Angiogenic Factors and Long-Term Cardiovascular Risk in Women That Developed Preeclampsia During Pregnancy.

Preeclampsia is caused by placental impairment with increased expression of sFlt-1 (soluble fms-like tyrosine kinase 1) and decreased PlGF (placental growth factor); it has been associated with cardiovascular morbidity and mortality later in life, but the underlying mechanism remains unknown. The aim of this study was to determine whether sFlt-1 and PlGF levels during preeclampsia are associated to long-term cardiovascular risk. We prospectively recruited 43 women with previous preeclampsia and 21 controls with uncomplicated pregnancies. Cardiovascular risk assessment ≈12 years later included maternal hemodynamic, cardiac function and structure, biomarker analysis, and carotid-intima thickness evaluation. Women with previous preeclampsia had higher prevalence of hypertensive disorders and dyslipidemia than controls. In addition, they had worse global longitudinal strain, thicker left ventricular septal and posterior walls, more myocardial mass and increased carotid intima-media thickness compared with controls. PlGF during pregnancy correlated positively with high-density lipoprotein (r=0.341; P=0.006), and negatively with global longitudinal strain (r=-0.581; P<0.001), carotid intima-media thickness (r=-0.251; P=0.045), and mean arterial blood pressure (r=-0.252; P=0.045), when adjusted by study group. sFlt correlated negatively with high-density lipoprotein (r=-0.372; P=0.002) and apolipoprotein A-1 (r=-0.257; P=0.040), and positively with carotid intima-media thickness (r=0.269; P=0.032) and left ventricular posterior wall thickness (r=0.368; P=0.003). The antiangiogenic state present in preeclampsia is related to greater prevalence of cardiovascular risk factors ≈12 years after delivery. The knowledge of altered angiogenic factors may help detect women with a higher risk for premature cardiovascular disease, who will require earlier follow-up after delivery.

TNFAIP3 haploinsufficiency is the cause of autoinflammatory manifestations in a patient with a deletion of 13Mb on chromosome 6.

There is scarce literature about autoinflammation in syndromic patients. We describe a patient who, in addition to psychomotor and growth delay, presented with fevers, neutrophilic dermatosis, and recurrent orogenital ulcers. Comparative Genomic Hybridization (CGH) array permitted to identify a 13.13Mb deletion on chromosome 6, encompassing 53 genes, and including TNFAIP3 gene (A20). A20 is a potent inhibitor of the NF-kB signalling pathway and restricts inflammation via its deubiquitinase activity. Western blotting and immunoprecipitation assays showed decreased A20 expression and increased phosphorylation of p65 and IkBa. Patient's cells displayed increased levels of total K63-linked ubiquitin and increased levels of ubiquitinated RIP and NEMO after stimulation with TNF. We describe the molecular characterization of an autoinflammatory disease due to a large chromosomal deletion and review the phenotypes of patients with A20 haploinsufficiency. CGH arrays should be the first diagnostic method for comprehensive analysis of patients with syndromic features and immune dysregulation.

Association of first-trimester angiogenic factors with placental histological findings in late-onset preeclampsia.

OBJECTIVE: To explore in women with late-onset preeclampsia (PE) the association between maternal levels of angiogenic/antiangiogenic factors in the first trimester of pregnancy and histological findings attributable to placental underperfusion (PUP). METHODS: A nested case-control cohort study was conducted in 73 women with pregnancies complicated by late-onset PE (>34 weeks at delivery) matched with controls. First trimester uterine artery Doppler (UtA); maternal levels of placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) were retrieved. Placentas were histologically evaluated using a hierarchical and standardized classification system. One-way ANOVA with linear polynomial contrast or linear-by-linear association test was performed to test the hypothesis of a linear association across study groups (controls, PE without PUP and PE with PUP). RESULTS: In 54 (74%) placentas, 89 placental histological findings qualifying for PUP were found. Across study groups, significant values were observed in maternal levels of decreased PlGF (MoM values: 1.53, 1.41 and 1.37; p < 0.001), increased sFlt-1 (MoM values: 3.11, 3.11 and 3.22; p = 0.002), increased sFlt-1/PlGF ratio (MoM values: 2.3, 2.3 and 2.44; p < 0.001), abnormal UtA Doppler (MoM values: 1, 1.26 and 1.32; p < 0.001), and worse perinatal outcomes in terms of gestational age at delivery, cesarean section for not reassuring fetal status, birth weight and neonatal acidosis. DISCUSSION: In late-onset PE an imbalance of circulating angiogenic and anti-angiogenic factors already present at 8-10 weeks of pregnancy was associated with histological findings reflecting placental insufficiency. An early first trimester screening by angiogenic factors might help to identify patients with placental involvement among late-onset PE cases. CONCLUSION: In late-onset preeclampsia, first-trimester uterine Doppler and circulating levels of angiogenic/antiangiogenic factors are associated with placental underperfusion.

Increased secretory sphingomyelinase activity in the first trimester of pregnancy in women later developing preeclampsia: a nested case-control study.

The pathogenic basis of abnormal placentation and dysfunction in preeclampsia (PE) is highly complex and incompletely understood. Secretory sphyngomyelinase activity (S-ASM) was analyzed in plasma samples from 158 pregnant women developing PE and 112 healthy pregnant controls. Serum PlGF, sFlt-1, s-Endoglin and sVCAM were measured. Results showed S-ASM activity to be higher in women who later developed PE than in those with uncomplicated pregnancies (40.6% and 28.8% higher in the late- and early-onset groups, respectively). Plasma S-ASM activity correlated significantly with circulating markers of endothelial damage in the late-PE group (endoglin and sVCAM-1), with plasma cholesterol and total lipid levels. However, these significant associations were not observed in the early-PE or control groups. This work provides the first evidence of significantly elevated circulating S-ASM activity in the first trimester of pregnancy in women who go on to develop PE; thus, it may be deduced that the circulating form of ASM is biologically active in PE and could contribute to promoting endothelial dysfunction and cardiovascular programming. Plasma S-ASM measurement may have clinical relevance as a further potential biomarker contributing to the earliest identification of women at risk of developing preeclampsia.

Angiogenic Factors and Doppler Evaluation in Normally Growing Fetuses at Routine Third-Trimester Scan: Prediction of Subsequent Low Birth Weight.

OBJECTIVE: To evaluate in normally growing fetuses at routine 32-36 weeks scan the performance of maternal angiogenic factors, Doppler and ultrasound indices in predicting smallness for gestational age (SGA) at birth. METHODS: A cohort of 1,000 singleton pregnancies with normal estimated fetal weight (EFW, ≥10th centile) at 32-36 weeks scan was included. At inclusion, Doppler indices (mean uterine artery pulsatility index [mUtA-PI], cerebroplacental ratio and normalized umbilical vein blood flow by EFW (ml/min/kg) were evaluated, and blood samples were collected and frozen. Nested in this cohort, maternal circulating placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) were assayed by enzyme-linked immunosorbent assay in all cases with a birth weight <10th centile by customized standards and in an equivalent number of controls (birth weight ≥10th centile). RESULTS: 160 cases were included (80 SGA and 80 controls). EFW (2,128 vs. 2,279 g, p < 0.001), mUtA-PI z-values (-0.25 vs. -0.65, p = 0.034) and sFlt-1/PlGF ratio (11.10 vs. 6.74, p < 0.005) were lower in SGA. The combination of sFlt-1/PlGF ratio and EFW resulted in a 66.3% detection rate for subsequent SGA, with 20% of false-positives. Fetal Doppler indices were not predictive of SGA. CONCLUSIONS: In normally growing fetuses, maternal angiogenic factors add to ultrasound parameters in predicting subsequent SGA at birth. This supports further research to investigate composite scores in order to improve the definition and identification of fetal growth restriction.

Intracellular oxidant activity, antioxidant enzyme defense system, and cell senescence in fibroblasts with trisomy 21.

Down's syndrome (DS) is characterized by a complex phenotype associated with chronic oxidative stress and mitochondrial dysfunction. Overexpression of genes on chromosome-21 is thought to underlie the pathogenesis of the major phenotypic features of DS, such as premature aging. Using cultured fibroblasts with trisomy 21 (T21F), this study aimed to ascertain whether an imbalance exists in activities, mRNA, and protein expression of the antioxidant enzymes SOD1, SOD2, glutathione-peroxidase, and catalase during the cell replication process in vitro. T21F had high SOD1 expression and activity which led to an interenzymatic imbalance in the antioxidant defense system, accentuated with replicative senescence. Intracellular ROS production and oxidized protein levels were significantly higher in T21F compared with control cells; furthermore, a significant decline in intracellular ATP content was detected in T21F. Cell senescence was found to appear prematurely in DS cells as shown by SA-ß-Gal assay and p21 assessment, though not apoptosis, as neither p53 nor the proapoptotic proteins cytochrome c and caspase 9 were altered in T21F. These novel findings would point to a deleterious role of oxidatively modified molecules in early cell senescence of T21F, thereby linking replicative and stress-induced senescence in cultured cells to premature aging in DS.

Molecular damage in Fabry disease: characterization and prediction of alpha-galactosidase A pathological mutations.

Loss-of-function mutations of the enzyme alpha-galactosidase A (GLA) causes Fabry disease (FD), that is a rare and potentially fatal disease. Identification of these pathological mutations by sequencing is important because it allows an early treatment of the disease. However, before taking any treatment decision, if the mutation identified is unknown, we first need to establish if it is pathological or not. General bioinformatic tools (PolyPhen-2, SIFT, Condel, etc.) can be used for this purpose, but their performance is still limited. Here we present a new tool, specifically derived for the assessment of GLA mutations. We first compared mutations of this enzyme known to cause FD with neutral sequence variants, using several structure and sequence properties. Then, we used these properties to develop a family of prediction methods adapted to different quality requirements. Trained and tested on a set of known Fabry mutations, our methods have a performance (Matthews correlation: 0.56-0.72) comparable or better than that of the more complex method, Polyphen-2 (Matthews correlation: 0.61), and better than those of SIFT (Matthews correl.: 0.54) and Condel (Matthews correl.: 0.51). This result is validated in an independent set of 65 pathological mutations, for which our method displayed the best success rate (91.0%, 87.7%, and 73.8%, for our method, PolyPhen-2 and SIFT, respectively). These data confirmed that our specific approach can effectively contribute to the identification of pathological mutations in GLA, and therefore enhance the use of sequence information in the identification of undiagnosed Fabry patients.

First trimester screening for early and late preeclampsia based on maternal characteristics, biophysical parameters, and angiogenic factors.

OBJECTIVE: The aim of this article is to develop the best first-trimester screening model for preeclampsia (PE) based on maternal characteristics, biophysical parameters, and angiogenic factors in a low-risk population. METHODS: A prospective cohort of 9462 pregnancies undergoing first-trimester screening is used. Logistic regression predictive models were developed for early and late PE (cut-off of 34 weeks' gestation at delivery). Data included the a priori risk (maternal characteristics), mean arterial pressure (MAP), and uterine artery (UtA) Doppler (11-13 weeks) in all cases. Plasma levels (8-11 weeks) of human chorionic gonadotrophin, pregnancy-associated plasma protein A, placental growth factor (PlGF), and soluble Fms-like tyrosine kinase-1 (sFlt-1) were analyzed using a nested case-control study design. RESULTS: The best model for early PE (n = 57, 0.6%) included a priori risk, MAP, UtA Doppler, PlGF, and sFlt-1 achieving detection rates of 87.7% and 91.2% for 5% and 10% false-positive rates, respectively (AUC: 0.98 [95% CI: 0.97-0.99]). For late PE (n = 246, 2.6%), the best model included the a priori risk, MAP, UtA Doppler, PlGF, and sFlt-1 achieving detection rates of 68.3% and 76.4% at 5% and 10% of false-positive rates, respectively (AUC: 0.87 [95% CI: 0.84-0.90]). CONCLUSION: Preeclampsia can be predicted with high accuracy in general obstetric populations with a low risk for PE, by combined algorithms. Angiogenic factors substantially improved the prediction.

Added value of angiogenic factors for the prediction of early and late preeclampsia in the first trimester of pregnancy.

OBJECTIVE: To explore the predictive role of angiogenic factors for the prediction of early and late preeclampsia (PE) in the first trimester. METHODS: A nested case-control study, within a cohort of 5,759 pregnancies, including 28 cases of early, 84 of late PE (cut-off 34 weeks) and 84 controls. Maternal characteristics, mean blood pressure (MAP), uterine artery (UtA) Doppler (11-13 weeks), vascular endothelial growth factor, placental growth factor (PlGF), soluble Fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (8-11 weeks) were measured/recorded. All parameters were normalized by logarithmic transformation; logistic regression analysis was used to predict PE. RESULTS: For early PE, significant contributions were chronic hypertension, previous PE, MAP, UtA Doppler, PlGF and sFlt-1. A model including these predictors achieved detection rates (DR) of 77.8 and 88.9% for 5 and 10% false-positive rates (FPR), respectively (AUC 0.958; 95% CI 0.920-0.996). For late PE, significant contributions were provided by body mass index, previous PE, UtA Doppler, PlGF and sFlt-1. The model including these factors achieved DR of 51.2 and 69% at 5 and 10% FPR, respectively (AUC 0.888; 95% CI 0.840-0.936). CONCLUSIONS: Among angiogenic factors, not only PlGF but also sFlt-1 substantially improve the prediction for early and late PE. The data need confirmation in larger studies.

Study of biomaterial-induced macrophage activation, cell-mediated immune response and molecular oxidative damage in patients with dermal bioimplants.

Several soft-tissue dermal fillers have been reported to provoke immunogenicity and may cause adverse reactions despite claims regarding their safety. This study aimed to assess biomaterial-induced macrophage activation, cell-mediated immune response and oxidative stress in 169 patients with dermal bioimplants. To this end, we analysed plasma concentrations of myeloperoxidase (MPO), the chitinase-like proteins chitotriosidase and YKL-40 and molecular oxidative damage. The present study shows, for the first time, that the components of innate immunity: chitotriosidase and YKL-40, are significantly higher in patients with certain bioimplants and these markers of monocyte/macrophage activation rose progressively as adverse reactions (AR) evolved. Plasma MPO levels increased 4-fold in filler users with AR and 3-fold in those without. Analysis by filler type showed subjects injected with calcium hydroxylapatite, methacrylate, acrylamides and silicone to have values significantly above those of non-filler subjects for at least two plasma biomarkers, probably because the afore-mentioned biomaterials are permanent and prone to trigger AR in the long term. By contrast, hyaluronic acid alone elicited little immune response. Plasma concentrations of markers of oxidative damage to lipids and proteins were found to be significantly higher in users of four of the nine dermal fillers studied. These diffusible products of molecular peroxidation would stem from the reaction catalysed by MPO that generates potent oxidants, leading to cell oxidative damage which, in turn, may exert deleterious effects on the organism. Overall, the results of this study on the effects of a range of dermal fillers point to chronic activation of the immune response mediated by macrophages and PMNs. The increases in plasma of MPO, chitotriosidase and YKL-40 proteins and products of macromolecular peroxidation suggests that these molecules could serve as blood-based biochemical markers and alert to the risk of chronic immune system activation and development of adverse events that may arise from the use of certain bioimplants.

A procedure for measuring triacylglyceride and cholesterol content using a small amount of tissue.

This paper describes the development of a procedure for measuring total lipid, triacylglyceride, and cholesterol content using small amounts of tissue (50 mg for liver samples and 10 mg for adipose tissue samples). In the first step, total lipids are extracted with an organic solvent mixture of hexane and isopropanol. In the second step, extracted lipids are emulsified by sonication in a buffer containing 1,4-piperazinediethanesulfonic acid (28.75 mM), magnesium chloride.6H(2)O (57.76 mM), free fatty acids-bovine serum albumin (8.76 microM), and sodium dodecyl sulfate (0.1%). Lipid concentrations from the resulting emulsion can then be assessed using commercial enzymatic kits. This method is also suitable for determining direct homogenate triacylglyceride and total cholesterol content in cases where a lipid percentage is not needed. The proposed method increases the yield of lipid recovery from small tissue samples and allows the measurement of both triacylglyceride and cholesterol content from a single starting sample. The methodology described here is the first to accomplish simultaneous determination of both parameters and is potentially useful for animal small tissue samples, particularly human biopsy samples.