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Simvastatin reduces leucocyte– and platelet–endothelial cell interaction in murine antigen-induced arthritis in vivo O Gottschalk1,2, ML Dao Trong1, P Metz2, J Wallmichrath2, S Piltz2, KW Jauch2, V Jansson3, M Schmitt-Sody1,3,4
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1 Walter Brendel Centre, 2Department of Surgery Großhadern, 3Department of Orthopaedic Surgery Großhadern, Ludwig Maximilians University of Munich, and 4Medical Park Chiemsee, Bernau-Felden, Germany
Objectives: The use of statins in the prevention and treatment of cardiovascular diseases is well established. Their use as anti-inflammatory and immunomodulatory agents in the treatment of rheumatoid arthritis (RA) has also been investigated, with several clinical and experimental studies indicating an anti-inflammatory effect of statins for RA, but other studies showing no effect or even the opposite. The current study was designed to examine the effect of simvastatin in an in vivo murine model of arthritis using intravital microscopy. Method: We assigned four groups (n = 7, female C57Bl6 mice), two with and two without antigen-induced arthritis (AiA), from which one of the non-AiA groups and one of the AiA groups were treated with simvastatin 40 mg/kg i.p. daily for 14 consecutive days after induction of arthritis. Platelet– and leucocyte–endothelial cell interaction was assessed by measurement of rolling and adherent fluorescence-labelled platelets and leucocytes, functional capillary density (FCD) was evaluated, and knee joint diameter was determined as a clinical parameter. Results: In arthritic mice treated with simvastatin, a significant reduction in platelet– and leucocyte–endothelial cell interaction was observed in comparison to arthritic mice treated with vehicle. In addition, a significant reduction in FCD was seen in arthritic mice treated with simvastatin, along with a reduction in knee joint swelling of the AiA mice. Conclusions: Treatment of AiA mice with simvastatin showed significant reductions in platelet– and leucocyte– endothelial cell interactions, in FCD, and in the swelling of the knee joint. These results support the hypothesis of the anti-inflammatory effects of statins in the treatment of RA.
Rheumatoid arthritis (RA) is a chronic inflammatory disease whose pathophysiology is not completely understood. An autoimmune reaction against synovial tissue leads consecutively to destruction of the entire joint. RA is the most common inflammatory disease in middle-aged adults, with a prevalence of about 0.5–1%; women are three to four times more commonly affected than men (1). Circulating mediators of inflammation and leucocyte– endothelial cell interaction in the synovial microcirculation are major factors involved in the course of the disease. Platelets seem to play a crucial role in this leucocyte–endothelial cell interaction and thus in the disease activity and progression of RA (2, 3). Current drug treatment protocols involve the use of non-steroidal anti-inflammatory drugs (NSAIDs), steroids, and disease-modifying anti-rheumatic drugs (DMARDs) (4). DMARDs influence the preservation of joint function by modifying and suppressing RA disease activity if early treatment is provided (5). Tumour necro-
Oliver Gottschalk, Department of Surgery, LMU – Campus Großhadern, Marchioninistrasse 15, 81377 Munich, Germany. E-mail: [email protected]
sis factor (TNF)-α inhibitors have an effect on disease activity and on the radiological evidence of progression, in addition to a lack of gastrointestinal sideeffects, compared to basic drugs (6). However, there are reports of a biologically related higher risk of rare serious infections or malignancies (7). Other therapy approaches such as inhibition of nitric oxide (NO) synthesis (8), inhibition of angiogenic pathways (9), and therapy with statins (10) have been discussed. Statins are well established in the prophylaxis of cardiovascular diseases by decreasing the morbidity/ mortality in a multifactorial manner (11, 12). The antiinflammatory role of statins was first investigated 1995 by Kobashigawa et al (13), who described a positive effect of pravastatin on the rejection rate after heart transplantation. A significant reduction in acute rejections after kidney transplantation was also described (14). Several in vitro studies have shown the immunomodulatory and anti-inflammatory effects of statins by induction of the expression of major histocompatibility complex (MHC)-II, thus inhibiting T-cell activation, reduction of the chemokine production involved in leucocyte migration, selective inhibition of leukocyte function antigen (LFA)-1-mediated adhesion and T-cell co-stimulation
Accepted 27 December 2013 © 2014 Informa Healthcare on license from Scandinavian Rheumatology Research Foundation
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DOI: 10.3109/03009742.2013.879606
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and reduction of interleukin (IL)-6 and IL-8 (15–19). Clinical studies support these findings by demonstrating significantly decreasing levels of C-reactive protein (CRP) (20, 21) and a significantly better 8-year survival rate with a lower incidence of transplant vasculopathies after heart transplantation (22). Leung et al showed that simvastatin reduces the production of pro-inflammatory cytokines [TNF-α, IL-6, IL-12, and interferon (IFN)-γ] through T-cell-activated macrophages in murine collagen-induced arthritis (23). Atorvastatin in combination with prednisolone had a beneficial effect on Freund’s adjuvant-induced arthritis in rats (24). Clinical improvement in RA patients was shown compared to therapy with chloroquine by decreases in CRP levels and erythrocyte sedimentation rate (ESR); reduction of the T-helper (Th)1/Th2 and CD4/CD8 ratios was described (25–27). A positive correlation between clinical improvement and IL-6 reduction was reported, and elevated baseline circulating IL-10 concentrations as a positive predictive factor for treatment response postulated (28). Kok et al (29) described a pathway showing that cysteine-rich protein 61 (CYR-61) plays an important role in the pathogenesis of RA and simvastatin induces an up-regulation of SIRT-1/FoxO3a signalling in synovial fibroblasts (SFs), which is important for the induction of CYR-61 in RA SFs. The aim of this study was to evaluate the effects of simvastatin on the leucocyte– and platelet–endothelial cell interaction as an inflammatory response and activity on the well-established in vivo murine model of AiA (30–32).
flank of 100 mg methylated bovine serum albumin (mBSA; Sigma, Deisenhofen, Germany), dissolved in 50 mL Freund’s complete adjuvant (Sigma), and supplemented with 2 mg/mL heat-killed Mycobacterium tuberculosis strain H37RA (Difco, Augsburg, Germany), and an additional intraperitoneal (i.p.) injection of 2 109 heatkilled Bortadella pertussis (Institute of Microbiology, Berlin, Germany). On day 0, arthritis was inducted by injection of 100 μg mBSA in 50 μL saline into the left knee joint. Control animals underwent the same procedure for pre-immuninization but received an equivalent volume of saline in the knee joint instead of mBSA.
Method
Surgical preparation
Animals and study design
In preparation for intravital fluorescence microscopy, on day 14, the mice were anaesthetized by inhalation of isoflurane 1.5% (Forence, Abbott, Wiesbaden, Germany) and a mixture of O2/N2O. They were fixed on a warmed plate on their back with the left knee embedded in a 45 flexion on a custom-made plexiglas block. Arterial and venous catheters were implanted in the tail. The intra-articular synovial tissue of the knee joint was then accessed through a 1-cm longitudinal incision over the knee, exposing the patellar tendon, which was then cut transversely at the tibial edge and flapped cranial (34).
We used female C57Bl6 mice (Charles-River, Sulzfeld, Germany) weighing between 18 and 24 g. The experiments were approved by, and performed according to, the German legislation for the protection of animals. The mice were randomly assigned to four groups (group 1: no arthritis, therapy with vehicle; group 2: with arthritis, therapy with vehicle; group 3: no arthritis, therapy with simvastatin; group 4: with arthritis, therapy with simvastatin), each containing seven valid animals (n = 7). In addition to the daily assessment of their weight, fur, and behaviour, we evaluated the swelling of the knee joint by measuring the transverse diameter to assess the severity of the AiA until the examination with intravital microscopy. Mice that showed alterations in their behaviour and fur were excluded from the study. AiA We used the well-developed model of Brackertz et al (32), which has been used previously in our department (33). Before the induction of arthritis, the mice were immunized on days –21 and –14 by a subcutaneous injection in the left
Preparation of simvastatin For the production of a 10 mM stock, 41.8 mg of simvastatin were dissolved in 1.5 mL 0.1 N NaOH and 1 mL ethanol and the solution incubated at 50 C for 2 h, which converted it from the inactive lactone form to the active acid form. After pH adjustment, the volume was corrected with phosphate-buffered saline (PBS) to 10 mL.
Treatment protocol We treated the mice in groups 3 and 4 with simvastatin i.p. for 14 consecutive days (40 mg/kg/day) and the mice in groups 1 and 2 with an equivalent volume of vehicle, starting on the day of arthritis induction (day 0) according to Leung et al (23).
Intravital fluorescence microscopy The microscopic set-up we used has been described previously (35). We investigated and stained platelets ex vivo from a donor mouse, leucocytes in vivo with rhodamine 6G, and plasma in vivo with fluorescein isothiocyanate (FITC)–Dextran (both Sigma, Deisenhofen, Germany) (36). To isolate platelets from a syngenic donor mouse we used cardiac puncture to obtain a 1-mL blood sample, which then was prepared with 0.2 mL of Alsevers puffer, 20 μL of prostaglandin E1 (PGE1, Serva, Heidelberg,
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Germany), and 0.5 mL of Dulbecco PBS (D-PBS, PANSystems, Aidenbach, Germany). The platelets were separated by centrifugation, resuspended with 1.5 mL D-PBS, 0.3 mL Alsevers, and 50 μL PGE1, labelled with the fluorescent marker carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; Molecular Probes, Eugene, OR, USA), and finally centrifuged and resuspended in 0.4 mL D-PBS. The purity of the sample and the platelet concentration were determined before application with a Coulter Counter (Coulter Corp, Miami, FL, USA). A total of 100 106 fluorescently labelled platelets were transfused through the lateral tail vein, corresponding to approximately 10% of all circulating platelets (37). Intravital microscopic images were recorded on videotape and data analysis was performed later offline using a computerassisted analysis system (CAP-Image, Dr Zeintl, Heidelberg, Germany).
Microhaemodynamic parameters Vessel diameter, venular red blood cell (RBC) velocity, and functional capillary density (FCD) were measured by injection of FITC-Dextran at the end of each experiment. FCD was defined as the length of respective capillaries vessels within the observation area (cm/cm2).
Microcirculatory parameters Platelet– and leucocyte–endothelial cell interactions were measured as rolling or adherent cells. Rolling cells were defined as cells that passed the vessel at a velocity significantly lower than the centreline velocity in the observed microvessel and were determined as the fraction of all platelets or leucocytes passing this predefined vessel segment within an observation interval of 30 s. Adherent cells were defined as cells that were attached to the endothelial lining and did not move within an observation interval of 30 s. Adherence was quantified as the number of cells per square millimetre of endothelial surface and calculated from the diameter and length of the observed vessel segment (1/mm2).
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Statistical analysis Quantitative data are expressed as means SEM. Comparison between multiple independent study groups was performed by a Kruskal–Wallis one-way analysis of variance (ANOVA) test on ranks. In case of significance (p < 0.05), a paired comparison was carried out by the Student–Newman–Keuls method. Analysis of repetitive measurement of multiple groups was performed using a two-way ANOVA test for combined samples.
Results Clinical parameters The induction of arthritis was monitored by swelling of the knee joint (Figure 1). After induction of arthritis on day 0, there was a significant increase in knee joint diameter in every group, confirming successful intraarticular injection. On day 2, the knee joint diameter in the AiA statin group was significantly higher than in the other groups (*p < 0.05). From day 3 to day 12, the knee joint diameter decreased back to baseline values in both non-arthritic groups compared to both arthritic groups (**p < 0.05). During the period of treatment, the increase in diameter persisted in the arthritic group treated with vehicle, whereas there was a significant decrease in knee joint diameter in the arthritic group treated with statin starting on day 9 (#p < 0.05). No deficits in the appearance and behaviour of the mice were observed. Body weight was stable at 18–21 g during the study period.
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All of the knee joints were collected together after intravital microscopy. They were fixated in paraformaldehyde and then decalcified [ethylenediaminetetraacetic acid, tetrasodium salt (EDTA-4Na) and 20% citric acid] for 72 h and embedded in paraffin. The samples were cut into slices of thickness 3 μm. Histological examination of the knee was performed with haematoxylin and eosin (H&E) staining and scored semiquantitatively following Brackertz et al (32) (0 = normal knee score, 1 = normal synovium, 2 = two or more lining cells and perivascular infiltrates, 4 = synovitis and pannus formation and cartilage/subchondral bone erosions).
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time Control vehicle Control statin AiA control AiA statin Figure 1. Change in knee joint diameter during the time of examination. Values given as mean SEM (n = 7). *p < 0.05 AiA statins vs. other groups; **p < 0.05 both AiA groups vs. non-arthritic groups; #p < 0.05 AiA statins vs. AiA control.
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Table 1. Microhaemodynamic parameters at the time of intravital microscopy. Group
Vessel diameter (μm)
RBC velocity (mm/s)
FCD (cm/cm2)
11.3 0.39 9.6 0.33 12.04 0.60 11.86 0.71
0.24 0.02 0.29 0.03 0.18 0.02 0.29 0.09
313.8 16.35 298.47 10.90 446.20 20.85 335 15.58*
Control vehicle Control statin AiA vehicle AiA statin
* Significant decrease for the AiA group treated with simvastatin compared to the AiA group treated with vehicle (p < 0.001).
No significant changes in vessel diameter and RBC velocity were seen between the different groups, apart from a slight decrease in RBC velocity in the AiA vehicle group (Table 1). With regard to the FCD (Figure 2), there was no significant difference between the non-arthritic groups (vehicle FCD = 313.8 6.4 cm/cm2, statin FCD = 298.3 9.6 cm/cm2) but we did observe a significant increase in the arthritic group treated with vehicle (FCD = 446.2 20.9 cm/cm2). In the AiA groups, the one treated with simvastatin (FCD = 338 15.6 cm/cm2; p < 0.001) showed a significant decrease in FCD compared to the vehicle group.
Histological results Histological analysis confirmed the inducation of arthritis by showing a severity score of 0 ¼ normal knee joint, 1 ¼ normal synovium with occasional mononuclear cells, 2 ¼ 2 or more synovial lining cells and perivascular infiltrates of leukocytes, 3 ¼ hyperplasia of synovium and dense infiltration, 4 ¼ synovitis, pannus formation, and cartilage/subchondral bone erosions.
*
500
Although the results were not significant, we obtained lower scores for the samples of AiA mice treated with simvastatin. Both control groups (no AiA) presented in all animals a normal knee score (severity score 0). The AiA group treated with vehicle showed mostly animals with a severity score of 3 or 4 whereas the statin-treated group presented a slight hyperplasia of the synovium and dense infiltration (score of 3), or scores of a lower level with some perivascular infiltrates of leukocytes (2 or smaller). Platelet–endothelial cell interaction in vivo A significant reduction in adherent platelets was seen in the AiA group treated with statins compared to the AiA vehicle group (AiA statin 170 71.6 cells/mm2 vs. AiA vehicle 502 102.1 cells/mm2; p < 0.05) (Figure 3A), almost down to the baseline values of the non-arthritic groups. The fraction of rolling platelets also decreased, although to a lesser extent, when treated with statins (AiA statins 0.027 0.007 vs. AiA vehicle 0.045 0.0045 (Figure 4A). The results are shown in Figures 5A–5C. Leucocyte–endothelial cell interaction in vivo The results for leucocyte–endothelial cell interaction were similar to those for the platelet–endothelial cell interaction in being favourable to simvastatin treatment (Figures 3B and 4B). A significant increase in the fraction of rolling and adherent leucocytes was observed in the AiA group treated with vehicle (0.095 0.021 cells/mm2 for rolling and 1125 139.5 cells/mm2 for adherent leucocytes) compared to both non-arthritic groups. There was a significant decrease in both rolling and adherent parameters in the AiA group treated with simvastatin (0.025 0.006; p < 0.05 and 443 97.8 cells/mm2; p < 0.001) compared to the arthritic vehicle group. Furthermore, the decrease went down to almost baseline values of the non-arthritic control group. The results are shown in Figures 6A–6C.
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Microhaemodynamic parameters
Figure 2. Functional capillary density (FCD), defined as the length of red blood cell (RBC)-perfused capillaries within the region of interest (ROI), for AiA and control groups. Values given as mean SEM (n = 7). *p < 0.05 AiA control vs. other groups.
Discussion Statins are used successfully in the primary and secondary prevention of cardiovascular diseases. They are also currently approved as prophylactic therapy for the higher comorbidity of cardiovascular diseases in rheumatic diseases (38). Their anti-inflammatory effects have been demonstrated in heart and kidney transplantations, in
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Figure 3. Number of (A) platelets and (B) leucocytes adherent (per mm2) to the endothelium in post-capillary venules in the synovium of the mouse knee joint (n = 7). Values given as mean SEM. *p < 0.05 A+B: AiA vehicle vs. other groups.
Figure 4. Fraction of (A) rolling platelets and (B) rolling leucocytes, given as the number of rolling cells divided by the sum of the rolling and non-adherent cells (n = 7). Values given as mean SEM. *p < 0.05 A: both AiA groups vs. non-arthritic groups; B: AiA vehicle vs. other groups.
atherosclerosis as an inflammatory disease, and within autoimmune diseases such as multiple sclerosis (39), systemic lupus erythematosus (40), and RA. These socalled pleiotropic effects are mainly explained by the inhibition of 3-hydroxy-3-methylglutaryl-coenzyme
A (HMG-CoA) reductase and thus the mevalonate pathway. At a cellular level, the anti-inflammatory effect has been explained by multiple pathways, including modulation of the expression, secretion, and function of a set of immunomediators that eventually leads to a reduction in
A
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Figure 5. Intravital microscopic visualization of CFDA-labelled platelets in (A) a non-arthritic mouse, (B) an arthritic mouse treated with vehicle, and (C) an arthritic mouse treated with simvastatin.
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Simvastatin reduces inflammation in AiA
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Figure 6. Intravital microscopic visualization of rhodamine 6G-labelled leucocytes in (A) a non-arthritic mouse, (B) an arthritic mouse treated with vehicle, and (C) an arthritic mouse treated with simvastatin.
CRP and pro-inflammatory cytokines, inhibition of INFγ-induced MHC-II expression, suppression of dendritic cell maturation, reduction of monocyte–endothelial cell adhesion (41), and many other inflammatory mechanisms (15, 27, 42, 43). RA is an MHC-II-associated, chronic inflammatory autoimmune disease, in which TNF-α has a crucial role because of its regulatory properties among the expression of other pro-inflammatory cytokines (44). It leads to activation of platelets and to a Th1-weighted immune response with activation and interaction between endothelial cells, macrophages, and B and T lymphocytes. This consequently leads to cell rolling through selectins and cell adhesion through LFA-1 integrins on leucocytes on the activated endothelium through intercellular adhesion molecule (ICAM)-1 adhesion molecules (45). Our results show significant decreases in platelet– and leucocyte–endothelial cell interaction following simvastatin treatment, representing the activity of inflammation. The FCD decreased as an expression of antiangiogenesis, thus supporting the hypothesis of the antiinflammatory effect of statins on RA. Additionally, we observed a slight reduction in all parameters in the nonarthritic group treated with simvastatin. These findings could indicate a protective character of statins for RA. The significant decrease in knee joint diameter in arthritic knee joints during the time of treatment with simvastatin compared to the treatment with vehicle strengthens the notion of an anti-inflammatory effect of simvastatin on clinical symptoms. In a study conducted on murine collagen-induced arthritis investigating the effects of pravastatin on clinical outcome, serum antiCII antibody levels, histology and immunohistochemistry, similar findings with a reduction in synovial tissue inflammation were described (46). A decrease in rolling platelets and leucocytes could be explained by the inhibition of platelet and endothelial P-selectins by statins as described by Eccles et al (47) and Oka et al (48). The importance of the P-selectins on these interactions has been shown previously in our AiA model (3). The reduction of adherent cells may be accounted for by the causal cohesion between rolling and adhering,
whereas another explanation could be the inhibition of the expression of the adhesion molecules ICAM-1 and LFA-1. Furthermore, it seems that statins stimulate the production of endothelial and platelet NO synthesis, and NO is a potent inhibitor of platelet function and aggregation (49). This effect is thought to be mediated by inhibition of Rho kinase geranylgeranyl phosphorylation by statins (50). Of note, recent studies postulate the opposite hypothesis, stating that statins accelerate the onset of collagen type II-induced arthritis in mice through induction of autoimmunity (51). Other studies also suggest a nonbeneficial effect of statins in RA (52–54). Further investigation is therefore required to explain why such significantly different results have been reported in the literature either in clinical or in experimental studies. We chose the AiA model because it enabled us to examine the local and controlled arthritis induction of a single joint. This allowed us to look specifically at the inflammatory process of the knee joint in comparison to the systemic analyses described in the above-mentioned murine studies, where an in vivo study with intravital microscopy at one joint was not possible. In conclusion, our results confirm the antiinflammatory effect of statins in murine AiA in vivo and therefore statins could be a new therapy option in the treatment of arthritis. References 1. Scott DL, Wolfe F, Huizinga TW. Rheumatoid arthritis. Lancet 2010;376:1094–108. 2. Milovanovic M, Nilsson E, Jaremo P. Relationships between platelets and inflammatory markers in rheumatoid arthritis. Clin Chim Acta 2004;343:237–40. 3. Schmitt-Sody M, Metz P, Gottschalk O, Birkenmaier C, Zysk S, Veihelmann A, et al. Platelet P-selectin is significantly involved in leukocyte-endothelial cell interaction in murine antigen-induced arthritis. Platelets 2007;18:365–72. 4. Rho YH, Oeser A, Chung CP, Milne GL, Stein CM. Drugs used in the treatment of rheumatoid arthritis: relationship between current use and cardiovascular risk factors. Arch Drug Inf 2009;2:34–40. 5. Saag KG, Teng GG, Patkar NM, Anuntiyo J, Finney C, Curtis JR, et al. American College of Rheumatology 2008 recommendations for the use of nonbiologic and biologic disease-modifying antirheumatic drugs in rheumatoid arthritis. Arthritis Rheum 2008;59:762–84.
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362 6. Maini RN, Breedveld FC, Kalden JR, Smolen JS, Furst D, Weisman MH, et al. Sustained improvement over two years in physical function, structural damage, and signs and symptoms among patients with rheumatoid arthritis treated with infliximab and methotrexate. Arthritis Rheum 2004;50:1051–65. 7. Bongartz T, Sutton AJ, Sweeting MJ, Buchan I, Matteson EL, Montori V. Anti-TNF antibody therapy in rheumatoid arthritis and the risk of serious infections and malignancies: systematic review and meta-analysis of rare harmful effects in randomized controlled trials. J Am Med Assoc 2006;295:2275–85. 8. Bhat AS, Tandan SK, Kumar D, Krishna V, Prakash VR. Interaction between inhibitors of inducible nitric oxide synthase and cyclooxygenase in adjuvant-induced arthritis in female albino rats: an isobolographic study. Eur J Pharmacol 2007;556:190–9. 9. Veale DJ, Fearon U. Inhibition of angiogenic pathways in rheumatoid arthritis: potential for therapeutic targeting. Best Pract Res Clin Rheumatol 2006;20:941–7. 10. Bansback N, Ara R, Ward S, Anis A, Choi HK. Statin therapy in rheumatoid arthritis: a cost-effectiveness and value-of-information analysis. Pharmacoeconomics 2009;27:25–37. 11. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet 1994;344:1383–9. 12. MRC/BHF Heart Protection Study of cholesterol lowering with simvastatin in 20,536 high-risk individuals: a randomised placebocontrolled trial. Lancet 2002;360:7–22. 13. Kobashigawa JA, Katznelson S, Laks H, Johnson JA, Yeatman L, Wang XM, et al. Effect of pravastatin on outcomes after cardiac transplantation. N Engl J Med 1995;333:621–7. 14. Katznelson S, Wilkinson AH, Kobashigawa JA, Wang XM, Chia D, Ozawa M, et al. The effect of pravastatin on acute rejection after kidney transplantation – a pilot study. Transplantation 1996; 61:1469–74. 15. Kwak B, Mulhaupt F, Myit S, Mach F. Statins as a newly recognized type of immunomodulator. Nat Med 2000;6:1399–402. 16. Lazzerini PE, Lorenzini S, Selvi E, Capecchi PL, Chindamo D, Bisogno S, et al. Simvastatin inhibits cytokine production and nuclear factor-kB activation in interleukin 1beta-stimulated synoviocytes from rheumatoid arthritis patients. Clin Exp Rheumatol 2007;25:696–700. 17. Romano M, Diomede L, Sironi M, Massimiliano L, Sottocorno M, Polentarutti N, et al. Inhibition of monocyte chemotactic protein-1 synthesis by statins. Lab Invest 2000;80:1095–100. 18. Weitz-Schmidt G, Welzenbach K, Brinkmann V, Kamata T, Kallen J, Bruns C, et al. Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site. Nat Med 2001;7:687–92. 19. Yokota K, Miyazaki T, Hirano M, Akiyama Y, Mimura T. Simvastatin inhibits production of interleukin 6 (IL-6) and IL-8 and cell proliferation induced by tumor necrosis factor-alpha in fibroblast-like synoviocytes from patients with rheumatoid arthritis. J Rheumatol 2006;33:463–71. 20. Albert MA, Danielson E, Rifai N, Ridker PM. Effect of statin therapy on C-reactive protein levels: the pravastatin inflammation/ CRP evaluation (PRINCE): a randomized trial and cohort study. J Am Med Assoc 2001;286:64–70. 21. Deng J, Wu Q, Liao Y, Huo D, Yang Z. Effect of statins on chronic inflammation and nutrition status in renal dialysis patients: a systematic review and meta-analysis. Nephrology (Carlton) 2012;17:545–51. 22. Wenke K, Meiser B, Thiery J, Nagel D, von Scheidt W, Krobot K, et al. Simvastatin initiated early after heart transplantation: 8-year prospective experience. Circulation 2003;107:93–7. 23. Leung BP, Sattar N, Crilly A, Prach M, McCarey DW, Payne H, et al. A novel anti-inflammatory role for simvastatin in inflammatory arthritis. J Immunol 2003;170:1524–30. 24. Abdin AA, Abd El-Halim MS, Hedeya SE, El-Saadany AA. Effect of atorvastatin with or without prednisolone on Freund’s adjuvant induced-arthritis in rats. Eur J Pharmacol 2012;676:34–40.
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O Gottschalk et al 25. Abud-Mendoza C, de la Fuente H, Cuevas-Orta E, Baranda L, CruzRizo J, Gonzalez-Amaro R. Therapy with statins in patients with refractory rheumatic diseases: a preliminary study. Lupus 2003; 12:607–11. 26. Kanda H, Yokota K, Kohno C, Sawada T, Sato K, Yamaguchi M, et al. Effects of low-dosage simvastatin on rheumatoid arthritis through reduction of Th1/Th2 and CD4/CD8 ratios. Mod Rheumatol 2007;17:364–8. 27. McCarey DW, McInnes IB, Madhok R, Hampson R, Scherbakov O, Ford I, et al. Trial of Atorvastatin in Rheumatoid Arthritis (TARA): double-blind, randomised placebo-controlled trial. Lancet 2004;363:2015–21. 28. Shirinsky IV, Zheltova OI, Solovyova NY, Kozlov VA, Shirinsky VS. Changes in disease activity, cytokine production, and proliferation of peripheral blood mononuclear cells in patients with rheumatoid arthritis after simvastatin treatment. Scand J Rheumatol 2009;38:23–7. 29. Kok SH, Lin LD, Hou KL, Hong CY, Chang CC, Hsiao M, et al. Simvastatin inhibits cysteine-rich protein 61 expression in rheumatoid arthritis synovial fibroblasts through the regulation of sirtuin-1/FoxO3a signaling. Arthritis Rheum 2013;65:639–49. 30. Brackertz D, Mitchell GF, Vadas MA, Mackay IR. Studies on antigen-induced arthritis in mice. III. Cell and serum transfer experiments. J Immunol 1977;118:1645–8. 31. Brackertz D, Mitchell GF, Vadas MA, Mackay IR, Miller JF. Studies on antigen-induced arthritis in mice. II. Immunologic correlates of arthritis susceptibility in mice. J Immunol 1977;118: 1639–44. 32. Brackertz D, Mitchell GF, Mackay IR. Antigen-induced arthritis in mice. I. Induction of arthritis in various strains of mice. Arthritis Rheum 1977;20:841–50. 33. Veihelmann A, Harris AG, Krombach F, Schutze E, Refior HJ, Messmer K. In vivo assessment of synovial microcirculation and leukocyte-endothelial cell interaction in mouse antigen-induced arthritis. Microcirculation 1999;6:281–90. 34. Veihelmann A, Szczesny G, Nolte D, Krombach F, Refior HJ, Messmer K. A novel model for the study of synovial microcirculation in the mouse knee joint in vivo. Res Exp Med (Berl) 1998;198:43–54. 35. Harris AG, Hecht R, Peer F, Nolte D, Messmer K. An improved intravital microscopy system. Int J Microcirc Clin Exp 1997; 17:322–7. 36. Schmitt-Sody M, Klose A, Gottschalk O, Metz P, Gebhard H, Zysk S, et al. Platelet-endothelial cell interactions in murine antigeninduced arthritis. Rheumatology (Oxford) 2005;44:885–9. 37. Tangelder GJ, Slaaf DW, Arts T, Reneman RS. Wall shear rate in arterioles in vivo: least estimates from platelet velocity profiles. Am J Physiol 1988;254:H1059–64. 38. Turesson C, Jacobsson LT, Matteson EL. Cardiovascular comorbidity in rheumatic diseases. Vasc Health Risk Manag 2008;4:605–14. 39. Youssef S, Stuve O, Patarroyo JC, Ruiz PJ, Radosevich JL, Hur EM, et al. The HMG-CoA reductase inhibitor, atorvastatin, promotes a Th2 bias and reverses paralysis in central nervous system autoimmune disease. Nature 2002;420:78–84. 40. Kotyla PJ, Sliwinska-Kotyla B, Kucharz EJ. Tumor necrosis factoralpha as a potential target in the treatment of systemic lupus erythematosus: a role for the HMG-CoA reductase inhibitor simvastatin. J Rheumatol 2006;33:2361–3. 41. He M, Liang X, He L, Wen W, Zhao S, Wen L, et al. Endothelial dysfunction in rheumatoid arthritis: the role of monocyte chemotactic protein-1-induced protein. Arterioscler Thromb Vasc Biol 2013;33:1384–91. 42. Kuipers HF, van den Elsen PJ. Immunomodulation by statins: inhibition of cholesterol vs. isoprenoid biosynthesis. Biomed Pharmacother 2007;61:400–7. 43. Rezaie-Majd A, Prager GW, Bucek RA, Schernthaner GH, Maca T, Kress HG, et al. Simvastatin reduces the expression of adhesion molecules in circulating monocytes from hypercholesterolemic patients. Arterioscler Thromb Vasc Biol 2003;23:397–403.
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Simvastatin reduces inflammation in AiA 44. Feldmann M. Pathogenesis of arthritis: recent research progress. Nat Immunol 2001;2:771–3. 45. Seidel MF, Keck R, Vetter H. ICAM-1/LFA-1 expression in acute osteodestructive joint lesions in collagen-induced arthritis in rats. J Histochem Cytochem 1997;45:1247–53. 46. Yamagata T, Kinoshita K, Nozaki Y, Sugiyama M, Ikoma S, Funauchi M. Effects of pravastatin in murine collagen-induced arthritis. Rheumatol Int 2007;27:631–9. 47. Eccles KA, Sowden H, Porter KE, Parkin SM, HomerVanniasinkam S, Graham AM. Simvastatin alters human endothelial cell adhesion molecule expression and inhibits leukocyte adhesion under flow. Atherosclerosis 2008;200:69–79. 48. Oka H, Ikeda S, Koga S, Miyahara Y, Kohno S. Atorvastatin induces associated reductions in platelet P-selectin, oxidized lowdensity lipoprotein, and interleukin-6 in patients with coronary artery diseases. Heart Vessels 2008;23:249–56. 49. Chou TC, Lin YF, Wu WC, Chu KM. Enhanced nitric oxide and cyclic GMP formation plays a role in the anti-platelet activity of simvastatin. Br J Pharmacol 2008;153:1281–7.
363 50. Laufs U, Liao JK. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem 1998;273:24266–71. 51. Vandebriel RJ, de Jong HJ, Gremmer ER, Klungel OH, Tervaert JW, Slob W, et al. Statins accelerate the onset of collagen type II-induced arthritis in mice. Arthritis Res Ther 2012;14:R90. 52. de Jong HJ, Klungel OH, van Dijk L, Vandebriel RJ, Leufkens HG, van der Laan JW, et al. Use of statins is associated with an increased risk of rheumatoid arthritis. Ann Rheum Dis 2012;71:648–54. 53. Lodi S, Evans SJ, Egger P, Carpenter J. Is there an antiinflammatory effect of statins in rheumatoid arthritis? Analysis of a large routinely collected claims database. Br J Clin Pharmacol 2010;69:85–94. 54. Palmer G, Chobaz V, Talabot-Ayer D, Taylor S, So A, Gabay C, et al. Assessment of the efficacy of different statins in murine collagen-induced arthritis. Arthritis Rheum 2004;50:4051–9.
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Scand J Rheumatol 2014;43:356–363
Simvastatin reduces leucocyte– and platelet–endothelial cell interaction in murine antigen-induced arthritis in vivo O Gottschalk1,2, ML Dao Trong1, P Metz2, J Wallmichrath2, S Piltz2, KW Jauch2, V Jansson3, M Schmitt-Sody1,3,4
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1 Walter Brendel Centre, 2Department of Surgery Großhadern, 3Department of Orthopaedic Surgery Großhadern, Ludwig Maximilians University of Munich, and 4Medical Park Chiemsee, Bernau-Felden, Germany
Objectives: The use of statins in the prevention and treatment of cardiovascular diseases is well established. Their use as anti-inflammatory and immunomodulatory agents in the treatment of rheumatoid arthritis (RA) has also been investigated, with several clinical and experimental studies indicating an anti-inflammatory effect of statins for RA, but other studies showing no effect or even the opposite. The current study was designed to examine the effect of simvastatin in an in vivo murine model of arthritis using intravital microscopy. Method: We assigned four groups (n = 7, female C57Bl6 mice), two with and two without antigen-induced arthritis (AiA), from which one of the non-AiA groups and one of the AiA groups were treated with simvastatin 40 mg/kg i.p. daily for 14 consecutive days after induction of arthritis. Platelet– and leucocyte–endothelial cell interaction was assessed by measurement of rolling and adherent fluorescence-labelled platelets and leucocytes, functional capillary density (FCD) was evaluated, and knee joint diameter was determined as a clinical parameter. Results: In arthritic mice treated with simvastatin, a significant reduction in platelet– and leucocyte–endothelial cell interaction was observed in comparison to arthritic mice treated with vehicle. In addition, a significant reduction in FCD was seen in arthritic mice treated with simvastatin, along with a reduction in knee joint swelling of the AiA mice. Conclusions: Treatment of AiA mice with simvastatin showed significant reductions in platelet– and leucocyte– endothelial cell interactions, in FCD, and in the swelling of the knee joint. These results support the hypothesis of the anti-inflammatory effects of statins in the treatment of RA.
Rheumatoid arthritis (RA) is a chronic inflammatory disease whose pathophysiology is not completely understood. An autoimmune reaction against synovial tissue leads consecutively to destruction of the entire joint. RA is the most common inflammatory disease in middle-aged adults, with a prevalence of about 0.5–1%; women are three to four times more commonly affected than men (1). Circulating mediators of inflammation and leucocyte– endothelial cell interaction in the synovial microcirculation are major factors involved in the course of the disease. Platelets seem to play a crucial role in this leucocyte–endothelial cell interaction and thus in the disease activity and progression of RA (2, 3). Current drug treatment protocols involve the use of non-steroidal anti-inflammatory drugs (NSAIDs), steroids, and disease-modifying anti-rheumatic drugs (DMARDs) (4). DMARDs influence the preservation of joint function by modifying and suppressing RA disease activity if early treatment is provided (5). Tumour necro-
Oliver Gottschalk, Department of Surgery, LMU – Campus Großhadern, Marchioninistrasse 15, 81377 Munich, Germany. E-mail: [email protected]
sis factor (TNF)-α inhibitors have an effect on disease activity and on the radiological evidence of progression, in addition to a lack of gastrointestinal sideeffects, compared to basic drugs (6). However, there are reports of a biologically related higher risk of rare serious infections or malignancies (7). Other therapy approaches such as inhibition of nitric oxide (NO) synthesis (8), inhibition of angiogenic pathways (9), and therapy with statins (10) have been discussed. Statins are well established in the prophylaxis of cardiovascular diseases by decreasing the morbidity/ mortality in a multifactorial manner (11, 12). The antiinflammatory role of statins was first investigated 1995 by Kobashigawa et al (13), who described a positive effect of pravastatin on the rejection rate after heart transplantation. A significant reduction in acute rejections after kidney transplantation was also described (14). Several in vitro studies have shown the immunomodulatory and anti-inflammatory effects of statins by induction of the expression of major histocompatibility complex (MHC)-II, thus inhibiting T-cell activation, reduction of the chemokine production involved in leucocyte migration, selective inhibition of leukocyte function antigen (LFA)-1-mediated adhesion and T-cell co-stimulation
Accepted 27 December 2013 © 2014 Informa Healthcare on license from Scandinavian Rheumatology Research Foundation
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DOI: 10.3109/03009742.2013.879606
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and reduction of interleukin (IL)-6 and IL-8 (15–19). Clinical studies support these findings by demonstrating significantly decreasing levels of C-reactive protein (CRP) (20, 21) and a significantly better 8-year survival rate with a lower incidence of transplant vasculopathies after heart transplantation (22). Leung et al showed that simvastatin reduces the production of pro-inflammatory cytokines [TNF-α, IL-6, IL-12, and interferon (IFN)-γ] through T-cell-activated macrophages in murine collagen-induced arthritis (23). Atorvastatin in combination with prednisolone had a beneficial effect on Freund’s adjuvant-induced arthritis in rats (24). Clinical improvement in RA patients was shown compared to therapy with chloroquine by decreases in CRP levels and erythrocyte sedimentation rate (ESR); reduction of the T-helper (Th)1/Th2 and CD4/CD8 ratios was described (25–27). A positive correlation between clinical improvement and IL-6 reduction was reported, and elevated baseline circulating IL-10 concentrations as a positive predictive factor for treatment response postulated (28). Kok et al (29) described a pathway showing that cysteine-rich protein 61 (CYR-61) plays an important role in the pathogenesis of RA and simvastatin induces an up-regulation of SIRT-1/FoxO3a signalling in synovial fibroblasts (SFs), which is important for the induction of CYR-61 in RA SFs. The aim of this study was to evaluate the effects of simvastatin on the leucocyte– and platelet–endothelial cell interaction as an inflammatory response and activity on the well-established in vivo murine model of AiA (30–32).
flank of 100 mg methylated bovine serum albumin (mBSA; Sigma, Deisenhofen, Germany), dissolved in 50 mL Freund’s complete adjuvant (Sigma), and supplemented with 2 mg/mL heat-killed Mycobacterium tuberculosis strain H37RA (Difco, Augsburg, Germany), and an additional intraperitoneal (i.p.) injection of 2 109 heatkilled Bortadella pertussis (Institute of Microbiology, Berlin, Germany). On day 0, arthritis was inducted by injection of 100 μg mBSA in 50 μL saline into the left knee joint. Control animals underwent the same procedure for pre-immuninization but received an equivalent volume of saline in the knee joint instead of mBSA.
Method
Surgical preparation
Animals and study design
In preparation for intravital fluorescence microscopy, on day 14, the mice were anaesthetized by inhalation of isoflurane 1.5% (Forence, Abbott, Wiesbaden, Germany) and a mixture of O2/N2O. They were fixed on a warmed plate on their back with the left knee embedded in a 45 flexion on a custom-made plexiglas block. Arterial and venous catheters were implanted in the tail. The intra-articular synovial tissue of the knee joint was then accessed through a 1-cm longitudinal incision over the knee, exposing the patellar tendon, which was then cut transversely at the tibial edge and flapped cranial (34).
We used female C57Bl6 mice (Charles-River, Sulzfeld, Germany) weighing between 18 and 24 g. The experiments were approved by, and performed according to, the German legislation for the protection of animals. The mice were randomly assigned to four groups (group 1: no arthritis, therapy with vehicle; group 2: with arthritis, therapy with vehicle; group 3: no arthritis, therapy with simvastatin; group 4: with arthritis, therapy with simvastatin), each containing seven valid animals (n = 7). In addition to the daily assessment of their weight, fur, and behaviour, we evaluated the swelling of the knee joint by measuring the transverse diameter to assess the severity of the AiA until the examination with intravital microscopy. Mice that showed alterations in their behaviour and fur were excluded from the study. AiA We used the well-developed model of Brackertz et al (32), which has been used previously in our department (33). Before the induction of arthritis, the mice were immunized on days –21 and –14 by a subcutaneous injection in the left
Preparation of simvastatin For the production of a 10 mM stock, 41.8 mg of simvastatin were dissolved in 1.5 mL 0.1 N NaOH and 1 mL ethanol and the solution incubated at 50 C for 2 h, which converted it from the inactive lactone form to the active acid form. After pH adjustment, the volume was corrected with phosphate-buffered saline (PBS) to 10 mL.
Treatment protocol We treated the mice in groups 3 and 4 with simvastatin i.p. for 14 consecutive days (40 mg/kg/day) and the mice in groups 1 and 2 with an equivalent volume of vehicle, starting on the day of arthritis induction (day 0) according to Leung et al (23).
Intravital fluorescence microscopy The microscopic set-up we used has been described previously (35). We investigated and stained platelets ex vivo from a donor mouse, leucocytes in vivo with rhodamine 6G, and plasma in vivo with fluorescein isothiocyanate (FITC)–Dextran (both Sigma, Deisenhofen, Germany) (36). To isolate platelets from a syngenic donor mouse we used cardiac puncture to obtain a 1-mL blood sample, which then was prepared with 0.2 mL of Alsevers puffer, 20 μL of prostaglandin E1 (PGE1, Serva, Heidelberg,
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Germany), and 0.5 mL of Dulbecco PBS (D-PBS, PANSystems, Aidenbach, Germany). The platelets were separated by centrifugation, resuspended with 1.5 mL D-PBS, 0.3 mL Alsevers, and 50 μL PGE1, labelled with the fluorescent marker carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; Molecular Probes, Eugene, OR, USA), and finally centrifuged and resuspended in 0.4 mL D-PBS. The purity of the sample and the platelet concentration were determined before application with a Coulter Counter (Coulter Corp, Miami, FL, USA). A total of 100 106 fluorescently labelled platelets were transfused through the lateral tail vein, corresponding to approximately 10% of all circulating platelets (37). Intravital microscopic images were recorded on videotape and data analysis was performed later offline using a computerassisted analysis system (CAP-Image, Dr Zeintl, Heidelberg, Germany).
Microhaemodynamic parameters Vessel diameter, venular red blood cell (RBC) velocity, and functional capillary density (FCD) were measured by injection of FITC-Dextran at the end of each experiment. FCD was defined as the length of respective capillaries vessels within the observation area (cm/cm2).
Microcirculatory parameters Platelet– and leucocyte–endothelial cell interactions were measured as rolling or adherent cells. Rolling cells were defined as cells that passed the vessel at a velocity significantly lower than the centreline velocity in the observed microvessel and were determined as the fraction of all platelets or leucocytes passing this predefined vessel segment within an observation interval of 30 s. Adherent cells were defined as cells that were attached to the endothelial lining and did not move within an observation interval of 30 s. Adherence was quantified as the number of cells per square millimetre of endothelial surface and calculated from the diameter and length of the observed vessel segment (1/mm2).
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Statistical analysis Quantitative data are expressed as means SEM. Comparison between multiple independent study groups was performed by a Kruskal–Wallis one-way analysis of variance (ANOVA) test on ranks. In case of significance (p < 0.05), a paired comparison was carried out by the Student–Newman–Keuls method. Analysis of repetitive measurement of multiple groups was performed using a two-way ANOVA test for combined samples.
Results Clinical parameters The induction of arthritis was monitored by swelling of the knee joint (Figure 1). After induction of arthritis on day 0, there was a significant increase in knee joint diameter in every group, confirming successful intraarticular injection. On day 2, the knee joint diameter in the AiA statin group was significantly higher than in the other groups (*p < 0.05). From day 3 to day 12, the knee joint diameter decreased back to baseline values in both non-arthritic groups compared to both arthritic groups (**p < 0.05). During the period of treatment, the increase in diameter persisted in the arthritic group treated with vehicle, whereas there was a significant decrease in knee joint diameter in the arthritic group treated with statin starting on day 9 (#p < 0.05). No deficits in the appearance and behaviour of the mice were observed. Body weight was stable at 18–21 g during the study period.
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All of the knee joints were collected together after intravital microscopy. They were fixated in paraformaldehyde and then decalcified [ethylenediaminetetraacetic acid, tetrasodium salt (EDTA-4Na) and 20% citric acid] for 72 h and embedded in paraffin. The samples were cut into slices of thickness 3 μm. Histological examination of the knee was performed with haematoxylin and eosin (H&E) staining and scored semiquantitatively following Brackertz et al (32) (0 = normal knee score, 1 = normal synovium, 2 = two or more lining cells and perivascular infiltrates, 4 = synovitis and pannus formation and cartilage/subchondral bone erosions).
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time Control vehicle Control statin AiA control AiA statin Figure 1. Change in knee joint diameter during the time of examination. Values given as mean SEM (n = 7). *p < 0.05 AiA statins vs. other groups; **p < 0.05 both AiA groups vs. non-arthritic groups; #p < 0.05 AiA statins vs. AiA control.
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Table 1. Microhaemodynamic parameters at the time of intravital microscopy. Group
Vessel diameter (μm)
RBC velocity (mm/s)
FCD (cm/cm2)
11.3 0.39 9.6 0.33 12.04 0.60 11.86 0.71
0.24 0.02 0.29 0.03 0.18 0.02 0.29 0.09
313.8 16.35 298.47 10.90 446.20 20.85 335 15.58*
Control vehicle Control statin AiA vehicle AiA statin
* Significant decrease for the AiA group treated with simvastatin compared to the AiA group treated with vehicle (p < 0.001).
No significant changes in vessel diameter and RBC velocity were seen between the different groups, apart from a slight decrease in RBC velocity in the AiA vehicle group (Table 1). With regard to the FCD (Figure 2), there was no significant difference between the non-arthritic groups (vehicle FCD = 313.8 6.4 cm/cm2, statin FCD = 298.3 9.6 cm/cm2) but we did observe a significant increase in the arthritic group treated with vehicle (FCD = 446.2 20.9 cm/cm2). In the AiA groups, the one treated with simvastatin (FCD = 338 15.6 cm/cm2; p < 0.001) showed a significant decrease in FCD compared to the vehicle group.
Histological results Histological analysis confirmed the inducation of arthritis by showing a severity score of 0 ¼ normal knee joint, 1 ¼ normal synovium with occasional mononuclear cells, 2 ¼ 2 or more synovial lining cells and perivascular infiltrates of leukocytes, 3 ¼ hyperplasia of synovium and dense infiltration, 4 ¼ synovitis, pannus formation, and cartilage/subchondral bone erosions.
*
500
Although the results were not significant, we obtained lower scores for the samples of AiA mice treated with simvastatin. Both control groups (no AiA) presented in all animals a normal knee score (severity score 0). The AiA group treated with vehicle showed mostly animals with a severity score of 3 or 4 whereas the statin-treated group presented a slight hyperplasia of the synovium and dense infiltration (score of 3), or scores of a lower level with some perivascular infiltrates of leukocytes (2 or smaller). Platelet–endothelial cell interaction in vivo A significant reduction in adherent platelets was seen in the AiA group treated with statins compared to the AiA vehicle group (AiA statin 170 71.6 cells/mm2 vs. AiA vehicle 502 102.1 cells/mm2; p < 0.05) (Figure 3A), almost down to the baseline values of the non-arthritic groups. The fraction of rolling platelets also decreased, although to a lesser extent, when treated with statins (AiA statins 0.027 0.007 vs. AiA vehicle 0.045 0.0045 (Figure 4A). The results are shown in Figures 5A–5C. Leucocyte–endothelial cell interaction in vivo The results for leucocyte–endothelial cell interaction were similar to those for the platelet–endothelial cell interaction in being favourable to simvastatin treatment (Figures 3B and 4B). A significant increase in the fraction of rolling and adherent leucocytes was observed in the AiA group treated with vehicle (0.095 0.021 cells/mm2 for rolling and 1125 139.5 cells/mm2 for adherent leucocytes) compared to both non-arthritic groups. There was a significant decrease in both rolling and adherent parameters in the AiA group treated with simvastatin (0.025 0.006; p < 0.05 and 443 97.8 cells/mm2; p < 0.001) compared to the arthritic vehicle group. Furthermore, the decrease went down to almost baseline values of the non-arthritic control group. The results are shown in Figures 6A–6C.
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Microhaemodynamic parameters
Figure 2. Functional capillary density (FCD), defined as the length of red blood cell (RBC)-perfused capillaries within the region of interest (ROI), for AiA and control groups. Values given as mean SEM (n = 7). *p < 0.05 AiA control vs. other groups.
Discussion Statins are used successfully in the primary and secondary prevention of cardiovascular diseases. They are also currently approved as prophylactic therapy for the higher comorbidity of cardiovascular diseases in rheumatic diseases (38). Their anti-inflammatory effects have been demonstrated in heart and kidney transplantations, in
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Figure 3. Number of (A) platelets and (B) leucocytes adherent (per mm2) to the endothelium in post-capillary venules in the synovium of the mouse knee joint (n = 7). Values given as mean SEM. *p < 0.05 A+B: AiA vehicle vs. other groups.
Figure 4. Fraction of (A) rolling platelets and (B) rolling leucocytes, given as the number of rolling cells divided by the sum of the rolling and non-adherent cells (n = 7). Values given as mean SEM. *p < 0.05 A: both AiA groups vs. non-arthritic groups; B: AiA vehicle vs. other groups.
atherosclerosis as an inflammatory disease, and within autoimmune diseases such as multiple sclerosis (39), systemic lupus erythematosus (40), and RA. These socalled pleiotropic effects are mainly explained by the inhibition of 3-hydroxy-3-methylglutaryl-coenzyme
A (HMG-CoA) reductase and thus the mevalonate pathway. At a cellular level, the anti-inflammatory effect has been explained by multiple pathways, including modulation of the expression, secretion, and function of a set of immunomediators that eventually leads to a reduction in
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Figure 5. Intravital microscopic visualization of CFDA-labelled platelets in (A) a non-arthritic mouse, (B) an arthritic mouse treated with vehicle, and (C) an arthritic mouse treated with simvastatin.
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Simvastatin reduces inflammation in AiA
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Figure 6. Intravital microscopic visualization of rhodamine 6G-labelled leucocytes in (A) a non-arthritic mouse, (B) an arthritic mouse treated with vehicle, and (C) an arthritic mouse treated with simvastatin.
CRP and pro-inflammatory cytokines, inhibition of INFγ-induced MHC-II expression, suppression of dendritic cell maturation, reduction of monocyte–endothelial cell adhesion (41), and many other inflammatory mechanisms (15, 27, 42, 43). RA is an MHC-II-associated, chronic inflammatory autoimmune disease, in which TNF-α has a crucial role because of its regulatory properties among the expression of other pro-inflammatory cytokines (44). It leads to activation of platelets and to a Th1-weighted immune response with activation and interaction between endothelial cells, macrophages, and B and T lymphocytes. This consequently leads to cell rolling through selectins and cell adhesion through LFA-1 integrins on leucocytes on the activated endothelium through intercellular adhesion molecule (ICAM)-1 adhesion molecules (45). Our results show significant decreases in platelet– and leucocyte–endothelial cell interaction following simvastatin treatment, representing the activity of inflammation. The FCD decreased as an expression of antiangiogenesis, thus supporting the hypothesis of the antiinflammatory effect of statins on RA. Additionally, we observed a slight reduction in all parameters in the nonarthritic group treated with simvastatin. These findings could indicate a protective character of statins for RA. The significant decrease in knee joint diameter in arthritic knee joints during the time of treatment with simvastatin compared to the treatment with vehicle strengthens the notion of an anti-inflammatory effect of simvastatin on clinical symptoms. In a study conducted on murine collagen-induced arthritis investigating the effects of pravastatin on clinical outcome, serum antiCII antibody levels, histology and immunohistochemistry, similar findings with a reduction in synovial tissue inflammation were described (46). A decrease in rolling platelets and leucocytes could be explained by the inhibition of platelet and endothelial P-selectins by statins as described by Eccles et al (47) and Oka et al (48). The importance of the P-selectins on these interactions has been shown previously in our AiA model (3). The reduction of adherent cells may be accounted for by the causal cohesion between rolling and adhering,
whereas another explanation could be the inhibition of the expression of the adhesion molecules ICAM-1 and LFA-1. Furthermore, it seems that statins stimulate the production of endothelial and platelet NO synthesis, and NO is a potent inhibitor of platelet function and aggregation (49). This effect is thought to be mediated by inhibition of Rho kinase geranylgeranyl phosphorylation by statins (50). Of note, recent studies postulate the opposite hypothesis, stating that statins accelerate the onset of collagen type II-induced arthritis in mice through induction of autoimmunity (51). Other studies also suggest a nonbeneficial effect of statins in RA (52–54). Further investigation is therefore required to explain why such significantly different results have been reported in the literature either in clinical or in experimental studies. We chose the AiA model because it enabled us to examine the local and controlled arthritis induction of a single joint. This allowed us to look specifically at the inflammatory process of the knee joint in comparison to the systemic analyses described in the above-mentioned murine studies, where an in vivo study with intravital microscopy at one joint was not possible. In conclusion, our results confirm the antiinflammatory effect of statins in murine AiA in vivo and therefore statins could be a new therapy option in the treatment of arthritis. References 1. Scott DL, Wolfe F, Huizinga TW. Rheumatoid arthritis. Lancet 2010;376:1094–108. 2. Milovanovic M, Nilsson E, Jaremo P. Relationships between platelets and inflammatory markers in rheumatoid arthritis. Clin Chim Acta 2004;343:237–40. 3. Schmitt-Sody M, Metz P, Gottschalk O, Birkenmaier C, Zysk S, Veihelmann A, et al. Platelet P-selectin is significantly involved in leukocyte-endothelial cell interaction in murine antigen-induced arthritis. Platelets 2007;18:365–72. 4. Rho YH, Oeser A, Chung CP, Milne GL, Stein CM. Drugs used in the treatment of rheumatoid arthritis: relationship between current use and cardiovascular risk factors. Arch Drug Inf 2009;2:34–40. 5. Saag KG, Teng GG, Patkar NM, Anuntiyo J, Finney C, Curtis JR, et al. American College of Rheumatology 2008 recommendations for the use of nonbiologic and biologic disease-modifying antirheumatic drugs in rheumatoid arthritis. Arthritis Rheum 2008;59:762–84.
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