PECAM-1 and Alveolization

描述（由申请人提供）：肺泡化是肺发育的最后阶段，在这个阶段，肺泡壁产生的隔膜向内突出，将大的初级肺泡空间分隔成较小的空气空间。在肺泡形成过程中，血管的扩张涉及血管生成的过程，在这个过程中，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依赖的配体相互作用，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募集到膜表面，在那里它介导局灶黏附蛋白的去磷酸化，导致局灶黏附的周转增加和内皮细胞运动性增强。这些PECAM-1依赖性活动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：表征PECAM-1缺失对小鼠肺泡化的影响。通过测定(i)肺形态学（ii）肺生理学和（iii）肺内皮细胞含量，PECAM-1缺失小鼠的肺泡化表型（维甲酸处理）将在形态学、细胞和功能水平上进行表征。具体目标2。明确SHP-2在介导中的作用

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.