Irreversible Electroporation of the Liver Increases the Transplant Engraftment of Hepatocytes.

INTRODUCTION: Irreversible electroporation (IRE) is a tissue ablation technology that kills cells with short electrical pulses that do not induce thermal damage, thereby preserving the extracellular matrix. Preclinical research suggests that IRE may be developed as a tool for regenerative surgery by clearing existing host cells within a solid organ and creating a supportive niche for new cell engraftment. We hypothesized that hepatocytes transplanted by injection into the portal circulation would preferentially engraft within liver parenchyma pretreated with IRE. METHODS: Transgene-positive ß-galactosidase-expressing hepatocytes were isolated from B6.129S7-Gt(ROSA)26Sor/J (ROSA26) mice and transplanted by intrasplenic injection into wild-type littermates that received liver IRE pretreatment or control sham treatment. Engraftment of donor hepatocytes in recipient livers was determined by X-gal staining. RESULTS: Significantly higher numbers of X-gal+ donor hepatocytes engrafted in the livers of IRE-treated mice as compared to sham-treated mice. X-gal+ hepatocytes persisted in IRE-treated recipients for at least 11 d post-transplant and formed clusters. Immunostaining demonstrated the presence of HNF4A/Ki67/ß-galactosidase triple-positive cells within IRE-ablation zones, indicating that transplanted hepatocytes preferentially engrafted in IRE-treated liver parenchyma and proliferated. CONCLUSIONS: IRE pretreatment of the liver increased engraftment of transplanted hepatocytes within the IRE-ablation zone. IRE treatment of the host liver may be developed clinically as a strategy to increase engraftment efficiency of primary hepatocytes and/or hepatocytes derived from stem cells in cell transplant therapies.

Normal and fibrotic liver parenchyma respond differently to irreversible electroporation.

BACKGROUND: The safety and efficacy of irreversible electroporation (IRE) in treating hepatic, biliary, and pancreatic malignancies are active areas of clinical investigation. In addition, recent studies have shown that IRE may enable regenerative surgery and in vivo tissue engineering. To use IRE effectively in these clinical applications, it is important to understand how different tissue microenvironments impact the response to IRE. In this study, we characterize the electrical and histological properties of non-fibrotic and fibrotic liver parenchyma before and after IRE treatment. METHODS: Electrical resistivity and histology of fibrotic liver from C57BL/6 mice fed a 0.1% 3,5-diethylcarbonyl-1,4-dihydrocollidine (DDC) diet were compared to those of non-fibrotic liver from matched control mice before and after IRE treatment. RESULTS: At baseline, the electrical resistivity of fibrotic liver was lower than that of non-fibrotic liver. Post-IRE, resistivity of non-fibrotic liver declined and then recovered back to baseline with time, correlating with hepatocyte repopulation of the ablated parenchyma without deposition of fibrotic scar. In contrast, resistivity of fibrotic liver remained depressed after IRE treatment, correlating with persistent inflammation. CONCLUSION: Non-fibrotic and fibrotic liver respond to IRE differently. The underlying tissue microenvironment is an important modifying factor to consider when designing IRE protocols for tissue ablation.

Neutrophils are important for the development of pro-reparative macrophages after irreversible electroporation of the liver in mice.

Irreversible electroporation (IRE) is a non-thermal tissue ablative technology that has emerging applications in surgical oncology and regenerative surgery. To advance its therapeutic usefulness, it is important to understand the mechanisms through which IRE induces cell death and the role of the innate immune system in mediating subsequent regenerative repair. Through intravital imaging of the liver in mice, we show that IRE produces distinctive tissue injury features, including delayed yet robust recruitment of neutrophils, consistent with programmed necrosis. IRE treatment converts the monocyte/macrophage balance from pro-inflammatory to pro-reparative populations, and depletion of neutrophils inhibits this conversion. Reduced generation of pro-reparative Ly6CloF4/80hi macrophages correlates with lower numbers of SOX9+ hepatic progenitor cells in areas of macrophage clusters within the IRE injury zone. Our findings suggest that neutrophils play an important role in promoting the development of pro-reparative Ly6Clo monocytes/macrophages at the site of IRE injury, thus establishing conditions of regenerative repair.

Liver epithelial focal adhesion kinase modulates fibrogenesis and hedgehog signaling.

Focal adhesion kinase (FAK) is an important mediator of extracellular matrix-integrin mechano-signal transduction that regulates cell motility, survival, and proliferation. As such, FAK is being investigated as a potential therapeutic target for malignant and fibrotic diseases, and numerous clinical trials of FAK inhibitors are underway. The function of FAK in nonmalignant, nonmotile epithelial cells is not well understood. We previously showed that hepatocytes demonstrated activated FAK near stiff collagen tracts in fibrotic livers. In this study, we examined the role of liver epithelial FAK by inducing fibrotic liver disease in mice with liver epithelial FAK deficiency. We found that mice that lacked FAK in liver epithelial cells developed more severe liver injury and worse fibrosis as compared with controls. Increased fibrosis in liver epithelial FAK-deficient mice was linked to the activation of several profibrotic pathways, including the hedgehog/smoothened pathway. FAK-deficient hepatocytes produced increased Indian hedgehog in a manner dependent on matrix stiffness. Furthermore, expression of the hedgehog receptor, smoothened, was increased in macrophages and biliary cells of hepatocyte-specific FAK-deficient fibrotic livers. These results indicate that liver epithelial FAK has important regulatory roles in the response to liver injury and progression of fibrosis.

Transgenerational effects of parental light environment on progeny competitive performance and lifetime fitness.

Plant and animal parents may respond to environmental conditions such as resource stress by altering traits of their offspring via heritable non-genetic effects. While such transgenerational plasticity can result in progeny phenotypes that are functionally pre-adapted to the inducing environment, it is unclear whether such parental effects measurably enhance the adult competitive success and lifetime reproductive output of progeny, and whether they may also adversely affect fitness if offspring encounter contrasting conditions. In glasshouse experiments with inbred genotypes of the annual plant Polygonum persicaria, we tested the effects of parental shade versus sun on (a) competitive performance of progeny in shade, and (b) lifetime reproductive fitness of progeny in three contrasting treatments. Shaded parents produced offspring with increased fitness in shade despite competition, as well as greater competitive impact on plant neighbours. Inherited effects of parental light conditions also significantly altered lifetime fitness: parental shade increased reproductive output for progeny in neighbour and understorey shade, but decreased fitness for progeny in sunny, dry conditions. Along with these substantial adaptive and maladaptive transgenerational effects, results show complex interactions between genotypes, parent environment and progeny conditions that underscore the role of environmental variability and change in shaping future adaptive potential. This article is part of the theme issue 'The role of plasticity in phenotypic adaptation to rapid environmental change'.