Akt Isoforms In the Pathogenesis of Alcoholic Liver Disease

此次修订的K01申请的目标是促进申请者发展成为多方面的 训练有素、独立的学术调查员。这个应用程序的综合优势是 定义在以下三个主要领域：1)凭据：Reyes-Gordillo的应用程序建立在她富有成效的轨道上- 建立她在酒精性肝病(ALD)领域的科学独立性的记录 自2015年7月1日起，GWU委任她为助理研究教授。申请者的目标包括 获得专业技能，并作为一名独立的终身教职学术研究人员而成熟。2)培训 环境：拉克什曼博士(导师)和高斌博士(共同导师)，这两位世界级的肝病专家，完全 致力于实施和完成必要的培训，使PI成为一名优秀的初级员工 教职员工。Kumar、Szabo、Schrum、Diehl和Casey博士拥有开发下一代 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奖励期间获得的奖励将帮助候选人成功获得R01奖励，以成为 独立终身调查员。3)创新模型和研究：Pi的主要假设是每个Akt 异构体在慢性乙醇/暴饮暴食/脂多糖(EBL)诱导的肝损伤中具有特异的调节功能， 她计划利用体外和体内方法来证明这一点，包括以下令人鼓舞的方法 初步结果：在体外：在乙醛(ACE)/脂多糖和/或乙醇/脂多糖培养模型中，siRNA- 定向沉默Akt2，但不抑制Akt1，显著抑制KC和HSC的细胞炎性标志物； (B)Akt1、Akt2抑制HSC细胞增殖；(C)Akt2单独抑制HSC细胞迁移；(D)Akt1、Akt2、 但AKT3不能抑制VL17A肝细胞和HSC的纤维化标志物。体内：EBL小鼠模型(A) 刺激所有Akt亚型，伴随着磷酸化PDK1、mTORC-2和PI3K的增加， 从而上调炎症、增殖和纤维化基因；(B)当Akt2， 但Akt1没有被药物阻断，而Akt1和Akt2的阻断都抑制了纤维化。圆周率将 使用Akt异构体特异性沉默的HSC/KC/肝细胞体外培养和肝细胞/HSC特异性Akt1/2- KO、野生型小鼠EBL模型及生化、分子生物学、免疫组织化学 实现以下具体目标的途径：具体目标1.具体的各自作用是什么 乙醇/脂多糖或血管紧张素转换酶/脂多糖介导的(A)NF-κB信号级联、肿瘤坏死因子α和白介素1-β，(B)细胞中的三种Akt亚型 增殖和迁移&(C)成纤维和成脂基因在人的HSC、KC和 肝细胞培养？具体目标2.(2A)EBL对Akt异构体的作用/S及其后续影响 对炎症、增殖、纤维化和成脂基因的影响？(2B)可以特异性的药物抑制 Akt1和/或Akt2对EBL肝损伤的保护作用？&(2C)EBL诱导的肝损伤可以通过以下方法预防 在肝细胞特异性或HSC特异性基因敲除小鼠中Akt1和/或Akt2基因的缺失？

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?