Regulation of mural cells during pulmonary capillary formation

描述（由申请人提供）： 项目概述：肺气肿时肺泡毛细血管的结构和功能紊乱。肺微血管的恢复也是间质纤维化和其他肺动脉高压原因的重要治疗目标。实现这一目标的关键一步是确定在肺泡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。在继发性中隔形成期间，PDGF介导的信号传导指导周细胞和MF以确保（a）这些细胞群的扩增和维持，（B）与内皮细胞的细胞间接触的迁移和建立，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增。具体目标1：为了（a）证明成纤维细胞和周细胞在分隔期间随着毛细血管网的形成而在空间上会聚在内皮上，以及（B）研究PDGF介导的信号传导如何调节小鼠中周细胞和肌成纤维细胞形成壁鞘。具体目标二：研究PDGF-A和PDGF-B通过增强细胞迁移和沿着内皮管形成细胞-细胞接触促进体外壁鞘形成的细胞机制。将使用遗传修饰小鼠来鉴定和定位肺泡细胞，其（a）表达PDGF受体-α（PDGF-R1）或（B）具有周细胞的特征。激光扫描共聚焦显微镜（LSCM）和体视学将用于分析LF和周细胞的侵入及其与肺泡内皮细胞在网状结构中的相关性。这些研究将确定毛细血管内皮与周围LF和周细胞之间的距离是否存在时间上的进行性减小。研究还将探讨如何破坏LF和周细胞的PDGF信号通路改变（a）毛细血管网和（B）周细胞和LF增殖和寿命。由PDGF启动的重要信号通路由Abelson激酶和Rac 1介导，并将检查改变周细胞或LF增殖和凋亡。第二个目的，我们将研究如何PDGF-A和PDGF-B直接迁移和协会的内皮细胞，LF，周细胞在文化。内皮细胞和壁细胞在毛细血管壁中结合。为了了解这些细胞是如何混合的，将比较壁细胞迁移时和壁与内皮基底膜结合时的细胞表面粘附分子。分子，包括nectin和钙粘蛋白，调节细胞间的相互作用，将被调查的细胞从迁移过渡到固着状态，并形成adherens和紧密连接。详细的延时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迁移。这些研究将提供新的信息，非萌芽血管生成（NSA）在肺。更好地了解NSA可能会发现在涉及其他微血管床（如肾脏和大脑）的疾病的发病机制和修复中也很重要的因素。微血管病是糖尿病、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，从而改善治疗肺气肿和血管疾病的结果并降低成本，这在退伍军人中很常见。 公共卫生相关性： 项目叙述：虽然吸烟的流行率已经下降，但慢性阻塞性肺疾病（COPD）及其伴随的病理性肺异常，肺气肿，是常见的，并且不成比例地导致退伍军人的住院和医疗保健费用。由于目前大多数可用的疗法仅针对症状，因此确定限制疾病进展的策略仍然是一个重要目标。该提案将研究调节支持结构细胞如何与肺毛细血管结合和修饰肺毛细血管的机制，以改善气体交换、增加血流量并提供血管稳定性。这些研究与其他血管疾病有关，包括中风，糖尿病和慢性肾功能不全，这些疾病在退伍军人中也很常见。

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. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE: Although the prevalence of smoking has declined, chronic obstructive pulmonary disease (COPD) and its attendant pathological pulmonary abnormality, emphysema, are common and disproportionately contribute to hospitalizations and health care costs among veterans. Because most of the currently available therapies only address symptoms, identification of strategies that limit disease progression remains an important goal. This proposal will investigate mechanisms that regulate how supporting structural cells associate with and modify lung capillaries, to improve gas exchange, increase blood flow, and provide vascular stability. These studies are relevant to other vascular diseases including stroke, diabetes, and chronic renal insufficiency, which are also common among veterans.