Matthew T. Rätsep

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a disease of neonatal lung development that is linked to impaired pulmonary vascularization, dysregulated TGF-β (transforming growth factor-β) signaling, and the accumulation of senescent cells. Despite the established role of TGF-β signaling in promoting vascular remodeling and suppressing the senolytic activity of natural killer (NK) cells, the contribution of NK cell TGF-β signaling to postnatal lung patterning and the pathogenesis of BPD remains unclear. METHODS: Mice bearing an NK cell-selective deletion of the type-II TGF-β receptor ( Tgfbr2 ^{NK− /−} ) were analyzed for vascular and alveolar structure, lung NK cell infiltration, senescence markers, and lung function testing across neonatal and adult time points. Lung tissue from both neonatal mice and human infants with BPD was assessed by single-cell RNA sequencing. The effects of enhanced NK cell activity in a hyperoxia-induced model of BPD were examined in Tgfbr2 ^NK−/− neonates, as well as pharmacologically, using the TGF-β ligand trap/IL-15 superagonist, HCW9218. RESULTS: Neonatal Tgfbr2 ^NK−/− mice exhibited a baseline reduction in distal arteriolar density, impaired alveolarization, and sex-specific deficits in long-term lung function. Single-cell RNA sequencing identified excessive clearance of senescent endothelial cells by TGF-β-insensitive NK cells in the lungs of Tgfbr2 ^NK−/− neonates. This enhanced NK cell activity promoted a BPD-like phenotype in naïve Tgfbr2 ^NK−/− mice but protected these animals from impaired lung development under hyperoxia. Sequencing of lung tissue from human infants with BPD confirmed excessive TGF-β signaling and cytotoxic impairment in NK cells. Treatment with HCW9218 prevented senescent cell accumulation and rescued lung development in the hyperoxia mouse model. CONCLUSIONS: TGF-β is a tunable regulator of NK cell senolytic activity that is essential for normal postnatal lung development. Excessive NK cell TGF-β signaling contributes to impaired lung development with neonatal hyperoxia and may serve as a viable therapeutic target in human BPD.

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 (PE) is a common pregnancy complication affecting 3–5% of women. PE 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 behaviour. 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.

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.

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.

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.

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.