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Published online September 5, 2007
Diabetes Care 30:3122-3124, 2007
DOI: 10.2337/dc07-0919
© 2007 by the American Diabetes Association
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Cardiovascular and Metabolic Risk
Original Research

Simvastatin Reduces Plasma Osteoprotegerin in Type 2 Diabetic Patients With Microalbuminuria

Birgitte Nellemann, RF1, Lars C. Gormsen, MD1, Jens Dollerup, MSC2, Ole Schmitz, MD3, Carl E. Mogensen, MD1, Lars M. Rasmussen, MD4,5 and Søren Nielsen, MD1

1 Medical Department M, Aarhus University Hospital, Aarhus, Denmark
2 Merck Research Laboratories, Copenhagen, Denmark
3 Pharmacology, Aarhus University Hospital, Aarhus, Denmark
4 Laboratory for Biochemical Pathology, Aarhus University Hospital, Aarhus, Denmark
5 Department of Clinical Biochemistry, Odense University Hospital, Odense, Denmark

Address correspondence and reprint requests to Søren Nielsen, MD, Aarhus University Hospital, Nørrebrogade 44, 8000 Aarhus C, Denmark. E-mail: nielsen.soren{at}dadlnet.dk

Abbreviations: CVD, cardiovascular disease • ICAM, intercellular adhesion molecule • OPG, osteoprotegerin • RANK, receptor activator of nuclear factor-{kappa}B • RANKL, RANK ligand • TNF, tumor necrosis factor • VCAM, vascular cell adhesion molecule


   INTRODUCTION
TOP
 INTRODUCTION
RESEARCH DESIGN AND METHODS--
 RESULTS--
 CONCLUSIONS--
 References
 
Osteoprotegerin (OPG), a secreted glycoprotein and member of the tumor necrosis factor (TNF) receptor superfamily, is a soluble receptor activator of nuclear factor-{kappa}B (RANK) ligand (RANKL) and TNF-related apoptosis-inducing ligand (1). OPG works as a decoy receptor preventing RANK/RANKL-induced osteoclast differentiation and activation (2). Moreover, the RANK/RANKL system has potential cardiovascular effects; the system induces vascular cell adhesion molecule (VCAM)-1 synthesis, prolongs endothelial cell survival, and promotes angiogenesis (3,4). Furthermore, OPG may be involved in cardiovascular disease (CVD). An epidemiological study identified OPG as an independent risk factor for CVD (5) and OPG is present in high concentrations in the arterial wall (6,7). Of note, diabetic patients are characterized by elevated OPG (3), which is associated with subclinical atherosclerosis in both type 1 (8) and type 2 (9) diabetes. Conversely, OPG may inhibit calcification in mice (10). Hence, it is possible that vascular calcification increases OPG, which then, in turn, is involved in calcification inhibition (4).

Development of atherosclerosis involves expression of adhesion molecules (e.g., VCAM-1 and intercellular adhesion molecule [ICAM]), allowing cellular attachment and migration of monocytes and macrophages into the vascular wall (11). Recent in vitro studies suggest that statins may suppress both OPG (12) and adhesion molecule (13) production.

Statin treatment reduces cardiovascular disease in type 2 diabetes (14). Moreover, additional so-called pleiotropic effects have also been proposed (15). Since both OPG and adhesion molecules are associated with CVD and potentially modifiable by statins, we examined the effect of simvastatin on OPG and adhesion molecules in type 2 diabetic patients at increased risk for CVD due to persistent microalbuminuria.


   RESEARCH DESIGN AND METHODS—
TOP
 INTRODUCTION
 RESEARCH DESIGN AND METHODS--
RESULTS--
 CONCLUSIONS--
 References
 
Informed consent was obtained from all participants, and the study received ethics committee approval. Eighteen type 2 diabetic patients were randomly recruited from the outpatient clinic (16). Inclusion criteria were microalbuminuria (overnight urinary albumin excretion 15–200 µg/min), plasma cholesterol ≥5.5 mmol/l, plasma triglyceride ≤4.5 mmol/l, A1C <10%, serum C-peptide >0.49 nmol/l, and blood pressure ≤160/95 mmHg.

The study design has previously been described (16). In brief, in a randomized, double-blind design, patients were allocated to treatment with 10 mg/day simvastatin or the placebo group for 18 weeks. If plasma cholesterol was ≥5.2 mmol/l at 6 weeks, the dose was doubled. Blood samples for OPG, VCAM-1, and ICAM were collected after an overnight fast at baseline and week 18. Sampling for OPG was insufficient for one patient in the placebo group. Sample size was based on previously decided main outcome measurements of renal function (16). Since no interventional studies describing changes in OPG could be identified from the literature, we were unable to perform a valid sample size calculation. We therefore included all patients from that study.

OPG was measured by a sandwich enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN) using a mouse anti-human OPG as capture antibody and a biotinylated goat anti-human OPG for detection. Recombinant human OPG was used for calibration. Samples were diluted and measured in duplicate (8). Serum ICAM and VCAM-1 were measured by monoclonal antibody–based enzyme-linked immunosorbent assays as described by the manufacturer (catalog nos. BBE1B, BBE3, and DY809, respectively; R&D Systems).

Data are presented as means ± SEM. Between-group differences were analyzed using Student's t test or the Mann-Whitney two-sample test. Changes were also evaluated as the 18 weeks–to–baseline ratio. Correlations were evaluated by Pearson's r.


   RESULTS—
TOP
 INTRODUCTION
 RESEARCH DESIGN AND METHODS--
 RESULTS--
CONCLUSIONS--
 References
 
The treatment groups were similar with respect to age, sex, diabetes duration, BMI, A1C, and serum C-peptide. The average simvastatin dose was 12.5 mg/day. Cholesterol was significantly reduced by simvastatin. A1C remained unchanged.

OPG levels were comparable at baseline (1,660 ± 161 vs. 1,961 ± 131 pg/ml for the placebo vs. simvastatin groups, respectively) and after 18 weeks (1,684 ± 154 vs. 1,816 ± 95 pg/ml), and within-group changes were not statistically significant. However, simvastatin treatment was associated with a significant reduction of baseline–to–18 weeks OPG ratio compared with that in the placebo group (Fig. 1).


Figure 1
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  		Figure 1— Baseline–to–18 weeks OPG ratio: mean ± SEM 1.021 ± 0.035 vs. 0.932 ± 0.024 for the placebo (•) vs. simvastatin ({square}) groups; P < 0.05. A relative reduction of 7% was observed in the simvastatin group (P < 0.05). n = 17 (9 in the placebo group and 8 receiving simvastatin treatment). Medians are represented by solid lines.

 
No significant differences were observed at baseline in VCAM-1 (755 ± 64 vs. 690 ± 51 ng/ml for the placebo vs. simvastatin groups, respectively) or ICAM (307 ± 83 vs. 336 ± 33 ng/ml). Moreover, the 18 weeks–to–baseline ratios were comparable between groups.

There was no significant correlation between the change in cholesterol and OPG ratio in the simvastatin group or between total cholesterol and OPG, VCAM-1, or ICAM at baseline in the combined group. OPG and A1C tended to correlate at baseline (r2 = 0.46, P = 0.06) and week 18 (r2 = 0.48, P = 0.07). Plasma insulin (picomoles per liter) correlated significantly with OPG at week 18 (r2 = 0.56, P < 0.05).


   CONCLUSIONS—
TOP
 INTRODUCTION
 RESEARCH DESIGN AND METHODS--
 RESULTS--
 CONCLUSIONS--
References
 
In this study, low-dose simvastatin treatment for 18 weeks reduced OPG levels in type 2 diabetic patients with microalbuminuria and mild hypercholesterolemia. To our knowledge, this has not previously been demonstrated in humans in vivo.

The cellular mechanism whereby statins affect OPG is unclear. Statins modulate inflammatory mediators on OPG secretion. Thus, simvastatin reduces TNF-{alpha}–induced OPG levels in vitro (12) and directly inhibits the nuclear factor-{kappa}B system (12,17). Moreover, OPG mRNA and RANKL mRNA were increased in mouse bone-cell cultures incubated with simvastatin for 7–16 days (18). These results seem contrary to ours, since we found decreased OPG levels. However, the different study design (in vitro vs. in vivo) and treatment duration (7 and 16 days vs. 18 weeks) may partly explain the differences. Moreover, since OPG is involved in calcification inhibition, at least in mice (10), our findings may reflect a simvastatin-mediated calcification reduction, which, in turn, might downregulate OPG. In type 2 diabetes, microalbuminuria is not known to be related to altered bone mineral metabolism.

Contrary to previous reports (19), we found no change in VCAM-1 or ICAM. These adhesion molecules are induced by inflammatory cytokines (i.e., TNF-{alpha} and interleukin-1), oxidative stress, and oxidized LDL (20). In theory, the anti-inflammatory and LDL-lowering effects of simvastatin should, therefore, exert an inhibitory effect on adhesion molecule expression (17,19). To our knowledge, this has never been shown in human in vivo studies. Mulder et al. (21) recently reported that switching to more aggressive lipid lowering in patients already on statin treatment may not have further effects on adhesion molecule expression. We included statin-näive patients in the present study. Although the inclusion criteria do not adhere to today's standards of clinical care, we think that the interpretations of the results are not hampered in a major way. Our inability to show changes in adhesion molecules may be due to the relatively low statin dose, the study duration, or the number of patients included.

In summary, 18 weeks of low-dose simvastatin treatment reduced circulating OPG levels in type 2 diabetic patients with microalbuminuria but had no effect on VCAM-1 or ICAM. The reduction of OPG was independent of cholesterol and suggests a pleiotropic effect of simvastatin per se. The OPG-lowering effect of simvastatin may signal diminished vascular calcification.


   Acknowledgments
 
This study was supported by Novo Nordisk Research Foundation, the Danish Medical Research Council, and Merck Sharp & Dohme.

We thank L. Larsen, A. Mengel, and M. Møller for technical assistance.


   Footnotes
 
Published ahead of print at http://care.diabetesjournals.org on 5 September 2007. DOI: 10.2337/dc07-0919. Clinical trial reg. no. NCT00471549, clinicaltrials.gov.

A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received for publication May 14, 2007. Accepted for publication August 27, 2008.


   References
TOP
 INTRODUCTION
 RESEARCH DESIGN AND METHODS--
 RESULTS--
 CONCLUSIONS--
 References
 

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