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The hallmark of liver fibrosis is an increased extracellular matrix deposition, caused by an activation of hepatic stellate cells (HSC). Therefore, this cell type is an important target for pharmacotherapeutic intervention. Antifibrotic drugs are not efficiently taken up by HSC or may produce unwanted side-effects outside the liver. Cell-specific delivery can provide a solution to these problems, but a specific drug carrier for HSC has not been described until now. The mannose 6-phosphate/insulin-like growth factor II (M6P/ IGF-II) receptor, which is expressed in particular upon HSC during fibrosis, may serve as a target-receptor for a potential carrier. The aim of the present study was to examine if human serum albumin (HSA) modified with mannose 6-phosphate (M6P) is taken up by HSC in fibrotic livers. A series of M6P x -modified albumins were synthetized: x ‫؍‬ 2, 4, 10, and 28. Organ distribution studies were performed to determine total liver uptake. The hepatic uptake of M6P x -HSA increased with increasing M6P density. M6P x -HSA with a low degree of sugar loading (x ‫؍‬ 2-10) remained in the plasma and accumulated for 9% ؎ 0.5% or less in fibrotic rat livers. An increase in the molar ratio of M6P: HSA to 28:1 caused an increased liver accumulation to 59% ؎ 9% of the administered dose. Furthermore, we determined quantitatively the in vivo intrahepatic distribution of M6P x -HSA using double-immunostaining techniques. An increased substitution of M6P was associated with an increased accumulation in HSC; 70% ؎ 11% of the intrahepatic staining for M6P 28

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Liver fibrogenesis is a process tightly controlled by endogenous anti-and pro-fibrogenic factors. Interferon gamma (IFNc) is a potent antifibrogenic cytokine in vitro and might therefore represent a powerful therapeutic entity. However, its poor pharmacokinetics and adverse effects, due to the presence of IFNc receptors on nearly all cells, prevented its clinical application so far. We hypothesized that delivery of IFNc specifically to the disease-inducing cells and concurrently avoiding its binding to nontarget cells might increase therapeutic efficacy and avoid side effects. We conjugated IFNc to a cyclic peptide recognizing the plateletderived growth factor beta receptor (PDGFbR) which is strongly up-regulated on activated hepatic stellate cells (HSC), the key effector cells responsible for hepatic fibrogenesis. The IFNc conjugates were analyzed in vitro for PDGFbR-specific binding and biological effects and in vivo in acute (early) and chronic (progressive and established) carbon-tetrachlorideinduced liver fibrosis in mice. The targeted-IFNc construct showed PDGFbR-specific binding to fibroblasts and HSC and inhibited their activation in vitro. In vivo, the targeted-IFNc construct attenuated local HSC activation in an acute liver injury model. In the established liver fibrosis model, it not only strongly inhibited fibrogenesis but also induced fibrolysis. In contrast, nontargeted IFNc was ineffective in both models. Moreover, in contrast to unmodified IFNc, our engineered targeted-IFNc did not induce IFNc-related side effects such as systemic inflammation, hyperthermia, elevated plasma triglyceride levels, and neurotropic effects. Conclusion: This study presents a novel HSC-targeted engineered-IFNc, which in contrast to systemic IFNc, blocked liver fibrogenesis and is devoid of side effects, by specifically acting on the key pathogenic cells within the liver. (HEPATOLOGY 2011;54:586-596)

Macrophages have been found to both promote liver fibrosis and contribute to its resolution by acquiring different phenotypes based on signals from the micro-environment. The best-characterized phenotypes in the macrophage spectrum are labeled M1 (classically activated) and M2 (alternatively activated). Until now the in situ localization of these phenotypes in diseased livers is poorly described. In this study, we therefore aimed to localize and quantify M1- and M2-dominant macrophages in diseased mouse and human livers. The scarred collagen-rich areas in cirrhotic human livers and in CCl4-damaged mouse livers contained many macrophages. Though total numbers of macrophages were higher in fibrotic livers, the number of parenchymal CD68-positive macrophages was significantly lower as compared to normal. Scar-associated macrophages were further characterized as either M1-dominant (IRF-5 and interleukin-12) or M2-dominant (CD206, transglutaminase-2, and YM-1) and significantly higher numbers of both of these were detected in diseased livers as compared to healthy human and mouse livers. Interestingly, in mouse, livers undergoing resolution of fibrosis, the total number of CD68+ macrophages was significantly lower compared to their fibrotic counterparts. M2-dominant (YM-1) macrophages were almost completely gone in livers undergoing resolution, while numbers of M1-dominant (IRF-5) macrophages were almost unchanged and the proteolytic activity (MMP9) increased. In conclusion, this study shows the distribution of macrophage subsets in livers of both human and murine origin. The presence of M1- and M2-dominant macrophages side by side in fibrotic lesions suggests that both are involved in fibrotic responses, while the persistence of M1-dominant macrophages during resolution may indicate their importance in regression of fibrosis. This study emphasizes that immunohistochemical detection of M1/M2-dominant macrophages provides valuable information in addition to widely used flow cytometry and gene analysis.

There is no effective therapy for advanced liver fibrosis. Angiotensin type 1 (AT1) receptor blockers attenuate liver fibrogenesis, yet their efficacy in reversing advanced fibrosis is unknown. We investigated whether the specific delivery of an AT1 receptor blocker to activated hepatic stellate cells (HSCs) reduces established liver fibrosis. We used a platinumbased linker to develop a conjugate of the AT1 receptor blocker losartan and the HSCselective drug carrier mannose-6-phosphate modified human serum albumin (losartan-M6PHSA). An average of seven losartan molecules were successfully coupled to M6PHSA.