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Topological Localization of Monomeric C-reactive Protein Determines Proinflammatory Endothelial Cell Responses*

  • Hai-Yun Li
    Affiliations
    Key Laboratory of Cell Activities and Stress Adaptations of Ministry of Education of China, School of Life Sciences, Lanzhou University, Lanzhou 730000, China
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  • Jing Wang
    Affiliations
    Key Laboratory of Cell Activities and Stress Adaptations of Ministry of Education of China, School of Life Sciences, Lanzhou University, Lanzhou 730000, China
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  • Yue-Xin Wu
    Affiliations
    Key Laboratory of Cell Activities and Stress Adaptations of Ministry of Education of China, School of Life Sciences, Lanzhou University, Lanzhou 730000, China
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  • Lin Zhang
    Affiliations
    Key Laboratory of Cell Activities and Stress Adaptations of Ministry of Education of China, School of Life Sciences, Lanzhou University, Lanzhou 730000, China
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  • Zu-Pei Liu
    Affiliations
    Key Laboratory of Cell Activities and Stress Adaptations of Ministry of Education of China, School of Life Sciences, Lanzhou University, Lanzhou 730000, China
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  • János G. Filep
    Affiliations
    the Research Center, Maisonneuve-Rosemont Hospital, University of Montréal, Montréal, Québec H1T 2M4, Canada
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  • Lawrence A. Potempa
    Affiliations
    Roosevelt University College of Pharmacy, Schaumburg, Illinois 60173
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  • Yi Wu
    Correspondence
    To whom correspondence may be addressed: MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China. Tel./Fax: 86-931-8914102
    Affiliations
    Key Laboratory of Cell Activities and Stress Adaptations of Ministry of Education of China, School of Life Sciences, Lanzhou University, Lanzhou 730000, China

    Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Lanzhou University, Lanzhou 730000, China
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  • Shang-Rong Ji
    Correspondence
    To whom correspondence may be addressed: MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China. Tel./Fax: 86-931-8914176
    Affiliations
    Key Laboratory of Cell Activities and Stress Adaptations of Ministry of Education of China, School of Life Sciences, Lanzhou University, Lanzhou 730000, China
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  • Author Footnotes
    * This work was supported by Ministry of Science and Technology of China grant 2011CB910500; by National Natural Science Foundation of China grants 30930024, 31222015, 31270813, 31170696; and by Ministry of Education of China grants PCSIRT: IRT1137, 121108, 20100211110006, and lzujbky-2010-k03.
Open AccessPublished:April 07, 2014DOI:https://doi.org/10.1074/jbc.M114.555318
      The activation of endothelial cells (ECs) by monomeric C-reactive protein (mCRP) has been implicated in contributing to atherogenesis. However, the potent proinflammatory actions of mCRP on ECs in vitro appear to be incompatible with the atheroprotective effects of mCRP in a mouse model. Because mCRP is primarily generated within inflamed tissues and is rapidly cleared from the circulation, we tested whether these discrepancies can be explained by topological differences in response to mCRP within blood vessels. In a Transwell culture model, the addition of mCRP to apical (luminal), but not basolateral (abluminal), surfaces of intact human coronary artery EC monolayers evoked a significant up-regulation of MCP-1, IL-8, and IL-6. Such polarized stimulation of mCRP was observed consistently regardless of EC type or experimental conditions (e.g. culture of ECs on filters or extracellular matrix-coated surfaces). Accordingly, we detected enriched lipid raft microdomains, the major surface sensors for mCRP on ECs, in apical membranes, leading to the preferential apical binding of mCRP and activation of ECs through the polarized induction of the phospholipase C, p38 MAPK, and NF-κB signaling pathways. Furthermore, LPS and IL-1β induction of EC activation also exhibited topological dependence, whereas TNF-α did not. Together, these results indicate that tissue-associated mCRP likely contributes little to EC activation. Hence, topological localization is an important, but often overlooked, factor that determines the contribution of mCRP and other proinflammatory mediators to chronic vascular inflammation.

      Introduction

      C-reactive protein (CRP)
      The abbreviations used are:
      CRP
      C-reactive protein
      mCRP
      monomeric C-reactive protein
      EC
      endothelial cell
      HAEC
      human aortic endothelial cell
      HCAEC
      human coronary artery endothelial cell
      MCP
      monocyte chemoattractant protein
      PLC
      phospholipase C.
      is a major human acute phase reactant that is highly conserved during evolution (
      • Pepys M.B.
      • Hirschfield G.M.
      C-reactive protein: a critical update.
      ). The serum level of CRP is closely associated with the risk and prognosis of many chronic inflammatory diseases, including cardiovascular disease (
      • Singh S.K.
      • Suresh M.V.
      • Voleti B.
      • Agrawal A.
      The connection between C-reactive protein and atherosclerosis.
      ) and cancer (
      • Allin K.H.
      • Nordestgaard B.G.
      Elevated C-reactive protein in the diagnosis, prognosis, and cause of cancer.
      ). However, it is still hotly debated whether this protein plays any significant role in the underlying pathological processes (
      • Pepys M.B.
      • Hirschfield G.M.
      C-reactive protein: a critical update.
      ,
      • Allin K.H.
      • Nordestgaard B.G.
      Elevated C-reactive protein in the diagnosis, prognosis, and cause of cancer.
      ,
      • Yousuf O.
      • Mohanty B.D.
      • Martin S.S.
      • Joshi P.H.
      • Blaha M.J.
      • Nasir K.
      • Blumenthal R.S.
      • Budoff M.J.
      High-sensitivity C-reactive protein and cardiovascular disease: a resolute belief or an elusive link?.
      ). This may be due to the following reasons. First, the 2-3 orders of nonspecific fluctuations in the serum level of CRP appear to be incompatible with a role of CRP in the fine regulation of chronic inflammation (
      • Pepys M.B.
      • Hirschfield G.M.
      C-reactive protein: a critical update.
      ). Second, large-scale genetic epidemiologic studies do not support a causal association of CRP with these diseases (
      • Allin K.H.
      • Nordestgaard B.G.
      • Zacho J.
      • Tybjaerg-Hansen A.
      • Bojesen S.E.
      C-reactive protein and the risk of cancer: a Mendelian randomization study.
      ,
      • Zacho J.
      • Tybjaerg-Hansen A.
      • Jensen J.S.
      • Grande P.
      • Sillesen H.
      • Nordestgaard B.G.
      Genetically elevated C-reactive protein and ischemic vascular disease.
      ). Third, contradictory findings have been reported in both in vitro and animal models regarding the proinflammatory and atherogenic activities of CRP (
      • Ma X.
      • Ji S.R.
      • Wu Y.
      Regulated conformation changes in C-reactive protein orchestrate its role in atherogenesis.
      ).
      Recently, we identified a recurrent promoter mutation and markedly enhanced expression of CRP in human cancers, indicating that CRP may be a potential driver in tumorigenesis and, hence, plays an important role in the regulatory network of inflammation (
      • Wang M.-Y.
      • Zhou H.-H.
      • Zhang S.-C.
      • Hui F.
      • Zhu W.
      • Su H.-X.
      • Guo H.-Y.
      • Li X.-W.
      • Ji S.-R.
      • Wu Y.
      Recurrent mutations at C-reactive protein promoter SNP position −286 in human cancers.
      ). Moreover, continuous efforts from several groups, including ours, have led to the notion that the discordant actions attributed to CRP can be explained by distinct activities and localizations of CRP isoforms as well as their interplay (
      • Ma X.
      • Ji S.R.
      • Wu Y.
      Regulated conformation changes in C-reactive protein orchestrate its role in atherogenesis.
      ,
      • Filep J.G.
      Platelets affect the structure and function of C-reactive protein.
      ,
      • Eisenhardt S.U.
      • Thiele J.R.
      • Bannasch H.
      • Stark G.B.
      • Peter K.
      C-reactive protein: how conformational changes influence inflammatory properties.
      ). Thus, circulating CRP is secreted by hepatocytes as a homopentamer and primarily exhibits anti-inflammatory activities (
      • Filep J.G.
      Platelets affect the structure and function of C-reactive protein.
      ). In inflammatory loci, however, pentameric CRP undergoes sequential conformational changes, including dissociation into subunits (i.e. mCRP) (
      • Molins B.
      • Peña E.
      • de la Torre R.
      • Badimon L.
      Monomeric C-reactive protein is prothrombotic and dissociates from circulating pentameric C-reactive protein on adhered activated platelets under flow.
      • Ji S.R.
      • Wu Y.
      • Zhu L.
      • Potempa L.A.
      • Sheng F.L.
      • Lu W.
      • Zhao J.
      Cell membranes and liposomes dissociate C-reactive protein (CRP) to form a new, biologically active structural intermediate: mCRP(m).
      ,
      • Eisenhardt S.U.
      • Habersberger J.
      • Murphy A.
      • Chen Y.C.
      • Woollard K.J.
      • Bassler N.
      • Qian H.
      • von Zur Muhlen C.
      • Hagemeyer C.E.
      • Ahrens I.
      • Chin-Dusting J.
      • Bobik A.
      • Peter K.
      Dissociation of pentameric to monomeric C-reactive protein on activated platelets localizes inflammation to atherosclerotic plaques.
      ,
      • Mihlan M.
      • Blom A.M.
      • Kupreishvili K.
      • Lauer N.
      • Stelzner K.
      • Bergström F.
      • Niessen H.W.
      • Zipfel P.F.
      Monomeric C-reactive protein modulates classic complement activation on necrotic cells.
      ), followed by reduction of the intrasubunit disulfide bond (
      • Wang M.Y.
      • Ji S.R.
      • Bai C.J.
      • El Kebir D.
      • Li H.Y.
      • Shi J.M.
      • Zhu W.
      • Costantino S.
      • Zhou H.H.
      • Potempa L.A.
      • Zhao J.
      • Filep J.G.
      • Wu Y.
      A redox switch in C-reactive protein modulates activation of endothelial cells.
      ), to express its full proinflammatory potential. Therefore, mCRP is proposed to be the major CRP isoform that functions as a regulator of local inflammation, whereas native pentameric CRP may serve as the precursor of mCRP and a systemic marker of inflammation (
      • Ma X.
      • Ji S.R.
      • Wu Y.
      Regulated conformation changes in C-reactive protein orchestrate its role in atherogenesis.
      ,
      • Filep J.G.
      Platelets affect the structure and function of C-reactive protein.
      ,
      • Eisenhardt S.U.
      • Thiele J.R.
      • Bannasch H.
      • Stark G.B.
      • Peter K.
      C-reactive protein: how conformational changes influence inflammatory properties.
      ).
      Because both reduced and Cys-mutated mCRP are potent activators of endothelial cells (ECs) (
      • Wang M.Y.
      • Ji S.R.
      • Bai C.J.
      • El Kebir D.
      • Li H.Y.
      • Shi J.M.
      • Zhu W.
      • Costantino S.
      • Zhou H.H.
      • Potempa L.A.
      • Zhao J.
      • Filep J.G.
      • Wu Y.
      A redox switch in C-reactive protein modulates activation of endothelial cells.
      ,
      • Khreiss T.
      • József L.
      • Potempa L.A.
      • Filep J.G.
      Conformational rearrangement in C-reactive protein is required for proinflammatory actions on human endothelial cells.
      ), it appears plausible that mCRP would contribute to the initiation of atherosclerosis, in which EC dysfunction is one of the earliest events (
      • Ross R.
      Atherosclerosis: an inflammatory disease.
      ). However, subcutaneous administration of Cys-mutated mCRP into ApoE−/− mice was found to reduce plaque formation (
      • Schwedler S.B.
      • Amann K.
      • Wernicke K.
      • Krebs A.
      • Nauck M.
      • Wanner C.
      • Potempa L.A.
      • Galle J.
      Native C-reactive protein increases whereas modified C-reactive protein reduces atherosclerosis in apolipoprotein E-knockout mice.
      ). ECs are highly polarized cells with compositionally and functionally distinct plasma membrane domains, i.e. apical and basolateral membranes (
      • Muller W.A.
      • Gimbrone Jr., M.A.
      Plasmalemmal proteins of cultured vascular endothelial cells exhibit apical-basal polarity: analysis by surface-selective iodination.
      ,
      • Mehta D.
      • Malik A.B.
      Signaling mechanisms regulating endothelial permeability.
      ). It is worth noting that, in in vitro experiments, mCRP is usually added to the apical surface of cultured EC. This would mimic the luminal localization of mCRP, whereas the generation of mCRP primarily occurs within inflamed tissues (
      • Ma X.
      • Ji S.R.
      • Wu Y.
      Regulated conformation changes in C-reactive protein orchestrate its role in atherogenesis.
      ,
      • Eisenhardt S.U.
      • Thiele J.R.
      • Bannasch H.
      • Stark G.B.
      • Peter K.
      C-reactive protein: how conformational changes influence inflammatory properties.
      ), wherein only the basolateral surface of ECs is accessible. Thus, we investigated the impact of distinct localizations of mCRP with respect to EC on EC function and detected profound EC responses only with apically applied, Cys-mutated mCRP.

      DISCUSSION

      The apical and basolateral surfaces of ECs face blood and tissues, respectively (
      • Muller W.A.
      • Gimbrone Jr., M.A.
      Plasmalemmal proteins of cultured vascular endothelial cells exhibit apical-basal polarity: analysis by surface-selective iodination.
      ,
      • Mehta D.
      • Malik A.B.
      Signaling mechanisms regulating endothelial permeability.
      ). ECs respond strongly to apical but weakly to basolateral stimulation of Cys-mutated or reduced mCRP, suggesting that tissue-resident mCRP may be inefficient in activating ECs. Because mCRP would be generated primarily within inflamed tissues (
      • Eisenhardt S.U.
      • Habersberger J.
      • Murphy A.
      • Chen Y.C.
      • Woollard K.J.
      • Bassler N.
      • Qian H.
      • von Zur Muhlen C.
      • Hagemeyer C.E.
      • Ahrens I.
      • Chin-Dusting J.
      • Bobik A.
      • Peter K.
      Dissociation of pentameric to monomeric C-reactive protein on activated platelets localizes inflammation to atherosclerotic plaques.
      ,
      • Mihlan M.
      • Blom A.M.
      • Kupreishvili K.
      • Lauer N.
      • Stelzner K.
      • Bergström F.
      • Niessen H.W.
      • Zipfel P.F.
      Monomeric C-reactive protein modulates classic complement activation on necrotic cells.
      ,
      • Wang M.Y.
      • Ji S.R.
      • Bai C.J.
      • El Kebir D.
      • Li H.Y.
      • Shi J.M.
      • Zhu W.
      • Costantino S.
      • Zhou H.H.
      • Potempa L.A.
      • Zhao J.
      • Filep J.G.
      • Wu Y.
      A redox switch in C-reactive protein modulates activation of endothelial cells.
      ,
      • Lauer N.
      • Mihlan M.
      • Hartmann A.
      • Schlötzer-Schrehardt U.
      • Keilhauer C.
      • Scholl H.P.
      • Charbel Issa P.
      • Holz F.
      • Weber B.H.
      • Skerka C.
      • Zipfel P.F.
      Complement regulation at necrotic cell lesions is impaired by the age-related macular degeneration-associated factor-H His402 risk variant.
      • Schwedler S.B.
      • Guderian F.
      • Dämmrich J.
      • Potempa L.A.
      • Wanner C.
      Tubular staining of modified C-reactive protein in diabetic chronic kidney disease.
      ,
      • Slevin M.
      • Matou-Nasri S.
      • Turu M.
      • Luque A.
      • Rovira N.
      • Badimon L.
      • Boluda S.
      • Potempa L.
      • Sanfeliu C.
      • de Vera N.
      • Krupinski J.
      Modified C-reactive protein is expressed by stroke neovessels and is a potent activator of angiogenesis in vitro.
      ,
      • Strang F.
      • Scheichl A.
      • Chen Y.C.
      • Wang X.
      • Htun N.M.
      • Bassler N.
      • Eisenhardt S.U.
      • Habersberger J.
      • Peter K.
      Amyloid plaques dissociate pentameric to monomeric C-reactive protein: a novel pathomechanism driving cortical inflammation in Alzheimer's disease?.
      ) and the half-life of blood-borne mCRP is rather short (Ref.
      • Motie M.
      • Schaul K.W.
      • Potempa L.A.
      Biodistribution and clearance of 125I-labeled C-reactive protein and 125I-labeled modified C-reactive protein in CD-1 mice.
      and this study), it appears conceivable that circulating mCRP does not play a significant role in direct induction of EC dysfunction, at least in the setting of chronic local inflammation (e.g. atherogenesis). Moreover, tissue-associated mCRP, irrespective of its redox state (
      • Wang M.Y.
      • Ji S.R.
      • Bai C.J.
      • El Kebir D.
      • Li H.Y.
      • Shi J.M.
      • Zhu W.
      • Costantino S.
      • Zhou H.H.
      • Potempa L.A.
      • Zhao J.
      • Filep J.G.
      • Wu Y.
      A redox switch in C-reactive protein modulates activation of endothelial cells.
      ), may exert antiatherosclerotic actions, in particular at initial stages of plaque development, by promoting opsonic activation of the early complement pathway and non-inflammatory clearance of damaged cells (
      • Mihlan M.
      • Blom A.M.
      • Kupreishvili K.
      • Lauer N.
      • Stelzner K.
      • Bergström F.
      • Niessen H.W.
      • Zipfel P.F.
      Monomeric C-reactive protein modulates classic complement activation on necrotic cells.
      ,
      • Lauer N.
      • Mihlan M.
      • Hartmann A.
      • Schlötzer-Schrehardt U.
      • Keilhauer C.
      • Scholl H.P.
      • Charbel Issa P.
      • Holz F.
      • Weber B.H.
      • Skerka C.
      • Zipfel P.F.
      Complement regulation at necrotic cell lesions is impaired by the age-related macular degeneration-associated factor-H His402 risk variant.
      ,
      • Ji S.R.
      • Wu Y.
      • Potempa L.A.
      • Liang Y.H.
      • Zhao J.
      Effect of modified C-reactive protein on complement activation: a possible complement regulatory role of modified or monomeric C-reactive protein in atherosclerotic lesions.
      ,
      • Mihlan M.
      • Stippa S.
      • Józsi M.
      • Zipfel P.F.
      Monomeric CRP contributes to complement control in fluid phase and on cellular surfaces and increases phagocytosis by recruiting factor H.
      ) and by inhibiting foam cell formation (
      • Schwedler S.B.
      • Hansen-Hagge T.
      • Reichert M.
      • Schmiedeke D.
      • Schneider R.
      • Galle J.
      • Potempa L.A.
      • Wanner C.
      • Filep J.G.
      Monomeric C-reactive protein decreases acetylated LDL uptake in human endothelial cells.
      ,
      • Ji S.R.
      • Wu Y.
      • Potempa L.A.
      • Qiu Q.
      • Zhao J.
      Interactions of C-reactive protein with low-density lipoproteins: implications for an active role of modified C-reactive protein in atherosclerosis.
      ). Overall, these would underlie the seemingly counterintuitive findings of the in vivo atheroprotective effects of Cys-mutated mCRP (
      • Schwedler S.B.
      • Amann K.
      • Wernicke K.
      • Krebs A.
      • Nauck M.
      • Wanner C.
      • Potempa L.A.
      • Galle J.
      Native C-reactive protein increases whereas modified C-reactive protein reduces atherosclerosis in apolipoprotein E-knockout mice.
      ).
      The polarized response of ECs to Cys-mutated mCRP may be due to the distinct cellular sensors engaged. However, our results are incompatible with such a scenario because Cys-mutated mCRP activates ECs through cholesterol-enriched lipid raft microdomains on both apical and basolateral membranes. By contrast, the asymmetric distribution of lipid rafts on EC surfaces seems to be the key determinant. Lipid rafts are enriched in the apical membranes but depleted in the basolateral membranes, tightly correlating with the preferential apical binding of Cys-mutated mCRP. Consequently, this may lead to differential induction of intracellular pathways as the result of distinct interaction strength and polarized localization of receptors (e.g. CD16 (
      • Khreiss T.
      • József L.
      • Potempa L.A.
      • Filep J.G.
      Conformational rearrangement in C-reactive protein is required for proinflammatory actions on human endothelial cells.
      )) or signaling components. Indeed, Cys-mutated mCRP signals through p38 MAPK and PLC to NF-κB from the apical surfaces but only activates the PLC → NF-κB pathway from the basolateral surfaces (Fig. 7). Therefore, the absence of the concurrent p38 MAPK signaling may account for the weak EC responses to basolaterally applied Cys-mutated mCRP.
      Besides Cys-mutated mCRP, LPS and IL-1β, but not TNF-α, also exhibited polarized EC stimulation. These data highlight the fact that topological localization of certain mediators within the vessels is an important factor that determines their contributions to vascular inflammation and, hence, may to some extent account for the inconsistencies between in vitro and in vivo findings. Our results identify spatial localization as a novel determinant for the highly context-dependent actions of CRP isoforms within the vessels. Tissue-resident mCRP may primarily act as a pattern recognition molecule to facilitate non-inflammatory complement activation (
      • Mihlan M.
      • Blom A.M.
      • Kupreishvili K.
      • Lauer N.
      • Stelzner K.
      • Bergström F.
      • Niessen H.W.
      • Zipfel P.F.
      Monomeric C-reactive protein modulates classic complement activation on necrotic cells.
      ,
      • Wang M.Y.
      • Ji S.R.
      • Bai C.J.
      • El Kebir D.
      • Li H.Y.
      • Shi J.M.
      • Zhu W.
      • Costantino S.
      • Zhou H.H.
      • Potempa L.A.
      • Zhao J.
      • Filep J.G.
      • Wu Y.
      A redox switch in C-reactive protein modulates activation of endothelial cells.
      ,
      • Ji S.R.
      • Wu Y.
      • Potempa L.A.
      • Liang Y.H.
      • Zhao J.
      Effect of modified C-reactive protein on complement activation: a possible complement regulatory role of modified or monomeric C-reactive protein in atherosclerotic lesions.
      ) and macrophage clearance (
      • Schwedler S.B.
      • Hansen-Hagge T.
      • Reichert M.
      • Schmiedeke D.
      • Schneider R.
      • Galle J.
      • Potempa L.A.
      • Wanner C.
      • Filep J.G.
      Monomeric C-reactive protein decreases acetylated LDL uptake in human endothelial cells.
      ,
      • Ji S.R.
      • Wu Y.
      • Potempa L.A.
      • Qiu Q.
      • Zhao J.
      Interactions of C-reactive protein with low-density lipoproteins: implications for an active role of modified C-reactive protein in atherosclerosis.
      ) within vessels undergoing chronic inflammation. However, in acute vascular inflammation, mCRP might be present in the circulation for an extended period of time as a consequence of endothelial damage, enhanced conversion, or transportation by microparticles (
      • Habersberger J.
      • Strang F.
      • Scheichl A.
      • Htun N.
      • Bassler N.
      • Merivirta R.M.
      • Diehl P.
      • Krippner G.
      • Meikle P.
      • Eisenhardt S.U.
      • Meredith I.
      • Peter K.
      Circulating microparticles generate and transport monomeric C-reactive protein in patients with myocardial infarction.
      ) or activated platelets (
      • Molins B.
      • Peña E.
      • de la Torre R.
      • Badimon L.
      Monomeric C-reactive protein is prothrombotic and dissociates from circulating pentameric C-reactive protein on adhered activated platelets under flow.
      ,
      • Eisenhardt S.U.
      • Habersberger J.
      • Murphy A.
      • Chen Y.C.
      • Woollard K.J.
      • Bassler N.
      • Qian H.
      • von Zur Muhlen C.
      • Hagemeyer C.E.
      • Ahrens I.
      • Chin-Dusting J.
      • Bobik A.
      • Peter K.
      Dissociation of pentameric to monomeric C-reactive protein on activated platelets localizes inflammation to atherosclerotic plaques.
      ,
      • de la Torre R.
      • Peña E.
      • Vilahur G.
      • Slevin M.
      • Badimon L.
      Monomerization of C-reactive protein requires glycoprotein IIb-IIIa activation: pentraxins and platelet deposition.
      ). Ultimately, these would lead to amplification of the inflammatory responses by further activating ECs (
      • Wang M.Y.
      • Ji S.R.
      • Bai C.J.
      • El Kebir D.
      • Li H.Y.
      • Shi J.M.
      • Zhu W.
      • Costantino S.
      • Zhou H.H.
      • Potempa L.A.
      • Zhao J.
      • Filep J.G.
      • Wu Y.
      A redox switch in C-reactive protein modulates activation of endothelial cells.
      ,
      • Khreiss T.
      • József L.
      • Potempa L.A.
      • Filep J.G.
      Conformational rearrangement in C-reactive protein is required for proinflammatory actions on human endothelial cells.
      ), platelets (
      • Molins B.
      • Peña E.
      • Vilahur G.
      • Mendieta C.
      • Slevin M.
      • Badimon L.
      C-Reactive protein isoforms differ in their effects on thrombus growth.
      ), and neutrophils (
      • Khreiss T.
      • József L.
      • Potempa L.A.
      • Filep J.G.
      Loss of pentameric symmetry in C-reactive protein induces interleukin-8 secretion through peroxynitrite signaling in human neutrophils.
      ). In summary, our data reveals an additional layer of regulation of the actions of mCRP on ECs by topological localization, consistent with the apical enrichment of lipid rafts, the primary endothelial sensors of mCRP.

      Acknowledgments

      We thank Jing Zhao and Li-Ping Guan for technical assistance.

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