Second-hit and sexual dimorphism effects in PAH

描述（由申请方提供）：肺动脉高压（PAH）是一种进行性疾病，发病率和死亡率较高。BMPRII的突变和/或单倍不足被认为是家族性肺动脉高压（FPAH）以及许多特发性PAH（IPAH）病例的基础。然而，即使在BMPRII中具有已知突变的FPAH中，疾病发展也具有低（10-15%）的突变率，导致搜索可能涉及的候选“二次打击”基因。此外，在这种疾病中存在性别二型性，女性的发病率约为男性的2.5倍。低的染色体畸变率和两性异形的潜在基础仍然未知。在亚细胞水平，PAH肺血管病变中的内皮细胞和平滑肌细胞具有细胞大小增加（“巨细胞症”）、特征性细胞质细胞器变化（高尔基体破碎、内质网囊性扩张）和无法处理蛋白质的细胞内运输。本项目是基于我们的意外发现，一个众所周知的转录因子STAT 5a“非基因组”与肺血管细胞的细胞质中的高尔基体膜。siRNA介导的STAT 5a/B基因敲低可导致原代培养的HPAEC和HPASMCs中高尔基体断裂、内质网囊状扩张、Nogo-B/RTN 4和atlastin-3（ATL 3）增加（分别为ER结构蛋白和ER驻留GT3），核的月形畸变，减少膜运输和继发性线粒体功能障碍和碎片化-所有这些变化，我们和其他人，（例如Smith和Heath，1977，1979）已经显示在PAH中发生-我们显示可以通过ER/高尔基体驻留的GTP酶atlastin-1的过表达来阻断的变化。此外，STAT 5a和BMPRII的双敲低组合抑制了EC中ER向质膜的运输，表明两者之间的“二次打击”效应。值得注意的是，众所周知，STAT 5物种是雌激素响应性的，并且已知介导性二型性表型。 在特定目的I中，我们提出研究亚细胞STAT 5a/B敲低信号在PAH肺组织血管病变细胞中的发生，鉴定特定细胞， 参与，并调查这些变化中基于性别的差异。在特定目标II中，我们建议研究PAH患者培养的内皮细胞和平滑肌细胞中亚细胞STAT 5a/B敲低特征的发生和潜在的性别差异，STAT 5a/B敲低和BMPRII变化的组合效应，以及在此类细胞中恢复野生型STAT 5a和atlastin亚型的治疗潜力。 该项目代表了对PAH新范例的评价，重点是新发现的STAT 5a/B的细胞质细胞器功能，作为潜在的“二次打击”靶点，潜在的性二态性有助于这种疾病的发展。 公共卫生相关性：肺动脉高压（PAH）是一种进行性致死性肺部疾病，我们提出了一种全新的思路来思考这一致命疾病的病因。我们认为，有一个协调的缺陷，在细胞内的细胞质细胞器在疾病病变，导致细胞的大小增加，以及增加数量。缺陷在于这些细胞处理细胞内蛋白质运动的能力。总的后果是阻塞血管。提出的机制开辟了新的方法来治疗这种疾病的基因治疗。(End摘要）

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)