Trophoblast Cell Function in the Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is a complex thrombo-inflammatory autoimmune disease characterized by the presence of antiphospholipid antibodies (aPL). Women with APS are at high risk of recurrent early pregnancy loss as well as late obstetrical complications-premature birth due to placental insufficiency or severe preeclampsia. Accumulating evidence implies that vascular thrombosis is not the only pathogenic mechanism in obstetric APS, and that the direct negative effect of aPL on the placental cells, trophoblast, plays a major role. In this review, we summarize the current findings regarding the potential mechanisms involved in aPL-induced trophoblast dysfunction. Introduction on the APS and aPL is followed by an overview of the effects of aPL on trophoblast-survival, cell function and aPL internalization. Finally, the implication of several non-coding RNAs in pathogenesis of obstetric APS is discussed, with special emphasis of their possible role in trophoblast dysfunction and the associated mechanisms.

Oleuropein Attenuates Oxidative Stress in Human Trophoblast Cells.

Olive-derived bioactive compound oleuropein was evaluated against damage induced by hydrogen peroxide in human trophoblast cells in vitro, by examining the changes in several markers implicated in oxidative stress interactions in the placenta. Trophoblast HTR-8/SVneo cells were preincubated with OLE at 10 and 100 µM and exposed to H2O2, as a model of oxidative stress. Protein and lipid peroxidation, as well as antioxidant enzymes' activity, were determined spectrophotometrically, and DNA damage was evaluated by comet assay. iNOS protein expression was assessed by Western blot, while the mRNA expression of pro- and anti-apoptotic genes BAX and BCL2 and transcription factor NFE2L2, as well as cytokines IL-6 and TNF α were determined by qPCR. Oleuropein demonstrated cytoprotective effects against H2O2 in trophoblast cells by significantly improving the antioxidant status and preventing protein and lipid damage, as well as reducing the iNOS levels. OLE reduced the mRNA expression of IL-6 and TNF α, however, it did not influence the expression of NFE2L2 or the BAX/BCL2 ratio after H2O2 exposure. Oleuropein per se did not lead to any adverse effects in HTR-8/SVneo cells under the described conditions, confirming its safety in vitro. In conclusion, it significantly attenuated oxidative damage and restored antioxidant functioning, confirming its protective role in trophoblast.

Oleuropein Stimulates Migration of Human Trophoblast Cells and Expression of Invasion-Associated Markers.

Successful pregnancy establishment requires highly synchronized cross talk between the invasive trophoblast cells and the receptive maternal endometrium. Any disturbances in this tightly regulated process may lead to pregnancy complications. Local factors such as nutrients, hormones, cytokines and reactive oxygen species modulate the invasion of extravillous trophoblasts through critical signaling cascades. Epidemiological studies strongly indicate that a Mediterranean diet can significantly impact molecular pathways during placentation. Therefore, the aim of the current study was to examine whether oleuropein (OLE), one of the main compounds of the Mediterranean diet, may influence trophoblast cell adhesion and migration, as well as the expression of invasion-associated molecular markers and inflammatory pathways fostering these processes. HTR-8/SVneo cells were incubated with OLE at selected concentrations of 10 and 100 µM for 24 h. Results showed that OLE did not affect trophoblast cell viability, proliferation and adhesion after 24 h in in vitro treatment. The mRNA expression of integrin subunits α1, α5 and ß1, as well as matrix-degrading enzymes MMP-2 and -9, was significantly increased after treatment with 10 µM OLE. Furthermore, OLE at a concentration of 10 µM significantly increased the protein expression of integrin subunits α1 and ß1. Also, OLE inhibited the activation of JNK and reduced the protein expression of COX-2. Finally, a lower concentration of OLE 10 µM significantly stimulated migration of HTR-8/SVneo cells. In conclusion, the obtained results demonstrate the effects of OLE on the function of trophoblast cells by promoting cell migration and stimulating the expression of invasion markers. As suggested from results, these effects may be mediated via inhibition of the JNK signaling pathway.

The Role of Dietary Polyphenols in Pregnancy and Pregnancy-Related Disorders.

Polyphenols are a group of phytochemicals with extensive biological functions and health-promoting potential. These compounds are present in most foods of plant origin and their increased widespread availability through the intake of nutritional supplements, fortified foods, and beverages, has also led to increased exposure throughout gestation. In this narrative review, we focus on the role of polyphenols in both healthy and pathological pregnancy. General information related to their classification and function is followed by an overview of their known effects in early-pregnancy events, including the current insights into molecular mechanisms involved. Further, we provide an overview of their involvement in some of the most common pregnancy-associated pathological conditions, such as preeclampsia and gestational diabetes mellitus. Additionally, we also discuss the estimated possible risk of polyphenol consumption on pregnancy outcomes. The consumption of dietary polyphenols during pregnancy needs particular attention considering the possible effects of polyphenols on the mechanisms involved in maternal adaptation and fetal development. Further studies are strongly needed to unravel the in vivo effects of polyphenol metabolites during pregnancy, as well as their role on advanced maternal age, prenatal nutrition, and metabolic risk of the offspring.

IL-6 and IL-8: An Overview of Their Roles in Healthy and Pathological Pregnancies.

Interleukin-6 (IL-6) is an acknowledged inflammatory cytokine with a pleiotropic action, mediating innate and adaptive immunity and multiple physiological processes, including protective and regenerative ones. IL-8 is a pro-inflammatory CXC chemokine with a primary function in attracting and activating neutrophils, but also implicated in a variety of other cellular processes. These two ILs are abundantly expressed at the feto-maternal interface over the course of a pregnancy and have been shown to participate in numerous pregnancy-related events. In this review, we summarize the literature data regarding their role in healthy and pathological pregnancies. The general information related to IL-6 and IL-8 functions is followed by an overview of their overall expression in cycling endometrium and at the feto-maternal interface. Further, we provide an overview of their involvement in pregnancy establishment and parturition. Finally, the implication of IL-6 and IL-8 in pregnancy-associated pathological conditions, such as pregnancy loss, preeclampsia, gestational diabetes mellitus and infection/inflammation is discussed.

Immunomodulatory components of Trichinella spiralis excretory-secretory products with lactose-binding specificity.

The immunomodulatory potential of Trichinella spiralis muscle larvae excretory-secretory products (ES L1) has been well documented in vitro on dendritic cells (DCs) and in animal models of autoimmune diseases. ES L1 products possess the potential to induce tolerogenic DCs and consequently trigger regulatory mechanisms that maintain immune homeostasis. The use of ES L1 as a potential treatment for various inflammatory disorders proved to be beneficial in animal models, although the precise immunomodulatory factors have not yet been identified. This study aimed at the isolation and characterization of ES L1 components that possess galectin family member properties. Galectin-1-like proteins (TsGal-1-like) were isolated from ES L1 based on the assumption of the existence of a lactose-specific carbohydrate-recognition domain and were recognized by anti-galectin-1 antibodies in Western blot. This TsGal-1-like isolate, similar to galectin-1, induced DCs with tolerogenic properties and hence, the capacity to polarize T cell response towards a regulatory type. This was reflected by a significantly increased percentage of CD4+CD25+Foxp3+ regulatory T cells and significantly increased expression of IL-10 and TGF-ß within this cell population. Proteomic analysis of TsGal-1-like isolate by mass spectrometry identified nineteen proteins, seven with annotated function after blast analysis against a database for T. spiralis and the UniProt database. To our surprise, none of the identified proteins possesses homology with known galectin family members. Nevertheless, the isolated components of ES L1 possess certain galectin-1 properties, such as specific lactose binding and the potential to elicit a regulatory immune response, so it would be worth further investigating the structure of sugar binding within isolated proteins and its biological significance.

Macrophage migration inhibitory factor in human early pregnancy events and association with placental pathologies.

Macrophage migration inhibitory factor (MIF) is a versatile cytokine acting as an important regulator of innate and adaptive immunity and implicated in many physiological and pathological processes. It is abundantly expressed at the feto-maternal interface and proposed to have a role in establishing and maintaining a healthy pregnancy. This review presents the current literature data regarding the MIF role in early pregnancy events and its association with some of the placental pathological conditions, including infection, preeclampsia, gestational diabetes mellitus and choriocarcinoma. General information regarding MIF structure and function is followed by an overview of its expression in reproductive tissues and in pregnancy. Futher, we discuss MIF's involvement in the survival of decidual stromal cells, placenta of the first trimester of pregnancy, and in trophoblast cell functions studied in vitro. Current findings associating this cytokine to placental infection, preeclampsia, gestational diabetes mellitus and choriocarcinoma are presented in the final part.

Galectins in Early Pregnancy and Pregnancy-Associated Pathologies.

Galectins are a family of conserved soluble proteins defined by an affinity for ß-galactoside structures present on various glycoconjugates. Over the past few decades, galectins have been recognized as important factors for successful implantation and maintenance of pregnancy. An increasing number of studies have demonstrated their involvement in trophoblast cell function and placental development. In addition, several lines of evidence suggest their important roles in feto-maternal immune tolerance regulation and angiogenesis. Changed or dysregulated galectin expression is also described in pregnancy-related disorders. Although the data regarding galectins' clinical relevance are still at an early stage, evidence suggests that some galectin family members are promising candidates for better understanding pregnancy-related pathologies, as well as predicting biomarkers. In this review, we aim to summarize current knowledge of galectins in early pregnancy as well as in pregnancy-related pathologies.

Analytical techniques for multiplex analysis of protein biomarkers.

INTRODUCTION: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. AREAS COVERED: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. EXPERT COMMENTARY: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine.

Anti-adhesive action of novel ruthenium(II) chlorophenyl terpyridine complexes with a high affinity for double-stranded DNA: in vitro and in silico.

Interactions of three Ru(II) chlorophenyl terpyridine complexes: [Ru(Cl-Ph-tpy)(en)Cl]Cl (1), [Ru(Cl-Ph-tpy)(dach)Cl]Cl (2) and [Ru(Cl-Ph-tpy)(bpy)Cl]Cl (3) (Cl-Ph-tpy = 4'-(4-chlorophenyl)-2,2':6',2''-terpyridine, en = 1,2-diaminoethane, dach = 1,2-diaminocyclohexane, bpy = 2,2'-bipyridine) with human serum albumin (HSA), calf thymus DNA and a double-helical oligonucleotide d(CGCGAATTCGCG)2 (1BNA) were examined. Fluorescence emission studies were used to assess the interactions of complexes with HSA, which were of moderate strength for 1 and 2. Molecular docking allowed us to predict mostly π-π stacking and van der Waals interactions between the complexes and the protein. We suggest that the complexes bind to a novel site on HSA, which is different from its druggable sites I, II or III. We suggest a partial intercalation of complexes through the minor groove as a possible mode of interaction with double-helical DNA. Finally, when applied to normal extravillous cell line HTR8/SVneo and JAr choriocarcinoma cell line, complexes 1 and 2 exerted anti-adhesive properties at very low doses, whereas complex 3 had a negligible effect. The obtained results are completion of our studies of Ru(II) terpyridyl complexes that carry N-N ancillary ligands. We suggest a new research direction towards studying the cellular effects of Ru(II) polypyridyl compounds.

Association of acute Babesia canis infection and serum lipid, lipoprotein, and apoprotein concentrations in dogs.

BACKGROUND: Babesia canis infection induces a marked acute phase response (APR) that might be associated with alteration in lipid and lipoprotein metabolism and disease prognosis. HYPOTHESIS: Dogs with B. canis-induced APR develop dyslipidemia with altered lipoprotein concentration and morphology. ANIMALS: Twenty-nine client-owned dogs with acute B. canis infection and 10 clinically healthy control dogs. METHODS: Observational cross-sectional study. Serum amyloid A (SAA) was measured using ELISA. Cholesterol, phospholipids, and triglycerides were determined biochemically. Lipoproteins were separated using agarose gel electrophoresis. Lipoprotein diameter was assessed by polyacrylamide gradient gel electrophoresis; correlation with ApoA-1 (radioimmunoassay) and SAA was determined. RESULTS: Dogs with B. canis infection had a marked APR (median SAA, 168.3 µg/mL; range, 98.1-716.2 µg/mL) compared with controls (3.2 µg/mL, 2.0-4.2 µg/mL) (P < .001). Dogs with B. canis infection had significantly lower median cholesterol (4.79 mmol/L, 1.89-7.64 mmol/L versus 6.15 mmol/L, 4.2-7.4 mmol/L) (P = .02), phospholipid (4.64 mmol/L, 2.6-6.6 mmol/L versus 5.72 mmol/L, 4.68-7.0 mmol/L) (P = .02), and α-lipoproteins (77.5%, 27.7%-93.5% versus 89.2%, 75.1%-93.5%) (P = .04), and higher ApoA-1 (1.36 U, 0.8-2.56 U versus 0.95 U, 0.73-1.54 U) concentrations (P = .02). Serum amyloid A correlated with high-density lipoproteins (HDLs) diameter (rho = .43; P = .03) and ApoA-1 (rho = .63, P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Major changes associated with B. canis-induced APR in dogs are related to concentration, composition, and morphology of HDL particles pointing to an altered reverse cholesterol transport. Parallel ApoA-1 and SAA concentration increase is a unique still unexplained pathophysiological finding.

Integrin ß1 is bound to galectin-1 in human trophoblast.

Interaction of sugar binding proteins-galectins, with glycoconjugates is considered relevant for various reproductive processes. Galectin-1 (gal-1) is a molecule involved in trophoblast cell invasion, which is accomplished through interaction with cell surface and/or extracellular matrix glycoproteins. A possibility of interaction of endogenous gal-1 and trophoblast ß1 integrins, both previously shown relevant for trophoblast invasion, was investigated. Confocal microscopy showed overlap in gal-1 and ß1 integrin localization at the plasma membrane of isolated cytotrophoblast, HTR-8/SVneo extravillous trophoblast cell line and JAr choriocarcinoma cells. Immunoprecipitation confirmed an interaction of gal-1 with integrin ß1, but not with α1 or α5 integrin subunits. Nondenaturing electrophoresis and subcellular fractionation suggested association of gal-1 with ß1 integrin in intracellular and plasma membrane compartments of HTR-8/SVneo cells. Gal-1/ß1 integrin complex was sensitive to chemical and enzyme treatments, indicating carbohydrate dependent interaction. Down-regulation of gal-1 by siRNA, however, had no effect on level or distribution of ß1 integrin, as determined by qPCR and flow cytometry. These results suggest complex lectin type interaction of gal-1 with ß1 integrin at the trophoblast cell membrane, which could influence trophoblast cell adhesion, migration and invasion.

Interaction of extravillous trophoblast galectin-1 and mucin(s)-Is there a functional relevance?

In the course of embryo implantation extensive interaction of the trophoblast with uterine tissue is crucial for adequate trophoblast invasion. This interaction is highly controlled, and it has been pointed out that a specific glycocode and changes in glycosylation may be important for successful implantation and maintenance of pregnancy. Both uterine and trophoblast cells have been shown to express cell surface glycoconjugates and sugar binding proteins, such as mucins (MUC) and galectins (gals). An increasing number of studies have investigated potential candidates interacting in this process. However, knowledge about the biochemical nature of the interactions and their importance for trophoblast cell function, and, consequently, for pregnancy outcome are still lacking. This review is aimed at deliberating the possibility that mucins, as heavily glycosylated proteins, might be among the functionally relevant galectin ligands in human trophoblast, based on both published data and our original research.

Galectin signature of the choriocarcinoma JAr cells: Galectin-1 as a modulator of invasiveness in vitro.

Our previous findings showed that galectin-1 (LGALS1) plays an important role in the in vitro invasion of normal human trophoblast cells. In the present study, choriocarcinoma JAr cells were found to express LGALS1, -2, -3, -8, -10, and -13 mRNA and at least LGALS1, -3, and -8 protein, as determined by reverse-transcriptase PCR and Western blot, respectively. The galectin mRNA signature of JAr cells thus differed from that of normal first-trimester extravillous trophoblasts. A Matrigel migration assay was also used to investigate and confirm the relevance and effect of LGALS1 on the invasive potential of JAr cells, as observed in other trophoblast models. This modulation in behavior was achieved by specific lectin-glycan binding.

Galectin-1 binds mucin in human trophoblast.

Mucins are multifunctional highly glycosylated proteins expressed by the female reproductive tract. Differential expression of MUC1 and MUC15 has been shown in trophoblast. This study was undertaken to establish the distribution of mucin(s) in cytotrophoblast cell cultures using anti-bovine submaxillary mucin (BSM) and to investigate the possibility of MUC1/mucin(s) being a binding partner of trophoblast galectin-1. MUC1 is demonstrated here using immunocytochemistry on isolated cytotrophoblast and the HTR-8/SVneo extravillous trophoblast cell line but detection of additional trophoblast mucins cannot be excluded. Western blot analysis showed similar bands ranging from 30 to >200 kDa with anti-BSM and the well-known mucin antibodies HMFG1 and B72.3. Immunocytochemistry and cell-based ELISA data were found to support that all of the antibodies used are reactive with BSM, suggesting the presence of shared epitopes between BSM and trophoblast mucin(s). Binding of galectin-1 to trophoblast MUC1/mucin(s) was analyzed using a solid-phase assay and co-immunoprecipitation. Recombinant galectin-1 binding to isolated trophoblast mucin in solid-phase assay was sensitive to lactose, a carbohydrate inhibitor of galectin binding. In whole HTR-8/SVneo lysates, ~200 kDa mucin was detected in galectin-1 immunoprecipitates, while endogenous galectin-1 was present in BSM-immunoprecipitates. Furthermore, double fluorescence immunocytochemistry showed overlap of galectin-1 and trophoblast mucins at the plasma membrane of HTR-8/SVneo cells. These results suggest that trophoblast mucin(s) could act as binding partners of galectin-1, in a carbohydrate-dependent manner.

Galectin-1 is part of human trophoblast invasion machinery--a functional study in vitro.

BACKGROUND: Interactions of glycoconjugates with endogenous galectins, have been long proposed to participate in several reproductive processes including implantation. In human placenta gal-1, gal-3, gal-8, and gal-13 proteins are known to be present. Each of them has been proposed to play multiple functions, but so far no clear picture has emerged. We hypothesized that gal-1 participates in trophoblast invasion, and conducted Matrigel invasion assay using isolated cytotrophoblast from first trimester placenta and HTR-8/SVneo cell line to test it. METHODS AND FINDINGS: Function blocking anti-gal-1 antibody was employed to assess participation of endogenous gal-1 in cell adhesion, cell invasion of HTR-8/SVneo cells. When gal-1 was blocked in isolated trophoblast cell invasion was reduced to 75% of control (SEM ± 6.3, P<0.001) and to 66% of control (SEM ± 1.7, P<0.001) in HTR-8/SVneo cell line. Increased availability of gal-1, as two molecular forms of recombinant human gal-1 (CS-gal-1 and Ox-gal-1), resulted in increased cell invasion by cytotrophoblast to 151% (SEM ± 16, P<0.01) with 1 ng/ml of CS-gal-1, and to 192% (SEM ± 51, P<0.05) with 1 µg/ml of Ox-gal-1. Stimulation was also observed in HTR-8/SVneo cells, to 317% (SEM ± 58, P<0.001) by CS-gal-1, and to 200% (SEM ± 24, P<0.001) by Ox-gal-1 at 1 µg/ml. Both sets of results confirmed involvement of gal-1 in trophoblast invasion. Galectin profile of isolated cytotrophoblast and HTR-8/SVneo cells was established using RT-PCR and real-time PCR and found to consist of gal-1, gal-3 and gal-8 for both cell types. Only gal-1 was located at the trophoblast cell membrane, as determined by FACS analysis, which is consistent with the results of the functional tests. CONCLUSION AND SIGNIFICANCE: These findings qualify gal-1 as a member of human trophoblast cell invasion machinery.