Recurrent venous and/or arterial thrombosis, pregnancy losses, and the presence of either medium or high levels of anticardiolipin (aCL) antibodies or positivity on the lupus anticoagulant test are the most significant features of the antiphospholipid syndrome (APS) (1, 2). Experimental evidence shows that antiphospholipid antibodies (aPL) not only are markers of the disease, but also may play a causative role in the development of vascular thrombosis and pregnancy morbidity (3–8). Nonetheless, the pathogenic mechanisms of aPL seem to be heterogeneous and far from being completely understood (9). Among the mechanisms suggested to explain the prothrombotic activity of aPL are the direct inhibition of the activated protein C pathway (10), abnormalities in platelet function (11, 12), up-regulation of the tissue factor pathway (13), and activation of endothelial cells (ECs) (14).
In particular, recent studies have shown that aPL or anti–β2-glycoprotein I (β2GPI) antibodies induce proadhesive, proinflammatory, and procoagulant molecules that provide a persuasive explanation for induction of thrombosis in APS (15). One marker for expression of EC activation is adherence of leukocytes to the vascular endothelium. This adherence is associated with the expression of adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1, and selectins (15). EC activation by aPL may result not only in expression of adhesion molecules, but also in production of cytokines and expression of tissue factor (13).
The 3-hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase inhibitors, or statins, are potent inhibitors of cholesterol synthesis. Clinical trials of statin therapy have demonstrated beneficial effects in primary and secondary prevention of coronary heart disease as well as ischemic stroke (16–21). Their beneficial effects are only partially explained by their ability to lower cholesterol levels. Statins have also been shown to modify the function of ECs, smooth muscle cells, platelets, and monocytes/macrophages (22). Their effects include decreasing the expression of adhesion molecules in monocytes and leukocyte–endothelial interactions (23–26), inhibiting platelet function (27), inhibiting tissue factor expression by mononuclear cells (28, 29), down-regulating inflammatory cytokines in ECs (30), increasing fibrinolytic activity (31, 32), and immunomodulation by decreasing the expression of class II major histocompatibility complex antigen (33).
Little is known about the effectiveness of statins in preventing the development of deep vein thrombosis (DVT). One recent observational study has addressed this question by showing that statins may play a role in the prevention of DVT (34, 35). In addition, Meroni and colleagues have shown that fluvastatin and simvastatin prevent expression of adhesion molecules and production of interleukin-6 by ECs. They reported that statins might act through inhibition of NF-κB activation (36).
The aim of this study was to determine whether peroral administration of fluvastatin to mice injected with purified IgG-aPL antibodies from patients with APS (IgG-APS) might result in reduced adherence of leukocytes to ECs and reduced formation of venous thrombus. To test this hypothesis, we used a previously devised mouse model of APS in which human polyclonal and monoclonal aCL antibodies have been demonstrated to increase the thrombus size induced by a standardized injury (4–6). We also used a microcirculation model of mouse cremaster muscle to study the effects of aPL on monocyte adherence to ECs (37). A secondary end point of this study was assessment of the ability of IgG-APS to increase levels of a marker of EC activation, namely soluble ICAM-1 (sICAM-1), and whether fluvastatin would inhibit levels of this marker.
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- MATERIALS AND METHODS
Monoclonal and polyclonal aPL antibodies have previously been demonstrated to have thrombogenic properties in mice (6, 37, 46, 48). Several studies have demonstrated that aPL activate ECs in vitro, suggesting a mechanism by which these antibodies exert procoagulant properties (15, 49, 50). Utilizing a mouse model, our group has previously demonstrated that aPL antibodies increase leukocyte adhesion to ECs of cremasteric muscle postcapillary venules, suggesting that these antibodies induce activation of ECs in vivo and that these effects correlate with enhanced thrombus formation (37). This was confirmed by subsequent studies showing that the effect of aPL on leukocyte adhesion was abrogated in ICAM and P-selectin knockout mice (15).
In this investigation, fluvastatin was found to significantly blunt the thrombogenic response (thrombus size) and inflammatory response (adhesion of leukocytes to postcapillary venules and levels of sICAM-1) elicited by injecting mice with IgG-APS. This confirms the observation of Meroni et al, who recently demonstrated with the use of in vitro experiments that fluvastatin modulates the expression of adhesion molecules and cytokine production by ECs activated by aPL (36).
There are several reports suggesting that statins such as simvastatin and fluvastatin may exert an antithrombotic effect. One possible mechanism of this effect includes the inhibition of tissue factor expression (51, 52). In a recent report, thrombogenicity of the arterial wall of hypercholesterolemic rabbits was decreased by fluvastatin and this was accompanied by a reduction in tissue factor expression and decrease in NF-κB activation in the aortic arch (53). The authors concluded that fluvastatin treatment reduced thrombogenesis by inhibiting tissue factor synthesis. The fact that aPL antibodies have recently been shown to up-regulate tissue factor expression, and the observation that lipophilic statins (fluvastatin, simvastatin) suppress tissue factor expression, offer a possible explanation for the observations of the present study, namely, that the inhibitory effects of fluvastatin on aPL-induced thrombus formation seem stronger when compared with similar effects of the drug on the aPL-induced adhesion of leukocytes to endothelium.
In this study, the effects of fluvastatin on thrombus formation and EC activation were independent of the cholesterol-lowering effect of the drug. This observation is not surprising. The enzyme HMG-CoA reductase is responsible for the conversion of HMG-CoA to mevalonate. Since mevalonic acid is the precursor not only of cholesterol, but also of many nonsteroidal isoprenoid compounds critical for several cellular processes of eukaryotic cells, inhibition of the mevalonate pathway by statins has pleiotropic effects (52). Many of these pleiotropic effects of statins are mediated by their ability to block the synthesis of important isoprenoid intermediates, which serve as lipid attachments for a variety of intracellular signaling molecules and have been shown to occur in the absence of a reduction of the cholesterol levels (54–56). In particular, the inhibition of small GTP-binding proteins, Rho, Ras, and Rac, whose proper membrane localization and function are dependent on isoprenylation, may play an important role in mediating the direct cellular effects of statins on the vascular wall (22, 52–56).
Based on available data, Fenton et al postulated that HMG-CoA reductase inhibitors may decrease tissue factor expression and down-regulate cell signaling following thrombin activation of protease-activated receptor 1, thus exerting an antithrombotic effect (56). These investigators proposed that statins may constitute a new class of antithrombotic drugs that are possibly effective in patients with a prothrombotic tendency (56). Statins have also been shown to decrease factor VII coagulant activity in patients with hyperlipidemia (57) and to enhance factor Va inactivation by activated protein C (58). These latter effects may also inhibit thrombus formation.
Most of the inhibitory effects of statins have been postulated to occur in the arterial circulation, but their actions in the venous circulation are unknown. It is possible that their effects are comparable in both arterial and venous ECs. Of relevance is a retrospective subgroup analysis by investigators of the Heart Estrogen Replacement Study showing that use of statins was associated with a 50% reduction in the risk of venous thromboembolism (34). More recently, a Canadian retrospective cohort study showed that use of these drugs was associated with a 22% reduction in the relative risk of DVT (35). Because these data do not come from randomized controlled trials, conclusions should be viewed with caution.
The findings of the present study support proposals that fluvastatin suppresses thrombus formation and IgG-APS–induced EC activation in the venous circulation. These effects were shown not to be related to lowered cholesterol levels. Interestingly, we showed that fluvastatin was effective at a dose that produces plasma concentrations close to the levels occurring in subjects taking this drug at therapeutic doses. In our study, and in order to validate the peroral feeding method, we examined the levels of fluvastatin in the blood of mice after 2 hours of ingestion of the drug. The levels of fluvastatin obtained, although higher than the values reported in humans receiving typical doses (52), are comparable with the levels obtained in humans after administration of high doses of the drug (45). These differences may be due to several factors, including differences in absorption, kinetics, and metabolism of the drug in rodents, and differences in food ingestion, among other factors. Importantly, our data confirm that fluvastatin was effectively administered and absorbed in our experiments in mice, after the animals were fed concentrations of the drug similar to the concentrations used to reduce cholesterol levels in humans.
To our knowledge, this is the first study to demonstrate an in vivo inhibitory effect of statins on thrombus formation induced by high titers of aCL antibodies. It is also the first to utilize this model to demonstrate that statins may prevent venous thrombosis. The exact mechanisms by which fluvastatin reverses the thrombogenic properties of aCL antibodies have yet to be fully elucidated.
One possible mechanism is suppression of adhesion molecules expressed on the surface of ECs by aPL antibodies, as our results demonstrated by the reduction in leukocyte adhesion to capillary ECs. In addition, we demonstrated that IgG-APS increased concentrations of sICAM-1, but pretreatment with fluvastatin suppressed this increase. Previous studies have demonstrated that increases in the circulating concentrations of these molecules are proportional to the increases in their cell membrane expression (59–63). Furthermore, the soluble phase of this molecule seems to have biologic activity. Soluble ICAM-1 has been shown to interact with the leukocyte β2 integrin (LFA-1), and it has been proposed as a useful marker of the inflammatory response (64). Closely related to this mechanism, statins have been shown to suppress the inflammatory response by binding directly to a regulatory site of the LFA-1 β2 integrin, which serves as a major counterreceptor for ICAM-1 on leukocytes (24).
It is conceivable that statins may be beneficial in a variety of circumstances in patients with APS. They might even replace warfarin in prevention of recurrent arterial and venous thrombosis (65, 66), thus eliminating the risk of the hemorrhagic complications associated with warfarin and enabling better lifestyles in these patients. Statins may also serve as an alternative treatment in APS patients who experience thrombosis despite adequate anticoagulation with warfarin, or in those with thrombocytopenia, in whom warfarin is contraindicated. Finally, statins would be an appealing alternative to warfarin as prophylactic therapy in patients with high levels of aCL antibodies and without a history of thrombosis. Although the latter group of patients may be at enhanced risk of thrombosis (67), many physicians believe the risk of prolonged warfarin therapy may outweigh any potential benefits. Unfortunately, statins are teratogenic, producing fetal skeletal abnormalities, and therefore their use in pregnancy is contraindicated (40). Thus, the prospective role of fluvastatin in the management of patients with APS will need to be further defined in clinical studies.