Second-hit and sexual dimorphism effects in PAH

PAH 的二次打击和性别二态性效应

• 批准号: 8350902
• 负责人: PRAVIN B SEHGAL
• 金额: $ 8.05万
• 依托单位: NEW YORK MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8350902
• 关键词: Binding; Biological Assay; Blood Vessels; Cell Nucleus; Cell Size; Cell membrane; Cells; Characteristics; Chronic; Collection; Cytoplasm; Cytoplasmic Organelle; Data; Defect; Development; Dilatation - action; Disease; Endoplasmic Reticulum; Endothelial Cells; Estrogen Receptors; Estrogens; Evaluation; Family; Functional disorder; Gender; Genes; Golgi Apparatus; Guanosine Triphosphate Phosphohydrolases; Human; Hypoxia; Image; Incidence; Lesion; Lung; Lung diseases; Mediating; Membrane; Membrane Protein Traffic; Mitochondria; Morbidity - disease rate; Movement; Mus; Mutation; Nuclear; Oils; Paraffin; Pathogenesis; Patients; Penetrance; Pharmaceutical Preparations; Phenotype; Predisposition; Progressive Disease; Protein Isoforms; Proteins; Regulation; STAT5A gene; STAT5a Transcription Factor; Semilunar Bone; Sex Characteristics; Small Interfering RNA; Smooth Muscle Myocytes; Structural Protein; Structure of parenchyma of lung; Subcellular structure; Therapeutic; Thinking; Transfection; VWF gene; Vesicular stomatitis Indiana virus; Woman; abstracting; base; combinatorial; gene therapy; human RTN4 protein; insight; man; men; mitochondrial dysfunction; mortality; mutant; novel; overexpression; protein transport; pulmonary arterial hypertension; reconstruction; response; sexual dimorphism; trafficking

DESCRIPTION (provided by applicant): Pulmonary arterial hypertension (PAH) is a progressive disease with high morbidity and mortality. Mutations and/or haploinsufficiency of BMPRII is considered to underlie familial pulmonary arterial hypertension (FPAH) as well as many instances of idiopathic PAH (IPAH). However, disease development, even in FPAH with known mutations in BMPRII, has low (10-15%) penetrance leading to searches for candidate "second-hit" genes that might be involved. Additionally, there is a sexual dimorphism in this disease with the incidence approximately 2.5-fold higher in women as in men. The underlying bases for low penetrance and sexual dimorphism remain unknown. At the subcellular level, endothelial and smooth muscle cells in the lung vascular lesions of PAH have an increase in cell size ("megalocytosis"), characteristic cytoplasmic organellar changes (Golgi apparatus fragmentation, cystic dilatation of the endoplasmic reticulum) and an inability to handle intracellular trafficking of proteins. The present project is based on our unexpected discovery that a well-known transcription factor STAT5a "nongenomically" associates with the Golgi membranes in the cytoplasm of pulmonary vascular cells. siRNA mediated knockdown of STAT5a/b in primary HPAECs and HPASMCs in culture produced Golgi fragmentation, cystic dilatation of endoplasmic reticulum (ER), increased Nogo-B/RTN4 and atlastin-3 (ATL3)(respectively an ER structural protein and an ER-resident GTPase), lunate distortion of nuclei, reduced membrane trafficking and secondary mitochondrial dysfunction and fragmentation - all changes that we and others (e.g. Smith & Heath, 1977, 1979) have shown to occur in PAH - changes that we show can be blocked by overexpression of the ER/Golgi-resident GTPase atlastin-1. Also, double knockdown of STAT5a and BMPRII combinatorially inhibited ER to plasma membrane trafficking in ECs suggesting "second-hit" effects between the two. Remarkably, it is well known that STAT5 species are estrogen-responsive and are known to mediate a sexual dimorphism phenotype. In Specific Aim I we propose to investigate the occurrence of the subcellular STAT5a/b knockdown signature in cells in vascular lesions in PAH lung tissues, identify the particular cells involved, and investigate gender-based differences in these changes. In Specific Aim II we propose to investigate endothelial and smooth muscle cells in culture derived from PAH patients for the occurrence of the subcellular STAT5a/b knockdown signature and potential gender-based differences, the combinatorial effects of STAT5a/b knockdown and BMPRII changes, and the therapeutic potential of restoring wt STAT5a and atlastin isoforms in such cells. This project represents evaluation of a new paradigm in PAH with a focus on the newly discovered cytoplasmic organellar functions of STAT5a/b as potential "second-hit" targets with underlying sexual dimorphism contributing to the development of this disease. PUBLIC HEALTH RELEVANCE: Pulmonary arterial hypertension (PAH) is a progressive fatal lung disease in man. We propose an all together novel way of thinking about the cause of this fatal disease. We suggest that there is a coordinated defect in the cytoplasmic organelles inside cells in disease lesions that leads to an increase in size of the cells as well as increase in number due. The defect lies in the ability of such cells to handle the movement of proteins inside the cells. The overall consequence is blockage of the blood vessels. The proposed mechanism opens up new ways to treat this disease using gene therapy. (End of Abstract)

描述(申请人提供)：肺动脉高压(PAH)是一种进展性疾病，发病率和死亡率都很高。BMPRII基因突变和/或单倍体功能不全被认为是家族性肺动脉高压(FPAH)和许多特发性肺动脉高压(IPAH)的基础。然而，疾病的发展，即使在BMPRII已知突变的FPAH中，外显率也很低(10%-15%)，这导致了对可能涉及的候选“二次命中”基因的搜索。此外，这种疾病具有性二型性，女性的发病率大约是男性的2.5倍。低外显性和性二型性的潜在基础仍不清楚。在亚细胞水平，PAH肺血管病变中的内皮细胞和平滑肌细胞体积增大(巨噬细胞增多)，细胞器发生特征性变化(高尔基体碎裂，内质网囊性扩张)，不能处理蛋白质在细胞内的运输。这个项目是基于我们出人意料的发现，即一个众所周知的转录因子Stat5a与肺血管细胞胞浆中的高尔基膜“非基因组”相关。在原代培养的HPAECs和HPASMCs中，siRNA介导的Stat5a/b的下调导致高尔基体碎裂，内质网(ER)囊性扩张，Nogo-B/RTN4和ATL3(分别是ER结构蛋白和ER驻留的GTP酶)增加，细胞核月形扭曲，膜转运减少，继发性线粒体功能障碍和碎裂-所有我们和其他人(例如Smith&Heath，1977,1979)已经证明在PAH中发生的变化-我们表明这些变化可以被ER/Golgi驻留的GTP酶atlastin-1的过表达所阻止。此外，Stat5a和BMPRII的双重敲除联合抑制了内皮细胞内质网到质膜的运输，表明两者之间存在“二次打击”效应。值得注意的是，众所周知，STAT5物种是雌激素敏感的，并已知介导性二型性表型。 在特定的目标I中，我们建议研究在PAH肺组织的血管病变中亚细胞Stat5a/b基因敲除信号的出现，鉴定特定的细胞 参与并调查这些变化中基于性别的差异。在特定的目的II中，我们建议研究PAH患者培养的内皮和平滑肌细胞中Stat5a/b亚细胞基因敲除特征的出现和潜在的性别差异，Stat5a/b基因敲除和BMPRII变化的组合效应，以及在这些细胞中恢复wt Stat5a和atlastin亚型的治疗潜力。 该项目代表了对PAH新范式的评估，重点是新发现的Stat5a/b的细胞质细胞器功能，这些功能是潜在的“二次打击”目标，其潜在的性别二型性有助于该病的发展。 公共卫生相关性：肺动脉高压(PAH)是一种进行性致命的人类肺部疾病。我们提出了一种全新的方式来思考这种致命疾病的原因。我们认为，在疾病皮损中，细胞内的细胞器存在协调缺陷，导致细胞大小和数量的增加。缺陷在于这些细胞处理细胞内蛋白质运动的能力。总的结果是血管堵塞。提出的机制为使用基因疗法治疗这种疾病开辟了新的途径。(摘要结束)