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To determine the effects of primary antiphospholipid syndrome (PAPS)‐derived anti‐β2GPI antibodies on gene expression in human umbilical vein endothelial cells (HUVEC) by gene profiling using microarrays.
Anti‐β2GPI antibodies purified from sera of patients with PAPS or control IgG isolated from normal subjects were incubated with HUVEC for 4 h before isolation of RNA and processing for hybridisation to Affymetrix Human Genome U133A‐2.0 arrays. Data were analysed using a combination of the MAS 5.0 (Affymetrix) and GeneSpring (Agilent) software programmes. For selected genes microarray data were confirmed by real‐time PCR analysis or at the protein level by ELISA.
A total of 101 genes were found to be upregulated and 14 genes were downregulated twofold or more in response to anti‐β2GPI antibodies. A number of novel genes not previously associated with APS were induced, including chemokines CCL20, CXCL3, CX3CL1, CXCL5, CXCL2 and CXCL1, the receptors Tenascin C, OLR1, IL‐18 receptor 1, and growth factors CSF2, CSF3 IL‐6, IL1β and FGF18. The majority of downregulated genes were transcription factors/signalling molecules including ID2. Quantitative real‐time RT‐PCR analysis confirmed the microarray results for selected genes (CSF3, CX3CL1, FGF18, ID2, SOD2, Tenascin C).
This study reveals a complex gene expression response in HUVEC to anti‐β2GPI antibodies with multiple chemokines, pro‐inflammatory cytokines, pro‐thrombotic and pro‐adhesive genes regulated by these antibodies in vitro. Some of these newly identified anti‐β2GPI antibody‐regulated genes could contribute to the vasculopathy associated with this disease.
Antiphospholipid syndrome (APS) is characterised by thrombosis, thrombocytopenia and recurrent foetal loss.1 Two forms of the syndrome have been described; the “primary” syndrome (PAPS), where there is no evidence of any other underlying disease and “secondary” syndrome that is mainly associated with systemic lupus erythematosus (SLE). Elevated serum titres of antiphospholipid antibodies (aPL) correlate with thrombotic events in APS2 and there is strong evidence that aPL display a pathogenic role in APS.3,4 β2‐glycoprotein I (β2GPI) binds to negatively charged phospholipids through a positively charged lysine‐rich sequence of amino acids in its fifth domain5 and is now recognised as the primary aPL target in APS.5,6,7,8 Anti‐β2GPI antibodies bind to the β2GPI protein adherent to the endothelial cell (EC) surface and induce EC activation.9
Anti‐β2GPI antibodies might exert a direct pathogenic effect in APS by perturbing homeostatic reactions that take place on the surface of EC.10 A number of in vitro studies have reported that anti‐β2GPI antibodies can activate EC as shown by early increases in monocyte adhesion and the expression of E‐selectin, vascular cell adhesion molecule‐1 (VCAM‐1), and intracellular adhesion molecule‐1 (ICAM‐1).9,11,12 In vivo, aPL infused into naïve mice caused increased adhesion of monocytes and formation of sustained and larger thrombi when compared to normal control IgG.13
In addition, recent studies have reported that nuclear factor kappa B (NF‐κB) translocation, the myeloid differentiation primary response gene 88 (MyD88) pathway and p38 mitogen‐activated protein kinase (MAPK) phosphorylation are involved in EC and monocyte activation by anti‐β2GPI antibodies.14,15,16 However, the extent and diversity of anti‐β2GPI‐mediated gene regulation in EC cells is not yet well characterised. The present study was undertaken to examine the profile and diversity of early gene regulation in EC in response to polyclonal patient‐derived anti‐β2GPI antibodies using Affymetrix microarray gene profiling.
Ethical approval for the collection of sera from PAPS patients was obtained prior to the initiation of the study from the St. Thomas' Hospital Research Ethics Committee. Following written patient consent, sera were collected from a total of five patients with PAPS. All 5 patients had high levels of IgG aPL and strong lupus anti‐coagulant activity. Anticardiolipin activity in the patients was β2GPI dependent (data not shown). The clinical profiles of patients from whom polyclonal anti‐β2GPI antibody preparations were isolated and used in this study are shown in table 11.. All 5 patients fulfilled the Sapporo classification criteria for definitive PAPS.17
IgG from patients or normal age and sex‐matched subjects were purified using a HiTrap Protein G HP affinity column (GE Healthcare, Buckinghamshire, UK) as per the manufacturer's instructions. Purified human β2GPI protein was purchased from SCIPAC Ltd. (Sittingborne, Kent, UK.) The protein was coupled to a HiTrap NHS‐activated HP column as recommended by the manufacturer (GE Healthcare). A 1/8 dilution of serum in starting buffer was applied to the column and affinity‐purified antibody was eluted in 0.1 M glycine‐HCL pH 2.7 and neutralised with 1 M Tris‐HCL pH 9.0. The purification was carried out on an AKTA prime 3 system (GE Healthcare). Protein concentration of IgG and affinity purified antibodies was determined by Bicinchoninic protein assay (Sigma).
Following isolation, patient‐derived anti‐β2GPI antibodies were tested for binding to β2GPI by enzyme‐linked immunosorbant assay (ELISA) using a previously described method.18 All antibodies were also tested in an anti‐cardiolipin ELISA.19
Human umbilical cords were obtained from the Labour Ward at St. Thomas' Hospital London following ethical approval and written patient consent. Human umbilical vein endothelial cells (HUVEC) were isolated from normal full term umbilical cord vein using collagenase enzyme (Sigma) and cultured as previously described at 37°C in a humidified incubator.20 For cell stimulation experiments HUVEC were incubated for 4 h with different antibody preparations. In all experiments, polymixin B (5 μg/ml) was included to exclude the possibility of endotoxin effects as previously described.15
Confluent HUVEC at passage 3 were incubated with the four PAP‐derived anti‐β2GPI antibody preparations (P1, P2, P3, P4, 50 μg/ml) preparations or four normal control IgG (N1, N2, N3, N4, 50 μg/ml) for 4 h at 37°C in a humidified incubator. Total HUVEC RNA was then extracted using the RNeasy Kit (Qiagen, Crawley, West Sussex, UK). The quality of the RNA was checked using a 1% agarose gel. Three independent experiments using HUVEC from three different donors were carried out on different occasions.
cRNA samples for microarray hybridisation were prepared following the manufacturer's instructions (Affymetrix, Santa Clara, California). Fragmented cRNA was hybridised overnight to gene chip arrays at 45°C for 18 hours. Control cRNAs were then added to the hybridisation mix. Human Genome U133A‐2.0 gene chips containing probe sets for 18 400 human transcripts were used. In one of the three independent experiments, one anti‐β2GPI antibody (P2) treated sample and one control IgG treated (N2) sample were not processed beyond initial RNA quantitation due to low RNA yield. Therefore, a total of 22 chips were hybridised and scanned. Gene chips were washed and stained on the Gene Chip Fluidics Station 400 (Affymetrix). Fluorescent signals were detected using the HP G2500A Gene Array Scanner.
After scanning the gene chips, images were analysed using the Affymetrix microarray suite (MAS) 5.0 (Affymetrix, Santa Clara, California, USA) to generate raw data in the form of “.cel” files. Further analysis was carried out using a combination of the MAS 5.0 and GeneSpring (Agilent Technologies, Santa Clara, California, USA) software programmes. The detection of a particular gene as “present, absent or marginal” was carried out using the MAS 5.0 software. The .cel files were imported into GeneSpring and normalised by GC‐Robust Multichip Average (GCRMA), an algorithm that normalises the data by quantile normalisation, in order to minimise the biological variation between samples. Further analysis was carried out on genes identified as present or marginal. Genes with statistically different expression between the control IgG and the anti‐β2GPI antibody treated cells (p<0.05) were identified by the Kruskal–Wallis test (non‐parametric one way analysis of variance (ANOVA)) with the Benjamin and Hochberg multiple testing correction.21 Filtering the gene list on the criteria of a twofold or more increase or decrease in expression identified a panel of genes that were significantly changed in HUVEC by anti‐β2GPI antibody treatment compared to normal control IgG treatment. Average‐linkage hierarchical clustering (using the Pearson Correlation) was carried out separately on the genes and the samples generating a genetree and condition tree, respectively, to highlight any distinct patterns in gene expression and the relationships between the samples.
Quantitative real time PCR was used to confirm the microarray results for the expression levels of selected genes. The primer pairs used for the following genes were: CSF3, forward 5′‐CGCTCCAGGAGAAGCTGT‐3′ and reverse 5′‐CCAGAGAGTGTCCGAGCAG‐3′, CX3CL1, forward 5′‐ATCTCTGTCGTGGCTGCTC‐3′ and reverse 5′‐TCACACCGTGGTGCTGTC‐3′, E‐selectin, forward 5′‐TGAAGCTCCCACTGAGTCCAA‐3′ and reverse 5′‐ GGTGCTAATGTCAGGAGGGAGA‐3′, FGF18, forward 5′‐CTCTACAGCCGGACCAGTG‐3′ and reverse 5′‐CCGAAGGTGTCTGTCTCCAC‐3′, ID2, forward 5′‐CAGCATCCTGTCCTTGCAG‐3′ and reverse 5′‐AAAGAAATCATGAACACCGCTTA‐3′, SOD2, forward 5′‐CAAATTGCTGCTTGTCCAAA‐3′ and reverse 5′‐CGTGCTCCCACACATCAAT‐3′, Tenascin C, forward 5′‐GCTCAAAGCAGCCACTCATT‐3′ and reverse, 5′‐CCCATATCTGGAACCTCCTCT‐3′, and β‐actin, forward 5′‐CCAACCGCGAGAAGATGA‐3′ and reverse 5′‐CCAGAGGCGTACAGGGATAG‐3′. β‐Actin was used as an internal control as no changes were found in levels of expression of this housekeeping gene when cells were treated with anti‐β2GPI antibodies in microarray experiments. Primers for the genes were designed using the Roche universal probe library. One µg of total RNA from HUVEC incubated for 4 h in medium alone (blank), or with 2 normal control IgG preparations (N3, N4, 50 μg/ml) or 4 anti‐β2GPI antibody preparations (P1, P2, P4, P5, 50 μg/ml), or with TNFα was reverse transcribed into cDNA with the Quantitect reverse transcription kit (Qiagen) using oligo‐dT primers. Antibody preparations N3, N4, P1, P2 and P4 were previously used in the microarray experiments. Quantitative real‐time PCR was carried out with the QuantiTect SYBR Green PCR Kit (Qiagen) in the ABI 7000 sequence detector (Applied Biosystems). Gene expression levels were calculated with the absolute quantitation method,22 and normalised to the β‐actin level. All PCR reactions were carried out in duplicate, and repeated at least twice for each gene. The specificity of the PCR reactions was verified with dissociation curve analysis.
E‐selectin cell surface expression was evaluated by a cell ELISA.9 Unstimulated cells were used as a negative control and TNFα (10 ng/ml, R&D Systems) was used a positive control stimulus.15 IL‐8 levels in cell supernatants were determined using a human IL‐8 ELISA kit according to the manufacturer's instructions (BD Biosciences, Cowley, Oxon, UK).
The non‐parametric Mann–Whitney U Test was used to compare E‐selectin and IL‐8 levels between cells incubated with anti‐β2GPI antibodies and normal control IgG preparations in ELISA experiments.
Sera were collected from four PAPs patients and anti‐β2GPI antibodies purified by protein G and β2GPI affinity column isolation. All patient derived anti‐β2GPI antibodies bound to β2GPI by ELISA and were also positive in a modified anti‐cardiolipin ELISA with but not without co‐factor, carried out as previously described (data not shown).8 Following gene chip hybridisation and scanning, HUVEC were found to express 13 727 out of 18 400 transcripts. Genes that were significantly changed (p<0.05, up or down) twofold or more were filtered and categorised as anti‐β2GPI antibody‐regulated genes. A total of 101 genes were upregulated by at least twofold or more by anti‐β2GPI antibodies (fig 11 and table 22).). Figure 11 shows a hierarchical cluster analysis of upregulated and downregulated genes. Genes were clustered according to their patterns of expression (vertical axis) and also per condition (similarities between total gene expression profiles in different samples). It is noteworthy that in the dendrogram, similarities in level of gene expression are grouped (see branching) per independent experiment (that is per separate HUVEC population) rather than per antibody preparation. This implies that the greatest source of variation, in terms of genes regulated by anti‐β2GPI antibody, is determined by individual HUVEC populations rather than between individual anti‐β2GPI antibody preparations. It is likely that inter‐experiment variation masked any subtle differences in the upregulation/downregulation of genes by anti‐β2GPI antibody from different patients.
Real‐time RT‐PCR analysis was carried out for selected novel anti‐β2GPI antibody regulated genes, covering a range of different levels of regulation. Genes included in this analysis were CSF3, CX3CL1, FGF18, SOD2 and Tenascin C plus E‐selectin as a positive control gene. We also included the downregulated gene ID2 in these experiments. The results of these experiments are shown in fig 22.. All six upregulated genes (CSF3, CX3CL1, E‐Selectin, FGF18, Tenascin C and SOD2) were also found to be upregulated by real time PCR analysis. Levels of upregulation were variable, but CX3CL1 was the highest‐fold upregulated gene of the six selected genes by microarray analysis (fig 2A2A).). ID2 was downregulated 2.3‐fold by microarray analysis and this was very similar to the level of downregulation by real‐time PCR analysis. (fig 2B2B).). In the present study increased mRNA levels of E‐selectin and IL‐8 following anti‐β2GPI antibody treatment and microarray analysis were consistent with increased protein levels following antibody treatment ((figsfigs 1, 2C, DD).
We assigned the 101 upregulated genes to the following APS relevant functional groups; cell receptors/adhesion molecules, cytokines/chemokines, coagulation genes, apoptosis/anti‐apoptosis, transcription factors/signalling, metabolism and miscellaneous genes (table 22).). Of particular note are the high level of induction of the chemokines CCL20, CXCL3, CX3CL1, CXCL5, CXCL2 and CXCL1 as well as genes classically associated with pro‐inflammatory cytokine TNFα signalling such as TNFAIP6, TNFAIP2, TNFAIP8, TNFAIP3 and the anti‐apoptotic gene BCL2A1. Cell receptors induced included Tenascin C, OLR1 (oxLDL receptor) and IL‐18 receptor 1. Other induced genes of interest included growth factors CSF2, CSF3, IL‐6, IL‐1β and FGF18. The list of upregulated genes includes some previously identified anti‐β2GPI‐induced genes such as E‐selectin, Tissue Factor (TF), ICAM‐1 and VCAM‐110 but the majority of the genes we have identified represent anti‐β2GPI‐induced genes not previously reported.
A smaller panel of anti‐β2GPI antibody‐regulated genes in EC were downregulated (fig 22 and table 33).). None of these genes has previously been reported to be anti‐β2GPI antibody‐regulated genes in EC. The majority of the 14 downregulated genes encode signalling and transcription factors/signalling molecules. Two receptor/adhesion molecules were also downregulated. GJA4 (connexin 37) is a gap junctional protein and OCLN (Occludin) is a structural protein of tight junctions.
The most striking feature of this study is the extent and diversity of anti‐β2GPI antibody regulated genes in EC. The results reveal induction of a complex pro‐inflammatory, as well as, a pro‐adhesive and pro‐coagulant milieu by these antibodies, which could potentially be involved in the pathogenesis of PAPs.
It is intriguing that many of the most highly upregulated genes in the present study are chemokines such as CCL20, CXCL3, CX3CL1 (fractalkine), CXCL5, CXCL2 and CXCL1, which are involved in recruitment, chemotaxis and proliferation of mononuclear cells and/or granulocytes. These findings are consistent with a number of in vitro and in vivo studies reporting that anti‐β2GPI antibodies increased monocyte adhesion to EC.9,11,12 Moreover, placental biopsies from APS patients had a higher concentration of inflammatory cells particularly macrophages23 and an association has been found between neutrophil recruitment and foetal loss in APS.24
CX3CL1 (fractalkine) and its receptor CX3CR1 are expressed in atherosclerotic lesions of humans and mice25 and in CX3CL1‐deficient mice there is a major reduction of atherosclerosis.26 Roughly one third of PAPS patients have atherosclerosis and a direct association of aPL with the pathogenesis of accelerated atherosclerosis in APS patients has been reported.27,28 aPLs are thought to accelerate this process by activating EC. β2GPI has also been demonstrated in high concentration in atherosclerotic plaque.29 Other cytokine and adhesion molecules found to be upregulated by anti‐β2GPI antibody could also have a role in the development of atherosclerosis. Monocytes have been shown to strongly express IL‐18 in atheromatous lesions in situ30 and EC expression of IL‐18R was increased 4.3‐fold in our study. Gerdes et al.31 suggested an IL‐18 mediated paracrine proinflammatory pathway involving monocytes ECs and smooth muscle cells in association with atherogenesis.
Expression of oxLDL receptor OLR1 was upregulated over fourfold in our study (table 22).). OLR1 expressed on vascular EC is involved in binding, internalisation and degradation of oxLDL and might therefore play a significant role in atherogenesis.32,33 Anti‐β2GPI antibodies bind to β2GPI‐oxLDL complexes and have been shown in vitro to enhance uptake into monocytes/macrophages potentially accelerating the lesion formation.34 GJA4 (connexin 37), a gap junction protein, was downregulated by anti‐β2GPI antibodies and polymorphisms in this protein have been associated with the development of arteriosclerotic plaques in human subjects.35
We have confirmed by gene microarray profiling anti‐β2GPI mediated upregulation of molecules, previously reported to be upregulated at the protein level, including TF, E‐selectin, ICAM, and VCAM‐1.9,11,12,36,37,38 It is tempting to speculate that a combination of increased adhesion molecules, pro‐inflammatory cytokines and chemokines in addition to increased TF expression could strongly support development of thrombosis and contribute to the advancement of atherosclerotic lesions in response to anti‐β2GPI antibodies.
A number of pro‐angiogenic cytokines/chemokines such as IL‐8 and fibroblast growth factor, which were upregulated by anti‐β2GPI antibodies in the present study, might contribute to the hyperplasia associated with PAPS. APS is associated with EC proliferation and fibrosis characterised by intimal hyperplasia within the lumen of micro‐capillaries typically within the kidney or skin.39 It is also associated with cardiac lesions involving thickening of heart valves with deposition of aPL in the subendothelial layers.40 Histologic examination of renal biopsies from 16 patients with PAPs showed small vessel vaso‐occlusive lesions associated with myofibroblastic intimal cellular proliferation and thrombosis, five patients showed endothelialised channels indicating recanalising thrombosis and EC proliferation.41
β2GPI has been reported to bind to cells in a number of different ways. For example, it can bind to anionic membrane molecules such as heparan sulphate and it has also been reported to bind as a ligand to the annexin II receptor.10,42 Intriguingly, an indirect mechanism for EC stimulation by anti‐β2GPI antibody could exist. Annexin II is also a high affinity receptor for Tenascin C, a component of the extracellular matrix that functions as an adhesion molecule, shown in this study to be upregulated by anti‐β2GPI antibody.43
One possibility that has to be considered, however, is that our regulated genes might include some or many genes not regulated by anti‐β2GPI antibody directly but rather indirectly by autocrine cytokine/chemokine production produced by EC cells shortly after anti‐β2GPI antibody exposure. We chose to study gene expression at 4 h after exposure to anti‐β2GPI antibody in order to study the early gene expression profile. However, many cytokines/chemokines are induced rapidly and they could themselves then induce gene expression in EC by binding to high affinity receptors on the EC. This possibility should be addressed in future studies aimed at investigating the signal transduction mechanisms responsible for anti‐β2GPI antibody mediated gene regulation in EC. The anti‐β2GPI antibody induced gene panel described here is largely distinct to those described in gene profiling studies on HUVEC with different cytokines or LPS.44,45,46 Nonetheless, a small subset of anti‐β2GPI antibody‐induced genes (for example; E‐selectin, IL‐8, VCAM‐1, TNFAIP6, TNFAIP2, TNFAIP8, TNFAIP3), have been shown to be induced by cytokine and/or LPS‐induced gene profiling of HUVEC.44,45,46 A study profiling monoclonal anti‐β2GPI antibody mediated gene regulation in human monocytes found upregulation of a number of genes using cDNA arrays, such as, IL‐1β and TF, which were also identified in our study.16 However many more anti‐β2GPI antibody regulated genes in HUVEC were found in the present study probably due to the much larger number of genes represented on the Affymetrix chips in comparison to the cDNA arrays used in the earlier study.16
An important question in relation to our findings is how they relate to the in vivo situation. A recent study, measuring a limited number of parameters of EC function, concluded that aPL were unable to support a full‐blown endothelial perturbation in vivo.47 There is evidence however from other studies for increased circulating levels of TF, IL‐6, TNFα48 and VCAM‐149 in APS patients. Our studies suggest increased levels of some cytokines might, at least in part, be EC derived and therefore evidence of endothelial perturbation in vivo.
In conclusion, global gene expression profiling using microarray technology has been used for the first time to examine the extent and diversity of PAPs patient‐derived anti‐β2GPI antibody mediated gene regulation in HUVEC. These studies have identified important anti‐β2GPI antibody regulated EC genes that might contribute to the vasculopathy in PAPs. Further studies on signal transduction mechanisms responsible for anti‐β2GPI antibody mediated gene regulation and the role of individual anti‐β2GPI antibody gene targets in APS pathogenesis should provide opportunities for new therapeutic strategies by either inhibiting the expression of particular (pathogenic) genes or the activation of corresponding signalling pathways. Moreover, these findings could have wider implications for other autoimmune diseases, where anti‐β2GPI antibodies have been described.
We would like to thank Beverley Hunt for providing sera samples from patients and Ewan Hunter for help and advice with analysis of microarray data. We also thank Pier‐Luigi Meroni for help and advice on isolating antibodies from patient sera, Phil Marsh for help and advice on real‐time PCR analysis and Helen Collins and Steve Thompson for helpful discussions on the manuscript.
This work was supported by Lupus UK and the Arthritis Research Campaign.
Competing interests: None.