TDK conference 2016

The matriptase-2 (MT-2) on the surface of the hepatocytes is a type II transmembrane serine protease, which regulates the production of the hepcidin responsible for the iron homeostasis. The hepatocyte mono- and the hepatocyte- Kupffer cell co-culture embedded in a hydrogel-based 3D structures could provide a great opportunity for studying the in vitro inhibition of matriptase. The advantage of the 3D cell culture versus the 2D techniques is that the characteristics of the in vivo microenvironment could be better maintained thus the phenotype and function of the cells could be provided to a greater extent.

In our experiments, the effects of two matriptase inhibtors (MI) with 3-amidinophenylalanine structures (MI-441 and MI-461) were investigated in hepatocyte mono- and hepatocyte- Kupffer cell co-cultures (with cell proportion 6:1). The localization pattern of a tight junction protein, occludin was detected with immunflourescent staining in these two cell cultures. The extracellular hydrogen peroxide levels were determined with Amplex Red reagent measurements. The interleukine- 6 and -8 (IL-6 and IL-8) levels and the effects of the inhibitors on the hepcidine levels were also determined by porcine specific ELISA kits.

According to our results, extracellular hydrogen peroxide levels were not elevated significantly after matriptase inhibition compared to the controls. Matriptase inhibitors did not cause increases in IL-6 levels in our hepatic models and the lack of IL-8 changes was also observed in the 3D monocultures. However, in the case of the inflammatory 3D co-culture model, MI-441 and MI-461 caused significant decrease in IL-8 levels. The hepcidin levels increased significantly in the cell culture containing only parenchymal cells in contrast to the co-cultures with Kupffer cells where surprisingly the hepcidin levels mainly decreased.

In summary it can be concluded that different results between 2D and 3D measurements necessitates further studies with the application of 3D hepatocye- Kupffer cell co-culture to investigate MI-441 and MI-461-caused pharmacological effects in-depth. Based on our in vitro experiments, treatment with MI-441 and MI-461 seems to be safe, they do not cause oxidative stress, and by the regulation of hepcidine levels they might have therapeutic values in the treatment of pathological iron metabolisms, and in case of mild types of hepatitis they might act as anti-inflammatory agents.