Atherosclerosis and Lipoproteins |
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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Key Words: extracellular matrix remodeling animal model atherosclerosis smooth muscle cells
| Introduction |
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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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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
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
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 antitype 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
/
-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 antigenpresenting 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
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 antitype VIII collagen monoclonal antibody (C8;
dilution 1:40; Medac) for
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
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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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 2A
). Concomitantly with time after
withdrawal of cholesterol, the number of RAM11-positive
macrophages decreased (Figure 2B
and 2C
). The relative
abundance and distribution of macrophages are summarized in the
Table
.
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Distribution Pattern of
1(VIII) Procollagen mRNA and
Type VIII Collagen
In carotid arteries of rabbits fed normal chow, in situ
hybridization with the procollagen
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 3A
). 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 3B
). 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 3C
).
Increased numbers of type VIII collagen mRNAexpressing SMCs were
observed throughout the entire media (Figure 3C
). With prolonged
time after cholesterol withdrawal, the number of type VIII
collagen mRNAexpressing cells increased (Figure 3D
). 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 mRNAexpressing SMCs was
increased, particularly in the adventitial part of the media and also
in the adventitia (Figure 3D
). Hybridization with the
procollagen
1(VIII) sense riboprobe generally
produced no background.
|
To correlate the procollagen
1(VIII) mRNA with
the type VIII collagen protein expression patterns, sections were
immunostained with the mouse antitype 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 4A
). 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 4B
). 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 4C
).
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 4D
), 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 5
), commonly in the form of "strings of
pearls" along fibrous material extending from the internal elastic
lamina (Figure 5
, inset).
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In Vitro RNA and Protein Analyses
Northern blot analyses of procollagen
1(VIII) mRNA levels during the course of the
experiment are shown in Figure 6
.
Cholesterol treatment resulted in a dramatic downregulation
of the procollagen
1(VIII) mRNA expression.
After withdrawal of cholesterol, the
1(VIII) procollagen mRNA level increased,
returning almost to control levels by 10 days after withdrawal (Figure 6
). The changes of type VIII collagen mRNA expression observed
by Northern blotting mirrored those of the corresponding protein
revealed by Western blot analyses (Figures 7
and 8
).
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
1(VIII) procollagen mRNA. The high levels were
subsequently maintained up to 12 weeks.
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| Discussion |
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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 typespecific 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 |
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Received August 25, 1998; accepted March 16, 1999.
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