CFD assessment of the effect of convective mass transport on the intracellular clearance of intracellular triglycerides in macrosteatotic hepatocytes.

Donor livers available to transplant for patients with end-stage liver disease are in severe shortage. One possible avenue to expand the donor pool is to recondition livers that would be otherwise discarded due to excessive fat content. Severely steatotic livers (also known as fatty livers) are highly susceptible to ischemia-reperfusion injury and as a result, primary liver non-function post-transplantation. Prior studies in isolated perfused rat livers suggest that "defatting" may be possible in a timeframe of a few hours; thus, it is conceivable that fatty liver grafts could be recovered by machine perfusion to clear stored fat from the organ prior to transplantation. However, studies using hepatoma cells and adult hepatocytes made fatty in culture report that defatting may take several days. Because cell culture studies were done in static conditions, we hypothesized that the defatting kinetics are highly sensitive to flow-mediated transport of metabolites. To investigate this question, we experimentally evaluated the effect of increasing flow rate on the defatting kinetics of cultured HepG2 cells and developed an in silico combined reaction-transport model to identify possible rate-limiting steps in the defatting process. We found that in cultured fatty HepG2 cells, the time required to clear stored fat down to lean control cells can be reduced from 48 to 4-6 h by switching from static to flow conditions. The flow required resulted in a fluid shear of .008 Pa, which did not adversely affect hepatic function. The reaction-transport model suggests that the transport of L-carnitine, which is the carrier responsible for taking free fatty acids into the mitochondria, is the key rate-limiting process in defatting that was modulated by flow. Therefore, we can ensure higher levels of L-carnitine uptake by the cells by choosing flow rates that minimize the limiting mass transport while minimizing shear stress.

Metabolic Flux Distribution during Defatting of Steatotic Human Hepatoma (HepG2) Cells.

Methods that rapidly decrease fat in steatotic hepatocytes may be helpful to recover severely fatty livers for transplantation. Defatting kinetics are highly dependent upon the extracellular medium composition; however, the pathways involved are poorly understood. Steatosis was induced in human hepatoma cells (HepG2) by exposure to high levels of free fatty acids, followed by defatting using plain medium containing no fatty acids, or medium supplemented with a cocktail of defatting agents previously described before. We measured the levels of 28 extracellular metabolites and intracellular triglyceride, and fed the data into a steady-state mass balance model to estimate strictly intracellular fluxes. We found that during defatting, triglyceride content decreased, while beta-oxidation, the tricarboxylic acid cycle, and the urea cycle increased. These fluxes were augmented by defatting agents, and even more so by hyperoxic conditions. In all defatting conditions, the rate of extracellular glucose uptake/release was very small compared to the internal supply from glycogenolysis, and glycolysis remained highly active. Thus, in steatotic HepG2 cells, glycolysis and fatty acid oxidation may co-exist. Together, these pathways generate reducing equivalents that are supplied to mitochondrial oxidative phosphorylation.

Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts.

During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. SB431542, an inhibitor of transforming growth factor-ß1 (TGF-ß1)-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-ß1 at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-ß1 is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia.

Fractional factorial design to investigate stromal cell regulation of macrophage plasticity.

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.

Elevated sensitivity of macrosteatotic hepatocytes to hypoxia/reoxygenation stress is reversed by a novel defatting protocol.

Macrosteatotic livers exhibit elevated intrahepatic triglyceride (TG) levels in the form of large lipid droplets (LDs), reduced adenosine triphosphate (ATP) levels, and elevated reactive oxygen species (ROS) levels, and this contributes to their elevated sensitivity to ischemia/reperfusion injury during transplantation. Reducing macrosteatosis in living donors through dieting has been shown to improve transplant outcomes. Accomplishing the same feat for deceased donor grafts would require ex vivo exposure to potent defatting agents. Here we used a rat hepatocyte culture system exhibiting a macrosteatotic LD morphology, elevated TG levels, and an elevated sensitivity to hypoxia/reoxygenation (H/R) to test for such agents and ameliorate H/R sensitivity. Macrosteatotic hepatocyte preconditioning for 48 hours with a defatting cocktail that was previously developed to promote TG catabolism reduced the number of macrosteatotic LDs and intracellular TG levels by 82% and 27%, respectively, but it did not ameliorate sensitivity to H/R. Supplementation of this cocktail with l-carnitine, together with hyperoxic exposure, yielded a similar reduction in the number of macrosteatotic LDs and a 57% reduction in intrahepatic TG storage, likely by increasing the supply of acetyl coenzyme A 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% (to levels near those of lean controls), and completely abolished H/R sensitivity as indicated by approximately 85% viability after H/R and the reduction of cytosolic lactate dehydrogenase release to levels seen in lean controls. Cultures maintained for 48 hours after H/R were approximately 83% viable and exhibited superior urea secretion and bile canalicular transport in comparison with untreated macrosteatotic cultures. In conclusion, these findings show that the elevated sensitivity of macrosteatotic hepatocytes to H/R can be overcome by defatting agents, and they suggest a possible route for the recovery of discarded macrosteatotic grafts.

Automated image analysis method for detecting and quantifying macrovesicular steatosis in hematoxylin and eosin-stained histology images of human livers.

Large-droplet macrovesicular steatosis (ld-MaS) in more than 30% of liver graft hepatocytes is a major risk factor for liver transplantation. An accurate assessment of the ld-MaS percentage is crucial for determining 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 relative size of the lipid droplets (LDs) and their propensity to displace a 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 and do not take into account LD-mediated nuclear displacement; this leads to a poor correlation with pathologists' assessments. Here we present an improved image analysis method that incorporates nuclear displacement as a key image feature for segmenting and classifying ld-MaS from H&E-stained liver histology slides. 52,000 LDs in 54 digital images from 9 patients were analyzed, and the performance of the proposed method was compared against the performance of current image analysis methods and the ld-MaS percentage evaluations of 2 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 pathologists' ld-MaS percentage assessments (linear regression coefficient of determination = 0.97). This performance vastly exceeds that of other automated image analyzers, which typically underestimate or overestimate pathologists' ld-MaS scores. 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 to standardize ld-MaS scores among centers and ultimately help in the process of determining liver graft transplantability.

Rat hepatocyte culture model of macrosteatosis: effect of macrosteatosis induction and reversal on viability and liver-specific function.

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. METHODS: Hepatocytes isolated from lean Zucker rats were cultured in a collagen sandwich, incubated for 6 days in fatty acid-supplemented medium to induce steatosis, and then switched for 2 days to medium supplemented with lipid metabolism promoting agents. Intracellular lipid droplet size distribution and triglyceride, viability, albumin and urea secretion, and bile canalicular function were measured. RESULTS: Fatty acid-supplemented medium induced microsteatosis in 3 days and macrosteatosis in 6 days, the latter evidenced by large lipid droplets dislocating the nucleus to the cell periphery. Macrosteatosis significantly impaired all functions tested. Macrosteatosis decreased upon returning hepatocytes to standard medium, and the rate of decrease was 4-fold faster with supplemented agents, yielding 80% reduction in 2 days. Viability of macrosteatosis reduced hepatocytes was similar to control lean cells. Accelerated macrosteatotic reduction led to faster recovery of urea secretion and bile canalicular function, but not of albumin secretion. CONCLUSIONS: Macrosteatosis reversibly decreases hepatocyte function and supplementary agents accelerate macrosteatosis reduction and some functional restoration with no effect on viability. This in vitro model may be useful to screen agents for macrosteatotic reduction in livers before transplantation.