G6pdN126D Variant Increases the Risk of Developing VEGFR (Vascular Endothelial Growth Factor Receptor) Blocker-Induced Pulmonary Vascular Disease.

BACKGROUND: G6PD (glucose-6-phosphate-dehydrogenase) is a key enzyme in the glycolytic pathway and has been implicated in the pathogenesis of cancer and pulmonary hypertension-associated vascular remodeling. Here, we investigated the role of an X-linked G6pd mutation (N126D polymorphism), which is known to increase the risk of cardiovascular disease in individuals from sub-Saharan Africa and many others with African ancestry, in the pathogenesis of pulmonary hypertension induced by a vascular endothelial cell growth factor receptor blocker used for treating cancer. METHODS AND RESULTS: CRISPR-Cas9 genome editing was used to generate the G6pd variant (N126D; G6pdN126D) in rats. A single dose of the vascular endothelial cell growth factor receptor blocker sugen-5416 (SU; 20 mg/kg in DMSO), which is currently in a Phase 2/3 clinical trial for cancer treatment, was subcutaneously injected into G6pdN126D rats and their wild-type littermates. After 8 weeks of normoxic conditions, right ventricular pressure and hypertrophy, pulmonary artery remodeling, the metabolic profile, and cytokine expression were assessed. Right ventricular pressure and pulmonary arterial wall thickness were increased in G6PDN126D+SU/normoxic rats. Simultaneously, levels of oxidized glutathione, inositol triphosphate, and intracellular Ca2+ were increased in the lungs of G6PDN126D+SU/normoxic rats, whereas nitric oxide was decreased. Also increased in G6PDN126D+SU/normoxic rats were pulmonary levels of plasminogen activator inhibitor-1, thrombin-antithrombin complex, and expression of proinflammatory cytokines CCL3 (chemokine [C-C motif] ligand), CCL5, and CCL7. CONCLUSIONS: Our results suggest G6PDN126D increases inositol triphosphate-Ca2+ signaling, inflammation, thrombosis, and hypertrophic pulmonary artery remodeling in SU-treated rats. This suggests an increased risk of vascular endothelial cell growth factor receptor blocker-induced pulmonary hypertension in those carrying this G6PD variant.

Loss-of-function G6PD variant moderated high-fat diet-induced obesity, adipocyte hypertrophy, and fatty liver in male rats.

Obesity is a major risk factor for liver and cardiovascular diseases. However, obesity-driven mechanisms that contribute to the pathogenesis of multiple organ diseases are still obscure and treatment is inadequate. We hypothesized that increased , glucose-6-phosphate dehydrogenase (G6PD), the key rate-limiting enzyme in the pentose shunt, is critical in evoking metabolic reprogramming in multiple organs and is a significant contributor to the pathogenesis of liver and cardiovascular diseases. G6PD is induced by a carbohydrate-rich diet and insulin. Long-term (8 months) high-fat diet (HFD) feeding increased body weight and elicited metabolic reprogramming in visceral fat, liver, and aorta, of the wild-type rats. In addition, HFD increased inflammatory chemokines in visceral fat. Interestingly, CRISPR-edited loss-of-function Mediterranean G6PD variant (G6PDS188F) rats, which mimic human polymorphism, moderated HFD-induced weight gain and metabolic reprogramming in visceral fat, liver, and aorta. The G6PDS188F variant prevented HFD-induced CCL7 and adipocyte hypertrophy. Furthermore, the G6PDS188F variant increased Magel2 - a gene encoding circadian clock-related protein that suppresses obesity associated with Prader-Willi syndrome - and reduced HFD-induced non-alcoholic fatty liver. Additionally, the G6PDS188F variant reduced aging-induced aortic stiffening. Our findings suggest G6PD is a regulator of HFD-induced obesity, adipocyte hypertrophy, and fatty liver.

Global and endothelial G-protein coupled receptor 75 (GPR75) knockout relaxes pulmonary artery and mitigates hypoxia-induced pulmonary hypertension.

RATIONALE: Pulmonary hypertension (PH) is a multifactorial disease with a poor prognosis and inadequate treatment options. We found two-fold higher expression of the orphan G-Protein Coupled Receptor 75 (GPR75) in leukocytes and pulmonary arterial smooth muscle cells from idiopathic PH patients and from lungs of C57BL/6 mice exposed to hypoxia. We therefore postulated that GPR75 signaling is critical to the pathogenesis of PH. METHODS: To test this hypothesis, we exposed global (Gpr75-/-) and endothelial cell (EC) GPR75 knockout (EC-Gpr75-/-) mice and wild-type (control) mice to hypoxia (10% oxygen) or normal atmospheric oxygen for 5 weeks. We then recorded echocardiograms and performed right heart catheterizations. RESULTS: Chronic hypoxia increased right ventricular systolic and diastolic pressures in wild-type mice but not Gpr75-/- or EC-Gpr75-/- mice. In situ hybridization and qPCR results revealed that Gpr75 expression was increased in the alveoli, airways and pulmonary arteries of mice exposed to hypoxia. In addition, levels of chemokine (CC motif) ligand 5 (CCL5), a low affinity ligand of GPR75, were increased in the lungs of wild-type, but not Gpr75-/-, mice exposed to hypoxia, and CCL5 enhanced hypoxia-induced contraction of intra-lobar pulmonary arteries in a GPR75-dependent manner. Gpr75 knockout also increased pulmonary cAMP levels and decreased contraction of intra-lobar pulmonary arteries evoked by endothelin-1 or U46619 in cAMP-protein kinase A-dependent manner. CONCLUSION: These results suggest GPR75 has a significant role in the development of hypoxia-induced PH.

Induction of aggressive arterial and venous dilation therapy in addition to pulmonary dilation therapy (super-Fontan strategy) improves Fontan circulation both at rest and during treadmill exercise.

Objective: Fontan circulation maintains preload and cardiac output by reducing venous capacitance and increasing central venous pressure (CVP). The resultant congestive end-organ damage affects patient prognosis. Therefore, a better circulatory management strategy to ameliorate organ congestion is required in patients with Fontan circulation. We sought to verify whether aggressive arterial and venous dilation therapy in addition to pulmonary dilation (super-Fontan strategy) can improve Fontan circulation and reduce congestion. Methods: Patients after Fontan surgery who received the super-Fontan strategy in a single center were recruited. Participants were examined using medical records between 2010 and 2018. We retrospectively analyzed the changes in hemodynamics at rest and during treadmill exercise before and after the introduction of this therapy. Results: The therapy significantly increased venous capacitance (3.21 ± 1.27 mL/kg/mm Hg to 3.79 ± 1.30 mL/kg/mm Hg, P = .017) and decreased total pulmonary resistance, leading to significantly reduced CVP (11.7 ± 2.4 mm Hg to 9.7 ± 2.2 mm Hg, P < .001) and increased cardiac index (CI) (3.09 ± 1.01 L/min/m2 to 3.54 ± 1.19 L/min/m2, P = .047). Furthermore, this strategy significantly reduced the elevations in CVP (19.6 ± 5.3 mm Hg to 15.4 ± 2.7 mm Hg, P = .002) with preserved CI in response to exercise. CVP at rest and during exercise was significantly positively correlated with serum markers of hepatic congestion and fibrosis, respectively. Conclusions: The super-Fontan strategy is a therapy that turns the heart failure condition of Fontan circulation into a more physiological condition. However, whether the strategy improves long-term prognosis warrants further studies.

Mediterranean G6PD variant rats are protected from Angiotensin II-induced hypertension and kidney damage, but not from inflammation and arterial stiffness.

RATIONALE: Epidemiological studies suggest that individuals in the Mediterranean region with deficiency of glucose-6-phosphate dehydrogenase (G6PD) are less susceptible to cardiovascular diseases. However, our knowledge regarding the effects of G6PD deficiency on pathogenesis of vascular diseases caused by factors, like angiotensin II (Ang-II), which stimulate synthesis of inflammatory cytokines and vascular inflammation, is lacking. Furthermore, to-date the effect of G6PD deficiency on vascular health has been controversial and difficult to experimentally prove due to a lack of good animal model. OBJECTIVE: To determine the effect of Ang-II-induced hypertension (HTN) and stiffness in a rat model of the Mediterranean G6PD (G6PDS188F) variant and in wild-type (WT) rats. METHODS AND RESULTS: Our findings revealed that infusion of Ang-II (490 ng/kg/min) elicited less HTN and medial hypertrophy of carotid artery in G6PDS188F than in WT rats. Additionally, Ang-II induced less glomerular and tubular damage in the kidneys - a consequence of elevated pressure - in G6PDS188F than WT rats. However, Ang-II-induced arterial stiffness increased in G6PDS188F and WT rats, and there were no differences between the groups. Mechanistically, we found aorta of G6PDS188F as compared to WT rats produced less sustained contraction and less inositol-1,2,3-phosphate (IP3) and superoxide in response to Ang-II. Furthermore, aorta of G6PDS188F as compared to WT rats expressed lower levels of phosphorylated extracellular-signal regulated kinase (ERK). Interestingly, the aorta of G6PDS188F as compared to WT rats infused with Ang-II transcribed more (50-fold) myosin heavy chain-11 (MYH11) gene, which encodes contractile protein of smooth muscle cell (SMC), and less (2.3-fold) actin-binding Rho-activating gene, which encodes a protein that enhances SMC proliferation. A corresponding increase in MYH11 and Leiomodin-1 (LMOD1) staining was observed in arteries of Ang-II treated G6PDS188F rats. However, G6PD deficiency did not affect the accumulation of CD45+ cells and transcription of genes encoding interleukin-6 and collagen-1a1 by Ang-II. CONCLUSIONS: The G6PDS188F loss-of-function variant found in humans protected rats from Ang-II-induced HTN and kidney damage, but not from vascular inflammation and arterial stiffness.

Perinatal factors associated with bubbly/cystic appearance in bronchopulmonary dysplasia: a nationwide, population-based cohort study in Japan.

BACKGROUND: Bronchopulmonary dysplasia (BPD) remains one of the most serious morbidities associated with preterm birth. Previous study reported that bubbly/cystic appearance on chest X-rays in the neonatal period is the strongest determinant of impaired lung function at school age in BPD patients. AIMS: To determine perinatal risk factors for bubbly/cystic appearance on chest X-rays in extremely premature infants with BPD exposed to histological chorioamnionitis histological chorioamnionitis (hCAM). STUDY DESIGN: Multicenter retrospective cohort study. SUBJECTS: We analyzed 1369 extremely premature infants with severe hCAM who were admitted to the neonatal intensive care units participating in the Neonatal Research Network, Japan (NRNJ) Neonatal research network Japan. OUTCOME MEASURES: Perinatal characteristics were compared and logistic regression analysis was performed for multivariate risk factor assessment. RESULT: Infants with bubbly/cystic appearance on chest X-rays underwent longer duration of invasive mechanical ventilation and required inhaled nitric oxide and home oxygen therapy more frequently. Low gestational age (odds ratio 1.244; 95% confidence interval 1.139-1.359) and preterm premature rupture of membranes (odds ratio 1.507; 95% confidence interval 1.200-1.893) were significant risk factors. CONCLUSION: Low gestational age and preterm premature rupture of membranes were independent risk factors for bubbly/cystic appearance in extremely premature infants with BPD following exposure to severe hCAM.

Is ibuprofen superior to indomethacin for patent ductus arteriosus in Japanese preterm infants?

BACKGROUND: Many clinical trials have indicated that ibuprofen (IBU) has similar effects to indomethacin (IND) on the closure of patent ductus arteriosus (PDA) with fewer adverse effects. Owing to the scarce evidence on IBU use in Japan because of its recent approval we performed this observational study to compare the efficacy and safety of IBU with the efficiency and safety of IND. METHODS: We included infants (gestational age < 30 weeks) with hemodynamically significant PDA under a prophylactic IND protocol for intraventricular hemorrhage who were treated with either IND (n = 30) or IBU (n = 30). We compared a PDA closing effect, changes in ultrasonography findings, and adverse effects between the groups. RESULTS: There was no significant difference in the rates of PDA closure in the first treatment course (IND vs IBU: 46.7% vs 50.0%, P = 0.796) and surgical closure (IND vs IBU: 20.0% vs 20.0%, P = 1.000) between the groups. Both groups showed significant oliguria (IND vs IBU: 30.0% vs 23.3%, P = 0.559) and increased serum creatinine levels after treatment. However, an increase in serum creatinine level by >0.3 mg/dL, a criterion for acute kidney injury, was less frequent in the IBU group (35.7%) compared with that in the IND group (84.2%, P = 0.004). There were no significant differences in echocardiographic changes and jaundice and hypoglycemia incidence rates between the groups. CONCLUSIONS: Except for an increase in serum creatinine levels by >0.3 mg/dL, which was less frequent with IBU, IBU had similar efficacy and safety as IND for preterm PDA. Ibuprofen and IND should be cautiously administered.

Difference in pyruvic acid metabolism between neonatal and adult mouse lungs exposed to hyperoxia.

OBJECTIVE: Neonatal lungs are more tolerant to hyperoxic injury than are adult lungs. This study investigated differences in the response to hyperoxic exposure between neonatal and adult mouse lungs using metabolomics analysis with capillary electrophoresis time-of-flight mass spectrometry (CE- TOFMS). METHODS: Neonatal and adult mice were exposed to 21% or 95% O2 for four days. Subsequently, lung tissue samples were collected and analyzed by CE-TOFMS. Pyruvate dehydrogenase (PDH) enzyme activity was determined using a microplate assay kit. PDH kinase (Pdk) 1, Pdk2, Pdk3, and Pdk4 mRNA expression levels were determined using quantitative reverse transcription-polymerase chain reaction. Pdk4 protein expression was quantified by Western blotting and Pdk4 protein localization was evaluated by immunohistochemistry. RESULTS: Levels of 3-phosphoglyceric acid, 2-phosphoglyceric acid, phosphoenolpyruvic acid, and lactic acid were significantly elevated in the lungs of hyperoxia-exposed versus normoxia-exposed adult mice, whereas no significant differences were observed with hyperoxia exposure in neonatal mice. PDH activity was reduced in the lungs of adult mice only. Pdk4 mRNA expression levels after hyperoxic exposure were significantly elevated in adult mice compared with that in neonatal mice. Conversely, gene expression levels of Pdk1, Pdk2, and Pdk3 did not differ after hyperoxic exposure in either neonatal or adult mice. Pdk4 protein levels were also significantly increased in adult mouse lungs exposed to hyperoxia and were localized mainly to the epithelium of terminal bronchiole. CONCLUSIONS: Specific metabolites associated with glycolysis and gluconeogenesis were altered after hyperoxia exposure in the lungs of adult mice, but not in neonates, which was likely a result of reduced PDH activity due to Pdk4 mRNA upregulation under hyperoxia.

Steroid-Refractory Protein-Losing Enteropathy with Gastrointestinal Bleeding in a Patient with Fontan Circulation.

Protein-losing enteropathy (PLE) is one of the major complications after a Fontan operation. Some PLE patients suffer from concurrent gastrointestinal bleeding. An effective treatment regimen for such patients has not been established yet. Further, it remains unknown whether PLE and gastrointestinal bleeding coexist independently, or protein losing is associated with gastrointestinal bleeding. We report a 7-year-old steroid-refractory post-Fontan PLE case suggesting the latter pathogenesis together with a literature review.

Attenuation of Hyperoxic Lung Injury in Newborn Thioredoxin-1-Overexpressing Mice through the Suppression of Proinflammatory Cytokine mRNA Expression.

The role of thioredoxin-1 (TRX), a small redox-active protein with antioxidant effects, during hyperoxic lung injury in newborns remains undetermined. We investigated TRX impact on hyperoxic lung injury in newborn TRX transgenic (TRX-Tg) and wildtype (WT) mice exposed to 21% or 95% O2 for four days, after which some mice were allowed to recover in room air for up to 14 days. Lung morphology was assessed by hematoxylin/eosin and elastin staining, as well as immunostaining for macrophages. The gene expression levels of proinflammatory cytokines were evaluated using quantitative real-time polymerase chain reaction. During recovery from hyperoxia, TRX-Tg mice exhibited an improved mean linear intercept length and increased number of secondary septa in lungs compared with the WT mice. Neonatal hyperoxia enhanced the mRNA expression levels of proinflammatory cytokines in the lungs of both TRX-Tg and WT mice. However, interleukin-6, monocyte chemoattractant protein-1, and chemokine (C-X-C motif) ligand 2 mRNA expression levels were reduced in the lungs of TRX-Tg mice compared with the WT mice during recovery from hyperoxia. Furthermore, TRX-Tg mice exhibited reduced macrophage infiltration in lungs during recovery. These results suggest that in newborn mice TRX ameliorates hyperoxic lung injury during recovery likely through the suppression of proinflammatory cytokines.

Safety and Feasibility of Intravenous Paracetamol for Patent Ductus Arteriosus in Indomethacin-/Ibuprofen-Resistant or -Contraindicated Preterm Infants: A Case Series.

Background Although indomethacin and ibuprofen are the standard treatments for hemodynamically significant patent ductus arteriosus (hsPDA), they are associated with renal impairment and gastrointestinal complications. Paracetamol for hsPDA closure does not provoke a peripheral vasoconstrictive effect and seems to have effects similar to those of indomethacin and ibuprofen. We have previously reported the safety of low-dose (7.5 mg/kg) intravenous paracetamol for preterm infants with hsPDA, who were indomethacin-resistant or -contraindicated but did not affect the need for surgical PDA ligation. However, reports considering the use of higher-dose (15 mg/kg) paracetamol for hsPDA have not been published in Japan. Cases In 16 premature infants in whom indomethacin or ibuprofen was contraindicated or ineffective, 15 mg/kg of paracetamol was intravenously administered every 6 hours for 3 days after obtaining parental consent. hsPDA closure or narrowing was observed in 14 infants (88%), with the need for surgical closure totally avoided in nine cases (56%). High plasma paracetamol levels were observed in three cases. No paracetamol-related side effects or adverse events were reported. Conclusion The intravenous administration of higher dose paracetamol was safe and feasible in premature infants with hsPDA. Future clinical trials to explore the optimized dose and timing of administration are needed.

Prevalence of Short Stature and Growth Hormone Deficiency and Factors Associated With Short Stature After Fontan Surgery.

Background: Fontan circulation is characterized by many features commonly observed in heart failure that may affect physical growth regardless of pituitary gland dysfunction status. The aims of the present study were to investigate the prevalence of short stature and growth hormone deficiency (GHD) and determine the factors associated with short stature after Fontan surgery. Methods and Results: On retrospective evaluation of 47 patients after Fontan surgery, a very high prevalence of short stature was observed (38.3%). In the short stature group, 5 patients were diagnosed with GHD (10.6% of patients after Fontan Surgery), which is much higher than the frequency of 1/10,000 in the general population. Central venous pressure (CVP) was significantly higher (14.6±4.5 vs. 12.2±1.9 mmHg, P<0.05) and the blood pressure and arterial oxygen saturation were significantly lower in the short stature group. Laboratory data also indicated volume retention and congestion in the short stature group. Mean change in stature from catheterization 1 year after Fontan surgery to the most recent visit was significantly lower in the short stature group (-1.1±1.1 SD vs. 0.0±0.8 SD, P<0.05) and significantly negatively correlated with CVP (r=-0.42, P<0.05). Conclusions: Volume retention and congestion, the prominent features of Fontan circulation, affect physical growth partly due to pituitary gland dysfunction, highlighting the need for the screening for and treatment of this condition after Fontan surgery.

Defining the reference range of regional cerebral tissue oxygen saturation using a new portable near-infrared spectroscopy device for term infants.

BACKGROUND: The arterial oxygen saturation of infants requiring resuscitation can be monitored using a pulse oximeter. However, the device cannot provide information about cerebral oxygenation. Thus, we used a new portable near-infrared spectroscopy (NIRS) device that can monitor regional cerebral tissue oxygen saturation (crSO2) with a probe attached to the examiner's finger. AIM: To identify the reference ranges for crSO2 within the first 10 min after birth in term infants who did not receive any medical intervention in the delivery room. RESULTS: crSO2 in the left frontoparietal area of the forebrain was measured within the first 10 min of life in 127 healthy term infants. The median gestational age and birth weight were 37.6 weeks and 2742 g, respectively, and 85% of the infants were delivered via a scheduled cesarean section. The 3rd-97th percentile values for crSO2 ranged from 33.1% to 56.7% at 1 min, from 33.2% to 59.5% at 2 min, and from 38.7% to 66.6% at 5 min after birth. A median of 3.5 min was required to achieve a crSO2 > 50%. CONCLUSIONS: This study showed the reference ranges for crSO2 measured with the new portable NIRS device within the first 10 min after birth in term infants.

Two Extremely Low Birth Weight Infants Who Survived Functional Pulmonary Atresia with Normal Intracardiac Anatomy.

For the first time, we report about two extremely low birth weight infants who were born at 25 and 22 weeks' gestation and who survived functional pulmonary atresia (fPA) with normal intracardiac anatomy. A slow, reflected, and bimodal blood flow pattern in the pulmonary artery (both cases) and the presence of pulmonary regurgitation (1 case) were useful for diagnosing fPA. Timely use of lipo-prostaglandin E1 to maintain adequate pulmonary flow and reduce pulmonary arterial resistance and sodium bicarbonate to improve acidosis were effective treatments to attain forward flow. As optimal management is essential for the intact survival of extremely early preterm infants and the accurate diagnosis of fPA is difficult without the awareness of the disease entity, our cases underline the importance of recognizing that fPA can occur even in extremely low birth weight infants with normal intracardiac anatomy.

Spontaneous regression of severe aortic coarctation in trisomy 18.

Spontaneous regression of severe aortic coarctation with ductus dependency has not been reported. We experienced a case of trisomy 18 with spontaneous regression of severe aortic coarctation complicated by ventricular septal defect and patent ductus arteriosus. The aortic isthmus diameter was 1.2 mm at birth. After 5 months, it increased to 4.5 mm, and the shape of the isthmus was fully normalised.

Low-Dose Intravenous Paracetamol for Patent Ductus Arteriosus in Indomethacin-Resistant or Contraindicated Preterm Infants: Three Cases Reports.

Background Although indomethacin (IND) is the standard treatment for hemodynamically significant patent ductus arteriosus (hsPDA) in Japan, it may be associated with renal impairment and gastrointestinal complications. The use of paracetamol for hsPDA closure has recently increased. Unlike IND, paracetamol does not have a peripheral vasoconstrictive effect and can be given to infants with contraindications to IND. Based on limited data available from randomized trials, paracetamol and IND seem to have similar effects. However, there have been no reports of the use of paracetamol for hsPDA in Japan. Cases Our drug administration protocol was approved by the institutional ethics committee after purchasing a clinical trial insurance. In three premature infants in whom IND was contraindicated or ineffective, a 7.5 mg/kg of paracetamol was intravenously administered every 6 hour for 3 days after obtaining parental consents. A temporary hsPDA closure was observed in two of the three infants. However, all three infants eventually needed surgical closure. No side effects, such as hepatic and renal dysfunctions, and adverse events were reported. Conclusion The intravenous administration of paracetamol was safe and feasible in premature infants with hsPDA. Future clinical trials with optimized dose and timing of administration are needed.

Intraspecific differentiation of Limonium wrightii (Plumbaginaceae) on northwestern Pacific Islands: rate heterogeneity in nuclear rDNA and its distance-independent geographic structure.

We conducted a molecular phylogenetic analysis of a coastal sea lavender in NW Pacific using ITS sequences of nrDNA. As a result, the ITS sequences of populations on the oceanic Daito Islands were considerably different from conspecific others in nine apomorphic substitutions. This finding indicates a distance-independent geographical structure of the genetic variation among islands in NW Pacific. The calibrated rate of nucleotide substitutions in the ITS regions within L. wrightii (average: 1.41x10(-8) substitutions per site per year) was higher than any other instances of substitution rates in flowering plants.

Intraspecific variation of flower colour and its distribution within a sea lavender, Limonium wrightii (Plumbaginaceae), in the northwestern Pacific Islands.

Differentiation of flower colour is thought to be one of the most important factors promoting plant speciation. We describe the intraspecific variation of flower colour and its distribution in Limonium wrightii. We conducted a survey on 36 islands in the northwestern Pacific and discriminated six morphs of flower colour variation. Two flower colour morphs, pink and yellow, were most frequently observed, and their geographical distributions were basically allopatric. These two morphs were in contact in a narrow zone on Okinoerabu Island, located in the middle region of the Ryukyu Archipelago. In addition, orange, white, and ivory flower morphs were also found in this zone. The geographical distribution of pink and yellow morphs showed a "leapfrog" pattern; the distribution of pink flowers was divided into two areas, intercalated by the distribution of the yellow flower morph. The orange morph may have resulted from hybridization between the pink and yellow flower morphs.