No abstract

To date, no study has described the effects of media flow load on a co-culture model of hepatocytes (HC), hepatic stellate cells (HSC) and endothelial cells (EC). Furthermore, no research has been reported regarding the influence of nitric oxide (NO) concentration in such a co-culture model. Therefore, we developed co-culture models that include two or three of these cell types, and assayed their hepatic functions both in static culture and under flow load. We also measured the NO concentration in each models and inhibited NO production of cells. In static culture, the HC+HSC and HC+HSC+EC models demonstrated higher hepatic function than in the model containing HC alone. Under flow load, all models exhibited higher hepatic function than in static culture. The HC+HSC and HC+HSC+EC models under flow demonstrated the highest hepatic function observed under any condition. In almost all models, NO concentration exhibited the same tendency to increase along with hepatic function, and NO improved hepatic function in vitro without in HC+HSC model under flow load. Inhibition of NO production decreased small levels of hepatic function in HC+HSC and HC+HSC+EC models under flow load. We conclude that co-culture and flow load positively impact hepatic function, and that HSC and NO are related to improvements in hepatic function. Furthermore, we consider that the presence of HSC is responsible for other aspects of improvement in hepatic function.

Hepatic functions, such as albumin secretion and ammonia metabolism, are upregulated in response to hepatocyte growth factor (HGF) produced by hepatic stellate cells (HSC), as well as nitric oxide (NO) produced by endothelial cells under shear stress. However, the simultaneous effect of HSC and NO has not been previously investigated in a tri-co-culture model containing hepatocytes with HSC and endothelial cells under shear stress. We hypothesized that NO inhibits HGF production from HSC. To test this idea, we constructed a mono-culture model of hepatocytes and a co-culture model of hepatocytes and HSC and measured ammonia decomposition and HGF production in each model under NO load. Ammonia decomposition was significantly higher in the co-culture model under 0 ppm NO load, but no significant increase was observed under NO load. In the co-culture model, HGF was produced at 1.0 ng/mL under 0 ppm NO load and 0.3 ng/mL under NO load. Ammonia decomposition was increased by 1.0 ng/mL HGF, but not by 0.3 ng/mL HGF. These results indicated that NO inhibits HGF production from HSC; consequently, the effects of NO and co-culture with HSC cannot improve hepatic function simultaneously. Instead, the simultaneous effect of 1.0 ng/mL HGF and NO may further enhance hepatic function in vitro.

When the liver is damaged, hepatic stellate cells (HSCs) can change into an activated, highly migratory state. The migration of HSCs may be affected by shear stress due not only to sinusoidal flow but also by the flow in the space of Disse because this space is filled with blood plasma. In this study, we evaluated the effects of shear stress on HSC migration in a scratch-wound assay with a parallel flow chamber. At regions upstream of the wound area, the migration was inhibited by 0.6 Pa and promoted by 2.0 Pa shear stress, compared to the static condition. The platelet-derived growth factor (PDGF)-BB receptor, PDGFR-β, was expressed in all conditions and the differences were not significant. PDGF increased HSC migration, except at 0.6 Pa shear stress, which was still inhibited. These results indicate that another molecular factor, such as PDGFR-α, may act to inhibit the migration under low shear stress. At regions downstream of the wound area, the migration was smaller under shear stress than under the static condition, although the expression of PDGFR-β was significantly higher. In particular, the migration direction was opposite to the wound area under high shear stress; therefore, migration might be influenced by the intercellular environment. Our results indicate that HSC migration was influenced by shear stress intensity and the intercellular environment.

A phenomenon has been observed in which intracellular Ca 2+ concentration in endothelial cells increases upon application of shear stress (Ca 2+ response). It is therefore assumed that Ca 2+ is the second messenger in the transfer of shear stress stimulation into cells. The Ca 2+ response is also known to spread to surrounding cells (Ca 2+ wave). We investigated the effects on Ca 2+ wave among cultured bovine aorta endothelial cells (BAECs) upon inhibiting the main intercellular signaling pathways, such as gap junction and paracrine pathways by inducing Ca 2+ wave using D-myo-inositol 1,4,5-trisphosphate, P4( 5)-(1-(2-nitrophenyl)ethyl) ester trisodium salt (Caged IP 3 ) due to an intracellular IP 3 elevation. In addition, we investigated the Ca 2+ wave among BAECs under shear stress loading. Using Caged IP 3 , local release of ATP from BAEC induced Ca 2+ wave. The Ca 2+ wave was inhibited by the inhibitors of paracrine pathways. Furthermore, the Ca 2+ response spread in the direction of the downstream under shear flow. These results suggest that paracrine pathway is dominant in both of flow and no flow conditions.