Akt Isoforms In the Pathogenesis of Alcoholic Liver Disease

酒精性肝病发病机制中的 Akt 亚型

基本信息

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
项目类别：
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

项目摘要

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?

该修订后的K01应用程序的目的是促进应用程序的开发，以成为多种多样的受过训练，独立的学术研究员。该应用程序的集体优势是在这三个主要领域中定义：1）凭据：雷耶斯·戈迪略的应用程序建立在其富有成效的轨道上 - 记录在酒精性肝病（ALD）领域建立了她的科学独立性 GWU自2015年7月1日起任命她为助理研究教授。申请人的目标包括获得专业技能，并成为一名独立的终身学术研究员。 2）培训环境：博士。 Lakshman（Mentor）和Bin Gao（Co-Insor），两位世界一流的肝病学家，完全是致力于实施并完成必要的培训，以推动PI作为未来的初级学院。博士。 Kumar，Szabo，Schrum，Diehl和Casey具有发展下一代的证书成功的学术科学家将担任咨询委员会成员。知识和经验在此K01颁奖期间获得的将有助于候选人成功赚取R01赠款，成为独立的终身调查员。 3）创新模型和研究：PI的主要假设是每个AKT 同工型在慢性乙醇（ETOH）/暴饮暴食/LPS（EBL）介导的肝损伤中具有特定的调节功能，她计划证明使用体外和体内方法，以下令人鼓舞初步结果：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，在KC和HSC中显着抑制了细胞炎症标记。（b）AKT1，AKT2抑制了HSC中的细胞增殖；（c）仅AKT2抑制HSC中的细胞迁移；（d）akt1，akt2，但是AKT3并未抑制VL17A肝细胞和HSC中的纤维化标记。体内：EBL鼠标模型（A）刺激所有磷酸化PDK1和MTORC-2和PI3K的AKT同工型，并同时增加从而上调炎症，增殖和纤维纤维基因；（b）当Akt2时，没有引起炎症但是AKT1没有物理阻塞，而AKT1和AKT2的阻塞都抑制了纤维化。 pi Will 在体外和肝细胞/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，免疫和组织化学化学化学完成以下特定目标的方法：特定目标1。 ETOH/LPS或ACE/LPS介导的（A）NFκB信号级联，TNFα和IL1β的三个AKT同工型，（b）细胞人类HSC，KC和肝细胞文化？特定目的2。（2a）EBL对Akt同工型的作用是什么和随之而来的效果关于炎症，增殖，纤维纤维和掺杂基因？（2b）可以特定的药物抑制作用 AKT1和/或AKT2防止EBL肝脏损害？＆（2C）可以防止EBL引起的肝脏损害使用肝细胞特异性或HSC特异性基因敲除小鼠的AKT1和/或Akt2基因的删除？