Lysophosphatidylcholine is a Major Component of Platelet Microvesicles Promoting Platelet Activation and Reporting Atherosclerotic Plaque Instability.

BACKGROUND: Microvesicles (MVs) are small cell-derived vesicles, which are mainly released by activated cells. They are part of a communication network delivering biomolecules, for example, inflammatory molecules, via the blood circulation to remote cells in the body. Platelet-derived MVs are known to induce vascular inflammation. Research on the mediators and mechanisms of their inflammatory effects has attracted major interest. We hypothesize that specific lipids are the mediators of vascular inflammation caused by platelet-derived MVs. METHODS AND RESULTS: Liquid chromatography electrospray ionization-tandem mass spectrometry was used for lipid profiling of platelet-derived MVs. Lysophosphatidylcholine (LPC) was found to be a major component of platelet-derived MVs. Investigating the direct effects of LPC, we found that it induces platelet activation, spreading, migration and aggregation as well as formation of inflammatory platelet-monocyte aggregates. We show for the first time that platelets express the LPC receptor G2AR, which mediates LPC-induced platelet activation. In a mouse model of atherosclerotic plaque instability/rupture, circulating LPC was detected as a surrogate marker of plaque instability. These findings were confirmed by matrix-assisted laser desorption ionization imaging, which showed that the LPC concentration of human plaques was highest in vulnerable plaque regions. CONCLUSION: LPC is a major component of platelet-derived MVs and via its interaction with G2AR on platelets contributes to platelet activation, spreading, migration and aggregation and ultimately to vascular inflammation. Circulating LPC reports on atherosclerotic plaque instability in mice and is significantly increased in unstable areas of atherosclerotic plaques in both mice and humans, linking LPC to plaque instability.

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.

Carotid artery stenting: a clinical update.

PURPOSE OF REVIEW: Carotid endarterectomy (CEA) has been shown to prevent stroke in patients with severe carotid stenosis. Carotid artery stenting (CAS) has emerged as a less invasive alternative technique. Data regarding comparative effectiveness of CAS and CEA are now available and merit review. RECENT FINDINGS: Four large randomized controlled trials (RCTs) comparing CAS and CEA have shown a higher rate of stroke in symptomatic patients. The largest and most recent trial reported a lower occurrence of myocardial infarction (MI) following CAS and showed overall comparability of CAS to CEA for both symptomatic and asymptomatic patients. Despite methodological differences, these RCTs are consistent in finding an interaction of patient age with outcomes. In younger patients, CAS appears equivalent or superior to CEA if considering the sum of death, stroke, and MI. In elderly patients, CEA appears to have a lower complication rate. For asymptomatic patients, reduction in event rates with current medical therapy may render previous trial results invalid. SUMMARY: CAS is an alternative to CEA in patients requiring carotid intervention. Comparison of both CAS and CEA with contemporary medical management will also be required before recommendations can be made regarding the optimal treatment of patients with asymptomatic carotid stenoses.

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.

Amyloid plaques dissociate pentameric to monomeric C-reactive protein: a novel pathomechanism driving cortical inflammation in Alzheimer's disease?

Beta-amyloid (Aß) plaques and local inflammation are central to the pathogenesis of Alzheimer's disease. Although an association between circulating pentameric C-reactive protein (pCRP) and Alzheimer's disease has been reported no pathomechanistic link has been established. We hypothesized that Aß plaques induce the dissociation of pCRP to individual monomers (mCRP), which possess strong pro-inflammatory properties not shared with pCRP and localizing inflammation to Alzheimer's plaques. pCRP was incubated with Aß plaques generated in vitro and with non-aggregated Aß(42) peptide. pCRP dissociation to mCRP was found only when co-incubated with Aß plaques. Furthermore, sections of frontal cortex from brains of patients with and without Alzheimer's disease were stained with antibodies specific for mCRP and pCRP. There was significantly more mCRP in the cortex of Alzheimer's disease patients (P ≤ 0.01). In contrast, there was no significant difference in pCRP staining. These findings establish that Aß plaques possess a previously unrecognized potential to dissociate pentameric CRP to monomeric CRP. The existence of mCRP but not pCRP in the brains of Alzheimer's disease patients strongly indicates that this newly described biological effect of Aß plaques is relevant in Alzheimer pathobiology; potentially localizing and amplifying inflammation via the strong pro-inflammatory effects of locally generated mCRP.

Measuring oxidative burden and predicting pharmacological response in coronary artery disease patients with a novel direct activator of haem-free/oxidised sGC.

OBJECTIVE: The soluble guanylate cyclase (sGC) activator Cinaciguat (BAY 58-2667) represents a novel class of drugs that selectively activate oxidised sGC. The extent of oxidised sGC depends on the patient's oxidative burden. We here describe two platelet-based assays that allow determining the extent of oxidised sGC and thus provide a basis for an individualised pharmacotherapy. METHODS/RESULTS: Platelets obtained from patients with (n=12) and without (n=12) coronary artery disease (CAD) were examined by flow cytometry (P-selectin expression), and Western blots (vasodilator associated phosphoprotein, VASP-phosphorylation). Results were compared to maximal oxidation of sGC achieved by the oxidising agent ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one). Treatment of platelets with Cinaciguat resulted in differential activation of oxidised sGC. Platelet P-selectin expression and VASP-phosphorylation revealed significant differences (p=0.012, p=0.039, respectively) between CAD and non-CAD patients. CONCLUSION: We describe platelet-based assays that allow the determination of patients' oxidative status and thus allow the prediction of pharmacological response to direct sGC activators.

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

BACKGROUND: C-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. METHODS: Protein 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. RESULTS: mCRP 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. CONCLUSION: The data obtained in this study support the hypothesis that isoform-specific effects of CRP may differentially regulate the phenotype of monocytes.

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 H(2)O(2)-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.

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.

Management of atrial fibrillation.

BACKGROUND: Atrial fibrillation affects a significant proportion of the Australian population, affecting approximately 5% of people over 65 years of age. OBJECTIVE: This article provides an approach to the management of this common arrhythmia. DISCUSSION: During initial assessment, it is important to identify and treat any comorbid conditions that may contribute to atrial fibrillation. The clinician must then determine an appropriate anticoagulation regimen and choice of either a rate or rhythm control approach. Anticoagulation can be with either warfarin or aspirin, with the decision based on an assessment of the patient's thromboembolic risk. Rate and/or rhythm control can be achieved pharmacologically, with device based therapy or with newer catheter ablation techniques. A combination of these differing approaches may be required in patients in whom it is difficult to achieve and maintain therapeutic goals.