Overwhelming evidence indicates that chronic inflammation plays a principal role in the pathophysiology of atherosclerosis.1-5 Immunohistochemical staining studies clearly identified inflammatory cells and C-reactive protein (CRP) in ruptured plaques of patients who have experienced acute coronary syndromes.6-8 Increased serum levels of CRP, an index of inflammation, have been found to be associated with increased risk of cardiovascular events.5,9-11 Additionally, CRP has recently emerged as one of the most important inflammatory mediators that can directly participate in pathogenesis of atherosclerosis by activating endothelial cells (ECs) and promoting the inflammatory component of atherosclerosis.12-14 Furthermore, CRP has been implicated in inducing adhesion molecules and chemokine expression in human ECs.14,15 Moreover CRP has been shown to facilitate EC apoptosis, inhibit angiogenesis, upregulate nuclear factor-κB signaling in ECs, downregulate endothelial NO synthase (eNOS) transcription in ECs, and destabilize eNOS mRNA, resulting in decreases in both basal and stimulated NO release.13,16 CRP therefore appears to function as an important circulating marker of endothelial dysfunction.
Growing evidence indicates that statin therapy strongly decreases CRP levels in patients with overt or without cardiovascular disease.14,17-19 Additionally, the antiinflammatory effects of statins have been recently confirmed by in vitro studies.14 Thus, statin therapy is believed to provide an antiatherosclerotic effect beyond lowering lipid levels.14,17-20
Losartan was the first orally administered selective antagonist of angiotensin II type 1 (AT1) receptor approved for treating hypertension.21 Additionally, studies have demonstrated that losartan is superior to atenolol for reducing stroke morbidity and mortality22 and reversing LV hypertrophy,23 a typical manifestation of target organ damage in hypertensive patients. Accumulating data demonstrate that losartan can reduce TXA2-dependent platelet activation and plasma levels of PAI-1 and improve endothelial function.24,25 One recent study demonstrated that losartan has antiinflammatory effects independent of AT1 receptor.25 However, the antiinflammatory properties of losartan itself remain currently uncertain. Therefore, we hypothesize that losartan, in addition to having an antihypertensive effect, has antiinflammatory properties that are compatible with those of simvastatin and is acting independently of AT1 receptor. Because no evidence supports that human umbilical vein endothelial cells (HUVECs) can express AT1 receptors, we tested our hypothesis by primary cell culture with HUVECs.
MATERIALS AND METHODS
Primary Cell Culture
HUVECs were harvested from umbilical veins by collagenase (Sigma) digestion. Cells were pooled from 3 to 6 prepared umbilical veins and were grown at 37°C in a 10 mm culture dish (Flacon) in M 199 (Gibco) medium with EC growth supplement, heparin, and 20% fetal bovine serum (Gibco). Third to fifth generation cells were used. Cell viability was assessed by Trypan blue exclusion. The HUVECs were then scraped with a cell scraper (Nunc) into a 12-well plate.
Recombinant human CRP and highly purified CRP from humans were purchased from Calbiochem; monoclonal antibody to vascular cell adhesion molecule-1 (VCAM-1) (anti-CD54) was obtained from BD (Bioscience). Fetal bovine serum was purchased from Gibco. Purity of CRP preparations was confirmed by 10% SDS-PAGE; no contaminated proteins were detected by silver staining of overloaded gels (a single band on silver staining of overloaded gels was obtained). Western blotting using HRP-conjugated IgG antibodies did not reveal any contamination of purified CRP with IgG. Preparation of CRP was under sterile conditions using endotoxin-removal columns (Pierce Biochemicals). All cell culture media were endotoxin-free.
Incubation of Endothelial Monolayers With CRP
The HUVECs at passages 3 through 5 (n = 6 per group) were incubated for 24 hours with various concentrations of CRP (from 25 μg/mL with stepwise increases to 50, 75, and 100 μg/mL). For inhibitory experiments, HUVECs were pretreated with various concentrations of simvastatin (Calbiochem) (from 25 μmol/L simvastatin increased stepwise to 50, 75, and 100 μmol/L) and losartan (MSD) (from 100 μmol/L losartan increased stepwise to 300, 500, and 750 μmol/L); or vehicle (0.1% DMSO or PBS) at indicated concentrations. After 4 hours, cells were incubated with CRP (25, 50, 75, and 100 μg/mL) for 24 hours.
Cell variability under condition of different concentrations of losartan plus CRP (after incubation for 28 h) was assessed by trypan blue exclusion. Cell variability was 96.3% (25 μg/mL CRP plus 750 μmol/L losartan), 94.7% (50 μg/mL CRP plus 750 μmol/L of losartan), 93.8% (75 μg/mL CRP plus 750 μmol/L losartan), and 95.7% (100 μg/mL CRP plus 750 μmol/L losartan), respectively.
Determination of Surface Expression of VCAM-1
Surface expression of VCAM-1 of HUVECs was stained using R-phycoerythrin (R-PE)-conjugated mouse anti-human monoclonal antibody (BD Bioscience) (anti-CD54 specific for VCAM-1), all at a dilution of 1:20. Cells were analyzed using a fluorescence-activated cell sorter (FACSCalibur system, Becton Dickinson). The fluorescence intensities of 10,000 cells for each sample were quantified, and unstained cells were used as control.
The percentage of ECs expressing VCAM-1 was defined as the fraction exhibiting specific binding (ie, anti-CD 54 positive) minus that exhibiting nonspecific binding (ie, the percentage defined with the IgG-PE conjugate) from the 10,000 stored ECs.
Enzyme-Linked Immunosorbent Assay
The supernatant concentrations of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) were determined in duplicate using a standard enzyme-linked immunosorbent assay (ELISA) and a commercial kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Intraindividual variability of MCP-1 and IL-6 levels was assessed. In our laboratory, the mean intraassay coefficients of variance for MCP-1 and IL-6 were <5%.
Determination of Surface Expression of AT1 Receptors
Immunohistochemical staining for AT1 receptor expression in human kidney, and immunocytochemical staining for possibility of AT1 receptor expression in HUVECs were performed in this study. Expression of AT1 receptors of these 2 tissues were stained using a commercially available primary mouse monoclonal antibody for AT1 receptor (Abcam, UK) to each section, all at a dilution of 1:20 for heart tissue and 1:100 for HUVECs. The supersensitive polymer-HRP conjugate was used as a detection system (Zymed Laboratories, San Francisco, CA). Hematoxylin (Sigma-Aldrich, St. Louis, MO) was added for a counterstain.
Data are expressed as mean ± SEM of 6 separate experiments for each group. Logarithm transformation was used to improve normality. Analysis of multiple paired data within the same group was utilized 2-way ANOVA. Statistical software SAS for Windows version 8.2 (SAS Institute, Cary, NC) was used for statistical analyses. P < 0.05 was considered significant.
CRP Induces Expression of VCAM-1 That Is Suppressed by Simvastatin
Figure 1 shows the effects of human recombinant CRP on expression of VCAM-1 on HUVEC surfaces. Cell viability by trypan blue exclusion was >95% in all experiments. Unstimulated HUVECs expressed very low levels of VCAM-1 (0.3% to 0.6% fraction of expression) (1st column of Fig. 1A through 1C). However, HUVECs cultured with recombinant CRP (25 μg/mL for 24 hours) had a markedly increased expression of VCAM-1 [from 0.3% at baseline to 34.1% fraction of expression (second column), P < 0.0001] (Fig. 1A) on HUVECs.
Figure 1 presents the dose-response relationship for the effect of CRP on VCAM-1 expression. Effect of CRP on VCAM-1 surface expression increased stepwise as the CRP concentration increased from 25 μg/mL and reached a significantly high surface expression of VCAM-1 as CRP concentration was increased to 75 μg/mL (P < 0.001) (second column of Fig. 1A through 1C).
Figure 1 shows simvastatin modulation of CRP effect on VCAM-1 surface expression. Stepwise suppression increased as the simvastatin dose increased from 25 to 100 μmol/L (Fig. 1A through 1C), and statistical significance was observed when the simvastatin dose was 50 μmol/L (P < 0.05).
CRP Induces HUVEC Secretions of MCP-1 and IL-6 That are Suppressed by Simvastatin
Figures 2 and 3 (A through C) show the effects of human recombinant CRP on induction of MCP-1 and IL-6 in HUVECs, respectively. The HUVECs cultured with recombinant CRP (25 μg/mL for 24 hours) showed remarkably increased supernatant levels of MCP-1 (from 1400 ± 246 pg/mL at baseline to 2481 ± 189 pg/mL, P < 0.001) (Fig. 2A, second column vs. baseline) and IL-6 (from 220 ± 28 pg/mL at baseline to 660 ± 50 pg/mL, P < 0.0001) (Fig. 3A, second column vs. baseline).
The dose-response relationship for effect of CRP on increasing supernatant levels of MCP-1 and IL-6 is shown in Figures 2 and 3. The effect of CRP on induction of supernatant levels of MCP-1 and IL-6 in HUVECs increased stepwise as the CRP concentration increased from 25 μg/mL and reached a maximum of 100 μg/mL (P < 0.0001 for both) (second column of Figs. 2A through 2C and second column of Fig. 3A through 3C).
Figures 2 and 3 show the effects of CRP on increased secretion of supernatant levels of MCP-1 and IL-6 that were modulated by simvastatin. Stepwise suppression increased as the simvastatin dose increased from 25 to 100 μmol/L (Fig. 2A through 2C and Fig. 3A through 3C), and a statistically significant increase was obtained when the simvastatin dose was only 25 μmol/L (P < 0.001 for all) (Figs. 2A and 3A, third column vs. second column).
Antiinflammatory Properties of Losartan
Low-dose losartan (100 μmol/L) did not suppress the effect of CRP (25 μg/mL) on expression of VCAM-1 on HUVEC surfaces (35 ± 2.8% vs. 36 ± 3.3%; P = 1) (Fig. 4A). Noticeably, even when losartan was increased to a very high dose (750 μmol/L), it did not significantly suppress the effect of CRP on HUVEC surface expression of VCAM-1 (35 ± 2.8% vs. 31 ± 2.8%; P = 0.768).
Low-dose losartan (100 μmol/L) did not suppress the effect of CRP (25 μg/mL) on HUVEC secretions of MCP-1 (2690 ± 188 pg/mL vs. 2750 ± 164 pg/mL; P = 0.892) (Fig. 4B) and IL-6 (613 ± 40 pg/mL vs. 600 ± 35 pg/mL; P = 0.887) (Fig. 4C). However, when the losartan dose was stepwise increased to 500 μmol/L, the effect of CRP on HUVEC secretion of MCP-1 was significantly suppressed (2690 ± 188 pg/mL vs. 500 ± 180 pg/mL; P < 0.0001) (Fig. 4B), while the effect of CRP on HUVEC secretion of IL-6 was still not significantly suppressed (613 ± 40 pg/mL vs. 580 ± 40 pg/mL; P = 0.667) (Fig. 4C). Moreover, when losartan dosage was increased to a maximal dose of 750 μmol/L, the effects of CRP on HUVEC secretions of MCP-1 (2690 ± 188 pg/mL vs. 128 ± 40 pg/mL; P < 0.0001) (Fig. 4B) and IL-6 (613 ± 40 pg/mL vs. 42 ± 18 pg/mL; P < 0.0001) (Fig. 4C) were noticeably suppressed.
Expression of AT1 Receptors on Human Tissues
Figure 5 shows immunohistochemical staining for human kidney and immunocytochemical staining for UHVECs. Angiotensin II type 1 receptors were identified on renal tubes (deep-brown color). However, the AT1 receptors were not identified on HUVECs.
This study, which investigated the inflammatory effect of CRP and the impact of antiinflammatory effects of simvastatin and losartan using primary cell culture obtained several striking findings. First, CRP directly participates in inflammatory processes, as reflected in the increased HUVEC surface expression of VCAM-1 and supernatant levels of MCP-1 and IL-6. Second, an inversed proportional correlation existed between increased concentrations of simvastatin, and reduced surface expression of VCAM-1 and supernatant levels of MCP-1 and IL-6 were observed. Conversely, losartan had no antiinflammatory effect unless a high dose of losartan was used in this in vitro experiment.
Experimental results of this study did not support the hypothesis that low-dose losartan has antiinflammatory properties; these properties only existed for very high doses of losartan. Conversely, low-dose simvastatin abrogated the inflammatory effects of CRP on HUVECs. Thus, results support that simvastatin has much stronger antiinflammatory properties than losartan. Both in vivo and ex vivo studies demonstrated that losartan improves endothelial function,24 inhibits platelet activation,25,26 reduces cardiovascular complications,22,23 and elicits anti-atherosclerotic features by blocking inflammatory mediators.25-27 We suggest that the discrepancies between this and previous studies25-27 likely have 2 explanations. First, losartan must be metabolized into biologically active forms before it can have antiinflammatory effects. Second, only hepatocytes, and not HUVECs, can metabolize losartan into active metabolites. Recent experimental studies25,28 demonstrated that the losartan metabolite EXP3179, which has no AT1 receptor blocking properties, suppressed tumor necrosis factor α-induced EC apoptosis and exhibited the antiinflammatory and anti-aggregatory properties, suggesting that the AT1 receptor-independent effects of losartan are mediated primarily by its metabolites. Thus, our suggestion, based on experimental findings, is supported by these recent studies.25,28
Atherosclerosis results from a chronic inflammatory disease. This concept is supported by findings from numerous studies showing that CRP, an inflammatory marker, directly participates in the pathogenesis of atherosclerosis by activating ECs and promoting the inflammatory component of atherosclerosis.12-15 Additionally, clinical observation studies have demonstrated that elevated CRP levels are associated with increased risk of cardiovascular events in various clinical settings.4,5,8-11 However, these clinical observational studies cannot completely exclude the possibility of the effects of other inflammatory mediators that exist in circulating blood and participate together with CRP to affect endothelial dysfunction and propagation of atherosclerosis. This experimental study, which used HUVECs for primary cell culturing, confirmed that CRP directly stimulated HUVEC surface expression of VCAM-1 and increased production of supernatant levels of MCP-1 and IL-6. Recently, another experimental study15 demonstrated that CRP directly mediated HUVEC surface expression of VCAM-1 and intercellular adhesion molecule-1. That finding15 is consistent with those obtained by this study. Furthermore, experimental results from these 2 studies15 further support the results obtained by those recent clinical observational studies.4,5,8-11
Importantly, this study identified a direct and proportional relationship between the increased CRP concentrations and the accentuated surface expression of VCAM-1 and increased supernatant levels of MCP-1 and IL-6. This finding of the association between increased CRP concentration and increased endothelial damage provides experimental evidence supporting that obtained by 2 recent studies showing that increased circulating levels of hs-CRP portend plaque to rupture in patients with acute coronary syndrome.4,6
Growing evidence shows that statins have an antiinflammatory effect beyond lowering the lipid levels.17-20,29 Moreover, current studies demonstratee that statin therapy strongly decreased CRP levels in patients in various clinical settings.17-19 The principal finding in this study was that simvastatin significantly suppressed the effects of CRP on surface expression of VCAM-1 and secretion of MCP-1 and IL-6 from HUVECs. Furthermore, associations were found between increased simvastatin concentration and reduced effects of CRP on surface expression of ICAM-1; reduced secretion of MCP-1 and IL-6 were also identified. The antiinflammatory effects of statins have been recently confirmed by in vitro studies.14 Therefore, the experimental results of this in vitro study further strengthen those obtained by recent in vitro studies14 and supports the findings from the clinical observational studies.17-20
It should be noted that experimental conclusions are based on a HUVEC culture. Therefore, caution should be used when extrapolating the results of this study to other in vitro vascular ECs. Furthermore, experimental results cannot be unequivocally extrapolated to in the vivo cardiovascular system. Moreover, this experimental study did not evaluate the antiinflammatory effect of losartan metabolite EXP3179. Thus, we could only provide the impact of losartan rather the impact of EXP3179 on inhibiting the inflammatory effects of CRP. However, the antiinflammatory effect of this metabolite has already been proved by recent investigations.25,28
In conclusion, experimental results of this in vitro study further reinforce that CRP plays a key role in the pathogenesis of the vascular inflammatory process. Additionally, results of this study demonstrate that simvastatin had stronger antiinflammatory properties than losartan, thus providing further insight into the novel mechanistic basis of the anti-atherosclerotic action of simvastatin.
1. van der Wal AC, Becker AE, van der Loos CM, et al. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of the dominant plaque morphology. Circulation
2. Libby P, Geng YJ, Sukhova GK, et al. Molecular determinants of atherosclerotic plaque vulnerability. Ann N Y Acad Sci
3. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med
4. Yip HK, Hung WC, Yang CH, et al. Serum concentrations of high-sensitivity C-reactive protein predict progressively obstructive lesions rather than late restenosis in patients with unstable angina undergoing coronary artery stenting. Cir J
5. Burke AP, Farb A, Malcom GT, et al. Coronary risk factors and plaque morphology in men with coronary disease who died suddenly. N Engl J Med
6. Burke AP, Tracy RP, Kologie F, et al. Elevated C-reactive protein values and atherosclerosis in sudden coronary death: association with different pathologies. Circulation
7. Ishikawa T, Hatakeyama K, Imamura T, et al. Involvement of C-reactive protein obtained by directional coronary atherectomy in plaque instability and developing restenosis in patients with stable or unstable angina pectoris. Am J Cardiol
8. Yip HK, Sun CK, Chang LT, et al. Strong correlation between serum levels of inflammatory mediators and their distribution in infarct coronary artery. Cir J
. 2006 (in press).
9. Koenig W, Sund M, Frohlich M, et al. C-reactive protein, a sensitive marker of inflammation, predicts future risk of coronary heart disease in initially healthy middle-aged men: results from the MONICA Augsburg Cohort Study, 1984 to 1992. Circulation
10. Mueller C, Buettner HJ, Hodgson JM, et al. Inflammation and long-term mortality after non-ST elevation acute coronary syndrome treated with a very early invasive strategy in 1042 consecutive patients. Circulation
11. Yip HK, Hang CL, Fang CY, et al. Level of high-sensitivity C-reactive protein is predictive of 30-day outcomes in patients with acute myocardial infarction undergoing primary coronary intervention. Chest
12. Verma S, Buchanan MR, Anderson TJ. Endothelial function testing as a biomarker of vascular disease. Circulation
13. Szmitko PE, Wang CH, Weisel RD, et al. New markers of inflammation and endothelial cell activation: part I. Circulation
14. Pasceri V, Chang J, Willerson JT, et al. Modulation of C-reactive protein-mediated monocyte chemoattractant protein-1 induction in human endothelial cell by anti-atherosclerosis drugs. Circulation
15. Pasceri V, Willerson JT, Yeh ET. Direct proinflammatory effect of C-reactive protein on human endothelial cells. Circulation
16. Vermas S, Wang CH, Li SH, et al. A self-fulfilling prophecy: C-reactive protein attenuates nitric oxide production and inhibits angiogenesis. Circulation
17. van de Ree MA, Huisman MV, Princen HMG, et al. Strong decrease of high sensitivity C-reactive protein with high-dose atorvastatin in patients with type 2 diabetes mellitus. Atherosclerosis
18. The Cholesterol and Recurrent Events (CARE) investigators, Ridker PM, Rifai N, Pfeffer MA, et al. Long-term effects of pravastatin on plasma concentration of C-reactive protein. Circulation
19. Strandberg TE, Vanhanen H, Tikkanen MJ. Association between change in C-reactive protein and serum lipids during statin treatment. Ann Med
20. Ridker PM, Rifai N, Pfeffer MA, et al. Inflammation, pravastatin, and the risk of coronary events after myocardial infarction in patients with average cholesterol levels. Circulation
21. Weber MA, Byyny RL, Pratt JH, et al. Blood pressure effects of the angiotensin II receptor blocker losartan. Arch Intern Med
22. Dahlof B, Burke TA, Krobot K, et al. Population impact of losartan use on stroke in the European Union (EU): Protections from the Losartan Intervention For Endpoint reduction in hypertension (LIFE) study. J Hum Hypertens
23. Okin PM, Devereux RB, Jern S, et al. for the Losartan Intervention For Endpoint reduction in hypertension (LIFE) Study Investigators. Regression of electrocardiographic left ventricular hypertrophy by losartan versus atenolol: the Losartan Intervention For Endpoint reduction in hypertension (LIFE) study. Circulation
24. Schiffrin EL, Park JB, Intengan HD, et al. Correction of arterial structure and endothelial dysfunction in human essential hypertension by the angiotensin receptor antagonist losartan. Circulation
25. Kramer C, Sunkomat J, Witte J, et al. Angiotensin II receptor-independent antiinflammatory and antiaggregatory properties of losartan: role of the active metabolite EXP3179. Cir Res
26. Monton M, Jimenez A, Nunez A, et al. Comparative effects of angiotensin II AT-1-type receptor antagonists in vitro on human platelet activation. J Cardiovasc Pharmacol
27. Strawn WB, Chappell MC, Dean RH, et al. Inhibition of early atherogenesis by losartan in monkeys with diet-induced hypercholesterolemia. Circulation
28. Watanabe T, Suzuki J, Yamawaki H, et al. Losartan metabolite EXP3179 activates Akt and endothelial nitric oxide synthase via vascular endothelial growth factor receptor-2 in endothelial cells: angiotensin II type 1 receptor-independent effects of EXP3179. Circulation
29. Bonetti PO, Lerman LO, Napoli C, et al. Statin effects beyond lipid lowering-are they clinical relevant. Eur Heart J