COL4A1 Mutations Cause Neuromuscular Disease with Tissue-Specific Mechanistic Heterogeneity.

Collagen type IV alpha 1 and alpha 2 chains form heterotrimers ([α1(IV)]2α2(IV)) that represent a fundamental basement membrane constituent. Dominant COL4A1 and COL4A2 mutations cause a multisystem disorder that is marked by clinical heterogeneity and variable expressivity and that is generally characterized by the presence of cerebrovascular disease with ocular, renal, and muscular involvement. Despite the fact that muscle pathology is reported in up to one-third of individuals with COL4A1 and COL4A2 mutations and in animal models with mutations in COL4A1 and COL4A2 orthologs, the pathophysiological mechanisms underlying COL4A1-related myopathy are unknown. In general, mutations are thought to impair [α1(IV)]2α2(IV) secretion. Whether pathogenesis results from intracellular retention, extracellular deficiency, or the presence of mutant proteins in basement membranes represents an important gap in knowledge and a major obstacle for developing targeted interventions. We report that Col4a1 mutant mice develop progressive neuromuscular pathology that models human disease. We demonstrate that independent muscular, neural, and vascular insults contribute to neuromyopathy and that there is mechanistic heterogeneity among tissues. Importantly, we provide evidence of a COL4A1 functional subdomain with disproportionate significance for tissue-specific pathology and demonstrate that a potential therapeutic strategy aimed at promoting [α1(IV)]2α2(IV) secretion can ameliorate or exacerbate myopathy in a mutation-dependent manner. These data have important translational implications for prediction of clinical outcomes based on genotype, development of mechanism-based interventions, and genetic stratification for clinical trials. Collectively, our data underscore the importance of the [α1(IV)]2α2(IV) network as a multifunctional signaling platform and show that allelic and tissue-specific mechanistic heterogeneities contribute to the variable expressivity of COL4A1 and COL4A2 mutations.

Advances in extracorporeal membrane oxygenator design for artificial placenta technology.

Extreme prematurity, defined as a gestational age of fewer than 28 weeks, is a significant health problem worldwide. It carries a high burden of mortality and morbidity, in large part due to the immaturity of the lungs at this stage of development. The standard of care for these patients includes support with mechanical ventilation, which exacerbates lung pathology. Extracorporeal life support (ECLS), also called artificial placenta technology when applied to extremely preterm (EPT) infants, offers an intriguing solution. ECLS involves providing gas exchange via an extracorporeal device, thereby doing the work of the lungs and allowing them to develop without being subjected to injurious mechanical ventilation. While ECLS has been successfully used in respiratory failure in full-term neonates, children, and adults, it has not been applied effectively to the EPT patient population. In this review, we discuss the unique aspects of EPT infants and the challenges of applying ECLS to these patients. In addition, we review recent progress in artificial placenta technology development. We then offer analysis on design considerations for successful engineering of a membrane oxygenator for an artificial placenta circuit. Finally, we examine next-generation oxygenators that might advance the development of artificial placenta devices.

Extracorporeal Membrane Oxygenation in Pediatric Pulmonary Hypertension.

OBJECTIVE: To describe the epidemiology, critical care interventions, and mortality of children with pulmonary hypertension receiving extracorporeal membrane oxygenation. DESIGN: Retrospective analysis of prospectively collected multicenter data. SETTING: Data entered into the Extracorporeal Life Support Organization database between January 2007 and November 2018. PATIENTS: Pediatric patients between 28 days and 18 years old with a diagnosis of pulmonary hypertension. MEASUREMENTS AND MAIN RESULTS: Six hundred thirty-four extracorporeal membrane oxygenation runs were identified (605 patients). Extracorporeal membrane oxygenation support type was pulmonary (43.1%), cardiac (40.2%), and extracorporeal cardiopulmonary resuscitation (16.7%). The majority of cannulations were venoarterial (80.4%), and 30% had a pre-extracorporeal membrane oxygenation cardiac arrest. Mortality in patients with pulmonary hypertension was 51.3% compared with 44.8% (p = 0.001) in those without pulmonary hypertension. In univariate analyses, significant predictors of mortality included age less than 6 months and greater than 5 years; pre-extracorporeal membrane oxygenation cardiac arrest; pre-extracorporeal membrane oxygenation blood gas with pH less than 7.12, PaCO2 greater than 75, PaO2 less than 35, and arterial oxygen saturation less than 60%; extracorporeal membrane oxygenation duration greater than 280 hours; extracorporeal cardiopulmonary resuscitation; and extracorporeal membrane oxygenation complications including cardiopulmonary resuscitation, inotropic support, myocardial stun, tamponade, pulmonary hemorrhage, intracranial hemorrhage, seizures, other hemorrhage, disseminated intravascular coagulation, renal replacement therapy, mechanical/circuit problem, and metabolic acidosis. A co-diagnosis of pneumonia was associated with significantly lower odds of mortality (odds ratio, 0.5; 95% CI, 0.3-0.8). Prediction models were developed using three sets of variables: 1) pre-extracorporeal membrane oxygenation (age, absence of pneumonia, and pH < 7.12; area under the curve, 0.62); 2) extracorporeal membrane oxygenation related (extracorporeal cardiopulmonary resuscitation, any neurologic complication, pulmonary hemorrhage, renal replacement therapy, and metabolic acidosis; area under the curve, 0.72); and 3) all variables combined (area under the curve, 0.75) (p < 0.001). CONCLUSIONS: Children with pulmonary hypertension who require extracorporeal membrane oxygenation support have a significantly greater odds of mortality compared with those without pulmonary hypertension. Risk factors for mortality include age, absence of pneumonia, pre-extracorporeal membrane oxygenation acidosis, extracorporeal cardiopulmonary resuscitation, pulmonary hemorrhage, neurologic complications, renal replacement therapy, and acidosis while on extracorporeal membrane oxygenation. Identification of those pulmonary hypertension patients requiring extracorporeal membrane oxygenation who are at even higher risk for mortality may inform clinical decision-making and improve prognostic awareness.

Disrupted NOS signaling in lymphatic endothelial cells exposed to chronically increased pulmonary lymph flow.

Associated abnormalities of the lymphatic circulation are well described in congenital heart disease. However, their mechanisms remain poorly elucidated. Using a clinically relevant ovine model of a congenital cardiac defect with chronically increased pulmonary blood flow (shunt), we previously demonstrated that exposure to chronically elevated pulmonary lymph flow is associated with: 1) decreased bioavailable nitric oxide (NO) in pulmonary lymph; and 2) attenuated endothelium-dependent relaxation of thoracic duct rings, suggesting disrupted lymphatic endothelial NO signaling in shunt lambs. To further elucidate the mechanisms responsible for this altered NO signaling, primary lymphatic endothelial cells (LECs) were isolated from the efferent lymphatic of the caudal mediastinal node in 4-wk-old control and shunt lambs. We found that shunt LECs (n = 3) had decreased bioavailable NO and decreased endothelial nitric oxide synthase (eNOS) mRNA and protein expression compared with control LECs (n = 3). eNOS activity was also low in shunt LECs, but, interestingly, inducible nitric oxide synthase (iNOS) expression and activity were increased in shunt LECs, as were total cellular nitration, including eNOS-specific nitration, and accumulation of reactive oxygen species (ROS). Pharmacological inhibition of iNOS reduced ROS in shunt LECs to levels measured in control LECs. These data support the conclusion that NOS signaling is disrupted in the lymphatic endothelium of lambs exposed to chronically increased pulmonary blood and lymph flow and may contribute to decreased pulmonary lymphatic bioavailable NO.

Pulmonary Hypertension.

OBJECTIVES: To review the clinical classification, diagnosis, and pathophysiology of pulmonary hypertension in children, emphasizing the role of right ventricular function, ventricular interaction, and congenital heart disease in the evolution and progression of disease, as well as management strategies and therapeutic options. DATA SOURCE: MEDLINE, PubMed. CONCLUSIONS: Critically ill children with pulmonary hypertension associated with congenital heart disease are a high-risk population. Congenital cardiac defects resulting in either increased pulmonary blood flow or impaired pulmonary venous drainage predispose patients to developing structural and functional aberrations of the pulmonary vasculature. Mortality from pulmonary hypertension is most directly related to right ventricular failure.

Right ventricular nitric oxide signaling in an ovine model of congenital heart disease: a preserved fetal phenotype.

We recently reported superior right ventricle (RV) performance in response to acute afterload challenge in lambs with a model of congenital heart disease with chronic left-to-right cardiac shunts. Compared with control animals, shunt lambs demonstrated increased contractility because of an enhanced Anrep effect (the slow increase in contractility following myocyte stretch). This advantageous physiological response may reflect preservation of a fetal phenotype, since the RV of shunt lambs remains exposed to increased pressure postnatally. Nitric oxide (NO) production by NO synthase (NOS) is activated by myocyte stretch and is a necessary intermediary of the Anrep response. The purpose of this study was to test the hypothesis that NO signaling is increased in the RV of fetal lambs compared with controls and shunt lambs have persistence of this fetal pattern. An 8-mm graft was placed between the pulmonary artery and aorta in fetal lambs (shunt). NOS isoform expression, activity, and association with activating cofactors were determined in fetal tissue obtained during late-gestation and in 4-wk-old juvenile shunt and control lambs. We demonstrated increased RNA and protein expression of NOS isoforms and increased total NOS activity in the RV of both shunt and fetal lambs compared with control. We also found increased NOS activation and association with cofactors in shunt and fetal RV compared with control. These data demonstrate preserved fetal NOS phenotype and NO signaling in shunt RV, which may partially explain the mechanism underlying the adaptive response to increased afterload seen in the RV of shunt lambs.

Altered reactivity and nitric oxide signaling in the isolated thoracic duct from an ovine model of congenital heart disease with increased pulmonary blood flow.

We have previously shown decreased pulmonary lymph flow in our lamb model of chronically increased pulmonary blood flow, created by the in utero placement of an 8-mm aortopulmonary shunt. The purpose of this study was to test the hypothesis that abnormal lymphatic function in shunt lambs is due to impaired lymphatic endothelial nitric oxide (NO)-cGMP signaling resulting in increased lymphatic vascular constriction and/or impaired relaxation. Thoracic duct rings were isolated from 4-wk-old shunt (n = 7) and normal (n = 7) lambs to determine length-tension properties, vascular reactivity, and endothelial NO synthase protein. At baseline, shunt thoracic duct rings had 2.6-fold higher peak to peak tension and a 2-fold increase in the strength of contractions compared with normal rings (P < 0.05). In response to norepinephrine, shunt thoracic duct rings had a 2.4-fold increase in vascular tone compared with normal rings (P < 0.05) and impaired relaxation in response to the endothelium-dependent dilator acetylcholine (63% vs. 13%, P < 0.05). In vivo, inhaled NO (40 ppm) increased pulmonary lymph flow (normalized for resistance) ∼1.5-fold in both normal and shunt lambs (P < 0.05). Inhaled NO exposure increased bioavailable NO [nitrite/nitrate (NOx); ∼2.5-fold in normal lambs and ∼3.4-fold in shunt lambs] and cGMP (∼2.5-fold in both) in the pulmonary lymph effluent (P < 0.05). Chronic exposure to increased pulmonary blood flow is associated with pulmonary lymphatic endothelial injury that disrupts NO-cGMP signaling, leading to increased resting vasoconstriction, increased maximal strength of contraction, and impaired endothelium-dependent relaxation. Inhaled NO increases pulmonary lymph NOx and cGMP levels and pulmonary lymph flow in normal and shunt lambs. Therapies that augment NO-cGMP signaling within the lymphatic system may provide benefits, warranting further study.

Adaptive right ventricular performance in response to acutely increased afterload in a lamb model of congenital heart disease: evidence for enhanced Anrep effect.

Patients with pulmonary hypertension associated with congenital heart disease survive longer with preserved right ventricular (RV) function compared with those with primary pulmonary hypertension. The purpose of this study was to test the hypothesis that superior RV performance can be demonstrated, at baseline and when challenged with increased RV afterload, in lambs with chronic left-to-right cardiac shunts compared with control lambs. A shunt was placed between the pulmonary artery and the aorta in fetal lambs (shunt). RV pressure-volume loops were obtained 4 wk after delivery in shunt and control lambs, before and after increased afterload was applied using pulmonary artery banding (PAB). Baseline stroke volume (8.7 ± 1.8 vs. 15.8 ± 2.7 ml, P = 0.04) and cardiac index (73.0 ± 4.0 vs. 159.2 ± 25.1 ml·min(-1)·kg(-1), P = 0.02) were greater in shunts. After PAB, there was no difference in the change in cardiac index (relative to baseline) between groups; however, heart rate (HR) was greater in controls (168 ± 7.3 vs. 138 ± 6.6 beats/min, P = 0.01), and end-systolic elastance (Ees) was greater in shunts (2.63 vs. 1.31 × baseline, P = 0.02). Control lambs showed decreased mechanical efficiency (71% baseline) compared with shunts. With acute afterload challenge, both controls and shunts maintained cardiac output; however, this was via maladaptive responses in controls, while shunts maintained mechanical efficiency and increased contractility via a proposed enhanced Anrep effect-the second, slow inotropic response in the biphasic ventricular response to increased afterload, a novel finding in the RV. The mechanisms related to these physiological differences may have important therapeutic implications.

Tezosentan increases nitric oxide signaling via enhanced hydrogen peroxide generation in lambs with surgically induced acute increases in pulmonary blood flow.

We have previously shown that acute increases in pulmonary blood flow (PBF) are limited by a compensatory increase in pulmonary vascular resistance (PVR) via an endothelin-1 (ET-1) dependent decrease in nitric oxide synthase (NOS) activity. The mechanisms underlying the reduction in NO signaling are unresolved. Thus, the purpose of this study was to elucidate mechanisms of this ET-1-NO interaction. Pulmonary arterial endothelial cells were acutely exposed to shear stress in the presence or absence of tezosentan, a combined ET(A) /ET(B) receptor antagonist. Shear increased NO(x) , eNOS phospho-Ser1177, and H(2) O(2) and decreased catalase activity; tezosentan enhanced, while ET-1 attenuated all of these changes. In addition, ET-1 increased eNOS phospho-Thr495 levels. In lambs, 4 h of increased PBF decreased H(2) O(2) , eNOS phospho-Ser1177, and NO(X) levels, and increased eNOS phospho-Thr495, phospho-catalase, and catalase activity. These changes were reversed by tezosentan. PEG-catalase reversed the positive effects of tezosentan on NO signaling. In all groups, opening the shunt resulted in a rapid increase in PBF by 30 min. In vehicle- and tezosentan/PEG-catalase lambs, PBF did not change further over the 4 h study period. PVR fell by 30 min in vehicle- and tezosentan-treated lambs, and by 60 min in tezosentan/PEG-catalase-treated lambs. In vehicle- and tezosentan/PEG-catalase lambs, PVR did not change further over the 4 h study period. In tezosentan-treated lambs, PBF continued to increase and LPVR to decrease over the 4 h study period. We conclude that acute increases in PBF are limited by an ET-1 dependent decrease in NO production via alterations in catalase activity, H(2) O(2) levels, and eNOS phosphorylation.

Rosiglitazone preserves pulmonary vascular function in lambs with increased pulmonary blood flow.

BACKGROUND: Pulmonary vascular function is impaired with increased pulmonary blood flow (PBF). We hypothesized that a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist would mitigate this effect. METHODS: An aorta-to-pulmonary-artery shunt was placed in 11 fetal lambs. Lambs received the PPAR-γ agonist rosiglitazone (RG, 3 mg/kg/d, n = 6) or vehicle (n = 5) for 4 wk. Lung tissue from five normal 4-wk-old lambs was used for comparisons. RESULTS: At 4 wk, pulmonary artery pressure (PAP) and vascular resistance (PVR) decreased with inhaled nitric oxide (NO) in RG- and vehicle-treated shunt lambs. PAP and PVR decreased with acetylcholine (Ach) in RG-treated, but not vehicle-treated, shunt lambs. In vehicle-treated shunt lambs, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, rac1, superoxide, and 3-nitrotyrosine (3-NT) levels were increased, and Ser1177 endothelial NO synthase (eNOS) protein was decreased as compared with normal lambs. In RG-treated shunt lambs, NOx, Ser1177 eNOS protein, and eNOS activity were increased, and NADPH activity, rac1, superoxide levels, and 3-NT levels were decreased, as compared with vehicle-treated shunt lambs. PPAR-γ protein expression was lower in vehicle-treated shunt lambs than in normal and RG-treated shunt lambs. CONCLUSION: The PPAR-γ agonist RG prevents the loss of agonist-induced endothelium-dependent pulmonary vascular relaxation in lambs with increased PBF, in part, due to decreased oxidative stress and/or increased NO production.

L-carnitine preserves endothelial function in a lamb model of increased pulmonary blood flow.

BACKGROUND: In our model of a congenital heart defect (CHD) with increased pulmonary blood flow (PBF; shunt), we have recently shown a disruption in carnitine homeostasis, associated with mitochondrial dysfunction and decreased endothelial nitric oxide synthase (eNOS)/heat shock protein (Hsp)90 interactions that contribute to eNOS uncoupling, increased superoxide levels, and decreased bioavailable nitric oxide (NO). Therefore, we undertook this study to test the hypothesis that L-carnitine therapy would maintain mitochondrial function and NO signaling. METHODS: Thirteen fetal lambs underwent in utero placement of an aortopulmonary graft. Immediately after delivery, lambs received daily treatment with oral L-carnitine or its vehicle. RESULTS: L-Carnitine-treated lambs had decreased levels of acylcarnitine and a reduced acylcarnitine:free carnitine ratio as compared with vehicle-treated shunt lambs. These changes correlated with increased carnitine acetyl transferase (CrAT) protein and enzyme activity and decreased levels of nitrated CrAT. The lactate:pyruvate ratio was also decreased in L-carnitine-treated lambs. Hsp70 protein levels were significantly decreased, and this correlated with increases in eNOS/Hsp90 interactions, NOS activity, and NOx levels, and a significant decrease in eNOS-derived superoxide. Furthermore, acetylcholine significantly decreased left pulmonary vascular resistance only in L-carnitine-treated lambs. CONCLUSION: L-Carnitine therapy may improve the endothelial dysfunction noted in children with CHDs and has important clinical implications that warrant further investigation.

Chronic inhibition of PPAR-γ signaling induces endothelial dysfunction in the juvenile lamb.

We have recently shown that the development of endothelial dysfunction in lambs with increased pulmonary blood flow (PBF) correlates with a decrease in peroxisome proliferator activated receptor-γ (PPAR-γ) signaling. Thus, in this study we determined if the loss of PPAR-γ signaling is necessary and sufficient to induce endothelial dysfunction by exposing lambs with normal PBF to the PPAR-γ antagonist, GW9662. Two-weeks of exposure to GW9662 significantly decreased both PPAR-γ protein and activity. In addition, although eNOS protein and nitric oxide metabolites (NO(x)) were significantly increased, endothelial dependent pulmonary vasodilation in response to acetylcholine was attenuated, indicative of endothelial dysfunction. To elucidate whether downstream mediators of vasodilation were impaired we examined soluble guanylate cyclase (sGC)-α and ß subunit protein, cGMP levels, and phosphodiesterase 5 (PDE5) protein and activity, but we found no significant changes. However, we found that peroxynitrite levels were significantly increased in GW9662-treated lambs and this correlated with a significant increase in protein kinase G-1α (PKG-1α) nitration and a reduction in PKG activity. Peroxynitrite is formed by the interaction of NO with superoxide and we found that there was a significant increase in superoxide generation in GW9662-treated lambs. Further, we identified dysfunctional mitochondria as the primary source of the increased superoxide. Finally, we found that the mitochondrial dysfunction was due to a disruption in carnitine metabolism. We conclude that loss of PPAR-γ signaling is sufficient to induce endothelial dysfunction confirming its important role in maintaining a healthy vasculature.

Pulmonary interstitial glycogenosis: an unrecognized etiology of persistent pulmonary hypertension of the newborn in congenital heart disease?

BACKGROUND: Pulmonary interstitial glycogenosis (PIG) arises from a developmental disorder of the pulmonary mesenchyme and presents clinically with reversible neonatal respiratory distress and/or persistent pulmonary hypertension of the newborn (PPHN). OBJECTIVE: We report two cases of PIG in patients with congenital heart disease (CHD) and evidence of PPHN. RESULTS: Both cases demonstrated the hallmark PIG histologic finding of diffuse, uniform interstitial thickening due to the presence of immature interstitial cells containing abundant cytoplasmic glycogen. CONCLUSIONS: We report the second and third patients with PIG associated with CHD. Because histologic examination is required to establish the diagnosis, we speculate that PIG, although rare, may be underrecognized in neonates presenting with PPHN in the setting of CHD.

Endostatin, an inhibitor of angiogenesis, decreases after bidirectional superior cavopulmonary anastamosis.

Pulmonary arteriovenous malformations (PAVMs) are a common source of morbidity after bidirectional superior cavopulmonary anastomosis (Glenn). The diversion of hepatic venous effluent away from the pulmonary circulation after Glenn appears to play a significant role in the pathogenesis of PAVMs. Although the liver is known to produce factors that regulate vascular development, specific hepatic inhibitors of angiogenesis have not been described in the post-Glenn population. Endostatin, produced from its precursor collagen XVIII, is a potent inhibitor of angiogenesis produced by the liver. This study aimed to investigate the hypothesis that endostatin levels decrease in patients after Glenn. Levels of endostatin and its precursor, long-type collagen XVIII, were determined by enzyme-linked immunoassay and immunoprecipitation, respectively, for serum samples from 38 patients undergoing Glenn, total cavopulmonary anastomosis (Fontan), or biventricular repair of cardiac defects. Samples were obtained before surgery and 24 h afterward. In patients undergoing a bidirectional Glenn procedure, endostatin levels decreased after surgery (n = 17; 4.42 vs 3.34 ng/ml; p < 0.001), and long type-collagen XVIII levels increased by 200 % (n = 10; p = 0.0001). However, endostatin levels did not change after surgery in patients undergoing Fontan (n = 13) or biventricular repair (n = 8). In patients undergoing Fontan, long-type collagen XVIII increased by 18 % (p < 0.01), whereas in control subjects, the levels were unchanged. These data suggest that the diversion of hepatic blood flow away from the pulmonary circulation in patients after the Glenn procedure inhibits endostatin production from collagen XVIII, resulting in decreased circulating serum endostatin levels. A decrease in endostatin may promote angiogenesis. The mechanism whereby the pulmonary circulation processes endostatin and its potential role in the pathogenesis of PAVMs warrant further study.

Altered lymphatics in an ovine model of congenital heart disease with increased pulmonary blood flow.

Abnormalities of the lymphatic circulation are well recognized in patients with congenital heart defects. However, it is not known how the associated abnormal blood flow patterns, such as increased pulmonary blood flow (PBF), might affect pulmonary lymphatic function and structure. Using well-established ovine models of acute and chronic increases in PBF, we cannulated the efferent lymphatic duct of the caudal mediastinal node and collected and analyzed lymph effluent from the lungs of lambs with acutely increased PBF (n = 6), chronically increased PBF (n = 6), and age-matched normal lambs (n = 8). When normalized to PBF, we found that lymph flow was unchanged following acute increases in PBF but decreased following chronic increases in PBF. The lymph:plasma protein ratio decreased with both acute and chronic increases in PBF. Lymph bioavailable nitric oxide increased following acute increases in PBF but decreased following chronic increases in PBF. In addition, we found perturbations in the transit kinetics of contrast material through the pleural lymphatics of lambs with chronic increases in PBF. Finally, there were structural changes in the pulmonary lymphatic system in lambs with chronic increases in PBF: lymphatics from these lambs were larger and more dilated, and there were alterations in the expression of vascular endothelial growth factor-C, lymphatic vessel endothelial hyaluronan receptor-1, and Angiopoietin-2, proteins known to be important for lymphatic growth, development, and remodeling. Taken together these data suggest that chronic increases in PBF lead to both functional and structural aberrations of lung lymphatics. These findings have important therapeutic implications that warrant further study.

Association of Ventilator Settings With Mortality in Pediatric Patients Treated With Extracorporeal Life Support for Respiratory Failure.

Extracorporeal life support (ECLS) is a treatment for acute respiratory failure that can provide extracorporeal gas exchange, allowing lung rest. However, while most patients remain mechanically ventilated during ECLS, there is a paucity of evidence to guide the choice of ventilator settings. We studied the associations between ventilator settings 24 hours after ECLS initiation and mortality in pediatric patients using a retrospective analysis of data from the Extracorporeal Life Support Organization Registry. 3497 patients, 29 days to 18 years of age, treated with ECLS for respiratory failure between 2015 and 2021, were included for analysis. 93.3% of patients on ECLS were ventilated with conventional mechanical ventilation. Common settings included positive end-expiratory pressure (PEEP) of 10 cm H 2 O (45.7%), delta pressure (ΔP) of 10 cm H 2 O (28.3%), rate of 10-14 breaths per minute (55.9%), and fraction of inspired oxygen (FiO 2 ) of 0.31-0.4 (30.3%). In a multivariate model, PEEP >10 cm H 2 O ( versus PEEP < 8 cm H 2 O, odds ratio [OR]: 1.53, 95% CI: 1.20-1.96) and FiO 2 ≥0.45 ( versus FiO 2 < 0.4; 0.45 ≤ FiO 2 < 0.6, OR: 1.31, 95% CI: 1.03-1.67 and FiO 2 ≥ 0.6, OR: 2.30; 95% CI: 1.81-2.93) were associated with higher odds of mortality. In a secondary analysis of survivors, PEEP 8-10 cm H 2 O was associated with shorter ECLS run times ( versus PEEP < 8 cm H 2 O, coefficient: -1.64, 95% CI: -3.17 to -0.11), as was ΔP >16 cm H 2 O ( versus ΔP < 10 cm H 2 O, coefficient: -2.72, 95% CI: -4.30 to -1.15). Our results identified several categories of ventilator settings as associated with mortality or ECLS run-time. Further studies are necessary to understand whether these results represent a causal relationship.

On-Hours Compared to Off-Hours Pediatric Extracorporeal Life Support Initiation in the United States Between 2009 and 2018-An Analysis of the Extracorporeal Life Support Organization Registry.

We aimed to investigate whether there are differences in outcome for pediatric patients when extracorporeal life support (ECLS) is initiated on-hours compared with off-hours. DESIGN: Retrospective cohort study. SETTING: Ten-year period (2009-2018) in United States centers, from the Extracorporeal Life Support Organization registry. PATIENTS: Pediatric (>30 d and <18 yr old) patients undergoing venovenous and venoarterial ECLS. INTERVENTIONS: The primary predictor was on versus off-hours cannulation. On-hours were defined as 0700-1859 from Monday to Friday. Off-hours were defined as 1900-0659 from Monday to Thursday or 1900 Friday to 0659 Monday or any time during a United States national holiday. The primary outcome was inhospital mortality. The secondary outcomes were complications related to ECLS and length of hospital stay. MEASUREMENTS AND MAIN RESULTS: In a cohort of 9,400 patients, 4,331 (46.1%) were cannulated on-hours and 5,069 (53.9%) off-hours. In the off-hours group, 2,220/5,069 patients died (44.0%) versus 1,894/4,331 (44.1%) in the on-hours group (p = 0.93). Hemorrhagic complications were lower in the off-hours group versus the on-hours group (hemorrhagic 18.4% vs 21.0%; p = 0.002). After adjusting for patient complexity and other confounders, there were no differences between the groups in mortality (odds ratio [OR], 0.95; 95% CI, 0.85-1.07; p = 0.41) or any complications (OR, 1.02; 95% CI, 0.89-1.17; p = 0.75). CONCLUSIONS: Survival and complication rates are similar for pediatric patients when ECLS is initiated on-hours compared with off-hours. This finding suggests that, in aggregate, the current pediatric ECLS infrastructure in the United States provides adequate capabilities for the initiation of ECLS across all hours of the day.

Delineating the angiogenic gene expression profile before pulmonary vascular remodeling in a lamb model of congenital heart disease.

Disordered angiogenesis is implicated in pulmonary vascular remodeling secondary to congenital heart diseases (CHD). However, the underlying genes are not well delineated. We showed previously that an ovine model of CHD with increased pulmonary blood flow (PBF, Shunt) has an "angiogenesis burst" between 1 and 4 wk of age. Thus we hypothesized that the increased PBF elicited a proangiogenic gene expression profile before onset of vessel growth. To test this we utilized microarray analysis to identify genes that could be responsible for the angiogenic response. Total RNA was isolated from lungs of Shunt and control lambs at 3 days of age and hybridized to Affymetrix gene chips for microarray analyses (n = 8/group). Eighty-nine angiogenesis-related genes were found to be upregulated and 26 angiogenesis-related genes downregulated in Shunt compared with control lungs (cutting at 1.2-fold difference, P < 0.05). We then confirmed upregulation of proangiogenic genes FGF2, Angiopoietin2 (Angpt2), and Birc5 at mRNA and protein levels and upregulation of ccl2 at mRNA level in 3-day Shunt lungs. Furthermore, we found that pulmonary arterial endothelial cells (PAEC) isolated from fetal lambs exhibited increased expression of FGF2, Angpt2, Birc5, and ccl2 and enhanced angiogenesis when exposed to elevated shear stress (35 dyn/cm²) compared with cells exposed to more physiological shear stress (20 dyn/cm²). Finally, we demonstrated that blocking FGF2, Angpt2, Birc5, or ccl2 signaling with neutralizing antibodies or small interfering RNA (siRNA) significantly decreased the angiogenic response induced by shear stress. In conclusion, we have identified a "proangiogenic" gene expression profile in a lamb model of CHD with increased PBF that precedes onset of pulmonary vascular remodeling. Our data indicate that FGF2, Angpt2, Birc5, and ccl2 may play important roles in the angiogenic response.