Adult Pgf-/- mice behaviour and neuroanatomy are altered by neonatal treatment with recombinant placental growth factor.

Offspring of preeclamptic pregnancies have cognitive alterations. Placental growth factor (PGF), is low in preeclampsia; reduced levels may affect brain development. PGF-null mice differ from normal congenic controls in cerebrovasculature, neuroanatomy and behavior. Using brain imaging and behavioral testing, we asked whether developmentally asynchronous (i.e. neonatal) PGF supplementation alters the vascular, neuroanatomic and/or behavioral status of Pgf-/- mice at adulthood. C57BL/6-Pgf-/- pups were treated intraperitoneally on postnatal days 1-10 with vehicle or PGF at 10 pg/g, 70 pg/g or 700 pg/g. These mice underwent behavioral testing and perfusion for MRI and analysis of retinal vasculature. A second cohort of vehicle- or PGF-treated mice was perfused for micro-CT imaging. 10 pg/g PGF-treated mice exhibited less locomotor activity and greater anxiety-like behavior relative to vehicle-treated mice. Depressive-like behavior showed a sex-specific, dose-dependent decrease and was lowest in 700 pg/g PGF-treated females relative to vehicle-treated females. Spatial learning did not differ. MRI revealed smaller volume of three structures in the 10 pg/g group, larger volume of seven structures in the 70 pg/g group and smaller volume of one structure in the 700 pg/g group. No cerebral or retinal vascular differences were detected. Overall, neonatal PGF replacement altered behavior and neuroanatomy of adult Pgf-/- mice.

Influences of placental growth factor on mouse retinal vascular development.

BACKGROUND: Placental growth factor (PGF) is important for wound-healing and vascular collaterogenesis. PGF deficiency is associated with preeclampsia, a hypertensive disease of human pregnancy. Offspring born to preeclamptic mothers display cognitive impairments and brain vascular and neurostructural deviations. Low PGF production during development may contribute to alterations in offspring cerebrovascular beds. Retina is a readily accessible part of the central nervous system with a well-described pattern of vascular development in mice. Impacts of PGF deficiency were addressed during mouse retinal vascularization. RESULTS: Retinal vessels were compared between Pgf-/- and congenic C57BL/6 (B6) mice. PGF deficiency altered neonatal retinal vascularization patterns. Some anatomic alterations persisted into adulthood, particularly in males. Greater arterial wall collagen IV expression was found in adult Pgf-/- females. Pregnancy (studied in adult females at gestational days 11.5 or 18.5) induced subtle changes upon the mother's retinal vasculature but these pregnancy-induced changes did not differ between genotypes. Significant sex-related differences occurred between adult male and female B6 although sexually dimorphic retinal vascular differences were absent in B6 neonates. CONCLUSIONS: Overall, PGF has a role in retinal vascular angiogenesis and vessel organization during development but does not affect retinal vessel adaptations in adult females during pregnancy. Developmental Dynamics 246:700-712, 2017. © 2017 Wiley Periodicals, Inc.

Localization of transient immature hematopoietic cells to two distinct, potential niches in the developing mouse placenta.

Previous studies have shown that human and mouse placentas have hematopoietic potential during mid-gestation. In this investigation, we used histological and immunohistological approaches to visualize hematopoietic cells in mouse placenta between 9.5 and 12.5 days of gestation (gd), identifying their topography and niche. Putative hematopoietic foci were present on 10.5 and 11.5 gd but not 9.5 or 12.5 gd and was restricted to the placental labyrinth. Two major niches each with distinctive hematopoietic cell clusters were present. One type of hematopoietic cell cluster involved the chorioallantoic vasculature and fetal vessels near the chorionic plate. These clusters resembled the hematopoietic stem cells produced by large embryonic arteries such as aorta that persist in postnatal marrow. The other type of hematopoietic cell cluster identified was at the opposite side of labyrinth next to the junctional zone and was composed of erythropoietic foci. Our results suggest that mouse placenta not only produces hematopoietic stem/progenitor cells but also a second wave of primitive erythrocytes that may support a rapid, mid-pregnancy, fetal growth trajectory. Our data also point to a close relationships in the origins of hematopoietic and endothelial cells within placenta.

Impacts of Preeclampsia on the Brain of the Offspring / Impactos da pré-eclâmpsia no cérebro de nascituros

Abstract Preeclampsia (PE) is a significant gestational disorder that causes complications in 3- 5% of all human pregnancies. Apart from the immediate risks and complications for mother and fetus, both additionally carry elevated lifelong risks for specific complications. Offspring of PE pregnancies (PE-F1) have higher risks for hypertension, stroke and cognitive impairment compared with well-matched offspring (F1) fromuncomplicated pregnancies. Prior to the clinical onset of PE, placental angiokines secreted into the maternal plasma are deviated. In many PE patients this includes deficits in placental growth factor (PGF). Our laboratory found that mice genetically-deleted for PGF (PGF - / -) have altered cerebrovascular and brain neurological development detectable from midgestation to adulthood. We hypothesized that the PGF deficits seen in human PE, deviate fetal cerebrovascular and neurological development in a manner that impairs cognitive functions and elevates stroke risk. Here we summarize the initial analytical outcomes from a pilot study of 8-10 year old male and female PE-F1s and matched controls. Our studies were the first to report magnetic resonance imaging (MRI), magnetic resonance angiography (MRA) and functional brain region assessment by eyemovement control and clinical psychometric testing in PE-F1s. Further studies in larger cohorts are essential to define whether there are image-based biomarkers that describe unique anatomical features in PE-F1 brains.

Resumo A pré-eclampsia (PE) é importante doença gravídica complicando 3-5% de todas as gestações humanas. Além dos riscos imediatos e complicações para a mãe e o feto, a PE associa-se a outros riscos materno-fetais elevados em longo prazo. Nascituros de gestações complicadas por PE (PE-F1) apresentam maiores riscos de desenvolver hipertensão, acidente vascular cerebral e disfunção cognitiva em comparação com prole (F1) de gestações sem complicações. Antes do aparecimento clínico da PE, angiocitocinas placentárias secretadas no plasma materno apresentam-se alteradas. Em muitos pacientes com PE, isso inclui valores plasmáticos reduzidos de Fator de Crescimento Placentário (PGF). Nosso laboratório identificou que camundongos geneticamente não produtores de PGF (PGF- / - ) apresentam alterações vasculares e de desenvolvimento cerebral detectáveis do período gestacional à idade adulta. Nossa hipótese é que os déficits de PGF identificados em mulheres que desenvolveram PE podem desviar o desenvolvimento neurológico e vascular cerebral fetal, de maneira a prejudicar funções cognitivas, elevando o risco de AVC. Aqui resumimos os resultados analíticos iniciais de um estudo piloto comcrianças do sexomasculino e feminino de 8- 10 anos de idade nascidas de mães que tiveram PE (PE-F1s) comparadas com crianças controle pareadas por idade e sexo. Nossos estudos são os primeiros a relatar a ressonância magnética (RNM), a angiorressonância e a avaliação funcional do cérebro pelo controle de movimento dos olhos e pelo teste clínico psicotécnico em PE-F1s. Estudos adicionais em coortes maiores são essenciais para definir se há biomarcadores com base em imagens que possam descrever características anatômicas únicas em cérebros de crianças PE-F1.

The Transcription Factor NFIL3 Is Essential for Normal Placental and Embryonic Development but Not for Uterine Natural Killer (UNK) Cell Differentiation in Mice.

Mice ablated for the gene encoding the transcription factor Nfil3 lack peripheral natural killer (NK) cells but retain tissue-resident NK cells, particularly in mucosal sites, including virgin uterus. We undertook a time course histological study of implantation sites from syngeneically (Nfil3(-/-)) and allogeneically (BALB/c) mated Nfil3(-/-) females. We also examined implantation sites from Rag2(-/-)Il2rg(-/-) females preconditioned by adoptive transfer of Nfil3(-/-) marrow or uterine cell suspensions to identify the Nfil3(-/-) pregnancy aberrations that could be attributed to nonlymphoid cells. Uterine NKs (UNKs) reactive and nonreactive with the lectin Dolichos biflorus agglutinin (DBA) differentiate, localize, and mature within Nfil3(-/-) implantation sites, although at reduced abundance. The DBA nonreactive UNK cells were enriched following Nfil3(-/-) marrow transplantation. Uterine lumen closure, early embryonic development, and differentiation of antimesometrial decidua were delayed in Nfil3(-/-) implantation sites. Major disturbances to the decidual-trophoblast interface that did not lead to fetal death were attributed to NFIL3 deficiency in trophoblast. At midgestation, vessels of the placental labyrinth were enlarged, suggestive of reduced branching morphogenesis. A major term complication in most Nfil3(-/-) × Nfil3(-/-) pregnancies but not Nfil3(-/-) × Nfil3(+/-) pregnancies was dystocia. These studies highlight the differentiation potential and functions of Nfil3(-/-) UNK cell progenitors and illustrate that much of the implantation site histopathology associated with this strain is due to Nfil3 deletion in nonlymphoid cell lineages.

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.

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.; Queen's 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.

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.

Identification of a novel neutrophil population: proangiogenic granulocytes in second-trimester human decidua.

The maternal leukocytes of the first-trimester decidua play a fundamental role in implantation and early development of the fetus and placenta, yet little is known regarding the second-trimester decidual environment. Our multicolor flow cytometric analyses of human decidual leukocytes detected an elevation in tissue resident neutrophils in the second trimester. These cells in both human and murine samples were spatially restricted to decidua basalis. In comparison with peripheral blood neutrophils (PMNs), the decidual neutrophils expressed high levels of neutrophil activation markers and the angiogenesis-related proteins: vascular endothelial growth factor-A, Arginase-1, and CCL2, similarly shown in tumor-associated neutrophils. Functional in vitro assays showed that second-trimester human decidua conditioned medium stimulated transendothelial PMN invasion, upregulated VEGFA, ARG1, CCL2, and ICAM1 mRNA levels, and increased PMN-driven in vitro angiogenesis in a CXCL8-dependent manner. This study identified a novel neutrophil population with a physiological, angiogenic role in human decidua.

Ly49 receptors activate angiogenic mouse DBA⁺ uterine natural killer cells.

In humans, specific patterns of killer immunoglobulin-like receptors (KIRs) expressed by uterine natural killer (uNK) cells are linked through HLA-C with pregnancy complications (infertility, recurrent spontaneous abortion, intrauterine growth restriction and preeclampsia). To identify mechanisms underpinning the associations between NK cell activation and pregnancy success, pregnancies were studied in mice with genetic knockdown (KD) of the MHC-activated Ly49 receptor gene family. B6.Ly49(KD) pregnancies were compared to normal control B6.Ly49(129) and C57BL/6 (B6) pregnancies. At mid-pregnancy (gestation day (gd9.5)), overall uNK cell (TCRß(-)CD122(+)DBA(+)DX5(-) (DBA(+)DX5(-))) and TCRß(-)CD122(+)DBA(-)DX5(+) (DBA(-)DX5(+))) frequencies in pregnant uterus were similar between genotypes. Ly49(KD) lowered the normal frequencies of Ly49(+) uNK cells from 90.3% to 47.8% in DBA(-)DX5(+) and 78.8% to 6.3% in DBA(+)DX5(-) uNK cell subtypes. B6.Ly49(KD) matings frequently resulted in expanded blastocysts that did not implant (subfertility). B6.Ly49(KD) mice that established pregnancy had gestational lengths and litter sizes similar to controls. B6.Ly49(KD) neonates, however, were heavier than controls. B6.Ly49(KD) implantation sites lagged in early (gd6.5) decidual angiogenesis and were deficient in mid-pregnancy (gd10.5) spiral arterial remodelling. Ultrastructural analyses revealed that B6.Ly49(KD) uNK cells had impaired granulogenesis, while immunocytochemistry revealed deficient vascular endothelial cell growth factor (VEGFA) production. Perforin and IFNG expression were normal in B6.Ly49(KD) uNK cells. Thus, in normal mouse pregnancies, Ly49 receptor signaling must promote implantation, early decidual angiogenesis and mid-pregnancy vascular remodelling. Disturbances in these functions may underlie the reported genetic associations between human pregnancy complications and the inability of specific conceptus MHCs to engage activating KIR on uNK cells.

Gestational modification of murine spiral arteries does not reduce their drug-induced vasoconstrictive responses in vivo.

Dynamic control of maternal blood flow to the placenta is critical for healthy pregnancy. In many tissues, microvasculature arteries control the flow. The uterine/endometrial vascular bed changes during pregnancy include physiological remodeling of spiral arteries from constricted artery-like structures to dilated vein-like structures between Gestation Day 8 (gd8) and gd12 in mice and wk 12-16 in humans. These changes occur, in part, due to local environmental changes such as decidualization, recruitment of maternal uterine natural killer cells, and invasion of conceptus-derived trophoblasts. No current preparations permit in vivo testing of decidual microvascular reactivity. We report an in vivo intravital fluorescence microscopy model that permits functional study of the entire uterine microvascular bed (uterine, arcuate, radial, basal, and spiral arteries) in gravid C57BL/6 mice. Vascular reactivities were measured at gd8 prespiral arterial remodeling and gd12 (postremodeling) to a range of concentrations of adenosine (10(-8)-10(-6) M), acetylcholine (10(-7)-10(-5) M), phenylephrine (10(-7)-10(-5) M), and angiotensin II (10(-8)-10(-6) M). At baseline, each arterial branch order was significantly more dilated on gd12 than gd8. Each microvascular level responded to each agonist on gd8 and gd12. At gd12, vasodilation to adenosine was attenuated in uterine, arcuate, and basal arteries, while constrictor activity to angiotensin II was enhanced in uterine and arcuate arteries. The tendency for increasing vasoconstriction between gd8 to gd12 and the constrictor responses of modified spiral arteries were unexpected findings that may reflect influences of the intact in vivo environment rather than inherent properties of the vessels and may be relevant to ongoing human pregnancies.

Hemodynamic and behavioral differences after administration of meloxicam, buprenorphine, or tramadol as analgesics for telemeter implantation in mice.

Cannulation of the common carotid artery for chronic, continuous radiotelemetric recording of aortic hemodynamic properties in mice is a highly invasive recovery surgery. Radiotelemetric recording, by its continuous nature, gives the most accurate measurements of hemodynamic variables in experimental animals, and is widely used in the study of cardiovascular diseases including hypertension. The American Heart Association has recommended data acquisition by radiotelemetric recording but did not provide guidelines regarding postoperative analgesic support. We assessed hemodynamic parameters, locomotor activity, food intake, and weight loss in radiotransmitter-implanted CD1 female mice receiving analgesic support during the first 48 h after surgery. The efficacy of analgesic support from the NSAID meloxicam was compared with that of the widely used opioid agonist buprenorphine and the related compound, tramadol. Meloxicam-treated mice recovered lost body weight more rapidly than did tramadol-or buprenorphine-treated mice. Furthermore, meloxicam-treated mice maintained circadian rhythm after surgery and had tighter regulation of mean arterial pressure than did tramadol- or buprenorphine-treated mice. Meloxicam was also superior with regard to food intake, locomotor activity, and limiting variance in hemodynamic parameters. This study indicates that when compared with buprenorphine and tramadol, meloxicam should be the postoperative analgesic of choice for radiotelemeter implantation in mice.

Gestational hypertension in atrial natriuretic peptide knockout mice and the developmental origins of salt-sensitivity and cardiac hypertrophy.

OBJECTIVE: To determine the effect of gestational hypertension on the developmental origins of blood pressure (BP), altered kidney gene expression, salt-sensitivity and cardiac hypertrophy (CH) in adult offspring. METHODS: Female mice lacking atrial natriuretic peptide (ANP-/-) were used as a model of gestational hypertension. Heterozygous ANP+/- offspring was bred from crossing either ANP+/+ females with ANP-/- males yielding ANP+/-(WT) offspring, or from ANP-/- females with ANP+/+ males yielding ANP+/-(KO) offspring. Maternal BP during pregnancy was measured using radiotelemetry. At 14weeks of age, offspring BP, gene and protein expression were measured in the kidney with real-time quantitative PCR, receptor binding assay and ELISA. RESULTS: ANP+/-(KO) offspring exhibited normal BP at 14weeks of age, but displayed significant CH (P<0.001) as compared to ANP+/-(WT) offspring. ANP+/-(KO) offspring exhibited significantly increased gene expression of natriuretic peptide receptor A (NPR-A) (P<0.001) and radioligand binding studies demonstrated significantly reduced NPR-C binding (P=0.01) in the kidney. Treatment with high salt diet increased BP (P<0.01) and caused LV hypertrophy (P<0.001) and interstitial myocardial fibrosis only in ANP+/-(WT) and not ANP+/-(KO) offspring, suggesting gestational hypertension programs the offspring to show resistance to salt-induced hypertension and LV remodeling. Our data demonstrate that altered maternal environments can determine the salt-sensitive phenotype of offspring.

DBA-lectin reactivity defines mouse uterine natural killer cell subsets with biased gene expression.

Endometrial decidualization, a process essential for blastocyst implantation in species with hemochorial placentation, is accompanied by an enormous but transient influx of natural killer (NK) cells. Mouse uterine NK (uNK) cell subsets have been defined by diameter and cytoplasmic granule number, reflecting stage of maturity, and by histochemical reactivity with Periodic Acid Schiff (PAS) reagent with or without co-reactivity with Dolichos biflorus agglutinin (DBA) lectin. We asked whether DBA- and DBA+ mouse uNK cells were equivalent using quantitative RT-PCR analyses of flow-separated, midpregnancy (Gestation Day [gd] 10) cells and immunohistochemistry. CD3E (CD3)-IL2RB (CD122)+DBA cells were identified as the dominant Ifng transcript source. Skewed IFNG production by uNK cell subsets was confirmed by analysis of uNK cells from eYFP-tagged IFNG-reporter mice. In contrast, CD3E-IL2RB+DBA+ uNK cells expressed genes compatible with significantly greater potential for IL22 synthesis, angiogenesis, and participation in regulation mediated by the renin-angiotensin system (RAS). CD3E-IL2RB+DBA+ cells were further divided into VEGFA+ and VEGFA- subsets. CD3E-IL2RB+DBA+ uNK cells but not CD3E-IL2RB+DBA- uNK cells arose from circulating, bone marrow-derived progenitor cells by gd6. These findings indicate the heterogeneous nature of mouse uNK cells and suggest that studies using only DBA+ uNK cells will give biased data that does not fully represent the uNK cell population.

Delta-like ligand (DLL)1 expression in early mouse decidua and its localization to uterine natural killer cells.

Uterine vascular changes, critical for pregnancy success, occur at each implant site during endometrial decidualization. Mesometrial decidualization recruits high numbers of angiogenic, uterine Natural Killer (uNK) cells that trigger midpregnancy spiral arterial remodeling. We postulated that uNK cells contribute to early decidual angiogenesis as endothelial-cell extrinsic sources of Delta-like ligand 1 (DLL1), a molecule that induces endothelial tip cell differentiation and orthogonal vascular growth in other tissues. Virgin uteri expressed Dll1 mesometrially and anti-mesometrially and relative expression increased in both anatomic regions as pregnancy progressed. Analyses of transcripts from gd10.5 uNK cells flow sorted on the basis of expression of Dolichos biflorus agglutinin (DBA) lectin revealed that DBA+ but not DBA- uNK cells expressed Dll1. Immunostaining at gd4.5 found DLL1-expressing cells rare. At gd6.5, DBA+ uNK cells at all stages of maturation expressed DLL1. By gd10.5, DLL1 immunoreactivity was strongly expressed by some but not all DBA+ uNK cells and more weakly by DBA- cells. DLL1+ cells were mesometrially stratified and concentrated within central decidua basalis. Our data suggest that uNK cells have the potential to induce endothelial tip cell differentiation and to promote non-planar vascular growth within early decidua basalis.

Endometrial decidualization does not trigger the blood pressure decline of normal early pregnancy in mice.

A drop in mean arterial pressure (MAP) characterizes early, normal pregnancies of humans and of inbred mice, species with hemochorial placentation. Murine MAP, assessed by continuous radiotelemetry, falls from implantation to Gestation Day 9 (GD9) and then recovers. The change in the trajectory of mouse MAP after GD9 coincides with full maturity of the placenta and onset of its circulation. To identify whether these early gestational changes in hemodynamic function are conceptus and/or maternally regulated, pseudopregnancy (conceptus absent) with endometrial decidualization was established in radio transmitter-implanted, randomly bred CD1 mice. To avoid destabilization of MAP by anesthesia and surgery, decidualization was induced by transcervical infusion of concanavalin A-coated Sepharose beads 48 h after the female had copulated with a vasectomized male. In comparison to the postimplantation drop in MAP recorded in CD1 females mated by fertile males, pseudopregnancy MAP was stable to Gestation-Equivalent Day 10 in mice with confirmed endometrial decidualization at euthanasia. Thus, decidualization, with its accompanying pregnancy-like endocrine environment and uterine neoangiogensis and immune cell recruitment, is inadequate to depress early postimplantation MAP. These data suggest that the physiological modulation of early gestational MAP is not driven by maternal changes but is altered through conceptus-based mechanisms.

Arteriolar reactivity in lymphocyte-deficient mice.

Mounting evidence suggests that lymphocytes have the capacity to contribute to the regulation of systemic circulatory control. We postulated that T and natural killer (NK) cells could modify basal microvascular activity under physiologically normal conditions. In situ intravital microscopy of mouse cremaster vasculature was used to evaluate arteriolar reactivities to the vasoconstrictors angiotensin II (ANG II) and phenylephrine (Phe) and the vasodilators acetylcholine (ACh) and adenosine (Ado) in normal [+/+; wild type (WT)] and genetically immunodeficient (T(-)B(-)NK(+) or T(-)B(-)\NK(-)) C57BL/6 and BALB/c mice, strain backgrounds with differentially polarized T cell cytokine production. Immunodeficient mice tended to have smaller baseline and maximal diameters of third-order cremaster arterioles than their congenic WT partners. In C57BL/6, baseline diameters were similar in T-B(-) mice without or with NK cells; in BALB/c, baseline diameters were larger in T-B-NK(-) mice than in T(-)B(-)NK(+) mice. Thus, at baseline, lymphocytes tended to promote vasodilation, except BALB/c NK cells, which mediated mild vasoconstriction. The presence of NK cells suppressed dilations to Ado in both strains, to ACh in the C57BL/6 strain, and dilatory responses to ANG II in C57BL/6 and to Phe in BALB/c. In the BALB/c strain, the presence of T and B cells promoted vasodilatory responses to Ado, attenuated dilations to low ACh concentrations, and exaggerated dilation and constriction responses to ANG II. Thus, under agonist challenge, NK cells generally promote constriction, whereas influences of T and B cells depend upon the stimulus. Therefore, lymphocytes or their products have physiological influences on microvascular arteriolar reactivity.

Expression of angiogenic basic fibroblast growth factor, platelet derived growth factor, thrombospondin-1 and their receptors at the porcine maternal-fetal interface.

BACKGROUND: Commercial swine breeds in North America undergo two waves of spontaneous fetal loss; one during peri-attachment and another during mid-gestation. Although an exact mechanism for this loss is not known, deficits in vasculature at the attachment sites appear to be a major cause. We hypothesized that a balance between pro-angiogenic and anti-angiogenic factors is needed at the maternal-fetal interface for successful conceptus development. Six selected members of the pro-angiogenic fibroblast growth factor (FGF) and platelet derived growth factor (PDGF) families and anti-angiogenic factor thrombospondin-1 (TSP-1) and its receptor CD36 were quantified and localized at the porcine maternal-fetal interface at early and midgestation time points. METHODS: Mesometrial endometrium was collected from non-pregnant gilts (n = 8). Endometrial and chorioallantoic membrane samples were collected from healthy and arresting conceptus attachment sites at gestation day (gd) 20 (n = 8) and gd 50 (n = 8). At gd20 arresting conceptus attachment sites were distinguished by decreased vasculature of the placental membranes and decreased conceptus size. At gd50 arresting conceptuses attachment sites were identified by smaller conceptus length and weight measurements. Quantitative real time PCR was used to determine relative transcript levels of genes of interest, and cellular localization was determined by immunohistochemistry in paraffin embedded endometrial sections. RESULTS: At gd20, endometrial samples from arresting conceptuses had elevated transcripts for bFGF, and PDGF-bb than healthy sites (p < 0.05). At gd50, bFGF, FGFR2, and CD36 were more abundant at arresting than at healthy conceptus attachment sites (p < 0.05). Chorioallantoic membrane from arresting conceptus attachment sites at gd20 had elevated transcripts for bFGF, FGFR1, FGFR2 and CD36 compared with healthy sites (p < 0.05). FGFR2 transcripts were more abundant in chorioallantoic membrane from arresting conceptuses at gd 50 (p < 0.05). Immunohistochemical localization of selected pro- and anti-angiogenic factors and receptors revealed their abundance in the luminal epithelium, uterine glands and perivascular areas of endometrium at gd20 and gd50. CONCLUSIONS: We provide comprehensive analysis of pro and anti-angiogenic factors at the porcine maternal fetal interface during early and mid-pregnancy. At mRNA levels, the majority of pro-angiogenic factors investigated were elevated at the sites of fetal arrest. These observations contrast with our previous findings of decreased Vascular Endothelial Growth Factor (VEGF) family members at arresting sites, and suggest that the bFGF family functions as a compensatory survival mechanism when major angiogenic proteins are decreasing at the sites of fetal arrest.

Angiogenic DC-SIGN(+) cells are present at the attachment sites of epitheliochorial placentae.

Spontaneous early and mid-gestation fetal losses occur in swine. At both stages, endometrial lymphocytes associated with smaller, paler conceptuses have fewer pro-angiogenic and more pro-inflammatory cytokine transcripts compared with robust conceptuses. We hypothesized that similar differences occur in conceptus-associated dendritic cells (DCs). Using laser capture-microdissection, dendritic cell-specific intercellular adhesion molecule-grabbing non-integrin (DC-SIGN)(+) cells were isolated from attachment sites of healthy and arresting conceptuses at gestation day (gd)20 and 50. DC-SIGN(+) cells were screened using real-time PCR for vascular endothelial cell growth factor (Vegf), its receptors, semaphorins (Sema) and plexins (Plxn), and for toll-like receptor (Tlr) transcripts to address potential activation pathways. Homogenized endometrial and trophoblast biopsies were quantified for type 1/type 2 cytokine transcripts/proteins. DC-SIGN(+) cells from healthy and arresting conceptuses had more Vegf transcripts at early than mid gestation whereas transcripts for Vegfr1 and Vegfr2 were stable. In gd20 arresting site DC-SIGN(+) cells, Neuropilin-2 transcripts were elevated, whereas at gd50 arresting sites, Plxn-A2 increased and Sema3A transcripts were lost. Tlr-1, Tlr-4 and Tlr-6 transcript abundance was independent of conceptus health. At gd20, type 1 cytokines were prevalent, whereas at gd50 type 2 cytokines predominated in endometrium and trophoblast. Thus, gestational features, characteristic of haemochorial placentation, are present in species with distinctly different placentation.

Cytokine array comparisons of plasma from cycling fertile women on cycle day 5 and ovulation.

PROBLEM: To identify plasma immuno-regulatory molecules up or down regulated between the follicular phase and ovulation of the human menstrual cycle. METHOD OF STUDY: RayBio cytokine arrays were used to screen 174 immuno-regulatory molecules in plasma collected during the follicular phase at menstrual cycle day 5 and at ovulation from five healthy, non-smoking, fertile women of reproductive age not using hormonal contraception. RESULTS: A total of 23 differentially expressed molecules were found: 10 molecules were differentially up-regulated and 13 down-regulated at ovulation compared with that at the follicular phase (alpha = 0.05, false discovery rate of 0.45). CONCLUSION: Circulating immuno-regulatory molecules fluctuate over the menstrual cycle in healthy women. The combination of differentially expressed molecules suggests roles in cyclical regulation of angiogenesis and immune cell trafficking.