INTRODUCTION. Congenital diaphragmatic hernia (CDH) combines a congenital malformation of the diaphragm with lung hypoplasia, leading to severe respiratory distress and intractable pulmonary hypertension of the newborn. Despite advances in prenatal diagnosis and neonatal intensive care, CDH is associated with high mortality and devastating morbidities. In the absence of curative treatment, numerous prenatal therapies have been used experimentally with varying success. So far, only fetal tracheal occlusion has been tested in clinical trials, but the consequences for the human lung are poorly known. AIMS. To further characterize the rabbit model of CDH, which was subsequently used to assess the effects of prenatal therapies on airway and pulmonary vascular development, including tracheal occlusion, and two novel approaches, perfluorooctylbromide and an activator of soluble guanylate cyclase (BAY 41–2272), which were given through tracheal instillation.METHODS. After a diaphragmatic incision during the pseudoglandular stage, fetal rabbits were randomized against placebo/sham operation during the saccular stage for tracheal occlusion, perfluorocarbon or BAY 41–2272. At term operated fetuses and controls were subject to evaluation of lung mechanics and/or hemodynamics as well as postmortem lung analyses. Human fetal and neonatal lung tissue, including controls and CDH with tracheal occlusion or expectant management, was analyzed histologically and biochemically.RESULTS. The rabbit model of CDH was characterized by reduced lung volumes and impaired compliance, disorders of elastin deposition within alveolar walls, and downregulation of elastogenesis-related genes. Moreover, this model reproduced features of pulmonary hypertension, including high right ventricular pressure and level of N-terminal-pro-B type natriuretic peptide, remodeling of pulmonary arterioles, decreased alveolar capillary density, and downregulation of vasodilation-related genes. In the rabbit model, lung distension caused by tracheal occlusion improved alveolar formation and elastogenesis, yet without correction of lung mechanical parameters. Tracheal occlusion increased also the expression of other extracellular matrix components, which reflected myofibroblast activity, and reduced the transcription of surfactant-associated proteins. Human neonatal lungs exposed to fetal tracheal occlusion displayed alveolar deposits of collagen and myofibroblasts. In human CDH as well as in the rabbit model of CDH, tracheal occlusion enhanced the pulmonary expression of transforming growth factor-β (TGFβ) and Rho kinase−associated proteins to the detriment of activation of SMAD2/3, which is normally detected in human lungs with advancing gestation. As an alternative to tracheal occlusion, pulmonary distension by perfluorocarbon in the fetal rabbit model of CDH improved lung mechanics and alveolar elastogenesis without transcriptional changes in extracellular matrix, surfactant protein genes or TGFβ. Finally, intratracheal instillation of BAY 41–2272 in the rabbit fetuses with CDH improved hemodynamics, reduced medial hypertrophy of pulmonary arterioles, and increased capillary bed formation by stimulating endothelial cell proliferation.CONCLUSIONS. In the fetal rabbit model of CDH, poor lung function after tracheal occlusion is compatible with activation of TGFβ and imbalance in extracellular matrix and epithelial homeostasis. In human CDH newborns treated by fetal tracheal occlusion, changes in the pulmonary interstitium and impaired TGFβ signaling raise the question of disturbances of postnatal lung development induced by tracheal occlusion. As potential alternatives to tracheal occlusion, prenatal perfluorocarbon improves lung hypoplasia, whereas prenatal BAY 41–2272 attenuates pulmonary hypertension. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

Nitric oxide (NO) is an important biological molecule within the body, which over production of this molecule in response to different stimulations can cause various inflammatory diseases. Over production of this molecule is caused by the induction of the inducible nitric oxide synthase (iNOS) enzyme. This enzyme uses L-arginine as a substrate and therefore the presence and transport of this amino acid into the cells can be a key factor in regulating NO over production. Different signalling mechanisms have been implicated in the regulation of this pathway and one of which involves the Mitogen Activated Protein Kinases (MAPK). This family of proteins respond to inflammatory conditions and may mediate effects induced by inflammatory mediators. Of the MAPKs, the role of the c-Jun-N-terminal kinase (JNK) pathway in the induction of iNOS is still controversial. JNK and its downstream target, the transcription factor Activator Protein-1 (AP-1), have shown contradictory effects on iNOS induction leading to controversies over their role in regulating iNOS expression in different cell systems or with various stimuli. The studies described in this thesis have determined the role of JNK/AP-1 on iNOS expression, NO production, L-arginine uptake and also on the transporters responsible for L-arginine transport into the cells. The studies were carried out in two different cell types: rat aortic smooth muscle cells (RASMCs) and J774 macrophages which are both critically associated with the over production of NO in vascular inflammatory disease states. The first approach was to block the expression of the inducible L-arginine-NO pathway using SP600125 and JNK Inhibitor VIII which are both pharmacological inhibitors of JNK. The results from these studies showed that the pharmacological intervention was without effect in RASMCs, but inhibited iNOS, NO and L-arginine transport in J774 macrophages. In contrast, the molecular approach employed using two dominant negative constructs of AP-1 (TAM-67 and a-Fos) revealed a different profile of effects in RASMCs, where a-Fos caused an induction in iNOS and NO while TAM-67 had an inhibitory effect on iNOS, NO, L-arginine transport and CAT-2B mRNA expression. The latter was unaffected in RASMCs but suppressed in J774 macrophages by SP600125. Examination of JNK isoforms expression showed the presence of JNK1 and 2 in both cell systems. Moreover, stimulation with LPS/IFN- or LPS alone resulted in JNK phosphorylation which did not reveal any difference between smooth muscle cells and macrophages. In contrast, expression and activation of AP-1 subunits revealed differences between the two cell systems. Activation of cells with LPS and IFN- (RASMCs) or LPS alone (J774 macrophages) resulted in changes in the activated status of the different AP-1 subunit which was different for the two cell systems. In both cell types c-Jun, JunD and Fra-1 were increased and in macrophages, FosB activity was also enhanced. Inhibition of JNK with SP600125 caused down-regulation in c-Jun in both cell types. Interestingly this down-regulation was in parallel with increases in the subunits JunB, JunD, c-Fos and Fra-1 in RASMCs or JunB and Fra-1 in J774 macrophages. Since, SP600125 was able to exert inhibitory effects in the latter cell type but not in RASMCs, it is possible that the compensatory up-regulation of certain AP-1 subunits in the smooth muscle cells may compensate for c-Jun inhibition thereby preventing suppression of iNOS expression. This notion clearly needs to be confirmed but it is potentially likely that hetero-dimers formed between JunB, JunD, c-Fos and Fra-1 could sustain gene transcription in the absence of c-Jun. The precise dimer required has not been addressed but unlikely to exclusively involve JunB and Fra-1 as these are up-regulated in macrophages but did not sustain iNOS, NO or induced L-arginine transport in the presence of SP600125. To further support the argument above, the dominant negatives caused varied effects on the activation of the different subunits. a-Fos down-regulated c-Jun, c-Fos, FosB, Fra-1 whereas TAM-67 reduced c-Jun and c-Fos but marginally induced Fra-1 activity. Associated with these changes was an up-regulation of iNOS-NO by a-Fos and inhibition by TAM-67. Taken together, the data proposes a complex mechanism(s) that regulate the expression of the inducible L-arginine-NO pathway in different cell systems and the complexity may reflect diverse intracellular changes that may be different in each cell type and not always be apparent using one experimental approach especially where this is pharmacological. Moreover, these findings strongly suggest exercising caution when interpreting pure pharmacological findings in cell-based systems particularly where these are inconsistent or contradictory.