Proteomic studies of VEGFR2 in human placentas reveal protein associations with preeclampsia, diabetes, gravidity, and labor.

VEGFR2 (Vascular endothelial growth factor receptor 2) is a central regulator of placental angiogenesis. The study of the VEGFR2 proteome of chorionic villi at term revealed its partners MDMX (Double minute 4 protein) and PICALM (Phosphatidylinositol-binding clathrin assembly protein). Subsequently, the oxytocin receptor (OT-R) and vasopressin V1aR receptor were detected in MDMX and PICALM immunoprecipitations. Immunogold electron microscopy showed VEGFR2 on endothelial cell (EC) nuclei, mitochondria, and Hofbauer cells (HC), tissue-resident macrophages of the placenta. MDMX, PICALM, and V1aR were located on EC plasma membranes, nuclei, and HC nuclei. Unexpectedly, PICALM and OT-R were detected on EC projections into the fetal lumen and OT-R on 20-150 nm clusters therein, prompting the hypothesis that placental exosomes transport OT-R to the fetus and across the blood-brain barrier. Insights on gestational complications were gained by univariable and multivariable regression analyses associating preeclampsia with lower MDMX protein levels in membrane extracts of chorionic villi, and lower MDMX, PICALM, OT-R, and V1aR with spontaneous vaginal deliveries compared to cesarean deliveries before the onset of labor. We found select associations between higher MDMX, PICALM, OT-R protein levels and either gravidity, diabetes, BMI, maternal age, or neonatal weight, and correlations only between PICALM-OT-R (p < 2.7 × 10-8), PICALM-V1aR (p < 0.006), and OT-R-V1aR (p < 0.001). These results offer for exploration new partnerships in metabolic networks, tissue-resident immunity, and labor, notably for HC that predominantly express MDMX.

Review of Prediabetes and Hypertensive Disorders of Pregnancy.

Obesity and diabetes increase hypertensive disorders of pregnancy (HDP) risk, thus preventive interventions are heavily studied. How pregestational prediabetes and related interventions impact HDP risk is less characterized. Therefore, we searched and reviewed the literature to assess the impact on HDP risk of prediabetes and varied interventions. We identified 297 citations related to pregnancy, prediabetes, and early pregnancy interventions. We also reviewed the references and citations of included articles. We included five studies assessing HDP outcomes in women with first trimester hemoglobin A1c in the prediabetes range (5.7-6.4%). One prospective observational study demonstrated first trimester hemoglobin A1c (5.9-6.4%) is associated with increased HDP risk, while another prospective observational study and one retrospective observational study had similar trends without statistical significance. A small and underpowered randomized controlled trial demonstrated initiating gestational diabetes mellitus treatment (i.e., diet, monitoring, ± insulin) in response to first trimester hemoglobin A1c (5.7-6.4%) did not statistically reduce HDP compared with standard care. One retrospective observational study suggested metformin, when started early, may reduce HDP risk in patients with prediabetes. Pregestational prediabetes appears to increase HDP risk. Interventions (i.e., metformin, diet/glucose monitoring, and/or exercise) to reduce HDP risk require additional study with long-term follow-up.

Decreased LIN28B in preeclampsia impairs human trophoblast differentiation and migration.

Preeclampsia (PE) is a common cause of maternal morbidity, characterized by impaired trophoblast invasion and spiral artery transformation resulting in progressive uteroplacental hypoxia. Given the primary role of LIN28A and LIN28B in modulating cell metabolism, differentiation, and invasion, we hypothesized that LIN28A and/or LIN28B regulates trophoblast differentiation and invasion, and that its dysregulation may contribute to PE. Here we show that LIN28B is expressed ∼1300-fold higher than LIN28A in human term placenta and is the predominant paralog expressed in primary human trophoblast cultures. The expression of LIN28B mRNA and protein levels are significantly reduced in gestational age-matched preeclamptic vs. normal placentas, whereas LIN28A expression is not different. First trimester human placental sections displayed stronger LIN28B immunoreactivity in extravillous (invasive) cytotrophoblasts and syncytial sprouts vs. villous trophoblasts. LIN28B overexpression increased HTR8 cell proliferation, migration, and invasion, whereas LIN28B knockdown in JEG3 cells reduced cell proliferation. Moreover, LIN28B knockdown in JEG3 cells suppressed syncytin 1 (SYN-1), apelin receptor early endogenous ligand (ELABELA), and the chromosome 19 microRNA cluster, and increased mRNA expression of ITGß4 and TNF-α. Incubation of BeWo and JEG3 cells under hypoxia significantly decreased expression of LIN28B and LIN28A, SYN-1, and ELABELA, whereas TNF-α is increased. These results provide the first evidence that LIN28B is the predominant paralog in human placenta and that decreased LIN28B may play a role in PE by reducing trophoblast invasion and syncytialization, and by promoting inflammation.-Canfield, J., Arlier, S., Mong, E. F., Lockhart, J., VanWye, J., Guzeloglu-Kayisli, O., Schatz, F., Magness, R. R., Lockwood, C. J., Tsibris, J. C. M., Kayisli, U. A., Totary-Jain, H. Decreased LIN28B in preeclampsia impairs human trophoblast differentiation and migration.

Estrogen Receptors and Estrogen-Induced Uterine Vasodilation in Pregnancy.

Normal pregnancy is associated with dramatic increases in uterine blood flow to facilitate the bidirectional maternal-fetal exchanges of respiratory gases and to provide sole nutrient support for fetal growth and survival. The mechanism(s) underlying pregnancy-associated uterine vasodilation remain incompletely understood, but this is associated with elevated estrogens, which stimulate specific estrogen receptor (ER)-dependent vasodilator production in the uterine artery (UA). The classical ERs (ERα and ERß) and the plasma-bound G protein-coupled ER (GPR30/GPER) are expressed in UA endothelial cells and smooth muscle cells, mediating the vasodilatory effects of estrogens through genomic and/or nongenomic pathways that are likely epigenetically modified. The activation of these three ERs by estrogens enhances the endothelial production of nitric oxide (NO), which has been shown to play a key role in uterine vasodilation during pregnancy. However, the local blockade of NO biosynthesis only partially attenuates estrogen-induced and pregnancy-associated uterine vasodilation, suggesting that mechanisms other than NO exist to mediate uterine vasodilation. In this review, we summarize the literature on the role of NO in ER-mediated mechanisms controlling estrogen-induced and pregnancy-associated uterine vasodilation and our recent work on a "new" UA vasodilator hydrogen sulfide (H2S) that has dramatically changed our view of how estrogens regulate uterine vasodilation in pregnancy.

Structural analysis of estrogen receptors: interaction between estrogen receptors and cav-1 within the caveolae†.

Pregnancy is a physiologic state of substantially elevated estrogen biosynthesis that maintains vasodilator production by uterine artery endothelial cells (P-UAECs) and thus uterine perfusion. Estrogen receptors (ER-α and ER-ß; ESR1 and ESR2) stimulate nongenomic rapid vasodilatory responses partly through activation of endothelial nitric oxide synthase (eNOS). Rapid estrogenic responses are initiated by the ∼4% ESRs localized to the plasmalemma of endothelial cells. Caveolin-1 (Cav-1) interactions within the caveolae are theorized to influence estrogenic effects mediated by both ESRs. Hypothesis: Both ESR1 and ESR2 display similar spatial partitioning between the plasmalemma and nucleus of UAECs and have similar interactions with Cav-1 at the plasmalemma. Using transmission electron microscopy, we observed numerous caveolae structures in UAECs, while immunogold labeling and subcellular fractionations identified ESR1 and ESR2 in three subcellular locations: membrane, cytosol, and nucleus. Bioinformatics approaches to analyze ESR1 and ESR2 transmembrane domains identified no regions that facilitate ESR interaction with plasmalemma. However, sucrose density centrifugation and Cav-1 immunoisolation columns uniquely demonstrated very high protein-protein association only between ESR1, but not ESR2, with Cav-1. These data demonstrate (1) both ESRs localize to the plasmalemma, cytosol and nucleus; (2) neither ESR1 nor ESR2 contain a classic region that crosses the plasmalemma to facilitate attachment; and (3) ESR1, but not ESR2, can be detected in the caveolar subcellular domain demonstrating ESR1 is the only ESR bound in close proximity to Cav-1 and eNOS within this microdomain. Lack of protein-protein interaction between Cav-1 and ESR2 demonstrates a novel independent association of these proteins at the plasmalemma.

Ovine uterine artery hydrogen sulfide biosynthesis in vivo: effects of ovarian cycle and pregnancy†.

Uterine vasodilation dramatically increases during the follicular phase of the estrous cycle and pregnancy, which are estrogen-dominant physiological states. Uterine vasodilation is believed to be mainly controlled by local uterine artery (UA) production of vasodilators and angiogenic factors. The extremely potent vasodilator and proangiogenic hydrogen sulfide (H2S) is synthesized via metabolizing L-cysteine by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH). This study was designed to determine if UA H2S production increases with augmented expression and/or activity of CBS and/or CTH during the ovarian cycle and pregnancy in sheep. Uterine arteries from intact nonpregnant (NP) luteal and follicular phase and late (130-135 days, term ≈ 145 days) pregnant (P) ewes were collected; endothelium-enriched proteins (UAendo) and endothelium-denuded smooth muscle (UAvsm) were mechanically prepared for accessing CBS and CTH proteins by immunoblotting; their cellular localization was determined by semi-quantitative immunofluorescence microscopy. H2S production was measured by the methylene blue assay. Immunoblotting revealed that CBS but not CTH protein was greater in P > > > NP follicular > luteal UAendo and UAvsm (P < 0.001). H2S production was greater in P > > > NP UAendo and UAvsm (P < 0.01). Pregnancy-augmented UAendo and UAvsm H2S production was inhibited by the specific CBS but not CTH inhibitor. CBS and CTH proteins were localized to both endothelium and smooth muscle; however, only CBS protein was significantly greater in P vs NP UA endothelium and smooth muscle. Thus, ovine UA H2S production is significantly augmented via selectively upregulating endothelium and smooth muscle CBS during the follicular phase and pregnancy in vivo.

E2ß stimulates ovine uterine artery endothelial cell H2S production in vitro by estrogen receptor-dependent upregulation of cystathionine ß-synthase and cystathionine γ-lyase expression†.

Endogenous hydrogen sulfide (H2S) is a potent vasodilator and proangiogenic second messenger synthesized from L-cysteine by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH). Estrogens are potent vasodilators that stimulate H2S biosynthesis in uterine arteries (UA) in vivo; however, the underlying mechanisms are unknown. We hypothesized that estrogens stimulate H2S biosynthesis in UA endothelial cells (UAEC) via specific estrogen receptor (ER)-dependent mechanisms. In cultured primary UAEC, treatment with estradiol-17ß (E2ß) stimulated CBS and CTH mRNAs and proteins in a time- and concentration-dependent fashion. As little as 0.1 nM E2ß was effective in increasing CBS and CTH expressions and these stimulatory effects maximized with 10-100 nM E2ß at 48-72 h. E2ß also activated CBS and CTH promoters in UAEC, leading to CBS and CTH expression. Treatment with E2ß stimulated H2S production, which was blocked by specific inhibitors of either CBS or CTH and their combination and the ER antagonist ICI 182780. Treatment with either specific agonist of ERα or ERß stimulated both CBS and CTH mRNA and protein expressions and H2S production to levels similar to that of E2ß. Specific antagonist of either ERα or ERß blocked E2ß-stimulated CBS and CTH mRNA and protein expressions and H2S production. Combinations of either ERα or ERß agonists or their antagonists had no additive effects. Thus, E2ß stimulates H2S production by upregulating CBS and CTH mRNA and protein expressions through specific ERα or ERß-dependent CBS and CTH transcription in UAEC in vitro.

Uteroplacental and Fetal 4D Flow MRI in the Pregnant Rhesus Macaque.

BACKGROUND: Pregnancy complications are often associated with poor uteroplacental vascular adaptation and standard diagnostics are unable to reliably quantify flow in all uteroplacental vessels and have poor sensitivity early in gestation. PURPOSE: To investigate the feasibility of using 4D flow MRI to assess total uteroplacental blood flow in pregnant rhesus macaques as a precursor to human studies. STUDY TYPE: Retrospective feasibility study. ANIMAL MODEL: Fifteen healthy, pregnant rhesus macaques ranging from the 1st trimester to 3rd trimester of gestation. FIELD STRENGTH/SEQUENCE: Abdominal 4D flow MRI was performed on a 3.0T scanner with a radially undersampled phase contrast (PC) sequence. Reference ferumoxytol-enhanced angiograms were acquired with a 3D ultrashort echo time sequence with a center-out radial trajectory. ASSESSMENT: Repeatability of flow measurements was assessed with scans performed same-day and on consecutive days in the uterine arteries and ovarian veins. In-flow was compared against out-flow in the uterus, umbilical cord, and fetal heart with a conservation of mass analysis. Conspicuity of uteroplacental vessels was qualitatively compared between PC angiograms derived from 4D flow data and ferumoxytol-enhanced angiograms. STATISTICAL TESTS: Bland-Altman analysis was used to quantify same-day and consecutive-day repeatability. RESULTS: Same-day flow measurements showed an average difference between scans of 13% in both the uterine arteries and ovarian veins, while consecutive-day measurements showed average differences of 22% and 24%, respectively. Comparisons of in-flow and out-flow showed average differences of 15% in the uterus, 8% in fetal heart, and 15% in the umbilical cord. PC angiograms showed similar depiction of main uteroplacental vessels as high-resolution, ferumoxytol-enhanced angiograms. DATA CONCLUSION: 4D flow MRI could be used in the rhesus macaque for repeatable flow measurements in the uteroplacental and fetal vasculature, setting the stage for future human studies. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2019;49:534-545.

Metformin's impact on statin-associated muscle symptoms: An analysis of ACCORD study data and research materials from the NHLBI Biologic Specimen and Data Repository Information Coordinating Center.

Statins are widely prescribed, yet statin muscle pain limits their use, leading to increased cardiovascular risk. No validated therapy for statin muscle pain exists. The goal of the study was to assess whether metformin was associated with reduced muscle pain. A secondary analysis of data from the ACCORD trial was performed. An ACCORD sub-study assessed patients for muscle cramps and leg/calve pain while walking, typical non-severe statin muscle pain symptoms. We compared muscle pain between patients using a statin (n = 445) or both a statin and metformin (n = 869) at baseline. Overall patient characteristics were balanced between groups. Unadjusted analysis showed fewer reports of muscle cramps (35%) and leg/calve pain while walking (40%) with statins and metformin compared to statin only (muscle cramps, 42%; leg/calve pain while walking, 47%). Multivariable regression demonstrated a 22% odds reduction for muscle cramps (P = 0.049) and a 29% odds reduction for leg/calve pain while walking (P = 0.01). Metformin appears to reduce the risk of non-severe statin muscle pain and additional research is needed to confirm the findings and assess metformin's impact on statin adherence and related cardiovascular outcomes.

Convergent ERK1/2, p38 and JNK mitogen activated protein kinases (MAPKs) signalling mediate catecholoestradiol-induced proliferation of ovine uterine artery endothelial cells.

KEY POINTS: The catechol metabolites of 17ß-oestradiol (E2 ß), 2-hydroxyoestradiol (2-OHE2 ) and 4-hydroxyoestradiol (4-OHE2 ), stimulate proliferation of pregnancy-derived ovine uterine artery endothelial cells (P-UAECs) through ß-adrenoceptors (ß-ARs) and independently of the classic oestrogen receptors (ERs). Herein we show that activation of ERK1/2, p38 and JNK mitogen activated protein kinases (MAPKs) is necessary for 2-OHE2 - and 4-OHE2 -induced P-UAEC proliferation, as well as proliferation induced by the parent hormone E2 ß and other ß-AR signalling hormones (i.e. catecholamines). Conversely, although 2-OHE2 and 4-OHE2 rapidly activate phosphatidylinositol 3-kinase (PI3K), its activation is not involved in catecholoestradiol-induced P-UAEC proliferation. We also show for the first time the signalling mechanisms involved in catecholoestradiol-induced P-UAEC proliferation; which converge at the level of MAPKs with the signalling mechanisms mediating E2 ß- and catecholamine-induced proliferation. The present study advances our understanding of the complex signalling mechanisms involved in regulating uterine endothelial cell proliferation during pregnancy. ABSTRACT: Previously we demonstrated that the biologically active metabolites of 17ß-oestradiol, 2-hydroxyoestradiol (2-OHE2 ) and 4-hydroxyoestradiol (4-OHE2 ), stimulate pregnancy-specific proliferation of uterine artery endothelial cells derived from pregnant (P-UAECs), but not non-pregnant ewes. However, unlike 17ß-oestradiol, which induces proliferation via oestrogen receptor-ß (ER-ß), the catecholoestradiols mediate P-UAEC proliferation via ß-adrenoceptors (ß-AR) and independently of classic oestrogen receptors. Herein, we aim to further elucidate the signalling mechanisms involved in proliferation induced by catecholoestradiols in P-UAECs. P-UAECs were treated with 2-OHE2 and 4-OHE2 for 0, 0.25, 0.5, 1, 2, 4, 12 and 24 h, to analyse activation of mitogen activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K)-AKT. Specific inhibitors for ERK1/2 MAPK (PD98059), p38 MAPK (SB203580), JNK MAPK (SP600125), or PI3K (LY294002) were used to determine the involvement of individual kinases in agonist-induced P-UAEC proliferation. 2-OHE2 and 4-OHE2 stimulated biphasic phosphorylation of ERK1/2, slow p38 and JNK phosphorylation over time, and rapid monophasic AKT phosphorylation. Furthermore, ERK1/2, p38 and JNK MAPKs, but not PI3K, were individually necessary for catecholoestradiol-induced proliferation. In addition, when comparing the signalling mechanisms of the catecholoestradiols, to 17ß-oestradiol and catecholamines, we observed that convergent MAPKs signalling pathways facilitate P-UAEC proliferation induced by all of these hormones. Thus, all three members of the MAPK family mediate the mitogenic effects of catecholoestradiols in the endothelium during pregnancy. Furthermore, the convergent signalling of MAPKs involved in catecholoestradiol-, 17ß-oestradiol- and catecholamine-induced endothelial cell proliferation may be indicative of unappreciated evolutionary functional redundancy to facilitate angiogenesis and ensure maintenance of uterine blood flow during pregnancy.

Uterine artery leptin receptors during the ovarian cycle and pregnancy regulate angiogenesis in ovine uterine artery endothelial cells†.

Leptin regulates body weight, reproductive functions, blood pressure, endothelial function, and fetoplacental angiogenesis. Compared to the luteal phase, the follicular phase and pregnancy are physiological states of elevated estrogen, angiogenesis, and uterine blood flow (UBF). Little is known concerning regulation of uterine artery (UA) angiogenesis by leptin and its receptors. We hypothesized that (1) ex vivo expression of leptin receptors (LEPR) in UA endothelium (UAendo) and UA vascular smooth muscle (UAvsm) is elevated in pregnant versus nonpregnant (Luteal and Follicular) sheep; (2) in vitro leptin treatments differentially modulate mitogenesis in uterine artery endothelial cells from pregnant (P-UAECs) more than in nonpregnant (NP-UAECs) ewes; and (3) LEPR are upregulated in P-UAECs versus NP-UAECs in association with leptin activation of phospho-STAT3 signaling. Local UA adaptations were evaluated using a unilateral pregnant sheep model where prebreeding uterine horn isolation (nongravid) restricted gravidity to one horn. Immunolocalization revealed LEPR in UAendo and UAvsm from pregnant and nonpregnant sheep. Contrary to our hypothesis, western analysis revealed that follicular UAendo and UAvsm LEPR were greater than luteal, nongravid, gravid, and control pregnant. Compared to pregnant groups, LEPR were elevated in renal artery endothelium of follicular and luteal sheep. Leptin treatment significantly increased mitogenesis in follicular phase NP-UAECs and P-UAECs, but not luteal phase NP-UAECs. Although UAEC expression of LEPR was similar between groups, leptin treatment only activated phospho-STAT3 in follicular NP-UAECs and P-UAECs. Thus, leptin may play an angiogenic role particularly in preparation for the increased UBF during the periovulatory period and subsequently to meet the demands of the growing fetus.

Ovine uterine space restriction causes dysregulation of the renin-angiotensin system in fetal kidneys.

In ovine pregnancy, uterine space restriction (USR) resulting from decreased space for placental attachment caused intrauterine growth restriction and impaired nephrogenesis. The fetal kidney renin-angiotensin system (RAS) is involved in nephrogenesis, fluid balance, and iron deposition. Angiotensin II exerts its effects via multiple receptors: angiotensin II 1-8 receptor type 1 (AT 1 R) and type 2 (AT 2 R), and angiotensin II 1-7 Mas receptor (MASR). Objective: : To test the hypothesis that ovine USR is associated with dysregulation of the fetal renal RAS. Methods: : Multiparous pregnant ewes (n = 32), 16 with surgical bifurcated disconnection of one uterine horn to further reduce placental attachment sites, were studied. USR (n = 31) ovine fetuses were compared to nonspace restricted (NSR) singleton controls (n = 22) on gestational day (GD) 120 or GD130, term GD147. Fetal plasma was collected to evaluate plasma renin activity and iron indices. Fetal kidney AT 1 R, AT 2 R, and MASR proteins were assessed by Western immunoblotting and immunohistochemistry. Results: : AT 1 R, AT 2 R, and MASR protein expression was higher in USR at GD130 than aged-matched NSR and USR at GD120, ( P < 0.05 all). AT 1 R and AT 2 R localization was homogenous throughout proximal and distal tubules in both USR and NSR at both gestational dates. MASR localization was punctate throughout renal cortical structures including tubules and glomeruli in both USR and NSR, shifted to intranuclear at GD130. Plasma renin activity was inversely related to plasma osmolarity ( P < 0.02) and was downregulated in USR at GD130 ( P < 0.05). Conclusions: : By late gestation, USR upregulated renal angiotensin receptor expression, an effect with potential functional implications.

Identification of Differential ER-Alpha Versus ER-Beta Mediated Activation of eNOS in Ovine Uterine Artery Endothelial Cells.

Endothelial nitric oxide (NO) production is partly responsible for maintenance of uterine vasodilatation during physiologic states of high circulating estrogen levels, e.g., pregnancy. Although 3%-5% of estrogen receptors (ER-alpha/beta) localize to the endothelial plasmalemma, these receptors are responsible for the nongenomic vasodilator responses. Estradiol induces endothelial NO synthase (eNOS) activation to increase NO production; however, it is unknown if eNOS regulation is dependent on both ERs. We hypothesize that ER-alpha and/or ER-beta are capable of changing eNOS phosphorylation and increasing NO production in uterine artery endothelial cells (UAECs). UAECs were 1) treated with vehicle or increasing concentrations (0.1-100 nM) or timed treatments (0-30 min) of estradiol and 2) pretreated with the inhibitors ICI 182,780 (nonspecific ER), 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP; ER-alpha specific), or 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP; ER-beta specific) followed by estradiol to analyze the changes in eNOS stimulatory (Ser1177)eNOS and (Ser635)eNOS versus inhibitory (Thr495)eNOS via Western blot analysis. UAECs were also pretreated with MPP, PHTPP, or MPP + PHTTP followed by estradiol or treated with the agonists estradiol, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol, 2,3-bis(4-hydroxyphenyl)-propionitrile, or ATP to quantify total NOx levels (NO2+NO3). Estrogen and ER-alpha activation induced an increase in (Ser1177)eNOS and (Ser635)eNOS, a decrease in (Thr495)eNOS, and an increase in NOx levels. In contrast, ER-beta activation only reduced (Thr495)eNOS without changes in (Ser1177)eNOS or (Ser635)eNOS. However, ER-beta activation increased NOx levels. Lastly, the antagonism of both receptors induced a reduction in basal and stimulated NOx levels in UAECs. These data demonstrate that 1) eNOS phosphorylation changes occur via ER-alpha- and ER-beta-dependent mechanisms and 2) ER-alpha and ER-beta can both increase NO levels independently from each other.

Dietary-induced gestational iron deficiency inhibits postnatal tissue iron delivery and postpones the cessation of active nephrogenesis in rats.

Gestational iron deficiency (ID) can alter developmental programming through impaired nephron endowment, leading to adult hypertension, but nephrogenesis is unstudied. Iron status and renal development during dietary-induced gestational ID (<6 mg Fe kg-1 diet from Gestational Day 2 to Postnatal Day (PND) 7) were compared with control rats (198 mg Fe kg-1 diet). On PND2-PND10, PND15, PND30 and PND45, blood and tissue iron status were assessed. Nephrogenic zone maturation (PND2-PND10), radial glomerular counts (RGCs), glomerular size density and total planar surface area (PND15 and PND30) were also assessed. Blood pressure (BP) was measured in offspring. ID rats were smaller, exhibiting lower erythrocyte and tissue iron than control rats (PND2-PND10), but these parameters returned to control values by PND30-PND45. Relative kidney iron (µg g-1 wet weight) at PND2-PND10 was directly related to transport iron measures. In ID rats, the maturation of the active nephrogenic zone was later than control. RGCs, glomerular size, glomerular density, and glomerular planar surface area were lower than control at PND15, but returned to control by PND30. After weaning, the kidney weight/rat weight ratio (mg g-1) was heavier in ID than control rats. BP readings at PND45 were lower in ID than control rats. Altered kidney maturation and renal adaptations may contribute to glomerular size, early hyperfiltration and long-term renal function.

Pregnancy induces persistent changes in vascular compliance in primiparous women.

OBJECTIVE: Pregnancy induces rapid, progressive, and substantial changes to the cardiovascular system. The low recurrence risk of preeclampsia, despite familial predisposition, suggests an adaptation associated with pregnancy that attenuates the risk for subsequent preeclampsia. We aimed to evaluate the persistent effect of pregnancy on maternal cardiovascular physiology. STUDY DESIGN: Forty-five healthy nulliparous women underwent baseline cardiovascular assessment before conception and repeated an average of 30 months later. After baseline evaluation, 17 women conceived singleton pregnancies and all delivered at term. The remaining 28 women comprised the nonpregnant control group. We measured mean arterial blood pressure, cardiac output, plasma volume, pulse wave velocity, uterine blood flow, and flow-mediated vasodilation at each visit. RESULTS: There was a significant decrease in mean arterial pressure from the prepregnancy visit to postpartum in women with an interval pregnancy (prepregnancy, 85.3±1.8; postpartum, 80.5±1.8 mm Hg), with no change in nonpregnant control subjects (visit 1, 80.3±1.4; visit 2, 82.8±1.4 mm Hg) (P=.002). Pulse wave velocity was significantly decreased in women with an interval pregnancy (prepregnancy, 2.73±0.05; postpartum, 2.49±0.05 m/s), as compared with those without an interval pregnancy (visit 1, 2.56±0.04; visit 2, 2.50±0.04 m/s) (P=.005). We did not observe a residual effect of pregnancy on cardiac output, plasma volume, uterine blood flow, or flow-mediated vasodilation. CONCLUSION: Our observations of decreased mean arterial pressure and reduced arterial stiffness following pregnancy suggest a significant favorable effect of pregnancy on maternal cardiovascular remodeling. These findings may represent a mechanism by which preeclampsia risk is reduced in subsequent pregnancies.

Gap junction regulation of vascular tone: implications of modulatory intercellular communication during gestation.

In the vasculature, gap junctions (GJ) play a multifaceted role by serving as direct conduits for cell-cell intercellular communication via the facilitated diffusion of signaling molecules. GJs are essential for the control of gene expression and coordinated vascular development in addition to vascular function. The coupling of endothelial cells to each other, as well as with vascular smooth muscle cells via GJs, plays a relevant role in the control of vasomotor tone, tissue perfusion and arterial blood pressure. The regulation of cell-signaling is paramount to cardiovascular adaptations of pregnancy. Pregnancy requires highly developed cell-to-cell coupling, which is affected partly through the formation of intercellular GJs by Cx43, a gap junction protein, within adjacent cell membranes to help facilitate the increase of uterine blood flow (UBF) in order to ensure adequate perfusion for nutrient and oxygen delivery to the placenta and thus the fetus. One mode of communication that plays a critical role in regulating Cx43 is the release of endothelial-derived vasodilators such as prostacyclin (PGI2) and nitric oxide (NO) and their respective signaling mechanisms involving second messengers (cAMP and cGMP, respectively) that are likely to be important in maintaining UBF. Therefore, the assertion we present in this review is that GJs play an integral if not a central role in maintaining UBF by controlling rises in vasodilators (PGI2 and NO) via cyclic nucleotides. In this review, we discuss: (1) GJ structure and regulation; (2) second messenger regulation of GJ phosphorylation and formation; (3) pregnancy-induced changes in cell-signaling; and (4) the role of uterine arterial endothelial GJs during gestation. These topics integrate the current knowledge of this scientific field with interpretations and hypotheses regarding the vascular effects that are mediated by GJs and their relationship with vasodilatory vascular adaptations required for modulating the dramatic physiological rises in uteroplacental perfusion and blood flow observed during normal pregnancy.

Ovine uterine space restriction alters placental transferrin receptor and fetal iron status during late pregnancy.

BACKGROUND: Fetal growth restriction is reported to be associated with impaired placental iron transport. Transferrin receptor (TfR) is a major placental iron transporter in humans but has not been studied in sheep. TfR is regulated by both iron and nitric oxide (NO), the molecule produced by endothelial nitric oxide synthase (eNOS). We hypothesized that limited placental development downregulates both placental TfR and eNOS expression, thereby lowering fetal tissue iron. METHODS: An ovine surgical uterine space restriction (USR) model, combined with multifetal gestation, tested the extremes of uterine and placental adaptation. Blood, tissues, and placentomes from non-space restricted (NSR) singletons were compared with USR fetuses at gestational day (GD) 120 or 130. RESULTS: When expressed proportionate to fetal weight, liver iron content did not differ, whereas renal iron was higher in USR vs. NSR fetuses. Renal TfR protein expression did not differ, but placental TfR expression was lower in USR fetuses at GD130. Placental levels of TfR correlated to eNOS. TfR was localized throughout the placentome, including the hemophagous zone, implicating a role for TfR in ovine placental iron transport. CONCLUSION: Fetal iron was regulated in an organ-specific manner. In USR fetuses, NO-mediated placental adaptations may prevent the normal upregulation of placental TfR at GD130.

Endothelial caveolar subcellular domain regulation of endothelial nitric oxide synthase.

Complex regulatory processes alter the activity of endothelial nitric oxide synthase (eNOS) leading to nitric oxide (NO) production by endothelial cells under various physiological states. These complex processes require specific subcellular eNOS partitioning between plasma membrane caveolar domains and non-caveolar compartments. Translocation of eNOS from the plasma membrane to intracellular compartments is important for eNOS activation and subsequent NO biosynthesis. We present data reviewing and interpreting information regarding: (i) the coupling of endothelial plasma membrane receptor systems in the caveolar structure relative to eNOS trafficking; (ii) how eNOS trafficking relates to specific protein-protein interactions for inactivation and activation of eNOS; and (iii) how these complex mechanisms confer specific subcellular location relative to eNOS multisite phosphorylation and signalling. Dysfunction in the regulation of eNOS activation may contribute to several disease states, in particular gestational endothelial abnormalities (pre-eclampsia, gestational diabetes etc.), that have life-long deleterious health consequences that predispose the offspring to develop hypertensive disease, Type 2 diabetes and adiposity.