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

颗粒酶 B 对 intersectin-1s 的裂解与肺动脉高压

• 批准号: 10302089
• 负责人: Sanda A Predescu
• 金额: $ 39.25万
• 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 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10302089
• 关键词: Address; Age; Animal Model; Arterial Disorder; Behavior; Biological Assay; Blood Vessels; Bromodeoxyuridine; CCND2 gene; Cell Cycle; Cell Cycle Progression; Cell Cycle Proteins; Cell Proliferation; Data; Diagnostic; Disease; Dosage Compensation (Genetics); Endothelial Cells; Endothelium; Estrogens; Ethnic Origin; Event; Failure; Female; Fibrinogen; Fluorescent in Situ Hybridization; Gene Expression; Genes; Genetic; Genetic Transcription; Germ Cells; Gonadal Steroid Hormones; Granzyme; Heart failure; Histology; Homeostasis; Human; Immunohistochemistry; Immunoprecipitation; In Vitro; Incidence; Inflammatory; Investigation; Label; Lesion; Link; Luciferases; Lung; Mass Spectrum Analysis; Measurement; Mediating; Metabolism; Modeling; Molecular; Mus; N-terminal; Nuclear; PECAM1 gene; Pathologic; Patients; Peptides; Phenotype; Physiologic pulse; Physiological; Population Study; Predisposition; Prevalence; Proteins; Publishing; Pulmonary artery structure; RNA; Race; Role; Sex Bias; Sex Chromosomes; Sex Differences; Sex Ratio; Signal Pathway; Small Interfering RNA; Specimen; Structure of parenchyma of lung; Therapeutic; Transcript; Untranslated RNA; Up-Regulation; Vascular Proliferation; Vascular remodeling; Woman; X Chromosome; X Inactivation; base; cyclin A1; elk-1 protein; endothelial dysfunction; genetic analysis; genome wide association study; hemodynamics; human disease; human female; human male; intersectin 1; knock-down; male; men; mouse model; novel; prevent; pulmonary arterial hypertension; pulmonary artery endothelial cell; response; sex; sexual dimorphism; therapeutic target; transcription factor

Despite some progress in understanding the role of the sex hormone estrogen in female susceptibility to pulmonary arterial hypertension (PAH), the female prevalence in PAH incidence remains largely unexplained. Investigations into the genetics of PAH in men and women are hampered by the failure to include the sex chromosomes in genome-wide association studies, to account for sex as variable in targeted genetic analyses, and even the focus on a single gene as cause of PAH. Moreover, an animal model that recapitulates the sex differences of PAH is missing. Our supportive data demonstrate that: a) our EHITSN-transduced intersectin-1s (ITSN) heterozygous mouse (EHITSN-K0ITSN+/-) model of PAH recapitulates most of the sex-specific differences of human disease; b) pulmonary artery endothelial cells (PAECs) are sex dimorphic in the proliferative potential; c) the expression and activity of the long non-coding RNA Xist is upregulated compared to baseline state in both, EHITSN-K0ITSN+/- and human PAH female lung specimens. The EHITSN is the N-terminal fragment of ITSN [a prominent protein of the lung], result of granzyme B cleavage under inflammatory conditions associated with PAH; EHITSN has endothelial cell proliferative potential and triggers a severe vascular arteriopathy in the lungs of EHITSN-K0ITSN+/- mice. Upregulation of Xist, essential for dosage compensation of chromosome X (ChrX)- linked genes expression leads to sex-specific upregulation of Elk1 transcription factor, a ChrX-linked PAH gene, and of cyclin A1, a cell cycle regulatory protein. The augmented Xist expression negatively regulates Klf2, a key transcription factor for endothelial homeostasis and quiescence. These molecular events are more prominent in female EHITSN-K0ITSN+/- mouse and human PAH lung specimens compared to males. Moreover, in vitro knockdown of Xist via siRNA or its inhibition via a specific EHITSN inhibitory peptide abolish the pathological manifestations. Based on these novel findings, we hypothesize that the increase in Xist expression in the female PAH specimens accounts at least in part, for the sex/ ratio imbalance in PAH. Two Specific Aims will address this hypothesis: SA #1 will demonstrate that Xist is a key regulator of the sexual dimorphism of PAECs in the proliferative response. Employing PAECs and lung tissue of PAH, male and female subjects, we will: 1a. Validate the increased Xist expression and transcriptional activity in female PAH specimens; 1b. Demonstrate that sex- specific upregulation of the Elk1/ cyclin A1 axis promotes progression of cell cycle and an increased proliferative response of female PAECs. 1c. Demonstrate that Xist upregulation and its sex-specific interactions with downstream partners (i.e., Klf2) contribute to the increased proliferative response of female PAECs. In SA #2 we propose to: 2a. Investigate the interactions of the EHITSN with nuclear factors; 2b. Explore the therapeutic potential of targeting Xist to inhibit female PAECs proliferation and ameliorate vascular remodeling in PAH. If successful, the studies will demonstrate that Xist upregulation accounts for female PAECs sexual dimorphism in the proliferative response and provide a better understanding of the origin of sex bias in PAH.

尽管在了解性激素雌激素在女性易患乳腺癌中的作用方面取得了一些进展， 肺动脉高压（PAH），PAH发病率中的女性患病率在很大程度上仍无法解释。 对男性和女性PAH遗传学的研究因未能纳入性别而受到阻碍 染色体在全基因组关联研究中，在有针对性的遗传分析中将性别作为变量， 甚至把注意力集中在单个基因上。此外，一种动物模型， PAH的差异缺失。我们的支持性数据表明：a）我们的EHITSN转导的intersectin-1 PAH的ITSN杂合子小鼠（EHITSN-K 0 ITSN +/-）模型重现了大多数性别特异性差异 B）肺动脉内皮细胞（PAEC）在增殖潜能方面具有性别二态性; c）长非编码RNA Xist的表达和活性与基线状态相比在两者中均上调， EHITSN-K 0 ITSN +/-和人PAH女性肺标本。EHITSN是ITSN的N-末端片段[a 肺的主要蛋白]，在与肺结核相关的炎症条件下颗粒酶B裂解的结果。 PAH; EHITSN具有内皮细胞增殖潜力，并引发肺部严重血管动脉病 的EHITSN-K 0 ITSN +/-小鼠。Xist的上调对于X染色体（ChrX）的剂量补偿至关重要- 连锁基因表达导致Elk 1转录因子，一种ChrX连锁PAH基因， 以及细胞周期蛋白A1，一种细胞周期调节蛋白。增强的Xist表达负调节Klf 2，这是一个关键的调节因子。 内皮稳态和静止的转录因子。这些分子事件在 与雄性相比，雌性EHITSN-K 0 ITSN +/-小鼠和人PAH肺标本。而且，体外 通过siRNA敲低Xist或通过特异性EHITSN抑制肽抑制Xist可消除病理性 表现。基于这些新的发现，我们假设在女性中Xist表达的增加， PAH标本至少部分解释了PAH的性别/比例失衡。两个具体目标将解决 这个假设：SA #1将证明Xist是PAEC性二态性的关键调节因子， 增殖反应使用PAH、男性和女性受试者的PAEC和肺组织，我们将：1a.验证 女性PAH标本中Xist表达和转录活性增加; 1b.证明性- Elk 1/细胞周期蛋白A1轴的特异性上调促进细胞周期的进展和细胞增殖的增加。 女性PAEC的反应。1c.证明Xist上调及其与性别特异性相互作用 下游伙伴（即，Klf 2）有助于增加雌性PAEC的增殖反应。在SA #2中 我们建议：2a.研究EHITSN与核因子的相互作用; 2b.探索治疗 靶向Xist抑制雌性PAEC增殖和改善PAH血管重塑的潜力。 如果成功的话，这些研究将证明Xist的上调可以解释女性PAEC的性行为。 在增殖反应的二型性，并提供了一个更好的理解PAH的性别偏见的起源。