Fetal Endothelial Dysfunction Research Articles

Abstract 4145829: Fatty Acid Binding Protein 4 as Potential Indicator for Gestational Diabetes Mellitus-Induced Fetal Endothelial Dysfunction

Introduction/Background: Gestational diabetes mellitus (GDM) is the most common complication during pregnancy. GDM is leading to an elevated risk for the development of cardiovascular diseases both in the mother and the child in later life. Previous studies have identified fatty acid-binding protein 4 (FABP4) as a prime candidate involved in the pathophysiology of GDM. Indeed, women with GDM exhibit elevated blood levels of FABP4, suggesting its potential role as a biomarker for endothelial dysfunction and cardiovascular risk assessment in GDM. Research Question/Hypothesis: Does FABP4 affect the function of human endothelial cells from patients with GDM? Goals/Aim: Our aim is a better understanding of the role of FABP4 in fetal endothelial dysfunction of patients with GDM and the development of potential therapeutic strategies. Methods/Approach: Primary human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords obtained from normoglycemic and GDM pregnancies. Total mRNA from HUVECs was used for bulk RNA sequencing to compare gene expression pattern. HUVECs were subjected to functional analyses assessing proliferation, migration, NO and insulin signaling. Finally, human endothelial cells were exposed to high laminar flow (30 dyn/cm 2 ) or simvastatin (10 nM) treatment to investigate their impact on FABP4 expression and endothelial function. Results/Data: Bulk RNA sequencing data analysis revealed FABP4 as a gene overexpressed in GDM HUVECs compared to normoglycemic controls. Gene set enrichment analysis showed in addition an enrichment of genes involved in angiogenesis and Notch signaling pathways in human endothelial cells from GDM patients. GDM HUVECs had a significantly higher wound healing capacity compared to normoglycemic controls. The PI3K/AKT/mTOR pathway was enriched in GDM HUVECs. GDM HUVECs displayed impaired activation of AKT and eNOS (expression and phosphorylation) after stimulation with insulin, suggesting a possible insulin resistance. Finally, subjecting HUVECs to high laminar shear stress or simvastatin treatment caused a reduction of FABP4 mRNA expression and an increase of eNOS expression, indicating potential therapeutic strategies to improve endothelial function. Conclusions: We provided evidence that FABP4 could be involved in fetal GDM-induced endothelial dysfunction. High laminar shear stress and medications activating eNOS signaling could protect the endothelium against the negative impact of FABP4 in GDM.

FABP4 as potential indicator for GDM-induced fetal endothelial dysfunction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG project no. 288034826—International Research Training Group (IRTG) 2251 Background Gestational diabetes mellitus (GDM) is linked to an elevated risk of developing cardiovascular diseases (CVD) both for the mother and the baby in later life. Previous studies have identified fatty acid-binding protein 4 (FABP4) as a prime candidate involved in the pathophysiology of GDM. FABP4 is known to promote an inflammatory response and vascular permeability, induce oxidative stress in endothelial cells (ECs), and impair the nitric oxide signaling pathway, thereby promoting endothelial dysfunction. Indeed, women with GDM exhibit elevated blood levels of FABP4, suggesting its potential as a biomarker for endothelial dysfunction and cardiovascular risk assessment in GDM. However, the role that FABP4 plays in the fetal endothelial function remains poorly understood. Purpose Thus, our aim is to understand the role of FABP4 in fetal endothelial dysfunction and its regulation to mitigate the impact of GDM on the vasculature. Methods Fetoplacental vessels and primary human umbilical vein endothelial cells (HUVECs) were isolated from placentas and umbilical cords obtained from both normoglycemic (NG) and GDM pregnancies. We also obtained the clinical data from the mothers and collected the cord blood. Total mRNA from HUVECs was used for bulk RNA sequencing to compare gene expression, while gene expression analysis in the vessels was conducted using RT-qPCR. Additionally, NG HUVECs were pooled and cultured under high laminar flow conditions (30 dyn/cm²) or subjected to various treatments, including insulin (5nM), glucose (25mM), simvastatin (10nm), or VEGF-A stimulation. Subsequent RT-qPCRs were performed to analyze the gene expression of several genes of interest. Results The bulk RNA sequencing data analysis revealed FABP4 as an overexpressed gene in GDM HUVECs compared to NG. Furthermore, the analysis of data through Gene Set Enrichment Analysis provided a list of different gene sets that are enriched in GDM samples. Among these gene sets are pathways or processes implicated in endothelial dysfunction, such as angiogenesis, PI3K/AKT/mTOR signaling, reactive oxygen species pathway, or inflammatory response. In vitro experiments provided evidence for an elevated FABP4 mRNA expression after VEGF treatment of HUVECs. Conversely, both simvastatin treatment and exposure to laminar flow reduced FABP4 mRNA expression and increased eNOS expression, suggesting a potential inhibitory effect of nitric oxide on FABP4. Conclusion We provided evidence that FABP4 could be involved in fetal GDM-induced endothelial dysfunction. Shear stress and statins might protect the endothelium against the negative impact of FABP4.

Abstract 12569: The Roles of microRNA in Preeclampsia Induced Fetal Sex-Specific Endothelial Dysfunction

Introduction: Preeclampsia (PE) is a hypertensive disorder that complicates 5-10% of human pregnancies and leads to high fetal morbidity and mortality. Maternal obesity increases the risks of PE by 3-fold in association with elevated circulating pro-inflammatory cytokines which are related to endothelial dysfunction observed in PE. MiR146a-5p is a cardiovascular disease-associated miRNA that regulates endothelial cell responses. We have previously reported fetal sex-specific fetal endothelial dysfunction in PE. Although miR146a-5p has been reported to be dysregulated in PE HUVECs, the role of miR146a-5p in PE-dysregulated fetal sex-specific endothelial dysfunction is unclear. Hypothesis: MiR146a-5p differently mediate PE-induced sex-specific fetal endothelial dysfunction in lean and obese pregnancies. Methods: Human umbilical vein endothelial cells (unpassaged, P0-HUVECs; 5-7 days of culture, P1-HUVECs) were isolated immediately after delivery from normotensive (NT; 39±0.5 wks) and PE (38±0.5 wks) pregnancies (lean and OB) with female (F) and male (M) fetuses. Expression of miR146a-5p was examined in P0-HUVECs using RT-qPCR (n=8-10/group). Bioinformatics analysis was performed with our existing RNAseq data to identify PE-dysregulated miR146a-5p target genes in HUVECs. Endothelial monolayer integrity (MI) responses to cytokines were examined in P1 HUVECs with and without miR146a-5p overexpression and knockdown (n=5-7/group/assay). Results: PE upregulated miR146a-5p in lean F-HUVECs, while downregulated miR146a-5p in obese M-HUVECs. PE differentially dysregulated vascular/endothelial function-associated miR146a-5p target genes (e.g., TNFα-/TGFβ-signaling pathways) in F&M HUVECs. PE further reduced the TNFα-weakened MI in F and M HUVECs in both lean and OB pregnancies. TGFβ1 strengthened MI only in F-HUVECs, which was abolished in lean PE-F-HUVECs and was further decreased in OB PE-F-HUVEC. Knockdown of miR146a-5p rescues the PE-impaired MI responses to TNF-α and TGF-β1 in lean F-HUVECs but only rescues PE-impaired MI responses to TGF-β1 in OB F-HUVECs. Conclusions: MiR146a-5p plays important roles in mediating PE-induced fetal sex-specific fetal endothelial dysfunction in lean and obese pregnancies.

The Procoagulant Phospholipid Dependent Clotting Time and the Initiation Phase of Thrombin Generation Test Are Mandatory for the Evaluation of the Risk for Severe Early Onset Preeclampsia

Bakground: Preeclampsia occurs in up to 7% of pregnancies in Western Countries and is a leading cause of maternal and fetal morbidity and mortality. Classification of preeclampsia is based on the timing of symptoms' onset and the severity of the clinical status. Accordingly, early and late preeclampsia are characterized by symptom onset before or after 32-34 weeks of gestation respectively. Pathogenesis of preeclampsia is characterized by abnormal placental vascular development with impaired invasion of the uterine spiral arteries by trophoblasts, resulting in fetal complications and maternal endothelial dysfunction. Intact antithrombotic properties of the endothelial cells and efficient regulation of thrombin generation and platelet activation at the microenvironment of the uterus are necessary conditions for successful trophoblast invasion and development of feto-maternal circulation during normal pregnancy. , Prompt identification of pregnant women at risk of preeclampsia worsening could help to anticipated therapeutic intervention and improvement of the clinical outcome. Aim: The ROADMAP-Early Onset Preeclampsia study (ROADMAP-EOP) aiming to respond to this unmet need assessed the clinical relevance of biomarkers of hypercoagulability and endothelial activation for prompt identification of pregnant women at high risk of preeclampsia. Materials and Methods: An observational single center retrospective case-control study was conducted in pregnant women diagnosed with preeclampsia from July 2020 to July 2021. Pregnant women were retrospectively enrolled upon EOP diagnosis and stratified in mild EOP group (n=34) and severe EOP group (n=15 according to the criteria of the International Society for the Study of Hypertension in Pregnancy ). Eligible women were included in the study before initiation of any treatment for preeclampsia. The control group (n=35) consisted of women with uncomplicated pregnancy. All women were assessed with thromboelastometry, Calibrated Automated Thrombo-gram®, tissue factor activity (TFa), Procoagulant phospholipid dependent clotting time (Procoag-PPL), Proteins S, TFPI, D-dimer, antithrombin, thrombomodulin, fibrinogen prothrombin time and activated partial thromboplastin time. Primary and secondary end points were severe EOP and EOP respectively. Principal component analysis (PCA) was performed to check for associations among the biomarkers and the clinical out-come. Results: There were no significant differences regarding age, gravidity, gestational age and the body mass index (BMI) between EOP groups and normal pregnancy as well as within the EOP groups. Platelet counts were similar in healthy pregnant women, and mild EOP group, and significantly lower in severe EOP (p<0.05). No other statistically significant differences were found between-groups. The PCA showed that the repartition of data allowed to discriminate the patients with EOP from healthy pregnant women as well as patients with mild EPO from those with severe EOP. When biomarkers of hypercoagulability were considered according to the severity of EOP in PCA analysis the lag-time and PPL clotting time were independently associated with the risk of severe EOP (Table 1and Figure 1)). The increase of the lag-time by 1 min was associated with a 20.2-fold increase of the risk of severe EOP. The marginal effects curve shows that for a lag-time longer than 2.6 min, the probability of severe EOP was higher than 50% .Longer Procoag-PPL clotting time was associated with lower risk of severe EOP. The marginal effects curve, showed that for Procoag-PPL clotting time longer than 58.75 sec the probability of severe EOP decreased by 50%. These data suggest that a biological score composed of the Procoag-PPL® clotting time and thrombin generation (Throbmogram-Thrombinoscope® PPP-reagent 1 pM-TF as-say) could be a useful tool to identify pregnant women with high probability of EOP Conclusion: the Procoag-PPL® clotting time and thrombin generation assay reflect cellular derived hypercoagulability and allow accurate screening of pregnant women for the prompt identification of those at risk of EOP and the evaluation of its severity. This concept has to be validated in a large prospective cohort trial. . An external validation of the proposed methodology needs to be performed in a large prospective observational study.

MicroRNA-29 differentially mediates preeclampsia-dysregulated cellular responses to cytokines in female and male fetal endothelial cells.

Preeclampsia (PE) differentially impairs female and male fetal endothelial cell function, which is associated with an increased risk of adult-onset cardiovascular disorders in children born to mothers with PE. However, the underlying mechanisms are poorly defined. We hypothesize that dysregulation of microRNA-29a-3p and 29c-3p (miR-29a/c-3p) in PE disturbs gene expression and cellular responses to cytokines in fetal endothelial cells in a fetal sex-dependent manner. RT-qPCR analysis of miR-29a/c-3p was performed on female and male unpassaged (P0) human umbilical vein endothelial cells (HUVECs) from normotensive (NT) pregnancies and PE. Bioinformatic analysis of an RNA-seq dataset was performed to identify PE-dysregulated miR-29a/c-3p target genes in female and male P0-HUVECs. Gain- and loss-of-function assays were conducted to determine the effects of miR-29a/c-3p on endothelial monolayer integrity and proliferation in response to transforming growth factor-β1 (TGFβ1) and tumour necrosis factor-α (TNFα) in NT and PE HUVECs at passage 1. We observed that PE downregulated miR-29a/c-3p in male and female P0-HUVECs. PE dysregulated significantly more miR-29a/c-3p target genes in female vs. male P0-HUVECs. Many of these PE-differentially dysregulated miR-29a/c-3p target genes are associated with critical cardiovascular diseases and endothelial function. We further demonstrated that miR-29a/c-3p knockdown specifically recovered the PE-abolished TGFβ1-induced strengthening of endothelial monolayer integrity in female HUVECs, while miR-29a/c-3p overexpression specifically enhanced the TNFα-promoted cell proliferation in male PE HUVECs. In conclusion, PE downregulates miR-29a/c-3p expression and differentially dysregulates miR-29a/c-3p target genes associated with cardiovascular diseases and endothelial function in female and male fetal endothelial cells, possibly contributing to the fetal sex-specific endothelial dysfunction observed in PE. KEY POINTS: Preeclampsia differentially impairs female and male fetal endothelial cell function in responses to cytokines. Pro-inflammatory cytokines are elevated in maternal circulation during pregnancy in preeclampsia. MicroRNAs are critical regulators of endothelial cell function during pregnancy. We have previously reported that preeclampsia downregulated microRNA-29a-3p and 29c-3p (miR-29a/c-3p) in primary fetal endothelial cells. However, it is unknown if PE differentially dysregulates the expression of miR-29a/c-3p in female and male fetal endothelial cells. We show that preeclampsia downregulates miR-29a/c-3p in male and female HUVECs and preeclampsia dysregulates cardiovascular disease- and endothelial function-associated miR-29a/c-3p target genes in HUVECs in a fetal sex-specific manner. MiR-29a/c-3p differentially mediate cell responses to cytokines in female and male fetal endothelial cells from preeclampsia. We have revealed fetal sex-specific dysregulation of miR-29a/c-3p target genes in fetal endothelial cells from preeclampsia. This differential dysregulation may contribute to fetal sex-specific endothelial dysfunction in offspring born to preeclamptic mothers.

Small extracellular vesicles from pregnant women with maternal supraphysiological hypercholesterolemia impair endothelial cell function in vitro

Maternal physiological hypercholesterolemia MPH, maternal total cholesterol (TC) levels at term of pregnancy ≤280 mg/dL) occurs to assure fetal development. Maternal supraphysiological hypercholesterolemia (MSPH, TC levels >280 mg/dL) is a pathological condition associated with maternal, placental, and fetal endothelial dysfunction and early neonatal atherosclerosis development. Small extracellular vesicles (sEVs) are delivered to the extracellular space by different cells, where they modulate cell functions by transporting active signaling molecules, including proteins and miRNA. AimTo determine whether sEVs from MSPH women could alter the function of endothelial cells (angiogenesis, endothelial activation and nitric oxide synthesis capacity). MethodsThis study included 24 Chilean women (12 MPH and 12 MSPH). sEVs were isolated from maternal plasma and characterized by sEV markers (CD9, Alix and HSP70), nanoparticle tracking analysis, transmission electron microscopy, and protein and cholesterol content. The endothelial cell line HMEC-1 was used to determine the uptake of labeled sEVs and the effects of sEVs on cell viability, endothelial tube formation, endothelial cell activation, and endothelial nitric oxide expression and function. ResultsIn MSPH women, the plasma concentration of sEVs was increased compared to that in MPH women. MSPH-sEVs were highly taken up by HMEC-1 cells and reduced angiogenic capacity and the expression and activity of eNOS without changing cell viability or endothelial activation compared to MPH-sEVs. ConclusionsEVs from MSPH women impair angiogenesis and nitric oxide synthesis in endothelial cells, which could contribute to MSPH-associated endothelial dysfunction.

Feto-placental endothelial dysfunction in Gestational Diabetes Mellitus under dietary or insulin therapy

ObjectiveGestational diabetes mellitus (GDM) is a serious complication in pregnancy. Despite controlling the plasma glucose levels with dietary intervention (GDM-D) or insulin therapy (GDM-I), children born of diabetic mothers suffer more long-term complications from childhood to early adulthood. Placental circulation and nutrient exchange play a vital role in fetal development. Additionally, placental endothelial function is an indicator of vascular health, and plays an important role in maintaining placental circulation for nutrient exchange. This study was conducted to assess changes in fetal endothelial dysfunction in GDM under different interventions during pregnancy.MethodsThe primary human umbilical vein endothelial cells (HUVECs) were obtained from normal pregnant women (n = 11), GDM-D (n = 14), and GDM-I (n = 12) patients. LC-MS/MS was used to identify differentially expressed proteins in primary HUVECs among the three groups, after which Bioinformatics analysis was performed. Glucose uptake, ATP level, apoptosis, and differentially expressed proteins were assessed to investigate changes in energy metabolism.ResultsA total of 8174 quantifiable proteins were detected, and 142 differentially expressed proteins were identified after comparing patients with GDM-D/GDM-I and healthy controls. Of the 142, 64 proteins were upregulated while 77 were downregulated. Bioinformatics analysis revealed that the differentially expressed proteins were involved in multiple biological processes and signaling pathways related to cellular processes, biological regulation, and metabolic processes. According to the results from KEGG analysis, there were changes in the PI3K/AKT signaling pathway after comparing the three groups. In addition, there was a decrease in glucose uptake in the GDM-I (P < 0.01) group. In GDM-I, there was a significant decrease in the levels of glucose transporter 1 (GLUT1) and glucose transporter 3 (GLUT3). Moreover, glucose uptake was significantly decreased in GDM-I, although in GDM-D, there was only a decrease in the levels of GLUT1. ATP levels decreased in GDM-I (P < 0.05) and apoptosis occurred in both the GDM-D and GDM-I groups. Compared to the normal controls, the levels of phosphate AKT and phosphate AMPK over total AKT and AMPK were reduced in the GDM-I group.ConclusionIn summary, endothelial dysfunction occurred in pregnancies with GDM even though the plasma glucose levels were controlled, and this dysfunction might be related to the degree of glucose tolerance. The energy dysfunction might be related to the regulation of the AKT/AMPK/mTOR signaling pathway.

Association of Umbilical Cord Serum Oxidative Stress Markers, ADMA and Adiponectin with Adverse Fetal Outcomes in Hypertensive Disorders of Pregnancy

Background: Limited studies are available on fetal oxidative stress and endothelial dysfunction and their association with adverse fetal outcomes in hypertensive disorders of pregnancy (HDP). Method: Umbilical cord blood samples were collected at delivery from 134 pregnant women with HDP and 59 controls. Markers of oxidative stress, endothelial dysfunction and inflammation and adipokines were analyzed. Results were correlated with adverse fetal outcomes. Results: Malondialdehyde, total antioxidant status(TAS), ADMA and hsCRP levels were increased in late and early onset preeclampsia. Adiponectin levels were decreased in early onset preeclampsia. High ADMA levels were positively associated with preterm births and fetal mortality and high TAS, protein carbonyl content(PC), ADMA and low adiponectin levels were positively associated with low birth weight babies. Conclusion: Fetal systemic oxidative stress, endothelial dysfunction and inflammation were altered in early and late onset preeclampsia. High TAS, PC and ADMA levels and low adiponectin levels were positively associated with adverse fetal outcomes in HDP.

Fetal inflammatory response and endothelial dysfunction in newborns from mothers with exacerbation of cytomegalovirus infection during the second trimester of pregnancy

Aim. To study the fetal inflammatory response and endothelial dysfunction in newborns from mothers with an exacerbation of cytomegalovirus infection (CMVI) in the second trimester of pregnancy. Materials and methods. We examined 90 newborns with an antenatal history, uncomplicated and aggravated by the acute phase of chronic CMVI, diagnosed in their mothers at 21-24 weeks of gestation. The concentration of TNFα, IL-6, and endothelin-1 was determined in cord blood serum. The control group consisted of 30 newborns whose intrauterine development proceeded against the background of physiological pregnancy seronegative for cytomegalovirus (CMV). The main group included 60 newborns, which were divided into 2 subgroups. Subgroup 1 included 30 newborns from mothers who had an acute phase of chronic CMVI, leading to the development of chronic compensated placental insufficiency, subgroup 2 consisted of 30 newborns from mothers with exacerbation of CMVI, initiating the formation of chronic subcompensated placental insufficiency. Results. In the control group, DNA of CMV, IgM antibodies to CMV and IgG antibodies to CMV were not detected. In the main group, in newborns of subgroup 1, compared with the control, DNA of CMV, both in the cord blood and in the nasopharyngeal aspirate, was isolated in 3.3 % of cases, and was not detected in scrapings of the buccal epithelium. The titers of IgG antibodies to CMV were determined in the ratio of 1: 100 in 36.7 %, 1: 200 in 43.3 %, 1: 400 in 13.3% and 1: 800 in 6.7 % of cases. At the same time, in mother-newborn pairs, higher titers of IgG antibodies to CMV in pregnant women, compared with those in their offspring, were detected in 18 cases, and equal titers of IgG antibodies to CMV were detected in 12 dyads. In the control group, the concentration of TNFα was equal to (Me) 16.2 (12.9-32.7) pg/ml, IL-6 – 1.93 (1.65-2.21) pg/mL, endothelin-1 – 0.52 (0.45–1.21) fmol/mL. In subgroup 1 of the main group, in comparison with the control one, there was an increase in the concentration of TNFα by 2.6 times (p=0.000001), IL-6 – by 2.1 times (p = 0.000001) and endothelin-1 – by 2.1 time (p = 0.000002). In newborns of subgroup 2, compared with subgroup 1, CMV antigens were identified in the blood in 6.7 % (p &gt; 0.05), and in nasopharyngeal aspirate – in 10% (p&gt;0.05) in the absence of identification of the CMV genome in the buccal epithelium. In 6.7%, IgM antibodies to CMV were detected. IgG antibodies to CMV 1:800 were detected more often (in 33.3%, p&lt;0.05), as well as equal titers of IgG antibodies to CMV in 20 mother-offspring pairs (p&lt;0.05). There was an increase in the concentration of TNFα by 1.47 times (p=0.0076), IL-6 - by 1.33 (p = 0.0016) and endothelin-1 - by 1.5 times (p = 0.0161), respectively. Conclusion. In the offspring of mothers who had an exacerbation of CMVI in the second trimester of pregnancy and with the presence of chronic subcompensated placental insufficiency, compared with newborns from women with the acute phase of chronic CMVI at similar gestation periods and formed chronic compensated placental insufficiency, higher levels of pro-inflammatory cytokines and endothelin-1 are recorded, indicating activation of the fetal immune system as a result of viral aggression and penetration of IgG to CMV of maternal origin.

Gestational Diabetes Mellitus Treatment Schemes Modify Maternal Plasma Cholesterol Levels Dependent to Women´s Weight: Possible Impact on Feto-Placental Vascular Function.

Gestational diabetes mellitus (GDM) associates with fetal endothelial dysfunction (ED), which occurs independently of adequate glycemic control. Scarce information exists about the impact of different GDM therapeutic schemes on maternal dyslipidemia and obesity and their contribution to the development of fetal-ED. The aim of this study was to evaluate the effect of GDM-treatments on lipid levels in nonobese (N) and obese (O) pregnant women and the effect of maternal cholesterol levels in GDM-associated ED in the umbilical vein (UV). O-GDM women treated with diet showed decreased total cholesterol (TC) and low-density lipoproteins (LDL) levels with respect to N-GDM ones. Moreover, O-GDM women treated with diet in addition to insulin showed higher TC and LDL levels than N-GDM women. The maximum relaxation to calcitonin gene-related peptide of the UV rings was lower in the N-GDM group compared to the N one, and increased maternal levels of TC were associated with even lower dilation in the N-GDM group. We conclude that GDM-treatments modulate the TC and LDL levels depending on maternal weight. Additionally, increased TC levels worsen the GDM-associated ED of UV rings. This study suggests that it could be relevant to consider a specific GDM-treatment according to weight in order to prevent fetal-ED, as well as to consider the possible effects of maternal lipids during pregnancy.

Supraphysiological Maternal Hypercholesterolemia Associates With Increased Maternal Levels Of Pcsk9 And With Changes In The Maternal And Neonatal Ldl And Hdl Profile

Background and Aims: Physiological maternal hypercholesterolemia (MPH; total cholesterol (TC)≤280 mg/dL) occurs during pregnancy. However, some women develop supraphysiological maternal hypercholesterolemia (MSPH; TC>280 mg/dL) which associates with increased levels of LDL and leads to fetal endothelial dysfunction and atherosclerosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates LDL levels. It is unknown whether (1) PCSK9 is modulated in MSPH and (2) if MSPH associates with changes in maternal and neonatal lipoproteins functions, favoring a pro-atherogenic profile.

MEG3 damages fetal endothelial function induced by gestational diabetes mellitus via AKT pathway.

To explore the role of maternally-expressed gene 3 (MEG3) in fetal endothelial dysfunction induced by gestational diabetes mellitus (GDM) and its underlying mechanism. Human umbilical vein endothelial cells (HUVECs) were extracted from GDM pregnancies and normal pregnancies. Cell proliferation, apoptosis, migration and angiogenesis of HUVECs were detected by cell counting kit-8 (CCK-8), enzyme-linked immunosorbent assay (ELISA), wound healing and tube formation assay, respectively. MEG3 expressions in HUVECs extracted from 16 GDM pregnancies and 18 normal pregnancies were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Besides, angiogenesis and MEG3 expression in HUVECs treated with glucose were detected, respectively. Proliferation, apoptosis, migration and angiogenesis were also detected after HUVECs were transfected with MEG3 lentivirus. Target genes of MEG3 were predicted by bioinformatics method and further verified by luciferase reporter gene assay. The protein expression of possible signaling pathway was detected by Western blot. HUVEC cells extracted from GDM pregnancies presented increased apoptosis and decreased proliferation, migration and angiogenesis compared with those from healthy pregnancies. Meanwhile, MEG3 was overexpressed in HUVECs extracted from GDM pregnancies compared with that of healthy pregnancies. High dose of glucose treatment led to reduced angiogenesis and elevated MEG3 expression in HUVECs. MEG3 overexpression further promoted apoptosis, but inhibited proliferation, migration and angiogenesis of HUVECs. By bioinformatics and luciferase reporter gene assay, microRNA-370-3p was found to be the target gene of MEG3 and directly targeted on AFF1. Moreover, MEG3 overexpression led to downregulated microRNA-370-3p and upregulated AFF1 mainly through inhibiting PI3K/AKT pathway. MEG3 is overexpressed in HUVECs extracted from GDM pregnancies. MEG3 damages fetal endothelial function through targeting microRNA-370-3p and AFF1 via PI3K/AKT pathway.

The use of folates for the prevention of fetal growth restriction during pregnancy

Purpose of the study: Rational for the use of folates for the prevention of fetal growth restriction on the basis of the identification of a MTHFR polymorphism.Material and methods: 200 patients (case-control) were enrolled in the study. The (main) group I included 100 patients with fetal growth restriction (FGR), the (control) group II included 100 conditionally healthy patients. The features of the anamnesis, the initial clinical characteristics, the course of pregnancy, childbirth and the postpartum period in patients with placental insufficiency were studied in detail, and the features of the early neonatal period were analysed comprehensively. At the second stage, the polymorphism of the MMP2, MMP3, MMP9, and MTHFR genes was studied and their dependence with the development of the FGR was established. Statistical processing of the results was carried out on a personal computer using the SPSS Statistics 17.0 for Windows software package.Results:The clinical risk factors for the development of FGR include: chronic pyelonephritis, medium degree myopia, varicose veins, threat of abortion, ultrasound-detected subchorionic hemorrhage in the I trimester of gestation, anemia. Along with clinical risk factors for ZRP, the carriage of MTGFR (677 C&gt; T) polymorphism is also considered, which justifies its identification in patients at risk.Conclusion: The data obtained make it possible to use the identification of MTHFR polymorphism as a predictor of placental abnormalities during pregnancy and to start taking folic acid supplements at the preconception stage in good time. Femibion 1 vitaminmineral complex has proved to be an effective product that is suitable for women in the period of preparation for pregnancy and in the first trimester of gestation, reducing the risk of fetal malformations and endothelial dysfunction caused by a violation of folic acid metabolism.

The effect of maternal type 2 diabetes on fetal endothelial gene expression and function.

Maternal type 2 diabetes (T2D) can result in adverse pathological outcomes to both the mother and fetus. The present study aimed to investigate the pathological effects of maternal T2D on the gene expression patterns and functions of fetal human umbilical vein endothelial cells (HUVECs), a representative of fetal vascular cells. Cell proliferation, apoptosis, mitochondrial ROS production and cell cycle were measured using flowcytometry. Genome-wide expression was measured using Affymetrix microarray. Gene expression of CCND2, STAT1, ITGB8, ALDH2, and ADAMTS5 was measured using real-time PCR. HUVECs derived from T2D mothers (T2D-HUVECs) showed elevated levels of mitochondrial superoxide anions, reduced cell proliferation, and increased apoptosis rates relative to HUVECs derived from healthy control mothers (C.HUVECs). In addition , T2D-HUVECs showed a decreased proportion of cells in G0/G1 and cell cycle arrest at the S phases relative to controls. Interestingly, microarray experiments revealed significant differences in genome-wide expression profiles between T2D-HUVECs and C.HUVECs. In particular, the analysis identified 90 upregulated genes and 42 downregulated genes. The upregulated genes CCND2, STAT1, ITGB8, ALDH2, and ADAMTS5 were validated as potential biomarkers for fetal endothelial dysfunction. Functional network analysis revealed that these genes are the important players that participate in the pathogenesis of endothelial dysfunction, which in turn influences the inflammatory response, cellular movement, and cardiovascular system development and function. Sustained alterations in the overall function of T2D-HUVEC and gene expression profiles provided insights into the role of maternal T2D on the pathophysiology of the fetal endothelial dysfunction.

Role for Tetrahydrobiopterin in the Fetoplacental Endothelial Dysfunction in Maternal Supraphysiological Hypercholesterolemia.

Maternal physiological hypercholesterolemia occurs during pregnancy, ensuring normal fetal development. In some cases, the maternal plasma cholesterol level increases to above this physiological range, leading to maternal supraphysiological hypercholesterolemia (MSPH). This condition results in endothelial dysfunction and atherosclerosis in the fetal and placental vasculature. The fetal and placental endothelial dysfunction is related to alterations in the L-arginine/nitric oxide (NO) pathway and the arginase/urea pathway and results in reduced NO production. The level of tetrahydrobiopterin (BH4), a cofactor for endothelial NO synthase (eNOS), is reduced in nonpregnant women who have hypercholesterolemia, which favors the generation of the superoxide anion rather than NO (from eNOS), causing endothelial dysfunction. However, it is unknown whether MSPH is associated with changes in the level or metabolism of BH4; as a result, eNOS function is not well understood. This review summarizes the available information on the potential link between MSPH and BH4 in causing human fetoplacental vascular endothelial dysfunction, which may be crucial for understanding the deleterious effects of MSPH on fetal growth and development.

The Role of Maternal Gestational Diabetes in Inducing Fetal Endothelial Dysfunction.

Gestational diabetes mellitus (GDM) is known to be associated with fetal endothelial dysfunction, however, the mechanisms are not fully understood. This study examines the effect of maternal diabetes on fetal endothelial function and gene expression under physiological glucose conditions (5 mM). Human umbilical vein endothelial cell (HUVEC) isolated from diabetic mothers (d.HUVEC) grew more slowly than HUVEC isolated from healthy mothers (c.HUVEC) and had delayed doubling time despite increased levels of total vascular endothelial growth factor (VEGF) expression and protein production as determined by real-time PCR and ELISA respectively. Using western blot, the levels of antiproliferative VEGF165b isoform were increased in d.HUVEC relative to c.HUVEC. Successful VEGF165b knockdown by small interfering RNA (siRNA) resulted in increased proliferation of d.HUVEC measured by MTT, compared with negative siRNA control, to similar levels measured in c.HUVEC. In addition, d.HUVEC generated excess levels of ROS as revealed by 2',7' Dichlorodihydrofluorescein Diacetate (DCFH-DA) and Nitrotetrazolium blue (NBT). Using microarray, 102 genes were differentially overexpressed between d.HUVEC versus c.HUVEC (>1.5-fold change; P < 0.05). Functional clustering analysis of these differentially expressed genes revealed participation in inflammatory responses (including adhesion) which may be related to pathological outcomes. Of these genes, ICAM-1 was validated as upregulated, confirming microarray results. Additional confirmatory immunofluorescence staining revealed increased protein expression of ICAM-1 compared with c.HUVEC which was reduced by vitamin C treatment (100 μM). Thus, maternal diabetes induces persistent alterations in fetal endothelial function and gene expression following glucose normalization and antioxidant treatment could help reverse endothelium dysfunction.

Heart disease link to fetal hypoxia and oxidative stress.

The quality of the intrauterine environment interacts with our genetic makeup to shape the risk of developing disease in later life. Fetal chronic hypoxia is a common complication of pregnancy. This chapter reviews how fetal chronic hypoxia programmes cardiac and endothelial dysfunction in the offspring in adult life and discusses the mechanisms via which this may occur. Using an integrative approach in large and small animal models at the in vivo, isolated organ, cellular and molecular levels, our programmes of work have raised the hypothesis that oxidative stress in the fetal heart and vasculature underlies the mechanism via which prenatal hypoxia programmes cardiovascular dysfunction in later life. Developmental hypoxia independent of changes in maternal nutrition promotes fetal growth restriction and induces changes in the cardiovascular, metabolic and endocrine systems of the adult offspring, which are normally associated with disease states during ageing. Treatment with antioxidants of animal pregnancies complicated with reduced oxygen delivery to the fetus prevents the alterations in fetal growth, and the cardiovascular, metabolic and endocrine dysfunction in the fetal and adult offspring. The work reviewed offers both insight into mechanisms and possible therapeutic targets for clinical intervention against the early origin of cardiometabolic disease in pregnancy complicated by fetal chronic hypoxia.

Maternal Hypercholesterolemia in Pregnancy Associates With Umbilical Vein Endothelial Dysfunction

Objective— Human pregnancy that courses with maternal supraphysiological hypercholesterolemia (MSPH) correlates with atherosclerotic lesions in fetal arteries. It is known that hypercholesterolemia associates with endothelial dysfunction in adults, a phenomenon where nitric oxide (NO) and arginase are involved. However, nothing is reported on potential alterations in the fetoplacental endothelial function in MSPH. The aim of this study was to determine whether MSPH alters fetal vascular reactivity via endothelial arginase/urea and l -arginine transport/NO signaling pathways. Approach and Results— Total cholesterol &lt;280 mg/dL was considered as maternal physiological hypercholesterolemia (n=46 women) and ≥280 mg/dL as MSPH (n=28 women). Maternal but not fetal total cholesterol and low-density lipoprotein-cholesterol levels were elevated in MSPH. Umbilical veins were used for vascular reactivity assays (wire myography), and primary cultures of umbilical vein endothelial cells to determine arginase, endothelial NO synthase (eNOS), and human cationic amino acid transporter 1 and human cationic amino acid transporter 2A/B expression and activity. MSPH reduced calcitonine gene–related peptide-umbilical vein relaxation and increased intima/media ratio (histochemistry), as well as reduced eNOS activity ( l -citrulline synthesis from l -arginine, eNOS phosphorylation/dephosphorylation), but increased arginase activity and arginase II protein abundance. Arginase inhibition increased eNOS activity and l -arginine transport capacity without altering human cationic amino acid transporter 1 or human cationic amino acid transporter 2A/B protein abundance in maternal physiological hypercholesterolemia and MSPH. Conclusions— MSPH is a pathophysiological condition altering umbilical vein reactivity because of fetal endothelial dysfunction associated with arginase and eNOS signaling imbalance. We speculate that elevated maternal circulating cholesterol is a factor leading to fetal endothelial dysfunction, which could have serious consequences to the growing fetus.

Contribution of PARP to endothelial dysfunction and hypertension in a rat model of pre‐eclampsia

Under conditions of increased oxidative stress, such as pre-eclampsia and diabetes, overstimulation of PARP leads to endothelial dysfunction. Inhibition of PARP has been demonstrated to reverse the vascular dysfunction associated with diabetes in vivo. The present study was carried out to investigate the role of PARP in mediating the endothelial dysfunction associated with pre-eclampsia. Uteroplacental perfusion was surgically reduced in pregnant rats to produce the reduced uterine perfusion pressure (RUPP) rat model of pre-eclampsia and the PARP inhibitor, PJ34, was administered either before or after surgery. Mean arterial BP and vascular function were measured in normal pregnant (NP) and both control and PJ34-treated RUPP rats. Mesenteric vessels from NP rats were incubated with either 3% RUPP or NP plasma alone or in combination with PJ34. Finally, immunohistochemical staining was carried out to measure nitrotyrosine (byproduct of peroxynitrite) immunoreactivity. RUPP rats were characterized by hypertension, fetal growth restriction and endothelial dysfunction when compared with NP rats. PJ34 administered in vivo before, but not after, surgery prevented the development of both endothelial dysfunction and hypertension. RUPP plasma-induced impaired vasorelaxation was prevented following co-incubation with PJ34 in vitro. Furthermore, the protective effect of PARP inhibition in vivo was accompanied by a reduction in nitrotyrosine immunoreactivity. PJ34 prevented the development of both endothelial dysfunction and hypertension and reduced vascular nitrotyrosine immunoreactivity, thus suggesting a role for oxidative-nitrosative stress/PARP activation in the aberration in both vascular and haemodynamic function in this rat model of pre-eclampsia.

Functional Link Between Adenosine and Insulin: A Hypothesis for Fetoplacental Vascular Endothelial Dysfunction in Gestational Diabetes

Gestational diabetes mellitus (GDM) is a syndrome compromising the health of the mother and the fetus. Endothelial damage and reduced metabolism of the vasodilator adenosine occur and fetal hyperinsulinemia associated with deficient insulin response and a metabolic rather than mitogenic phenotype is characteristic of this pathology. These phenomena lead to endothelial dysfunction of the fetoplacental unit. Major databases were searched for the relevant literature in the field. Special attention was placed on publications related with diabetes and hormone/metabolic disorders. We aimed to summarize the information regarding insulin sensitivity changes in GDM and the role of adenosine in this phenomenon. Evidence supporting the possibility that fetal endothelial dysfunction involves a functional link between adenosine and insulin signaling in the fetal endothelium from GDM pregnancies is summarized. Since insulin acts via membrane receptors type A (preferentially associated with mitogenic responses) or type B (preferentially associated with metabolic responses), a differential activation of these receptors in this syndrome is proposed.