Rebecca G. Wells

The role of matrix stiffness in regulating cell behavior.

Matrix stiffness (resistance to deformation), one of the many mechanical forces acting on cells, is increasingly appreciated as an important mediator of cell behavior. It regulates cell signaling broadly, with effects on growth, survival, and motility. Although the stiffness optima for different kinds of adherent cells vary widely, it is generally true that cell proliferation and differentiation increase with the stiffness of the matrix. This review summarizes recent data exploring the nature of matrix stiffness, mechanotransducers, and the many effects of changes in stiffness on cell function. Particular mention is made of data suggesting that cells of the liver are mechanosensitive, highlighting the potential importance of these findings in understanding the biology of normal and diseased liver. (HEPATOLOGY 2008.)

Transforming growth factor‐β and substrate stiffness regulate portal fibroblast activation in culture

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 α‐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‐β (TGF‐β): cells remained quiescent in the presence of a TGF‐β receptor kinase inhibitor, whereas exogenous TGF‐β1 enhanced portal fibroblast α‐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‐β and mechanical tension. (HEPATOLOGY 2007.)

Matrix stiffness modulates proliferation, chemotherapeutic response, and dormancy in hepatocellular carcinoma cells

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;)

Robust cellular reprogramming occurs spontaneously during liver regeneration

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.

Portal fibroblasts: Underappreciated mediators of biliary fibrosis†

Portal fibroblasts are an important yet often overlooked nonparenchymal cell population in the liver. They are distinct from hepatic stellate cells, yet like stellate cells differentiate in the setting of chronic injury to fibrogenic myofibroblasts, playing an important role in collagen production in the fibrotic liver. Portal fibroblasts (PFs) are located adjacent to bile duct epithelia and thus play a particularly significant role in biliary fibrosis. New data suggest that they may also have key functions independent of fibrogenesis. This review addresses the definition and characteristics of PFs as well as their signaling pathways, interactions with the biliary epithelium, and contributions to liver pathobiology. Conclusion: PFs are an important and multifunctional nonparenchymal cell population in need of further study. (HEPATOLOGY 2010.)

Hepatic stellate cells require a stiff environment for myofibroblastic differentiation

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.

Smads 2 and 3 Are Differentially Activated by Transforming Growth Factor-β (TGF-β) in Quiescent and Activated Hepatic Stellate Cells CONSTITUTIVE NUCLEAR LOCALIZATION OF Smads IN ACTIVATED CELLS IS TGF-β-INDEPENDENT

Hepatic stellate cells are the primary cell type responsible for matrix deposition in liver fibrosis, undergoing a process of transdifferentiation into fibrogenic myofibroblasts. These cells, which undergo a similar transdifferentiation process when cultured in vitro, are a major target of the profibrogenic agent transforming growth factor-β (TGF-β). We have studied activation of the TGF-β downstream signaling molecules Smads 2, 3, and 4 in hepatic stellate cells (HSC) cultured in vitro for 1, 4, and 7 days, with quiescent, intermediate, and fully transdifferentiated phenotypes, respectively. Total levels of Smad4, common to multiple TGF-β superfamily signaling pathways, do not change as HSC transdifferentiate, and the protein is found in both nucleus and cytoplasm, independent of treatment with TGF-β or the nuclear export inhibitor leptomycin B. TGF-β mediates activation of Smad2 primarily in early cultured cells and that of Smad3 primarily in transdifferentiated cells. The linker protein SARA, which is required for Smad2 signaling, disappears with transdifferentiation. Additionally, day 7 cells demonstrate constitutive phosphorylation and nuclear localization of Smad 2, which is not affected by pretreatment with TGF-β-neutralizing antibodies, a type I TGF-β receptor kinase inhibitor, or activin-neutralizing antibodies. These results demonstrate essential differences between TGF-β-mediated signaling pathways in quiescent and in vitro transdifferentiated hepatic stellate cells.

Lineage tracing demonstrates no evidence of cholangiocyte epithelial‐to‐mesenchymal transition in murine models of hepatic fibrosis

Whether or not cholangiocytes or their hepatic progenitors undergo an epithelial‐to‐mesenchymal transition (EMT) to become matrix‐producing myofibroblasts during biliary fibrosis is a significant ongoing controversy. To assess whether EMT is active during biliary fibrosis, we used Alfp‐Cre × Rosa26‐YFP mice, in which the epithelial cells of the liver (hepatocytes, cholangiocytes, and their bipotential progenitors) are heritably labeled at high efficiency with yellow fluorescent protein (YFP). Primary cholangiocytes isolated from our reporter strain were able to undergo EMT in vitro when treated with transforming growth factor‐β1 alone or in combination with tumor necrosis factor‐α, as indicated by adoption of fibroblastoid morphology, intracellular relocalization of E‐cadherin, and expression of α‐smooth muscle actin (α‐SMA). To determine whether EMT occurs in vivo, we induced liver fibrosis in Alfp‐Cre × Rosa26‐YFP mice using the bile duct ligation (BDL) (2, 4, and 8 weeks), carbon tetrachloride (CCl4) (3 weeks), and 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC; 2 and 3 weeks) models. In no case did we find evidence of colocalization of YFP with the mesenchymal markers S100A4, vimentin, α‐SMA, or procollagen 1α2, although these proteins were abundant in the peribiliary regions. Conclusion: Hepatocytes and cholangiocytes do not undergo EMT in murine models of hepatic fibrosis. (Hepatology 2011;)

Matrix Elasticity, Cytoskeletal Tension, and TGF-β: The Insoluble and Soluble Meet

Soluble growth factors are potent regulators of normal and pathological processes. Mechanical factors are emerging as similarly important, but there has been no obvious mechanism linking the different factors. A recent report now demonstrates that cell-generated mechanical tension results in release of active transforming growth factor–β from stiff extracellular matrix, providing a mechanism for differentiation and maintenance of myofibroblasts in processes like fibrosis. More broadly, the work suggests that matrix stiffness could regulate the equilibrium between storage and release of a host of matrix-bound growth factors.

Foxl1-Cre-marked adult hepatic progenitors have clonogenic and bilineage differentiation potential

Isolation of hepatic progenitor cells is a promising approach for cell replacement therapy of chronic liver disease. The winged helix transcription factor Foxl1 is a marker for progenitor cells and their descendants in the mouse liver in vivo. Here, we purify progenitor cells from Foxl1-Cre; RosaYFP mice and evaluate their proliferative and differentiation potential in vitro. Treatment of Foxl1-Cre; RosaYFP mice with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet led to an increase of the percentage of YFP-labeled Foxl1(+) cells. Clonogenic assays demonstrated that up to 3.6% of Foxl1(+) cells had proliferative potential. Foxl1(+) cells differentiated into cholangiocytes and hepatocytes in vitro, depending on the culture condition employed. Microarray analyses indicated that Foxl1(+) cells express stem cell markers such as Prom1 as well as differentiation markers such as Ck19 and Hnf4a. Thus, the Foxl1-Cre; RosaYFP model allows for easy isolation of adult hepatic progenitor cells that can be expanded and differentiated in culture.