Hypoxia-Inducible Factor-1α in SM22α-Expressing Cells Modulates Alveolarization.

Worldwide, the incidence of both preterm births and chronic lung disease of infancy, or bronchopulmonary dysplasia, remains high. Infants with bronchopulmonary dysplasia have larger and fewer alveoli, a lung pathology that can persist into adulthood. Although recent data point to a role for hypoxia-inducible factor-1α (HIF-1α) in mediating pulmonary angiogenesis and alveolarization, the cell-specific role of HIF-1α remains incompletely understood. Thus, we hypothesized that HIF-1α, in a distinct subset of mesenchymal cells, mediates postnatal alveolarization. To test the hypothesis, we generated mice with a cell-specific deletion of HIF-1α by crossing SM22α promoter-driven Cre mice with HIF-1αflox/flox mice (SM22α-HIF-1α-/-), determined SM-22α-expressing cell identity using single-cell RNA sequencing, and interrogated samples from preterm infants. Deletion of HIF-1α in SM22α-expressing cells had no effect on lung structure at day 3 of life. However, at 8 days, there were fewer and larger alveoli, a difference that persisted into adulthood. Microvascular density, elastin organization, and peripheral branching of the lung vasculature were decreased in SM22α-HIF-1α-/- mice, compared with control mice. Single-cell RNA sequencing demonstrated that three mesenchymal cell subtypes express SM22α: myofibroblasts, airway smooth muscle cells, and vascular smooth muscle cells. Pulmonary vascular smooth muscle cells from SM22α-HIF-1α-/- mice had decreased angiopoietin-2 expression and, in coculture experiments, a diminished capacity to promote angiogenesis that was rescued by angiopoietin-2. Angiopoietin-2 expression in tracheal aspirates of preterm infants was inversely correlated with overall mechanical ventilation time, a marker of disease severity. We conclude that SM22α-specific HIF-1α expression drives peripheral angiogenesis and alveolarization in the lung, perhaps by promoting angiopoietin-2 expression.

Pulmonary artery smooth muscle cell HIF-1α regulates endothelin expression via microRNA-543.

Pulmonary artery smooth muscle cells (PASMCs) express endothelin (ET-1), which modulates the pulmonary vascular response to hypoxia. Although cross-talk between hypoxia-inducible factor-1α (HIF-1α), an O2-sensitive transcription factor, and ET-1 is established, the cell-specific relationship between HIF-1α and ET-1 expression remains incompletely understood. We tested the hypotheses that in PASMCs 1) HIF-1α expression constrains ET-1 expression, and 2) a specific microRNA (miRNA) links HIF-1α and ET-1 expression. In human (h)PASMCs, depletion of HIF-1α with siRNA increased ET-1 expression at both the mRNA and protein levels ( P < 0.01). In HIF-1α-/- murine PASMCs, ET-1 gene and protein expression was increased ( P < 0.0001) compared with HIF-1α+/+ cells. miRNA profiles were screened in hPASMCs transfected with siRNA-HIF-1α, and RNA hybridization was performed on the Agilent (Santa Clara, CA) human miRNA microarray. With HIF-1α depletion, miRNA-543 increased 2.4-fold ( P < 0.01). In hPASMCs, miRNA-543 overexpression increased ET-1 gene ( P < 0.01) and protein ( P < 0.01) expression, decreased TWIST gene expression ( P < 0.05), and increased ET-1 gene and protein expression, compared with nontargeting controls ( P < 0.01). Moreover, we evaluated low passage hPASMCs from control and patients with idiopathic pulmonary arterial hypertension (IPAH). Compared with controls, protein expression of HIF-1α and Twist-related protein-1 (TWIST1) was decreased ( P < 0.05), and miRNA-543 and ET-1 expression increased ( P < 0.001) in hPASMCs from patients with IPAH. Thus, in PASMCs, loss of HIF-1α increases miRNA-543, which decreases Twist expression, leading to an increase in PASMC ET-1 expression. This previously undescribed link between HIF-1α and ET-1 via miRNA-543 mediated Twist suppression represents another layer of molecular regulation that might determine pulmonary vascular tone.

Emergency Severity Index Version 4 and Triage of Pediatric Emergency Department Patients.

Importance: Most emergency departments (EDs) across the US use the Emergency Severity Index (ESI) to predict acuity and resource needs. A comprehensive assessment of ESI accuracy among pediatric patients is lacking. Objective: To assess the frequency of mistriage using ESI (version 4) among pediatric ED visits using automated measures of mistriage and identify characteristics associated with mistriage. Design, Setting, and Participants: This cohort study used operational measures for each ESI level to classify encounters as undertriaged, overtriaged, or correctly triaged to assess the accuracy of the ESI and identify characteristics of mistriage. Participants were pediatric patients at 21 EDs within Kaiser Permanente Northern California from January 1, 2016, to December 31, 2020. During that time, version 4 of the ESI was in use by these EDs. Visits with missing ESI, incomplete ED time variables, patients transferred from another ED, and those who left against medical advice or without being seen were excluded. Data were analyzed between January 2022 and June 2023. Exposures: Assigned ESI level. Main Outcomes and Measures: Rates of undertriage and overtriage by assigned ESI level based on mistriage algorithm, patient and visit characteristics associated with undertriage and overtriage. Results: This study included 1 016 816 pediatric ED visits; the mean (SD) age of patients was 7.3 (5.6) years, 479 610 (47.2%) were female, and 537 206 (52.8%) were male. Correct triage occurred in 346 918 visits (34.1%; 95% CI, 34.0%-34.2%), while overtriage and undertriage occurred in 594 485 visits (58.5%; 95% CI, 58.4%-58.6%) and 75 413 visits (7.4%; 95% CI, 7.4%-7.5%), respectively. In adjusted analyses, undertriage was more common among children at least 6 years old compared with those younger 6 years; male patients compared with female patients; patients with Asian, Black, or Hispanic or other races or ethnicities compared with White patients; patients with comorbid illnesses compared with those without; and patients who arrived by ambulance compared with nonambulance patients. Conclusions and Relevance: This multicenter retrospective study found that mistriage with ESI version 4 was common in pediatric ED visits. There is an opportunity to improve pediatric ED triage, both in early identification of critically ill patients (limit undertriage) and in more accurate identification of low-acuity patients with low resource needs (limit overtriage). Future research should include assessments based on version 5 of the ESI, which was released after this study was completed.

Embedding time-limited laboratory orders within computerized provider order entry reduces laboratory utilization.

OBJECTIVES: To test the hypothesis that limits on repeating laboratory studies within computerized provider order entry decrease laboratory utilization. DESIGN: Cohort study with historical controls. SETTING: A 20-bed PICU in a freestanding, quaternary care, academic children's hospital. PATIENTS: This study included all patients admitted to the pediatric ICU between January 1, 2008, and December 31, 2009. A total of 818 discharges were evaluated prior to the intervention (January 1, 2008, through December 31, 2008) and 1,021 patient discharges were evaluated postintervention (January 1, 2009, through December 31, 2009). INTERVENTION: A computerized provider order entry rule limited the ability to schedule repeating complete blood cell counts, chemistry, and coagulation studies to a 24-hour interval in the future. The time limit was designed to ensure daily evaluation of the utility of each test. MEASUREMENTS AND MAIN RESULTS: Initial analysis with t tests showed significant decreases in tests per patient day in the postintervention period (complete blood cell counts: 1.5 ± 0.1 to 1.0 ± 0.1; chemistry: 10.6 ± 0.9 to 6.9 ± 0.6; coagulation: 3.3 ± 0.4 to 1.7 ± 0.2; p < 0.01, all variables vs. preintervention period). Even after incorporating a trend toward decreasing laboratory utilization in the preintervention period into our regression analysis, the intervention decreased complete blood cell counts (p = 0.007), chemistry (p = 0.049), and coagulation (p = 0.001) tests per patient day. CONCLUSIONS: Limits on laboratory orders within the context of computerized provider order entry decreased laboratory utilization without adverse affects on mortality or length of stay. Broader application of this strategy might decrease costs, the incidence of iatrogenic anemia, and catheter-associated bloodstream infections.

Inhibiting NF-κB in the developing lung disrupts angiogenesis and alveolarization.

Bronchopulmonary dysplasia (BPD), a chronic lung disease of infancy, is characterized by arrested alveolar development. Pulmonary angiogenesis, mediated by the vascular endothelial growth factor (VEGF) pathway, is essential for alveolarization. However, the transcriptional regulators mediating pulmonary angiogenesis remain unknown. We previously demonstrated that NF-κB, a transcription factor traditionally associated with inflammation, plays a unique protective role in the neonatal lung. Therefore, we hypothesized that constitutive NF-κB activity is essential for postnatal lung development. Blocking NF-κB activity in 6-day-old neonatal mice induced the alveolar simplification similar to that observed in BPD and significantly reduced pulmonary capillary density. Studies to determine the mechanism responsible for this effect identified greater constitutive NF-κB in neonatal lung and in primary pulmonary endothelial cells (PEC) compared with adult. Moreover, inhibiting constitutive NF-κB activity in the neonatal PEC with either pharmacological inhibitors or RNA interference blocked PEC survival, decreased proliferation, and impaired in vitro angiogenesis. Finally, by chromatin immunoprecipitation, NF-κB was found to be a direct regulator of the angiogenic mediator, VEGF-receptor-2, in the neonatal pulmonary vasculature. Taken together, our data identify an entirely novel role for NF-κB in promoting physiological angiogenesis and alveolarization in the developing lung. Our data suggest that disruption of NF-κB signaling may contribute to the pathogenesis of BPD and that enhancement of NF-κB may represent a viable therapeutic strategy to promote lung growth and regeneration in pulmonary diseases marked by impaired angiogenesis.

Hypoxia-inducible factor-1α regulates KCNMB1 expression in human pulmonary artery smooth muscle cells.

Previously, we observed that hypoxia increases the expression of the ß1-subunit (KCNMB1) of the calcium-sensitive potassium channel (BK(Ca)). Herein, we elucidate the mechanism whereby hypoxia increases KCNMB1 expression in human pulmonary artery smooth muscle cells (hPASMC). In response to hypoxia, the expression of both the transcription factor hypoxia-inducible factor 1-α (HIF-1α) and KCNMB1 are increased. Knockdown of HIF-1α using a shRNA plasmid blocked the hypoxic induction of KCNMB1 expression. Chromatin immunoprecipitation (ChIP) demonstrated HIF-1α binding to three discrete regions of the human KCNMB1 promoter known to contain hypoxia response elements (HREs). A KCNMB1 promoter reporter assay combined with site-directed mutagenesis identified two adjacent HREs located between -3,540 bp and -3,311 bp that are essential for the hypoxic induction of KCNMB1 promoter activity. Furthermore, additional ChIP assays demonstrated recruitment of the HIF-1α transcriptional coactivator, p300, to this same promoter region. Treatment of hPASMC with the histone deacetylase inhibitor, trichostatin, prolonged the increase in KCNMB1 observed with hypoxia, suggesting that alterations in chromatin remodeling function to limit the hypoxic induction of KCNMB1. Finally, KCNMB1 knockdown potentiated the hypoxia-induced increase in cytosolic calcium in hPASMC, highlighting the contribution of the ß1-subunit in modulating vascular SMC tone in response to acute hypoxia. In conclusion, HIF-1α increases KCNMB1 expression in response to hypoxia in hPASMC by binding to two HREs located at -3,540 to -3,311 of the KCNMB1 promoter. We speculate that selective modulation of KCNMB1 expression may serve as a novel therapeutic approach to address diseases characterized by an increase in vascular tone.

A critical appraisal of "transfusion strategies for patients in pediatric intensive care units" by Lacroix J, Hebert PC, Hutchison, et al (N Engl J Med 2007; 356:1609-1619).

OBJECTIVE: To review the findings and discuss the implications of transfusion strategies in stable critically ill children. DESIGN: A critical appraisal of the article "Transfusion strategies for patients in pediatric intensive care units" by Lacroix J, Hebert PC, Hutchison, et al, published in the N Engl J Med in 2007 with literature review. FINDINGS: In this prospective, randomized, controlled, noninferiority trial the authors compared a liberal transfusion strategy, using a transfusion threshold of 9 g/dL, to a conservative transfusion strategy, using a transfusion threshold of 7 g/dL. The primary end point was multiple organ dysfunction syndrome (MODS) or progression of MODS. The authors found that when comparing the restrictive transfusion strategy to the liberal strategy, the absolute risk reduction for developing new or progressive MODS was only 0.4% (95% confidence interval, -4.6 -5.5). Using the restrictive protocol, the number needed to treat to prevent one red blood cell (RBC) transfusion was only two. The number of RBC units per patient in the restrictive group was 0.9, and in the liberal group was 1.7 (p < 0.001). When comparing the two strategies there was a relative reduction of 96% in the number of patients who had any transfusion exposure and a relative decrease of 44% in the number of transfusions administered in the restrictive strategy. CONCLUSIONS: Using a restrictive transfusion protocol with a transfusion threshold of 7 g/dL in stable critically ill children is as safe as using a liberal protocol and can decrease the number of patients exposed to RBC transfusions.

Percutaneous closure of recanalised ductus arteriosus--a single-centre experience.

INTRODUCTION: Restoration of blood flow through a previously occluded ductus arteriosus may occur in some patients. Treatment strategy in patients with such residual shunts has not yet been uniformly established. AIM: To present single-centre experience and to attempt to establish a strategy of management of patients with residual ductus arteriosus shunts following percutaneous closure. METHODS: Of 352 patients who underwent percutaneous closure of ductus arteriosus, in 13 subjects complete closure failed (coils and Rashkind occluders were used in 10 and 3 patients, respectively). In these patients other percutaneous interventions aiming at total closure of residual shunt were attempted. RESULTS: In 12 patients coils were inserted (one patient received two coils). Introduction of implant in one patient failed, but total occlusion of the shunt was confirmed one day after the procedure. Trivial residual shunt was observed in one patient after one-year follow-up. CONCLUSIONS: Percutaneous treatment of residual shunts within the ductus arteriosus is an effective and safe procedure. In our opinion identifying and treating such leaks is important, as it prevents complications and long-term need for antibiotic prevention of infective endocarditis. In the case of a small residual shunt, insertion of a coil seems to be the optimal therapy due to the low cost of the device, favourable design and high effectiveness. For patients in whom anatomy of the ductus arteriosus has been significantly changed, particularly in previously treated subjects, techniques using vascular loops or insertion using a catheter wedge may be helpful.

Clinical Decision Support for Pediatric Blood Product Prescriptions.

Since the beginning of the 20th century, blood products have been used to effectively treat life-threatening conditions. Over time, we have come to appreciate the many benefits along with significant risks inherent to blood product transfusions. As such, recommendations for the safe and effective use of blood products have evolved over time. Current evidence supports the use of restrictive transfusion strategies that can avoid the risks of unnecessary transfusions. In spite of good evidence, there is a considerable amount of variability in transfusion practices across providers. Clinical decision support (CDS) is an effective tool capable of increasing adherence to evidence-based practices. CDS has been used successfully to improve adherence to transfusion guidelines. Pediatric literature demonstrates strong evidence for the use of CDS to improve appropriateness of red blood cell and plasma transfusion utilization. Further studies in more diverse settings with more standardized reporting are needed to provide more clarity around the effectiveness of CDS in blood product prescriptions.

Computerized physician order entry with decision support decreases blood transfusions in children.

OBJECTIVE: Timely provision of evidence-based recommendations through computerized physician order entry with clinical decision support may improve use of red blood cell transfusions (RBCTs). METHODS: We performed a cohort study with historical controls including inpatients admitted between February 1, 2008, and January 31, 2010. A clinical decision-support alert for RBCTs was constructed by using current evidence. RBCT orders resulted in assessment of the patient's medical record with prescriber notification if parameters were not within recommended ranges. Primary end points included the average pretransfusion hemoglobin level and the rate of RBCTs per patient-day. RESULTS: In total, 3293 control discharges and 3492 study discharges were evaluated. The mean (SD) control pretransfusion hemoglobin level in the PICU was 9.83 (2.63) g/dL (95% confidence interval [CI]: 9.65-10.01) compared with the study value of 8.75 (2.05) g/dL (95% CI: 8.59-8.90) (P < .0001). The wards' control value was 7.56 (0.93) g/dL (95% CI: 7.47-7.65), the study value was 7.14 (1.01) g/dL (95% CI: 6.99-7.28) (P < .0001). The control PICU rate of RBCTs per patient-day was 0.20 (0.11) (95% CI: 0.13-0.27), the study rate was 0.14 (0.04) (95% CI: 0.11-0.17) (P = .12). The PICU's control rate was 0.033 (0.01) (95% CI: 0.02-0.04), and the study rate was 0.017 (0.007) (95% CI: 0.01-0.02) (P < .0001). There was no difference in mortality rates across all cohorts. CONCLUSIONS: Implementation of clinical decision-support alerts was associated with a decrease in RBCTs, which suggests improved adoption of evidence-based recommendations. This strategy might be widely applied to promote timely adoption of scientific evidence.