statins for the treatment of antiphospholipid syndrome?

CONTEMPORARY CHALLENGES IN AUTOIMMUNITY

Statins for the Treatment of AntiphospholipidSyndrome?

Praveen Jajoria,a Vijaya Murthy,a Elizabeth Papalardo,b

Zurina Romay-Penabad,b Caroline Gleason,c

and Silvia S. Pierangelib

aDepartment of Internal Medicine, bDivision of Rheumatology, Department of InternalMedicine, Antiphospholipid Standardization Laboratory, University of Texas Medical

Branch, Galveston, Texas, USAcRheumatology Associates of Louisville, Louisville, Kentucky, USA

Fluvastatin has been shown to revert proinflammatory/prothrombotic effects of an-tiphospholipid antibodies (aPL) in vitro and in mice. Here, we examined whether fluvas-tatin affects the levels of proinflammatory/prothrombotic markers in antiphospholipidsyndrome (APS) patients. Vascular endothelial growth factor (VEGF), soluble tissue fac-tor (sTF), tumor necrosis factor-α (TNF-α), soluble intercellular adhesion molecule-1(sICAM-1), sE-selectin (E-sel), C-reactive protein (CRP), and soluble vascular cell adhe-sion molecule (sVCAM-1), were measured in the sera of 93 APS patients and 60 controlsand in the sera of nine patients with APS before and after 30 days of treatment withfluvastatin. Elevated levels of VEGF, sTF, and TNF-α were found in APS patients. Flu-vastatin significantly reduced those markers in the majority of treated subjects. Thedata from this study show that statins may be beneficial in aPL-positive patients andwarrant larger clinical trials to confirm the efficacy of the drug for the treatment of APSclinical manifestations.

Key words: anticardiolipin antibodies; antiphospholipid antibodies; antiphospholipidsyndrome; statins; proinflammatory markers

Introduction

Antiphospholipid syndrome (APS) is an au-toimmune and multisystem disorder of throm-bosis and pregnancy loss associated with thepersistent presence of antiphospholipid anti-bodies (aPL).1,2 APS was first described in pa-tients with systemic lupus erythematosus (SLE),more specifically, in a subset of patients withSLE who had an abnormal lupus anticoag-ulant (LA) test.3 APS was later classified as“secondary” (SAPS) in the presence of SLEand “primary” (PAPS) in the absence of SLE

Address for correspondence: Silvia S. Pierangeli, Ph.D., Division ofRheumatology, Department of Internal Medicine, University of TexasMedical Branch, 301 University Boulevard, Galveston, TX 77555-0883,USA. Voice: +409-772-0222; fax: +409-772-0223. st

or other autoimmune disorders. APS is nowrecognized as the most common cause of ac-quired hypercoagulability in the general popu-lation4 and the most important treatable causeof recurrent miscarriage.5–8 APL can be de-tected in up to 40% of SLE patients and af-fects disease morbidity by being associated withrecurrent thrombosis, pregnancy loss, throm-bocytopenia, and worse lupus nephritis andkidney transplantation outcomes.9,10 Cardio-vascular morbidity and mortality is a frequentcomplication in patients with SLE, in whomthe risk of myocardial infarction is raised 50-fold. In addition to traditional risk factors suchas hypertension or diabetes, several factorsspecifically related to lupus are proposed tobe of importance, including inflammation andaPL.11,12

Contemporary Challenges in Autoimmunity: Ann. N.Y. Acad. Sci. 1173: 736–745 (2009).doi: 10.1111/j.1749-6632.2009.04815.x c© 2009 New York Academy of Sciences.

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Praveen et al.: Statins and APS 737

There is strong evidence from studies in ani-mal models that aPL are pathogenic in vivo.13–16

Investigators have shown that endothelial cells(EC) express significantly higher amounts ofcellular adhesion molecules (CAMs) such as in-tercellular cell adhesion molecule-1 (ICAM-1),vascular cell adhesion molecule-1 (VCAM-1)and E-selectin (E-sel) when incubated with aPLin vitro.17–20 Our group has shown that aPLactivate endothelium in vitro and in mousemodels and this correlated with enhance-ment of thrombus formation in vivo.15 UtilizingICAM-1, E-sel, and P-selectin knock-out miceand specific anti-VCAM-1 monoclonal anti-bodies, we demonstrated that EC-activatingproperties of aPL are mediated by theseCAMs.21,22 Tissue factor (TF) upregulation hasbeen advocated as an important mechanismto explain the prothrombotic effects of aPL.23

Studies have shown up-regulation of TF ex-pression and function in EC and monocytestreated with aPL.22–28 Monocyte procoagulantactivity (PCA) is increased in patients withAPS.24 We showed that aPL induce significantTF transcription, expression, and function andincrease proinflammatory cytokines (IL-6 andIL-8) on EC.29 In another recent study, weshowed that treatment of mice with aPL in-duced significantly increased TF function inperitoneal cells and in carotid artery, whencompared to control mice, and that this cor-related with enhanced thrombosis and EC ac-tivation in vivo.30 In addition, some investi-gators have shown increased levels of soluble(s)VCAM-1 in patients with aPL and thrombo-sis.31 Others have demonstrated a significantincrease of soluble vascular endothelial growthfactor (sVEGF) and sTF in patients with aPLand thrombosis and elevated levels of IL-6 andTNF-α. VEGF may stimulate monocyte TFexpression through its receptor, the tyrosine ki-nase Flt-1.32–35 Given that increased TF ac-tivity, expression of CAMs, and upregulationof proinflammatory cytokines are known to berelated to thrombosis in APS, using pharmaco-logical agents that block TF activity or expres-sion of CAMs or inflammatory cytokines may

be a novel and attractive therapeutic approachin APS.

Recent studies have suggested that fluvas-tatin has beneficial effects on aPL-mediatedpathogenic effects. First, one study showed thatfluvastatin prevented the expression of CAMsand IL-6 in EC treated with aPL.36 Subse-quently, we showed that the thrombogenic andproinflammatory effects of aPL in vivo couldbe abrogated in mice fed with fluvastatin for15 days,37 and this effect was independent ofthe cholesterol-lowering effects of the drug. Wethen showed that fluvastatin inhibited the ef-fects of aPL Abs on TF expression on EC in vitro

at doses utilized to reduce cholesterol levels inpatients.38

We hypothesize that patients with APShave increased levels of the proinflammatory/prothrombotic biomarkers when comparedwith healthy controls and that can be an in-dication of their predisposition to thromboticand other clinical manifestations of APS. Here,we first examined changes in the level proin-flammatory and prothrombotic markers—sICAM-1, E-Sel, sTF, VEGF, interleukin (IL)-1β, IL-6, IL-8, C-reactive protein (CRP) andtumor necrosis factor (TNF)-α—in previouslyidentified APS patients who were positivefor aPL in comparison with normal controls.Then, we determined whether treatment ofAPS patients with fluvastatin has an effect onthe levels of those biomarkers.

Materials and Methods

Patients

Ninety-three patients with APS were selectedfrom the Antiphospholipid Syndrome Collab-orative Registry (APSCORE). APS was diag-nosed in these patients according to the re-vised Sapporo criteria.2 They were comparedwith 60 healthy controls. For the fluvastatin pi-lot study, nine APS patients were selected, ofwhom eight had primary APS (PAPS) and onehad concomitant SLE (SAPS). Patients were

738 Annals of the New York Academy of Sciences

35 to 65 years of age, with a male:female ratio of1:2. Six subjects were from Dr. Gleason’s officein Kentucky, and the rest of the 9 subjects werefrom the Rheumatology Clinic at the Univer-sity of Texas Medical Branch. All patients andcontrols provided a written, informed consentand the study was approved by the InstitutionalReview Board (IRB) of the University of TexasMedical Branch.

Inclusion Criteria

Only patients who had anticardiolipin (aCL)titers more than 40 G phospholipid (GPL) orM phospholipid (MPL) units and anti-β2-GPImore than the 99th percentile of normal con-trols were included in the APS group.2,39

Exclusion Criteria

Patients with liver problems, on “pulse”steroid therapy, pregnant, or less than 18 yearsof age were excluded from the pilot study. Atthe screening visit, pregnancy tests were ad-ministered to all women of child-bearing age.If patients became pregnant during the study,they were required to be withdrawn from thestudy. Liver function tests were performed atbaseline and those having elevated liver en-zymes (AST/ALT) were excluded. Those whowere already on a statins other than fluvastatinwere considered eligible for the study only ifstatin use was discontinued at least 2 weeksprior to the onset of the study. Those takinghydrochloroquine (Plaquenil), aspirin, heparin,warfarin, or low-dose prednisone (5–10 mg perday) were not excluded. Other medications thatpatients were taking at the time of recruitmentwere not discontinued.

Fluvastatin Pilot Study Intervention

Fluvastatin 40 mg per day (LescolTM,Norvartis) was given to nine patients for30 days. The patients were not responsible forthe medication costs. Blood samples were col-lected at baseline and then again after 30 daysof treatment. Patients were monitored for APS-related events and side effects of the drug such

as muscle aches, fatigue, and abdominal pain.At each visit, blood was drawn, not only to mo-nitor aPL antibodies, but also to monitor liverenzymes, lipid profiles, and creatinine levels.

Determination of Biomarkers

Anticardiolipin antibodies and anti-β2-GPItiters were determined using the standard aCLELISA40 and the Quanta-LiteTM kit (INOVA,San Diego, CA, USA), respectively. CRP levelswere measured at the central laboratory at theUniversity of Texas Medical Branch (UTMB),Galveston, Texas. VEGF, sTF, sVCAM-1,sICAM-1, sE-selectin, IL-6, IL-8, IL-1β, andTNF-α levels were measured using ELISA (Rand D systems kit, Minneapolis, MN, USA).IMUBIND Tissue Factor ELISA kit of Ameri-can Diagnostic Inc. was used to analyze sTF inserum. All assays were carried out according tothe manufacturers’ instructions.

Statistical Methods

A paired t test was used to determine if was astatistically significant change in laboratory pa-rameters, and a 95% confidence interval wascalculated for the change in laboratory param-eters (before and after fluvastatin treatment).P values of ≤ 0.05 were considered a priori sta-tistically significant.

Results

APS versus Controls

Mean levels of TNF-α, sTF, and VEGF weresignificantly higher in the sera of aPL-positivepatients than in controls (Table 1). TNF-α lev-els in the APS group were 281.6 ± 0.31, com-pared with 51.9 ± 0.19 in the control group(P = 0.0413). Significantly higher sTF levelswere also seen in APS patients (417.8 ± 139 vs.46.7 ± 25 in controls, P = 0.0209), and VEGFlevels averaged 740.2 ± 88.2 in APS patientsand 450 ± 27.9 in controls (P = 0.027). No


TABLE 1. Levels of Proinflammatory/Prothrombotic Markers in APS and in Controls

TNF-α (mean ± SD) TF (mean ± SD) VEGF (mean ± SD)Sample type pg/mL pg/mL pg/mL

APS 281.6 ± 0.31 417.8 ± 139 740.2 ± 88.2Control 51.9 ± 0.19 46.7 ± 25 450 ± 27.9

P values: 0.0413, 0.0209, and 0.027 for TNF-α, TF, and VEGF respectively.

significant differences between APS patientsand controls were observed in the mean val-ues of sICAM-1, sE-sel, sVCAM-1, or CRP.All APS patient samples showed medium-highpositivity for aCL and anti-β2-GPI antibodytiters.

Fluvastatin Pilot Study

After 30 days of treatment with fluvastatin,seven out of nine, three out of nine, and sixout of nine patients showed variable but sig-nificant decreases in VEGF, sTF, and TNF-αtiters, respectively (Table 2). No significant dif-ference was seen in aCL or anti-β2-GPI anti-body titers after treatment with fluvastatin (datanot shown). Also none of the nine APS patientstreated with fluvastatin had elevated CRP, andthere were no significant changes in the lev-els of soluble adhesion molecules. Of the nineAPS patients included for the fluvastatin pilotstudy, one could not continue the study beyondthe initial 30 days due to side effects of mus-cle pain and elevated CPK levels and anothercould not continue due to side effects of nauseaand vomiting.

Discussion

In this study we first showed that certainproinflammatory and prothrombotic markers(VEGF, sTF, and TNF-α) are elevated inpatients with APS compared with controls.These results are in agreement with other pub-lished studies.31–35 Recently, Cuadrado and col-leagues showed that monocytes isolated fromAPS patients showed higher levels of VEGFthan healthy donors, which further corre-

lated with IgG aCL titers and TF expres-sion rank. Moreover, monocyte VEGF andFlt-1 levels were significantly higher in pa-tients with previous thrombosis than in patientswithout.35 In vitro, IgG from APS patients in-creased monocyte VEGF expression in a dose-dependent manner. The authors concludedthat VEGF might act as a regulatory factorin aPL-mediated monocyte activation and TFexpression, thereby contributing to the proin-flammatory/prothrombotic phenotype of APSpatients.

The statins are potent inhibitors of choles-terol synthesis in the mevalonate pathway. Clin-ical trials of statin therapy have demonstratedbeneficial effects in primary and secondaryprevention of coronary heart disease as wellas ischemic stroke.41 However, their beneficialeffects are only partially explained by theirability to lower cholesterol levels. Pleiotropiceffects of statins have been reported, includ-ing decreasing the expression of CAMs inmonocytes and affecting leukocyte–endothelialinteractions, downregulating inflammatory cy-tokines in EC, and increasing fibrinolytic ac-tivity.42–47 Colli and colleagues demonstratedthat simvastatin and fluvastatin decrease TFmRNA expression and activity in cultured hu-man monocytes obtained from healthy indi-viduals.48 Treatment with cerivastatin, simvas-tatin, pravastatin, and fluvastatin substantiallyreduced TF expression in atherosclerotic le-sions, along with suppression of inflammationin atheroma, independently of lipid lowering,in animal models.49–52 These findings havebeen corroborated in humans by the findingsof the double-blind, placebo-controlled Ator-vastatin and Thrombogenicity of the CarotidAtherosclerotic Plaque (ATROCAP) study, in


TABLE 2. Effects of Fluvastatin on Proinflammatory and Prothrombotic Markers

Patient # (diagnosis) % decrease VEGF % decrease sTF % decrease TNF-α

A (SAPS) 42 83 42B (PAPS) 51 NS 62C (PAPS) 8 NS 62D (PAPS) 18 ND 76E (PAPS) 30 30 NSF (PAPS) NS NS NSG (PAPS) NS NS NSH (PAPS) 100 NS 9I (PAPS) 70 70 42

7 out of 9, 3 out of 9, and 6 out of 9 patients showed variable but significant decrease in VEGF, sTF, and TNF-αtiters respectively. NS, no significant decrease; ND, not determined.

which 4 to 6 months of treatment with ator-vastatin (20 mg/d) was associated with 29%lower TF antigen levels and 56% lower TFactivity in atherosclerotic plaques comparedwith subjects receiving placebo.53 Taken to-gether, currently available data point to a sig-nificant downregulation of TF expression onmonocytes and various vascular cells inducedby statins.54–56 Hence, cardiovascular benefitis provided by lowering raised cholesterol levelsand by modulation of the inflammatory compo-nent of this disease. Such an anti-inflammatoryeffect may also benefit patients with autoim-mune rheumatic diseases. Statins also directlyinhibit interferon (IFN)-γ-induced major histo-compatibility complex class II (MHC-II) ex-pression in vitro, thus preventing subsequentT cell activation.42,57 These findings suggesta possible modulatory role in Th1-driven au-toimmunity. More recently, the administrationof atorvastatin to lupus-prone New ZealandBlack (NZB)/W F1 mice resulted in a signif-icant reduction in anti-dsDNA antibodies andproteinuria.58 Simvastatin induced a rapid andsignificant reduction of the proteinuria leveland of the expression of lymphocyte activationmarkers in a small series of SLE patients.59

In this study, we showed for the first time asignificant decrease in the titers of three of thebiomarkers studied—VEGF, sTF, and TNF-α—in the blood of the majority of the APS(PAPS and SAPS) subjects after 30 days of treat-ment with fluvastatin. Fluvastatin did not seem

to affect the level of aPL in the sera of thepatients, hence indicating that the effects ob-served are not due to a decrease in the titerof the autoantibodies. In this study, which in-cluded nine APS patients treated with fluvas-tatin for 30 days, none of the patients experi-enced any aPL-related clinical event. This maydue to the relatively short monitoring periodand the fact that the patients were not requiredto discontinue—for obvious ethical reasons—any other antithrombotic or prophylactic treat-ments. However, with the exception of the twopatients who had to be discontinued from thestudy due to adverse side effects, the rest ofthe subjects will continue to receive fluvastatinfor up to 5 months, and we will monitor thelevels of biomarkers and for the presence ofany aPL-related clinical event throughout thatperiod. Importantly, the significant reductionin proinflammatory/prothrombotic markers inthis group of patients may indicate a protectiveeffect of fluvastatin. Furthermore, the findingsin this study confirm our previous-publishedobservations that showed antithrombotic andanti-inflammatory effects of fluvastatin in amouse model of thrombosis and effects onTF expression on cultured EC.37,38 Similarly,Meroni and coworkers showed a reduction inaPL-induced expression of adhesion moleculeson EC.36

A premature atherosclerosis has been pre-sumed in patients with APS. The potentialrole of aPL in the development of atheroma is


rather controversial.60 In a recent study, Beliznaand colleagues tested the hypothesis that aPLantibodies could induce atherosclerosis via vas-cular functional changes. CD1 mice receiveda single injection of aPL monoclonal antibod-ies derived from male (BXSB x NZW) F1 micewith a lupus-like disease associated with APSand coronary artery disease. One week later,first-order mesenteric arteries were isolated andmounted on a small-vessel myograph for themeasurement of the relaxation responses toacetylcholine or the NO donor nitroprusside af-ter precontraction by phenylephrine. Five outof eight aPL monoclonal antibodies reducedthe response to acetylcholine compared withcontrol mice, and this effect was especiallymarked with one of them. No change in theresponse to nitroprusside was observed. Theimpairment was maintained after 3 weeks oftreatment and appeared related to a moder-ate decrease in NO-mediated responses and amarked decrease in prostanoid-mediated relax-ations. Importantly, these vascular functionalchanges could be prevented by chronic treat-ment with statins.61 The data indicate for thefirst time that statins may be beneficial alsoin the prevention of accelerated atherosclero-sis induced by aPL. Finally, statins may havebeen shown to be beneficial in other autoim-mune diseases where accelerated atherosclero-sis is also an important morbidity feature.62,63

Fetal loss induced by aPL in mice is acomplement-driven inflammatory condition.64

Engagement of the complement receptorC5aR on neutrophils induces expression ofTF, the principal initiator of the blood clot-ting mechanism, and blocking this down-stream event of complement activation pre-vents antibody-induced fetal loss.65 Recently,Redecha and colleagues showed that in micethe contribution of TF to this pathogenic mech-anism is independent of its role in coagula-tion and thrombosis, but involves inflamma-tory signaling through the receptor PAR2. Thestudy not only underlines a critical effectormechanism of aPL-induced fetal loss, but alsosuggests that treatment with statins (specif-

ically fluvastatin), which decreases TF andPAR2 expression, may hold promise as a thera-peutic approach to APS-associated pregnancycomplications.65

The treatment of thrombosis in APS hasbeen focused on utilizing antithrombotic med-ications or modulating the immune responseitself. Recurrences, in spite of treatment, havebeen reported, and the use of oral anticoagula-tion at a relative high international normalizedratio (INR) for a long period of time has beenassociated with a high risk of bleeding, with theneed for frequent monitoring and patient com-pliance with diet and lifestyle prescriptions tooptimize the therapy.66–69 Moreover, still de-bated is the approach to patients with aPLAbs without a previous thrombotic event. Somephysicians would recommend prophylaxis withlow dose aspirin—although no controlled dataexist to support this approach—while otherswould advise a more aggressive treatment andstill others would recommend no treatment atall. It is well-known that aPL Abs might bepersistently present in the serum of APS pa-tients for long periods of time, but thromboticevents occur only occasionally. It has been sug-gested that aPL (first hit) increase the throm-bophilic threshold (i.e., induce a prothromboticand proinflammatory phenotype in EC), butthat clotting takes place only in the presence of asecond hit or triggering event (such as an infec-tion, a surgical procedure, use of estrogens, orprolonged immobilization).70,71 Current treat-ments of thrombosis in APS are directed tomodulate the final event or “second hit.” Treat-ments that modulate early effects of aPL Abson target cells—such as monocytes or EC—(first hit) would be more beneficial and poten-tially less harmful than current treatments. Thedata indicate that statins may be good candi-dates to be used in treatment and prevention ofthrombosis in APS. Based on this study and thedata available, it is conceivable that statins maybe beneficial in reversing upregulation of TF,CAMs, and inflammatory cytokines in EC andmonocytes (first hit). Upon successful comple-tion of clinical trials, statins might even replace


warfarin and antiplatelet agents in preventionof recurrent arterial and venous thrombosis,thus eliminating the risk of hemorrhagic com-plications associated with warfarin and en-abling better life style in these patients. Statinsmay also serve as an alternative treatment inAPS patients who experience thrombosis de-spite adequate anticoagulation with warfarinor with antiplatelet agents, or in those withthrombocytopenia in whom warfarin is con-traindicated. Finally, statins would be an ap-pealing prophylactic therapy in patients withhigh levels of aPL Abs without a history ofthrombosis. Unfortunately, statins are terato-genic, producing fetal skeletal abnormalities,and therefore their use in pregnancy is con-traindicated. The prospective use of statins inthe management of patients with APS needsto be further delineated in well-designed clin-ical studies. As a first step, in this pilot study,we found that aPL-induced proinflammatoryand prothrombotic effects are affected by flu-vastatin in aPL-positive patients with PAPS orSAPS. The data from this study undoubtedlyprovide the basis for future and larger random-ized, controlled trials to examine the effects ofstatins on thrombosis and possibly other clinicalmanifestations in patients with APS.

Acknowledgments

These studies were partially funded withsources from the Antiphospholipid Standard-ization Laboratory (UTMB, Galveston, TX)and with funds from an Arthritis FoundationGrant (Texas Chapter) and an American HeartAssociation Grant.

Conflicts of Interest

The authors declare no conflicts of interest.

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