The lab is part of PSOC@Penn, an NCI-funded Physical Sciences in Oncology Center that takes a soft matter physics approach to understanding hepatocellular carcinoma (HCC). HCC is a devastating cancer that most often occurs in the setting of a stiff, cirrhotic liver. As part of PSOC@Penn Project 1 (with Paul Janmey), we are studying the impact of matrix and mechanical changes on the function of hepatocytes and other cells of the liver – cells from normal livers, fatty livers, and cirrhotic livers. We use a variety of cell culture substrates including hydrogels (such as polyacrylamide gels of different stiffnesses, coated with different matrix proteins including cell-derived matrices) as well as decellularized human and rodent livers; we are particularly interested in differences in hepatocyte mechanotransduction pathways and phenotype in response to matrix and mechanical changes. Ongoing work with the Janmey lab includes a study of the role of hyaluronic acid in hepatocyte mechanotransduction and cell behavior in fatty liver disease, cirrhosis, and HCC.
The Wells lab also hosts the Cell and Tissue Core of PSOC@Penn. Led by technical director Likang Chin, PhD, the core procures human liver tissue, maintains primary and passaged cell lines, and develops animal models of HCC. As part of the core's service to the center, we carry out detailed rheology measurements and matrix analyses of most human samples, and have embarked on a study to define and compare the 3D architecture and matrix composition of livers with cirrhosis of metabolic, biliary, and viral etiologies.
Biliary Atresia and Bile Duct Biology
The lab has an active program investigating biliary atresia and cholangiocyte biology. Biliary atresia is a rare disease of unknown etiology characterized by the development of extrahepatic biliary fibrosis and duct obstruction in previously healthy neonates. We are part of an international group that identified biliatresone, a previously unknown plant isoflavonoid, as a biliary toxin that causes a biliary atresia-like syndrome in neonatal livestock and larval zebrafish. Biliatresone causes loss of cell polarity, increased monolayer permeability, and lumen obstruction in cholangiocyte organoids; in neonatal mouse bile duct explants (see figure below), it causes cholangiocyte monolayer disruption and submucosal fibrosis. We have shown that biliatresone causes rapid decreases in levels of reduced glutathione in cholangiocytes, and that other agents that alter glutathione levels mimic biliatresone in in vitro culture systems. Additionally, biliatresone causes reductions in the transcription factor SOX17, and artificial knockdown of SOX17 disrupts cholangiocyte organoids in a manner similar to biliatresone. Ongoing work is focused on determining the mechanism of specific neonatal susceptibility to biliatresone-induced injury. (See Waisbourd-Zinman O et al, Hepatology 2016; and Lorent K et al, Science Translational Medicine 2015.)
The lab has also collaborated with Neil Theise and Petros Benias (Mt. Sinai Beth Israel Medical Center, New York City) to study the submucosa of the extrahepatic bile duct. New work suggests that this structure is an interstitial space filled with fluid, dense collagen bundles, elastin fibers, and fibroblastic cells (with some endothelial cell markers) which adhere directly to one side of the collagen bundles. Our group is particularly interested in the role of this space in extrahepatic bile duct fibrosis, for example in settings of increased cholangiocyte monolayer permeability and leakage of bile into the space. We have begun to isolate and characterize the fibroblastic cells and find that they differentiate to myofibroblasts in culture; we hypothesize that they are precursors to the fibrogenic cells of the extrahepatic bile duct. Ongoing work is focused on further characterization of these cells in the healthy and diseased bile duct.
Matrix and Mechanical Factors in Fibrosis
Our lab has extensively characterized the tissue mechanics of the normal and fibrotic liver. We have shown that injured livers stiffen before becoming fibrotic, and that this is due in part to lysyl oxidase-mediated collagen cross-linking. Hepatic stellate cells and portal fibroblasts, the two major fibrogenic cell precursors of the liver, require a stiff environment in order to differentiate to fibrogenic myofibroblasts, and it is thus likely that early liver stiffening facilitates the deposition of abnormal matrix proteins. The liver, like other tissues, demonstrates complex tissue mechanics, with marked shear strain softening and compression stiffening that are a function of cells, matrix, and the interactions between them. Proteoglycans appear to play a particularly important role in compression stiffening, which may have relevance in understanding liver physiology in the setting of portal hypertension. (See Perepelyuk M et al, PLOS One, 2016.)
We are currently studying the role of mechanical factors in large scale tissue rearrangements, most notably bridging fibrosis. In collaboration with Vivek Shenoy's group in the School of Engineering and Applied Sciences, we have studied long-range force transmission across collagen and collagen/fibronectin matrices in vitro, and shown that contractile cells can compact and align matrix proteins, yielding collagen "lines" similar in appearance to bridging fibrosis. In vivo, administration of ROCK inhibitors prevents bridging but not matrix deposition, suggesting that similar long-range force transmission determines the formation of bridges.
As part of lab work on fibronectin, we have shown that the cellular fibronectin splice variant EIIIA is not required for liver fibrosis (in contrast to fibrosis in other organs), but that it plays a role in early liver regeneration, particularly at the level of the sinusoids. Interestingly, there are marked differences between males and females as far as the role of fibronectin EIIIA in both fibrosis and regeneration. The presence of EIIIA in complex matrices renders them stiffer; we are currently investigating the impact of EIIIA on the magnitude of long-range force transmission.
Center for Engineering Mechanobiology
The lab is part of the Center for Engineering Mechanobiology (CEMB), a 7-institution, $24 million dollar NSF-funded center with the goal of developing a research and educational program integrating concepts in the physical sciences, engineering, and biology. Please visit our website www.cemb.org for more information!