Dr. SU. Eisenhardt
Dr. Eisenhardt, in the most recent issue of Circulation Research you and your colleagues tell a very fascinating story about monomeric C-reactive protein (CRP) and its potential pathophysiological role in atherosclerosis. Would you mind briefly recapitulating this story for us?
A: Thank you very much for the opportunity to discuss our work.Briefly, my coworkers and I found deposition of monomeric, but not pentameric C-reactive protein in samples of human atherosclerotic plaques, which triggered the question: What is it doing there and where is it coming from? As CRP circulates as a pentamer (pCRP) and is known to be remarkably stable in the circulation, we assumed that a local process of dissociation must take place. We could show, that indeed activated, but not non-activated platelets are able to dissociate pentameric to monomeric CRP via a mechanism dependent on the exposure of bioactive lipids, such as lysophosphatidylcholine. Furthermore, we could show that this platelet derived monomeric CRP (mCRP) exerts pro-inflammatory functions. Compared to the native pCRP, mCRP leads to increased monocyte activation and adhesion under static and shear flow conditions and it stimulates the production of reactive oxygen species in monocytes. All these characteristics can be interpreted as pro-atherosclerotic effects. Interestingly, we could show that mCRP effects on monocytes are mainly mediated by a lipid raft signaling mechanism involving PI3-kinases. In conclusion, we identified a mechanism by which pCRP is converted to a highly pro-inflammatory isoform that mediates and enhances pro-inflammatory and thus pro-atherogenic properties in a localized setting.The schematic drawing given as Figure 7 in our article illustrates our findings and conclusions. Q: What sparked the hypothesis that monomeric but not pentameric CRP is deposited in atherosclerosis plaques?A: The deposition of CRP in atherosclerotic plaques has been described previously. Previously published reports on the very distinct in vitro activity of monomeric and pentameric CRP by Janos Filep and Lawrence Potempa and their coworkers were intriguing, however they did not answer the question whether mCRP does exist in vivo. Thus, this was the first question to be answered.Q: How do you interpret previous studies showing immunoreactivity for CRP in atherosclerotic plaques? Were these performed with antibodies that would detect both types of CRP? As monomeric CRP was not known at the time, was it assumed that it'll be pentameric CRP and hence an example of the “ice-berg effect”?A: Most of the studies that investigated human atherosclerotic plaque material indeed used antibodies that show a strong reactivity for both isoforms and I think your interpretation is correct. In contrast, we used a conformation specific set of anti-CRP antibodies provided by our close collaborator Lawrence Potempa. However, I would like to point out that mCRP has been known for some time, however the lack of a proof of its in vivo existence and even more the missing concept of where it could come from lead to a strange orphan existence as it just did not fit into the concept of research at the time. We hope that our data might help to interpret some findings obtained in the past in CRP research, as some data will have to be re-evaluated in regard to the question whether the findings are due to pCRP or potential contaminations with mCRP or even dissociation processes occurring in the course of the respective experiments.Q: How exactly does monomeric CRP get deposited in the atherosclerotic plaque?A: Our current manuscript does not fully answer this question and I will have to speculate on this one. I believe that mCRP is presented on the surface of platelets and localizes inflammation to the area were it has been dissociated. It then triggers the aforementioned inflammatory cascade by interacting with circulating monocytes and inducing rolling, adhesion and transmigration. The platelets are either phagocytosed with the mCRP or mCRP hitchhikes on the monocytes into the intima of the vessel. This would also explain the co-localization of mCRP and CD68 positive cells in the atherosclerotic plaque.Q: You performed a number of studies on both types of CRP. How do they differ with regards to the effects on atherosclerosis?A: We investigated the effects of both types of CRP on monocytes and could show that mCRP triggers increased activation, adhesion under static and shear flow conditions, production of reactive oxygen species and chemotaxis. pCRP show only weak pro-inflammatory effects in nonphysiologically high concentrations. However it needs to be mentioned that other studies have also identified pro-inflammatory effects for pCRP in other in vitro studies. This either suggests that the bioactivity of the two CRP isoforms is more complex than we are currently aware of, or it highlights that the reaction conditions of previously published experiments have to be reevaluated carefully for a potential contamination with mCRP. As our study shows, exposure of bioactive lipids in the experimental setting could potentially lead to pCRP dissociation and thus influence the outcome of the study. Interestingly, we also see this in our shear-flow experiments. pCRP stimulates monocyte adhesion on a monolayer of adhering platelets (albeit much weaker than mCRP), but hardly under static conditions on a fibrinogen matrix, which might be due to the dissociation induced by the adhering and thus activated platelets. The relevance of our and previous findings of the effects of p- and mCRP for research on the pathogenesis of atherosclerosis has been discussed in more detail in our manuscript, as well as in the accompanying editorial by Dr. Janos Filep.Q: Do these effects require receptor mediation? If so, via which receptor, and if not, what is the intracellular signaling pathway?A: The CRP-receptor question has been an ongoing debate and despite remarkable work by Terry duClos and coworkers I believe that it is far from being answered. Our data suggests that FcgammaReceptors are partially involved in mediating CRP effects, but the majority is mediated by lipid raft signaling and Pi3-kinase and src-kinase dependent intracellular pathways. It has been shown previously that mCRP is integrated into lysophospatidylcholine containing membranes so a receptor independent signaling mechanism as suggested by our results is a potential explanation. However our data cannot answer this question fully.
Q: How does this translate to the results obtained by the high-sensitive CRP assay? Does this assay detect pentameric CRP, monomeric CRP, or both? Could we conclude though that in either way, the results indicate the available load for monomeric CRP in any given patient? What is your opinion?A: This is indeed an interesting and important question. At this stage we have not been able to detect mCRP in the circulation. The high sensitivity CRP assay only detects pCRP, but not mCRP. As the dissociation process is a localized mechanism on the surface of cells it probably will not be detectable in the circulation but will be deposited locally. However it is reasonable to believe that the more pCRP is circulating, the more can be dissociated to mCRP and fuel the localized inflammatory reaction.Q: Activated platelets seem to be a crucial component to the story. Do you think this explains some of the cardiovascular benefits of aspirin and Plavix? For this matter, should all patients with CAD be on both drugs long-term.A: The risk reduction for cardiovascular events seen with aspirin and/or clopidogrel may indeed be partially caused by reduction in mCRP generation. Inhibition of platelet activation may thus prevent proinflammatory effects of mCRP and thereby reduce the risk and/or extent of plaque rupture and arterial thrombosis. Following this line of argumentation you would expect that the combination of aspirin and clopidogrel is more effective in the inhibition of pCRP to mCRP conversion compared to both drugs alone. But so far these are only fascinating hypotheses, which have yet to be proven. Only large scale clinical trials can answer the question whether the benefits associated with the combination of aspirin and clopidogrel can outweigh the increased risk of bleeding complications associated with this drug combination.Q: The other facilitating cell for the conversion of pentameric to monomeric CRP seems to be the apoptotic cell. How do these cells interact with CRP in the atherosclerotic plaque?A: We only investigated the deposition of monomeric CRP in fatty streaks and fibro-fatty lesions, which only represent timepoints in the formation of the atherosclerotic plaque. We don’t have any information on whether all of the pCRP is transferred to mCRP before it is deposited in the lesion or whether some pCRP residues are still interacting with apoptotic cells in the course of the formation of the plaque. However, the identification of other cells that potentially can facilitate the dissociation process allows for the conclusion that this mechanism might be of relevance in a much broader context than the one under investigation in our manuscript. We speculate that pCRP dissociation might be an ubiquitous activation signal for inflammatory and phagocytotic cells in all sorts of inflammatory reactions were bioactive lipids are exposed, be it by platelets or apoptotic/necrotic cells.Q: In your article you point to lipoprotein phospholipase A2 - how does it link into this? Will inhibitors of this enzyme be effective on monomeric CRP generation? In other words, is this enzyme one of the main, if not the central generator?A: Yes, we certainly believe so. We are currently working on the identification of the exact mechanism, but we already know that blocking the enzyme completely abolishes the dissociation process on activated platelets.Q: Finally, given the mentioning of the JUPITER trial in your manuscript, how does statin therapy play into this?A: As mentioned above, we believe that lowering the levels of circulating pCRP would certainly reduce the amount of pCRP dissociated in the localized inflammation reaction and reduces the load of mCRP. It is reasonable to believe that pleiotropic effects of statins leading to reduced activation of platelets would also inhibit the dissociation process and thus might have direct effects on the local inflammation contributing to the formation of the atherosclerotic plaques.Conclusion:Q: Thank you so much for this interview and this discussion which will hopefully spark further interest and research in this area! In case someone would like to take it further, may they contact you via E-mailA: We appreciate the opportunity to elaborate on our work further in this interview. We hope that our work stimulates more research in this field and that the identification of mCRP as a major mediator of atherosclerosis and inflammation will result in therapeutic approaches targeting mCRP.
Of course we are happy to discuss our findings in moredetail ( firstname.lastname@example.org and email@example.com ).
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