PECAM-1 and Alveolization

描述（由申请人提供）：肺泡化代表肺发育的最后阶段，其中大的初级肺泡空间被源自肺泡壁的隔膜向内突起分隔成较小的空气空间。 在肺泡化过程中，血管系统的扩张涉及血管生成过程，其中新血管从先前存在的血管中产生。 这表明调节血管生成所需的内皮细胞活性的分子，如PECAM-1，可能对肺泡形成很重要。 我们的假设是，在肺发育过程中，血管生成刺激启动PECAM-1依赖性配体相互作用，触发PECAM-1酪氨酸磷酸化和内皮PECAM-1与SHP-2的关联。 这促进了SHP-2向膜表面的募集，在膜表面SHP-2介导粘着斑蛋白的去磷酸化，导致粘着斑的周转增加和内皮细胞运动性增强。 这些PECAM-1依赖性活性促进有效的血管生成，从而促进肺泡形成。 提出了三个具体目标：具体目标1。 表征PECAM-1缺失对小鼠肺泡形成的影响。 通过测定（i）肺形态学（ii）肺生理学和（iii）肺内皮细胞含量，在形态学、细胞和功能水平上表征PECAM-1敲除小鼠中肺泡化（维甲酸处理）的表型。具体目标2。确定SHP-2在调解中的作用 PECAM-1依赖性细胞运动。 将用腺病毒载体转导表达PECAM-1的稳定细胞转染子，所述腺病毒载体编码在催化结构域、介导与支架蛋白相互作用的C-末端结构域或在这两个区域中具有突变的三种SHP-2显性阴性构建体之一。 将使用已建立的细胞迁移和管形成体外模型分析转导细胞的（i）粘着斑的数量和分布;（ii）关键粘着斑酪氨酸磷酸化事件的发生;和（iii）细胞运动性。具体目标3。 确定PECAM-1酪氨酸磷酸化在体内血管生成和肺泡形成过程中的作用。 转基因小鼠将在PECAM无效背景下产生，其以内皮限制性方式表达野生型PECAM-1或其中Y663和Y686已突变的PECAM-1。 然后将在这些动物中研究体内血管生成和肺泡化。 这些研究将进一步加深我们对肺泡形成的分子基础的理解，并可能为支气管肺发育不良的早产儿和肺气肿或纤维化肺病的成人再生功能性肺泡提供新的方法。

DESCRIPTION (provided by applicant): Alveolization represents the final phase of lung development in which large primary alveolar spaces are partitioned into smaller air spaces by inward protrusions of septae derived from the alveolar walls. During alveolization, expansion of the vasculature involves the process of angiogenesis in which new vessels arise from preexisting ones. This suggests that molecules, such PECAM-1, which regulate endothelial cell activity required for angiogenesis are likely to be important for alveolization. Our hypothesis is that during lung development, angiogenic stimuli initiate PECAM- 1 dependent ligand interactions that trigger PECAM-1 tyrosine phosphorylation and an association of endothelial PECAM-1 with SHP-2. This facilitates the recruitment of SHP-2 to the membrane surface, where it mediates the dephosphorylation of focal adhesion proteins, leading to increased turnover of focal adhesions and enhanced endothelial cell motility. These PECAM-1 dependent activities facilitate efficient angiogenesis and thus alveolization. Three specific aims are proposed: Specific Aim 1. Characterize the effect of the loss of PECAM-1 on murine alveolization. The phenotype of alveolization (¿ retinoic acid treatment) in PECAM-1 null mice will be characterized at a morphological, cellular, and functional level by determination of (i) lung morphology (ii) lung physiology and (iii) lung endothelial cell content. Specific Aim 2. Define the role of SHP-2 in mediating PECAM-1-dependent cell motility. Stable cell transfectants expressing PECAM-1 will be transduced with adenoviral vectors encoding one of three SHP-2 dominant negative constructs bearing mutations in the catalytic domain, the C-terminal domain that mediates interactions with scaffold proteins or in both regions. Transduced cells will be analyzed for (i) the number and distribution of focal adhesions; (ii) the occurrence of critical focal adhesion tyrosine phosphorylation events; and (iii) cell motility using established in vitro models of cell migration and tube formation. Specific Aim 3. Determine the role of PECAM-1 tyrosine phosphorylation during in vivo angiogenesis and alveolization. Transgenic mice will be generated on the PECAM-null background that express in an endothelial-restricted manner, either wild type PECAM-1, or PECAM-1 in which Y663 and Y686 have been mutated. In vivo angiogenesis and alveolization will then be studied in these animals. These studies will further our understanding of molecular basis of alveolization and may provide insights into new approaches for regenerating functional alveoli in premature infants with bronchopulmonary dysplasia and adults with emphysema or fibrotic lung disease.