Steffen U. Eisenhardt

Dissociation of Pentameric to Monomeric C-Reactive Protein on Activated Platelets Localizes Inflammation to Atherosclerotic Plaques

C-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentamer (pentameric CRP) in plasma. The in vivo existence of monomeric (m)CRP has been postulated, but its function and source are not clear. We show that mCRP is deposited in human aortic and carotid atherosclerotic plaques but not in healthy vessels. pCRP is found neither in healthy nor in diseased vessels. As source of mCRP, we identify a mechanism of dissociation of pCRP to mCRP. We report that activated platelets, which play a central role in cardiovascular events, mediate this dissociation via lysophosphatidylcholine, which is present on activated but not resting platelets. Furthermore, the dissociation of pCRP to mCRP can also be mediated by apoptotic monocytic THP-1 and Jurkat T cells. The functional consequence is the unmasking of proinflammatory effects of CRP as demonstrated in experimental settings that are pathophysiologically relevant for atherogenesis: compared to pCRP, mCRP induces enhanced monocyte chemotaxis; monocyte activation, as determined by conformational change of integrin Mac-1; generation of reactive oxygen species; and monocyte adhesion under static and physiological flow conditions. In conclusion, we demonstrate mCRP generation via pCRP dissociation on activated platelets and H2O2-treated apoptotic THP-1 and Jurkat T cells, thereby identifying a mechanism of localized unmasking of the proinflammatory properties of CRP. This novel mechanism provides a potential link between the established cardiovascular risk marker, circulating pCRP, and localized platelet-mediated inflammatory and proatherogenic effects.

C-reactive protein: How conformational changes influence inflammatory properties

Recent evidence suggests that the prototypic acute phase reactant C-reactive protein (CRP) is not only a marker but also a potential contributor to inflammatory diseases. CRP belongs to the family of pentraxins and as such consists of five identical non-covalently linked subunits. Contradictory data on the characteristics of CRP as either being pro- or anti-inflammatory may be explained by the existence of two conformations of the protein: the circulating native, pentameric CRP (pCRP) and the monomeric isoform (mCRP), formed as a result of a dissociation process of pCRP. In vitro both isoforms exhibit a very distinct inflammatory profile. We recently identified a localized, physiologically relevant pCRP dissociation mechanism by activated platelets and apoptotic cells and showed the deposition of mCRP in inflamed tissue. Here we review the literature on the causal role of p- and mCRP in the light of our findings and critically analyze the current controversies around CRP. The novel understanding of the localized dissociation of circulating pentameric CRP to the distinctively pro-inflammatory monomeric CRP allows for a new view on CRP in inflammatory reactions and further highlights mCRP and the pCRP dissociation process as a potential therapeutic target.

Targeting Ligand-Induced Binding Sites on GPIIb/IIIa via Single-Chain Antibody Allows Effective Anticoagulation Without Bleeding Time Prolongation

Objective—Therapeutic anticoagulation is widely used, but limitations in efficacy and bleeding complications cause an ongoing search for new agents. However, with new agents developed it seems to be an inherent problem that increased efficiency is accompanied by an increase in bleeding complications. We investigate whether targeting of anticoagulants to activated platelets provides a means to overcome this association of potency and bleeding. Methods and Results—Ligand-induced binding sites (LIBS) on fibrinogen/fibrin-binding GPIIb/IIIa represent an abundant clot-specific target. We cloned an anti-LIBS single-chain antibody (scFvanti-LIBS) and genetically fused it with a potent, direct factor Xa (fXa) inhibitor, tick anticoagulant peptide (TAP). Specific antibody binding of fusion molecule scFvanti-LIBS-TAP was proven in flow cytometry; anti-fXa activity was demonstrated in chromogenic assays. In vivo anticoagulative efficiency was determined by Doppler-flow in a ferric chloride–induced carotid artery thrombosis model in mice. ScFvanti-LIBS-TAP prolonged occlusion time comparable to enoxaparine, recombinant TAP, and nontargeted mutant-scFv-TAP. ScFvanti-LIBS-TAP revealed antithrombotic effects at low doses at which the nontargeted mutant-scFv-TAP failed. In contrast to the other anticoagulants tested, bleeding times were not prolonged by scFvanti-LIBS-TAP. Conclusions—The novel clot-targeting approach of anticoagulants via single-chain antibody directed against a LIBS-epitope on GPIIb/IIIa promises effective anticoagulation with reduced bleeding risk.

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

Background— The relevance of the dissociation of circulating pentameric C-reactive protein (pCRP) to its monomeric subunits (mCRP) is poorly understood. We investigated the role of conformational C-reactive protein changes in vivo. Methods and Results— We identified mCRP in inflamed human striated muscle, human atherosclerotic plaque, and infarcted myocardium (rat and human) and its colocalization with inflammatory cells, which suggests a general causal role of mCRP in inflammation. This was confirmed in rat intravital microscopy of lipopolysaccharide-induced cremasteric muscle inflammation. Intravenous pCRP administration significantly enhanced leukocyte rolling, adhesion, and transmigration via localized dissociation to mCRP in inflamed but not noninflamed cremaster muscle. This was confirmed in a rat model of myocardial infarction. Mechanistically, this process was dependent on exposure of lysophosphatidylcholine on activated cell membranes, which is generated after phospholipase A2 activation. These membrane changes could be visualized intravitally on endothelial cells, as could the colocalized mCRP generation. Blocking of phospholipase A2 abrogated C-reactive protein dissociation and thereby blunted the proinflammatory effects of C-reactive protein. Identifying the dissociation process as a therapeutic target, we stabilized pCRP using 1,6-bis(phosphocholine)-hexane, which prevented dissociation in vitro and in vivo and consequently inhibited the generation and proinflammatory activity of mCRP; notably, it also inhibited mCRP deposition and inflammation in rat myocardial infarction. Conclusions— These results provide in vivo evidence for a novel mechanism that localizes and aggravates inflammation via phospholipase A2–dependent dissociation of circulating pCRP to mCRP. mCRP is proposed as a pathogenic factor in atherosclerosis and myocardial infarction. Most importantly, the inhibition of pCRP dissociation represents a promising, novel anti-inflammatory therapeutic strategy.Background— The relevance of the dissociation of circulating pentameric C-reactive protein (pCRP) to its monomeric subunits (mCRP) is poorly understood. We investigated the role of conformational C-reactive protein changes in vivo. Methods and Results— We identified mCRP in inflamed human striated muscle, human atherosclerotic plaque, and infarcted myocardium (rat and human) and its colocalization with inflammatory cells, which suggests a general causal role of mCRP in inflammation. This was confirmed in rat intravital microscopy of lipopolysaccharide-induced cremasteric muscle inflammation. Intravenous pCRP administration significantly enhanced leukocyte rolling, adhesion, and transmigration via localized dissociation to mCRP in inflamed but not noninflamed cremaster muscle. This was confirmed in a rat model of myocardial infarction. Mechanistically, this process was dependent on exposure of lysophosphatidylcholine on activated cell membranes, which is generated after phospholipase A2 activation. These membrane changes could be visualized intravitally on endothelial cells, as could the colocalized mCRP generation. Blocking of phospholipase A2 abrogated C-reactive protein dissociation and thereby blunted the proinflammatory effects of C-reactive protein. Identifying the dissociation process as a therapeutic target, we stabilized pCRP using 1,6-bis(phosphocholine)-hexane, which prevented dissociation in vitro and in vivo and consequently inhibited the generation and proinflammatory activity of mCRP; notably, it also inhibited mCRP deposition and inflammation in rat myocardial infarction. Conclusions— These results provide in vivo evidence for a novel mechanism that localizes and aggravates inflammation via phospholipase A2–dependent dissociation of circulating pCRP to mCRP. mCRP is proposed as a pathogenic factor in atherosclerosis and myocardial infarction. Most importantly, the inhibition of pCRP dissociation represents a promising, novel anti-inflammatory therapeutic strategy. # CLINICAL PERSPECTIVE {#article-title-40}

A Mechanistic Model for Paradoxical Platelet Activation by Ligand-Mimetic αIIbβ3 (GPIIb/IIIa) Antagonists

Objective—Integrins are attractive therapeutic targets. Inhibition of integrin &agr;IIb&bgr;3 effectively blocks platelet aggregation. However, limitations with intravenous &agr;IIb&bgr;3 antagonists and failure of oral &agr;IIb&bgr;3 antagonists prompted doubts on the current concept of ligand-mimetic integrin blockade. Methods and Results—Evaluating P-selectin expression on platelets by flow cytometry, we report a mechanism of paradoxical platelet activation by ligand-mimetic &agr;IIb&bgr;3 antagonists and define three requirements: (1) Induction of ligand-bound conformation of &agr;IIb&bgr;3, (2) receptor clustering, (3) prestimulation of platelets. Conformational change is inducible by clinically used ligand-mimetic &agr;IIb&bgr;3 antagonists, RGD-peptides, and anti-LIBS antibodies. In a mechanistic experimental model, clustering is achieved by crosslinking integrins via antibodies, and preactivation is induced by low-dose ADP. Finally, we demonstrate that platelet adhesion on collagen represents an in vivo correlate of platelet prestimulation and receptor clustering, in which the presence of ligand-mimetic &agr;IIb&bgr;3 antagonists results in platelet activation as detected by P-selectin, CD63, and CD40L expression as well as by measuring Ca2+-signaling. Blockade of the ADP receptor P2Y12 by AR-C69931MX and clopidogrel inhibits &agr;IIb&bgr;3 antagonist-induced platelet activation. Conclusion—These findings can explain limitations of ligand-mimetic anti-&agr;IIb&bgr;3 therapy. They describe potential benefits of concomitant ADP receptor blockade and support a shift in drug development from ligand-mimetic toward allosteric or activation-specific integrin antagonists.

Subtractive single-chain antibody (scFv) phage-display: tailoring phage-display for high specificity against function-specific conformations of cell membrane molecules

Phage-display of antibody libraries is a powerful tool to select antibodies for specific epitopes. We describe a strategy for selecting highly specific scFv-clones that discriminate between various conformational states of cell surface receptors. This approach adapts the M13 pIII phage-display technology toward a cell suspension-based strategy, which allows panning against complex, multimeric, fully functional cell membrane epitopes without alteration of structure due to purification or immobilization. As the functional properties are preserved, phage can be specifically depleted or selected for neo-epitopes exposed after physiological alterations of the targeted molecules. This subtractive strategy allows highly specific selection for single-chain antibodies directed against functionally regulated epitopes on the cell surface molecules that can be tailored for diagnostic and therapeutic applications. Using this protocol, activation-specific single-chain antibodies can be obtained within 4–6 weeks.

A proteomic analysis of C-reactive protein stimulated THP-1 monocytes

BackgroundC-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentameric protein in plasma. Recently, a potential dissociation mechanism from the disc-shaped pentameric CRP (pCRP) into single monomers (monomeric or mCRP) has been described. It has been shown that mCRP has strong pro-inflammatory effects on monocytes. To further define the role of mCRP in determining monocyte phenotype, the effects of CRP isoforms on THP-1 protein expression profiles were determined. The hypothesis to be tested was that mCRP induces specific changes in the protein expression profile of THP-1 cells that differ from that of pCRP.MethodsProtein cell lysates from control and mCRP, pCRP or LPS-treated THP-1 cells were displayed using 2-dimensional SDS PAGE and compared. Differentially expressed proteins were identified by MALDI-TOF MS and confirmed by Western blotting.ResultsmCRP significantly up-regulates ubiquitin-activating enzyme E1, a member of the ubiquitin-proteasome system in THP-1 monocytes. Furthermore, HSP 70, alpha-actinin-4 (ACTN4) and alpha-enolase/enolase 1 were upregulated. The proteomic profile of LPS and pCRP treated monocytes differ significantly from that of mCRP.ConclusionThe data obtained in this study support the hypothesis that isoform-specific effects of CRP may differentially regulate the phenotype of monocytes.

Monomeric C-Reactive Protein Generation on Activated Platelets: The Missing Link Between Inflammation and Atherothrombotic Risk

C-reactive protein (CRP) belongs to the family of pentraxins and as such consists of five identical non-covalently linked subunits. Recent evidence links CRP to the pathogenesis of atherosclerosis. We recently identified a dissociation mechanism on activated platelets that leads to a conformational change from the circulating native, pentameric CRP (pCRP) to its monomeric subunits (mCRP). This dissociation changes the proinflammatory profile of the protein and might be of causal relevance in the pathogenesis of atherosclerosis. Here, we review our results in the light of the recent literature with emphasis on the role of activated platelets, of different CRP isoforms, and of the CRP dissociation process in atherosclerotic plaque formation.

Pathophysiological Levels of Soluble P-Selectin Mediate Adhesion of Leukocytes to the Endothelium Through Mac-1 Activation

Plasma soluble P-selectin (sP-selectin) levels are increased in pathologies associated with atherosclerosis, including peripheral arterial occlusive disease (PAOD). However, the role of sP-selectin in regulating leukocyte–endothelial adhesion is unclear. The aim of this study was to assess the ability of exogenous and endogenous sP-selectin to induce leukocyte responses that promote their adhesion to various forms of endothelium. In flow chamber assays, sP-selectin dose-dependently increased neutrophil adhesion to resting human iliac artery endothelial cells. Similarly, sP-selectin induced neutrophil adhesion to the endothelial surface of murine aortae and human radial venous segments in ex vivo flow chamber experiments. Using intravital microscopy to examine postcapillary venules in the mouse cremaster muscle, in vivo administration of sP-selectin was also found to significantly increase leukocyte rolling and adhesion in unstimulated postcapillary venules. Using a Mac-1–specific antibody and P-selectin knockout mouse, it was demonstrated that this finding was dependent on a contribution of Mac-1 to leukocyte rolling and endothelial P-selectin expression. This was confirmed in an ex vivo perfusion model using viable mouse aorta and human radial vessels. In contrast, with tumor necrosis factor-&agr;–activated endothelial cells and intact endothelium, where neutrophil adhesion was already elevated, sP-selectin failed to further increase adhesion. Plasma samples from PAOD patients containing pathologically elevated concentrations of sP-selectin also increased neutrophil adhesion to the endothelium in a sP-selectin–dependent manner, as demonstrated by immunodepletion of sP-selectin. These studies demonstrate that raised plasma sP-selectin may influence the early progression of vascular disease by promoting leukocyte adhesion to the endothelium in PAOD, through Mac-1–mediated rolling and dependent on endothelial P-selectin expression.

Negative pressure wound therapy reduces the ischaemia/reperfusion-associated inflammatory response in free muscle flaps☆

BACKGROUND We recently established negative pressure wound therapy (NPWT) as a safe postoperative care concept for free muscle flaps; however, the molecular effects of NPWT on free muscle flaps remain elusive. Here we investigated the effects of NPWT on pathological changes associated with ischaemia/reperfusion injury in free flap tissue. METHODS From July 2008 to September 2010, 30 patients receiving skin-grafted free muscle transfer for defect coverage were randomly assigned to two treatment groups: In one group the skin-grafted free flap was covered by a vacuum dressing (NPWT); in the second group, flaps were covered by conventional petroleum gauze dressings (conv). Biopsies were taken intra-operatively prior to clipping of the pedicle and on postoperative day 5. Samples were analysed by immunohistochemistry for infiltration of inflammatory cells, real-time polymerase chain reaction (RT-PCR) for the analysis of expression levels of interleukin-1β (IL-1β) and tumour necrosis factor (TNF)-alpha as markers of inflammation. Histological samples were also examined for interstitial oedema formation, and apoptosis was detected by a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. RESULTS NPWT leads to a significantly reduced tissue infiltration of CD68 + macrophages and reduced expression of the inflammatory cytokines IL-1β and TNFα. None of these parameters was significantly elevated in the pre-ischaemic biopsies. Furthermore, NPWT reduced the interstitial oedema formation and the number of apoptotic cells in free flap tissue. CONCLUSION NPWT of skin-grafted free muscle flaps leads to a reduced inflammatory response following ischaemia/reperfusion, resulting in reduced oedema formation improving the microcirculation and ultimately reduced tissue damage. We thereby deliver new insight into the effects of NPWT.