At birth, ventilation and oxygenation immediately produce a dramatic decrease in pulmonary vascular resistance and an increase in pulmonary blood flow with more gradual changes occurring over the next several hours. Nitric oxide (NO), produced from L-arginine by endothelial nitric oxide synthase (eNOS), appears to mediate, at least in part, these gradual changes. Aberrations in NO activity have also been implicated in the pathophysiology of disease states such as persistent pulmonary hypertension of the newborn (PPHN). The mechanism producing the increased NO activity at birth remains unknown. Our overall goals are to determine the factors that regulate pulmonary blood flow and pulmonary vascular resistance at the fetal to newborn transition. The increases in pulmonary blood flow occurring at birth will likely produce an increase in the shear forces on the pulmonary vascular endothelium. NO production, NOS activity, eNOS mRNA and protein expression are increased when endothelial cells (ECS) are exposed to increased shear stress. We hypothesize that the increased shear forces to which the pulmonary system is exposed to after birth will produce an increase in eNOS gene expression; subsequently, this leads to the gradual decrease in PVR and the increased PBF seen in the first several hours after birth. To allow a molecular analysis of the effects of birth on the fetal pulmonary endothelium we have isolated and cultured the relevant late gestation ovine pulmonary arterial ECs. To mimic the effects of increased blood flow we have built a cone-plate viscometer which allows us to expose the EC monolayer to a controlled level of shear stress. We have also cloned the promoter region of the human eNOS gene upstream of a Luciferase reporter. We will use this to identify the DNA sequences within the eNOS promoter (cis-elements) that are activated by shear stress in fetal pulmonary arterial ECs. This may eventually lead to the identification of transcription (transacting) factors responsible for the increase in eNOS gene expression during the transition from fetal to neonatal life.
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