Expression Of Human C-reactive Protein Research Articles

Excess ischemic tachyarrhythmias trigger protection against myocardial infarction in hypertensive rats.

Increased level of C-reactive protein (CRP) is a risk factor for cardiovascular diseases, including myocardial infarction and hypertension. Here, we analyzed the effects of CRP overexpression on cardiac susceptibility to ischemia/reperfusion (I/R) injury in adult spontaneously hypertensive rats (SHR) expressing human CRP transgene (SHR-CRP). Using an in vivo model of coronary artery occlusion, we found that transgenic expression of CRP predisposed SHR-CRP to repeated and prolonged ventricular tachyarrhythmias. Excessive ischemic arrhythmias in SHR-CRP led to a significant reduction in infarct size (IS) compared with SHR. The proarrhythmic phenotype in SHR-CRP was associated with altered heart and plasma eicosanoids, myocardial composition of fatty acids (FAs) in phospholipids, and autonomic nervous system imbalance before ischemia. To explain unexpected IS-limiting effect in SHR-CRP, we performed metabolomic analysis of plasma before and after ischemia. We also determined cardiac ischemic tolerance in hearts subjected to remote ischemic perconditioning (RIPer) and in hearts ex vivo. Acute ischemia in SHR-CRP markedly increased plasma levels of multiple potent cardioprotective molecules that could reduce IS at reperfusion. RIPer provided IS-limiting effect in SHR that was comparable with myocardial infarction observed in naïve SHR-CRP. In hearts ex vivo, IS did not differ between the strains, suggesting that extra-cardiac factors play a crucial role in protection. Our study shows that transgenic expression of human CRP predisposes SHR-CRP to excess ischemic ventricular tachyarrhythmias associated with a drop of pump function that triggers myocardial salvage against lethal I/R injury likely mediated by protective substances released to blood from hypoxic organs and tissue at reperfusion.

Human C-reactive protein aggravates osteoarthritis development in mice on a high-fat diet

C-reactive protein (CRP) levels can be elevated in osteoarthritis (OA) patients. In addition to indicating systemic inflammation, it is suggested that CRP itself can play a role in OA development. Obesity and metabolic syndrome are important risk factors for OA and also induce elevated CRP levels. Here we evaluated in a human CRP (hCRP)-transgenic mouse model whether CRP itself contributes to the development of 'metabolic' OA. Metabolic OA was induced by feeding 12-week-old hCRP-transgenic males (hCRP-tg, n=30) and wild-type littermates (n=15) a 45kcal% high-fat diet (HFD) for 38 weeks. Cartilage degradation, osteophytes and synovitis were graded on Safranin O-stained histological knee joint sections. Inflammatory status was assessed by plasma lipid profiling, flow cytometric analyses of blood immune cell populations and immunohistochemical staining of synovial macrophage subsets. Male hCRP-tg mice showed aggravated OA severity and increased osteophytosis compared with their wild-type littermates. Both classical and non-classical monocytes showed increased expression of CCR2 and CD86 in hCRP-tg males. HFD-induced effects were evident for nearly all lipids measured and indicated a similar low-grade systemic inflammation for both genotypes. Synovitis scores and synovial macrophage subsets were similar in the two groups. Human CRP expression in a background of HFD-induced metabolic dysfunction resulted in the aggravation of OA through increased cartilage degeneration and osteophytosis. Increased recruitment of classical and non-classical monocytes might be a mechanism of action through which CRP is involved in aggravating this process. These findings suggest interventions selectively directed against CRP activity could ameliorate metabolic OA development.

Hepatic but Not CNS-Expressed Human C-Reactive Protein Inhibits Experimental Autoimmune Encephalomyelitis in Transgenic Mice

We recently demonstrated that human C-reactive protein (CRP), expressed hepatically in transgenic mice (CRPtg), improved the outcome of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). The liver is the primary site of CRP synthesis in humans and in CRPtg mice but is also expressed by both at low levels in the CNS. To determine if CNS expression of human CRP is sufficient to impact EAE, we generated neuronal CRP transgenic mice (nCRPtg) wherein human CRP expression is driven by the neuron-specific Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) gene promoter. We found that hepatically expressed/blood-borne CRP, but not CNS expressed CRP, lessened EAE severity. These outcomes indicate that the protective actions of human CRP in EAE are manifested in the periphery and not in the CNS and reveal a previously unappreciated site specificity for the beneficial actions of CRP in CNS disease.

Central nervous system-specific expression of C-reactive protein exacerbates experimental autoimmune encephalomyelitis. (BA3P.202)

Abstract Long considered merely a marker of inflammation, new evidence indicates that C-reactive protein (CRP) may also affect the maintenance of health and propagation of disease. Our lab previously showed that human CRP, hepatically synthesized by human CRP transgenic mice (CRPtg), delays onset and reduces severity of Experimental Autoimmune Encephalomyelitis (EAE; a model of Multiple Sclerosis). Though no causal data are available for humans, research suggests that unlike CRPtg, expression/deposition of CRP in the CNS associates with disease, rather than protection. Though human CRP expression in both man and CRPtg mouse is largely hepatic, studies have shown that it is also expressed by multiple cells of the CNS, particularly during neuroinflammation. To test CNS-expressed CRP’s impact on EAE, we made transgenic mice wherein the neuron-specific Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) promoter drives human CRP expression. We confirmed that human CRP expression is restricted to the CNS in these neuronal CRPtg (nCRPtg) mice. In contrast to CRPtg, CNS-specific expression of CRP neither delays EAE onset nor improves its symptoms. As in humans, CNS-expressed CRP associates with more severe disease. In summary, observations made with CRPtg vs. nCRPtg reveal a previously unappreciated site-specificity to the impact of CRP on the CNS. The possibility that CRP mediates beneficial vs. detrimental actions depending on the local cellular environment will be investigated.

Endothelial C-reactive protein increases platelet adhesion under flow conditions

While data regarding the pathogenetic role of C-reactive protein (CRP) in atherothrombosis are accumulating, it is still controversial whether local CRP secretion is of any pathobiological significance. The present study examined whether endothelial-derived CRP modulates autocrine prothrombotic activity. Endothelial cells were isolated from hearts of mice transgenic to human CRP and grown in primary cultures. Human CRP expression was confirmed in these cells compared with no expression in cultures derived from wild-type congenes. Adhesion of human platelets to endothelial cells was studied in the "cone and plate" flow system. Platelet adhesion to cells expressing CRP was significantly increased compared with that in controls (n = 6, P < 0.01). The proadhesive effect of CRP was significantly suppressed in mouse heart endothelial cells and in human umbilical vein endothelial cells following treatment with small interfering RNA for human CRP. Adhesion was modulated by an increase in P-selectin. P-selectin expression correlated with a proadhesive phenotype, and blocking P-selectin with neutralizing antibody significantly decreased the adhesion of platelets to CRP-expressing cells (40.4 ± 10.5 to 9.4 ± 6.9 platelets/high-power field, n = 5 to 6, P < 0.01). In conclusion, human CRP that is locally produced in endothelial cells increases platelet adhesion to endothelial cells under normal shear flow conditions. These findings indicate that CRP exerts a local effect on endothelial cells via P-selectin expression, which promotes platelet adhesion and subsequent thrombus formation.

Anti-inflammatory, anti-proliferative and anti-atherosclerotic effects of quercetin in human in vitro and in vivo models

ObjectivePolyphenols such as quercetin may exert several beneficial effects, including those resulting from anti-inflammatory activities, but their impact on cardiovascular health is debated. We investigated the effect of quercetin on cardiovascular risk markers including human C-reactive protein (CRP) and on atherosclerosis using transgenic humanized models of cardiovascular disease. MethodsAfter evaluating its anti-oxidative and anti-inflammatory effects in cultured human cells, quercetin (0.1%, w/w in diet) was given to human CRP transgenic mice, a humanized inflammation model, and ApoE*3Leiden transgenic mice, a humanized atherosclerosis model. Sodium salicylate was used as an anti-inflammatory reference. ResultsIn cultured human endothelial cells, quercetin protected against H2O2-induced lipid peroxidation and reduced the cytokine-induced cell-surface expression of VCAM-1 and E-selectin. Quercetin also reduced the transcriptional activity of NFκB in human hepatocytes. In human CRP transgenic mice (quercetin plasma concentration: 12.9±1.3μM), quercetin quenched IL1β-induced CRP expression, as did sodium salicylate. In ApoE*3Leiden mice, quercetin (plasma concentration: 19.3±8.3μM) significantly attenuated atherosclerosis by 40% (sodium salicylate by 86%). Quercetin did not affect atherogenic plasma lipids or lipoproteins but it significantly lowered the circulating inflammatory risk factors SAA and fibrinogen. Combined histological and microarray analysis of aortas revealed that quercetin affected vascular cell proliferation thereby reducing atherosclerotic lesion growth. Quercetin also reduced the gene expression of specific factors implicated in local vascular inflammation including IL-1R, Ccl8, IKK, and STAT3. ConclusionQuercetin reduces the expression of human CRP and cardiovascular risk factors (SAA, fibrinogen) in mice in vivo. These systemic effects together with local anti-proliferative and anti-inflammatory effects in the aorta may contribute to the attenuation of atherosclerosis.

Expression of human c-reactive protein in different systems and its purification from Leishmania tarentolae

With its homo-pentameric structure and calcium-dependent specificity for phosphocholine (PCh), human c-reactive protein (CRP) is produced by the liver and secreted in elevated quantities in response to inflammation. CRP is widely accepted as a cardiac marker, e.g. in point-of-care diagnostics, however, its heterologous expression has proven difficult. Here, we demonstrate the expression of CRP in different Escherichia coli strains as well as by in vitro transcription/translation. Although expression in these systems was straightforward, most of the protein that accumulated was insoluble. We therefore expanded our study to include the expression of CRP in two eukaryotic hosts, namely the yeast Kluyveromyces lactis and the protozoon Leishmania tarentolae. Both expression systems are optimized for secretion of recombinant proteins and here allowed successful expression of soluble CRP. We also demonstrate the purification of recombinant CRP from Leishmania growth medium; the purification of protein expressed from K. lactis was not successful. Functional and intact CRP pentamer is known to interact with PCh in Ca 2+-dependent manner. In this report we verify the binding specificity of recombinant CRP from L. tarentolae (2 μg/mL culture medium) for PCh.

Increased C-reactive protein expression exacerbates left ventricular dysfunction and remodeling after myocardial infarction

We previously reported serum C-reactive protein (CRP) elevation after acute myocardial infarction (MI) to be associated with adverse outcomes including cardiac rupture, left ventricular (LV) remodeling, and cardiac death. Experimental studies have indicated that CRP per se has various biological actions including proinflammatory and proapoptotic effects, suggesting a pathogenic role of CRP in the post-MI remodeling process. We tested the hypothesis that increased CRP expression would exacerbate adverse LV remodeling after MI via deleterious effects of CRP. Transgenic mice with human CRP expression (CRP-Tg) and their transgene-negative littermates (control) underwent left coronary artery ligation. There was no apparent difference in phenotypic features between CRP-Tg and control mice before MI. Although mortality and infarct size were similar in the two groups, CRP-Tg mice showed more LV dilation and worse LV function with more prominent cardiomyocyte hypertrophy and fibrosis in the noninfarcted regions after MI than controls. Histological evaluation conducted 1 wk post-MI revealed a higher rate of apoptosis and more macrophage infiltration in the border zones of infarcted hearts from CRP-Tg mice in relation to increased monocyte chemotactic protein (MCP)-1 expression and matrix metalloproteinase (MMP)-9 activity. Increased CRP expression exacerbates LV dysfunction and promotes adverse LV remodeling after MI in mice. The deleterious effect of CRP on post-MI LV remodeling may be associated with increased apoptotic rates, macrophage infiltration, MCP-1 expression, and MMP-9 activity in the border zone.

Letter by Nakajima and Saito Regarding Article, “Human C-Reactive Protein Does Not Promote Atherosclerosis in Transgenic Rabbits”

To the Editor: Koike et al1 recently reported in Circulation that the transgenic expression of human C-reactive protein (CRP) in rabbits fed a cholesterol-rich diet did not promote the pathogenesis of atherosclerosis despite its detection in atherosclerotic plaque. This result supports some animal studies cited in the article and several epidemiological studies that indicate no associations between elevated circulating CRP and cardiovascular disease.2 However, in clinical practice, CRP is often elevated in people with excess visceral fat, independent of genetic influences,3 and predisposes them to critical metabolic dysfunctions related to insulin resistance, type 2 diabetes mellitus, and the metabolic syndrome (MetS). …

MIF Deficiency Reduces Chronic Inflammation in White Adipose Tissue and Impairs the Development of Insulin Resistance, Glucose Intolerance, and Associated Atherosclerotic Disease

Chronic inflammation in white adipose tissue (WAT) is positively associated with obesity, insulin resistance (IR) and the development of type 2 diabetes. The proinflammatory cytokine MIF (macrophage migration inhibitory factor) is an essential, upstream component of the inflammatory cascade. This study examines whether MIF is required for the development of obesity, IR, glucose intolerance, and atherosclerosis in the LDL receptor-deficient (Ldlr(-/-)) mouse model of disease. Ldlr(-/-) mice develop IR and glucose intolerance within 15 weeks, whereas Mif(-/-)Ldlr(-/-) littermates are protected. MIF deficiency does not affect obesity and lipid risk factors but specifically reduces inflammation in WAT and liver, as reflected by lower plasma serum amyloid A and fibrinogen levels at baseline and under inflammatory conditions. Conversely, MIF stimulates the in vivo expression of human C-reactive protein, an inflammation marker and risk factor of IR and cardiovascular disease. In WAT, MIF deficiency reduces nuclear c-Jun levels and improves insulin sensitivity; MIF deficiency also reduces macrophage accumulation in WAT and blunts the expression of two proteins that regulate macrophage infiltration (intercellular adhesion molecule-1, CD44). Mechanistic parallels to WAT were observed in aorta, where the absence of MIF reduces monocyte adhesion, macrophage lesion content, and atherosclerotic lesion size. These data highlight the physiological importance of chronic inflammation in development of IR and atherosclerosis and suggest that MIF is a potential therapeutic target for reducing the inflammatory component of metabolic and cardiovascular disorders.

Abstract 5393: Increased C-Reactive Protein Expression Exacerbates Left Ventricular Dysfunction and Remodeling after Myocardial Infarction

We have previously reported that elevated serum C-reactive protein (CRP) level after acute myocardial infarction (MI) is associated with adverse outcomes including cardiac rupture, left ventricular (LV) remodeling and cardiac death. Recent experimental studies have shown that CRP per se has some biological properties including proinflammatory and proapoptotic effects, suggesting a pathogenetic role of CRP in the remodeling process after MI. We tested the hypothesis that increased CRP expression would exacerbate adverse LV remodeling after MI through some deleterious effects of CRP. Transgenic mice with human CRP expression (CRP-Tg) and their nontransgenic littermates (Control) underwent proximal ligation of the left coronary artery. Despite increased serum CRP level and cardiac CRP expression in CRP-Tg mice, there was no difference in phenotype between CRP-Tg and control mice before MI. Mortality at five weeks after MI was not different between groups (CRP-Tg: 49%, n=35; Control: 38%, n=40, P =0.28). Five weeks after MI, echocardiography showed that CRP-Tg mice had more LV dilation (LVEDD, CRP-Tg: 5.8 ± 0.1 mm, n=14; Control: 5.2 ± 0.1 mm, n=17, P =0.002) and worse LV function (EF, CRP-Tg: 13 ± 2%, n=14; Control: 19 ± 1%, n=17, P =0.01). Hemodynamic studies indicated that LV +dP/dt (CRP-Tg: 2,947 ± 480 mmHg/s, n=9; Control: 3,788 ± 656 mmHg/s, n=10, P =0.02) and -dP/dt (CRP-Tg: −2,230 ± 48 mmHg/s, n=9; Control: −2,890 ± 161 mmHg/s, n=10, P =0.003) were lower in the CRP-Tg group than in the Control group, although infarct size was comparable. Histological evaluation at one week after MI showed a higher rate of apoptosis in the border zone of infarcted hearts from CRP-Tg mice (CRP-Tg: 1,434 ± 322 per 10 5 nuclei; Control: 596 ± 112 per 10 5 nuclei, n=6 for each, P =0.03). Quantitative RT-PCR showed that angiotensin II type 1a receptor and interleukin-6 were upregulated in viable LV samples from CRP-Tg mice compared with controls. Increased CRP expression exacerbates LV dysfunction and remodeling after MI, associated with increased apoptotic rates, increased angiotensin II receptor expression and exaggerated inflammatory response.

Perplexity of monocyte responses to C-reactive protein (CRP)

Thromb Haemost 2008; 99: 461–462 C-reactive protein (CRP), a member of the pentraxin family, is one of the prototypic plasma biomarkers of inflammation (1, 2). The physiological roles of CRP in the innate immune response have, however, remained elusive as it possesses both proand anti-inflammatory actions (1, 2). Inflammation is pivotal in all phases of atherosclerosis from the nascent lesion through progression to the culmination in acute coronary syndromes (3). To date, over two dozen large-scale prospective clinical studies have reported that moderate elevations in base-line CRP levels predict, albeit to varying degrees, future cardiovascular disease and death across all levels of lowdensity lipoprotein (LDL) cholesterol and of the Framingham Risk Score (4, 5). CRP has been implicated inmultiple aspects of atherogenesis and plaque vulnerability; however, whether CRP plays a direct causal role in these events remains controversial (reviewed in [6]). For example, transgenic expression of human CRP in apolipoprotein E-deficient mice has been reported to accelerate atherogenesis (7, 8), to show no effect (9) or even to slow atherosclerosis development (10). In vitro, CRP induces release of proinflammatory interleukins IL-1, IL-6, IL-8 and tumor necrosis factor-α NF (11, 12) from human monocytes; promotes monocye adhesion to endothelial cells through upregulation of endothelial adhesion molecules (13, 14) and enhances phagocytosis of oxidized LDL (6). These observations are consistent with the role of monocytes in the initiation and progression of atherosclerosis (3). However, monocytes may have a more complex role in atherogenesis than previously thought. In this issue of Thrombosis and Haemostasis, Hanriot et al. (15) show that CRP modulates expression of genes known to mediate both proand anti-inflammatory responses. These novel observations raise the intriguing possibility that CRP could activate yet unidentified pathways in human monocytes. Using a custom-made 250 oligonucleotide microarray for screening CRP regulated genes in monocytes isolated from apparently healthy men and women (aged 25 to 35 years), Hanriot et al. detected changes in approximately 10% of the genes represented on the microarray. The authors confirm previous studies on CRP induction of pro-inflammatory cytokine gene expression with the exception of TNF, which was unaffected by CRP, and show increased mRNA expression of monocyte chemotactic protein-1 (MCP-1), Gro-β, the chemokine receptors CCR-8 and CXCR-4, and plasminogen activator inhibitor type 2 (PAI-2). In concert with IL-8,MCP-1 andGro-β facilitatemonocyte recruitment and adhesion to endothelial cells, whereas CCR-8 and CXCR-4 mediate monocyte (and macrophage) chemotaxis. The function of PAI-2 appears to be more complex. PAI-2 promotes fibrin deposition in the arterial wall, thereby contributing to progression of the initial lesion, whereas it may stabilize more advanced plaques by inhibiting activation of matrix metalloproteinases, enzymes overexpressed in human atheromata (3). While the concept of CRP’s anti-inflammatory role is not new (reviewed in [1, 2]), Hanriot et al. also report CRP-induced changes in monocyte gene expression that are consistent with suppression of inflammation. Thus, the authors show that CRP attenuates expression of the monocyte/lymphocyte chemotactic proteins MIP-1α and MIP-1β and α2-macroglobulin, which antagonize the actions of the anti-inflammatory cytokine transforming growth factor-β. Protection of mice overexpressing human CRP from allergic encephalomyelitis is mediated through reduction of MIP-1 (16). The most striking finding is that CRP increases expression of the nuclear receptor liver X receptor-α (LXR-α) and one of its target genes the ATP-binding cassette transporter A1 (ABCA1). ABCA1 promotes reverse cholesterol transport from macrophages in atherosclerotic plaques to high density lipoprotein (HDL) and suppresses genes involved in macrophage inflammatory signaling and apoptosis, thereby inhibiting atherosclerosis (17). CRP induction of LXR-α expression and activation is mediated through the high affinity IgG receptor FcγR1 (CD64) and, to lesser a degree, the low affinity IgG receptor FcγRII (CD32), and involves activation of both p42/44MAPK and phosphatidylinositol 3-kinase signaling pathways. A critical issue whether CRP could effectively facilitate reverse cholesterol transport in monocytes (and macrophages) remains to be elucidated. The complexity of LXR-α responses makes it difficult to extrapolate the beneficial or deleterious effects of a target gene in isolation to human pathology when target genes are likely beingmodulated in concert. For example, an-

Transgenic human CRP is not pro-atherogenic, pro-atherothrombotic or pro-inflammatory in apoE−/− mice

The pathogenic significance, if any, of the epidemiological association between baseline C-reactive protein (CRP) values and future atherothrombotic events is not known. We therefore investigated spontaneous atherosclerosis and atherothrombosis, and systemic markers of inflammation (acute phase proteins), in aged, normal diet-fed, male apolipoprotein E deficient (apoE−/−) mice with and without transgenic expression of human CRP. At 18 months of age, aortic atherosclerosis was extensive but with no significant difference in plaque size between C57BL/6apoE−/− mice with (apoE−/−-hCRP+) and without transgenic human CRP (apoE−/−). Atherosclerotic lesions in brachiocephalic arteries were typically complex and layered, with extensive fibrotic-cholesterol deposits, calcification and occasional recent intraplaque haemorrhage and thrombus, but with no significant overall differences between apoE−/− and apoE−/−-hCRP+ animals. Concentrations of mouse serum amyloid P component (SAP) were essentially normal throughout and did not differ between apoE−/− and apoE−/−-hCRP+ mice, or between wild-type (apoE+/+) and apoE−/− mice, regardless of human CRP expression. Mouse serum amyloid A protein (SAA), and human CRP concentrations were modestly but significantly higher in apoE−/−-hCRP+ than in apoE+/+-hCRP+ animals, but mouse SAA values were unaffected by transgenic expression of human CRP in either background. Thus, there was no evidence in this 18 month study of apoE−/−, and control apoE+/+ mice, that transgenic human CRP was pro-atherogenic, pro-inflammatory or pro-atherothrombotic.

C-Reactive Protein and Reendothelialization

See related article, pages 1452–1459 Disruption of the endothelium and its subsequent dysfunction appears to set the stage for atherogenesis.1 The maintenance of vascular homeostasis depends in part on a balance between endothelium-derived relaxing and contracting factors. Alterations in this balance lead to inflammation and the formation of fatty streaks and fibrous plaques. The diminished production or availability of nitric oxide (NO) appears paramount to setting the stage for inflammation and vascular injury. In addition to its vasodilatory effect on the vasculature, endothelium-derived NO promotes endothelial cell growth, survival and migration,2 stimulates angiogenesis and neovascularization in part by endothelial progenitor cell (EPC) recruitment,3 inhibits leukocyte adhesion to the endothelium,4 maintains vascular smooth muscle in a nonproliferative state,5 and limits platelet aggregation and thrombosis.6 Maintenance of an intact and functional endothelium protects against vascular disease while its disruption is detrimental. The study by Schwartz et al,7 in this issue of Circulation Research , adds to our understanding of how C-reactive protein (CRP) works to promote vascular pathology by inhibiting NO synthesis. CRP, long regarded as an acute phase protein and an active participant in the innate immune system, has more recently been linked to the development of cardiovascular disease.8 On the basis of several prospective epidemiologic studies, circulating high sensitivity CRP has emerged as an independent predictor of cardiovascular disease risk in various subgroups of patients, including those without overt cardiovascular disease, patients with stable angina or acute coronary syndromes, and in those with the metabolic syndrome.9–12 In primary prevention, CRP confers additional prognostic value at all levels of Framingham risk score and blood pressure.13,14 However, whether or not CRP plays a direct biological role in the initiation and progression of atherosclerosis is controversial.15,16 …

Transgenic expression of human C-reactive protein suppresses endothelial nitric oxide synthase expression and bioactivity after vascular injury

C-reactive protein (CRP) is a risk marker and a potential modulator of vascular disease. Whether CRP modulates nitric oxide (NO) synthase (NOS) activity and NO metabolism remains unclear. We studied the effect of CRP on NO metabolism in transgenic mice that express human CRP (CRPtg). CRPtg and wild-type mice were subjected to controlled femoral artery wire injury. CRP serum levels at baseline and 6 and 24 h after injury were 12.4 +/- 9, 18.6 +/- 6.9, and 58.4 +/- 13 mg/l, respectively, in CRPtg mice but were undetectable at all time points in wild-type mice. Endothelial NOS protein and mRNA expression were significantly suppressed in the injured arteries of CRPtg mice (n = 5, P < 0.05). A similar reduction in eNOS expression was observed in the distant lung and heart. NO release after injury was significantly lower in CRPtg mice, as measured by nitrate and nitrite breakdown products, with a concomitant suppression of cGMP NO signaling after injury. Endothelial NOS and NO expression after vascular injury are locally and systemically suppressed in mice that express human CRP. These in vivo observations support the hypothesis that CRP modulates NO metabolism and may have implications regarding the mechanisms by which CRP modulates vascular disease.

Transgenic human C-reactive protein is not proatherogenic in apolipoprotein E-deficient mice

The association between circulating concentrations of C-reactive protein (CRP) and future atherothrombotic events has provoked speculation about a possible pathogenetic role of CRP. However, we show here that transgenic expression of human CRP had no effect on development, progression, or severity of spontaneous atherosclerosis, or on morbidity or mortality, in male apolipoprotein E (apoE)-deficient C57BL/6 mice up to 56 weeks, despite deposition of human CRP and mouse complement component 3 in the plaques. Although female apoE knockouts develop atherosclerosis more rapidly than males, the human CRP transgene is under sex hormone control and is expressed at human levels only in males. We therefore studied only male mice. The concentration of mouse serum amyloid P component, an extremely sensitive systemic marker of inflammation, remained normal throughout except for transient spikes in response to fighting in a few animals, indicating that atherogenesis in this model is not associated with an acute-phase response. However, among human CRP transgenic mice, the circulating CRP concentration was higher in apoE knockouts than in wild-type controls. The higher CRP values were associated with substantially lower estradiol concentrations in the apoE-deficient animals. Human CRP transgene expression is thus up-regulated in apoE-deficient mice, apparently reflecting altered estrogen levels, despite the absence of other systemic signs of inflammation. Extrapolation to human pathology from this xenogeneic combination of human CRP with apoE deficiency-mediated mouse atherosclerosis must be guarded. Nevertheless, the present results do not suggest that human CRP is either proatherogenic or atheroprotective in vivo.

CRP—Marker or Maker of Cardiovascular Disease?

C-reactive protein (CRP) has emerged as an interesting novel and potentially clinically useful marker for increased cardiovascular risk.1,2 This is an attractive concept because atherosclerosis is a disease characterized by chronic arterial inflammation3,4 and suggests the possibility that subclinical states of atherosclerosis can be identified by an increase in circulating markers of inflammation before acute events occur. Based on data obtained primarily from in vitro studies it has also been proposed that CRP in itself is actively contributing to disease progression and that it should be considered as true risk factor and consequently as a target for intervention. However, in the present issue of Arteriosclerosis, Thrombosis, and Vascular Biology , two independent reports demonstrate that transgenic overexpression of CRP does not affect the development of atherosclerosis in mice, suggesting that this is not the case. See pages 1635 and 1641 The association between moderately elevated CRP levels and an increased risk for development of cardiovascular disease is well established. Although the increase in risk may have been overestimated in initial studies, recent meta-analysis comprising >7000 patients with coronary events shows that subjects with CRP in the upper tertile have a 50% increased risk for development of acute cardiovascular events.2 There are several possible explanations for this association (Figure). (1) The inflammatory process in arterial tissue affected by atherosclerosis results in release of cytokines into the circulation that subsequently activates expression of CRP in the liver. Plasma CRP levels would then reflect the severity of atherosclerosis and, as a consequence, the risk for development of clinical events. If CRP in addition …

C-reactive protein is an important immunoregulatory molecule that decreases systemic inflammatory response and ameliorates autoimmune disease

C-reactive protein (CRP) is an ancient member of the innate immune system. CRP is also a member of the pentraxin family of proteins and is a strong acute phase reactant in man. CRP has been shown to bind to phagocytes through interaction with Fcγ receptors, to affect the production of cytokines, and to enhance phagocytosis. CRP has been shown to protect mice from infection, to bind to nuclear antigens and to delay the onset of systemic lupus erythematosus (SLE) in autoimmune mice. The mechanism for these diverse effects has remained obscure. Both CRP and serum amyloid P (SAP) have been shown to bind to nuclear antigens that are targets of autoantibodies in patients with SLE. Additional studies have shown that CRP and SAP can mediate the uptake and clearance of apoptotic cells. These studies suggested that the pentraxins might prevent exposure and reactivity of the immune system to self-antigens. Our studies have shown that CRP and SAP can delay the onset of autoimmune disease accelerated by the injection of chromatin into autoimmune mice. More recently, it has been demonstrated that expression of human CRP as a transgene on autoimmune disease can delay the onset of autoimmune disease. We report now that a single injection of CRP can delay the onset of proteinuria in NZB × NZW female mice. In all three cases, CRP failed to substantially decrease anti-DNA autoantibodies yet protected mice from glomerular injury and death. We suggest that CRP delays the onset of autoimmune disease by modulating the effect of immune complexes on inflammatory cells. As we have shown recently, the anti-inflammatory effect of CRP in the lipopolysaccharide challenge model requires Fcγ receptor expression. It is proposed that CRP mediates protection from nephritis in SLE by the induction of anti-inflammatory cytokines and altering the reactivity of macrophages to inflammatory stimuli.

Experimental allergic encephalomyelitis is inhibited in transgenic mice expressing human C-reactive protein.

We show here using a transgenic model that human C-reactive protein (CRP) protects against experimental allergic encephalomyelitis (EAE) in C57BL/6 mice. In transgenic compared with wild-type females, the duration of the human CRP acute phase response that accompanies the inductive phase of active EAE correlates with a delay in disease onset. In transgenic males, which have higher human CRP expression than females do, EAE is delayed, and its severity is reduced relative to same-sex controls. Furthermore, in male transgenics, there is little or no infiltration of the spinal cord by CD3(+) T cells and CD11b(+) monocytes and macrophages, and EAE is sometimes prevented altogether. CRP transgenics also resist EAE induced passively by transfer of encephalitogenic T cells from wild-type donors. Human CRP has three effects on cultured encephalitogenic cells that could contribute to the protective effect observed in vivo: 1) CRP inhibits encephalitogenic peptide-induced proliferation of T cells; 2) CRP inhibits production of inflammatory cytokines (TNF-alpha, IFN-gamma) and chemokines (macrophage-inflammatory protein-1alpha, RANTES, monocyte chemoattractant protein-1); and 3) CRP increases IL-10 production. All three of these actions are realized in vitro only in the presence of high concentrations of human CRP. The combined data suggest that during the acute phase of inflammation accompanying EAE, the high level of circulating human CRP that is achieved in CRP-transgenic mice inhibits the damaging action of inflammatory cells and/or T cells that otherwise support onset and development of EAE.

C-reactive protein: structural biology, gene expression, and host defense function.

Over the past few years substantial insight was gained into the biology and biochemistry of human C-reactive protein (CRP). X-ray crystallography in conjunction with mutational analyses, generated the three-dimensional structure of the protein and indicated the topology and structure of ligand-binding sites. Using human CRP transgenic mice infected with Streptococcus pneumoniae, we obtained data that clearly established CRP as an important host defense molecule. Studies using the same mice revealed a previously unknown testosterone-dependence of constitutive expression of human CRP. In this article we provide a brief overview of these recent findings.