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Wiebke Bartels, Simon Adamson, Lisa Leung, Don D Sin, Stephan F van Eeden Centre for Heart Lung Innovation, Department of Medicine, University of British Columbia, Vancouver, BC, Canada Rationale: Readmissions are common following acute exacerbations of chronic obstructive pulmonary disease (AECOPD) and are partially responsible for increased morbidity and mortality in COPD. Numerous factors have been shown to predict readmission of patients previously admitted to hospital for AECOPD; however, factors related to readmission in patients who are triaged in emergency departments (EDs) and sent directly home are poorly understood. We postulate that patients seen in the ED for AECOPD and directly sent home have a high readmission rate, and we suspect that inadequate management and follow-up contribute to this high readmission rate. Methods: We conducted a 1-year retrospective study of all patients seen in the ED for AECOPD at an inner-city tertiary care hospital; 30- and 90-day readmission rates for COPD and all-cause admissions to the ED and hospital were determined. Patients discharged directly home from the ED were compared with those admitted to hospital for management. Patient, treatment, and system variables that could potentially impact readmission were documented. Multivariate Poisson regression models were used to determine which factors predicted readmissions. Results: The readmission rates in the ED group (n=240) were significantly higher than that in the hospitalized group (n=271): 1) the 90-day ED readmissions (1.29 vs 0.51, p

Simon L Adamson,1 Jane Burns,1,2 Pat G Camp,1,2 Don D Sin,1,3 Stephan F van Eeden1,31The Centre for Heart Lung Innovation, St Paul’s Hospital, University of British Columbia, 2Department of Physical Therapy, 3Division of Respirology, Department of Medicine, University of British Columbia, Vancouver, BC, CanadaBackground: Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) increase COPD morbidity and mortality and impose a great burden on health care systems. Early readmission following a hospitalization for AECOPD remains an important clinical problem. We examined how individualized comprehensive care influences readmissions following an index hospital admission for AECOPD.Methods: We retrospectively reviewed data of patients admitted for AECOPD to two inner-city teaching hospitals to determine the impact of a comprehensive and individualized care management strategy on readmissions for AECOPD. The control group consisted of 271 patients whose index AECOPD occurred the year before the comprehensive program, and the experimental group consisted of 191 patients who received the comprehensive care. The primary outcome measure was the total number of readmissions in 30- and 90-day postindex hospitalizations. Secondary outcome measures included the length of time between the index admission and first readmission and all-cause mortality.Results: The two groups were similar in terms of age, sex, forced expiratory volume in 1 second, body mass index (BMI), pack-years, and the number and types of comorbidities. Comprehensive care significantly reduced 90-day readmission rates in females (P=0.0205, corrected for age, BMI, number of comorbidities, substance abuse, and mental illness) but not in males or in the whole group (P>0.05). The average times between index admission and first readmission were not different between the two groups. Post hoc multivariate analysis showed that substance abuse (P

Preeclampsia is a life-threatening disorder characterized by maternal gestational hypertension and proteinuria that results from placental dysfunction. Placental abnormalities include abnormal syncytiotrophoblast and a 50% reduction in placental expression of the transcription factor Gcm1. In mice, homozygous deletion of Gcm1 prevents syncytiotrophoblast differentiation and is embryonic lethal. We used heterozygous Gcm1 mutants (Gcm1(+/-)) to test the hypothesis that hypomorphic expression of placental Gcm1 causes defective syncytiotrophoblast differentiation and maternal and placental phenotypes that resemble preeclampsia. We mated wild-type female mice with Gcm1(+/-) fathers to obtain wild-type mothers carrying ≈50% Gcm1(+/-) conceptuses. Gcm1(+/-) placentas had syncytiotrophoblast abnormalities including reduced gene expression of Gcm1-regulated SynB, elevated expression of sFlt1, a thickened interhemal membrane separating maternal and fetal circulations, and electron microscopic evidence in syncytiotrophoblast of necrosis and impaired maternal-fetal transfer. Fetoplacental vascularity was quantified by histomorphometry and microcomputed tomography imaging. In Gcm1(+/-), it was ≈30% greater than wild-type littermates, whereas placental vascular endothelial growth factor A (Vegfa) expression and fetal and placental weights did not differ. Wild-type mothers carrying Gcm1(+/-) conceptuses developed late gestational hypertension (118±2 versus 109.6±0.7 mm Hg in controls; P<0.05). We next correlated fetoplacental vascularity with placental Gcm1 expression in human control and pathological pregnancies and found that, as in mice, fetoplacental vascularity increased when GCM1 protein expression decreased (R(2)=-0.45; P<0.05). These results support a role for reduced placental Gcm1 expression as a causative factor in defective syncytiotrophoblast differentiation and maternal and placental phenotypes in preeclampsia in humans. [ABSTRACT FROM AUTHOR]

Background: Many stillbirths of normally formed fetuses in the third trimester could be prevented via delivery if reliable means to anticipate this outcome existed. However, because the etiology of these stillbirths is often unexplained and although the underlying mechanism is presumed to be hypoxia from placental insufficiency, the placentas often appear normal on histopathological examination. Gestational age is a risk factor for antepartum stillbirth, with a rapid rise in stillbirth rates after 40 weeks' gestation. We speculate that a common mechanism may explain antepartum stillbirth in both the late-term and postterm periods. Mice also show increasing rates of stillbirth when pregnancy is artificially prolonged. The model therefore affords an opportunity to characterize events that precede stillbirth., Objective: The objective of the study was to prolong gestation in mice and monitor fetal and placental growth and cardiovascular changes., Study Design: From embryonic day 15.5 to embryonic day 18.5, pregnant CD-1 mice received daily progesterone injections to prolong pregnancy by an additional 24 hour period (to embryonic day 19.5). To characterize fetal and placental development, experimental assays were performed throughout late gestation (embryonic day 15.5 to embryonic day 19.5), including postnatal day 1 pups as controls. In addition to collecting fetal and placental weights, we monitored fetal blood flow using Doppler ultrasound and examined the fetoplacental arterial vascular geometry using microcomputed tomography. Evidence of hypoxic organ injury in the fetus was assessed using magnetic resonance imaging and pimonidazole immunohistochemistry., Results: At embryonic day 19.5, mean fetal weights were reduced by 14% compared with control postnatal day 1 pups. Ultrasound biomicroscopy showed that fetal heart rate and umbilical artery flow continued to increase at embryonic day 19.5. Despite this, the embryonic day 19.5 fetuses had significant pimonidazole staining in both brain and liver tissue, indicating fetal hypoxia. Placental weights at embryonic day 19.5 were 21% lower than at term (embryonic day 18.5). Microcomputed tomography showed no change in quantitative morphology of the fetoplacental arterial vasculature between embryonic day 18.5 and embryonic day 19.5., Conclusion: Prolongation of pregnancy renders the murine fetus vulnerable to significant growth restriction and hypoxia because of differential loss of placental mass rather than any compromise in fetoplacental blood flow. Our data are consistent with a hypoxic mechanism of antepartum fetal death in human term and postterm pregnancy and validates the inability of umbilical artery Doppler to safely monitor such fetuses. New tests of placental function are needed to identify the late-term fetus at risk of hypoxia to intervene by delivery to avoid antepartum stillbirth., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Introduction: The fetoplacental vasculature network is essential for the exchange of nutrients, gases and wastes with the maternal circulation and for normal fetal development. The present study quantitatively compares arterial and venous morphological and functional differences in the mouse fetoplacental vascular network., Methods: High resolution X-ray micro-computed tomography was used to visualize the 3D geometry of the arterial and venous fetoplacental vasculature in embryonic day 15.5 CD-1 mice (n = 5). Automated image analysis was used to measure the vascular geometry of the approximately 4100 arterial segments and 3200 venous segments per specimen to simulate blood flow through these networks., Results: Both the arterial and venous trees demonstrated a hierarchical branching structure with 8 or 9 (arterial) or 8 (venous) orders. The venous tree was smaller in volume and overall dimensions than the arterial tree. Venous vessel diameters increased more rapidly than arteries with each successive order, leading to lower overall resistance, although the umbilical vein was notably smaller and of higher resistance than these scaling relationships would predict. Simulation of blood flow for these vascular networks showed that 57% of total resistance resides in the umbilical artery and arterial tree, 17% in the capillary bed, and 26% in the venous tree and umbilical vein., Discussion: A detailed examination of the mouse fetoplacental arterial and venous tree revealed features, such as the distribution of resistance and the dimension of the venous tree, that were both morphologically distinct from other vascular beds and that appeared adapted to the specialized requirements of sustaining a fetus., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Miscarriage and intrauterine growth restriction (IUGR) are devastating complications in fetal/neonatal alloimmune thrombocytopenia (FNAIT). We previously reported the mechanisms for bleeding diatheses, but it is unknown whether placental, decidual immune cells or other abnormalities at the maternal-fetal interface contribute to FNAIT. Here we show that maternal immune responses to fetal platelet antigens cause miscarriage and IUGR that are associated with vascular and immune pathologies in murine FNAIT models. Uterine natural killer (uNK) cell recruitment and survival beyond mid-gestation lead to elevated NKp46 and CD107 expression, perforin release and trophoblast apoptosis. Depletion of NK cells restores normal spiral artery remodeling and placental function, prevents miscarriage, and rescues hemorrhage in neonates. Blockade of NK activation receptors (NKp46, FcɣRIIIa) also rescues pregnancy loss. These findings shed light on uNK antibody-dependent cell-mediated cytotoxicity of invasive trophoblasts as a pathological mechanism in FNAIT, and suggest that anti-NK cell therapies may prevent immune-mediated pregnancy loss and ameliorate FNAIT.Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a gestational disease caused by maternal immune responses against fetal platelets. Using a FNAIT mouse model and human trophoblast cell lines, here the authors show that uterine natural killer cell-mediated trophoblast apoptosis contributes to FNAIT pathogenesis.

Introduction: Placental examination is recommended when genetic mutations cause fetal lethality in mice. But how fetal death alters histomorphology of the surviving mouse placenta is not known., Methods: Placentas were examined at E17.5 after fetectomy of 1-2 fetal mice per pregnancy at either embryonic day (E) 15.5 (N = 8; Fx-2 group) or E13.5 (N = 5; Fx-4 group), which left 12 ± 2 surviving fetuses per litter., Results: Fetectomy caused no changes in placental weights and no increases in placental hypoxia (pimonidazole staining). The size and cell morphology of the decidua and junctional zone regions were unchanged and, in the Fx-2 group, these regions became significantly less hypoxic. Significant changes in labyrinth volume included a 30% increase in the Fx-2 group and in both groups, a >50% decrease in % fetal blood space and >40% increase in % labyrinth tissue. Maternal blood sinusoid volume was unchanged. Cell death in the labyrinth was significantly increased (22-fold increase in TUNEL staining) whereas placental mRNA expression of the proliferation marker Mki67 was unchanged. mRNA expression of sFlt1 and Prl3b1 (mPL-II) was unchanged in the labyrinth and junctional zone tissues in the Fx-2 group and in whole placental tissue in the Fx-4 group., Discussion: Placental examination of the junctional zone and decidual regions after spontaneous fetal death in late gestation is likely to yield useful phenotypic information and abnormalities that may contribute to fetal death. In contrast, labyrinth abnormalities including increased tissue volume and reduced fetoplacental vascularity may not be due to genetic perturbation nor predate fetal death., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The purpose of this study was to establish the time course and hemodynamic significance of de novo formed and enlarged uteroplacental arteries during pregnancy. Using x-ray microcomputed tomography (n = 4-7 placentas from 2-4 dams/gestational group), uteroplacental arterial vascular dimensions were measured at individual implantation sites. Dimensions and topology were used to compute total and vessel-specific resistances and cross-sectional areas. Diameter enlargement of the uterine artery (+55% by Embryonic Day 5.5 [E5.5]) and preplacental radial arteries (+30% by E8.5) was significant only in early gestation. Formation of spiral arteries (E9.5-E11.5), maternal canals, and canal branches (E11.5-E13.5) during midgestation was followed by enlargement of these vessels such that, from E9.5 to E17.5 (near term), spiral artery resistance dropped 9-fold, and canal resistance became negligible. A 12-fold increase in terminal vessel cross-sectional area was nearly sufficient to offset known increases in flow so that blood velocity entering the exchange region was predicted to increase by only 2-fold. The calculated 47% decrease in total resistance downstream of the uterine artery, determined from vascular geometry, was in accord with prior uterine blood flow data in vivo and was due to enlarging spiral artery diameters. Interestingly, radial artery resistance was unchanged after E9.5 so that radial arteries accounted for 91% of resistance and pressure drop in the uteroplacental arterial network by E17.5. These findings led us to propose functional roles for the three morphologically defined vessel types: radial arteries to reduce pressure, spiral artery enlargement to increase flow with gestation, and maternal canal elaboration and enlargement to maintain low exit velocities into the exchange region., (© 2016 by the Society for the Study of Reproduction, Inc.)

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disease in which intracranial hemorrhage (ICH) is the major risk. Although thrombocytopenia, which is caused by maternal antibodies against β3 integrin and occasionally by maternal antibodies against other platelet antigens, such as glycoprotein GPIbα, has long been assumed to be the cause of bleeding, the mechanism of ICH has not been adequately explored. Utilizing murine models of FNAIT and a high-frequency ultrasound imaging system, we found that ICH only occurred in fetuses and neonates with anti-β3 integrin-mediated, but not anti-GPIbα-mediated, FNAIT, despite similar thrombocytopenia in both groups. Only anti-β3 integrin-mediated FNAIT reduced brain and retina vessel density, impaired angiogenic signaling, and increased endothelial cell apoptosis, all of which were abrogated by maternal administration of intravenous immunoglobulin (IVIG). ICH and impairment of retinal angiogenesis were further reproduced in neonates by injection of anti-β3 integrin, but not anti-GPIbα antisera. Utilizing cultured human endothelial cells, we found that cell proliferation, network formation, and AKT phosphorylation were inhibited only by murine anti-β3 integrin antisera and human anti-HPA-1a IgG purified from mothers with FNAIT children. Our data suggest that fetal hemostasis is distinct and that impairment of angiogenesis rather than thrombocytopenia likely causes FNAIT-associated ICH. Additionally, our results indicate that maternal IVIG therapy can effectively prevent this devastating disorder.

The sites of elevated vascular resistance that impede placental perfusion in pathological pregnancies are unknown. In the current study, we identified these sites in a knockout mouse model (eNOS(-/-)) with reduced uterine (-55%) and umbilical (-29%) artery blood flows caused by endothelial nitric oxide synthase deficiency. Uteroplacental and fetoplacental arterial vascular trees of pregnant mice near term were imaged using x-ray microcomputed tomography (n = 5-10 placentas from 3-5 dams/group). The resulting three-dimensional images were analyzed to assess vessel geometry and vascular resistance. In control and eNOS(-/-) trees, ∼90% of total uteroplacental vascular resistance was located in the radial arteries. Changes in eNOS(-/-) vessel geometry, including 30% reductions in uterine, radial, and spiral artery diameters, were calculated to increase arterial resistance downstream of the uterine artery by 2.3-fold, predicting a 57% decrease in uterine blood flow. Despite large reductions in eNOS(-/-) spiral arteries (-55% by volume) and maternal canals (-67% by volume), these vessels were relatively minor contributors to resistance. In the eNOS(-/-) fetoplacental tree, the number of arterioles (50-75 μm diameter) increased by 26%. Nevertheless, calculated resistance rose by 19%, predominantly because arteries near the periphery of the tree selectively exhibited a 7%-9% diameter reduction. We conclude that previously observed decreases in uterine and umbilical blood flows in eNOS(-/-) pregnancies are associated with markedly divergent structural changes in the uteroplacental versus fetoplacental circulations. Results showed the radial arteries were critical determinants of uteroplacental resistance in mice and therefore warrant greater attention in future studies in pathological human pregnancies., (© 2015 by the Society for the Study of Reproduction, Inc.)

Preeclampsia, a life-threatening complication of human pregnancy, has a spectrum of clinical signs and is likely caused by an array of pathological mechanisms. However, elevated levels of soluble fms-like tyrosine kinase-1 (sFLT1) in the placenta and in the maternal circulation has emerged as a common finding in women with preeclampsia and likely is a causative factor in this disorder. In this issue of the JCI, Fan and colleagues provide experimental evidence from both humans and mice that suggests placental trophoblast cells overexpress sFLT1 in self defense against excessive VEGFA produced by maternal decidual cells. The authors' work thus implicates the decidual cells of the mother as the culprit responsible for increased placental expression of sFLT1, a VEGFA antagonist that enters the maternal circulation and consequently induces the clinical signs of preeclampsia.

Given the angiogenic function of vascular endothelial growth factor A (VEGFA), the function of its expression by trophoblast in the avascular placental junctional zone is unknown. In mice, cells from the trophoblast-specific protein alpha (Tpbpa) lineage populate this zone and, in late gestation, some of these cells invade the decidual layer. To diminish Vegfa expression in Tpbpa cells, we crossed Vegfa(flox/flox) females with males carrying Tpbpa-Cre. For single deletion (sd) of Vegfa in Tpbpa cells in 100% of conceptuses (SD100 pregnancies, sd conceptuses) we crossed homozygous lines. For double deletion (dd) of both Vegfa alleles in 50% of the conceptuses (DD50 pregnancies, 50% dd conceptuses and 50% no deletion [nd]), we crossed homozygous Vegfa(flox/flox) females with males heterozygous for Tpbpa-Cre and homozygous for Vegfa(flox/flox). Controls were Vegfa(flox/flox) females bred to wild-type males (V-CTRL pregnancies). In SD100 pregnancies, maternal plasma immunoreactive VEGFA significantly increased and arterial blood pressure decreased, whereas fetal body weight and placental Flt1, sFlt1, and Prl3b1 mRNA were unchanged. In DD50, maternal immunoreactive VEGFA and arterial pressures were unaltered, but both dd and nd conceptuses exhibited significantly increased embryonic lethality, altered expression of Flt1, sFlt1, and Prl3b1 mRNA in the decidual layer, and decreased fetal body weight relative to V-CTRL. Maternal cardiac output significantly increased in proportion to dd conceptuses in the pregnancy. In DD50, results are consistent with altered maternal function beginning in early gestation and adversely impacting both conceptus genotypes. We conclude that maternal function is influenced by Vegfa expression in trophoblast cells at the maternal-fetal interface, likely via an endocrine mechanism., (© 2014 by the Society for the Study of Reproduction, Inc.)

The fetoplacental arterial tree is critical for efficient distribution of arterial blood to capillaries throughout the placental exchange region; yet, little is known about the factors and mechanisms that control its development. Advances in micro-CT imaging and analysis, and available mutant mouse strains, are facilitating rapid progress. Indeed, micro-CT studies show that genetic differences between the CD1 and C57Bl/6 mouse strains, and between Gcm1 heterozygotes and wild-type littermates alter the developmental trajectory of the fetoplacental arterial tree as do environmental factors including maternal exposure to toxins in cigarette smoke and malarial infection. Relative to other vascular beds, the fetoplacental arterial tree is particularly tractable because veins can more easily be excluded when infusing the contrast agent and because of the placenta's small size, which means that the whole organ can be imaged (maintaining connectivity) and that the tree is simpler (fewer branching generations). Despite these differences, measured parameters were found to be similar to arterial trees in other adult rodent organs. Thus, micro-CT analysis provides a means for advancing of our understanding of the mechanisms controlling development of the fetoplacental arterial tree. Results will likely have relevance to other arterial vasculatures as well., (© 2013 John Wiley & Sons Ltd.)

End-tidal breath carbon monoxide (CO) is abnormally low in women with preeclampsia (PE), while women smoking during pregnancy have shown an increase in CO levels and a 33% lower incidence of PE. This effect may be, in part, due to lowered sFLT1 plasma levels in smokers, and perhaps low-level CO inhalation can attenuate the development of PE in high-risk women. Our previous work showed maternal chronic CO exposure (<300 ppm) throughout gestation had no maternal or fetal deleterious effects in mice. Our current study evaluated the uteroplacental vascular effects in CD-1 maternal mice that inhaled CO (250 ppm) both chronically, gestation day (GD) 0.5 to 18.5, and acutely, 2.5 h on each of GD 10.5 and 14.5. We demonstrated, using microultrasound measurements of blood velocity and microcomputed tomography imaging of the uteroplacental vasculature, that chronic maternal exposure to CO doubled uterine artery blood flow and augmented uteroplacental vascular diameters and branching. This finding may be of benefit to women with PE, as they exhibit uteroplacental vascular compromise. The ratio of VEGF protein to its FLT1 receptor was increased in the placenta, suggesting a shift to a more angiogenic state; however, maternal circulating levels of VEGF, sFLT1, and their ratio were not significantly changed. Doppler blood velocities in the maternal uterine artery and fetal umbilical artery and vein were unaltered. This study provides in vivo evidence that chronic inhalation of 250 ppm CO throughout gestation augments uterine blood flow and uteroplacental vascular growth, changes that may protect against the subsequent development of preeclampsia.

Utilizing ENU mutagenesis, we identified a mutant mouse with elevated platelets. Genetic mapping localized the mutation to an interval on chromosome 19 that encodes the Jak2 tyrosine kinase. We identified a A3056T mutation resulting in a premature stop codon within exon 19 of Jak2 (Jak2(K915X)), resulting in a protein truncation and functionally inactive enzyme. This novel platelet phenotype was also observed in mice bearing a hemizygous targeted disruption of the Jak2 locus (Jak2(+/-)). Timed pregnancy experiments revealed that Jak2(K915X/K915X) and Jak2(-/-) displayed embryonic lethality; however, Jak2(K915X/K915X) embryos were viable an additional two days compared to Jak2(-/-) embryos. Our data suggest that perturbing JAK2 activation may have unexpected consequences in elevation of platelet number and correspondingly, important implications for treatment of hematological disorders with constitutive Jak2 activity.

Background: The increasing use of the ketogenic diet (KD), particularly by women of child-bearing age, raises a question about its suitability during gestation. To date, no studies have thoroughly investigated the direct implications of a gestational ketogenic diet on embryonic development., Methods: To fill this knowledge gap we imaged CD-1 mouse embryos whose mothers were fed either a Standard Diet (SD) or a KD 30 days prior to, as well as during gestation. Images were collected at embryonic days (E) 13.5 using Optical Projection Tomography (OPT) and at E17.5 using Magnetic Resonance Imaging (MRI)., Results: An anatomical comparison of the SD and KD embryos revealed that at E13.5 the average KD embryo was volumetrically larger, possessed a relatively larger heart but smaller brain, and had a smaller pharynx, cervical spinal cord, hypothalamus, midbrain, and pons, compared with the average SD embryo. At E17.5 the KD embryo was found to be volumetrically smaller with a relatively smaller heart and thymus, but with enlarged cervical spine, thalamus, midbrain and pons., Conclusion: A ketogenic diet during gestation results in alterations in embryonic organ growth. Such alterations may be associated with organ dysfunction and potentially behavioral changes in postnatal life.

Objectives: Intrauterine growth restriction (IUGR) and pre-eclampsia are severe and clinically important manifestations of placental insufficiency. In the mouse, dual specificity phosphatase 9 (DUSP9) is critical to the normal development of the placenta, where knock-outs are growth restricted and have a placental phenotype similar to that seen in syndromes of human placental insufficiency. Our purpose was to characterize DUSP9 expression in normal human pregnancy and in cases of placental insufficiency., Study Design: We used RT-PCR, immuno-histochemistry and Western blotting to characterize DUSP9 gene expression and protein levels across human gestation and in pregnancies complicated by severe IUGR and/or severe pre-eclampsia. DUSP9 promoter methylation was studied in pathologic and pre-term control placentas to investigate potential epigenetic regulation. First trimester villous explants and BeWo cells were treated with DUSP9 silencing RNA to determine the effect on downstream pathways. Placental hypoxia is a hallmark of pre-eclampsia; therefore explants were subjected to hypoxic culture conditions to determine the effect of oxygen on DUSP9 expression in vitro., Results: DUSP9 expression was evident in villous trophoblast and declined during development. DUSP9 protein was significantly lower in severe pre-eclamptic placentas compared to severe growth restriction. This was not epigenetically mediated by promoter hyper-methylation, and the downstream pathway ERK1/2 was not significantly affected. DUSP9 expression in first trimester explants was significantly decreased by 74 ± 20% in hypoxic (3% oxygen) culture conditions. In BeWo cells and explanted placental villi treated with DUSP9 silencing RNA, expression of DUSP9 was down-regulated by 61% and 62% respectively. There was a trend to increased phosphorylation of the downstream target ERK1/2 in DUSP9 down-regulated BeWo cells and explanted placental villi., Conclusion: DUSP9 protein levels were markedly suppressed in severe pre-eclampsia, but not in severe IUGR. This suppression might be attributable to the prolonged hypoxic conditions found in pre-eclampsia., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Vascular endothelial growth factor A (VEGF) is highly expressed in adipose tissue. Its role, however, has not been fully elucidated. Here, we reveal the metabolic role of adipose-VEGF by studying mice with deletion (VEGF(AdΔ)) or doxycycline-inducible overexpression of a VEGF transgene (VEGF(AdTg)) in the adipose tissue. VEGF(AdΔ) mice have reduced adipose vascular density and show adipose hypoxia, apoptosis, inflammation, and metabolic defects on a high-fat diet. In contrast, induction of VEGF expression in VEGF(AdTg) mice leads to increased adipose vasculature and reduced hypoxia. The latter changes are sufficient to counteract an established compromising effect of high-fat diet on the metabolism, indicating that metabolic misbalance is reversible by adipose vessel density increase. Our data clearly show the essential role of VEGF signaling for adequate adipose function. Besides revealing insights into the molecular mechanisms of obesity-related metabolic diseases, this study points to the therapeutic potential of increased adipose angiogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

KLF3 is a Krüppel family zinc finger transcription factor with widespread tissue expression and no previously known role in heart development. In a screen for dominant mutations affecting cardiovascular function in N-ethyl-N-nitrosourea (ENU) mutagenized mice, we identified a missense mutation in the Klf3 gene that caused aortic valvular stenosis and partially penetrant perinatal lethality in heterozygotes. All homozygotes died as embryos. In the first of three zinc fingers, a point mutation changed a highly conserved histidine at amino acid 275 to arginine (Klf3(H275R) ). This change impaired binding of the mutant protein to KLF3's canonical DNA binding sequence. Heterozygous Klf3(H275R) mutants that died as neonates had marked biventricular cardiac hypertrophy with diminished cardiac chambers. Adult survivors exhibited hypotension, cardiac hypertrophy with enlarged cardiac chambers, and aortic valvular stenosis. A dominant negative effect on protein function was inferred by the similarity in phenotype between heterozygous Klf3(H275R) mutants and homozygous Klf3 null mice. However, the existence of divergent traits suggested the involvement of additional interactions. We conclude that KLF3 plays diverse and important roles in cardiovascular development and function in mice, and that amino acid 275 is critical for normal KLF3 protein function. Future exploration of the KLF3 pathway provides a new avenue for investigating causative factors contributing to cardiovascular disorders in humans., Competing Interests: The authors have declared that no competing interests exist.

It is not known whether eNOS deficiency in the mother or the conceptus (ie, placenta and fetus) causes fetal growth restriction in mice lacking the endothelial NO synthase gene (eNOS knockout [KO]). We hypothesized that eNOS sustains fetal growth by maintaining low fetoplacental vascular tone and promoting fetoplacental vascularity and that this is a conceptus effect and is independent of maternal genotype. We found that eNOS deficiency blunted fetal growth, and blunted the normal increase in umbilical blood flow and umbilical venous diameter and the decrease in umbilical arterial Resistance Index in late gestation (14.5-17.5 days) in eNOS KO relative to C57Bl/6J controls. On day 17.5, fetoplacental capillary lobule length and capillary density in vascular corrosion casts were reduced in eNOS KO placentas. Reduced vascularization may be a result of decreased vascular endothelial growth factor mRNA and protein expression in eNOS KO placentas at this stage. These factors, combined with significant anemia found in eNOS KO fetuses, would be anticipated to reduce fetal oxygen delivery and contribute to the fetal tissue hypoxia that was detected in the heart, lung, kidney, and liver by immunohistochemistry using pimonidazole. Although maternal eNOS deficiency impairs uteroplacental adaptations to pregnancy, maternal genotype was not a significant factor affecting growth in heterozygous conceptuses. This indicates that fetal growth restriction was primarily caused by conceptus eNOS deficiency. In mice, placental hemodynamic and vascular changes with gestation and growth restriction showed strong parallels with human pregnancy. Thus, the eNOS KO model could provide insights into the pathogenesis of human intrauterine growth restriction.

X-linked hypophosphatemic rickets (XLH) is a dominantly inherited disease characterized by renal phosphate wasting, aberrant vitamin D metabolism, and defective bone mineralization. It is known that XLH in humans and in certain mouse models is caused by inactivating mutations in PHEX/Phex (phosphate-regulating gene with homologies to endopeptidases on the X chromosome). By a genome-wide N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen in mice, we identified a dominant mouse mutation that exhibits the classic clinical manifestations of XLH, including growth retardation, skeletal abnormalities (rickets/osteomalacia), hypophosphatemia, and increased serum alkaline phosphatase (ALP) levels. Mapping and sequencing revealed that these mice carry a point mutation in exon 14 of the Phex gene that introduces a stop codon at amino acid 496 of the coding sequence (Phex(Jrt) also published as Phex(K496X) [Ichikawa et al., 2012]). Fgf23 mRNA expression as well as that of osteocalcin, bone sialoprotein, and matrix extracellular phosphoglycoprotein was upregulated in male mutant long bone, but that of sclerostin was unaffected. Although Phex mRNA is expressed in bone from mutant hemizygous male mice (Phex(Jrt)/Y mice), no Phex protein was detected in immunoblots of femoral bone protein. Stromal cultures from mutant bone marrow were indistinguishable from those of wild-type mice with respect to differentiation and mineralization. The ability of Phex(Jrt)/Y osteoblasts to mineralize and the altered expression levels of matrix proteins compared with the well-studied Hyp mice makes it a unique model with which to further explore the clinical manifestations of XLH and its link to FGF23 as well as to evaluate potential new therapeutic strategies., (Copyright © 2012 Wiley Periodicals, Inc.)

Preeclampsia is associated with impaired uteroplacental adaptations during pregnancy and abnormalities in the endothelial NO synthase (eNOS)-NO pathway, but whether eNOS deficiency plays a causal role is unknown. Thus, the objective of the current study was to determine the role of eNOS in the mother and/or conceptus in uteroplacental changes during pregnancy using eNOS knockout mice. We quantified uterine artery blood flow using microultrasound, visualized the uteroplacental vasculature using vascular corrosion casts, and used pimonidazole and hypoxia-inducible factor 1α immunohistochemistry as markers of hypoxia in the placentas of eNOS knockout mice versus the background strain, C57Bl/6J (wild type). We found that increases in uteroplacental blood flow, uterine artery diameter, and spiral artery length were reduced, and markers of placental hypoxia in the junctional zone were elevated in late gestation in eNOS knockout mice. Both maternal and conceptus genotypes contributed to changes in uterine artery diameter and flow. Despite placental hypoxia, placental soluble fms-like tyrosine kinase 1 and tumor necrosis factor-α mRNA, and in maternal plasma, soluble fms-like tyrosine kinase 1 were not elevated in eNOS knockout mice. Thus, our results show that both eNOS in the mother and the conceptus contribute to uteroplacental vascular changes and increased uterine arterial blood flow in normal pregnancy.

The placental microvasculature is essential for efficient transfer of gases, nutrients and waste between the mother and fetus. Microvascular hypoplasia of the terminal villi is a common pathology in severe Intra Uterine Growth Restriction (IUGR). We used novel methods to obtain placental micro-vascular endothelial cells (PlMEC) from preterm control placentas (n = 3) and placentas from pregnancies with severe IUGR (n = 6) with absent or reversed end-diastolic velocity in the umbilical artery. Distal placental villous tissue was collected to enrich for intermediate and terminal villi. Tissue was digested and PlMEC positively selected using tocosylated magnetic Dynabeads labeled with Human Endothelial Antigen lectin. The purity of the PlMEC (94 ± 2 SD %) was assessed by CD31 and vimentin immunocytochemistry. RNA was extracted from the PlMEC samples and subjected to Affymetrix microarray analysis (U133Plus2 array chips). Comparison of preterm and IUGR PlMEC gene expression profiles identified BTNL9 and NTRK2 transcripts to be upregulated and SAA1 and SLAMF1 transcripts to be downregulated in all 6 IUGR cases relative to preterm controls. A third downregulated gene GNAS was identified to be near significance. Changes were demonstrated to be significant at the mRNA level by Real Time PCR in the PlMEC samples. Changes in the IUGR endothelium were confirmed at the protein level by immunohistochemistry for the 3 with available antibodies. We used a tissue microarray constructed from an independent cohort of placental samples from severe IUGR (n = 7), preeclamptic (n = 7), preterm control (n = 6) and term control (n = 6) pregnancies. Results confirmed differential endothelial expression of BTNL9, NTRK2 and SLAMF1 in IUGR versus preterm and term samples. These studies are the first to characterize PlMEC gene expression profiles thus we have advanced our understanding of the molecular basis of placental micro-vascular pathophysiology in fetal growth restriction., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

How the fetoplacental arterial tree grows and expands during late gestational development is largely unknown. In this study, we quantified changes in arterial branching in the fetal exchange region of the mouse placenta during late gestation, when capillarization increases rapidly. We studied two commonly used mouse strains, CD1 and C57Bl/6 (B6), at embryonic days (E)13.5, 15.5, and 17.5. B6 mice differ from CD1 mice by exhibiting a blunted fetal weight gain in late gestation. We found that B6 capillarization and interhemal membrane thinning were reduced and placental hypoxia-inducible factor-1α and VEGF-A expression were higher than CD1 near term. Automated vascular segmentation of microcomputed tomography data sets revealed that the number of arterial vessels ≥50 μm remained constant during late gestation in both strains, despite large increases in downstream capillary volume quantified by stereology (+65% in B6 mice and +200% in CD1 mice). Arterial diameters expanded in both strains from E13.5 to E15.5; however, diameters continued to expand to E17.5 in B6 mice only. The diameter scaling coefficient at branch sites was near optimal (-3.0) and remained constant in CD1 mice, whereas it decreased, becoming abnormal, in B6 mice at term (-3.5 ± 0.2). Based on arterial tree geometry, resistance remained constant throughout late gestation (∼0.45 mmHg·s·μl(-1)) in CD1 mice, whereas it decreased by 50% in late gestation in B6 mice. Quantification of the fetoplacental vasculature revealed significant strain-dependent differences in arterial and capillary expansion in late gestation. In both strains, enlargement of the fetoplacental arterial tree occurred primarily by increased arterial diameters with no change in segment numbers in late gestation.

The ability of random mutagenesis techniques to annotate the mammalian genome can be hampered due to genetic redundancy and compensatory pathways that mask heterozygous mutations under homeostatic conditions. The objective of this study was to devise a pharmacologically sensitized screen using the chemotherapeutic drug, 5-fluorouracil (5FU), to induce cytopenia. 5FU dose was optimized in the 129/SvImJ, C57BL/6J, BALB/cJ, and C3H/HeJ strains of laboratory mice. N-ethyl-N-nitrosourea (ENU) mutagenesis was performed on 129/SvImJ males and phenotypic variants were identified by backcrossing on to the C57BL/6J background. G1 animals were challenged with 100 μg/g 5FU and phenodeviants with altered platelet recovery were monitored. Of 546 G1 animals tested, 15 phenodeviants were identified that displayed increased baseline platelet number, a platelet overshoot, or delayed platelet recovery, thereby demonstrating the utility of this approach for uncovering mutations in megakaryocyte and platelet development. Four G1 mice were selected for further analysis. The phenotypes were heritable in all four strains and genetic mapping identified a chromosome location in two of the three G2 lines tested. In conclusion, our group has developed a sensitized random mutagenesis screen utilizing 5FU and has shown that the strain combination of 129/SvImJ × C57BL/6J is robust for identification of founder lines with defects in megakaryocyte and platelet development., (Copyright © 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Preeclampsia (PE) adversely impacts ~5% of pregnancies. Despite extensive research, no consistent biomarkers or cures have emerged, suggesting that different molecular mechanisms may cause clinically similar disease. To address this, we undertook a proteomics study with three main goals: (1) to identify a panel of cell surface markers that distinguish the trophoblast and endothelial cells of the placenta in the mouse; (2) to translate this marker set to human via the Human Protein Atlas database; and (3) to utilize the validated human trophoblast markers to identify subgroups of human preeclampsia. To achieve these goals, plasma membrane proteins at the blood tissue interfaces were extracted from placentas using intravascular silica-bead perfusion, and then identified using shotgun proteomics. We identified 1181 plasma membrane proteins, of which 171 were enriched at the maternal blood-trophoblast interface and 192 at the fetal endothelial interface with a 70% conservation of expression in humans. Three distinct molecular subgroups of human preeclampsia were identified in existing human microarray data by using expression patterns of trophoblast-enriched proteins. Analysis of all misexpressed genes revealed divergent dysfunctions including angiogenesis (subgroup 1), MAPK signaling (subgroup 2), and hormone biosynthesis and metabolism (subgroup 3). Subgroup 2 lacked expected changes in known preeclampsia markers (sFLT1, sENG) and uniquely overexpressed GNA12. In an independent set of 40 banked placental specimens, GNA12 was overexpressed during preeclampsia when co-incident with chronic hypertension. In the current study we used a novel translational analysis to integrate mouse and human trophoblast protein expression with human microarray data. This strategy identified distinct molecular pathologies in human preeclampsia. We conclude that clinically similar preeclampsia patients exhibit divergent placental gene expression profiles thus implicating divergent molecular mechanisms in the origins of this disease.

Cells sense and respond to changes in oxygen concentration through gene regulatory processes that are fundamental to survival. Surprisingly, little is known about how anemia affects hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular responses to acute hypoxia, we defined the effects of NOS deficiency in acute anemia. In contrast to endothelial NOS or inducible NOS deficiency, neuronal NOS (nNOS)(-/-) mice demonstrated increased mortality during anemia. Unlike wild-type (WT) animals, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS(-/-) mice. At the cellular level, anemia increased expression of HIF-1α protein and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK1) in the brain of WT, but not nNOS(-/-) mice, despite comparable reductions in tissue PO(2). Paradoxically, nNOS(-/-) mice survived longer during hypoxia, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. Real-time imaging of transgenic animals expressing a reporter HIF-α(ODD)-luciferase chimeric protein confirmed that nNOS was essential for anemia-mediated increases in HIF-α protein stability in vivo. S-nitrosylation effects the functional interaction between HIF and pVHL. We found that anemia led to nNOS-dependent S-nitrosylation of pVHL in vivo and, of interest, led to decreased expression of GSNO reductase. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo and provide essential mechanistic insight into the differences between anemia and hypoxia.

Over the past decade, non-invasive preclinical imaging has emerged as an important tool to facilitate biomedical discovery. Not only have the markets for these tools accelerated, but the numbers of peer-reviewed papers in which imaging end points and biomarkers have been used have grown dramatically. High frequency 'micro-ultrasound' has steadily evolved in the post-genomic era as a rapid, comparatively inexpensive imaging tool for studying normal development and models of human disease in small animals. One of the fundamental barriers to this development was the technological hurdle associated with high-frequency array transducers. Recently, new approaches have enabled the upper limits of linear and phased arrays to be pushed from about 20 to over 50 MHz enabling a broad range of new applications. The innovations leading to the new transducer technology and scanner architecture are reviewed. Applications of preclinical micro-ultrasound are explored for developmental biology, cancer, and cardiovascular disease. With respect to the future, the latest developments in high-frequency ultrasound imaging are described.

A mouse founder with high bone mineral density and an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) screen. It was found to carry a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit of the vacuolar type H(+)-ATPase (V-ATPase), resulting in replacement of a highly conserved amino acid (R740S). The +/R740S mice have normal appearance, size, and weight but exhibit high bone density. Osteoblast parameters are unaffected in bones of +/R740S mice, whereas osteoclast number and marker expression are increased, concomitant with a decrease in the number of apoptotic osteoclasts. Consistent with reduced osteoclast apoptosis, expression of Rankl and Bcl2 is elevated, whereas Casp3 is reduced. Transmission electron microscopy revealed that unlike other known mutations in the a3 subunit of V-ATPase, polarization and ruffled border formation appear normal in +/R740S osteoclasts. However, V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport, whereas ATP hydrolysis is not significantly affected. We show for the first time that a point mutation within the a3 subunit, R740S, which is dominant negative for proton pumping and bone resorption, also uncouples proton pumping from ATP hydrolysis but has no effect on ruffled border formation or polarization of osteoclasts. These results suggest that the V(0) complex has proton-pumping-independent functions in mammalian cells., (Copyright © 2011 American Society for Bone and Mineral Research.)

Objective: Hereditary spherocytosis (HS) is a heterogeneous group of spontaneously arising and inherited red blood cell disorders ranging from very mild subclinical cases to severe and life-threatening cases, with symptoms linked directly to the severity of the mutation at the molecular level. We investigated a novel mouse model in which the heterozygotes present with the diagnostic hallmarks of mild HS and surviving homozygotes phenocopy severe hemolytic HS., Materials and Methods: We used N-ethyl-N-nitrosourea mutagenesis to generate random point mutations in the mouse genome and a dominant screen to identify mouse models of human hematopoietic disease. Gene mapping of the HS strain revealed a unique in-frame nonsense mutation arising from a single base transversion in exon 27 of Ank1 (strain designation: Ank1(E924X)). Employing conventional hematopoietic, pathological, biochemical, and cell biology assays, we characterized heterozygous and homozygous Ank1(E924X) mice at the biochemical, cellular, and pathophysiological levels., Results: Although Ank1(E924X/E924X) red blood cell ghosts lack abundant full-length ankyrin-1 isoforms, N-terminal epitope ankyrin-1 antibodies reveal a band consistent with the theoretical size of a truncated mutant ankyrin-1. Using domain-specific antibodies, we further show that this protein lacks both a spectrin-binding domain and a C-terminal regulatory domain. Finally, using antisera that detect C-terminal residues of the products of alternative Ank1 transcripts, we find unique immunoreactive bands not observed in red blood cell ghosts from wild-type or Ank1(E924X) heterozygous mice, including a band similar in size to full-length ankyrin-1., Conclusions: The Ank1(E924X) strain provides a novel tool to study Ank1 and model HS., (Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and the main toxicants found in cigarettes. Women are often exposed to PAHs before pregnancy, typically via prepregnancy smoking. To determine how prepregnancy exposure affects the fetoplacental vasculature of the placenta, we exposed female mice to PAHs before conception, perfused the fetoplacental arterial trees with X-ray contrast agent, and imaged the vasculature ex vivo by microcomputed tomography (micro-CT) at embryonic day 15.5. Automated vascular segmentation and flow calculations revealed that in control trees, <40 chorionic plate vessels (diameter>180 μm) gave rise to ∼1,300 intraplacental arteries (50-180 μm), predicting an arterial vascular resistance of 0.37±0.04 mmHg·s·μl(-1). PAH exposure increased vessel curvature of chorionic plate vessels and significantly increased the tortuousity ratio of the tree. Intraplacental arteries were reduced by 17%, primarily due to a 27% decrease in the number of arteriole-sized (50-100 μm) vessels. There were no changes in the number of chorionic vessels, the depth or span of the tree, the diameter scaling coefficient, or the segment length-to-diameter ratio. PAH exposure resulted in a tree with a similar size and dichotomous branching structure, but one that was comparatively sparse so that arterial vascular resistance was increased by 30%. Assuming the same pressure gradient, blood flow would be 19% lower. Low flow may contribute to the 23% reduction observed in fetal weight. New insights into the specific effects of PAH exposure on a developing arterial tree were achieved using micro-CT imaging and automated vascular segmentation analysis.

Inhibiting renal glucose transport is a potential pharmacologic approach to treat diabetes. The renal tubular sodium-glucose transporter 2 (SGLT2) reabsorbs approximately 90% of the filtered glucose load. An animal model with sglt2 dysfunction could provide information regarding the potential long-term safety and efficacy of SGLT2 inhibitors, which are currently under clinical investigation. Here, we describe Sweet Pee, a mouse model that carries a nonsense mutation in the Slc5a2 gene, which results in the loss of sglt2 protein function. The phenotype of Sweet Pee mutants was remarkably similar to patients with mutations in the Scl5a2 gene. The Sweet Pee mutants had improved glucose tolerance, higher urinary excretion of calcium and magnesium, and growth retardation. Renal physiologic studies demonstrated a prominent distal osmotic diuresis without enhanced natriuresis. Sweet Pee mutants did not exhibit increased KIM-1 or NGAL, markers of acute tubular injury. After induction of diabetes, Sweet Pee mice had better overall glycemic control than wild-type control mice, but had a higher risk for infection and an increased mortality rate (70% in homozygous mutants versus 10% in controls at 20 weeks). In summary, the Sweet Pee model allows study of the long-term benefits and risks associated with inhibition of SGLT2 for the management of diabetes. Our model suggests that inhibiting SGLT2 may improve glucose control but may confer increased risks for infection, malnutrition, volume contraction, and mortality.

Trophoblast invasion likely depends on complex cross talk between the fetal and maternal tissues and may involve the modulation of phosphatidylinositol 3-kinase (PI3K)/AKT signaling activity in maternal decidual cells. In this report, we studied implantation in Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) mice, which lack the PI3K signaling antagonist gene Pten in myometrial and stromal/decidual cells. Primiparous Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) mice were found to be subfertile because of increased fetal mortality at e11.5. Histopathological analyses revealed a failure of decidual regression in these mice, accompanied by reduced or absent invasion of fetal trophoblast glycogen cells and giant cells, abnormal development of the placental labyrinth, and frequent apparent intrauterine fetal growth restriction. Unexpectedly, the loss of phosphate and tensin homolog deleted on chromosome 10 (PTEN) expression in Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) decidual cells was not accompanied by a detectable increase in AKT phosphorylation or altered expression or activation of PI3K/AKT downstream effectors such as mammalian target of rapamycin or glycogen synthase kinase-3β. Terminal deoxynucleotidyl transferase-mediated nick end labeling and bromodeoxyuridine incorporation analyses attributed to the lack of decidual regression mainly to decreased apoptosis in Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) decidual cells, rather than to increased proliferation. Remodeling of the maternal vasculature was delayed in Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) uteri at e11.5, as evidenced by persistence of vascular smooth muscle and decreased infiltration of uterine natural killer cells. In addition, thickening of the myometrium and disorganization of the muscle fibers were observed before and throughout gestation. Almost all Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) mice failed to carry a second litter to term, apparently attributable to endometrial hyperplasia and uterine infections. Together, these data demonstrate novel roles of PTEN in the mammalian uterus and its requirement for proper trophoblast invasion and decidual regression.

Missense mutations in the Fibroblast Growth Factor Receptor 2 (FGFR2) have been identified in human craniosynostotic syndromes such as Crouzon (CS) and Pfeiffer (PS). FGFR2 has two major isoforms, IIIb and IIIc, generated through alternative splicing with their own temporal, spatial, and ligand-binding specificities. In this study, we report the identification and characterization of a missense mutation in codon 290 of murine Fgfr2 (W290R). The defects in W290R mutants are suggestive of disruption of signalling in both IIIb and IIIc isoforms of the Fgfr2 gene. Heterozygous mutants presented with features resembling those found in patients with CS. Fgfr2(W290R) homozygotes displayed constitutive FGFR2 activation with increased, but correct tissue-specific, expression of the IIIb and IIIc isoforms in many of the defective organs. Our Fgfr2(W290R) mouse model thus represents an excellent mouse model of CS to probe the many questions around the pathogenesis of craniosynostotic birth defects consequent to defects in FGF signaling.

Adverse developmental environments can increase the risk of adult cardiovascular disease, but not all individuals are affected, suggesting the importance of genotype. Genetically distinct mouse strains allow the genetic dissection of complex traits; however, they have not been used to evaluate the developmental origins of adult cardiovascular disease. Our objective was to determine the effect of prenatal nutrient restriction (R) on adult cardiovascular function in A/J (AJ) and C57BL/6J (B6) mice and whether a postnatal high-fat (HF) diet exacerbates these effects. Pregnant AJ and B6 mice underwent a 30% R or ad libitum diet, and their offspring underwent a HF or control diet. Hypertension (+17 mmHg; P<0.001) was observed in B6R mice at 9 wk, and their arterial pressure tended to remain high at 25 wk (+13 mmHg; not significant). In AJR mice, the normal decrement in arterial pressure over this age range in this strain was abolished. Heart rate prematurely increased in B6R and decreased in AJR (all; P<0.05) mice from 9 to 25 wk. There was no effect of postnatal HF diet on these relationships. The Tei index (from a 26-wk microultrasound) was increased in both AJR and B6R mice (all; P<0.05), suggesting an improved global myocardial performance. Neither R nor HF alone changed diastolic (ratio of E wave to A wave) or systolic (%fractional shortening) function in either strain; however, R and HE combined improved diastolic function in B6 (P<0.05) but not in AJ mice. Therefore, there are strain-dependent alterations in adult cardiovascular function in response to prenatal nutrient restriction. Unexpectedly, a postnatal HF diet did not exacerbate the effects of prenatal nutrient restriction on postnatal cardiovascular outcomes.

Placental abnormalities are associated with two of the most common and serious complications of human pregnancy, maternal preeclampsia (PE) and fetal intrauterine growth restriction (IUGR), each disorder affecting approximately 5% of all pregnancies. An important question for the use of the mouse as a model for studying human disease is the degree of functional conservation of genetic control pathways from human to mouse. The human and mouse placenta show structural similarities, but there have been no systematic attempts to assess their molecular similarities or differences. We collected protein and mRNA expression data through shot-gun proteomics and microarray expression analysis of the highly vascular exchange region, microdissected from the human and mouse near-term placenta. Over 7000 ortholog genes were detected with 70% co-expressed in both species. Close to 90% agreement was found between our human proteomic results and 1649 genes assayed by immunohistochemistry for expression in the human placenta in the Human Protein Atlas. Interestingly, over 80% of genes known to cause placental phenotypes in mouse are co-expressed in human. Several of these phenotype-associated proteins form a tight protein-protein interaction network involving 15 known and 34 novel candidate proteins also likely important in placental structure and/or function. The entire data are available as a web-accessible database to guide the informed development of mouse models to study human disease.

Background: In mice the exchange of oxygen and nutrients between mother and fetus occurs in the chorioallantoic placenta where fetal capillaries come in close proximity with maternal blood perfusing trophoblast-lined sinusoids. Despite its critical importance, quantitative in vivo gene expression over the initial stages of chorioallantoic placental development has not been described, nor are there in vitro systems recapitulating the critical syncytiotrophoblast differentiation step in its formation. Here we describe molecular events that occur during the onset of chorioallantoic morphogenesis in mice in vivo, and in placental explant and whole conceptus cultures in vitro., Results: Chorioallantoic morphogenesis began immediately following allantoic fusion with the chorion in vivo, and was associated with significant upregulation of syncytiotrophoblast associated mRNA (Gcm1 and Syncytin A). However mouse placentas with chorioallantoic point attachment cultured with the allantois or as whole conceptuses did not upregulate Gcm1 and/or Syncytin A, suggesting that syncytiotrophoblast differentiation did not occur in vitro. Failure of morphogenesis appeared to be due to failure to sustain in vitro the chorionic trophoblast cells from which the syncytiotrophoblast cells are derived. In vitro culture conditions did support the upregulation of ectoplacental cone marker Tpbpalpha, maintenance of giant cell marker Pl1, and maintenance of Fgfr2 expression; all of which mimicked in vivo events observed over this developmental interval., Conclusions: We conclude that chorionic trophoblast maintenance and the early events that occur in vivo between chorioallantoic point attachment and primary villous formation are dependent on undefined intrauterine factors that were not present in the in vitro culture system. Nevertheless, in vitro culture conditions were appropriate to reproduce in vivo expression levels of Fgfr2, Pl1, and Tpbpalpha in placental explants.

Background: Non-invasive micro-ultrasound was evaluated as a method to quantify intrauterine growth phenotypes in mice. Improved methods are required to accelerate research using genetically-altered mice to investigate the interactive roles of genes and environments on embryonic and placental growth. We determined (1) feasible age ranges for measuring specific variables, (2) normative growth curves, (3) accuracy of ultrasound measurements in comparison with light microscopy, and (4) weight prediction equations using regression analysis for CD-1 mice and evaluated their accuracy when applied to other mouse strains., Methods: We used 30-40 MHz ultrasound to quantify embryonic and placental morphometry in isoflurane-anesthetized pregnant CD-1 mice from embryonic day 7.5 (E7.5) to E18.5 (full-term), and for C57Bl/6J, B6CBAF1, and hIGFBP1 pregnant transgenic mice at E17.5., Results: Gestational sac dimension provided the earliest measure of conceptus size. Sac dimension derived using regression analysis increased from 0.84 mm at E7.5 to 6.44 mm at E11.5 when it was discontinued. The earliest measurement of embryo size was crown-rump length (CRL) which increased from 1.88 mm at E8.5 to 16.22 mm at E16.5 after which it exceeded the field of view. From E10.5 to E18.5 (full term), progressive increases were observed in embryonic biparietal diameter (BPD) (0.79 mm to 7.55 mm at E18.5), abdominal circumference (AC) (4.91 mm to 26.56 mm), and eye lens diameter (0.20 mm to 0.93 mm). Ossified femur length was measureable from E15.5 (1.06 mm) and increased linearly to 2.23 mm at E18.5. In contrast, placental diameter (PD) and placental thickness (PT) increased from E10.5 to E14.5 then remained constant to term in accord with placental weight. Ultrasound and light microscopy measurements agreed with no significant bias and a discrepancy of less than 25%. Regression equations predicting gestational age from individual variables, and embryonic weight (BW) from CRL, BPD, and AC were obtained. The prediction equation BW = -0.757 + 0.0453 (CRL) + 0.0334 (AC) derived from CD-1 data predicted embryonic weights at E17.5 in three other strains of mice with a mean discrepancy of less than 16%., Conclusion: Micro-ultrasound provides a feasible tool for in vivo morphometric quantification of embryonic and placental growth parameters in mice and for estimation of embryonic gestational age and/or body weight in utero.

Maternal cigarette smoking is considered an important risk factor associated with fetal intrauterine growth restriction (IUGR). Polycyclic aromatic hydrocarbons (PAHs) are well-known constituents of cigarette smoke, and the effects of acute exposure to these chemicals at different gestational stages have been well established in a variety of laboratory animals. In addition, many PAHs are known ligands of the aryl hydrocarbon receptor (AhR), a cellular xenobiotic sensor responsible for activating the metabolic machinery. In this study, we have applied a chronic, low-dose regimen of PAH exposure to C57Bl/6 female mice before conception. This treatment caused IUGR in day 15.5 post coitum (d15.5) fetuses and yielded abnormalities in the placental vasculature, resulting in significantly reduced arterial surface area and volume of the fetal arterial vasculature of the placenta. However, examination of the small vasculature within the placental labyrinth of PAH-exposed dams revealed extensive branching and enlargement of these vessels, indicating a possible compensatory mechanism. These alterations in vascularization were accompanied by reduced placental cell death rates, increased expression levels of antiapoptotic Xiap, and decreased expression of proapoptotic Bax, cleaved poly(ADP-ribose) polymerase-1, and active caspase-3. AhR-deficient fetuses were rescued from PAH-induced growth restriction and exhibited no changes in the labyrinthine cell death rate. The results of this investigation suggest that chronic exposure to PAHs is a contributing factor to the development of IUGR in human smokers and that the AhR pathway is involved.

To elucidate the role of endothelial NO synthase (eNOS)-derived NO during mammalian embryogenesis, we assessed the expression of the eNOS gene during development. Using transgenic eNOS promoter/reporter mice (with beta-galactosidase and green fluorescent protein reporters), in situ cRNA hybridization, and immunohistochemistry to assess transcription, steady-state mRNA levels, and protein expression, respectively, we noted that eNOS expression in the developing cardiovascular system was highly restricted to endothelial cells of medium- and large-sized arteries and the endocardium. The onset of transcription of the native eNOS gene and reporters coincided with the establishment of robust, unidirectional blood flow at embryonic day 9.5, as assessed by Doppler ultrasound biomicroscopy. Interestingly, reporter transgene expression and native eNOS mRNA were also observed in discrete regions of the developing skeletal musculature and the apical ectodermal ridge of developing limbs, suggesting a role for eNOS-derived NO in limb development. In vitro studies of promoter/reporter constructs indicated that similar eNOS promoter regions operate in both embryonic skeletal muscle and vascular endothelial cells. In summary, transcriptional activity of the eNOS gene in the murine circulatory system occurred following the establishment of embryonic blood flow. Thus, the eNOS gene is a late-onset gene in endothelial ontogeny.