Cleavage of Intersectin-1s by Granzyme B and Pulmonary Arterial Hypertension

颗粒酶 B 对 Intersectin-1 的裂解与肺动脉高压

基本信息

批准号：
9282794
负责人：
Sanda A Predescu
金额：
$ 38.75万
依托单位：
RUSH UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9282794
关键词：
Abnormal Endothelial Cell Address Animal Model Arterial Disorder Biological C-terminal CD8-Positive T-Lymphocytes Cell Proliferation Cell physiology Cells Cessation of life Chemicals Cleaved cell Data Development Disease Dominant-Negative Mutation ERG gene Elements Endothelial Cells Event Experimental Animal Model FOS gene Failure Functional disorder Generations Genetic Transcription Granzyme Growth Heart Hypertrophy Human Hypertrophy Impairment Incidence Inflammatory Lead Length Lesion Lung MAPK14 gene Mediating Molecular Mus N-terminal Pathogenesis Patients Peptide Hydrolases Peptides Phenotype Phosphorylation Phosphotransferases Physiological Protein Fragment Proteins Pulmonary Inflammation Pulmonary Vascular Resistance Pulmonary artery structure Reaction Role Signal Pathway Signal Transduction Smooth Muscle Myocytes Structure of parenchyma of lung Sum Systolic Pressure Testing Vascular Proliferation Vascular remodeling Vascular resistance Ventricular cytotoxic elk-1 protein expectation immunoreactivity in vivo intersectin 1 knock-down mouse model novel therapeutics perforin prevent public health relevance pulmonary arterial hypertension pulmonary artery endothelial cell response small molecule therapeutic target

项目摘要

DESCRIPTION (provided by applicant): Plexogenic human pulmonary arterial hypertension (hPAH) is a severe disease with no cure and nearly always lethal. While it is evident that plexiform lesions (PLs) contain predominantly phenotypically altered, proliferative and dysfunctional endothelial cells (ECs), the cellular and molecular factors that contribute to abnormal proliferation of ECs are poorly understood. The objective for this application is to understand how cleavage of intersectin-1s (ITSN) by granzyme B (GrB) during pulmonary inflammation associated with PAH contributes to EC proliferation and formation of PLs. ITSN, a major pro-survival protein of lung ECs, is a recently identified GrB substrate. GrB cleaves ITSN and generates two biologically active protein fragments with the N-terminal fragment (GrB-EHITSN) possessing EC proliferative potential, mediated via phosphorylation of p38MAPK and Elk- 1 transcription factor, and abolished by chemical inhibition of p38 kinase. Moreover, lung tissue of PAH animal models and hPAH lungs with PLs, as well as pulmonary artery ECs of PAH subjects, contain the GrB- EHITSN and express lower levels of full-length ITSN compared to controls. GrB immunoreactivity is associated with PLs in hPAH lungs. The C-terminal fragment (GrB-SH3A-EITSN) has dominant negative effects on Erk1/2 signaling and the concurrent expression of the two GrB/ITSN fragments results in a high p38 to Erk1/2 activity ratio, critical for the EC proliferative phenotype associated with hPAH. Furthermore, mice transduced with the GrB-EHITSN show a PAH phenotype (i.e. increased lung EC proliferation, elevated right ventricular systolic pressure, right heart hypertrophy, pulmonary arteriopathy) which is ameliorated by treatment with a GrB-EHITSN inhibitory peptide. Thus, we hypothesize that decrease of full-length ITSN expression due to GrB cleavage during pulmonary inflammation associated with hPAH and the high p38 to Erk1/2 activity ratio caused by the two GrB/ITSN cleavage products lead to EC proliferation and selection of a proliferative/plexiform EC phenotype. Three specific aims (SA) will test this hypothesis: SA1 will demonstrate that decrease of full-length ITSN expression due to GrB cleavage and the presence of GrB-EHITSN account for the EC proliferative phenotype in hPAH lungs. SA2 will demonstrate that GrB-EHITSN-induced p38 activation favors transcriptional activity of Elk-1 and preferential transcription of c-fos immediate early response gene leading to EC proliferation and overgrowth, in vivo. SA3 will demonstrate that GrB-EHITSN elicits a mouse lung EC proliferative response and vascular remodeling that can be ameliorated via a GrB-EHITSN inhibitory peptide. Successful completion of the aims will identify the GrB-EHITSN as the "trigger" in the emergence of the proliferative/plexiform EC phenotype and as a critical factor of a patho-physiological mechanism for PAH initiation and progression. The findings are central for understanding the mechanisms responsible for abnormal EC proliferation in severe hPAH and they may provide therapeutic targets for a new generation of small molecules that, by inhibiting the proliferative effect of GrB EHITSN can effectively ameliorate EC proliferation and halt PAH progression, making survival a realistic expectation for PAH patients.

描述（由申请人提供）：丛源性人肺动脉高压（hPAH）是一种无法治愈且几乎总是致命的严重疾病，但很明显，丛状病变（PL）主要包含表型改变、增殖和功能障碍的内皮细胞（EC）。，导致 EC 异常增殖的细胞和分子因素知之甚少。本应用的目的是了解 intersectin-1 的裂解方式。在与 PAH 相关的肺部炎症过程中，颗粒酶 B (GrB) 产生的 ITSN 有助于 EC 增殖和 PL 的形成，ITSN 是肺 EC 的主要促存活蛋白，是最近发现的 GrB 底物，可裂解 ITSN 并产生两种生物学活性。活性蛋白片段，其 N 端片段 (GrB-EHITSN) 具有 EC 增殖潜力，通过 p38MAPK 磷酸化介导和Elk-1转录因子，并通过p38激酶的化学抑制而被消除。此外，PAH动物模型的肺组织和具有PL的hPAH肺，以及PAH受试者的肺动脉EC，含有GrB-EHITSN并表达较低水平的GrB-EHITSN。与对照相比，全长 ITSN 与 hPAH 肺中的 PL 相关，C 端片段 (GrB-SH3A-EITSN) 占主导地位。对 Erk1/2 信号传导的负面影响以及两个 GrB/ITSN 片段的同时表达导致 p38 与 Erk1/2 的高活性比，这对于与 hPAH 相关的 EC 增殖表型至关重要。此外，用 GrB-EHITSN 转导的小鼠显示。 PAH 表型（即肺 EC 增殖增加、右心室收缩压升高、右心肥大、肺动脉病），可通过以下药物治疗得到改善因此，我们研究了与 hPAH 相关的肺部炎症过程中 GrB 裂解导致全长 ITSN 表达的减少，以及两种 GrB/ITSN 裂解产物引起的高 p38 与 Erk1/2 活性比。 EC 增殖和增殖/丛状 EC 表型的选择三个特定目标 (SA) 将检验这一假设： SA1 将证明全长 ITSN 表达的减少。 GrB 裂解和 GrB-EHITSN 的存在解释了 hPAH 肺中 EC 增殖表型，将证明 GrB-EHITSN 诱导的 p38 激活有利于 Elk-1 的转录活性和 c-fos 立即早期反应基因的优先转录。 SA3 将证明 GrB-EHITSN 会引发小鼠肺 EC 增殖反应和血管反应。可以通过 GrB-EHITSN 抑制肽改善的重塑成功完成目标将确定 GrB-EHITSN 是增殖/丛状 EC 表型出现的“触发因素”，也是病理生理机制的关键因素。这些发现对于理解严重 hPAH 异常 EC 增殖的机制至关重要，并且可能为新一代小分子提供治疗靶点。抑制GrB的增殖作用 EHITSN 可以有效改善 EC 增殖并阻止 PAH 进展，使生存成为 PAH 患者的现实期望。