The pulmonary vasculature of infants with persistent pulmonary hypertension is characterized by excessive smooth muscle cell (SMC replication and matrix deposition. These structural alterations may exacerbate, maintain, or directly cause the hypertensive state. The control of SMC replication appears to be intricately coupled to the character of the extracellular matrix: heparin-like glycosaminoglycans, for example, have been shown to be potent inhibitors of pulmonary SMC growth. This inhibition may result from an interaction of heparin with the growth-facilitative glycoprotein thrombospondin (TS), or from the induction of synthesis of an uncharacterized 60,000kD short-chain collagen. We hypothesize that heparin-regulated extracellular matrix molecules (TS and the 60,000kD collagen) play important, yet undefined, roles in the pathophysiology of hypoxic pulmonary hypertension of the newborn by virtue of their involvement in the regulation of SMC replication. The research proposed in this application centers on i) the mechanistic role played by TS in the potentiation of SMC growth, ii) the role of 60kD heparin-inducible collagen in vascular SMC biology, and iii) the roles played by these molecules in the pulmonary vasculature in normal development and during persistent pulmonary hypertension. The pattern of expression of TS in the pulmonary vasculature during development and under hypertensive conditions will be determined and correlated with increased SMC replication. The molecular mechanisms underlying the ability of TS to facilitate SMC replication will be investigated using synthetic or recombinant peptides; in particular, we will examine the effects of exogenous purified TS or TS peptides on second messenger pathways, EGF receptors, and extracellular proteolysis. We also plan to construct and characterize TS-less SMC lines using antisense technologies. The factors which control the synthesis of the novel, uncharacterized, heparin-regulated 60kD collagen will be determined in cultured SMC of different developmental ages. We will isolate the protein from pepsin digests of cultured SMC and prepare antibodies. The antibodies will be used to determine the pattern of expression of this protein in the pulmonary vascular bed under normal developmental and hypertensive conditions. Finally, we will obtain a CDNA for the 60kD collagen, derive the complete amino acid sequence of the protein, and study the distribution and regulation of the MRNA. Taken together, the data should contribute substantially to our evolving knowledge of how extracellular matrix molecules regulate gene expression and cell function, and how these interactions contribute to the development of pulmonary hypertension and other vascular disease states.
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