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（通过应用提供）：丛生的人类肺动脉高压（HPAH）是一种严重的疾病，无法治愈，几乎总是致命的。虽然有证据表明，丛状病变（PLS）主要含有表型改变，增殖和功能障碍的内皮细胞（EC），但促成EC异常增殖的细胞和分子因子是鲜为人知的。该应用的目的是了解与PAH相关的肺部炎症期间，谷物酶B（GRB）对相交1S（ITS）的切割有助于EC的增殖和PL的形成。 ITS是肺ECS的主要促生存蛋白，是最近鉴定出的GRB底物。 GRB裂解ITS并产生两个生物活性的蛋白质片段，该蛋白质片段具有N末端片段（GRB-EHITSN）潜在的EC增殖潜力，通过p38mapk和Elk-1转录因子的磷酸化介导，并通过化学抑制p38激酶消除。此外，与对照组相比，PAH动物模型的肺组织和带有PLS的HPAH肺以及PAH受试者的肺动脉EC含有GRB-EHITSN，并表现出较低水平的全长ITN水平。 GRB免疫反应性与HPAH肺中的PLS有关。 C末端片段（GRB-SH3A-EITSN）对ERK1/2信号传导具有显着的负面影响，并且两个GRB/ITSN片段的同时表达会导致p38与ERK1/2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转移的小鼠表现出PAH表型（即增加肺EC增殖，右心室收缩压升高，右心肥力，肺动脉乳腺病）通过GRB-EHITSN抑制性肽来改善。这是我们假设，由于与HPAH相关的肺部注射过程中的GRB裂解以及高p38与ERK1/2活性比相关的GRB裂解引起的全长ITS表达降低，这是由两种GRB/ITSN裂解产物引起的，导致EC的增殖和选择增殖/PLEX COLLISTIST/PLECISTION EC EC型。三个具体目标（SA）将检验以下假设：SA1将证明由于GRB裂解而导致的全长ITS表达降低，并且GRB-Ehitsn的存在是HPAH肺中EC增殖表型的占EC增殖表型的降低。 SA2将证明GRB-EHITSN诱导的p38激活有利于ELK-1的转录活性和C-FOS的首选转录，立即在体内导致EC增殖和过度生长，并立即在体内进行EC响应基因。 SA3将证明GRB-Ehitsn会引起小鼠肺EC增殖反应和血管重塑，可以通过GRB-EHITSN抑制性肽来改善。成功完成目标将确定Grb-ehitsn是增殖/丛状EC表型出现的“触发”，并且是PAH启动和进展的病理生理机制的关键因素。这些发现是理解严重HPAH异常EC增殖的机制的中心 Ehitsn可以有效地改善EC的增殖并停止PAH的进展，使生存成为PAH患者的现实期望。