Glucose, Insulin and Oxygen Modulate Expression of Serotonin-Regulating Genes in Human First-Trimester Trophoblast Cell Line ACH-3P.

Serotonin signaling plays an important role in regulating development and functions of the placenta. We hypothesized that metabolic disturbances associated with maternal obesity and/or gestational diabetes mellitus (GDM) affect placental serotonin homeostasis. Therefore, we examined the effects of high glucose (25 mM) and insulin (10 nM)-two hallmarks of maternal obesity and GDM-on mRNA expression of key regulators of serotonin homeostasis, including serotonin transporter (SERT), tryptophan hydroxylase 1 (TPH1), and monoamine oxidase A (MAOA), in the first-trimester trophoblast cell line ACH-3P, focusing on oxygen levels characteristic of early human placental development. Glucose downregulated expression of SERT and MAOA independently of oxygen level and upregulated expression of TPH1 at 6.5% oxygen but not at 2.5% oxygen. Compared to 6.5% oxygen, 2.5% oxygen upregulated SERT and downregulated TPH1 expression, with no effect on MAOA expression. Insulin upregulated SERT only at 2.5% oxygen but had no effect on TPH1 and MAOA expression. These results suggest that maternal metabolic alterations in early pregnancy may be a driving force for changes in placental serotonin homeostasis.

Distinct maternal metabolites are associated with obesity and glucose-insulin axis in the first trimester of pregnancy.

BACKGROUND/OBJECTIVES: Obesity in pregnancy associates with changes in the glucose-insulin axis. We hypothesized that these changes affect the maternal metabolome already in the first trimester of human pregnancy and, thus, aimed to identify these metabolites. PATIENTS/METHODS: We performed untargeted metabolomics (HPLC-MS/MS) on maternal serum (n = 181, gestational weeks 4+0-11+6). For further analysis, we included only non-smoking women as assessed by serum cotinine levels (ELISA) (n = 111). In addition to body mass index (BMI) and leptin as measures of obesity and adiposity, we metabolically phenotyped women by their fasting glucose, C-peptide and insulin sensitivity (ISHOMA index). To identify metabolites (outcome) associated with BMI, leptin, glucose, C-peptide and/or ISHOMA (exposures), we used a combination of univariable and multivariable regression analyses with multiple confounders and machine learning methods (Partial Least Squares Discriminant Analysis, Random Forest and Support Vector Machine). Additional statistical tests confirmed robustness of results. Furthermore, we performed network analyses (MoDentify package) to identify sets of correlating metabolites that are coordinately regulated by the exposures. RESULTS: We detected 2449 serum features of which 277 were annotated. After stringent analysis, 15 metabolites associated with at least one exposure (BMI, leptin, glucose, C-peptide, ISHOMA). Among these, palmitoleoyl ethanolamine (POEA), an endocannabinoid-like lipid endogenously synthesized from palmitoleic acid, and N-acetyl-L-alanine were consistently associated with C-peptide in all the analyses (95% CI: 0.10-0.34; effect size: 21%; p < 0.001; 95% CI: 0.04-0.10; effect size: 7%; p < 0.001). In network analysis, most features correlating with palmitoleoyl ethanolamide and N-acetyl-L-alanine and associated with C-peptide, were amino acids or dipeptides (n = 9, 35%), followed by lipids (n = 7, 27%). CONCLUSIONS: We conclude that the metabolome of pregnant women with overweight/obesity is already altered early in pregnancy because of associated changes of C-peptide. Changes of palmitoleoyl ethanolamide concentration in pregnant women with obesity-associated hyperinsulinemia may reflect dysfunctional endocannabinoid-like signalling.

Maternal Smoking in the First Trimester and its Consequence on the Early Placenta.

Smoking during pregnancy increases the risk of adverse pregnancy outcomes, such as stillbirth and fetal growth restriction. This suggests impaired placental function and restricted nutrient and oxygen supply. Studies investigating placental tissue at the end of pregnancy have revealed increased DNA damage as a potential underlying cause, which is driven by various toxic smoke ingredients and oxidative stress induced by reactive oxygen species (ROS). However, in the first trimester, the placenta develops and differentiates, and many pregnancy pathologies associated with reduced placental function originate here. Therefore, we determined DNA damage in a cohort of first-trimester placental samples of verified smokers and nonsmokers. In fact, we observed an 80% increase in DNA breaks (P < .001) and shortened telomeres by 5.8% (P = .04) in placentas exposed to maternal smoking. Surprisingly, there was a decrease in ROS-mediated DNA damage, ie, 8-oxo-guanidine modifications, in placentas of the smoking group (-41%; P = .021), which paralleled the reduced expression of base excision DNA repair machinery, which restores oxidative DNA damage. Moreover, we observed that the increase in placental oxidant defense machinery expression, which usually occurs at the end of the first trimester in a healthy pregnancy as a result of the full onset of uteroplacental blood flow, was absent in the smoking group. Therefore, in early pregnancy, maternal smoking causes placental DNA damage, contributing to placental malfunction and increased risk of stillbirth and fetal growth restriction in pregnant women. Additionally, reduced ROS-mediated DNA damage along with no increase in antioxidant enzymes suggests a delay in the establishment of physiological uteroplacental blood flow at the end of the first trimester, which may further add to a disturbed placental development and function as a result of smoking in pregnancy.

Cord Blood Advanced Lipoprotein Testing Reveals an Interaction between Gestational Diabetes and Birth-Weight and Suggests a New Early Biomarker of Infant Obesity.

Abnormal lipid metabolism is associated with gestational diabetes mellitus (GDM) and is observed in neonates with abnormal fetal growth. However, the underlying specific changes in the lipoprotein profile remain poorly understood. Thus, in the present study we used a novel nuclear magnetic resonance (NMR)-based approach to profile the umbilical cord serum lipoproteins. Two-dimensional diffusion-ordered 1H-NMR spectroscopy showed that size, lipid content, number and concentration of particles within their subclasses were similar between offspring born to control (n = 74) and GDM (n = 62) mothers. Subsequent data stratification according to newborn birth-weight categories, i.e., small (n = 39), appropriate (n = 50) or large (n = 49) for gestational age (SGA, AGA and LGA, respectively), showed an interaction between GDM and birth-weight categories for intermediate-density lipoproteins (IDL)-cholesterol content and IDL- and low-density lipoproteins (LDL)-triglyceride content, and the number of medium very low-density lipoproteins (VLDL) and LDL particles specifically in AGA neonates. Moreover, in a 2-year follow-up study, we observed that small LDL particles were independently associated with offspring obesity at 2 years (n = 103). Collectively, our data demonstrate that GDM disturbs triglyceride and cholesterol lipoprotein content across birth-weight categories, with AGA neonates born to GDM mothers displaying a profile more similar to that of adults with dyslipidemia. Furthermore, an altered fetal lipoprotein pattern was associated with the development of obesity at 2 years.

Type 1 Diabetes Mellitus and the First Trimester Placenta: Hyperglycemia-Induced Effects on Trophoblast Proliferation, Cell Cycle Regulators, and Invasion.

Type 1 diabetes mellitus (T1DM) is associated with reduced fetal growth in early pregnancy, but a contributing role of the placenta has remained elusive. Thus, we investigated whether T1DM alters placental development in the first trimester. Using a protein array, the level of 60 cell-cycle-related proteins was determined in human first trimester placental tissue (gestational week 5-11) from control (n = 11) and T1DM pregnancies (n = 12). Primary trophoblasts (gestational week 7-12, n = 32) were incubated in the absence (control) or presence of hyperglycemia (25 mM D-glucose) and hyperosmolarity (5.5 mM D-glucose + 19.5 mM D-mannitol). We quantified the number of viable and dead trophoblasts (CASY Counter) and assessed cell cycle distribution (FACS) and trophoblast invasion using a transwell assay. T1DM was associated with a significant (p < 0.05) downregulation of Ki67 (-26%), chk1 (-25%), and p73 (-26%). The number of viable trophoblasts was reduced under hyperglycemia (-23%) and hyperosmolarity (-18%), whereas trophoblast invasion was increased only under hyperglycemia (+6%). Trophoblast cell death and cell cycle distribution remained unaffected. Collectively, our data demonstrate that hyperglycemia decreases trophoblast proliferation as a potential contributing factor to the reduced placental growth in T1DM in vivo.

Maternal C-Peptide and Insulin Sensitivity, but Not BMI, Associate with Fatty Acids in the First Trimester of Pregnancy.

Maternal obesity in pregnancy is a pro-inflammatory condition exposing the fetus to an adverse environment. Here, we tested associations of maternal obesity (primary exposures: BMI, leptin) and metabolic parameters (secondary exposures: glucose, C-peptide, and insulin sensitivity) with total serum concentrations of fatty acids in the first trimester of human pregnancy. This cross-sectional study included 123 non-smoking women with singleton pregnancy. In maternal serum, cotinine, leptin, and C-peptide (ELISA), glucose (hexokinase-based test) and fatty acids (gas chromatography) were quantified, and the insulin sensitivity index (ISHOMA) was calculated. Concentrations of fatty acid classes and total fatty acids did not differ between BMI or leptin categories. However, n-3 polyunsaturated fatty acids (PUFA) were decreased in the category with the highest C-peptide concentration (n-3 PUFA: CI -35.82--6.28, p < 0.006) and in the lowest ISHOMA category (n-3 PUFA: CI -36.48--5.61, p < 0.008). In a subcohort, in which fetal sex was determined (RT-qPCR of placental tissue), C-peptide was significantly associated with docosahexaenoic acid (DHA) in mothers bearing a female (n = 46), but not male (n = 37) fetus. In conclusion, pregnant women with high fasting C-peptide and low ISHOMA had decreased n-3 PUFA, and DHA was lower with higher C-peptide only in mothers bearing a female fetus.

Design and Synthesis of Water-Soluble and Potent MMP-13 Inhibitors with Activity in Human Osteosarcoma Cells.

Osteoarthritis is a degenerative disease, often resulting in chronic joint pain and commonly affecting elderly people. Current treatments with anti-inflammatory drugs are palliative, making the discovery of new treatments necessary. The inhibition of matrix metalloproteinase MMP-13 is a validated strategy to prevent the progression of this common joint disorder. We recently described polybrominated benzotriazole derivatives with nanomolar inhibitory activity and a promising selectivity profile against this collagenase. In this work, we have extended the study in order to explore the influence of bromine atoms and the nature of the S1' heterocyclic interacting moiety on the solubility/selectivity balance of this type of compound. Drug target interactions have been assessed through a combination of molecular modeling studies and NMR experiments. Compound 9a has been identified as a water-soluble and highly potent inhibitor with activity in MG-63 human osteosarcoma cells.

Different regulation of IRE1α and eIF2α pathways by oxygen and insulin in ACH-3P trophoblast model.

Endoplasmic reticulum (ER)-stress activates the unfolded protein response (UPR), which plays a (patho)physiological role in the placenta. Oxygen and hyperinsulinemia are major regulators of placental development. Thus, we hypothesized that oxygen, insulin and their interplay modulate ER-stress in early pregnancy. Using the human first-trimester trophoblast cell line ACH-3P, we quantified mRNA and protein of several members of UPR by RT-qPCR and Western blotting, respectively. ER-stress induction using tunicamycin and brefeldin A resulted in increased CHOP (4.6-fold change; P ≤ 0.001), XBP1 expression (1.7- and 1.3-fold change, respectively; P ≤ 0.001 and P < 0.05) and XBP1 splicing (7.9- and 12.8-fold change, respectively; P ≤ 0.001). We subsequently analyzed the effect of oxygen (6.5%, 2.5%), insulin (0.1-10 nM) and their interaction using ANCOVA adjusted for cell passage as co-variate. Although GRP78 protein remained unaffected, low oxygen (2.5% O2) increased IRE1α phosphorylation (+52%; P < 0.05) and XBP1 splicing (1.8-fold change; P ≤ 0.001) after 24 h, while eIF2α protein and CHOP expression were downregulated (-28%; P < 0.05 and -24%; P ≤ 0.001; respectively). eIF2α phosphorylation was also reduced after 48 h by low oxygen (-61%; P < 0.05) but increased in the presence of insulin (+46%; P ≤ 0.01). These changes were not PERK-mediated, since PERK phosphorylation and total protein were not altered. Overall, our results suggest that IRE1α and eIF2α UPR-pathways are differentially regulated by oxygen and insulin in early pregnancy.

Sex matters: XIST and DDX3Y gene expression as a tool to determine fetal sex in human first trimester placenta.

Fetal sex influences placental function as well as maternal and fetal health, being an important factor to consider in pregnancy studies. However, fetal sex determination in the first trimester of pregnancy still faces some technical limitations. Here we describe an RT-qPCR technique to determine fetal sex based on X-inactive specific transcript (XIST) and DEAD-Box helicase 3 Y-linked (DDX3Y) gene expression. This method is straightforward, reliable, fast and applicable on both, placental tissue and primary cells.

Matrix metalloproteinase 15 plays a pivotal role in human first trimester cytotrophoblast invasion and is not altered by maternal obesity.

Adequate anchoring of the placenta in the uterus through invasion of first trimester cytotrophoblasts (CTB) is required for a successful pregnancy. This process is mediated by matrix metalloproteinases (MMPs) and regulated by the maternal environment. Obesity is known to alter the intrauterine milieu and has been related to impaired invasion. We hypothesized that placental MMP15, a novel membrane-type MMP, is involved in CTB invasion and regulated by maternal obesity in early pregnancy. Thus, in this study MMP15 was immunolocalized to invasive extravillous and interstitial CTB. MMP15 silencing in chorionic villous explants using two different siRNAs reduced trophoblast outgrowth length (-35%, P ≤ .001 and -26%, P < .05) and area (-43%, P ≤ .001 and -36%, P ≤ .01) without altering trophoblast proliferation or apoptosis. Short-term treatment of primary first trimester trophoblasts with IL-6 (10 ng/mL), interleukin 10 (IL-10) (50 ng/mL), and tumor necrosis factor α (TNF-α) (10 ng/mL) did not affect MMP15 protein levels. Likewise, MMP15 mRNA and protein levels were unaltered between human first trimester placentas from control pregnancies vs those complicated with maternal obesity. Overall, our results suggest that the role of MMP15 in placental development and function in early pregnancy is limited to CTB invasion without being affected by short- and long-term inflammation.

Maternal Obesity Alters Placental Cell Cycle Regulators in the First Trimester of Human Pregnancy: New Insights for BRCA1.

In the first trimester of pregnancy, placental development involves a wide range of cellular processes. These include trophoblast proliferation, fusion, and differentiation, which are dependent on tight cell cycle control. The intrauterine environment affects placental development, which also includes the trophoblast cell cycle. In this work, we focus on maternal obesity to assess whether an altered intrauterine milieu modulates expression and protein levels of placental cell cycle regulators in early human pregnancy. For this purpose, we use first trimester placental tissue from lean and obese women (gestational week 5+0-11+6, n = 58). Using a PCR panel, a cell cycle protein array, and STRING database analysis, we identify a network of cell cycle regulators increased by maternal obesity in which breast cancer 1 (BRCA1) is a central player. Immunostaining localizes BRCA1 predominantly to the villous and the extravillous cytotrophoblast. Obesity-driven BRCA1 upregulation is not able to be explained by DNA methylation (EPIC array) or by short-term treatment of chorionic villous explants at 2.5% oxygen with tumor necrosis factor α (TNF-α) (50 mg/mL), leptin (100 mg/mL), interleukin 6 (IL-6) (100 mg/mL), or high glucose (25 nM). Oxygen tension rises during the first trimester, but this change in vitro has no effect on BRCA1 (2.5% and 6.5% O2). We conclude that maternal obesity affects placental cell cycle regulation and speculate this may alter placental development.

Expression of matrix metalloproteinase 12 is highly specific for non-proliferating invasive trophoblasts in the first trimester and temporally regulated by oxygen-dependent mechanisms including HIF-1A.

During first trimester pregnancy, trophoblast cells invade from the placenta into the maternal decidua where they anchor the placenta and remodel luminal structures like spiral arteries. This process depends on proteases secreted by invading trophoblasts, which degrade extracellular matrix (ECM). We here aimed to identify proteases particularly important for trophoblast invasion. We generated a list of proteases capable of degrading decidual ECM and trophoblast integrins using MEROPS database and compared expression of these proteases between primary trophoblasts isolated from first trimester placenta (FT, n = 3), representing an invasive phenotype, vs trophoblasts isolated from term pregnancy (TT, n = 3), representing a non-invasive trophoblast phenotype. Matrix metalloproteinase 12 (MMP12) revealed highest expression levels in FT, with absent expression in TT. In situ hybridisation and immunofluorescence localised MMP12 specifically to extravillous trophoblasts (evCT) whilst Ki67 co-staining revealed that proliferating trophoblasts of the cell columns were almost negative for MMP12. Quantification revealed a decline in MMP12 positive evCT at the end of first trimester, when oxygen levels start rising. MMP12 promoter analysis identified potential binding sites for hypoxia-inducible factor (HIF-1) and other oxygen-sensitive transcription factors. Moreover, MMP12 protein was increased by low oxygen in FT in vitro and by addition of a HIF-1α activator. Collectively, MMP12 is a highly expressed protease specific for invasive evCT during the first trimester. MMP12 down regulation by increasing oxygen concentration enables temporal expression control of MMP12 and involves several mechanisms including HIF-1α. These findings suggest MMP12 involved in trophoblast invasion during the first trimester.

Correction to: Expression of matrix metalloproteinase 12 is highly specific for non-proliferating invasive trophoblasts in the first trimester and temporally regulated by oxygen-dependent mechanisms including HIF-1A.

In the original publication, the contribution of Dr. Christian Eyth as equal first author was not indicated. This has been corrected confirming that U. Hidden and C. Eyth contributed equally to this work.

Maternal Type 1 diabetes activates stress response in early placenta.

INTRODUCTION: Human pregnancy and in particular the first trimester, is a period highly susceptible towards adverse insults such as oxidative stress, which may lead to inadequate embryonic and feto-placental development. Diabetes mellitus is associated with increased oxidative stress caused by hyperglycemia, reactive oxygen species (ROS) production and inflammatory signals. In pregnancy, diabetes elevates the risk for early pregnancy loss, preeclampsia and fetal growth restriction, pathologies that origin from early placental maldevelopment. We hypothesized that maternal Type 1 diabetes mellitus (T1DM) induces oxidative stress in the first trimester human placenta. METHODS: We quantified stress induced, cytoprotective proteins, i.e. heat shock protein (HSP)70 and heme oxygenase (HO)-1 and determined protein modifications as markers for oxidation and glycation, i.e. levels of 4-hydroxynonenal (HNE) or Nε-(carboxymethyl)lysine (CML) modified proteins. Moreover, we measured expression levels of enzymes involved in antioxidant defense in the first trimester (week 7-9) placenta of normal and T1DM women by immunoblot and real-time qPCR. Primary human trophoblasts were isolated from first trimester placenta and the effects of oxygen, hyperglycemia and the pro-inflammatory cytokine tumor necrosis factor (TNF)-α on levels of HSP70 and HO-1 were analyzed. RESULTS: HSP70 (+19.9± 10.1%) and HO-1 (+63.5± 14.5%) were elevated (p < 0.05) in first trimester placenta of T1DM women when compared to normal women. However, levels of HNE or CML modified proteins were unchanged. Also, expression of most antioxidant enzymes was unchanged, with only superoxide dismutase 3 (SOD3) being upregulated by 3.0-fold (p < 0.05). In isolated primary trophoblasts, HSP70 and HO-1 were upregulated by increasing oxygen tension, but not by hyperglycemia or TNF-α. CONCLUSION: Although protein oxidation and glycation was not elevated, we infer that T1DM increases placental cellular stress in the first trimester. Elevated stress in early placenta of T1DM women may contribute to disturbances in placental development.

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

STUDY QUESTION: Does endothelin-1 (ET-1) regulate matrix metalloproteinase (MMP) 14 and 15 production and invasion of human first trimester trophoblasts? 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. 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. STUDY DESIGN, SIZE, DURATION: In vitro study using primary human trophoblasts from 50 first trimester placentas (gestational week 7-12). 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. MAIN RESULTS AND THE ROLE OF CHANCE: ET-1 down-regulated MMP14 and MMP15 mRNA (-21% and -26%, respectively, P < 0.05) and protein levels (-18% and -22%, respectively, P < 0.05). This effect was mediated via ETRB. ET-1 decreased trophoblast outgrowth in placental explants (-24%, P < 0.05) and trophoblast invasion (-26%, P ≤ 0.01). TNF-α enhanced ET-1 mediated MMP15 down-regulation (by 10%, P < 0.05), whereas hypoxia abolished the effect of ET-1 on both MMPs. LARGE SCALE DATA: N/A. 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. 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. STUDY FUNDING/COMPETING INTERESTS: 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). The authors have no conflict of interest.

Pigment epithelium-derived factor (PEDF): a novel trophoblast-derived factor limiting feto-placental angiogenesis in late pregnancy.

The rapidly expanding feto-placental vasculature needs tight control by paracrine and endocrine mechanisms. Here, we focused on paracrine influence by trophoblast, the placental epithelium. We aimed to identify differences in regulation of feto-placental angiogenesis in early versus late pregnancy. To this end, the effect of conditioned media (CM) from early and late pregnancy human trophoblast was tested on network formation, migration and proliferation of human feto-placental endothelial cells. Only CM of late pregnancy trophoblast reduced network formation and migration. Screening of trophoblast transcriptome for anti-angiogenic candidates identified pigment epithelium-derived factor (PEDF) with higher expression and protein secretion in late pregnancy trophoblast. Addition of a PEDF-neutralizing antibody restored the anti-angiogenic effect of CM from late pregnancy trophoblast. Notably, human recombinant PEDF reduced network formation only in combination with VEGF. Also in the CAM assay, the combination of PEDF with VEGF reduced branching of vessels below control levels. Analysis of phosphorylation of ERK1/2 and FAK, two key players in VEGF-induced proliferation and migration, revealed that PEDF altered VEGF signaling, while PEDF alone did not affect phosphorylation of ERK1/2 and FAK. These data suggest that the trophoblast-derived anti-angiogenic molecule PEDF is involved in restricting growth and expansion of the feto-placental endothelium predominantly in late pregnancy and targets to modulate the intracellular effect of VEGF.

Placental membrane-type metalloproteinases (MT-MMPs): Key players in pregnancy.

Membrane-type matrix metalloproteinases (MT-MMPs) are a sub-family of zinc-dependent endopeptidases involved in the degradation of the extracellular matrix. Although MT-MMPs have been mainly characterized in tumor biology, they also play a relevant role during pregnancy. Placental MT-MMPs are required for cytotrophoblast migration and invasion of the uterine wall and in the remodeling of the spiral arteries. They are involved in the fusion of cytotrophoblasts to form the syncytiotrophoblast as well as in angiogenesis. All these processes are crucial for establishing and maintaining a successful pregnancy and, thus, MT-MMP activity has to be tightly regulated in time and space. Indeed, a de-regulation of MT-MMP expression has been linked with pregnancy complications such as preeclampsia (PE), fetal growth restriction (FGR), gestational diabetes mellitus (GDM) and was also found in maternal obesity. Here we review what is currently known about MT-MMPs in the placenta, with a focus on their general features, their localization and their involvement in pregnancy disorders.

Different Preference of Degradome in Invasion versus Angiogenesis.

Proteases are required for a multitude of cellular processes including homeostatic tissue remodelling, invasion and angiogenesis. The physiological function of a cell or tissue is reflected by the set of proteases expressed, also termed degradome. The role of proteases in invasion and angiogenesis has been studied intensively, mostly in cancer. We aimed to compare the set of proteases required for physiological invasion versus physiological angiogenesis from cells deriving from the same organ, and to identify the proteases specific for each process. The human placenta comprises trophoblasts that invade the maternal uterus in a regulated, physiological manner, and it is the source of primary endothelial cells. We isolated the trophoblasts and endothelial cells and verified their invasive phenotype and angiogenic properties, respectively. We then performed gene expression analysis of the degradome, e.g. cysteine, metallo, serine, threonine and aspartic proteases, identified the differentially expressed proteases among the trophoblasts and endothelial cells, and clustered them hierarchically. The results revealed that the set of proteases in trophoblasts versus in endothelial cells overlaps, with a total of 69% in common. Nevertheless, 42% of the studied degradomes differed, with a fold change ≥2. For instance, metalloproteinases were predominantly expressed in trophoblasts, and 31% of the proteases were exclusively expressed in either trophoblasts or endothelial cells; this suggests particular roles for these proteases in either invasion or angiogenesis. Our data identify common and distinct proteases in cells capable of performing invasion and angiogenesis, and may provide basic information for the design of techniques to specifically investigate invasion or angiogenesis.