Akt Isoforms In the Pathogenesis of Alcoholic Liver Disease

酒精性肝病发病机制中的 Akt 亚型

• 批准号: 9754569
• 负责人: Karina Reyes-Gordillo
• 金额: $ 13.68万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2020-06-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9754569
• 关键词: 1-Phosphatidylinositol 3-Kinase; AKT1 gene; AKT2 gene; Acetaldehyde; Address; Advisory Committees; Alcohol dehydrogenase; Alcoholic Liver Diseases; Applications Grants; Area; B Cell Proliferation; Biochemical; Biological; C57BL/6 Mouse; CYP2E1 gene; Cell Culture Techniques; Cell Proliferation; Cells; Chemicals; Chronic; Collagen; Committee Members; Complex; Development; Endotoxins; Environment; Epigenetic Process; Ethanol; Extracellular Matrix Proteins; FRAP1 gene; Faculty; Fibronectins; Fibrosis; Generations; Genes; Goals; Grant; Hepatic; Hepatic Stellate Cell; Hepatocyte; Human; In Vitro; Individual; Inflammation; Inflammatory; Interleukin-1 beta; Intestinal permeability; Investigation; Knockout Mice; Knowledge; Kupffer Cells; Lead; Lipids; Lipopolysaccharides; Liver Fibrosis; Mediating; Mentored Research Scientist Development Award; Mentors; Mentorship; Methyl-CpG-Binding Protein 2; Modeling; Molecular; NF-kappa B; Oxidative Stress; Oxides; PDPK1 gene; PPAR gamma; Pathogenesis; Pathogenicity; Peroxisome Proliferator-Activated Receptors; Pharmacology; Phenotype; Phosphotransferases; Platelet-Derived Growth Factor beta Receptor; Play; Prevention; Process; Production; Protein Isoforms; Protein-Serine-Threonine Kinases; Reactive Oxygen Species; Research; Research Assistant; Research Personnel; Role; Scientist; Signal Transduction; Small Interfering RNA; Smooth Muscle Actin Staining Method; TNF gene; Training; Up-Regulation; Vitamin A; Wild Type Mouse; base; career development; cell motility; cytokine; experience; fibrogenesis; in vivo; inflammatory marker; inhibitor/antagonist; innovation; liver injury; migration; mouse model; new therapeutic target; next generation; phosphoinositide-dependent kinase 1; prevent; professor; skills; stellate cell; tenure track; transdifferentiation

The goal of this revised K01 application is to promote the development of the applicant to become a multi- disciplinarily trained, and independent academic investigator. The collective strengths of this application are defined in these 3 major areas: 1) Credentials: Reyes-Gordillo’s application builds upon her productive track- record establishing her scientific independence in the field of Alcoholic Liver Disease (ALD) based on which GWU appointed her as Assistant Research Professor since 07/01/2015. The applicant’s goals include achieving professional skills, and maturing as an independent tenure-track academic researcher. 2) Training Environment: Drs. Lakshman (mentor) and Bin Gao (co-mentor), the two world class hepatologists, are fully committed to implement and complete the training necessary to advance the PI as an outstanding junior faculty. Drs. Kumar, Szabo, Schrum, Diehl, and Casey with credentials in developing the next generation of successful academic scientists will serve as Advisory Committee members. The knowledge and experience gained during this K01 award period will facilitate the candidate to successfully earn a R01 grant to become an independent tenured investigator. 3) Innovative Models and Research: PI’s main hypothesis is that each Akt isoform has a specific regulatory function in chronic Ethanol(EtOH)/Binge/LPS (EBL)-mediated liver injury, which she plans to prove utilizing both in vitro and in vivo approaches with the following encouraging preliminary results: In vitro: In the acetaldehyde (ACE)/LPS and/or EtOH/LPS human culture models, siRNA- directed silencing of (A) Akt2, but not Akt1, significantly suppressed cell inflammatory markers in KC and HSC; (B) Akt1, Akt2 inhibited cell proliferation in HSC; (C) Akt2 alone inhibited cell migration in HSC; (D) Akt1, Akt2, but not Akt3 inhibited the fibrogenic markers in VL17A hepatocytes and HSC. In vivo: EBL mouse model (a) stimulated all Akt isoforms with concomitant increases in phosphorylated PDK1 and mTORC-2, and PI3K, thereby up regulating inflammatory, proliferative, and fibrogenic genes; (b) caused no inflammation when Akt2, but not Akt1 was pharmacologically blocked, whereas blocking of both Akt1 and Akt2 inhibited fibrosis. PI will use Akt-isoform-specific silenced HSC/KC/hepatocyte cultures in vitro and hepatocyte/HSC-specific Akt1/2- KO, and wild type mice in the EBL model, and biochemical, molecular biological, immuno- & histo-chemical approaches to accomplish the following specific aims: Specific Aim 1. What are the specific respective roles of the three Akt isoforms in EtOH/LPS or ACE/LPS-mediated (A) NFκB signaling cascade, TNFα and IL1β, (B) cell proliferation and migration & (C) fibrogenic and adipogenic gene cascades in individual human HSC, KC and hepatocyte cultures? Specific Aim 2. (2A) What are the action/s of EBL on Akt isoforms and consequent effects on inflammatory, proliferative, fibrogenic and adipogenic genes? (2B) Could specific pharmacological inhibition of Akt1 and/or Akt2 protect against EBL liver damage? & (2C) Could EBL-induced liver damage be prevented by the deletion of Akt1 and/or Akt2 gene using hepatocyte-specific or HSC-specific knockout mice?

此次修订的K01申请的目标是促进申请人发展成为一名多才多艺的人。 受过严格训练的独立学术研究者。该应用程序的集体优势是 定义在这 3 个主要领域：1) 证书：Reyes-Gordillo 的应用程序建立在她的生产轨迹之上 - 记录确立了她在酒精性肝病（ALD）领域的科学独立性 自2015年7月1日起，乔治城大学任命她为助理研究教授。申请人的目标包括 获得专业技能，并作为一名独立的终身教授走向成熟。 2）培训 环境：博士。拉克什曼（导师）和高斌（联合导师）两位世界级肝病专家，完全 致力于实施并完成将 PI 提升为优秀初级人员所需的培训 学院。博士。 Kumar、Szabo、Schrum、Diehl 和 Casey 拥有开发下一代技术的资历 成功的学术科学家将担任顾问委员会成员。知识和经验 在此 K01 奖励期内获得的奖励将有助于候选人成功获得 R01 补助金，成为一名 独立终身研究员。 3）创新模型和研究：PI的主要假设是每个Akt 同种型在慢性乙醇（EtOH）/暴食/LPS（EBL）介导的肝损伤中具有特定的调节功能， 她计划通过以下令人鼓舞的方法利用体外和体内方法来证明这一点 初步结果：体外：在乙醛 (ACE)/LPS 和/或 EtOH/LPS 人类培养模型中，siRNA- (A) Akt2（而非 Akt1）的定向沉默可显着抑制 KC 和 HSC 中的细胞炎症标志物； (B) Akt1、Akt2抑制HSC细胞增殖； (C) Akt2 单独抑制 HSC 中的细胞迁移； (D) Akt1、Akt2、 但 Akt3 不抑制 VL17A 肝细胞和 HSC 中的纤维化标志物。体内：EBL小鼠模型（a​​） 刺激所有 Akt 同工型，同时磷酸化 PDK1 和 mTORC-2 以及 PI3K 增加， 从而上调炎症、增殖和纤维化基因； (b) Akt2 时不引起炎症， 但 Akt1 并未被药物阻断，而同时阻断 Akt1 和 Akt2 则可抑制纤维化。 PI将 使用 Akt 异构体特异性沉默 HSC/KC/肝细胞体外培养物和肝细胞/HSC 特异性 Akt1/2- EBL 模型中的 KO 和野生型小鼠，以及生化、分子生物学、免疫和组织化学 实现以下具体目标的方法： 具体目标 1. 各自的具体作用是什么 EtOH/LPS 或 ACE/LPS 介导的三种 Akt 亚型 (A) NFκB 信号级联，TNFα 和 IL1β，(B) 细胞 增殖和迁移 & (C) 人类 HSC、KC 和 KC 中的纤维形成和脂肪形成基因级联 肝细胞培养？具体目标 2. (2A) EBL 对 Akt 同工型的作用和后续影响是什么 炎症、增殖、纤维形成和脂肪形成基因？ (2B) 能否特异性药理抑制 Akt1 和/或 Akt2 可以预防 EBL 肝损伤吗？ & (2C) EBL 引起的肝损伤可以通过以下方式预防吗 使用肝细胞特异性或 HSC 特异性敲除小鼠删除 Akt1 和/或 Akt2 基因？

项目成果

Thymosin β4 inhibits PDGF-BB induced activation, proliferation, and migration of human hepatic stellate cells via its actin-binding domain.

• DOI: 10.1080/14712598.2018.1478961
• 发表时间: 2018-07
• 期刊: Expert opinion on biological therapy
• 影响因子: 4.6
• 作者: Shah R;Reyes-Gordillo K;Rojkind M
• 通讯作者: Rojkind M