Designing a fibrotic microenvironment to investigate changes in human liver sinusoidal endothelial cell function

Abstract

The deposition of extracellular matrix (ECM) proteins by hepatic cells during fibrosis leads to the stiffening of the organ and perturbed cellular functions. Changes in the elasticity of liver tissue are manifested by altered phenotype in hepatic cells. We have investigated changes in human liver sinusoidal endothelial cells (hLSECs) that occur as the elastic modulus of their matrix transitions from healthy (6kPa) to fibrotic (36kPa) conditions. We have also investigated the role played by Kupffer cells in the dedifferentiation of hLSECs. We report the complete loss of fenestrae and the expression of CD31 at the surface as a result of increasing elastic moduli. LSECs exhibited a greater number of actin stress fibers and vinculin focal adhesion on the stiffer substrate, as well. A novel finding is that these identical trends can be obtained on soft (6kPa) substrates by introducing an inflamed microenvironment through the addition of Kupffer cells. hLSEC monocultures on 6kPa gels exhibited fenestrae that were 140.7±52.6nm in diameter as well as a lack of surface CD31 expression. Co-culturing hLSECs with rat Kupffer cells (rKCs) on 6kPa substrates, resulted in the complete loss of fenestrae, an increase in CD31 expression and in a well-organized cytoskeleton. These results demonstrate that the increasing stiffness of liver matrices does not solely result in changes in hLSEC phenotype. Even on soft substrates, culturing hLSECs in an inflamed microenvironment can result in their dedifferentiation. Our findings demonstrate the interplay between matrix elasticity and inflammation in the progression of hepatic fibrosis.

The hepatic “matrisome” responds dynamically to injury: Characterization of transitional changes to the extracellular matrix in mice

“…For example, ECM/integrin interactions mediate rapid and flexible responses to changes in the environment. It is known that fibrotic ECM is known to impact cell phenotype, inflammation, and metastasis in the liver . The impact of the ECM changes observed here on earlier stages of liver injury should be determined.…”

Sinusoidal communication in liver fibrosis and regeneration

“…LSEC seeded on 6 kPa substrates exhibited well-defined fenestrae arranged in sieve plate structures, and underwent pseudocapillarization after 96h. However, as a result of increasing elastic modulus, LSEC seeded on 36 kPa substrates completely lose fenestrae and express CD31 at the surface just after 24 h [39], thus confirming the importance of HSC-derived ECM on LSEC phenotype [18]. …”

A microfluidically perfused three dimensional human liver model

“…This likely affects liver cell function and contributes to experimental bias. To overcome these limitations, we used freshly isolated human umbilical vein endothelial cells (HUVEC) instead of liver sinusoidal endothelial cells (LSEC), since LSEC rapidly tend to dedifferentiate in vitro, which is associated with a loss of fenestrae and re-organization of the cytoskeleton [22]. This dedifferentiation process is difficult to monitor or control, and potentially adds an additional bias in day-to-day experimentation.…”