This proposal aims to understand endothelial cell (EC)-smooth muscle cell (SMC) interactions in normal and abnormal pulmonary artery development. Prior evidence suggest that ECs inhibit SMC growth; that heparin, a non- physiologic compound, is an effective inhibitor of SMC growth in vivo and in vitro; that HS proteoglycans are molecules that contain heparin-like glycosaminoglycan (GAG) chains; and that endothelial cells produce various HS proteoglycans. It is unclear whether one or more of these EC derived HS proteoglycans can inhibit SMC growth. Heparan sulfate proteoglycans, proteins containing covalently attached HS chains, are grouped together on the basis of the HS chain, but represent a variety of gene products. These are currently defined on the basis of molecular mass, buoyant density, HS chain size, and cellular location. It is not known whether the HS chains are distinct on the different gene products made by the same cell. The only way to unequivocally identify distinct HS proteoglycans is to clone and sequence the core protein and map the HS chain. The normal formation of the pulmonary vascular tree requires EC-SMC interaction. If the EC and the basement membrane is removed from the pulmonary vasculature, abnormal muscularization occurs. If the basement membrane is left intact or the vasculature treated wit heparin, this abnormal muscularization does not occur. The infant born with this abnormal muscularization shows neonatal pulmonary hypertension, which results in the right-to-left shunting of pulmonary blood flow. This disease is of great concern to physicians, due to the high (50%) mortality rate, and significant morbidity (seizures, development delay, thromboembolic complications). The proposed work will establish new insights into the molecular basis of the EC-SMC interaction during pulmonary artery morphogenesis, and result in rational prevention and therapy for neonatal pulmonary hypertension. This project will also establish new insights into the post-translational modification of HS proteoglycans.
The specific aims of this research are to: (i) isolate and characterize the Hs proteoglycans produced by cultured pulmonary microvascular ECs by preparing monoclonal antibodies against their core proteins and by isolating and sequencing their core protein cDNAs; (ii) establish which HS proteoglycans inhibit growth by adding purified HS proteoglycans to pulmonary artery SMC cultures and attempting to inhibit the expression of specific HS proteoglycans in pulmonary microvascular EC cultures; (iii) establish that the structural characteristics of the HS chain convey the growth inhibitory activity by comparing the oligosaccharide maps of highly active and less active HS proteoglycans; and (iv) assess the role of the core protein in specifying the structural characteristics of the HS chains by comparing the oligosaccharide maps of the HS proteoglycans.
