Analysis of Airway Serpins in Baboon Models of BPD

描述(由申请人提供)： 尽管最近在新生儿重症监护方面取得了进展，但支气管肺发育不良(BPD)仍然是婴儿发病率和死亡率的主要原因。BPD的发生机制是多因素的，尚未明确界定。炎症是BPD的常见特征。呼吸道炎症与炎症细胞及其产物的涌入有关。中性粒细胞、巨噬细胞和呼吸道上皮细胞在炎性级联反应中释放有效的蛋白酶。这些蛋白参与多种生理和病理过程，如细胞外基质重塑、血管生成、细胞凋亡和先天免疫。在健康的肺中以及在正常的发育过程中，蛋白水解酶的活性受到局部和系统抗蛋白水解酶的严格调节。在局部的蛋白酶抑制剂中，Ov-serpin家族的成员(卵清蛋白相关的serpins)是一组新出现的蛋白质，在肺的几种细胞类型中都有丰富的表达，包括呼吸道上皮细胞、内皮细胞和炎症细胞。这些Ov-serp包括serpinB1、-B2、-B3、-B4、B6和B9。Ov-serpins抑制了一系列在肺损伤中发挥重要作用的蛋白酶。根据它们的定位、调节和生化特性，我们假设Over-Serpins理想地定位于肺组织和炎症细胞中，以调节炎症过程中释放的蛋白酶的活性，例如BPD。未成熟肺中Ov-serpin表达的转录或翻译后变化可能与非对抗性蛋白酶活性相关，从而增加了BPD的易感性。为了验证我们的假设，我们建议使用BPD的特征很好的狒狒模型。本项目的具体目标是：1)通过定量RT-PCR、免疫印迹和免疫组织化学的方法，将恒河猴肺组织中Over-serpin基因和蛋白的表达与BPD的发生发展联系起来；2)通过动力学方法表征BPD患者和非BPD患者呼吸道中作为Over-serpin靶标的丝氨酸蛋白酶和半胱氨酸蛋白酶的活性，并通过免疫共沉淀和纳米毛细管高效液相色谱-离子捕获质谱仪(LC-MS/MS)分析气管吸出液，鉴定Over-Serpins的体内靶标蛋白。3)观察重组SCCA1(SERPINB3)的临床、生化指标及肺组织病理学变化，以确定重组SCCA1(SERPINB3)是否能改变BPD的发生。这些研究将加深我们对BPD潜在的细胞和分子机制的理解，并促进BPD新的预防和治疗策略的发展。

DESCRIPTION (provided by applicant): Despite recent advances in neonatal intensive care, bronchopulmonary dysplasia (BPD) remains a major cause of infant morbidity and mortality. The mechanisms for the development of BPD are multifactorial and not yet clearly defined. Inflammation is a common feature of BPD. Airway inflammation is associated with an influx of inflammatory cells and their products. Neutrophils, macrophages and airway epithelial cells release potent proteinases during the inflammatory cascade. These proteinases are involved in diverse physiologic and pathologic processes, such as extracellular matrix remodeling, angiogenesis, apoptosis and innate immunity. In the healthy lung as well as during normal development, the activity of proteinases are tightly regulated by local and systemic anti-proteinases. Among the local proteinase inhibitors, members of the ov-serpin family (ovalbumin-related serpins) are an emerging group of proteins that are abundantly expressed by several cell types in the lung, including airway epithelial cells, endothelial cells and inflammatory cells. These ov-serpins include SERPINB1, -B2, -B3, -B4, B6 and B9. Ov-serpins inhibit an array of proteinases that play significant roles in lung injury. Based on their localization, regulation and biochemical properties, we hypothesize that ov-serpins are ideally localized in the lung tissue and inflammatory cells to regulate the activity of proteinases released during inflammation, such as occurs in BPD. Transcriptional or post-translational alterations in ov-serpin expression in the immature lung can be associated with unopposed proteinase activity and thus, increased susceptibility to BPD. In order to investigate our hypothesis, we propose to utilize the well-characterized baboon models of BPD. The specific aims of this project are to: 1) correlate ov-serpin mRNA and protein expression in baboon lungs with the development of BPD by quantitative RT-PCR, immunoblotting, and immunohistochemistry, 2) characterize the activity of serine- and cysteine proteinases as ov-serpin targets in the airways of baboons with and without BPD by kinetic assays and identify in vivo target proteinases of ov-serpins by analyzing tracheal aspirate fluids by co-immunoprecipitation and nano-capillary HPLC-ion trap mass spectrometry (LC-MS/MS), 3) determine whether administration of recombinant SCCA1 (SERPINB3) alters the development of BPD in baboon models by monitoring clinical and biochemical parameters, and lung histopathology. These studies will enhance our understanding of the cellular and molecular mechanisms underlying BPD and facilitate development of novel preventive and therapeutic strategies for BPD.