BIOLOGY OF REPRODUCTION (2013) 89(4):88, 1–11 Published online before print 21 August 2013. DOI 10.1095/biolreprod.113.109637
Potential New Mechanisms of Placental Damage in Celiac Disease: AntiTransglutaminase Antibodies Impair Human Endometrial Angiogenesis1 Nicoletta Di Simone,2,3 Marco De Spirito,4 Fiorella Di Nicuolo,3 Chiara Tersigni,3 Roberta Castellani,3 Marco Silano,5 Giuseppe Maulucci,4 Massimiliano Papi,4 Riccardo Marana,6 Giovanni Scambia,3 and Antonio Gasbarrini7 3
Department of Obstetrics and Gynecology, Policlinico A. Gemelli, Universita` Cattolica Del Sacro Cuore, Rome, Italy Institute of Physics, Universita` Cattolica Del Sacro Cuore, Rome, Italy 5 Unit of Human Nutrition and Health, Istituto Superiore di Sanita`, Rome, Italy 6 International Scientific Institute Paolo VI, Universita` Cattolica Del Sacro Cuore, Rome, Italy 7 Department of Internal Medicine, Policlinico A. Gemelli, Universita` Cattolica Del Sacro Cuore, Rome, Italy 4
genetically susceptible individuals. CD affects as many as 1% of the population worldwide [1–5]. It is well known that in CD, the interplay of four components—gluten/gliadin, glutenspecific T cells, the major histocompatibility complex locus HLA-DQ, and the endogenous tissue enzyme transglutaminase type 2 (TG2)—induces the enteropathy [6]. TG2 can deamidate glutamine to glutamic acid, producing the negatively charged residues necessary for efficient binding to DQ2 and the subsequent T-cell activation. Genetic, molecular, and functional studies have clarified the powerful Th1-dominated proinflammatory response that characterizes the reaction in the small intestine of patients with active CD [7]. It has also been proposed that TG2-mediated cross-linking between gliadin peptides and the enzyme itself leads to the formation of TG2gliadin complexes that are processed by B cells and presented to gliadin-specific T cells that, in turn, provide the help necessary to induce the production of anti-TG2 antibodies [8]. Patients with active CD have immunoglobulin (Ig) A and IgG anti-TG2 antibodies. The determination of the serum concentration of the anti-TG2 antibodies provides a powerful tool to screen the general population for CD and to identify the individuals to be evaluated by endoscopy [9]. When CD goes into complete remission on a gluten-free diet, anti-TG2 antibodies are no longer detectable in the serum. Although the disease primarily involves the small intestine, CD is a classic example of a systemic disorder involving several organs, such as the skin, thyroid, pancreas, liver, and heart, as well as the reproductive system [10,11]. Previous studies have suggested that CD may be associated with reduced fertility and an increased risk of adverse pregnancy-related events like intrauterine growth restriction, low-birth-weight babies, or miscarriages [12–16]. Among women with untreated CD, the risk of multiple miscarriages has been reported to be eight- to ninefold higher than that for celiac women on a gluten-free diet [17]. Even if a definitive explanation for these associations has not yet been proposed, we have recently described that circulating anti-TG2 antibodies might be responsible for this association by impairing the placental function [18]. In particular, anti-TG2 antibodies isolated from sera of patients with untreated CD were able to bind to trophoblast cells, affecting invasiveness through apoptotic damage. Endometrial angiogenesis and decidualization, as well as trophoblast invasion, are prerequisites for a successful implantation and a good outcome of pregnancy. In the uterus during pregnancy, critical angiogenic signals likely are produced by the decidualizing endometrial cells acting on the endothelial cells to promote their proliferation and differenti-
ABSTRACT Celiac disease (CD) is an autoimmune enteropathy triggered by gluten ingestion and characterized by circulating antitransglutaminase type 2 (anti-TG2) autoantibodies. An epidemiological link between maternal CD and increased risk of pregnancy failure has been established; however, the mechanism underlying this association is still poorly understood. Because proper endometrial angiogenesis and decidualization are prerequisites for placental development, we investigated the effect of anti-TG2 antibodies on the process of endometrial angiogenesis. Binding of anti-TG2 antibodies to human endometrial endothelial cells (HEECs) was evaluated by ELISA. Angiogenesis was studied in vitro on HEECs and in vivo in a murine model. In particular, we investigated the effect of antiTG2 antibodies on HEEC matrix metalloprotease-2 (MMP-2) activity by gelatin zymography, cytoskeletal organization and membrane properties by confocal microscopy, and activation of extracellular signal-regulated kinases (ERKs) and focal adhesion kinase (FAK) by Western blot analysis. Anti-TG2 antibodies bound to HEECs and decreased newly formed vessels both in vitro and in vivo. Anti-TG2 antibodies impaired angiogenesis by inhibiting the activation of MMP-2, disarranging cytoskeleton fibers, changing the physical and mechanical properties of cell membranes, and inhibiting the intracellular phosphorylation of FAK and ERK. Anti-TG2 antibodies inhibit endometrial angiogenesis affecting the TG2-dependent migration of HEECs and extracellular matrix degradation, which are necessary to form new vessels. Our results identify pathogenic mechanisms of placental damage in CD. angiogenesis, celiac disease, cytoskeleton, endometrium, reproductive immunology
INTRODUCTION Celiac disease (CD) is a small intestinal enteropathy triggered by ingestion of cereal prolamins (gluten) in 1
Supported by a research grant from the Universita` Cattolica del Sacro Cuore (D1, 2011), Rome, Italy (N.D.S., M.D.S., M.P.). 2 Correspondence: Nicoletta Di Simone, Department of Obstetrics and Gynecology, Universita` Cattolica del Sacro Cuore, Largo Agostino Gemelli 8, 00168 Rome, Italy. E-mail:
[email protected] Received: 4 April 2013. First decision: 24 April 2013. Accepted: 25 July 2013. Ó 2013 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363
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ation. After stimulation of the endothelial cells by angiogenic factors, the basement membrane is degraded by proteolytic enzymes, particularly matrix metalloproteases (MMPs). Then, the cells invade, migrate, and proliferate into the underlying interstitial matrix and form new capillary structures [19, 20]. Together with steroid hormones, angiogenesis induces the changes in the endometrium that enable it to accept the blastocyst and initiate the process of implantation. To date, only a few reports concerning the isolation and characterization of the endothelial cells of the endometrium have appeared [21, 22]. The aim of the present study was to investigate whether antibodies directed against TG2 affect human endometrial angiogenesis in vitro on HEECs and in vivo in a mice model. Because we found a significant anti-TG2 antibody-mediated inhibition of endometrial angiogenesis, we hypothesized that its effect could probably be exerted by interaction with TG2 on cell surfaces. Because the actin fibers of the cytoskeleton (F-actin) interact with cell membranes and mediate a variety of cellular functions, such as cell-substrate or cell-cell adhesions, which are fundamental for cell replication and migration, we also investigated whether anti-TG2 antibodies could damage HEEC F-actin fibers by phalloidinfluorescein isothiocyanate (FITC) staining. In addition, we analyzed anti-TG2 antibody-mediated impairment of HEEC structure by studying mechanical properties of the membranes by atomic force microscopy (AFM) and confocal fluorescent microscopy. Finally, we evaluated the effects of anti-TG2 antibodies on the activation of extracellular signal-regulated kinases (ERKs) and focal adhesion kinase (FAK), the key kinases of the intracellular pathway involved in endothelial cells differentiation and migration during the angiogenic process.
HEEC Isolation and Purification Endometrial samples (3–4 g each) were minced and incubated in M199/ penicillin/streptomycin containing 0.2% collagenase type II at 378C for 2 h, after which all remaining tissue was dissolved by powerful resuspension of digested endometrial tissue by pipetting up and down, resulting in a homogenous solution. After centrifugation (1200 rpm for 5 min at room temperature), the pellet obtained was resuspended in culture medium (Medium 199 [Sigma-Aldrich] þ Fetal bovine serum [FBS] 10% þ Hepes 20 mM þ antibiotic/antimycotic solution 2% þ Endothelial Cell Growth Supplement [ECGS, 50 mg/ml, BD Biosciences] þ Heparin 5 U/ml) and transferred into a fibronectin-coated culture dish. After 24 h, the nonadhered cells were removed, and the adherent cells were cultured in hEMVEC (Human Microvascular Endothelial Cells) culture medium. The primary heterogeneous cell population was cultured until confluence, and then endothelial cells were selected by anti-human CD31- and CD105coated microbead antibodies. Briefly, after detachment by trypsin and centrifugation, the cells were immunolabeled with CD31 or CD105 MicroBeads (20 ll per 1 3 107 cells; 130-091-935 or 130-051-201; Miltenyi Biotec S.r.l.), then loaded onto a column placed in the magnetic field of a MACS Separator (Miltenyi Biotec). The magnetically labeled CD31 þ/CD105 þ cells were retained on the column. The unlabeled cells ran through, and the cell fraction was depleted of CD31 þ/CD105 þ retained cells. After removing the column from the magnetic field, the magnetically retained CD31 þ/CD105 þ cells were eluted and taken directly into culture or analyzed for purity by flow cytometry.
Flow Cytometry Characterization of the isolated HEECs was performed by flow cytometry. Briefly, aliquots of HEECs were incubated for 15 min at room temperature with specific endothelial markers: FITC- or phycoerythrin (PE)-conjugated monoclonal antibodies against CD105 (1:20 dilution; ab11415; Millipore) and/or Kinase insert Domain Receptor (KDR; 1:10 dilution, 130-093-598; Miltenyi Biotec). Appropriate fluorochrome-conjugated, isotype-matched irrelevant monoclonal antibody was used as negative control for background staining. Cells were run through a flow cytometer (FACS Canto Flow Cytometer; BD Biosciences). A minimum of 10 000 events were collected and acquired in list mode with the accompanying software. Human umbilical vein endothelial cells (HUVECs) from LGC Standards were used as positive control.
MATERIALS AND METHODS Purification of IgA and IgG Autoantibodies
Binding Assay by ELISA
Total IgA fraction of serum samples from seven women with CD on a gluten-containing diet positive for anti-TG2 antibodies (IgA titer: median, 100 U/ml; minimum, 56.4 U/ml; maximum, 263 U/ml; IgG titer: median, .100 U/ ml; minimum, 48.6 U/ml; maximum, 228 U/ml) and from three normal healthy women (normal healthy subjects [NHS]) negative for anti-TG2 antibodies (,5 U/ml) was purified by a commercial kit according to the manufacturer’s instructions hIgA purification kit (Affiland). Whole IgG fraction was purified on protein G-Sepharose (mAbTrap kit; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) as previously described [23]. The final protein concentration (IgG or IgA) was evaluated by nephelometry, and the specific reactivity with anti-TG2 antibody-coated plates was confirmed as previously described [24]. Using ELISA, we also tested IgA and IgG fractions for beta2GPI (beta2glycoprotein I, Lupus anticoagulant), cardiolipin, and LAC specificity to exclude an angiogenesis inhibition mediated by antiphospholipid antibodies, finding no significant binding of our polyclonal fractions. The sterile-filtered IgG or IgA fractions were determined to be endotoxin-free by the limulus amebocyte lysate assay (E-TOXATE kit; Sigma-Aldrich). The study protocol was approved by the Human Investigation Committee of the Universita` Cattolica del Sacro Cuore. All patients enrolled in the study signed a written informed consent.
Cell cultures were carried out for 24 h in standard medium, washed three times with HBSS at room temperature for 5 min, and incubated with serial concentrations of different antibody preparations: monoclonal IgG anti-TG2 antibodies (3–50 lg/ml; CUB 7402; Bio-Optica), polyclonal IgG or IgA antiTG2 antibodies from seven patients’ serum (3–50 lg/ml), or polyclonal IgG or IgA from three NHS (5–50 lg/ml), separately, in complete medium at a final volume of 100 ll. After a 2-h incubation followed by three washes with PBS (phosphate buffer saline) at room temperature for 5 min, the plates were incubated with alkaline phosphatase-conjugated secondary antibodies for 90 min. After two further washes with PBS at room temperature for 5 min, pnitrophenylphosphate (1 mg/ml) in 10% diethanolamine buffer (pH 9.8) was added to each well and incubated for 30 min. Optical density was read at 405 nm using a microplate photometer (Titertek Multiscan Plus; ICN Flow, Thermo Fisher Scientific Inc.).
TG2 Activity For the detection of TG2 enzymatic activity, endothelial cells were treated for 2 h with CUB 7402 or polyclonal IgG or IgA anti-TG2 antibodies at a concentration of 50 lg/ml on coated slides. Next, we added biotinylated monodansylcadaverine and then 1 ml of streptavidin conjugated with Alexa Flour 488 (1:100; Life Technologies) for 1 h at room temperature. The reaction was stopped with 25 mmol/L of ethylenediaminetetra-acetic acid (EDTA; Sigma) for 5 min; the slides were then fixed in 4% paraformaldehyde (PFA) for 10 min. Control experiments included the omission of biotinylated monodansylcadaverine and replacement of 200 mmol/L of CaCl2 with 200 mmol/L of EDTA.
Tissue Collection Endometrial tissues were obtained from five fertile women undergoing hysterectomy for fibroid uterus and biopsy for benign diseases in the midsecretory phase. Informed consent was obtained from each patient enrolled. The day of the menstrual cycle was set according to the patient’s menstrual history and was verified through histological examination of the endometrium according to Noyes criteria [25]. Estradiol and progesterone levels were determined in the serum to confirm the midsecretory phase. The tissues were placed in Hank balanced salt solution (HBSS) and carried to the laboratory for HEEC isolation and culture. Each experimental setup was repeated on at least five occasions using cells obtained from different patients.
Binding Assay by Immunofluorescence Staining The HEECs were rinsed twice in PBS and then fixed with 4% PFA (5 min). After rinsing with PBS, cells were incubated with CUB 7402 (50 lg/ml), polyclonal IgG or IgA anti-TG2 antibodies (50 lg/ml), or polyclonal IgG or
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CELIAC DISEASE AND HUMAN ENDOMETRIAL ANGIOGENESIS IgA (50 lg/ml) from three NHS, as control, for 1 h at room temperature. Then, the specific secondary FITC-conjugated antibody (1:500 dilution; Novus Biologicals, Inc.) was added for 1 h at 378C. HEECs were then rinsed twice in PBS, mounted under microscopic glass coverslips in a Gel/Mount (Permanent Aqueous Mounting Medium; Biomeda Corporation), and evaluated by an inverted-phase fluorescent microscope (2003; Axiover 35, Zeiss). Images were acquired with a digital camera (Nikon Coolpix 4500).
determined by FITC-lectin staining. After staining, the vessels were washed with PBS at room temperature for 5 min, and the fluorescence was measured in 96-multiwell plates using spectrofluorometer (Twinkle LB 970, Berthold Technologies).
F-Actin Immunofluorescent Staining To stain F-actin, HEECs were seeded on Matrigel-coated glass bottom dishes (2 3 104 cell/dish; MatTek Corporation) in endothelial cell differentiation culture medium (EBM-2) MV SingleQuots containing monoclonal or polyclonal anti-TG2 antibodies (50 lg/ml) for 24 h. After treatment, cells were rinsed twice in PBS, fixed with 4% PFA for 10 min at room temperature, and permeabilized with 0.1% Triton-X for 5 min. Next, HEECs were incubated with primary antibodies against FITC-labeled phalloidin for 30 min at room temperature. F-actin fibers were visualized by an inverted confocal microscope (DMIRE2; Leica Microsystems) fitted with a 633 oil immersion objective (NA 1.4) and LCS 2.61 acquisition Software (Leica Microsystems).
Gelatin Zymography Levels of MMP-2 activity in the supernatant of HEEC cultures were measured by gelatin zymography. Samples were electrophoresed on SDSPAGE containing 0.3% gelatin. Following electrophoresis, gels were washed three times for 10 min at room temperature in 2.5% Triton X-100 to remove SDS. After overnight incubation at 378C in 50 mM Tris-HCl (pH 7.4; containing 5 mM CaCl2, 0.15 M NaCl, and 0.02% NaN3), gels were stained with 0.5% Coomassie Brilliant Blue (Sigma-Aldrich) for 30 min and destained in 20% methanol and 10% acetic acid. Gelatinolytic activity was observed as a clear band of digested gelatin on a blue background. Images were acquired with a digital camera (Nikon Coolpix 4500), and bands were analyzed by Image Analysis System Gel Doc 200 System (Bio-Rad Laboratories) using the Quantity One Quantitation Software (Bio-Rad Laboratories).
Two-Photon Microscopy Laurdan (Life Technologies) is a fluorescent molecule that detects changes in cell membrane phase properties (gel phase to liquid crystalline) through its sensitivity to the polarity of the bilayer environment. Polarity changes are shown by a shift in the Laurdan emission spectrum and quantified by calculating the generalized polarization (GP). Laurdan labeling was performed directly in HEEC culture. After 30 min of incubation in the dark at room temperature, the cover glass was washed once with PBS at room temperature for 5 min and mounted upon a microscope slide. Laurdan fluorescence image were obtained with an inverted confocal microscope (DMIRE2) using a 633 oil immersion objective (NA 1.4) under excitation at 800 nm with a mode-locked titanium-sapphire laser. Laurdan intensity images were recorded simultaneously with emission in the ranges of 400–460 and 470–530 nm, and imaging was performed at room temperature. The GP, defined as
In Vitro Angiogenesis Assay Endothelial cell differentiation into capillary-like tube structures was monitored by the BD BioCoat Angiogenesis System. HEECs (or HEECs transfected by small interfering RNA [siRNA] oligos) were seeded on Matrigel-coated plates (2 3 104 cell/wells; Matrigel, BD Biosciences) in endothelial cell differentiation culture medium (EBM-2) MV SingleQuots (Lonza) containing monoclonal CUB 7402 (50 lg/ml), polyclonal IgG or IgA anti-TG2 antibodies (50 lg/ml), or polyclonal IgG or IgA (50 lg/ml) from NHS or suramin (40 lM) as a negative control, and incubated for 22 h at 378C in a 5% CO2 atmosphere. Following incubation, the plates were washed twice with HBSS at room temperature for 5 min, and tube formation was observed using an inverted-phase optical microscope (Olympus IX50). Images were acquired with a digital camera (Nikon Coolpix 4500) and quantified by Photoshop (Adobe), measuring the number and the total length of tubules within each well.
GP ¼
Ið400460Þ GIð470530Þ ; Ið400460Þ þ GIð470530Þ
was calculated for each pixel using the two Laurdan intensity images (I(400–460) and I(470–530)) [26, 27]. The calibration factor G was obtained from the GP values of Laurdan solutions in dimethyl sulfoxide. The G factor has approximately 2% variation across the imaging area. GP images were pseudocolored in Image-J software (http://rsbweb.nih.gov/ij/). Background values (defined as intensities , 7% of the maximum intensity) were set to zero and colored black. GP histograms values were determined within multiple regions of interest (single cells) for each sample.
TG2 Knock-Down by siRNA Commercial oligonucleotide specific for siRNA-TG2 (5 0 -AAGGGCGAACCACCTGAACAA-3 0 ; Qiagen Sciences) was used. To control general toxicity of siRNA, a negative oligonucleotide control sequence, not homologous to any human mRNA (5 0 -AATTCTCCGAACGTGTCACGT3 0 ), was used. Transfection of siRNA oligos was carried out using HiPerFect Transfection Reagent (Qiagen) according to the manufacturer’s instructions. Silencing efficiency of TG2 was determined by Western blot analysis.
Atomic Force Microscopy Imaging and force spectroscopy, a well-established tool to investigate the surface morphology [28] at the nanoscale together with the mechanical properties [29], were performed by an atomic force microscope (NanoWizard II; JPK Instruments) combined with an optical microscope (Axio Observer; Zeiss). To evaluate the correlation between HEEC stiffness, adhesion, and the cytoskeletal network structure, force spectroscopy experiments were performed in standard-culture conditions and after incubation with polyclonal IgA and IgG anti-TG2 antibodies or CUB 7402 and with IgA or IgG from NHS. Cantilevers with a silica conical tip (end radius of approximately 10 nm, an half conical angle of 208; CSC16; Micromesh) were used for all the samples studied. All cantilevers, with a nominal spring constant of approximately k ¼ 0.01 N/m, were accurately calibrated using the thermal method [30]. All images were processed by using Gwyddion software (http://gwyddion.net/).
Angiogenesis by Direct In Vivo Assay Five-week-old CD1 female nude mice, obtained from an outbreeding background, were purchased (Charles River Laboratories, Inc.). The housing and handling of mice were checked to be consistent to requirements of Commission Directive 86/609/EEC concerning the protection of animals used for experimental and other scientific purposes. All experimental procedures were approved by the Ethics Committee on preclinical studies of Universita` Cattolica del Sacro Cuore. The mice were allowed to acclimate to their new environment for 1 wk after arrival. They were housed in disinfected polycarbonate mouse cages, maintained in cabinet with laminar flow at 288C under controlled artificial lighting (12L:12D), and given ad libitum access to rodent chow and water during the study. We performed the experiments on groups of 10 animals. For direct angiogenesis assay, a commercial direct in vivo assay kit (DIVAA; Trevigen) was used. Briefly, angioreactors were filled with Matrigel with or without the angiogenic factor (fibroblast growth factor-2 [FGF-2]), CUB 7402 or polyclonal IgG or IgA anti-TG2 antibodies (50 lg/ml), or polyclonal IgG or IgA from NHS (50 lg/ ml). They were incubated at 378C for 1 h to allow gel formation before subcutaneous implantation into the dorsal flank of mice. In each mice, two angioreactors were implanted: the positive control with an angioreactor coated with FGF-2 in the left flank and an angioreactor with FGF-2 plus monoclonal or polyclonal anti-TG2 antibodies in the right flank. After 14 days, the angioreactors were collected. The new vessels formed by mice vascular endothelial cells that migrated and proliferated in the Matrigel were
SDS-PAGE and Immunoblotting To perform Western blot analysis, HEEC lysates were separated by 10% SDS-PAGE electrophoresis under reducing conditions. After gel electrophoresis and transfer of proteins to a nitrocellulose membrane, nitrocellulose sheets were blocked at room temperature for 1 h in 5% nonfat dry milk and incubated overnight at 48C with CUB 7402. Immunoreactivity was detected by sequential incubation of membranes with appropriate horseradish peroxidase (HRP)conjugated secondary antibody for 1 h at room temperature and visualized using a chemiluminescence detection system. The level of anti-TG2 antibodies
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FIG. 1. Binding to HEECs of human IgG anti-TG2 monoclonal antibody (CUB 7402) and polyclonal IgG and IgA fractions isolated from the sera of patients with CD or from NHS. A) Cell ELISA assay. Cell cultures were incubated with monoclonal or polyclonal IgG and IgA anti-TG2 antibodies (3–50 lg/ml) for 24 h. Binding values are expressed as the mean 6 SEM of optical density (O.D.) units. Dose-dependent binding to HEECs was observed with both monoclonal and polyclonal IgG and IgA anti-TG2 antibodies at concentrations of 3–6 lg/ml. Binding values in the presence of IgG from NHS were the same as those observed in untreated HEECs. The differences between treatment groups were examined using one-factor ANOVA followed by a posthoc test (Bonferroni test, *P , 0.005). B) Immunofluorescence assay. The presence of a fine line surrounding the cells was observed after incubation of HEECs in the presence of CUB 7402, polyclonal IgG and IgA anti-TG2 antibodies, and anti-human secondary antibody conjugated with FITC. In contrast, polyclonal IgG or IgA fractions obtained from NHS showed no fluorescent staining as seen with untreated cells (CTR). Original magnification 3200. was estimated versus the constant level of a 42-kDa protein present in the cytosolic extract (b-actin). To study ERK and FAK activation, HEECs were plated in six-well plates at 2 3 106 cells/well in EBM-2 medium lacking SingleQuots growth supplements and containing 0.1% FBS. After 24 h, cells were treated with Vascular Endothelial Growth Factor (VEGF)165 with or without monoclonal or polyclonal anti-TG2 antibodies (50 lg/ml) for 30 min. After stimulation, cell lysates were prepared in Cell Lysis Buffer (Cell Signaling Technology) supplemented with sodium orthovanadate (1 mM), PMSF (1 mM), phosphatase inhibitor cocktail 1, and phosphatase inhibitor cocktail 2. Eighty micrograms of protein were separated by SDS-PAGE and blotted to polyvinylidene fluoride membrane. Membranes were blocked with 10% nonfat dried milk in PBS supplemented with Tween-20 (PBST) for 1 h, followed by incubation with the primary antibodies overnight at 48C. Monoclonal anti-phospho-ERK1/2 and polyclonal anti-ERK1/2 (both from Santa Cruz Biotechnologies, Inc.) as well as monoclonal anti-phospho-FAK and polyclonal anti-FAK (both from Millipore) were used. The membranes were washed with PBST at room temperature for 15 min and incubated 2 h at room temperature in HRP-conjugated goat anti-rabbit or anti-mouse IgG diluted 1:2000 in 5% nonfat dried milk in PBST. Bound secondary antibody was detected by chemiluminescence. Densitometric analysis was applied to each blot to quantify the intensity of bands, followed by normalization against loading controls.
Statistical Analyses The results are presented as the mean 6 SEM. Data were analyzed using one-way ANOVA followed by a post-hoc test (Bonferroni test). Statistical significance was considered to be P , 0.05.
RESULTS Flow Cytometry Endometrial samples were subjected to CD31 and CD105 cell immunomagnetic isolation. Cell viability after immunoselection exceeded 98% in all the cases. The cells recovered were analyzed for the presence of endothelial markers (vascular endothelial growth factor receptor-2 or KDR and CD105) by flow cytometry in dual-color analysis (KDR-PE þ/CD105FITC þ, 93.69% 6 2.5%). Anti-TG2 Antibodies Bind to HEECs In Figure 1A, the quantitative analysis of polyclonal IgA and IgG or monoclonal CUB 7402 binding to HEECs is described. CUB 7402 displayed a clear binding at a protein 4
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FIG. 2. A) Polyclonal or monoclonal anti-TG2 antibodies reduce HEEC differentiation into tube-like structures. Quantitative analysis of number and total length of tube-like structures after treatment with monoclonal or polyclonal anti-TG2 antibodies and polyclonal IgA or IgG from NHS was performed. Results are presented as the mean 6 SEM of five experiments and are expressed as a percentage of control (CTR; untreated cells). *P , 0.05 vs. CTR. B) Polyclonal or monoclonal anti-TG2 antibodies inhibit the angiogenesis process in vivo. Endothelial cells in the angioreactors were incubated with FITC-lectin, recovered, and analyzed for FITC-lectin by fluorescence spectrometry. The analysis demonstrated a reduced fluorescence (70% reduction) in reactors containing FGF-2 plus polyclonal IgG or IgA or monoclonal anti-TG2 antibodies compared with the positive CTR (FGF-2 alone) or with FGF-2 plus IgA or IgG antibodies from NHS. Results are presented as the mean 6 SEM of four experiments and are expressed as relative fluorescent units (RFUs). *P , 0.05 vs. CTR.
FIG. 3. A) Evaluation of TG2 knock-down expression in HEECs. Representative Western blot for relative amount of TG2 expression in untransfected endothelial cells (HEECs) as well as those transfected with control siRNA (negative control) or siRNA-TG2 at 24 h after transfection. Control (CTR), untreated cells; negative control, cells transfected with nonsilencing siRNA oligonucleotides; siRNA-TG2, cells transfected with TG2-specific siRNA oligonucleotides. Data are presented as the mean 6 SEM of three independent experiments and are expressed as a percentage of control. *P , 0.05 vs. CTR. B) Assessment of tube-like structure number after TG2 silencing. HEECs not transfected or transfected with siRNA-TG2 (100 nM) were treated with and without monoclonal anti-TG2 antibodies (CUB 7402, 50 lg/ml), and tube formation was evaluated as described in Materials and Methods. Data are presented as the mean 6 SEM of three independent experiments repeated in duplicate. *P , 0.05 vs. untreated cells.
concentration of 3 lg/ml. Polyclonal IgA and IgG antibodies displayed significant binding activity from a concentration of 6 and 12 lg/ml, respectively. In contrast, IgA or IgG fractions obtained from NHS showed negligible background values. Anti-TG2 antibody (both monoclonal and polyclonal) binding to HEEC cell membrane decreased the cellular TG2 activity (data not shown). The immunofluorescence analysis of the HEECs confirmed anti-TG2 antibody binding to cellular membranes. No reactivity was observed in untreated cells or in cells treated with polyclonal fractions obtained from NHS (Fig. 1B).
after implantation, and the newly formed vessels were quantified. Measurement of FITC-lectin binding to the murine endothelial cells in the angioreactors demonstrated reduced fluorescence (70% reduction) in the presence of polyclonal or monoclonal anti-TG2 antibodies (50 lg/ml, P , 0.01) compared with the positive controls (FGF-2 alone) (Fig. 2B). The respective negative control (IgA and IgG from NHS) did not modify in vitro or in vivo angiogenesis (Fig. 2). Down-Regulation of TG2 by siRNA Blocks the Inhibition of Angiogenesis by Anti-TG2 Antibodies
Angiogenesis Is Inhibited by Polyclonal or Monoclonal Anti-TG2 Antibodies
To demonstrate the specific role of TG2 on anti-TG2 antibody-mediated inhibition of HEEC differentiation, the effect of its down-regulation by siRNA was investigated. Figure 3A shows the down-regulation of TG2 after 24 h of silencing, when the level of the protein was reduced by 50% with respect to cells transfected with control siRNA (scrambled siRNA) or untreated cells. We found that the treatment of TG2silenced HEECs with polyclonal or monoclonal anti-TG2 antibodies did not affect cell differentiation (Fig. 3B). These results, showing no antibody-mediated inhibition of HEEC angiogenesis in the presence of ‘‘silenced’’ TG2, demonstrated the role of TG2 on HEEC membranes as a target for anti-TG2 antibodies.
To investigate whether anti-TG2 antibodies affect the ability of HEECs to form capillary-like tubes, HEECs were cultured in endothelial cell differentiation culture medium (EBM-2) MV SingleQuots containing 50 lg/ml of polyclonal IgA and IgG anti-TG2 antibodies, CUB 7402, or IgA and IgG from NHS. Cells were seeded in extracellular matrix gels and examined for tube formation microscopically. After 18 h of culture, both CUB 7402 and polyclonal anti-TG2 antibodies induced a significant decrease in number and total length of the tube-like structures (Fig. 2A). To study angiogenesis in vivo, two angioreactors were implanted into the dorsal flanks of mice as described in Materials and Methods. Angioreactors were dissected 14 days 5
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Anti-TG2 Antibodies Reduce MMP-2 Secretion A key enzyme involved in cell invasion, MMP-2 is constitutively secreted by endothelial cells. HEEC incubation with polyclonal anti-TG2 antibodies or CUB 7402 (50 lg/ml), but not with IgA or IgG (50 lg/ml) from NHS, significantly reduced active and pro-MMP-2 protein levels in HEEC culture media (evaluated by gelatin zymography) in comparison to control cells (P , 0.05) (Fig. 4). Anti-TG2 Antibodies Disarrange HEEC Cytoskeleton Cytoskeleton rearrangement of endothelial cells plays a pivotal role in the angiogenic process. We performed phalloidin-FITC staining to visualize the F-actin cytoskeleton in HEECs to study whether the inhibited cell differentiation could be due to a disarrangement of the cytoskeleton. The staining clearly showed a disorganization of the actin stress fibers in cells treated with anti-TG2 antibodies compared to untreated cells (Fig. 5, A and D–F). Polyclonal IgA or IgG from NHS did not modify actin fiber organization (Fig. 5, B and C).
FIG. 4. MMP-2 levels in the supernatant of HEEC cultures. MMP-2 levels were measured by gelatin zymography. A) Gelatinolytic activities were observed as a white band of digested gelatin on a blue background that was subsequently acquired with a digital camera and analyzed on the Image Analysis System Gel Doc 200 System. B) Effect of monoclonal or polyclonal anti-TG2 antibodies and polyclonal IgA or IgG antibodies from NHS on active and pro-MMP2 gelatinolytic capacity in the supernatant of HEECs. Results are presented as the mean 6 SEM of five experiments and are expressed as a percentage of untreated cells (CTR). O.D., optical density. *P , 0.05 vs. CTR.
Anti-TG2 Antibodies Reduce Plasma Membrane Fluidity To assess changes in the physical state of cell membranes after polyclonal IgA and IgG treatment or CUB 7402, we analyzed Laurdan fluorescence using two-photon microscopy. Laurdan is an environmentally sensitive, fluorescent probe that exhibits an emission spectral shift depending on the lipid phase state: bluish in ordered, gel phases and greenish in disordered, liquid-crystalline phases. Laurdan distributes equally between lipid phases and does not
FIG. 5. Polyclonal or monoclonal anti-TG2 antibodies induce a decrease of fiber orientation in F-actin cytoskeleton. A–F) Representative images of Factin organization in HEECs. A) Untreated cells. B and C) Cells treated with polyclonal IgA or IgG from NHS. F-actin is organized in fibers, with a welldefined orientation. D–F) Cells treated with polyclonal or monoclonal anti-TG2 antibodies. Fibers are less structured, with a shorter average length. Bar ¼ 20 lm.
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FIG. 6. Polyclonal or monoclonal anti-TG2 antibodies induce plasma membrane rigidification. A) GP images of control cells revealed in a pseudocolored rainbow scale. High-GP regions are evident at the plasma membrane, whereas low-GP regions were mainly found at internal membranes. A1) GP histogram of the images in A. A two-logistic deconvolution of the histogram (blue line) was employed to separate low-GP (red line) and high-GP (green line) regions. A2) Separated high-GP regions corresponding to the plasma membrane (pseudocolored in green) and low-GP regions corresponding to endomembranes (pseudocolored in purple). A3 and A4) GP images of cells treated with polyclonal IgA or IgG fractions from NHS. B) GP images of cells treated with IgA anti-TG2 antibody fraction. The whole membranes became more ordered than in the controls. B1) GP histogram of the images in B. An average shift toward higher GP is evident. B2) Separated high-GP regions corresponding to the plasma membrane (pseudocolored in green) and low-GP regions corresponding to endomembranes (pseudocolored in purple). C) GP images of cells treated with IgG anti-TG2 antibody fraction. C1) GP histogram of the images shown in C. C2) Separated high-GP regions corresponding to the plasma membrane (pseudocolored in green) and low-GP regions corresponding to endomembranes (pseudocolored in purple). D) GP images of cells treated with monoclonal anti-TG2 antibodies (CUB 7402). D1) GP histogram of the images shown in D. D2) Separated high-GP regions corresponding to the plasma membrane (pseudocolored in green) and low-GP regions corresponding to endomembranes (pseudocolored in purple). E) Peaks of the logistic functions represent GP values of the two separated regions. Plasma membrane becomes more rigid in the IgA anti-TG2 antibody-treated sample, together with the internal membranes. An analog effect, with less rigidification, was found in HEECs treated with IgG anti-TG2 antibody fraction and CUB 7402 (*P , 0.05). Bar ¼ 20 lm.
associate preferentially with specific fatty acids or phospholipid headgroups. As a normalized ratio of the intensity at the two emission wavelength regions, the GP provides a measure of membrane order, in the range between 1 (liquid crystalline) and þ1 (gel). In our experiments with Laurdanlabeled cells, GP images of control cells (Fig. 6A) revealed high-GP regions at the plasma membrane, whereas low-GP regions were mainly found at internal membranes, consistent with previous findings in various cell types [31]. In Figure
6A1, the GP histogram of Figure 6A is shown. A twologistic deconvolution of the histogram (blue line) was employed following to separate low-GP (red line) and highGP (green line) regions [32]. The threshold chosen after the deconvolution process is used to generate the images in Figure 6A2, which represent the separated high-GP regions corresponding to the plasma membrane (pseudocolored in green) from low-GP regions corresponding to endomembranes (pseudocolored in purple). The peaks of the logistic 7
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FIG. 7. Anti-TG2 antibody modification of HEEC adhesion and stiffness. A) Representative optical image of the atomic force microscope cantilever during force measurements on the control sample. Bar ¼ 20 lm. B) Representative force-distance curve performed on a control cell: approaching (red) and retracting (blue) parts of the force curve and the adhesion energy (gray) are reported. C–D) Histograms of the adhesion work and the cell stiffness evaluated by single cell atomic force spectroscopy (AFM). Treatment with polyclonal IgG and IgA or monoclonal anti-TG2 antibodies strongly reduces the adhesion energy increasing the cell stiffness (Young Modulus). *P-value , 0.05 was considered significant.
NHS. Interaction forces between AFM tip and cell surface were measured in force-distance cycles. At a fixed position, the tip was brought to the cell surface (Fig. 7A) and successively withdrawn. During this cycle, the bending of the cantilever, which is proportional to the force, was continuously measured and plotted versus cell-tip separation (Fig. 7B). Upon approaching the cell surface, the cantilever bends upward (red line), consistent with a repulsive force (stiffness), characterized by the Young modulus, which increases with the indentation. Subsequent tip retraction (blue line) first leads to relaxation of the cantilever bending until the repulsive force drops to zero. Upon further retraction, the cantilever progressively bends downward, reflecting an attractive force (adhesion) that increases with increasing cell-tip separation. The gray area in Figure 7B represents the energy that would be exerted to detach the tip from the surface (adhesion energy). It is well established that cell stiffness, in absence of any specific interaction, is directly affected by the functional state of the cytoskeletal network and that changing in the cell-tip adhesion is associated to the attachment of membrane nanotubes to the AFM probe [33], cell membrane composition [34, 35], and cytoskeleton state and actin polymerization [36].
functions represent GP values of the two separated regions, which are reported in Figure 6E for each sample. When cells were treated with IgA, the membranes became more ordered than in the controls (Fig. 6B), and this resulted in an average shift toward higher GP values of the relative GP histogram (Fig. 6B1). Logistic-deconvolution analysis demonstrated that plasma membrane becomes more rigid, together with the internal membranes (Fig. 6, B2 and E). An analog effect, with less rigidification, was found in polyclonal IgG- and CUB 7204-treated cells (Fig. 6E), the GP maps of which are shown in Figure 6, C and D, respectively, together with the GP histograms (Fig. 6, C1 and C2) and the separated regions (Fig. 6, D1 and D2). Anti-TG2 Antibodies Deeply Modify HEEC Adhesion and Stiffness To evaluate the correlation between HEEC stiffness, adhesion, and the cytoskeletal network structure, force spectroscopy experiments were performed in standard culture conditions and after incubation with polyclonal IgA and IgG anti-TG2 antibodies or CUB 7402 and with IgA or IgG from 8
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FIG. 8. Western blot analysis of phosphorylated (p-) and total FAK and ERK-1/2 expression in HEEC treated with anti-TG2 antibodies. Representative Western Blot for phosphorylated and total FAK (A) and phosphorylated and total ERK (B). The levels of p- and total FAK (C) or ERK (D) protein expression after treatment were evaluated by comparing the constant level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as O.D. (Optical Density) (CTR: cells in medium alone). Polyclonal or monoclonal anti-TG2 antibodies, significantly reduced FAK and ERK activation and expression in comparison to controls or to polyclonal anti IgA or IgG fractions from NHS. Results are means 6 SEM of five independent experiments. (*P , 0.05 compared with CTR).
DISCUSSION
In Figure 7, C and D, we report the treatment-induced modifications of adhesion energy and the Young modulus, respectively, of HEECs. Histograms show that both parameters are strongly affected by polyclonal or monoclonal anti-TG2 antibodies, but a statistically significant difference between polyclonal IgA and IgG was not observed. Notably, the HEEC adhesion energy (Fig. 7D) was much more sensitive than the cell mechanical stiffness (Fig. 7C) to anti-TG2 antibodies. IgG from NHS did not modify mechanical or physical properties of HEEC membranes.
Transglutaminase type 2 belongs to a family of Ca2þ dependent enzymes responsible for catalyzing posttranslational modification of proteins [40, 41]. It has been shown to be involved in the molecular mechanisms responsible for CD, which is characterized in part by the presence of TG2-modified proteins and in part by aberrant TG2 activity [42]. Indeed, TG2 in patients with CD is able to deamidate gliadin-derived peptides, eliciting a Th1 response characterized by the production of proinflammatory cytokines and of autoantibodies directed against TG2. Transglutaminase type 2 is abundantly expressed in many organs, including the liver, heart, intestine, and blood cells [43], as well as the placenta [44]. It is found in both intracellular and extracellular locations [45]. Extracellular TG2 in particular is involved in cell adhesion [46, 47], matrix assembly [48, 49], wound healing [50–52], receptor signaling [53], and a variety of cellular behaviors, including proliferation, invasion, and survival [54, 55]. In active CD, anti-TG2 antibodies, both class IgG and class IgA, are produced and are responsible for most of the disease’s systemic manifestation. It has been shown that anti-TG2 antibodies bind to cell surfaces, interfering with mediation of out-in cell signaling [18, 56, 57]. Extraintestinal manifestation of CD includes iron-deficiency anaemia, osteoporosis, dermatitis herpetiformis, neurologic disorders, and adverse pregnancy outcomes like miscarriage, intrauterine growth restriction, and low-birth-weight babies
Anti-TG2 Antibodies Decrease FAK and ERK1/2 Phosphorylation Incubation of HEECs with endothelial cell culture medium (EBM-2) containing FGF-2 leads to phosphorylation of multifunctional protein kinases closely associated with angiogenesis and cell differentiation [37, 38]. FAK is believed to be a primary signaling mediator of the dynamic changes in cytoskeletal reorganization [39]. FAK phosphorylation on specific residues initiates a intracellular signaling cascade that involves ERK1/2 activation and results in cell spreading and migration. As shown in Figure 8, we found a significant decrease of total FAK as well as phosphorylated FAK and ERK1/2 proteins after treatment with monoclonal or polyclonal anti-TG2 antibodies (50 lg/ml) in comparison with untreated cells. Polyclonal IgG and IgA from NHS (50 lg/ml) did not reduce FAK and ERK activation. 9
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In conclusion, we demonstrated in the present study that antigenic structures for anti-TG2 antibodies are present on HEECs. In addition, we showed, to our knowledge for the first time, that the binding of autoantibodies to endometrial endothelial cells and their consequent functional inhibition might represent a key mechanism by which anti-TG2 antibodies could affect embryo implantation and placentation. Because endometrial angiogenesis is essential for placental development and fetal growth, these data provide a novel way to explain early pregnancy losses and intrauterine growth retardation related to CD.
[12–16]. Obstetric complications can sometimes be the only feature of the disease. To understand the fundamental pathogenic mechanism of the increased occurrence of placenta-related obstetric complications in CD, we demonstrated previously that anti-TG2 antibodies are able to bind to human trophoblast cells in vitro and to affect cell invasiveness through an apoptotic mechanism [18]. In that study, we provided an initial model of immunemediated placental damage at the embryo’s interface, partially explaining the increased risk of placental-related adverse pregnancy outcomes in women with CD but without a gluten-free diet. The aim of the present study was to investigate a possible CD-related damage at the fetomaternal interface, studying the effect of anti-TG2 antibodies on human endometrial angiogenesis. A successful placentation requires several events, including trophoblast invasion and endometrial neoangiogenesis [58]. The angiogenic process, together with steroid hormones, induces the endometrial changes that enable the endometrium to accept the blastocyst and to initiate the process of implantation [58–60]. In the present study, we have shown that anti-TG2 antibodies could be able to impair human endometrial angiogenesis, as suggested by the in vitro findings on HEECs and confirmed by the parallel experiments performed in a murine model. Because a proper actin dynamic is a prerequisite for the migration of cells and anti-TG2 antibodies have a negative effect on HUVEC cytoskeletal organization [61], we hypothesized that a specific autoantibody-induced disarrangement of actin fibers might explain the inhibited angiogenesis of HEECs observed in vitro and in vivo after exposure to anti-TG2 antibodies. Interestingly, the disarrangement of F-actin cytoskeleton has been clearly documented on anti-TG2 antibodytreated HEECs both directly, by visualization, and indirectly, by detecting a stiffening of the cell membrane, which represents the functional counterpart of a cytoskeleton modification. The damaging effect of anti-TG2 antibodies on HEEC differentiation was confirmed by the demonstration of a dramatic disarrangement of the F-actin cytoskeleton, and a reduction of cell membrane fluidity and adhesiveness has been detected. Because membrane fluidity reflects the structure of lipids in the membrane whereas adhesiveness strongly depends on the cytoskeleton architecture, our results shed light on a functional interplay between cytoskeleton and membrane lipids [62, 63]. Accordingly, the anti-TG2 antibodies disarrange the cytoskeleton, which in turn causes an increase in the cytoskeleton stiffness and a decrease in the cell adhesiveness. Overall, this mechanism represents a chemomechanical signaling pathway. This is a significant issue and provides further evidence for the close connection between cell mechanics and chemical stimuli in regulating cell signaling. To better understand the intracellular mechanisms regulating the changes in TG2-mediated HEEC motility and cytoskeletal organization during the process of angiogenesis, we also examined the effect of anti-TG2 antibodies on FAK and ERK activation, the key kinases of intracellular pathway promoting proteins regulating cytoskeleton arrangement and the transcription of proangiogenic factors [64, 65]. We first demonstrated that anti-TG2 antibodies carry out their antiangiogenic activity on HEECs through a direct inhibition of the intracellular proangiogenic signal mediators ERK and FAK. Additional evidence for the role of extracellular TG2 on HEECs as a target for anti-TG2 antibodies in determining angiogenesis inhibition was provided by experiments of TG2 gene-silencing that showed no antibody-mediated inhibition of HEEC angiogenesis in absence of TG2.
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