Endothelin-1 down-regulates matrix metalloproteinase 14 and 15 expression in human first trimester trophoblasts via endothelin receptor type B

$\textbf{STUDY QUESTION}$: Does endothelin-1 (ET-1) regulate matrix metalloproteinase (MMP) 14 and 15 production and invasion of human first trimester trophoblasts? $\textbf{SUMMARY ANSWER}$: ET-1 in pathophysiological concentrations down-regulates MMP14 and MMP15 expression via endothelin receptor (ETR) type B and decreases trophoblast migration and invasion. $\textbf{WHAT IS KNOWN ALREADY}$: MMP14 and MMP15 are involved in trophoblast invasion. Impairment of invasion has been linked to pregnancy complications such as pre-eclampsia (PE). ET-1 is up-regulated in PE. $\textbf{STUDY DESIGN, SIZE, DURATION}$: $\textit{In vitro}$ study using primary human trophoblasts from 50 first trimester placentas (gestational week 7-12). $\textbf{PARTICIPANTS/MATERIALS, SETTING, METHODS}$: Trophoblasts were cultured in the absence or presence of 10-100 nM ET-1. MMP14 and MMP15 mRNA and protein were quantified by RT-qPCR and Western blotting, respectively. Selective antagonists for ETRA (BQ-123) or ETRB (BQ-788) were used to identify ETR subtypes involved. Functional ET-1 effects were tested in first trimester chorionic villous explants and transwell invasion assays. The roles of tumor necrosis factor (TNF)-α (25 ng/ml) and oxygen (1%) in ET-1 regulation of MMP14 and 15 expression were assessed by Western blotting. $\textbf{MAIN RESULTS AND THE ROLE OF CHANCE}$: ET-1 down-regulated MMP14 and MMP15 mRNA (-21% and -26%, respectively, $\textit{P}$ < 0.05) and protein levels (-18% and -22%, respectively, $\textit{P}$ < 0.05). This effect was mediated via ETRB. ET-1 decreased trophoblast outgrowth in placental explants (-24%, $\textit{P}$ < 0.05) and trophoblast invasion (-26%, $\textit{P}$ ≤ 0.01). TNF-α enhanced ET-1 mediated MMP15 down-regulation (by 10%, $\textit{P}$ < 0.05), whereas hypoxia abolished the effect of ET-1 on both MMPs. $\textbf{LARGE SCALE DATA}$: N/A. $\textbf{LIMITATIONS, REASONS FOR CAUTION}$: Only primary trophoblasts were used in this study. Since trophoblast yield from first trimester placental material is limited, further aspects of MMP14 and 15 regulation could not be characterized. Other anti-invasive factors may be altered by ET-1 in trophoblasts and, thus, contribute to the reduced invasion, but have not been investigated. Oxygen levels similar to those found in the decidua (5-8% O2) were not analyzed in this study. $\textbf{WIDER IMPLICATIONS OF THE FINDINGS}$: ET-1 modifies placental function already during the first trimester of pregnancy, the time-window when the placental changes implicated in PE occur. Thus, our results improve the understanding of the placental mechanisms underlying trophoblast invasion and PE.The study was funded by the Oesterreichische Nationalbank (Anniversary Fund, project number: 14796) and the Herzfelder'sche Familienstiftung (to J.P.; number: 00685). AMM received funding from the Austrian Science Fund FWF (W1241) and the Medical University Graz through the PhD Program Molecular Fundamentals of Inflammation (DK-MOLIN)

Control of human endometrial stromal cell motility by PDGF-BB, HB-EGF and trophoblast-secreted factors

Human implantation involves extensive tissue remodeling at the fetal-maternal interface. It is becoming increasingly evident that not only trophoblast, but also decidualizing endometrial stromal cells are inherently motile and invasive, and likely contribute to the highly dynamic processes at the implantation site. The present study was undertaken to further characterize the mechanisms involved in the regulation of endometrial stromal cell motility and to identify trophoblast-derived factors that modulate migration. Among local growth factors known to be present at the time of implantation, heparin-binding epidermal growth factor-like growth factor (HB-EGF) triggered chemotaxis (directed locomotion), whereas platelet-derived growth factor (PDGF)-BB elicited both chemotaxis and chemokinesis (non-directed locomotion) of endometrial stromal cells. Supernatants of the trophoblast cell line AC-1M88 and of first trimester villous explant cultures stimulated chemotaxis but not chemokinesis. Proteome profiling for cytokines and angiogenesis factors revealed neither PDGF-BB nor HB-EGF in conditioned media from trophoblast cells or villous explants, while placental growth factor, vascular endothelial growth factor and PDGF-AA were identified as prominent secretory products. Among these, only PDGF-AA triggered endometrial stromal cell chemotaxis. Neutralization of PDGF-AA in trophoblast conditioned media, however, did not diminish chemoattractant activity, suggesting the presence of additional trophoblast-derived chemotactic factors. Pathway inhibitor studies revealed ERK1/2, PI3 kinase/Akt and p38 signaling as relevant for chemotactic motility, whereas chemokinesis depended primarily on PI3 kinase/Akt activation. Both chemotaxis and chemokinesis were stimulated upon inhibition of Rho-associated, coiled-coil containing protein kinase. The chemotactic response to trophoblast secretions was not blunted by inhibition of isolated signaling cascades, indicating activation of overlapping pathways in trophoblast-endometrial communication. In conclusion, trophoblast signals attract endometrial stromal cells, while PDGF-BB and HB-EGF, although not identified as trophoblast-derived, are local growth factors that may serve to fine-tune directed and non-directed migration at the implantation site

Методика автоматизированного флуоресцентного контроля загрязнения водных объектов нефтепродуктами

В работе предложена методика автоматизированного флуоресцентного контроля загрязнения водных объектов нефтепродуктами, основанная на скрининговом подходе к измерению содержания загрязняющих веществ в воде. Скрининговый подход предполагает выявление значимого превышения нормативного (фонового) диапазона значений определяемого параметра (в данном случае – флуоресцентного отклика нефти и нефтепродуктов) объекта окружающей среды. Предложенная методика автоматизированного флуоресцентного контроля позволит выявлять загрязнение поверхностных водных объектов нефтью и нефтепродуктами на раннем этапе (в ходе прорывов нефтепроводов и небольших утечек), предотвращая развитие аварийных ситуаций и причинение значительного ущерба окружающей среде.The paper proposes a method of automated fluorescent control of pollution of water bodies with oil products, based on a screening approach to measuring the content of pollutants in water. The screening approach involves identifying a significant excess of the normative (background) range of values of the parameter being defined (in this case, the fluorescent response of oil and oil products) of the environment object. The proposed method of automated fluorescence control will allow detecting pollution of surface water bodies with oil and oil products at an early stage (during breakthroughs of oil pipelines and small leaks), preventing the development of emergency situations and causing significant environmental damage

One Gene, Many Facets: Multiple Immune Pathway Dysregulation in SOCS1 Haploinsufficiency.

BACKGROUND: Inborn errors of immunity (IEI) present with a large phenotypic spectrum of disease, which can pose diagnostic and therapeutic challenges. Suppressor of cytokine signaling 1 (SOCS1) is a key negative regulator of cytokine signaling, and has recently been associated with a novel IEI. Of patients described to date, it is apparent that SOCS1 haploinsufficiency has a pleiotropic effect in humans. OBJECTIVE: We sought to investigate whether dysregulation of immune pathways, in addition to STAT1, play a role in the broad clinical manifestations of SOCS1 haploinsufficiency. METHODS: We assessed impacts of reduced SOCS1 expression across multiple immune cell pathways utilizing patient cells and CRISPR/Cas9 edited primary human T cells. RESULTS: SOCS1 haploinsufficiency phenotypes straddled across the International Union of Immunological Societies classifications of IEI. We found that reduced SOCS1 expression led to dysregulation of multiple intracellular pathways in immune cells. STAT1 phosphorylation is enhanced, comparably with STAT1 gain-of-function mutations, and STAT3 phosphorylation is similarly reduced with concurrent reduction of Th17 cells. Furthermore, reduced SOCS1 E3 ligase function was associated with increased FAK1 in immune cells, and increased AKT and p70 ribosomal protein S6 kinase phosphorylation. We also found Toll-like receptor responses are increased in SOCS1 haploinsufficiency patients. CONCLUSIONS: SOCS1 haploinsufficiency is a pleiotropic monogenic IEI. Dysregulation of multiple immune cell pathways may explain the variable clinical phenotype associated with this new condition. Knowledge of these additional dysregulated immune pathways is important when considering the optimum management for SOCS1 haploinsufficient patients

Impact of osteosynthesis in fracture care : a cost comparison study

Aim: To estimate the health economic impact of osteosynthesis (OS) in fracture care over six decades in 17 high-income countries. Patients & methods: Applying a decision tree model, we assumed a hypothetical absence of OS and compared OS (intervention) with conservative treatment (CONS; comparator). We included patients with femur, tibia and radius fractures (age <65 years) and for proximal femur fractures also elderly patients (≥70 years). Results: We estimated savings in direct and indirect costs of 855 billion Swiss francs in the working age population in addition to 4.6 million years of life gained. In the elderly population, 69 billion Swiss francs were saved in direct costs of proximal femur fractures in addition to 73 million years of life gained. Conclusion: OS contributed to maximize health gains of society

The term basal plate of the human placenta as a source of functional extravillous trophoblast cells

Background\ud Extravillous trophoblast (EVT) cells are of pivotal importance in human embryo implantation and homeostasis of the maternal fetal interface. Invasion of the endometrium by EVT contributes to placental anchorage, spiral artery remodeling, immunological defense, tolerogenic responses, and several collaborative cross talks involved in establishing and maintaining a successful pregnancy. We report here an improved protocol for the isolation of fully differentiated EVT cells from the basal plate of the human term placenta.\ud \ud \ud Methods\ud The basal plate was carefully dissected from the villous tissue and the amniochorion membrane prior to enzymatic digestion. Term basal EVT cells were isolated using a 30 and 60% Percoll gradient. A panel of markers and characteristics of the isolated cells were used to confirm the specificity and efficiency of the method so that their potential as an investigative tool for placental research could be ascertained.\ud \ud \ud Results\ud Isolated cells were immunoreactive for cytokeratin-7 (CK-7), placental growth factor, placental alkaline phosphatase, human leukocyte antigen G1 (HLA-G1), and α1 and α5 integrins, similarly to the EVT markers from first trimester placental villi. Around 95% of the isolated cells labeled positively for CK-7 and 82% for HLA-G1. No significant change in viability was observed during 48 h of EVT culture as indicated by propidium iodide incorporation and trypan blue test exclusion. Genes for metalloproteinases MMP-2 and MMP9 (positive regulators of trophoblast invasiveness) were expressed up to 48 h of culturing, as also the gelatinolytic activity of the isolated cells. Transforming growth factor (TGF)-beta, which inhibits proliferation, migration, and invasiveness of first-trimester EVT cells, also reduced invasion of isolated term EVT cells in transwell assays, whereas epidermal growth factor was a positive modulator.\ud \ud \ud Conclusions\ud Term basal plate may be a viable source of functional EVT cells that is an alternative to villous explant-derived EVT cells and cell lines. Isolated term EVT cells may be particularly useful in investigation of the role of trophoblast cells in pathological gestations, in which the precise regulation and interactive ability of extravillous trophoblast has been impaired.Fundação de Amparo à Pesquisa do Estado de São Paulo [2009/05354-0; 2013/12243-5]Conselho Nacional de Desenvolvimento Científico e Tecnológico [40088/2010-5]Coordenação de Aperfeiçoamento de Pessoal de Nível Superior [4178-11-4]Austrian Science Funds [P-22687-B13

Regulated Expression of ADAMTS-12 in Human Trophoblastic Cells: A Role for ADAMTS-12 in Epithelial Cell Invasion?

Metastatic carcinoma cells exploit the same molecular machinery that allows human placental cytotrophoblasts to develop an invasive phenotype. As altered expression levels of ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin repeats) subtypes have been associated with cancer progression, we have examined the function and regulation of members of this gene family in epithelial cell invasion using cultures of highly invasive extravillous cytotrophoblasts and the poorly invasive JEG-3 cytotrophoblast cell line as model systems. Of the multiple ADAMTS subtypes identified in first trimester human placenta and these two trophoblastic cell types, only ADAMTS-12 was preferentially expressed by extravillous cytotrophoblasts. Transforming growth factor-β1 and interleukin-1β, two cytokines that promote and restrain cytotrophoblast invasion in vitro, were also found to differentially regulate trophoblastic ADAMTS-12 mRNA levels. Loss- or gain-of-function studies confirmed that ADAMTS-12, independent of its proteolytic activity, plays a specific, non-redundant role in trophoblast invasion. Furthermore, we demonstrated that ADAMTS-12 regulated cell-extracellular matrix adhesion and invasion through a mechanism involving the αvβ3 integrin heterodimer. This study identifies a novel biological role for ADAMTS-12, and highlights the importance and complexity of its non-proteolytic domain(s) pertaining to its function

Senescent Syncytiotrophoblast Secretion During Early Onset Preeclampsia.

BACKGROUND: Preeclampsia is a severe hypertensive disorder in pregnancy that causes preterm delivery, maternal and fetal morbidity, mortality, and life-long sequelae. Understanding the pathogenesis of preeclampsia is a critical first step toward protecting mother and child from this syndrome and increased risk of cardiovascular disease later in life. However, effective early predictive tests and therapies for preeclampsia are scarce. METHODS: To identify novel markers and signaling pathways for early onset preeclampsia, we profiled human maternal-fetal interface units (fetal villi and maternal decidua) from early onset preeclampsia and healthy controls using single-nucleus RNA sequencing combined with spatial transcriptomics. The placental syncytiotrophoblast is in direct contact with maternal blood and forms the barrier between fetal and maternal circulation. RESULTS: We identified different transcriptomic states of the endocrine syncytiotrophoblast nuclei with patterns of dysregulation associated with a senescence-associated secretory phenotype and a spatial dysregulation of senescence in the placental trophoblast layer. Elevated senescence markers were validated in placental tissues of clinical multicenter cohorts. Importantly, several secreted senescence-associated secretory phenotype factors were elevated in maternal blood already in the first trimester. We verified the secreted senescence markers, PAI-1 (plasminogen activator inhibitor 1) and activin A, as identified in our single-nucleus RNA sequencing model as predictive markers before clinical preeclampsia diagnosis. CONCLUSIONS: This indicates that increased syncytiotrophoblast senescence appears weeks before clinical manifestation of early onset preeclampsia, suggesting that the dysregulated preeclamptic placenta starts with higher cell maturation resulting in premature and increased senescence-associated secretory phenotype release. These senescence-associated secretory phenotype markers may serve as an additional early diagnostic tool for this syndrome