Enhanced Interleukin-1β in Hypercholesterolemia
Effects of Simvastatin and Low-Dose Aspirin
Background— This study was aimed at verifying whether activation of platelets might represent a source of interleukin (IL)-1β levels in hypercholesterolemia. To this purpose, we compared the effects of a short-term treatment with simvastatin or low-dose aspirin on circulating levels of this cytokine.
Methods and Results— Fifty patients with hypercholesterolemia were randomly allocated to receive an 8-week therapeutic course of simvastatin 20 mg daily (n=25) or aspirin 100 mg daily (n=25). Baseline soluble (s) P-selectin directly correlated with IL-1β (P<0.0001) and C-reactive protein (CRP) (P<0.05) but not with von Willebrand factor, total cholesterol, or LDL cholesterol levels. Furthermore, sP-selectin (P<0.02) and IL-1β (P<0.0001) levels were independently related to CRP by multiple regression analysis. Both drugs were associated with comparable, significant reductions in IL-1β and sP-selectin. Simvastatin, but not aspirin treatment, significantly lowered CRP levels (P<0.05). The change in IL-1β levels correlated with the change in sP-selectin in patients randomized to either simvastatin (Rho, 0.42; P<0.05) or aspirin (Rho, 0.42; P<0.05). In contrast, the simvastatin-induced change in IL-1β did not correlate with the change in CRP levels.
Conclusions— This study suggests that platelets might contribute to IL-1β production in hypercholesterolemia, thus providing an additional link between inflammation and the prothrombotic state in this setting.
Received June 3, 2003; revision received August 8, 2003; accepted August 9, 2003.
High cholesterol levels are frequently associated with increased soluble markers of systemic inflammation, such as C-reactive protein (CRP),1 sCD40L,2 and interleukin-1β (IL-1β).3 Enhanced thromboxane (Tx)-dependent platelet activation is present in hypercholesterolemic patients, as shown by increased urinary excretion of 11-dehydro-TxB2 (a major enzymatic metabolite of TxA2).4 Besides this Tx-dependent pathway, other mechanisms may be involved in the occurrence of in vivo platelet activation in hypercholesterolemia, as suggested by the increased levels of soluble (s) P-selectin.3,5
A few years ago, platelets were thought to play a minor role, if any, in ongoing inflammation or vascular injury and repair beyond initial adhesion, secretion, and eicosanoid synthesis. However, increasing evidence is accumulating suggesting that platelets have inflammatory actions and are a rich source of chemokines (eg, platelet factor-4 and RANTES)6,7 and inflammatory cytokines (eg, IL-1β and sCD40L)8,9 that are preformed and packaged in storage granules. In hypercholesterolemia, increased sCD40L levels are associated with in vivo platelet activation (as reflected by sP-selectin levels and urinary 11-dehydro-TxB2 excretion) and with procoagulant state (as reflected by FVIIa and F1+2), and statin therapy may significantly reduce both sCD40L and prothrombotic state.2 Stimulated platelets also release and actively synthesize IL-1β.8 However, no evidence links platelet activation and IL-1β production in vivo.
Because simvastatin is able to reverse platelet activation9 as well as plasma CRP levels10 in hypercholesterolemia, we sought to verify whether activation of platelets might represent a source of IL-1β levels by comparing the effects of a short-term treatment with simvastatin or low-dose aspirin on circulating levels of this cytokine.
Fifty patients with hypercholesterolemia (baseline LDL cholesterol levels >130 mg/dL) entered in the study. Clinical features of patients are summarized in Table 1.
Participants were on an American Heart Association step I diet and were free of lipid-lowering drugs during the 2-month period before enrollment. No subject was taking nonsteroidal antiinflammatory drugs or antiplatelet agents in the 2 weeks preceding the study. The local ethics committee approved the study, and patients provided written, informed consent.
The participants, selected on the basis of their willingness to participate to the study, were randomly allocated to receive an 8-week therapeutic course of simvastatin 20 mg daily (n=25) or aspirin 100 mg daily (n=25).
Patients attended the Department of Medical Therapy after an overnight fast. Serum and plasma samples were frozen and stored at −80°C until assayed. For all analyses, samples were handled in blinded fashion such that investigators had no knowledge of randomization status.
Total cholesterol, HDL cholesterol, and triglycerides were immediately measured using an automated analyzer (CX5 Beckman). LDL cholesterol was calculated by the Friedenwald’s formula.
Plasma sP-selectin and IL-1β were determined by ELISA (both by R&D Systems) according to the manufacturer’s instructions. Plasma von Willebrand factor (vWF) antigen was measured by ELISA (Imubind vWF ELISA, American Diagnostica). Serum CRP levels were measured with a highly sensitive immunoassay.
Student’s t test and Pearson Product moment correlation analyses were used for normally distributed variables. Mann-Whitney U test and Spearman rank correlation test were used for nonparametric variables. A sample size of 25 patients participated in the study with 80% power to detect a 30% reduction in LDL cholesterol after lipid-lowering treatment. The differences between baseline and posttreatment values were analyzed with the Wilcoxon signed-rank test. Only 2-tailed probabilities were used for testing statistical significance. Data are presented as mean±SD. Only P<0.05 was regarded as statistically significant. All calculations were made using a computer software package (Statistica 5.5, StatSoft).
Mean baseline levels of total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides did not differ between patients randomized to either simvastatin or aspirin (Table 2). Among those randomized to low-dose aspirin, total and LDL cholesterol did not change over the 8-week time course (Table 2). As expected, simvastatin therapy significantly lowered both total cholesterol (−25%, P<0.0001) and LDL cholesterol (−31%, P<0.0001) levels.
Analysis of the correlation among the analytical variables analyzed in the study demonstrated that baseline sP-selectin directly correlated with IL-1β (Rho, 0.64; P<0.0001) and CRP (Rho, 0.31; P<0.05) but not with vWF (P=0.32), total cholesterol (P=0.39), or LDL cholesterol (P=0.91) levels. Thus, to additionally analyze the relationship between inflammation and platelet activation in these patients, a multiple regression analysis including all clinical and laboratory variables was carried out. The final model obtained by stepwise regression analysis revealed that both sP-selectin (regression coefficient, 0.30; P<0.02) and IL-1β (regression coefficient, 0.50; P<0.0001) levels were independently related to CRP.
Randomization to either simvastatin or aspirin treatment caused a significant reduction in both sP-selectin and IL-1β levels (Table 2). Simvastatin treatment significantly lowered CRP levels by approximately 17% (P<0.05), whereas aspirin therapy did not cause any change over the 8-week time course (Table 2). No significant changes in vWF levels were observed after either simvastatin or aspirin.
The change in IL-1β levels correlated significantly with the change in sP-selectin in patients randomized to either simvastatin (Rho, 0.42; P<0.05) or aspirin (Rho, 0.42; P<0.05). In contrast, the simvastatin-induced change in IL-1β did not correlate with the change in CRP levels (data not shown).
The findings that stimulated platelets actively synthesize IL-1β8 and that IL-1β production is enhanced in hypercholesterolemia3 lead us to hypothesize that IL-1β upregulation may be related to the occurrence of in vivo platelet activation in this setting.
In this report, the association between IL-1β and platelet activation is supported by the downregulation of this protein by aspirin therapy and by the relationship between this cytokine and sP-selectin. P-selectin is an adhesion molecule whose basal level is essentially endothelial cell–derived,11 whereas its increase above the basal level is primarily attributable to platelet activation.12 Because sP-selectin is enhanced in hypercholesterolemia,3,5 it has been hypothesized that the platelet is the origin of this increment in this setting.3,5 In our study, an endothelial origin of P-selectin is excluded by the fact that either simvastatin or aspirin did not reduce vWF levels. Because P-selectin and vWF are coexpressed by endothelial cells,13 one would expect a concomitant decrease of both proteins, provided that sP-selectin was derived from endothelial cells rather than platelets. Therefore, the significant correlation between sP-selectin and IL-1β is suggestive of the platelet origin of the latter. Conversely, the nonsignificant correlation between IL-1β and total or LDL cholesterol suggests that the impaired lipid profile per se is unlikely to be the major determinant of IL-1β production in hypercholesterolemia.
In the present study, either simvastatin or aspirin decreased IL-1β levels and platelet activation. Indeed, a 2-month treatment with simvastatin (20 mg daily) caused a downregulation of plasma IL-1β with a concomitant reduction of sP-selectin. This finding, along with the similar effects observed after a 2-month treatment with low-dose aspirin, corroborates the hypothesis that platelets are likely to be a relevant source of IL-1β in hypercholesterolemia.
These effects could contribute to explain the early reduction in cardiovascular events observed in clinical trials with simvastatin. It is well-known that IL-1β activates monocytes or endothelial cells, causing a switch in their procoagulant activity. Thus, the reduction in platelet-derived IL-1β in hypercholesterolemia might lower, almost in part, the associated prothrombotic state. This may be obtained with drugs, such as low-dose aspirin or simvastatin, that reduce platelet activation with rather different mechanisms.9,14
One limitation to this study derives from the fact that many cell types involved in the inflammatory response can produce IL-1β. Indeed, we should consider that P-selectin–dependent adhesion of activated platelets to circulating monocytes might induce the latter to synthesize and release IL-1β, as previously shown for other cytokines.15 Therefore, it may seem questionable whether a concomitant reduction after treatment of IL-1β and sP-selectin does justify the conclusion of causality. However, in our study, the contribution of platelets to IL-1β production in hypercholesterolemia is strengthened by the observation that low-dose aspirin, which is intended to act as an antiplatelet drug, did not cause any reduction in CRP levels but was able to concomitantly decrease both IL-1β and sP-selectin.
In conclusion, we believe that the observed downregulation of both IL-1β production and platelet activation with either simvastatin or low-dose aspirin provides an additional link between inflammation and the prothrombotic state in the setting of hypercholesterolemia. Additional studies will be required to better characterize the inflammatory activity of platelets in the pathophysiology of atherosclerosis.
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