Treatment of Mid‐Pregnant Mice with KRN633, an Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinase,Induces Abnormal Retinal Vascular Patterning in Their Newborn Pups

We previously reported that treatment of mid‐pregnant mice with KRN633, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, caused fetal growth restriction resulting from diminished vascularization in the placenta and fetal organs. In this study, we examined how the treatment of mid‐pregnant mice with KRN633 affects the development and morphology of vascular components (endothelial cells, pericytes, and basement membrane) in the retinas of their newborn pups. Pregnant mice were treated with KRN633 (5 mg/kg) once daily from embryonic day 13.5 until the day of delivery. Vascular components were examined using immunohistochemistry with specific markers for each component. Radial vascular growth in the retina was slightly delayed until postnatal day 4 (P4) in the newborn pups of KRN633‐treated mothers. On P8, compared with the pups of control mothers, the pups of KRN633‐treated mothers exhibited decreased numbers of central arteries and veins and abnormal branching of the central arteries. No apparent differences in pericytes or basement membrane were observed between the pups of control and KRN633‐treated mothers. These results suggest that a critical period for determining retinal vascular patterning is present at the earliest stages of retinal vascular development, and that the impaired vascular endothelial growth factor signaling during this period induces abnormal architecture in the retinal vascular network     

Induction of intrauterine growth restriction by reducing placental vascular growth with the angioinhibin TNP-470

The placenta is a specialized vascular interface between the maternal and fetal circulations that increases in size to accommodate the nutritional and metabolic demands of the growing fetus. Vascular proliferation and expansion are critical components of placental development and, consequently, interference with vascular growth has the potential to severely restrict concurrent development of both the placenta and fetus. In this study, we describe the effects of an antiangiogenic agent, TNP-470, on placental vascular development and the induction of a form of intrauterine growth restriction (IUGR) in mice. Administration of TNP-470 to dams in the second half of pregnancy resulted in a smaller maternal weight gain accompanied by decreased placental and fetal sizes in comparison with control animals. Total numbers of fetuses per litter were not affected significantly. Stereological analysis of placentas revealed no changes in the combined lengths of vessels. However, the mean cross-sectional areas of maternal and fetal vessels in the labyrinth of TNP-470-treated mice were reduced at Embryonic Day 13.5 (E13.5) but not at E18.5. Further analysis showed reduced placental endothelial proliferation at E13.5 and E18.5 in TNP-470-treated animals. No other structural or morphometric differences in placentas were detected between TNP-470-treated and control mice at E18.5. This study provides conclusive evidence that administration of TNP-470 interferes with placental vascular proliferation and vessel caliber and results in a reproducible model of IUGR.     

血管紧张素AT1受体抗体阳性孕鼠后代高盐饮食后血管功能障碍

血管紧张素AT1受体抗体(AT1-Ab)可损伤胎盘发育,进而导致胎儿宫内生长受限(intrauterine growth restriction,IUGR).根据胎儿源性成人疾病学说,IUGR会明显增加成人后患心血管疾病的几率.本研究旨在观察AT1-Ab阳性孕鼠后代生长至成年后血管功能有无异常.24只雌性Wistar大...     

血管生长异常与流产的关系研究进展

微血管密度异常、血管生长因子(VEGF、PDGF等)及其受体表达异常通过一系列级联反应导致血管异常生长的结果。众多因子均和血管形成有关,在妊娠过程中对胎盘的血管发育有着重要的作用,导致滋养细胞的表型转换障碍、血管结构发育不良、血管生成受阻、血管数目减少,引起胎盘血管重铸障碍,胎儿胎盘单位灌注不足发生流产。研究表明许多自然流产的发生与胎盘组织中血管增生平衡和胎儿血液供应不足有密切关系,从而认为血管生长异常是导致流产的又一重要因素。随着研究的深入进展血管的异常生长与流产的关系是有确定关系的,对于血管生长异常所致的流产,抑制血管各种血管因子的形成、阻止其与受体结合,从而抑制血管的异常生长最终达到克服流产的发展,无异于把幸福带给更多的家庭,不仅是妇产科发展的里程碑,更是人类医学发展史上光辉的一笔。     

Vascular endothelial growth factor and placenta growth factor in intrauterine growth-restricted fetuses and neonates

The angiogenic factors vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) are respectively up- and downregulated by hypoxia. We aimed to study circulating levels of the above factors in intrauterine growth restriction (IUGR) and to correlate their levels with the customized centiles of the infants. The study included 25 IUGR and 25 appropriate for gestational age (AGA) full-term, singleton infants and their mothers. Maternal (MS), fetal (UC), and neonatal day 1 (N1) and 4 (N4) blood was examined. MS and N1 PlGF, as well as UC VEGF levels correlated with the customized centiles of the infants (r= 0.39, P=.007, r=0.34, P=.01, and r= -0.41, P=.004, resp). Furthermore, UC, N1, and N4 VEGF levels were higher in girls (r=0.36, P=.01, r=0.33, P=.02, and r=0.41, P=.005 resp). In conclusion, positive and negative correlations of examined factors with the customized centiles of the infant could rely on placental function and intrauterine oxygen concentrations-both being usually lower in IUGR cases-while higher VEGF levels in girls should possibly be attributed to the stimulating action of estrogens.     

PDGFB regulates the development of the labyrinthine layer of the mouse fetal placenta.

PDGFB is a growth factor which is vital for the completion of normal prenatal development. In this study, we report the phenotypic analysis of placentas from mouse conceptuses that lack a functional PDGFB or PDGFRbeta gene. Placentas of both types of mutant exhibit changes in the labyrinthine layer, including dilated embryonic blood vessels and reduced numbers of both pericytes and trophoblasts. These changes are seen from embryonic day (E) 13.5, which coincides with the upregulation of PDGFB mRNA levels in normal placentas. By E17, modifications in shape, size, and number of the fetal blood vessels in the mutant placentas cause an abnormal ratio of the surface areas between the fetal and the maternal blood vessels in the labyrinthine layer. Our data suggest that PDGFB acts locally to contribute to the development of the labyrinthine layer of the fetal placenta and the formation of a proper nutrient-waste exchange system during fetal development. We point out that the roles of PDGFB/Rbeta signaling in the placenta may be analogous to those in the developing kidney, by controlling pericytes in the labyrinthine layer and mesangial cells in the kidney.     

Interactions between trophoblast cells and the maternal and fetal circulation in the mouse placenta

Mammalian embryos have an intimate relationship with their mothers, particularly with the placental vasculature from which embryos obtain nutrients essential for growth. It is an interesting vascular bed because maternal vessel number and diameter change dramatically during gestation and, in rodents and primates, the terminal blood space becomes lined by placental trophoblast cells rather than endothelial cells. Molecular genetic studies in mice aimed at identifying potential regulators of these processes have been hampered by lack of understanding of the anatomy of the vascular spaces in the placenta and the general nature of maternal-fetal vascular interactions. To address this problem, we examined the anatomy of the mouse placenta by preparing plastic vascular casts and serial histological sections of implantation sites from embryonic day (E) 10.5 to term. We found that each radial artery carrying maternal blood into the uterus branched into 5-10 dilated spiral arteries located within the metrial triangle, populated by uterine natural killer (uNK) cells, and the decidua basalis. The endothelial-lined spiral arteries converged together at the trophoblast giant cell layer and emptied into a few straight, trophoblast-lined "canals" that carried maternal blood to the base of the placenta. Maternal blood then percolated back through the intervillous space of the labyrinth toward the maternal side of the placenta in a direction that is countercurrent to the direction of the fetal capillary blood flow. Trophoblast cells were found invading the uterus in two patterns. Large cells that expressed the trophoblast giant cell-specific gene Plf (encoding Proliferin) invaded during the early postimplantation period in a pattern tightly associated with spiral arteries. These peri/endovascular trophoblast were detected only approximately 150-300 microm upstream of the main giant cell layer. A second type of widespread interstitial invasion in the decidua basalis by glycogen trophoblast cells was detected after E12.5. These cells did not express Plf, but rather expressed the spongiotrophoblast-specific gene Tpbp. Dilation of the spiral arteries was obvious between E10.5 and E14.5 and was associated with a lack of elastic lamina and smooth muscle cells. These features were apparent even in the metrial triangle, a site far away from the invading trophoblast cells. By contrast, the transition from endothelium-lined artery to trophoblast-lined (hemochorial) blood space was associated with trophoblast giant cells. Moreover, the shaping of the maternal blood spaces within the labyrinth was dependent on chorioallantoic morphogenesis and therefore disrupted in Gcm1 mutants. These studies provide important insights into how the fetoplacental unit interacts with the maternal intrauterine vascular system during pregnancy in mice.     

阻断VEGF旁分泌通路抑制乳腺癌血管生成与肿瘤生长

以人乳腺癌细胞株MCF 7为研究对象 ,通过构建有义与反义血管内皮生长因子 (VEGF)基因表达质粒 ,并转染MCF 7细胞 ,建立了高与低水平表达VEGF的细胞克隆。稳定转染反义VEGF表达质粒的细胞产生和分泌VEGF的能力明显下降 ,尽管在体外培养条件下细胞的增殖速度与未经转染的对照相比不是减慢而是略有增快 ,但在体内的成瘤能力、生长速度和转移能力等却明显低于未经转染的对照细胞或稳定转染有义VEGF表达质粒高水平表达VEGF的细胞克隆。通过体内电穿孔技术介导反义VEGF12 1及可溶性VEGF受体sFlk 1表达质粒转移至荷瘤鼠肿瘤组织内 ,反义VEGF12 1及sFlk 1的表达能显著抑制肿瘤的生长。研究结果证实了VEGF旁分泌通路在诱导乳腺癌肿瘤血管生成、促进肿瘤生长和转移方面起重要作用 ,阻断VEGF旁分泌通路能有效抑制乳腺癌的生长     

Exposure to high‐concentration oxygen in the neonatal period induces abnormal retinal vascular patterning in mice

The interruption of vascular development could cause structural and functional abnormalities in tissues. We have previously reported that short‐term treatment of newborn mice with vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitors induces abnormal retinal vascular growth and patterns. An exposure of neonatal mice to high‐concentration oxygen disturbs normal retinal vascular development. The present study aimed to determine (1) whether vascular abnormalities are observed in the retina of newborn mice exposed to high concentrations of oxygen, and (2) how astrocyte network formation is affected following the exposure to hyperoxia. Newborn (postnatal day 0) mice were exposed to 75% oxygen for 48 or 96 hr. During hyperoxia exposure, VEGF expression decreased, and the onset of retinal vascularization was completely suppressed. After completion of the hyperoxic period, retinal vascularization occurred, but it was delayed in a hyperoxic exposure duration‐dependent manner. In retinas of hyperoxia‐exposed mice, dense capillary plexuses were found, and the number of arteries and veins decreased. The astrocyte network formation was slightly delayed under hyperoxic conditions, and the network became denser in retinas of mice with an episode of hyperoxia. Expression of VEGF levels in the avascular retina of mice that were exposed to hyperoxia was higher than that of control mice. These results suggest that short‐term interruption of the onset of vascular development resulting from the reduction in VEGF signals induces abnormal vascular patterns in the mouse retina. The abnormalities in retinal astrocyte behavior might contribute to the formation of an abnormal retinal vascular growth.     

Thrombophilic-type placental pathologies and skeletal growth delay following maternal administration of angiostatin4.5 in mice

During placentation, the concentration of fibrinous deposits on the surfaces of maternal vasculature plays a role in villous development and has been strongly implicated in the pathophysiology of human fetal growth restriction (FGR). Fibrinous deposits are conspicuous sites of platelet aggregation where there is local activation of the hemostatic cascade. During activation of the hemostatic cascade, a number of pro- and antiangiogenic agents may be generated at the cell surface, and an imbalance in these factors may contribute to the placental pathology characteristic of FGR. We tested the hypothesis that angiostatin(4.5) (AS(4.5)), a cleavage fragment of plasminogen liberated at the cell surface, is capable of causing FGR in mice. Increased maternal levels of AS(4.5) in vivo result in reproducible placental pathology, including an altered vascular compartment (both in decidual and labyrinthine layers) and increased apoptosis throughout the placenta. In addition, there is significant skeletal growth delay and conspicuous edema in fetuses from mothers that received AS(4.5). Maternally generated AS(4.5), therefore, can access maternal placental vasculature and have a severe effect on placental architecture and inhibit fetal development in vivo. These findings strongly support the hypothesis that maternal AS(4.5) levels can influence placental development, possibly by directly influencing trophoblast turnover in the placenta, and contribute to fetal growth delay in mice.     

Genetic insights into trophoblast differentiation and placental morphogenesis

The placenta is comprised of an inner vascular network covered by an outer epithelium, called trophoblast, all designed to promote the delivery of nutrients to the fetus. Several specialized trophoblast cell subtypes arise during development to promote this function, including cells that invade the uterus to promote maternal blood flow to the implantation site, and other cells that fuse into a syncytium, expand and fold to increase the surface area for efficient transport. Mutation of many genes in mice results in embryonic mortality or fetal growth restriction due to defects in placental development. Several important principles about placental development have emerged from these studies. First, distinct molecular pathways regulate the differentiation of the various trophoblast cell subtypes. Second, trophoblast proliferation, differentiation and morphogenesis are highly regulated by interactions with adjacent cell types. Finally, the specific classes of mutant phenotypes observed in the placenta of knockout mice resemble those seen in humans that are associated with preeclampsia and intrauterine growth restriction.     

VEGF modulates synaptic activity in the developing spinal cord

Although it has been documented that the nervous and the vascular systems share numerous analogies and are closely intermingled during development and pathological processes, interactions between the two systems are still poorly described. In this study, we investigated whether vascular endothelial growth factor (VEGF), which is a key regulator of vascular development, also modulates neuronal developmental processes. We report that VEGF enhances the gamma‐aminobutyric acid (GABA)/glycinergic but not glutamatergic synaptic activity in embryonic spinal motoneurons (MNs), without affecting MNs excitability. In response to VEGF, the frequency of these synaptic events but not their amplitude was increased. Blocking endogenous VEGF led to an opposite effect by decreasing frequency of synaptic events. We found that this effect occurred specifically at early developmental stages (E13.5 and E15.5) and vanished at the prenatal stage E17.5. Furthermore, VEGF was able to increase vesicular inhibitory amino acid transporter density at the MN membrane. Inhibition of single VEGF receptors did not modify electrophysiological parameters indicating receptor combinations or an alternative pathway. Altogether, our findings identify VEGF as a modulator of the neuronal activity during synapse formation and highlight a new ontogenic role for this angiogenic factor in the nervous system. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1110–1122, 2014     

A Bayesian Latent Variable Mixture Model for Longitudinal Fetal Growth

Summary Fetal growth restriction is a leading cause of perinatal morbidity and mortality that could be reduced if high‐risk infants are identified early in pregnancy. We propose a Bayesian model for aggregating 18 longitudinal ultrasound measurements of fetal size and blood flow into three underlying, continuous latent factors. Our procedure is more flexible than typical latent variable methods in that we relax the normality assumptions by allowing the latent factors to follow finite mixture distributions. Using mixture distributions also permits us to cluster individuals with similar observed characteristics and identify latent classes of subjects who are more likely to be growth or blood flow restricted during pregnancy. We also use our latent variable mixture distribution model to identify a clinically meaningful latent class of subjects with low birth weight and early gestational age. We then examine the association of latent classes of intrauterine growth restriction with latent classes of birth outcomes as well as observed maternal covariates including fetal gender and maternal race, parity, body mass index, and height. Our methods identified a latent class of subjects who have increased blood flow restriction and below average intrauterine size during pregnancy. These subjects were more likely to be growth restricted at birth than a class of individuals with typical size and blood flow.     

Infant growth restriction is associated with distinct patterns of DNA methylation in human placentas

The placenta acts not only as a conduit of nutrient and waste exchange between mother and developing fetus, but also functions as a regulator of the intrauterine environment. Recent work has identified changes in the expression of candidate genes, often through epigenetic alteration, which alter the placenta''s function and impact fetal growth. In this study, we used the Illumina Infinium HumanMethylation27 BeadChip array to examine genome-wide DNA methylation patterns in 206 term human placentas. Semi-supervised recursively partitioned mixture modeling was implemented to identify specific patterns of placental DNA methylation that could differentially classify intrauterine growth restriction (IUGR) and small for gestational age (SGA) placentas from appropriate for gestational age (AGA) placentas, and these associations were validated in a masked testing series of samples. Our work demonstrates that patterns of DNA methylation in human placenta are reliably and significantly associated with infant growth and serve as a proof of principle that methylation status in the human term placenta can function as a marker for the intrauterine environment, and could potentially play a critical functional role in fetal development.Key words: epigenetics, DNA methylation, placenta, intrauterine growth restriction, small for gestational age, development, human     

Infant growth restriction is associated with distinct patterns of DNA methylation in human placentas

The placenta acts not only as a conduit of nutrient and waste exchange between mother and developing fetus, but also functions as a regulator of the intrauterine environment. Recent work has identified changes in the expression of candidate genes, often through epigenetic alteration, which alter the placenta's function and impact fetal growth. In this study, we used the Illumina Infinium HumanMethylation27 BeadChip array to examine genome-wide DNA methylation patterns in 206 term human placentas. Semi-supervised recursively partitioned mixture modeling was implemented to identify specific patterns of placental DNA methylation that could differentially classify intrauterine growth restriction (IUGR) and small for gestational age (SGA) placentas from appropriate for gestational age (AGA) placentas, and these associations were validated in a masked testing series of samples. Our work demonstrates that patterns of DNA methylation in human placenta are reliably and significantly associated with infant growth and serve as a proof of principle that methylation status in the human term placenta can function as a marker for the intrauterine environment, and could potentially play a critical functional role in fetal development.     

Developmental changes in hemodynamics of uterine artery, utero- and umbilicoplacental, and vitelline circulations in mouse throughout gestation

In human pregnancy, abnormal placental hemodynamics likely contribute to the etiology of early-onset preeclampsia and fetal intrauterine growth restriction. The mouse is increasingly being deployed to study normal and abnormal mammalian placental development, yet the placental hemodynamics in normal pregnancy in mice is currently unknown. We used ultrasound biomicroscopy to noninvasively image and record Doppler blood velocity waveforms from the maternal and embryonic placental circulations in mice throughout gestation. In the uterine artery, peak systolic velocity (PSV) increased significantly from 23+/-2 (SE) to 59+/-3 cm/s, and end-diastolic velocity (EDV) increased from 7+/-1 to 28+/-2 cm/s in nonpregnant versus full-term females so that the uterine arterial resistance index (RI) decreased from 0.70+/-0.02 to 0.53+/-0.02. Velocities in the maternal arterial canal in the placenta were low and nearly steady and increased from 0.9+/-0.03 cm/s at embryonic day 10.5 (E10.5) to 2.4+/-0.07 cm/s at E18.5. PSV in the umbilical artery increased steadily from 0.8+/-0.1 cm/s at E8.5 to 15+/-0.6 cm/s at E18.5, whereas PSV in the vitelline artery increased from 0.6+/-0.1 cm/s at E8.5 to 4+/-0.2 cm/s at E13.5 and then remained stable to term. In the umbilical artery, the EDV detection rate was 0% at 相似文献   

Effects of Hyperthyroidism on Expression of Vascular Endothelial Growth Factor (VEGF) and Apoptosis in Fetal Adrenal Glands

This study investigated the expression of vascular endothelial growth factor (VEGF), vascular density, and apoptosis in fetal rat adrenal glands with hyperthyroidism in late gestation. Twelve mature female Wistar albino rats with the same biological and physiological features were used for this study. Rats were divided into two groups: control and hyperthyroidism. Hyperthyroidism was induced by daily subcutaneous injections of L-thyroxine (250 µg/kg) before pregnancy for 21 days and during pregnancy. Rats in the control and hyperthyroidism groups were caged according to the number of male rats. Zero day of pregnancy (Day 0) was indicated when the animals were observed to have microscopic sperm in vaginal smears. Pregnant rats were sacrificed on the 20th day of pregnancy; blood from each animal was collected to determine the concentrations of maternal adrenocorticotropic hormone and thyroxine. Rat fetuses were then quickly removed from the uterus, and the adrenal glands of the fetuses were dissected. VEGF expression, vascular density, and apoptosis were analyzed in fetal rat adrenal glands. Maternal serum levels of the ACTH and free thyroxine were significantly higher in the hyperthyroidism group than in the control group. Immunohistochemistry revealed that the number of VEGF positive cells and vessel density significantly increased in the hyperthyroidism rat fetal adrenal group compared with the control group. Hyperthyroidism did not change the fetal and placental weights and the number of fetuses. This study demonstrates that hyperthyroidism may have an effect on the development of rat adrenal glands mediated by VEGF expression, angiogenesis, and apoptosis.     

Sildenafil Citrate Increases Fetal Weight in a Mouse Model of Fetal Growth Restriction with a Normal Vascular Phenotype

Fetal growth restriction (FGR) is defined as the inability of a fetus to achieve its genetic growth potential and is associated with a significantly increased risk of morbidity and mortality. Clinically, FGR is diagnosed as a fetus falling below the 5^th centile of customised growth charts. Sildenafil citrate (SC, Viagra™), a potent and selective phosphodiesterase-5 inhibitor, corrects ex vivo placental vascular dysfunction in FGR, demonstrating potential as a therapy for this condition. However, many FGR cases present without an abnormal vascular phenotype, as assessed by Doppler measures of uterine/umbilical artery blood flow velocity. Thus, we hypothesized that SC would not increase fetal growth in a mouse model of FGR, the placental-specific Igf2 knockout mouse, which has altered placental exchange capacity but normal placental blood flow. Fetal weights were increased (by 8%) in P0 mice following maternal SC treatment (0.4 mg/ml) via drinking water. There was also a trend towards increased placental weight in treated P0 mice (P = 0.056). Additionally, 75% of the P0 fetal weights were below the 5^th centile, the criterion used to define human FGR, of the non-treated WT fetal weights; this was reduced to 51% when dams were treated with SC. Umbilical artery and vein blood flow velocity measures confirmed the lack of an abnormal vascular phenotype in the P0 mouse; and were unaffected by SC treatment. ¹⁴C-methylaminoisobutyric acid transfer (measured to assess effects on placental nutrient transporter activity) per g placenta was unaffected by SC, versus untreated, though total transfer was increased, commensurate with the trend towards larger placentas in this group. These data suggest that SC may improve fetal growth even in the absence of an abnormal placental blood flow, potentially affording use in multiple sub-populations of individuals presenting with FGR.