The Role of Matrix Stiffness in Hepatic Stellate Cell Activation and Liver Fibrosis

Transdifferentiation of hepatic stellate cells into proliferative and fibrogenic myofibroblasts is a critical event in the development of liver fibrosis. The causes of HSC transdifferentiation are not well understood, although the extracellular matrix has been implicated as one possible factor. We hypothesized that HSC transdifferentiation is determined by the mechanical properties of the surrounding matrix, and that the diseased liver demonstrates an increased elastic modulus that contributes to the perpetuation of HSC transdifferentiation and the progression of fibrosis. To test this hypothesis in an in vitro system, primary rat HSC were plated on inert polyacrylamide supports of variable but precisely defined elastic modulus ranging from 100 to 12 K Pa. These were coated with different matrices (type I collagen, fibronectin, or Matrigel™) in layers thin enough not to alter the mechanical properties of the system. As determined by morphology, degree of cell spreading, and expression of α-smooth muscle actin, cells became progressively transdifferentiated as substrate stiffness (elastic modulus) increased, independent of the chemical identity of the coating matrix. The degree rather than speed of HSC transdifferentiation correlated with substrate stiffness, with cells cultured on supports of intermediate stiffness adopting stable intermediate phenotypes. These changes were independent of the addition or removal of TGF-β. This demonstrated in an in vitro system that HSC transdifferentiation is primarily a function of the physical rather than the chemical properties of the substrate. As a preliminary to determining the role of stiffness in vivo, we determined the elastic modulus of normal and progressively fibrotic rat liver. Rats were injected twice weekly with carbon tetrachloride or vehicle, and were sacrificed in groups of 7–8 (2 controls and 5–6 CCl4) at time periods ranging from four days to ten weeks after the first injection. Control rats demonstrated a G’(elastic modulus) of 460 +/− 25 Pa. The mean elastic modulus of CCl4-treated livers increased progressively, beginning at the four day point, 525 +/− 39 Pa, and reaching 1617 +/− 170 Pa at 10 weeks. These values are consistent with the values at which we saw intermediate degrees of HSC transdifferentiation in vitro, and suggest that alterations in liver stiffness are a key factor driving the progression of fibrosis.