Matthew T. Rätsep

Uterine natural killer cells: supervisors of vasculature construction in early decidua basalis

Mammalian pregnancy involves tremendous de novo maternal vascular construction to adequately support conceptus development. In early mouse decidua basalis (DB), maternal uterine natural killer (uNK) cells oversee this process directing various aspects during the formation of supportive vascular networks. The uNK cells recruited to early implantation site DB secrete numerous factors that act in the construction of early decidual vessels (neoangiogenesis) as well as in the alteration of the structural components of newly developing and existing vessels (pruning and remodeling). Although decidual and placental development sufficient to support live births occur in the absence of normally functioning uNK cells, development and structure of implantation site are optimized through the presence of normally activated uNK cells. Human NK cells are also recruited to early decidua. Gestational complications including recurrent spontaneous abortion, fetal growth restriction, preeclampsia, and preterm labor are linked with the absence of human NK cell activation via paternally inherited conceptus transplantation antigens. This review summarizes the roles that mouse uNK cells normally play in decidual neoangiogenesis and spiral artery remodeling in mouse pregnancy and briefly discusses changes in early developmental angiogenesis due to placental growth factor deficiency.

Brain Structural and Vascular Anatomy Is Altered in Offspring of Pre-Eclamptic Pregnancies: A Pilot Study

The authors assessed the brain structural and vascular anatomy in 7- to 10-year-old offspring of pre-eclamptic pregnancies compared with matched controls (n=10 per group). TOF-MRA and a high-resolution anatomic T1-weighted MPRAGE sequence were acquired for each participant. Offspring of pre-eclamptic pregnancies exhibited enlarged brain regional volumes of the cerebellum, temporal lobe, brain stem, and right and left amygdalae. These offspring displayed reduced cerebral vessel radii in the occipital and parietal lobes. The authors conclude that these structural and vascular anomalies may underlie the cognitive deficits reported in the pre-eclamptic offspring population. BACKGROUND AND PURPOSE: Pre-eclampsia is a serious clinical gestational disorder occurring in 3%–5% of all human pregnancies and characterized by endothelial dysfunction and vascular complications. Offspring born of pre-eclamptic pregnancies are reported to exhibit deficits in cognitive function, higher incidence of depression, and increased susceptibility to stroke. However, no brain imaging reports exist on these offspring. We aimed to assess brain structural and vascular anatomy in 7- to 10-year-old offspring of pre-eclamptic pregnancies compared with matched controls. MATERIALS AND METHODS: Offspring of pre-eclamptic pregnancies and matched controls (n = 10 per group) were recruited from an established longitudinal cohort examining the effects of pre-eclampsia. Children underwent MR imaging to identify brain structural and vascular anatomic differences. Maternal plasma samples collected at birth were assayed for angiogenic factors by enzyme-linked immunosorbent assay. RESULTS: Offspring of pre-eclamptic pregnancies exhibited enlarged brain regional volumes of the cerebellum, temporal lobe, brain stem, and right and left amygdalae. These offspring displayed reduced cerebral vessel radii in the occipital and parietal lobes. Enzyme-linked immunosorbent assay analysis revealed underexpression of the placental growth factor among the maternal plasma samples from women who experienced pre-eclampsia. CONCLUSIONS: This study is the first to report brain structural and vascular anatomic alterations in the population of offspring of pre-eclamptic pregnancies. Brain structural alterations shared similarities with those seen in autism. Vascular alterations may have preceded these structural alterations. This pilot study requires further validation with a larger population to provide stronger estimates of brain structural and vascular outcomes among the offspring of pre-eclamptic pregnancies.

Placental growth factor deficiency is associated with impaired cerebral vascular development in mice

STUDY HYPOTHESIS Placental growth factor (PGF) is expressed in the developing mouse brain and contributes to vascularization and vessel patterning. STUDY FINDING PGF is dynamically expressed in fetal mouse brain, particularly forebrain, and is essential for normal cerebrovascular development. WHAT IS KNOWN ALREADY PGF rises in maternal plasma over normal human and mouse pregnancy but is low in many women with the acute onset hypertensive syndrome, pre-eclampsia (PE). Little is known about the expression of PGF in the fetus during PE. Pgf  (-/-) mice appear normal but recently cerebral vascular defects were documented in adult Pgf  (-/-) mice. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Here, temporal-spatial expression of PGF is mapped in normal fetal mouse brains and cerebral vasculature development is compared between normal and congenic Pgf  (-/-) fetuses to assess the actions of PGF during cerebrovascular development. Pgf/PGF, Vegfa/VEGF, Vegf receptor (Vegfr)1 and Vegfr2 expression were examined in the brains of embryonic day (E)12.5, 14.5, 16.5 and 18.5 C57BL/6 (B6) mice using quantitative PCR and immunohistochemistry. The cerebral vasculature was compared between Pgf  (-/-) and B6 embryonic and adult brains using whole mount techniques. Vulnerability to cerebral ischemia was investigated using a left common carotid ligation assay. MAIN RESULTS AND THE ROLE OF CHANCE Pgf/PGF and Vegfr1 are highly expressed in E12.5-14.5 forebrain relative to VEGF and Vegfr2. Vegfa/VEGF is relatively more abundant in hindbrain (HB). PGF and VEGF expression were similar in midbrain. Delayed HB vascularization was seen at E10.5 and 11.5 in Pgf  (-/-) brains. At E14.5, Pgf  (-/-) circle of Willis showed unilateral hypoplasia and fewer collateral vessels, defects that persisted post-natally. Functionally, adult Pgf  (-/-) mice experienced cerebral ischemia after left common carotid arterial occlusion while B6 mice did not. LIMITATIONS, REASONS FOR CAUTION Since Pgf  (-/-) mice were used, consequences of complete absence of maternal and fetal PGF were defined. Therefore, the effects of maternal versus fetal PGF deficiency on cerebrovascular development cannot be separated. However, as PGF was strongly expressed in the developing brain at all timepoints, we suggest that local PGF has a more important role than distant maternal or placental sources. Full PGF loss is not expected in PE pregnancies, predicting that the effects of PGF deficiency identified in this model will be more severe than any effects in PE-offspring. WIDER IMPLICATIONS OF THE FINDINGS These studies provoke the question of whether PGF expression is decreased and cerebral vascular maldevelopment occurs in fetuses who experience a preeclamptic gestation. These individuals have already been reported to have elevated risk for stroke and cognitive impairments. LARGE SCALE DATA N/A. STUDY FUNDING AND COMPETING INTERESTS This work was supported by awards from the Natural Sciences and Engineering Research Council, the Canada Research Chairs Program and the Canadian Foundation for Innovation to B.A.C. and by training awards from the Universidade Federal de Pernambuco and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil to R.L.L.; Queens University to V.R.K. and the Canadian Institutes of Health Research to M.T.R. The work of P.C. is supported by the Belgian Science Policy BELSPO-IUAP7/03, Structural funding by the Flemish Government-Methusalem funding, and the Flemish Science Fund-FWO grants. There were no competing interests.

Impact of placental growth factor deficiency on early mouse implant site angiogenesis

Effects of placental growth factor (PGF), an angiokine product of fetal trophoblasts and maternal decidual cells, on early decidual angiogenesis are undefined. We used whole-mount immunofluorescence analyses to compare uterus and gestation day 4.5-9.5 mouse implantation sites that differed genetically in fetal or maternal PGF deficiency. Implant site number and embryonic development were similar in Pgf(-/-) and Pgf(+/+) females although Pgf(-/-) lymphatic vessels were anomalous. Correct, fine branching angiogenesis of anti-mesometrial vessels required both conceptus and maternal PGF; correct mesometrial branching angiogenesis depended solely upon conceptus PGF. Thus, PGF is non-redundant for optimizing branching angiogenesis in early decidua.

Impact of preeclampsia on cognitive function in the offspring

Preeclampsia (PE) is a significant clinical disorder occurring in 3-5% of all human pregnancies. Offspring of PE pregnancies (PE-F1s) are reported to exhibit greater cognitive impairment than offspring from uncomplicated pregnancies. Previous studies of PE-F1 cognitive ability used tests with bias that do not assess specific cognitive domains. To improve cognitive impairment classification in PE-F1s we used standardized clinical psychometric testing and eye tracking studies of saccadic eye movements. PE-F1s (n=10) and sex/age matched control participants (n=41 for psychometrics; n=59 for eye-tracking) were recruited from the PE-NET study or extracted from the NeuroDevNet study databases. Participants completed a selected array of psychometric tests which assessed executive function, working memory, attention, inhibition, visuospatial processing, reading, and math skills. Eye-tracking studies included the prosaccade, antisaccade, and memory-guided tasks. Psychometric testing revealed an impairment in working memory among PE-F1s. Eye-tracking studies revealed numerous impairments among PE-F1s including additional saccades required to reach the target, poor endpoint accuracy, and slower reaction time. However, PE-F1s made faster saccades than controls, and fewer sequence errors in the memory-guided task. Our study provides a comprehensive assessment of cognitive function among PE-F1s. The development of PE may be seen as an early predictor of reduced cognitive function in children, specifically in working memory and oculomotor control. Future studies should extended to a larger study populations, and may be valuable for early studies of children born to pregnancies complicated by other disorders, such as gestational diabetes or intrauterine growth restriction.

The Elsevier trophoblast research award lecture: Impacts of placental growth factor and preeclampsia on brain development, behaviour, and cognition.

Preeclampsia (PE) is a significant gestational disorder affecting 3-5% of all human pregnancies. In many PE pregnancies, maternal plasma is deficient in placental growth factor (PGF), a placentally-produced angiokine. Beyond immediate fetal risks associated with acute termination of the pregnancy, offspring of PE pregnancies (PE-F1) have higher long-term risks for hypertension, stroke, and cognitive impairment compared to F1s from uncomplicated pregnancies. At present, mechanisms that explain PE-F1 gains in postpartum risks are poorly understood. Our laboratory found that mice genetically-deleted for Pgf have altered fetal and adult brain vascular development. This is accompanied by sexually dimorphic alterations in anatomic structure in the adult Pgf-/- brain and impaired cognitive functions. We hypothesize that cerebrovascular and neurological aberrations occur in fetuses exposed to the progressive development of PE and that these brain changes impair cognitive functioning, enhance risk for stroke, elevate severity of stroke, and lead to worse stroke outcomes. These brain and placental outcomes may be linked to down-regulated PGF gene expression in early pre-implantation embryos, prior to gastrulation. This review explores our hypothesis that there are mechanistic links between low PGF detection in maternal plasma prodromal to PE, PE, and altered brain vascular, structural, and functional development amongst PE-F1s. We also include a summary of preliminary outcomes from a pilot study of 7-10 year old children that is the first to report magnetic resonance imaging, magnetic resonance angiography, and functional brain region assessment by eye movement control studies in PE-F1s.

Neurological function in children born to preeclamptic and hypertensive mothers – A systematic review

BACKGROUND Offspring whose mothers developed preeclampsia (PE-F1s) show developmental effects that are now being identified, such as cognitive, behavioural, and mood differences compared to offspring from non-complicated pregnancies. We hypothesize that the progressive angiokine dysregulation associated with development of preeclampsia (PE) reflects gene dysregulation in pre-implantation conceptuses, and manifests in all developing fetal tissues rather than exclusively to the placenta. This hypothesis predicts that fetal cerebrovascular and brain development are deviated by fetal-intrinsic, brain-based mechanisms during what is currently considered a placentally-induced maternal disease. Due to our initial results from brain-imaging and cognitive screening in a child pilot PE-F1 cohort, we developed this systematic review to answer the question of whether any consistent neurological measurements have been found to discriminate between brain functions in offspring of mothers who experienced a hypertensive pregnancy vs. offspring of mothers that did not. METHODS Relevant studies were searched systematically up to June 2017 in MEDLINE, PsycINFO, EMBASE and the grey literature. RESULTS Following predetermined inclusion and exclusion criteria, our search identified 27 out of 464 studies reporting on neurological function in offspring born to preeclamptic and hypertensive mothers. CONCLUSION The current literature strongly supports the conclusion of the behavioural and cognitive deviations in PE-F1s. However, only three studies associated their findings with brain measurements via magnetic resonance imaging (MRI) in both healthy and at-risk pediatric populations. PE-F1s should be identified as an at-risk pediatric population during brain development and studied further as a defined group, perhaps stratified by maternal plasma angiokine levels.

Diffusion Tensor Imaging of White Matter in Children Born from Preeclamptic Gestations

BACKGROUND AND PURPOSE: Individuals born from pregnancies complicated by preeclampsia have an elevated risk for cognitive impairment. Deviations in maternal plasma angiokines occur for prolonged intervals before clinical signs of preeclampsia. We hypothesized that fetal brain vascular and nervous tissue development become deviated during maternal progression toward preeclampsia and that such deviations would be detectable by MR imaging. MATERIALS AND METHODS: In this pilot study, 10 matched (gestational and current ages) pairs (5 boys/5 girls, 7–10 years of age) from preeclampsia or control pregnancies were examined by using diffusion tensor MR imaging. An unbiased voxel-based analysis was conducted on fractional anisotropy and mean diffusivity parametric maps. Six brain ROIs were identified for subsequent analysis by tractography (middle occipital gyrus, caudate nucleus and precuneus, cerebellum, superior longitudinal fasciculus, and cingulate gyrus). RESULTS: Statistical differences were present between groups for fractional anisotropy in the caudate nucleus (offspring from preeclamptic gestation > controls), volume of the tract for the superior longitudinal fasciculus (offspring from preeclamptic gestation > controls) and the caudate nucleus (offspring from preeclamptic gestation > controls), and for parallel diffusivity of the cingulate gyrus (offspring from preeclamptic gestation > controls). CONCLUSIONS: These novel preliminary results along with previous results from the same children that identified altered cerebral vessel calibers and increased regional brain volumes justify fully powered MR imaging studies to address the impact of preeclampsia on human fetal brain development.

Whole Mount In Situ Immunohistochemistry

Chapter Summary Whole mount in situ immunohistochemistry is a simple technique that can be customized very easily to the investigator’s interests. It permits acquisition of multidimensional information on cellular relationships in intact, live tissue. By varying the antibodies used and strains of pregnant mouse and their manipulations, biologically accurate data concerning the maternal–fetal interface are obtained.

Changes in Structures and Relationships of Trophoblasts, Leukocytes, and Maternal Vessels in Implantation Sites to Midpregnancy

Chapter Summary Change is the only “true” constant during mouse implantation site development. Changes occur to the uterine stroma (including its vasculature), leukocytes are recruited, the conceptus enlarges during development and growth, trophoblast cells invade, and the placenta forms. These complex changes are most accurately found in intact tissue and are described through serial time course analyses. Whole mount in situ immunohistochemistry of gd4.5–9.5 implant sites is a simple imaging technique applicable to almost any mouse pregnancy model and research question. Fluorochrome-tagged antibodies to cell surface molecules are applied to intact, viable implant sites. Specimens are then studied using fluorescence photomicroscopy. Combined with the use of genetic reporter molecules expressing fluorescent gene-tracking tags and embryo or adoptive cell transfers, applications of this technique in mouse pregnancy research are almost limitless. Baseline findings in early implant sites for genetically normal mice are described in this chapter, as are example studies that address the roles of lymphocytes and their activation in genetically modified mice.