Regulation of mural cells during pulmonary capillary formation

描述(由申请人提供)： 项目摘要：肺气肿患者肺泡毛细血管结构和功能紊乱。恢复肺微血管系统也是间质纤维化和其他原因引起的肺动脉高压的重要治疗目标。实现这一目标的关键一步是确定在肺泡间隔形成过程中调节毛细血管扩张和成熟的因素。假设：肺泡毛细血管网络的形成和扩张需要内皮细胞、周细胞和肺成纤维细胞(LF)之间的合作。在次级间隔形成过程中，PDGF介导的信号转导周细胞和MF确保(A)这些细胞群体的扩张和维持，(B)迁移和建立与内皮的细胞间联系，以及(C)现有内皮管的细分，以允许毛细血管表面积的快速扩张。具体目标1：(A)证明在间隔形成过程中，随着毛细血管网络的形成，成纤维细胞和周细胞在空间上聚集在内皮细胞上；(B)研究PDGF介导的信号如何调节小鼠周细胞和肌成纤维细胞壁鞘的形成。具体目的2：研究PDGF-A和PDGF-B通过促进细胞迁移和沿内皮管形成细胞-细胞接触促进体外壁鞘形成的细胞机制。转基因小鼠将被用于鉴定和定位具有周细胞特征的(A)表达PDGF受体-α(PDGF-R1)或(B)具有周细胞特征的肺泡细胞。激光扫描共聚焦显微镜(LSCM)和体视学将被用来分析LF和周细胞的进入及其与网络中的肺泡内皮细胞的关系。这些研究将确定毛细血管内皮细胞与周围的LF和周细胞之间的距离是否有时间上的进行性减少。研究还将研究破坏周细胞和周细胞中的PDGF信号通路如何改变(A)毛细血管网络和(B)周细胞和周细胞的增殖和寿命。重要的信号通路是由PDGFs启动的，由Abelson激酶和rac1介导，并改变周细胞或LF的增殖和凋亡。第二个目标，我们将研究PDGF-A和PDGF-B如何在培养中指导内皮细胞、LF和周细胞的迁移和联合。毛细血管壁内皮细胞和壁细胞结合在一起。为了了解这些细胞是如何混合在一起的，我们将比较当壁细胞迁移到壁细胞与内皮基底膜结合时细胞表面的黏附分子。调节细胞间相互作用的分子，包括胶凝素和钙粘附素，将随着细胞从迁移状态转变为静止状态而被研究，并形成贴壁和紧密连接。详细的时间推移显微镜研究将揭示层粘连蛋白和整合素如何促进PDGF-A或PDGF-B刺激的成纤维细胞和周细胞的迁移。这些研究将提供有关肺内非发芽血管生成(NSA)的新信息。更好地了解NSA可能会确定一些因素，这些因素在涉及肾脏和大脑等其他微血管床的疾病的发病和修复中也是重要的。微血管病变是糖尿病、中风和慢性肾功能不全的核心，这些疾病在退伍军人中越来越普遍。。对退伍军人医疗保健的潜在影响：这些研究可能会促进新疗法的开发，从而改善结果并降低治疗肺气肿和血管疾病的成本，这些疾病在退伍军人中很常见。

DESCRIPTION (provided by applicant): PROJECT SUMMARY: The structure and function of alveolar capillaries are deranged in pulmonary emphysema. Restoration of the pulmonary microvasculature is also an important therapeutic goal for interstitial fibrosis and other causes of pulmonary hypertension. A critical step towards achieving this goal is to identify factors which regulate capillary expansion and maturation during alveolar septal formation. Hypothesis: Formation and expansion of the alveolar capillary network requires cooperation among endothelial cells, pericytes, and lung fibroblasts (LF). During secondary septal formation, PDGF-mediated signaling directs pericytes and MF to ensure (a) expansion and maintenance of these cell populations, (b) migration and establishment of intercellular contacts with the endothelium, and (c) sub-division of the existing endothelial tubes to allow rapid expansion of capillary surface area. Specific Aim 1: To (a) demonstrate that fibroblasts and pericytes spatially converge on the endothelium as the capillary meshwork forms during septation and (b) investigate how PDGF-mediated signaling regulates formation of the mural sheath by pericytes and myofibroblasts in mice. Specific Aim 2: Examine cellular mechanisms whereby PDGF-A and PDGF-B promote formation of the mural sheath in vitro by enhancing cell migration and the formation of cell- cell contacts along endothelial tubes. Genetically modified mice will be used to identify and localize alveolar cells which (a) express PDGF receptor-alpha (PDGF-R1) or (b) have characteristics of pericytes. Laser scanning confocal microscopy (LSCM) and stereology will be used to analyze ingress of LF and pericytes and their association with alveolar endothelial cells in the meshwork. These studies will ascertain if there is a temporally progressive decrease in the distance between the capillary endothelium and surrounding LF and pericytes. Studies will also examine how disrupting PDGF-signaling pathways in LF and pericytes alters (a) the capillary meshwork and (b) pericyte and LF proliferation and longevity. Important signaling pathways, which are initiated by PDGFs, are mediated by Abelson kinase and Rac1, and alter pericyte or LF proliferation and apoptosis will be examined. The second aim we will examine how PDGF-A and PDGF-B direct the migration and association of endothelial cells, LF, and pericytes in culture. Endothelial and mural cells coalesce in the capillary wall. To learn how these cells intermingle, cell surface adhesion molecules will be compared when mural cells migrate to when the mural coalesce with the endothelial basement membrane. Molecules, including nectins and cadherins, which regulate cell-cell interactions will be investigated as cells transition from a migratory to a sessile state, and form adherens and tight junctions. Detailed time-lapse microscopic studies will reveal how laminin and integrins promote PDGF-A or PDGF-B-stimulated migration of fibroblasts and pericytes. These studies will provide novel information about non-sprouting angiogenesis (NSA) in the lung. A better understanding of NSA may identify factors that are also important in the pathogenesis and repair of diseases involving other microvascular beds such as the kidneys and brain. Microangiopathies are central to diabetes, stroke, and chronic renal insufficiency, which are becoming more prevalent among veterans. . Potential impact for veterans' health care: These studies may foster the development of new therapies resulting in improved outcomes and lower costs for treating pulmonary emphysema and vascular diseases, which is are common among veterans.