There is a growing literature of clinical studies of bone marrow (BM) cell therapy for liver cirrhosis. At present, the optimum choice of cell type(s) and the mechanism(s) of effect remain undefined. Cells of the monocyte-macrophage lineage have key roles in the development and resolution of liver fibrosis. Therefore, I tested the therapeutic effects of these cells in the context of experimental murine liver fibrosis. The effects of unmanipulated, syngeneic macrophages, their specific BM precursors and unfractionated (whole) BM cells were examined in the iterative carbon tetrachloride model of liver fibrosis. BM-derived macrophage (BMM) delivery resulted in early chemokine upregulation with the hepatic recruitment of endogenous macrophages and neutrophils. These cells delivered matrix metalloproteinases-13 and -9 respectively, into the hepatic scar. The effector cell infiltrate was accompanied by increased levels of the anti-inflammatory cytokine IL-10. A reduction in hepatic myofibroblasts was followed by reduced fibrosis detected 4 weeks after macrophage infusion. Serum albumin levels were elevated at this time. Upregulation of the liver progenitor cell mitogen TWEAK preceded expansion of the progenitor cell compartment. BMM delivery increased hepatic expression of cytokines with reparative effects (including colony stimulating factor-1, insulin-like growth factor-1 and vascular endothelial growth factor). In contrast to the effects of differentiated macrophages, liver fibrosis was not significantly improved by the application of macrophage precursors and was exacerbated by whole BM. BMMs did not affect liver fibrosis or regeneration in the 1% DDC model of biliary disease. These effects were only detected following the intraportal delivery of BM cells. The peripheral (tail) vein administration of BMMs, either singly or repeatedly did not recapitulate the therapeutic phenotype. This was investigated by in vivo tracking of BMMs constitutively expressing green fluorescent protein (GFP). The peripheral administration route resulted in the early (1 hour) accumulation of BMMs within the pulmonary system. This was followed by delayed hepatic engraftment, which was also numerically reduced (< 30%) compared with intraportal administration. Macrophage cell therapy improves clinically relevant parameters in experimental chronic liver injury. Paracrine signalling to endogenous cells amplifies the effect. The benefits from this single, defined cell type suggest clinical potential.

University of Macau / Institute of Chinese Medical Sciences

The purpose of the investigation was the design and development of an oxygen carrier system for oxygenation of liver cells (hepatocytes) in a bio-artificial liver support system. Acute liver failure is a devastating condition with higher than 80% mortality. Currently the only successful treatment is orthotopic liver transplantation. The high mortality rate could be reduced if a system could be developed that could bridge the patient either until recovery (due to the liver’s well-known regeneration ability) or until transplantation. Such a system requires a bioreactor with a high density of cultured cells. Sufficient oxygen delivery to the cells is critical to ensure efficient cell function. The CSIR and University of Pretoria (UP) have designed and developed a novel bio-artificial liver support system (BALSS) that utilizes perfluorooctyl bromide (PFOB) as artificial oxygen carrier. As the PFOB is not miscible with water, it needs to be emulsified. To enable the use of the PFOB emulsion in the UP-CSIR BALSS, a study was carried out to investigate relevant aspects relating to the PFOB emulsion, i.e. the formulation, manufacturing procedure, stability, rheology and mass transfer characteristics. The study results are reported in this dissertation, including a proposed mass transfer model for describing oxygen mass transfer to and from the PFOB emulsions. Emulsion stability can be improved through control of the droplet size and size distribution, limiting Ostwald ripening, and control of zeta potential of the dispersed phase droplets. PFOB emulsions with dispersed phase (PFOB) volume fractions between 0.4 and 0.5 and Sauter mean droplet diameter between 100 and 200 nm were found to be optimal for oxygen mass transfer in cell culture systems. The PFOB emulsion in the UP-CSIR BALSS can be concentrated and recirculated using ultrafiltration. Quantitative recovery of PFOB from its emulsions can be carried out using distillation with orthophosphoric acid. Experimental overall mass transfer coefficients for membrane oxygenators obtained without PFOB compared well with literature reported values of 2.5x10-5 m/s by Goerke et al. (2002) and 1 – 3x10-5 m/s by Schneider et al. (1995) for similar systems. The addition of 0.2 v/v PFOB leads to an increase in the membrane oxygenator mass transfer coefficient by a factor of about 30, and an increase in oxygen carrying capacity by a factor of about 4.5. It was also shown that suitable PFOB emulsions can have a significant impact on the growth and function of hepatocytes in a BALSS. / Thesis (PhD (Chemical Engineering))--University of Pretoria, 2005. / Chemical Engineering / unrestricted

Recent reports in the literature suggest that oral contraceptive steroid therapy may be implicated in the development of benign hepatic adenomas in women. Since estrogens and progestins are known to affect liver function, we studied effects of chronic administration of the oral contraceptive agents mestranol and norethindrone on various indices of hepatic integrity. Several hepatic mixed function oxidase activities were measured: benzo(a)pyrene hydroxylase, epoxide hydrase, aniline hydroxylase (Appendix II) and aminopyrlne N-demethylase (Appendix II). In addition, benzo(a)pyrene hydroxylase activity in lung tissue was measured. As an indication of whether metabolites of the contraceptive steroids were bound to liver macromolecules, irreversible binding of [³H]-benzopyrene was measured. Hepatic histopathology (light microscopy using hematoxylin and eosin stain and oil red 0 stain) was carried out to determine if functional alterations correlated with pathological changes in the liver. Comparisons were also made between ethanol treated and non-ethanol treated groups to determine if contraceptive steroid-associated hepatotoxiclty was enhanced by or would enhance, the hepatotoxiclty of ethanol administration. Female and male Wistar rats were pair-fed a nutritional liquid diet, Sustacal[sup R] (Mead Johnson) to which was added either sucrose or ethanol as 40% of calories. Oral contraceptive steroids were administered daily in the liquid, diet in the following doses: mestranol, 0.6 mg/kg per day, alone or in combination with norethindrone, 5.0 mg/kg per day. Initial short-term studies showed that the ethanol plus Sustacal[sup R] diet generally caused enzyme induction compared to the plain Sustacal[sup R] diet or the sucrose plus Sustacal[sup R] diet in animals treated for up to 6 weeks. Animals that were administered the contraceptive steroids for a similar time period also demonstrated hepatic microsomal enzyme induction. Enzyme activity in animals that received ethanol plus the contraceptive steroids was increased above that seen for each agent alone. Chronic studies showed that ethanol administration for 6 months produced hepatotoxiclty in both male and female rats. Hepatotoxiclty was observed functionally as decreased hepatic benzo(a)pyrene hydroxylase activity and histopathologically as increased fat accumulation in zone 3 of the liver lobules. It was found that administration of the contraceptive steroids to female rats tended to protect against ethanol-associated hepatotoxiclty. The protective effect was observed functionally as maintenance of control levels of hepatic benzo(a)pyrene hydroxylase activity and morphologically as lesser amounts of fat accumulation ln the liver. That is, there tended to be a correlation between the level of hepatic benzo(a)pyrene hydroxylase activity and histological fat accumulation as an indication of ethanol-associated hepatotoxiclty. A Sustacal associated phenomenon was evident in all animals in which hlstopathology was carried out. The "Sustacal effect" was observed, as mlcrodroplet fat accumulation ln zone 1 of the liver lobule. Contraceptive steroid treated females showed the least "Sustacal effect". Microsomal enzyme activity did not appear to be affected by the "Sustacal effect". It was concluded that the contraceptive steroids administered did not increase ethanol hepatotoxicity. Instead, it appeared that female sex steroids tended to attenuate ethanol-assoclated hepatotoxicity. There was no evidence to suggest that the oral contraceptive steroids were directly associated with overt hepatotoxicity. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22302号 / 医博第4543号 / 新制||医||1040(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 妹尾 浩, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13355号 / 論医博第2201号 / 新制||医||1044(附属図書館) / (主査)教授 妹尾 浩, 教授 羽賀 博典, 教授 武藤 学 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The metabolism of lignocaine to monoethylglycinexylidide has been found useful as an indicator of hepatic function in association with liver transplantation. It has been postulated that this might be due to the common effect of hypoxic damage on liver function and lignocaine metabolism. The aim of this work was to establish whether hepatic lignocaine elimination was impaired by hypoxia and whether lignocaine extraction ratio and clearance could be used as an indicator of hepatic function. This was studied using the isolated pig liver perfused via the hepatic artery and portal vein. To establish whether the pig liver could be used as a possible human model for this investigation and whether lignocaine had any detrimental effects on liver function and blood flow in vivo, hepatic lignocaine elimination and the effects of lignocaine administration on hepatic function and blood flow were studied in the anaesthetized pig, surgically prepared to allow sampling across the liver and direct hepatic blood flow measurement. Hepatic lignocaine elimination was then studied in the isolated perfused liver to determine whether this was similar to that found in vivo. The definitive studies required preliminary investigations not available from the literature to determine the feasibility of comparing in vivo and ex vivo hepatic function using the same liver. In addition, by studying the decay of lignocaine after bolus dose administration the necessary pharmacokinetic parameters to achieve similar constant hepatic affluent lignocaine concentrations in vivo and in the isolated preparation could be determined. The preliminary investigations showed that a sequential experiment using the same liver to compare in vivo and ex vivo function was inappropriate as the energy state of isolated perfused livers previously studied in vivo was significantly different from that in livers perfused immediately. The decay of lignocaine after a bolus dose in vivo and ex vivo could be described by a two-compartment open model and in both preparations the derived pharmacokinetic parameters from this analysis were used to achieve similar constant hepatic affluent concentrations over the study period used to determine hepatic lignocaine elimination. Lignocaine extraction ratio by the in situ pig liver was similar to that reported in man and together with hepatic clearance and intrinsic clearance was similar to that determined in the isolated state when different livers were used for this comparison. There was no detrimental effect of lignocaine administration on hepatic function and blood flow In vivo. Lignocaine extraction ratio and clearance and monoethylglycinexylidide formation were significantly impaired in livers subjected to hypoxia. Lignocaine elimination correlated strongly with hepatic cellular ATP, energy charge and ATP/ ADP ratio as well as with hepatic potassium release but less strongly with aspartate aminotransferase release when this relationship was tested using the combined data from hypoxic and normoxic livers ex vivo. These correlations were positive for hepatic adenine nucleotide status and negative for hepatic potassium and aspartate aminotransferase release. Neither hepatic alanine aminotransferase release nor lactate utilization were significantly affected by hypoxia. Lignocaine extraction ratio, hepatic oxygen consumption, ATP content, bile flow and potassium release were shown to be equivalent, more highly sensitive, and earlier indicators of hypoxic hepatic injury than hepatic aspartate aminotransferase release in the isolated perfused pig liver.

The worldwide prevalence of obesity and obesity-associated liver diseases have attracted great attention in the past decades. Obesity is an increasing health problem, which can induce a series of metabolic syndrome associated diseases, such as fatty liver disease, type 2 diabetes. The conventional causes for obesity, such as over-eating, sedentary life-style, and genetic factors, cannot fully explain the global rapid increase of obese population in the last few decades. It was found that the production of persistent organic pollutants (POPs) in the industry was closely correlated with the prevalence of obesity. POPs are organic chemicals that are resisted to degrade by various processes and widely applied in daily products to improve the quality of our life. 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) is the most abundant and toxic congener in the family of polybrominated diphenyl ethers (PBDEs), which are the commonly used flame retardants and listed as POPs in 2009. High concentration of BDE-47 has been found in indoor dust and marine fish in Hong Kong. Owing to their high lipophilic and persistent characters, BDE-47 is mainly accumulated in adipose tissue. Epidemiological data indicates that exposure to BDE-47 is associated with obesity and obesity-associated liver diseases. Therefore, based on published research, we hypothesize that BDE-47 exposure may increase the occurrence of obesity and aggravate the progression of obesity-associated fatty liver disease through promoting adipocyte differentiation and impairing lipid metabolism. To verify this hypothesis, mouse preadipocytes (3T3-L1 cells) were exposed to BDE-47 and differentiated into adipocytes. Excitedly, with BDE-47 exposure, more lipid droplets were formed and accumulated in the treated cells than that in untreated adipocytes (without BDE-47 exposure). Along with the increased content of triglyceride accumulation, augmented gene and protein levels of transcription factors (PPARγ and PGC-1α), and related genes (FABP4 and C/EBPα) were also detected in BDE-47 treated cells. In addition, the total production of reactive oxygen species (ROS), contents of lipid peroxidation and DNA oxidation were obviously increased in adipocytes treated with BDE-47 (10 μM). To explore how BDE-47 regulated the oxidative stress signal pathways, antioxidants of ROS sources were employed with BDE-47 exposure during adipocyte differentiation. Notably, mitochondrial respiration, xanthine oxidase and NADPH pathway were significantly influenced by BDE-47 exposure to generate ROS in the treated adipocytes. The effects of BDE-47 on mitochondrial respiration were also determined for further exploring the relationship between mitochondrial ROS and adipocyte differentiation. Significant elevation of mitochondrial ROS was detected in adipocytes exposed with BDE-47 (10 μM). Furthermore, to support the energy requirements for the growth of adipocytes during differentiation process, BDE-47 improved the mitochondrial metabolism for ATP production via increasing the spare mitochondrial respiration capacity. Inhibiting the mitochondrial ROS generation in BDE-47-treated adipocytes with antioxidant attenuated the generation of ROS and reduced the accumulation of lipid droplets as well. This phenomenon indicated that the ROS-induced by BDE-47 through mitochondrial chain was critical for adipocyte differentiation. Global metabolomic profiling based on high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was performed on differentiated 3T3-L1 cells to reveal the metabolic changes induced by BDE-47. Twenty three significantly changed metabolites were identified in the adipocytes after BDE-47 exposure. The results of pathway analysis showed that purine and glutathione metabolism were the main impacted pathways and upregulated by BDE-47 treatment. In purine metabolism, increasing levels of adenosine monophosphate (AMP) and guanosine monophosphate (GMP) induced by BDE-47 led to the increment of inosine 5'-monophosphate (IMP) in adipocytes. These increases forwarded the pathway and caused high production of uric acid along with hydrogen peroxide, which contributed to the elevation of ROS after exposure to BDE-47. Inhibiting the synthesis of uric acid with antioxidant could significantly decrease the production of ROS, the levels of adipogenesis-related genes, and the accumulation of lipid droplets in BDE-47 exposed adipocytes. These results further demonstrated that exposure to BDE-47 promoted adipocyte differentiation via causing oxidative stress, upregulating purine metabolism, and increasing production of uric acid. Subsequently, C57BL/6J mouse model with diet interaction was employed to explore the obesogenic effects of BDE-47. Male C57BL/6J mice were fed with either a low-fat diet (LFD, 10% fat) or high-fat diet (HFD, 60% fat) for 15 weeks and subcutaneously injected with BDE-47 (7mg/kg [Low dose, L] or 70mg/kg [High dose, H]) or the vehicle weekly. It was found that exposure to BDE-47 (H) significantly led to the elevation of body weight and serum triglyceride content in HFD fed mice. Besides, the combination of BDE-47 and HFD also significantly increased the weight of white adipose tissue (WAT) and augmented the size of adipocytes in WAT. These have confirmed the obesogenic effects of BDE-47 in vivo. Additionally, BDE-47 (H) exposure significantly increased the accumulation of hepatic triglyceride content and lipid droplets accompanying with elevated inflammation in HFD fed mice, indicating the deterioration of hepatic steatosis in BDE-47 treated mice. Moreover, the integration analysis of lipidomic and gene expression revealed that BDE-47 up-regulated triglyceride synthesis but suppressed lipid exportation and β oxidation to impair the lipid metabolism and worsen the accumulation of hepatic lipid in HFD fed mice. In addition, the increase of liver fibrosis scars (the protein level of αSMA and collagens), serum transaminase levels, as well as lipid peroxidation have been detected in the mice with co-treatment of BDE-47 and HFD. BDE-47 exposure also increased the production of ROS and the levels of fibrotic genes in hepatocytes. However, in LFD with BDE-47 exposed mouse liver, we cannot observe such changes compared with the control (LFD-DMSO). Interestingly, the application of antioxidants reversed the BDE-47-induced fibrotic responses (the expression of αSMA and col3) in hepatocytes, which indicated that the increase of liver fibrosis scars was tightly associated with the level of oxidative stress. In conclusion, these results offered a new insight of lipid toxicities and underlying mechanism of BDE-47 induced obesity-related liver fibrosis. As far as we know, this is the first systematic study of the obesogenic effects and underlying mechanisms of BDE-47 in diet-induced mouse model. These results have showed the pathological roles of BDE-47 in the development of obesity and related liver diseases by an integration analysis of omics study and biological analysis in vivo and in vitro. Meanwhile, inhibitors were applied to investigate the mechanism of BDE-47-induced toxicity. Taken together, our results indicated that BDE-47 exposure could accelerate the development of obesity and aggravate the progression of fatty liver in obese mice via causing oxidative stress. This study may shed a light for an explanation for the worldwide prevalence of obesity and related liver diseases. Furthermore, this work reflects the potential of omics study and biological methods for toxicity assessment of environmental pollutants on human health. It would be helpful for the clinical diagnose and treatment.

The burden of liver diseases is increasing worldwide, accounting for two million deaths annually. In the past decade, tremendous progress has been made in the basic and translational research of liver tissue engineering, which seeks to build physiologically relevant liver models to better understand liver diseases, accelerate drug development, and advance regenerative medicine. Liver microtissues are small, three-dimensional (3D) hepatocyte cultures that recapitulate liver physiology and have been used in many biomedical applications. However, sourcing of high-quality human hepatocytes for microtissue fabrication poses a significant challenge. Since the inception of induced pluripotent stem cell (iPSC) technology, iPSC-derived hepatocyte-like cells (HLCs) have demonstrated significant improvement over other hepatocyte cell sources in many studies. Despite their promising potential, HLCs face certain challenges: they resemble fetal hepatocytes rather than adult hepatocytes; they undergo dedifferentiation quickly after reaching maturity; they are produced on a small scale; and they exhibit large donor-to-donor and batch-to-batch variability. This doctoral thesis focuses on engineering patient-specific liver microtissues with prolonged phenotypic maintenance and disease modeling potential. Chapter 1 provides a review of recent advances, challenges, and future directions in liver microtissue research. 3D microtissues can be generated by scaffold-free assembly or scaffold-assisted methods using macroencapsulation, droplet microfluidics, and bioprinting. Optimization of the hepatic microenvironment entails incorporating the appropriate cell composition for enhanced cell-cell interactions and niche-specific signals, and creating scaffolds with desired chemical, mechanical and physical properties. Perfusion-based culture systems such as bioreactors and microfluidic systems are used to achieve efficient exchange of nutrients and soluble factors in the microtissues. Chapter 2 describes our efforts in optimizing methods of generating human HLCs from the peripheral blood of selected donors. Peripheral blood mononuclear cells (PBMCs) were first reprogrammed to iPSCs using Sendai viruses carrying the four Yamanaka factors. We developed an optimized protocol for hepatocyte differentiation from iPSCs, and obtained HLCs that exhibited hepatocyte-specific phenotypes and functions that were comparable to other reports. We then demonstrated the one-step generation of homogeneous, microencapsulated liver microtissues in Chapter 3. Droplet microfluidics was used to produce double emulsion droplets that served as individual microenvironments where HLCs were encapsulated in methylated collagen and alginate. The cells self-assembled in <16 hours through dynamic interactions with methylated collagen, and individual spheroids were encapsulated in polymerized alginate gel to prevent cell fusion and attachment. HLC spheroids remained viable and functional for >24 days, whereas 2D HLCs underwent dedifferentiation within 7 days of reaching maturity. The spheroids showed further maturation compared to the 2D HLCs at peak maturity. Co-culture of HLCs with human endothelial cells was also investigated in the 3D system, but no improvement was observed over monoculture spheroids with our current methods. To our knowledge, this is the first study to utilize droplet microfluidics to generate homogeneous, compartmentalized droplets that serve as optimized 3D microenvironments for HLC aggregation and maturation. It demonstrated the potential of using high-throughput droplet microfluidics to produce and encapsulate mature, functional human HLCs for long-term applications. In Chapter 4, we developed a TM6SF2 knockout and overexpression model in iPSCs to investigate its molecular function and potential role in nonalcoholic fatty liver disease (NAFLD). Transmembrane 6 superfamily member 2 (TM6SF2) is a protein of unknown function, and analysis from our model suggested that TM6SF2 dysregulation has a biphasic response. Our data showed that both knockout and overexpression can result in the upregulation of cholesterol biosynthesis and a defect in the proper processing of lipid droplets. Additionally, high expression of the TM6SF2 rs58542926 variant has an increased risk for cholesterol upregulation, compared to the major allele. Future works will focus on generating liver microtissues from the TM6SF2 knockout and transgene-expressing cells using droplet microfluidics, and validating our hypotheses with established biochemical and functional assays.

There are limited data on the outcomes of chronic liver disease (CLD) patients admitted for percutaneous coronary intervention (PCI). All PCI hospitalizations from the Nationwide Inpatient Sample (2004 to 2015) were analyzed and stratified by the presence, cause and severity of CLD, as well as the indication for PCI. Multivariable logistic regression analysis was performed to determine the adjusted odds ratios (aOR) of in-hospital adverse outcomes in patients with CLD compared with those without CLD. Among 7,296,679 PCI admissions, 54,368 (0.7%) had a CLD diagnosis. Among patients with CLD, 36,853 (67.8%) had severe CLD. Patients with CLD had higher likelihood of adverse outcomes including major adverse cardiovascular and cerebrovascular events (MACCE) (aOR 1.25, 95%CI 1.20 to 1.30), mortality (aOR 1.43, 95%CI 1.35 to 1.51), major bleeding (aOR 2.22, 95%CI 2.12 to 2.32). When accounting for severity, only severe CLD subgroup was more likely to have MACCE and all-cause mortality compared to no-CLD patients (p <0.001). Among CLD etiologic subgroups, those with ‘alcohol-related liver disease’ and ‘other CLD’ were consistently more likely to develop MACCE, all-cause mortality and major bleeding in comparison to no-CLD patients, while ‘chronic viral hepatitis’ subgroup had only increased odds of major bleeding (p <0.001). In conclusion, CLD patients admitted for PCI are more likely to have worse in-hospital outcomes, particularly in the severe CLD subgroup and ‘alcohol-related liver disease’ and ‘other CLD’ etiologic subgroups.