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Regular Article
The role of fibrin matrices and tissue factor in early-term trophoblastproliferation and spreading
Ayelet Snir a,d,⁎, Benjamin Brenner c,d, Baram Paz b, Gonen Ohel b, Naomi Lanir c
a Division of Allergy and Clinical Immunology, Bnai-Zion Medical Center, Haifa, Israelb Department of Obstetrics and Gynecology, Bnai-Zion Medical Center, Haifa, Israelc Department of Hematology and Bone Marrow Transplantation, Rambam Health Care Campus, Haifa, Israeld Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel
⁎ Corresponding author at: Division of Allergy and CMedical Center, 47 Golomb Street, Haifa 31048, Israel. Tel.4 845 9961.
Introduction: Fibrin deposition in placenta is a commonphenomenonwhich canbe triggered by villous injury andcoagulation activation. Fibrin abnormalities (hypo/dysfibrinogenemia) and factor XIII deficiency are associatedwith infertility and pregnancy loss. While trophoblasts are known to grow on fibrin matrices, the role of thisprotein in trophoblast repair processes remains unclear. We hypothesize that fibrin may have an essential rolein trophoblast remodeling.Methods:Morphology and spreading of primary early-term human trophoblasts and villi explants were investi-gated on various fibrin components. Cross-linking of matrices was evaluated by D-dimer assay. TF procoaguantactivity, protein andmRNA levels in cells and villi were determined by chromogenic assay, ELISA, immunohisto-chemistry and reverse-transcription PCR (RT-PCR).Results: Fibrin but not fibrinogen, thrombin or fibronectin caused increased trophoblast proliferation and spread-
ing. Trophoblasts cultured on factor XIII (FXIII) depletedfibrin caused their increased proliferation and spreading,associatedwith cross-linking. FXIII addition further increased this effect, while cell culturing on active FXIII with-out fibrin retained cellular proliferation. Decreased TF activity, antigen and RNA expression were demonstratedin fibrin-cultured trophoblasts and villi explants, compared to matrigel explants.Conclusion: Results obtained demonstrate distinct mechanisms underlying fibrin cross-linking, which can affecttrophoblast proliferation. The excess of fibrin deposits may be limited by the decrease in TF levels, thus enablingadequate placental perfusion.These findings demonstrate fibrin importance for placental repair and may partly explain poor pregnancy out-come associated with certain fibrinogen/fibrin abnormalities and FXIII deficiency.
Cytotrophoblasts are specialized epithelial cells of human placentathat invade the uterus and acquire endothelial-like characteristics [1].Similar to endothelial blood vessel cells, exposure of the trophoblastbasementmembrane can stimulate thrombin generation and fibrin for-mation through the abundant TF available on cytotrophoblasts facingvillous blood [2]. As a consequence, part of the matrix contains fibrin[3], which is found to be more prevalent in placentas from complicatedpregnancies [4]. The physiological role and clinical significance of fibrinaccumulation in placenta are not entirely clear. While some reportshave attributed placenta injury and pregnancy complications to tropho-blasts cell death and apoptosis [5], others have pointed out that fibrinenhanced differentiation but not apoptosis in cultured term humantrophoblasts [6]. Cellular denudation in the placental villus is usually
repaired by trophoblasts overgrowing on fibrin which is later replacedby connective tissue [7,8].
Fibrin may stimulate trophoblast proliferation through growth fac-tor binding [9] and intergrins upregulation, as demonstrated in our pre-vious study [10].
Abnormalities in fibrin formation and afibringnemia are associatedwith early spontaneous abortions [11,12], and lack of cross-linkedfibrin,resulting from factor XIII deficiency, is associated with early miscar-riages, bleedings and disturbed wound healing [13]. The current studyaimed to explore the influence of fibrin matrices on early-term culturedhuman throphoblasts in order to elucidate the role of placental fibrindeposition in physiological and pathological pregnancy.
Materials and methods
Cells, Ex vivo villous explants and spheroid cultures
First trimester trophoblasts were obtained from placental tissues ofnormal pregnancies which were interrupted due to social reasons at
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week 8-13 of gestation. The use of the placentas was approved by theInstitutional Review Board (IRB) of the Rambam Medical Center. Cellswere separated and cultured according to published guidelines [14].Briefly, placental tissues were separated from placental membranes,digested by 0.125% trypsin (Sigma–Aldrich, Israel) and purified on percollgradient (Amersham Biosciences, Sweden). Following washings, cellswere cultured in 6-well plates pre-coated with fibronectin (10 μg/ml)with DMEM:F12 (1:1) medium supplemented with 15% FCS and antibi-otics (trophoblast medium, Biological Industries, Beth Haemek, Israel).The identity and purity of cell cultures were assessed by positive stainingfor cytokeratin 7 andhPL, andwere found to be 96% and91%, respectively.
Villous explant cultures were obtained from first trimester(7–13 weeks of pregnancy) human pooled placentas, as previouslydescribed [15]. Briefly, placental tissue was placed on ice-cold salineand processed within 2 h of collection. The tissue was aseptically dis-sected to remove decidual tissue and fetal membranes. Small fragmentsof placental villi were placed on 4-well plates (Nunclon®, Nalge Nunc,Denmark), pre-coated with matrigel (BD biosciences, NJ, USA), fibrin(fibrinogen 2.5 mg/ml and 1u/ml thrombin) or collagen (all fromSigma-Aldrich, Rehovot, Israel). Explants were cultured in the tropho-blast medium at 37 °C in 5% CO2.
Trophoblast spheroids were formed by placing 750 cells in round-bottom 96-well plates in 0.25% methyl cellulose and culture mediumfor 24 h. Following cells aggregation, every 8 -10 spheroids were trans-ferred to a 24-well plate in matrix containing 0.5% methyl cellulose inculture medium and fibrin (2.5 mg/ml) or collagen (1 mg/ml) and cov-ered with medium.
Matrices coating
Wells were coated with fibrin as follows: 2.5 mg/ml fibrinogen(Sigma–Aldrich,) or 2.5 mg/ml FXIII-depleted fibrinogen (Americandiagnostica, Stamford, CT, USA) were diluted with PBS and mixed with1U/ml thrombin (Sigma–Aldrich) on ice. A thin layer was spreadon wells and allowed to clot in 37 °C incubator for 30 minutes. FXIII(50 μg/ml; Fibrogammin-P, Aventis Behring) was added to FXIII-depleted fibrin. For coating wells with fibronectin 50 μg/ml (BiologicalIndustries, Beth Haemek, Israel), fibrinogen 2.5 mg/ml, thrombin1U/ml, FXIII and FXIIIa 50 μg/ml, wells were incubated with indicatedmaterials at the above concentrations for 1 hour, then washed 3 timeswith PBS. FXIIIawas kindly donated by Prof. A. Inbal, Tel Aviv University,Israel, and was activated as described by Dardik et al. [16].
Proliferation and spreading
Proliferation was assessed by cell nuclei counting. Cells were fixedwith 3% formaldehyde, stained with DAPI, captured, and an averagenumber of nuclei per well was evaluated using the Image Pro software.For determination of cell area, cells were fixed and stained with 0.1%crystal-violet. Cells were captured, and an average cell area was calcu-lated using Image Pro software.
Immunofluorescent staining
Trophoblasts were seeded on cover-glasses coated with fibrin orfibronectin, allowed to adhere and then submerged in medium. Immu-nofluorescent staining was performed on adherent cells, using mouseanti-human vinculin (Santa Cruz Biotechnology, Santa Cruz, Ca, USA)as a primary antibody and Cy3 conjugated goat anti-mouse IgG (JacksonImmunoRresearch, Pennsylvania USA) as second antibody. Fluorescenceimages were captured by confocal microscopy.
D-Dimer analysis
Trophoblasts were cultured in wells coated with FXIII-depletedfibrin and FXIII-depleted fibrin with a supplement of FXIII. Identically
Please cite this article as: Snir A, et al, The role of fibrin matrices and tissueRes (2013), http://dx.doi.org/10.1016/j.thromres.2013.08.023
coated wells were incubated with medium only. Following 3 days ofculture, fibrin coating was lysed by plasminogen 0.5 μg/ml (Sigma –
Aldrich) and t-PA 580 μg/ml for two hours. D-Dimer level in lysateswas determined by ELISA (American Diagnostica, Stamford, CT, USA).
TF activity assay
TF procoagulant activity in whole cell lysate was quantified usingthe chromogenic assay. Trophoblasts were cultured in 96-wells coatedwith fibrin or fibronectin. To obtain cell lysate, cells underwent threefreeze-thaw cycles. A mixture of human factor (F) VII, CaCl2, factor (F)X and chromogenic substrate s-7265 (Chromogenix, Milano, Italy) wasadded to the lysates. The amount of generated FXa was quantified bymeasuring the absorbance of the cleaved substrate at 405 nm.
Human recombinant TF (Innovin, Dade BehringDiagnostic, Germany)was used for standard curve formation. The results were expressed inarbitrary units (AU).
TF expression
Total RNA was isolated utilizing TRI reagent (Molecular ResearchCenter Inc. OH, USA). cDNA was synthesized using EZ-First StrandcDNA Synthesis kit (Biological Industries, Beth Haemek, Israel).
Real time-PCR was performed with Rotor-Gene 3000, (Korbettresearch, Australia), Detection System using ABsolute™ QPCR Mas-ter Mix (Abgene, Epsom, UK), Assays-on-Demand Gene Expressionprobes (Applied Biosystems, CA, USA), The following conditionswere ap-plied for TF and GAPDH (control gene) PCR amplification: 2 minutes at50 °C, 10 minutes at 95 °C, 40 cycles of 15 seconds at 95 °C and1 minuteat 60 °C.
Total protein concentration in cell and villi lysates was measuredby the Bradford method (Sigma –Aldrich). TF protein levels in cells andvilli explants lysates were determined by TF ELISA (Imubind, AmericanDiagnostica Stamford).
Immunohistochemistry of ex-vivo villous explants
Explants obtained from placentas from 8 and 13 weeks of gestationwere cultured in fibrin or matrigel; frozen sectins of villous explantswere fixed and blocked, stained with anti-TF (American Diagnosticainc. Stamford, CT, USA) and processed with second antibody and per-oxidase substrate using the Super Picture kit (Zymed, San Francisco,CA, USA).
Statistical analysis
The data were analyzed by Student T-test. P b 0.05 was consideredsignificant.
Results
Fibrin affects cellular spreading and proliferation
When trophoblasts isolated from first trimester placenta were cul-tured on fibrin, they appeared to be rounded and flattened and theaverage cell area was significantly larger compared to cells cultured onfibronectin. This effect was studied in other relevant three-dimensionmodels: spheroids composed of trophoblasts that were cultured on fi-brin or collagen, and villi explantswhichwere grown infibrin comparedto matrigel. In both models, the same phenomenon was documented:when cultured on fibrin, edges of villi and spheroids appeared spreadand flattened (Fig. 1A and B).
The effect of fibrin on cell proliferation was studied using tropho-blasts cultured on fibrin or fibronectin for 24, 72 and 96 hours. Whencultured on fibrin, a significant increase in nuclei number was observedat 72 hours and 96 hours (3.5-fold and 4.7-fold, respectively), while on
factor in early-term trophoblast proliferation and spreading, Thromb
Fig. 1. Fibrin induces early trophoblasts, spheroids and villi explant spreading andproliferation. (A)Cells (EST) cultured onfibrin or fibronectin (fn)werefixed and stainedwithmethyleneblue. (B) Edges of villi and spheroids appeared spread andflattened as compared tomatrigel cultured villi. (C)Number of nuclei of early trophoblasts (EST) cultured onfibrin for 24, 72 and96 hours as compared to cells cultured on fn. (D)Villi explants sprouts in fibrin andmatrigel following 24 hours and 3 days of culturing. (E) Significantly increased cell area in fibrin culturedearly trophoblasts (EST) as compared to full-term trophoblasts (term). Statistical significance * (C:72 h: p b 0.001, 96 h p b 0.001, D: 24 h: p b 0.01, 3 days p b 0.03, E: p b 0.001).
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fibronectin, only a 2.5-fold increase was seen at 72 hours, whichremained unchanged at 96 hours (Fig. 1C). As the same nuclei num-ber was observed at 24 hours of culture on fibronectin and fibrin, in-creased proliferation rather than preferred cell adhesion to fibrinwas suggested. The effect of fibrin on trophoblast proliferation wasalso studied in villi explants by evaluation of the sprouts number inmatrigel or fibrin following 24 hours and 3 days of culture. The sproutsnumber in fibrin was increased by 1.7-fold compared to matrigel fol-lowing 24 hours (p b 0.01) and 3 days of culturing (p b 0.03) (Fig. 1D).
Remarkably, when primary trophoblasts obtained from termplacentas were cultured on fibrin, no changes in cell morphology ornumber were observed in comparison to cells cultured on fibronectin,implicating a selective effect of fibrin on early trophoblasts (Fig. 1E).
Fibrin concentration was found to affect both cell number andarea. When cells were cultured at different concentrations (0.5, 1.25,2.5 mg/ml) of fibrin, formed from fibrinogen, the highest cell numberwas observed with the fibrin matrix of 2.5 mg/ml, while the maximumcell area was recorded at a concentration of 1.25 mg/ml (Fig. 2A,B).
Fibrin composition was further investigated by culturing cells onfibrin,fibrinogen, thrombin andfibronectin. Nuclei number and averagecell area in trophoblasts cultured on fibrin were increased as comparedto those cultured on fibronectin, fibrinogen or thrombin, implying thatthis effect is exclusive for fibrin clot (Fig. 2C and D).
Changes occurring in cell spreading often involve cell skeleton andfocal adhesion reorganization. Assessment of vinculin localization in
Please cite this article as: Snir A, et al, The role of fibrin matrices and tissueRes (2013), http://dx.doi.org/10.1016/j.thromres.2013.08.023
trophoblasts cultured on fibrin or fibronectin revealed that vinculinwas localized in the periphery of cells cultured on fibrin, correspondingto focal adhesion, while it was distributed evenly in cells cultured onfibronectin (Fig. 2E).
Potential role of cross-linking and factor XIII in fibrin effect on trophoblasts
Cells were cultured in wells coated with: FXIII, FXIIIa, a specificpreparation of fibrin without FXIII (FXIII-depleted), FXIII-depleted fibrinwith addition of FXIII or fibrin alone, with fibronectin used as a control.The number and area of cells cultured on FXIIIa, FXIII-depleted fibrinand FXIII-depleted fibrin + FXIII, were increased similar to fibrin-cultured cells. No changes in cell number and area were observedwhen cells were cultured on FXIII compared to fibronectin (Fig. 3A,B and C).
Activated factor XIII (FXIIIa) was found to induce cell proliferationthrough a fibrin-independent mechanism (Fig. 3B). As inhibition ofactive factor XIII by iodoacetamide in the presence of cells, resultedin decreased cell viability, the identity of a possible effect of cellulartransglutaminase could not be assessed.
The contribution of trophoblasts to fibrin cross-linking was furtherinvestigated by quantification of the D-Dimer level in fibrin lysates,since it is known to correlate with the cross-linking degree of γchains within the clot. Wells were coated with FXIII-depleted fibrinor fibrin + 50 μg/ml FXIII. Trophoblasts were cultured in half of the
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Fig. 2. Fibrin, but not fibrinogen or thrombin caused an increase in cell number and area. (A)Cell number and (B) cell area of trophoblasts cultured on fibrinmatrices composed of 0.5, 1.25,2.5 mg/ml fibrinogen (fng). Cell number (C) and cell area (D)weremeasuredwhen cells were cultured on fibrin, fibrinogen and thrombin. (E) Focal adhesion and peripheral localizationwere demonstrated by immunofluorescent staining for vinculin, when EST were cultured on fibrin or fibronectin. Statistical significance * (A: p b 0.005, B: p b 0.001, C: p b 0.003,D: p b 0.0001).
4 A. Snir et al. / Thrombosis Research xxx (2013) xxx–xxx
wells, whereas the other half was incubated with the medium only.While in FXIII-depleted fibrin without cells, D-Dimer level was un-detectable, in presence of cells, it was high and similar to that ob-served in fibrin + FXIII without cells. When cells were cultured inwells coated with fibrin supplemented with FXIII, a further increasein D-Dimer level (1.8-fold) was documented (Fig. 3C).
Fibrin is associated with decreased TF level
Trophoblasts were cultured on fibrin- or fibronectin-coated wellsand villi explants were cultured on fibrin or matrigel. TF procoagulantactivity, protein level and TF mRNA expression were evaluated in cellextracts. Presence and localization of TF were confirmed by immuno-staining of placental villi explants. As shown in Fig. 4, TF procoagulantactivity, protein and mRNA levels were all significantly reduced in tro-phoblasts cultured on fibrin compared to fibronectin (Fig. 4, A: p =0.01, B: p = 0.009, C: p = 0.05), while TF protein in villi explants wasnot affected by fibrin (data not shown). Immunostaining of villi ex-plants cultured on matrigel or fibrin, demonstrated high expression ofTF in the trophoblast layer within villi explants cultured on matrigel,whereas TF was poorly stained in the trophoblast layer within the villiexplants cultured on fibrin (Fig. 4). This observation is in agreementwith the reduced TF expression in trophoblasts cultured on fibrin. Theunaffected TF level in villi explants could be related to the presence ofparenchimal TF expressing cells within the villous tissue.
Please cite this article as: Snir A, et al, The role of fibrin matrices and tissueRes (2013), http://dx.doi.org/10.1016/j.thromres.2013.08.023
Discussion
The results obtained in the current study support the hypothesisthat early-term trophoblasts may respond to fibrin by increased cellproliferation and spreading. Fibrin cross-linking mediated either byadded factor XIII or by cellular TtG was found to be associated withthis proliferation. In addition, the level of cellular TF, the main activatorof fibrin formation, appeared to be restricted by fibrin.
We have utilized relevant three-dimension models which mimicthe phenotype of human trophoblasts [15,17]. In fibrin-cultured cells,spheroids composed of trophoblasts, and villi explants, the phenome-non of increased proliferation and spreading was documented. Theobserved elevated number of cells cultured onfibrinmay result from in-creased trophoblast proliferation ormay reflect augmented adhesion tofibrin, or both. The similarity of nuclei numbers documented in 24-hourfibrin and fibronectin cultures, suggesting a comparable adhesion rate,is confirmed by results of our previous studies of trophoblast adhesionindicating that fibrin did not cause an increased cell adhesion comparedto fibronectin (data not shown). These findings, along with increasedsprouting into fibrin compared to collagen or matrigel, in the villi ex-plants model, suggest extensive proliferation rather than preferred celladhesion to fibrin.
High expression of anti-apoptotic Bcl-2 was observed in syncytio-trophoblasts when fibrin deposits were located in the sub-trophoblastregion in first trimester and term placentas [18,19]. These results
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Fig. 3. Effects of factor XIII and cross-linked fibrin on trophoblasts. (A) Average numberand (B) area of cells cultured on fibrin, FXIIIa, FXIII-depleted fibrin and FXIII-depletedfibrin with added FXIII, FXIII and fibronectin cultured cells. (C) D-Dimer assay was usedto detect cross-linked fibrin following plasmin degradation. D-Dimer level in lysates wasdetermined by ELISA (American Diagnostica) in wells coated with factor XIII-depletedfibrin and fibrin supplemented with 100u/ml FXIII both with and without cells. Statisticalsignificance * (A: p b 0.001, B: p b 0.05).
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confirm a favorable role of fibrin in trophoblasts viability and prolifera-tion at an early stage of pregnancy. Moreover, fibrin was widely usedas matrix scaffold in cell and tissue models where cell proliferationwas essential [20] and induction of proliferation by fibrin was reportedin progenitor endothelial cells and other cell types [21].
The distance and dispersion of ligand molecules were shown tobe pivotal for cell adhesion and formation of focal adhesion [22].Our results point to an essential role of fibrin cross-linking in cellproliferation and spreading. Decreased cell proliferation associatedwith low fibrin concentration may reflect the effect of reduced cross-linking and increased dispersion of ligand molecules such as RGD. Thefinding of peripheral localization of vinculin in fibrin cultured cellsfurther confirmed the fibrin effect on cell spreading via focal adhesion(Fig. 2E).
Please cite this article as: Snir A, et al, The role of fibrin matrices and tissueRes (2013), http://dx.doi.org/10.1016/j.thromres.2013.08.023
As factor XIII presence in trophoblasts is still debatable, it seems thatin its absence in the matrix, trophoblasts are likely to participate incross-linking generation via tissue transglutaminase (tTG) [23].
The mechanism through which fibrin affects the trophoblast cyto-skeleton and induces proliferation is not clear. Belkin et al reportedthat cellular tTG may form this fibrin cross-linking via endothelial cellintegrins and promote cell adhesion and signaling [24]. Our previousstudy [10] demonstrated that presence of alpha2beta3, a fibrinogenbinding integrin, on early trophobalsts, was increased by fibrin as com-pared to fibronectin. A complex formedwith tTG and VEGFR-2 in endo-thelial cells (EC) was shown to modulate the angiogenic effect of VEGF[25]. Growth factors such as VEGF were found to preferentially affectEC through binding to fibrin [9]. It is likely that tTG- or factor XIIIcross-linked-fibrin binds to cellular integrins and influences cell spread-ing and proliferation through growth factors signaling. Following integrinactivation, other proteins, such as vinculin and focal adhesion kinase(FAK), are recruited, linking integrins to the cytoskeleton and thus pro-moting cellular signaling pathways.
Given that FXIIIa alonewas found to induce trophoblats proliferation,we assume that it may affect cells by a fibrin-independent mechanism.Proangiogenic effects of factor XIIIa on endothelial cells proliferationand migration were previously described by Dardik et al. [26].
States of impaired fibrin formation (i.e., afibrogenemia, FXIII defi-ciency) are associated with early pregnancy loss in humans andmice, suggesting a role of fibrin in implantation and early pregnancymaintenance. Fibrinogen and fibrin are required to support pregnan-cies by preserving hemostatic balance and stabilizing utero-placentalattachment at the fibrinoid layer found in the fetal-maternal junction[13].
While fibrin contributes to trophoblasts remodeling, TF, which isabundant on trophoblasts [2], may play a crucial role in fibrin accumu-lation. We therefore explored the effect of fibrin on TF and foundreduced TF activity and expression in early-term trophoblasts and villiexplants.
Excessive fibrin deposition is considered to be associated with preg-nancy complications; therefore, strict control of fibrin synthesis isrequired. Our findings suggest a possible feedback inhibition mecha-nismwhere trophoblast TF expression and activity are limited by fibrinaccumulation, hence leading to coagulation reduction. Isermann et alsuggested the protein C system as the main haemostatic control mech-anism in placenta [27]. TFPI, the main inhibitor of TF/FVIIa complex,could also serve as a control mechanism for coagulation, as humanTFPI is expressed by trophoblasts from 10 weeks of gestation to term[2,27,28].
In conclusion, the current study suggests a sequence by which fibrindeposition induces trophoblast proliferation, while TF level is limitedby fibrin. This delicate balance of fibrin deposition may affect the abilityof early placenta to remodel, thus potentially influencing pregnancyoutcome.
Author contribution statement
AS: designed and performed research, wrote the paper, approvedthe final version of the paper
BB: designed research, wrote the paper, approved the final versionof the paper
BP: participated in research, approved the final version of the paperGO: participated in research, approved the final version of the paperNL: designed research, wrote the paper, approved the final version
of the paper
Conflict of interest statement
The authors have no conflicts to declare.
factor in early-term trophoblast proliferation and spreading, Thromb
Fig. 4.Reduced TFwas demonstrated in fibrin-cultured trophoblasts. (A) TF procoagulant activity, (B) protein level and (C)mRNA level in cell extracts of trophoblasts cultured on fibrin ascompared to fibronectin. (D) IHC staining of frozen sections demonstrates poorly stained TF in the trophoblast layer within villi explants cultured on fibrin compared to TF in matrigelcultured villi. Statistical significance * (A: p b 0.01, B: p b 0.009, C: p b 0.05).
6 A. Snir et al. / Thrombosis Research xxx (2013) xxx–xxx
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