Sodium-coupled neutral amino acid transporter 1 (SNAT1) modulates L-citrulline transport and nitric oxide (NO) signaling in piglet pulmonary arterial endothelial cells

Rationale There is evidence that impairments in nitric oxide (NO) signaling contribute to chronic hypoxia-induced pulmonary hypertension. The L-arginine-NO precursor, L-citrulline, has been shown to ameliorate pulmonary hypertension. Sodium-coupled neutral amino acid transporters (SNATs) are involved in the transport of L-citrulline into pulmonary arterial endothelial cells (PAECs). The functional link between the SNATs, L-citrulline, and NO signaling has not yet been explored. Objective We tested the hypothesis that changes in SNAT1 expression and transport function regulate NO production by modulating eNOS coupling in newborn piglet PAECs. Methods and Results A silencing RNA (siRNA) technique was used to assess the contribution of SNAT1 to NO production and eNOS coupling (eNOS dimer-to-monomer ratios) in PAECs from newborn piglets cultured under normoxic and hypoxic conditions in the presence and absence of L-citrulline. SNAT1 siRNA reduced basal NO production in normoxic PAECs and prevented L-citrulline-induced elevations in NO production in both normoxic and hypoxic PAECs. SNAT1 siRNA reduced basal eNOS dimer-to-monomer ratios in normoxic PAECs and prevented L-citrulline-induced increases in eNOS dimer-to-monomer ratios in hypoxic PAECs. Conclusions SNAT1 mediated L-citrulline transport modulates eNOS coupling and thus regulates NO production in hypoxic PAECs from newborn piglets. Strategies that increase SNAT1-mediated transport and supply of L-citrulline may serve as novel therapeutic approaches to enhance NO production in patients with pulmonary vascular disease

Free radical formation in cerebral cortical astrocytes in culture induced by methylmercury

Abstract Oxidative stress has been implicated in neurotoxic damage associated with various metals, including methylmercury (MeHg). Although the mechanism(s) of MeHg-induced neurotoxicity remains unclear, evidence supports a mediatory role for astrocytes, a cell type that preferentially accumulates MeHg. Using scanning confocal microscopy (LSCM), the present study was undertaken to examine the role of astrocytes as the site of reactive oxygen species (ROS). Three redox-sensitive fluorescent probes were used for ROS analysis, (a) CM-H 2 DCFDA (chloromethyl derivative of dichlorodihydrofluorescein diacetate), a probe for intracellular hydrogen peroxide (H 2 O 2 ); (b) hydroethidine (HETH), a probe for superoxide anion (ÁO 2 À ), and (c) CM-H 2 XRos (chloromethyl derivative of dihydro X-rosamine), and a probe that is selective for mitochondrial reactive oxygen intermediates. Astrocytes were treated with 10 AM MeHg for 30 min, following which the various fluorescent probes were added; 20 min later LSCM images were collected. Astrocytes loaded with CM-H 2 DCFDA and HE demonstrated a significant MeHg-induced increase in fluorescence intensity indicative of increased intracellular H 2 O 2 and ÁO 2 À , respectively. Similar results were obtained with the mitotracker dye, CM-H 2 XRos. Additionally, exposure of astrocytes for 24 h to 100 AM buthionine-Lsulfoxane (BSO), a glutathione (GSH) synthesis inhibitor, caused a significant increase in ROS formation. Furthermore, BSO pretreatment significantly enhanced the MeHg-induced formation of ÁO 2 À , indicating an important role for GSH in the maintenance of optimal cellular redox status. Time-course experiments performed in the simultaneous presence of CM-H 2 XRos and CM-H 2 DCFDA demonstrated that the MeHg-induced CM-H 2 XRos fluorescence changes preceded those of CM-H 2 DCFDA, suggesting that the mitochondria represent an early primary site for ROS formation. Taken together, these studies illustrate that MeHg induces the generation of astrocyte-derived ROS and support a role for astrocytic ROS in MeHg-associated neurotoxic damage.

Associations of Neighborhood Opportunity and Social Vulnerability With Trajectories of Childhood Body Mass Index and Obesity Among US Children

IMPORTANCE: Physical and social neighborhood attributes may have implications for children\u27s growth and development patterns. The extent to which these attributes are associated with body mass index (BMI) trajectories and obesity risk from childhood to adolescence remains understudied. OBJECTIVE: To examine associations of neighborhood-level measures of opportunity and social vulnerability with trajectories of BMI and obesity risk from birth to adolescence. DESIGN, SETTING, AND PARTICIPANTS: This cohort study used data from 54 cohorts (20 677 children) participating in the Environmental Influences on Child Health Outcomes (ECHO) program from January 1, 1995, to January 1, 2022. Participant inclusion required at least 1 geocoded residential address and anthropometric measure (taken at the same time or after the address date) from birth through adolescence. Data were analyzed from February 1 to June 30, 2022. EXPOSURES: Census tract-level Child Opportunity Index (COI) and Social Vulnerability Index (SVI) linked to geocoded residential addresses at birth and in infancy (age range, 0.5-1.5 years), early childhood (age range, 2.0-4.8 years), and mid-childhood (age range, 5.0-9.8 years). MAIN OUTCOMES AND MEASURES: BMI (calculated as weight in kilograms divided by length [if aged \u3c2 \u3eyears] or height in meters squared) and obesity (age- and sex-specific BMI ≥95th percentile). Based on nationwide distributions of the COI and SVI, Census tract rankings were grouped into 5 categories: very low (\u3c20th \u3epercentile), low (20th percentile to \u3c40th \u3epercentile), moderate (40th percentile to \u3c60th \u3epercentile), high (60th percentile to \u3c80th \u3epercentile), or very high (≥80th percentile) opportunity (COI) or vulnerability (SVI). RESULTS: Among 20 677 children, 10 747 (52.0%) were male; 12 463 of 20 105 (62.0%) were White, and 16 036 of 20 333 (78.9%) were non-Hispanic. (Some data for race and ethnicity were missing.) Overall, 29.9% of children in the ECHO program resided in areas with the most advantageous characteristics. For example, at birth, 26.7% of children lived in areas with very high COI, and 25.3% lived in areas with very low SVI; in mid-childhood, 30.6% lived in areas with very high COI and 28.4% lived in areas with very low SVI. Linear mixed-effects models revealed that at every life stage, children who resided in areas with higher COI (vs very low COI) had lower mean BMI trajectories and lower risk of obesity from childhood to adolescence, independent of family sociodemographic and prenatal characteristics. For example, among children with obesity at age 10 years, the risk ratio was 0.21 (95% CI, 0.12-0.34) for very high COI at birth, 0.31 (95% CI, 0.20-0.51) for high COI at birth, 0.46 (95% CI, 0.28-0.74) for moderate COI at birth, and 0.53 (95% CI, 0.32-0.86) for low COI at birth. Similar patterns of findings were observed for children who resided in areas with lower SVI (vs very high SVI). For example, among children with obesity at age 10 years, the risk ratio was 0.17 (95% CI, 0.10-0.30) for very low SVI at birth, 0.20 (95% CI, 0.11-0.35) for low SVI at birth, 0.42 (95% CI, 0.24-0.75) for moderate SVI at birth, and 0.43 (95% CI, 0.24-0.76) for high SVI at birth. For both indices, effect estimates for mean BMI difference and obesity risk were larger at an older age of outcome measurement. In addition, exposure to COI or SVI at birth was associated with the most substantial difference in subsequent mean BMI and risk of obesity compared with exposure at later life stages. CONCLUSIONS AND RELEVANCE: In this cohort study, residing in higher-opportunity and lower-vulnerability neighborhoods in early life, especially at birth, was associated with a lower mean BMI trajectory and a lower risk of obesity from childhood to adolescence. Future research should clarify whether initiatives or policies that alter specific components of neighborhood environment would be beneficial in preventing excess weight in children

Opportunities for understanding the COVID-19 pandemic and child health in the United States: the Environmental influences on Child Health Outcomes (ECHO) program

Objective Ongoing pediatric cohort studies offer opportunities to investigate the impact of the COVID-19 pandemic on children's health. With well-characterized data from tens of thousands of US children, the Environmental influences on Child Health Outcomes (ECHO) Program offers such an opportunity. Methods ECHO enrolled children and their caregivers from community- and clinic-based pediatric cohort studies. Extant data from each of the cohorts were pooled and harmonized. In 2019, cohorts began collecting data under a common protocol, and data collection is ongoing with a focus on early life environmental exposures and five child health domains: birth outcomes, neurodevelopment, obesity, respiratory, and positive health. In April of 2020, ECHO began collecting a questionnaire designed to assess COVID-19 infection and the pandemic's impact on families. We describe and summarize the characteristics of children who participated in the ECHO Program during the COVID-19 pandemic and novel opportunities for scientific advancement. Results This sample (n = 13,725) was diverse by child age (31% early childhood, 41% middle childhood, and 16% adolescence up to age 21), sex (49% female), race (64% White, 15% Black, 3% Asian, 2% American Indian or Alaska Native, <1% Native Hawaiian or Pacific Islander, 10% Multiple race and 2% Other race), Hispanic ethnicity (22% Hispanic), and were similarly distributed across the four United States Census regions and Puerto Rico. Conclusion ECHO data collected during the pandemic can be used to conduct solution-oriented research to inform the development of programs and policies to support child health during the pandemic and in the post-pandemic era

Requirement of argininosuccinate lyase for systemic nitric oxide production

Nitric oxide (NO) is crucial in diverse physiological and pathological processes. We show that a hypomorphic mouse model of argininosuccinate lyase (encoded by Asl) deficiency has a distinct phenotype of multiorgan dysfunction and NO deficiency. Loss of Asl in both humans and mice leads to reduced NO synthesis, owing to both decreased endogenous arginine synthesis and an impaired ability to use extracellular arginine for NO production. Administration of nitrite, which can be converted into NO in vivo, rescued the manifestations of NO deficiency in hypomorphic Asl mice, and a nitric oxide synthase (NOS)-independent NO donor restored NO-dependent vascular reactivity in humans with ASL deficiency. Mechanistic studies showed that ASL has a structural function in addition to its catalytic activity, by which it contributes to the formation of a multiprotein complex required for NO production. Our data demonstrate a previously unappreciated role for ASL in NOS function and NO homeostasis. Hence, ASL may serve as a target for manipulating NO production in experimental models, as well as for the treatment of NO-related diseases

Association of Severe Bronchiolitis during Infancy with Childhood Asthma Development: An Analysis of the ECHO Consortium

Objective: Many studies have shown that severe (hospitalized) bronchiolitis during infancy is a risk factor for developing childhood asthma. However, the population subgroups at the highest risk remain unclear. Using large nationwide pediatric cohort data, namely the NIH Environmental influences on Child Health Outcomes (ECHO) Program, we aimed to quantify the longitudinal relationship of bronchiolitis hospitalization during infancy with asthma in a generalizable dataset and to examine potential heterogeneity in terms of major demographics and clinical factors. Methods: We analyzed data from infants (age <12 months) enrolled in one of the 53 prospective cohort studies in the ECHO Program during 2001–2021. The exposure was bronchiolitis hospitalization during infancy. The outcome was a diagnosis of asthma by a physician by age 12 years. We examined their longitudinal association and determined the potential effect modifications of major demographic factors. Results: The analytic cohort consisted of 11,762 infants, 10% of whom had bronchiolitis hospitalization. Overall, 15% subsequently developed asthma. In the Cox proportional hazards model adjusting for 10 patient-level factors, compared with the no-bronchiolitis hospitalization group, the bronchiolitis hospitalization group had a significantly higher rate of asthma (14% vs. 24%, HR = 2.77, 95%CI = 2.24–3.43, p < 0.001). There was significant heterogeneity by race and ethnicity (Pinteraction = 0.02). The magnitude of the association was greater in non-Hispanic White (HR = 3.77, 95%CI = 2.74–5.18, p < 0.001) and non-Hispanic Black (HR = 2.39, 95%CI = 1.60–3.56; p < 0.001) infants, compared with Hispanic infants (HR = 1.51, 95%CI = 0.77–2.95, p = 0.23). Conclusions: According to the nationwide cohort data, infants hospitalized with bronchiolitis are at a higher risk for asthma, with quantitative heterogeneity in different racial and ethnic groups

Characterizing Long COVID in Children and Adolescents

ImportanceMost research to understand postacute sequelae of SARS-CoV-2 infection (PASC), or long COVID, has focused on adults, with less known about this complex condition in children. Research is needed to characterize pediatric PASC to enable studies of underlying mechanisms that will guide future treatment.ObjectiveTo identify the most common prolonged symptoms experienced by children (aged 6 to 17 years) after SARS-CoV-2 infection, how these symptoms differ by age (school-age [6-11 years] vs adolescents [12-17 years]), how they cluster into distinct phenotypes, and what symptoms in combination could be used as an empirically derived index to assist researchers to study the likely presence of PASC.Design, setting, and participantsMulticenter longitudinal observational cohort study with participants recruited from more than 60 US health care and community settings between March 2022 and December 2023, including school-age children and adolescents with and without SARS-CoV-2 infection history.ExposureSARS-CoV-2 infection.Main outcomes and measuresPASC and 89 prolonged symptoms across 9 symptom domains.ResultsA total of 898 school-age children (751 with previous SARS-CoV-2 infection [referred to as infected] and 147 without [referred to as uninfected]; mean age, 8.6 years; 49% female; 11% were Black or African American, 34% were Hispanic, Latino, or Spanish, and 60% were White) and 4469 adolescents (3109 infected and 1360 uninfected; mean age, 14.8 years; 48% female; 13% were Black or African American, 21% were Hispanic, Latino, or Spanish, and 73% were White) were included. Median time between first infection and symptom survey was 506 days for school-age children and 556 days for adolescents. In models adjusted for sex and race and ethnicity, 14 symptoms in both school-age children and adolescents were more common in those with SARS-CoV-2 infection history compared with those without infection history, with 4 additional symptoms in school-age children only and 3 in adolescents only. These symptoms affected almost every organ system. Combinations of symptoms most associated with infection history were identified to form a PASC research index for each age group; these indices correlated with poorer overall health and quality of life. The index emphasizes neurocognitive, pain, and gastrointestinal symptoms in school-age children but change or loss in smell or taste, pain, and fatigue/malaise-related symptoms in adolescents. Clustering analyses identified 4 PASC symptom phenotypes in school-age children and 3 in adolescents.Conclusions and relevanceThis study developed research indices for characterizing PASC in children and adolescents. Symptom patterns were similar but distinguishable between the 2 groups, highlighting the importance of characterizing PASC separately for these age ranges

Methylmercury alters the activities of Hsp90 client proteins, prostaglandin E synthase/p23 (PGES/23) and nNOS.

Methylmercury (MeHg) is a persistent pollutant with known neurotoxic effects. We have previously shown that astrocytes accumulate MeHg and play a prominent role in mediating MeHg toxicity in the central nervous system (CNS) by altering glutamate signaling, generating oxidative stress, depleting glutathione (GSH) and initiating lipid peroxidation. Interestingly, all of these pathways can be regulated by the constitutively expressed, 90-kDa heat shock protein, Hsp90. As Hsp90 function is regulated by oxidative stress, we hypothesized that MeHg disrupts Hsp90-client protein functions. Astrocytes were treated with MeHg and expression of Hsp90, as well as the abundance of complexes of Hsp90-neuronal nitric oxide synthase (nNOS) and Hsp90-prostaglandin E synthase/p23 (PGES/p23) were assessed. MeHg exposure decreased Hsp90 protein expression following 12 h of treatment while shorter exposures had no effect on Hsp90 protein expression. Interestingly, following 1 or 6 h of MeHg exposure, Hsp90 binding to PGES/p23 or nNOS was significantly increased, resulting in increased prostaglandin E2 (PGE2) synthesis from MeHg-treated astrocytes. These effects were attenuated by the Hsp90 antagonist, geldanmycin. NOS activity was increased following MeHg treatment while cGMP formation was decreased. This was accompanied by an increase in •O2- and H2O2 levels, suggesting that MeHg uncouples NO formation from NO-dependent signaling and increases oxidative stress. Altogether, our data demonstrates that Hsp90 interactions with client proteins are increased following MeHg exposure, but over time Hsp90 levels decline, contributing to oxidative stress and MeHg-dependent excitotoxicity

Pharmacotherapy for Pulmonary Hypertension in Infants with Bronchopulmonary Dysplasia: Past, Present, and Future

Approximately 8–42% of premature infants with chronic lung disease of prematurity, bronchopulmonary dysplasia (BPD), develop pulmonary hypertension (PH). Infants with BPD-PH carry alarmingly high mortality rates of up to 47%. Effective PH-targeted pharmacotherapies are desperately needed for these infants. Although many PH-targeted pharmacotherapies are commonly used to treat BPD-PH, all current use is off-label. Moreover, all current recommendations for the use of any PH-targeted therapy in infants with BPD-PH are based on expert opinion and consensus statements. Randomized Control Trials (RCTs) are needed to determine the efficacy of PH-targeted treatments in premature infants with or at risk of BPD-PH. Prior to performing efficacy RCTs, studies need to be conducted to obtain pharmacokinetic, pharmacodynamic, and safety data for any pharmacotherapy used in this understudied and fragile patient population. This review will discuss current and needed treatment strategies, identify knowledge deficits, and delineate both challenges to be overcome and approaches to be taken to develop effective PH-targeted pharmacotherapies that will improve outcomes for premature infants with or at risk of developing BPD-PH

Mitochondrial-dependent manganese neurotoxicity in rat primary astrocyte cultures

Chronic exposure to excessive levels of Mn results in a movement disorder termed manganism, which resembles Parkinson's disease (PD). The pathogenic mechanisms underlying this disorder are not fully understood. Several lines of evidence implicate astrocytes as an early target of Mn neurotoxicity. In the present study, we investigated the effects of Mn on mitochondrial function. Primary astrocyte cultures were prepared from cerebral cortices of one-day-old Sprague-Dawley rats. We have examined the cellular toxicity of Mn and its effects on the phosphorylation of extracellular signal-regulated kinase (ERK) and activation of the precursor protein of caspase-3. The potentiometric dye, tetramethyl rhodamine ethyl ester (TMRE), was used to assess the effect of Mn on astrocytic mitochondrial inner membrane potential (Delta psi(m)). Our studies show that, in a concentration- dependent manner, Mn induces significant (p 0.05) activation of astrocyte caspase-3 and phosphorylated extracellular signal-regulated kinase (p-ERK). Mn treatment (1 and 6 h) also significantly (p 0.01) dissipates the Delta psi(m) in astrocytes as evidenced by a decrease in mitochondrial TMRE fluorescence. These results suggest that activations of astrocytic caspase-3 and ERK are involved in Mn-induced neurotoxicity via mitochondrial-dependent pathways. (c) 2008 Elsevier B.V. All rights reserved