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From the Department of Cardiology (A.T., J.W.J., A.v.d.L.), Leiden University Medical Center, the Netherlands; Biomedical Research (A.T., M.P.M.d.M., M.C.M., E.H.O., L.M.H., H.M.G.P., J.J.E.), TNO, Leiden, the Netherlands; the Department of Hematology (M.P.M.d.M.), Erasmus Medical Center, Rotterdam, the Netherlands; and the Department of Medicine (A.J.S.), University of Alabama at Birmingham.
Correspondence to J.J. Emeis, Biomedical Research, TNO, Gaubius Bldg PO Box 2215, 2301 CE Leiden, The Netherlands. E-mail jj.emeis@pg.tno.nl
Abstract
Objective— C-reactive protein (CRP) has been associated with risk of cardiovascular disease. It is not clear whether CRP is causally involved in the development of atherosclerosis. Mouse CRP is not expressed at high levels under normal conditions and increases in concentration only several-fold during an acute phase response. Because the dynamic range of human CRP is much larger, apolipoprotein E*3-Leiden (E3L) transgenic mice carrying the human CRP gene offer a unique model to study the role(s) of CRP in atherosclerosis development.
Methods and Results— Atherosclerosis development was studied in 15 male and 15 female E3L/CRP mice; E3L transgenic littermates were used as controls. The mice were fed a hypercholesterolemic diet to induce atherosclerosis development. Cholesterol exposure did not differ between E3L/CRP and E3L mice. Plasma CRP levels were on average 10.2±6.5 mg/L in male E3L/CRP mice, 0.2±0.1 mg/L in female E3L/CRP mice, and undetectable in E3L mice. Quantification of atherosclerosis showed that lesion area in E3L/CRP mice was not different from that in E3L mice.
Conclusion— This study demonstrates that mildly elevated levels of CRP in plasma do not contribute to the development of early atherosclerosis in hypercholesterolemic E3L/CRP mice.
C-reactive protein (CRP) is associated with incidence and severity of cardiovascular disease (CVD). Whether CRP is causally involved in atherosclerosis, the underlying cause of CVD, is unclear. This study in apolipoprotein E*3-Leiden transgenic mice demonstrated that mildly elevated levels of CRP in plasma did not contribute to atherosclerosis development.
Key Words: atherosclerosis ? C-reactive protein ? inflammation ? mouse ? transgene
Introduction
Cardiovascular disease attributable to atherosclerosis is a major cause of mortality and morbidity in Western society. In recent years, inflammation has emerged as a key factor in atherosclerosis development.1 The acute phase protein C-reactive protein (CRP) has been shown to predict the risk of cardiovascular events.2 Because the plasma concentration of CRP reflects the inflammatory condition, it has been hypothesized that CRP levels reflect the inflammatory condition of the vascular wall. However, whether CRP itself contributes to atherosclerosis development is still unknown.
See page 1527
CRP is a member of the highly conserved pentraxin family and is produced mainly by hepatocytes in response to infection or inflammation. Its production is stimulated by cytokines such as interleukin-6 (IL-6), IL-1, and tumor necrosis factor- (TNF-). CRP binds to phosphatidylcholine in cell membranes and plasma lipoproteins in a Ca2+-dependent manner and has a role in opsonization of infectious agents and damaged cells.3 In vitro studies have reported numerous effects of CRP on endothelial cells, smooth muscle cells, and monocytes. CRP treatment often elicits proinflammatory and proatherosclerotic effects.4 For instance, CRP activates endothelial cells to produce adhesion molecules, induces monocyte chemoattractant chemokine-1 (MCP-1) production facilitating leukocyte adhesion and diapedesis, contributes to the migration of smooth muscle cells, enhances uptake of native low-density lipoprotein by macrophages, and activates complement.5–11 Furthermore, local CRP production by cells in the atherosclerotic lesion has been reported.12
Apolipoprotein E*3-Leiden (E3L) mice exhibit elevated plasma cholesterol and triglyceride levels,13 resembling familial dysbetalipoproteinemia in humans. Plasma cholesterol levels in these mice can easily be titrated by diet.14–16 E3L mice develop various stages of atherosclerosis, varying from mild types I to III foamy lesions to severe type IV to V lesions, depending on plasma cholesterol levels and duration of exposure.14,16 Because CRP is a major acute phase reactant in man but not in mice, the human CRP gene was introduced into E3L mice to enable us to study the contribution of human CRP to the development of early atherosclerosis in hypercholesterolemic mice.
Materials and Methods
The detailed materials and methods used in this study are described in the online supplement (available at http://atvb.ahajournals.org).
E3L+/–/CRP+/– mice were obtained by cross-breeding female E3L mice13–16 with heterozygous male CRP transgenic mice.17–19
Four groups of mice (15 male and 15 female E3L/CRP transgenic mice and 15 male and 15 female E3L transgenic littermates ) were used. Mice were matched on the basis of age (average 14 weeks) and plasma cholesterol level. The institutional animal care and use committee approved all animal experiments.
Females were fed a semisynthetic diet supplemented with 0.5% (w/w) cholesterol (Table I, available online at http://atvb.ahajournals.org); males20 were fed a diet supplemented with 1% cholesterol and 0.05% sodium cholate (w/w; Table I). In addition, fructose was added to the drinking water of male mice, which resulted in an additional plasma cholesterol increase of 3 mmol/L. Female and male mice were kept on these diets for 25 and 30 weeks, respectively. Body weight and food intake were monitored every 4 weeks.
Blood samples were obtained by tail incision at baseline, at t=3, 8, 12, 16, 20, 24, 28 weeks and at death. Total plasma cholesterol and triglyceride levels were measured enzymatically. Lipoprotein distribution was determined for each group in pooled plasma by fast protein liquid chromatography.
Human CRP concentrations in plasma were measured using an in-house enzyme immunoassay21 (lower limit of sensitivity 0.05 mg/L). Serum amyloid A (SAA) and von Willebrand factor (vWF) were determined by ELISA.22
After 25 or 30 weeks of diet feeding, mice were euthanized. Serial paraffin-embedded cross-sections were obtained from the aortic root area, and stained with hematoxylin-phloxin-saffron. For each mouse, 4 sections (5 μm thick) at intervals of 50 μm, representing that stretch of the aortic root where the aortic valves are clearly visible, were used for quantification and typing of atherosclerotic lesions. Total lesion area was determined using Leica Qwin image analysis software. To determine the severity of atherosclerosis, the lesions were classified into 5 categories as described before:14,16 type I) early fatty streak, type II) regular fatty streak, type III) mild plaque, type IV) moderate plaque, and type V) severe plaque.
Immunohistochemistry was performed using primary antibodies specific for intercellular adhesion molecule-1 (ICAM-1), monocytes and macrophages, and CRP, followed by biotinylated secondary antibody, avidin-biotin–conjugated horseradish peroxidase and NovaRed as substrate.
All data are presented as mean±SD or median (95% CI). For statistical analysis, SPSS 10.0 for Windows was used. Effects of the CRP transgene were tested statistically for female and male mice separately. P values <0.05 were regarded as significant.
Results
Body Weight and Food Intake
Body weight (Table) and food intake (data not shown) of E3L/CRP mice did not differ significantly from body weight and food intake of E3L mice at any time point.
Plasma Levels of Lipids, Inflammatory Markers, and Lesion Area at Death
Inflammation Markers
CRP could only be detected in the plasma of E3L/CRP transgenic mice. Plasma CRP levels were on average 10.2±6.5 mg/L in male mice. In female E3L/CRP mice, plasma CRP levels were much lower at 0.2±0.1 mg/L (Table).
Data on SAA and plasma vWF levels are shown in the Table. SAA levels were considerably higher in male mice compared with female mice. Even although the SAA levels were lower in the E3L/CRP transgenic mice compared with the gender-matched E3L mice, these differences were not statistically significant. vWF was significantly higher in the male mice compared with the females, but there were no significant differences between E3L/CRP mice and E3L mice. CRP levels were not significantly correlated with either SAA or vWF levels.
Plasma Cholesterol Levels, Cholesterol Exposure, and Lipoprotein Composition
Plasma cholesterol levels, as measured at 8 time points (t=3, 8, 12, 16, 20, 24, 28, and 30 weeks), were on average 13.3±3.8 mmol/L for female E3L/CRP mice, 13.6±3.1 mmol/L for female E3L mice, 17.2±6.2 mmol/L for male E3L/CRP mice, and 14.7±5.7 mmol/L for male E3L mice (Table). There was no significant difference in cholesterol exposure (plasma cholesterolxweeks of exposure) between the 2 male groups, nor between the 2 female groups (Table; Figure 1A). Thus, the presence of the human CRP gene did not modify plasma cholesterol levels in E3L mice.
Figure 1. A. Total cholesterol exposure (mmol/Lxweeks) of the study groups (n=13 per group). Data are presented as mean±SD. There are no significant differences in cholesterol exposure between the 2 male groups, nor between the 2 female groups. B, Cholesterol profiles of plasma lipoproteins. Lipoprotein distribution was determined for each group in a pooled plasma sample by fast protein liquid chromatography. VLDL indicates very low–density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein.
Triglyceride levels did not differ significantly between the female groups, but the male E3L/CRP mice had higher triglyceride levels than male E3L mice (Table). Lipoprotein profiles at 20 weeks of high-cholesterol feeding did not differ between groups of the same sex (Figure 1B).
Initially, alanine aminotansferase (ALAT) activities in plasma were 50 U/L at t=0. In the female groups, plasma ALAT levels gradually increased to 150 U/L at the end of the study. In the male groups, plasma ALAT levels increased more, to final values of 350 U/L, presumably because of the use of 0.05% cholate in the diet.
Atherosclerosis Development
Atherosclerotic lesion area and the severity (types I to V) of the lesions were quantified in 4 cross-sections of the aortic valve area for each individual mouse. Representative photomicrographs of atherosclerotic lesions present in the 4 groups are shown in Figure 2.
Figure 2. A through C, Representative photomicrographs of mild atherosclerotic lesions (types I to III) as observed in the different study groups. D, A more severe type IV lesion (hematoxylin-phloxin-saffron staining).
No significant differences in average lesion size were observed between the E3L/CRP and E3L mice, either in male or female mice (Table; Figure 3A). In the male mice, there was no correlation between lesion size and plasma CRP concentration (rs=0.56; NS).
Figure 3. Atherosclerotic lesion development. A, Shown are individual values () for average atherosclerotic lesion area per section per mouse; dash indicates the median value for each study group. There are no significant differences in lesion size between the male groups or the female groups. B, Distribution of the severity of atherosclerotic lesions, as determined by the percentage of lesions classified as type I to III lesions (fatty dots and streaks and mild plaques) and type IV to V lesions (moderate and severe plaques). Data are presented as mean±SD. See Table II for a statistical evaluation.
Lesions were somewhat larger in female mice (with minimal expression of CRP) than in male mice (which did express CRP), despite a larger cholesterol exposure in the male groups. This sex difference, which differs from that in most strains of mice, is in agreement with previous observations in this strain of mice20 and with observations in unpublished studies (J.J. Emeis, 2004) that lesion development in males is slower than in females in E3L mice.
Assessment of lesion severity (scored as type I to V lesions) showed that in female mice, 69% of the lesions found were mild (type I to III lesions), and 29% were more severe (type IV and V lesions; Figure 3B; Table II, available online at http://atvb.ahajournals.org). In the male mice, 83% of the lesions found were of type I to III and 17% of types IV to V. No differences in lesion severity distribution were observed between E3L/CRP mice and E3L mice of the same sex, nor between male and female mice (Figure 3B; Table II).
Characterization of Vessel Wall and Lesions
Because no differences in lesion area had been observed, we sought to determine whether CRP was associated with any differences in the composition of the lesions. This was determined by assessment of endothelial activation (ie, ICAM staining) by assessing the number of adhering monocytes and by the determination of the macrophage-containing area of the lesion.
The endothelium of the vessel wall was found to be ICAM positive. However, no differences in staining intensity of ICAM-1 in the lesions were observed whether mice expressed CRP or not, either in male or in female mice.
The number of monocytes adhering to the endothelium was counted in the same slides used for quantification of atherosclerosis. Monocyte adhesion to the endothelium was not significantly different between the female E3L/CRP and female E3L mice, nor between the male E3L/CRP and male E3L mice (Figure 4A).
Figure 4. Effect of CRP on monocyte attachment to the endothelium and macrophage area. Adherent monocytes were counted in the same sections used for quantification of atherosclerosis. A, Average number of monocytes per section adhering to the endothelium. No significant differences in monocyte adhesion were observed between the E3L/CRP and E3L mice of the same sex (n=5 per group). B, Macrophage area as percentage of total lesion area. No significant differences in macrophage area were observed between the E3L/CRP and E3L mice of the same sex (n=5 per group).
In general, the female mice had a larger macrophage-containing area than the males, which is in agreement with the fact that the lesion area of the females was larger (Table). When macrophage area was corrected for total lesion area, no differences were observed between female E3L/CRP and female E3L mice (Figure 4B). In male E3L/CRP mice, macrophage-containing area was 1.8-fold (NS) increased when compared with E3L mice of the same sex (Figure 4B).
We were unable to demonstrate the presence of CRP in the atherosclerotic lesions using the same antibody technique as described by Paul et al.23
Discussion
Although numerous in vitro studies have suggested a possible causal involvement of CRP in atherosclerosis, in vivo evidence for a proatherogenic role of CRP is scarce. In the present study, we demonstrated that in E3L mice, the presence of CRP in the blood did not modify the extent nor the severity of atherosclerosis development. Also, the effect of CRP on ICAM-1 expression observed in vitro7 could not be confirmed in vivo in the present study.
The E3L transgenic mice are an established model for diet-induced atherosclerosis and a useful model for drug-induced effects on lesion formation.24,25 The lesions found in this study were mostly mild because we wanted to study the earlier stages of lesion development. Also, lesions in male mice were smaller than lesions in female mice, in agreement with previous (unpublished, 2004) observations that male E3L mice develop lesions more slowly. However, more severe lesions were also present in male and female mice, demonstrating that these mice have the potential to develop more extensive and more complex lesions, as we have also observed in other studies (compare also van Vlijmen et al20). Lesion severity did not differ between male and female mice, nor between E3L and E3L/CRP mice (Table II).
Inflammation has been implicated in atherosclerosis development, and plasma levels of inflammatory markers can be used as predictors of the risk for cardiovascular events.26,27 Therefore, we measured, in addition to CRP, also SAA and vWF at the end point of the study, with vWF serving as a marker of endothelial cell activation. SAA was used as a second, general murine inflammation marker because it is present in all mice. There were no significant differences in SAA and vWF levels between the E3L/CRP and E3L mice. Male mice had higher levels of SAA and vWF than the females. This is probably a result of the cholate, which had been added to the diet of the males only, and may produce inflammatory responses.28,29 The difference in plasma CRP level between male and female mice is attributable to the testosterone dependence of CRP synthesis in this species.19 Therefore, there was no evidence of a difference in inflammation status between the 2 male study groups or between the 2 female study groups.
Recently, Paul et al23 reported a significant increase in lesion size in CRP/apolipoprotein E knockout mice. They also detected CRP in the atherosclerotic lesions, in which it was associated with increased complement C3 deposition, suggesting that CRP stimulated activation of complement within these lesions. Paul et al concluded that CRP has a proatherogenic role in vivo. However, the plasma levels of CRP in these mice were extremely high, with a basal expression of 120±77 mg/L. In the general population, presymptomatic, baseline plasma CRP levels >3 mg/L are associated with an increased risk of heart attack and stroke.30 Among patients with acute coronary syndromes, CRP values >3 mg/L are associated with increased risk of coronary events. However, CRP values >10 mg/L can reflect a wide range of pathologies, and therefore, if patients are presenting with CRP levels >10 mg/L, CRP can no longer be used in the prediction of risk of atherothrombotic events. In the study reported here, the plasma CRP level in the male mice was 10 mg/L, much lower than the very high CRP levels in the mice studied by Paul et al.23
We were not able to demonstrate the presence of CRP in lesions described by Paul et al,23 despite the fact that we used exactly the same technique. Maier et al31 suggested recently that in unstable human coronary lesions, SAA and IL-6 were locally produced, but CRP was not. If that would also be the case in mice, the lower CRP levels in our mice could explain our inability to demonstrate CRP in lesions because CRP diffusion into the lesion would be less than in the mice described by Paul et al.23
In vitro studies have shown numerous effects of CRP on endothelial cells, smooth muscle cells, and monocytes, usually eliciting proinflammatory and proatherosclerotic effects.7–11,32 However, in those studies, the concentrations of CRP used were usually very high, ranging up to 100 mg/L, whereas significant effects were generally observed only at CRP levels >10 mg/L.
Another point of uncertainty in the in vitro experiments relates to the purity of the CRP preparations used. Nagoshi et al33 have shown that even recombinant CRP preparations can be contaminated with endotoxin. Removal of endotoxin blocked the ability of recombinant CRP to induce the secretion of IL-6 and MCP-1 by human coronary artery endothelial cells. In vivo, this problem obviously plays no role.
The results reported here do not support a role of CRP in atherosclerosis development in the E3L mouse. However, the association between CRP and cardiovascular disease (CVD) is well established because elevated CRP levels do predict future cardiovascular risk.2,34,35 Possibly, CRP is implicated in other processes than atherosclerosis development that contribute to the incidence of CVD, such as thrombosis or revascularization after ischemia,32,36–38 worsening the prognosis of CVD through these mechanisms. Danenberg et al36 used human CRP transgenic mice to investigate whether CRP has a prothrombotic effect. They demonstrated that at 4 weeks after transluminal wire injury, 75% of the femoral arteries were occluded in CRP transgenic mice compared with 17% in wild-type mice.36 However, these results can only be extrapolated to the human situation with care because CRP levels in these mice (18±6 mg/L at baseline to 56±5 mg/L after surgery) were higher than in humans.
CRP has been found to be deposited in the infarcted area together with complement.39 Griselli et al40 demonstrated in an animal model that human CRP and complement activation are major mediators of ischemic myocardial injury. In rats injected with CRP, infarct size was increased by 40% compared with mice not injected with CRP. So although CRP does not appear to be involved in atherosclerosis directly, it may well have other effects that may adversely influence the outcome of a cardiovascular event.
Also, protective functions for CRP in atherosclerosis have been reported recently in in vitro studies. On incubation with CRP, endothelial cells from human coronary artery or human saphenous vein show increased expression of complement inhibitory factors.41 However, again, the issue of possible artifacts attributable to contamination of the CRP preparation used is raised because van den Berg and Taylor demonstrated that removal of azide from the CRP preparations used inhibited the CRP-induced decay-accelerating factor expression reported by Li et al.42 CRP was also reported to protect cells against assembly of the terminal complement complex,43,44 and native CRP (but not monomeric CRP) was shown to inhibit platelet activation.45,46 However, in the present study, we could not demonstrate a protective effect of CRP.
In conclusion, the present study does not support a direct role of CRP in atherosclerosis development in E3L transgenic mice. No differences in lesion area or lesion severity could be observed between mice with and mice without CRP in this study. We conclude that moderately increased levels of plasma CRP do not affect the development of atherosclerosis in hypercholesterolemic E3L/CRP transgenic mice.
Acknowledgments
The research described in this article is supported by an unrestricted research grant from Pfizer Inc.
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