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Cardiovascular Research 2005 68(1):47-55; doi:10.1016/j.cardiores.2005.05.010
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Copyright © 2005, European Society of Cardiology

C-reactive protein induces tissue factor expression and promotes smooth muscle and endothelial cell proliferation

Plinio Cirilloa, Paolo Golinob,*, Paolo Calabròa, Gaetano Calìc, Massimo Ragnia, Salvatore De Rosaa, Giovanni Cimminob, Mario Pacileoa, Raffaele De Palmad, Lavinia Forteb, Annarita Gargiuloa, Fabio Granato Coriglianoa, Valeria Angria, Raffaele Spagnuoloa, Lucio Nitsche and Massimo Chiarielloa

aFrom the Division of Cardiology, University of Naples "Federico II", Italy
bDivision of Cardiology, Second University of Naples, Italy
cIEOS "G. Salvatore" - National Council of Research, Naples, Italy
dDepartment of Clinical and Experimental Medicine, Second University of Naples, Italy
eDepartment of Molecular and Cellular Biology and Pathology, University of Naples "Federico II", Naples, Italy

* Corresponding author. Tel.: +39 81 7064050; fax: +39 81 7064275. Email address: paolo.golino{at}unina2.it

Received 20 January 2005; revised 25 April 2005; accepted 3 May 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Objective: Inflammation plays a pivotal role in atherothrombosis. In addition to being a prognostic marker for major cardiovascular events, recent data indicate that C-reactive protein (CRP) might directly promote atherothrombosis by exerting direct effects on vascular cells. The aim of the present study was to determine whether CRP might affect the prothrombotic and proliferative characteristics of endothelial (ECs) and smooth muscle cells (SMCs).

Methods and results: Incubation of ECs and SMCs with CRP resulted in a dose-dependent activation of cell proliferation, which was mediated by activation of the p44/42 MAP Kinase (ERK 1/2) pathway. In addition, CRP also induced tissue factor (TF) expression in both cell types in a dose-dependent fashion, exerting its effect at the transcriptional level, as demonstrated by semiquantitative and by real time PCR. Activation of the transcription factor, NF-{kappa}B, by CRP was demonstrated by EMSA and by suppression of TF expression by the NF-{kappa}B inhibitor, pyrrolidine-dithio-carbamate ammonium.

Conclusions: These data indicate that CRP exerts direct effects on ECs and SMCs by promoting proliferation and TF expression and support the notion that CRP, besides representing a marker of inflammation, is an effector molecule able to induce a pro-atherothrombotic phenotype in cells of the vessel wall.

KEYWORDS C-reactive protein; Tissue factor; Thrombosis; Cell proliferation

This article is referred to in the Editorial by A. Pandolfi (pages 3–4) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Several experimental and clinical studies indicate that tissue factor (TF) plays a pivotal role in the pathophysiology of acute coronary syndromes by triggering the formation of intracoronary thrombi following endothelial injury [1–4]. In this respect, cells normally not exposed to the flowing blood, such as smooth muscle cells, constitutively express TF on their surface [5,6], while cells exposed to the blood stream, such as endothelial cells, express TF on their membrane only when activated after exposure to specific stimuli, such as LPS [7], certain cytokines [7], and oxygen free radicals [8].

In recent years, an impressive mass of data have assigned a central role to inflammation as a phenomenon linked to the clinical occurrence of acute coronary syndromes [9,10]. Furthermore, several epidemiological studies have demonstrated that C-reactive protein (CRP), a marker of inflammation, is an important prognostic factor for the future occurrence of major cardiovascular events, both in patients with known cardiovascular disease and apparently healthy subjects [11–15]. However, despite this impressive mass of clinical data, to date the possible links existing between high plasma levels of CRP and the occurrence of major cardiovascular events are not completely understood. Thus, in the present study we have tested the hypothesis that CRP might affect the prothrombotic and proliferative status of two cell populations, such as endothelial and smooth muscle cells, which are known to be involved in the pathophysiology of atherothrombosis.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

2.1 Vascular smooth muscle and endothelial cells isolation and culture
Smooth muscle cells (SMCs) and endothelial cells (ECs) were isolated from the thoracic aortas and the coronary circulation of New Zealand white rabbits and cultivated as previously described [16,17]. Cells were enzymatically harvested and counted in a hemocytometer and subcultured in 24-well plates at an initial density of about 5 x 104 cells per well. At confluence, cell density was of about 8.5 x 104 cells/well. All experiments on SMCs were performed under conditions of serum starvation for 48 h. This step was necessary because it is known that quiescent SMCs express TF at low level [18].

Human purified CRP (Sigma Chemical Co.) was used in all studies described. Given the concern surrounding the potential contamination of CRP with endotoxin, we analysed our CRP and found endotoxin level to be <0.125 EU/mL (<12.5 pg/mL) by Limulus assay (BioWhittaker). All media and water were also tested and endotoxin level was found to be <0.125 EU/mL.

2.2 Effects of C-reactive protein on TF transcription
Quiescent SMCs and ECs were incubated with CRP (100 µg/ml). Thirty minutes after the addition of CRP, cells were washed with PBS and fresh medium (DMEM containing 0.1% FCS) was added. TF mRNA levels were examined by real-time reverse transcription (RT) and polymerase chain reaction (PCR) by LightCycler (Roche Diagnostics, Basel, Switzerland) and by semiquantitative PCR. Total RNA was extracted according to the methods of Cromzynsky and Sacchi [19] at baseline, 30, 60 and 120 min. cDNA was obtained by RT-PCR using 2 µg of total RNA and esanucleotide random primers; specific TF cDNA was obtained by PCR (35 cycles) using the following specific primers: forward 5'-CTACTGTTTCAGTGTCCAAGCA-3', reverse 3'-GGCGATGTTCAGGGGGGAGCTCTC-5', corresponding to a 300 bp-length fragment of TF. Each cycle consisted of a denaturation step at 96 °C for 1 min, an annealing step at 62 °C for 1.5 min and an elongation step at 72 °C for 4 min, as described elsewhere [20]. RT-PCR products were separated by electrophoresis on a 1% ethidium bromide-agarose gel and their intensities were quantified by a laser densitometry. Housekeeping GAPDH mRNA was also simultaneously amplified as internal control. Three different experiments were performed for each experimental condition.

2.3 Real-time quantitative RT-PCR
Total mRNA was extracted from cell cultures using TRIzol reagent (GIBCO), according to the manufacturer's instructions. Reverse transcription was performed using mMLV (GIBCO) and 100 ng of the RNA samples from each culture conditions. Samples were run in triplicate in 50 µl reactions by using an ABI PRISM 5700 sequence detector system (Applied Biosystems). Samples were incubated at 50 °C for 2 min, 95 °C for 10 min and then underwent 40 cycles at 95 °C for 15 s and 60 ° C for 1 min. Specific oligonucleotides for rabbit GAPDH and rabbit CRP were designed on the basis of published sequences using PRIMER EXPRESS Software (Applied Biosystems) and validated for their specificity. SYBR-green chemistry was used to detect fluorescence and an internal standard (Applied Biosystems) was used for quantization of the message. Three different experiments were performed for each experimental condition.

2.4 Dose–response effects of C-reactive protein on TF activity
SMCs and ECs were incubated with increasing concentration of CRP (5, 10, 20, 50, 100 and 200 µg/ml) for 6 h. TF activity was determined by a two-step colorimetric assay, based on the ability of TF to promote generation of coagulation FXa, as previously described [21]. Briefly, cells were incubated with 1 nmol of recombinant human FVIIa (Novo Nordisk A/S Gentofte, Denmark), followed by 100 nmol of purified human factor X (Calbiochem-Navobiochem, La Jolla, CA, USA) and 5mM CaCl2 for 15 min at 37 °C. A chromogenic substrate, specific for factor X (Cromozym X, Roche Diagnostics, Mannheim, Germany, 0.5 mmol/l) was then added and incubated for 30 min at 37 °C. The reaction was stopped by adding 200 µl/ml of sample of a 30% solution of acetic acid. The change in optical density at 405 nm was quantified with a spectrophotometer and converted to nanograms of TF using standard curves obtained with standard dilutions of recombinant human, re-lipidated TF [21].

Since previous studies indicated that cell-associated TF exists in three different pools of which two are stored in an encrypted form [22], additional experiments were designed to evaluate whether CRP-induced expression of TF resulted from de novo synthesis or from exposure of these cryptic TF sites. Thus, cells were pre-incubated with cycloheximide (10 µg/ml), an inhibitor of protein synthesis, or with 5, 6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB, 10 µg/ml), an inhibitor of DNA transcription, before adding CRP (100 µg/ml). In addition, to demonstrate that CRP-induced TF expression resulted in a maximal or nearly maximal TF exposure on the cell membrane, TF activity was also measured in unstimulated, quiescent cells incubated with the calcium ionophore, A23187 [GenBank] (20 µM), a compound known to induce exposure of TF from the cryptic cellular sites to the cell membrane [23], alone or added at the end of the 6 h period of CRP stimulation.

Additional control experiments included cells pre-incubated with AP-1 (5 µg/ml), a monoclonal antibody against rabbit TF [1]. Positive control experiments included SMCs and ECs incubated for 6 h with LPS (100 µg/ml). Six different experiments were performed for each experimental condition.

2.5 Effects of C-reactive protein on SMC and EC proliferation
To investigate the effects of CRP on SMC and EC proliferation, cells were seeded at the same density in 24-well plates; 48 h following serum deprivation, quiescent cells were stimulated with CRP at increasing concentrations (5, 10, 20, 50, 100 and 200 µg/ml). The growth effects of CRP were quantified by measuring the extent of 3H-thymidine incorporation into cell DNA. Thirty minutes after the addition of CRP (as described above), cells were washed and fresh medium containing 0.1% FCS was added. Twenty-four hours later, cells were pulsed with 1 µCi 3H-thymidine and, after 24 additional hours, the culture medium was discarded, the cells were rinsed twice with PBS and lysed with 0.2% perchloric acid. The contents of the wells were aspirated and the acid-precipitated cellular material was solubilized with 0.5 ml 0.01 N sodium Hydroxide–0.1% sodium dodecyl sulfate. The content of each well was added to 7 ml of Optifluor and radioactivity was measured with a Beckman beta-scintillator. Incorporation of 3H-thymidine was expressed as counts per minute per well. Negative control experiments included both SMCs and ECs incubated under conditions of serum deprivation, while positive control experiments included cells incubated with 10% FCS. Parallel experiments were performed as above described, except that proliferation was evaluated by directly counting the number of cells with a hemocytometer after enzymatic dissociation. Six different experiments were performed for each experimental condition.

2.6 CRP and cell signaling
To elucidate the mechanisms by which CRP exerts its effects on ECs and SMCs, we tested the hypothesis that Mitogen-Activated Protein Kinases (MAPKs) and Nuclear Factor-kappa B (NF-{kappa}B) may be involved in mediating proliferation and TF expression, respectively.

2.6.1 ERK Western blotting
The following primary antibodies were used: a rabbit polyclonal anti ERK-1/2 and a mouse monoclonal anti phospho-ERK (E4) from Santa Cruz Biothecnology, Inc. (Santa Cruz, CA). SMCs and ECs were seeded onto 100 mm diameter plastic dishes and starved in serum-free medium as described above. Then, in a first set of experiments, the time course of activation of ERK was investigated as previously described [24]; ECs and SMCs were washed and incubated with 100 µg/ml of CRP for 2, 10, 15, 20, and 30 min and then processed to evaluate the phosphorylation/activation of ERK [24]. Additionally, in another set of experiments, the effects of different CRP doses on ERK activation was investigated by stimulating ECs and SMCs with CRP at increasing concentrations (5, 10, 20, 50, 100, 200 µg/ml) for 15 min. Three different experiments were performed for each experimental condition.

2.7 NF-{kappa}B activation
The levels of NF-{kappa}B proteins in nuclear extracts from ECs and SMCs were analyzed by electrophoretic mobility shift assay (EMSA). SMCs and ECs starved in serum-free medium as described above were washed and incubated with CRP at increasing concentrations (5, 10, 20, 50, 100 µg/ml) for 30 min. Cells incubated with 100 µg/ml of LPS served as positive control. Nuclear proteins from these cells were isolated as previously described [25] and were subjected to EMSA using 32P-labeled NF-{kappa}B double-strand oligonucleotide (5'-ACTTGAGGGGACTTTCCCAGGC-3'). Nuclear proteins were incubated with oligonucleotide for 30 min, subjected to gel electrophoresis and finally autoradiographed.

2.8 Three different experiments were performed for each experimental condition
2.8.1 Statistical analysis
Data are presented as mean ± SD. Differences in terms of cell proliferation and TF expression between cell types and different concentrations of CRP were determined by a two-way ANOVA followed, if an F value was found to be significant, by a Student's t test with Bonferroni's correction. A p value<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1 Effects of C-reactive protein on TF transcription
Semiquantitative RT-PCR showed that TF mRNA was expressed at low levels in serum-deprived, quiescent SMCs, while it was almost undetectable in ECs, as expected [8]. Thirty minutes of incubation with CRP (100 µg/ml) did not cause any significant increase in TF mRNA levels in both cells, as compared to unstimulated cells. A significant increase in TF mRNA levels was observed at 60 min after stimulation with CRP, and at 120 min, TF mRNA levels progressively decreased (Fig. 1A). Real time PCR experiments showed similar results, with undetectable levels of TF mRNA under baseline conditions and a significant increase in both cell types after CRP stimulation. (Fig. 1B).


Figure 1
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  		Fig. 1 Effects of C-reactive protein (CRP) on TF transcription in endothelial cells (ECs) and smooth muscle cells (SMCs) assessed by semiquantitative PCR (panel A) and Real Time quantitative PCR (panel B). Thirty minutes of incubation with CRP did not cause any significant increase in TF mRNA levels in both cells, as compared to unstimulated cells. An increase in TF mRNA levels was observed at 60 min while at 120 min TF mRNA levels progressively decreased. Each bar represents the mean ± SD of 3 different experiments.

 
3.2 Dose–response effects of C-reactive protein on TF activity
A low TF activity was observed in quiescent, unstimulated SMCs. In these cells, low concentrations of CRP (5, 10, and 20 µg/ml) did not induce any significant increase in TF activity, while a progressive increase was observed when SMCs were incubated with higher CRP concentrations (Fig. 2). ECs showed undetectable TF activity at baseline, but differently from SMCs, these cells showed a progressive increase in TF activity even at low CRP concentrations, suggesting a higher sensitivity of these cells to CRP as compared to SMCs (Fig. 2).


Figure 2
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  		Fig. 2 Dose–response effects of C-Reactive protein (CRP, 5, 10, 20, 50, 100 and 200 µg/ml for 6 h) on TF activity in smooth muscle cells (SMCs) and endothelial cells (ECs), determined by a two-step colorimetric assay based on the ability of TF/FVIIa to promote generation of coagulation FXa. CRP induced a dose–response increase in TF activity in both ECs and SMCs, with ECs being more sensitive than SMCs at low CRP concentrations. Control experiments, performed by incubating ECs and SMCs with AP-1, just prior TF activity assay, confirmed that the procoagulant activity measured was really due to TF expression on cell surface after CRP induction (Panel A). Additional controls, performed by pre-incubating ECs and SMCs with cycloheximide and DRB or with the calcium ionophore A23187 (Panel B), showed that CRP-induced TF expression required de novo mRNA transcription and protein synthesis and that CRP did not exert significant effects on encrypted TF. Each bar represents the mean ± SD of 6 different experiments. *p<0.05 vs corresponding value at baseline.

 
Control experiments, performed by pre-incubating ECs and SMCs with AP-1, a monoclonal antibody against rabbit TF, confirmed that the procoagulant activity measured was actually due to TF expression on cell surface after CRP induction (Fig. 2). To test the hypothesis that CRP induced de novo synthesis of TF as opposed to exposure of encrypted TF, cells were pre-incubated with cycloheximide or DRB, an inhibitor of protein synthesis and mRNA transcription, respectively. In addition, to evaluate the relative contribution of TF encrypted pools in the observed phenomenon [22,23], another set of experiments were performed in which the calcium ionophore, A23187 [GenBank] , was added at baseline and at the end of the 6 h period of CRP stimulation. As shown in Fig. 2, both cycloheximide and DRB completely inhibited CRP-induced TF expression; in addition, A23187 [GenBank] added at the end of the incubation period with CRP only modestly increased TF expression on cell surface.

3.3 Effects of C-reactive protein on SMC and EC proliferation
Results are expressed as a percent of 3H-thymidine incorporation with respect to negative control experiments, represented by unstimulated cells kept under conditions of serum deprivation.

Stimulation of SMCs and ECs with CRP caused a dose-dependent increase in 3H-thymidine incorporation, reflecting cell proliferation. Low concentrations of CRP (i.e., 5 µg/ml) were unable to induce cell proliferation (Fig. 3) and a progressive increase in 3H-thymidine incorporation could be observed only with higher concentrations of CRP in both cell types (Fig. 3). Similar results were obtained by direct cell counting (data not shown).


Figure 3
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  		Fig. 3 Dose–response effects of increasing concentrations of CRP (5, 10, 20, 50, 100 and 200 µg/ml) on EC and SMC proliferation, as assessed by 3H-thymidine incorporation. Thirty minutes after the addition of CRP, cells were washed and fresh medium was added. Twenty-four hours later, cells were pulsed with 1 µCi 3H-thymidine and, after 24 additional hours, they were lysed and radioactivity counted in the cell lysates. Negative control experiments included unstimulated ECs and SMCs incubated under conditions of serum deprivation. Data are expressed as delta percent of quiescent, serum-deprived cells. CRP induced a dose-dependent increase in EC and SMC proliferation which was almost completely blocked by UO126, a selective inhibitor of ERK phosphorilation. Each bar represents the mean ± SD of 6 experiments. *p<0.05 vs corresponding value at baseline.

 
3.4 CRP and cell signaling
3.4.1 CRP-induced SMC proliferation and ERK activation
We used a specific antibody against the phosphorylated (activated) form of ERK to determine whether CRP-induced cell proliferation was associated with activation of the ERK pathway. Fig. 4 shows Western blots of cell lysates from SMCs and ECs exposed to 100 µg/ml CRP for different time periods. ERK activation in both cell types was evident at 2 min and peaked at 15 min. In addition, the magnitude of ERK activation showed a different pattern in the two cell population and was related with CRP concentrations (Fig. 4). Interestingly, CRP-induced cell proliferation in SMCs and in ECs was completely inhibited by preicubation with UO126, a selective inhibitor of ERK activation (Fig. 3).


Figure 4
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  		Fig. 4 Panel (A) Time-course of ERK activation in ECs and SMCs stimulated with CRP (100 µg/ml). Filters were probed with an anti phospho-ERK, and an antibody directed against the two ERK isoforms (ERK 1/2). Control experiments included ECs and SMCs pre-incubated with UO126 (10 µM), a selective inhibitor of ERK phosphorilation. Three different experiments were performed for each experimental condition. A time-dependent activation of ERK was observed, starting at 2 min and peaking at about 10 min. Pretreatment with UO126, did not result in any appreciable activation of ERK. Panel (B) Dose–response effects of CRP on ERK activation in ECs and SMCs. A significant activation of ERK was observed in ECs at CRP concentrations as low as 10 µg/ml, while SMCs required higher concentrations. Bars represent mean ± SD of three different experiments. *p<0.05 vs corresponding baseline values.

 
3.5 CRP-induced TF expression and activation of NF-{kappa}B
To investigate whether CRP may lead to activation of the NF-{kappa}B pathway, we employed EMSA on nuclear extracts obtained from cells stimulated with CRP. As shown in Fig. 5, NF-{kappa}B was activated after incubation with CRP. In particular, CRP was able to induce a distinct shifted band in both cell populations, while no shift could be observed in unstimulated, control cells. Interestingly, these effects of CRP on NF-{kappa}B activation were dose-related, and a more interestingly, ECs and SMCs showed a different sensitivity in terms of activation of this transcription factor, with ECs being responsive to lower CRP concentrations than SMCs. In addition, CRP-induced TF expression in SMCs was significantly reduced when cells where pre-incubated with pyrrolidine dithio carbamate ammonium (PDTC), an inhibitor of NF-{kappa}B activation (Fig. 5).


Figure 5
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  		Fig. 5 Panel (A) Effects of CRP on nuclear translocation of NF-kB. Nuclear extracts were prepared from untreated ECs and SMCs or cells treated with CRP (100 µg/ml, 60' and 120') or LPS as a positive control. The NF-kB activation was determined by electrophoretic mobility shift assay. Data are representative of 3 separate experiments. Panel (B) Dose–response effects of CRP on nuclear translocation of NF-kB. Nuclear extracts were prepared from untreated ECs and SMCs or cells treated with increasing concentrations of CRP (5, 10, 20, 50 100 µg/ml, 30') or LPS as a positive control. Data are representative of 3 separate experiments. Panel (C) CRP-induced TF expression in SMCs was significantly reduced when cells where preincubated with pyrrolidine dithio carbamate ammonium (PDTC), an inhibitor of NF-kB activation.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
Over the past few years it has become clear that inflammation plays an important pathophysiological role not only in the development of atherosclerosis, but also of its complications, such as the occurrence of acute coronary syndromes [9,10]. In this respect, increasing importance has been assigned to selected markers of systemic inflammation, such as CRP, as predictors of major cardiovascular events [11–15]; however, our understanding about the pathophysiological links between increased CRP serum levels and the occurrence of acute coronary syndromes still remains largely incomplete. In particular, at present it is not completely clear whether CRP is a simple marker of systemic inflammation, or may itself represent an effector molecule able to mediate important biological effects on several cell types. To date, previous studies have already underlined that CRP can exert direct biological effects on cells involved in the atherosclerotic milieu. It has been demonstrated that CRP acts on endothelial cells inducing expression of monocyte chemoattractant protein-1 [26], adhesion molecules [27], and PAI-1 [28], while it downregulates NO production [29]. In addition, CRP has been demonstrated to induce expression of TF in monocytes [30], to upregulate angiotensin type-1 receptors in SMC [31], to promote SMC apoptosis [32], and to induce several inflammatory genes via NF-{kappa}B activation [33].

In the present study we have demonstrated that CRP induces TF expression in two cell populations widely represented in the arterial wall, such as endothelial and smooth muscle cells. In particular, CRP exerts its effects on TF expression in a dose-dependent fashion. This phenomenon appears to be mainly related to the synthesis of new TF molecules, since cycloheximide and DRB, an inhibitor of protein synthesis and mRNA transcription, respectively, completely inhibited the CRP effects on TF expression. On the other hand, experiments performed with the calcium ionophore, A23187 [GenBank] , which is known to decrypt TF from its cellular pool [34], showed that A23187 [GenBank] only modestly increased TF expression on cell surface after stimulation with CRP, providing further evidence for the concept that CRP-induced TF expression requires de novo protein synthesis.

Of particular pathophysiological interest was the finding that these newly formed TF molecules were functionally active, as demonstrated by the parallel increase in TF-procoagulant activity, which was detectable on the surface of stimulated cells. Interestingly, ECs and SMCs respond to CRP, in terms of TF expression, with a different sensitivity, ECs being significantly more responsive than SMCs to lower concentrations of CRP. This observation might have important pathophysiological consequences, considering that ECs are at the interface between the vessel wall and circulating blood, thus being exposed to CRP concentrations in the 1–10 µg/ml order of magnitude. Thus, these CRP concentrations are able to induce a "procoagulant" phenotype in ECs. CRP, on the other hand, tends to accumulate within the atherosclerotic plaque, thus making SMCs easily exposed to much higher concentrations of CRP, as previously suggested [35].

Although in the present study, the effects of CRP on ECs and SMCs in terms of TF expression seem to occur directly, other studies have suggested that TF expression by human monocytes may require cell–cell interactions, particularly with other leukocytes [36]. This finding has suggested the possibility that CRP is an indirect inducer of TF expression in monocytes, probably via IFN-{gamma} secretion by activated T-lymphocytes [36]. Our data however, demonstrate that CRP-induced TF expression in ECs and SMCs does not require the interaction with other cell types.

Another finding of the present study is that, in both cell types, CRP-induced TF expression was mediated via activation of the transcription factor, NF-{kappa}B; indeed, PDTC, a selective NF-{kappa}B inhibitor, significantly reduced CRP-induced TF expression. Interestingly, low concentrations of CRP were able to activate NF-{kappa}B in ECs, while in SMCs this phenomenon occurred only at higher CRP concentrations. This different pattern of NF-{kappa}B activation in the two cell population might explain why they respond to CRP, in terms of TF expression, with a different sensitivity. Although recent evidence have suggested that CRP activates the NF-{kappa}B pathway in SMCs [33] and ECs [37], the present study is the first one in demonstrating that CRP-induced TF expression is caused by activation of the NF-{kappa}B pathway.

Furthermore, another interesting finding of the present study is that CRP stimulates proliferation of ECs and SMCs in a dose-dependent manner, which, in turn, seems to be mediated via a dose-dependent activation of the p44/42 MAPK (ERK 1/2) pathway. Again, previous different studies have already separately demonstrated that CRP stimulates SMC migration and proliferation [31] and that it activates MAPKs [33]; our study, however, for the first time demonstrates that CRP-induced cell proliferation is indeed mediated via p44/p42 MAPK activation.

The data of our study are in line with the recent observation that transgenic mice expressing high levels of human CRP showed much faster and higher rates of complete thrombotic events using a transluminal wire injury model than their wild-type counterparts [38]. In fact, it is tempting to speculate that high serum CRP levels might stimulate TF expression in the arterial wall, thus shifting toward a prothrombotic phenotype. In addition, considering also that NF-{kappa}B has been demonstrated to be activated in the peripheral monocytes [39], as well as within the unstable plaques of patients with acute coronary syndromes [40], the finding observed in our study that CRP induces TF expression via activation of NF-kB might explain, at least in part, why patients with acute coronary syndromes and high CRP serum levels have a worse clinical outcome than patients with normal CRP serum levels. Furthermore, since it is well established that migration and proliferation of SMCs in the vessel wall play an important role in the development of atherosclerosis [41], data of the present study might also suggest that CRP can actively contribute to this phenomenon by inducing EC and SMC proliferation. On the other hand, it should be emphasized that other studies have demonstrated that certain inflammatory cytokines, might result in EC apoptosis, suggesting a general role for inflammation in promoting plaque instability [42]. Although in that study the role of CRP in promoting EC apoptosis was not tested, it might be hypothesized that CRP may have a role in this phenomenon. Very recently indeed, Blaschke et al. have shown that CRP may induce apoptosis of SMCs in culture via activation of GADD153 gene [32]. We do not know the reasons for the discrepancy between our study and that of Blaschke et al., although differences in culture conditions (proliferating vs quiescent cells), or duration of the stimulation with CRP (72 vs 24 h) might well account for it. It is important to stress, however, that because cell proliferation and apoptosis might coexist in the same atherosclerotic lesion [43], the overall effect of CRP on cell proliferation or apoptosis within atherosclerotic lesions, ultimately leading to plaque stabilization or instabilization, might result from the ultimate balance between these phenomena.

4.1 Potential limitations of the present study
The present study, although in vitro, describes a potential new link between inflammation (as reflected by high CRP plasma levels), thrombosis, and cell proliferation. The evidence that CRP exerts direct effects on ECs and SMCs, leading to proliferation and TF expression on their surface, might explain, at least in part, why patients with high CRP plasma levels have a worse clinical outcome than patients with normal CRP levels, via a broad activation of the coronary endothelial cells, ultimately increasing the risk to develop new coronary thrombotic events. These findings clearly indicate a direct effect of CRP in promoting atherothrombosis, but the clinical relevance of this pathophysiological mechanism might appear questionable, considering that some of the experiments performed in the present study employed very high CRP concentrations. This discrepancy, however, is only apparent as it should be emphasized that the transcriptional factor, NF-{kappa}B, was activated and TF expression was significantly induced in ECs at CRP concentrations as low as 5 µg/ml, which are well within the range typically observed in patients with an increased cardiovascular risk. Furthermore, it should be also kept in mind that plasma CRP concentrations might only loosely reflect real tissue CRP levels and that locally, i.e., within the arterial wall, CRP might be present in amounts sufficiently high to exert relevant cellular effects [35].


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
In conclusion, the present study underlines the close relationship between inflammation, CRP levels, and atherothrombosis, providing further evidence for CRP not only as a marker of inflammation, but also as a possible direct mediator of this disease. Further studies are warranted to clarify whether these mechanisms are also important in the clinical setting.


   Notes
 
Time for primary review 24 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

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