Angiogenic Function of Human Placental Endothelial Cells in Severe Fetal Growth Restriction Is Not Rescued by Individual Extracellular Matrix Proteins.

Severe fetal growth restriction (FGR) is characterized by increased placental vascular resistance resulting from aberrant angiogenesis. Interactions between endothelial cells (ECs) and the extracellular matrix (ECM) are critical to the complex process of angiogenesis. We have previously found that placental stromal abnormalities contribute to impaired angiogenesis in severe FGR. The objective of this research is to better characterize the effect of individual ECM proteins on placental angiogenic properties in the setting of severe FGR. ECs were isolated from human placentae, either control or affected by severe FGR, and subjected to a series of experiments to interrogate the role of ECM proteins on adhesion, proliferation, migration, and apoptosis. We found impaired proliferation and migration of growth-restricted ECs. Although individual substrates did not substantially impact migratory capacity, collagens I, III, and IV partially mitigated proliferative defects seen in FGR ECs. Differences in adhesion and apoptosis between control and FGR ECs were not evident. Our findings demonstrate that placental angiogenic defects that characterize severe FGR cannot be explained by a singular ECM protein, but rather, the placental stroma as a whole. Further investigation of the effects of stromal composition, architecture, stiffness, growth factor sequestration, and capacity for remodeling is essential to better understand the role of ECM in impaired angiogenesis in severe FGR.

Mechanistic insights into the development of severe fetal growth restriction.

Fetal growth restriction (FGR), which most commonly results from suboptimal placental function, substantially increases risks for adverse perinatal and long-term outcomes. The only "treatment" that exists is delivery, which averts stillbirth but does not improve outcomes in survivors. Furthermore, the potential long-term consequences of FGR to the fetus, including cardiometabolic disorders, predispose these individuals to developing FGR in their future pregnancies. This creates a multi-generational cascade of adverse effects stemming from a single dysfunctional placenta, and understanding the mechanisms underlying placental-mediated FGR is critically important if we are to improve outcomes and overall health. The mechanisms behind FGR remain unknown. However, placental insufficiency derived from maldevelopment of the placental vascular systems is the most common etiology. To highlight important mechanistic interactions within the placenta, we focus on placental vascular development in the setting of FGR. We delve into fetoplacental angiogenesis, a robust and ongoing process in normal pregnancies that is impaired in severe FGR. We review cellular models of FGR, with special attention to fetoplacental angiogenesis, and we highlight novel integrin-extracellular matrix interactions that regulate placental angiogenesis in severe FGR. In total, this review focuses on key developmental processes, with specific focus on the human placenta, an underexplored area of research.

Umbilical Venous Volume Flow in Late-Onset Fetal Growth Restriction.

OBJECTIVES: Umbilical vein flow (UVF) is reduced in fetal growth restriction (FGR). We compared absolute and size-adjusted UVF (estimated fetal weight [EFW] and abdominal circumference [AC]) and rates of abnormal UVF parameters (<10th percentile) among FGR fetuses meeting Delphi criteria (FGR-D) against small for gestational age (SGA) fetuses and appropriate for gestational age (AGA) controls. METHODS: Absolute UVF, UVF/EFW, and UVF/AC were compared between 73 FGR pregnancies (35 FGR-D, 38 SGA) and 108 AGA controls. Rates of abnormal UVF were compared to abnormal umbilical artery pulsatility index (UAPI). Independent samples t-tests, Mann-Whitney U, odds ratio (OR), chi-squared, and Fisher's exact tests were used as appropriate. RESULTS: Mean absolute UVF was significantly decreased in FGR-D compared to AGA (P = .0147), but not between SGA and AGA fetuses. The incidence of both abnormal absolute UVF and UVF/AC values (<10th centile) was higher among late-onset FGR fetuses versus AGA fetuses (UVF: OR 2.7, confidence interval [CI] 1.37-5.4; UVF/AC: OR 2.73, CI 1.37-5.4). UVF was more frequently abnormal than UAPI and in only two fetuses were both Doppler values abnormal. CONCLUSION: Absolute UVF is altered in late-onset FGR, and most pronounced among FGR-D. UVF may provide additional insight into fetal compromise in those affected by growth restriction.

Dysregulation of integrin αvß3 and α5ß1 impedes migration of placental endothelial cells in fetal growth restriction.

Placentas from pregnancies complicated by severe early-onset fetal growth restriction (FGR) exhibit diminished vascular development mediated by impaired angiogenesis, but underlying mechanisms remain unknown. In this study, we show that FGR endothelial cells demonstrate inherently reduced migratory capacity despite the presence of fibronectin, a matrix protein abundant in placental stroma that displays abnormal organization in FGR placentas. Thus, we hypothesized that aberrant endothelial-fibronectin interactions in FGR are a key mechanism underlying impaired FGR endothelial migration. Using human fetoplacental endothelial cells isolated from uncomplicated term control and FGR pregnancies, we assessed integrin α5ß1 and αvß3 regulation during cell migration. We show that endothelial integrin α5ß1 and αvß3 interactions with fibronectin are required for migration and that FGR endothelial cells responded differentially to integrin inhibition, indicating integrin dysregulation in FGR. Whole-cell expression was not different between groups. However, there were significantly more integrins in focal adhesions and reduced intracellular trafficking in FGR. These newly identified changes in FGR endothelial cellular processes represent previously unidentified mechanisms contributing to persistent angiogenic deficiencies in FGR.

Maternal Amino Acid Profiles to Distinguish Constitutionally Small versus Growth-Restricted Fetuses Defined by Doppler Ultrasound: A Pilot Study.

OBJECTIVE: Fetuses measuring below the 10th percentile for gestational age may be either constitutionally small for gestational age (SGA) or have pathologic fetal growth restriction (FGR). FGR is associated with adverse outcomes; however, identification of low-risk SGA cases is difficult. We performed a pilot study evaluating maternal markers of pathologic FGR, hypothesizing there are distinct amino acid signatures that might be used for diagnosis and development of new interventions. STUDY DESIGN: This was a cohort study of healthy women with sonographic fetal estimated fetal weight <5th percentile divided into two groups based upon umbilical artery (UmA) Doppler studies or uterine artery (UtA) Doppler studies. We collected maternal blood samples prior to delivery and used ion pair reverse phase liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry to assess 44 amino acids. RESULTS: Among 14 women included, five had abnormal UmA, and three had abnormal UtA Doppler results. Those with abnormal UmA showed elevated ornithine. Those with abnormal UtA had lower dimethylglycine, isoleucine, methionine, phenylalanine, and 1-methylhistidine. CONCLUSION: We found several amino acids that might identify pregnancies affected by pathologic FGR. These findings support the feasibility of future larger studies to identify maternal metabolic approaches to accurately stratify risk for small fetuses.

Uteroplacental Ischemia Is Associated with Increased PAPP-A2.

Residence at high altitude (> 2500 m) has been associated with an increased frequency of preeclampsia. Pappalysin-2 (PAPP-A2) is an insulin-like growth factor binding protein-5 (IGFBP-5) protease that is elevated in preeclampsia, and up-regulated by hypoxia in placental explants. The relationships between PAPP-A2, altitude, and indices of uteroplacental ischemia are unknown. We aimed to evaluate the association of altitude, preeclampsia, and uterine artery flow or vascular resistance with PAPP-A2 levels. PAPP-A2, uterine artery diameter, volumetric blood flow, and pulsatility indices were measured longitudinally in normotensive Andean women residing at low or high altitudes in Bolivia and in a separate Andean high-altitude cohort with or without preeclampsia. PAPP-A2 levels increased with advancing gestation, with the rise tending to be greater at high compared to low altitude, and higher in early-onset preeclamptic compared to normotensive women at high altitude. Uterine artery blood flow was markedly lower and pulsatility index higher in early-onset preeclamptic normotensive women compared to normotensive women. PAPP-A2 was unrelated to uterine artery pulsatility index in normotensive women but positively correlated in the early-onset preeclampsia cases. We concluded that PAPP-A2 is elevated at high altitude and especially in cases of early-onset preeclampsia with Doppler indices of uteroplacental ischemia.

Size and shape of the four-chamber view of the fetal heart in fetuses with an estimated fetal weight less than the tenth centile.

BACKGROUND: Fetuses with an estimated fetal weight below the 10th centile have an increased risk of adverse perinatal and long-term outcomes as well as increased rates of cardiac dysfunction, which often alters cardiac size and shape of the 4-chamber view and the individual ventricles. As a result, a simple method has emerged to screen for potential cardiac dysfunction in fetuses with estimated fetal weights <10th centile by measuring the size and shape of the 4-chamber view and the size of the ventricles. OBJECTIVE: To determine the number of fetuses with an abnormal size and shape of the 4-chamber view and size of the ventricles in fetuses with an estimated fetal weight <10th centile. MATERIALS AND METHODS: This was a retrospective study of 50 fetuses between 25 and 37 weeks of gestation with an estimated fetal weight <10th centile. Data from their last examination were analyzed. From an end-diastolic image of the 4-chamber view, the largest basal-apical length and transverse width were measured from their corresponding epicardial borders. This allowed the 4-chamber view area and global sphericity index (4-chamber view length/4-chamber view width) to be computed. In addition, tracing along the endocardial borders with speckle tracking software enabled measurements of the right and left ventricular chamber areas and the right ventricle/left ventricle area ratios to be computed. Doppler waveform pulsatility indices from the umbilical (umbilical artery pulsatility index) and middle cerebral arteries (middle cerebral artery pulsatility index) were analyzed, and the cerebroplacental ratio (middle cerebral artery pulsatility index/umbilical artery pulsatility index) computed. Umbilical artery pulsatility indices >90th and cerebroplacental ratios <10th centile were considered abnormal. Using data from the control fetuses, the centile for each of the cardiac measurements was categorized by whether it was <10th or >90th centile, depending upon the measurement. RESULTS: Of the 50 fetuses with estimated fetal weight <10th centile, 50% (n = 25) had a normal umbilical artery pulsatility index and cerebroplacental ratio. These fetuses had significantly more (P < 0.02 to <0.0001) abnormalities of the size and shape of the 4-chamber view than controls. In all, 44% had a 4-chamber view area >90th centile, 32% had a 4-chamber view global sphericity index <10th centile, 56% had a 4-chamber view width >90th centile, and 80% had 1 or more abnormalities of size and/or shape. The remaining 50% of fetuses (n = 25) had abnormalities of 1 or both for the umbilical artery pulsatility index and/or cerebroplacental ratio. These fetuses had significantly higher rates of abnormalities (P <0.05 to <0.0001) than controls for the following 4-chamber view measurements: 36% had a 4-chamber view area >90th centile; 28% had a 4-chamber view global sphericity index <10th centile; and 68% had a 4-chamber view width >90th centile. Only those fetuses with an abnormal umbilical artery pulsatility index had significant changes in ventricular size; 56% had a left ventricular area <10th centile; 28% had a right ventricular area <10th centile; 36% had right ventricular/left ventricular area ratio >90th centile. One or more of the above abnormal measurements were present in 92% of the fetuses. CONCLUSION: Higher rates of abnormalities of cardiac size and shape of the 4-chamber view were found in fetuses with an estimated fetal weight <10th centile, regardless of their umbilical artery pulsatility index and cerebroplacental ratio measurements. Those with a normal umbilical artery pulsatility index and an abnormal cerebroplacental ratio had larger and wider measurements of the 4-chamber view. In addition, the shape of the 4-chamber view was more globular or round than in controls. These fetuses may have an increased risk of perinatal complications and childhood and/or adult cardiovascular disease. Screening tools derived from the 4-chamber view, acting as surrogates for ventricular dysfunction, may identify fetuses who could benefit from further comprehensive testing and future preventive interventions.

Assessment of ventricular contractility in fetuses with an estimated fetal weight less than the tenth centile.

OBJECTIVE: To determine whether abnormal global, transverse, and longitudinal ventricular contractility of the heart in fetuses with an estimated fetal weight <10th centile is present, irrespective of Doppler studies of the umbilical artery and cerebroplacental ratio. STUDY DESIGN: This was a retrospective study of 50 fetuses with an estimated fetal weight <10th centile that were classified based on Doppler results from the pulsatility indices of the umbilical artery and middle cerebral artery, and the calculated cerebroplacental ratio (pulsatility indices of the umbilical artery/middle cerebral artery). Right and left ventricular measurements were categorized into 3 groups: (1) global ventricular contractility (fractional area change), (2) transverse ventricular contractility (24-segment transverse fractional shortening), and (3) basal-apical longitudinal contractility (longitudinal strain, longitudinal displacement fractional shortening, and basal lateral and septal wall annular plane systolic excursion). Z scores for the above measurements were computed for fetuses with an estimated fetal weight <10th centile using the mean and standard deviation derived from normal controls. Ventricular contractility measurements were considered abnormal if their Z score values were <5th centile (z score <-1.65) or >95th centile (Z score >1.65), depending on the specific ventricular measurement. RESULTS: The average gestational age at the time of the examination was 32 weeks 4 days (standard deviation 3 weeks 4 days). None of the 50 study fetuses demonstrated absent or reverse flow of the umbilical artery Doppler waveform. Eighty-eight percent (44/50) of fetuses had one or more abnormal measurements of cardiac contractility of 1 or both ventricles. Analysis of right ventricular contractility demonstrated 78% (39/50) to have 1 or more abnormal measurements, which were grouped as follows: global contractility 38% (19/50), transverse contractility 66% (33/50); and longitudinal contractility 48% (24/50). Analysis of left ventricular contractility demonstrated 1 or more abnormal measurements in 58% (29/50) that were grouped as follows: global contractility 38% (19/50); transverse contractility 40% (20/50); and longitudinal contractility 40% (20/50). Of the 50 study fetuses, 25 had normal pulsatility index of the umbilical artery and cerebroplacental ratios, 80% of whom had 1 or more abnormalities of right ventricular contractility and 56% of whom had 1 or more abnormalities of left ventricular contractility. Abnormal ventricular contractility for these fetuses was present in all 3 groups of measurements; global, transverse, and longitudinal. Those with an isolated abnormal pulsatility index of the umbilical artery (n=11) had abnormalities of transverse contractility of the right ventricular and global contractility in the left ventricle. When an isolated cerebroplacental ratio abnormality was present, the right ventricle demonstrated abnormal global, transverse, and longitudinal contractility, with the left ventricle only demonstrating abnormalities in transverse contractility. When both the pulsatility index of the umbilical artery and cerebroplacental ratio were abnormal (3/50), transverse and longitudinal contractility measurements were abnormal for both ventricles, as well as abnormal global contractility of the left ventricle. CONCLUSIONS: High rates of abnormal ventricular contractility were present in fetuses with an estimated fetal weight <10th centile, irrespective of the Doppler findings of the pulsatility index of the umbilical artery, and/or cerebroplacental ratio. Abnormalities of ventricular contractility were more prevalent in transverse measurements than global or longitudinal measurements. Abnormal transverse contractility was more common in the right than the left ventricle. Fetuses with estimated fetal weight less than the 10th centile may be considered to undergo assessment of ventricular contractility, even when Doppler measurements of the pulsatility index of the umbilical artery, and cerebroplacental ratio are normal.

Placental lipoprotein lipase activity is positively associated with newborn adiposity.

INTRODUCTION: Recent data suggest that in addition to glucose, fetal growth is related to maternal triglycerides (TG). To reach the fetus, TG must be hydrolyzed to free fatty acids (FFA) and transported across the placenta, but regulation is uncertain. Placental lipoprotein lipase (pLPL) hydrolyzes TG, both dietary chylomicron TG (CM-TG) and very-low density lipoprotein TG (VLDL-TG), to FFA. This may promote fetal fat accretion by increasing the available FFA pool for placental uptake. We tested the novel hypothesis that pLPL activity, but not maternal adipose tissue LPL activity, is associated with newborn adiposity and higher maternal TG. METHODS: Twenty mothers (n = 13 normal-weight; n = 7 obese) were prospectively recruited. Maternal glucose, insulin, TG (total, CM-TG, VLDL-TG), and FFA were measured at 14-16, 26-28, and 36-37 weeks, and adipose tissue LPL was measured at 26-28 weeks. At term delivery, placental villous biopsies were immediately analyzed for pLPL enzymatic activity. Newborn percent body fat (newborn %fat) was assessed by skinfolds. RESULTS: Placental LPL activity was positively correlated with birthweight (r = 0.48;P = 0.03) and newborn %fat (r = 0.59;P = 0.006), further strengthened by correcting for gestational age at delivery (r = 0.75;P = 0.0001), but adipose tissue LPL was not. Maternal TG and BMI were not correlated with pLPL activity. Additionally, pLPL gene expression, while modestly correlated with enzymatic activity (r = 0.53;P < 0.05), was not correlated with newborn adiposity. DISCUSSION: This is the first study to show a positive correlation between pLPL activity and newborn %fat. Placental lipase regulation and the role of pLPL in pregnancies characterized by nutrient excess and fetal overgrowth warrant further investigation.

Endothelial Progenitor Cells of the Human Placenta and Fetoplacental Circulation: A Potential Link to Fetal, Neonatal, and Long-term Health.

The fetoplacental circulation plays a key role in both short- and long-term outcomes, and aberrant flow indices as manifested by abnormal fetal Doppler velocimetry within this compartment have been associated with significant adverse consequences. These include fetal growth restriction, which often coexists with preeclampsia, and long-lasting medical issues as a result of both the underlying pathology and prematurity such as bronchopulmonary dysplasia, chronic lung disease, and neurodevelopmental delay. Furthermore, it is also clear that exposure to an abnormal in utero environment increases risk for long-term, adulthood issues such as cardiovascular disease. Endothelial progenitor cells (EPCs) have been implicated in vasculogenesis and angiogenesis, and they have been isolated from both human placenta and umbilical cord blood. This review outlines the extensive nomenclature of EPCs, summarizes existing literature surrounding human placental and umbilical cord blood EPCs, explores their potential role in pregnancy complications and adverse perinatal outcome, and highlights key areas where future investigations are needed.

Umbilical Cord Blood Circulating Progenitor Cells and Endothelial Colony-Forming Cells Are Decreased in Preeclampsia.

Preeclampsia (PE) is a pregnancy-specific disease characterized by the new onset of hypertension and proteinuria. Mothers with PE are known to develop endothelial dysfunction, but its effect on infants has been understudied, as newborns are often asymptomatic. Recent studies indicate that infants born from preeclamptic pregnancies develop endothelial dysfunction including higher blood pressure during childhood and an increased risk of stroke later in life. We hypothesize that PE reduces the number and function of fetal angiogenic progenitor cells and may contribute to this increased risk. We quantified 2 distinct types of angiogenic progenitors, pro-angiogenic circulating progenitor cells (CPCs) and endothelial colony-forming cells (ECFCs), from the umbilical cord blood of preeclamptic pregnancies and normotensive controls. Pro-angiogenic and nonangiogenic CPCs were enumerated via flow cytometry and ECFCs by cell culture. Additionally, we studied the growth, migration, and tube formation of ECFCs from PE and gestational age-matched normotensive control pregnancies. We found that PE resulted in decreased cord blood pro-angiogenic CPCs and ECFCs. Nonangiogenic CPCs were also decreased. Preeclamptic ECFCs demonstrated decreased growth and migration but formed tube-like structures in vitro similar to controls. Our results suggest that the preeclamptic environment alters the number and function of angiogenic progenitor cells and may increase the risk of later vascular disease.

Activation of protein kinase Cα increases phosphorylation of the UT-A1 urea transporter at serine 494 in the inner medullary collecting duct.

Hypertonicity increases urea transport, as well as the phosphorylation and membrane accumulation of UT-A1, the transporter responsible for urea permeability in the inner medullary collect duct (IMCD). Hypertonicity stimulates urea transport through PKC-mediated phosphorylation. To determine whether PKC phosphorylates UT-A1, eight potential PKC phosphorylation sites were individually replaced with alanine and subsequently transfected into LLC-PK1 cells. Of the single mutants, only ablation of the S494 site dampened induction of total UT-A1 phosphorylation by the PKC activator phorbol dibutyrate (PDBu). This result was confirmed using a newly generated antibody that specifically detected phosphorylation of UT-A1 at S494. Hypertonicity increased UT-A1 phosphorylation at S494. In contrast, activators of cAMP pathways (PKA and Epac) did not increase UT-A1 phosphorylation at S494. Activation of both PKC and PKA pathways increased plasma membrane accumulation of UT-A1, although activation of PKC alone did not do so. However, ablating the PKC site S494 decreased UT-A1 abundance in the plasma membrane. This suggests that the cAMP pathway promotes UT-A1 trafficking to the apical membrane where the PKC pathway can phosphorylate the transporter, resulting in increased UT-A1 retention at the apical membrane. In summary, activation of PKC increases the phosphorylation of UT-A1 at a specific residue, S494. Although there is no cross talk with the cAMP-signaling pathway, phosphorylation of S494 through PKC may enhance vasopressin-stimulated urea permeability by retaining UT-A1 in the plasma membrane.

Vasopressin regulation of multisite phosphorylation of UT-A1 in the inner medullary collecting duct.

Vasopressin signaling is critical for the regulation of urea transport in the inner medullary collecting duct (IMCD). Increased urea permeability is driven by a vasopressin-mediated elevation of cAMP that results in the direct phosphorylation of urea transporter (UT)-A1. The identification of cAMP-sensitive phosphorylation sites, Ser(486) and Ser(499), in the rat UT-A1 sequence was the first step in understanding the mechanism of vasopressin action on the phosphorylation-dependent modulation of urea transport. To investigate the significance of multisite phosphorylation of UT-A1 in response to elevated cAMP, we used highly specific and sensitive phosphosite antibodies to Ser(486) and Ser(499) to determine cAMP action at each phosphorylation site. We found that phosphorylation at both sites was rapid and sustained. Furthermore, the rate of phosphorylation of the two sites was similar in both mIMCD3 cells and rat inner medullary tissue. UT-A1 localized to the apical membrane in response to vasopressin was phosphorylated at Ser(486) and Ser(499). We confirmed that elevated cAMP resulted in increased phosphorylation of both sites by PKA but not through the vasopressin-sensitive exchange protein activated by cAMP pathway. These results elucidate the multisite phosphorylation of UT-A1 in response to cAMP, thus providing the beginning of understanding the intracellular factors underlying vasopressin stimulation of urea transport in the IMCD.