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Cardiovascular Research 1999 43(3):744-754; doi:10.1016/S0008-6363(99)00148-0
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Inducible nitric oxide synthase colocalizes with signs of lipid oxidation/peroxidation in human atherosclerotic plaques

Kristel M Cromheekea, Mark M Kockxb,*, Guido R.Y De Meyerc, Johan M Bosmansa, Hidde Bultc, Walter J.F Beelaertsb, Chris J Vrintsa and Arnold G Hermanc

aDepartment of Cardiology, University Hospital, Antwerp, Belgium
bDepartment of Pathology, AZ Middelheim, Antwerp, Belgium
cDivision of Pharmacology University of Antwerp (UIA) Wilrijk, Belgium

* Tel.: +32-3-280-4816; fax: +32-3-280-4816 mark.kockx{at}uia.ua.ac.be

Received 7 December 1998; accepted 13 April 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Advanced human atherosclerotic plaques are characterized by the abundant presence of the autofluorescent non-soluble lipid pigment ceroid, consisting of oxidized lipoproteins. The aim of the present study was to examine the topographical and cellular distribution of inducible nitric oxide synthase (iNOS or NOS II) within different stages of atherosclerosis and its colocalization with ceroid deposits and nitrotyrosine. Methods and results: Different stages of atherosclerosis were studied by immunohistochemistry on whole-mount longitudinal sections of carotid endarterectomy specimens. In the adaptive intimal thickening the predominant cell type were smooth muscle cells. The fatty streaks contained both smooth muscle cells and macrophages with an extremely low NOS II immunoreactivity. The advanced atherosclerotic plaques however, showed a very dense infiltration by macrophages, of which a subpopulation expressed NOS II as a vesicular immunoreactivity in their cytoplasm. These were mainly present around the necrotic core, in association with ceroid accumulation and nitrotyrosine. Fluorescence quenching microscopy showed the presence of NOS II on autofluorescent ceroid vesicles in the macrophages. Large extracellular ceroid granules were not NOS II immunoreactive. NOS II mRNA was detected by RT-PCR and the protein by Western blot in the plaque tissue but not in mammary arteries used as controls. Conclusion: Ceroid, nitrotyrosine and NOS II colocalized in late stages of atherosclerosis and were found around the necrotic core in the plaque. This could suggest that NOS II expression in macrophages is involved in oxidation and peroxidation of lipids, leading to ceroid formation.

KEYWORDS Atherosclerosis; Cholesterol; Macrophages; Nitric oxide; Platelets


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Advanced atherosclerotic plaques are characterized by the presence of ceroid [1–3]. Ceroid is an insoluble material composed of complexes between oxidized lipids, like polyunsaturated fatty acids, and proteins. Ceroid is initially present in the cytoplasm of macrophages but is later found as larger extracellular deposits in the necrotic core. The term ceroid is given to autofluorescent insoluble material, and is found in a variety of mammalian tissue, mainly in disease states. It retains the ability to stain with lipid-soluble dyes, such as oil red O. It is usually acid-fast and can be stained with the Ziehl-Neelsen method [1]. Ceroid is related to the lipofuscin group of pigments, and originates of oxidation and peroxidation of phospholipids and unsaturated fatty acids. Ceroid is found in all plaques but not in areas of diffuse intimal thickening of human aorta and coronary arteries. It marks sites of oxidative events [1]. The insoluble lipid is present as membrane-bound vesicles in macrophage-like cells, showing a characteristic ring structure suggesting that membrane-associated oxidative systems might be responsible for rendering the lipid insoluble [3].

Recent immunohistochemical studies demonstrated the expression of inducible nitric oxide synthase (NOS II) in human [4–6] and experimental [6,7] atherosclerosis. NOS II was found in macrophage-derived foam cells in fatty streaks of the human aorta, and particularly in macrophages around the necrotic core of advanced plaques of human aorta and carotid artery [4,5]. Although NO is known as an important vasodilator, its role in atherogenesis is controversial. NO may exert anti-atherogenic properties through inhibition of oxidative processes, inhibition of monocyte recruitment and inhibition of the proliferation of T cells and smooth muscle cells, but high doses of NO could be atherogenic via stimulation of apoptosis and matrix breakdown, and by the formation of the cytotoxic peroxynitrite (reviewed in [8]). The strong oxidant peroxynitrite, which is formed when NO reacts with superoxide anion, could be involved in ceroid formation. Peroxynitrite can indeed initiate peroxidation of polyunsaturated fatty acids, as indicated by the formation of F2 isoprostanes when plasma or human low-density lipoproteins are exposed to peroxynitrite [9–11].

The purpose of this study was therefore to examine the colocalization of ceroid and NOS II in different stages of the atherosclerotic plaque. Moreover, since peroxynitrite may nitrate tyrosine residues in proteins [12], the presence of nitrotyrosine residues was investigated as well. Nitrotyrosine residues have been documented in atherosclerotic plaques and are considered as signs of both NO and superoxide anion. For this purpose longitudinal sections of whole-mount carotid endarterectomy specimens were investigated since they have the advantage that different stages of atherosclerosis are present and can be compared within the same patient [13].


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
Human carotid endarterectomy specimens were obtained from patients (n=10) (Table 1) with a carotid stenosis >70%, as demonstrated by digital subtraction angiography and Duplex. The patients had a mean age of 67 years, 60% were male, 70% of them had neurological disorders (cerebrovascular accident or transient ischemic attack), 40% hypertension, 40% hypercholesterolemia and 30% diabetes mellitus. At this age advanced atherosclerotic plaques alternate with less advanced and even early stages of atherosclerosis in the same specimen. The specimens were opened along their longitudinal axis, and within 2 min of surgical removal one half of the specimen was fixed in 4% formalin and complete longitudinal sections of the paraffin embedded specimens were mounted on 3-aminopropyltriethoxysilane (APES) precoated slides. These whole-mount sections contained the inner wall of the distal common carotid artery, the proximal part of the external carotid artery and the carotid sinus (Fig. 1). Haematoxylin/eosin and Trichrome-Masson were performed on two adjacent sections of all specimens. A prolonged Ziehl-Neelsen stain was performed on the same section as the immunohistochemical stainings (see Section 2.3).


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  		Table 1 Summary of the samples and techniques used in this study

 

Figure 1
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  		Fig. 1 Projected tissue sections of a whole-mount carotid endarterectomy specimen used in this study. The specimen contains the atherosclerotic plaques of the arteria carotis communis (cc), the bifurcation with the sinus caroticus (sc), the arteria carotis interna (ci) and externa (ce). The tissue section was projected on a paper and the contours were traced. Each specimen contained remnants of media (M) where the specimen was cleaved by the surgeon, intimal thickening (IT), fatty streak (FS) and advanced atherosclerotic plaques (AP), which were mostly found in the sinus caroticus. Scale bar 0.5 cm.

 
The other half of six carotid endarterectomy specimens, as well as four mammary arteries obtained during bypass surgery (Table 1) were immediately frozen in liquid nitrogen for frozen sections, RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) to detect NOS II messenger RNA (mRNA), and Western blotting. Sudan IV (Scarlet Red Michaelis, BDH) was done on frozen sections, to stain the lipids in the fatty streak and the atherosclerotic plaque.

The investigation conforms with the principles outlined in the Declaration of Helsinki.

2.1 Classification of the different stages within the atherosclerotic plaques
The classification was based on the definitions of the American Heart Association [14–16].

2.1.1 Adaptive intimal thickening
This was defined as a region of the arterial wall from and including the endothelial surface at the lumen to the luminal margin of the media. The arterial intima is composed of smooth muscle cells and the interstitium contains mainly proteoglycans (Fig. 1) [14]. Lipids (Sudan IV stain) could not be detected.

2.1.2 Fatty streaks
They were classified as intimal thickenings with lipid accumulation, as demonstrated with Sudan IV. They were mostly characterized by layers of macrophage foam cells and lipid droplets within intimal smooth muscle cells and minimal coarse-grained particles and heterogeneous droplets of extracellular lipid [15]. This in contrast with the atherosclerotic plaque that contained regions of tissue damage (necrotic core). However, as stated by Wissler et al. [17], "The problem with this definition is the precise way of differentiating the fatty streak, defined by the AHA as a type I lesion with its abundant macrophages, from a type II lesion." Some fatty streaks were almost devoid of macrophages and were composed almost completely of fat-filled smooth muscle cells, as also demonstrated by Wissler et al. [17].

2.1.3 Atherosclerotic plaques
These regions were defined by the presence of foci of pronounced cell loss (necrotic or lipid cores), corresponding with type IV, V and VI lesions of the classification of the American Heart Association [16]. The region between the endothelium and the necrotic core was defined as the fibrous cap.

2.2 Immunohistochemistry
The following primary monoclonal antibodies were used: NOS II from Transduction Laboratories, {alpha}-smooth muscle actin from Sigma (St. Louis, MO, USA) CD68 (anti-macrophage) from Dako (Glostrup, Denmark).

The following polyclonal antibodies were used: NOS II from Affinity Bioreagents (USA) nitrotyrosine from Upstate Biotechnology, CD3 (anti-T lymphocyte), and myeloperoxidase from Dako. H2O2 (3%) was used for 15 min to inactivate the endogenous peroxidase activity. Trypsin (Fluka, 1.U./ml in 50 mM Tris–HCl) and citrate (1.8x10–3M citric acid and 8.2x10–3M sodium citrate) microwave (480 W) pre-treatment were performed for 10 and 15 min respectively. All antibodies were diluted in phosphate buffered saline. The monoclonal antibodies were detected by an indirect peroxidase antibody conjugate technique; the sections were incubated with a goat anti-mouse peroxidase antibody (Jackson) for 45 min. The polyclonal antibodies were detected by a swine anti-rabbit peroxidase-conjugated secondary antibody (Dako) and a peroxidase anti-peroxidase complex. For demonstration of the complex, 0.1% H2O2 was used as a substrate and 3-amino-9-ethyl carbazole (AEC) as a chromogen.

Double labeling immunohistochemistry was performed to demonstrate the colocalization of CD68 and NOS II, nitrotyrosine and NOS II, nitrotyrosine and CD68, CD3 and NOS II, {alpha}-smooth muscle cell actin and NOS II. The double staining was performed with a peroxidase detection system and an alkaline phosphatase with fast blue and fast red system.

2.3 Colocalization studies: successive immunohistochemistry
The sections were first stained for {alpha}-smooth muscle actin and digital images were captured. The sections were then destained (with 1% hydrochloric acid in 70% ethanol), as described previously [18]. Then the same sections were stained with a monoclonal antibody against CD68. By this method a perfect colocalization could be obtained. To check the destaining procedure, controls with omission of the primary antibody were included. After acquisition of the CD68 images, the sections were destained again, and then stained with the polyclonal antibody against NOS II. In this way, the secondary antibody (swine anti-rabbit) did not interfere with antibodies (mouse monoclonal) used in previous runs. Furthermore, negative controls with omission of the primary anti NOS II antibody were included. Finally, after acquisition of the NOS II images, the sections were destained again, and then stained for ceroid with the prolonged Ziehl-Neelsen procedure.

2.4 Quantification
The images were analyzed using a color image analysis system (PC Image Color, Foster Findlay Associates, Newcastle-upon-Tyne, UK). The whole-mount carotid endarterectomy specimen, consisting of a complete cast of the atherosclerotic carotid bifurcation, was projected on paper with an end magnification of 5x, and the contours of regions with the three different stages of atherosclerosis were traced for each specimen. This allows a mapping of the atherosclerotic plaques present in each specimen (Fig. 1). Subsequently, the immunoreactive area in eight different regions of 100x65 µm was quantitatively measured. A systematic procedure was used for the random selection of different regions. A numbered grid was placed over the projected carotid specimens, and using random numbers, regions from the adaptive intimal thickening (n=2), the fatty streak (n=2), and the atherosclerotic plaque (n=4) were selected to acquire images of the section stained for {alpha}-smooth muscle cell actin. Subsequently, images of the same regions were acquired after staining for CD68, NOS II and ceroid respectively. Thus, in addition to being a random procedure, the selection was blind with respect to the presence or absence of CD68 or NOS II reactive material. The segmentation of the immunoreactive area was done by interactive selection of the gray level corresponding with the brown color of the immunoreactive regions as described previously [19]. The diameter of the ceroid vesicles was measured with a computer analyzing system which gives the diameter of the vesicles on the images captured on the computer screen.

2.5 Fluorescence and quenching microscopy
Since ceroid is strongly autofluorescent, quenching by immunostaining for NOS II was used as an indication for colocalization. Therefore the sections were immunohistochemically stained for NOS II and examined with fluorescence microscopy (Olympus fluorescence microscope excitation filter BP360–370) and light microscopy. If a colocalization exists the dark brown reaction product would quench the autofluorescence of ceroid. The immunostain was then destained with 1% hydrochloric acid in 70% ethanol and the autofluorescence was examined again.

2.6 Transmission electron microscopy
Fragments from the fibrous cap and necrotic core were removed from the paraffin blocks, deparaffinated and washed twice in toluol, rehydrated in a gradient of ethanol. They were fixed and embedded as described previously [19].

Sections (50-nm thick) were cut. They were stained for 30 min at 40°C with uranyl acetate and for 15 min at 20°C with lead citrate in an Ultrostainer 2168 (LKB, Bromma, Sweden). The sections were examined in a Jeol-1200 EX transmission electron microscope at 80 kV. Photographs were made with electron microscopy film 4489 Estar thick base (Kodak).

2.7 RNA extraction and RT-PCR analysis
Total RNA was isolated using the guanidinium isothiocyanate and phenol–chloroform extraction method. RNA was isolated from six carotid endarterectomy specimens and four mammary arteries. The latter were obtained from patients undergoing bypass surgery, and did not contain atherosclerotic lesions. RT-PCR was performed with an automatic thermal cycler (MultiCycler PTC-200, MJ Research, Watertown, MA, USA) using a one-step RT-PCR system (Titan, Boehringer Mannheim, Mannheim, Germany). The AMV-reverse transcriptase was applied for first strand synthesis and an enzyme blend, which consisted of Taq DNA polymerase and Pwo DNA polymerase for the PCR part. The following specific primers were used: human NOS II mRNA sense (5'-ATG CCA GAT GGC AGC ATC AGA-3', exon 8), human NOS II mRNA antisense (5'-ACT TCC TCC AGG ATG TTG TA-3', exon 11) [20]. Human β-actin mRNA sense (5'-CAG GCA CCA GGG CGT-3' (155–169)) and human β-actin mRNA antisense (5'-ATG GCT GGG GTG TTG AAG-3' (419–436)) primers were used as controls [21]. The final concentrations in the RT-PCR mixture (50 µl) were: sense- and anti-sense primers 0.4 µM each, dNTP 0.2 mM each, dithiothreitol 5 mM, MgCl2 20 1.5 mM, RNAse inhibitor 8 U, total RNA 1 µg and enzyme mix 1 µl. Reverse transcription was performed at 50°C for 30 min. The thermocycling parameters were: denaturation of the template at 94°C for 2 min, ten cycles consisting of incubations at 94°C for 30 s, 55°C for 30 s, 68°C for 45 s, 25 cycles consisting of incubations at 94°C for 30 s, 55°C for 30 s, 68°C for 45 s plus cycle elongation of 5 s for each cycle, followed by a prolonged elongation time for 7 min at 68°C. Products were analyzed by agarose gel electrophoresis (2.5%, Gibco BRL Life Technologies, Belgium) and visualized by Sybr Green I nucleic acid gel stain (FMC BioProducts, Rockland, ME, USA) under ultraviolet light. Negative controls with omission of AMV reverse transcriptase were included.

2.8 Western blot analysis
Carotid endarterectomy segments (n=6) (100 mg tissue/segment) and mammary arteries (n=4) (100 mg tissue/segment) were pulverized separately in liquid nitrogen and homogenized in Tris buffered saline (sodium chloride 0.9%, 20 mM Tris–HCl, pH 7) containing Triton X-100 1% and phenylmethylsulphonyl fluoride 1 mM. Extracts containing 50 µg of total protein were loaded on a 12% SDS–polyacrylamide gel and the separated proteins were electrophoretically transferred to nitrocellulose membranes (Hybond Ecl, Amersham). The membrane was blocked in Tris buffered saline with 0.1% Tween, 5% non-fat dry milk and probed with the polyclonal antibody to NOS II (1/1000 dilution, Affinity Bioreagents) overnight, followed by a peroxidase-linked secondary antibody (1/1000 dilution, Dako). Detection was performed with electrochemical luminescence detection reagents (Amersham) by autoradiography.

2.9 Statistical analysis
The immunoreactive area of {alpha}-smooth muscle cell actin, CD68, NOS II and ceroid per fixed cross-sectional area (65 000 µm2) was compared between the three types of vascular lesions (intimal thickening, fatty streak, atherosclerotic plaque) using one-way analysis of variance (ANOVA). Multiple comparisons were made with the Bonferroni test. If the variances were not homogenous, transformed data (logarithm or square root) were used. Fisher exact probability test was used to compare the presence of NOS II mRNA in mammary arteries and carotid endarterectomy specimens. The SPSS package for Windows (SPSS, Chicago, IL, USA) was applied for these purposes. A 5% level of significance was selected.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Adaptive intimal thickening
This stage was often present in the communis and the distal part of the interna (distally from the carotid sinus, Fig. 1), regions which also contained adjacent fragments of the inner media. The predominant cell type in the adaptive intimal thickening were smooth muscle cells (8.5±1.8% immunoreactive (IR) area) that strongly expressed {alpha}-smooth muscle actin (Fig. 2). Sudanophilia was almost not found in these regions.


Figure 2
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  		Fig. 2 Representative digital images of three stages of atherosclerosis, present in a single section of an endarterectomy specimen. The tissue section was successively stained for {alpha}-smooth muscle cell actin (first column), CD68 (second column), NOS II (third column) and finally acid-fast ceroid (fourth column). First row: adaptive intimal thickening; this region which was mainly present in the proximal carotis communis regions and the carotis interna regions, consisted almost exclusively of smooth muscle cells. Macrophages (CD68), NOS II, and ceroid were not present. Second row: fatty streak; the fatty streak, which was randomly distributed and alternated with advanced atherosclerotic plaques, contained besides lipid laden smooth muscle cells (arrow head), a layer of foam cells of macrophage origin (arrow). The presence of lipid was demonstrated by Sudan IV (not shown). Third row: advanced atherosclerotic plaques; the atherosclerotic plaques were mainly found at the sinus caroticus. These regions did not show {alpha}-smooth muscle actin expression. The gray-brown color (*) represented endogenous pigment, but does not correspond with immunoreactivity, since this pigment is also present in unstained sections. Macrophages were abundant. A giant cell of macrophage origin which is CD68 immunoreactive, shows a strong signal for NOS II and contains acid fast material, compatible with ceroid accumulation (arrow). Some macrophages (arrow head) were CD68 immunoreactive but did not show NOS II expression. Scale bar: 48 µm.

 
Macrophages, as detected by immunoreactivity for CD68 (0.03±0.01% IR area), were absent. NOS II immunoreactivity (0.05±0.02% IR area) was also absent. Ceroid was not found in this region (0.4±0.2% stained area) (Fig. 2 first row, Fig. 3). In addition to the negative controls (not shown) the lack of immunoreactivity of CD68 and NOS II further proved that the destaining procedure caused a complete wash out of the AEC deposits and that the primary antibody used in the previous run, did not interfere.


Figure 3
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  		Fig. 3 Quantification of the relative immunoreactive areas of {alpha}-smooth muscle cell actin, CD68, NOS II and acid fast ceroid in the three different stages of atherosclerosis (intimal thickening, fatty streak, atherosclerotic plaque) are shown. In intimal thickening and fatty streak, smooth muscle cells predominated, whereas macrophages with NOS II immunoreactivity predominated in the atherosclerotic plaque. *P<0.05 vs. intimal thickening, +P<0.05 vs. fatty streak (one way ANOVA, followed by the Bonferroni test).

 
3.2 Fatty streaks
These regions, which were randomly distributed and alternated with the advanced atherosclerotic plaques, were composed of smooth muscle cells that express {alpha}-smooth muscle cell actin (4.0±1.4% IR area) with layers of macrophage foam cells and lipid droplets. A significant fraction of these smooth muscle cells showed intracellular fat accumulation as shown by the sudanophilia. The layers of macrophages were present, as shown by the CD68 immunoreactivity (1.2±0.3% IR area) (Fig. 2, row 2). In these regions NOS II was barely detectable (0.4±0.1% IR area) (Fig. 2, row 2), although occasionally some smooth muscle cells and macrophages with a faint vesicular reactivity for NOS II were found. Fatty streaks were generally devoid of immunoreactivity for nitrotyrosine, and ceroid was not found by a prolonged Ziehl-Neelsen stain (0.3±0.08% stained area) (Fig. 2 row 2, Fig. 3). The first observation, together with the negative control (omission of the primary antibody for NOS II) again demonstrated the effectiveness of the wash out procedure for the antibodies, also controls concerning the maintenance of the antigenicity of the antibody were included, and were positive.

3.3 Advanced atherosclerotic plaques
Most advanced plaques were located at the outer wall of the carotid sinus, opposite the bifurcation flow divider (Fig. 1). The plaques showed a shoulder, a fibrous cap and a necrotic core. Around the necrotic core macrophages were found which were mostly foam cells (12.7±1.3% IR area) (Fig. 2 row 3, Fig. 3). In these regions {alpha}-smooth muscle actin immunoreactive cells (1.7±0.5% IR area) were nearly absent. The macrophages contained ceroid (5.5±1.5% stained area) and a subpopulation was strongly immunoreactive for NOS II (5.2±2.1% IR area). The use of a H2O2 pre-treatment was not responsible for the oxidation of the lipids with ceroid formation, since specimens without any treatment did show the presence of ceroid. NOS II was present as a vesicular immunoreactivity within the macrophages. A fraction of these macrophages consisted of giant cells (Fig. 2 row 3). The NOS II immunoreactive vesicles were of a variable diameter ranging from 0.5 to 7 µm. NOS II immunoreactivity was often present as a ring at the periphery of these vesicles, whereas their lipid rich center was not immunoreactive. Extracellular ceroid deposits in the necrotic core were not associated with NOS II or nitrotyrosine immunoreactivity.

3.4 Double labeling immunohistochemistry
The colocalization of NOS II in the macrophages was also demonstrated by this method. NOS II was not detectable in the smooth muscle cells by our methods. Nitrotyrosine, a marker of oxidative stress, was present in the same region as the NOS II immunoreactive macrophages (Fig. 4). The immunoreactive vesicular aspect of nitrotyrosine in the cytoplasm of the macrophages was almost identical to that of NOS II. T lymphocytes (CD3) were abundantly present in the regions of the NOS II containing macrophages, but did not contain ceroid or NOS II themselves. Myeloperoxidase was present in the granulocytes around the ceroid containing macrophages, but not in the macrophages themselves. (Fig. 4).


Figure 4
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  		Fig. 4 Double immunohistochemical staining. (A) NOS II (blue) is present in the macrophages (CD68), a subpopulation of the macrophages show the granular NOS II immunoreactivity (arrows), some macrophages do not contain NOS II (arrow heads). (B) There is a strong colocalization between nitrotyrosine (brown) and NOS II (blue) (arrows). However, a small fraction of the NOS II immunoreactive cells do not show nitrotyrosine (arrow heads) (C) Also a colocalization of macrophages (CD68, red) and nitrotyrosine (brown) is present (arrows). (D) T lymphocytes (CD3, brown) (arrows) are present in the vicinity of the macrophages, but do not contain NOS II (blue). (E) NOS II (blue) (arrows) is not present in the smooth muscle cells (brown) present in the fibrous cap overlying the necrotic core. (F) Myeloperoxidase (arrows) is present in granulocytes around the ceroid containing macrophages, but not in the macrophages themselves. Scale bar: 34 µm.

 
The monoclonal and the polyclonal antibody against NOS II gave an identical staining pattern.

3.5 Fluorescence and quenching microscopy
Ceroid was strongly autofluorescent but when immunohistochemically stained for NOS II, the fluorescence of the intracellular ceroid vesicles was quenched by the dark red-brown AEC deposits. To prove that the dark areas were not due to the absence of ceroid, the sections were destained and the autofluorescence of ceroid reappeared (Fig. 5). This quenching and dequenching demonstrated the strong colocalization between the vesicular autofluorescent intracellular ceroid and NOS II expression in human advanced atherosclerotic plaques. This was in strong contrast with the extracellular ceroid pigment which was autofluorescent but did not show quenching by the NOS II antibody. The extracellular ceroid was located in the acellular regions of the necrotic core.


Figure 5
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  		Fig. 5 Quenching fluorescence microscopy: since ceroid is strongly autofluorescent, the amount of quenching by the dark brown reaction product of the immunohistochemical staining for NOS II, is used as an indication for colocalization. The first column (A,D,G) shows the brown-red immunoreactive staining for NOS II, with light microscopy. The second column (B,E,H) shows the same images as in the first column, but with fluorescence microscopy. The third column (C,F,I) are the same images with fluorescence microscopy but after destaining the NOS II immunoreactivity. The first row shows the fatty streak, the second row the atherosclerotic plaque, and the third row the a higher magnification of the NOS II immunoreactive macrophages, containing ceroid. FC, fibrous cap; NC, necrotic core. Arrow: immunoreactivity in D, respectively G, quenches fluorescence in E, respectively H. This autofluorescence reappears after destaining of NOS II stain in F, respectively I. Scale bars: A–F: 180 µm, G–I: 18 µm.

 
Most of the intracellular ceroid vesicles that were quenched by the NOS II antibody show a similar quenching by the nitrotyrosine antibody, only rare intracellular ceroid vesicles were not quenched by these antibodies. This was in strong contrast with the extracellular ceroid deposits which were not quenched by either the NOS II and the nitrotyrosine antibody.

The quenching of the autofluorescence in the cytoplasm of the macrophages with the antibody against CD68 was less complete since most of the ceroid vesicles did not stain. The antibodies against {alpha}-smooth muscle cell actin and CD3, which recognize different cell types like smooth muscle cells and lymphocytes did not quench the ceroid vesicles at all.

3.6 Transmission electron microscopy
The ceroid containing macrophages were cut and the paraffin section and examined by TEM. The macrophages were easily recognized by pronounced accumulation of multilaminated structures [1] in their cytoplasm (Fig. 6). These structures show a labyrinth of membranes. In the center of these membranous structure a lipid vacuole of variable size can be recognized (Fig. 6). These multilaminated bodies are packed together and piled in the cytoplasm of the macrophages and form the ceroid content of the macrophage/foam cell.


Figure 6
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  		Fig. 6 Transmission electronmicroscopic photograph of one single ceroid vesicle in the cytoplasm of a macrophage in the atherosclerotic plaque. (A) Magnification: 15 000x. CB, ceroid bodies; N, nucleus. The ceroid vesicles consisted often of a central lipid droplet surrounded by multilaminated membranous structures (60 000x).

 
3.7 RT-PCR
Total RNA from the advanced atherosclerotic plaques was extracted and RT-PCR was performed with human NOS II primers. The NOS II message was represented by a 371-bp band (Fig. 7). In each carotid endarterectomy specimen (six out of six advanced atherosclerotic plaques) NOS II mRNA was detected. In the mammary artery specimens (n=4), NOS II mRNA was not found (P=0.005 Fisher’s exact probability test). The RNA extracts of all specimens produced a strong positive reaction for β-actin mRNA represented by a 282-bp band, indicating that the isolation of mRNA from all arteries had been successful (Fig. 7). The negative controls (omission of the AMV reverse transcriptase) did not yield a signal (results not shown).


Figure 7
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  		Fig. 7 Representative example of RT-PCR for NOS II and β-actin mRNA: the first lane represents the message in a mammary artery in which NOS II was not detectable. Lane 2 represents a carotid endarterectomy specimen in which the NOS II message (371-bp band) is seen. Lanes 3 and 4 represent the β-actin message (282 bp) in, respectively, a mammary artery and a carotid endarterectomy specimen. The last lane represents a DNA ladder.

 
3.8 Western blot
Atherosclerotic plaque tissues of the same carotid endarterectomy specimens showed a specific band at Mr 130 000, corresponding with NOS II. The antibody used for this Western blotting was the same as used for immunohistochemistry which demonstrated its specificity. In the mammary artery NOS II protein was not detectable (Fig. 8).


Figure 8
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  		Fig. 8 Western blot of NOS II in a mammary artery (lane 1), and in atherosclerotic endarterectomy specimens (lanes 2–4). The atherosclerotic arteries show a band at Mr 130 000 (lanes 2–4) which is specific for NOS II, the mammary artery (lane 1) is strictly negative.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The present study compared the distribution of NOS II, nitrated proteins and ceroid, a complex of oxidized lipids with proteins, in different stages of atherosclerotic plaque formation. Complete specimens of human atherosclerotic plaques and adjacent non-atherosclerotic intimal thickening obtained during carotid endarterectomy, were examined. By this approach we could compare different stages of atherosclerosis in one plaque of the same patient, with respect to expression of NOS II, nitrated proteins and ceroid. The early stages of atherosclerosis in these older patients were comparable with early stages of atherosclerosis in younger persons [22].

The data clearly demonstrate that NOS II and nitrotyrosine are predominantly present in the advanced atherosclerotic plaque, absent from the intimal thickening, and almost absent from the fatty streak. The immunoreactivity of both NOS II and nitrotyrosine was found in the macrophages and foam cells around the necrotic core of advanced atherosclerotic plaques.

T cells were often found around NOS II expressing macrophages but we were not able to detect ceroid or NOS II immunoreactivity in the cytoplasm of T cells themselves. This is in contrast with the findings of Esaki et al. who found NOS II in macrophages and T cells of rabbit atherosclerotic plaques [7], and with Sakuma et al. who found ceroid in circulating T lymphocytes in Hermansky–Pudlak syndrome [23]. The presence of T cells in the vicinity of the NOS II expressing macrophages points to a complex interplay between both cell types in the regulation of NOS II in human plaques. The very low expression of NOS II and nitrotyrosine residues in the smooth muscle cells of the normal vessel constituents (media and intimal thickening) and of the early atherosclerotic lesions (fatty streak) is in accordance with the report by Wilcox et al. [5], but is different from the results of Buttery et al. [4]. The reasons for the latter discrepancy are unclear, but might be due to differences in sensitivity and/or specificity of the polyclonal antisera; which were raised against murine NOS II [present study] or a synthetic peptide from human hepatocyte NOS II [4]. An alternative explanation, that NOS II was present in smooth muscle cells, which had lost expression of {alpha}-smooth muscle actin, seems less likely in view of the different topographical distributions of smooth muscle cells and NOS II expressing cells.

In extension to the work of Wilcox et al. [5], who used in situ hybridization with a probe directed against the 5' end of cDNA of a NOS II isolated from human hepatocytes and could not detect NOS II mRNA in atherosclerotic plaques, we detected NOS II mRNA by means of RT-PCR. The difference could be due to a greater sensitivity of the latter technique, or a shorter delay between tissue removal and fixation. The size of the RT-PCR transcript matched the length reported by Vouldoukis et al. [20], who used the same primers for human macrophage NOS II. The protein NOS II was detected by Western blot and indicated the specificity of the antibody used.

We focused on the colocalization of NOS II with markers of nitration and oxidative stress. NOS II was present in the advanced plaques in macrophages that also contained nitrated proteins. Nitrotyrosine and nitrated proteins are considered as signs of the formation and activity of the NO-derived oxidant peroxynitrite [4,12]. Colocalization of NOS II and nitrotyrosine indicates that NOS II was enzymatically active and that the produced NO reacts with superoxide (O2–) to form peroxynitrite (OONO–) which then nitrates tyrosine residues of proteins [24]. The presence of hypochlorous acid, a reaction product catalyzed by myeloperoxidase may also catalyze oxidative reactions in the vascular wall [25,26]. Although we found myeloperoxidase only in the granulocytes in regions around the ceroid containing macrophages, the presence of myeloperoxidase in the macrophages has been described Daugherty et al. [25]. The nitrite–hypochlorous mixture acid demonstrated the initiation of lipid oxidation and nitration [26]. This suggests that NOS II metabolites secreted into atherosclerotic environment may contribute to lipid peroxidation, and is a conceivable explanation for the presence of myeloperoxidase, NOS II and nitrotyrosine in the same regions in the atherosclerotic plaque.

This also explains the novel finding of this study that the NOS II expressing macrophages also contained ceroid. This insoluble material consists of complexes of oxidized lipids with proteins and is considered as a marker of oxidative stress [1–3]. Ceroid was characterized ultrastructurally by rings of multilaminated membranes around soluble lipid droplets, confirming the studies of Mitchinson et al. [3]. Oxidative mechanisms of macrophages are known to be membrane-associated [27]. Multilaminated membranous structures were also described in foam cells after platelet phagocytosis [28]. In that study the cytoplasm of the macrophages contained various amounts of phospholipids, that were derived from platelet membranes and had a myelin-like aspect. Thus phagocytosis of platelets followed by oxidation and peroxidation of phospholipids or unsaturated fatty acids could be responsible for the ceroid formation. In the present study NOS II was often seen as a pronounced ring at the periphery of the vesicles containing the multilaminated ceroid, whereas an unstained halo marked the central area of these vesicles in which lipid droplets reside. This points to the role of NOS II in the active process of ceroid formation in the macrophages, since extracellular ceroid, present in the necrotic core did not show NOS II immunoreactivity. Interestingly a close colocalization was recently reported between iron and ceroid in human atherosclerotic plaques [29]. The electron dense iron deposits were present in the lipid ring structures of the ceroid pigment [29]. It is well known that oxidation of LDL and lipids is greatly enhanced in the presence of hemoglobin/iron [30], leading to the accumulation of ceroid in these cell. Iron accumulation in the atherosclerotic plaque could indeed be derived from hemoglobin as a result of red blood cell phagocytosis, into the macrophages via the scavenger receptor [31], also explaining the abundancy of nitrated proteins in the ceroid containing macrophages since heme proteins, are easily nitrated by enzymatically synthesized NO [32].

The autofluorescence of ceroid was quenched by the NOS II immunoreactivity. This indicates that NOS II was present on the ceroid vesicles in the macrophages. However, the exact subcellular localization has to be investigated further. This colocalization of NOS II and ceroid could suggest that NOS II is involved in the oxidation and peroxidation of the lipids leading to the formation of ceroid.

Another explanation for the colocalization of NOS II and ceroid could be that oxidative stress can induce NOS II. NOS II is a nuclear factor {kappa}B (NF{kappa}B)-dependent gene [33] that may be upregulated by oxidative stress. Work by our group has shown that oxidized LDL can induce NOS II in macrophages [34], though less efficiently than the combination of lipopolysaccharides and interferon-{gamma} [34,35]. Furthermore, ceroid itself could cause upregulation of NOS II as a foreign body reaction of the macrophage to the presence of insoluble ceroid. Upregulation of NOS II in response to phagocytosis of microorganisms like intracellular bacteria and foreign materials by the macrophages has been described [36,37].

The presence of NOS II and nitrotyrosine are strong indicators that high levels of NO and superoxide anion were formed at those sites. NO or peroxynitrite could lead to plaque destabilization by the induction of cell death [5,13] and matrix breakdown [38]. Indeed recently the presence of smooth muscle cell apoptosis in the vicinity of activated macrophages of human atherosclerotic plaques, has been demonstrated [13,39]. Although the nature of the macrophage derived factor responsible for smooth muscle cell killing is not known yet, the formation of high levels of NO by NOS II could be involved.

In conclusion, NOS II mRNA and protein were expressed in the advanced plaques. A novel repetitive staining technique was used to study the cellular distribution of NOS II. NOS II was predominantly present in a subpopulation of macrophages, where it showed a strong colocalization with intracellular ceroid and nitrotyrosine. The latter finding suggests that NOS II contributes to lipid peroxidation and oxidative stress in those cells, and could form an explanation for the deposition of ceroid in atherosclerosis.

Time for primary review 35 days.


   Acknowledgements
 
The authors wish to thank Johannes Muhring, Michiel Knaapen (APCAM), and Anne-Elise Van Hoydonck for their expert technical assistance. M.M.K. is a holder of a fund for fundamental clinical research of the Flemish Fund for Scientific Research (FWO), G.R.Y.De M. is a research associate of the Flemish Fund for Scientific Research (FWO), this work was supported by a research grant (1998) of the Bekales foundation and the FWO levenslijn project 7.0014.98.


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

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