The antiphospholipid syndrome (APS) is a recently described autoimmune disease characterized by the presence of elevated antiphospholipid antibodies (aPL) accompanied by the occurrence of recurrent arterial/venous thrombosis and fetal loss (1). The aPL appear to play a pathogenic role, rather than just being a simple diagnostic marker of the syndrome (2, 3). They constitute a heterogeneous family of autoantibodies with different specificities that are mainly directed against phospholipid-binding plasma proteins, of which β2-glycoprotein I (β2GPI) is now believed to represent the most important antigen target for aPL (2–7).
In both in vitro and in vivo models, aPL can react with β2GPI expressed on the endothelial cell surface, and this results in a proadhesive and a proinflammatory endothelial phenotype (7–9). The antibody-mediated crosslinking of β2GPI has been suggested to play an important role in inducing a cell signal that ends in adhesion molecule expression, cytokine secretion, accelerated prostacyclin metabolism, and tissue factor (TF) synthesis (10–12). Endothelial activation itself was suggested to be crucial in inducing the thrombophilic diathesis of APS (3, 8, 9).
Early atherosclerosis is now becoming recognized as one of the most serious complications in the management of patients with systemic autoimmune diseases, and a close link between atheroma and aPL recently has been pointed out (13, 14). The hypothesis that aPL, besides their ability to mediate a thrombophilic diathesis and an adverse pregnancy outcome, may have a potential role in the accelerated arterial disease observed in APS is related to their ability to induce endothelial activation, which could be relevant in the onset of the atherosclerotic process (15). Additional findings appear to support such a hypothesis: β2GPI has been shown to be localized in atherosclerotic plaques (16), and mice lacking the low-density lipoprotein (LDL) receptor develop accelerated atherosclerosis after active immunization with β2GPI (17). Moreover, in vitro studies have shown that aPL cross-react with oxidized LDL (ox-LDL) (18), and that they can even enhance the uptake of ox-LDL by macrophages (19).
Statins are cholesterol-lowering drugs that act by a competitive inhibition of the hydroxymethylglutaryl–coenzyme A (HMG-CoA) reductase enzyme (20). Large clinical studies have shown that, besides its cholesterol-lowering activity, statin treatment is associated with regression of atherosclerotic lesions and with a reduction of cardiovascular complications (21–24). Experimental evidence suggests that statins may influence several events in the vessel wall that are relevant for the progression of atherosclerosis, such as smooth muscle cell migration and proliferation (25), monocyte activation in terms of cytokine and metalloprotease synthesis (26–28), and TF expression (29). More interestingly, statins can also affect endothelial cell function by preventing the reduction of endothelial cell nitric oxide synthase (NOS) induced by ox-LDL (30), by increasing tissue-type plasminogen activator synthesis (31), by inhibiting TF expression after cytokine or lipopolysaccharide (LPS) activation (32), and by promoting angiogenesis (33). All these effects appear to be primarily related to HMG-CoA reductase inhibition, since intermediates of the mevalonate pathway might reverse such an effect (27, 31–33).
The aim of the present study was to investigate whether statins are able to affect endothelial activation induced by anti-β2GPI antibodies and to elucidate the mechanism by which the drugs are active. From our results, it appears that statins might inhibit the anti-β2-GPI–induced proadhesive and proinflammatory endothelial phenotype, and that such an inhibition occurs at the nuclear level. Taken together, these findings suggest the potential use of statins as an additional tool for the treatment of the clinical manifestations related to aPL.
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- MATERIALS AND METHODS
Fluvastatin inhibits U937 adhesion to HUVEC. Endothelial activation by human affinity-purified polyclonal anti-β2GPI IgG (100 μg/ml) increased U937 adhesion to HUVEC monolayers (71 ± 4%, 77 ± 3%, and 14 ± 2%, mean ± SD percentage of adherent U937 for affinity-purified anti-β2GPI IgG samples 1 and 2 and NHS IgG, respectively), as previously reported (39). The human anti-β2GPI IgM mAb (GR1D5) gave comparable increases in U937 adhesion (data not shown). This rules out the possibility that the engagement of the Fcγ receptor on U937 was responsible for the increased adhesion to HUVEC. LPS (20 ng/ml), TNFα (10 ng/ml), and IL-1β (50 units/ml) exerted similar effects (81 ± 3%, 79 ± 4%, and 84 ± 2%, respectively).
Preincubation of HUVECs for 16 hours with fluvastatin (1–10 μM) reduced, in a concentration-dependent manner, the adhesion of U937 induced by polyclonal anti-β2GPI antibodies, with maximal effect at the 5-μM concentration (Figure 1A). Similarly, fluvastatin reduced, in a concentration-dependent manner, the adhesion of U937 to HUVEC activated by LPS, IL-1β, and TNFα (Figure 1A). Mevalonate (100 μM) prevented the effects of fluvastatin (Figure 1B), which suggests that fluvastatin inhibits monocyte adhesion to HUVECs with a mechanism involving the inhibition of the HMG-CoA reductase enzyme.
Figure 1. Inhibition of U937 adherence to human umbilical vein endothelial cells (HUVECs) by fluvastatin. A, Effect of serial concentrations of fluvastatin on the percentage of U937 cells adhering to HUVEC monolayers incubated with lipopolysaccharide (LPS) (20 ng/ml, ▪), interleukin-1β (IL-1β) (50 units/ml, ▴), tumor necrosis factor α (TNFα) (10 ng/ml, •), affinity-purified (a.p.) IgG fraction 1 (×) and fraction 2 (□) (100 μg/ml), normal human serum (NHS) IgG (100 μg/ml, ▵), and medium alone (+). B, Effect of fluvastatin (5 μM) (□) and fluvastatin (5 μM) + mevalonate (100 μM) (▓) on the percentage of U937 cells adhered to HUVEC monolayers incubated with LPS (20 ng/ml), affinity-purified IgG fractions 1 and 2 (100 μg/ml), NHS IgG (100 μg/ml), and medium alone. Values are the mean and SEM of 3 different sets of experiments. ∗ = P < 0.05.
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Statins inhibit E-selectin and ICAM-1 expression in HUVECs. To explore the mechanisms responsible for the inhibition of monocyte adhesion exerted by fluvastatin, we investigated its effects on E-selectin and ICAM-1 expression by HUVECs exposed to the different stimuli. Incubation of HUVECs with human affinity-purified polyclonal IgG anti-β2GPI antibodies increased E-selectin and ICAM-1 expression, and these effects were comparable with those exerted by IL-1β, TNFα, or LPS. Similar results were found when HUVECs were activated by the human anti-β2GPI IgM mAb (GR1D5) (data not shown); adhesion molecule expression was not influenced by polyclonal NHS IgG (Figures 2A and 3) or by the irrelevant human IgM mAb (data not shown).
Figure 2. Inhibition of E-selectin expression on HUVECs by fluvastatin. A, Effect of fluvastatin (5 μM) (□), fluvastatin (5 μM) + mevalonate (100 μM) (▓), and medium alone (░) on the E-selectin expression in HUVEC monolayers incubated with LPS (20 ng/ml), TNFα (10 ng/ml), IL-1β (50 units/ml), affinity-purified IgG fractions 1 and 2 (100 μg/ml), NHS IgG (100 μg/ml), and medium alone. ∗ = P < 0.05. B, Effect of serial concentrations of fluvastatin on the E-selectin expression in HUVEC monolayers incubated with LPS (20 ng/ml, ▪), IL-1β (50 units/ml, ▴), TNFα (10 ng/ml, •), IgM monoclonal antibody (mAb) GR1D5 (100 μg/ml, ×), irrelevant IgM mAb TM1B9 (100 μg/ml, ○), and medium alone (+). Values are the mean and SEM of 3 different sets of experiments. O.D. = optical density (see Figure 1 for other definitions).
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Figure 3. Inhibition of intercellular adhesion molecule 1 (ICAM-1) expression on HUVECs by fluvastatin. Effect of fluvastatin (5 μM) (□), fluvastatin (5 μM) + mevalonate (100 μM) (▓), and medium alone (░) on ICAM-1 expression in HUVEC monolayers incubated with LPS (20 ng/ml), TNFα (10 ng/ml), IL-1β (50 units/ml), affinity-purified IgG fractions 1 and 2 (100 μg/ml), NHS IgG (100 μg/ml), and medium alone. Values are the mean and SEM of 3 different sets of experiments. ∗ = P < 0.05. O.D. = optical density (see Figure 1 for other definitions).
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Preincubation of HUVECs with fluvastatin reduced, in a concentration-dependent manner, the expression of E-selectin induced by anti-β2GPI antibodies, with a maximal effect (50% reduction) at the 5-μM concentration (Figure 2B). Similarly, the drug reduced the up-regulation of E-selectin induced by 50 units/ml IL-1β, 10 ng/ml TNFα, or 20 ng/ml LPS (Figure 2B). Mevalonate completely prevented the inhibition of E-selectin expression induced by the tested stimuli (Figure 2A).
Incubation of HUVECs with the above stimuli also increased the expression of ICAM-1. Fluvastatin at the 5-μM concentration reduced ICAM-1 expression by almost 50%. As for E-selectin expression, mevalonate prevented the effect of fluvastatin by >70% under all conditions tested (Figure 3). Similarly, simvastatin (5 μM), a semisynthetic statin with lipophilic properties, reduced E-selectin and ICAM-1 expression in HUVECs exposed to all the above stimuli. The effect of simvastatin, however, was lower compared with that of fluvastatin (Figures 4A and B).
Figure 4. Inhibition of E-selectin by simvastatin (A) and inhibition of intercellular adhesion molecule 1 (ICAM-1) expression by HUVECs (B). Cells were incubated with LPS (20 ng/ml), TNFα (10 ng/ml), IL-1β (50 units/ml), affinity-purified IgG fractions 1 and 2 (100 μg/ml), NHS IgG (100 μg/ml), and medium alone in the absence (▓) or the presence (□) of simvastatin (5 μM). Values are the mean and SEM of 3 different sets of experiments. ∗ = P < 0.05. O.D. = optical density (see Figure 1 for other definitions).
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Fluvastatin impairs NF-κB translocation. E-selectin gene expression is mainly regulated at the transcriptional level. Exposure of endothelial cells to inflammatory mediators, such as TNFα, IL-1β, or LPS, leads to a rapid increase in transcription rate through activation of the NF-κB transcription factor, which recognizes multiple regulatory elements in the E-selectin promoter region. We therefore evaluated whether fluvastatin affected the activation of this transcription factor in HUVECs. Unstimulated HUVECs exhibited no NF-κB binding, whereas binding of the E-selectin–specific NF-κB was observed in response to human anti-β2GPI polyclonal IgG or TNFα (Figure 5). Fluvastatin (5 μM) prevented NF-κB translocation. Similar results were obtained by using human anti-β2GPI mAb to activate HUVEC monolayers (data not shown). Competition experiments performed with a 100-fold excess of unlabeled κB oligonucleotide demonstrated the specificity of DNA binding activity.
Figure 5. Prevention of DNA-binding activity of nuclear factor κB (NF-κB) by fluvastatin in stimulated HUVECs. HUVECs were either left untreated and then stimulated with medium alone (lane 1), TNFα (10 ng/ml) (lane 2), affinity-purified human anti–β2-glycoprotein I (anti-β2GPI) polyclonal IgG (100 μg/ml) (lane 3), or NHS IgG (100 μg/ml) (lane 4) for 1 hour, or similar experiments were performed with HUVECs incubated for 16 hours with fluvastatin (5 μM) and then with medium alone (lane 5), TNFα (10 ng/ml) (lane 6), affinity-purified human anti-β2GPI polyclonal IgG (100 μg/ml) (lane 7), or NHS IgG (100 μg/ml) (lane 8); lane 9 represents the result of the competition with cold oligonucleotide. Three separate experiments yielded similar results. See Figure 1 for other definitions.
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Fluvastatin inhibits IL-6 mRNA expression in HUVECs. IL-6 mRNA expression was evaluated by RNA protection assay experiments. Human affinity-purified polyclonal IgG antibodies and TNFα up-regulated IL-6 mRNA expression in HUVECs (Figure 6). Human anti-β2GPI mAb GR1D5, but not the irrelevant control TM1B9, gave comparable results (data not shown). Fluvastatin markedly reduced the IL-6 mRNA expression under all conditions tested (Figure 6).
Figure 6. Inhibition of endothelial IL-6 mRNA expression by fluvastatin. IL-6 mRNA expression was evaluated by RNA protection assay experiments. When HUVECs were incubated with TNFα (10 ng/ml) (lane 2) or with the polyclonal affinity-purified anti-β2GPI IgG (100 μg/ml) (lane 3), an up-regulation of IL-6 mRNA was observed. The negative controls were represented by HUVECs incubated with medium alone (lane 1) or with NHS IgG (100 μg/ml) (lane 4). The addition of fluvastatin (5 μM) induced a clear inhibition of the IL-6 mRNA expression in parallel cultures (lane 6 for TNFα and lane 7 for affinity-purified anti-β2GPI–activated HUVECs), while the expression was unchanged in the controls (lane 5 for cells in medium alone and lane 8 for HUVECs incubated with NHS IgG). Three separate experiments yielded similar results. See Figure 1 for definitions.
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- MATERIALS AND METHODS
This study shows for the first time that fluvastatin and simvastatin prevent endothelial cell adhesive properties induced by anti-β2GPI antibodies and that this effect is a consequence of inhibition of NF-κB binding to DNA. The β2GPI-dependent aPL are widely accepted to play a pathogenic role in APS and have been shown to activate endothelial cells in different experimental models, both in vitro and in vivo (8, 9). Up-regulation of endothelial adhesion molecule expression has been particularly closely related to a procoagulant state in in vivo models (9). It is also worth mentioning that aPL detected by the anti-β2GPI assay appear to display a higher diagnostic specificity than the antibodies detected by the anticardiolipin assay (43). In this regard, the antibody preparations used in this study are representative of the pathogenic autoantibodies spontaneously occurring in the syndrome, further stressing the relevance of our data (34–36, 44).
We previously reported that anti-β2GPI antibodies increase cytokine secretion by HUVECs (10). We now show that the same antibodies also increase the expression of IL-6 mRNA in HUVECs and that the effect is prevented by fluvastatin. Thus, statins are capable of controlling both expression of adhesion molecules and cytokine production by vascular endothelium activated by anti-β2GPI antibodies. Such a protective effect of statins on the endothelium might consequently prevent monocyte adhesion/activation, a pathogenic mechanism relevant both in the induction of a procoagulant phenotype and in the early steps of the atherosclerotic process.
Interestingly, statins also prevent endothelial cell activation induced by cytokines or bacterial LPS. This observation is consistent with results of several studies that show that both fluvastatin and simvastatin can down-regulate endothelial adhesion molecule expression in different experimental models (45–48), but it is in contrast with an apparent potentiation reported by Sadeghi et al in which lower concentrations of the drug and submaximal amounts of IL-1β and TNFα were used (49). As a whole, these findings suggest that statins affect mechanisms common to different agonists, which operate downstream of the interaction of anti-β2GPI antibodies with the endothelial cells. Accordingly, fluvastatin impairs NF-κB translocation induced by TNFα as well as by monoclonal or polyclonal anti-β2GPI antibodies. We have previously shown that statins reduce TF expression by influencing RelC/p65 binding to DNA in human macrophages (32). Activation of the NF-κB/Rel transcription factor family by nuclear translocation of cytoplasmic complexes plays a central role in inflammation through its ability to induce transcription of proinflammatory genes. This pathway is activated upon appropriate cellular stimulation, most often by signals related to pathogens and stress (50).
In our study, anti-β2GPI antibodies are shown to induce this pathway, which, in turn, results in an activation of endothelial cells similar to that occurring with cytokines or bacterial LPS. Overexpression of NF-κB has been reported in diverse inflammatory diseases and, in particular, in inflammation associated with atherosclerosis (51). Thus, statins, through the control of inflammatory gene activation, may be effective in several conditions in which inflammatory endothelial activation is present. A newly discovered effect of statins as an effective repressor of major histocompatibility complex II expression has been reported, which suggests that statins may also act as immunomodulators (52). The high degree of patient tolerance of statins makes them a potentially welcome addition to the currently limited arsenal of immunosuppressive agents.
The activity of fluvastatin is reversed to a great extent by mevalonate, which suggests a link between the inhibition of HMG-CoA reductase enzyme and the activity of this drug. The inhibitory effects of statins reported here, however, are not dependent upon inhibition of cholesterol biosynthesis since, in our experimental conditions, exogenous cholesterol was provided to cultured cells. Thus, inhibition of endothelial proadhesive and proinflammatory phenotypes is another feature of these drugs, which have been previously reported to display a variety of pleiotropic effects independent of their capacity to lower cholesterol biosynthesis. These include inhibition of smooth muscle cell proliferation, TF, and matrix metalloproteinase expression in monocytes (53). In addition, statins have been reported to up-regulate plasminogen activator and constitutive NOS in endothelial cells (53). Some of these effects have been shown to be dependent upon inhibition of Rho family proteins. Interestingly, expression of Rho proteins has been shown to be required in endothelial cells for the assembly of stable adhesion with monocytes via the clustering of monocyte-binding receptors and their association with the actin cytoskeleton, independent of stress fiber formation (54).
Endothelial cell activation mediated by antiβ2GPI antibodies may result not only in the expression of an adhesive and inflammatory state but also in the development of procoagulant features, in part due to increased TF expression (12). Data reported here strongly suggest that statins may counteract endothelial activation induced by anti-β2GPI antibodies, at least in vitro; the finding that statin therapy may control both the proinflammatory and the procoagulant properties of vascular endothelium raises the possibility that it could be used in conjunction with standard therapy focused on the prevention of recurrences by oral anticoagulants and/or antiplatelet treatment. Interestingly, in our study, statins are effective at concentrations close to the levels occurring in vivo after their administration at therapeutic doses.
The endothelial activation mediated by antiβ2GPI antibodies has also been thought to have a potential role in the accelerated atherosclerosis associated with APS (14). These findings, together with the high degree of patient tolerance of statins, make them a potentially welcome addition to the current arsenal of pharmacologic agents in the treatment of the whole clinical spectrum of APS.