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

婴幼儿持续性肺病的肺血管构筑 高血压的特征是平滑肌细胞(SMC)过多 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。这个 对SMC复制的控制似乎与 细胞外基质的特征：类肝素糖胺聚糖， 例如，已被证明是肺SMC的有效抑制剂 成长。这种抑制可能是由于肝素与 促生长糖蛋白凝血酶敏感蛋白(TS)，或来自 6万kD短链的诱导合成 胶原蛋白。我们假设肝素调节的细胞外基质 分子(TS和6万kD胶原蛋白)发挥着重要的作用，但还没有确定， 缺氧性肺动脉高压的病理生理机制 通过参与SMC的调节而产生的新生儿 复制。本申请中提出的研究主要集中在：1) TS在促进SMC生长中的机制作用，II) 60kD肝素诱导的胶原在血管SMC生物学中的作用 三)这些分子在#年的肺血管系统中所起的作用 正常发育和持续性肺动脉高压期间。这个 血管紧张素转换酶在肺血管中的表达 发展和高血压的情况将被确定和 与SMC复制增加相关。分子机制 TS促进SMC复制的基础能力将是 使用合成或重组多肽进行研究；尤其是，我们 将研究外源纯化的TS或TS多肽对 第二信使通路、EGF受体和胞外蛋白分解。 我们还计划构建和表征无TS的SMC系 反义技术。控制合成的因素 新型的、未特化的、受肝素调节的60kD胶原蛋白将被 在不同发育年限的培养的SMC中测定。我们会 从培养的SMC胃酶消化液中分离蛋白质并制备 抗体。抗体将被用来确定 该蛋白在正常肺血管床中的表达 发育和高血压状况。最后，我们将获得一个 为60kD的胶原基因，推导出其完整的氨基酸序列 并对该基因的分布和调控进行研究。 综上所述，这些数据应该会对我们的发展做出重大贡献 关于细胞外基质分子如何调控基因表达的知识 和细胞功能，以及这些相互作用如何有助于 发展为肺动脉高压和其他血管疾病状态。

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.