Wolfgang Moritz

Central necrosis in isolated hypoxic human pancreatic islets: evidence for postisolation ischemia.

A variety of explanations have been provided to elucidate the requirement of the large islet mass that is essential for a successful treatment of patients with type I diabetes by intrahepatic transplantation. The purpose of this study was to investigate islet cell survival under the effect of prolonged hypoxia and/or nutrient withdrawal, which mimics posttransplantation environment of transplanted islets in the liver. We studied the influence of 24 h of hypoxia (1% O2) in intact isolated human and rat islets as well as the effect of combined oxygen/nutrient deprivation in a mouse insulinoma cell line (MIN6). In intact human islets, 24 h of hypoxia led to central necrosis combined with apoptotic features such as nuclear pyknosis and DNA fragmentation. In the course of hypoxic treatment, ultrastructural analysis demonstrated a gradual transition from an apoptotic to a necrotic morphology particularly pronounced in central areas of large islets. In MIN6 cells, on the other hand, hypoxia led to a twofold (p < 0.01) increase in caspase-3 activity, an indicator of apoptosis, but not to necrosis, as determined by release of lactate dehydrogenase (LDH). Only in combination with nutrient/serum deprivation was a marked increase in LDH release observed (sixfold vs. control, p < 0.01). We therefore conclude that, similar to MIN6 cells, central necrosis in isolated hypoxic islets is the result of the combined effects of hypoxia and nutrient/serum deprivation, most likely due to limited diffusion. Provided that transplanted islets undergo a similar fate as shown in our in vitro study, future emphasis will require the development of strategies that protect the islet graft from early cell death and accelerate the revascularization process.

Serotonin promotes tumor growth in human hepatocellular cancer

In addition to its function as a neurotransmitter and vascular active molecule, serotonin is also a mitogen for hepatocytes and promotes liver regeneration. A possible role in hepatocellular cancer has not yet been investigated. Human hepatocellular cancer cell lines Huh7 and HepG2 were used to assess the function of serotonin in these cell lines. Characteristics of autophagy were detected with transmission electron microscopy, immunoblots of microtubule‐associated protein light chain 3(LC3) and p62 (sequestosome 1). Immunoblots of the mammalian target of rapamycin (mTOR) and its downstream targets p70S6K and 4E‐BP1 were used to investigate signaling pathways of serotonin. Two different animal models served as principle of proof of in vitro findings. Clinical relevance of the experimental findings was evaluated with a tissue microarray from 168 patients with hepatocellular carcinoma. Serotonin promotes tumor growth and survival in starved hepatocellular carcinoma cells. During starvation hepatocellular carcinoma cells exhibited characteristics of autophagy, which disappeared in serotonin‐treated cells. Rapamycin, an inhibitor of mTOR, is known to induce autophagy. Serotonin could override rapamycin by an mTOR‐independent pathway and activate common downstream signals such as p70S6K and 4E‐BP1. In two tumor models of the mouse, inhibition of serotonin signaling consistently impaired tumor growth. Human biopsies revealed expression of the serotonin receptor HTR2B, correlating with downstream signals, e.g., phosphorylated p70S6K and proliferation. Conclusion: This study provides evidence that serotonin is involved in tumor growth of hepatocellular cancer by activating downstream targets of mTOR, and therefore serotonin‐related pathways might represent a new treatment strategy. (HEPATOLOGY 2010.)

Towards automated production and drug sensitivity testing using scaffold-free spherical tumor microtissues

Although the relevance of three-dimensional (3-D) culture has been recognized for years and exploited at an academic level, its translation to industrial applications has been slow. The development of reliable high-throughput technologies is clearly a prerequisite for the industrial implementation of 3-D models. In this study the robustness of spherical microtissue production and drug testing in a 96-well hanging-drop multiwell plate format was assessed on a standard 96-well channel robotic platform. Microtissue models derived from six different cell lines were produced and characterized according to their growth profile and morphology displaying high-density tissue-like reformation and growth over at least 15 days. The colon cancer cell line HCT116 was chosen as a model to assess microtissue-based assay reproducibility. Within three individual production batches the size variations of the produced microtissues were below 5%. Reliability of the microtissue-based assay was tested using two reference compounds, staurosporine and chlorambucil. In four independent drug testings the calculated IC(50) values were benchmarked against 2-D multiwell testings displaying similar consistency. The technology presented here for the automated production of a variety of microtissues for efficacy testing in a standard 96-well format will aid the implementation of more organotypic models at an early time point in the drug discovery process.

Prevention of reperfusion injury and microcirculatory failure in macrosteatotic mouse liver by omega‐3 fatty acids

Macrovesicular hepatic steatosis has a lower tolerance to reperfusion injury than microvesicular steatosis with an abnormally high ratio of omega‐6 (n‐6): omega‐3 (n‐3) polyunsaturated fatty acids (PUFAs). We investigated the influence of PUFAs on microcirculation in steatotic livers and the potential to minimize reperfusion injury in the macrosteatotic liver by normalization of PUFAs. Ob/ob mice were used as a model of macrovesicular hepatic steatosis and C57/Bl6 mice fed a choline‐deficient diet for microvesicular steatosis. Steatotic and lean livers were subjected to 45 minutes of ischemia and 3 hours of reperfusion. Hepatic content of omega‐3 and omega‐6 PUFAs was determined. Microcirculation was investigated using intravital fluorescence microscopy. A second group of ob/ob mice was supplemented with dietary omega‐3 PUFAs and compared with the control diet–fed group. Microcirculation, AST, and Kupffer cell activity were assessed. Macrosteatotic livers had significant microcirculatory dysfunction correlating with high omega‐6: omega‐3 PUFA ratio. Dietary omega‐3 PUFA resulted in normalization of this ratio, reduction of intrahepatic lipids, and decrease in the extent of macrosteatosis. Defective microcirculation was dramatically ameliorated with significant reduction in Kupffer cell activity and protection against hepatocellular injury both before ischemia and after reperfusion. Conclusion: Macrosteatotic livers disclosed an abnormal omega‐6: omega‐3 PUFA ratio that correlates with a microcirculatory defect that enhanced reperfusion injury. Thus, protective strategies applied during or after ischemia are unlikely to be useful. Preoperative dietary omega‐3 PUFAs protect macrosteatotic livers against reperfusion injury and might represent a valuable method to expand the live liver donor pool. (HEPATOLOGY 2007;45:855–863.)

Bile salt toxicity aggravates cold ischemic injury of bile ducts after liver transplantation in Mdr2+/− mice

Intrahepatic bile duct strictures are a serious complication after orthotopic liver transplantation (OLT). We examined the role of endogenous bile salt toxicity in the pathogenesis of bile duct injury after OLT. Livers from wild‐type mice and mice heterozygous for disruption of the multidrug resistance 2 Mdr2 gene (Mdr2+/−) were transplanted into wild‐type recipient mice. Mdr2+/− mice secrete only 50% of the normal amount of phospholipids into their bile, leading to an abnormally high bile salt/phospholipid ratio. In contrast to homozygous Mdr2−/− mice, the Mdr2+/− mice have normal liver histology and function under normal conditions. Two weeks after OLT, bile duct injury and cholestasis were assessed by light and electron microscopy, as well as through molecular and biochemical markers. There were no signs of bile duct injury or intrahepatic cholestasis in liver grafts from wild‐type donors. Liver grafts from Mdr2+/− donors, however, had enlarged portal tracts with cellular damage, ductular proliferation, biliostasis, and a dense inflammatory infiltrate after OLT. Parallel to this observation, recipients of Mdr2+/− livers had significantly higher serum transaminases, alkaline phosphatase, total bilirubin, and bile salt levels, as compared with recipients of wild‐type livers. In addition, hepatic bile transporter expression was compatible with the biochemical and histological cholestatic profile found in Mdr2+/− grafts after OLT. In conclusion, toxic bile composition, due to a high biliary bile salt/phospholipid ratio, acted synergistically with cold ischemia in the pathogenesis of bile duct injury after transplantation. (HEPATOLOGY 2006;43:1022–1031.)

Successful Simultaneous Islet-Kidney Transplantation using a Steroid-free Immunosuppression: Two-Year Follow-up

We report on the feasibility of a glucocorticoid‐free immunosuppression (sirolimus, low‐dose tacrolimus, and daclizumab) in simultaneous islet‐kidney transplantation in nine patients with type 1 diabetes. There was one renal primary nonfunction. Renal function (n = 8) as assessed by creatinine and creatinine clearance over time was 103 ± 6 μmol/L and 64 ± 6 mL/min/1.73 m2, respectively. Five out of six patients with ≥ 2 islet transplantations became insulin independent. The mean HbA1c during the follow‐up period for all patients after transplantation is 6.2 ± 0.9% as compared with 8.7 ± 1.9% prior to transplant. These results in patients with a median follow‐up of 2.3 years suggest that kidney transplantation under a glucocorticoid‐free immunosuppression is feasible, and that the rate of insulin independence of 80% can be achieved not only in patients with no or minimal diabetes complications, but also in patients with more advanced late complications and in conjunction with kidney transplantation.

Activation of serotonin receptor-2B rescues small-for-size liver graft failure in mice.

The implantation of grafts below 30% of the normal liver volume is associated with a high risk of failure known as small‐for‐size (SFS) syndrome. Strategies to rescue small grafts may have a dramatic impact on organ shortage. Serotonin is a potent growth factor for the liver. The goal of this study was to determine whether enhanced serotonin signaling could prevent the deleterious effects of SFS syndrome. We performed 30% normal liver volume transplantations in wild‐type C57/BL6 and interleukin‐6 (IL‐6)−/− mice. Some animals received α‐methyl‐5‐HT (DOI), an agonist of serotonin receptor‐2 (5‐HT2B). Endpoints included long‐term survival, serum and hepatic markers of liver injury and regeneration, assessment of hepatic microcirculation by intravital fluorescence microscopy and scanning electron microscopy, and transcript levels of a variety of serotonin receptors, tumor necrosis factor α, and IL‐6. All recipients of small grafts (controls) died within 2‐4 days of transplantation, whereas half of those receiving DOI survived permanently. Control animals disclosed major liver injury, including diffuse microvesicular steatosis in hepatocytes, impairment of microcirculation, and a failure of regeneration, whereas these parameters were dramatically improved in animals subjected to DOI. Blockage of 5‐HT2B blunted the protective effects of DOI. Whereas IL‐6 levels were higher in DOI‐treated animals, IL‐6−/− mice were still protected by DOI, suggesting a protective pathway independent of IL‐6. Conclusion: Serotonin through its action on receptor‐2B protects SFS liver grafts from injury and prevents microcirculation and regeneration. The mechanism of hepato‐protection is independent of IL‐6. (Hepatology 2011;)

Development and Characterization of a Scaffold-Free 3D Spheroid Model of Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes.

Cardiomyocytes (CMs) are terminally differentiated cells in the adult heart, and ischemia and cardiotoxic compounds can lead to cell death and irreversible decline of cardiac function. As testing platforms, isolated organs and primary cells from rodents have been the standard in research and toxicology, but there is a need for better models that more faithfully recapitulate native human biology. Hence, a new in vitro model comprising the advantages of 3D cell culture and the availability of induced pluripotent stem cells (iPSCs) of human origin was developed and characterized. Human CMs derived from iPSCs were studied in standard 2D culture and as cardiac microtissues (MTs) formed in hanging drops. Two-dimensional cultures were examined using immunofluorescence microscopy and western blotting, while the cardiac MTs were subjected to immunofluorescence, contractility, and pharmacological investigations. iPSC-derived CMs in 2D culture showed well-formed myofibrils, cell-cell contacts positive for connexin-43, and other typical cardiac proteins. The cells reacted to prohypertrophic growth factors with a substantial increase in myofibrils and sarcomeric proteins. In hanging drop cultures, iPSC-derived CMs formed spheroidal MTs within 4 days, showing a homogeneous tissue structure with well-developed myofibrils extending throughout the whole spheroid without a necrotic core. MTs showed spontaneous contractions for more than 4 weeks that were recorded by optical motion tracking, sensitive to temperature and responsive to electrical pacing. Contractile pharmacology was tested with several agents known to modulate cardiac rate and viability. Calcium transients underlay the contractile activity and were also responsive to electrical stimulation, caffeine-induced Ca(2+) release, and extracellular calcium levels. A three-dimensional culture using iPSC-derived human CMs provides an organoid human-based cellular platform that is free of necrosis and recapitulates vital cardiac functionality, thereby providing a new and promising relevant model for the evaluation and development of new therapies and detection of cardiotoxicity.

Has Time Come for New Goals in Human Islet Transplantation

The enthusiasm regarding clinical islet transplantation has been dampened by the long‐term results. Concerns about the associated risks of life‐long immunosuppression and the striking imbalance between potential recipients and available donor pancreata warrant changes in some of the current goals.

Serotonin protects mouse liver from cholestatic injury by decreasing bile salt pool after bile duct ligation

Obstructive cholestasis induces liver injury, postoperative complications, and mortality after surgery. Adaptive control of cholestasis, including bile salt homeostasis, is necessary for recovery and survival. Peripheral serotonin is a cytoprotective neurotransmitter also associated with liver regeneration. The effect of serotonin on cholestatic liver injury is not known. Therefore, we tested whether serotonin affects the severity of cholestatic liver injury. We induced cholestasis by ligation of the bile duct (BDL) in either wild‐type (WT) mice or mice lacking peripheral serotonin (Tph1−/− and immune thrombocytopenic [ITP] mice). Liver injury was assessed by the levels of plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT) and tissue necrosis. Bile salt–regulating genes were measured by quantitative polymerase chain reaction and confirmed by western blotting and immunohistochemistry. Tph1−/− mice displayed higher levels of plasma AST, ALT, bile salts, and hepatic necrosis after 3 days of BDL than WT mice. Likewise, liver injury was disproportional in ITP mice. Moreover, severe cholestatic complications and mortality after prolonged BDL were increased in Tph1−/− mice. Despite the elevation in toxic bile salts, expression of genes involved in bile salt homeostasis and detoxification were not affected in Tph1−/− livers. In contrast, the bile salt reabsorption transporters Ostα and Ostβ were up‐regulated in the kidneys of Tph1−/− mice, along with a decrease in urinary bile salt excretion. Serotonin reloading of Tph1−/− mice reversed this phenotype, resulting in a reduction of circulating bile salts and liver injury. Conclusion: We propose a physiological function of serotonin is to ameliorate liver injury and stabilize the bile salt pool through adaptation of renal transporters in cholestasis. (HEPATOLOGY 2012;56:209–218)