PhD Student Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Abstract: Connexin (Cx)-formed gap junctions mediates important physiological and pathological events in the liver. However, little is known about the impact of liver fibrogenesis on gap junctions. To address this issue, a TGF-β induced in vitro fibrogenesis model was applied on multicellular spheroids (MS) obtained from HepG2, GRX (liver cell lines) and menstrual blood-derived mesenchymal cells CeSaM (n=10, Clementino Fraga Filho University Hospital Ethics Committee approval 056/09). Before, and after 7 and 14 days in culture, control and fibrotic (treated with 10 ng/mL of TGF-β) MS were submitted to volumetric (V=4/3лR3) and phase contrast analyses. The presence of Cx26, Cx32, Cx43, albumin, αSMA, COL I and III, Ki67 and Cyp3a4 was evaluated by immunofluorescence analyses. Confocal and transmission electron microscopy were performed to detect structural alterations on MS. To evaluate functionality, MS (n=5) were treated with different concentrations of acetaminophen (1, 5, 25, 50, 60, 70, 80 mM) for 2, 5, and 24h. MS viability was accessed by PrestoBlue assay. Intracellular Ca2+ signalization assay was performed to investigated whether fibrogenesis impacts on MS physiology. Volumetric analysis revealed significant changes when comparing MS obtained from different cell densities (2 and 4x10^4 cells, p< 0.01). Fibrotic MS differed from control in size (smaller at 7 and 14d; p< 0.05). Histological analysis revealed a spherical and uniform MS. Sirius red staining highlighted collagen distribution on fibrotic MS. Immunofluorescence analyses showed differences between Cx26, Cx32, Cx43 and COL I & III distribution between control and fibrotic spheroids. TEM showed morphological changes between control and fibrotic MS and revealed that fibrogenesis impairs architecture of liver organelles. Dose-response assay shows that acetaminophen was metabolized and contributed to a significant decrease in viability at 2 and 24h. Sequential confocal images obtained in Fluo-4/AM loaded SM showed that fibrogenesis promoted a significant impact on Ca2+ signaling amplitude and ATP responsiveness when compared to control MS. Taken together, these data suggest that liver fibrogenesis can impact on gap junctions promoting structural alterations that impairs on liver physiology.
