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

• 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
• 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

 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)：多发性人类肺动脉高压(HPAH)是一种严重的疾病，无法治愈，几乎总是致命的。虽然丛状病变(plexiform disks，PLs)主要包含表型改变、增殖和功能障碍的内皮细胞(ECs)，但导致ECs异常增殖的细胞和分子因素却知之甚少。该应用的目的是了解颗粒酶B(GRB)在与PAH相关的肺部炎症过程中对交叉素-1s(ITSN)的切割如何促进EC的增殖和pls的形成。ITSN是肺内皮细胞的主要促生存蛋白，是最近发现的一种GRB底物。GRB裂解ITSN产生两个具有生物活性的蛋白质片段(GRB-EHITSN)，具有EC增殖潜能，通过p38MAPK和ELK-1转录因子的磷酸化介导，并被p38激酶的化学抑制所消除。此外，PAH动物模型和高血压性肺出血合并PLS的肺组织以及PAH受试者的肺动脉内皮细胞均含有GRB-EHITSN，且全长ITSN的表达水平低于对照组。HPAH肺组织中GRB免疫反应与PLS相关。C末端片段(GRB-SH3A-EITSN)对ERK1/2信号转导具有明显的负性作用，两个GRB/ITSN片段的同时表达导致高p38/ERK1/2活性比率，这对hPAH相关的EC增殖表型至关重要。此外，转导GRB-EHITSN的小鼠表现出PAH表型(即肺内皮细胞增殖增加，右室收缩压升高，右心肥大，肺动脉病变)，该表型可通过GRB-EHITSN抑制肽治疗而改善。因此，我们推测，在hPAH相关的肺部炎症过程中，由于GRB裂解导致全长ITSN表达的减少，以及两种GRB/ITSN裂解产物导致的高p38/ERK1/2活性比导致EC增殖和选择增殖/丛状EC表型。三个特定的AIMS(SA)将验证这一假设：SA1将证明GRB切割导致的全长ITSN表达的减少以及GRB-EHITSN的存在是hPAH肺EC增殖表型的原因。SA2将证明GRB-EHITSN诱导的p38激活在体内有利于ELK-1的转录活性和c-fos即刻早期反应基因的优先转录，从而导致EC的增殖和过度生长。SA3将证明GRB-EHITSN可以引起小鼠肺内皮细胞的增殖反应和血管重塑，这可以通过GRB-EHITSN抑制肽来改善。AIMS的成功完成将确认GRB-EHITSN是增殖性/丛状EC表型出现的“触发器”，也是PAH启动和进展的病理生理机制的关键因素。这些发现对于理解重度hPAH中EC异常增殖的机制是至关重要的，它们可能为新一代小分子提供治疗靶点，这些小分子通过抑制GRB的增殖效应 EHITSN可以有效地改善EC的增殖，阻止PAH的进展，使生存成为PAH患者的现实期望。