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Clinical Chemistry 54: 814-823, 2008. First published March 13, 2008; 10.1373/clinchem.2007.095547
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Right arrow Lipids, Lipoproteins, and Cardiovascular Risk Factors
(Clinical Chemistry. 2008;54:814-823.)
© 2008 American Association for Clinical Chemistry, Inc.

Lipids, Lipoproteins, and Cardiovascular Risk Factors

Tissue Factor and Monocyte Chemoattractant Protein-1 Expression in Hypertensive Individuals with Normal or Increased Carotid Intima-Media Wall Thickness

Maria A. Sardo1, Salvatore Campo2, Giuseppe Mandraffino1, Carlo Saitta1, Antonio Bonaiuto1, Maria Castaldo1, Maurizio Cinquegrani1, Giovanni Pizzimenti3 and Antonino Saitta1,a

1 Department of Internal Medicine, 2 Department of Biochemical, Physiological and Nutritional Sciences, 3 Department of Physics, University of Messina, Messina, Italy.

aAddress correspondence to this author at: Department of Internal Medicine, Via Camiciotti, 82-98123-Messina, Italy. Fax 39-090-2213900; e-mail asaitta{at}unime.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: People with hypertension display an inflammatory pattern that includes increased plasma concentrations of monocyte chemoattractant protein 1 (MCP-1) and C-reactive protein (CRP) and enhanced expression of tissue factor (TF) mRNA in blood monocytes.

Methods: In this study, we investigated the relationship between CRP concentrations and TF and MCP-1 mRNA expression in unstimulated and lipopolysaccharide (LPS)-stimulated monocytes isolated from hypertensives with or without an increase in carotid intima-media thickness (IMT). We also investigated the expression of TF and MCP-1 mRNA and MCP-1 protein after in vitro addition of CRP to monocytes. We measured CRP (by immunonephelometry) and monocyte expression of TF and MCP-1 (by real-time PCR) in 80 untreated hypertensive patients without clinical cardiovascular disease (CVD) or additional risk factors for CVD compared with 41 controls. Based on IMT measured by carotid Doppler ultrasonography, patients were classified into the categories of normal (≤1 mm) or abnormal (>1 mm). TF and MCP-1 mRNA and MCP-1 protein (by Western blotting) were measured after in vitro addition of CRP to monocytes from 10 randomized controls as well as 10 hypertensives with IMT ≤1 mm and 10 with IMT >1 mm.

Results: CRP and TF and MCP-1 mRNA concentrations were significantly higher in IMT >1 mm hypertensives vs those with IMT ≤1 mm and controls. CRP had no effect on monocyte TF mRNA from either hypertensives or controls. CRP-stimulated monocytes from hypertensives, however, showed increased MCP-1 mRNA and protein expression compared with controls and LPS-stimulated cells.

Conclusions: Our findings suggest that the inflammatory response of blood monocytes plays an important role in the development of atherosclerosis and hypertension.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic vessel wall inflammation is a critical event in initiating and developing cardiovascular disease (CVD).1 Several investigators have shown a significant association between inflammation and risk factors for atherosclerosis. Healthy participants with arterial hypertension, a major risk factor for CVD, display signs of vascular and systemic inflammation, including elevation of serum monocyte chemoattractant protein 1 (MCP-1)(1) and C-reactive protein (CRP)(2) concentrations as well as an activation of peripheral blood monocyte cells(3)(4). These findings suggest that inflammation may play a role in hypertension and related atherothrombotic complications.

MCP-1 is a secreted C-C chemokine, having a strong chemotactic activity on inflammatory cells(5) that mediate the recruitment of blood monocytes into the vessel wall, one of the earliest events in atherosclerosis. Enhanced expression of MCP-1 also leads to the progression of vascular damage in animal models(6). In contrast, gene transfer of a dominant-negative MCP-1 mutant attenuated atherosclerotic lesion development in apolipoprotein E–deficient mice(7). In humans, MCP-1 has been detected in atheromatous plaques(8), and MCP-1 plasma concentrations have been found to be highly associated with clinical outcomes in individuals with coronary syndromes(9). Furthermore, high concentrations of circulating MCP-1 have been found in patients with postangioplasty restenosis(10) and with risk factors for CVD(11), suggesting that MCP-1 plays a critical role at multiple stages in atherosclerosis.

The systemic response to inflammation is associated with a significant increase of CRP, a highly conserved pentameric plasma protein synthesized in the liver that contributes to host defense. Slight increase of plasma CRP is currently thought to be associated with the risk of developing CVD including arterial hypertension(2). Additionally, CRP has been postulated to have a causal role in atherosclerosis(12)(13)(14) through the induction of MCP-1(15) and tissue factor (TF) expression(16). In contrast, no direct activity of CRP has been found in other studies(17)(18); therefore, whether the functional effects of CRP are rather the result of further factors that may lead to the progression of the atherosclerotic disease remains to be clarified.

Marked expression of TF, a transmembrane glycoprotein that is able to initiate coagulation in vivo and induce thrombus formation in the plaque, has been also demonstrated in patients with atherosclerosis(19). TF production has been reported to be regulated by numerous stimuli, including MCP-1(20), CRP(16), and hemodynamic forces(21). Recently, we showed that TF mRNA expression was increased in monocyte cells isolated from hypertensives with carotid atherosclerosis(22). In the present study, we evaluated plasma CRP concentration and monocyte expression of MCP-1 mRNA in hypertensives with and without increased intima-media thickness (IMT). We also studied the in vitro effects of adding CRP to activated monocytes from hypertensive individuals on the cellular expression of TF- and MCP-1 mRNA and MCP-1 protein.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
participants
We examined 80 patients with essential hypertension [41 men and 39 women, mean age 41 (SD 12) years] and 41 normotensive controls matched for sex and age. Hypertensives were newly diagnosed outpatients, and controls were recruited from hospital personnel. All participants were enrolled in our previous work, which investigated TF mRNA expression and activity in monocytes(22). TF values were considered together with the present data. In this study, we examined fresh samples collected at the time of diagnosis and stored samples of cDNA for each individual.

The characteristics of the study population have been previously described(22). Briefly, only individuals with systolic blood pressure (SBP) ≥140 mmHg and/or diastolic blood pressure (DBP) ≥90 mmHg were enrolled. Individuals were excluded if they had secondary hypertension or body mass index (BMI) ≥30; if they smoked or consumed alcohol; or if they had abnormal electrocardiographic and echocardiographic (left ventricular ejection function, left ventricular regional function) patterns, a clinical history of CVD or diabetes, serum cholesterol concentrations >6 mmol/L and triglyceride concentrations >2.5 mmol/L, or thyroid, liver, or kidney disease. Informed consent was obtained from all participants, and the study was approved by the Ethics Committee of the University of Messina.

At the time of sample collection, none of the participants were receiving medications(22). Antihypertensive therapy was not initiated in the hypertensives until after sample collection. All participants underwent carotid Doppler ultrasonography and clinical chemistry testing as well as having blood monocytes isolated. We measured fasting plasma lipid and glucose concentrations were measured by routine methods. We measured HDL cholesterol after precipitation of the apolipoprotein B–containing lipoproteins with magnesium phosphotungstate and calculated LDL cholesterol by the Friedewald formula. We measured high-sensitivity CRP (hsCRP) plasma concentration using a sensitive immunonephelometry assay.

ultrasound scanning procedure
We used high-resolution B-mode ultrasound images (Vivid 3 Expert; General Electric Company) with a 10.0-MHz linear array transducer to measure IMT.

The carotid arteries were examined bilaterally in the areas of the common carotid (1 cm proximal to the carotid bulb), the carotid bifurcation (1 cm proximal to the flow divider), and the internal carotid artery (1 cm distal to the flow divider). All measurements were determined manually in longitudinal and transverse planes with anterior, lateral, and posterior approaches. From B-mode images, we selected single video frames for IMT measurements.

The IMT was defined as the distance between the lumen/intima and the media/adventitia interfaces. For each parameter, the mean value was calculated. The mean IMT was used as the key variable for statistical analysis, because of its strong association with cardiovascular disease(23). Two independent readers, who were blinded from participants’ clinical and laboratory results, made the measurements. Participants were classified as having a normal (≤1 mm) or an abnormal (>1 mm) IMT, including atheromatous plaques. We evaluated interobserver variability on the measurements obtained from all participants of the study. The interobserver variability of IMT measurements, as evaluated by comparing values obtained by 2 sets of scans performed by each reader, was 0.03 mm (CV 2.15%). The intraobserver variability was 0.02 mm (CV 3.59%).

monocyte isolation
We isolated peripheral blood mononuclear cells from heparinized venous blood after overnight fasting. Blood samples were centrifugated immediately at 500g for 10 min at 4 °C to obtain platelet-rich plasma. After removal of the plasma, the platelet-free pellet was suspended in RPMI 1640 (1:3, vol:vol) (ICN Biomedicals) supplemented with 2 mmol/L glutamine, 0.5% streptomycin-penicillin-Fungizone, and 10% fetal calf serum and centrifuged over Ficoll-Paque (Pharmacia, Fine Chemicals) gradient at 1000g for 20 min.

Mononuclear cells were collected from the interface of the Ficoll/medium, suspended in RPMI 1640, and incubated in 35-mm plastic dishes for 90 min at 37 °C in 5% CO2 humid atmosphere. The monocytes were collected by their adherence to dishes, whereas the lymphocytes (nonadherent cells) were removed by aspiration with Pasteur pipette and washing of the dishes with RPMI 1640. Cell preparations were >90% monocytes, as determined by nonspecific esterase staining. After isolation, cells were incubated at 1 x 106 cells/mL in RPMI 1640 at 37 °C, 5% CO2 for 6 h.

Cultures were incubated in presence or absence of bacterial lipopolysaccharide (LPS) (Escherichia coli OB11:B4; Sigma) at a final concentration of 0.1 µg/mL (in vitro addition of LPS was used as a standard proinflammatory stimulus)(4). At the end of the incubation period, the cells (5 x 105 cells/mL) were washed, and mRNA and protein were extracted.

induction of mcp-1 and tf expression by human crp
An aliquot of monocytes (5 x 105 cells/mL), isolated from 30 randomized individuals (10 controls, 10 IMT ≤1 mm and 10 IMT >1 mm hypertensives), was incubated for 12 h in RPMI 1640 at 37 °C, 5% CO2, in plastic dishes and cultured in the presence or absence of highly purified human CRP (100 µg/mL) or LPS (0.1 µg/mL) (both from Sigma Aldrich). CRP was purified of LPS by passing through a detoxigel column (Pierce Biotechnology Inc.) and eluted with Tris/NaCl/CaCl2 buffer. To remove azide contamination, eluted CRP was dialyzed against Tris/NaCl/CaCl2 buffer at 4 °C using a dialysis slide (Pierce Biotechnology Inc.) with a cutoff of 10 kDa(24). After extraction of total RNA and proteins, we measured the TF and MCP-1 mRNA and MCP-1 protein.

mcp-1 and tf expression by real-time pcr
Total RNA was extracted from 5 x 105 cells using a commercial kit (Trizol Reagent; Invitrogen) according to the manufacturer’s instruction and spectrophotometrically quantified (Biomate 3; Thermo Electron Corp.). Total RNA (1 µg) was reverse transcribed using a high-capacity cDNA Archive Kit (Applied Biosystems) and random primers according to manufacturer’s instructions. In addition, we used 0.25 µg total cDNA to quantify the amount of MCP-1 and TF cDNA by real-time PCR. We used β-actin cDNA as endogenous control for final normalization. We carried out the reaction for each cDNA and endogenous controls in the same tube (biplex) by Taq Man Universal PCR master mix and Assays on Demand ready-to-use primers and probes with different reporter dyes (Applied Biosystems).

We monitroed the progression of PCR using 7500 real-time PCR System (Applied Biosystems) and determined the relative quantification by a standard curve method for both the target and endogenous reference. To obtain the standard curves, we performed a series of dilutions of cDNA of MCP-1, TF, and β-actin clones. Each clone was obtained by a Gene Race method (Invitrogen) according to the manufacturer’s instructions. After normalization, the median of MCP-1 and TF target levels in unstimulated monocytes from control participants was considered as the calibrator (1x sample), and the results were expressed as an n-fold difference relative to normal controls (relative expression levels)(25). We confirmed the nucleotide sequence of some PCR products by direct sequencing using Big Dye cycle sequencing kit v. 1.1 and model 310 ABI Prism sequencer analyzer (Applied Biosystems) with a 5' internal primer.

mcp-1 protein expression by western blotting
We measured the MCP-1 protein expression of the 30 randomized individuals (10 controls and 10 uncomplicated and 10 complicated hypertensive participants) by Western blotting. Approximately 105 cells for each sample were lysed by a cell extraction buffer (Biosource) in the presence of 1 nmol/L phenylmethylsulfonyl fluoride and protease inhibitors cocktail. After centrifugation at 9800g for 10 min at 4 °C, we measured the protein content of the supernatant using a DC protein assay kit (Bio-Rad). A volume of supernatant containing 40 µg of protein was diluted with the same volume of Laemmli sample buffer (Bio-Rad). Each sample was treated with chondroitinase ABC, 1 unit dissolved in 20 mL Tris-HCl, pH 8. Digestion was carried out at 37 °C for 2 h, and the enzymes were inactivated by heating in a boiling water bath for 10 min. After denaturation at 95 °C for 10 min, the samples were applied to a SDS 12% polyacrylamide gel, together with 100 ng MCP-1 recombinant protein (RMCP120; Pierce Endogen), and electrophoresed at the constant voltage of 160V. After separation, proteins were electroblotted on nitrocellulose polyvinylidene difluoride (Amersham) overnight at 30V. The membrane was treated at room temperature for 1 h with 10 mmol/L Tris, 50 mmol/L NaCl buffer, pH 7.4 (Tris-buffered saline [TBS]), containing 7% nonfat dry milk and 0.1% Tween-20. The membrane was incubated for 2 h at room temperature with the same TBS/Tween 20 buffer, containing 5% nonfat dry milk and antihuman MCP-1 antibody (mouse [S14], ab18677; Abcam plc). After washing with TBS/Tween-20 buffer to eliminate excess antibody, the membrane was treated for 1 h at room temperature with antirabbit secondary polyclonal goat antibody conjugated with peroxidase in TBS/Tween-20 buffer containing 5% nonfat dry milk. After washing, the membrane was treated with ECL (Amersham Biosciences) for 5 min, and chemiluminescence was measured by autoradiography on Fuji Medical x-ray film. Results were expressed as semiquantitative relative amounts (using β-actin as endogenous control) detected on membrane by specific antibody and revealed by chemiluminescence. After normalization, we considered the median of MCP-1 target concentrations in unstimulated monocytes from control participants as the calibrator (1x sample) and expressed the results as n-fold differences relative to normal controls (relative expression levels).

statistical methods
Data are expressed as mean (SD). Examined variables were not normally distributed as verified by Kolmogorov-Smirnov tests (SPSS statistical package); consequently, we used the nonparametric approach. Because sample numbers were low and data distributions were unknown, we chose a permutation tests-based analysis(26). This statistical approach enables a more effective statistical design(27). In particular, we compared controls and hypertensives using the nonparametric multivariate ANOVA, nonparametric combination (NPC) test. Once the significance of the test was verified, we performed pairwise comparisons between the mean data from hypertensives with IMT ≤1 mm and IMT >1 mm and controls(27). The Bonferroni correction method was performed to control for the effects of multiple testing. In line with the chosen statistical approach, we compared unstimulated and stimulated monocytes using NPC tests for repeated measures. For the MANOVA NPC procedure, pairwise comparisons, and the NPC test for repeated measures, we used the NPC test statistical package by Methodologica S.R.L. (Italy). The correlation among the examined variables was tested by Spearman test. We used a step-by-step regression based on permutation testing (MATLAB statistical package)(28) to assess the contribution of each study variable (sex, age, BMI, IMT, SBP, DBP, total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, hsCRP, fibrinogen, glucose) in determining variation on response variables (TF mRNA and MCP-1 mRNA). The same regression model was also performed to verify the influence of each variable on IMT and hsCRP. In the described models, we evaluated interactions among the explanatory variables in assessing the effect of the independent variables on the dependent variable. To avoid the effects of colinearity, variables that were highly correlated were not entered into the regression models simultaneously. Values of P < 0.05 were considered statistically significant, and P values were calculated by 2-sided test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
characteristic of the study participants
The baseline characteristics of the study groups are shown in Table 1 . Hypertensive participants with IMT >1 mm showed significantly higher SBP and fibrinogen values than hypertensives with IMT ≤1 mm. No differences in DBP were found in the 2 hypertensive groups. Age, sex, BMI, DBP, and lipid and glucose concentrations were comparable in all participants.


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  		Table 1. Characteristics of the study participants.

Plasma hsCRP was significantly higher in hypertensives [1.72 (1.04) mg/L] compared with controls. Comparing the 2 hypertensive subgroups, the IMT >1 mm hypertensive participants showed a concentration of hsCRP significantly higher than that of IMT ≤1 mm hypertensives.

evaluation of tf and mcp-1 Mrna expression
In IMT ≤1 mm hypertensives, basal and LPS-stimulated expression of TF and MCP-1 mRNA (Table 1Up ) was not found to be significantly different from control participants. In contrast, in hypertensives with IMT >1 mm, basal and LPS-stimulated expression of TF and MCP-1 mRNA (Table 1Up ) were significantly higher than in IMT ≤1 mm hypertensive participants and controls.

effect of lps on monocyte tf Mrna expression and mcp-1Mrna and protein expression
TF and MCP-1 mRNA values in monocytes isolated from 10 controls and 10 IMT ≤1 mm and 10 IMT >1 mm hypertensive participants at baseline and after 4 and 8 h of incubation with LPS are shown in Figure 1 .


Figure 1
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  		Figure 1. Time course of TF (A) and MCP-1 (B) mRNA expression after 4 and 8 h of LPS stimulation.

{blacksquare}, Control participants (10); {blacktriangleup}, hypertensive participants with IMT ≤1 mm (10); •, hypertensive participants with IMT >1 mm (10). Values are expressed as n-fold variation vs controls’ basal mean (SD). *P < 0.001 vs both controls and IMT ≤1 mm hypertensives. No significant difference between IMT ≤1 mm hypertensives and controls. All stimulated TF and MCP-1 mRNA values are significant vs baseline levels (P < 0.001). Numbers in brackets indicate participants of each group.

Addition of LPS to monocytes isolated from controls and hypertensives resulted in a higher expression of TF mRNA, which peaked at 4–8 h, compared with baseline values (Fig. 1AUp ). Similarly, monocytes from control and hypertensive participants showed a significant increase in MCP-1 mRNA expression after LPS stimulation (Fig. 1BUp ). The relative increases seen in IMT >1 mm hypertensives were significantly higher than those seen in the controls and IMT ≤1 mm hypertensive participants.

effect of crp on monocyte tf Mrna expression and mcp-1 Mrna and protein expression
TF and MCP-1 mRNA values in monocytes isolated from 10 controls and 10 IMT ≤1 mm and 10 IMT >1 mm hypertensives at baseline and after 4 and 8 h of incubation with CRP are shown in Fig. 2 .


Figure 2
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  		Figure 2. Time course of TF (A) and MCP-1 (B) mRNA expression after 4 and 8 h of CRP stimulation.

{blacksquare}, Control participants (10); {blacktriangleup}, hypertensive participants with IMT ≤1 mm (10); •, hypertensive participants with IMT >1 mm (10). Values are expressed as n-fold variation vs controls’ basal mean (SD). *P < 0.001 vs both controls and IMT ≤1 mm hypertensives; **P < 0.001 vs controls; {infty}P < 0.001 vs baseline levels. Numbers in brackets indicate participants of each group. NS, not significant.

Addition of CRP to monocytes isolated from controls and hypertensives did not increase TF mRNA (Fig. 3A ). Similarly, in controls, CRP did not induce a significant increase of MCP-1 mRNA, whereas in IMT ≤ and >1 mm hypertensives, it induced an increase in MCP-1 mRNA (Fig. 3B ).


Figure 3
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  		Figure 3. Time course of MCP-1 protein expression in LPS or CRP stimulated circulating monocytic cells.

Straight lines, CRP stimulation; dotted lines, LPS stimulation (4 and 8 h); {blacksquare}, control participants (10); {blacktriangleup}, hypertensive participants with IMT ≤1 mm (10); •, hypertensive participants with IMT >1 mm (10). Values are expressed as n-fold variation vs controls’ basal mean (SD). *P < 0.001 vs both controls and IMT ≤1 mm hypertensives (MCP-1); **P < 0.001 vs controls (MCP-1); {infty}P < 0.001 vs both controls and IMT ≤1 mm hypertensives (TF). Numbers in brackets indicate participants of each group.

The expression of monocyte MCP-1 protein in the study groups is shown in Fig. 3Up . Monocyte MCP-1 protein did not increase after CRP stimulation in controls, but only after LPS stimulation. In IMT ≤ and >1 mm hypertensives, the incubation of monocytes with CRP significantly increased MCP-1 protein compared with the values measured after LPS stimulation. Furthermore, in IMT >1 mm hypertensives, the increase was higher than that seen in hypertensives with IMT ≤1 mm.

interdependence and dependence analysis
On examining the pairwise interdependence among the considered variables, we observed a strong correlation between IMT and both MCP-1 (rs = 0.265, P = 0.014) and TF (rs = 0.836, P = 0.000) mRNA basal expression. Moreover, the data revealed a strong correlation between hsCRP and SBP (rs = 0.448, P = 0.001) in the combined hypertensive group. When the groups of hypertensives with IMT ≤1 mm and IMT >1 mm were separated, we found a significant correlation (rs = 0.195, P = 0.037) only for the hypertensives with IMT >1 mm.

When we evaluated the contribution of the study variables (age, sex, BMI, SBP, DBP, total cholesterol, HDL cholesterol, triglycerides, LDL cholesterol, glucose, fibrinogen, hsCRP, MCP-1, TF, IMT) in predicting TF- and MCP-1 mRNA concentrations, we observed a significant dependence of TF mRNA on IMT (P = 0.000) and of MCP-1 on IMT (P = 0.003). Moreover, when estimating a dependence model for IMT, there was a significant dependence of IMT on TF mRNA (P = 0.000). These findings confirm the hypothesis that CRP, TF, and MCP-1 are highly influenced by carotid atherosclerosis (Table 2 ).


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  		Table 2. Nonparametric regression based on permutation test.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we observed that plasma CRP and TF and MCP-1 expression were not increased in monocytes isolated from hypertensive participants who had no other risk factor for CVD and IMT <1 mm. By contrast, hypertensives with IMT >1 mm had significantly higher CRP concentrations and monocyte TF and MCP-1 expression.

Previous studies reported a significant relationship between increased blood pressure, IMT, and plasma CRP values(29). A positive association of CRP with blood pressure and IMT was also found in our study population, but adjustment for potential confounding variables by regression analysis strongly attenuated the CRP association with carotid atherosclerosis. This suggests that different inflammatory pathways may be involved in the development of carotid lesions in hypertension(30).

In a previous study, hypertensive individuals with IMT >1 mm were found to have increased TF mRNA compared with controls, whereas no significant differences were observed between hypertensive individuals with IMT ≤1 mm and controls, suggesting a modulation of TF mRNA in monocytes(22). The increased MCP-1 expression in hypertensive participants with IMT >1 mm observed in the present study strengthens this suggestion. An enhanced MCP-1 expression has been previously observed in human monocytes after stimulation with leukotriene B4, a molecule implicated in arterial tone and pressure regulation(31). A critical role of CCR2 (chemokine [C-C motif] receptor-2), a MCP-1 receptor on monocytes, has been also seen in hypertensives(32). Overexpression of MCP-1 was recently found in circulating endothelial cells from hypertensives carrying a polymorphic mutation in MCP-1 gene, now officially named CCL2, chemokine (C-C motif) ligand 2(1). Thus, blood pressure is not the primary determinant of TF and MCP-1 expression, but rather other factors, including carotid atherosclerosis, may significantly influence the production of such molecules from monocytes.

We also investigated if CRP may, in vitro, induce TF- and MCP-1 expression in monocytes isolated from hypertensives. It has been assumed that CRP may modulate TF expression in cultured blood cells(15)(16) and upregulate TF in endothelial and smooth muscle cells(33). A remarkable influence of CRP on TF expression was found in monocytes from patients with inflammatory rheumatic diseases(34). Furthermore, CRP has been reported to promote monocyte chemotactic activity in response to MCP-1 via upregulation of monocyte CCR2(35). By contrast, CRP had no effects in stimulating TF mRNA in highly purified monocytes from healthy patients(36) or angina patients(37), indicating that TF stimulation by CRP may be favored by the presence of other circulating cells and inflammatory mediators.

By analyzing circulating monocytes, an enhanced CRP-mediated expression of MCP-1 in hypertensives was found in this study, but there was no significant effect of CRP in inducing TF. A recent study demonstrated that CRP may activate nuclear factor-{kappa}B in human monocytes(38) and may be hypothesized as a new mechanism for CRP in regulating the generation of inflammatory mediators. However, CRP did not influence TF expression in the present study, suggesting that the proinflammatory phenotype of monocytes could not be directly modulated by CRP. Indeed, multiple mediators(39) and genetic factors(40) may be involved in triggering vascular injury and monocyte activation in hypertension(3)(4). Otherwise, carotid atherosclerosis might itself sustain monocyte inflammatory response, amplifying vascular damage and its related complications.

Although LPS and azide have been reported to contaminate commercial CRP preparations(17)(18), the human CRP used in the present experiments has been shown to have no effect on saphenous vein endothelial cells(18). In the control population of the present study, monocyte MCP-1 was not influenced by CRP stimulation. Conversely, the hypertensives in the present study demonstrated increased monocyte MCP-1 expression following CRP stimulation, likely because the monocytes in their blood were already activated.

In conclusion, the data in this study provide evidence that MCP-1 expression is enhanced in monocytes of hypertensives with increased IMT, suggesting that in essential hypertension, the activation of circulating monocytes is associated with the extent of atherosclerosis and inflammation. Further studies are needed to better understand the mechanisms that regulate overexpression of functional molecules, including MCP-1, in hypertensive patients. We postulate that the inhibition of such factors might be useful for the treatment of vascular lesion associated with hypertension.


   Acknowledgments
 
Grant/Funding Support: This work was supported by a grant of the University of Messina (Fondo d’Ateneo PRA 2004).

Financial Disclosures: None declared.

Acknowledgments: The authors thank to the hospital personnel used as control participants.


   Footnotes
 
1 Nonstandard abbreviations: CVD, cardiovascular disease; MCP-1, monocyte chemoattractant protein 1; CRP, C-reactive protein; TF, tissue factor; IMT, intima-media thickness; SBP, systolic blood pressure; DBP, diastolic blood pressure; BMI, body mass index; hsCRP, high-sensitivity CRP; LPS, lipopolysaccharide; TBS, Tris-buffered saline; NPC, nonparametric combination.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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