Journal of Diabetes and Its Complications 23 (2009) 214–218WWW.JDCJOURNAL.COM

The effect of simvastatin on the serum monocyte chemoattractantprotein-1 and intracellular adhesion molecule-1 levels in diabetic rats

Yang Lina, Shandong Yea,⁎, Yan Chenb, Xiucai Lia, Guang-wei Yanga,Aihong Fanb, Yingxin Wangc

aDepartment of Endocrinology, Anhui Provincial Hospital affiliated to Anhui Medical University, Hefei, 230001, ChinabEndocrinological Laboratory of Anhui Provincial Hospital, Hefei, 230001, China

cDepartment of Pharmacology, Anhui Medical College, Hefei, China

Received 28 July 2007; accepted 6 September 2007

Abstract

Objective: This study aimed to observe the effect of simvastatin on the serum monocyte chemoattractant protein-1 (MCP-1) andintracellular adhesion molecule-1 (ICAM-1) levels and to probe its protective mechanisms on macroangiopathy in diabetic rats. Methods:Twenty-four Wistar rats were randomly assigned to a normal control group (Group A, n=8), and STZ-induced diabetic group (Group B,n=8), or a simvastatin-treated diabetic group (Group C, n=8). Rats in Group C were treated with simvastatin (20 mg kg−1 day−1) 1 weekafter the establishment of the diabetic model. Groups A and B were treated with corresponding sodium chloride. Peripheral blood glucosewas tested weekly; serum MCP-1, ICAM-1, and HbA1c levels were tested at the eighth week. Results: At the second, fourth, and eighthweek, peripheral blood glucose levels in Group B were similar to those of Group C, which were much higher than those of Group A. SerumMCP-1 and ICAM-1 levels in Groups B and C were higher than those of Group A (Pb.01), and serum MCP-1 and ICAM-1 levels in Group Cwere lower than those of Group B (Pb.01); HbA1c was not significantly different between Group C and Group B. Conclusion: Simvastatinhas the effect of anti-inflammation, which may play some protection against the progress of atherosclerosis in diabetic rats.© 2009 Elsevier Inc. All rights reserved.

Keywords: Simvastatin; Diabetes mellitus; Macroangiopathy; MCP-1; ICAM-1

1. Introduction

The chronic complications of diabetes spread all over thebody; the prevalence rate of cardiovascular disease of thepatients with diabetes increases by two to four timescompared with that of healthy adults. Monocyte chemoat-tractant protein-1 (MCP-1) is a member of chemotacticcytokine (CC) subfamily chemokines and plays a pivotal rolein the development of atherosclerosis. Atherosclerosis is nowrecognized as a disease of arterial inflammation that arisesfrom the interactions of migratory leukocytes with residentvascular endothelial cells (ECs), smooth muscle cells, andfibroblasts. At the molecular level, interactions among these

⁎ Corresponding author. Tel.: +86 551 2283301; fax: +86 551 2283292.E-mail address: ysd6464@yahoo.com.cn (S. Ye).

1056-8727/07/$ – see front matter © 2009 Elsevier Inc. All rights reserved.doi:10.1016/j.jdiacomp.2007.09.003

cell types are regulated by cytokines, adhesion molecules,and chemoattractants. MCP-1 induces CCR2+ monocytesfrom the circulation to the artery endothelium and promotesmonocyte to differentiate to lipid-laden macrophages. MCP-1 also contributes to the proliferation of arterial smoothmuscle cells, which constitutes the key cellular componentsof atherosclerotic plaques (Han et al., 2005). Intracellularadhesion molecule-1 (ICAM-1) induces the adhesion ofleukocyte-endothelial cells, the activation of EC, thereinforcement of vasopermeability, which is known as theearly stage of inflammatory reaction, and the pathophysio-logic basis of atherosclerosis.

Statins, potent inhibitors of 3-hydroxy-3-methylglutarycoenzyme A (HMG-CoA) reductase, inhibit the transfor-mation from HMG-CoA to mevalonic acid, lower theserum levels of cholesterol, and are successfully used to

Table 1Serum MCP-1 and ICAM-1 levels among Groups A, B, and C

Group n Time (weeks) MCP-1 (pg/ml) ICAM-1 (pg/ml) Blood glucose (mmol/l) HbA1c (%)

A 8 2 – – 5.707±0.572 –8 4 – – 6.105±0.428 –8 8 3.817±0.046 4.918±0.347 5.878±0.541 5.463±0.101

B 8 2 – – 30.672±2.301 ⁎ –7 4 – – 29.545±2.883 ⁎ –6 8 4.752±0.066 † 6.301±0.368 † 31.529±2.067 ⁎ 9.174±1.329 ⁎

C 8 2 – – 29.925±3.417 ⁎ –7 4 – – 28.705±2.510 ⁎ –7 8 4.015±0.049 ‡ 5.580±0.433 ‡ 28.714±2.393 ⁎ 8.870±1.258 ⁎

⁎ Compared with Group A, Pb.01; compared with Group B, Pb.01.† Compared with Group A, Pb.01; compared with Group C, Pb.01.‡ Compared with Group A, Pb.01.

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treat hypercholesterolemia. Moreover, the ability of statinsto reduce the mortality and morbidity of cardiovasculardiseases has been ascribed not only to their cholesterol-lowering activities but also to a number of additionaleffects, including improved EC function, enhanced fibri-nolysis, antioxidation, and antithrombotic activity. Inaddition, important anti-inflammatory effects of statinshave been reported. In this experiment, we try to addressthe hypothesis that simvastatin exerts an anti-inflammatoryeffect in diabetic rats, which may play some protectionagainst the progress of atherogenesis.

Fig. 1. Serum MCP-1 level among the three groups.

2. Subjects and methods

Twenty-four maleWistar rats (2 months old, weighing 200±20 g) supplied by the Experimental Animal Center of AnhuiMedical University were randomly assigned to a normalcontrol group (GroupA, n=8), STZ-induced diabetesmellitusgroup (Group B, n=8), and a simvastatin-treated group(Group C, n=8). Peripheral blood glucose of every rat wasnormal before this experiment. Each rat in Groups B and Creceived an intraperitoneal injection of STZ (65 mg/kg;Sigma Chemical, St. Louis, MO) in 0.1 mol/l citrate buffer(pH 4.3). After 48 h, peripheral blood was harvested fromvena caudalis to assess the blood glucose. Animals with ablood glucose level higher than 16.9 mmol/l were considereddiabetic. All rats were kept in an air-conditioned room with a12-h light–12-h dark cycle and were given free access towater and food until they were used for the experiments. Toprevent sudden death, such as that due to diabeticketoacidosis, all of the rats with a blood sugar level higherthan 33.3 mmol/l were subcutaneously injected with 0.5 Ulong-acting insulins two to three times for 1 week accordingto the blood sugar level. Rats in Group Awere dealt with thesame dose of the buffer solution of citric acid. Rats in GroupC were lavaged with simvastatin (20 mg kg−1 day−1,MERCK & CO., Inc., China) 1 week after the establishmentof the diabetic model. Rats in Groups A and B were treatedwith corresponding sodium chloride. Fasting plasma glucose

was measured by an Accu-Chek Active System (RocheDiagnostics GmbH, Germany). All rats were killed whileunder anesthesia by intraperitoneal injection of sodiumpentobarbital (50 mg/kg body weight). Experiments wereconducted in accordance with institutional and NationalInstitutes of Health guidelines.

Peripheral blood glucose levels were tested weekly.Blood was harvested from the femoral artery to assess theserum MCP-1, ICAM-1, and HbA1c levels at the eighthweek. Serum MCP-1 and ICAM-1 levels were measuredvia quantitative sandwich ELISA using a commercial kit(Biosource Inc., Camarillo, CA) according to themanufacturer's instructions. The assay was performed induplicate and the intensity of the color was measured inan ELISA reader at 450 nm. HbA1c was measured bymicrocolumn chromatography.

2.1. Statistical analysis

Data were presented as means±S.D. and analyzed usingStatistical Package for the Social Sciences 10.0. Two-groupcomparisons were performed using the Student's t test. Therelationship between serum MCP-1 and ICAM-1 in diabeticrats was determined via linear correlation. In all cases, aP value ≤.05 was considered significant.

Fig. 3. Relationship between serum MCP-1 and ICAM-1 in Group A.

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3. Results

One rat from Group B died due to tussling at the thirdweek, one rat died from tail infection at the seventhweek, and one rat from Group C died due to tussling atthe third week.

At the second, fourth, and eighth week, blood glucoselevels in Group B were similar to those of Group C, whichwere much higher than those of Group A. At the eighthweek, serum MCP-1, ICAM-1, and HbA1c levels in GroupsB and C were higher than those of Group A(Pb.01), andserum MCP-1 and ICAM-1 levels in Group C were lowerthan those of Group B (Pb.01); HbA1c levels were notsignificantly different between Group B and Group C, asshown in Table 1 and in Figs. 1 and 2.

Linear relationship showed that serum MCP-1 level hadsignificant positive relationship with serum ICAM-1 level inall rats (Group A: r=.766, Pb.05; Group B: r=.863, Pb.05;Group C: r=.816, Pb.05; Figs. 3, 4, and 5).

4. Discussion

MCP-1 is expressed by a variety of cell types, includingmonocyte, macrophage, EC, vascular smooth muscle cell(VSMC), fibroblast, and so forth. Under normal physiologi-cal condition, the expression level of MCP-1 in the above-mentioned cells is low. Once given the stimulations, such ashyperglycemia, advanced glycation end products, angioten-sin II, oxidative stress, interleukin-1 (IL-1), tumor necrosisfactor-α, and so forth, the expression level of MCP-1 isobviously up-regulated (Ehlermann et al., 2006; Lee et al.,2001; Zhan et al., 2005; Zhu, 2006). MCP-1 recruitsmonocytes, leukocytes, and other inflammatory cells intoartery subendarterium in response to an inflammatorychallenge. In humans, circulating MCP-1 has been found tobe associated with cardiovascular disease (De Lemos et al.,2003) and is elevated in type 2 diabetic patients comparedwith nondiabetics (Nomura, Shouzu, Omoto, Nishikawa, &Fukuhara, 2000). MCP-1 recruits monocytes from the bloodinto atherosclerotic lesions; then, it promotes them to foamcells. MCP-1 also promotes the VSMC to move into theinner membrane of the artery.

Fig. 2. Serum ICAM-1 level among the three groups.

Increased expression of ICAM-1 and its ligand onleukocytes and ECs by inflammatory cytokines mediatesthe adhesion, recruitment, and migration of white blood cellsthrough vascular surfaces, thus providing an essential step inatherogenesis (Jang, Lincoff, Plow, & Topol, 1994). Focalincreased expression of leukocyte/endothelial-bound ICAM-1 has been found in human atherosclerotic lesions (DeGrabaet al., 1998). The ligand of ICAM-1-lymphocyte function-associated antigen-1 (LFA-1 or CD11a/CD18) is a hetero-dimeric glycoprotein belonging to the β2-integrin family.LFA-1 is also constitutively expressed on the surface ofinactive leukocytes, which appears to be the most importantadhesion molecule expressed on activated leukocytes.Hence, these integrins mediate spreading and firm adhesionof leukocytes, including peripheral blood mononuclear cells(PBMCs), followed by transendothelial migration (Carlos &Harlan, 1994). The binding of ICAM-1 and its ligand is thebasis of the adhesion of monocyte-endothelial cells; MCP-1promotes the monocyte to move into the inner membrane ofthe artery and to differentiate to lipid-laden macrophages.ICAM-1 strengthened monocyte transmigration in responseto MCP-1. ICAM-1 is highly expressed by activated EC,which induces monocyte to adhere to EC; then, inflamma-

Fig. 4. Relationship between serum MCP-1 and ICAM-1 in Group B.

Fig. 5. Relationship between serum MCP-1 and ICAM-1 in Group C.

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tory cytokines are excreted from the activated monocyte.These inflammatory cytokines also promote the smoothmuscle cell to express vascular cell adhesion molecule-1(VCAM-1); the interaction of VCAM-1 and its receptorintensifies the adhesion and transmigration of monocyte. Theinteraction of adhesion molecule and MCP-1 accentuates therecruitment of monocyte into the vascular wall and thencontributes, in some extent, to the development andprogression of atherogenesis.

An important anti-inflammatory effect of statins hasbeen reported recently. It has been suggested that statinsmay have additive beneficial effects on atherosclerosisindependently of their lipid-lowering effect (Sowers, 2003).Koh et al. (2004) reported that 47 hypertensive hyperch-olesterolemic patients were given simvastatin 20 mg andplacebo for 2 months. Simvastatin significantly decreasedplasma MCP-1 levels relative to baseline value by 7±3%.Han et al. (2005) showed that treatment of simvastatin(20 mg/day for 2 weeks) in 21 normocholesterolemic mendecreased CCR2 protein and mRNA expression incirculating monocytes and reduced MCP-1-mediated mono-cyte transmigration to the arterial wall. Romano et al.(2000) reported that simvastatin could inhibit dose-dependently MCP-1 production in PBMCs exposed tolipopolysaccharide or inactivated Streptococcus hemolyticusand in human ECs exposed to IL-1β. Elevated levels ofICAM-1 have been reported in diabetic patients. Cerielloet al. (2004) showed that plasma ICAM-1 levels weredecreased significantly by simvastatin (40 mg/day for12 weeks) in 30 type 2 diabetic patients. In our presentexperimental study, serum MCP-1 and ICAM-1 levels inSTZ-induced diabetic rats were significantly increasedcompared to the normal control group, which suggestedthat a low degree of inflammation existed in diabetic stateand that treatment with simvastatin (20 mg kg−1 day−1 for8 weeks) could markedly reduce serum MCP-1 and ICAM-1 levels. Our results further identified the effect of anti-inflammation of simvastatin. In addition, a positiverelationship between serum MCP-1 and ICAM-1 in diabeticrats was also found in this study.

The mechanisms of simvastatin that reduce MCP-1and ICAM-1 are not clear, but it can be proven throughthe following:

1. MCP-1 and ICAM-1 have been shown to be producedby macrophages and ECs via an activation of thenuclear factor kappa B (NF-κB) pathway. Recently,statins were shown to prevent activation of NF-κB bythe up-regulation of the NF-κB inhibitory protein IκB(Dichtl et al., 2003; Kleemann et al., 2004). Thismechanism was primarily dependent on the suppres-sion of mevalonate-derived products.

2. Simvastatin may work as an intracellular antioxidant(Takemoto et al., 2001). Li et al. (2002) showed thatox-LDL up-regulated the expression of ICAM-1 inhuman coronary artery ECs and that simvastatincould attenuate ox-LDL-induced activation of LOX-1and subsequent up-regulation of expression of adhe-sion molecules.

3. Statins have been demonstrated to have directprotective effects on ECs, reducing their response tovarious stimuli (März & Köenig, 2003; Wierzbicki,Poston, & Ferro, 2003).

To summarize, statins such as simvastatin can reduceserum MCP-1 and ICAM-1 levels and attenuate theinflammatory reaction in diabetic state, which may con-tribute, in some extent, to their protection against athero-sclerosis, but the serum MCP-1 and ICAM-1 levels insimvastatin-treated diabetic rats were still higher than thosein normal control rats; this means that we should take someoverall and integrated therapeutic management to lessenmore effectively the degree of inflammation in order toprevent and treat diabetes-related vascular complications.In addition, the mechanisms of statins in inhibiting MCP-1and ICAM-1 expression and production need to beevaluated further.

5. Conclusion

This study showed that simvastatin can reduce serumMCP-1 and ICAM-1 levels; it has been suggested that statinsmay have additive beneficial effects on atherosclerosisindependently of their lipid-lowering effect.

References

Carlos, T. M., & Harlan, J. M. (1994). Leukocyte-endothelial adhesionmolecules. Blood, 84, 2068−2102.

Ceriello, A., Quagliaro, L., Piconi, L., Assaloni, R., Ros, R. D., Maier, A.,Esposito, K., & Giugliano, D. (2004). Effect of postprandial hyper-triglyceridemia and hyperglycemia on circulating adhesion moleculesand oxidative stress generation and the possible role of simvastatin intreatment. Diabetes, 53, 701−710.

DeGraba, T. J., Siren, A. L., Penix, L., McCarron, R. M., Hargraves, R.,Sood, S., Karen, D., Pettigrew, K. D., & Hallenbeck, J. M. (1998).Increased endothelial expression of intercellular adhesion molecule-1 in

218 Y. Lin et al. / Journal of Diabetes and Its Complications 23 (2009) 214–218

symptomatic versus asymptomatic human carotid atherosclerotic plaque.Stroke, 29, 1405−1410.

De Lemos, J. A., Morrow, D. A., Sabatine, M. S., Murphy, S. A., Gibson,C. M., Antman, E. M., McCabe, C. H., Cannon, C. P., & Braunwald,E. (2003). Association between plasma levels of monocyte chemoat-tractant protein-1 and long-term clinical outcomes in patients withacute coronary syndromes. Circulation, 107, 690−695.

Dichtl, W., Dulak, J., Frick, M., Alber, H. F., Schwarzacher, S. P., Ares,M. P., Nilsson, J., Pachinger, O., & Weidinger, F. (2003). HMG-CoAreductase inhibitors regulate inflammatory transcription factors inhuman endothelial and vascular smooth muscle cells. Arteriosclerosis,Thrombosis, and Vascular, 23, 58−63.

Ehlermann, P., Eggers, K., Bierhaus, A.,Most, P.,Weichenhan, D., Greten, J.,Nawroth, P. P., Katus, H. A., & Remppis, A. (2006). Increasedproinflammatory endothelial response to S100A8/A9 after preactivationthrough advanced glycation end products. Cardiovascular Diabetolo-gica, 5, 1−9.

Han, K. H., Ryu, J., Hong, K. H., Ko, J., Pak, Y. K., Kim, J. B., Park, S. W.,& Kim, J. J. (2005). HMG-CoA reductase inhibition reduces monocyteCC chemokine receptor 2 expression and monocyte chemoattractantprotein-1-mediated monocyte recruitment in vivo. Circulation, 111,1439−1447.

Jang, Y., Lincoff, A. M., Plow, E. F., & Topol, E. J. (1994). Cell adhesionmolecules in coronary artery disease. Journal of the American Collegeof Cardiology, 24, 1591−1601.

Kleemann, R., Verschuren, L., de Rooij, B. J., Lindeman, J., de Maat, M. M.,Szalai, A. J., Princen, H. M., & Kooistra, T. (2004). Evidence for anti-inflammatory activity of statins and PPAR-α activators in human C-reactive protein transgenic mice in vivo and in cultured humanhepatocytes in vitro. Blood, 103, 4188−4194.

Koh, K. K., Quon, M. J., Han, S. H., Chung, W. J., Ahn, J. Y., Seo, Y. H.,Kang, M. H., Ahn, T. H., Choi, I. S., & Shin, E. K. (2004). Additivebeneficial effects of losartan combined with simvastatin in the treatmentof hypercholesterolemic, hypertensive patients. Circulation, 110,3687−3692.

Lee, S., Kim, B., Sik, Y., Kim, S. B., Park, S. K., & Park, J. S. (2001). Highglucose inducesMCP-1 expression partly via tyrosine kinase-AP-1 pathwayin peritoneal mesothelial cells. Kidney International, 60, 55−64.

Li, D., Chen, H., Romeo, F., Sawamura, T., Saldeen, T., & Mehta, J.L. (2002). Statins modulate oxidized low-density lipoprotein-mediated adhesion molecule expression in human coronary arteryendothelial cells: Role of LOX-1. Pharmacology & Therapeutics,302, 601−605.

März, W., & Köenig, W. (2003). HMG-CoA reductase inhibition: Anti-inflammatory effects beyond lipid lowering? Journal of CardiovascularRisk, 10, 169−179.

Nomura, S., Shouzu, A., Omoto, S., Nishikawa, M., & Fukuhara, S.(2000). Significance of chemokines and activated platelets inpatients with diabetes. Clinical and Experimental Immunology,121, 437−443.

Romano, M., Diomede, L., Sironi, M., Massimiliano, L., Sottocorno, M.,Polentarutti, N., Guqlielmotti, A., Albani, D., Bruno, A., Fruscella, P.,Salmona, M., Vecchi, A., Pinza, M., & Mantovani, A. (2000). Inhibitionof monocyte chemotactic protein-1 synthesis by statins. LaboratoryInvestigation, 80, 1095−1100.

Sowers, J. R. (2003). Effects of statins on the vasculature: Implications foraggressive lipid management in the cardiovascular metabolic syndrome.American Journal of Cardiology, 91, 14B−22B.

Takemoto, M., Node, K., Nakagami, H., Liao, Y., Grimm, M., Takemoto, Y.,Kitakaze, M., & Liao, J. K. (2001). Statins as antioxidant therapy forpreventing cardiac myocyte hypertrophy. Journal of Clinical Investiga-tion, 108, 1429−1437.

Wierzbicki, A. S., Poston, R., & Ferro, A. (2003). The lipid and non-lipideffects of statins. Pharmacology & Therapeutics, 99, 95−112.

Zhan, Y., Brown, C., Maynard, E., Anshelevich, A., Ni, W., Ho, I. C., &Oettgen, P. (2005). Ets-1 is a critical regulator of Ang II-mediatedvascular inflammation and remodeling. Journal of Clinical Investiga-tion, 115, 2508−2516.

Zhu, D. L. (2006). The relationship between inflammation and type 2diabetes. Chinese Journal of Investigative, 14, 73−74.