HEPARIN-REGULATED ECM MOLECULES IN CONTROL OF PULMONARY VASCULAR SMOOTH MUSCLE

婴幼儿持续性肺动脉高压的肺血管 高血压以平滑肌细胞（SMC）过多为特征 复制和基质沉积。这些结构变化可能 加重、维持或直接导致高血压状态。的 SMC复制的控制似乎与 细胞外基质的特征：肝素样糖胺聚糖， 例如，已被证明是肺SMC的有效抑制剂 增长这种抑制作用可能是由于肝素与 生长促进糖蛋白血小板反应蛋白（TS），或来自 诱导合成未表征的60，000 kD短链 胶原我们假设肝素调节的细胞外基质 分子（TS和60，000 kD胶原蛋白）起着重要的作用，但尚未确定， 在缺氧性肺动脉高压病理生理学中的作用 新生儿由于参与SMC的调节 复制的本申请中提出的研究集中在i） TS在增强SMC生长中发挥的机械作用，ii） 60 kD肝素诱导型胶原在血管SMC生物学中的作用，以及 iii）这些分子在肺血管系统中的作用， 正常发育和持续性肺动脉高压期间。的 期间TS在肺血管中的表达模式 发展和高血压条件下将被确定， 与SMC复制增加相关。的分子机制 TS促进SMC复制的能力的基础将是 研究使用合成或重组肽;特别是，我们 将检查外源性纯化TS或TS肽对 第二信使途径，EGF受体和细胞外蛋白水解。 我们还计划构建和表征TS-少SMC线使用 反义技术控制合成的因素 新的，未表征的，肝素调节的60 kD胶原蛋白将被 在不同发育年龄的培养SMC中测定。我们将 从培养SMC的胃蛋白酶消化物中分离蛋白， 抗体的这些抗体将用于确定 正常肺血管床中该蛋白的表达 发育和高血压状况。最后，我们将获得一个 cDNA的60 kD胶原蛋白，推导出完整的氨基酸序列， 蛋白质，并研究mRNA的分布和调控。 总之，这些数据应该有助于我们不断发展 了解细胞外基质分子如何调节基因表达 和细胞功能，以及这些相互作用如何有助于 肺动脉高压和其他血管疾病状态的发展。

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.