Macrovesicular steatosis in greater than 30% of hepatocytes is a significant risk factor for primary graft nonfunction due to increased sensitivity to ischemia reperfusion (I/R) injury. The growing prevalence of hepatic steatosis due to the obesity epidemic, in conjunction with an aging population, may negatively impact the availability of suitable deceased liver donors. Some have suggested that metabolic interventions could decrease the fat content of liver grafts prior to transplantation. This concept has been successfully tested through nutritional supplementation in a few living donors. Utilization of deceased donor livers, however, requires defatting of explanted organs. Animal studies suggest that this can be accomplished by ex vivo warm perfusion in a time scale of a few hours. We estimate that this approach could significantly boost the size of the donor pool by increasing the utilization of steatotic livers. Here we review current knowledge on the mechanisms whereby excessive lipid storage and macrosteatosis exacerbate hepatic I/R injury, and possible approaches to address this problem, including ex vivo perfusion methods as well as metabolically induced defatting. We also discuss the challenges ahead that need to be addressed for clinical implementation.

Understanding the regulatory networks which control specific macrophage phenotypes is essential in identifying novel targets to correct macrophage mediated clinical disorders, often accompanied by inflammatory events. Since mesenchymal stromal cells (MSCs) have been shown to play key roles in regulating immune functions predominantly via a large number of secreted products, we used a fractional factorial approach to streamline experimental evaluation of MSC mediated inflammatory macrophage regulation. Our macrophage reprogramming metrics, human bone marrow MSC attenuation of macrophage pro-inflammatory M1 TNFα secretion and simultaneous enhanced expression of the M2 macrophage marker, CD206, were used as analysis endpoints. Objective evaluation of a panel of MSC secreted mediators indicated that PGE2 alone was sufficient in facilitating macrophage reprogramming, while IL4 only provided partial reprogramming. Inhibiting stromal cell PGE2 secretion with Indomethacin, reversed the macrophage reprogramming effect. PGE2 reprogramming was mediated through the EP4 receptor and indirectly through the CREB signaling pathway as GSK3 specific inhibitors induced M1 macrophages to express CD206. This reprogramming pathway functioned independently from the M1 suppression pathway, as neither CREB nor GSK3 inhibition reversed PGE2 TNF-α secretion attenuation. In conclusion, fractional factorial experimental design identified stromal derived PGE2 as the factor most important in facilitating macrophage reprogramming, albeit via two unique pathways.

Background & Aims-A common cause of liver donor ineligibility is macrosteatosis. Recovery of such livers could enhance donor availability. Living donor studies have shown diet-induced reduction of macrosteatosis enables transplantation. However, cadaveric liver macrosteatotic reduction must be performed ex vivo within hours. Towards this goal, we investigated the effect of accelerated macrosteatosis reduction on hepatocyte viability and function using a novel system of macrosteatotic hepatocytes.

Macrosteatotic livers exhibit elevated intrahepatic triglyceride (TG) content in the form of large lipid droplets (LDs), reduced ATP, and elevated reactive oxygen species (ROS), contributing to their elevated sensitivity to ischemia-reperfusion injury during transplantation. Decreasing macrosteatosis in living donors through dieting has been shown to improve transplantation outcome. Accomplishing the same feat in deceased donor grafts would require ex-vivo exposure to potent defatting agents. Herein, we used a rat hepatocyte culture system exhibiting macrosteatotic LD morphology, elevated TG levels, and elevated sensitivity to hypoxia and reoxygenation (H/R), to test for such agents and ameliorate H/R sensitivity. Macrosteatotic hepatocyte preconditioning for 48h with a defatting cocktail, previously developed to promote TG catabolism, reduced the number of macrosteatotic LDs and intracellular TG levels by 82% and 27%, respectively, but did not ameliorate sensitivity to H/R. L-carnitine supplementation to this cocktail, together with hyperoxic exposure, yielded a similar reduction in macrosteatotic LD numbers, and to a 57% reduction in intrahepatic TG storage, likely by increasing the supply of acetyl-CoA to mitochondria, as indicated by a 70% increase in ketone body secretion. Furthermore, this treatment reduced ROS levels by 32%, increased ATP levels by 27%, nearing ATP levels of lean controls, and completely abolished H/R sensitivity as indicated by ~85% viability post H/R and return of cytosolic lactate dehydrogenase release down to levels seen in lean controls. Cultures maintained for 48h post H/R were ~83% viable and exhibited superior urea secretion and bile canalicular transport compared to untreated macrosteatotic cultures. These findings show that the elevated sensitivity of macrosteatotic hepatocytes to H/R can be overcome by defatting agents, suggesting a possible route for the recovery of discarded macrosteatotic grafts.

Large-droplet macrovesicular steatosis (ld-MaS) in over 30% of the liver graft hepatocytes is a major risk factor in liver transplantation. An accurate assessment of ld-MaS percentage is crucial to determine liver graft transplantability, which is currently based on pathologists’ evaluations of hematoxylin and eosin (H&E) stained liver histology specimens, with the predominant criteria being the lipid droplets’ (LDs) relative size and their propensity to displace the hepatocyte’s nucleus to the cell periphery. Automated image analysis systems aimed at objectively and reproducibly quantifying ld-MaS do not accurately differentiate large LDs from small-droplet macrovesicular steatosis (sd-MaS) and do not take into account LD-mediated nuclear displacement, leading to poor correlation with pathologists’ assessment. Here we present an improved image analysis method that incorporates nuclear displacement as a key image feature to segment and classify ld-MaS from H&E stained liver histology slides. More than 52,000 LDs in 54 digital images from 9 patients were analyzed, and the performance of the proposed method was compared against that of current image analysis methods and the ld-MaS percentage evaluations of two trained pathologists from different centers. We show that combining nuclear displacement and LD size information significantly improves the separation between large and small macrovesicular LDs (specificity=93.7%, sensitivity=99.3%) and the correlation with the pathologists’ ld-MaS percentage assessment (R2=0.97). This performance vastly exceeds that of other automated image analyzers, which typically underestimate or overestimate the pathologists’ ld-MaS score. This work demonstrates the potential of automated ld-MaS analysis in monitoring the steatotic state of livers. The image analysis principles demonstrated here may help standardize ld-MaS scores among centers and ultimately help in the process of determining liver graft transplantability.