Arginase upregulation and eNOS uncoupling contribute to impaired endothelium-dependent vasodilation in a rat model of intrauterine growth restriction.

Individuals born after intrauterine growth restriction (IUGR) are at increased risk of developing cardiovascular diseases in adulthood, notably hypertension (HTN). Alterations in the vascular system, particularly impaired endothelium-dependent vasodilation, may play an important role in long-term effects of IUGR. Whether such vascular dysfunction precedes HTN has not been fully established in individuals born after IUGR. Moreover, the intimate mechanisms of altered endothelium-dependent vasodilation remain incompletely elucidated. We therefore investigated, using a rat model of IUGR, whether impaired endothelium-dependent relaxation precedes the development of HTN and whether key components of the l-arginine-nitric oxide (NO) pathway are involved in its pathogenesis. Pregnant rats were fed with a control (CTRL, 23% casein) or low-protein diet (LPD, 9% casein) to induce IUGR. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography in 5- and 8-wk-old male offspring. Aortic rings were isolated to investigate relaxation to acetylcholine, NO production, endothelial NO synthase (eNOS) protein content, arginase activity, and superoxide anion production. SBP was not different at 5 wk but significantly increased in 8-wk-old offspring of maternal LPD (LP) versus CTRL offspring. In 5-wk-old LP versus CTRL males, endothelium-dependent vasorelaxation was significantly impaired but restored by preincubation with l-arginine or the arginase inhibitor S-(2-boronoethyl)-l-cysteine; NO production was significantly reduced but restored by l-arginine pretreatment; total eNOS protein, dimer-to-monomer ratio, and arginase activity were significantly increased; superoxide anion production was significantly enhanced but normalized by pretreatment with the NO synthase inhibitor Nω-nitro-l-arginine. In this model, IUGR leads to early-impaired endothelium-dependent vasorelaxation, resulting from arginase upregulation and eNOS uncoupling, which precedes the development of HTN.

Erythropoietin protects newborn rat against sevoflurane-induced neurotoxicity.

INTRODUCTION: Recent data on newborn animals exposed to anesthetics have raised safety concerns regarding anesthesia practices in young children. Indeed, studies on rodents have demonstrated a widespread increase in brain apoptosis shortly after exposure to sevoflurane, followed by long-term neurologic impairment. In this context, we aimed to evaluate the protective effect of rh-EPO, a potent neuroprotective agent, in rat pups exposed to sevoflurane. MATERIAL AND METHODS: At postnatal day 7, 75 rat pups were allocated into three groups: SEVO + EPO (n = 27) exposed to sevoflurane 2 vol% (0.5 MAC) for 6 h in an air/O2 mixture (60/40) + 5000 UI.kg(-1) rh-EPO IP; SEVO (n = 27) exposed to sevoflurane + vehicle IP; and CONTROL (n = 21) exposed to the mixture without sevoflurane + vehicle IP. Three days after anesthesia (D10), apoptosis was quantified on brain extract with TUNEL method and caspase 3. NGF and BDNF expression was determined by Western blotting. Rats reaching adulthood were evaluated in terms of exploration capacities (object exploration duration) together with spatial and object learning (water maze and novel object test). RESULTS: Sevoflurane exposure impaired normal behavior in adult rats by reducing the exploratory capacities during the novel object test and impaired both spatial and object learning capacities in adult rats (water maze, ratio time to find platform 3rd trial/1st trial: 1.1 ± 0.2 vs 0.4 ± 0.1; n = 9, SEVO vs CONTROL; P = 0.01). Rh-EPO reduced sevoflurane-induced behavior and learning abnormalities in adult rats (water maze, ratio time to find platform 3rd trial/1st trial: 0.3 ± 0.1 vs 1.1 ± 0.2; n = 9, SEVO + EPO vs SEVO; P = 0.01). Three days after anesthesia, rh-EPO prevented sevoflurane-induced brain apoptosis (5 ± 3 vs 35 ± 6 apoptotic cells·mm(-2) ; n = 6, SEVO + EPO vs SEVO; P = 0.01) and elevation of caspase three level and significantly increased the brain expression of BDNF and NGF (n = 6, SEVO + EPO vs SEVO; P = 0.01). CONCLUSION: Six hours of sevoflurane anesthesia in newborn rats induces significant long-term cognitive impairment. A single administration of rh-EPO immediately after postnatal exposure to sevoflurane reduces both early activation of apoptotic phenomenon and late onset of neurologic disorders.

Impact of cardiac surgical timing on the neurodevelopmental outcomes of newborns with Complex congenital heart disease (CHD).

Background: More than half of infants with complex congenital heart disease (CHD) will have a neurodevelopmental disorder of multifactorial causes. The preoperative period represents a time-window during which neonates with complex CHD are in a state of hypoxia and hemodynamic instability, which fosters the emergence of brain injuries and, thus, affects early brain networks and neurodevelopmental outcomes. Currently, there is no consensus regarding the optimal age for cardiac surgery in terms of neurodevelopmental outcomes, and its definition is a real challenge. Our aim is to determine the relationship between cardiac surgical timing and long-term neurodevelopmental outcomes for various types of complex CHD. Methods: We hypothesize that earlier surgical timing could represent a neuroprotective strategy that reduces perioperative white matter injuries (WMIs) and postoperative morbidity, leading to improved neurodevelopmental outcomes in infants with complex CHD. Firstly, our prospective study will allow us to determine the correlation between age at the time of surgery (days of life) and neurodevelopmental outcomes at 24 months. We will then analyze the correlation between age at surgery and (i) the incidence of WMIs (through pre- and postoperative MRIs), (ii) postoperative morbidity, and (iii) the duration of the hospital stay. Implications and Dissemination: This research protocol was registered in the Clinical Trial Registry (National Clinical Trial: NCT04733378). This project aims to help launch the first Neurocardiac Investigation Clinic in Marseille - AP-HM - to propose an overall personalized monitoring and treatment program for patients operated on for complex CHD.

Clinical research in newborn infants: difficulties and specificity.

BACKGROUND: Clinical trials are essential in neonates to evaluate scientifically the efficacy and safety of drugs. However, major specificities condition clinical research in human neonates. OBJECTIVE: To review specific constraints to be taken into account in neonatal research studies. METHODS: A review of the literature and contribution of authors' opinions was carried out. RESULTS AND CONCLUSION: Neonatal specificities that induce obstacles in neonatal studies and proposals are detailed. This review also looks at recommendations recently developed by the European Commission to promote safe and ethical research in neonatology.

[Early origins of arterial hypertension and cardiovascular diseases]. / Les origines précoces de l'hypertension artérielle et des maladies cardio-vasculaires.

Research has shown that most of the principal causes of mortality in industrialized countries have their roots in early development. Thus, the period from conception through pregnancy to early infancy is uniquely sensitive to long-lasting effects of environmental insults, potentially leading to physiological malprogramming and lifelong disease. Beyond the classical association between low birth weight and increased coronary mortality in adulthood, peri-conceptional and perinatal insults can set the scene for later obesity, cancer and behavioral disorders. Taking the developmental programming of hypertension as an example, this review addresses the remarkably similar mechanisms of early programming, particularly those involving the kidney and vasculature ; the continuum between normality and disease ; the role of early and later nutrition ; and early biomarkers and epigenetic mechanisms of later cardiovascular and metabolic disorders. The developmental origins of adult health and disease represent a global research challenge as emerging countries undergo major nutritional and environmental upheavals. Much research is focused on the benefits of early nutritional and lifestyle interventions, in both animal models and human studies.

Case Report: Clostridium neonatale Bacteremia in a Preterm Neonate With Necrotizing Enterocolitis.

Necrotizing enterocolitis is a life-threatening acquired gastrointestinal disorder among preterm neonates and is associated with a high mortality rate and long-term neurodevelopmental morbidity. No etiologic agent has been definitively established; nonetheless, the most implicated bacteria include members of the Clostridium genus. We reported here on a case of Clostridium neonatale bacteremia in a preterm neonate with necrotizing enterocolitis, providing more information regarding the potential role of this bacterium in pathogenesis of necrotizing enterocolitis. We emphasized the sporulating form of C. neonatale that confers resistance to disinfectants usually applied for the hospital environmental cleaning. Further works are needed to establish the causal relationship between the occurrence of NEC and the isolation of C. neonatale, with promising perspectives in terms of diagnostic and therapeutic management.

Long-Term Recovery After Endothelial Colony-Forming Cells or Human Umbilical Cord Blood Cells Administration in a Rat Model of Neonatal Hypoxic-Ischemic Encephalopathy.

Neonatal hypoxic-ischemic encephalopathy (NHIE) is a dramatic perinatal complication, associated with poor neurological prognosis despite neuroprotection by therapeutic hypothermia, in the absence of an available curative therapy. We evaluated and compared ready-to-use human umbilical cord blood cells (HUCBC) and bankable but allogeneic endothelial progenitors (ECFC) as cell therapy candidate for NHIE. We compared benefits of HUCBC and ECFC transplantation 48 hours after injury in male rat NHIE model, based on the Rice-Vannucci approach. Based on behavioral tests, immune-histological assessment and metabolic imaging of brain perfusion using single photon emission computed tomography (SPECT), HUCBC, or ECFC administration provided equally early and sustained functional benefits, up to 8 weeks after injury. These results were associated with total normalization of injured hemisphere cerebral blood flow assessed by SPECT/CT imaging. In conclusion, even if ECFC represent an efficient candidate, HUCBC autologous criteria and easier availability make them the ideal candidate for hypoxic-ischemic cell therapy. Stem Cells Translational Medicine 2017;6:1987-1996.

Altered angiogenesis in low birth weight individuals: a role for anti-angiogenic circulating factors.

OBJECTIVE: Low birth weight (LBW) is a risk factor for hypertension at adulthood. Endothelial progenitor cells (EPCs) dysfunction has been characterized in LBW neonates. We hypothesized that changes in soluble, plasma pro- or anti-angiogenic factors are associated with EPCs dysfunction and impaired angiogenesis in LBW neonates. METHOD: Venous umbilical cord blood was collected from 42 normal, term neonates and 75 LBW neonates. Cord blood endothelial colony forming cells (ECFC) from control patients were cultured in the presence of 10% of serum obtained from both groups. RESULTS: The proliferation and the migration of ECFC were significantly reduced when cultured with 10% of serum of LBW neonates compared to serum of control neonates. Matrigel invasion assay was not significantly altered. Umbilical vein plasma VEGF concentration was significantly reduced in LBW neonates while that of sVEGFR and PF4 were significantly higher. Addition of VEGF corrected the inhibitory effect of LBW serum on normal ECFC proliferation. CONCLUSIONS: Serum obtained from LBW babies contains factors that exhibit an antiangiogenic effect on ECFC proliferation and migration. VEGF/sVEGF/PF4 pathway seems to be involved in the EPCs dysfunction in LBW neonates.

Effect of two models of intrauterine growth restriction on alveolarization in rat lungs: morphometric and gene expression analysis.

Intrauterine growth restriction (IUGR) in preterm infants increases the risk of bronchopulmonary dysplasia, characterized by arrested alveolarization. We evaluated the impact of two different rat models (nitric oxide synthase inhibition or protein deprivation) of IUGR on alveolarization, before, during, and at the end of this postnatal process. We studied IUGR rat pups of dams fed either a low protein (LPD) or a normal diet throughout gestation and pups of dams treated by continuous infusion of Nω-nitro-L-arginine methyl ester (L-NAME) or its diluent on the last four days of gestation. Morphometric parameters, alveolar surface (Svap), mean linear intercept (MLI) and radial alveolar count (RAC) and transcriptomic analysis were determined with special focus on genes involved in alveolarization. IUGR pups regained normal weight at day 21 in the two treated groups. In the LPD group, Svap, MLI and RAC were not different from those of controls at day 4, but were significantly decreased at day 21, indicating alveolarization arrest. In the L-NAME group, Svap and RAC were significantly decreased and MLI was increased at day 4 with complete correction at day 21. In the L-NAME model, several factors involved in alveolarization, VEGF, VEGF-R1 and -R2, MMP14, MMP16, FGFR3 and 4, FGF18 and 7, were significantly decreased at day 4 and/or day 10, while the various factors studied were not modified in the LPD group. These results demonstrate that only maternal protein deprivation leads to sustained impairment of alveolarization in rat pups, whereas L-NAME impairs lung development before alveolarization. Known growth factors involved in lung development do not seem to be involved in LPD-induced alveolarization disorders, raising the question of a possible programming of altered alveolarization.

Ibubrofen in the treatment of patent ductus arteriosus in preterm infants: what we know, what we still do not know.

The patency of the ductus arteriosus has ever been considered as a pathological situation in preterm infants and one likely cause of mortality and morbidity, including broncho-pulmonary dysplasia, necrotizing enterocolitis, intraventricular haemorrhage, retinopathy of prematurity. The incidence of patent ductus arteriosus is inversely proportional to gestational age and infants with the lowest gestational ages are the most exposed to the complications of prematurity. So, associations between patent ductus arteriosus and the other morbidities may not be causative and patent ductus arteriosus could be more a sign of immaturity and severity of disease than the cause of these problems. Non-steroidal anti-inflammatory agents, such as indomethacin or ibuprofen, have been shown to be effective in closing or preventing patent ductus arteriosus, with differences in side effects. However nearly all randomized controlled trials have been designed with the closure of the ductus arteriosus, not mortality or morbidity, as the main endpoint. Thus, evidence is still lacking on the eventual benefits for the patient of pharmacological or surgical intervention on PDA. Moreover, both ibuprofen and indomethacin efficacy seems markedly reduced in extremely low gestational age infants, who are the most likely to benefit from such intervention. The explanation of the reduced pharmacodymanic effect in such population is unclear; so far, studies using increased dosing of ibuprofen have failed to show a clear benefit. Prophylaxis with indomethacin or ibuprofen has failed to show sustained benefits on neurodevelopment at 2 years of age in low gestational age infants. New curative trials may aim at investigating the effects of early curative administration of ibuprofen, which has reduced side effects compared to indomethacin, on immature kidney function, on mortality and morbidity in very low gestational age infants, ideally with a combined endpoint such as survival in the absence of severe neurodevelopmental alteration at 2 years age. Despite an understandable reluctance given the historical background of systematic, therapeutic closure of ductus arteriosus in preterm infants, there are no definite ethical obstacles to a placebo-controlled design.

The intensity of IUGR-induced transcriptome deregulations is inversely correlated with the onset of organ function in a rat model.

A low-protein diet applied during pregnancy in the rat results in intrauterine growth restricted (IUGR) fetuses. In humans, IUGR is associated with increased perinatal morbidity, higher incidence of neuro-developmental defects and increased risk of adult metabolic anomalies, such as diabetes and cardiovascular disease. Development and function of many organs are affected by environmental conditions such as those inducing fetal and early postnatal growth restriction. This phenomenon, termed "fetal programming" has been studied unconnectedly in some organs, but very few studies (if any) have investigated at the same time several organs, on a more comparative basis. However, it is quite probable that IUGR affects differentially most organ systems, with possible persistent changes in gene expression. In this study we address transcriptional alterations induced by IUGR in a multi-organ perspective, by systematic analysis of 20-days rat fetuses. We show that (1) expressional alterations are apparently stronger in organs functioning late in foetal or postnatal life than in organs that are functioning early (2) hierarchical classification of the deregulations put together kidney and placenta in one cluster, liver, lungs and heart in another; (3) the epigenetic machinery is set up especially in the placenta, while its alterations are rather mild in other organs; (4) the genes appear deregulated in chromosome clusters; (5) the altered expression cascades varies from organ to organ, with noticeably a very significant modification of the complement and coagulation cascades in the kidney; (6) we found a significant increase in TF binding site for HNF4 proteins specifically for liver genes that are down-regulated in IUGR, suggesting that this decrease is achieved through the action of HNF transcription factors, that are themselves transcriptionnally induced in the liver by IUGR (x 1.84 fold). Altogether, our study suggests that a combination of tissue-specific mechanisms contributes to bring about tissue-driven modifications of gene cascades. The question of these cascades being activated to adapt the organ to harsh environmental condition, or as an endpoint consequence is still raised.

Low birth weight infants and the developmental programming of hypertension: a focus on vascular factors.

Low birth weight infants, in particular those born preterm, have been shown to develop increased arterial blood pressure and hypertension at adulthood. Three main systems are involved in the developmental programming of hypertension: the kidney, the neuroendocrine system, and the vascular tree. This review focuses on vascular dysfunction and discusses clinical and experimental evidence that relates low birth weight and the risk for hypertension at adulthood. Recent studies demonstrate an impairment of vascular structure and function. Both arterial vessels, through altered arterial stiffness and endothelium-dependent vasodilation, and the capillary bed, through microvascular rarefaction, are involved in the early pathogenesis of hypertension. The key role of the endothelium, as shown by altered vasodilatation, angiogenesis, endothelial progenitor cells, and microparticle number and function, is discussed as a possible explanatory mechanism.