure 4DDown), the distribution patterns of type VIII collagen in part resembled those of the controls, with stronger labeling in the adventitial facing zone of the media and in the adventitia itself, although irreversible features were the deposition of type VIII collagen in the intimal space, its association with the internal elastic lamina, and small deposits of type VIII collagen beneath the internal elastic lamina. Consistent with these findings, immunogold labeling at the electron-microscopic level demonstrated clusters of gold marker within the intima (Figure 5Down), commonly in the form of "strings of pearls" along fibrous material extending from the internal elastic lamina (Figure 5Down, inset).



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  		Figure 4. Modulation of type VIII collagen distribution in response to cholesterol-supplemented diet and cholesterol deprivation. Immunoconfocal visualization of type VIII collagen using mouse anti–bovine type VIII collagen antibody and donkey anti-mouse conjugated with Cy3 as secondary antibody. A, Occurrence of type VIII collagen in carotid arteries of rabbits fed normal chow. Type VIII collagen was located in the endothelium, media (m), and adventitia (a). B, After cholesterol diet, the signal dramatically decreased. However, type VIII collagen accumulated in the intimal space. C, After withdrawal of cholesterol, the first changes were observed in the 10-day postwithdrawal group, with strong signal in the intimal space and adventitia. D, At 12 weeks after withdrawal, the distribution of type VIII collagen almost returned to the control patterns. Magnification x400. Arrowheads indicate internal and external elastic laminae; lu, lumen.



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  		Figure 5. Preembedding immunogold labeling (arrows) of type VIII collagen in the intima (i) of a hypercholesterolemic rabbit fed a 1% cholesterol diet for 5 weeks. Inset, In the electron micrographs, the gold marker is frequently seen as "strings of pearls" associated with fibrils extending from the surface of the elastica interna (e). ec indicates endothelium; lu, blood vessel lumen. Magnifications: x52 000; inset, x108 000; bars: 500 nm; inset, 200 nm.

In Vitro RNA and Protein Analyses
Northern blot analyses of procollagen {alpha}1(VIII) mRNA levels during the course of the experiment are shown in Figure 6Down. Cholesterol treatment resulted in a dramatic downregulation of the procollagen {alpha}1(VIII) mRNA expression. After withdrawal of cholesterol, the {alpha}1(VIII) procollagen mRNA level increased, returning almost to control levels by 10 days after withdrawal (Figure 6Down). The changes of type VIII collagen mRNA expression observed by Northern blotting mirrored those of the corresponding protein revealed by Western blot analyses (Figures 7Down and 8Down). The level of type VIII collagen was reduced after cholesterol treatment and remained so after 1 day of cholesterol withdrawal (P<0.005 versus control levels). After 10 days of withdrawal, protein synthesis increased, reaching the control level, in a pattern similar to that observed for {alpha}1(VIII) procollagen mRNA. The high levels were subsequently maintained up to 12 weeks.



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  		Figure 6. Top, Northern blot probed with {alpha}1(VIII) procollagen cRNA probes. RNA (4 µg) was derived from carotid arteries of normocholesterolemic rabbits (control), hypercholesterolemic rabbits (6w chol), and 10 days (10d), 5 weeks (5w), and 12 weeks (12w) after cholesterol withdrawal. The lower Northern blot shows the expression of G3PDH mRNA. The {alpha}1(VIII) procollagen cRNA probes detected 3 mRNA species, with lengths of 4.2, 3.6, and 2.8 kb. The majority of the mRNA transcripts were 2.8 kb. Transcription of {alpha}1(VIII) procollagen mRNA was markedly downregulated after cholesterol diet. Although expression of {alpha}1(VIII) procollagen mRNA was dramatically decreased after cholesterol diet, levels in the 10-day up to 12-week postwithdrawal groups were comparable to control levels.



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  		Figure 7. Western blot containing arterial extracts from control carotid arteries (normocholesterolemic rabbits), carotid arteries harvested after 1% cholesterol-supplemented diet for 6 weeks (6w chol) and 1 day (1d), 10 days (10d), and 12 weeks (12w) after withdrawal of cholesterol. Total protein (20 µg) was added to each lane. The antibody raised against bovine type VIII collagen bound to 3 bands, with molecular masses of 125, 65, and 50 kD. Type VIII collagen levels decreased in response to cholesterol diet. Cholesterol deprivation restimulated the synthesis of type VIII collagen, with control levels being reestablished after 10 days.



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  		Figure 8. Relative expression of type VIII collagen in carotid arteries of rabbits after they had received normal chow (control), after 1% cholesterol-enriched diet for 6 weeks (6wchol), and after cessation of cholesterol (1d, 10d, and 12w). Top, Relative expression of type VIII collagen. The absorbance units obtained from control arteries were set to 100%. Bottom, Membranes (n=4) were scanned and absorbance units evaluated by use of Image Quant and spreadsheet and statistical analysis software. Expression of type VIII collagen mRNA was significantly downregulated after cholesterol feeding and in the 1-day group (*P<0.05). After 10 days of withdrawal, protein synthesis increased to approach that of the control level. The high level was subsequently maintained up to 12 weeks.


*    Discussion
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The present study has examined the influence of cholesterol diet on the expression and deposition of type VIII collagen in rabbit carotid arteries. The principal novel findings to emerge are that (1) the transcription and synthesis of type VIII collagen in the vessel wall is modulated in response to a cholesterol-rich diet, (2) distinct patterns of spatial distribution arise from this process, and (3) the changed pattern of expression is reversible only in part on withdrawal of cholesterol diet. These conclusions are based on comprehensive experimental data from in situ hybridization and immunohistochemistry backed by Northern as well as Western blot analyses.

Previous studies have established that lipids have the capacity to modulate the synthesis of specific matrix molecules, notably fibrillar collagens.22 23 24 The present findings show that feeding of a cholesterol-rich diet leads to a highly regulated pattern of altered type VIII collagen expression and deposition in the vessel wall. Specifically, type VIII collagen is downregulated in the media and adventitia, but it accumulates in the intimal space. These opposite changes in the vascular ECM composition may potentially have a significant influence on the initial deposition of lipids in the vessel wall and thereby contribute to the progression of atherosclerosis. In this context, the altered expression and deposition of type VIII collagen represents an early atherosclerotic event, leading to the characteristic feature of a prominent layer of type VIII collagen in the intimal space.

The intimal ECM, a dense meshwork consisting of collagens, fibronectin, laminin, and proteoglycans, has previously been shown to be important in lipid and lipoprotein retention.33 34 Furthermore, several studies have provided evidence of the subendothelial deposition of lipoproteins for the development of atherosclerotic lesions.35 36 The vascular ECM involved in this process is produced by both ECs and subendothelial SMCs. The close spatial relationship to the endothelium and the lack of intimal SMCs allow us to suggest that deposition of type VIII collagen in the intimal space is due to enhanced secretory activities of ECs. As previously shown, EC-derived proteoglycans participate in the retention of lipoproteins by the subendothelial space.35 36 37 Our present data, combined with these earlier reports, raise the possibility that in the endothelium, type VIII collagen may be expressed concomitantly with the proteoglycans and both matrix components may have similar functions.

The association of type VIII collagen with microfibrillar components of many but not all reactive elastic fiber systems has been demonstrated by immunocytochemistry.38 Consistent with the observations of Sawada and Konomi38 and Kittelberger et al,39 in our model, elastic fibers of carotid arteries of rabbits fed normal chow show no significant labeling in association with the elastic fiber system. However, in rabbits fed a cholesterol-supplemented diet, we found that type VIII collagen was associated with microfibrillar components at the surface of elastic fibers, mainly at the luminal aspect of the internal elastic lamina. Sensitivity of type VIII collagen to neutrophil elastase has been reported. Kittelberger and coworkers40 concluded that the "inherited" susceptibility of type VIII collagen to proteinases may result in the disadvantageous susceptibility of the vascular endothelium to lytic attack by activated neutrophils.

Ultrastructural studies have previously indicated an association of lipoproteins with collagen fibers and elastic tissues in the intima.18 19 Trapping mechanisms of lipoproteins in the ECM by means of collagen, particularly type I and type III collagen, have been suggested.12 41 Although our present ultrastructural studies do not directly demonstrate association of lipoproteins specifically with type VIII collagen, these previous studies raise the possibility that such an association might occur.

In experimental hypercholesterolemia, the subendothelial aspect of the media reportedly contains elevated levels of oxidized LDL.1 5 6 42 43 44 In SMC culture experiments, it has been demonstrated that modified lipoproteins stimulate the synthesis of interstitial collagen, ie, type I and type III collagen.23 24 In contrast, expression of type VIII collagen mRNA by cultured human and porcine SMCs was downregulated after treatment with modified lipoproteins (data not shown). This in vitro effect was directly reflected by the missing label for type VIII collagen in the media immediately after cholesterol diet. Furthermore, the opposite effect of lipids on the expression of type VIII collagen by ECs and SMCs may indicate varying cell type–specific regulatory pathways. In addition, our findings show that cessation of cholesterol led to deposition of type VIII collagen in the luminal aspect of the media, directly adjacent to the internal elastic lamina. In rabbits, deposition and accumulation of fibrillar collagen after withdrawal of cholesterol has been described. Further studies will clarify the mechanisms that regulate the deposition of type VIII collagen in the media in response to cholesterol diet and after cessation of cholesterol and the role of physiologically different types of contractile SMCs in this context.

In our experiments, macrophages were observed only in the adventitia. Their number was inversely correlated with the levels of type VIII collagen. In contrast to other studies that suggested combined injury and hypercholesterolemia46 47 or inflammation48 as stimulators of the infiltration of monocytes and macrophages into the adventitia, our data give evidence that deposition of lipids in the adventitia might directly attract inflammatory cells. However, indirect mechanisms, such as lipid-induced ECM remodeling, might facilitate adventitial infiltration by inflammatory cells as well. In experimental aneurysm, the accumulation of monocytes/macrophages in the adventitia is reportedly associated with ECM remodeling, both by the synthesis of ECM-degrading enzymes and by the release of cytokines regulating the turnover of ECM components.49

In contrast to the studies of Bendeck et al50 and Sibinga et al51 in the rat balloon injury model demonstrating upregulation of type VIII collagen expression in the media in response to injury and platelet-derived growth factor, our studies in the rabbit model revealed a marked downregulation of type VIII collagen in the media due to cholesterol diet. Under our experimental conditions, we found no evidence for migratory activity of SMCs, and in particular, no intimal thickening. As reported by others,52 lipids even downregulate the migratory activity of SMCs in vitro, and a close relation of this effect to the ECM environment has been suggested. Nevertheless, our data are not in conflict with the hypothesis of Bendeck et al50 and Sibinga et al51 that upregulation of type VIII collagen in the media and intima is associated with increased migratory activity of SMCs and that coating with type VIII collagen supported a higher level of migration toward a source of platelet-derived growth factor in vitro.51 However, their data reveal very limited expression of type VIII collagen in noninjured rat carotids, whereas our study in the rabbit model demonstrated a marked expression of either mRNA or protein in the controls. This discrepancy might be due to differences in the vessel structure between rats and rabbits. Alternatively, traumatization of the media and endothelial injury induced by balloon denudation may render the 2 models not strictly comparable.

Taken together, our findings demonstrate that type VIII collagen is an ECM component that becomes reorganized in clearly defined patterns during early experimental atherosclerosis. Type VIII collagen expression is modulated in the presence of cholesterol and is likely to be a key player in the structural and functional remodeling of the newly growing intima.


*    Acknowledgments
 
This study was supported by the Innovative Medizinische Forschung (IMF) program, Münster, and the Heart Center, Muenster. We wish to thank Brigitta Milskemper, Sezan Maleki, Marianne Opalka, and Daniel Ziomeck for their expert technical assistance.

Received August 25, 1998; accepted March 16, 1999.


*    References
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