Date on Master's Thesis/Doctoral Dissertation

5-2021

Document Type

Master's Thesis

Degree Name

M.S.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, MS

Committee Chair

Gobejishvili, Leila

Committee Co-Chair (if applicable)

Barve, Shirish

Committee Member

Barve, Shirish

Committee Member

McClain, Craig

Committee Member

Hein, David

Committee Member

Feng, Wenke

Committee Member

Maldonado, Claudio

Author's Keywords

PDE4 inhibition; liver fibrosis; hepatic stellate cells; CCl4; LX2 cells; proteomic analysis

Abstract

Background: Liver fibrosis is accumulation of extracellular matrix (ECM) proteins due to chronic liver injury. Chronic hepatic damage can occur due to multiple causes including, alcohol, non-alcoholic steatohepatitis and chronic viral hepatitis. Liver fibrosis is a critical problem worldwide due to the extremely high incidence of alcohol associated liver damage and chronic viral hepatitis. Moreover, there is no FDA approved therapy for liver fibrosis. Activation and transdifferentiation of quiecent hepatic stellate cells (HSCs) to myofibroblasts (MFBs) is the main event contributing to liver fibrosis. This process involves two phases, the initiation and the perpetuation phases. Hepatocyte injury and inflammatory response result in the initiation phase. During this phase, several profibrotic mediators (e.g.trasforming growth factor β1 (TGFβ1)) initiate the activation of HSCs. Activated MFBs deposit extracellular matrix proteins (ECM), including collagen and fibronectin. In perpetuation phase, MFBs proliferate and acquire a contractile/motile phenotype. Earlier work done by our group showed that spontaneous in culture HSC activation was accompanied by an increase in phosphodiesterase 4 (PDE4), a cAMP degrading enzyme. We also showed that PDE4 inhibition by Rolipram attenuated fibrogenic signaling in a bile duct ligation liver fibrosis rat model. More recent studies done by our group showed that PDE4 inhibition by Rolipram attenuates CCl4 induced liver fibrosis in C57Bl/6 mice. Rolipram decreased collagen deposition as demonstrated by Sirius red staining and hydroxyproline assay. We also observed attenuation of markers of HSC activation (α smooth muscle actin) and motility/contractility (pMLC and EDN1). Importantly, downstream cAMP effectors including, protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) were shown to have significant antifibrotic effects. Hence, we hypothesized that PDE4 inhibition prevents development of liver fibrosis by attenuating TGFβ1-induced activation of HSCs. Methods: For in vivo studies, C57Bl6/J mice were subjected to a repeated CCl4 injections twice a week for the duration of 4 weeks. One group of mice received Rolipram twice a week, day after CCl4 administration. For in vitro studies, LX2 human HSC line was used. A group of cells was pretreated with Rolipram and then, recombinant human TGFβ1 was added. Cells were collected at early time points (30 and 90 minutes) and late timepoint (24 hours). RNA was extracted by trizol method from LX2 HSCs and, expression of several fibrotic genes was assessed using quintitative RT qPCR. We also made whole cell lysates from LX2 cells (late timepoint) using Radioimmunoprecipitation assay buffer (RIPA) buffer as well as nuclear and cytoplasmic lysates from early time points. Protein lysates were used in western blot analysis to assess expression of relevant proteins as well as early TGFβ1 signaling changes. We also performed scratch assay to examine the effect of Rolipram on HSC motility/contractility. In chronic CCl4 mouse model, We performed proteomic analysis to examine the affected proteins and pathways by PDE4 inhibition in liver fibrosis. Statistical analysis was done using one-way ANOVA and unpaired t-test (*p < 0.05, **p < 0.01 and ***p < 0.001). Results: Proteomic analysis showed a significant effect of PDE4 inhibition on liver fibrosis pathways as well as pathways related to cytoskeleton remodeling, cell adhesion and motility/contractility. In LX2 cells, Rolipram attenuated TGFβ1-induced HSC activation by decreasing phosphorylation of SMAD3 and HSC activation marker (αSMA). Rolipram also attenuated TGFβ1-induced HSC motility/contractility by decreasing phosphorylated myosin light chain (pMLC) and endothelin-1 expression. Importantly, the effect of Rolipram on cell motility was validated by the results of the scratch assay which showed a significant decrease in percentage of wound closure at 24 hours. Conclusions: These results demonstrate that the PDE4 inhibition attenuates the initiation and perpetuation of liver fibrosis thorugh reduction of TGFβ1-mediated HSC activation and motility/contractility

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