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Fluvastatin increases the expression of adhesion molecules, monocyte chemoattractant protein-1 and tissue factor in HUVEC

stimulated by patient IgG fractions containing antiphospholipid antibodies

GEINDRE, Sylvie Françoise, et al.

Abstract

The presence of antiphospholipid antibodies (APLA) is associated with an increased risk of recurrent thrombosis and pregnancy loss. APLA are able to activate endothelial cells (EC) and induce an increase in the expression of inflammatory marker proteins, such as leukocyte adhesion molecules, tissue factor or the monocyte chemoattractant protein-1 (MCP-1). Our objective was to investigate the effect of statins on EC activation induced by APLA in vitro.

IgG was purified from the plasma of six patients with APLA and from healthy controls. EC were incubated with patient IgG or with control IgG, in the presence or absence of 5microM of fluvastatin, and expression of the leukocyte adhesion molecules, VCAM-1 and E-selectin, analyzed by flow cytometry and by quantitative reverse transcriptase-PCR (QRT-PCR). The expression of tissue factor and the chemokine MCP-1 was analyzed by QRT-PCR alone.

Incubation of EC with patient IgG increased the expression of VCAM-1, E-selectin, tissue factor and MCP-1. Prior treatment of the cells with fluvastatin further increased the expression of these proteins. The fluvastatin effect was reversed [...]

GEINDRE, Sylvie Françoise, et al . Fluvastatin increases the expression of adhesion molecules, monocyte chemoattractant protein-1 and tissue factor in HUVEC stimulated by patient IgG fractions containing antiphospholipid antibodies. Thrombosis and Haemostasis , 2005, vol.

93, no. 2, p. 339-45

DOI : 10.1267/THRO05020339 PMID : 15711752

Available at:

http://archive-ouverte.unige.ch/unige:36193

Disclaimer: layout of this document may differ from the published version.

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Fluvastatin increases the expression of adhesion molecules,

monocyte chemoattractant protein-1 and tissue factor in HUVEC stimulated by patient IgG fractions containing antiphospholipid antibodies

Sylvie Dunoyer-Geindre,Yordanka Dimitrova, Richard J. Fish, Nathalie Satta, Guido Reber, Egbert K.O. Kruithof, Philippe de Moerloose

Division of Angiology and Haemostasis, University Hospital, Geneva, and Faculty of Medicine, Geneva, Switzerland Summary

The presence of antiphospholipid antibodies (APLA) is associ- ated with an increased risk of recurrent thrombosis and preg- nancy loss.APLA are able to activate endothelial cells (EC) and induce an increase in the expression of inflammatory marker proteins, such as leukocyte adhesion molecules, tissue factor or the monocyte chemoattractant protein-1 (MCP-1). Our objec- tive was to investigate the effect of statins on EC activation in- duced by APLAin vitro. IgG was purified from the plasma of six patients with APLA and from healthy controls. EC were incu- bated with patient IgG or with control IgG, in the presence or absence of 5μM of fluvastatin, and expression of the leukocyte adhesion molecules,VCAM-1 and E-selectin, analyzed by flow cytometry and by quantitative reverse transcriptase-PCR (QRT- Keywords

Antiphospholipid antibodies, endothelial cells, statins, tissue fac- tor, cell adhesion molecules

PCR).The expression of tissue factor and the chemokine MCP-1 was analyzed by QRT-PCR alone. Incubation of EC with patient IgG increased the expression ofVCAM-1, E-selectin, tissue fac- tor and MCP-1. Prior treatment of the cells with fluvastatin further increased the expression of these proteins.The fluvasta- tin effect was reversed by co-incubation with mevalonate or ge- ranylgeranylpyrophosphate and mimicked by the geranylgeranyl transferase inhibitor GGTI-286. Our results show that in cul- tured human EC,statins increase the extent of inflammatory ac- tivation induced by APLA.This effect appears to be mediated by an inhibitory effect of statins on one or more geranylgeranylated protein(s).

Thromb Haemost 2005; 93: 339-45

Endothelium andVascular Development

Correspondence to:

Philippe de Moerloose Unit of Haemostasis University Hospital of Geneva 24, Rue Micheli-du-Crest 1211 Geneva 14, Switzerland

Tel.: 0041–22–37 29 751, Fax: 0041–22–37 29 777 E-mail: philippe.demoerloose@hcuge.ch

Received May 14, 2004 Accepted after resubmission November 25, 2004 Financial support This work was supported by the Swiss National Science Foundation (3200–067746.02).

Prepublished online January 7, 2005 DOI: 10.1160/TH04–05–0297

Introduction

The vascular endothelium represents a dynamic barrier between blood and surrounding tissue and is a major regulator of haemos- tasis, leukocyte migration and vascular tone. In response to acti- vation by inflammatory cytokines, endothelial cells (EC) in- crease the expression of proteins involved in leukocyte recruit- ment, adhesion and transmigration leading to a proinflammatory phenotype. Among these proteins, adhesion molecules such as VCAM-1, ICAM-1 and E-selectin, or chemokines, such as monocyte chemoattractant protein-1 (MCP-1) play an important role (1, 2). In addition, expression of tissue factor is increased in activated EC (3).

In patients, the presence of antiphospholipid antibodies (APLA) is associated with an increased risk of thrombosis and

recurrent fetal loss. The occurrence ofAPLA with clinical mani- festations characterizes the antiphospholipid syndrome (APS).

Several studies have shown that incubation of EC with APLA leads to an increased expression of leukocyte adhesion mol- ecules and of tissue factor, and enhance monocyte adhesionin vitro(4–6). Furthermore,in vivo,APLA increase the adhesion of leukocytes to the endothelium in the cremaster muscle microcir- culation model in mice (7, 8). In patients withAPS, plasma levels of circulating markers of EC activation are increased (9, 10).

These observations suggest thatin vivoandin vitroAPLA induce EC activation, which may contribute to their pathological ef- fects.

Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) are cholesterol-lowering drugs and are widely used in the treatment of hyperlipidemia to prevent cardiovascular com-

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Dunoyer-Geindre, et al.: Statins, antiphospholipid antibodies, and endothelial cells plications.They inhibit the formation of mevalonate, a rate limit-

ing intermediate in the biosynthesis not only of cholesterol but also of farnesyl- and geranylgeranyl-pyrophosphate, which pro- vide lipid anchors that are required for the biological activity of small GTP binding proteins. More than one hundred small GTP binding proteins have been identified. They are classified into five families, including the Ras and Rho protein families, which play key roles in the regulation of gene expression and of the or- ganization of the cytoskeleton. The inhibition of the farnesyl- ation or geranylgeranylation of small GTP binding proteins may underlie many of the pleiotropic effects of statins (11). The anti- inflammatory effect of statins has been reported in several studies, and may contribute to their beneficial effects. However, the anti-inflammatory effects of statins on cytokine-mediated in- duction of leukocyte adhesion molecule expression in EC re- main controversial. Several groups reported that statins inhibit adhesion molecule expression induced by proinflammarory cy- tokinesin vitro(5, 12). In contrast, others have shown that statins increased adhesion molecule expression induced by TNFα (13–16). Until now, few studies focused on the possible effect of statins on EC activation induced byAPLA.In vitro, it was shown that fluvastatin, and to a lesser extent simvastatin, reduced both the adhesion of U937 monocyte-like cells to APLA-treated human umbilical vein endothelial cells (HUVEC) and the ex- pression of E-selectin and ICAM-1 (5). Furthermore, in anin vivo study, fluvastatin treatment reduced thrombus size, the number of adherent leukocytes and soluble ICAM-1 concen- trations in mice treated with APLA (17).

The aim of our study was to investigate the influence of sta- tins on the synthesis and expression of several inflammation-re- lated proteins by APLA-activated EC. We show that, in EC treated with IgG from patients with APLA, statins further in- crease the expression of the leukocyte adhesion molecules VCAM-1 and E-selectin, of the monocyte chemoattractant pro- tein MCP-1 and of tissue factor. The statin-mediated increase was prevented by the addition of mevalonate or GGPP and mi- micked by the addition of the geranylgeranyl transferase in- hibitor GGTI- 286.

Material and methods

Reagents

Fluvastatin and the geranylgeranyl transferase inhibitor- 286 (GGTI-286) were obtained from Calbiochem (San Diego, CA, USA). Mevalonic acid lactone and geranylgeranyl-pyrop- hosphate (GGPP) were obtained from Sigma (St. Louis, MO, USA). Mevalonic acid was activated by treatment with 0.1 M NaOH at 50°C for 2 hours; then the pH was adjusted to 7.4 with HCl. Monoclonal anti-VCAM-1 (anti-CD106), and anti- E-selectin (anti-CD62E/CD62P) antibodies were purchased from Serotec (Oxford, UK). FITC-conjugated goat anti-mouse antibodies used as secondary antibodies for flow cytometry analysis, was from Cappel Organon Technika (Durham NC, USA).

Patients, blood samples and isolation of IgG

Six patients with APLA, and suffering from thrombotic compli- cations and/or pregnancy morbidity, were chosen among a group

of patients evaluated in a previous study for the ability of their IgG to induce cell activationin vitro(18). The clinical and sero- logical characteristics of these patients are summarized in table 1. IgGs were isolated from the patient plasmas on Protein A CL-4B Sepharose (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Protein levels were measured by the bicinchoninic acid protein assay (BCA Protein assay, Pierce, Rockford IL, USA).

Endotoxin levels, measured by the Limulus Amebocyte Lysate EndochromeAssay (Charles River Laboratories, Charleston SC, USA), were found to be below the detection limit (0.25 EU/ml) for all IgG samples at the concentration used in the activation as- says. The negative control antibody consisted of IgG prepared from a pool of ten healthy donors.

APLA assays

The titers of anticardiolipin IgG in the patient plasmas were de- termined as previously described and higher than 5 GPL Units (19). Levels of anti-β2GP1 IgG in plasma were evaluated by ELISA (20) and were positive in all patients. Lupus anticoagu- lant was evaluated according to the International Society of Thrombosis and Haemostasis criteria using aPTT-LA and Sta- clot LA (Stago, Anières, France) and dilute Russell's viper venom tests (Dade Behring, Marburg, Germany). Lupus anti- coagulant was present in four out of 6 patients. All APLA assays were repeated at least once at an interval of at least six weeks.

The purified IgG fractions were analyzed for anticardiolipin and anti-β2GP1 activity and found to be positive.

Isolation and culture of endothelial cells

Endothelial cells (HUVEC) were isolated from umbilical cord veins and cultured as described previously (21, 22). Cells were passaged by 0.05% trypsin-0.02%EDTA (Seromed Biochrom KG, Berlin, Germany) treatment at a split ratio of 1:2, and used between passages 1 to 4. The cell culture medium was RPMI 1640 supplemented with 10% fetal calf serum (FCS) (Invit- rogen, Carlsbad, CA, USA), 90μg/ml heparin (Roche, Reinach,

Table 1: Clinical and laboratory profile of the patients.

Patient Sex/Age Clinical manifes- tations

IgGACL* IgG

antiβ2GP1 LA

P1 F/14 SLE, throm-

boembolism 24 + +

P2 F/39 Fetal loss >60 + -

P3 F/54 Thromboem-

bolism, fetal loss

>60 + +

P4 F/44 Thromboem-

bolism, throm- bocytopenia

>60 + -

P5 F/47 Thromboem-

bolism >60 + +

P6 F/30 Fetal loss, skin

lesion 36 + +

* ACL IgG are expressed as GPL Units (normal values<5GPL) ; SLE for systemic lupus erythemato- sus, LA for lupus anticoagulant.

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Switzerland), 15 μg/ml EC growth supplement (Upstate Bio- technology, Lake Placid NY, USA), 10 mM HEPES, 100 IU/ml penicillin and 100μg/ml streptomycin (Gibco BRL-Life Tech- nologies, Rockville MD, USA). The FCS was decomplemented by treatment for one hour at 56°C.

Stimulation of endothelial cells with APLA and treatment with fluvastatin and inhibitors

HUVEC were grown in 24-well plates. After reaching con- fluence, the cells were incubated in complete medium overnight with 5μM fluvastatin with or without mevalonate (400μM) or GGPP (15μM). Then, culture medium was replaced by RPMI 1640, 20% FCS, 10mM Hepes, 100 UI/ml penicillin and 100μg/

ml streptomycin, containing, where appropriate, fluvastatin, me- valonate and GGPP. Cells were stimulated with purified IgG- APLA (0.5 mg/ml). For negative controls cells were incubated in the same conditions with medium alone or normal human serum IgG (0.5 mg/ml). Twenty-four hours after stimulation with IgG- APLA or control IgG, cells were isolated for flow cytometry analysis or mRNA extraction.

Similar experiments were performed with HUVEC after prior incubation for 48 hours with GGTI-286 (10 μM). Fresh GGTI-286 solutions were added after 24 hours. Thereafter, cells were treated with IgG-APLA or control IgG for 24 hours and cells or mRNA isolated for further analysis.

Analysis of adhesion molecule expression on EC by flow cytometry

Cells were examined for surface antigen expression by flow cy- tometry as previously described (15). The cells were washed in RPMI and incubated for 1 hour at 4°C with mouse monoclonal antibody (anti-E-selectin or anti-VCAM-1) at 10μg/ml in PBS (Gibco, BRL-Life Technologies, U.K.), 5% FCS and 0.02% so-

dium azide. Cells were washed in PBS-FCS-sodium azide and incubated for 1 hour at 4°C with polyclonal FITC-conjugated goat anti-mouse antibodies, dissolved in PBS/FCS/sodium azide. Cells were washed in PBS-sodium azide, harvested with trypsin-EDTA and, after centrifugation at 800 g for 5 minutes, fixed in 2.5% formaldehyde, 2% glucose, 0.02% sodium azide in PBS. Propidium iodide (10μl of 50μg/ml) was added to each sample. Non-viable propidium iodide-positive cells were gated out. Cell fluorescence was measured on a Becton Dickinson FACScan flow cytometer (San Jose CA, USA). A total of 10000 viable cells were analysed per experimental sample. Data were analysed using Cellquest software (Becton Dickinson).

RNA extraction and reverse transcription

Total RNA was extracted from cells, and treated with DNaseI, using RNeasykits (Qiagen GmbH, Hilden, Germany). The RNA yield and purity were assessed by absorbance measure- ments at 260 and 280 nm. From each sample, 1μg of RNA was used as template for cDNA synthesis using the ImPromIITMRe- verse transcription system (Promega, Madison WI, USA).

Quantitative real time reverse transcriptase PCR assay Relative mRNA levels of VCAM-1, E-selectin, MCP-1, and tis- sue factor were quantified by real-time PCR using the cDNA templates, with an Applied Biosystems Prism 7000 instrument and SYBR-greenPCR master mix reagent (Applied Biosys- tems, Warrington, UK). Specific forward and reverse primers used were respectively:

VCAM-1: CATGGAATTCGAACCCAAACA and GACCAA- GACGGTTGTATCTCTGG;

E-selectin: ATCATCCTGCAACTTCACC and ACACCTCAC- CAAACCCTTC;

MCP-1: GTCACCTGCTGCTATAACTTC and TGCTGCTGGTGATTCTTCTA;

tissue factor: CTACTGTTTCAGTGTTCAAGCAGTGA and CAGTGCAATATAGCATTTGCAGTAGC. Glyceralde- hyde-3-phosphate dehydrogenase, used as the control gene, was amplified and quantified using as a forward primer

GGTGAAGGTCGGAGTCAAC and reverse primer

CCATGGGTGGAATCATATTG.

Data were analyzed using the∆CTmethod and Applied Bio- systems Prism software, according to the manufacturers instruc- tions and expressed as fold increase over control cells to which no IgG or other agents had been added.

Statistical analysis

The effect of fluvastatin on the cell surface expression of VCAM-1 and E-selectin and on mRNA synthesis of VCAM-1, E-selectin, tissue factor and MCP-1 was assessed by the paired student’s t-test. The statistical analysis of the effect of mevalon- ate was also performed by the paired student’s t-test.

Results

Effect of fluvastatin on adhesion molecules and tissue factor expression in non stimulated HUVEC

Fluvastatin alone had no effect on VCAM-1 or E selectin ex- pression as measured by flow cytometry analysis. We made a Figure 1: Effect of fluvastatin onVCAM-1, E-selectin, MCP-1

and tissue factor mRNAs in HUVEC.Confluent cells were incu- bated overnight with 5μM fluvastatin. Relative mRNA levels were evalu- ated by quantitative RT-PCR and are expressed relative to mRNA ex- pression in control cells. White bars represent control cells, grey bars represent cells incubated overnight with fluvastatin.

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Dunoyer-Geindre, et al.: Statins, antiphospholipid antibodies, and endothelial cells time course on the effect of 5μM fluvastatin on mRNA levels of

inflammatory marker proteins.The maximum effect was observ- ed at 16 h: indeed 5μM fluvastatin induces a 1.6 to 3.6-fold in- crease in VCAM-1, E selectin, MCP-1 and tissue factor mRNAs (Fig. 1). The increase was reversed by cotreatment with meval- onate (not shown).

Fluvastatin increases the surface expression ofVCAM-1 and E-selectin on HUVEC stimulated by IgG from patients with APLA

IgG was isolated from six patients positive for APLA (IgG- APLA). In preliminary experiments these IgGs were shown to induce a significant increase in VCAM-1 expression as com- pared with control IgG.Treatment of HUVEC for 16h with 5μM fluvastatin prior to IgG-APLA stimulation for 24h resulted in in- creased expression of VCAM-1 or E-selectin. Figure 2 shows a representative flow cytometry profile of VCAM-1 (A) and E-se- lectin (B) expression obtained with cells incubated with 500μg/

ml of IgG-APLA from patient 1 in the presence or absence of flu- vastatin. For all six IgG-APLA samples, we observed an increase in VCAM-1 and in E-selectin expression in fluvastatin-treated cells, compared with cells treated with IgG-APLA alone (p <

0.01 fig 2C and p < 0.01 fig 2D, respectively, paired student’s t-test). Fluvastatin was used at 5μM, a concentration that is not cytotoxic (5).

Effect of fluvastatin on the expression ofVCAM-1, E-selectin, MCP-1 and tissue factor mRNA by IgG-APLA-activated HUVEC

We further investigated the effect of fluvastatin on IgG-APLA -treated HUVEC by measuring the mRNA levels of various in- flammation-related proteins. HUVEC were incubated overnight with or without fluvastatin and then stimulated with IgG-APLA for 24h (from 4 out of the 6 IgG-APLA samples). Total RNA samples were isolated, reverse transcribed and quantitative RT- PCR analysis performed for VCAM-1, E-selectin, MCP-1 and tissue factor. Levels of each mRNA were similar in non-treated HUVEC and in HUVEC treated with control IgG. As shown in Figure 3, mRNA levels ofVCAM-1, E-selectin, tissue factor and MCP-1 increased after incubation with IgG-APLA from pa-

Figure 2: Flow cytometry analysis of IgG-APLA-induced VCAM-1 and E-selectin expression in HUVEC and the effect of fluvastatin and GGTI-286 on IgG-APLA-inducedVCAM-1 and E-selectin expression and its reversal by mevalonate and GGPP.

Confluent HUVEC were pretreated overnight with 5μM fluvastatin, with 5μM fluvastatin plus 400μM mevalonate, with 5μM fluvastatin plus 15μM GGPP or with 10μM GGTI-286. IgG-APLA, isolated from the plasma of patients with APLA (P1 to P6, 500μg/ml), was added to the medium for 24 hours andVCAM-1 or E-selectin expression at the cell surface was measured by flow cytometry. A: (VCAM-1) and 2B (E-selectin): Representative flow cytometry profile obtained with IgG- APLA from patient 1: I represents expression in control HUVEC; II in HUVEC incubated for 24 h with IgG-APLA (500μg/ml); III-V in HUVEC pretreated for 18h with fluvastatin (5μM) (III), fluvastatin (5μM) and me- valonate (400μM)(IV) or GGPP (15μM)(V) and then for 24 h with IgG- APLA (500μg/ml); andVI in HUVEC pretreated for 48h with GGTI (10μM) and then for 24 h with IgG-APLA (500μg/ml). C-D: Flow cyto- metry analysis ofVCAM-1 (C) and E-selectin (D) expression obtained with HUVEC treated with IgG-APLA from the 6 patients: control cells (white bars), HUVEC incubated with IgG-APLA (500μg/ml) from pa- tients P1 to P6 (black bars), HUVEC preincubated for 18h with 5μM flu- vastatin (diagonally striped bars), 5μM fluvastatin and 400μM meval- onate (dotted bars) or 5μM fluvastatin and 15 μM GGPP (gray bars) and then with IgG-APLA (500μg/ml), or preincubated for 48h with 10μM GGTI-286 (horizontally striped bars) and then with IgG-APLA (500μg/

ml).VCAM-1 and E-selectin expression assessed by a paired student’s t-test differs significantly between cells treated with APLA alone and cells pretreated with fluvastatin prior to IgG-APLA stimulation (p<0.01 forVCAM-1 and p<0.01 for E-selectin). The reversal effect of meval- onate was assessed by a paired student’s t-test: p<0.005 forVCAM-1 ex- pression and p<0.001 for E-selectin expression.

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tients, compared to untreated HUVEC. Simultaneous stimu- lation of HUVEC with fluvastatin and IgG-APLA further increased mRNA expression of the four molecules (respectively p < 0.05 for VCAM-1, p < 0.01 for E-selectin, p < 0.005 for MCP-1 and p < 0.025 for tissue factor, paired student’s t-test, n=4).

The effect of fluvastatin on IgG-APLA-induced overexpression of adhesion molecules, tissue factor and MCP-1 are related to the isoprenoid pathway The addition of 400μM mevalonate partially reversed the over- expression of VCAM-1 and E- selectin observed by flow cyto- metry in fluvastatin- and IgG-APLA -treated HUVEC (Fig. 2).

This confirms that the effect of fluvastatin is due to the inhibition of HMG-CoA reductase. The difference between mean fluor- escence values obtained with the combination IgG-APLA and fluvastatin with or without mevalonate assessed by a paired stu- dent’s t-test had a p value <0.005 for VCAM-1 and p < 0.001 for E-selectin (n=6).

In a previous study, we have shown that the increasing effect of statins (simvastatin and fluvastatin) on adhesion molecule ex- pression induced by TNFαwas completely reversed by GGPP (15μM) whereas FPP had only a partial effect, even when higher doses were used (15). Based on this result, we studied the effect of GGPP on the fluvastatin-mediated increase inVCAM-1, E-se-

lectin, MCP-1 and tissue factor expression induced by IgG- APLA. Fig 2A and B shows the results obtained by flow cyto- metry analysis with IgG-APLA from one of the patients. (Patient 1). These results show that 15μM GGPP reverses the effect of fluvastatin to a similar extent as mevalonate. Treatment of HUVEC with GGTI-286 induced a significant increase in E- selectin or VCAM-1 expression induced by IgG-APLA. Similar results were obtained for the six IgG-APLA samples (Fig 2C and D).

We studied the effect of mevalonate, the isoprenoid inter- mediate GGPP as well as GGTI-286 on the expression of VCAM-1, E-selectin, MCP-1 and tissue factor mRNA after stimulation of EC with IgG-APLA (Fig. 3).The enhancing effect of fluvastatin on the mRNA levels was reverted by coincubation with 400μM mevalonate or 15μM GGPP. Similar profiles were obtained for the four IgG-APLA -positive patient IgG samples tested. Coincubation with GGTI-286 had a similar effect as flu- vastatin on the IgG-APLA induced mRNA overexpression of the four molecules tested.

Discussion

In this study we show that APLA from patients increase the ex- pression of leukocyte adhesion molecules, of MCP-1 and of tis- sue factor and that fluvastatin further enhances this stimulatory Figure 3: Effect of fluvastatin and GGTI onVCAM-1, E-selectin,

MCP-1 and tissue factor mRNAs in IgG-APLA-treated HUVEC, and its reversal by mevalonate and GGPP.Relative mRNA levels were evaluated by quantitative RT-PCR. Results are ex- pressed relative to mRNA expression in control cells. Measurements were made in triplicate and the results are given as the mean and the standard deviation for each IgG-APLA. Control cells (white bars), HUVEC incubated with 500μM IgG-APLA from patients P1 to P4 (black bars), HUVEC preincubated with 5μM fluvastatin (diagonally striped

bars), HUVEC preincubated with 5μM fluvastatin and 400μM meval- onate (dotted bars), HUVEC preincubated with 5μM fluvastatin and 15 μM GGPP (gray bars), HUVEC preincubated with 10μM GGTI-286 be- fore APLA stimulation (horizontally striped bars). The increasing effect of fluvastatin on APLA induced mRNA expression ofVCAM-1, E selectin, MCP-1 and tissue factor was assessed by a paired student’s t-test: re- spectively p<0.05 forVCAM-1, p<0.01 for E-selectin, p<0.005 for MCP-1 and p<0.025 for tissue factor (n=4).

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Dunoyer-Geindre, et al.: Statins, antiphospholipid antibodies, and endothelial cells effect. The effects observed for APLA on protein expression in

EC confirm the results of previous studies (4–6, 23). Induction of an adhesive phenotype on endothelial cell is not attributable to contaminating endotoxins since purified IgG from healthy do- nors do not induce adhesion molecule expression and Limulus assays on sample preparations were confirmed to be endotoxin negative. We therefore conclude that the effect ofAPLA on EC is antibody specific.

As the so-called pleiotropic „anti-inflammatory“ activity of statins represent an attractive mechanism for their beneficial clinical effects in addition to their well known cholesterol lower- ing effects (11, 24–26), we examined whether fluvastatin modi- fied the APLA-induced up-regulation of VCAM-1, E-selectin, tissue factor and MCP-1. We observed that preincubation with fluvastatin led to an additional increase in each mRNA in re- sponse to IgG-APLA treatment of HUVEC. The effects of flu- vastatin on the expression of VCAM-1 and E-selectin, induced by IgG-APLA, were comparable with our previous observations on the effect of simvastatin and fluvastatin on the expression of adhesion molecules induced by TNFαor LPS (15).

Several studies have been conducted to evaluate the effect of statins on EC activation induced by inflammatory cytokines with conflicting results. Part of these studies reported that statins were able to decrease adhesion molecule expression and macrophage adhesion on EC stimulated by TNFα or LPS (5,12). Fur- thermore, statins were reported to decrease the expression of tis- sue factor on macrophages stimulated with LPS (27). From this, an attractive mechanism for a protective effect of statins has been proposed, in which statins decrease the inflammatory response of the endothelium resulting in a lower leukocyte adhesion and a lower tissue factor expression both on the endothelium and the attached leukocytes. These effects would then lead to a lower procoagulant activity in inflammatory regions. In support for this hypothesis, fluvastatin was reported to reduce tissue factor expression and macrophage accumulation in carotid lesions of cholesterol-fed rabbit (28). In contrast, several other studies rather observed that statins or geranylgeranyltransferase in- hibitors rather potentiated the cytokine-mediated increase in ad- hesion molecules (13–16). Several reasons for these discrep- ancies were put forward without finding a definitive expla- nation: the use of different compounds of the statin family, the di- verse agonists employed in activating the cells or differences in the techniques of evaluating adhesion molecules expression (29). One recent study reported results that may reconcile, at least in part, the discrepancies (16). In this study statins did in- crease adhesion molecule expression onTNF-treated EC, but de- creased adhesion molecule clustering and monocyte-like cell ad- hesion. It is known that adhesion molecule clustering and leuko- cyte adhesion are dependent on members of the Rho family of GTP-binding proteins (30), which require geranylgeranylation for expression of their activities. In this model the effect of sta- tins or geranylgeranyltransferase inhibitors on leukocyte ad- hesion is due to their inhibition of these small GTP-binding pro- teins, rather than to an inhibition of adhesion molecule responses to inflammatory mediators.

The effect of statins on the inflammatory properties ofAPLA was investigated in two previous studies: in anin vitroexperi-

ment, Meroni (5) reported that simvastatin and fluvastatin de- creased the EC expression of E-selectin and ICAM-1 in response to immunopurified APLA. In a microcirculation model of ex- posed cremaster muscle, mice receiving intraperitoneally APLA and treated with fluvastatin showed smaller thrombi, a reduced number of adherent leukocytes and decreased levels of circu- lating soluble ICAM-1 compared with animals receiving APLA without fluvastatin (17). These two results implied a beneficial effect of statins on thein vivoandin vitroresponse toAPLA. Our findings contradict these results. We observed that fluvastatin alone induced a small increase in VCAM-1, E-selectin, MCP-1 and tissue factor mRNA. However, this effect was not detected at the cell surface by flow cytometry. Kiener et al (31) found that treatment of human monocytes with lipophilic statins alone stimulated the production of MCP-1, IL8, TNFαand IL1βand markedly sensitized the cells to subsequent challenge with in- flammmatory agents. Thus one explanation could be that statins

“pre-stimulate” the cells leading to an enhanced response to agonists such as cytokines or APLA. The enhanced adhesion molecule response of statin pre-treated cells not necessarily translates into an increased inflammatory responsein vivo. In- deed, Bernot et al. (16) observed that statins increased the ad- hesion molecule response of TNF-treated EC, but that leukocyte adhesion was decreased due to an inhibition of Rho geranylger- anylation, which block the clustering of adhesion molecules at the EC surface.

The effect of fluvastatin was HMG-CoA-reductase depend- ent since it was reversed by mevalonate. In addition, the effect of fluvastatin was inhibited by the isoprenoid intermediate GGPP and mimicked by the geranylgeranyl-transferase inhibitor GGTI-286, which suggests that the observed effect is due to an inhibition of protein geranylgeranylation.These results are simi- lar to our previous findings that the enhancing effect of simvas- tatin or fluvastatin on TNFα- or LPS-induced E-selectin ex- pression was reversed by GGPP and mimicked by GGTI-286.

This indicates that the inhibition of protein geranylgeranylation, but not of cholesterol synthesis, is associated with the effect of these statins (15). More than one hundred small GTP-binding proteins are known that require post-translational geranylger- anylation for their anchoring to the cell membrane, which is es- sential for their activity. Our data suggest that one or more of these geranylgeranylated proteins exert a negative retrocontrol on EC activation induced by APLA, TNFαor LPS.

In conclusion, our results show that statins potentiate the ac- tivation of endothelial cells in response to APLA, leading to an increased expression of leukocyte adhesion molecules, of a monocyte chemoattractant chemokine and of tissue factor. In combination with an increased expression of tissue factor by ad- herent activated monocytes, these changes alone would be ex- pected to lead to an increased tendency for local thrombus formation. This implies that the well-known clinical benefits of statins are not due to a decrease in the inflammatory response of endothelial cells to cytokines or autoantibodies. In addition, our findings suggest that a geranylgeranylated protein limits the re- sponse of endothelial cells to APLA.

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