The hypothesis to be tested in this study was that mCRP induces specific changes in the protein expression profile of THP-1 cells that potentially represent a pro-inflammatory phenotype. An in vitro treatment-control experimental design was used to determine CRP isoform-specific effects on proteins isolated and displayed using 2D SDS PAGE and MALDI Tof/Tof analysis. The three major findings of this study are: (1) mCRP and pCRP both induce changes in the protein expression profile of THP-1 monocytes that can be interpreted as pro-inflammatory. (2) mCRP induces changes that are distinct from pCRP, further supporting the concept of a distinct role of mCRP and pCRP in vascular inflammatory diseases, including atherosclerosis. (3) LPS induced proteomic changes and LPS induced signaling pathways differ from mCRP and/or pCRP induced changes.
The causal role of CRP in the pathophysiology of atherosclerosis is currently an area of great controversy. In particular the existence of two isoforms mCRP and pCRP have complicated the analysis and interpretation of experimental findings. In vitro, pCRP has been reported to increase IL-8 production in monocytes, and to increase monocyte adhesion to human aortic and umbilical vein endothelial cells under static and flow conditions[10, 11]. However, several studies have also identified possible anti-inflammatory effects of pCRP , and anti-atherosclerotic actions for mCRP . In contrast to pCRP, mCRP induces IL-8 secretion in neutrophils  and human coronary artery endothelial cells (HCAEC) , promotes neutrophil-endothelial cell adhesion , delays apoptosis of human neutrophils . Additionally, both CRP conformations interact differently with components of the complement cascade .
Recently, we identified a mechanism by which pentameric CRP is dissociated to monomeric CRP on activated platelets, which is then deposited in atherosclerotic plaques [1, 17, 18]. These findings support the concept of a causal role for CRP in the development of atherosclerosis that might be independent of CRP as a risk factor in atherosclerosis associated cardiovascular events . In addition, we could show that mCRP induces monocyte activation and adhesion to a far greater extent than pCRP. Based on our previous findings we have investigated the intracellular mechanisms involved resulting in this current study.
The result of our proteomic analysis suggests that mCRP has pro-inflammatory properties at the proteomic level, recognizing the limitations of proteomic data in predicting the functional relevance of up-regulation of single proteins. One of our key findings is the up-regulation of the ubiquitin-activating enzyme E1 (UAE1) by mCRP, but not pCRP or LPS. This is of particular interest as the role and the stimulation of the ubiquitin-proteasome system (UPS) in atherosclerosis has been suggested previously . The UPS consists of several enzymes which function to degrade proteins that are presented by helper proteins, so called chaperones. These chaperones assist in the post-translational folding of proteins to their correct 3-D-structure, but also recognize mis-folded proteins and present them for degradation by the UPS [20, 21]. Thus, the UPS-chaperone system assures the quality of intracellular proteins. The coronary artery plaques associated with fatal acute myocardial infarction are characterized by increased expression of ubiquitinated proteins . Similarly, carotid artery plaques from patients with symptoms of focal cerebral ischemia display a higher level of ubiquitinated proteins than those obtained from asymptomatic patients [23, 24].
The up-regulation of UAE1 in mCRP stimulated monocytes may also be a sign of the induction of the macrophagic differentiation process induced by mCRP, as a crucial role for UAE1 in the differentiation process and the associated nucleotide excision repair has been recently described .
Both LPS  and mCRP  can induce reactive oxygen species (ROS) in monocytes or THP-1 monocytes. Therefore it is unlikely that the proteomic changes observed in the treatment groups are mediated by ROS as there is a substantial difference in the proteomic profile of THP-1 cells treated with LPS and those treated with mCRP. However, the induced oxidative stress can lead to an increase in the expression of chaperones, including those of the family of heat shock proteins (HSPs). One of the HSPs that is most closely associated with atherosclerosis is HSP 70, which is expressed in atherosclerotic lesions . In our proteomic analysis we found a significantly increased expression of HSP70 in monocytes stimulated with mCRP compared to the pCRP treated cells and PBS treated control cells. In experimental models of ischemia and reperfusion injury it has been suggested that HSP 70 mediates myocardial protection [28, 29]. Furthermore, HSP70 has been found to be up-regulated in the proteome of monocytes in acute coronary syndrome (ACS) , a disease in which circulating CRP levels are increased as well. Our results suggest that there is a potential interaction of HSP 70 with CRP that warrants further research.
Other HSPs differentially expressed are members of the HSP 105 family. HSP 105 alpha and beta are alternatively spliced products derived from the HSP 105 gene transcript. HSP 105 alpha is constitutively expressed but transcription is increased in response to various stressors. In contrast HSP 105 beta, which lacks 44 amino acids from HSP 105 alpha, is specifically expressed during mild heat shock . These proteins can exist as complexes associated with HSP 70 in mammalian cells . Similar to HSP 70, HSP 105 alpha and beta suppress the aggregation of denatured proteins caused by heat shock in vitro. It also has been suggested that HSP 105 alpha and beta can function as a substitute for HSP 70 to suppress the aggregation of denatured proteins in cells under severe stress, in which cellular ATP levels are elevated . Overall, the up-regulation of these proteins could potentially indicate cellular stress, induced by CRP stimulation of THP-1 monocytes. Interestingly, the differential expression of HSP 105 alpha is induced by mCRP, whereas pCRP induces expression of HSP 105 beta, further supporting our concept of discrete function and effects of mCRP and pCRP.
Other proteins increased in the treatment groups that are associated with the inflammatory response include alpha-enolase, alpha-actinin 4 (ACTN4) and EH domain containing protein 1 (EHD1 = Testilin). Alpha-enolase is significantly and uniquely increased after mCRP treatment. Alpha-enolase serves as a plasminogen receptor on macrophages and therefore has an important function in inflammatory cell recruitment . In respect to the development of atherosclerosis, cholesterol loading of macrophages induces expression of alpha-enolase in mice .
ACTN4, as a non-muscle alpha-actinin, is functionally important for leukocyte diapedesis in endothelial cells  and as such important for cell motility in inflammation. In addition, it has been detected in lipid rafts of THP-1 monocytes, and an involvement in vesicular trafficking and phagosome formation has been suggested .
EHD1 regulates the endosomal transport of beta-1 integrins , which are important cell adhesion receptors involved in adhesion and transmigration of inflammatory cells . It is significantly up-regulated in LPS stimulated THP-1 monocytes, but not in cells incubated with CRP isoforms further underlining the differentiated proteomic response of these cells to LPS versus CRP co-incubation that cannot be attributed to the contamination of the CRP preparations with LPS, as suggested for other experiments .
Overall, the proteins up-regulated by the CRP isoforms potentially represent the induction of a pro-inflammatory monocytic phenotype for adhesion cell recruitment and transmigration into the area of inflammation.
A recent comprehensive genomic study of human primary monocytes by Hanriot et al.  demonstrated complex cellular responses, both pro- and anti-inflammatory, following stimulation by (p)CRP. Amongst other findings chemokines (Gro protein alpha and beta, Interleukin (IL)-8), cytokines (IL-1 alpha and beta, IL-6, IL-19), integrins (Integrin alpha 6 and beta 1), Liver X receptor alpha and MAPK 8 were all up-regulated. Interestingly these authors also found a down regulation of several pro-inflammatory chemokines, suggesting potential anti-inflammatory effects of (p)CRP in primary monocytes at the genomic level.
Our proteomic dataset derived from a monocytic cell line complements these findings. The increased expression of Alpha-enolase and ACTN4 identified in our work is consistent with the up-regulation of several genes discussed above that encode for proteins involved in leukocyte trafficking, adhesion and transmigration by Hanriot et al. . The increased expression of these genes and proteins potentially characterizes a pro-inflammatory phenotype induced by (p)CRP.
CRP is generally believed to bind to receptors of the Fc gamma Receptor family . Recently it has been shown that lipid rafts are involved in mCRP signal transduction [1, 42]. The proteomic expression model used in this study involving prolonged incubation periods was not able to identify a discrete receptor ligand interaction. Successful resolution of this question will most likely require development of dedicated cell models and remains an area of active ongoing research.
One potential limitation of our study is the use of THP-1 cells, instead of primary monocytes. THP-1 cells could potentially reveal different properties compared to the primary cells. However, THP-1 cells have been previously used as a surrogate for primary monocytes, particularly in proteome research, and their use has so far contributed towards several pathophysiologically important findings, specifically in the field of atherosclerosis research . Therefore, although our findings must be interpreted with this in mind it does not invalidate this data.
Compared to our previous findings, in which pCRP showed significant less pro-inflammatory properties compared to mCRP, our findings in this proteomic study are surprising, as pCRP induces some pro-inflammatory proteomic changes as well. This may be due to one of the following factors. In our previous work we examined the short-term effects of the two isoforms on monocytes, which is fundamentally different from the 18 hours incubation period that is necessary to evaluate proteomic changes in the set of experiments described herein. Secondly, as the incubation was carried out in polystyrene tissue culture dishes spontaneous dissociation of pCRP to mCRP may occur over the course of the experiment. Indeed such dissociation has been described previously on polystyrene materials  and was confirmed by us. Therefore some effects seen in pCRP treated cells may indeed be effects caused by mCRP, which could potentially influence the outcome of the experiments and warrants further investigations into the distinct properties of the two isoforms.