doi: 10.1161/CIRCULATIONAHA.105.611350
(Circulation. 2006;113:2128-2151.)
© 2006 American Heart Association, Inc.
Controversies in Cardiovascular Medicine |
Is C-reactive protein an innocent bystander or proatherogenic culprit?
The Verdict Is Still Out
From the TIMI Study Group, Cardiovascular Division, Department of Medicine, Brigham & Women’s Hospital and Harvard Medical School, Boston, Mass (B.S, D.M.); Division of Cardiac Surgery, St. Michael’s Hospital, University of Toronto, Toronto, Canada (S.V.); Laboratory for Artherosclerosis and Metabolic Research, Department of Medical Pathology and Laboratory Medicine (S.D., I.J.), and Division of Endocrinology, Clinical Nutrition and Vascular Medicine, Department of Medicine (I.J.), University of California, Davis Medical Center, Sacramento.
Correspondence to David A. Morrow, MD, MPH, TIMI Study Group, Cardiovascular Division, Brigham and Women’s Hospital, 75 Francis St, Boston, MA 02115 (e-mail dmorrow{at}partners.org); Dr S. Verma, Division of Cardiac Surgery, St. Michael’s Hospital, 30 Bond St, Toronto, Canada (e-mail subodh.verma@sympatico.ca); or Dr I. Jialal, Laboratory for Artherosclerosis and Metabolic Research, University of California, Davis Medical Center, Sacramento, CA (e-mail ishwarlal.jialal@ucdmc.ucdavis.edu).
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Introduction |
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 Top Introduction CRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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Presence at the scene of a crime is not itself necessarily compelling evidence of guilt.— Mark B. Pepys
The recognition of inflammation as a central contributor to atherothrombosis has engendered a sustained effort to characterize the specific participants and pathways and to identify noninvasive markers that enable detection of underlying inflammatory activation for the purpose of assessing cardiovascular risk. C-reactive protein (CRP), an acute-phase reactant, has been investigated in the pursuit of both of these objectives. Epidemiological studies have demonstrated an increased risk of cardiovascular events in patients with elevated levels of CRP.2–5 When considered together with experimental evidence placing CRP within arterial atheroma6–8 and clinical data revealing lowering of CRP with some preventive therapies, this strong base of epidemiological evidence has led to the hypothesis that CRP is both a marker of and a causal agent in the development of atherosclerosis.9,10 In other words, CRP may be both a "marker" and a "maker" of atherothrombosis.11 This hypothesis carries substantial clinical implications in that it forms the basis for both development of potential therapeutic agents that directly target CRP and consideration of CRP itself as a modifiable cardiovascular risk factor.
This unifying theory regarding CRP, while appealing, is not yet established by the available evidence.1,11 We will review the in vitro and in vivo data that support the assertion that CRP is itself pathogenic and the conflicting findings that render this conclusion premature.
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CRP as a Marker of Clinical Risk |
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TopIntroductionCRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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To understand the basis for the intense investigation of CRP as a proatherogenic agent, it is worthwhile to trace the epidemiological evidence available to date. In 1944, Löfström12 described concordant rises in body temperature and white blood cell count, along with an increase in the concentration of serum capsular swelling protein (later named CRP), in a patient with acute myocardial infarction. It was not until 4 decades later that this increase in CRP in unstable coronary artery disease was recognized as potentially reflecting more than an inflammatory response to necrosis.13,14 Since then, >30 epidemiological studies have demonstrated a significant association between elevated serum or plasma concentrations of CRP and the prevalence of underlying atherosclerotic vascular disease, the risk of recurrent cardiovascular events among those with established disease, or the incidence of first cardiovascular events among those at risk.
In particular, at least 24 prospective studies of testing for high-sensitivity CRP (hsCRP) have shown a consistent and robust relationship between levels of hsCRP and the risk of future cardiovascular events.3,4,15,16 Meta-analysis of 11 of the earlier prospective studies indicated a 2-fold higher relative risk (95% confidence interval, 1.6 to 2.5) for major coronary events between the upper and lower tertiles of hsCRP independent of clinical risk assessment or lipid profiles. More recent updates that include a revised meta-analysis of 22 studies15 and 2 additional studies provide a modestly attenuated estimate of the relative risk, on the order of 1.5 to 1.8 after adjustment for traditional risk factors.17,18 Nevertheless, in these analyses, CRP adds to traditional risk factors for prediction of major coronary events, including at least 7 studies in which CRP was added to the Framingham Risk Score as a tool for global assessment of cardiovascular risk (Figure 1). Notably, the strength of the risk relationship appears at least as strong as a well-established marker such as hypertension, supporting the clinical relevance of CRP. From the available data, the Centers for Disease Control and Prevention and the American Heart Association Scientific Statement on Markers of Inflammation and Cardiovascular Disease has recommended that hsCRP may be measured at the physician’s discretion in asymptomatic people with an intermediate risk of coronary heart disease (Class IIa recommendation) to optimize the global assessment of cardiovascular risk. Patients can be categorized using CRP-based risk categories of low (<1 mg/L), average (2 to 3 mg/L), and high (>3 mg/L) on the basis of the average of 2 measurements taken optimally at least 2 weeks apart.19
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In addition, specific therapies that are effective for primary and secondary prevention have been shown to lower levels of CRP and/or to mitigate the risk associated with elevated levels of this marker.20 These observations provide indirect evidence that has fueled interest in CRP as a direct contributor to atherothrombosis.
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CRP as a Potential Proatherogenic Agent |
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TopIntroductionCRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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Why CRP?
With the sound epidemiological association between CRP and cardiovascular risk, in conjunction with the growing laboratory research placing inflammation at the center of atherogenesis, it is reasonable to cast CRP as a potential causal agent. However, before we review the experimental data, it is worth a brief digression to ask why CRP became the focus of such intense investigation.
The emergence of CRP as the most extensively clinically studied of the inflammatory markers is due largely to its preanalytical and analytical properties. The biological properties and kinetics of CRP—a relatively long half-life of 18 hours, no relationship to fasting state or diurnal patterns—make it a relatively stable serum protein compared with many other markers. In addition, assays for CRP are sensitive, reproducible, internationally standardized, relatively inexpensive, and widely available.21 Moreover, the availability of preparations of CRP for calibration of these assays also has provided a convenient source for exogenous CRP for in vitro and animal experiments. Despite these favorable properties for experimentation, CRP as an acute-phase reactant is a nonspecific indicator of inflammation and thus a priori might be less likely than other inflammatory proteins (eg, metalloproteinases and cellular adhesion molecules) to be a direct participant in atherogenesis.
Moreover, recent genetic analysis in humans has highlighted the limitation of deducing causality from observational clinical studies. Specifically, elevated levels of CRP have been associated with the development of the metabolic syndrome, which has therefore been proposed to result from chronic systemic inflammation. A sophisticated analysis that compared the genetic polymorphisms in the genes that encode CRP, serum concentrations of CRP, and components of the metabolic syndrome reported that there was no causal association between CRP and the metabolic syndrome, suggesting that the association with serum concentrations of CRP was due to either residual confounding or reverse causation (ie, the metabolic syndrome increases concentrations of CRP).22,23 Similar analyses may follow for the role of CRP in atherogenesis.
Laboratory Evidence
The experimental evidence implicating CRP as a potent stimulus of atherogenesis rests predominantly on experiments that have demonstrated CRP within the atheroma and inflammatory changes in cells and animals exposed to exogenous CRP. CRP is a highly conserved, 5-subunit protein produced predominantly in the liver. It is believed to play an important role as an acute-phase reactant to tissue damage, infection, inflammation, and malignant neoplasia,24 and its production is stimulated largely by levels of interleukin-6. CRP binds many ligands, including phosphocholine residues, very low-density lipoprotein, and low-density lipoprotein (LDL; especially oxidized LDL25), damaged cell membranes, and complement. Histological staining of atherosclerotic lesions consistently places CRP within the lesion, and there is emerging evidence that CRP is produced in smooth muscle cells and macrophages in the atheroma.26,27 Proximity, however, does not prove culpability. Other putative pathogenic agents, particularly viruses or bacteria, have been identified within atherosclerotic lesions but have not been demonstrated to have a definitive causal role, nor did targeted therapy to eradicate infection provide clinical benefit.28
In vitro experiments testing the addition of exogenous CRP to cultured endothelial cells, smooth muscle cells, and monocytes/macrophages have identified several potential proinflammatory mechanisms by which CRP may promote atherosclerosis. Exogenous CRP induced the expression of adhesion molecules such as intracellular and vascular cellular adhesion molecules and E-selectin,29,30 known to promote adhesion of monocytes to the endothelial cells during the earliest stages of atherogenesis. Exogenous CRP also has been shown to decrease levels endothelial nitric oxide synthase31 (NOS) and prostacyclin32 while increasing levels of endothelin-1,33 all critical regulators of arterial vasodilatation. In response to exposure to exogenous CRP, smooth muscle cells upregulated angiotensin I receptors, thereby increasing reactive oxygen species and proliferation.34 In addition, monocytes/macrophages exposed to CRP increased release of tissue factor, potentially stimulating cell migration and adhesion to endothelial cells35 and promoting the uptake of oxidized LDL.25
Although these observations are intriguing, important questions have arisen regarding the purity of the exogenous CRP commonly used in these experiments. CRP can be produced in several ways. Purification of malignant ascites and generation of CRP using recombinant techniques are both time consuming. The most convenient and therefore most commonly used preparations of CRP are from commercial sources that produce recombinant CRP using predominantly Escherichia coli. The principal use of commercially available CRP is to calibrate CRP assays. Therefore, although the concentration undergoes thorough quality control, the purity of the preparation is less important and can be compromised by contaminants such as bacterial lipopolysaccharide (LPS) and preservatives (in particular, azide). There is concern that both LPS and azide, independently of CRP, will produce many of the proinflammatory and prothrombotic responses seen in prior studies and ascribed to CRP.1,11
Several studies have systematically evaluated the effects of different preparations of CRP on a variety of cell lines to determine whether inflammatory changes are due to CRP or contaminant. Studies that used either local preparations of recombinant CRP or specific techniques to purify commercial CRP have not reported similar inflammatory reactions.36–38 For example, Taylor and colleagues38 compared a commercially available CRP preparation, an ascites-derived CRP preparation, a nonbacterial recombinant preparation, and solutions of azide and LPS alone. The commercial CRP produced results similar to those in prior studies in terms of impaired cellular viability, decreased endothelial NOS activity, and increased expression of vascular adhesion molecules. In stark contrast, however, the CRP prepared from ascites and the nonbacterial recombinant methods failed to induce similar changes except when exogenous azide or LPS was added. In fact, the only solution that matched the effects of the commercial CRP was a combination of azide and LPS (Figure 2). In another series of experiments, Lafuente and colleagues36 reported a reduction in the production of the antiinflammatory enzyme inducible NOS in vascular smooth muscle cells exposed to commercially prepared CRP and a medium containing only azide. In contrast, when azide was then removed from the commercial CRP, the exogenous CRP did not cause a reduction in inducible NOS. In another report, Pepys and colleagues37 added human malignant ascites–derived CRP and commercial CRP to endothelial cell lines and demonstrated that the commercial CRP induced the inflammatory marker tumor necrosis factor-
production and activated nuclear factor-
B, a key inflammatory regulatory gene, whereas the ascites-derived CRP did not, again suggesting that a contaminant of the commercial preparation is a probable cause of the inflammatory stimulation rather than CRP itself.
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Contamination of CRP preparations also may explain some of the apparently contradictory results observed in previous studies. For example, 2 studies that compared 2 different conformational shapes of CRP reported opposite results. One suggested that the pentameric CRP was proinflammatory39; the other implicated a monomeric form of CRP.40 The divergent results are not easily explained. It is possible, however, that the discordance may have arisen from differences in the separation and purification of CRP preparations that removed contaminant in a subset of preparations.
Animal Models
Several animal models and 1 human experiment have tested the proinflammatory effects of CRP as an in vivo pathological link to atherothrombosis. Bisoendial and colleagues40a infused CRP into 7 healthy humans and provoked an immediate and marked inflammatory response. Because commercial CRP was used, however, the possibility remains that the CRP was contaminated. Pepys et al37 repeated the experiment in mice with several CRP preparations. The ascites-derived CRP and the control buffer did not elicit any increase in complement activation or inflammatory response, whereas injections with commercially available CRP created a robust response (Figure 3).
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Mouse models with CRP are inherently difficult to interpret because CRP does not appear to be an acute-phase reactant in mice. Thus, that it is possible to induce atherosclerosis in mice poses a challenge to (but cannot dispel completely) the notion of CRP as a primary contributor to atherogenesis in humans. This fact alone poses a challenge to the theory that CRP is a primary causal agent in atherothrombosis. Several groups have transgenetically bred mice that produce human CRP (CRPtg) and reported conflicting results. Paul and colleagues41 bred a mouse that was both a CRPtg "knock-in" and apolipoprotein E (ApoE) deficient, or "knockout" (ApoE–/–), to measure the expression of vascular adhesion molecules and complement deposition in atherosclerosis both under basal conditions and after infusion of turpentine. Male mice, but not females, developed larger atherosclerotic lesions in CRPtg+ compared with CRPtg– mice. However, 2 subsequent reports of ApoE–/– mice that express human CRP failed to demonstrate any relationship between the degree of atherosclerosis and the presence of human CRP. Hirschfield and colleagues42 limited their investigation to male mice, the group in which Paul et al41 found an association, and found no difference at 1 year with respect to the size of atherosclerotic lesions or complement deposition between mice that did or did not produce CRP (Figure 4). In addition, CRPtg mice had low levels of other circulating inflammatory markers, suggesting that circulating CRP did not induce a systemic inflammatory state.37 Trion and colleagues43 also examined atherosclerotic lesion size in CRPtg/ApoE–/– mice. Despite higher levels of CRP in the CRPtg male mice and a mild increase in female mice, there was no increase in lesion size compared with mice that did not produce CRP. In addition, there were no differences in other proinflammatory or thrombotic markers between the groups. In contrast to the findings of Paul and colleagues, CRP also was not isolated in atherosclerotic plaques, despite the use of similar techniques.
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The conflicting data derived from the CRPtg mouse may be manifesting several fundamental deficiencies with the CRPtg model. First, CRP is a foreign protein to the mouse; therefore, its effects on atherosclerosis in the mouse may be completely unrelated to its actions in humans.44 Second, the serum levels of CRP generated in CRPtg mice are much higher than associated with coronary artery disease in humans. In the positive study by Paul and colleagues,41 for example, the level of CRP was >100 mg/L in male CRPtg mice, ie, 30- to 100-fold higher than the relevant range in humans (<3 mg/L). Many proteins could induce inflammatory responses at such supraphysiological levels.
A better animal model with which to investigate the role of CRP and atherosclerosis may be that using Watanabe heritable hyperlipidemic rabbit, which, unlike mice, produce a native CRP that is 70% homologous to humans, responsive to inflammatory stimuli, and elevated in the setting of high cholesterol.44 Furthermore, serum levels of CRP correlate with atherosclerotic burden in normal and Watanabe heritable hyperlipidemic rabbits. As in humans, CRP can be found within atherosclerotic plaques in the Watanabe heritable hyperlipidemic rabbit.45 However, no evidence to date has demonstrated that CRP is pathogenic in rabbits; experimental data place CRP "at the scene of the crime" without providing any evidence of guilt. Further investigation should be planned in this model to evaluate the role of CRP in atherosclerothrombosis.44
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CRP as a Target for Therapy |
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TopIntroductionCRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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The investigation of CRP as a target for therapy is likely to provide insight toward resolving whether CRP is directly proatherogenic. The most extensively studied agents proven to lower levels of CRP are the statins.46–48 Statins reduced the concentration of CRP by 13% to 50% compared with placebo in 13 controlled studies.49 Moreover, in statin-treated patients, the achieved level of CRP correlates with subsequent risk of major coronary events independently of LDL.48 These clinical observations, along with experimental work pointing toward immunoregulatory properties of statins, have supported the hypothesis that the well-proven clinical benefit of statins is related in part to non–lipid-lowering, so-called "pleiotropic," effects. Nevertheless, given the influence of LDL cholesterol on inflammatory processes, it remains challenging to unravel the lipid- and nonlipid-related effects of this class of agents.50–52 Ongoing studies that specifically target statin therapy in patients with high CRP and low cholesterol53 are likely to contribute valuable information with respect to the clinical relevance of nonlipid effects but are not designed to determine whether any observed influence of statin therapy is mediated directly through CRP or other antiinflammatory actions that manifest with diminution of the acute-phase response.
In contrast, investigation of agents that interfere directly with CRP, without other antiinflammatory effects, may provide critical evidence to support or refute a direct pathogenic role of CRP. There are at least 4 potential targets for inhibition CRP inhibition: (1) transcriptional inhibition of hepatic CRP synthesis, (2) antisense therapy, (3) blockade of CRP-mediated complement activation, and (4) blockade of CRP receptors.44 The development of a specific anti-CRP therapy would help resolve the 2 important questions regarding CRP and atherosclerosis: Does blocking or inhibiting CRP delay or prevent atherothrombosis; does blocking CRP reduce cardiovascular risk? The first question can be examined through in vitro and animal experiments; the second will require clinical trials. The potential for parallel pathways and redundancy of inflammatory contributors, however, may limit the ability to conclude on the basis of negative results that there is no participation of CRP.
Additional research at the basic and clinical levels is required to complete the story regarding CRP. To establish conclusively that CRP is a direct modulator of atherosclerosis, each of "modified" Koch’s postulates must be addressed. Specifically, research should demonstrate that (1) CRP is identified in related stages of atherosclerotic lesions; (2) the activation of CRP ligands promotes atherosclerosis; (3) the addition of purified, exogenous CRP promotes atherosclerosis; and (4) the disruption or blockade of CRP or its actions inhibits the development of atherosclerosis in animal and human studies.
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Conclusions |
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TopIntroductionCRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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In conclusion, CRP is a well-proven clinical marker of increased cardiovascular risk. The experimentation that links CRP as a causal agent in atherogenesis is still a work in progress. In particular, challenges to the validity of supportive evidence must be addressed (Table). The investigation of agents that directly inhibit CRP is likely to provide the most compelling evidence in this case. For now, CRP remains innocent until proven guilty.
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Acknowledgments |
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Disclosures
Dr Morrow has received research grant support from Bayer Healthcare Diagnostics, Beckman-Coulter, Biosite, Dade-Behring, and Roche Diagnostics. He has received honoraria for educational presentations from Bayer Healthcare Diagnostics, Beckman-Coulter, and Dade-Behring. He is a consultant to OrthoClinical Diagnostics. Dr Scirica has no conflicts.
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References |
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TopIntroductionCRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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49. Balk EM, Lau J, Goudas LC, Jordan HS, Kupelnick B, Kim LU, Karas RH. Effects of statins on nonlipid serum markers associated with cardiovascular disease: a systematic review. Ann Intern Med. 2003; 139: 670–682.
50. Baigent C, Keech A, Kearney PM, Blackwell L, Buck G, Pollicino C, Kirby A, Sourjina T, Peto R, Collins R, Simes R. Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins. Lancet. 2005; 366: 1267–1278.[CrossRef][Medline] [Order article via Infotrieve]
51. Robinson JG, Smith B, Maheshwari N, Schrott H. Pleiotropic effects of statins: benefit beyond cholesterol reduction? A meta-regression analysis. J Am Coll Cardiol. 2005; 46: 1855–1862.
52. Nissen SE. High-dose statins in acute coronary syndromes: not just lipid levels. JAMA. 2004; 292: 1365–1367.
53. Ridker PM. Rosuvastatin in the primary prevention of cardiovascular disease among patients with low levels of low-density lipoprotein cholesterol and elevated high-sensitivity C-reactive protein: rationale and design of the JUPITER trial. Circulation. 2003; 108: 2292–2297.
When Tillett and Francis discovered C-reactive protein (CRP) in 1930, few could have predicted that this acute-phase reactant would emerge as a powerful marker and partaker of atherothrombosis. Over the past few years, we have witnessed an explosive amount of information linking CRP as an independent biomarker of atherosclerosis and cardiovascular death in diverse patient populations.1 Although initial reports suggested a role of CRP as a surrogate of the underlying inflammatory process of atherothrombosis, accumulating evidence from in vitro and in vivo studies in clinical and experimental models strongly point toward a role of CRP as a proatherogenic factor.2–4 In this point-counterpoint article, we review the available evidence that implicates CRP as a key autocrine and paracrine factor involved in the development and progression of atherothrombosis via effects on endothelial cell regulation, alterations in vascular smooth muscle and monocyte/macrophage function, changes in matrix biology, and effects to promote coagulation, which may serve to sustain a proinflammatory, proatherosclerotic, and prothrombotic environment.
C-Reactive Protein Promotes Atherothrombosis
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CRP Promotes Endothelial Cell Activation and Dysfunction |
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TopIntroductionCRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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The endothelium is the monolayer of cells lining the lumen of all blood vessels. These cells function as a protective biocompatible barrier between all tissues and the circulating blood. Endothelial cells also function as a selective sieve to facilitate bidirectional passage of macromolecules and blood gases to and from tissues and blood. The strategic location of the endothelium allows it to "sense" changes in hemodynamic forces and blood-borne signals and "respond" by releasing a number of autocrine and paracrine substances.5–7 A balanced release of these bioactive factors facilitates vascular homeostasis. Endothelial cell dysfunction disrupts this balance, thereby predisposing the vessel wall to vasoconstriction, leukocyte adherence, platelet activation, mitogenesis, pro-oxidation, thrombosis, vascular inflammation, and atherosclerosis.
An impressive amount of data now implicates CRP as a direct regulator of endothelial cell activation and dysfunction. Earlier observations demonstrating that CRP levels correlated inversely with endothelial vasoreactivity8–10 were followed by data demonstrating that incubation of human umbilical vein endothelial cells and human coronary artery endothelial cells with CRP induced expression of intercellular adhesion molecules, vascular cell adhesion molecules, and E-selectin, in addition to the chemokine monocyte chemoattractant protein-1 (MCP-1).11,12 They also demonstrated that this increase in adhesion molecule and chemokine expression translated into a biological effect, with evidence of increased adhesion of U937 cells to human umbilical vein endothelial cells. The most compelling data implicating CRP as a determinant of endothelial dysfunction came from studies demonstrating that human recombinant CRP reduced basal and stimulated nitric oxide (NO) release from arterial and venous endothelial cells (Figure 1). In human aortic endothelial cells (HAECs), CRP resulted in a significant reduction in mRNA and protein for endothelial NO synthase (eNOS). Furthermore, CRP reduced eNOS activity (ie, conversion of L-arginine to L-citrulline) and bioactivity (secretion of cGMP), in part through decreasing eNOS mRNA stability.13,14 By virtue of inhibiting eNOS expression and NO release, CRP was demonstrated to reduce NO-dependent processes such as angiogenesis while promoting endothelial cell apoptosis. Recently, Qamirani et al15 showed that CRP inhibits endothelium-dependent NO-mediated dilation in coronary arterioles by producing superoxide from NAD(P)H oxidase via p38 kinase activation. More recently, we showed that the inhibition of eNOS by CRP was mediated via the Fc
receptors, and data suggest that FcRIIB mediates CRP inhibition of endothelial NO synthase via protein phosphatase 2A.16,17 The authors demonstrated that in cultured endothelium, highly purified CRP prevents eNOS activation by diverse agonists, resulting in the promotion of monocyte adhesion. Furthermore, CRP antagonism of eNOS occurred nongenomically and was attributable to blunted eNOS phosphorylation at Ser1179. Heterologous expression studies revealed that CRP antagonism of eNOS requires FcRIIB. In FcRIIB+/+ mice, CRP blunted acetylcholine-induced increases in carotid artery vascular conductance; in contrast, CRP enhanced acetylcholine responses in FcRIIB–/– mice, providing firm evidence that FcRIIB mediates CRP inhibition of eNOS.
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14 hours in CRP-treated vs >24 hours in untreated endothelial cells). E, CRP (25 µg/mL, 24 hours) inhibits eNOS protein expression. Representative Western blotting data of immunoblotting of eNOS (with a monoclonal eNOS antibody). Con indicates control. F, Bottom panel describes the statistical summary of densitometric analysis of 4 separate experiments. *P<0.01. Reprinted with permission from Circulation.
P<0.05 vs no CRP. Fc
), and 10 minutes after the administration of L-NAME (
). Values are mean±SEM (n=3 for control, n=7 for CRP). *P<0.05 vs baseline. Reprinted with permission from Circulation Research.
Another important product of endothelial cells is prostacyclin, a potent vasodilator, inhibitor of platelet aggregation, and inhibitor of smooth muscle cell proliferation. CRP in concentrations as low as 10 µg/mL resulted in a decrease in the release of prostaglandin F-1, the stable metabolite of prostacyclin, in both HAECs and human coronary artery endothelial cells, an effect that was mediated through increased nitration of prostacyclin synthase activity.18
CRP has been shown in venous endothelium to promote the release of the potent endothelium-derived contracting factor endothelin-1. Endothelin-1 not only is a potent vasoconstrictor but also appears to be a mediator of CRP-induced upregulation of adhesion molecules and MCP-1 in endothelial cells.19
Several reports suggest that CRP activates the nuclear factor-B (NF-B) signal transduction pathway in endothelial cells.20,21 Degradation of IB-, but not IB-ß, seems to be the major pathway leading to NF-B nuclear translocation and activation induced by CRP.
CRP has been demonstrated to promote monocyte-endothelium interaction. CRP promotes the endothelial release of the chemoattractant chemokine interleukin (IL)-8 in an NF-B–dependent fashion. Furthermore, the increased adhesion of monocytes to endothelium in the presence of CRP was attenuated 30% by preincubating the cells with IL-8 antibodies.20 We have recently demonstrated not only that CRP promotes NF-B–dependent adhesion of monocytes to endothelial cells under static conditions but also that this effect occurs under more clinically relevant shear flow conditions and is inhibited by antibodies to CD32 and CD64.21
A major advance in the field was the demonstration that binding and internalization of CRP by Fc receptors mediates biological effects in endothelial cells16 (Figure 2). Binding studies were performed by incubation of endothelial cells with biotinylated CRP (25 to 200 µg/mL) for 30 to 180 minutes. Biotinylated CRP binding was quantified with streptavidin-fluorescein isothiocyanate, followed by flow cytometry. Saturable binding of CRP was obtained at 60 minutes with a CRP concentration between 100 and 150 µg/mL and Kd of 88 nM. CRP binding was inhibited by 10x cold CRP (58%). CRP (100 µg/mL) significantly upregulated surface expression of the Fc receptors CD32 and CD64 on HAECs. Also, preincubation with anti-CD32 and CD64 antibodies significantly inhibited maximal binding of CRP to HAECs by 64% and 30%, respectively, whereas antibodies to CD16 had no effect. Internalization of CRP, as determined by loss of surface expression, was 50%. Also, binding and internalization of biotinylated CRP were confirmed by fluorescence microscopy, and CRP colocalized with CD32 and CD64. Most importantly, we showed that a stimulatory effect of CRP, ie, the increase in IL-8, and an inhibitory effect of CRP, ie, a decrease in prostacyclin, were abrogated with antibodies to CD32 and CD64. Taken together, these data provide critical insight that CRP mediates its biological effects in endothelial cells via binding and internalization through the Fc receptors CD32 and CD64.
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Receptor for advanced glycation end products (RAGE) has been implicated in the development of endothelial dysfunction and atherosclerosis, especially in diabetes. We have recently demonstrated that CRP upregulates RAGE protein and mRNA expression and that RNA interference (small interfering RNA) with RAGE gene expression significantly decreased the level of MCP-1, a key chemoattractant and downstream mediator of CRP (S. Verma, unpublished data, 2006). CRP also amplifies the effects of hyperglycemia on endothelial cell activation, an effect that is attenuated with the PPAR agonist rosiglitazone.22
CRP enhances lectinlike oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) expression in HAECs.23 LOX-1 is a newly identified endothelial receptor for oxidized LDL that plays a pivotal role in oxidized LDL–induced endothelial dysfunction. Incubation of endothelial cells with CRP enhanced, in a dose- and time-dependent manner, LOX-1 mRNA and protein levels at concentrations as low as 5 µg/mL. This effect was reduced by antibodies against CD32/CD64, endothelin-1, and IL-6. The extent of stimulation of LOX-1 achieved by CRP was comparable to that elicited by high glucose and IL-6, and CRP increased, through LOX-1, both human monocyte adhesion to endothelial cells and oxidized LDL uptake by these cells.
The CD40/CD40 ligand signaling dyad has emerged as a critical cellular hub for oxidative stress, matrix degradation, and plaque rupture. Recently, Lin and colleagues24 have demonstrated a direct effect of CRP to upregulate CD40/CD40 ligand expression in endothelial cells.
Myocardial ischemia provides a potent stimulus to angiogenesis, and the mobilization and differentiation of endothelial progenitor cells (EPCs) have been shown to be important in this process. We have recently evaluated the effects of CRP on human EPC survival and function. CRP at concentrations >15 µg/mL significantly reduced EPC number; inhibited the expression of the endothelial cell–specific markers Tie-2, EC-lectin, and VE-cadherin; significantly increased EPC apoptosis; and impaired EPC-induced angiogenesis.25 EPC-induced angiogenesis was dependent on the presence of NO, and CRP treatment caused a decrease in eNOS mRNA expression by EPCs. We have recently extended these observations and demonstrated that highly purified azide- and endotoxin-free CRP impairs EPC antioxidant defenses and promotes EPC sensitivity toward oxidant-mediated apoptosis and telomerase inactivation, suggesting that CRP not only partakes in the development of endothelial dysfunction but also inhibits the compensatory mechanisms of EPC-mediated endothelial repair and regeneration (S. Verma, unpublished data, 2005).
In addition to the effects of CRP on endothelial NO and PGI2 release, which are known to be inhibitors of coagulation and thrombosis, CRP has a direct effect on inhibiting fibrinolysis via effects on plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA).26,27 We demonstrated that CRP increases PAI-1 expression in HAECs (Figure 3). Incubation of endothelial cells with CRP resulted in a time- and dose-dependent increase in secreted PAI-1 antigen, PAI-1 activity, intracellular PAI-1 protein, and PAI-1 mRNA. CRP stabilized PAI-1 mRNA and caused an additional increase in PAI-1 under hyperglycemic conditions. This was confirmed in bovine aortic endothelial cells in a study in which CRP activated Rho/Rho-kinase signaling, which in turn activated NF-B activity, resulting in increased PAI-1 expression.28 More recently, we have demonstrated that CRP decreases tPA in aortic endothelial cells27 (Figure 3). Endothelial cells exposed to CRP exhibited a profound reduction in tPA antigen and activity. CRP increased IL-1ß and tumor necrosis factor- (TNF-). Neutralization of both IL-1ß and TNF- reversed the inhibition of tPA by CRP. Furthermore, in volunteers who have high CRP levels, euglobulin clot lysis time is significantly increased compared with those who have low CRP levels, providing further evidence that high CRP levels are associated with a procoagulant state. In human coronary artery endothelial cells, Nan et al29 showed that CRP significantly decreased expression of thrombomodulin and endothelial protein C receptor, thereby promoting thrombogenic conditions. This effect was partially mediated by CD32. Human CRP transgenic mice also have been demonstrated to exhibit increased thrombotic occlusion in the femoral artery after injury.30
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CRP Effects Are Not Related to Contamination With Azide and Lipopolysaccharide |
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TopIntroductionCRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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Recent reports suggest that CRP-induced effects are artifacts attributable to the presence of contaminants (lipopolysaccharide [LPS] and azide) in commercial CRP. Because we have previously shown that endotoxin-purified CRP inhibits NO release via eNOS downregulation, we were prompted to reexamine the effect of endotoxin-purified azide-free native CRP on eNOS. We showed that in HAECs this CRP (25 µg/mL) decreased cGMP release (72%; P<0.05) and increased IL-8 (2.1-fold; P<0.01).16,26 Furthermore, using different strategies, we carefully ruled out the possibility that contamination by LPS or NaN3 could be a mediator of the proatherosclerotic effects of CRP. Strategies used to determine whether the effect on tPA was attributed to CRP and not a contaminant included the following (Figure 4). First, CRP was trypsinized by incubating with trypsin-coated beads (TPCK-Trypsin, Pierce Biotechnology, Inc, Rockford, Ill), followed by centrifugation to sediment the trypsin-coated beads. Proteolytic digestion of CRP was confirmed by SDS-PAGE. Second, CRP was incubated in a boiling water bath for 1 hour. Third, CRP was removed by preabsorbing CRP on plates coated with anti-CRP IgG (Alpco Diagnostics, Salem, NH) to serve as negative control, and the corresponding positive control was to incubate CRP in a regular 96-well tissue culture plate without anti-CRP IgG and with the supernatant. Fourth, cells were pretreated with polymyxin B for 30 minutes before CRP challenge to rule out any possible LPS-mediated effect. Fifth, cells also were treated with different doses of LPS (50 and 1000 pg/mL) alone to determine its effect on tPA activity. Sixth, cells were treated with monoclonal antibody (mAb) to CD32 (2.5 µg/mL, BD PharMingen, San Diego, Calif) for 1 hour before CRP challenge. Our experiments document that mAb to CD32 significantly reversed azide-free and LPS-free CRP-mediated tPA inhibition, further supporting our hypothesis that CRP per se inhibits tPA. We have now used similar strategies to demonstrate that the effects of CRP on endothelial progenitor cells and endothelial RAGE expression are not related to contamination with azide or LPS (S. Verma, unpublished data, 2005, 2006). Furthermore, we have isolated ultrapure CRP from human pleural fluid and subjected it to extensive dialysis to confirm the specificity of CRP on endothelial activation and cytokine elaboration (I. Jialal, unpublished data, 2006). Furthermore, Nakakuki et al,28 using an azide-free CRP with an endotoxin concentration of <0.0008 endotoxin units (EU)/mL, convincingly demonstrated increased PAI-1 release in bovine aortic endothelial cells while azide actually inhibited PAI-1 release in this system. Thus, the effects of CRP on eNOS, prostacyclin, PAI-1, and IL-8 have been shown to be independent of endotoxin and azide contamination.16,26–28,31 Also recently, Montero et al31 reported that the effects of CRP on matrix metalloproteinase (MMP)-1 and MMP-10 are specific to CRP and not mediated by endotoxin and that azide failed to have any effects on MMP secretion. The effects of CRP on endothelial cells are specific and receptor-mediated events and not a result of commercial contamination.
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In summary, accumulating evidence suggests that the endothelium is a target for the biological effects of CRP (Figure 5); these effects are specific to CRP, are not related to contamination with either endotoxin or azide, and occur at concentrations observed to predict future vascular events.
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Effects of CRP on Vascular Smooth Muscle Cells and Neointimal Formation |
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TopIntroductionCRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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Given the central importance of angiotensin type 1 receptor (AT1-R) in the pathogenesis of atherosclerosis, we examined the effects of CRP on AT1-R expression and kinetics in vascular smooth muscle cells (VSMCs).32 In addition, the effects of CRP on VSMC migration, proliferation, and reactive oxygen species production were evaluated in the presence and absence of the angiotensin receptor blocker losartan. Finally, the effects of CRP (and losartan) on neointimal formation were examined in vivo in a rat carotid angioplasty model. CRP markedly upregulated AT1-R mRNA and protein expression and increased AT1-R number on VSMCs (Figure 6). CRP promoted VSMC migration and proliferation in vitro and increased reactive oxygen species production. Furthermore, CRP potentiated the effects of angiotensin II on these processes. In the rat carotid artery angioplasty model, exposure to CRP resulted in an increase in cell migration and proliferation, collagen and elastin content, and AT1-R expression, as well as an increase in neointimal formation (Figure 6); these effects were attenuated by losartan. These data point to an important role of CRP as a mediator of adverse vascular remodeling via direct effects on smooth muscle migration and proliferation in vitro and in vivo.
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P<0.01, different from the CRP group. Arrows indicate the position of the internal elastic lamina. Reprinted with permission from Circulation.Blaschke et al33 recently reported that CRP induced caspase-mediated apoptosis of human coronary VSMCs. DNA microarray analysis was used to identify CRP-regulated genes. The growth arrest– and DNA damage–inducible gene 153 (GADD153) mRNA expression was prominently upregulated by CRP. CRP regulation of GADD153 mRNA expression in VSMCs occurred primarily at the posttranscriptional level by mRNA stabilization. Small interfering RNA specifically targeted to GADD153 reduced CRP-induced apoptosis. GADD153 also specifically colocalized to apoptotic VSMCs in human coronary lesions, further supporting a functional role for GADD153 in CRP-induced smooth muscle death.
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Effects of CRP on Monocyte and Macrophage Activity |
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TopIntroductionCRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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Cermak et al34 were the first to demonstrate that CRP induced monocyte tissue factor secretion. Subsequently, Paffen et al35 showed that CRP does not directly induce tissue factor in purified human monocytes. However, when they cultured the peripheral mononuclear cells in which the monocyte population was 10% to 30% with CRP, there was significant upregulation in tissue factor secretion, suggesting that other types of leukocytes may be needed for activation of monocytes to secrete CRP. In monocyte macrophages, after internalization and degradation, CRP has been shown to induce production of hydrogen peroxide at concentrations >10 µg/mL. Ballou and Lozanski36 conducted a study in which they incubated human monocytes with CRP at different doses for 16 hours and were able to demonstrate significantly increased levels of IL-1ß, TNF-
, and IL-6 at concentrations of CRP >5 µg/mL. This induction of cytokine release was unaffected by polymixin B but was completely abrogated by boiling of CRP, confirming that this effect of CRP was not caused by LPS contamination. A single report has shown increased CD11b expression on monocytes incubated with CRP, and this resulted in increased adhesion of these monocytes to LPS-activated human umbilical vein endothelial cells.37 CRP induced phosphorylation of Syk and an increase in [Ca2+](i), both of which were inhibitable by the Syk specific antagonist piceatannol. Piceatannol also inhibited the CRP-induced increase in surface CD11b. In addition, pretreatment of primary monocytes with the Ca2+ mobilizer thapsigargin increased CD11b expression; this effect was accentuated in the presence of CRP but was abolished in the presence of the [Ca2+](i) chelator BAPTA. CRP also increased cytosolic peroxide levels; this effect was attenuated by antioxidants (ascorbate, -tocopherol), with expression of surface CD11b not being inhibited by antioxidants alone. These data suggest that CRP induces CD11b expression in monocytes through a peroxide-independent pathway involving both Syk phosphorylation and [Ca2+](i) release. CRP has been shown to activate complement and stimulate human monocyte chemotaxis. There has been a report that CRP promotes uptake of native LDL. However, this has been brought into question by the Witztum group,37a who showed recently in an elegant study that CRP promotes the uptake of oxidized but not native LDL because of certain unexposed phosphocholine epitopes on oxidized LDL.
Recently, van Tits et al38 have demonstrated that CRP protein and annexin A5 bind to distinct sites of negatively charged phospholipids present in oxidized LDL. The authors report that CRP and annexin A5 at physiological concentrations bind Ca2+ dependently to oxidized phosphatidylcholine present in oxidized LDL but not to native LDL. Binding of CRP to oxidized LDL did not interfere with binding of annexin A5 and vice versa. In the presence of 2 to 10 mg/L CRP, binding of 125I-labeled oxidized LDL to undifferentiated U937 cells increased 50% to 100%. This effect was independent of the presence of complement and could be inhibited by irrelevant IgG and by antibodies to CD64 but not by annexin A5. Annexin A5 alone had no effect on binding of oxidized LDL to the cells. These findings provide conclusive evidence that CRP and annexin A5 at physiological concentrations bind to distinct sites of negatively charged phospholipids present in oxidized LDL and that CRP enhances binding of oxidized LDL to monocyte/macrophage-like cells via Fc receptors. Lim and colleagues39 have demonstrated that p38 MAP inhibition attenuates the proinflammatory response to CRP by human peripheral blood mononuclear cells. CRP-induced p38 kinase activity in human mononuclear cells was blocked by treatment with an inhibitor of p38 kinase, SD-282. CRP induced the expression of tissue factor protein and the secretion of IL-6, IL-8, IL-1ß, TNF-, and PGE.2 Coexposure to CRP and SD-282 blocked the secretion of these proinflammatory and prothrombotic mediators. CRP treatment elevated IL-6, IL-8, IL-1ß, TNF-, COX-2, and tissue factor mRNA expression. These effects of CRP also required p38 activity because SD-282 blocked mRNA induction of each. These results indicate an important relationship between p38 MAPK signaling and CRP-induced proinflammatory and prothrombotic activities in human mononuclear cells.
In human monocytes, Han et al40 demonstrated that CRP upregulated MCP-1–mediated chemotaxis through upregulating CC chemokine receptor 2 expression in human monocytes. Additionally, CRP has been shown to alter the balance of inflammatory cytokines released from monocytes/macrophages. We have recently demonstrated an important effect of CRP on the antiinflammatory cytokine IL-10. Because monocytes/macrophages are the major source of IL-10, we tested the effect of CRP on LPS-induced IL-10 secretion in human monocyte-derived macrophages. Incubation of human monocyte-derived macrophages with azide-free CRP (25 µg/mL) significantly decreased LPS (500 ng/mL)–induced IL-10 mRNA and intracellular and secreted IL-10 in vitro via inhibition of adenyl cyclase. Furthermore, human CRP delivered to Sprague-Dawley rats decreased plasma IL-10 levels. These are the most cogent data that CRP has proinflammatory effects that are independent of LPS or azide.41 In addition to reducing the expression of antiinflammatory cytokines, CRP promotes the release of proinflammatory cytokines.
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Effects of CRP on Matrix Metalloproteinases |
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TopIntroductionCRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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MMPs have been widely implicated in the development of plaque instability and rupture. Recently, Montero et al31 demonstrated that CRP increased levels of MMP-1 and MMP-10 in human endothelial cells without any significant change in the tissue inhibitor of MMP. They also showed that CRP treatment resulted in an increase in MMP activity. Furthermore, specific inhibition of p38MAPK or MEK abolished the CRP induction of MMP-1, whereas blockade of MMP-10 induction required the simultaneous blockade of p38MAPK and JNK pathways. In patients with CRP >3 mg/L compared with patients with lower levels of CRP, both MMP-1 and MMP-10 levels were elevated after adjustment for confounding variables. Finally, CRP and MMP colocalized in the endothelial layer and macrophage-rich areas of advanced atherosclerotic plaques. Similar findings have been reported in macrophages in which CRP increased production of MMP-1 through the Fc
receptor II CD32 via ERK activation. Recently, Doronzo et al42 evaluated the effects of CRP on the synthesis and release of MMP-2, which are known to play a critical role in plaque instabilization and vascular remodeling. CRP upregulated MMP-2 mRNA expression. MMP-2 synthesis and activity were increased by 1 to 10 mg/L CRP starting from an 8-hour incubation. The effect was prevented by exposure to PD98059. CRP did not modify the tissue inhibitor of MMP-2 mRNA expression, protein synthesis, and secretion. Extracellular MMP inducer and MMP-9 have been reported to be expressed at the macrophage-rich area in human coronary atherosclerotic plaque. Abe and colleagues43 have recently demonstrated that CRP at 5 µg/mL increased the gene expression of extracellular MMP inducer in human macrophages. Furthermore, CRP increased gene expression and activity of MMP-9 with no effect on the tissue inhibitor of MMP-1. Boiled CRP at 5 µg/mL for 48 hours had no effect on MMP-9 activity. Taken together, CRP may directly influence the integrity of the extracellular matrix and tip the balance in favor of matrix degradation with eventual predisposition to rupture. |
Autocrine and Paracrine Role of CRP in Atherosclerosis |
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TopIntroductionCRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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An emerging body of evidence suggests that CRP may alter vascular homeostasis through an autocrine and/or paracrine mechanism. Although initially thought to be produced solely from the liver, recent evidence suggests that CRP may be released from endothelial cells, VSMCs, and even adipocytes.44 Yasojima et al45 provided cogent evidence that CRP mRNA in atheroma was 10 times greater than in the normal vessel. Using an anti-sense riboprobe, Kobayashi et al46 have shown in 39 directed coronary atherectomy samples that CRP is present in the coronary atheroma. This has been confirmed by other groups.44 Sattler et al47 have recently documented CRP protein and mRNA using techniques of Western blotting, immunohistochemistry, and real-time reverse transcriptase–polymerase chain reaction (RT-PCR) in plaques obtained from patients undergoing carotid endarterectomy (n=41). Furthermore, the CRP staining was in plaque shoulders, microvessels, or borders, mainly in foam cells and endothelial cells. Inoue et al48 demonstrated local release of CRP from vulnerable plaques and coronary arterial wall injured by stenting. In that study, CRP levels in coronary arterial blood sampled just distal and proximal to the culprit lesions in 36 patients with stable angina and 13 patients with unstable angina were examined. CRP levels were higher in distal blood than proximal blood in both stable and unstable angina. The translesional CRP gradient and the proximal and distal CRP were higher in unstable angina than in stable angina. Furthermore, the transcardiac CRP gradient (coronary sinus minus peripheral blood) and activated Mac-1 increased gradually after stenting, reaching a maximum at 48 hours, with a positive correlation between the transcardiac CRP gradient and activated Mac-1. These data suggest that CRP is released locally from the coronary vasculature and may partake in an autocrine/paracrine fashion to impair endothelial function and thrombogenicity.
The role of CRP as a local factor involved in atherothrombosis was strengthened by observations indicating that human arterial endothelial cells produce CRP (Figure 7). We detected the presence of CRP mRNA by RT-PCR and in situ hybridization, intracellular protein by Western blot, and secreted protein by enzyme-linked immunoassay.49 Coincubation with the cytokines IL-1, IL-6, and TNF alone and in combination showed that the most potent agonist for CRP production from HAECs is the combination of IL-1 and IL-6 (P<0.05). To mimic the in vivo situation, we examined whether VSMC- and/or macrophage-conditioned media (MCM) could augment CRP production by HAECs. Although VSMC-conditioned media had no effect, incubation with MCM resulted in a significant 2-fold increase in the synthesis of both intracellular and secreted CRP. The effect of MCM could be reversed by inhibiting both IL-1 and IL-6. The recent observations that CRP is produced in aortic endothelial cells and that secreted CRP could be augmented 100-fold with human MCM incubated with endothelial cells argue for paracrine and autocrine loops in the atheroma that could result in exceedingly high CRP concentration in microdomains. Indeed, plasma CRP levels ranging from 20 to 64 mg/L have been reported in patients with acute coronary syndrome, and levels appear to be higher in aortic sinus samples and predict poorer outcomes.50
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In Vivo Role of CRP to Promote Endothelial Dysfunction, Extend Myocardial Infarction, and Increase Atherothrombosis |
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TopIntroductionCRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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An in vivo role for CRP in the development of endothelial dysfunction and inflammation has been suggested by elegant experiments. Bisoendial et al51 demonstrated a marked activation of inflammation and coagulation after infusion of CRP in humans (Figure 8). Male volunteers received an infusion on 2 occasions of 1.25 mg/kg recombinant human CRP (rhCRP) or diluent, respectively. CRP concentrations rose after rhCRP infusion from 1.9 mg/L (0.3 to 8.5 mg/L) to 23.9 mg/L (20.5 to 28.1 mg/L); subsequently, both inflammation and coagulation were activated. CRP concentrations rose on infusion of rhCRP from 1.9 mg/L (0.3 to 8.5 mg/L) to 23.9 mg/L (20.5 to 28.1 mg/L). After an initial fall, concentrations rose again to 29.0 mg/L (17.2 to 48.9 mg/L) 24 hours after infusion. Hemodynamic measurements and temperature recordings were stable throughout the infusion studies. There was no record of any adverse effect in the volunteers. Hematology indexes remained stable, except for transient increases in neutrophil counts. CRP infusion resulted in an increase in von Willebrand factor antigen, E-selectin, IL-6, and IL-8 with a trend toward monocytic CD11b and CD18 upregulation. After 8 hours, both serum amyloid A protein and sPLA2 concentrations rose significantly. There was also a 3-fold increase in prothrombin F1+2 concentrations 4 hours after rhCRP infusion and a marked increase in D-dimer concentrations and PAI-1 release. The authors conclude that these findings strongly suggest that CRP activates several pathways with known consequences for cardiovascular events. Even a short-term increase from a single bolus, obtaining concentrations that are pathophysiologically relevant, induces endothelial cell activation, elicits an acute systemic inflammatory response, and activates the coagulation cascade. This striking sequence of events indicates that CRP, beyond its predictive value, probably also has a causal relation to the occurrence of cardiovascular events. The study was criticized by Pepys and colleagues,52 who suggested that endotoxin contamination may have mediated these effects. However, as asserted by Bisoendial et al,53 there were errors in the interpretation of the data by Pepys et al. In fact, compared with the Pepys et al observations, endotoxin activity was 30 times lower (<1.5 EU/mL), which resulted in <1.6 EU per 1 mg of rhCRP (at a CRP concentration of 0.91 mg/mL). This trace amount was similar to that reported by Pepys et al in their natural human CRP solution, (0.9 EU/mg of natural human CRP). Pepys et al also failed to acknowledge that the residual endotoxin levels in the rhCRP have been proved insufficient to cause any bioactivity in vivo and in vitro. Bisoendial and colleagues directly refuted the claims of Pepys et al by demonstrating that infusion of LPS (Escherichia coli LPS, lot G2B274, United States Pharmacopeial Convention Inc, Rockville, Md) into 2 healthy volunteers at a dose (1.5 EU/kg) that equaled the mean coinfused dose during the CRP infusion experiments did not result in any change in TNF.53 Thus, the results of Bisoendial and colleagues demonstrating direct endothelial and inflammatory activation by CRP in humans in vivo cannot be explained by endotoxin contamination. In fact, using their method for endotoxin purification, Bisoendial et al54 showed that their final purified product was >97% pure by high-pressure size-exclusion chromatography and reverse-phase high-performance liquid chromatography, whereas time-of-flight mass spectrometry provided supporting data for high purity, showing no other protein fractions besides the rhCRP.
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Additional evidence for an in vivo role of CRP in cardiovascular disease stems from studies demonstrating the ability of CRP to induce myocardial infraction in a rat coronary ligation model, to induce increased susceptibility to simulated cardiomyocyte ischemia and reperfusion (S. Verma, unpublished data, 2003), to induce increased cerebral infarct size in rats after middle cerebral artery occlusion, and to promote neointimal formation after balloon angioplasty in a rat model. In the hypercholesterolemic pig model, Turk et al55 showed that serum CRP correlated with macrophage accumulation and coronary artery disease; these researchers immunohistochemically demonstrated costaining for CRP in the macrophage foam cells in the intima.
Recently, Sun et al56 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 (Figure 9). 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 and stable and unstable plaques (Figure 10). CRP was present extracellularly and colocalized with apolipoprotein B but was rarely associated with the cytoplasm of macrophages and foam cells, further pointing to a role of CRP in the pathophysiology of atherothrombosis. Also, mRNA for CRP has been found in lesions (Figure 10).
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If CRP contributes to plaque instability and the genesis of acute coronary syndromes, then modulating CRP in the setting of the acute coronary syndrome may prove beneficial. In this regard, exciting new data are emerging. In both Pravastatin or Atorvastatin Evaluation and Infection Therapy–Thrombolysis in Myocardial Infarction 22 (PROVE IT-TIMI 22) and the Aggrastat to Zocor (A to Z) study,57,58 concomitant reduction of LDL and CRP with statin therapy resulted in a greater benefit in cardiovascular end points. In addition, in patients with chronic coronary artery disease (as demonstrated by the REVERSAL [Reversal of Atherosclerosis With Aggressive Lipid Lowering] study), intensive treatment with statin resulted in the greatest benefit, and reductions in high-sensitivity CRP and LDL cholesterol below the median were associated with slower disease progression.59 These studies further support the notion that CRP might indeed be an active participant in atherothrombosis and the genesis of acute coronary syndromes. However, these exciting preliminary findings need to be confirmed in future studies.
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Pitfalls of Mice Models of CRP and Atherosclerosis |
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TopIntroductionCRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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As stated by Torzewski,60 it is important to note that the most broadly available animal model, ie, the mouse, is considered useless for the study of CRP functions because CRP is not an acute-phase reactant in mice. The development of a transgenic CRP-overexpressing mouse to study atherogenesis is also fraught with problems and has resulted in conflicting observations. Although Paul et al61 demonstrated increased atherosclerosis in CRP transgenic apolipoprotein E–deficient mice, Hirschfield et al62 and Trion et al63 were unable to uncover a similar phenotype. As elegantly stated by Torzewski, it is difficult to determine which of the reports is right or wrong, valid or invalid, because the model itself encounters several problems, as human CRP is a foreign antigen in the mouse, with many uncertainties concerning its functional role in the immune system of these animals. Reifenberg et al64 also cautioned against overinterpretation of the atherosclerotic effects of CRP in transgenic mice models and stated that the interactions among CRP, complement, and LDL, as delineated in humans, may not exist similarly in mice and that the lack of effect noted may be due to inactivity of CRP in mice. The fact that CRP transgenic mice are incapable of mounting one of the most fundamental effects of CRP, ie, lipoprotein-dependent complement activation, argues strongly against the validity of this model for physiological and pathophysiological assessments.
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Future Directions |
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TopIntroductionCRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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Given the limitations of the CRP transgenic mouse model, it is imperative to develop in vivo antisense oligonucleotides directed against CRP and test them in experimental and clinical models. Such directed antisense oligonucleotide therapies are currently being developed (ISIS Pharmaceuticals, oral communication, October, 2005); the results of these studies are eagerly awaited. The importance of modified monomeric CRP requires clinical validation.65–67 Although monomeric CRP appears to exert greater proinflammatory effects in endothelial cells and leukocytes, it remains to be seen whether monomeric CRP is expressed to a greater degree in atherosclerotic plaques relative to pentameric CRP. Paradoxically, a recent article by Schwedler et al68 reported that native CRP promoted but modified CRP reduced atherosclerosis in apolipoprotein E knockout mice, further questioning the in vivo relevance of monomeric CRP.
In summary, the data suggesting that CRP incites vascular disease continue to evolve. At present, the balance of published information supports a role of CRP as a partaker of endothelial dysfunction, vascular remodeling, and atherothrombosis. Studies demonstrating CRP release from endothelial cells suggest that local concentrations of CRP may be much higher than systemic levels evaluated in clinical trials and may serve to amplify atherothrombosis.
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Acknowledgments |
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TopIntroductionCRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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This work was supported by a grant-in-aid from Heart and Stroke Foundation and Canadian Institutes of Health Research (Dr Verma) and NIH K24 AT 00596 and NIH HL07436 (Dr Jialal).
Disclosures
None.
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References |
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TopIntroductionCRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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Response to Verma et al
We read with great interest the excellent review by Verma, Deveraj, and Jialal and compliment them on both their comprehensive overview and their contributions to the field. Inflammation, and in particular C-reactive protein (CRP), has taken center stage in our view of atherothrombosis. As emphasized by both of our reviews, investigation regarding the potential role of CRP in atherosclerosis is rapidly evolving, with important clinical implications. Before we can achieve our ultimate goal of translating this research into interventions that improve the care of patients, several important and outstanding questions must be settled. The recent reports of possible contamination of CRP preparations used for in vitro and in vivo experiments must be addressed, and we look forward to the forthcoming data that Verma and colleagues cite in their review. We all agree that there is a need for development of an animal model other than the murine model used frequently in this field so that the potential pathogenic properties of native CRP can be adequately tested. Last, we believe that the most convincing test of the allegation that CRP is proatherogenic will come with investigation of agents that interfere directly with CRP without modulation of other contributors to atherogenesis, such as lipids. The data elaborated by Verma and others has supported the mounting case for a proatherogenic role of CRP. However, conflicting evidence remains and must be adequately "cross-examined" before this case can be closed.
Response to Scirica and Morrow
In our previous article in this section, we presented a cogent argument that C-reactive protein (CRP) is an active participant in atherothrombosis. However, Scirica and Morrow argue that CRP is an innocent bystander and failed to critically appraise the recent studies with regard to potential contaminants in CRP. As detailed in our article, numerous groups have shown that CRP indeed stimulates plasminogen activator inhibitor-1, interleukin-8, matrix metalloproteinases, and monocyte-endothelial cell adhesion and inhibits endothelial nitric oxide synthase and tissue plasminogen activator in endothelial cells, independently of contaminants. Furthermore, these effects could be reversed by blocking Fc receptors.
In addition, Scirica and Morrow argue that one of the effects of CRP, ie, a decrease in inducible nitric oxide synthase, is due to contaminants and quote LaFuente et al.1 However, Venugopal et al2 have clearly shown that purified CRP indeed stimulates inducible nitric oxide synthase, resulting in nitration of prostacyclin synthase. This clearly separates a direct effect of CRP from contaminants. We also present data that in relevant animal models (rat, rabbit, and pig), CRP levels correlate with atherothrombosis.
Finally, delivery of CRP to humans induces a proinflammatory, procoagulant effect that does not appear to be due to endotoxin or other contaminants. Thus, several lines of evidence support a role of CRP in atherothrombosis. However, much more research is needed before it can be used as a target for treatment.
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Footnotes |
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The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.
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TopIntroductionCRP as a Marker...CRP as a Potential...CRP as a Target...ConclusionsReferencesCRP Promotes Endothelial Cell...CRP Effects Are Not...Effects of CRP on...Effects of CRP on...Effects of CRP on...Autocrine and Paracrine Role...In Vivo Role of...Pitfalls of Mice Models...Future DirectionsAcknowledgments References References |
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1. Lafuente N, Azcutia V, Matesanz N, Cercas E, Rodriguez-Manas L, Sanchez-Ferrer CF, Peiro C. Evidence for sodium azide as an artifact mediating the modulation of inducible nitric oxide synthase by C-reactive protein. J Cardiovasc Pharmacol. 2005; 45: 193–196.[CrossRef][Medline] [Order article via Infotrieve]
2. Venugopal SK, Devaraj S, Jialal I. C-reactive protein decreases prostacyclin release from human aortic endothelial cells. Circulation. 2003; 108: 1676–1678.
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 H. Schunkert and N. J. Samani Elevated C-Reactive Protein in Atherosclerosis -- Chicken or Egg? N. Engl. J. Med., October 30, 2008; 359(18): 1953 - 1955. [Full Text] [PDF]
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 P. G. Surtees, N. W. J. Wainwright, S. M. Boekholdt, R. N. Luben, N. J. Wareham, and K.-T. Khaw Major Depression, C-Reactive Protein, and Incident Ischemic Heart Disease in Healthy Men and Women Psychosom Med, October 1, 2008; 70(8): 850 - 855. [Abstract] [Full Text] [PDF]
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 C J Currie, C D Poole, and P Conway Evaluation of the association between the first observation and the longitudinal change in C-reactive protein, and all-cause mortality Heart, April 1, 2008; 94(4): 457 - 462. [Abstract] [Full Text] [PDF]
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 S. K. Singh, M. V. Suresh, D. C. Prayther, J. P. Moorman, A. E. Rusinol, and A. Agrawal C-Reactive Protein-Bound Enzymatically Modified Low-Density Lipoprotein Does Not Transform Macrophages into Foam Cells J. Immunol., March 15, 2008; 180(6): 4316 - 4322. [Abstract] [Full Text] [PDF]
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 E. A. Van Vre, H. Bult, V. Y. Hoymans, V. F.I. Van Tendeloo, C. J. Vrints, and J. M. Bosmans Human C-Reactive Protein Activates Monocyte-Derived Dendritic Cells and Induces Dendritic Cell-Mediated T-Cell Activation Arterioscler Thromb Vasc Biol, March 1, 2008; 28(3): 511 - 518. [Abstract] [Full Text] [PDF]
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M. Salio, S. Chimenti, N. De Angelis, F. Molla, V. Maina, M. Nebuloni, F. Pasqualini, R. Latini, C. Garlanda, and A. Mantovani Cardioprotective Function of the Long Pentraxin PTX3 in Acute Myocardial Infarction Circulation, February 26, 2008; 117(8): 1055 - 1064. [Abstract] [Full Text] [PDF]
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 D. Xing, F. G. Hage, Y.-F. Chen, M. A. McCrory, W. Feng, G. A. Skibinski, E. Majid-Hassan, S. Oparil, and A. J. Szalai Exaggerated Neointima Formation in Human C-Reactive Protein Transgenic Mice Is IgG Fc Receptor Type I (Fc{gamma}RI)-Dependent Am. J. Pathol., January 1, 2008; 172(1): 22 - 30. [Abstract] [Full Text] [PDF]
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 S. M. Nelson, N. Sattar, D. J. Freeman, J. D. Walker, and R. S. Lindsay Inflammation and Endothelial Activation Is Evident at Birth in Offspring of Mothers With Type 1 Diabetes Diabetes, November 1, 2007; 56(11): 2697 - 2704. [Abstract] [Full Text] [PDF]
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P. M. Ridker C-Reactive Protein and the Prediction of Cardiovascular Events Among Those at Intermediate Risk: Moving an Inflammatory Hypothesis Toward Consensus J. Am. Coll. Cardiol., May 29, 2007; 49(21): 2129 - 2138. [Abstract] [Full Text] [PDF]
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E. Arbustini Total Erythrocyte Membrane Cholesterol: An Innocent New Marker or an Active Player in Acute Coronary Syndromes? J. Am. Coll. Cardiol., May 29, 2007; 49(21): 2090 - 2092. [Full Text] [PDF]
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S. Lavi, J. P. McConnell, C. S. Rihal, A. Prasad, V. Mathew, L. O. Lerman, and A. Lerman Local Production of Lipoprotein-Associated Phospholipase A2 and Lysophosphatidylcholine in the Coronary Circulation: Association With Early Coronary Atherosclerosis and Endothelial Dysfunction in Humans Circulation, May 29, 2007; 115(21): 2715 - 2721. [Abstract] [Full Text] [PDF]
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P. E. Szmitko and S. Verma C-Reactive Protein and Reendothelialization: NO Involvement Circ. Res., May 25, 2007; 100(10): 1405 - 1407. [Full Text] [PDF]
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R. Schwartz, S. Osborne-Lawrence, L. Hahner, L. L. Gibson, A. K. Gormley, W. Vongpatanasin, W. Zhu, R. A. Word, D. Seetharam, S. Black, et al. C-Reactive Protein Downregulates Endothelial NO Synthase and Attenuates Reendothelialization In Vivo in Mice Circ. Res., May 25, 2007; 100(10): 1452 - 1459. [Abstract] [Full Text] [PDF]
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W. Koenig Serum Amyloid P Component and Cardiovascular Disease: Is There a Sensible Link? Arterioscler Thromb Vasc Biol, April 1, 2007; 27(4): 698 - 700. [Full Text] [PDF]
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NACB WRITING GROUP MEMBERS, D. A. Morrow, C. P. Cannon, R. L. Jesse, L. K. Newby, J. Ravkilde, A. B. Storrow, A. H.B. Wu, R. H. Christenson, NACB COMMITTEE MEMBERS, et al. National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines: Clinical Characteristics and Utilization of Biochemical Markers in Acute Coronary Syndromes Clin. Chem., April 1, 2007; 53(4): 552 - 574. [Full Text] [PDF]
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N. Sattar, H. M. Murray, A. McConnachie, G. J. Blauw, E. L.E.M. Bollen, B. M. Buckley, S. M. Cobbe, I. Ford, A. Gaw, M. Hyland, et al. C-Reactive Protein and Prediction of Coronary Heart Disease and Global Vascular Events in the Prospective Study of Pravastatin in the Elderly at Risk (PROSPER) Circulation, February 27, 2007; 115(8): 981 - 989. [Abstract] [Full Text] [PDF]
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C. Rocker, D. E. Manolov, E. V. Kuzmenkina, K. Tron, H. Slatosch, J. Torzewski, and G. U. Nienhaus Affinity of C-Reactive Protein toward Fc{gamma}RI Is Strongly Enhanced by the {gamma}-Chain Am. J. Pathol., February 1, 2007; 170(2): 755 - 763. [Abstract] [Full Text] [PDF]
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S. Blankenberg and S. Yusuf The Inflammatory Hypothesis: Any Progress in Risk Stratification and Therapeutic Targets? Circulation, October 10, 2006; 114(15): 1557 - 1560. [Full Text] [PDF]
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 S. Devaraj, B. Davis, S. I. Simon, and I. Jialal CRP promotes monocyte-endothelial cell adhesion via Fc{gamma} receptors in human aortic endothelial cells under static and shear flow conditions Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1170 - H1176. [Abstract] [Full Text] [PDF]
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D. A. Morrow, J. A. de Lemos, M. S. Sabatine, S. D. Wiviott, M. A. Blazing, A. Shui, N. Rifai, R. M. Califf, and E. Braunwald Clinical Relevance of C-Reactive Protein During Follow-Up of Patients With Acute Coronary Syndromes in the Aggrastat-to-Zocor Trial Circulation, July 25, 2006; 114(4): 281 - 288. [Abstract] [Full Text] [PDF]
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G. Davey Smith, N. Timpson, and D. A. Lawlor C-Reactive Protein and Cardiovascular Disease Risk: Still an Unknown Quantity? Ann Intern Med, July 4, 2006; 145(1): 70 - 72. [Full Text] [PDF]
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