1 Hepatic Stellate Cells Require a Stiff Environment for 1 Myofibroblastic Differentiation

45 46 The myofibroblastic differentiation of hepatic stellate cells (HSC) is a critical event in liver 47 fibrosis and is part of the final common pathway to cirrhosis in chronic liver disease from 48 all causes. The molecular mechanisms driving HSC differentiation are not fully 49 understood. Because macroscopic tissue stiffening is a feature of fibrotic disease, we 50 hypothesized that mechanical properties of the underlying matrix are a principal 51 determinant of HSC activation. Methods: Primary rat HSC were cultured on inert 52 polyacrylamide supports of variable but precisely defined shear modulus (stiffness) 53 coated with different extracellular matrix proteins or poly-L-lysine. HSC differentiation 54 was determined by cell morphology, immunofluorescence staining, and gene 55 expression. Results: HSC became progressively myofibroblastic as substrate stiffness 56 increased on all coating matrices, including Matrigel. The degree rather than speed of 57 HSC activation correlated with substrate stiffness, with cells cultured on supports of 58 intermediate stiffness adopting stable intermediate phenotypes. Quiescent cells on soft 59 supports were able to undergo myofibroblastic differentiation with exposure to stiff 60 supports. Stiffness-dependent differentiation required adhesion to matrix proteins and 61 the generation of mechanical tension. Transforming growth factor-β treatment enhanced 62 differentiation on stiff supports, but was not required. Conclusions: HSC differentiate to 63 myofibroblasts in vitro primarily as a function of the physical rather than the chemical 64 properties of the substrate. HSC require a mechanically stiff substrate, with adhesion to 65 matrix proteins and the generation of mechanical tension, in order to differentiate. These 66 findings suggest that alterations in liver stiffness are a key factor driving the progression 67 of fibrosis. 68 69