Blebbistatin inhibits contraction and accelerates migration in mouse hepatic stellate cells

Abstract

Background and purpose: Blebbistatin, an inhibitor of myosin-II-specific ATPase, has been used to inhibit contraction of invertebrate and mammalian muscle preparations containing non-muscle myosin. Activated hepatic stellate cells have contractile properties and play an important role in the pathophysiology of liver fibrosis and portal hypertension. Therefore, hepatic stellate cells are considered as therapeutic target cells. In the present study, we studied the effect of blebbistatin during the transition of mouse hepatic stellate cells into contractile myofibroblasts. Experimental approach: Effects of blebbistatin on cell morphology were evaluated by phase contrast microscopy. Cell stress fibres and focal adhesions were investigated by dual immunofluorescence staining and visualized using fluorescence microscopy. Contractile force generation was examined by silicone wrinkle formation assays and collagen gel contraction assays. Intracellular Ca 2+ release in response to endothelin-1 was measured by using Fluo-4. Cell migration was measured by wound healing experiments. Key results: In culture-activated hepatic stellate cells, blebbistatin was found to change both cell morphology and function. In the presence of blebbistatin, stellate cells became smaller, acquired a dendritic morphology and had less myosin IIAcontaining stress fibres and vinculin-containing focal adhesions. Moreover, blebbistatin impaired silicone wrinkle formation, reduced collagen gel contraction and blocked endothelin-1-induced intracellular Ca 2+ release. Finally, it promoted woundinduced cell migration. Conclusions and implications: By inhibiting myosin II ATPase, blebbistatin has profound effects on the morphology and function of activated hepatic stellate cells. Our data suggest that myosin II could be a therapeutic target in the treatment of liver fibrosis and portal hypertension.

Activation of Rac by Asef2 promotes myosin II-dependent contractility to inhibit cell migration on type I collagen

“…Treatment of GFP control cells with blebbistatin led to an increase in migration speed (Fig. 6A,B), which is consistent with previous studies (Even-Ram et al, 2007;Liu et al, 2010;Niggli et al, 2006). At the end of the migration assay, we performed washout experiments to show that the effects on migration were due to blebbistatin.…”

The Rho family GEF Asef2 regulates cell migration in three dimensional (3D) collagen matrices through myosin II

“…This augmentation of migration by blebbistatin treatment has been previously reported by us as well as by other groups. 13,[23][24][25] Importantly, the migration speed of GFP-Asef2 cells was significantly decreased compared with that observed with GFP cells, and blebbastatin treatment abolished this Asef2 effect on migration (Fig. 2D).…”

Control of microenvironmental cues with a smart biomaterial composite promotes endothelial progenitor cell angiogenesis

“…3) Reports on the effects of blebbistatin over cell chemotaxis are contradictory. Some authors find enhanced motility (Liu et al, 2010;Niggli et al, 2006), while others inhibition (Wang et al, 2008). These discrepancies might arise from the different cell types and concentrations employed.…”

“…For this purpose blebbistatin, a specific nonmuscle myosin II inhibitor, was used (Engler et al, 2006). Blebbistatin can cause a number of secondary effects on cells, depending on dose and cell type (Goeckeler et al, 2008;Liu et al, 2010;Wang et al, 2008). We characterised the EPC response to blebbistatin to make sure that the effects observed were not the consequence of toxicity (data not shown).…”