IC100, a humanized therapeutic monoclonal anti-ASC antibody alleviates oxygen-induced retinopathy in mice.

BACKGROUND: Retinopathy of prematurity (ROP), which often presents with bronchopulmonary dysplasia (BPD), is among the most common morbidities affecting extremely premature infants and is a leading cause of severe vision impairment in children worldwide. Activations of the inflammasome cascade and microglia have been implicated in playing a role in the development of both ROP and BPD. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. Utilizing mouse models of both oxygen-induced retinopathy (OIR) and BPD, this study was designed to test the hypothesis that hyperoxia induces ASC speck formation, which leads to microglial activation and retinopathy, and that inhibition of ASC speck formation by a humanized monoclonal antibody, IC100, directed against ASC, will ameliorate microglial activation and abnormal retinal vascular formation. METHODS: We first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR. RESULTS: ASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2. CONCLUSION: These data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.

Chorioamnionitis accelerates granule cell and oligodendrocyte maturation in the cerebellum of preterm nonhuman primates.

BACKGROUND: Preterm birth is often associated with chorioamnionitis and leads to increased risk of neurodevelopmental disorders, such as autism. Preterm birth can lead to cerebellar underdevelopment, but the mechanisms of disrupted cerebellar development in preterm infants are not well understood. The cerebellum is consistently affected in people with autism spectrum disorders, showing reduction of Purkinje cells, decreased cerebellar grey matter, and altered connectivity. METHODS: Preterm rhesus macaque fetuses were exposed to intra-amniotic LPS (1 mg, E. coli O55:B5) at 127 days (80%) gestation and delivered by c-section 5 days after injections. Maternal and fetal plasma were sampled for cytokine measurements. Chorio-decidua was analyzed for immune cell populations by flow cytometry. Fetal cerebellum was sampled for histology and molecular analysis by single-nuclei RNA-sequencing (snRNA-seq) on a 10× chromium platform. snRNA-seq data were analyzed for differences in cell populations, cell-type specific gene expression, and inferred cellular communications. RESULTS: We leveraged snRNA-seq of the cerebellum in a clinically relevant rhesus macaque model of chorioamnionitis and preterm birth, to show that chorioamnionitis leads to Purkinje cell loss and disrupted maturation of granule cells and oligodendrocytes in the fetal cerebellum at late gestation. Purkinje cell loss is accompanied by decreased sonic hedgehog signaling from Purkinje cells to granule cells, which show an accelerated maturation, and to oligodendrocytes, which show accelerated maturation from pre-oligodendrocytes into myelinating oligodendrocytes. CONCLUSION: These findings suggest a role of chorioamnionitis on disrupted cerebellar maturation associated with preterm birth and on the pathogenesis of neurodevelopmental disorders among preterm infants.

Protective role of CXCR7 activation in neonatal hyperoxia-induced systemic vascular remodeling and cardiovascular dysfunction in juvenile rats.

Neonatal hyperoxia induces long-term systemic vascular stiffness and cardiovascular remodeling, but the mechanisms are unclear. Chemokine receptor 7 (CXCR7) represents a key regulator of vascular homeostasis and repair by modulating TGF-ß1 signaling. This study investigated whether pharmacological CXCR7 agonism prevents neonatal hyperoxia-induced systemic vascular stiffness and cardiac dysfunction in juvenile rats. Newborn Sprague Dawley rat pups assigned to room air or hyperoxia (85% oxygen), received CXCR7 agonist, TC14012 or placebo for 3 weeks. These rat pups were maintained in room air until 6 weeks when aortic pulse wave velocity doppler, cardiac echocardiography, aortic and left ventricular (LV) fibrosis were assessed. Neonatal hyperoxia induced systemic vascular stiffness and cardiac dysfunction in 6-week-old rats. This was associated with decreased aortic and LV CXCR7 expression. Early treatment with TC14012, partially protected against neonatal hyperoxia-induced systemic vascular stiffness and improved LV dysfunction and fibrosis in juvenile rats by decreasing TGF-ß1 expression. In vitro, hyperoxia-exposed human umbilical arterial endothelial cells and coronary artery endothelial cells had increased TGF-ß1 levels. However, treatment with TC14012 significantly reduced the TGF-ß1 levels. These results suggest that dysregulation of endothelial CXCR7 signaling may contribute to neonatal hyperoxia-induced systemic vascular stiffness and cardiac dysfunction.

GSDMD gene knockout alleviates hyperoxia-induced hippocampal brain injury in neonatal mice.

BACKGROUND: Neonatal hyperoxia exposure is associated with brain injury and poor neurodevelopment outcomes in preterm infants. Our previous studies in neonatal rodent models have shown that hyperoxia stimulates the brain's inflammasome pathway, leading to the activation of gasdermin D (GSDMD), a key executor of pyroptotic inflammatory cell death. Moreover, we found pharmacological inhibition of caspase-1, which blocks GSDMD activation, attenuates hyperoxia-induced brain injury in neonatal mice. We hypothesized that GSDMD plays a pathogenic role in hyperoxia-induced neonatal brain injury and that GSDMD gene knockout (KO) will alleviate hyperoxia-induced brain injury. METHODS: Newborn GSDMD knockout mice and their wildtype (WT) littermates were randomized within 24 h after birth to be exposed to room air or hyperoxia (85% O2) from postnatal days 1 to 14. Hippocampal brain inflammatory injury was assessed in brain sections by immunohistology for allograft inflammatory factor 1 (AIF1) and CD68, markers of microglial activation. Cell proliferation was evaluated by Ki-67 staining, and cell death was determined by TUNEL assay. RNA sequencing of the hippocampus was performed to identify the transcriptional effects of hyperoxia and GSDMD-KO, and qRT-PCR was performed to confirm some of the significantly regulated genes. RESULTS: Hyperoxia-exposed WT mice had increased microglia consistent with activation, which was associated with decreased cell proliferation and increased cell death in the hippocampal area. Conversely, hyperoxia-exposed GSDMD-KO mice exhibited considerable resistance to hyperoxia as O2 exposure did not increase AIF1 + , CD68 + , or TUNEL + cell numbers or decrease cell proliferation. Hyperoxia exposure differentially regulated 258 genes in WT and only 16 in GSDMD-KO mice compared to room air-exposed WT and GSDMD-KO, respectively. Gene set enrichment analysis showed that in the WT brain, hyperoxia differentially regulated genes associated with neuronal and vascular development and differentiation, axonogenesis, glial cell differentiation, hypoxia-induced factor 1 pathway, and neuronal growth factor pathways. These changes were prevented by GSDMD-KO. CONCLUSIONS: GSDMD-KO alleviates hyperoxia-induced inflammatory injury, cell survival and death, and alterations of transcriptional gene expression of pathways involved in neuronal growth, development, and differentiation in the hippocampus of neonatal mice. This suggests that GSDMD plays a pathogenic role in preterm brain injury, and targeting GSDMD may be beneficial in preventing and treating brain injury and poor neurodevelopmental outcomes in preterm infants.

GSDMD gene knockout alleviates hyperoxia-induced hippocampal brain injury in neonatal mice.

Background: Neonatal hyperoxia exposure is associated with brain injury and poor neurodevelopment outcomes in preterm infants. Our previous studies in neonatal rodent models have shown that hyperoxia stimulates the brain's inflammasome pathway, leading to the activation of gasdermin D (GSDMD), a key executor of pyroptotic inflammatory cell death. Moreover, we found inhibition of GSDMD activation attenuates hyperoxia-induced brain injury in neonatal mice. We hypothesized that GSDMD plays a pathogenic role in hyperoxia-induced neonatal brain injury and that GSDMD gene knockout (KO) will alleviate hyperoxia-induced brain injury. Methods: Newborn GSDMD knockout mice and their wildtype (WT) littermates were randomized within 24 h after birth to be exposed to room air or hyperoxia (85% O2) from postnatal day 1 to 14. Hippocampal brain inflammatory injury was assessed in brain sections by immunohistology for allograft inflammatory factor 1 (AIF1), a marker of microglial activation. Cell proliferation was evaluated by Ki-67 staining, and cell death was determined by TUNEL assay. RNA sequencing of the hippocampus was performed to identify the transcriptional effects of hyperoxia and GSDMD-KO, and qRT-PCR was performed to confirm some of the significantly regulated genes. Results: Hyperoxia-exposed WT mice had increased microglia consistent with activation, which was associated with decreased cell proliferation and increased cell death in the hippocampal area. Conversely, hyperoxia-exposed GSDMD-KO mice exhibited considerable resistance to hyperoxia as O2 exposure failed to increase either AIF1+ or TUNEL+ cell numbers, nor decrease cell proliferation. Hyperoxia exposure differentially regulated 258 genes in WT and only 16 in GSDMD-KO mice compared to room air- exposed WT and GSDMD-KO, respectively. Gene set enrichment analysis showed that in the WT brain, hyperoxia differentially regulated genes associated with neuronal and vascular development and differentiation, axonogenesis, glial cell differentiation, and core development pathways hypoxia-induced factor 1, and neuronal growth factor pathways. These changes were prevented by GSDMD-KO. Conclusion: GSDMD-KO alleviates hyperoxia-induced inflammatory injury, cell survival and death, and alterations of transcriptional gene expression of pathways involved in neuronal growth, development, and differentiation in the hippocampus of neonatal mice. This suggests that GSDMD plays a pathogenic role in preterm brain injury, and targeting GSDMD may be beneficial in preventing and treating brain injury and poor neurodevelopmental outcomes in preterm infants.

Extracellular vesicles: pathogenic messengers and potential therapy for neonatal lung diseases.

Extracellular vesicles (EVs) are a heterogeneous group of nano-sized membranous structures increasingly recognized as mediators of intercellular and inter-organ communication. EVs contain a cargo of proteins, lipids and nucleic acids, and their cargo composition is highly dependent on the biological function of the parental cells. Their cargo is protected from the extracellular environment by the phospholipid membrane, thus allowing for safe transport and delivery of their intact cargo to nearby or distant target cells, resulting in modification of the target cell's gene expression, signaling pathways and overall function. The highly selective, sophisticated network through which EVs facilitate cell signaling and modulate cellular processes make studying EVs a major focus of interest in understanding various biological functions and mechanisms of disease. Tracheal aspirate EV-miRNA profiling has been suggested as a potential biomarker for respiratory outcome in preterm infants and there is strong preclinical evidence showing that EVs released from stem cells protect the developing lung from the deleterious effects of hyperoxia and infection. This article will review the role of EVs as pathogenic messengers, biomarkers, and potential therapies for neonatal lung diseases.

Maternal SARS-CoV-2, Placental Changes and Brain Injury in 2 Neonates.

Long-term neurodevelopmental sequelae are a potential concern in neonates following in utero exposure to severe acute respiratory syndrome coronavirus disease 2 (SARS-CoV-2). We report 2 neonates born to SARS-CoV-2 positive mothers, who displayed early-onset (day 1) seizures, acquired microcephaly, and significant developmental delay over time. Sequential MRI showed severe parenchymal atrophy and cystic encephalomalacia. At birth, neither infant was SARS-CoV-2 positive (nasopharyngeal swab, reverse transcription polymerase chain reaction), but both had detectable SARS-CoV-2 antibodies and increased blood inflammatory markers. Placentas from both mothers showed SARS-CoV-2-nucleocapsid protein and spike glycoprotein 1 in the syncytiotrophoblast, fetal vascular malperfusion, and significantly increased inflammatory and oxidative stress markers pyrin domain containing 1 protein, macrophage inflammatory protein 1 ßη, stromal cell-derived factor 1, interleukin 13, and interleukin 10, whereas human chorionic gonadotropin was markedly decreased. One infant (case 1) experienced sudden unexpected infant death at 13 months of age. The deceased infant's brain showed evidence of SARS-CoV-2 by immunofluorescence, with colocalization of the nucleocapsid protein and spike glycoprotein around the nucleus as well as within the cytoplasm. The constellation of clinical findings, placental pathology, and immunohistochemical changes strongly suggests that second-trimester maternal SARS-CoV-2 infection with placentitis triggered an inflammatory response and oxidative stress injury to the fetoplacental unit that affected the fetal brain. The demonstration of SARS-CoV-2 in the deceased infant's brain also raises the possibility that SARS-CoV-2 infection of the fetal brain directly contributed to ongoing brain injury. In both infants, the neurologic findings at birth mimicked the presentation of hypoxic-ischemic encephalopathy of newborn and neurologic sequelae progressed well beyond the neonatal period.

GSDMD deficiency ameliorates hyperoxia-induced BPD and ROP in neonatal mice.

Bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP) are among the most common morbidities affecting extremely premature infants who receive oxygen therapy. Many clinical studies indicate that BPD is associated with advanced ROP. However, the mechanistic link between hyperoxia, BPD, and ROP remains to be explored. Gasdermin D (GSDMD) is a key executor of inflammasome-induced pyroptosis and inflammation. Inhibition of GSDMD has been shown to attenuate hyperoxia-induced BPD and brain injury in neonatal mice. The objective of this study was to further define the mechanistic roles of GSDMD in the pathogenesis of hyperoxia-induced BPD and ROP in mouse models. Here we show that global GSDMD knockout (GSDMD-KO) protects against hyperoxia-induced BPD by reducing macrophage infiltration, improving alveolarization and vascular development, and decreasing cell death. In addition, GSDMD deficiency prevented hyperoxia-induced ROP by reducing vasoobliteration and neovascularization, improving thinning of multiple retinal tissue layers, and decreasing microglial activation. RNA sequencing analyses of lungs and retinas showed that similar genes, including those from inflammatory, cell death, tissue remodeling, and tissue and vascular developmental signaling pathways, were induced by hyperoxia and impacted by GSDMD-KO in both models. These data highlight the importance of GSDMD in the pathogenesis of BPD and ROP and suggest that targeting GSDMD may be beneficial in preventing and treating BPD and ROP in premature infants.

c-Myc Drives inflammation of the maternal-fetal interface, and neonatal lung remodeling induced by intra-amniotic inflammation.

Background: Intra-amniotic inflammation (IAI) is associated with increased risk of preterm birth and bronchopulmonary dysplasia (BPD), but the mechanisms by which IAI leads to preterm birth and BPD are poorly understood, and there are no effective therapies for preterm birth and BPD. The transcription factor c-Myc regulates various biological processes like cell growth, apoptosis, and inflammation. We hypothesized that c-Myc modulates inflammation at the maternal-fetal interface, and neonatal lung remodeling. The objectives of our study were 1) to determine the kinetics of c-Myc in the placenta, fetal membranes and neonatal lungs exposed to IAI, and 2) to determine the role of c-Myc in modulating inflammation at the maternal-fetal interface, and neonatal lung remodeling induced by IAI. Methods: Pregnant Sprague-Dawley rats were randomized into three groups: 1) Intra-amniotic saline injections only (control), 2) Intra-amniotic lipopolysaccharide (LPS) injections only, and 3) Intra-amniotic LPS injections with c-Myc inhibitor 10058-F4. c-Myc expression, markers of inflammation, angiogenesis, immunohistochemistry, and transcriptomic analyses were performed on placenta and fetal membranes, and neonatal lungs to determine kinetics of c-Myc expression in response to IAI, and effects of prenatal systemic c-Myc inhibition on lung remodeling at postnatal day 14. Results: c-Myc was upregulated in the placenta, fetal membranes, and neonatal lungs exposed to IAI. IAI caused neutrophil infiltration and neutrophil extracellular trap (NET) formation in the placenta and fetal membranes, and neonatal lung remodeling with pulmonary hypertension consistent with a BPD phenotype. Prenatal inhibition of c-Myc with 10058-F4 in IAI decreased neutrophil infiltration and NET formation, and improved neonatal lung remodeling induced by LPS, with improved alveolarization, increased angiogenesis, and decreased pulmonary vascular remodeling. Discussion: In a rat model of IAI, c-Myc regulates neutrophil recruitment and NET formation in the placenta and fetal membranes. c-Myc also participates in neonatal lung remodeling induced by IAI. Further studies are needed to investigate c-Myc as a potential therapeutic target for IAI and IAI-associated BPD.

Neonatal intermittent hypoxia persistently impairs lung vascular development and induces long-term lung mitochondrial DNA damage.

Adults born preterm have an increased risk of pulmonary vascular disease. Extreme preterm infants often require supplemental oxygen but they also exhibit frequent intermittent hypoxemic episodes (IH). Here, we test the hypothesis that neonatal IH induces lung endothelial cell mitochondrial DNA (mitDNA) damage and contributes to long-term pulmonary vascular disease and pulmonary hypertension (PH). Newborn C57BL/6J mice were assigned to the following groups: 1) normoxia, 2) hyperoxia (O2 65%), 3) normoxia cycling with IH (O2 21% + O2 10%), and 4) hyperoxia cycling with IH (O2 65% + O2 10%) for 3 wk. IH episodes were initiated on postnatal day 7. Lung angiogenesis, PH, and mitDNA lesions were assessed at 3 wk and 3 mo. In vitro, the effect of IH on tubule formation and mitDNA lesions was evaluated in human pulmonary microvascular endothelial cells (HPMECs). Data were analyzed by ANOVA. In vitro, IH exposure reduced tubule formation and increased mitDNA lesions in HPMECs. This was most marked in HPMECs exposed to hyperoxia cycling with IH. In vivo, neonatal IH increased lung mitDNA lesions, impaired angiogenesis, and induced PH in 3-wk-old mice. These findings were pronounced in mice exposed to hyperoxia cycling with IH. At 3 mo follow-up, mice exposed to neonatal IH had persistently increased lung mitDNA lesions and impaired lung angiogenesis, even without concomitant hyperoxia exposure. Neonatal IH induces lung endothelial cell mitDNA damage and causes persistent impairment in lung angiogenesis. These findings provide important mechanistic insight into the pathogenesis of pulmonary vascular disease in preterm survivors.NEW & NOTEWORTHY Our current study demonstrates that neonatal intermittent hypoxia (IH) alters lung endothelial cell function, induces mitochondrial DNA lesions, and impairs lung vascular growth into adulthood. Moreover, when superimposed on hyperoxia, neonatal IH induces a severe lung vascular phenotype that is seen in preterm infants with PH. These findings suggest that neonatal IH contributes to PH in adults born preterm and importantly, that mitochondrial protection strategies may mitigate these deleterious effects.

Educational Review: The Impact of Perinatal Oxidative Stress on the Developing Kidney.

Oxidative stress occurs when there is an imbalance between reactive oxygen species/reactive nitrogen species and antioxidant systems. The interplay between these complex processes is crucial for normal pregnancy and fetal development; however, when oxidative stress predominates, pregnancy related complications and adverse fetal programming such as preterm birth ensues. Understanding how oxidative stress negatively impacts outcomes for the maternal-fetal dyad has allowed for the exploration of antioxidant therapies to prevent and/or mitigate disease progression. In the developing kidney, the negative impact of oxidative stress has also been noted as it relates to the development of hypertension and kidney injury mostly in animal models. Clinical research addressing the implications of oxidative stress in the developing kidney is less developed than that of the neurodevelopmental and respiratory conditions of preterm infants and other vulnerable neonatal groups. Efforts to study the oxidative stress pathway along the continuum of the perinatal period using a team science approach can help to understand the multi-organ dysfunction that the maternal-fetal dyad sustains and guide the investigation of antioxidant therapies to ameliorate the global toxicity. This educational review will provide a comprehensive and multidisciplinary perspective on the impact of oxidative stress during the perinatal period in the development of maternal and fetal/neonatal complications, and implications on developmental programming of accelerated aging and cardiovascular and renal disease for a lifetime.

Mesenchymal Stem Cell-derived Extracellular Vesicles Prevent Experimental Bronchopulmonary Dysplasia Complicated By Pulmonary Hypertension.

Mesenchymal stem cell (MSC) extracellular vesicles (EVs) have beneficial effects in preclinical bronchopulmonary dysplasia and pulmonary hypertension (BPD-PH) models. The optimal source, dosing, route, and duration of effects are however unknown. The objectives of this study were to (a) compare the efficacy of GMP-grade EVs obtained from Wharton's Jelly MSCs (WJ-MSCs) and bone marrow (BM-MSCs), (b) determine the optimal dosing and route of administration, (c) evaluate its long-term effects, and (d) determine how MSC EVs alter the lung transcriptome. Newborn rats exposed to normoxia or hyperoxia (85% O2) from postnatal day (P)1-P14 were given (a) intra-tracheal (IT) BM or WJ-MSC EVs or placebo, (b) varying doses of IT WJ-MSC EVs, or (c) IT or intravenous (IV) WJ-MSC EVs on P3. Rats were evaluated at P14 or 3 months. Early administration of IT BM-MSC or WJ-MSC EVs had similar beneficial effects on lung structure and PH in hyperoxia-exposed rats. WJ-MSC EVs however had superior effects on cardiac remodeling. Low, medium, and high dose WJ-MSC EVs had similar cardiopulmonary regenerative effects. IT and IV WJ-MSC EVs similarly improved vascular density and reduced PH in hyperoxic rats. Gene-set enrichment analysis of transcripts differentially expressed in WJ-MSC EV-treated rats showed that induced transcripts were associated with angiogenesis. Long-term studies demonstrated that a single early MSC EV dose has pulmonary vascular protective effects 3 months after administration. Together, our findings have significant translational implications as it provides critical insight into the optimal source, dosing, route, mechanisms of action, and duration of effects of MSC-EVs for BPD-PH.

Comparative Effects of Bone Marrow-derived Versus Umbilical Cord Tissue Mesenchymal Stem Cells in an Experimental Model of Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is a life-threatening condition in preterm infants with few effective therapies. Mesenchymal stem or stromal cells (MSCs) are a promising therapeutic strategy for BPD. The ideal MSC source for BPD prevention is however unknown. The objective of this study was to compare the regenerative effects of MSC obtained from bone marrow (BM) and umbilical cord tissue (UCT) in an experimental BPD model. In vitro, UCT-MSC demonstrated greater proliferation and expression of anti-inflammatory cytokines as compared to BM-MSC. Lung epithelial cells incubated with UCT-MSC conditioned media (CM) had better-wound healing following scratch injury. UCT-MSC CM and BM-MSC CM had similar pro-angiogenic effects on hyperoxia-exposed pulmonary microvascular endothelial cells. In vivo, newborn rats exposed to normoxia or hyperoxia (85% O2) from postnatal day (P) 1 to 21 were given intra-tracheal (IT) BM or UCT-MSC (1 × 106 cells/50 µL), or placebo (PL) on P3. Hyperoxia PL-treated rats had marked alveolar simplification, reduced lung vascular density, pulmonary vascular remodeling, and lung inflammation. In contrast, administration of both BM-MSC and UCT-MSC significantly improved alveolar structure, lung angiogenesis, pulmonary vascular remodeling, and lung inflammation. UCT-MSC hyperoxia-exposed rats however had greater improvement in some morphometric measures of alveolarization and less lung macrophage infiltration as compared to the BM-MSC-treated group. Together, these findings suggest that BM-MSC and UCT-MSC have significant lung regenerative effects in experimental BPD but UCT-MSC suppresses lung macrophage infiltration and promotes lung epithelial cell healing to a greater degree.

Circulating extracellular vesicles activate the pyroptosis pathway in the brain following ventilation-induced lung injury.

BACKGROUND: Mechanical ventilation of preterm newborns causes lung injury and is associated with poor neurodevelopmental outcomes. However, the mechanistic links between ventilation-induced lung injury (VILI) and brain injury is not well defined. Since circulating extracellular vesicles (EVs) are known to link distant organs by transferring their cargos, we hypothesized that EVs mediate inflammatory brain injury associated with VILI. METHODS: Neonatal rats were mechanically ventilated with low (10 mL/kg) or high (25 mL/kg) tidal volume for 1 h on post-natal day 7 followed by recovery for 2 weeks. Exosomes were isolated from the plasma of these rats and adoptively transferred into normal newborn rats. We assessed the effect of mechanical ventilation or exosome transfer on brain inflammation and activation of the pyroptosis pathway by western blot and histology. RESULTS: Injurious mechanical ventilation induced similar markers of inflammation and pyroptosis, such as increased IL-1ß and activated caspase-1/gasdermin D (GSDMD) in both lung and brain, in addition to inducing microglial activation and cell death in the brain. Isolated EVs were enriched for the exosomal markers CD9 and CD81, suggesting enrichment for exosomes. EVs isolated from neonatal rats with VILI had increased caspase-1 but not GSDMD. Adoptive transfer of these EVs led to neuroinflammation with microglial activation and activation of caspase-1 and GSDMD in the brain similar to that observed in neonatal rats that were mechanically ventilated. CONCLUSIONS: These findings suggest that circulating EVs can contribute to the brain injury and poor neurodevelopmental outcomes in preterm infants with VILI through activation of GSDMD.

Hyperoxia-activated circulating extracellular vesicles induce lung and brain injury in neonatal rats.

Hyperoxia-induced lung injury plays a key role in the development of bronchopulmonary dysplasia (BPD), characterized by inflammatory injury and impaired lung development in preterm infants. Although BPD is a predictor of poor neurodevelopmental outcomes, currently it is uncertain how lung injury contributes to brain injury in preterm infants. Extracellular vesicles (EVs) are a heterogeneous group of cell-derived membranous structures that regulate intercellular and inter-organ communications. Gasdermin D (GSDMD) has emerged as a key executor of inflammasome-mediated cell death and inflammation. In this study, we utilized a neonatal rat model of BPD to assess if hyperoxia stimulates lung release of circulating EVs and if these EVs induce lung and brain injury. We found that hyperoxia-exposed rats had elevated numbers of plasma-derived EVs compared to rats maintained in room air. These EVs also had increased cargos of surfactant protein C, a marker of type II alveolar epithelial cells (AEC), and the active (p30) form of GSDMD. When these EVs were adoptively transferred into normal newborn rats via intravenous injection, they were taken up both by lung and brain tissues. Moreover, EVs from hyperoxic animals induced not only the pathological hallmarks of BPD, but also brain inflammatory injury in recipient rats, as well as inducing cell death in cultured pulmonary vascular endothelial cells and neural stem cells (NSC). Similarly, hyperoxia-exposed cultured AEC-like cells released EVs that also contained increased GSDMD-p30 and these EVs induced pyroptotic cell death in NSC. Overall, these data indicate that hyperoxia-activated circulating EVs mediate a lung to brain crosstalk resulting in brain injury and suggest a mechanism that links lung injury and neurodevelopmental impairment in BPD infants.

Long-term consequences of prematurity.

Due to a shortened period of in utero organ development, premature infants are at higher risk of chronic respiratory, cardiac, renal, and endocrine system disorders later in life. With more premature babies being born and more of them surviving, internists and primary care practitioners should be aware of their patient's birth history and of the potential long-term effects of prematurity. Such understanding can lead to early detection of disease and targeted lifestyle modifications.

Soluble Klotho, a biomarker and therapeutic strategy to reduce bronchopulmonary dysplasia and pulmonary hypertension in preterm infants.

Preterm infants with bronchopulmonary dysplasia (BPD) and pulmonary hypertension (PH) have accelerated lung aging and poor long-term outcomes. Klotho is an antiaging protein that modulates oxidative stress, angiogenesis and fibrosis. Here we test the hypothesis that decreased cord Klotho levels in preterm infants predict increased BPD-PH risk and early Klotho supplementation prevents BPD-like phenotype and PH in rodents exposed to neonatal hyperoxia. In experiment 1, Klotho levels were measured in cord blood of preterm infants who were enrolled in a longitudinal cohort study. In experiment 2, using an experimental BPD-PH model, rat pups exposed to room air or hyperoxia (85% O2) were randomly assigned to receive every other day injections of recombinant Klotho or placebo. The effect of Klotho on lung structure, PH and cardiac function was assessed. As compared to controls, preterm infants with BPD or BPD-PH had decreased cord Klotho levels. Early Klotho supplementation in neonatal hyperoxia-exposed rodents preserved lung alveolar and vascular structure, attenuated PH, reduced pulmonary vascular remodeling and improved cardiac function. Together, these findings have important implications as they suggest that perinatal Klotho deficiency contributes to BPD-PH risk and strategies that preserve Klotho levels, may improve long-term cardiopulmonary outcomes in preterm infants.

Effects of Klotho supplementation on hyperoxia-induced renal injury in a rodent model of postnatal nephrogenesis.

BACKGROUND: Hyperoxia (HO) causes kidney injury in preterm infants; however, whether these effects are modifiable is unknown. We hypothesized that administration of exogenous soluble Klotho, a kidney-derived antioxidant, would attenuate HO-induced kidney injury during postnatal nephrogenesis in rats. METHODS: Sprague Dawley neonatal rats assigned to normoxia (21% O2) or HO (85% O2) groups from postnatal day (P) 1 to 21 were randomly assigned to receive alternate day intraperitoneal injections of recombinant Klotho or placebo for 3 weeks. They were recovered in normoxia for an additional 3 weeks and sacrificed at 6 weeks. Renal artery resistance and pulsatility indices, tubular injury scores, glomerular area, and renal antioxidant capacity were assessed. RESULTS: Rodents exposed to HO during postnatal nephrogenesis had reduced kidney Klotho expression, glomerulomegaly, and higher tubular injury scores. Exogenous Klotho administration improved renal perfusion as indicated by decreases in both resistance and pulsatility indices and increased antioxidant enzyme expression. CONCLUSIONS: HO exposure during postnatal nephrogenesis in rodents results in a decline in kidney Klotho expression, decreased renal perfusion, enlarged glomerular size, and tubular injury. The exogenous administration of Klotho attenuated HO-induced kidney injury and augmented antioxidant capacity.

Neonatal hyperoxia exposure induces aortic biomechanical alterations and cardiac dysfunction in juvenile rats.

Supplemental oxygen (O2 ) therapy in preterm infants impairs lung development, but the impact of O2 on long-term systemic vascular structure and function has not been well-explored. The present study tested the hypothesis that neonatal O2 therapy induces long-term structural and functional alterations in the systemic vasculature, resulting in vascular stiffness observed in children and young adults born preterm. Newborn Sprague-Dawley rats were exposed to normoxia (21% O2 ) or hyperoxia (85% O2 ) for 1 and 3 weeks. A subgroup exposed to 3 weeks hyperoxia was recovered in normoxia for an additional 3 weeks. Aortic stiffness was assessed by pulse wave velocity (PWV) using Doppler ultrasound and pressure myography. Aorta remodeling was assessed by collagen deposition and expression. Left ventricular (LV) function was assessed by echocardiography. We found that neonatal hyperoxia exposure increased vascular stiffness at 3 weeks, which persisted after normoxic recovery at 6 weeks of age. These findings were accompanied by increased PWV, aortic remodeling, and altered LV function as evidenced by decreased ejection fraction, cardiac output, and stroke volume. Importantly, these functional changes were associated with increased collagen deposition in the aorta. Together, these findings demonstrate that neonatal hyperoxia induces early and sustained biomechanical alterations in the systemic vasculature and impairs LV function. Early identification of preterm infants who are at risk of developing systemic vascular dysfunction will be crucial in developing targeted prevention strategies that may improve the long-term cardiovascular outcomes in this vulnerable population.