(717g) Imparting Hepatoprotection to Engineered Liver Tissues Using Anti-Microbial Peptides

Every day the liver is confronted with a wide range of toxins resulting in the apoptosis of hepatic cells which if left uncontrolled, could potentially cause significant liver failure.  While antimicrobial peptides (AMP) are typically known for their bactericidal activity, recent studies have shown they also possess a hepatoprotective effect [1].  However, there is limited information on how this effect is conferred by the administration of AMPs.  Human cathelicidins, such as LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), are peptide sequences that also exhibit anti-microbial properties.  In this study, we have investigated the hepatoprotective effect of LL-37 on organotypic liver models by administration with transforming growth factor β (TGF-β), a pro-inflammatory signaling molecule typically associated with the activation of liver fibrosis and hepatocyte apoptosis.  To the best of our knowledge, this is the first study that demonstrates that the human cathelicidin, LL-37, can prevent hepatic cell death.  Our goal is to obtain information on its protective effect on each of three major hepatic cell types (hepatocytes, liver sinusoidal endothelial cells, and Kupffer cells) as well as changes in their inter-cellular communications.

Organotypic liver models were assembled using primary hepatic cells obtained through the surgical excision of rat livers.  Cellular models that closely mimic the three dimensional architecture and in vivo composition of the liver were assembled using detachable polyelectrolyte multilayers that serve as a mimic for the space of disse [2].  Hepatocyte monolayers (HMs) served as controls.  All hepatic cultures were maintained for up to 12 days.  TGF-β was added to cultures at different time points and its effects on cell death and hepatic phenotype were monitored.  LL-37 was administered at a physiological concentration of 1 μg/mL at these same time points.  Apoptosis was monitored through fluorescence imaging for FITC-Annexin V and assaying for caspase 3 activity. 

Studies conducted so far have demonstrated that LL-37 alone does not result in significant cell death. Furthermore, apoptosis that results due to the addition of exogenous TGF-β can be reduced by administering LL-37. When LL-37 was administered to HMs 30 min prior to TGF-β, the protective effect was maximized when investigated by FITC-Annexin staining. This was further confirmed by caspase 3 activity where TGF-β treatment exhibited a decrease only when hepatocytes had already been exposed to LL-37. However, this trend was not observed at later time points in HMs, possibly due to dedifferentiation of hepatocytes.  .  

Since the organotypic liver model is a multi-cellular system, our current efforts are focused on unearthing the functional changes of each hepatic cell type and perturbations in inter-cellular signaling.. We have demonstrated for the first time that human cathelicidins such as LL-37 confer a protective effect against TGF-β induced apoptosis in hepatocytes. 

References:

1.         Lee, W.R., and coworkers, Toxicology and Applied Pharmacology, 2011. 256(2): p. 209-215.

2.         Larkin A. L. and coworkers, Tissue Engineering Part C: Methods, In Press, 2013.