Hepatic stellate cells require a stiff environment for myofibroblastic differentiation

Abstract

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

Hepatic stellate cells and portal fibroblasts are the major cellular sources of collagens and lysyl oxidases in normal liver and early after injury

“…4) and that hepatic stellate cells and portal fibroblasts require increased mechanical tension to differentiate to myofibroblasts (19,25) suggest a model whereby increased stiffness shortly after injury drives the myofibroblastic and then the fibrotic response. Potential causes of increased liver stiffness include changes in the matrix (cross-linking or increased matrix deposition) and tension due to edema or vascular pressure.…”

Matrix Stiffness–Induced Myofibroblast Differentiation Is Mediated by Intrinsic Mechanotransduction

“…Recent studies suggest that matrix stiffness, a measure of matrix resistance to mechanical deformation, regulates myofibroblast differentiation (3). Stiff matrix-induced myofibroblast differentiation has been extensively reported in fibroblasts isolated from heart (4), aortic valves (5), lung (6)(7)(8), liver (9,10), and gingiva (11). Despite this, the molecular mechanisms by which matrix stiffness regulates myofibroblast differentiation are not well understood.…”

Versican: a novel modulator of hepatic fibrosis

“…[6][7][8][9][10] The accumulation of fibrotic tissue results mainly from sustained activation of hepatic stellate cells (HSCs) to a fibroblastic phenotype, in response to many factors, including stimulation by proinflammatory and fibrogenic cytokines (particularly TGF-β), tissue hypoxia, reactive oxygen species, innate immune responses via interaction with Toll-like receptors (TLRs), the stiffness of the ECM, and ECM/fibroblast interactions. 5,[11][12][13][14][15][16][17] During the fibroproliferative stage, treatment of the underlying cause of liver disease results in the best prognostic outcome with disease reversal occurring in many patients. [18][19][20][21] Matrix metalloproteinase (MMP)-mediated degradation of fibrillar collagens is a well-established aspect of recovery from liver fibrosis in both experimental models and human disease, with a majority of the MMPs being synthesized by HSCs.…”