Simvastatin Reduces Circulating Plasminogen Activator Inhibitor 1 Activity in Volunteers with the Metabolic Syndrome

Abstract

Background

The Metabolic Syndrome (MS) confers an increased risk for diabetes and cardiovascular disease. We previously showed that simvastatin has concomitant benefits in reducing low-density lipoprotein (LDL)–cholesterol and inflammation in MS subjects. The levels of plasminogen activator inhibitor 1(PAI-1), soluble P-selectin (sP-selectin), and soluble CD40 ligand (sCD40L) play an important role in the development and progression of atherosclerosis. Their levels are increased in the MS. The current study was to investigate the effects of simvastatin on PAI-1, sP-selectin, and sCD40 ligand.

Methods

Fifty subjects with MS were randomized into either placebo or simvastatin (40 mg/day) group for 8 weeks. Blood samples were obtained at baseline and at the end of the study. PAI-1 activity and sP-selectin and sCD40L levels were measured by enzyme-linked immunosorbent assay (ELISA).

Results

There was no baseline difference in any of the parameters studied. Compared to baseline, simvastatin significantly reduced (P < 0.05) the circulating PAI-1 activity (24.3 ± 5.2 IU/mL at baseline vs. 21.4 ± 3.9 IU/mL after 8 weeks of treatment). Simvastatin did not alter (P < 0.05) the levels of sP-selectin (111.4 ± 35.9 ng/mL at baseline vs. 118.5 ± 71.2 ng/mL after 8 weeks) or sCD40L (2.0 ± 1.6 ng/mL at baseline vs. 1.5 ± 1.0 ng/mL after 8 weeks).

Conclusion

Our data indicate that simvastatin therapy has significant effects on the fibrinolytic system in MS subjects as evidenced in a reduction in PAI-1 activity.

Introduction

The metabolic syndrome (MS) affects approximately one quarter of the US population.¹ It confers a two-fold increase in risk for cardiovascular disease (CVD) and a fivefold increase in risk for type 2 diabetes mellitus (T2DM).² The low-grade inflammation state in MS is pivotal in the development and progression of atherosclerotic plaques.³ CD40 ligand (CD40L) binds to CD40 and leads to downstream pathways that are proatherosclerotic.⁴ Soluble CD40 ligand (sCD40L) is elevated in MS.⁵ P-selectin is a cell adhesion molecule that mediates the initial recruitment of monocytes in plaque formation.⁶ Soluble P-selectin is increased in MS.⁵ Plasminogen activator inhibitor 1 (PAI-1) inhibits fibrinolysis, the physiological process that degrades blood clots.⁷ Increased PAI-1 is observed in obese subjects with MS as well as in patients with T2DM.^7,8 We have previously shown that simvastatin therapy (40 mg/day for 8 weeks) concomitantly reduced low-density lipoprotein (LDL) cholesterol levels and inflammation in subjects with MS when compared to placebo, as evidenced by decreased inflammation markers such as high-sensitivity C-reactive protein (hsCRP), tumor necrosis factor α (TNF-α), and monocyte interleukin 6 (IL-6) in volunteers with MS.⁹ but failed to have impact on adiponectin levels or insulin sensitivity.¹⁰ The objective of the current study was to investigate the effects of simvastatin on plasma sCD40L and sP-selectin levels as well as PAI-1 activity.

Subjects and Methods

All protocols were approved by the Institutional Review Board at the University of California, Davis, Medical Center. Informed consent was obtained for all volunteers. Fifty volunteers with MS, defined by the criteria of the National Cholesterol Education Panel Adult Treatment Panel III,¹⁰ were randomized into either placebo (n=25) or simvastatin (40 mg/d) group (n=25) for 8 weeks. Fasting blood was obtained at baseline and at the end of the study.

The effects on inflammation markers, adiponectin, and insulin sensitivity have been previously reported.^9,10 Plasma sP-selectin and sCD40L levels were determined using ELISA kits from R&D System (Minneapolis, MN) following the manufacturer's instructions. Plasma PAI-1 activity was determined using ELISA kit from American Diagnostic (Stamford, CT) following manufacturer's instructions. The coefficient of variation for sCD40L was <9%, for sP-selectin <8%, and for PAI-1 <10%.

Two-tail t test was performed using Microsoft Excel 2003 (Seattle, WA). Data were expressed as means ± SD. Significance was defined as P<0.05.

Results

As previously reported, there was no difference in baseline characteristics between the placebo and simvastatin group. Simvastatin significantly lowered LDL-cholesterol, hsCRP, and IL-6 levels, but failed to affect adiponectin level or insulin sensitivity.^9,10 There was no difference between the placebo and simvastatin group in any of the parameters studied at baseline (P>0.05). As depicted in Figure 1, an 8-week simvastatin therapy (40mg/d) significantly decreased plasma PAI-1 activity (P<0.05) at the end of the study compared to baseline and to placebo. As shown in Table 1, simvastatin therapy did not affect the plasma levels of sP-selectin or sCD40L (P>0.05).

FIG. 1.

Simvastatin therapy (40mg/d for 8 weeks) decreased plasma PAI-1 activity in subjects with the MS compared to baseline and placebo. Data were mean ± SD. *P < 0.05 compared to baseline and placebo

Table 1.

Effect of Simvastatin Therapy on Plasma Sp-Selectin and Scd40 Ligand Levels in Subjects with the MS

	Placebo		Simvastatin
Variable	Baseline	Week 8	Baseline	Week 8
sP-selectin (ng/mL)	119.9 ± 39.8	117.3 ± 41.7	111.4 ± 35.9	118.5 ± 71.2
sCD40 ligand (ng/mL)	2.6 ± 2.4	2.4 ± 2.1	2.0 ± 1.6	1.5 ± 1.0

Simvastatin therapy (40mg/d for 8 weeks) did not affect plasma levels of sCD40L or sP-selectin (P ≥ 0.05). Data were expressed as mean ±SD.

Discussion

The metabolic syndrome (MS) confers an increased risk for atherosclerosis and the proinflammatory state in MS is pivotal in all phases of atherosclerosis from the formation of nascent lesion to plaque rupture.³ Statin therapy has been shown to reduce the levels of LDL-cholesterol and proinflammatory cytokines.^9,12 The current study investigated the effect of simvastatin therapy (40 mg/d for 8 weeks) on three markers, PAI-1, sCD40L, and sP-selectin, which play an important role in fibrinolysis and coagulation, and therefore in development and progression of atherosclerosis.

In blood, PAI-1 inhibits tissue plasminogen activator and urokinase, and hence inhibits fibrinolysis, the physiological process that degrades blood clots.¹³ There is clinical evidence that elevated PAI-1 levels are associated with atherothrombosis in human.^14,15 PAI-1 is elevated in MS.^16,17 An animal study revealed that statins increased local fibrinolysis by reducing PAI-1 production, which may be partially mediated by intermediates of the cholesterol biosynthetic pathway that regulate Rho protein signaling.¹⁸ Data from in vitro studies indicated that simvastatin down regulated PAI-1 expression at both the transcriptional and translational levels in aortic endothelial cells, which was mediated by the PI3K pathway.¹⁹ The current study measured the actual circulating activity of PAI-1, which is more relevant with regard to the net effect of simvastatin therapy on PAI-1. The significant decrease in PAI-1 activity indicated the fibrinolysis in the simvastatin group added one more piece of evidence to the protective effects of simvastatin in people with MS.

CD40L is a member of the TNF family of molecules. It can exist in the soluble form as sCD40L. The CD40-CD40L dyad triggers downstream pathways that lead to the development and progression of atherosclerosis.⁴ Complete inhibition of the CD40L pathway led to a decrease in atherosclerosis and induced the phenotype of a stable atherosclerotic plaque.^20,21 Statin therapy has been shown to reduce oxidized LDL-induced CD40/CD40L overexpression and sCD40L in patients with coronary artery disease.²² In our group, a low-dose simvastatin therapy (20 mg/day for 3 months) reduced sCD40L in people with T1DM.¹² In the current study, however, sCD40L was not affected by simvastatin therapy in people with MS. The baseline sCD40L level in this MS study was approximately one-third of the T1DM study, which we believe is the main reason for lack of effect. Moreover, the shorter therapy duration in the current study might also have contributed to the lack of effect on sCD40L level.

P-selectin is a cell adhesion molecule that mediates rolling of monocytes on activated endothelial cells,⁶ the first step in the cell adhesion cascade.^23,24 It can exist in soluble form as sP-selectin. Plasma sP-selectin correlates with parameters of glycemia and HbA_1C in T2DM patients.²⁵ In a six-month study, statins have been shown to reduce sP-selectin in CVD patients and the reduction was inversely correlated with the progress of CVD.²⁶ In the current study which lasted only 8 weeks, simvastatin therapy did not affect the plasma sP-selectin levels in MS volunteers. We believe that dose of medication, duration of therapy, and baseline condition of the volunteers in this study contributed to the lack of significant difference.

In summary, our data demonstrates that simvastatin reduced circulating PAI-1 activity in volunteers with MS, indicating that the fibrinolytic system is one of the mechanisms through which simvastatin exerts its protective effects in the metabolic syndrome.

Acknowledgements

The authors would like to sincerely thank all the volunteers and their families for participation in and support of this study.

This work was supported by NIH K24 AT00596 and Merck Medical School Grant.

This study was partially presented as a poster at Experimental Biology 2008 at San Diego, CA in April 2008.