M echanical forces have long been a focus of research and treatment in hepatology. Portal pressure is recognized as a critical determinant of a patient's functional state and is the target of many interventions in end-stage liver disease. Liver stiffness, now measured by increasingly sophisticated elastography techniques, has been an important diagnostic consideration for generations of physicians palpating the hard edge of the cirrhotic liver. Only recently, however, have the effects of mechanical forces at the cellular level been appreciated, in the liver and elsewhere in the body. There are many such forces, including stretch, compression, shear stress from fluid flow, and static mechanical forces. These forces impact multiple aspects of cell function, including differentiation state, growth and survival, motility, adhesion, and contractility. Mechanical forces may be particularly important in the liver, where a number of common pathological conditions including fibrosis and nonalcoholic fatty liver disease result in significant mechanical changes at whole organ, regional, and cellular levels.This review focuses specifically on the role of 1 mechanical force, extracellular matrix (ECM) stiffness (resistance to deformation), in regulating cell behavior. Recent work from a number of laboratories has demonstrated that matrix mechanics broadly impact the in vitro behaviors of many kinds of cells, including cells of the liver. The challenge of the future is to clarify the role of these findings in vivo.
Cellular reprogramming-the ability to interconvert distinct cell types with defined factors-is transforming the field of regenerative medicine. However, this phenomenon has rarely been observed in vivo without exogenous factors. Here, we report that activation of Notch, a signaling pathway that mediates lineage segregation during liver development, is sufficient to reprogram hepatocytes into biliary epithelial cells (BECs). Moreover, using lineage tracing, we show that hepatocytes undergo widespread hepatocyte-to-BEC reprogramming following injuries that provoke a biliary response, a process requiring Notch. These results provide direct evidence that mammalian regeneration prompts extensive and dramatic changes in cellular identity under injury conditions.
There is increasing evidence that the physical environment is a critical mediator of tumor behavior. Hepatocellular carcinoma (HCC) develops within an altered biomechanical environment, and increasing matrix stiffness is a strong predictor of HCC development. The aim of this study was to establish whether changes in matrix stiffness, which are characteristic of inflammation and fibrosis, regulate HCC cell proliferation and chemotherapeutic response. Using an in vitro system of “mechanically tunable” matrix‐coated polyacrylamide gels, matrix stiffness was modeled across a pathophysiologically relevant range, corresponding to values encountered in normal and fibrotic livers. Increasing matrix stiffness was found to promote HCC cell proliferation. The proliferative index (assessed by Ki67 staining) of Huh7 and HepG2 cells was 2.7‐fold and 12.2‐fold higher, respectively, when the cells were cultured on stiff (12 kPa) versus soft (1 kPa) supports. This was associated with stiffness‐dependent regulation of basal and hepatocyte growth factor–stimulated mitogenic signaling through extracellular signal‐regulated kinase, protein kinase B (PKB/Akt), and signal transducer and activator of transcription 3. β1‐Integrin and focal adhesion kinase were found to modulate stiffness‐dependent HCC cell proliferation. Following treatment with cisplatin, we observed reduced apoptosis in HCC cells cultured on stiff versus soft (physiological) supports. Interestingly, however, surviving cells from soft supports had significantly higher clonogenic capacity than surviving cells from a stiff microenvironment. This was associated with enhanced expression of cancer stem cell markers, including clusters of differentiation 44 (CD44), CD133, c‐kit, cysteine‐X‐cysteine receptor 4, octamer‐4 (CXCR4), and NANOG. Conclusion: Increasing matrix stiffness promotes proliferation and chemotherapeutic resistance, whereas a soft environment induces reversible cellular dormancy and stem cell characteristics in HCC. This has implications for both the treatment of primary HCC and the prevention of tumor outgrowth from disseminated tumor cells. (HEPATOLOGY 2011;)
Liver fibrosis, the response to chronic liver injury, results from the activation of mesenchymal cells to fibrogenic myofibroblasts. We have recently shown that two key myofibroblast precursor populations, hepatic stellate cells and portal fibroblasts, undergo activation in culture in response to increasing substrate stiffness. We therefore hypothesized that alterations in liver stiffness precede myofibroblast activation and fibrosis in vivo as well. To test this hypothesis, we induced fibrosis in rats by twice weekly injections of carbon tetrachloride (CCl4) and then killed the animals at various time points ranging from 3 to 70 days after the initiation of injury. The shear storage modulus of the whole liver was measured on fresh tissue; fixed and frozen tissue from the same livers was used to quantify fibrosis. We observed that liver stiffness increased immediately and continued to increase, leveling out by day 28. Fibrosis, measured histologically by trichrome staining as well as by quantitative sirius red staining, increased with time, although these increases were delayed relative to changes in stiffness. There was no direct correlation between stiffness and fibrosis at early or late time points. Treatment of a second cohort of rats with the lysyl oxidase inhibitor, β-aminopropionitrile (BAPN), partially prevented early increases in liver stiffness. We concluded that increases in liver stiffness precede fibrosis and potentially myofibroblast activation. Liver stiffness appears to result from matrix cross-linking and possibly other unknown variables in addition to matrix quantity. We suggest that increased liver stiffness may play an important role in initiating the early stages of fibrosis.
The mechanical properties of extracellular matrices can control the function of cells. Studies of cellular responses to biomimetic soft materials have been largely restricted to hydrogels and elastomers that have stiffness values independent of time and extent of deformation, so the substrate stiffness can be unambiguously related to its effect on cells. Real tissues, however, often have loss moduli that are 10 to 20% of their elastic moduli and behave as viscoelastic solids. The response of cells to a time-dependent viscous loss is largely uncharacterized because appropriate viscoelastic materials are lacking for quantitative studies. Here we report the synthesis of soft viscoelastic solids in which the elastic and viscous moduli can be independently tuned to produce gels with viscoelastic properties that closely resemble those of soft tissues. Systematic alteration of the hydrogel viscosity demonstrates the time dependence of cellular mechanosensing and the influence of viscous dissipation on cell phenotype.
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.
Myofibroblasts derived from portal fibroblasts are important fibrogenic cells in the early stages of biliary fibrosis. In contrast to hepatic stellate cells, portal fibroblasts have not been well studied in vitro, and little is known about their myofibroblastic differentiation. In this article we report the isolation and characterization of rat portal fibroblasts in culture. We demonstrate that primary portal fibroblasts undergo differentiation to ␣-smooth muscle actin-expressing myofibroblasts over 10 -14 days. Marker analysis comparing portal fibroblasts to hepatic stellate cells demonstrated that these are distinct populations and that staining with elastin and desmin can differentiate between them. Portal fibroblasts expressed elastin at all stages in culture but never expressed desmin, whereas hepatic stellate cells consistently expressed desmin but never elastin. Immunostaining of rat liver tissue confirmed these results in vivo. Characterization of portal fibroblast differentiation in culture demonstrated that these cells required transforming growth factor- (TGF-): cells remained quiescent in the presence of a TGF- receptor kinase inhibitor, whereas exogenous TGF-1 enhanced portal fibroblast ␣-smooth muscle actin expression and stress fiber formation. In contrast, platelet-derived growth factor inhibited myofibroblastic differentiation. Portal fibroblasts were also dependent on mechanical tension for myofibroblastic differentiation, and cells cultured on polyacrylamide supports of variable stiffness demonstrated an increasingly myofibroblastic phenotype as stiffness increased. Conclusion: Portal fibroblasts are morphologically and functionally distinct from hepatic stellate cells. Portal fibroblast myofibroblastic differentiation can be modeled in culture and requires both TGF- and mechanical tension. (HEPATOLOGY 2007;46:1246-1256
Mechanical forces are essential to the development and progression of fibrosis, and are likely to be as important as soluble factors. These forces regulate the phenotype and proliferation of myofibroblasts and other cells in damaged tissues, the activation of growth factors, the structure and mechanics of the matrix, and, potentially, tissue patterning. Better understanding of the variety and magnitude of forces, the characteristics of those forces in biological tissues, and their impact on fibrosis in multiple tissues is needed and may lead to identification of important new therapeutic targets.
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