(162f) Mechanotransduction of Hepatocytes Drives Hepatocytes-Stellate Cell Communication during Liver Fibrosis Development

Background: Chronic liver diseases affect over 35 million Americans with estimated health care costs of $10 billion per year. Irrespective of the etiology, liver fibrosis is a ubiquitous response with no FDA-approved interventions. Fibroscan measurements have indicated a graded change in liver stiffness at various stages of fibrosis (2-4kPa: healthy liver, 8-10kPa: fibrosis stage of F0-1, 12-25kPa: F2-4 fibrotic liver, and >55kPa: cirrhosis). The increase in liver stiffness is attributed to perpetual activation of hepatic stellate cells (HSC). In response to fibrogenic cues from hepatocytes, HSCs alter their phenotypes resulting in altered liver matrix and increase in liver stiffness. The impact of stiffness on hepatocytes-HSC crosstalk is a gap in knowledge. The overall goal of our study is the development and implementation of a biomimetic platform that enables the convergence of engineered cell microenvironments with the phenotypic and functional analysis of hepatocytes-stellate cell crosstalk. Using our innovative biomimetic liver fibrosis model that allows modulation of substrate stiffness, we investigated the role of liver matrix stiffness in modulating hepatocytes-HSC communication in fibrotic-like microenvironment.

Materials and Methods: Direct contact patterned co-cultures of primary hepatocytes and LX2 (stellate cells) were cultured on our innovative biomimetic platform named “BEASTS (Bio-Engineered Adhesive Siloxane substrate with Tunable Stiffness)” based on polydimethylsiloxane (PDMS) substrate in combination with our patented polyelectrolyte multilayer film (PEM)-coating technology to engineer mechanically tunable substrates to mimic healthy (2 kPa), fibrotic (25 kPa), and extremely fibrotic substrates (55 kPa). A wide range of functional analysis were performed: urea/albumin assay, transporter genes.

Results: We demonstrated that stiffness impedes hepatic urea and albumin production, expression of drug transporter gene and epithelial cell phenotype marker, hepatocyte nuclear factor 4 alpha (HNF4a). We also demonstrated that hepatocytes cultured with LX2 cells performed better than mono-culture cells. We also observed decrease in hepatocytes function (urea, albumin) when in co-culture with stellate cells with increasing stiffness. These data suggest a plausible mechanism that increased stiffness modulates hepatocyte-LX2 cross-talk causing liver functional failure.

Conclusions: Together, all these data demonstrates the plausible role of stiffness in regulating hepatocytes-HSC communication and contribute to liver dysregulation during fibrosis.