Analysis of Airway Serpins in Baboon Models of BPD

描述（由申请人提供）： 尽管新生儿重症监护取得了进展，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发生的机制是多因素的，尚未明确定义。 炎症是BPD的共同特征。 气道炎症与炎性细胞及其产物的流入有关。 中性粒细胞、巨噬细胞和气道上皮细胞在炎症级联反应期间释放强效蛋白酶。 这些蛋白酶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。 在健康的肺以及正常发育过程中，蛋白酶的活性受到局部和全身抗蛋白酶的严格调节。 在局部蛋白酶抑制剂中，ov-serpin家族的成员（卵清蛋白相关的serpin）是一组新兴的蛋白质，其由肺中的几种细胞类型大量表达，包括气道上皮细胞、内皮细胞和炎性细胞。 这些ov-serpin包括SERPINB 1、SERPINB 2、SERPINB 3、SERPINB 4、SERPINB 6和SERPINB 9。 Ov-serpins抑制一系列在肺损伤中起重要作用的蛋白酶。 基于它们的定位、调节和生化性质，我们假设ov-serpins理想地定位于肺组织和炎症细胞中以调节炎症期间释放的蛋白酶的活性，例如在BPD中发生的。 未成熟肺中ov-serpin表达的转录或翻译后改变可与未对抗的蛋白酶活性相关，从而增加对BPD的易感性。 为了研究我们的假设，我们建议利用特征良好的狒狒模型的BPD。 该项目的具体目标是：1）通过定量RT-PCR、免疫印迹和免疫组织化学将狒狒肺中ov-serpin mRNA和蛋白表达与BPD的发展相关联，2）通过动力学测定表征丝氨酸和半胱氨酸蛋白酶作为ov-serpin靶标在具有和不具有BPD的狒狒的气道中的活性，并鉴定ov-serpin的体内靶蛋白酶。通过免疫共沉淀和纳米毛细管HPLC-离子阱质谱法（LC-MS/MS）分析气管抽吸液来测定丝氨酸蛋白酶抑制剂，3）通过监测临床和生化参数以及肺组织病理学来确定重组SCCA 1（SERPINB 3）的施用是否改变狒狒模型中BPD的发展。 这些研究将加深我们对BPD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.