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bioRxiv · 10.1101/2020.01.27.921353

Matrix Stiffness Regulates Liver Sinusoidal Endothelial Cell Function Mimicking Responses in Fatty Liver Disease

Natarajan, V.·Moeller, M.·Casey, C.·Harris, E.·Kidambi, S.
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Abstract

Liver sinusoidal endothelial cells (LSECs) are a highly specialized endothelial cell that participates in numerous liver metabolic activities and collectively function as a scavenger system in the liver by removing waste macromolecules playing a vital role in the balance of lipids, cholesterol, and vitamins. Prior to hepatic fibrosis, independent of their etiology, LSECs become highly pro-inflammatory, capillarized (loss in fenestrations), and loss in specialized receptors (Stabilin-1, Stabilin-2, CD31 and SE-1). Liver fibrosis also leads to significant loss in the endocytosis function of LSECs. Thus understanding regulation of LSEC phenotype may be critical to understanding fibrosis. Extensive remodeling of the extracellular matrix occurs during fibrosis that leads to liver stiffening. The role of matrix stiffness as related to subtle but pivotal changes in LSECs physiology is under explored. The overall goal of our study is the development and implementation of a platform that enables the convergence of engineered cell microenvironments with the phenotypic and functional analysis of LSECs. Using our innovative biomimetic liver fibrosis model that allows modulation of substrate stiffness, we investigated the role of liver matrix stiffness in modulating LSECs function in fibrotic-like microenvironment. Primary LSECs were cultured on our novel polymer film coated polydimethylsiloxane (PDMS) gels with 2 kPa, 9 kPa 25 kPa and 55 kPa elastic modulus mimicking healthy, early fibrotic, fibrotic and extremely fibrotic substrates. SEM was used to image to fenestrations of LSECs and HA endocytosis assay was performed to measure the LSECs function. LSECs cultured on stiffer environment had significant remodeling of cytoskeletal proteins and morphology indicated of stress fibers. Also we observed that LSECs on fibrotic substrates resulted in loss of fenestrations (capillarization). This is critical as capillarization has been show to precede hepatic fibrosis and "capillarized" LSECs lose the ability to promote hepatic stellate cell (HSC) quiescence. LSECs on stiffer environment also had higher expression of cell adhesion molecules, VCAM-1 and ICAM-1 indicating the loss of phenotype of the cells. Fibrotic stiffness also impeded the HA endocytosis in LSECs, one of the main functions of the cells. These data suggest a plausible mechanism that increased stiffness modulates hepatocyte and LSEC function causing liver functional failure. Similar effect was observed in LSECs isolated from Non-Alcoholic Fatty Liver Disease (NAFLD) rat models indicating correlation to physiological conditions. Together, all these data demonstrates the plausible role of stiffness in regulating LSECs function and contribute to HSC activation and progression of liver fibrosis.

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Natarajan, V., Moeller, M., Casey, C., Harris, E., Kidambi, S.. 2020-01-28. Matrix Stiffness Regulates Liver Sinusoidal Endothelial Cell Function Mimicking Responses in Fatty Liver Disease. https://doi.org/10.1101/2020.01.27.921353

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