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1Department of Biomedical Sciences, 2Animal Physiology Research Unit, Agricultural Research Service, US Department of Agriculture, 3Dalton Cardiovascular Research Center, 4Department of Nutritional Sciences, and 5Departments of Medical Pharmacology and Physiology and Internal Medicine, University of Missouri, Columbia, Missouri 65211
Submitted 7 April 2003 ; accepted in final form 8 May 2003
ABSTRACT
A growing body of evidence supports the hypothesis that C-reactive protein (CRP) is a marker of inflammation in coronary artery disease. The purpose of the present study was to test the hypothesis that CRP correlates with macrophage accumulation during the initial stages of coronary vascular disease. Adult male pigs were fed a normal chow (NF) or a high-fat high-cholesterol (HF) diet for 20 wk. After 20 wk, blood was collected for analyses of interleukin-6 (IL-6), CRP, and lipids. After blood collection, the pigs were euthanized and the right coronary arteries (RCA) were harvested and fixed in neutral buffered formalin. Paraffin-embedded sections of RCA were stained immunohistochemically for CRP, scavenger receptor A (SRA), and monocyte chemoattractant protein-1 (MCP-1). All cholesterol fractions were elevated in the HF vs. the NF group (P < 0.05). There was little or no positive staining for CRP, SRA, or MCP-1 in the RCA of NF pigs, but there was extensive staining in lipidladen macrophage foam cells in the HF pigs. Double staining revealed colocalization of CRP with SRA and CRP with MCP-1 in foam cells. Serum IL-6 was below the assay detection limit in all pigs. Serum CRP correlated directly with plasma total cholesterol (R = 0.727, P = 0.041) and accumulation of SRA-positive macrophages (R = 0.938, P < 0.001) in RCA of HF pigs. We conclude that serum CRP correlates with macrophage accumulation and coronary artery disease in hypercholesterolemic pigs.
hypercholesterolemia; monocyte chemoattractant protein; scavenger receptor A
METHODS
Animals, diets, and biochemistry. All procedures involving animals were approved by the Animal Care and Use Committee of the University of Missouri and complied fully with those approved by the American Veterinary Medical Association Panel on Euthanasia. Sexually mature gonadally intact male Yucatan pigs (n = 16) between the ages of 9 and 12 mo were housed in a temperature-controlled room (20-22°C) with a 12:12-h light-dark cycle. Pigs were divided into body mass-matched groups (n = 8) that were fed once daily for 20 wk Purina minipig chow (NF) or the HF diet, which consisted of minipig chow supplemented to contain 2% cholesterol, 17.1% coconut oil, 2.3% corn oil, and 0.7% sodium cholate (3). Pigs were fed to maintain matched body mass throughout the study. At the end of 20 wk, blood was collected by syringe from the jugular vein into tubes without anticoagulant for analyses of interleukin-6 (IL-6; R & D Systems) and CRP (Tridelta Diagnostics) using solid-phase sandwich immunoassays. Analyses were conducted as outlined by the manufacturer, and all serum samples were analyzed within one assay. Anticoagulated blood was collected into a Vacutainer containing EDTA for analysis of triglycerides, total cholesterol, LDL cholesterol, and HDL cholesterol by methods previously reported (16).
Immunohistochemistry. After blood collection, the pigs were
anesthetized with intramuscular atropine, ketamine, and xylazine and
intravenous pentobarbital sodium, and the chest was opened to achieve
euthanasia. Samples of the right coronary artery (RCA) were fixed in neutral
buffered 10% formalin for 
24 h, embedded routinely in paraffin, and
sectioned serially at 5-µm thickness. Immunohistochemistry was performed as
reported previously (7) by
using primary mouse monoclonal antibodies to SRA (1:100; SRA-E5, Cosmo Bio)
(17) and CRP (1:500; Sigma
Chemical) (18) or rabbit
polyclonal antibody to MCP-1 (1:100; Peprotech). For double labeling, sections
stained for SRA or MCP-1 were stained sequentially for CRP, and the VIP
substrate kit for peroxidase (Vector) was used to visualize the second
reaction product before the section was counterstained
(18). Sections were
photographed with an Olympus BX40 photomicroscope and Spot Insight Color
camera (Diagnostic Instruments). The area of positive staining for SRA was
calculated as a percentage of total section area utilizing ImagePro Plus
(Media Cybernetics). Coronary lesions were graded according to Stary stage
(14).
Statistical analysis. Values are means ± SE. Differences were analyzed by Student's paired t-test. The association between CRP and total cholesterol and SRA staining was analyzed by univariate linear regression analysis. P < 0.05 was accepted as statistically different. All calculations were performed by using a standard statistical package (Sigma Stat).
RESULTS
All cholesterol fractions were elevated in HF vs. NF pigs as reported previously (16). Body mass (42.5 ± 2.2 vs. 43.4 ± 0.73 kg) was not different between groups at the end of the feeding period. There was little or no positive staining for CRP, SRA, or MCP-1 in RCA of NF pigs (data not shown).
Stary type I-III lesions developed in RCA of HF pigs. Foam cells in these lesions stained positively for CRP, SRA, and MCP-1 (Fig. 1). Small numbers of macrophages in the adventitia stained positively for SRA (Fig. 1B). The endothelium stained positively for all three proteins. CRP colocalized with SRA (Fig. 1C) and MCP-1 (Fig. 1D) in foam cells in doubly stained sections. There was multifocal positive staining for CRP and MCP-1 in smooth muscle cells of the media (Fig. 1, C and D).
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Serum IL-6 was below the 4.7 pg/ml detection limit of the assay in all pigs. Serum CRP was detected in all pigs and ranged from 0.8 to 35.2 mg/l. There was a trend (P = 0.16) for serum CRP to be higher in the HF than in the NF pigs (10.7 ± 4.3 vs. 3.7 ± 0.8 mg/l; Fig. 2A). Serum CRP and total cholesterol were unrelated in normocholesterolemic NF pigs (R = 0.223, P = 0.631). Serum CRP and total cholesterol were correlated when NF and HF groups were combined (R = 0.66, P = 0.01). The strongest correlations were between serum CRP and total cholesterol (R = 0.727, P < 0.041; Fig. 2B) and the accumulation of SRA-positive macrophages (R = 0.938, P < 0.001; Fig. 2C) in RCA of hypercholesterolemic HF pigs.
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DISCUSSION
The role of inflammation and inflammatory mediators in atherogenesis is gradually being elucidated (8). CRP was once thought to be produced solely by the liver (6); however, CRP mRNA recently has been detected in smooth muscle-like cells and macrophages in atherosclerotic arteries (21). We detected CRP in endothelium, macrophages, and smooth muscle cells in RCA of HF pigs.
Serum IL-6 is an acute-phase inflammatory cytokine that stimulates hepatic CRP synthesis (6). Serum IL-6 was below the detection limit of the assay in all pigs. These data suggest an absence of subclinical bacterial infection and are used for herd health monitoring (4). CRP was detected in the serum of normocholesterolemic NF pigs and hypercholesterolemic HF pigs in this study. Because serum CRP correlates positively with body mass (2), we controlled for this potentially confounding factor by feeding to maintain similar body mass in the two groups of pigs. Serum CRP recently has been reported to be detectable in 14% of adult pigs fed a normal diet and 75% of pigs fed a hypercholesterolemic diet for 37 wk (21). Failure to detect CRP in the serum of some pigs could have been the result of differences in diets, time frame (37 vs. 20 wk in the present study), body mass (unreported), or the use of a less-sensitive assay for CRP (detection limit = 3 mg/l).
The present literature does not establish whether CRP is a risk factor or a risk marker for CAD (11). We observed a higher correlation between serum CRP and macrophage accumulation in RCA than between serum CRP and total cholesterol. These results and a previous report that CRP deposition precedes the appearance of monocytes in early atherosclerotic lesions suggest that elevated CRP is a risk factor for CAD (18).
In the present study, serum CRP correlated directly with total cholesterol, an observation that has been reported previously in patients with the metabolic syndrome (5). Elevation of plasma CRP has been reported to be a stronger predictor of cardiovascular disease than LDL cholesterol level (12). Indeed, 40-50% of patients with CAD have normal or mildly increased cholesterol levels (2), suggesting that inflammation may be a more important risk factor than blood lipids. Recently, patients with high plasma CRP levels on first admission were at higher risk of rapidly progressive CAD and restenosis after percutaneous coronary intervention (22).
The high correlation of CRP with accumulation of SRA-positive macrophages (R = 0.938) and total cholesterol (R = 0.727) suggests that inflammation plays an active role in disease induced by hypercholesterolemia. Verhamme et al. (19) recently reported that withdrawal of cholesterol feeding for 26 wk after 37 wk of cholesterol feeding lowered blood cholesterol, serum CRP, and macrophage content in advanced (Stary stage IV-V) coronary lesions in pigs. The present data suggest that elevated CRP may participate actively in the accumulation of macrophages in early stage I-III lesions of CAD, whereas the data of Verhamme et al. suggest that lowering serum CRP may reduce macrophage content in advanced stage V-VI lesions of CAD. The examination of a single death time point in the present study precludes the study of temporal evolution of the lesions.
Human endothelial cells and murine macrophages exposed to CRP in vitro express the chemokine MCP-1 (1, 10). MCP-1 is believed to play a critical role in monocyte recruitment and lesion development in atherosclerosis (8). We report for the first time colocalization of CRP, MCP-1, and SRA in macrophage foam cells. These observations support a potential proatherogenic role for CRP in early atherosclerosis.
DISCLOSURES
This work was supported by National Heart, Lung, and Blood Institute Grant HL-52490.
Mention of a trade name or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.
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FOOTNOTES |
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FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
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