Stretch and Vitamin A Effects on Alveolar Formation

描述(申请人提供)：人类研究表明，早产儿的视黄醇治疗可以改善早产儿和常规机械通气的病态后果，包括肺泡简化和随后的慢性肺部疾病(CLD)。动物研究进一步揭示视黄醇信号调节决定肺泡形成的过程，但人类和动物研究均未涉及CLD中视黄醇改善或调节的分子机制。利用早产、常规机械通气和随后的肺泡简化的羔羊模型，我们建立了常规机械通气早产羔羊(VITA+CV)的视黄醇疗法(VITA+CV)允许肺泡形成，类似于接受持续气道正压治疗(CPAP；早产对照)的早产羔羊和足月新生羔羊(妊娠对照)的肺泡形成。此外，VITA+CV或CPAP治疗与CV治疗相比，允许表面活性蛋白B(SP-B)、血管内皮生长因子(VEGF)和p53(细胞凋亡的标志)的表达。我们小组的一个主要新进展是，在我们的CLD模型中，一种特定的RARA激动剂在面对常规机械通气时刺激正常肺泡形成。我们的重点将放在维生素A对SP-B(直接调控)、VEGF(间接调控)和P53(间接调控)基因的调控上。肺泡II型细胞共同表达SP-B和血管内皮生长因子；间质表达P53。通过以下3个具体目标验证的总体假设是，维生素A治疗通过经典的维甲酸信号通路允许适当的肺泡形成，并导致对下游基因产物的直接和间接调节。具体目标1将确定维生素A对慢性呼吸机早产羔羊肺泡形成的挽救作用。特定目标2将比较使用或不使用RAR/RXR激动剂的CV与使用或不使用RAR/RXR拮抗剂的CPAP，以确定维生素A的作用机制。这一目标将检验我们模型中的维生素A信号通过RAR(/RXR)发生的假设。具体目标3将确定维生素A影响SP-B、血管内皮生长因子和p53启动子功能的分子机制。我们将用从羔羊身上分离的肺细胞来总结体内的研究结果，并瞬时转染稳定的细胞系来分离特定的机制。我们将测试假设，我们的3个目标基因的启动子区域是维甲酸反应的。因此，该项目将为慢性阻塞性肺疾病的肺泡形成及其失调提供新的机制见解。

DESCRIPTION (provided by applicant): Human studies suggest that retinol therapy in preterm infants ameliorates the morbid consequences of prematurity and conventional ventilation, which include alveolar simplification and subsequent chronic lung disease (CLD). Animal studies further reveal that retinol signaling regulates the processes that determine alveolar formation, but neither human nor animal studies have addressed the molecular mechanism by which either retinol amelioration or regulation occurs in CLD. Using a lamb model of prematurity, conventional ventilation, and subsequent alveolar simplification, we established that retinol therapy of conventionally ventilated preterm lambs (vitA+CV) permits alveolar formation that resembles the lungs of preterm lambs treated with continuous positive airway pressure (CPAP; preterm control), and term newborn lambs (gestation control). Furthermore, vitA+CV or CPAP treatment permits expression of surfactant protein B (SP-B), vascular endothelial growth factor (VEGF), and p53 (a marker of apoptosis) versus CV treatment. A major novel advancement by our group is that a specific RARa agonist stimulates normal alveolar formation in the face of conventional ventilation in our CLD model. Our focus on molecular mechanisms will be on vitamin A regulation of genes for SP-B (direct regulation), VEGF (indirect regulation), and p53 (indirect regulation). SP-B and VEGF are co-expressed by alveolar type II cells in the lung; p53 is expressed by mesenchyme. The overall hypothesis, tested by the following 3 specific aims, is that vitamin A therapy permits appropriate alveolar formation through the classical retinoid signaling pathway and results in both direct and indirect regulation of downstream gene products. Specific Aim 1 will determine the rescue of alveolar formation by vitamin A in chronically ventilated preterm lambs. Specific Aim 2 will compare CV with or without RAR/RXR agonists versus CPAP with or without RAR/RXR antagonists to identify mechanisms of action of vitamin A. This aim will test the hypothesis that vitamin A signaling in our model occurs through RAR(/RXR. Specific Aim 3 will identify the molecular mechanisms by which vitamin A impacts SP-B, VEGF, and p53 promoter function. We will use lung cells isolated from lambs to recapitulate the in vivo findings, and transient transfection of stable cell lines to isolate specific mechanisms. We will test the hypothesis that the promoter regions of our 3 target genes are retinoid responsive. Thus, this project will provide novel mechanistic insights about alveolar formation and its dysregulation in CLD.