Analysis of Airway Serpins in Baboon Models of BPD

狒狒BPD模型中气道丝氨酸蛋白酶抑制剂分析

• 批准号: 6948181
• 负责人: SULE CATALTEPE
• 金额: $ 18.91万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2006-11-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6948181
• 关键词: baboons; bronchopulmonary dysplasia; cooperative study; disease /disorder model; enzyme activity; histopathology; immunocytochemistry; immunoprecipitation; inflammation; liquid chromatography mass spectrometry; molecular pathology; polymerase chain reaction; protease inhibitor; recombinant proteins; serine proteinases; western blottings

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.

描述（由申请人提供）： 尽管新生儿重症监护术的最新进展，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开发的机制是多因素的，尚未明确定义。 炎症是BPD的常见特征。 气道炎症与炎症细胞及其产物的流入有关。 在炎症性级联反应期间，中性粒细胞，巨噬细胞和气道上皮细胞释放有效的蛋白酶。 这些蛋白酶参与了多种生理和病理过程，例如细胞外基质重塑，血管生成，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（与卵蛋白相关的丝氨酸）的成员是一组新兴的蛋白质，这些蛋白质由肺中的几种细胞类型（包括气道上皮细胞，内皮细胞和炎症细胞）大量表达。 这些OV -Serpins包括Serpinb1，-b2，-b3，-b4，b6和b9。 OV-serpins抑制一系列在肺损伤中起重要作用的蛋白酶。 根据它们的定位，调节和生化特性，我们假设OV-serpins在肺组织和炎症细胞中理想地定位于炎症过程中释放的蛋白酶活性，例如BPD中发生的蛋白酶的活性。 未成熟肺中OV-serpin表达的转录或后翻译变化可能与无反抗的蛋白酶活性有关，从而增加了对BPD的敏感性。 为了调查我们的假设，我们建议利用BPD的狒狒模型。 该项目的具体目的是：1）将狒狒肺中的OV-serpin mRNA和蛋白质表达与定量RT-PCR，免疫印迹和免疫组织化学的BPD的发展相关联，2）表征丝氨酸和囊这氏蛋白酶的活性。通过动力学测定法和不带有BPD的狒狒气道中的OV-serpin靶标，并通过共同免疫沉淀和纳米含量的HPLC-ion trap Masseptry（LC-MS/ LC-MS/ LC-MS/ LC-MS/ LC-MS/ LC-MS/ LC-MS/ LC-MS/ LC-MS/ LC-MS/ LC-MS/ nano-complations）通过分析气管抽吸流体来鉴定OV-serpins的体内靶蛋白酶。 MS），3）确定重组SCCA1（SERPINB3）是否通过监测临床和生化参数以及肺组织病理学来改变狒狒模型中BPD的发展。 这些研究将增强我们对BPD潜在的细胞和分子机制的理解，并促进BPD的新型预防和治疗策略的发展。