MicroRNA-218-5p Promotes Endovascular Trophoblast Differentiation and Spiral Artery Remodeling.

Preeclampsia (PE) is the leading cause of maternal and neonatal morbidity and mortality. Defects in trophoblast invasion, differentiation of endovascular extravillous trophoblasts (enEVTs), and spiral artery remodeling are key factors in PE development. There are no markers clinically available to predict PE, leaving expedited delivery as the only effective therapy. Dysregulation of miRNA in clinical tissues and maternal circulation have opened a new avenue for biomarker discovery. In this study, we investigated the role of miR-218-5p in PE development. miR-218-5p was highly expressed in EVTs and significantly downregulated in PE placentas. Using first-trimester trophoblast cell lines and human placental explants, we found that miR-218-5p overexpression promoted, whereas anti-miR-218-5p suppressed, trophoblast invasion, EVT outgrowth, and enEVT differentiation. Furthermore, miR-218-5p accelerated spiral artery remodeling in a decidua-placenta co-culture. The effect of miR-218-5p was mediated by the suppression of transforming growth factor (TGF)-ß2 signaling. Silencing of TGFB2 mimicked, whereas treatment with TGF-ß2 partially reversed, the effects of miR-218-5p. Taken together, these findings demonstrate that miR-218-5p promotes trophoblast invasion and enEVT differentiation through a novel miR-218-5p-TGF-ß2 pathway. This study elucidates the role of an miRNA in enEVT differentiation and spiral artery remodeling and suggests that downregulation of miR-218-5p contributes to PE development.

Cytoplasmic mislocalization of p27 and CDK2 mediates the anti-migratory and anti-proliferative effects of Nodal in human trophoblast cells.

p27(Kip1), a cyclin-dependent kinase (CDK) inhibitor, is a multi-functional protein that regulates various cellular activities. Trophoblast proliferation, migration and invasion are some of the key processes of placental development. We have recently reported that Nodal, a member of the transforming growth factor-ß (TGF-ß) superfamily, inhibits human trophoblast cell proliferation, migration and invasion. In the present study, we investigated the mechanism by which Nodal regulates trophoblast activities. We found that Nodal increased p27 mRNA and protein levels by enhancing their stability. Interestingly, Nodal signaling also induced nuclear export of p27 and CDK2. Cytoplasmic translocation of p27 induced by Nodal requires p27 phosphorylation at S10. In addition, Nodal enhanced the association of p27 with CDK2, CDK5 and a microtubule-destabilizing protein, stathmin, and induced stathmin phosphorylation at S25 and S38. Furthermore, Nodal increased tubulin stability as revealed by immunofluorescent staining of acetylated tubulin. Finally, silencing of p27 reversed the inhibitory effect of Nodal on trophoblast cell proliferation, migration and invasion. Taken together, our findings revealed a novel function of simultaneous p27 and CDK2 cytoplasmic mislocalization in mediating growth-factor-regulated cell proliferation, migration and invasion.

MicroRNAs in Human Placental Development and Pregnancy Complications.

MicroRNAs (miRNAs) are small non-coding RNAs, which function as critical posttranscriptional regulators of gene expression by promoting mRNA degradation and translational inhibition. Placenta expresses many ubiquitous as well as specific miRNAs. These miRNAs regulate trophoblast cell differentiation, proliferation, apoptosis, invasion/migration, and angiogenesis, suggesting that miRNAs play important roles during placental development. Aberrant miRNAs expression has been linked to pregnancy complications, such as preeclampsia. Recent research of placental miRNAs focuses on identifying placental miRNA species, examining differential expression of miRNAs between placentas from normal and compromised pregnancies, and uncovering the function of miRNAs in the placenta. More studies are required to further understand the functional significance of miRNAs in placental development and to explore the possibility of using miRNAs as biomarkers and therapeutic targets for pregnancy-related disorders. In this paper, we reviewed the current knowledge about the expression and function of miRNAs in placental development, and propose future directions for miRNA studies.

miR-218-5p Induces Interleukin-1ß and Endovascular Trophoblast Differentiation by Targeting the Transforming Growth Factor ß-SMAD2 Pathway.

The acquisition of an endovascular trophoblast (enEVT) phenotype is essential for normal placental development and healthy pregnancy. MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in regulating gene expression. We have recently reported that miR-218-5p promotes enEVT differentiation and spiral artery remodeling in part by targeting transforming growth factor ß2 (TGFß2). We also identified IL1B, which encodes interleukin 1ß (IL1ß), as one of the most highly upregulated genes by miR-218-5p. In this study, we investigated how miR-218-5p regulates IL1B expression and IL1ß secretion and the potential role of IL1ß in enEVT differentiation. Using two cell lines derived from extravillous trophoblasts (EVTs), HTR-8/SVneo and Swan 71, we found that stable overexpression of miR-218-5p precursor, mir-218-1, or transient transfection of miR-218-5p mimic, significantly increased IL1B mRNA and IL1ß protein levels in cells and conditioned media. We also showed that miR-218-5p directly interacted with SMAD2 3'UTR and reduced SMAD2 at mRNA and protein levels. Knockdown of SMAD2 induced IL1B expression and attenuated the inhibitory effect of TGFß2 on IL1B expression. On the other hand, overexpression of SMAD2 reduced IL1ß levels and blocked the stimulatory effects of miR-218-5p on IL1B expression, trophoblast migration and endothelial-like network formation. In addition, treatment of trophoblasts with IL1ß induced the formation of endothelial-like networks and the expression of enEVT markers in a dose-dependent manner. These results suggest that miR-218-5p inhibits the TGFß/SMAD2 pathway to induce IL1ß and enEVT differentiation. Finally, low doses of IL1ß also inhibited the expression of miR-218-5p, suggesting the existence of a negative feedback regulatory loop. Taken together, our findings suggest a novel interactive miR-218-5p/TGFß/SMAD2/IL1ß signaling nexus that regulates enEVT differentiation.

Fibrinogen is required for maintenance of platelet intracellular and cell-surface P-selectin expression.

Platelet P-selectin plays important roles in inflammation and contributes to thrombosis and hemostasis. Although it has been reported that von Willebrand factor (VWF) affects P-selectin expression on endothelial cells, little information is available regarding regulation of platelet P-selectin expression. Here, we first observed that P-selectin expression was significantly decreased on platelets of fibrinogen and VWF double-deficient mice. Subsequently, we identified this was due to fibrinogen deficiency. Impaired P-selectin expression on fibrinogen-deficient platelets was further confirmed in human hypofibrinogenemic patients. We demonstrated that this impairment is unlikely due to excessive P-selectin shedding, deficient fibrinogen-mediated cell surface P-selectin binding, or impaired platelet granule release, but rather is due to decreased platelet P-selectin content. Fibrinogen transfusion completely recovered this impairment in fibrinogen-deficient (Fg(-/-)) mice, and engagement of the C-terminus of the fibrinogen gamma chain with beta3 integrin was required for this process. Furthermore, Fg(-/-) platelets significantly increased P-selectin expression following transfusion into beta3 integrin-deficient mice and when cultured with fibrinogen. These data suggest fibrinogen may play important roles in inflammation, thrombosis, and hemostasis via enhancement of platelet P-selectin expression. Since human fibrinogen levels vary significantly in normal and diseased populations, P-selectin as an activation marker on platelets should be used with caution.

Two Cases of Hidradenitis Suppurativa Treated with Adalimumab at the Department of Dermatology and Venereology, Clinical Hospital Mostar.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease primarily affecting apocrine gland-rich areas of the body and presenting with painful nodules, abscesses, sinus tracts, and scarring (1). HS is a defect of the follicular epithelium; some have therefore called for the naming the disease acne inversa instead of hidradenitis suppurativa. The term acne inversa links the pathogenesis to acne and reflects the fact that it is an expression of follicular occlusion in localizations inverse to acne vulgaris (2). HS typically occurs after puberty. Studies have shown that the average onset is in the second or third decades of life (3). One of the most frequently cited risk factors for HS is cigarette smoking. Another significant risk factor for HS is obesity. About one-third of patients with HS have reported a family history of the disease (4). A clinically relevant staging and disease severity assessment is essential for the development of evidence-based treatments. There are several scoring systems for the assessment of disease severity of HS, including Hurley staging, HS Physician's Global Assessment (PGA), the modified Sartorius score (MSS), and the HS Severity Index (HSSI). Each of these assessments has both advantages and limitations in daily practice; there is currently no gold standard (5-8). The Hurley staging system is the simplest and most widely used instrument for HS classification in routine clinical practice. It classifies HS into three stages. HS-PGA is relatively easy to apply and is frequently used to measure clinical improvement in clinical trials of medical treatments (5). The system describes six disease stages, increasing in severity on a scale from 1 to 6 (9). MSS is a more detailed and dynamic classification system based on the counting of individual nodules and fistulas within seven anatomical regions. The system, which was developed by Sartorius et al. and later modified, is the first disease-specific instrument for dynamically measuring clinical severity of HS (10). The treatment of HS includes topical clindamycin, triamcinolone acetonide, clobetasol, topical resorcinol, oral antibiotics, hormonal therapy, oral retinoids, and biologic therapies (11). Biologic therapies are increasingly used in patients who fail to sufficiently respond to antibiotic and hormonal treatments. Adalimumab, infliximab, and etanercept have all been tested in the treatment of HS but vary in effectiveness and in how well they have been studied. Subcutaneous weekly adalimumab (160 mg at week 0, 80 mg at week 2, and 40 mg each week thereafter) is the only biologic agent approved by the US Food and Drug Administration (FDA) and the European Medicine Agency (EMA) for the treatment of HS, and it is recommended as first-line therapy for patients with moderate-to-severe disease who are intolerant or unresponsive to oral antibiotics (12). The first male patient aged 59 years was referred to our Department with very long history of HS. The first symptoms had been unrecognized and presented as a pilonidal cyst 25 years ago as well as cysts on the intergluteal region treated with multiple surgical interventions and systemic antibiotics. The first hospitalization at our Department was in 2016. In addition to HS, the patient had diabetes mellitus (DM) type II and hypertension. A physical examination showed multiple abscesses, fistulas, and nodules in the axillary, inguinal, perianal, gluteal, and intergluteal regions; Hurley staging: stage II, PGA staging: IV, DLQI: 24 (Figure 1, Figure 2). Microbiological repeated swabs showed numerous bacteria such as Esch.coli, S.aureus, Serratia.spp, Enterococcus spp, St.epidermidis, and Proteus mirabilis. Laboratory tests which included complete blood cell count, biochemistry, serology for syphilis, HIV, and hepatitis B and C infection together with chest X-rays were all within normal limits. Abdominal ultrasound examination found no abnormalities. Quantiferon test was positive. After the monotherapy with isoniazid, a repeated Quantiferon test two months later was negative. The patient was treated with betadine solution and pus drainage until 2018, when at the Department of Dermatology and Venerology prescribed adalimumab in doses of 80 mg initially, 40 mg ×2 on the first day and the day after that, then 80 mg after fifteen days followed by 40 mg every ten days. After 16 weeks of treatment with adalimumab, Hurley staging was II, PGA IV, DLQI 3. The second male patient aged 28 years was referred to our Department with a shorter history: the first symptoms were presented as pilonidal sinus in 2012, after that in 2015 as inflamed nodules and fistulas in the axillary and inguinal regions. In 2018, physical examination showed the same nodules with a more intense character as well as furuncles on the scalp and skin of the back, with Hurley staging stage II, PGA staging III, DLQI 14 (Figure 3, Figure 4). Until the disease was diagnosed, the patient was treated several times with peroral antibiotics, while laboratory tests which included complete blood cell count, biochemistry, serology for syphilis, HIV, and hepatitis B and C infection together with chest X-rays were all within normal limits with the exception of elevated cholesterol (6.1). Abdominal ultrasound examination found no abnormalities. Quantiferon test was negative. The following therapy was administered during hospitalization: Humira (adalimumab) initial dose 160 mg, a dose of 80 mg after 14 days, and after 7 days 40 mg, in addition to local therapy with 10% resorcinol solution at the location of the skin changes. After 16 weeks of treatment with adalimumab, Hurley staging was II, PGA staging was III, and DLQI index was 3. Hidradenitis suppurativa is a chronic, recurrent inflammatory and debilitating skin disease of the terminal hair follicle that usually presents after puberty with painful, deep seated, inflamed lesions in the apocrine gland-bearing areas of the body, most commonly the axillary, inguinal, and anogenital region (3). Biological therapies have been increasingly used for patients who failed to sufficiently respond to antibiotics and hormonal treatments. Adalimumab, infliximab, and etanercept have all been tested in the treatment of hidradenitis suppurativa but vary in effectiveness and in how well they have been studied. Subcutaneous weekly adalimumab (160 mg at week, 80 mg at week 2, and 40 mg each week thereafter) is the only biologic agent approved by the US Food and Drug Administration (FDA) and the European Medicine Agency (EMA) for the treatment of HS and is recommended as first-line therapy for patients who moderate-to-severe disease and who are intolerant or unresponsive to oral antibiotics (5,12). Treatment of hidradenitis suppurativa remains a considerable challenge and should be individualized according to the state and extent of the disease. Therapeutic options for hidradenitis suppurativa were long restricted to the use of local disinfectants and systemic antibiotics as well as repeated incisions and drainage, which produce only short-term benefits. Our patients showed regression of lesions after sixteen weeks of biological therapy.

Differential Role of Smad2 and Smad3 in the Acquisition of an Endovascular Trophoblast-Like Phenotype and Preeclampsia.

During placental development, cytotrophoblast progenitor cells differentiate into the syncytiotrophoblast and invasive extravillous trophoblasts (EVTs). Some EVTs further differentiate into endovascular trophoblasts (enEVTs) which exhibit endothelial-like properties. Abnormal placental development, including insufficient enEVT-mediated remodeling of the uterine spiral arteries, is thought to be a precipitating factor in the onset of preeclampsia (PE), a pregnancy-related hypertensive disorder. Several members of the transforming growth factor-ß (TGF-ß) superfamily, such as TGF-ßs, Nodal, and Activin have been reported to either promote or inhibit the invasive EVT pathway. These ligands signal through serine/threonine receptor complexes to activate downstream signaling mediators, Smad2 and Smad3. In this study, we determined Smad2 and Smad3 expression pattern in placenta and their effects on trophoblast invasion and differentiation. Total Smad2/3 levels were relatively constant across gestation while the ratio of active phosphorylated forms to their total levels varied with gestational stages, with a higher pSmad2/total Smad2 in later gestation and a higher pSmad3/total Smad3 in early gestation. Immunofluorescent staining revealed that pSmad3 was localized in nuclei of EVTs in anchoring villi. On the other hand, pSmad2 was mostly absent in this invasive EVT population. In addition, pSmad3/total Smad3, but not pSmad2/total Smad2, was significantly lower in both early onset and late onset PE cases, as compared to gestational age-matched controls. Functional studies carried out using a first trimester trophoblast cell line, HTR-8/SVneo, and first trimester human placental explants showed that Smad2 and Smad3 had differential roles in the invasive pathway. Specifically, siRNA-mediated knockdown of Smad2 resulted in an increase in trophoblast invasion and an upregulation of mRNA levels of enEVT markers while the opposite was observed with Smad3 knockdown. In addition, Smad2 siRNA accelerated the EVT outgrowth in first trimester placental explants while the Smad3 siRNA reduced the outgrowth of EVTs when compared to the control. Furthermore, knockdown of Smad2 enhanced, whereas overexpression of Smad2 suppressed, the ability of trophoblasts to form endothelial-like networks. Conversely, Smad3 had opposite effects as Smad2 on network formation. These findings suggest that Smad2 and Smad3 have opposite functions in the acquisition of an enEVT-like phenotype and defects in Smad3 activation are associated with PE.

DNA-Conjugated Gold Nanoparticles as High-Mass Probes in Imaging Mass Cytometry.

We employ imaging mass cytometry (IMC) to investigate in vitro uptake and cellular distribution of DNA-functionalized gold nanoparticles (AuNPs). IMC enables the multiparametric imaging of cell components and allows for the detection of AuNPs in cells with >100 times higher sensitivity than conventional confocal fluorescence imaging, as each nanoparticle contains thousands of atoms for signal amplification. Changes in the accumulation of nanoparticles in cells due to oligonucleotide sequence-dependent interactions are exploited to examine a model biomarker for hypoxia-microRNA-210. We find that AuNPs functionalized with microRNA-210-targeting sequence accumulate in hypoxic cells 3 to 4-fold compared to normoxic cells. The work examines the potential use of DNA-AuNP as high-mass probes for the analysis of nonabundant nucleic acids.

miR-590-3p Promotes Ovarian Cancer Growth and Metastasis via a Novel FOXA2-Versican Pathway.

miRNAs play important roles in gene regulation, and their dysregulation is associated with many diseases, including epithelial ovarian cancer (EOC). In this study, we determined the expression and function of miR-590-3p in EOC. miR-590-3p levels were higher in high-grade carcinoma when compared with low-grade or tumors with low malignant potential. Interestingly, plasma levels of miR-590-3p were significantly higher in patients with EOC than in subjects with benign gynecologic disorders. Transient transfection of miR-590-3p mimics or stable transfection of mir-590 increased cell proliferation, migration, and invasion. In vivo studies revealed that mir-590 accelerated tumor growth and metastasis. Using a cDNA microarray, we identified forkhead box A2 (FOXA2) and versican (VCAN) as top downregulated and upregulated genes by mir-590, respectively. miR-590-3p targeted FOXA2 3' UTR to suppress its expression. In addition, knockdown or knockout of FOXA2 enhanced cell proliferation, migration, and invasion. Overexpression of FOXA2 decreased, whereas knockout of FOXA2 increased VCAN mRNA and protein levels, which was due to direct binding and regulation of the VCAN gene by FOXA2. Interrogation of the TCGA ovarian cancer database revealed a negative relationship between FOXA2 and VCAN mRNA levels in EOC tumors, and high FOXA2/low VCAN mRNA levels in tumors positively correlated with patient survival. Finally, overexpression of FOXA2 or silencing of VCAN reversed the effects of mir-590. These findings demonstrate that miR-590-3p promotes EOC development via a novel FOXA2-VCAN pathway.Significance: Low FOXA2/high VCAN levels mediate the tumor-promoting effects of miR-590-3p and negatively correlate with ovarian cancer survival. Cancer Res; 78(15); 4175-90. ©2018 AACR.

Activated NK cells cause placental dysfunction and miscarriages in fetal alloimmune thrombocytopenia.

Miscarriage and intrauterine growth restriction (IUGR) are devastating complications in fetal/neonatal alloimmune thrombocytopenia (FNAIT). We previously reported the mechanisms for bleeding diatheses, but it is unknown whether placental, decidual immune cells or other abnormalities at the maternal-fetal interface contribute to FNAIT. Here we show that maternal immune responses to fetal platelet antigens cause miscarriage and IUGR that are associated with vascular and immune pathologies in murine FNAIT models. Uterine natural killer (uNK) cell recruitment and survival beyond mid-gestation lead to elevated NKp46 and CD107 expression, perforin release and trophoblast apoptosis. Depletion of NK cells restores normal spiral artery remodeling and placental function, prevents miscarriage, and rescues hemorrhage in neonates. Blockade of NK activation receptors (NKp46, FcɣRIIIa) also rescues pregnancy loss. These findings shed light on uNK antibody-dependent cell-mediated cytotoxicity of invasive trophoblasts as a pathological mechanism in FNAIT, and suggest that anti-NK cell therapies may prevent immune-mediated pregnancy loss and ameliorate FNAIT.Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a gestational disease caused by maternal immune responses against fetal platelets. Using a FNAIT mouse model and human trophoblast cell lines, here the authors show that uterine natural killer cell-mediated trophoblast apoptosis contributes to FNAIT pathogenesis.

Placental trophoblast cell differentiation: physiological regulation and pathological relevance to preeclampsia.

The placenta is a transient organ that forms during pregnancy to support the growth and development of the fetus. During human placental development, trophoblast cells differentiate through two major pathways. In the villous pathway, cytotrophoblast cells fuse to form multinucleated syncytiotrophoblast. In the extravillous pathway, cytotrophoblast cells acquire an invasive phenotype and differentiate into either (1) interstitial extravillous trophoblasts, which invade the decidua and a portion of the myometrium, or (2) endovascular extravillous trophoblasts, which remodel the maternal vasculature. These differentiation events are tightly controlled by the interplay of oxygen tension, transcription factors, hormones, growth factors, and other signaling molecules. More recently, microRNAs have been implicated in this regulatory process. Abnormal placental development, particularly the limited invasion of trophoblast cells into the uterus and the subsequent failure of the remodeling of maternal spiral arteries, is believed to cause preeclampsia, a severe pregnancy related disorder characterized by hypertension and proteinuria. Oxidative stress, the abnormal production and/or function of signaling molecules, as well as aberrant microRNAs expression have been suggested to participate in the pathogenesis of preeclampsia. Several potential biomarkers for preeclampsia have been identified, creating new opportunities for the development of strategies to diagnose, prevent, and treat this disorder.