PECAM-1 and Alveolization

描述(申请人提供)：肺泡化代表肺发育的最后阶段，大的初级肺泡腔被来自肺泡壁的向内突出的隔膜分割成较小的空气空间。在肺泡化过程中，血管系统的扩张涉及到血管生成的过程，在这个过程中，新的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动的分子，如PECAM-1，可能对肺泡化很重要。我们的假设是，在肺发育过程中，血管生成刺激启动了PECAM-1依赖的配体相互作用，从而触发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在膜表面的募集，在那里它介导了焦点黏附蛋白的去磷酸化，导致焦点黏附的周转增加和内皮细胞的运动性增强。这些依赖于PECAM-1的活性促进了有效的血管生成，从而促进了肺泡化。提出了三个具体目标：具体目标1.研究PECAM-1缺失对小鼠牙槽骨形成的影响。PECAM-1基因缺失小鼠的肺泡化表型(维甲酸治疗)将通过测定(I)肺形态(II)肺生理学和(III)肺内皮细胞含量在形态、细胞和功能水平上进行表征。具体目标2.确定SHP-2在调解中的作用 PECAM-1依赖的细胞运动。稳定表达PECAM-1的细胞将被编码三个SHP-2显性负结构之一的腺病毒载体转导，该负结构在催化区域、介导与支架蛋白相互作用的C-末端区域或在两个区域都有突变。转导细胞将被分析：(I)局部粘连的数量和分布；(Ii)关键的局部粘连酪氨酸磷酸化事件的发生；以及(Iii)使用已建立的细胞迁移和管形成的体外模型来分析细胞的运动性。具体目的3.确定PECAM-1酪氨酸磷酸化在体内血管生成和肺泡化过程中的作用。转基因小鼠将产生在PECAM缺失的背景下以内皮受限的方式表达的转基因小鼠，要么是野生型PECAM-1，要么是Y663和Y686突变的PECAM-1。然后将在这些动物身上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化的分子基础的理解，并可能为早产儿支气管肺发育不良和成人肺气肿或纤维化肺疾病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.