Nicole Bassler

Microparticles: major transport vehicles for distinct microRNAs in circulation

Aims Circulating microRNAs (miRNAs) have attracted major interest as biomarkers for cardiovascular diseases. Since RNases are abundant in circulating blood, there needs to be a mechanism protecting miRNAs from degradation. We hypothesized that microparticles (MP) represent protective transport vehicles for miRNAs and that these are specifically packaged by their maternal cells. Methods and results Conventional plasma preparations, such as the ones used for biomarker detection, are shown to contain substantial numbers of platelet-, leucocyte-, and endothelial cell-derived MP. To analyse the widest spectrum of miRNAs, Next Generation Sequencing was used to assess miRNA profiles of MP and their corresponding stimulated and non-stimulated cells of origin. THP-1 (monocytic origin) and human umbilical vein endothelial cell (HUVEC) MP were used for representing circulating MP at a high purity. miRNA profiles of MP differed significantly from those of stimulated and non-stimulated maternal THP-1 cells and HUVECs, respectively. Quantitative reverse transcription–polymerase chain reaction of miRNAs which have been associated with cardiovascular diseases also demonstrated significant differences in miRNA profiles between platelets and their MP. Notably, the main fraction of miRNA in plasma was localized in MP. Furthermore, miRNA profiles of MP differed significantly between patients with stable and unstable coronary artery disease. Conclusion Circulating MP represent transport vehicles for large numbers of specific miRNAs, which have been associated with cardiovascular diseases. miRNA profiles of MP are significantly different from their maternal cells, indicating an active mechanism of selective ‘packaging’ from cells into MP. These findings describe an interesting mechanism for transferring gene-regulatory function from MP-releasing cells to target cells via MP circulating in blood.

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.

CD40 Ligand Mediates Inflammation Independently of CD40 by Interaction With Mac-1

Background— Strong evidence supports a role for CD40 ligand (CD40L) as marker and mediator of inflammatory diseases such as atherosclerosis. Despite extensive characterization of CD40, the classic receptor of CD40L, its role in immune defense against inflammatory diseases remains uncertain. The present study aimed to characterize the contribution of CD40 signaling to atherogenesis. Methods and Results— Surprisingly, mice deficient in both CD40 and the low-density lipoprotein receptor did not develop smaller lesions in the aortic arch, root, and thoracoabdominal aorta compared with mice deficient only in the low-density lipoprotein receptor that consumed an atherogenic diet for 8 and 16 weeks. By flow cytometry, radioactive binding assays, and immunoprecipitation, we demonstrate that CD40L interacts with the integrin Mac-1, which results in Mac-1–dependent adhesion and migration of inflammatory cells as well as myeloperoxidase release in vitro. Furthermore, mice deficient in CD40L show significantly reduced thioglycolate-elicited invasion of inflammatory cells into the peritoneal cavity compared with mice deficient in CD40 and wild-type controls. Inhibition of Mac-1 in low-density lipoprotein receptor–deficient mice attenuates lesion development and reduces lesional macrophage accumulation. Conclusions— These observations identify the interaction of CD40L and Mac-1 as an alternative pathway for CD40L-mediated inflammation. This novel mechanism expands understanding of inflammatory signaling during atherogenesis.

Magnetic Resonance Imaging Contrast Agent Targeted Toward Activated Platelets Allows In Vivo Detection of Thrombosis and Monitoring of Thrombolysis

Background— Platelets are the key to thrombus formation and play a role in the development of atherosclerosis. Noninvasive imaging of activated platelets would be of great clinical interest. Here, we evaluate the ability of a magnetic resonance imaging (MRI) contrast agent consisting of microparticles of iron oxide (MPIOs) and a single-chain antibody targeting ligand-induced binding sites (LIBS) on activated glycoprotein IIb/IIIa to image carotid artery thrombi and atherosclerotic plaques. Methods and Results— Anti-LIBS antibody or control antibody was conjugated to 1-&mgr;m MPIOs (LIBS MPIO/control MPIO). Nonocclusive mural thrombi were induced in mice with 6% ferric chloride. MRI (at 9.4 T) was performed once before and repeatedly in 12-minute-long sequences after LIBS MPIO/control MPIO injection. After 36 minutes, a significant signal void, corresponding to MPIO accumulation, was observed with LIBS MPIOs but not control MPIOs (P<0.05). After thrombolysis, in LIBS MPIO–injected mice, the signal void subsided, indicating successful thrombolysis. On histology, the MPIO content of the thrombus, as well as thrombus size, correlated significantly with LIBS MPIO–induced signal void (both P<0.01). After ex vivo incubation of symptomatic human carotid plaques, MRI and histology confirmed binding to areas of platelet adhesion/aggregation for LIBS MPIOs but not for control MPIOs. Conclusions— LIBS MPIOs allow in vivo MRI of activated platelets with excellent contrast properties and monitoring of thrombolytic therapy. Furthermore, activated platelets were detected on the surface of symptomatic human carotid plaques by ex vivo MRI. This approach represents a novel noninvasive technique allowing the detection and quantification of platelet-containing thrombi.

Conformation-Specific Blockade of the Integrin GPIIb/IIIa: A Novel Antiplatelet Strategy That Selectively Targets Activated Platelets

Platelet activation causes conformational changes of integrin GPIIb/IIIa (&agr;IIb&bgr;3), resulting in the exposure of its ligand-binding pocket. This provides the unique possibility to design agents that specifically block activated platelets only. We used phage display of single-chain antibody (scFv) libraries in combination with several rounds of depletion/selection to obtain human scFvs that bind specifically to the activated conformation of GPIIb/IIIa. Functional evaluation of these scFv clones revealed that fibrinogen binding to human platelets and platelet aggregation can be effectively inhibited by activation-specific scFvs. In contrast to clinically used GPIIb/IIIa blockers, which are all conformation unspecific, activation-specific GPIIb/IIIa blockers do not induce conformational changes in GPIIb/IIIa or outside-in signaling, as evaluated by ligand-induced binding-site (LIBS) exposure in flow cytometry or P-selectin expression in immunofluorescence microscopy, respectively. In contrast to the conformation-unspecific blocker abciximab, activation-specific scFvs permit cell adhesion and spreading on immobilized fibrinogen, which is mediated by nonactivated GPIIb/IIIa. Mutagenesis studies and computer modeling indicate that exclusive binding of activation-specific scFv is mediated by RXD motifs in the heavy-chain complementary-determining region (CDR) 3 of the antibodies, which in comparison with other antibodies forms an exceptionally extended loop. In vivo experiments in a ferric-chloride thrombosis model of the mouse carotid artery demonstrate similar antithrombotic potency of activation-specific scFv, when compared with the conformation-unspecific blockers tirofiban and eptifibatide. However, in contrast to tirofiban and eptifibatide, bleeding times are not prolonged with the activation-specific scFvs, suggesting lower bleeding risks. In conclusion, activation-specific GPIIb/IIIa blockade via human single-chain antibodies represents a promising novel strategy for antiplatelet therapy.

Single-chain antibodies for the conformation-specific blockade of activated platelet integrin αIIbβ3 designed by subtractive selection from naive human phage libraries

Binding of fibrinogen to platelet integrin αIIbβ3 mediates platelet aggregation, and thus inhibition of αIIbβ3 represents a powerful therapeutic strategy in cardiovascular medicine. However, the currently used inhibitors of αIIbβ3 demonstrate several adverse effects like thrombocytopenia and bleeding, which are associated with their property to bind to non‐activated αIIbβ3. To circumvent these problems, we designed blocking single‐chain antibody‐fragments (scFv) that bind to αIIbβ3 exclusively in its activated conformation. Two naïve phage libraries were created: a natural phage library, based on human lymphocyte cDNA, and a synthetic library, with randomized VHCDR3. We performed serial rounds of subtractive panning with depletion on non‐activated and selection on activated αIIbβ3, which were provided on resting and ADP‐stimulated platelets and CHO cells, expressing wild‐type or mutated and thereby activated αIIbβ3. In contrast to isolated, immobilized targets, as generally used for phage display, this unique cell‐based approach for panning allowed the preservation of functional integrin conformation. Thereby, we obtained several scFv‐clones that demonstrated exclusive binding to activated platelets and complete inhibition of fibrinogen binding and platelet aggregation. Interestingly, all activation‐specific clones contained an RXD pattern in the HCDR3. Binding studies on transiently expressed point mutants and mouse‐human domain‐switch mutants of αIIbβ3 indicate a binding site similar to fibrinogen. In conclusion, we generated human activation‐specific scFvs against αIIbβ3, which bind selectively to activated αIIbβ3 and thereby potently inhibit fibrinogen binding to αIIbβ3 and platelet aggregation.

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.

Circulating microparticles generate and transport monomeric C-reactive protein in patients with myocardial infarction

AIMS Elevated serum C-reactive protein (CRP) following myocardial infarction (MI) is associated with poor outcomes. Although animal studies have indicated a direct pathogenic role of CRP, the mechanism underlying this remains elusive. Dissociation of pentameric CRP (pCRP) into pro-inflammatory monomers (mCRP) may directly link CRP to inflammation. We investigated whether cellular microparticles (MPs) can convert pCRP to mCRP and transport mCRP following MI. METHODS AND RESULTS MPs enriched in lysophosphatidylcholine were obtained from cell cultures and patient whole-blood samples collected following acute MI and control groups. Samples were analysed by native western blotting and flow cytometry. MPs were loaded with mCRP in vitro and incubated with endothelial cells prior to staining with monoclonal antibodies. In vitro experiments demonstrated that MPs were capable of converting pCRP to mCRP which could be inhibited by the anti-CRP compound 1,6 bis-phosphocholine. Significantly more mCRP was detected on MPs from patients following MI compared with control groups by western blotting and flow cytometry (P = 0.0005 for association). MPs containing mCRP were able to bind to the surface of endothelial cells and generate pro-inflammatory signals in vitro, suggesting a possible role of MPs in transport and delivery of pro-inflammatory mCRP in vascular disease. CONCLUSION Circulating MPs can convert pCRP to pro-inflammatory mCRP in patients following MI, demonstrating for the first time mCRP generation in vivo and its detection in circulating blood. MPs can bind to cell membranes and transfer mCRP to the cell surface, suggesting a possible mCRP transport/delivery role of MPs in the circulation.

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.

Binding of CD40L to Mac-1’s I-domain involves the EQLKKSKTL motif and mediates leukocyte recruitment and atherosclerosis – but does not affect immunity and thrombosis in mice

Rationale: CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and hemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor. Objective: Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo. Methods and Results: CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high-affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the &agr;1 helix and the &bgr;-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. A cyclisized version optimized for in vivo use, cM7, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr−/− mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo. Conclusions: We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L.