Impaired Interleukin-15 Signaling via BMPR2 Loss Drives Natural Killer Cell Deficiency and Pulmonary Hypertension.

BACKGROUND: Natural killer (NK) cell impairment is a feature of pulmonary arterial hypertension (PAH) and contributes to vascular remodeling in animal models of disease. Although mutations in BMPR2, the gene encoding the BMP (bone morphogenetic protein) type-II receptor, are strongly associated with PAH, the contribution of BMPR2 loss to NK cell impairment remains unknown. We explored the impairment of IL (interleukin)-15 signaling, a central mediator of NK cell homeostasis, as both a downstream target of BMPR2 loss and a contributor to the pathogenesis of PAH. METHODS: The expression, trafficking, and secretion of IL-15 and IL-15Rα (interleukin 15 α-type receptor) were assessed in human pulmonary artery endothelial cells, with or without BMPR2 silencing. NK cell development and IL-15/IL-15Rα levels were quantified in mice bearing a heterozygous knock-in of the R899X-BMPR2 mutation (bmpr2+/R899X). NK-deficient Il15-/- rats were exposed to the Sugen/hypoxia and monocrotaline models of PAH to assess the impact of impaired IL-15 signaling on disease severity. RESULTS: BMPR2 loss reduced IL-15Rα surface presentation and secretion in human pulmonary artery endothelial cells via impaired trafficking through the trans-Golgi network. bmpr2+/R899X mice exhibited a decrease in NK cells, which was not attributable to impaired hematopoietic development but was instead associated with reduced IL-15/IL-15Rα levels in these animals. Il15-/- rats of both sexes exhibited enhanced disease severity in the Sugen/hypoxia model, with only male Il15-/- rats developing more severe PAH in response to monocrotaline. CONCLUSIONS: This work identifies the loss of IL-15 signaling as a novel BMPR2-dependent contributor to NK cell impairment and pulmonary vascular disease.

Preeclampsia may influence offspring neuroanatomy and cognitive function: a role for placental growth factor†.

Preeclampsia (PE) is a common pregnancy complication affecting 3-5% of women. Preeclampsia is diagnosed clinically as new-onset hypertension with associated end organ damage after 20 weeks of gestation. Despite being diagnosed as a maternal syndrome, fetal experience of PE is a developmental insult with lifelong cognitive consequences. These cognitive alterations are associated with distorted neuroanatomy and cerebrovasculature, including a higher risk of stroke. The pathophysiology of a PE pregnancy is complex, with many factors potentially able to affect fetal development. Deficient pro-angiogenic factor expression is one aspect that may impair fetal vascularization, alter brain structure, and affect future cognition. Of the pro-angiogenic growth factors, placental growth factor (PGF) is strongly linked to PE. Concentrations of PGF are inappropriately low in maternal blood both before and during a PE gestation. Fetal concentrations of PGF appear to mirror maternal circulating concentrations. Using Pgf-/- mice that may model effects of PE on offspring, we demonstrated altered central nervous system vascularization, neuroanatomy, and behavior. Overall, we propose that development of the fetal brain is impaired in PE, making the offspring of preeclamptic pregnancies a unique cohort with greater risk of altered cognition and cerebrovasculature. These individuals may benefit from early interventions, either pharmacological or environmental. The early neonatal period may be a promising window for intervention while the developing brain retains plasticity.

Spontaneous pulmonary hypertension in genetic mouse models of natural killer cell deficiency.

Natural killer (NK) cells are cytotoxic innate lymphoid cells with an established role in the regulation of vascular structure in pregnancy and cancer. Impaired NK cell function has been identified in patients with pulmonary arterial hypertension (PAH), a disease of obstructive vascular remodeling in the lungs, as well as in multiple rodent models of disease. However, the precise contribution of NK cell impairment to the initiation and progression of PAH remains unknown. Here, we report the development of spontaneous pulmonary hypertension in two independent genetic models of NK cell dysfunction, including Nfil3-/- mice, which are deficient in NK cells due to the absence of the NFIL3 transcription factor, and Ncr1-Gfp mice, which lack the NK activating receptor NKp46. Mouse models of NK insufficiency exhibited increased right ventricular systolic pressure and muscularization of the pulmonary arteries in the absence of elevated left ventricular end-diastolic pressure, indicating that the development of pulmonary hypertension was not secondary to left heart dysfunction. In cases of severe NK cell impairment or loss, a subset of mice failed to develop pulmonary hypertension and instead exhibited reduced systemic blood pressure, demonstrating an extension of vascular abnormalities beyond the pulmonary circulation into the systemic vasculature. In both mouse models, the development of PAH was linked to elevated interleukin-23 production, whereas systemic hypotension in Ncr1-Gfp mice was accompanied by a loss of angiopoietin-2. Together, these results support an important role for NK cells in the regulation of pulmonary and systemic vascular function and the pathogenesis of PAH.

Effects of placental growth factor deficiency on behavior, neuroanatomy, and cerebrovasculature of mice.

Preeclampsia, a hypertensive syndrome occurring in 3-5% of human pregnancies, has lifelong health consequences for fetuses. Cognitive ability throughout life is altered, and adult stroke risk is increased. One potential etiological factor for altered brain development is low concentrations of proangiogenic placental growth factor (PGF). Impaired PGF production may promote an antiangiogenic fetal environment during neural and cerebrovascular development. We previously reported delayed vascularization of the hindbrain, altered retinal vascular organization, and less connectivity in the circle of Willis in Pgf-/- mice. We hypothesized Pgf-/- mice would have impaired cognition and altered brain neuroanatomy in addition to compromised cerebrovasculature. Cognitive behavior was assessed in adult Pgf-/- and Pgf+/+ mice by four paradigms followed by postmortem high-resolution MRI of neuroanatomy. X-ray microcomputed tomography imaging investigated the three-dimensional cerebrovascular geometry in another cohort. Pgf-/- mice exhibited poorer spatial memory, less depressive-like behavior, and superior recognition of novel objects. Significantly smaller volumes of 10 structures were detected in the Pgf-/- compared with Pgf+/+ brain. Pgf-/- brain had more total blood vessel segments in the small-diameter range. Lack of PGF altered cognitive functions, brain neuroanatomy, and cerebrovasculature in mice. Pgf-/- mice may be a preclinical model for the offspring effects of low-PGF preeclampsia gestation.

Resting-state functional connectivity in children born from gestations complicated by preeclampsia: A pilot study cohort.

BACKGROUND: Individuals (PE-F1s) born from preeclampsia (PE)-complicated pregnancies have elevated risks for cognitive impairment. Intervals of disturbed maternal plasma angiokines precede clinical signs of PE. We hypothesized pan-blastocyst dysregulation of angiokines underlies altered PE-F1 brain vascular and neurological development. This could alter brain functional connectivity (FC) patterns at rest. MATERIALS AND METHODS: Resting-state functional MRI datasets of ten, matched child pairs (5 boys and 5 girls aged 7-10 years of age) from PE or control pregnancies were available for study. Seed-based analysis and independent component analysis (ICA) methodologies were used to assess whether differences in resting-state functional connectivity (rs-FC) were present between PE-F1s and controls. Bilateral amygdala, bilateral hippocampus, and medial prefrontal cortex (MPFC) were selected as regions of interest (ROI) for the seed-based analysis based on previous imaging differences that we reported in this set of children. RESULTS: Compared to controls, PE-F1 children had increased rs-FC between the right amygdala and left frontal pole, the left amygdala and bilateral frontal pole, and the MPFC and precuneus. PE-F1 children additionally had decreased rs-FC between the MPFC and the left occipital fusiform gyrus compared to controls. CONCLUSION: These are the first reported rs-FC data for PE-F1s of any age. Theysuggest that PE alters FC during human fetal brain development. Altered FC may contribute to the behavioural and neurological alterations reported in PE-F1s. Longitudinal MRI studies with larger sample sizes are required to confirm these novel findings.

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.

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.

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.

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.

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.

ETA and ETB receptors are expressed in vascular adventitial fibroblasts.

The adventitia has been recognized to play important roles in vascular oxidative stress, remodeling, and contraction. We recently demonstrated that adventitial fibroblasts are able to express endothelin (ET)-1 in response to ANG II. However, it is unclear whether ET-1 receptors are expressed in the adventitia. We therefore investigated the expression and roles of both ET(A) and ET(B) receptors in collagen synthesis and ET-1 clearance in adventitial fibroblasts. Adventitial fibroblasts were isolated and cultured from the mouse thoracic aorta by the explant method. Cultured cells were treated with ANG II (100 nmol/l) or ET-1 (10 pM) in the presence or absence of the ANG II type 1 receptor antagonist losartan (100 µM), the ET-1 receptor antagonists BQ-123 (ET(A) receptor, 1 µM) and BQ-788 (ET(B) receptor, 1 µM), and the ET(B) receptor agonist sarafotoxin 6C (100 nM). ET-1 peptide levels were determined by ELISA, whereas ET(A), ET(B), and collagen levels were determined by Western blot analysis. ANG II increased ET-1 peptide levels in a time-dependent manner. ANG II increased ET(A) and ET(B) receptor protein levels as well as collagen in a similar fashion. ANG II-induced collagen was reduced while in the presence of BQ-123, suggesting a role for the ET(A) receptor in the regulation of the extracellular matrix. ANG II treatment in the presence of BQ-788 significantly increased ET-1 peptide levels. Conversely, the ET(B) receptor agonist sarafotoxin 6C significantly decreased ET-1 peptide levels. These data implicate a role for the ET(B) receptor in the clearance of the ET-1 peptide. In conclusion, both ET(A) and ET(B) receptors are expressed in adventitial fibroblasts, which paves the ground for the biological significance of adventitial ET-1. The ET(A) receptor subtype mediates collagen I expression, whereas the ET(B) receptor subtype may play a protective role through increasing the clearance of the ET-1 peptide.

Adventitial fibroblasts in vascular structure and function: the role of oxidative stress and beyond.

The vascular adventitia, defined as the area between the external elastic lamina and the outermost edge of the blood vessel, is composed primarily of fibroblasts and for years was thought to be merely a passive structural support for the blood vessel. Consequently, studies pertaining to the role of the adventitia in regulating vascular function have been far outnumbered by those regarding the vascular endothelium. However, recent work has begun to reveal the dynamic properties of the adventitia. It was therefore the aim of this review to provide an overview of the existing knowledge demonstrating the role of the adventitia in regulating vessel structure and function. The main topics covered in this review include the cellular composition of the adventitia and the role of the adventitia in vascular oxidative stress, vasomotor responses, extracellular matrix protein expression, growth factor expression, and endothelin-1 (ET-1) expression. Recent evidence suggests that the adventitia is a major producer of vascular reactive oxygen species. It displays a distinct response to injury, hypoxia, and pulmonary hypertension, mediating vascular remodelling, repair, and extracellular matrix deposition. It may also play a role in regulating vascular tone. More recently, it has been reported that adventitial fibroblasts can produce ET-1 after Ang II treatment. Additionally, emerging evidence suggests that the adventitia may be a potent source of vasoactive hormones such as growth factors and ET-1, which may regulate vascular structure and function via autocrine or paracrine signalling mechanisms. Despite these findings, many important questions regarding the role of the vascular adventitia remain unanswered, suggesting the need for further research to determine its exact function in health and disease.