Cotyledon-Specific Flow Evaluation of Rhesus Macaque Placental Injury Using Ferumoxytol Dynamic Contrast-Enhanced MRI.

BACKGROUND: Recently, dynamic contrast-enhanced (DCE) MRI with ferumoxytol as contrast agent has recently been introduced for the noninvasive assessment of placental structure and function throughout. However, it has not been demonstrated under pathological conditions. PURPOSE: To measure cotyledon-specific rhesus macaque maternal placental blood flow using ferumoxytol DCE MRI in a novel animal model for local placental injury. STUDY TYPE: Prospective animal model. SUBJECTS: Placental injections of Tisseel (three with 0.5 mL and two with 1.5 mL), monocyte chemoattractant protein 1 (three with 100 µg), and three with saline as controls were performed in a total of 11 rhesus macaque pregnancies at approximate gestational day (GD 101). DCE MRI scans were performed prior (GD 100) and after (GD 115 and GD 145) the injection (term = GD 165). FIELD STRENGTH/SEQUENCE: 3 T, T1-weighted spoiled gradient echo sequence (product sequence, DISCO). ASSESSMENT: Source images were inspected for motion artefacts from the mother or fetus. Placenta segmentation and DCE processing were performed for the dynamic image series to measure cotyledon specific volume, flow, and normalized flow. Overall placental histopathology was conducted for controls, Tisseel, and MCP-1 animals and regions of tissue infarctions and necrosis were documented. Visual inspections for potential necrotic tissue were conducted for the two Tisseelx3 animals. STATISTICAL TESTS: Wilcoxon rank sum test, significance level P < 0.05. RESULTS: No motion artefacts were observed. For the group treated with 1.5 mL of Tisseel, significantly lower cotyledon volume, flow, and normalized flow per cotyledon were observed for the third gestational time point of imaging (day ~145), with mean normalized flow of 0.53 minute-1 . Preliminary histopathological analysis shows areas of tissue necrosis from a selected cotyledon in one Tisseel-treated (single dose) animal and both Tisseelx3 (triple dose) animals. DATA CONCLUSION: This study demonstrates the feasibility of cotyledon-specific functional analysis at multiple gestational time points and injury detection in a placental rhesus macaque model through ferumoxytol-enhanced DCE MRI. LEVEL OF EVIDENCE: NA TECHNICAL EFFICACY: Stage 2.

Src kinase partially mediates cytokine-induced endothelial dysfunction.

OBJECTIVES: Endothelial dysfunction is known to be a key characteristic of preeclampsia (PE) and can contribute to progression of symptoms and injury to multiple organ systems. Delivery is the only treatment for progression of PE, but development of an endothelial-based therapy for PE presents a promising strategy. Growth factors and cytokines are dysregulated in PE and can impact endothelial function, manifesting changes in Ca2+ signaling and interruptions in monolayer barrier function that contribute to symptoms of hypertension, proteinuria, and edema. In this study, we highlight Src kinase as a partial mediator of growth factor and cytokine mediated endothelial dysfunction. STUDY DESIGN: Fura-2 Ca2+ imaging and Electrical Cell Impedance Sensing (ECIS) assays are performed on growth factor or cytokine exposed human umbilical vein endothelial cells (HUVECs). Inhibitors to MEK/ERK (U0126) or Src (PP2) are used to determine the contribution of kinase signaling pathways. MAIN OUTCOME MEASURES: Decreases in HUVEC Ca2+ signaling or monolayer resistance measure endothelial dysfunction. Reversal of endothelial dysfunction by kinase inhibitors reveals the respective contibutions of MEK/ERK and Src kinase. RESULTS: We show that Src inhibition protects Ca2+ signaling responses against insults induced by VEGF165, bFGF, PlGF, TNFα, and IL-1ß. Additionally, we show that Src inhibition protects the endothelial monolayer from the full impact of TNFα insult. Further, we find that MEK/ERK inhibition does not offer protection from growth factor-mediated endothelial dysfunction. CONCLUSIONS: The results of this study suggest cytokine and growth factor-stimulated Src kinase plays a partial role on promoting endothelial dysfunction in HUVECs.

Ferumoxytol dynamic contrast enhanced magnetic resonance imaging identifies altered placental cotyledon perfusion in rhesus macaques†.

Identification of placental dysfunction in early pregnancy with noninvasive imaging could be a valuable tool for assessing maternal and fetal risk. Dynamic contrast enhanced (DCE) magnetic resonance imaging (MRI) can be a powerful tool for interrogating placenta health. After inoculation with Zika virus or sham inoculation at gestation age (GA) 45 or 55 days, animals were imaged up to three times at GA65, GA100, and GA145. DCE MRI images were acquired at all imaging sessions using ferumoxytol, an iron nanoparticle-based contrast agent, and analyzed for placental intervillous blood flow, number of perfusion domains, and perfusion domain volume. Cesarean section was performed at GA155, and the placenta was photographed and dissected for histopathology. Photographs were used to align cotyledons with estimated perfusion domains from MRI, allowing comparison of estimated cotyledon volume to pathology. Monkeys were separated into high and low pathology groups based on the average number of pathologies present in the placenta. Perfusion domain flow, volume, and number increased through gestation, and total blood flow increased with gestation for both low pathology and high pathology groups. A statistically significant decrease in perfusion domain volume associated with pathology was detected at all gestational ages. Individual perfusion domain flow comparisons demonstrated a statistically significant decrease with pathology at GA100 and GA145, but not GA65. Since ferumoxytol is currently used to treat anemia during human pregnancy and as an off-label MRI contrast agent, future transition of this work to human pregnancy may be possible.

Cytokine concentrations direct endothelial function in pregnancy and preeclampsia.

Endothelial dysfunction is a prominent feature of preeclampsia, a hypertensive disorder of pregnancy, and contributes to multiple symptoms characteristic of the syndrome. A myriad of growth factors and cytokines are dysregulated in preeclampsia as compared to normal pregnancy, however, a complete appreciation of the effect of changing concentrations of these factors on endothelial function is lacking. In this study, we evaluate the effect of a variety of growth factors and cytokines on Ca2+ signaling and monolayer integrity. We report that VEGF165, TNFα, EGF, and IL-1ß either improve or inhibit Ca2+ signaling depending on dose, whereas TNFα and IL-1ß reduce monolayer integrity and bFGF increases monolayer integrity. Additionally, to model the effects of combinations of growth factors and cytokines, we screened for Ca2+ signaling changes in response to 16 dose combinations of VEGF165 and TNFα together. This revealed an optimal combination capable of supporting pregnancy-adapted Ca2+ signaling, and that changes in either VEGF165 or TNFα dose would result in a shift toward suppressed function. This study shows in detail how growth factor or cytokine concentration effects endothelial cell function. Such data can be used to model how changing growth factor and cytokine levels in normal pregnancy may contribute to healthy endothelial function and in preeclampsia may promote endothelial dysfunction. The results of VEGF165 and TNFα combination treatments suggest that more complex growth factor and cytokine combination modeling may be important in order to more accurately understand the effects of circulating factors on the endothelial function.

Conjugated linoleic acid improves endothelial Ca2+ signaling by blocking growth factor and cytokine-mediated Cx43 phosphorylation.

Sustained Ca2+ burst signaling is crucial for endothelial vasodilator production and is disrupted by growth factors and cytokines. Conjugated linoleic acid (CLA), a Src inhibitor in certain preparations, is generally regarded as safe during pregnancy by the FDA. Multiple CLA preparations; t10, c12 or c9, t11 CLA, or a 1:1 mixture of the two were administered before growth factor or cytokine treatment. Growth factors and cytokines caused a significant decrease in Ca2+ burst numbers in response to ATP stimulation. Both t10, c12 CLA and the 1:1 mixture rescued VEGF165 or TNFα inhibited Ca2+ bursts and correlated with Src-specific phosphorylation of connexin 43. VEGF165, TNFα, and IL-6 in combination at physiologic concentrations revealed IL-6 amplified the inhibitory effects of lower dose of VEGF165 and TNFα. Again, the 1:1 CLA mixture was most effective at rescue of function. Therefore, CLA formulations may be a promising treatment for endothelial dysfunction in diseases such as preeclampsia.

Quantitative ferumoxytol-enhanced MRI in pregnancy: A feasibility study in the nonhuman primate.

OBJECTIVES: To assess the feasibility of ferumoxytol-enhanced MRI in pregnancy with a nonhuman primate model. MATERIALS AND METHODS: In this prospective study, eleven pregnant rhesus macaques at day 98 ±â€¯5 of gestation were divided into three groups, untreated control (UC) (n = 3), saline control (SC) (n = 4) and interleukin 1 beta (IL-1ß) treated (IT) (n = 4), which were administered with either saline or IL-1ß into the amniotic fluid. All animals were imaged at multiple time points before and after ferumoxytol administration (4 mg/kg). Longitudinal R2* and susceptibility of tissues were obtained using region-of-interest analysis and the longitudinal changes were assessed using linear mixed models and Student's t-test. RESULTS: In fetuses, a slope of 0.3 s-1/day (P = 0.008), 0.00 ppm/day (P = 0.699) and - 0.2 s-1/day (P = 0.023) was observed in liver R2*, liver susceptibility, and lung R2*, respectively. In placentas, R2* and susceptibility increased immediately after ferumoxytol administration (P < 0.001) and decreased to baseline within two days. The mean change from baseline showed no significant difference between the SC group and the IT group at all scan time points. In maternal livers, R2* increased immediately after ferumoxytol administration, further increased at one-day, and then decreased but remained elevated (P < 0.001). The mean change from baseline showed no significant difference between the SC group and the IT group at all scan time points. CONCLUSIONS: This work demonstrates the feasibility of quantitative ferumoxytol-enhanced MRI to measure dynamics of ferumoxytol delivery and washout in the placenta. Stable MRI measurements indicated no evidence of iron deposition in fetal tissues of nonhuman primates after maternal ferumoxytol exposure.

Evaluation of a motion-robust 2D chemical shift-encoded technique for R2* and field map quantification in ferumoxytol-enhanced MRI of the placenta in pregnant rhesus macaques.

BACKGROUND: 3D chemical shift-encoded (CSE)-MRI techniques enable assessment of ferumoxytol concentration but are unreliable in the presence of motion. PURPOSE: To evaluate a motion-robust 2D-sequential CSE-MRI for R2* and B0 mapping in ferumoxytol-enhanced MRI of the placenta. STUDY TYPE: Prospective. ANIMAL MODEL: Pregnant rhesus macaques. FIELD STRENGTH/SEQUENCE: 3.0T/CSE-MRI. ASSESSMENT: 2D-sequential CSE-MRI was compared with 3D respiratory-gated CSE-MRI in placental imaging of 11 anesthetized animals at multiple timepoints before and after ferumoxytol administration, and in ferumoxytol phantoms (0 µg/mL-440 µg/mL). Motion artifacts of CSE-MRI in 10 pregnant women without ferumoxytol administration were assessed retrospectively by three blinded readers (4-point Likert scale). The repeatability of CSE-MRI in seven pregnant women was also prospectively studied. STATISTICAL TESTS: Placental R2* and boundary B0 field measurements (ΔB0) were compared between 2D-sequential and 3D respiratory-gated CSE-MRI using linear regression and Bland-Altman analysis. RESULTS: In phantoms, a slope of 0.94 (r2 = 0.99, concordance correlation coefficient ρ = 0.99), and bias of -4.8 s-1 (limit of agreement [LOA], -41.4 s-1 , +31.8 s-1 ) in R2*, and a slope of 1.07 (r2 = 1.00, ρ = 0.99) and bias of 11.4 Hz (LOA -12.0 Hz, +34.8 Hz) in ΔB0 were obtained in 2D CSE-MRI compared with 3D CSE-MRI for reference R2* ≤390 s-1 . In animals, a slope of 0.92 (r2 = 0.97, ρ = 0.98) and bias of -2.2 s-1 (LOA -55.6 s-1 , +51.3 s-1 ) in R2*, and a slope of 1.05 (r2 = 0.95, ρ = 0.97) and bias of 0.4 Hz (LOA -9.0 Hz, +9.7 Hz) in ΔB0 were obtained. In humans, motion-impaired R2* maps in 3D CSE-MRI (Reader 1: 1.8 ± 0.6, Reader 2: 1.3 ± 0.7, Reader 3: 1.9 ± 0.6), while 2D CSE-MRI was motion-free (Reader 1: 2.9 ± 0.3, Reader 2: 3.0 ± 0, Reader 3: 3.0 ± 0). A mean difference of 0.66 s-1 and coefficient of repeatability of 9.48 s-1 for placental R2* were observed in the repeated 2D CSE-MRI. DATA CONCLUSION: 2D-sequential CSE-MRI provides accurate R2* and B0 measurements in ferumoxytol-enhanced placental MRI of animals in the presence of respiratory motion, and motion-robustness in human placental imaging. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2020;51:580-592.

Perfusion of the placenta assessed using arterial spin labeling and ferumoxytol dynamic contrast enhanced magnetic resonance imaging in the rhesus macaque.

PURPOSE: To investigate the correspondence between arterial spin labeling (ASL) flow-sensitive alternating inversion recovery (FAIR) and ferumoxytol DCE MRI for the assessment of placental intervillous perfusion. METHODS: Ten pregnant macaques in late second trimester were imaged at 3 T using a 2D ASL FAIR, with and without outer-volume saturation pulses used to control the bolus width, and a 3D ferumoxytol DCE-MRI acquisition. The ASL tagged/control pairs were averaged, subtracted, and normalized to create perfusion ratio maps. Contrast arrival time and uptake slope were estimated by fitting the DCE data to a sigmoid function. Macaques (N = 4) received interleukin-1ß to induce inflammation and disrupt perfusion. RESULTS: The FAIR tag modification with outer-volume saturation reduced the median ASL ratio percentage compared with conventional FAIR (0.64% ± 1.42% versus 0.71% ± 2.00%; P < .05). Extended ferumoxytol arrival times (34 ± 25 seconds) were observed across the placenta. No significant DCE signal change was measured in fetal tissue ( - 0.6% ± 3.0%; P = .52) or amniotic fluid (1.9% ± 8.8%; P = .59). High ASL ratio was significantly correlated with early arrival time and high uptake slope (P < .05), but ASL signal was not above noise in late-DCE-enhancing regions. No significant differences were observed in perfusion measurements between the interleukin-1ß and controls (P > .05). CONCLUSION: The ASL-FAIR and ferumoxytol DCE-MRI methods are feasible to detect early blood delivery to the macaque placenta. Outer volume saturation reduced the high macrovascular ASL signal. Interleukin-1ß exposure did not alter placental intervillous perfusion. An endogenous-labeling perfusion technique is limited due to extended transit times for flow within the placenta beyond the immediate vicinity of the maternal spiral arteries.

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.

Pharmacokinetics of Hydroxyprogesterone Caproate and its Primary Metabolites during Pregnancy.

Objective To measure pharmacokinetics of hydroxyprogesterone caproate (OHPC) and its major metabolites throughout pregnancy. Study Design Thirty women were prescribed OHPC for recurrent preterm birth prevention. Three cohorts of subjects had blood drawn for 7 consecutive days at one of three times: cohort 1 ( n = 6) after the first dose (weeks 16-20), cohort 2 ( n = 8) between weeks 24 and 28, and cohort 3 ( n = 16) between weeks 32 and 36. We measured serum trough levels after week 1 in cohort 1 or after two consecutive weekly doses in cohorts 2 and 3. In 10 subjects, we estimated OHPC terminal half-life at 28 days after their last dose. Results In cohorts 1, 2, and 3, the areas under curve (ng × h/mL) for OHPC were 571.4 ± 195.2, 1,269.6 ± 285.0, and 1,268.0 ± 511.6, respectively. Maximum OHPC levels (ng/mL) were 5.0 ± 1.5, 12.5 ± 3.9, and 12.3 ± 4.9, respectively. The areas under the curve for mono-hydroxylated metabolites were 208.5 ± 92.4, 157.1 ± 64.6, and 211.2 ± 113.1, and maximum concentrations were 1.9 ± 0.7, 1.5 ± 0.7, and 1.8 ± 1.0, respectively. Di-hydroxylated metabolite levels were significantly lower than mono-hydroxylated metabolites. Estimated terminal half-life of OHPC was 16.3 ± 3.6 days and 19.7 ± 6.2 days for the mono-hydroxylated metabolites. Conclusion After the first injection, OHPC maximum serum level was approximately half steady-state level. Measurable metabolites of unknown activity were detected.

Renin-angiotensin system transgenic mouse model recapitulates pathophysiology similar to human preeclampsia with renal injury that may be mediated through VEGF.

Using a transgenic cross, we evaluated features of preeclampsia, renal injury and the sFlt1/VEGF changes. Transgenic hAGT and hREN, or wild-type (WT) C57Bl/6 mice were cross-bred: female hAGT × male hREN for preeclampsia (PRE) model and female WT × male WT for pregnant controls (WTP). Samples were collected for plasma VEGF, sFlt1, and urine albumin. Blood pressures (BP) were monitored by telemetry. Vascular reactivity was investigated by wire myography. Kidneys and placenta were immunostained for sFlt1 and VEGF. Eleven PRE and 9 WTP mice were compared. PRE more frequently demonstrated albuminuria, glomerular endotheliosis (80% vs. 11%; P = 0.02), and placental necrosis (60% vs. 0%; P < 0.01). PRE group demonstrated declining BPs with advancing gestation. Plasma sFlt1 increased across pregnancy in PRE; VEGF did not vary. IHC demonstrated the presence of sFlt1 in glomeruli, lymphatics, and collecting tubules of PRE kidneys, suggesting excretion. VEGF immunostaining was increased specifically in the glomeruli of PRE kidneys. Placenta in PRE showed marked immunostaining for sFlt1. We conclude that this transgenic model of preeclampsia recapitulates human preeclamptic state with high fidelity, and that, vascular adaptation to pregnancy is suggested by declining BPs and reduced vascular response to PE and increased response to acetylcholine. Placental damage with resultant increased release of sFlt1, proteinuria, deficient spiral artery remodeling, and glomerular endotheliosis were observed in this model of PRE. Increased VEGF binding to glomerular endothelial cells in this model of PRE is similar to human PRE and leads us to hypothesize that renal injury in preeclampsia may be mediated through local VEGF.

Positive versus negative effects of VEGF165 on Ca2+ signaling and NO production in human endothelial cells.

The role increased vascular endothelial growth factor (VEGF) plays in vascular function during normal vs. preeclamptic pregnancy has been a source of some controversy of late. In this study, we seek to understand how VEGF165 influences vasodilator production via Ca2+ signaling mechanisms in human endothelial cells. We utilize human umbilical vein endothelial cells (HUVEC) as well as intact ex vivo human umbilical vein (HUV Endo) to address direct stimulation of Ca2+ and NO by VEGF165 alone, as well as the effect of VEGF165 on subsequent ATP-stimulated Ca2+ signaling and NO production. We show that VEGF165 stimulates Ca2+ responses in both HUVEC and HUV Endo, which results in a corresponding increase in NO production in HUV Endo. Longer-term VEGF165 pretreatment then inhibits sustained Ca2+ burst responses to ATP in HUVEC and HUV Endo. This is paralleled by a corresponding drop in ATP-stimulated NO production in HUV Endo, likely through inhibition of Cx43 gap-junction function. Thus, although VEGF165 makes a small initial positive impact on vasodilator production via direct stimulation of Ca2+ responses, this is outweighed by the greater subsequent negative impact on Ca2+ bursts and vasodilator production promoted by more potent agonists such as ATP. Overall, elevated levels of VEGF165 associated with preeclampsia could contribute to the endothelial dysfunction by preventing Ca2+ bursts to other agonists including but not limited to ATP. NEW & NOTEWORTHY: In this manuscript, we show that VEGF levels associated with preeclampsia are a net negative contributor to potential vasodilator production in both a human ex vivo and in vitro endothelial cell model. Therefore, pharmacological targeting of VEGF-stimulated signaling pathways could be a novel treatment modality for preeclampsia-related hypertension.

Establishment of the Human Uteroplacental Circulation: A Historical Perspective.

The uterine vasculature undergoes marked changes during pregnancy in order to provide the necessary increase in blood flow to support growth and nutrition of the uterus, placenta, and developing fetus. Pregnancy-associated uterine vascular transformations are orchestrated by a complex array of endocrine and cellular mechanisms to bring about structural modifications at the maternal-fetal interface, which collectively lead to development of the uteroplacental circulation. Understanding intrinsic uterine vascular remodeling in pregnancy is essential for understanding the physiologic and pathophysiologic regulation of maternal uterine blood flow. Aberrations of uterine vascular remodeling are potentially involved in the etiology of several pregnancy disorders, for example, preeclampsia, fetal growth restriction, and preterm labor; therefore, it is essential for subspecialist clinicians and investigators interested in reproductive physiology to fully understand the establishment of uteroplacental circulation. The foundational literature in this area is extensive; thus, a succinct review is likely to be a useful resource. Herein, we present and discuss a historical perspective on uterine vascular anatomy, maternal vascular growth associated with decidualization, trophoblast invasion, intervillous circulation, aberrations in uterine vascular modeling, and the clinical implications of improper development of the uteroplacental circulation.

Changes in Ca2+ Signaling and Nitric Oxide Output by Human Umbilical Vein Endothelium in Diabetic and Gestational Diabetic Pregnancies.

Diabetes (DM) complicates 3%-10% of pregnancies, resulting in significant maternal and neonatal morbidity and mortality. DM pregnancies are also associated with vascular dysfunction, including blunted nitric oxide (NO) output, but it remains unclear why. Herein we examine changes in endothelial NO production and its relationship to Ca(2+) signaling in endothelial cells of intact umbilical veins from control versus gestational diabetic (GDM) or preexisting diabetic subjects. We have previously reported that endothelial cells of intact vessels show sustained Ca(2+) bursting in response to ATP, and these bursts drive prolonged NO production. Herein we show that in both GDM and DM pregnancies, the incidence of Ca(2+) bursts remains similar, but there is a reduction in overall sustained phase Ca(2+) mobilization and a reduction in NO output. Further studies show damage has occurred at the level of NOS3 protein itself. Since exposure to DM serum is known to impair normal human umbilical vein endothelial cell (HUVEC) function, we further studied the ability of HUVEC to signal through Ca(2+) after they were isolated from DM and GDM subjects and maintained in culture for several days. These HUVEC showed differences in the rate of Ca(2+) bursting, with DM > GDM = control HUVEC. Both GDM- and DM-derived HUVEC showed smaller Ca(2+) bursts that were less capable of NOS3 activation compared to control HUVEC. We conclude that HUVEC from DM and GDM subjects are reprogrammed such that the Ca(2+) bursting peak shape and duration are permanently impaired. This may explain why ROS therapy alone is not effective in DM and GDM subjects.

The loss of sustained Ca(2+) signaling underlies suppressed endothelial nitric oxide production in preeclamptic pregnancies: implications for new therapy.

Approximately 8% of pregnancies are complicated by preeclampsia (PE), a hypertensive condition characterized by widespread endothelial dysfunction. Reduced nitric oxide (NO) output in PE subjects has been inferred but not directly measured, and there is little understanding of why this occurs. To address this we have used direct imaging of changes in intracellular Ca(2+) concentration ([Ca(2+)]i) and NO in umbilical vein endothelium of normal and PE subjects that is still intact and on the vessel luminal surface. This was achieved by dissection and preloading with fura 2 and DAF-2 imaging dyes, respectively, before subsequent challenge with ATP (100 µM, 30 min). As a control to reveal the content of active endothelial nitric oxide synthase (eNOS) per vessel segment, results were compared with a maximal stimulus with ionomycin (5 µM, 30 min). We show for the first time that normal umbilical vein endothelial cells respond to ATP with sustained bursting that parallels sustained NO output. Furthermore, in subjects with PE, a failure of sustained [Ca(2+)]i bursting occurs in response to ATP and is associated with blunted NO output. In contrast, NO responses to maximal [Ca(2+)]i elevation using ionomycin and the levels of eNOS protein are more similar between groups than the responses to ATP. When the endothelial cells from PE subjects are isolated and allowed to recover in culture, they regain the ability under fura 2 imaging to show multiple [Ca(2+)]i bursts otherwise seen in the cells from normal subjects. Thus novel clinical therapy aimed at restoring function in vivo may be possible.

Technical challenges of a term delivery in a separated ischiopagus twin.

BACKGROUND: Conjoined twins occur in one in 100,000 live births. Successful term pregnancy in a separated conjoined twin is rare. CASE: We present a 27-year-old woman, gravida 2 para 0, former ischiopagus conjoined twin with successful separation at 12 days of life. We report a successful term gestation delivered by cesarean without complications. CONCLUSION: Term pregnancy is possible in a previous conjoined twin patient having undergone surgical separation. We recommend a multidisciplinary approach with close evaluation of maternal anatomy to achieve a successful pregnancy outcome while minimizing the risk of complications.

Curcumin down-regulates Ets-1 and Bcl-2 expression in human endometrial carcinoma HEC-1-A cells.

OBJECTIVE: Curcumin has been demonstrated to have an anti-tumor activity but the underlying molecular mechanisms are not fully uncovered. The present study was undertaken to determine the effect of curcumin on the expression of the proto-oncogene Ets-1 and the anti-apoptotic molecule Bcl-2 in human endometrial adenocarcinoma HEC-1-A cells. METHODS: Confluent HEC-1-A cells were treated with curcumin at various doses for 16 h or at 60 microM for various time points. At the end of the designated treatments, changes in cell morphology, DNA fragmentation and protein contents of Ets-1 and Bcl-2 were determined, respectively, by light microscopy, DNA laddering assay and Western blot analysis. As an initial step towards understanding whether Ets-1 was a possible up-stream regulator of Bcl-2 expression in HEC-1-A cells and if so, whether curcumin could attenuate the Ets-1-induced up-regulation of Bcl-2 expression, cells were transiently transfected with an Ets-1/GFP (Green Fluorescence Protein) fusion construct and the transfectants were treated with 60 microM curcumin for 16 h, followed by whole cell lysate preparation for Western blot analysis of Bcl-2 protein contents. RESULTS: Curcumin induced apoptosis-like morphological changes and DNA degradation and decreased basal levels of Ets-1 and Bcl-2 protein contents in HEC-1-A cells in a time- and dose-dependent manner. Overexpression of Ets-1 in the cell resulted in an increase in Bcl-2 protein contents and that increase was attenuated by curcumin treatment. CONCLUSIONS: Curcumin down-regulates Ets-1 and Bcl-2 expression and induces apoptosis in HEC-1-A cells, suggesting a novel molecular mechanism for the anti-tumor activity of curcumin.

Preeclampsia: new insights.

PURPOSE OF REVIEW: Preeclampsia is a disorder of gestation characterized by hypertension and proteinuria and can be complicated by eclamptic seizures. This review describes recent advances in the role of the renin-angiotensin system and angiogenic and anti-angiogenic factors of placental origin in its pathogenesis. RECENT FINDINGS: Deficient uteroplacental perfusion has been recognized to be a feature in all preeclampsia syndromes. Increased renin expression observed in humans and animal models supports the concept that activation of the decidual renin-angiotensin system may mediate the pathogenesis of preeclampsia. Novel angiotensin II-related biomolecular mechanisms, angiotensin II type 1-B2 receptor heterodimerization and autoantibody against angiotensin II type 1 have recently been described in preeclampsia. New evidence suggests that vascular endothelial growth factor and its receptors, antagonists, and reduced placental growth factor may play a role in the development of proteinuria and other renal injury-mediated manifestations in preeclampsia. SUMMARY: Vascular maladaptation, with increased vasomotor tone, endothelial dysfunction, increased sensitivity to angiotensin II and norepinephrine, and multiorgan dysfunction seen in preeclampsia, may be explained by angiotensin II-mediated mechanisms. Future investigations need to define the mechanism of activation of the decidual renin-angiotensin system and the release of placental factors in the pathogenesis of preeclampsia.

The role of RAS in the pathogenesis of preeclampsia.

Preeclampsia is a hypertensive disorder that is unique to pregnancy, with consistent involvement of the kidney. The renin-angiotensin system (RAS) has been implicated in the pathogenesis of preeclampsia. In the gravid state, in addition to the RAS in the kidney, there is a tissue-based RAS in the uteroplacental unit. Increased renin expression in human preeclampsia and in transgenic mouse models with a human preeclampsia-like syndrome shows that activation of the uteroplacental RAS, with angiotensin II entering the systemic circulation, may mediate the pathogenesis of preeclampsia. Vascular maladaptation in preeclampsia with increased vasomotor tone, endothelial dysfunction, and increased sensitivity to angiotensin II and norepinephrine in manifest preeclampsia may be explained on the basis of angiotensin II-mediated mechanisms through angiotensin receptor type I (AT1) activation. Recently, novel angiotensin II-related biomolecular mechanisms have been described in preeclampsia. These include AT1 and bradykinin B2 receptor heterodimerization and the production of autoantibody against AT1. Various organ systems with predilection for involvement in preeclampsia are sites of tissue-based RAS. Angiotensin II-mediated mechanisms may explain the primary clinicopathologic features of preeclampsia. In this review, these various aspects are critically examined and an integrated concept on the role of RAS in preeclampsia is presented.

Role of the renin-angiotensin system in the pathogenesis of preeclampsia.

Preeclampsia is a hypertensive disorder unique to pregnancy with consistent involvement of the kidney. The renin-angiotensin system (RAS) has been implicated in the pathogenesis of preeclampsia. In the gravid state, in addition to the RAS in the kidney, there is a tissue-based RAS in the uteroplacental unit. Increased renin expression observed both in human preeclampsia and in a transgenic mouse model with a human preeclampsia-like syndrome supports the concept that activation of the uteroplacental RAS, with angiotensin II entering the systemic circulation, may mediate the pathogenesis of preeclampsia. A novel disease paradigm of the two-kidney one-clip (2K-1C) Goldblatt model is presented for preeclampsia, wherein the gravid uterus is the clipped "kidney" and the two maternal kidneys represent the unclipped kidney. Validation of the 2K-1C Goldblatt model analogy requires evidence of elevated angiotensin II in the peripheral circulation before vascular maladaptation in preeclampsia. Convincing evidence of the elevation of angiotensin II in preeclampsia does not exist despite the fact that much of vascular pathogenesis appears to be due to angiotensin type I (AT(1)) receptor activation. Vascular maladaptation with increased vasomotor tone, endothelial dysfunction, and increased sensitivity to angiotensin II and norepinephrine in manifest preeclampsia may be explained on the basis of angiotensin II-mediated mechanisms. Recently, novel angiotensin II-related biomolecular mechanisms have been described in preeclampsia. These include AT(1) and bradykinin B(2) receptor heterodimerization and the production of an autoantibody against AT(1). Various organ systems with a predilection for involvement in preeclampsia are each a site of a tissue-based RAS. How angiotensin II-mediated mechanisms may explain the primary clinical-pathological features of preeclampsia is described. Future investigations are proposed to more precisely define the role of activation of the uteroplacental RAS in the mechanisms underlying preeclampsia.