BMP9 (Bone Morphogenetic Protein 9) et BMP10 sont deux facteurs de croissance de la famille du TGFβ qui partagent le fait d'être les ligands du récepteur Alk1 (Activin receptor-like kinase 1). Les mutations de ce récepteur sont la cause de 2 pathologies vasculaires, la maladie de Rendu Osler et l'Hypertension Artérielle pulmonaire. L'invalidation de BMP10 a été décrite, elle entraine une létalité embryonnaire du fait de graves défauts cardiaques. L'invalidation de BMP9, un facteur de quiescence de l'endothélium vasculaire, n'avait pas encore été réalisée. Dans ce contexte l'objectif de cette thèse a été d'étudier les rôles respectifs de BMP9 et de BMP10 dans les remodelages vasculaires. Nous avons pour cela utilisé les souris invalidées pour BMP9, obtenues dans le cadre d'une collaboration avec l'équipe de S-J Lee (Baltimore, USA). Bien que les souris Bmp9-KO soient viables et fertiles, notre travail montre qu'elles présentent un réseau lymphatique anormal, avec un élargissement des vaisseaux et une réduction du nombre de valves dans leurs collecteurs. Les défauts observés ont un retentissement fonctionnel sur l'efficacité du drainage lymphatique. En accord avec ces observations, nous avons pu montrer que BMP9 régule un certain nombre de gènes impliqués dans le développement du réseau lymphatique. En contraste avec les résultats précédents, la vascularisation de la rétine est normale chez les souriceaux Bmp9-KO. Cette absence de phénotype est causée par une redondance entre BMP9 et BMP10. En effet, la neutralisation de BMP10 chez les souriceaux Bmp9-KO entraine une absence de maturation de la vascularisation de leur rétine. De plus, ces souriceaux présentent un défaut de fermeture de leur canal artériel. Ce vaisseau permet de dévier le sang hors des poumons fœtaux non fonctionnels et sa fermeture au moment de la naissance est nécessaire à la survie postnatale. Au total, ce travail met en exergue les rôles de BMP9 et de BMP10 dans les remodelages vasculaires périnataux lymphatiques et sanguins. / BMP9 (Bone Morphogenetic Protein 9) and BMP10 are two growth factors of the TGFβ family. They are both high affinity ligands for the receptor Alk1 (Activin receptor-like kinase 1), whose mutations are responsible of two vascular pathologies: Rendu-Osler disease and Pulmonary arterial hypertension. BMP10 invalidation has been described as embryonically lethal due to serious cardiac defects. BMP9 has been revealed as circulating vascular quiescence factor, but its inactivation has not been described before. In this context, the aim of my thesis was to evaluate the respective roles of BMP9 and BMP10 in vascular remodeling. We used Bmp9-KO mice for this purpose, which were provide by S-J Lee's team from Baltimore (USA). Although these mice are viable and fertile, we showed that they displayed abnormal lymphatic vessels characterized by vessel enlargement and reduction in number of valves, leading to a decreased lymphatic draining efficiency. Consistent with these data, we showed that BMP9 regulates expression of several genes known to be involved in lymphatic development. In contrast, BMP9 loss-of-function didn't affect blood vessels. We demonstrated that this was due to BMP9 and BMP10 redundancy, as BM10 neutralization in Bmp9-KO pups led to a dramatic decrease in retinal vascular expansion. These pups also display an abnormal closure of their ductus arteriosus. This vessel diverts blood away from the non-functional lungs during fetal life and its closure is essential for postnatal survival. Taken together, our results underlie the implication of BMP9 and BMP10 in lymphatic and blood postnatal vascular remodeling.

Os polifenois são normalmente considerados compostos que apresentam atividades biológicas promissoras, em especial pelos seus efeitos antioxidantes e anti-inflamatórios. No entanto, estudos recentes têm demonstrado que o consumo materno de alimentos ricos em polifenois (ARP) durante a gestação interfere na dinâmica de fluxo do ductus arteriosus (DA) no coração fetal de humanos, provavelmente pelo efeito anti-inflamatório dos polifenois, e também tem sido demonstrado que a restrição da ingestão de ARP é capaz de reverter a constrição ductal. Neste trabalho, um estudo experimental foi desenvolvido com ovelhas prenhas, no qual os animais receberam suplementação oral de polifenois durante 14 dias. Realizou-se ecocardiografia fetal e a análise de amostras de sangue e urina para investigar biomarcadores de estresse oxidativo e inflamação além da excreção de polifenois totais na urina. Houve aumento nas velocidades sistólicas (VS) e diastólicas (VD) e uma diminuição no índice de pulsatilidade (IP), o que indica uma constrição prematura do DA após o consumo de polifenois. Houve diminuição da peroxidação lipídica, determinada pelos níveis de TBARS, e nos níveis de tióis reduzidos não proteicos após o tratamento. Houve um aumento das atividades das enzimas catalase (CAT) e glutationa peroxidase (GPx) após o tratamento. Apesar do não envolvimento de dano lipídico na constrição ductal, observou-se um aumento no dano proteico através da dosagem de proteínas carboniladas (PCO). O efeito vasoconstritor e anti-inflamatório foi verificado pela diminuição nos níveis de nitritos/nitratos (NOx) após o consumo de polifenois. O estresse oxidativo estava associado com parâmetros de constrição ductal, através das correlações de dano protéico (PCO) com VS (r=0,629, p=0,028), VD (r=0,905, p=0,0001) e IP (r=-0,772, p= 0,003). Ainda, VS foi correlacionada com catalase (r=0,672, p=0,033) assim como IP com GPx (r=-0,629, p= 0,05). A constrição ductal estava ainda associada com o parâmetro inflamatório, sendo VS e VD correlacionadas com NOx (r=-0,853, p=0,0004 e r=-0,705, p=0,010, respectivamente) além da correlação entre IP e NOx (r=0,599, p=0,039). Além disso, ambos os mecanismos anti-inflamatórios e antioxidantes estavam correlacionados: NOx e GPx (r=-0,755, p=0,004) e entre NOx e catalase (r=-0,812, p=0,001), confirmando a ocorrência de ambos efeitos atribuíveis aos 10 polifenois. Neste estudo, foi possível perceber que um elevado consumo de polifenois induziu constrição ductal em ovelhas prenhas com uma excreção urinária aumentada de polifenois totais e alterações em biomarcadores de estresse oxidativo e inflamação. Estes resultados ressaltam a necessidade de uma orientação dietética ao final da gestação com relação ao consumo de alimentos ricos em polifenois devido à possibilidade de indução de constrição ductal através da ação anti-inflamatória em fetos expostos. / Polyphenols are often referred to as compounds with promising biological activities, especially antioxidant and anti-inflammatory effects. However, it has been recently reported that maternal consumption of polyphenol-rich foods (PRF) interferes with ductus arteriosus (DA) flow in human fetuses’ hearts, probably by an anti-inflammatory effect and it has also been shown that restriction of PRF ingestion reverses ductal constriction. In this work, an experimental study was carried out with pregnant sheep, in which the animals received oral polyphenol supplementation for 14 days. Fetal echocardiography was performed along with blood and urine analysis to investigate antioxidant and anti-inflammatory biomarkers and total polyphenol (TP) urinary excretion. We found a decrease in lipid peroxidation by TBARS levels and a decrease in non-protein reduced thiols after treatment. In addition, an increase in enzymatic activities of catalase (CAT) and glutathione peroxidase (GPx) was observed. Despite that lipid peroxidation was not involved in ductal constriction, protein damage by enhanced protein carbonyls (PCO) were found. Anti-inflammatory and vasoconstrictive effects were observed by a decrease in nitrites/nitrates (NOx) in pregnant sheep after polyphenol consumption. Oxidative stress was associated to ductal constriction parameters, according to the correlations.of protein damage marker PCO to SV (r=0.629, p=0.028), VD (r=0.905, p=0.0001) and IP (r=-0.772, p=0.003). Also, SV was positively correlated to CAT (r=0.672, p=0.033) and IP negatively correlated to GPx (r=-0.629, p=0.05). Ductal constriction was also associated to the inflammatory parameter, due to the correlations of SV and DV to NOx (r=-0.853, p=0.0004 and r=-0.705, p=0.010, respectively) as well as the correlation between IP and NOx (r=0.599, p=0.039). Besides, association of both inflammatory and antioxidant mechanisms were found: NOx vs. GPx (r=-0.755, p=0.004) and NOx vs. CAT (r=-0.812, p=0.001), confirming the presence of both effects attributed to polyphenols. We report that high polyphenol intake induced fetal DA constriction in pregnant sheep followed by an increased TP excretion and alterations in inflammatory and oxidative biomarkers. These results highlight the need for a dietary orientation in late-pregnancy regarding maternal intake of foods with high polyphenol contents in light of the possible induction of ductal constriction through an anti-inflammatory action of polyphenols in exposed fetuses.

There is no consensus about the hemodynamic significance and, therefore, the need to treat a persistent ductus arteriosus in preterm newborns. Since the diagnosis of a hemodynamically significant persistent ductus arteriosus (hsPDA) is made by a summary of non-uniform echo-criteria in combination with the clinical deterioration of the preterm neonate, standardized clinical and ultrasound scoring systems are needed. The objective of this study was the development of a clinical score for the detection and follow-up of hsPDA. In this observational cohort study of 154 preterm neonates (mean gestational age 28.1 weeks), clinical signs for the development of hsPDA were recorded in a standardized score and compared to echocardiography. Analyzing the significance of single score parameters compared to the diagnosis by echocardiography, we developed a short clinical score (calculated sensitivity 84% and specificity 80%). In conclusion, this clinical diagnostic PDA score is non-invasive and quickly to implement. The continuous assessment of defined clinical parameters allows for a more precise diagnosis of hemodynamic significance of PDA and, therefore, should help to detect preterm neonates needing PDA-treatment. The score, therefore, allows a more targeted use of echocardiography in these very fragile preterm neonates.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: References located at the end of each chapter.

The chicken ductus arteriosi (DA) are two embryonic blood vessels that shunt blood away from the non-ventilated lungs and towards the body and chorioallantoic membrane. I show that prostaglandins have a diminished role in maintaining chicken DA patency and nitric oxide inhibits oxygen induced contraction of the day 19 proximal DA in a time dependent manner. The pathways governing oxygen induced contraction in the chicken DA are similar to those found in mammals and include contributions from ROS, Kv channels, L-type Ca2+ channels, and the Rho kinase pathway. Longer exposure to high oxygen generates increased oxygen induced constriction of the day 19 DA that may be mediated through the Rho kinase pathway.

Background Modern neonatal care has improved the survival rate of extremely preterm infants. These infants are at high risk of malnutrition and growth failure during 3-4 months of hospital care. The objectives of this study was to investigate nutritional intakes during hospitalization and explore associations between nutritional intakes, postnatal growth and retinopathy of prematurity (ROP). Perioperative nutrition in infants undergoing surgery for patent ductus arteriosus (PDA) was also investigated. Methods This is a population-based study of Swedish extremely preterm infants (<27 weeks) born during 2004-2007 (n=602). Detailed data on nutritional supply and anthropometric measurements during hospitalization were retrospectively retrieved from hospital records. Comprehensive data on cohort characteristics, neonatal morbidity and infant mortality were obtained from the Extremely Preterm Infants in Sweden Study (EXPRESS). Results During the first 70 days of life, intakes of energy, protein and several micronutrients, with the exception of iron and some vitamins, were less than estimated requirements, and infants showed severe postnatal growth failure. Energy and protein intake predicted growth in all anthropometric outcomes even when adjusting for severity of illness, and fat intake was positively associated with head growth. Low folate intake was positively correlated with poor weight and length gain while high iron intake, mainly explained by blood transfusions, was negatively associated with poor length gain. Furthermore, a low energy intake was associated with severe ROP (stage 3-5). An increased energy intake of 10 kcal/kg/d was associated with 24% decrease in severe ROP (p=0.01). During the first month, 99% of the infants were exclusively fed human milk. Infants who underwent surgery for PDA (n=140) were malnourished, with energy and macronutrient intakes below minimum estimated requirements before, during and after surgery. Conclusions The severe postnatal growth failure observed in Swedish extremely preterm infants may be prevented by improved intakes of energy, protein, fat and folate and a reduction of the number of blood transfusions. Human milk is the main enteral food source and analyses of human milk macronutrient contents facilitates individualized fortification. Provision of adequate energy intakes during the first four weeks of life may be an effective way to reduce the risk of severe ROP. Perioperative nutrition in infants undergoing PDA surgery needs to be improved. The study results have important implications for nutritional regimens, postnatal growth and health outcome in this new generation of survivors.

En dépit du nombre croissant d’études cliniques sur le canal artériel (CA), des failles méthodologiques entretiennent plusieurs incertitudes concernant l’efficacité et la sécurité des traitements chez les bébés nés prématurés. L’objectif de cette recherche était de comparer les concentrations de prostaglandine E2 (PGE2) et les mesures du flux mésentérique par échographie Doppler chez les enfants nés prématurément et ayant un canal artériel traité à l’ibuprofène par voie intraveineuse ou entérale, en utilisant la méthodologie randomisée contrôlée et à double insu. Dans notre étude pilote, 20 nouveau-nés prématurés de moins de 34 semaines ayant un CA symptomatique confirmé par échocardiographie, furent randomisés au traitement à l’ibuprofène par voie intraveineuse ou entérale. La voie d’administration fut maintenue à l’insu de l’équipe traitante, des cardiologues et des investigateurs. Des dosages des prostaglandines plasmatiques ont été mesurés avant le début du traitement ainsi que 3, 24 et 48 h après le début du traitement. Les mesures du flux mésentérique ont été effectuées avant le traitement à l’ibuprofène ainsi que 1 h et 3 h après le traitement. Nous avons démontré à partir de nos observations que les niveaux plasmatiques de prostaglandines E2 diminuent chez les patients ayant répondu au traitement à l’ibuprofène, indépendamment de la voie d’administration. Nous n’avons pas observé de changement dans l’évolution des dosages de PGE2 chez les patients qui n’ont pas répondu au traitement. Les paramètres mesurés par échographie Doppler au niveau de l’artère mésentérique supérieure n’étaient pas affectés par la voie d’administration du traitement à l’ibuprofène, intraveineuse ou entérale. La présente étude suggère ainsi que le traitement du CA par ibuprofène intraveineux ou entéral n’influe pas sur le flux sanguin mesuré par échographie Doppler. La baisse de la prostaglandine E2 coïncide avec la fermeture du CA, et son dosage pourrait jouer un rôle dans la gestion du traitement. Nous avons démontré la faisabilité d’une étude clinique randomisée à double insu dans le traitement du canal artériel; une méthodologie qui devrait désormait être employé dans la recherche clinique sur les traitements de la persistance du CA. / Despite the growing body of research on the patent ductus arteriosus (PDA), issues with clinical research methodology impairs much of our understanding regarding treatment efficacy and safety in the preterm population. The purpose of this study was to determine plasma prostaglandin E2 (PGE2) concentrations in preterm infants with symptomatic persistence of the ductus arteriosus treated with IV and oral ibuprofen, and measure Doppler flow parameters in the superior mesenteric artery, utilizing randomized controlled and double-blind methodology. Twenty patients age < 34 wks with a symptomatic PDA confirmed by echocardiography randomized to oral vs intravenous ibuprofen regimen. Treating physician, cardiologists and study investigators were blinded to treatment allocation. Plasma PGE2 levels were measured prior to ibuprofen treatment and at 3, 24 and 48 h after treatment. Mesenteric Doppler measurements were taken prior to ibuprofen treatment, and 1 h and 3 h after treatment. Our results showed that plasma PGE2 levels decreased over time in patients that exhibited ductal closure after IV or oral ibuprofen treatment; no time-dependent changes in PGE2 were seen in subjects that failed to respond to ibuprofen. Superior mesenteric artery Doppler flow measurements were not affected by ibuprofen treatment (IV or oral), regardless of efficacy on ductal closure and of PGE2 changes. We conclude that treatment with oral or intravenous ibuprofen does not impact on superior mesenteric artery blood flow measured by Doppler ultrasound. Decreases in plasma PGE2 concentrations coincide with ibuprofen efficacy, and may be more cost-effective to monitor than ultrasound. This study also demonstrated the successful use of double blinded randomized controlled research methodology, which should be more strictly applied in future clinical research on PDA treatment.

En dépit du nombre croissant d’études cliniques sur le canal artériel (CA), des failles méthodologiques entretiennent plusieurs incertitudes concernant l’efficacité et la sécurité des traitements chez les bébés nés prématurés. L’objectif de cette recherche était de comparer les concentrations de prostaglandine E2 (PGE2) et les mesures du flux mésentérique par échographie Doppler chez les enfants nés prématurément et ayant un canal artériel traité à l’ibuprofène par voie intraveineuse ou entérale, en utilisant la méthodologie randomisée contrôlée et à double insu. Dans notre étude pilote, 20 nouveau-nés prématurés de moins de 34 semaines ayant un CA symptomatique confirmé par échocardiographie, furent randomisés au traitement à l’ibuprofène par voie intraveineuse ou entérale. La voie d’administration fut maintenue à l’insu de l’équipe traitante, des cardiologues et des investigateurs. Des dosages des prostaglandines plasmatiques ont été mesurés avant le début du traitement ainsi que 3, 24 et 48 h après le début du traitement. Les mesures du flux mésentérique ont été effectuées avant le traitement à l’ibuprofène ainsi que 1 h et 3 h après le traitement. Nous avons démontré à partir de nos observations que les niveaux plasmatiques de prostaglandines E2 diminuent chez les patients ayant répondu au traitement à l’ibuprofène, indépendamment de la voie d’administration. Nous n’avons pas observé de changement dans l’évolution des dosages de PGE2 chez les patients qui n’ont pas répondu au traitement. Les paramètres mesurés par échographie Doppler au niveau de l’artère mésentérique supérieure n’étaient pas affectés par la voie d’administration du traitement à l’ibuprofène, intraveineuse ou entérale. La présente étude suggère ainsi que le traitement du CA par ibuprofène intraveineux ou entéral n’influe pas sur le flux sanguin mesuré par échographie Doppler. La baisse de la prostaglandine E2 coïncide avec la fermeture du CA, et son dosage pourrait jouer un rôle dans la gestion du traitement. Nous avons démontré la faisabilité d’une étude clinique randomisée à double insu dans le traitement du canal artériel; une méthodologie qui devrait désormait être employé dans la recherche clinique sur les traitements de la persistance du CA. / Despite the growing body of research on the patent ductus arteriosus (PDA), issues with clinical research methodology impairs much of our understanding regarding treatment efficacy and safety in the preterm population. The purpose of this study was to determine plasma prostaglandin E2 (PGE2) concentrations in preterm infants with symptomatic persistence of the ductus arteriosus treated with IV and oral ibuprofen, and measure Doppler flow parameters in the superior mesenteric artery, utilizing randomized controlled and double-blind methodology. Twenty patients age < 34 wks with a symptomatic PDA confirmed by echocardiography randomized to oral vs intravenous ibuprofen regimen. Treating physician, cardiologists and study investigators were blinded to treatment allocation. Plasma PGE2 levels were measured prior to ibuprofen treatment and at 3, 24 and 48 h after treatment. Mesenteric Doppler measurements were taken prior to ibuprofen treatment, and 1 h and 3 h after treatment. Our results showed that plasma PGE2 levels decreased over time in patients that exhibited ductal closure after IV or oral ibuprofen treatment; no time-dependent changes in PGE2 were seen in subjects that failed to respond to ibuprofen. Superior mesenteric artery Doppler flow measurements were not affected by ibuprofen treatment (IV or oral), regardless of efficacy on ductal closure and of PGE2 changes. We conclude that treatment with oral or intravenous ibuprofen does not impact on superior mesenteric artery blood flow measured by Doppler ultrasound. Decreases in plasma PGE2 concentrations coincide with ibuprofen efficacy, and may be more cost-effective to monitor than ultrasound. This study also demonstrated the successful use of double blinded randomized controlled research methodology, which should be more strictly applied in future clinical research on PDA treatment.

Abstract The ductus arteriosus connects the pulmonary artery and the descending aorta in the foetus. In normal neonatal transition, the ductus closes soon after birth. If the duct remains significantly open after birth, it may complicate the recovery of a very preterm infant. Present treatments of patent ductus arteriosus (PDA) are either medical (ibuprofen or indomethacin) or surgical (ligation). However, these treatments can have serious side effects, especially in the most immature infants. This doctoral thesis studied the potential role of intravenous paracetamol for PDA treatment in very preterm infants born before 32 weeks of gestation. Consequences of the PDA treatments in an epidemiological birth cohort were also studied. In retrospective Study I stated that treatments of PDA decreased after the introduction of IV paracetamol for early pain management in preterm infants. Study II showed in a randomised clinical trial for the first time that paracetamol has a biological effect on the ductus arteriosus in preterm infants soon after birth. The ductus closed significantly earlier in the paracetamol group than in the placebo group. The epidemiological cohort Study III showed evidence that both medical and surgical treatment of PDA associated with severe bronchopulmonary dysplasia in infants born very preterm. Additionally, surgical PDA ligation was associated with increased risk of necrotising enterocolitis and intraventricular haemorrhage. Study IV showed that treatment of PDA was not associated with increased mortality, even in the most immature preterm infants born before 28 weeks of gestation. / Tiivistelmä Valtimotiehyt on sikiöaikana avoimena oleva suoni, joka yhdistää keuhkovaltimon laskevaan aorttaan ja ohjaa vähähappisen veren istukkaan. Yhdessä soikean aukon kanssa suoni takaa sikiön verenkierron normaalin toiminnan ennen keuhkojen avautumista. Mikäli valtimotiehyt jää syntymän jälkeen pitkittyneesti auki, muuttaa se keskosen verenkiertoa siten, että osa aortan verenkiertoa ohjautuu keuhkoverenkiertoon vaikeuttaen pienen keskosen toipumista. Nykyhoitoina käytetään joko lääkkeellistä (ibuprofeeni tai indometasiini) tai kirurgista sulkua. Lääkkeellinen hoito ei ole kovin tehokas kaikista epäkypsimmillä keskosilla ja hoitoihin liittyy vakaviakin sivuvaikutuksia. Väitöskirjassa tutkittiin parasetamolilääkityksen vaikutusta hyvin pienen keskosen avoimen valtimotiehyen sulkeutumiseen. Epidemiologisessa osiossa tutkittiin nykyhoitojen sivuvaikutuksia hyvin pienillä keskosilla. Osatyössä I todettiin, että avoimen valtimotiehyen hoidon tarve väheni merkittävästi sen jälkeen kun parasetamoli oli otettu käyttöön kivun hoidossa vastasyntyneiden teholla. Osatyö II oli satunnaistettu ja sokkoutettu hoitotutkimus, jossa todettiin alkuperäishavaintona, että parasetamolilla on biologinen vaikutus keskosen avoimeen valtimotiehyeen. Parasetamolia saaneilla keskosilla valtimotiehyt sulkeutui aikaisemmin kuin verrokeilla. Hoidolla ei todettu merkittäviä sivuvaikutuksia. Osatöissä III ja IV tutkittiin kaikkien vuosina 2005−2013 Suomessa syntyneiden hyvin pienten keskosten avoimen valtimotiehyen hoitoja. Lääkehoidolla (ibuprofeeni ja indometasiini) ja kirurgisella hoidolla todettiin olevan yhteys keskosen kroonisen keuhkotaudin (BPD) vaikeimpaan muotoon. Kirurgisella hoidolla oli yhteys keskosen vaikeaan suolitulehdukseen ja vaikeaan aivoverenvuotoon. Kuolleisuuden riskin ei kuitenkaan todettu lisääntyneen valtimotiehyen hoitoihin liittyen.

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