Fibronectin Extra Domain A Promotes Liver Sinusoid Repair following Hepatectomy.

Liver sinusoidal endothelial cells (LSECs) are the main endothelial cells in the liver and are important for maintaining liver homeostasis as well as responding to injury. LSECs express cellular fibronectin containing the alternatively spliced extra domain A (EIIIA-cFN) and increase expression of this isoform after liver injury, although its function is not well understood. Here, we examined the role of EIIIA-cFN in liver regeneration following partial hepatectomy. We carried out two-thirds partial hepatectomies in mice lacking EIIIA-cFN and in their wild type littermates, studied liver endothelial cell adhesion on decellularized, EIIIA-cFN-containing matrices and investigated the role of cellular fibronectins in liver endothelial cell tubulogenesis. We found that liver weight recovery following hepatectomy was significantly delayed and that sinusoidal repair was impaired in EIIIA-cFN null mice, especially females, as was the lipid accumulation typical of the post-hepatectomy liver. In vitro, we found that liver endothelial cells were more adhesive to cell-deposited matrices containing the EIIIA domain and that cellular fibronectin enhanced tubulogenesis and vascular cord formation. The integrin α9ß1, which specifically binds EIIIA-cFN, promoted tubulogenesis and adhesion of liver endothelial cells to EIIIA-cFN. Our findings identify a role for EIIIA-cFN in liver regeneration and tubulogenesis. We suggest that sinusoidal repair is enhanced by increased LSEC adhesion, which is mediated by EIIIA-cFN.

Gradually softening hydrogels for modeling hepatic stellate cell behavior during fibrosis regression.

The extracellular matrix (ECM) presents an evolving set of mechanical cues to resident cells. We developed methacrylated hyaluronic acid (MeHA) hydrogels containing both stable and hydrolytically degradable crosslinks to provide cells with a gradually softening (but not fully degradable) milieu, mimicking physiological events such as fibrosis regression. To demonstrate the utility of this cell culture system, we studied the phenotype of rat hepatic stellate cells, the major liver precursors of fibrogenic myofibroblasts, within this softening environment. Stellate cells that were mechanically primed on tissue culture plastic attained a myofibroblast phenotype, which persisted when seeded onto stiff (∼20 kPa) hydrogels. However, mechanically primed stellate cells on stiff-to-soft (∼20 to ∼3 kPa) hydrogels showed reversion of the myofibroblast phenotype over 14 days, with reductions in cell area, expression of the myofibroblast marker alpha-smooth muscle actin (α-SMA), and Yes-associated protein/Transcriptional coactivator with PDZ-binding motif (YAP/TAZ) nuclear localization when compared to stellate cells on stiff hydrogels. Cells on stiff-to-soft hydrogels did not fully revert, however. They displayed reduced expression of glial fibrillary acidic protein (GFAP), and underwent abnormally rapid re-activation to myofibroblasts in response to re-stiffening of the hydrogels through introduction of additional crosslinks. These features are typical of stellate cells with an intermediate phenotype, reported to occur in vivo with fibrosis regression and re-injury. Together, these data suggest that mechanics play an important role in fibrosis regression and that integrating dynamic mechanical cues into model systems helps capture cell behaviors observed in vivo.

Stiffening hydrogels for investigating the dynamics of hepatic stellate cell mechanotransduction during myofibroblast activation.

Tissue fibrosis contributes to nearly half of all deaths in the developed world and is characterized by progressive matrix stiffening. Despite this, nearly all in vitro disease models are mechanically static. Here, we used visible light-mediated stiffening hydrogels to investigate cell mechanotransduction in a disease-relevant system. Primary hepatic stellate cell-seeded hydrogels stiffened in situ at later time points (following a recovery phase post-isolation) displayed accelerated signaling kinetics of both early (Yes-associated protein/Transcriptional coactivator with PDZ-binding motif, YAP/TAZ) and late (alpha-smooth muscle actin, α-SMA) markers of myofibroblast differentiation, resulting in a time course similar to observed in vivo activation dynamics. We further validated this system by showing that α-SMA inhibition following substrate stiffening resulted in attenuated stellate cell activation, with reduced YAP/TAZ nuclear shuttling and traction force generation. Together, these data suggest that stiffening hydrogels may be more faithful models for studying myofibroblast activation than static substrates and could inform the development of disease therapeutics.

Prevalence and classification of pathogenic Escherichia coli isolated from fresh beef, poultry, and pork in Korea.

Foodborne diseases occur worldwide, including through the consumption of contaminated meat. This study was conducted to investigate the prevalence of Escherichia coli contamination in fresh beef, poultry, and pork, and to determine whether any isolated E. coli possessed genes associated with pathogenicity. Three thousand meat samples were collected from 2004 to 2006 and were tested for the presence of E. coli. Two hundred and seventy-three E. coli isolates were obtained from beef, poultry, and pork, resulting in an overall isolation rate of 9.1%. Of these isolates, 201 were obtained from 1350 pork samples (14.9%), followed by 41 of 900 poultry samples (4.6%) and 31 of 750 beef samples (4.1%). A total of 39 pathogenic E. coli isolates from the three meat types were categorized into three virulence groups, namely enterotoxigenic E. coli (43.6%), enterohemorrhagic E. coli (EHEC) (35.9%; 22.6% of beef, 7.3% of poultry, and 2.0% of pork), and enteropathogenic E. coli (20.5%). Fourteen strains were identified as belonging to the EHEC, which included O18, O136, O119, O86, O8, O111, O15, O128, and O6. This study demonstrated that pathogenic E. coli are found in meat in Korea, and could act as a transmission vehicle for human infection as suggested by the occurrence and classification of pathogenic E. coli in retail meats. Furthermore, the data from this study could be used in the risk assessment of foodborne illnesses linked to meat consumption.