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.

Retromer complex mediates the sorting of cargo from the endosome to the Golgi apparatus. At the endosome, recognition of Ypt7 (Rab7) by the Vps35 retromer subunit is essential for the cargo export step of the retromer functional cycle. Retromer also controls Ypt7-regulated fusion dynamics of the late endovacuolar system.

Background & Aims Myofibroblasts are the primary cell type involved in physiologic wound healing and its pathologic counterpart, fibrosis. Cellular fibronectin that contains the alternatively spliced extra domain A (EIIIA) is upregulated during these processes, and is believed to promote myofibroblast differentiation. We sought to determine the requirement for EIIIA in fibrosis and differentiation of myofibroblasts in rodent livers. Methods We used a mechanically tunable hydrogel cell culture system to study differentiation of primary hepatic stellate cells and portal fibroblasts from rats into myofibroblasts. Liver fibrosis was induced in mice by bile duct ligation or administration of thioacetamide. Results EIIIA was not required for differentiation of rat hepatic stellate cells or portal fibroblasts into fibrogenic myofibroblasts. Instead, hepatic stellate cells cultured on EIIIA-containing cellular fibronectin formed increased numbers of lamellipodia; their random motility and chemotaxis also increased. These increases required the receptor for EIIIA, the integrin α9β1. In contrast, the motility of portal fibroblasts did not increase on EIIIA and these cells expressed little α9β1. Male EIIIA−/− mice were modestly protected from thioacetamide-induced fibrosis, which requires motile hepatic stellate cells, but not from bile duct ligation-induced fibrosis, in which portal fibroblasts are more important. Notably, myofibroblasts developed during induction of fibrosis with either thioacetamide or bile duct ligation in EIIIA−/− mice. Conclusions EIIIA is dispensable for differentiation of hepatic stellate cells and portal fibroblasts to myofibroblasts, both in culture and in mouse models of fibrosis. These findings indicate a role for EIIIA in promoting stellate cell motility and parenchymal liver fibrosis.

Background & Aims IκB kinase-β (IKKβ) mediates activation of the nuclear factor-κB (NFκB), which regulates immune and inflammatory responses. Although NFκB is activated in cells from patients with inflammatory diseases or cancer, little is known about its roles in development and progression of esophageal diseases. We investigated whether mice that express an activated form of IKKβ in the esophageal epithelia develop esophageal disorders. Methods We generated ED-L2-Cre/Rosa26-IKK2caSFL mice, in which the ED-L2 promoter activates expression of Cre in the esophageal epithelia, leading to expression of a constitutively active form of IKKβ (IKKβca) in epithelial cells but not inflammatory cells or the surrounding stroma (IKKβca mice). Mice lacking the Cre transgene served as controls. Some mice were given intraperitoneal injections of neutralizing antibodies against granulocyte macrophage colony-stimulating factor (GMCSF) or tumor necrosis factor (TNF), or immunoglobulin G1 (control), starting at 1 month of age. Epithelial tissues were collected and analyzed by immunofluorescence, immunohistochemical, and quantitative real-time PCR assays. Transgenes were overexpressed from retroviral vectors in primary human keratinocytes. Results IKKβca mice developed esophagitis and had increased numbers of blood vessels in the esophageal stroma, compared with controls. Esophageal tissues from IKKβca mice had increased levels of GMCSF. Expression of IKKβca in primary human esophageal keratinocytes led to 11-fold overexpression of GMCSF and 200-fold overexpression of TNF. Incubation of human umbilical vein endothelial cells with conditioned media from these keratinocytes increased endothelial cell migration by 42% and promoted formation of capillary tubes; these effects were blocked by a neutralizing antibody against GMCSF. Injections of anti-GMCSF reduced angiogenesis and numbers of CD31+ blood vessels in esophageal tissues of IKKβca mice but did not alter the esophageal vasculature of control mice and did not alter recruitment of intraepithelial leukocytes to esophageal tissues of IKKβca mice. Injections of anti-TNF prevented development of esophagitis in IKKβca mice. Conclusions Constitutive activation of IKKβ in the esophageal epithelia of mice leads to inflammation and angiogenesis, mediated by TNF and GMCSF, respectively.