Prostacyclin (PGI2) and prostaglandin E2 (PGE2) are critical mediators of vasomotor tone, airway reactivity, and surfactant synthesis in the developing lung. They are produced primarily in vascular cells by cyclooxygenase (COX), which exists in two isoforms, COX-1 and COX-2. We have demonstrated O2 modulation of PGI2 and PGE2 synthesis in ovine fetal and newborn intrapulmonary arteries (PA), with hypoxia causing decreased synthesis in fetal PA and increased synthesis in newborn PA. We have also demonstrated a 32-fold maturational rise in PGI2 synthesis. Both the O2- and age-associated alterations in synthesis are related to changes in COX protein expression. The OVERALL OBJECTIVE of this proposal is to determine the molecular mechanisms by which COX gene expression in pulmonary vascular cells is modified by changes in O2 and by maturation, focusing the latter studies on modulation by hormonal factors and shear stress.
Aim No. 1 is to determine the effects of O2 on COX expression. Fetal and newborn ovine PA endothelial cells (PAEC) and vascular smooth muscle (VSM) cells will be subjected to varying O2 and changes in PGI2 and PGE2 synthesis, and COX-1 and COX-2 protein and mRNA levels will be assessed. COX protein synthesis and degradation, and COX gene transcription and mRNA stability will also be evaluated. We hypothesize that hypoxia downregulates COX expression in fetal cells and upregulates expression in newborn cells.
Aim No. 2 is to determine the effects of estrogen and glucocorticoids on COX expression in fetal PAEC and VSM, using a similar approach. Estrogen enhances and glucocorticoids attenuate PGI2 synthesis in other cell types, and estrogen levels rise in the fetus and cortisol levels fall postnatally as PA COX protein increases. As such, we hypothesize that COX expression is upregulated by estrogen and downregulated by glucocorticoids.
Aim No. 3 is to determine if COX expression is upregulated by prolonged shear stress, which increases PG2 synthesis in nonpulmonary endothelium. Fetal PAEC will be subjected to 24 h of shear, and changes in COX function, protein and mRNA expression will be assessed.
Aim No. 4 is to test the hypothesis that there is cell- specific developmental regulation of COX expression. Immunohistochemical analyses and in situ hybridization for COX-1 and COX-2 will be performed in fetal, newborn and adult ovine lung. By revealing the molecular processes modulating PGI2 and PGE2 synthesis in the developing lung, these studies will increase our understanding of the mechanisms underlying successful pulmonary vascular, airway and alveolar function in the neonatal period.
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