Protein Therapy for Acute Lung Injury

描述（由申请人提供）：目的：本项目的主要目的是开发一种基于蛋白质的治疗方法，以增强内皮屏障特性。虽然急性肺损伤（ALI）是发病率和死亡率的主要原因，但没有治愈性治疗。ALI的主要病理学是肺水肿，这是肺中内皮（EC）屏障恶化的直接结果。在这个项目中，我们将纯化其生物活性形式的屏障保护蛋白，Rac 1，β-辅肌动蛋白和黏着斑蛋白。使用我们新开发的方法，我们将这些屏障保护蛋白质连接到细胞渗透性肽达特上。将通过小鼠尾静脉注射给予蛋白质，以确定它们在保护免受ALI方面的功效。操作步骤：我们将利用杆状病毒表达载体系统（BEVS）构建表达目的蛋白Rac 1、黏着斑蛋白和β-辅肌动蛋白的重组病毒。该扩增的病毒将用于感染Sf 9昆虫细胞以表达这些蛋白质中的每一种。使用固定化金属亲和色谱法（IMAC）从细胞裂解物中纯化表达的蛋白质。我们将应用我们新开发的方法来非共价复合的细胞渗透性肽，达特，组氨酸标记的蛋白质。我们将培养的内皮细胞暴露于复合物。我们将在小鼠体内静脉注射复合物。为了确定摄取，我们将通过实时荧光成像对内皮细胞和肺微血管进行成像。意义：目前尚无针对ALI的治疗方法。我们的研究将首次建立治疗这种严重疾病的新治疗方法。我们提供治疗性蛋白质的方法是新颖的，并且可能上级现有方法。这项研究将确定潜在适用于人类应用的治疗的临床前可行性。 公共卫生相关性：尽管急性肺损伤（ALI）是发病率和死亡率的主要原因，但目前尚无治愈性治疗方法。ALI的主要病理学是肺水肿，其由肺中的内皮屏障恶化引起。因此，我们的目标是开发针对内皮屏障增强的疗法。我们建议在肺微血管内皮细胞中引入屏障增强蛋白。这种方法的可行性得到了我们未发表的纯化粘着斑激酶（FAKp）研究的支持。我们已经开发了一种新的化学方法，非共价连接FAKp的细胞渗透性肽，达特。TAT-FAKp 1静脉内注射的TAT-FAKp在肺微血管中被吸收。通过检测肺内皮细胞通透性、血管外肺水（EVLW）含量以及支气管肺泡灌洗液（BAL）中白细胞和蛋白质含量，证实FAKp对酸诱导的ALI具有保护作用。我们提供治疗性蛋白质的方法是新颖的，并且可能上级现有方法。这项研究将确定潜在适用于人类应用的治疗的临床前可行性。

DESCRIPTION (provided by applicant): Aims: The main objective of this project is to develop a protein based therapy to enhance the endothelial barrier properties. Although acute lung injury (ALI) is a major cause of morbidity and mortality, no curative therapies are available. The main pathology in ALI is attributed to pulmonary edema, a direct result of endothelial (EC) barrier deterioration in the lung. In this project we will purify in their biologically active forms the barrier-protective proteins, Rac1, (-actinin and vinculin. Using our newly developed approach, we will attach these barrier-protective proteins to the cell-permeable peptide, TAT. The proteins will be given by mouse tail vein injection to determine their efficacy in protection against ALI. Procedures: We will use the Baculovirus Expression Vector System (BEVS) to develop the recombinant virus expressing the cDNA of the targeted proteins Rac1, vinculin and (-actinin as a His-tagged protein. This amplified virus will be used to infect the Sf9 insect cells to express each of these proteins. The expressed proteins will be purified from the cell lysates using Immobilized Metal Affinity Chromatography (IMAC). We will apply our newly developed approach to non-covalently complex the cell-permeable peptide, TAT, to His-tagged protein. We will expose cultured endothelial cells to the complexes. We will inject the complexes intravenously in mice. To determine uptake, we will image endothelial cells and lung microvessels by real-time fluorescence imaging. Significance: No therapy exists for ALI. Our research will establish for the first time, novel therapeutic modalities for curing this severe disease. Our approach for delivering therapeutic proteins is novel and potentially, superior to existing approaches. This research will determine preclinical feasibility of therapy that is potentially suitable for human application. PUBLIC HEALTH RELEVANCE: Although acute lung injury (ALI) is a major cause of morbidity and mortality, no curative therapies are available. Major pathology in ALI is attributable to pulmonary edema, which results from endothelial barrier deterioration in the lung. Hence, our aim is to develop therapy directed at endothelial barrier enhancement. We propose to introduce barrier-enhancing proteins in endothelial cells of lung microvessels. Feasibility of this approach is supported by our unpublished studies with purified focal adhesion kinase (FAKp). We have developed a new approach for chemically, non-covalently linking FAKp to the cell permeable peptide, TAT. The TAT-FAKp l Intraveously injected TAT-FAKp is taken up in lung microvessels. Intraveously injected FAKp protects against acid-induced ALI as determined by assays of lung endothelial permeability, extravascular lung water (EVLW) content and leukocyte and protein contents of the broncho alveolar lavage (BAL).We will apply our novel TAT linkage approach to develop intracellular delivery of three barrier-protective proteins, namely, vinculin, (-actinin and Rac1. Our approach for delivering therapeutic proteins is novel and potentially, superior to existing approaches. This research will determine preclinical feasibility of therapy that is potentially suitable for human application.