Arterio-venous fetoplacental vascular geometry and hemodynamics in the mouse placenta.

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.

Activated NK cells cause placental dysfunction and miscarriages in fetal alloimmune thrombocytopenia.

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.

A mouse model of antepartum stillbirth.

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.

Effect of selective fetectomy on morphology of the mouse placenta.

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.

Quantification of Gestational Changes in the Uteroplacental Vascular Tree Reveals Vessel Specific Hemodynamic Roles During Pregnancy in Mice.

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.

Maternal anti-platelet ß3 integrins impair angiogenesis and cause intracranial hemorrhage.

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 ß3 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-ß3 integrin-mediated, but not anti-GPIbα-mediated, FNAIT, despite similar thrombocytopenia in both groups. Only anti-ß3 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-ß3 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-ß3 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.

Site-specific increases in utero- and fetoplacental arterial vascular resistance in eNOS-deficient mice due to impaired arterial enlargement.

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.

sFLT1 in preeclampsia: trophoblast defense against a decidual VEGFA barrage?

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.

Trophoblast-specific reduction of VEGFA alters placental gene expression and maternal cardiovascular function in mice.

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.

Effects of genes and environment on the fetoplacental arterial microcirculation in mice revealed by micro-computed tomography imaging.

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.

Enu mutagenesis identifies a novel platelet phenotype in a loss-of-function Jak2 allele.

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.

Chronic carbon monoxide inhalation during pregnancy augments uterine artery blood flow and uteroplacental vascular growth in mice.

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.

ENU-induced mutation in the DNA-binding domain of KLF3 reveals important roles for KLF3 in cardiovascular development and function in mice.

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.

Adipose vascular endothelial growth factor regulates metabolic homeostasis through angiogenesis.

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.

Endothelial NO synthase augments fetoplacental blood flow, placental vascularization, and fetal growth in mice.

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.

Endothelial nitric oxide synthase deficiency reduces uterine blood flow, spiral artery elongation, and placental oxygenation in pregnant mice.

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.

A novel Phex mutation in a new mouse model of hypophosphatemic rickets.

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.

Expansion of the fetoplacental vasculature in late gestation is strain dependent in mice.

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.

Effects of reduced Gcm1 expression on trophoblast morphology, fetoplacental vascularity, and pregnancy outcomes in mice.

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.