(200c) Multi-Scale Control of Liver Regeneration: Integrating Molecular Regulation, Cell Phenotype Dynamics, and Physiological Response

Following damage, the liver initiates a recovery program inducing hepatocytes to enter the cell cycle and recover lost mass. As early as 30 seconds after injury, signaling cascades become active within the liver. Dynamic molecular changes continue for approximately 1 week following injury, restoring liver mass to the pre-injury levels. The dynamics of several molecular mediators of this process has been investigated previously to identify transient activation of inflammatory molecules for a few hours and a sustained activation of growth factors over several days. Yet much remains to be understood as to how the regulation of multiple molecular factors is coordinated to control liver repair mechanisms. Additionally, cell-types within the liver can each take on multiple distinct phenotypes either contributing to or inhibiting repair. The contributions of these phenotypes to liver repair and disease progression are just beginning to be appreciated. The present study takes a systems-based approach to investigate how multi-scale balances in cell phenotypes and molecular regulation impact liver regeneration.

We developed a computational model to synthesize the intrinsically multi-scale nature of liver regeneration by simulating connections between physiological-scale dynamics, activation phenotypes of non-parenchymal cells, and molecular signaling networks. Model analysis showed that shifting balances between populations of non-parenchymal cell activation phenotypes was sufficient to alter regeneration dynamics and overall tissue recovery following partial hepatectomy. As a perturbation to regeneration phenotype, we simulated alcohol-mediated suppression of liver regeneration by fitting our model to experimental data of liver recovery following chronic alcohol consumption and partial hepatectomy. Based on the model simulations, we predict that chronic alcohol consumption acts at a cellular-scale by shifting Kupffer cells from an M1 phenotype to an M2 phenotype following partial hepatectomy and by shifting hepatic stellate cells from a pro-regenerative phenotype to an anti-proliferative phenotype. At a molecular-scale, these changes in cell phenotypes are paralleled by dynamic increases in anti-inflammatory cytokine production and high levels of the anti-regenerative molecules such as fibrous collagens and TGFβ.

We tested these predictions at multiple-scales using high-throughput mRNA and protein measurements following partial hepatectomy in alcohol-fed rats and controls. We used laser capture microdissection to collect individual hepatic stellate cells from the livers of chronic alcohol-fed animals and controls before and after hepatectomy. We then used a high-throughput gene expression platform to quantify mRNA levels of ~100 genes across these individual cells and used multivariate statistics to identify clusters of hepatic stellate cell activation phenotypes. Our experimental results indicate that multiple activation of phenotypes of hepatic stellate cells arise following partial hepatectomy and alcohol exposure, consistent with model predictions. We followed a high-throughput ELISA-based approach to measure the cytokine microenvironment of the regenerating liver and how chronic alcohol consumption modulates the inflammatory milieu at the tissue-scale. Our results thus far indicate that anti-inflammatory cytokines increase in alcohol-fed animals prior to hepatocyte replication thus impairing hepatocyte priming, matching model predictions.

Taken together, these results provide novel insights into how molecular regulation during liver regeneration influences multiple scales to regulate non-parenchymal cell activation phenotype and tissue recovery.

Funding: R01 AA018873, T32 AA007463.