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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1233-1238.)
© 1997 American Heart Association, Inc.

Articles

Increased Immunolocalization of Paraoxonase, Clusterin, and Apolipoprotein A-I in the Human Artery Wall With the Progression of Atherosclerosis

Bharti Mackness; Roger Hunt; Paul N. Durrington; ; Michael I. Mackness

From the Departments of Medicine (B.M., P.N.D., M.I.M.) and Pathology (R.H.), University of Manchester, The Royal Infirmary, UK.

Correspondence to Bharti Mackness, University Department of Medicine, The Royal Infirmary, Manchester M13 9WL, UK.

	Abstract

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Abstract Using immunolocalization techniques, we have shown that paraoxonase (Pon), clusterin, and apolipoprotein (apo) A-I accumulate in the artery wall during the development of atherosclerosis. In normal aortas (n=6) there were low levels of extracellular Pon, clusterin, and apoA-I immunoreactivity. The cytoplasm of smooth muscle cells in the media showed granular positivity for both Pon and apoA-I, indicating that these proteins were undergoing lysosomal degradation. This activity was also indicated by the presence of both intact and degradation products of Pon in smooth muscle cells as shown by Western blotting. With the progression of disease from fatty streaks (n=3) to advanced atherosclerosis (n=8) there was an increase in Pon, apoA-I, and clusterin immunoreactivity, indicating the increasing presence of these proteins with disease progression. These proteins are the components of a specific HDL subspecies that has been implicated in the prevention of peroxidative damage to phospholipids in LDL and membranes. The increase in Pon, clusterin, and apoA-I during the development of atherosclerosis may therefore represent a protective response to the oxidative stress associated with the development of atherosclerosis.

Key Words: atherosclerosis • paraoxonase • clusterin • high-density lipoprotein

	Introduction

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The accumulation of lipid-laden foam cells, derived from macrophages and SMCs, is one of the characteristic early changes in the arterial intima at the site of developing atherosclerotic plaques.¹ Although a detailed understanding of the process leading to the transformation of macrophages and SMCs into foam cells is currently lacking, one theory suggests that the oxidative modification of LDL is critical.^{2 3} Several cell types have been shown to oxidize LDL in vitro, including endothelial cells, SMCs, and macrophages, all of which are present in the arterial wall. Monocyte-derived macrophages of the type present in the subintimal space avidly take up oxidized LDL in vitro to become foam cells, but this uptake is not mediated through the LDL receptor. In fact, as LDL becomes oxidatively modified, it loses its ability to bind to these receptors. Instead, macrophage uptake involves different receptors. These are the scavenger receptors,⁴ which also recognize LDL modified by acetylation, and the oxidized-LDL receptors.^{5 6}

Several previous immunolocalization studies have identified the presence of elevated levels of both normal and oxidatively modified LDL and HDL in atherosclerotic lesions.^{7 8 9 10} Levels of other macromolecules such as lipoprotein(a) and fibrinogen also accumulate in atherosclerotic regions of arteries.^{9 10 11}

Previous reports from our and other laboratories have shown that the HDL-associated enzyme Pon can prevent the accumulation of lipid peroxides in LDL incubated under oxidizing conditions.^{12 13 14} Clusterin is a glycoprotein found in many biological fluids. It is induced in cells surrounding several kinds of pathological lesions and is believed to protect cell membranes from damage and to participate in wound-repair processes.^{15 16} Pon is associated with a specific HDL particle also containing clusterin and apoA-I,¹⁷ and it has been suggested that this HDL particle may have a specific protective role in decreasing the accumulation of lipid peroxides in membranes and lipoproteins.^{18 19}

To be effective in preventing lipid peroxidation of LDL and its damaging consequences to cells of the artery wall, the Pon-containing HDL particle would be expected to be present in tissues in which LDL is subjected to increased oxidative stress, eg, within the artery wall. Herein we report the colocalization of Pon, clusterin, and apoA-I within the artery wall and an increase in these HDL components in the artery as atherosclerosis becomes more advanced.

	Methods

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Human Tissue Procurement
Normal aortic tissue was obtained from six people (three male; three female, aged 6 to 26 years) who died as the result of road traffic accidents. Specimens of diseased aortas were obtained from 11 patients (8 male; 3 female, aged 52 to 81 years) undergoing coronary artery bypass grafting or aortic bypass grafting surgery at the Manchester Royal Infirmary. All tissues were washed by flushing with PBS and fixed within 8 hours in 4% neutral buffered formalin for a minimum of 12 hours at 4°C before being embedded in paraffin wax.

Histological Examination
Serial sections (4 µm thick) of the wax-embedded samples were dewaxed. Every sixth section was stained with hematoxylin and eosin to permit the evaluation of the overall morphology of the specimens (R. Hunt). The five sections between these were used for immunostaining. Specimens stained with hematoxylin and eosin were classified as normal, having fatty streaks/intimal fibrosis, or having advanced lesions with a cholesterol-rich core.

Antibodies
Sheep polyclonal antibody directed against human apoA-I was obtained from Immunodiagnostics and used at a dilution of 1:50. Monoclonal antibody directed against the NH₂-terminal synthetic decapeptide of -smooth muscle actin was obtained from Sigma Chemical Co. and used at a dilution of 1:400. Monoclonal antibody directed against human clusterin was a gift from Dr Richard James (University of Geneva) and used at a dilution of 1:10. Sheep polyclonal antibody to human apoB was affinity purified using protein A–Sepharose²⁰ and used at a dilution of 1:100. Rabbit polyclonal antibody to human serum Pon was affinity purified using protein A–sepharose and used at a dilution of 1:10. Details of the specificity of these last two antibodies have been described previously.^{21 22}

The monospecificity of our polyclonal antibody to human serum Pon was established by the presence of a single immunoprecipitation arc on crossed immunoelectrophoresis of human serum and by the lack of immunoreactivity against human serum albumin or pure apoA-I (both obtained from Sigma), using Western blotting. In this system, the antibody reacted only with the Pon doublet of HDL prepared by ultracentrifugation (Fig 1) and with no other HDL protein. The rabbit polyclonal anti-Pon antibody also gave an identical pattern of immunostaining to that of the anti-Pon monoclonal antibody F41F2-K on Western blotting of purified Pon after two-dimensional gel electrophoresis.²³

View larger version (116K): [in this window] [in a new window]   Figure 1. Immunolocalization of Pon, clusterin, and apoA1 in normal and atherosclerotic human aortas. Details of the immunostaining procedures are given in "Methods." Normal human aorta stained for Pon (A), apoA-I (B), and clusterin (C); fatty streak stained for Pon (D), apoA-I (E), and clusterin (F); and advanced atherosclerosis stained for Pon (G), apoA-I (H), and clusterin (I) are shown. Magnification x75.

Affinity-purified biotinylated goat anti-rabbit IgG and rabbit anti-goat IgG (heavy and light chain) were obtained from Sigma.

Immunohistochemistry
Serial sections (4 µm thick) of the wax-embedded samples were dewaxed in xylene and rehydrated through a series of graded alcohols. The sections were washed in TBS, pH 7.4, and incubated with goat or sheep serum as appropriate to the secondary antibody for 10 minutes to block nonspecific binding. Excess serum was removed by washing in TBS, and the primary antibody was applied overnight at 4°C. After three 10-minute washes in TBS, samples were incubated with the appropriate secondary IgG conjugated to biotin for 1 hour at room temperature. The sections were washed three times for 10 minutes each in TBS and then incubated with avidin-biotin alkaline phosphatase reaction complex (Dako). After three 10-minute washes with TBS, antibody binding was visualized by using a naphthyl phosphate/fast red substrate mixture (Sigma). Endogenous alkaline phosphatase activity was blocked using levamisole (Sigma). Sections were counterstained lightly with Mayer's hematoxylin to permit evaluation of the overall morphology of the specimens. Negative controls included (1) omission of the primary antibody and (2) substitution of the primary antibody with an affinity-purified anti-IgG (Sigma) appropriate to the species used to raise the primary antibody at the same concentration as the primary antibody.

Assessment of Immunostaining
The degree and pattern of immunostaining both with and between specimens was assessed by standard light microscopy. The intensity of staining was graded minimal (+), moderate (++), intense (+++), or very intense (++++), the latter corresponding to the highest level of immunoreactivity observed in the positive control. The observer (M.I. Mackness) was blinded to the source of the specimens, which were arranged in random order (B. Mackness) before grading.

Preparation of Vascular Cells and HDL
HDL (d=1.063 to 1.21) was prepared by sequential ultracentrifugation.²⁴ Lymphocytes were prepared from peripheral human blood by Ficoll gradient centrifugation with lymphocyte separation medium (Flow Laboratories) according to the manufacturer's instructions. HUVECs were a gift from Dr B. Edwards (Department of Renal Medicine, Manchester Royal Infirmary) and human aortic SMCs were a gift from Dr M. Patel (Department of Clinical Pharmacology and Therapeutics, St Mary's Hospital, London, UK). Both endothelial cells and SMCs were frozen immediately after isolation and washing in PBS.

Western Blotting
The different cell types were solubilized in PBS containing 1% Triton X-100 by sonication (3x20 seconds) at 4°C. The amount of protein in each lysate was quantitated by the bicinchoninic acid method.²⁵ Cell lysates and HDL were subjected to 12.5% SDS–polyacrylamide gel electrophoresis, each well containing 15 µg of protein. After electrotransfer to nitrocellulose membrane under a constant current of 80 mA for 1 hour, the membrane was blocked with 3% bovine serum albumin and 0.05% Tween 20 in TBS. Then the membrane was incubated with 106 µg/mL rabbit anti-human Pon IgG. Subsequently, the membrane was incubated with a secondary horseradish peroxidase–conjugated anti-rabbit IgG antibody, and Pon bands were visualized in 0.3% hydrogen peroxide with freshly prepared 3,3'-diamino-benzidine tetrahydrochloride as substrate.

	Results

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Controls
No immunostaining was found in sections processed with omission of the primary antibody or with its substitution with the appropriate anti-IgG (results not shown).

Normal Aorta
In the normal human aortic tissue, there were low levels of immunostaining for apoB, apoA-I, clusterin, and Pon (Fig 2, Table). The cytoplasm of SMCs in the media showed granular positivity for both apoA-I and Pon, indicating the possibility that these proteins were undergoing lysosomal degradation. ApoA-I and Pon had both an intracellular and extracellular distribution, whereas apoB and clusterin appeared to have an extracellular distribution only.

View larger version (94K): [in this window] [in a new window]   Figure 2. Immunolocalization of Pon, clusterin, and apoA1 in vasa vasorum. Details of the immunostaining procedures are given in "Methods." Shown are immunostaining for Pon (A), apoA-I (B), and clusterin (C). Magnification x75.

View this table: [in this window] [in a new window]   Table 1. Immunolocalization of HDL/LDL Components in Human Aortic Tissue

SMCs in the vasa vasorum from normal aortas also showed low levels of cytoplasmic staining for apoA-I and Pon. These proteins were also present in the interstitial fluid between the cells, as was apoB. However, clusterin was not detectable in the vasa vasorum (Fig 3).

View larger version (148K): [in this window] [in a new window]   Figure 3. Western blot analysis for the presence of Pon in human HDL, blood lymphocytes, aortic SMCs, and umbilical vein endothelial cells, using a rabbit anti-human Pon antibody. Lane 1 shows molecular weight standards; lane 2, lymphocytes; lane 3, SMCs; lane 4, endothelial cells; and lane 5, HDL. The analysis was performed as detailed under "Methods."

Fatty Streaks
The sections from the aortas with fatty streaks had intimal fibrosis, with lipid deposition in the cytoplasm of SMCs of the subintimal media. The architecture of the media in this location had been lost, while that of the outer media was intact. More intense immunostaining was evident for all the proteins studied than in normal aortas. This was particularly evident for apoB, apoA-I, and Pon (Fig 2, Table). All four antigens (apoB, apoA-I, Pon, and clusterin) appeared to have both an intracellular and extracellular distribution, although it was difficult to determine whether cells were intact in these sections.

Advanced Atherosclerosis
In the aortas with advanced atherosclerosis, there was intimal fibrosis, with a complete loss of the architecture of the artery wall and large areas of cholesterol deposition. There was very intense noncellular immunostaining of apoB, apoA-I, Pon, and clusterin (Fig 2, Table), indicating massive accumulation of these proteins in atherosclerotic tissue.

Western Blot
We determined whether HUVECs and SMCs contained Pon by Western blot analysis by using a rabbit anti-human Pon antibody. HDL contained two immunoreactive bands of Pon (Fig 1). The doublet pattern is typical for human serum Pon and appears to represent two oxidation states of the enzyme.²⁶ Low levels of Pon staining, corresponding to the position of the bands from HDL, were found in both HUVECs and SMCs. However, several bands of lower molecular weight could also be seen in both cell types. These bands could either represent cellular forms of Pon, which cross-react with the antibody, or be degradation products of serum and interstitial fluid Pon. Little or no reactivity toward the anti-Pon antibody was found in the blood lymphocytes studied.

	Discussion

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Several previous studies have shown components of both LDL and HDL to be present in the artery wall. Both of these lipoproteins can diffuse through the endothelium into the subintimal space. HDL, because of its smaller size, is present in the vessel wall at higher concentrations than LDL.²⁷ This is, however, the first investigation to immunolocalize apoA-I, Pon, and clusterin in normal and atherosclerotic aortas. Kaesberg et al⁸ have previously shown that in normal aortas SMCs have weak, diffuse labelling for both apoB and apoA-I and more intense immunostaining of the extracellular matrix. Previous studies have also indicated increases in normal and modified apoB (probably reflecting increases in oxidized LDL), lipoprotein(a), apoA-I, and clusterin in atherosclerotic artery walls.^{7 8 9 10 11 15 16 28}

In normal aortas, we found low levels of immunostaining for apoB, apoA-I, Pon, and clusterin. ApoA-I and Pon were found in the cytoplasm of SMCs, indicating that they were internalized and possibly undergoing degradation within lysosomes. However, whereas apoA-I and Pon also showed an extracellular distribution, neither apoB nor clusterin showed an intracellular distribution. This observation could indicate a dissociation of clusterin in the artery wall from the specific HDL subspecies in which apoA-I, Pon, and clusterin are believed to be associated in the circulation.^{17 26} Clusterin was also not present with Pon and apoA-I in the vasa vasorum. Clusterin in atherosclerotic plaques is believed to be derived from stores in platelet granules released into the extracellular fluid after platelet activation.²⁸ However, local synthesis by vascular cells cannot be excluded. Therefore, the different distribution of clusterin in normal aorta and its absence from the vasa vasorum could be indicative of either a lack of local synthesis or low levels of platelet activation in nondiseased vessel walls. It is also possible that clusterin associates with apoA-I and Pon in HDL only after it has been released into the interstitial fluid in the vessel wall.

When we used Western blot analysis with a monospecific anti-Pon antibody, degradation products of Pon appeared to be present in vascular endothelial cells and SMCs but not in peripheral blood monocytes. This finding suggests that there is uptake of Pon by vascular endothelial cells and SMCs. The possibility also exists that vascular endothelial cells and SMCs synthesize Pon. Pon mRNA has thus far been found only in human liver cells,²⁹ but vascular cells have not been previously investigated in this respect. Several tissues are known to contain Pon activity; however, their identity with serum Pon is unknown.³⁰ Recently, Primo-Parmo et al³¹ have shown that Pon is a member of a multigene family and, using a sensitive reverse transcription–polymerase chain reaction technique, have shown that mRNA for Pon 1 (human serum Pon) is present in a number of tissues in the mouse apart from the liver. However, whether these tissues synthesize active Pon is not known. These authors did not investigate artery wall cells, although, interestingly, white blood cells did not contain Pon 1 mRNA in their investigation and did not stain for Pon in the present investigation. The identity of the Pon reactive bands found in SMCs and endothelial cells in our study (Fig 1) remains to be established; however, whole homogenates of these cells hydrolyzed paraoxon at a low rate (8.05 and 26.3 pmol · min^-1 · mg^-1 protein for SMCs and endothelial cells, respectively). At present, we are unable to determine whether this activity is due to serum Pon or cell-specific Pon. This is the subject of further investigation in our laboratory. The local synthesis of some HDL components has also been found previously.^{32 33 34} ApoE is the only apolipoprotein known to be synthesized by macrophages in the vessel wall³² ; however, it can also be synthesized by aortic SMCs.³³ In the cornea, which is an avascular tissue, stromal keratocytes show positive immunostaining for apoA-I, which, it has been suggested, is due to local synthesis.³⁴ However, it is also possible that HDL can diffuse into the corneal stroma from the limbic vasculature.³⁴ It is therefore possible that vascular endothelial cells and SMCs could synthesize Pon in response to certain stimuli such as oxidative stress. It seems likely that the Pon deposited in the artery wall is serum Pon, due to the relatively high concentration of HDL in the interstitial fluid. However, we cannot dismiss the possibility that some of the Pon comes from vascular cells. We have shown vascular endothelial cells and SMCs to contain active Pon, and these may contribute to the Pon deposited in the artery wall. Other cells present in atherosclerotic lesions, such as macrophages or mast cells, may also contribute; however, this is unknown at present. In addition, several tissues have been shown to synthesize clusterin,^{15 16} possibly in response to cytotoxic stimuli.

In our study, apoB, apoA-I, Pon, and clusterin all showed increased intensity of immunostaining with the progression of atherosclerotic disease. These HDL components may have accumulated in atherosclerotic tissue as the result of increased quantities of HDL being trapped in the artery wall. Pon and apoA-I are known to aggregate due to the very hydrophobic N-terminal end of Pon, which results in tight association of the two proteins. It is therefore possible that the Pon, apoA-I, and clusterin are deposited in the artery wall of diseased vessels due to simple chemical reactions between these proteins, leading to the formation of insoluble complexes. However, Pon has been shown in vitro to decrease the oxidative modification of LDL, possibly by a mechanism, which appears to involve the removal, by hydrolysis, of the damaging lipid peroxides formed by the oxidation of the polyunsaturated fatty acyl groups of phospholipid.^{13 14} It has also been suggested that clusterin functions as an acceptor of oxidatively damaged components of cell membranes, perhaps as a stage in their removal for disposal.¹⁵ It is an attractive hypothesis, therefore, that under normal circumstances, the specific Pon/clusterin HDL particle may serve to protect against the damaging consequences of lipid peroxidation and that elevated levels of Pon and clusterin in atherosclerotic tissue may be a response to increased oxidative stress. However, the system is presumably overwhelmed by increasing LDL oxidation in the vessel wall of arteries in which atherosclerosis progresses. This hypothesis, however, depends on the presence of active Pon in atherosclerotic tissue. The technology to answer the question is not available at the present time; therefore, the hypothesis will remain unproven until we have the ability to colocalize Pon activity and protein in the same vascular regions.

In conclusion, we have shown that Pon, apoA-I, and clusterin accumulate in the artery wall during the progression of atherosclerosis. These components are associated with a specific HDL subspecies in the circulation, which is believed to prevent lipid peroxidation. It is therefore possible that this HDL subspecies accumulates in the artery wall during the progression of atherosclerosis or that its components are elaborated there in response to an increase in oxidative stress.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein HUVEC = human umbilical vein endothelial cell Pon = paraoxonase SMC = smooth muscle cell TBS = Tris-buffered saline

	Acknowledgments

 
Monoclonal antibody directed against human clusterin was a gift from Dr Richard James (University of Geneva, Switzerland). HUVECs were a gift from Dr B. Edwards (Department of Renal Medicine, Manchester Royal Infirmary, UK) and human aortic SMCs were a gift from Dr M. Patel (Department of Clinical Pharmacology and Therapeutics, St Mary's Hospital. We thank M.G. Walker and B. Chapman for the provision of aortic tissue.

Received May 29, 1996; accepted September 25, 1996.

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