P-L14 Ex-vivo normothermic perfusion of an abattoir-derived porcine hepatic segment as a model for scientific research

Background The human immune response to bacterial and viral pathogens has been the focus of intense research, but details on the earliest phases of infection remain unclear. Better knowledge regarding the response of immune cells in the liver is important for the treatment of severe bacterial disease. Ex vivo perfusion which mimics physiological conditions of the liver may provide useful models for this research. An ex vivo model which perfuses hepatic segments would allow translational research on a physiological and reliable model in the scarce resource of human organs. Here we describe the extra-corporeal perfusion of a porcine hepatic segment. Methods Whole porcine livers were retrieved from animals slaughtered according to UK laws for food production and connected to a normothermic extracorporeal perfusion circuit. Constant perfusion via the hepatic artery and portal vein with heparinized autologous blood was established. Sodium bicarbonate, epoprostanol sodium and calcium chloride were also added to the perfusate to regulate acid-base status and reduce vasospasm. Functionality was assessed by monitoring blood-gases, perfusion pressures and flow rate. A segmental ex-vivo liver resection was performed to leave hepatic segment IV in circuit and isolated segment IV perfusion was maintained for one hour. Results Portal venous pressure was maintained between 8-16mmHg and hepatic arterial pressure between 80-90mmHg. Metabolic acidosis resolved with addition of sodium bicarbonate to the circuit with a pH of 7.42 after segmental perfusion for 1 hour. The lactate increased over the course of the perfusion to 20mmol/L after 1 hour of perfusion, however glucose levels were found to improve with the addition of sodium bicarbonate to the circuit. Conclusions Isolated segmental perfusion via ex-vivo resection of porcine hepatic segments is technically challenging. Ischaemic-reperfusion injury coupled with progressive metabolic acidosis may limit model viability. However, addition of sodium bicarbonate to the perfusate aids reduction of glucose levels and improves acidosis. Successful perfusion of a porcine hepatic segment provides the potential for segmental perfusion of human hepatic segments such as those resected during hemi-hepatectomies (HRA approved; REC 21/PR/0287). This is an important milestone leading to the creation of a model to study the early changes in human liver tissue for example during infection.

Number of detected proteins as the function of the sensitivity of proteomic technology in human liver cells

The main goal of the Russian part of C-HPP is to detect and functionally annotate missing proteins (PE2-PE4) encoded by human chromosome 18. However, identifying such proteins in a complex biological mixture using mass spectrometry (MS)-based methods is difficult due to the insufficient sensitivity of proteomic analysis methods. In this study, we determined the proteomic technology sensitivity using a standard set of UPS1 proteins as an example. The results revealed that 100% of proteins in a mixture could only be identified at a concentration of at least 10-9 M. The decrease in concentration leads to protein losses associated with technology sensitivity, and no UPS1 protein is detected at a concentration of 10-13 M. Therefore, two-dimensional fractionation of samples was applied to improve sensitivity. The human liver tissue was examined by selected reaction monitoring and shotgun methods of MS analysis using one-dimensional and two-dimensional fractionation to identify the proteins encoded by human chromosome 18. A total of 134 proteins were identified. The overlap between proteomic and transcriptomic data in human liver tissue was ~50%. This weak convergence is due to the low sensitivity of proteomic technology compared to transcriptomic approaches. Data is available via ProteomeXchange with identifier PXD026997.

Ruxolitinib Suppresses Liver Fibrosis Progression and Accelerates Fibrosis Reversal via Selectively Targeting Janus Kinase 1/2

BackgroundJAK1 and JAK2 have been implicated in fibrosis and cancer as a fibroblast-related marker; however, their role in liver fibrosis has not been elucidated. Here, we aim to determine the effect and underlying mechanism of JAK1/2 inhibition on liver fibrosis and hepatic stellate cells (HSCs) and further explore the therapeutic efficacy of Ruxolitinib, a JAK1/2 selective inhibitor, on preventing and reversing liver fibrosis in mice. MethodsImmunohistochemistry staining of JAK1 and JAK2 were performed on liver tissue in mice with hepatic fibrosis and human liver tissue microarray of liver cirrhosis and liver cancer. LX-2 cells treated with specific siRNA of JAK1 and JAK2 were used to analysis activation, proliferation and migration of HSCs regulated by JAK1/2. The effects of Ruxolitinib (JAK1/2 inhibitor) on liver fibrosis were studied in LX-2 cells and two progressive and reversible fibrosis animal models (carbon tetrachloride (CCl4), Thioacetamide (TAA)). ResultsWe found that JAK1/2 expression was positively correlated with the progression of HCC in humans and the levels of liver fibrosis in mice. Silencing of JAK1/2 down-regulated their downstream signaling and inhibited proliferation, migration, and activation of HSCs in vitro, while Ruxolitinib had similar effects on HSCs. Importantly, Ruxolitinib significantly attenuated fibrosis progression, improved cell damage, and accelerated fibrosis reversal in the liver of mice treated with CCl 4 or TAA. ConclusionsJAK1/2 regulates the function of HSCs and plays an essential role in liver fibrosis and HCC development. Its inhibitor, Ruxolitinib, may be an effective drug for preventing and treating liver fibrosis.

Using a Human Liver Tissue Equivalent (hLTE) Platform to Define the Functional Impact of Liver-Directed AAV Gene Therapy

Clinical trials employing AAV vectors for hemophilia A have been hindered by unanticipated immunological and/or inflammatory responses in some of the patients. Also, these trials have often yielded lower levels of transgene expression than were expected based upon preclinical studies, highlighting the poor correlation between the transduction efficiency observed in traditional 2D cultures of primary cells in vitro, and that observed in those same cell types in vivo. It has been also recognized that there are marked species-specific differences in AAV-vector tropism, raising the critical question of the accuracy with which various animal models will likely predict tropism/vector transduction efficiency, and eventual treatment success in humans. Human liver tissue equivalents (hLTEs) are comprised of major cell types in the liver in physiologically relevant frequencies and possess the ability to recapitulate the biology and function of native human liver. Here, we hypothesize that hLTEs can be used as a better model to predict the efficacy and safety of AAV gene therapy in humans. We fabricated hLTEs using 75% hepatocytes, 10% stellate cells, 10% Kupffer cells, and 5% liver sinusoid-derived endothelial cells in 96-well Elplasia plates with 79 microwells per well. hLTEs were transduced at an MOI of 10 5vg/cell, on the day of fabrication, with the clinically relevant serotypes AAV5 (hLTE-5) or AAV3b (hLTE-3b), both encoding a GFP reporter. After 4 days of self-aggregation, live/dead assay was performed to confirm viability. Non-transduced hLTEs served as negative controls (hLTE(-)), and hLTEs exposed to 20 mM acetaminophen were used as positive controls for liver inflammation/damage. Incucyte® Live-Cell Imaging system was used to track the aggregation and GFP expression of hLTEs. Over the course of the next 5 days, media was collected to determine hepatic functionality, RNA was isolated to assess dysregulation of genes involved in inflammation and fibrosis, DNA was isolated to determine whether AAV vectors integrate into the genome of human hepatocytes and, if so, to define the frequency at which this occurs and the genomic loci of integration, and hLTEs were fixed and processed at appropriate times for histological analyses and transmission electron microscopy (TEM). TEM analysis revealed that all groups exhibited microvilli and bile-canaliculus-like structures, demonstrating the formation of a rudimentary biliary system and, more importantly, proving that hLTEs resemble native liver structure. Incucyte® imaging showed that AAV5 and AAV3b transduction impaired formation of hLTEs (57.57 ± 2.42 and 24.57 ± 4.01 spheroids/well, respectively) in comparison with hLTE(-) (74.86 ± 3.8 spheroids/well). Quantification of GFP expression demonstrated that AAV5 yielded the most efficient transduction of hLTEs (fold change in GFP expression compared to control: 2.73 ± 0.09 and 1.19 ± 0.03 for hLTE-5 and hLTE-3b, respectively). Chromogenic assays showed decreased urea production in cell culture supernatants of AAV transduced groups compared to the non-transduced hLTEs on days 6 and 10 of culture, demonstrating decreased hepatocyte functionality. However, ALT and AST levels were similar in all groups. On day 10, hLTEs were either used for RNA isolation or fixed in 4% PFA and processed for histology. Masson's Trichrome and Alcian Blue/Sirius Red staining was performed to detect fibrosis, which was then quantified using ImageJ. These analyses showed no significant increase in fibrosis in either hLTE-5 or hLTE-3b compared to hLTE(-). Nevertheless, RT 2 PCR Array for Human Fibrosis detected dysregulation of several genes involved in fibrosis/inflammation in both hLTE-5 and hLTE-3b (16/84 and 26/84, respectively). In conclusion, data collected thus far show successful recapitulation of native liver biology and demonstrate that AAV5 transduces hLTEs more efficiently than AAV3b. However, impaired self-aggregation and decreased hepatocyte functionality was observed in both AAV-transduced groups. Studies to address the incidence and location(s) of AAV integration are ongoing. We have thus shown that the hLTE system can provide critical new knowledge regarding the efficacy and safety of AAV gene therapy in the human liver. Disclosures No relevant conflicts of interest to declare.

Long-term maintenance of functional primary human hepatocytes in 3D gelatin matrices produced by solution blow spinning

Solution blow spinning (SBS) has recently emerged as a novel method that can produce nano- and microfiber structures suitable for tissue engineering. Gelatin is an excellent precursor for SBS as it is derived mainly from collagens that are abundant in natural extracellular matrices. Here we report, for the first time the successful generation of 3D thermally crosslinked preforms by using SBS from porcine gelatin. These SBS mats were shown to have three-dimensional fibrous porous structure similar to that of mammalian tissue extracellular matrix. In pharma industry, there is an urgent need for adequate 3D liver tissue models that could be used in high throughput setting for drug screening and to assess drug induced liver injury. We used SBS mats as culturing substrates for human hepatocytes to create an array of 3D human liver tissue equivalents in 96-well format. The SBS mats were highly cytocompatible, facilitated the induction of hepatocyte specific CYP gene expression in response to common medications, and supported the maintenance of hepatocyte differentiation and polarization status in long term cultures for more than 3 weeks. Together, our results show that SBS-generated gelatin scaffolds are a simple and efficient platform for use in vitro for drug testing applications.

Fat causes necrosis and inflammation in parenchymal cells in human steatotic liver

Adapted fixation methods for electron microscopy allowed us to study liver cell fine structure in 217 biopsies of intact human livers over the course of 10 years. The following novel observations and concepts arose: single fat droplets in parenchymal cells can grow to a volume four times larger than the original cell, thereby extremely marginalizing the cytoplasm with all organelles. Necrosis of single parenchymal cells, still containing one huge fat droplet, suggests death by fat in a process of single-cell steatonecrosis. In a later stage of single-cell steatonecrosis, neutrophils and erythrocytes surround the single fat droplet, forming an inflammatory fat follicle indicating the apparent onset of inflammation. Also, fat droplets frequently incorporate masses of filamentous fragments and other material, most probably representing Mallory substance. No other structure or material was found that could possibly represent Mallory bodies. We regularly observe the extrusion of huge fat droplets, traversing the peripheral cytoplasm of parenchymal cells, the Disse space and the endothelium. These fat droplets fill the sinusoid as a sinusoidal lipid embolus. In conclusion, adapted methods of fixation applied to human liver tissue revealed that single, huge fat droplets cause necrosis and inflammation in single parenchymal cells. Fat droplets also collect Mallory substance and give rise to sinusoidal fat emboli. Therefore, degreasing of the liver seems to be an essential therapeutic first step in the self-repairing of non-alcoholic fatty liver disease. This might directly reduce single-cell steatotic necrosis and inflammation as elements in non-alcoholic steatohepatitis progression.

Multi-trait GWAS of atherosclerosis detects novel pleiotropic loci

Rationale: Although affecting different arterial territories, the related atherosclerotic vascular diseases coronary artery disease (CAD) and peripheral artery disease (PAD) share similar risk factors and have shared pathobiology. Analysis of their shared genetic architecture, along with that of common risk factors, may identify novel common biology. Objective: To identify novel pleiotropic genetic loci associated with atherosclerosis and provide a better understanding of biological pathways underlying atherosclerosis. Methods and Results: Summary statistics from genome wide association studies (GWAS) of nine known atherosclerotic (CAD, PAD) or atherosclerosis risk factors (body mass index, smoking initiation, type 2 diabetes, low density lipoprotein (LDL), high density lipoprotein, total cholesterol, and triglycerides) were combined to perform 15 separate multi-trait genetic association scans which resulted in 31 unique novel pleiotropic loci not yet reported as genome-wide significant for their respective traits. Colocalization with single-tissue eQTLs identified 34 candidate causal genes across 14 of the detected signals. Notably, the signal between PAD and CAD at the VDAC2 locus (rs7088974) colocalized with VDAC2 expression in aorta and tibial artery tissues. Additionally, the signal between PAD and LDL at the PCSK6 locus (rs1531817) affects PCSK6 splicing in human liver tissue and induced pluripotent derived hepatocyte like cells. Conclusions: Joint analysis of related atherosclerotic disease traits and their risk factors allowed identification of unified biology that may offer the opportunity for therapeutic manipulation. VDAC2 and PCSK6 represent possible shared causal biology where existing inhibitors may be able to be leveraged for novel therapies.

XIAP Knockdown in Alcohol-Associated Liver Disease Models Exhibits Divergent in vitro and in vivo Phenotypes Owing to a Potential Zonal Inhibitory Role of SMAC

Alcohol-associated liver disease (ALD) has been recognized as the most common cause of advanced liver disease worldwide, though mechanisms of pathogenesis remain incompletely understood. The X-linked inhibitor of apoptosis (XIAP) protein was originally described as an anti-apoptotic protein that directly binds and inhibits caspases-3, 7, and 9. Here, we investigated the function of XIAP in hepatocytes in vitro using gain and loss-of-function approaches. We noted an XIAP-dependent increase in caspase activation as well as increased inflammatory markers and pro-inflammatory EV release from hepatocytes in vitro. Primary hepatocytes (PMH) from XiapAlb.Cre and XiaploxP mice exhibited higher cell death but surprisingly, lower expression of inflammation markers. Conditioned media from these isolated Xiap deleted PMH further decrease inflammation in bone marrow-derived macrophages. Also, interestingly, when administered an ethanol plus Fas-agonist-Jo2 model and an ethanol plus CCl4 model, these animals failed to develop an exacerbated disease phenotype in vivo. Of note, neither XiapAlb.Cre nor XiapAAV8.Cre mice presented with aggravated liver injury, hepatocyte apoptosis, liver steatosis, or fibrosis. Since therapeutics targeting XIAP are currently in clinical trials and caspase-induced death is very important for development of ALD, we sought to explore the potential basis of this unexpected lack of effect. We utilized scRNA-seq and spatially reconstructed hepatocyte transcriptome data from human liver tissue and observed that XIAP was significantly zonated, along with its endogenous inhibitor second mitochondria-derived activator of caspases (SMAC) in periportal region. This contrasted with pericentral zonation of other IAPs including cIAP1 and Apollon as well as caspases 3, 7, and 9. Thus providing a potential explanation for compensation of the effect of Xiap deletion by other IAPs. In conclusion, our findings implicate a potential zonallydependent role for SMAC that prevented development of a phenotype in XIAP knockout mice in ALD models. Targeting SMAC may also be important in addition to current efforts of targeting XIAP in treatment of ALD.

Transcriptomic profiles of human livers undergoing rewarming machine perfusion before transplantation—first insights

Machine perfusion by controlled oxygenated rewarming (COR) is feasible and safe in clinical application and result in a promising outcome. This study utilizes next-generation sequencing (NGS) to investigate the transcriptome of human liver tissue undergoing COR before liver transplantation. Cold-stored livers were subjected to machine-assisted slow COR for ~120 min before transplantation. Biopsies were taken before (preCOR) and after COR (postCOR) and 1 h after reperfusion (postRep). The samples were sequenced, using RNA-seq to analyze differential transcriptional changes between the different stages and treatments of the grafts. Comparison of differential gene expression preCOR and postCOR demonstrated 10 upregulated genes. postRep 97 and 178 genes were upregulated and 7 and 13 downregulated compared to preCOR and postCOR, respectively. A shift of gene expressions by machine perfusion to the TGF-beta pathway was observed. The present study demonstrates distinct transcriptome profiles associated with machine perfusion by COR and transplantation of human livers. Such data provide a deeper understanding of the molecular mechanisms of machine perfusion technology in human liver transplantation.