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(Circulation. 1996;94:3103-3108.)
© 1996 American Heart Association, Inc.

Articles

C-Type Natriuretic Peptide in Human Coronary Atherosclerotic Lesions

Takahiko Naruko, MD; Makiko Ueda, MD; Allard C. van der Wal, MD; Chris M. van der Loos, PhD; Hiroshi Itoh, MD; Kazuwa Nakao, MD; Anton E. Becker, MD

the Department of Cardiovascular Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands (T.N., A.C.W., C.M.L., A.E.B.); the Department of Pathology, Osaka City University Medical School, Osaka (M.U.); and the Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Japan (H.I., K.N.).

Correspondence to Prof Anton E. Becker, Department of Cardiovascular Pathology, Academic Medical Center, University of Amsterdam, PO Box 22700, 1100 DE Amsterdam, the Netherlands.

	Abstract

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Background C-type natriuretic peptide (CNP) belongs to the natriuretic peptide family and is considered to have regulatory effects on vascular tone and smooth muscle growth. Since these features play a role in atherogenesis, the presence of CNP at such sites was studied.

Methods and Results Thirty-three coronary artery segments were harvested at autopsy: 10 normal, with diffuse intimal thickening, and 23 atherosclerotic lesions. Samples were snap-frozen and processed for immunohistochemical staining. For the identification of CNP, a mouse monoclonal antibody (KY-CNP-1) was used. 1A4, EBM-11 (CD68), and von Willebrand factor antibodies were used to stain smooth muscle cells, macrophages, and endothelial cells, respectively. CNP is present in several cell types. Normal arterial segments show CNP-positive endothelial cells. Hypercellular atherosclerotic lesions show distinct CNP positivity of smooth muscle cells and macrophages but a decrease in positivity of endothelial cells. Advanced atherosclerotic lesions contain CNP-positive macrophages, but the smooth muscle cells within the fibrous cap and the surface endothelial cells are almost all CNP-negative.

Conclusions These observations suggest that CNP has functional significance in atherogenesis.

Key Words: natriuretic peptides • muscle, smooth • atherosclerosis

	Introduction

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The natriuretic peptide family consists of three components: ANP,^{1 2} BNP,³ and CNP.⁴ The major production sites for ANP and BNP are in the heart.^{5 6 7 8} CNP was initially found mainly in the central nervous system and considered to act principally as a neuropeptide.^{9 10 11} Recently, however, CNP production has been found also in bovine and human endothelial cells,^{12 13 14} and gene expression of CNP and its specific receptor (ANP-B) have been detected in the human aorta.¹⁵ Moreover, other studies indicate that CNP is expressed also by monocytes/macrophages.^{16 17}

It is presently well established that ANP and BNP act as cardiac hormones, which play an important role in regulating vascular tone and blood pressure.^{1 2 3 5 6 7 8} The function of CNP, on the other hand, is less well known. Initially, CNP was shown to act as a vasoactive peptide, like ANP and BNP, causing a decrease in blood pressure.⁴ More recently, Furuya et al^{18 19} and Porter et al²⁰ demonstrated that CNP stimulates intracellular levels of cGMP in cultured vascular SMCs and thus acts as a potent inhibitor of SMC proliferation. Suga et al^{12 21} found that cytokines (ie, transforming growth factor-ß [TGF-ß], interleukin-1 [IL-1], and tumor necrosis factor- [TNF-]) stimulate the production of CNP from bovine endothelial cells. Ishizaka et al¹⁶ demonstrated the production of CNP in a human monocyte cell line and its augmentation after transformation into macrophages.

It thus appears that CNP could act as another hormone with regulatory effects on vascular tone and SMC growth. This is of interest, since accumulation of SMCs is an important feature of growth of atherosclerotic plaques.²² Moreover, the vasomotor activity of the arterial wall at the site of atherosclerotic lesions is altered.^{23 24} Under these circumstances, SMC function is under the influence of secretory products of both endothelial cells, and macrophages and several growth factors and vasoactive substances have been identified.^{22 25 26 27} The role of CNP has thus far not been evaluated in this setting, and it is not known whether this hormone is present at sites of atherosclerotic plaques.

	Methods

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Thirty-three native coronary artery segments were harvested at the time of autopsy (16 patients; age range, 11 to 77 years). Ten segments contained diffuse intimal thickening but otherwise were normal. Twenty-three contained atherosclerotic lesions. The latter were characterized histologically as hypercellular lesions (n=7) or advanced atherosclerotic lesions (n=16). The hypercellular lesion was defined as a cell-rich intimal lesion, predominantly composed of SMCs and macrophages but without an extracellular lipid core. The advanced atherosclerotic lesions were further divided into fibrous plaques (n=5), intermediate (fibro-lipid) plaques (n=8), or lipid-rich plaques (n=3).²⁸

The coronary arteries were removed from the epicardial surface, and a 2-mm slice from each segment was snap-frozen and stored at -80°C.

The snap-frozen samples were subsequently sectioned serially at 6-µm thickness and fixed in acetone. Every first section was stained with hematoxylin and eosin; the other sections were used for immunocytochemical staining.

Immunohistochemical Staining
The source, specificity, and working dilution of all antibodies used in this study are summarized in the Table. For the identification of CNP, a mouse monoclonal antibody (KY-CNP-1) was used that was obtained by immunizing BALB/c mice with purified CNP. The method of antibody production and specificity testing have been reported.²⁹

View this table: [in this window] [in a new window]   Table 1. Monoclonal Antibodies Used in the Study

The sections were then subjected to a three-step staining procedure, with the use of streptavidin-biotin complex with HRP for color detection. Tris-buffered saline was used for washing between the subsequent incubation steps. HRP activity was visualized with 3-amino-9-ethylcarbazole, and the sections were faintly counterstained with hematoxylin.

The double immunostaining was based on either two primary antibodies of different IgG subclasses (1A4/CD68) or one primary unlabeled antibody and one biotinylated antibody (CNP/CD68-biotin), according to modifications of procedures previously reported.³⁰ For double staining with CNP/1A4, a new method has been developed.³¹ In short, the procedure involved the use of one unlabeled antibody (CNP) and one directly HRP-labeled antibody (1A4/EPOS-HRP). The following incubations were performed in subsequent order: CNP antibody, biotinylated goat anti-mouse immunoglobulin (Dako), alkaline phosphatase-conjugated streptavidin (Dako), normal mouse serum (Dako), 1A4/EPOS-HRP (Dako), rabbit anti-HRP (Dako), and ß-galactosidase–conjugated goat anti-rabbit immunoglobulin (Southern Biotechnology Associates). Finally, the enzymatic activities of ß-galactosidase and alkaline phosphatase were visualized in turquoise (BioGenex kit, San Ramon) and in red (New Fuchsin kit, Dako), respectively.

The specificity and results obtained with KY-CNP-1 were checked by omission of the primary antibodies and use of a nonimmune mouse IgG antibody (Dako) as negative control. Rat pituitary gland served as a positive control.¹⁰ Moreover, KY-CNP-1 neutralization with a relevant peptide was performed on frozen sections of human coronary arteries with advanced atherosclerosis and on frozen sections of rat pituitary.

Area Quantification of CNP-Positive Endothelial Cells
The number of CNP-positive endothelial cells was quantified with the use of serial sections stained with vWf and KY-CNP, respectively, followed by computer-aided planimetry. The immunostained areas were outlined, and the amount of CNP-positive endothelial cells was calculated as a percentage of the total endothelial cell area and expressed as a "CNP-endothelial score."

Statistical Analysis
Results are expressed as mean±SD. Statistical comparisons between groups were performed by one-way ANOVA and post hoc multiple comparison with the use of Scheffe's test. Values of P<.05 were considered significant.

	Results

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In this study, CNP reactivity could be detected in endothelial cells, macrophages, and SMCs, although in highly variable proportions for each type of lesion. In all cases, however, the anti-CNP staining resulted in a characteristic finely granular intracytoplasmic staining pattern that was also found in the positive control section of rat pituitary gland. A weak cellular staining pattern resulted from a low number of granules in the cytoplasm, and strong reactivity resulted from high numbers of granules per cell and some diffuse cytoplasmic staining, but the granular pattern remained (see Fig 1). Neutralization experiments and incubation with nonimmune mouse serum gave a negative result (see Fig 2).

View larger version (517K): [in this window] [in a new window]   Figure 1. Sections illustrating the granular staining pattern obtained with KY-CNP-1. A, Endothelial cells (diffuse intimal thickening); B, macrophages (advanced atherosclerotic lesion); and C, smooth muscle cells (hypercellular atherosclerotic lesion). Despite differences in staining intensity, the granular appearance remains clearly visible. Magnification x1060.

View larger version (662K): [in this window] [in a new window]   Figure 2. Display of control sections. A and B, Rat pituitary gland: A, positive staining with KY-CNP-1; B, absent staining after KY-CNP-1 neutralization. Magnification x640. C and D, Segment of an atherosclerotic lesion in a coronary artery: C, positive staining with KY-CNP-1; D, absent staining after use of a nonimmune mouse IgG antibody. Magnification x80.

Diffuse Intimal Thickening
CNP staining was observed primarily in the endothelial cells lining the luminal surface (n=10; 5 patients) (Fig 3). The mean CNP endothelial score (±SD) was 0.64 (±0.16). The double-stain method (CNP/1A4) revealed occasional weak positivity of SMCs in the intima.

View larger version (460K): [in this window] [in a new window]   Figure 3. Sections of a normal coronary artery with diffuse intimal thickening. A, Hematoxylin-eosin stain; B, adjacent section stained with vWf reveals positive staining of endothelial cells; C, staining with KY-CNP-1 shows positive staining of endothelial cells; no positivity in the intimal layer. Magnification x510.

Hypercellular Atherosclerotic Lesions
The mean CNP endothelial score (±SD) was 0.52 (±0.22) (n=7; 4 patients). Three hypercellular lesions contained only few macrophages, scattered throughout the intima, and these showed CNP positivity of >60% of the surface endothelial cells. Double immunostaining revealed that only a few SMCs and macrophages were CNP-positive. The four remaining hypercellular lesions contained numerous foci of macrophages. These lesions showed little CNP positivity of the surface endothelial cells at sites of atherosclerotic lesions. In two of these, both obtained from a 28-year-old patient, endothelial cells in the plaque-free wall stained positive for CNP, while endothelial cells at the site of the atherosclerotic plaque showed little CNP positivity (Fig 4). Double immunostaining revealed that most macrophages and some SMCs within the plaque were positive for CNP (Fig 4).

View larger version (829K): [in this window] [in a new window]   Figure 4. Sections of a hypercellular atherosclerotic lesion. A, Cross section through a coronary artery with an eccentric atherosclerotic plaque. Magnification x28. The area indicated by the arrow is shown in detail in B and C. The area indicated by the asterisk is shown in detail in D and E. B, Staining with vWf; endothelial cells are positive. The right-hand side corresponds with the plaque free wall; the left-hand side corresponds with the atherosclerotic lesion. C, Staining with CNP; the endothelial cells lining the plaque-free wall stain positive. Those lining the atherosclerotic lesion show little or no staining. D, Double immunostaining (CD68, blue/KY-CNP-1, red); most cells show double staining (purple), indicating CNP positivity of macrophages. E, Double immunostaining (1A4, turquoise/KY-CNP-1, red); a few smooth muscle cells show double staining (purple), indicating CNP positivity. Macrophages stain red. Magnification x280.

Advanced Atherosclerotic Lesions
In these lesions the mean CNP endothelial score (±SD) was 0.16±0.09 (n=16; 8 patients). In fibro-lipid plaques and in lipid-rich plaques, many CNP-positive cells were found in the lesions with preference for the shoulder area of the fibro-lipid plaque or for the fibrous cap of the lipid-rich plaque (Fig 5). Double immunostaining revealed that these CNP-positive cells were predominantly macrophages. In the fibrous plaques there were only a few clustered macrophages, some of which were CNP-positive.

View larger version (440K): [in this window] [in a new window]   Figure 5. Sections of an advanced atherosclerotic plaque. A, Double immunostaining (1A4, turquoise/CD68, red); the fibrous cap contains massive infiltration with macrophages (red) and only sparse smooth muscle cells (turquoise). B, Adjacent section stained with KY-CNP-1; the area that corresponds with the macrophage-rich fibrous cap zone in A, contains many CNP-positive cells. Surface endothelial cells do not stain. C, Adjacent section stained with vWf; the surface endothelial cells are positive. Calcifications are present that appear brown in A and blue in B and C. Magnification x44.

Area Quantification
Fig 6 illustrates CNP-positive endothelial scores in the three groups. CNP-positive endothelial score was significantly (P<.01) lower in advanced atherosclerotic lesions (0.16±0.09) than in diffuse intimal thickening (0.64±0.16) and hypercellular lesions (0.52±0.22).

View larger version (12K): [in this window] [in a new window]   Figure 6. CNP endothelial score for each case in each of the three groups. Mean±SD is indicated.

	Discussion

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To the best of our knowledge, this is the first study to demonstrate CNP-positive cells in coronary atherosclerotic lesions. The findings, moreover, suggest that the cellular components involved in CNP positivity in early and advanced atherosclerotic lesions are different. At early stages, endothelial cells, SMCs, and macrophages appear to be involved, whereas in advanced stages, only macrophages remain as the predominant source. Once clustered macrophages are present in the lesions, these macrophages become distinctly CNP-positive, while endothelial cell positivity for CNP diminishes.

What Are Potential Functional Implications?
Cell culture studies have shown that CNP inhibits proliferation of vascular SMCs.^{18 19 20} Moreover, intimal thickening of the carotid artery, after balloon injury in rats and rabbits, could be inhibited by CNP infusion.^{32 33} Atherosclerotic lesions show an increase in SMCs, and it is widely accepted that these growth processes are controlled by both growth-promoting and growth-inhibitory factors.^{22 26 27} The observation that early atherosclerotic lesions with an inflammatory component show less production of endothelial cell–dependent CNP, therefore, could indicate that this delicate balance is disrupted. In light of the above, it is of interest that a recent study has shown that oxidized low-density lipoprotein—considered a key factor in atherogenesis—suppresses the secretion of CNP by endothelial cells.³⁴ This raises the possibility that the effects of oxidized low-density lipoprotein, which results in augmentation of SMCs in atherosclerotic lesions, relate—at least in part—to its inhibitory effect on the endothelial cell–dependent production of CNP.

Our observations that SMCs present CNP in early atherosclerotic lesions suggest that SMCs themselves are capable of producing CNP. This observation certainly needs further verification since, to the best of our knowledge, the production of CNP by SMCs in humans has not been reported. Recently, however, CNP positivity of SMCs has been documented in the neointima of carotid arteries in the rat after balloon angioplasty.³⁵ Therefore, the question is raised as to whether CNP, apart from its potential paracrine effects, also may have an autocrine effect on SMCs.

It is of interest also that coronary arteries with early atherosclerotic lesions show a tendency to have enhanced vasomotor activity. It is known that this relates to a decreased production of endothelial cell–derived relaxation factor (NO). The finding of a decreased expression of CNP, however, raises the question of whether this substance could be involved also.

Recently, it has been shown that CNP can be secreted in vitro by human monocytes¹⁶ and by mouse macrophages.¹⁷ Our observations show distinct positivity of CNP on macrophages involved in the process of atherogenesis.

Since it is widely accepted that the chronic inflammatory process present in atherosclerotic lesions has a modulating effect on plaque composition, the potential for CNP to be involved functionally seems likely and certainly should be further explored.

Limitations of the Study
This immunocytochemical study cannot determine with certainty whether CNP observed in various cells represents production of CNP or receptor binding of circulating CNP. On the other hand, the granular cytoplasmic appearance strongly suggests that storage and/or synthesis of CNP has taken place in these cells. It is mandatory, therefore, to study the CNP receptor in order to evaluate whether the presence of CNP could have a biological effect on the tissues examined. We realize also that only a limited number of sections has been studied. We should not forget that the process of growth promotion and growth inhibition is a complex phenomenon that involves interaction of many factors. CNP is only one of these, and its functional significance cannot be ascertained without taking other factors into consideration. The present study, nevertheless, provides sufficient data to include CNP as one of the actors in the delicate interplay between growth-promoting and growth-inhibitory factors involved in atherogenesis and, hence, to warrant further studies.

	Selected Abbreviations and Acronyms

 

ANP = atrial natriuretic peptide BNP = brain natriuretic peptide CNP = C-type natriuretic peptide EPOS = enhanced polymer one-step HRP = horseradish peroxidase SMC(s) = smooth muscle cell(s) vWf = von Willebrand factor

	Acknowledgments

 
During this study, Dr T. Naruko was a research fellow from the Osaka City University Medical School, Osaka, Japan.

Received April 24, 1996; revision received July 15, 1996; accepted August 4, 1996.

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