Novel therapeutic strategies for the treatment of liver diseases
Eline Geervliet is a PhD student in the Department Medical Cell Biophysics. Promotors are prof.dr. L.W.M.M. Terstappen and dr. R. Bansal from the Faculty of Science & Technology and prof.dr. R. Weiskirchen from RWTH Aachen University.
Liver disease is the 11th leading cause of death worldwide and accounts for more than two million deaths annually (1 out of 25 deaths). The major causes of liver disease are metabolic dysfunction (metabolic associated fatty liver disease, MAFLD), alcohol (alcohol liver disease, ALD), and viral infection (viral hepatitis). Despite high morbidity and mortality rates, there is no pharmacological treatment available for liver diseases. In this dissertation, we evaluated different disease targets and designed novel therapeutics against acute or chronic liver injury.
Design and development of new therapeutics require an understanding of the underlying disease mechanisms. Hepatocyte damage, upon acute or chronic liver injury, instigate liver inflammation by triggering the infiltration and activation of immune cells particularly monocytes and macrophages. Ongoing liver inflammation activate hepatic stellate cells (HSCs) which upon activation secrete excessive amounts of extracellular matrix (ECM) components especially collagens. Excessive ECM accumulation results in distortion of liver architecture resulting in the loss of liver function, portal hypertension and hepatic decompensation leading to death or hepatocellular carcinoma (HCC, primary liver cancer). Here below, we summarize the key findings of this dissertation.
In chapter 1, we demonstrated that fibroblast growth factor 7 (FGF7) and fibroblast growth factor receptor 2b (FGFR2b) were upregulated in liver injury, and that FGF7 via FGFR2b promotes hepatocyte proliferation and survival. FGF7 activated AKT and ERK pathways while reducing p27, ensuring controlled hepatocyte proliferation. Moreover, FGF7 enhanced drug detoxification genes and inhibited liver inflammation and fibrosis through paracrine mechanisms. Importantly, FGF7 treatment showed no severe adverse effects, suggesting its safety. These findings underscored FGF7's therapeutic potential in liver injury by regulating hepatocyte proliferation and survival ameliorating liver inflammation and fibrosis.
In chapter 2, we validated that increased chemokine receptor CCR8 expression, especially on hepatic macrophages, correlated with liver injury. We designed and characterized four CCR8 antagonizing peptides, identifying AP8ii as the most promising candidate. In vitro experiments confirmed AP8ii's specific binding to CCR8 and its ability to inhibit CCL1-induced migration of macrophages and monocytes. In an acute liver injury mouse model, AP8ii reduced intrahepatic infiltration of monocyte-derived macrophages and alleviated inflammation and early fibrosis, demonstrating its potential as a therapeutic agent. Lastly, AP8ii showed a favorable safety profile, indicating its potential as a safe and effective treatment for liver injury.
In chapter 3, we confirmed the upregulation of chemokine receptor CCR2 and chemokine ligand CCL2 in acute and chronic liver injury. Our novel in silico designed CCR2 antagonizing peptide (AP2) showed promising results in inhibiting CCL2-driven monocyte and macrophage migration in vitro. In vivo studies using acute and chronic liver injury models revealed that AP2 treatment significantly reduced inflammation and fibrosis, improving liver function without adverse effects. For clinical applicability of AP2, we cyclized AP2 and observed comparable efficacy between linear and cyclic AP2 peptides. These findings suggested that targeting the CCR2/CCL2 axis using linear or cyclic peptides could be a promising therapeutic strategy for liver diseases.
In chapter 4, we present the current knowledge about ECM remodeling and role of the different matrix metalloproteinases (MMPs) in liver diseases. MMP expression patterns in different stages of liver diseases have also been reviewed to determine their role as biomarkers. Finally, we highlight MMPs as promising therapeutic targets for the resolution of liver diseases.
In chapter 5, we demonstrated the fabrication, purification, and storage of MMPsomes, engineered for drug delivery, integrating MMP1 into polymersomes, offering MMP1 stability and enzymatic activity. Characterization studies confirmed MMP-1's locations within MMPsomes, primarily on the surface and partially integrated into the membrane. In vitro studies demonstrated MMPsomes' efficacy in inhibiting collagen I expression and cell migration. In vivo studies in a liver fibrosis model showed MMPsomes' potential in reducing fibrosis and inflammation. Overall, polymersomes offer a versatile platform for delivering MMPs or (matrix-degrading) enzymes, for mitigating liver inflammation and fibrosis.
Finally, in chapter 6, we elucidated the role of CCL2 and TGFβ-activated fibroblasts in macrophage recruitment, indicating a synergistic effect in liver inflammation. Additionally, CVC and MMP1 inhibited macrophage migration and collagen-I expression in fibroblasts, with a combined treatment showing superior efficacy. In vivo, CVC+MMP1 treatment reduced liver weight, improved liver function, attenuated monocyte recruitment and liver inflammation, while also ameliorating liver fibrogenesis. These findings underscored the potential therapeutic benefits of targeting both chemokine signaling and fibroblast activation pathways in liver fibrosis.
We truly believe that the insights from this dissertation can contribute to the better understanding of the liver diseases and to the development of effective and safe therapies for patients with liver disease.
