CDC42调节肺血管内皮细胞迁移在支气管肺发育不良中的作用及机制研究

Bronchopulmonary dysplasia (BPD), the chronic lung disease associated with preterm birth, results from disruption of normal pulmonary vascular and alveolar growth. Vascular endothelial growth factor (VEGF) is a critical agent for vascular development enabling formation and remodeling of pulmonary vessels and alveolar structures, and the VEGF pathway is of importance in promoting angiogenesis in the developing lung. In developing lung, distal airspace epithelial-expressed VEGF attract developing capillaries to the epithelial basement membrane. A comparison of autopsy specimens taken from infants dying from BPD compared to infants dying without lung disease at similar post-conceptional ages and BPD animal models demonstrated that the lungs of infants with BPD or BPD animal models had an overall reduction in immunostaining for the endothelial specific marker CD31, suggesting a decrease in pulmonary microvascular density. Moreover, the pulmonary capillaries, when present, appeared to be abnormally dilated and frequently located within thickened alveolar septa, rather than immediately adjacent to the alveolar epithelium. We have demonstrated that intra-amniotic injection of LPS lead to reduced VEGF expression, and up-regulated RhoA activity, which negatively correlated with CDC42. These results suggest the abnormal migration of pulmonary vascular endothelial cells may contribute to arrest pulmonary vascular development. However, it remains unclear how VEGF influences the migration of pulmonary vascular endothelial cells. Each guidance molecule must activate a cascade of cytoplasmic effectors that eventually results in the cytoskeleton rearrangement. CDC42 has emerged as key regulators of cytoskeletal rearrangements. We thus examined the involvement of CDC42 in the regulation of pulmonary vascular endothelial cells migration by VEGF. We will study whether VEGF/VEGFR2 signaling induces spatiotemporal dynamics of CDC42 activity through inhibition of phosphorylation of IQGAP1. Furthermore, the lungs of mice were carried out to observe the migration behavior of pulmonary vascular endothelial cells with time-lapse imaging. We also investigated the spatial distribution of CDC42-GTP in the lung induced by LPS, and then disrupt direction of cell migration, leading to pulmonary vascular developmental arrest in BPD. It is beneficial to understand the pathogenesis of BPD patients and lung development.

支气管肺发育不良（BPD）和肺血管发育受阻有关。血管内皮生长因子（VEGF）可以诱导血管内皮细胞定向迁移，在肺血管发育过程中起到至关重要的作用。有研究发现在BPD动物模型以及患者肺部组织中可以观察到血管内皮细胞分布异常，项目组前期研究发现在LPS诱导宫内感染的BPD大鼠模型中，VEGF表达明显减少，且Western Blot检测发现报道与CDC42活性负相关的RhoA活性升高。据此，我们提出了BPD肺血管发育受阻可能的新机制：肺血管发育过程中肺血管内皮细胞定向迁移发生异常。本项目将探索VEGF影响肺血管内皮细胞定向迁移的机制，检测调节细胞骨架的中心环节—CDC42在VEGF引发的迁移变化中的信号传导通路，即VEGF/VEGFR2是否通过抑制IQGAP1磷酸化，使CDC42活性发生变化，致血管内皮细胞迁移方向发生改变，进而影响肺血管发育。研究结果有助理解BPD发病机制及更清楚解读肺发育。

气管肺发育不良(BPD)主要表现为肺泡化阻滞以及肺血管发育异常，本研究在前期研究基础上，通过构建的BPD动物模型和细胞实验对肺泡和肺血管发育受阻的机制进行了进一步的研究。结果发现， (1)LPS通过TGF-α /EGFR/14-3-3β信号通路提高了RhoA的活性，进而干扰肺肌成纤维细胞的极化和定向迁移，从而影响肺泡化过程中次级分隔的延伸，导致肺泡化阻滞。(2)血管内皮生长因子(VEGF)可以诱导血管内皮细胞定向迁移，在肺血管发育过程中起到至关重要的作用。在孕鼠羊膜腔注射LPS的BPD模型中，我们发现VEGF表达增加，同时在细胞实验中我们也发现LPS可以使人肺上皮细胞(BEAS-2B)和人脐静脉内皮细胞(HUVEC)VEGF表达增加，而且LPS和VEGF均可以干扰HUVEC的定向迁移。（3）有研究报道血管紧张素II (Ang II)可以诱导的VEGF及其受体表达增加。我们的研究发现LPS通过其受体TLR4激活TACE，导致血管紧张素转换酶2（ACE2）的胞外域脱落而使得ACE2活性下降，使Ang II表达增加。（4）此外我们还研究了文献报道的可能影响VEGF表达的CCR5，研究发现LPS诱导的新生大鼠BPD模型中，肺组织标本中CCR5表达明显增多，同时进一步的研究还发现LPS通过CCR5促进了巨噬细胞趋化能力，进而使得肺中巨噬细胞的募集增加，影响BPD的发生发展。综合上述研究结果，本研究探索了LPS干扰肺肌成纤维细胞和血管内皮细胞的定向迁移的分子机制，揭示了肺泡和肺血管发育的新机制，为深入理解BPD的分子病理机制和发现新的干预方法提供新思路和新靶点，并有助于人们更清楚地解读肺发育过程。

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