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) NF?B signaling cascade, TNF? and IL1?, (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?
This exploratory K01 grant application will provide mentorship and focused training to facilitate the successful transition of the candidate to an independent research scientist. The proposed plan of this K01 is to identify what specific roles each of the isoforms of Akt play in alcoholic liver injury utilizing in vitro human VL17A hepatocytes, Kupffer cells, and hepatic stellate cell cultures, as well as an in vivo chronic ethanol-Binge-LPS mouse model. The results from this exploratory study may lead to novel therapeutic targets for the prevention and treatment of alcoholic liver disease.
|Arellanes-Robledo, Jaime; Reyes-Gordillo, Karina; Ibrahim, Joseph et al. (2018) Ethanol targets nucleoredoxin/dishevelled interactions and stimulates phosphatidylinositol 4-phosphate production in vivo and in vitro. Biochem Pharmacol 156:135-146|
|Shah, Ruchi; Reyes-Gordillo, Karina; Rojkind, Marcos (2018) Thymosin ?4 inhibits PDGF-BB induced activation, proliferation, and migration of human hepatic stellate cells via its actin-binding domain. Expert Opin Biol Ther 18:177-184|
|Shah, Ruchi; Reyes-Gordillo, Karina; Cheng, Ying et al. (2018) Thymosin ?4 Prevents Oxidative Stress, Inflammation, and Fibrosis in Ethanol- and LPS-Induced Liver Injury in Mice. Oxid Med Cell Longev 2018:9630175|
|Casas-Grajales, Sael; Alvarez-Suarez, Diana; Ramos-Tovar, Erika et al. (2018) Stevioside inhibits experimental fibrosis by downregulating profibrotic Smad pathways and blocking HSC activation. Basic Clin Pharmacol Toxicol :|