Morphogenesis of the pulmonary artery smooth muscle layer

描述(申请人提供)：血管平滑肌细胞(VSMCs)的去分化、增殖和运动能力增强是许多血管疾病发病的关键因素。因此，对正常血管的VSMC层发育的详细了解有望为血管疾病提供关键的见解。然而，任何特定的血管或血管床的形态发生的潜在过程还不是很清楚。这项建议的中心目标是阐明在小鼠肺发育的最初四天中围绕左肺动脉平滑肌细胞(SMC)层形态发生的关键分子和细胞事件。这一目标是迈向长期目标的第一步，长期目标是描述在整个发育过程中建立所有层的肺动脉的关键事件。 实现这些目标的一个基本要素是确定特定的信号通路在肺动脉发育中所起的作用。血小板衍生生长因子(PDGF)诱导的信号转导与冠状动脉成形术后肺动脉高压和再狭窄的发病机制有关，然而，PDGF信号在这些疾病或大血管形态发生中的具体作用仍不清楚。我推测，通过PDGF途径的信号对多点早期肺间充质细胞群的维持、增殖和/或募集至关重要，该细胞群具有分化为左肺动脉SMC层的能力。 该建议有三个具体目标：1)建立分子标志物和细胞事件的时间表，以识别小鼠肺间充质细胞向分化的左肺动脉SMC转变的特定阶段，并将该时间表与内皮细胞和非血管组织的发育联系起来；2)确定PDGF受体介导的信号通路在左肺动脉SMC发育中的激活模式和作用；3)通过谱系分析确定早期PDGF受体β阳性的间充质细胞在肺中的命运。为了实现这些目标，将从野生型和转基因小鼠胚胎中解剖肺，并进行整体免疫染色、原位杂交或X-Gal染色。在选定的实验中，胚胎肺将在分析之前进行培养。共聚焦显微镜将被用来在细胞水平上分析荧光染色。 总而言之，这项拟议的工作将使左肺动脉成为了解特定血管形态发生的最好例子。这些研究将对冠状动脉动脉粥样硬化等常见血管疾病的潜在机制产生关键的见解。此外，这项工作有望推进对肺内严重缺乏研究的平滑肌细胞生理学疾病的研究，如肺动脉高压和淋巴管肌瘤病。

DESCRIPTION (provided by applicant): Dedifferentiation and enhanced proliferation and motility of vascular smooth muscle cells (VSMCs) are key elements in the pathogenesis of many vascular diseases. Thus, a detailed understanding of the development of the VSMC layer of normal blood vessels promises to provide critical insights into vascular disease. However, the processes underlying the morphogenesis of any specific vessel or vascular bed are not well understood. The central goal of this proposal is to elucidate the key molecular and cellular events encompassing the morphogenesis of the smooth muscle cell (SMC) layer of the left pulmonary artery during the initial four days of murine lung development. This goal is the first step towards the long term objective of delineating the critical events in the building of all layers of the pulmonary artery throughout development. An essential element of achieving these goals is to characterize the roles specific signaling pathways play in pulmonary artery development. Platelet derived growth factor (PDGF)-induced signaling has been implicated in the pathogenesis of pulmonary artery hypertension and restenosis following coronary artery angioplasty; yet, the specific effects of PDGF signaling in these diseases or in the morphogenesis of large vessels remain elusive. I hypothesize that signaling through the PDGF pathway is critical for the maintenance, proliferation and/or recruitment of a multipoint early lung mesenchymal cell population that has the capacity to differentiate into the SMC layer of the left pulmonary artery. The proposal has three specific aims: 1) establish a timeline of molecular markers and cellular events that identify specific stages during the transition of a murine lung mesenchymal cell into a differentiated left pulmonary artery SMC and relate this timeline to'endothelial cell and non-vascular tissue development; 2) identify the pattern and effects of activation of PDGF receptor-mediated signaling pathways in left pulmonary artery SMC development; 3) determine the fate in the lung of early PDGF receptor-beta-positive mesenchymal cells through lineage analysis. To achieve these aims, lungs will be dissected from wildtype and transgenic mouse embryos and subjected to whole mount immunostaining, in situ hybridization or X-gal staining. In selected experiments, embryonic lungs will be cultured prior to analysis. Confocal microscopy will be used to analyze fluorescent stains on a cellular level. In sum, the proposed work will make the left pulmonary artery the best understood example of morphogenesis of a specific blood vessel. These studies will yield critical insights into the mechanisms underlying prevalent vascular diseases such as coronary artery atherosclerosis. In addition, this work promises to advance the investigation of critically understudied disorders of smooth muscle cell physiology in the lung, such as pulmonary artery hypertension and lymphangioleiomyomatosis.