Monomeric CRP: A NEW PLAYER IN EVALUATION OF CARDIOVASCULAR RISK?

BACKGROUND: CRP AND MONOMERIC CRP (mCRP) IN INFLAMMATION

Although CRP is an independent risk factor for CVD and offers a prognostic advantage over measurement of lipids alone, the precise mechanism by which CRP is related to CVD pathogenesis is poorly understood. It is generally accepted, that CRP plays an active role in endothelial dysfunction, and induces complement activation. However, there is evidence that natural CRP is not a direct mediator of cardiovascular events.  The modest association between risk evaluation and CVD was inappropriately conflated with causality, and it has been claimed that CRP is proatherogenic. The reported proinflammatory effects of human CRP in-vitro or in-vivo resulted from impurities of CRP preparations and above that, it was revealed that pharmaceutical graded natural CRP is not proinflammatory in healthy human adults.

There are distinct isoforms of CRP, pCRP (pentameric CRP) and mCRP (monomeric CRP), and the pCRP isoform can irreversibly dissociate at sites of inflammation, tissue damage, and infection into five mCRP subunits. Evidence indicates that pCRP often tends to exhibit more anti-inflammatory activities compared to mCRP, which contrary shows pro-inflamatory and pro-thrombotic effects. The pCRP isoform activates the complement pathway, induces phagocytosis, and promotes apoptosis, whereas mCRP promotes the chemotaxis and recruitment of circulating leukocytes to areas of inflammation and can delay apoptosis. In terms of pro-inflammatory cytokine production, mCRP increases IL-8 and MCP-1 production, while pCRP has no detectable effect on their levels. These findings suggest the differential roles of each CRP isoform in inflammation and infection.

C-reactive protein (CRP) undergoes conformational changes between circulating native pentameric CRP (pCRP), pentameric symmetrical forms (pCRP*) and monomeric CRP (mCRP) forms. mCRP exhibits strong pro-inflammatory activity and activates platelets, leukocytes, and endothelial cells. Abundant deposition of mCRP in inflamed tissues plays a role in several disease conditions, such as ischemia/reperfusion injury, Alzheimer’s disease, and cardiovascular disease.

Conversion of pCRP to mCRP induces inflammatory signalling.  Monoacyl phosphatidylcholine, generated by PLA₂, or by oxidation lipid acyl chains, promotes binding and dissociation of pCRP to mCRP, which exposes cholesterol binding sequence. The hydrophobic element allows traffic through the plasmatic membrane into cells and activates NF-κβ signaling pathway. mCRP gains functionally active neoepitopes that carry out highly pro-inflammatory and pro-thrombotic features. Deposition of mCRP, which has significantly lower water solubility than pCRP, has been demonstrated in the brain in infarcted areas of Alzheimer’s disease and in areas of amyloid burden, in atherosclerotic plaques in vascular disease and in other foci of inflammatory tissue damage.

References
Rajab IM, Hart PC, Potempa LA. How C-Reactive Protein Structural Isoforms With Distinctive Bioactivities Affect Disease Progression. Front Immunol. Vol 11:2126 (2020)
Braig D. et al.: Transitional changes in the CRP structure lead to the exposure of proinflammatory binding sites. Nat Commun. Vol 23;8:14188 (2017)

Cardiovascular risk and mCRP

The cardiovascular risk that persists despite aggressive lipid-lowering therapy -such as anti-PCSK-9 therapy – and correction of modifiable risk factors is called “residual cardiovascular risk” [1]. One of its main types is the residual inflammatory risk resulting from low-grade inflammation in atherosclerotic plaques [2].  It is determined by the level of the main inflammatory biomarker C-reactive protein (CRP), measured using a high-sensitivity assay (hsCRP), with a value of 2.0 mg/L or more [3]. The hsCRP assay measures the level of the pentameric form of CRP (pCRP), which is produced in the liver under the stimulation by interleukin (IL)-6 [4]. The USPSTF meta-analysis that explored studies published from 1966 to 2007 demonstrated that relative cardiovascular risk is 1.58-fold higher in individuals with a CRP level more than 3.0 mg/L than in those with a CRP level less than 1.0 mg/L [5].

Recent in-vitro and animal-model studies have suggested a task for mCRP in cardiovascular risk initiation and development, and show its active role in platelet activation, adhesion, and aggregation; endothelial activation; leukocyte recruitment and polarization; foam-cell formation; and neovascularization. mCRP contributes to the complex interplay between blood coagulation and inflammation, which is called thromboinflammation [6]. Bound on a collagen substrate, mCRP substantially increases platelet adhesion and thrombus growth rate. Unlike pCRP, mCRP induces platelet glycoprotein (GP) IIb/IIIa activation in a dose-dependent manner, and facilitates platelet adhesion via activation of GP IIb/IIIa receptors. Additionally, mCRP stimulates platelet adhesion to the endothelial cells [7] and induces tissue-factor expression and fibrin formation on endothelial cells [8]. When dissociated on platelets and adhering to the vessel wall, mCRP enhances endothelial activation and neutrophil attachment to the endothelium [7,9]; monocyte adhesion to the collagen [10], fibrinogen [11], and fibronectin matrix [12]; and T-lymphocyte extravasation [13]. In vitro, mCRP decreased nitric-oxide release and increased production of proinflammatory IL-8 and monocyte chemoattractant protein-1 by endothelial cells via the NF-kB pathway [14]. Moreover, mCRP stimulated leukocyte recruitment to the vessel wall, inducing the expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin, as well as the production of IL-6 and IL-8 by the endothelium [7,14,15]. mCRP can also stimulate oxidized LDL uptake by macrophages [16]. The in vivo evidence that mCRP can stimulate monocyte infiltration into damaged tissues was obtained from recent animal studies [17]. In addition, mCRP has been shown to stimulate neoangiogenesis and stabilize novel microvessels [18,19]. mCRP deposition into atherosclerotic plaques has been addressed in several immunohistochemical studies. In human tissues, mCRP deposits have been detected in atherosclerotic plaques of the aorta [10], carotid [10,11,20], coronary [21,22], and femoral arteries [23], as well as diseased coronary artery venous bypass grafts [24] or infarcted myocardium [11]. In contrast, no mCRP deposits have been found in intact arteries or fibrous or calcific plaques [10,11,20,21,23,24]. mCRP can cross the endothelial barrier after dissociation [11] or be synthesized locally. Nevertheless, it is still unclear the contribution of local synthesis to the total concentration of mCRP in the tissues and bloodstream. The studies clearly distinguishing between the two forms of CRP confirmed that mCRP, but not pCRP, was deposited into damaged tissues [10,11,22], whereas other studies did not discriminate between CRP forms [20,21,23,24].

References
1/ Lawler, P.R. et al.: Targeting cardiovascular inflammation: Next steps in clinical translation. Eur. Heart J., Vol 42, ehaa099 (2020)
2/ Ridker, P.M. Residual inflammatory risk: Addressing the obverse side of the atherosclerosis prevention coin. Eur. Heart J., Vol 37, 1720–1722 (2016)
3/  Arnett, D.K. et al.: 2019 ACC/AHA Guideline on the Primary Prevention of Cardiovascular Disease: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. Circulation, Vol 140, e596–e646 (2019)
4/  McFadyen, J.D et al.: Dissociation of C-Reactive Protein Localizes and Amplifies Inflammation: Evidence for a Direct Biological Role of C-Reactive Protein and Its Conformational Changes. Front. Immunol., Vol 9, 1351 (2018)
5/  Buckley, D.I et al.: C-Reactive Protein as a Risk Factor for Coronary Heart Disease: A Systematic Review and Meta-analyses for the U.S. Preventive Services Task Force. Ann. Intern. Med., Vol 151, 483 (2009)
6/  d’Alessandro, E. et al.: Thrombo-Inflammation in Cardiovascular Disease: An Expert Consensus Document from the Third Maastricht Consensus Conference on Thrombosis. Thromb. Haemost., Vol 120, 538–564 (2020)
7/  Khreiss, T. et al.: Conformational Rearrangement in C-Reactive Protein Is Required for Proinflammatory Actions on Human Endothelial Cells. Circulation, Vol 109, 2016–2022 (2004)
8/  Li, R. et al.: Monomeric C-reactive protein alters fibrin clot properties on endothelial cells. Thromb. Res., Vol 129, e251–e256 (2012)
9/  Zouki, C et al.: Loss of Pentameric Symmetry of C-Reactive Protein Is Associated with Promotion of Neutrophil-Endothelial Cell Adhesion. J. Immunol., Vol 167, 5355–5361 (2001)
10/  Eisenhardt, S.U. et al.: Dissociation of Pentameric to Monomeric C-Reactive Protein on Activated Platelets Localizes Inflammation to Atherosclerotic Plaques. Circ. Res., Vol 105, 128–137 (2009)
11/  Thiele, J.R. et al.: Dissociation of Pentameric to Monomeric C-Reactive Protein Localizes and Aggravates Inflammation: In Vivo Proof of a Powerful Proinflammatory Mechanism and a New Anti-Inflammatory Strategy. Circulation, Vol 130, 35–50 (2014)
12/  Ullah, N. et al.: Monomeric C-reactive protein regulates fibronectin mediated monocyte adhesion. Mol. Immunol., Vol 117, 122–130 (2020)
13/  Zhang, Z. et al.: Monomeric C-reactive protein via endothelial CD31 for neurovascular inflammation in an ApoE genotype-dependent pattern: A risk factor for Alzheimer’s disease? Aging Cell, Vol 20, e13501 (2021)
14/  Li, H.-Y. et al.: Topological Localization of Monomeric C-reactive Protein Determines Proinflammatory Endothelial Cell Responses. J. Biol. Chem., Vol 289, 14283–14290 (2014)
15/  Ji, S.-R. et al.: Monomeric C-reactive protein activates endothelial cells via interaction with lipid raft microdomains. FASEB J., Vol 23, 1806–1816 (2009)
16/  Ji, S. et al.: Interactions of C-reactive protein with low-density lipoproteins: Implications for an active role of modified C-reactive protein in atherosclerosis. Int. J. Biochem. Cell Biol., Vol 38, 648–661 (2006)
17/  Thiele, J.R. et al.: A Conformational Change in C-Reactive Protein Enhances Leukocyte Recruitment and Reactive Oxygen Species Generation in Ischemia/Reperfusion Injury. Front. Immunol., Vol 9, 675 (2018)
18/  Boras, E. et al.: Monomeric C-reactive protein and Notch-3 co-operatively increase angiogen through PI3K signalling pathway. Cytokine, Vol 69, 165–179 (2014)
19/  Turu, M.M. et al.: C-reactive protein exerts angiogenic effects on vascular endothelial cells and modulates associated signalling pathways and gene expression. BMC Cell Biol., Vol 9, 47 (2008)
20/  Krupinski, J. et al.:  Endogenous Expression of C-Reactive Protein Is Increased in Active (Ulcerated Noncomplicated) Human Carotid Artery Plaques. Stroke, Vol 37, 1200–1204 (2006)
21/  Kobayashi, S et al.: Interaction of Oxidative Stress and Inflammatory Response in Coronary Plaque Instability: Important Role of C-Reactive Protein. ATVB 2003, 23, 1398–1404 (2003)
22/  Melnikov, I.S et al.: Monomeric C-reactive protein and local inflammatory reaction in the wall of the coronary arteries in patients with stable coronary artery disease. Russ. J. Cardiol., Vol 24, 56–61 (2019)
23/  Vainas, T.; Stassen, F.R.M.; de Graaf, R.; Twiss, E.L.L.; Herngreen, S.B.; Welten, R.J.T.J.; van den Akker, L.H.J.M.; van Dieijen-Visser, M.P.; Bruggeman, C.A.; Kitslaar, P.J.E.H.M. C-reactive protein in peripheral arterial disease: Relation to severity of the disease and to future cardiovasc events. J. Vasc. Surg., Vol 42, 243–251 (2005)
24/  Jabs, W.J. et al.: Local Generation of C-Reactive Protein in Diseased Coronary Artery Venous Bypass Grafts and Normal Vascular Tissue. Circulation, Vol 108, 1428–1431 (2003)

ANTI-INFLAMMATORY THERAPY AND mCRP

In the future, tailored antibodies for inhibiting transformation of pCRP into mCRP or selective inhibition of deposition of mCRP in the injured myocardium could be a promising method for minimizing ischaemia–reperfusion injury in patients with elevated serum CRP. A small-molecule inhibitors of pCRP (e.g. 1,6-bis(phosphocholine)-hexane), which blocks the pCRP–microvesicle interactions, abrogates its proinflammatory effects. The inhibition of the conformational change generating pro-inflammatory CRP isoforms via phosphocholine-mimicking compounds represents a promising, potentially broadly applicable anti-inflammatory therapy, improving the outcome of myocardial infarction, stroke and other inflammatory conditions.

Recently, Zeller et al designed a low molecular weight compound that targets the PC/PE (phosphatidylcholine/ phosphatidylethanolamine) binding pocket on pCRP and thereby has the potential to prevent the formation of the pro‐inflammatory pCRP* and mCRP species. The compound labelled C10M (3‐(dibutylamino)propyl)phosphonic acid) did not show immunosuppression activities, and might represents a successful anti-inflammatory treatment strategy.

References
Zeller J. et al.: A novel phosphocholine-mimetic inhibits a pro-inflammatory conformational change in C-reactive protein. EMBO Mol Med. Vol 11;15(1):e16236 (2023)
Filep JG.: Targeting conformational changes in C-reactive protein to inhibit pro-inflammatory actions. EMBO Mol Med. Vol 11;15(1):e17003 (2023)
PMID: 36465053

POPULATION AND CLINICAL DATA

Typical mCRP values in human serum were obtained with BioLab Assays Human Monomeric CRP (mCRP) ELISA kit

According to the health status, mCRP next to the ratio mCRP/pCRP should be consider for a risk value assessment.