Attenuation of fibrogenesis by PDE4 inhibitor via decreased cytoskeleton remodeling, activation and contractility of hepatic stellate cells
Authors: Elnagdy Mohamed , Rodriguez-Alvarez Walter , Wang Yali , Merchant Michael L. , Maldonado Claudio , Barve Shirish , McClain Craig , Gobejishvili Leila 
Published: April 2020

Liver fibrosis is characterized by accumulation of extracellular matrix (ECM) proteins that occurs due to chronic liver injury. Activated hepatic stellate cells (HSCs) are main source of ECM in the liver. Rho‐associated kinase (Rho‐kinase/ROCK) promotes the assembly of focal adhesions and actin stress fibers by regulating the organization of the cytoskeleton in HSCs. Exchange protein directly regulated by cAMP 1 (EPAC1) attenuated Rho kinase signaling in HSCs and alleviated the development of liver fibrosis. We previously showed that cAMP degrading phosphodiesterase 4 (PDE4) enzymes play a critical role in the pathogenesis of liver fibrosis suggesting that hepatic cAMP signaling is compromised during fibrogenesis. Based on these observations, we hypothesize that preserving cAMP/EPAC1 signaling by inhibiting PDE4 will attenuate HSC activation and fibrotic signaling.C57Bl/6 mice were subjected to repeated carbon tetrachloride (CCl4) injection for 4 weeks to estabilesh liver fibrosis. One group of mice received PDE4 inhibitor, Rolipram (targeted to the liver) twice a week. To examine the direct effect of EPAC1/PDE4 pathway on HSCs, in vitro studies using human HSC cell line (LX2) were also performed. Activation of LX2 HSCs was achieved by using recombinant TGFβ1. Liver tissue proteomic analysis was performed. Pathway enrichment analysis was done using Clarivate analytics software, MetaCore. Sirius Red staining and hydroxyproline (HYP) assay was performed to assess collagen deposition. Real time qPCR and Western blot were performed to validate the findings from proteomic analysis. Statistical analysis was performed using ANOVA.CCl4 administration resulted in a significant accumulation of collagen confirmed by Sirius red staining and hydroxyproline (HYP) content in mice. Importantly, Rolipram prevented HYP accumulation and the expression of genes involved in collagen synthesis (Serpinh1) and crosslinking (Loxl1 and Lox). Additionally, Smad3 (central modulator of canonical TGFβ1 signaling) activation and mRNA levels of Mmp2 and Timp2 were markedly decreased by Rolipram. Pathway enrichment analysis of proteomic data identified several fibrotic pathways impacted by Rolipram, including HSC activation, integrin signaling and cytoskeleton remodeling regulated by Rho‐kinase. Western blot analyses confirmed that Rolipram significantly decreased phosphorylation of myosin light chain (MLC), target of Rho‐associated protein kinase. The effect of PDE4 inhibition on TGFβ1 inducible Smad3 and MLC phosphorylation was recapitulated in LX2 cells by using Rolipram and EPAC1 specific agonist. Moreover, Rolipram decreased Endothelin 1 (ET1) levels both in vivo and in vitro LX2 cells indicating a reduced contractility of HSCs. In LX2 cells TGFβ1 inducible nuclear translocation of Yes‐associated protein (YAP) was also inhibited by Rolipram suggesting that PDE4 inhibition promotes the crosstalk between TGFβ1 and YAP pathway to repress TGFβ1 signaling.These results demonstrate that PDE4 inhibition attenuates liver fibrosis by affecting multiple pathways involved in HSC activation and perpetuation of fibrosis.Support or Funding InformationNIH P20GM113226