C-reactive protein in atherosclerotic lesions: its origin and pathophysiological significance

Abstract

C-reactive protein (CRP) is frequently deposited in the lesions of the arterial intima; however, the origin and pathological significance of CRP in these lesions are not completely understood. In this study, we measured CRP levels in the plasma of hypercholesterolemic rabbits and investigated CRP expression at both the mRNA and protein levels using rabbit and human atherosclerotic specimens. CRP levels were significantly elevated in both cholesterol-fed and Watanabe heritable hyperlipidemic rabbits, and CRP levels were clearly correlated with aortic atherosclerotic lesion size. Immunohistochemical staining coupled with Western blotting analysis revealed that CRP-immunoreactive proteins were found at all stages of atherosclerosis from the early to advanced lesions. CRP was present extracellularly and co-localized with apolipoprotein B but was rarely associated with the cytoplasm of macrophages and foam cells. Real-time reverse transcriptase-polymerase chain reaction analysis revealed that CRP mRNA in atherosclerotic lesions was barely detectable, and isolated macrophages did not express CRP mRNA, suggesting that CRP proteins found in the lesions were essentially derived from the circulation rather than synthesized de novo by vascular cells. These results suggest that there is a link between plasma CRP and the degree of atherosclerosis and that inhibition of plasma CRP may represent a therapeutic modality for the treatment of cardiovascular disease.

Figure 1

A: Increased plasma CRP levels in cholesterol-fed and WHHL rabbits. The values are expressed as mean ± SE. **P < 0.01 versus normal rabbits or cholesterol-fed rabbits versus WHHL rabbits. Average levels of plasma total cholesterol are 30 ± 9 mg/dl in normal rabbits, 860 ± 53 mg/dl in cholesterol-fed rabbits, and 459 ± 21 mg/dl in WHHL rabbits. Total en face lesion area of the aorta is 14 ± 3.3% in cholesterol-fed rabbits and 42.3 ± 5.9% in WHHL rabbits. B and C: Correlations of plasma CRP and aortic atherosclerosis gross size (B) and plasma cholesterol levels (C).

Figure 2

Demonstration of CRP deposition in atherosclerotic lesions of cholesterol-fed rabbits. Two representative lesions were selected from cholesterol-fed rabbits and stained with H&E or mAbs against rabbit CRP, macrophages (Mφ), SMCs, and apoB. A: Early-stage lesion, which is composed of a single layer of macrophage-derived foam cells. B: Advanced lesion, which is covered by a fibrotic cap. CRP is co-localized with apoB staining. See supplemental data for sections stained with CRP Abs shown in higher magnification at http://ajp.amjpathol.org.

Figure 3

Demonstration of CRP deposition and apoB in the early-stage lesions of WHHL rabbits. Three representative lesions with different features were selected and stained with H&E or mAbs against CRP, apoB, macrophages (Mφ), and SMCs. CRP is deposited on the surface (A) or under foam cells (B) and co-localized with apoB and does not overlap with macrophage-rich area (C). See supplemental data for sections stained with CRP Abs shown in higher magnification at http://ajp.amjpathol.org.

Figure 4

Demonstration of CRP deposition and apoB in advanced lesions of WHHL rabbits. Two representative lesions with a necrotic core (A) or calcification (B) were selected and both lesions show CRP deposition overlapping with apoB. See supplemental data for sections stained with CRP Abs shown in higher magnification at http://ajp.amjpathol.org.

Figure 5

Immunoblotting analysis of tissue CRP from WHHL rabbits. Aortic lesions along with isolated alveolar and peritoneal macrophages were analyzed for the presence of CRP protein as described in Materials and Methods. Liver and plasma were used as positive controls. Note that CRP bands in macrophages are smaller than other CRP bands.

Figure 6

CRP was closely associated with unstable vulnerable plaque (A) or ruptured plaques (B) in human coronary arteries. Human coronary arteries, obtained from patients who died of MI, were used for the detection of CRP immunoreactive proteins. Macrophage-rich areas (stained by CRP, Mφ, SMCs, or C3), boxed area, were shown in higher magnification (B). Note that CRP is concentrated on the surface of macrophages of both lesions. See supplemental data for sections stained with CRP Abs shown in higher magnification at http://ajp.amjpathol.org.

Figure 7

Detection of CRP deposition in stable plaques of human coronary arteries. Two macrophage-rich lesions were selected and both show CRP deposition. CRP was either located on the top (A) or the bottom (B) of the lesions. See supplemental data for sections stained with CRP Abs shown in higher magnification at http://ajp.amjpathol.org.

Figure 8

Demonstration of CRP protein in human aorta by Western blotting. The samples were from eight autopsy cases and all of them contained CRP proteins detected by Western blotting. The lesions were divided grossly: DIC, diffuse intimal thickening and plaques.

Figure 9

Northern blot (A) and real-time RT-PCR (B) analysis of CRP expression in rabbit tissues. A: The liver was the only organ that expresses detectable CRP by Northern blotting. B: Real-time RT-PCR analysis showed that compared to the liver, aortic lesions expressed very small amounts (∼1/100th of the liver) of CRP mRNA whereas macrophages did not produce detectable CRP mRNA.