David N. Cornfield

Pediatric Pulmonary Hypertension Guidelines From the American Heart Association and American Thoracic Society

Pulmonary hypertension is associated with diverse cardiac, pulmonary, and systemic diseases in neonates, infants, and older children and contributes to significant morbidity and mortality. However, current approaches to caring for pediatric patients with pulmonary hypertension have been limited by the lack of consensus guidelines from experts in the field. In a joint effort from the American Heart Association and American Thoracic Society, a panel of experienced clinicians and clinician-scientists was assembled to review the current literature and to make recommendations on the diagnosis, evaluation, and treatment of pediatric pulmonary hypertension. This publication presents the results of extensive literature reviews, discussions, and formal scoring of recommendations for the care of children with pulmonary hypertension.

Wnt5a Is Required for Cardiac Outflow Tract Septation in Mice

Lack of septation of the cardiac outflow tract (OFT) results in persistent truncus arteriosus (PTA), a form of congenital heart disease. The outflow myocardium expands through addition of cells originating from the pharyngeal mesoderm referred to as secondary/anterior heart field, whereas cardiac neural crest (CNC) cell–derived mesenchyme condenses to form an aortopulmonary septum. We show for the first time that a mutation in Wnt5a in mice leads to PTA. We provide evidence that Wnt5a is expressed in the pharyngeal mesoderm adjacent to CNC cells in both mouse and chicken embryos and in the myocardial cell layer of the conotruncus at the time when CNC cells begin to form the aortopulmonary septum in mice. Although expression domains of secondary heart field markers are not altered in Wnt5a mutant embryos, the expression of CNC cell marker PlexinA2 is significantly reduced. Stimulation of CNC cells with Wnt5a protein elicits Ca2+ transients, suggesting that CNC cells are capable of responding to Wnt5a. We propose a novel model in which Wnt5a produced in the OFT by cells originating from the pharyngeal mesoderm signals to adjacent CNC cells during formation of the aortopulmonary septum through a noncanonical pathway via localized intracellular increases in Ca2+.

Effects of Inhaled Nitric Oxide and Oxygen in High-Altitude Pulmonary Edema

BACKGROUND High-altitude pulmonary edema (HAPE) is characterized by pulmonary hypertension, increased pulmonary capillary permeability, and hypoxemia. Treatment is limited to descent to lower altitude and administration of oxygen. METHODS AND RESULTS We studied the acute effects of inhaled nitric oxide (NO), 50% oxygen, and a mixture of NO plus 50% oxygen on hemodynamics and gas exchange in 14 patients with HAPE. Each gas mixture was given in random order for 30 minutes followed by 30 minutes washout with room air. All patients had severe HAPE as judged by Lake Louise score (6.4+/-0.7), PaO2 (35+/-3. 1 mm Hg), and alveolar to arterial oxygen tension difference (AaDO2) (26+/-3 mm Hg). NO had a selective effect on the pulmonary vasculature and did not alter systemic hemodynamics. Compared with room air, pulmonary vascular resistance fell 36% with NO (P<0.001), 23% with oxygen (P<0.001 versus air, P<0.05 versus NO alone), and 54% with NO plus 50% oxygen (P<0.001 versus air, P<0.005 versus oxygen and versus NO). NO alone improved PaO2 (+14%) and AaDO2 (-31%). Compared with 50% oxygen alone, NO plus 50% oxygen had a greater effect on AaDO2 (-18%) and PaO2 (+21%). CONCLUSIONS Inhaled NO may have a therapeutic role in the management of HAPE. The combined use of inhaled NO and oxygen has additive effects on pulmonary hemodynamics and even greater effects on gas exchange. These findings indicate that oxygen and NO may act on separate but interactive mechanisms in the pulmonary vasculature.

Quantitative Analysis of the Human Airway Microbial Ecology Reveals a Pervasive Signature for Cystic Fibrosis

Deep sequencing of microbial marker genes in lung secretions revealed individualized microbiomes in healthy subjects that differed from stereotyped microbiomes of cystic fibrosis patients. There is There, There Although the absence of evidence is not necessarily evidence of absence,* scientists have generally considered the inability to culture microbes from tissues or other medical specimens to be evidence of sterility. For example, the health of the human lung has been defined in part by whether or not microbes are present. Now, Blainey et al. use high-throughput sequencing to survey microbes in sputum from control subjects and patients with the genetic disease cystic fibrosis (CF) and show that both groups have diverse, albeit different, microbial communities. Patients with CF suffer severe lung dysfunction and often die from respiratory failure. Mucus build-up in the lungs can cause life-threatening infections, which are treated by long courses of antibiotics. However, sometimes patients benefit clinically from this therapy even while their lungs continue to house bacteria. When the authors performed high-throughput pyrosequencing of 16S ribosomal DNA on sputum from 16 CF patients and 9 control individuals, they found that both groups of samples contained multiple microbial species. But CF patients displayed a different microbial ecology, most notably when one considered bacterial phylotypes that currently cannot be cultured in the lab. Although all patient samples did not contain an identical complement of organisms, all displayed a subset that constituted a microbial profile characteristic of CF. In fact, even when typical CF pathogens were excluded from the analysis, the microbial profiles still distinguished between control-subject and CF-patient samples. Similar findings were obtained from lung tissue taken from CF patients versus patients with other lung diseases. To bring the sequencing findings back to the clinic, the authors found that decreased microbial diversity correlated with the extent of inflammation in CF lungs. *Often attributed to cosmologist Martin Rees. Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene encoding the CF transmembrane conductance regulator. Disruption of electrolyte homeostasis at mucosal surfaces leads to severe lung, pancreatic, intestinal, hepatic, and reproductive abnormalities. Loss of lung function as a result of chronic lung disease is the primary cause of death from CF. Using high-throughput sequencing to survey microbes in the sputum of 16 CF patients and 9 control individuals, we identified diverse microbial communities in the healthy samples, contravening conventional wisdom that healthy airways are not significantly colonized. Comparing these communities with those from the CF patients revealed significant differences in microbial ecology, including differential representation of uncultivated phylotypes. Despite patient-specific differences, our analysis revealed a focal microbial profile characteristic of CF. The profile differentiated case and control groups even when classically recognized CF pathogens were excluded. As a control, lung explant tissues were also processed from a group of patients with pulmonary disease. The findings in lung tissue corroborated the presence of taxa identified in the sputum samples. Comparing the sequencing results with clinical data indicated that diminished microbial diversity is associated with severity of pulmonary inflammation within our adult CF cohort.

Randomized, controlled trial of low-dose inhaled nitric oxide in the treatment of term and near-term infants with respiratory failure and pulmonary hypertension.

Recent reports indicate that inhaled nitric oxide (iNO) causes selective pulmonary vasodilation, increases arterial oxygen tension, and may decrease the use of extracorporeal membrane oxygenation (ECMO) in infants with persistent pulmonary hypertension of the newborn (PPHN). Despite these reports, the optimal dose and timing of iNO administration in PPHN remains unclear. Objectives. To test the hypotheses that in PPHN 1) iNO at 2 parts per million (ppm) is effective at acutely increasing oxygenation as measured by oxygenation index (OI); 2) early use of 2 ppm of iNO is more effective than control (0 ppm) in preventing clinical deterioration and need for iNO at 20 ppm; and 3) for those infants who fail the initial treatment protocol (0 or 2 ppm) iNO at 20 ppm is effective at acutely decreasing OI. Study Design. A randomized, controlled trial of iNO in 3 nurseries in a single metropolitan area. Thirty-eight children, average gestational age of 37.3 weeks and average age <1 day were enrolled. Thirty-five of 38 infants had echocardiographic evidence of pulmonary hypertension. On enrollment, median OI in the control group, iNO at 0 ppm, (n = 23) was 33.1, compared with 36.9 in the 2-ppm iNO group (n = 15). Results. Initial treatment with iNO at 2 ppm for an average of 1 hour was not associated with a significant decrease in OI. Twenty of 23 (87%) control patients and 14 of 15 (92%) of the low-dose iNO group demonstrated clinical deterioration and were treated with iNO at 20 ppm. In the control group, treatment with iNO at 20 ppm decreased the median OI from 42.6 to 23.8, whereas in the 2-ppm iNO group with a change in iNO from 2 to 20 ppm, the median OI did not change (42.6 to 42.0). Five of 15 patients in the low-dose nitric oxide group required ECMO and 2 died, compared with 7 of 23 requiring ECMO and 5 deaths in the control group. Conclusion. In infants with PPHN, iNO 1): at 2 ppm does not acutely improve oxygenation or prevent clinical deterioration, but does attenuate the rate of clinical deterioration; and 2) at 20 ppm acutely improves oxygenation in infants initially treated with 0 ppm, but not in infants previously treated with iNO at 2 ppm. Initial treatment with a subtherapeutic dose of iNO may diminish the clinical response to 20 ppm of iNO and have adverse clinical sequelae.

Extubation failure in pediatric intensive care incidence and outcomes

Objectives: To evaluate the hypotheses that children requiring reintubation are at an increased risk of prolonged hospitalizations, congenital heart disease, and death compared with age- and disease-severity-matched control patients. Design: Prospective decision to evaluate all children undergoing extubation over a 5-yr time interval (1997–2001) with retrospective analysis of all failed extubation patients. Setting: A large multidisciplinary, dual-site, single-system pediatric intensive care unit caring for critically ill and injured children. Patients: All children intubated and ventilated during the study period (1997–2001). Interventions: None. Measurements and Main Results: Failed extubation was defined as the unanticipated requirement to replace an endotracheal tube within 48 hrs of extubation. One hundred thirty children of 3,193 pediatric intensive care unit patients failed extubation (4.1%). The median age of children who failed extubation was 6.5 months, compared with a median age of 21.3 months in the control population. The median age of failed extubation in children with cardiac disease was 9.3 months. Failed extubation patients had lengthier hospital and pediatric intensive care unit stays, longer duration of mechanical ventilation, and a higher rate of tracheostomy placement than nonfailed extubation patients (p < .001). Children with congenital heart disease who failed extubation had the longest duration of hospitalization (40.0 ± 5.4 days). Conversely, cardiac patients who did not fail extubation had the shortest length of stay (11.2 ± 0.4 days). Conclusions: In the present trial, 4.1% of mechanically ventilated children failed extubation. Pediatric intensive care unit patients with failed extubation have longer hospital, pediatric intensive care unit, and ventilator courses but are not at increased risk of death relative to nonfailed extubation patients.

Noninvasive monitoring of infection and rejection after lung transplantation.

Significance Over 3,500 patients receive life-saving lung transplants every year. Nonetheless, complications due to infection and rejection occur frequently and undermine the long-term benefits of lung transplantation. Although clinicians strive to carefully monitor patients, diagnostic options are often limited. Rejection monitoring currently relies on invasive tissue biopsies, and tests of infection are predominately limited to testing one pathogen at a time. This manuscript describes a noninvasive assay based on sequencing of circulating cell-free DNA that simultaneously enables diagnosis of rejection and broad screening of infections. The survival rate following lung transplantation is among the lowest of all solid-organ transplants, and current diagnostic tests often fail to distinguish between infection and rejection, the two primary posttransplant clinical complications. We describe a diagnostic assay that simultaneously monitors for rejection and infection in lung transplant recipients by sequencing of cell-free DNA (cfDNA) in plasma. We determined that the levels of donor-derived cfDNA directly correlate with the results of invasive tests of rejection (area under the curve 0.9). We also analyzed the nonhuman cfDNA as a hypothesis-free approach to test for infections. Cytomegalovirus is most frequently assayed clinically, and the levels of CMV-derived sequences in cfDNA are consistent with clinical results. We furthermore show that hypothesis-free monitoring for pathogens using cfDNA reveals undiagnosed cases of infection, and that certain infectious pathogens such as human herpesvirus (HHV) 6, HHV-7, and adenovirus, which are not often tested clinically, occur with high frequency in this cohort.

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 in Pulmonary Artery Smooth Muscle Cells Lowers Vascular Tone by Decreasing Myosin Light Chain Phosphorylation

Rationale: Hypoxia-inducible factor-1&agr; (HIF-1&agr;), an oxygen (O2)-sensitive transcription factor, mediates transcriptional responses to low-O2 tension states. Although acute hypoxia causes pulmonary vasoconstriction and chronic hypoxia can cause vascular remodeling and pulmonary hypertension, conflicting data exist on the role of HIF-1&agr; in modulating pulmonary vascular tone. Objective: To investigate the role of smooth muscle cell (SMC)–specific HIF-1&agr; in regulating pulmonary vascular tone. Methods and Results: Mice with an SMC-specific deletion of HIF-1&agr; (SM22&agr;-HIF-1&agr;−/−) were created to test the hypothesis that pulmonary artery SMC (PASMC) HIF-1&agr; modulates pulmonary vascular tone and the response to hypoxia. SM22&agr;-HIF-1&agr;−/− mice exhibited significantly higher right ventricular systolic pressure compared with wild-type littermates under normoxia and with exposure to either acute or chronic hypoxia in the absence of histological evidence of accentuated vascular remodeling. Moreover, myosin light chain phosphorylation, a determinant of SMC tone, was higher in PASMCs isolated from SM22&agr;-HIF-1&agr;−/− mice compared with wild-type PASMCs, during both normoxia and after acute hypoxia. Further, overexpression of HIF-1&agr; decreased myosin light chain phosphorylation in HIF-1&agr;–null SMCs. Conclusions: In both normoxia and hypoxia, PASMC HIF-1&agr; maintains low pulmonary vascular tone by decreasing myosin light chain phosphorylation. Compromised PASMC HIF-1&agr; expression may contribute to the heightened vasoconstriction that characterizes pulmonary hypertension.

NO causes perinatal pulmonary vasodilation through K+-channel activation and intracellular Ca2+release

Evidence suggests that nitric oxide (NO) causes perinatal pulmonary vasodilation through K+-channel activation. We hypothesized that this effect worked through cGMP-dependent kinase-mediated activation of Ca2+-activated K+ channel that requires release of intracellular Ca2+ from a ryanodine-sensitive store. We studied the effects of 1) K+-channel blockade with tetraethylammonium, 4-aminopyridine, a voltage-dependent K+-channel blocker, or glibenclamide, an ATP-sensitive K+-channel blocker; 2) cyclic nucleotide-sensitive kinase blockade with either KT-5823, a guanylate-sensitive kinase blocker, or H-89, an adenylate-sensitive kinase blocker; and 3) blockade of intracellular Ca2+ release with ryanodine on NO-induced pulmonary vasodilation in acutely prepared late-gestation fetal lambs. N-nitro-L-arginine, a competitive inhibitor of endothelium-derived NO synthase, was infused into the left pulmonary artery, and tracheotomy was placed. The animals were ventilated with 100% oxygen for 20 min, followed by ventilation with 100% oxygen and inhaled NO at 20 parts/million (ppm) for 20 min. This represents the control period. In separate protocols, the animals received an intrapulmonary infusion of the different blockers and were ventilated as above. Tetraethylammonium (n = 6 animals) and KT-5823 (n = 4 animals) attenuated the response, whereas ryanodine (n = 5 animals) blocked NO-induced perinatal pulmonary vasodilation. 4-Aminopyridine (n = 5 animals), glibenclamide (n = 5 animals), and H-89 (n = 4 animals) did not affect NO-induced pulmonary vasodilation. We conclude that NO causes perinatal pulmonary vasodilation through cGMP-dependent kinase-mediated activation of Ca2+-activated K+ channels and release of Ca2+ from ryanodine-sensitive stores.Evidence suggests that nitric oxide (NO) causes perinatal pulmonary vasodilation through K+-channel activation. We hypothesized that this effect worked through cGMP-dependent kinase-mediated activation of Ca2+-activated K+ channel that requires release of intracellular Ca2+ from a ryanodine-sensitive store. We studied the effects of 1) K+-channel blockade with tetraethylammonium, 4-aminopyridine, a voltage-dependent K+-channel blocker, or glibenclamide, an ATP-sensitive K+-channel blocker; 2) cyclic nucleotide-sensitive kinase blockade with either KT-5823, a guanylate-sensitive kinase blocker, or H-89, an adenylate-sensitive kinase blocker; and 3) blockade of intracellular Ca2+ release with ryanodine on NO-induced pulmonary vasodilation in acutely prepared late-gestation fetal lambs. N-nitro-l-arginine, a competitive inhibitor of endothelium-derived NO synthase, was infused into the left pulmonary artery, and tracheotomy was placed. The animals were ventilated with 100% oxygen for 20 min, followed by ventilation with 100% oxygen and inhaled NO at 20 parts/million (ppm) for 20 min. This represents the control period. In separate protocols, the animals received an intrapulmonary infusion of the different blockers and were ventilated as above. Tetraethylammonium ( n = 6 animals) and KT-5823 ( n = 4 animals) attenuated the response, whereas ryanodine ( n = 5 animals) blocked NO-induced perinatal pulmonary vasodilation. 4-Aminopyridine ( n = 5 animals), glibenclamide ( n = 5 animals), and H-89 ( n = 4 animals) did not affect NO-induced pulmonary vasodilation. We conclude that NO causes perinatal pulmonary vasodilation through cGMP-dependent kinase-mediated activation of Ca2+-activated K+ channels and release of Ca2+ from ryanodine-sensitive stores.