Immune-Deficient Pfp/Rag2-/- Mice Featured Higher Adipose Tissue Mass and Liver Lipid Accumulation with Growing Age than Wildtype C57BL/6N Mice.

Aging is a risk factor for adipose tissue dysfunction, which is associated with inflammatory innate immune mechanisms. Since the adipose tissue/liver axis contributes to hepatosteatosis, we sought to determine age-related adipose tissue dysfunction in the context of the activation of the innate immune system fostering fatty liver phenotypes. Using wildtype and immune-deficient mice, we compared visceral adipose tissue and liver mass as well as hepatic lipid storage in young (ca. 14 weeks) and adult (ca. 30 weeks) mice. Adipocyte size was determined as an indicator of adipocyte function and liver steatosis was quantified by hepatic lipid content. Further, lipid storage was investigated under normal and steatosis-inducing culture conditions in isolated hepatocytes. The physiological age-related increase in body weight was associated with a disproportionate increase in adipose tissue mass in immune-deficient mice, which coincided with higher triglyceride storage in the liver. Lipid storage was similar in isolated hepatocytes from wildtype and immune-deficient mice under normal culture conditions but was significantly higher in immune-deficient than in wildtype hepatocytes under steatosis-inducing culture conditions. Immune-deficient mice also displayed increased inflammatory, adipogenic, and lipogenic markers in serum and adipose tissue. Thus, the age-related increase in body weight coincided with an increase in adipose tissue mass and hepatic steatosis. In association with a (pro-)inflammatory milieu, aging thus promotes hepatosteatosis, especially in immune-deficient mice.

Assessment of the hepatocytic differentiation ability of human skin-derived ABCB5+ stem cells.

The continuously decreasing willingness for liver donation aggravates treatment of end-stage liver diseases requiring organ transplantation as the only curative strategy. Cell therapy approaches using human hepatocytes or stem cell-derived hepatocyte-like cells may be a therapeutic option out of this dilemma. ABCB5-positive mesenchymal stromal cells from human skin featured promising potential to treat immune-mediated diseases. Since most of chronic liver diseases involve exaggerating immune mechanisms, it was the aim to demonstrate in this study, whether ABCB5+ stem cells may serve as a resource to generate hepatocytic cells for application in liver cell transplantation. Using an established single-step protocol, which had been successfully applied to differentiate mesenchymal stromal cells into the hepatocytic lineage, ABCB5+ skin-derived stem cells did not gain significant characteristics of hepatocytes. Yet, upon culture in hepatocytic differentiation medium, ABCB5+ stem cells secreted immunomodulatory and anti-fibrotic factors as well as proteins, which may prompt hepatic morphogenesis besides others. Hepatic transplantation of ABCB5+ stem cells, which had been prior cultured in hepatocytic differentiation medium, did not cause any obvious deterioration of liver architecture suggesting their safe application. Thus, human ABCB5+ skin-derived stem cells secreted putative hepatotropic factors after culture in hepatocytic differentiation medium.

Improvement of portal venous pressure in cirrhotic rat livers by systemic treatment with adipose tissue-derived mesenchymal stromal cells.

BACKGROUND AIMS: Portal hypertension is the main cause of complications in cirrhosis caused primarily by extensive fibrosis. Both anti-fibrotic and pro-fibrotic properties of mesenchymal stromal cells (MSCs) have been described in various animal models of liver fibrosis. Therefore, the impact of MSCs on portal hypertension and fibrosis should be investigated in an animal model of liver cirrhosis. METHODS: The effect of systemic treatment with adipose tissue-derived MSCs, pre-differentiated into hepatocytic cells, was investigated in a rat model of liver cirrhosis induced by chronic inhalation of carbon tetrachloride. RESULTS: Chronic intoxication with carbon tetrachloride increased the portal venous pressure, which was significantly attenuated by the treatment with MSCs. Consistent with the increase in portal and sinusoidal resistance in the cirrhotic liver, the splenic weight increased, which was again attenuated by the MSCs. The cells had no impact on the spontaneous improvement of liver dysfunction after cessation of treatment with carbon tetrachloride. However, fibrosis was significantly improved as assessed by image quantification of collagen stained with Sirius red. However, hydroxyproline was unchanged indicating that fibrillary collagen content was not affected. That was in line with the finding that the activation of hepatic stellate cells, mainly contributing to excess collagen production in liver cirrhosis, was not affected by the MSCs. The expression of metalloproteinases and their inhibitors did also not change. DISCUSSION: It is suggested that hepatocytic differentiated MSCs improved portal blood flow in the cirrhotic liver rather by the physical reestablishment of liver architecture than by biochemical repair.

Mesenchymal stem cells correct haemodynamic dysfunction associated with liver injury after extended resection in a pig model.

In patients, acute kidney injury (AKI) is often due to haemodynamic impairment associated with hepatic decompensation following extended liver surgery. Mesenchymal stem cells (MSCs) supported tissue protection in a variety of acute and chronic diseases, and might hence ameliorate AKI induced by extended liver resection. Here, 70% liver resection was performed in male pigs. MSCs were infused through a central venous catheter and haemodynamic parameters as well as markers of acute kidney damage were monitored under intensive care conditions for 24 h post-surgery. Cytokine profiles were established to anticipate the MSCs' potential mode of action. After extended liver resection, hyperdynamic circulation, associated with hyponatraemia, hyperkalaemia, an increase in serum aldosterone and low urine production developed. These signs of hepatorenal dysfunction and haemodynamic impairment were corrected by MSC treatment. MSCs elevated PDGF levels in the serum, possibly contributing to circulatory homeostasis. Another 14 cytokines were increased in the kidney, most of which are known to support tissue regeneration. In conclusion, MSCs supported kidney and liver function after extended liver resection. They probably acted through paracrine mechanisms improving haemodynamics and tissue homeostasis. They might thus provide a promising strategy to prevent acute kidney injury in the context of post-surgery acute liver failure.

Identification of Pathways in Liver Repair Potentially Targeted by Secretory Proteins from Human Mesenchymal Stem Cells.

BACKGROUND: The beneficial impact of mesenchymal stem cells (MSC) on both acute and chronic liver diseases has been confirmed, although the molecular mechanisms behind it remain elusive. We aim to identify factors secreted by undifferentiated and hepatocytic differentiated MSC in vitro in order to delineate liver repair pathways potentially targeted by MSC. METHODS: Secreted factors were determined by protein arrays and related pathways identified by biomathematical analyses. RESULTS: MSC from adipose tissue and bone marrow expressed a similar pattern of surface markers. After hepatocytic differentiation, CD54 (intercellular adhesion molecule 1, ICAM-1) increased and CD166 (activated leukocyte cell adhesion molecule, ALCAM) decreased. MSC secreted different factors before and after differentiation. These comprised cytokines involved in innate immunity and growth factors regulating liver regeneration. Pathway analysis revealed cytokine-cytokine receptor interactions, chemokine signalling pathways, the complement and coagulation cascades as well as the Januskinase-signal transducers and activators of transcription (JAK-STAT) and nucleotide-binding oligomerization domain-like receptor (NOD-like receptor) signalling pathways as relevant networks. Relationships to transforming growth factor ß (TGF-ß) and hypoxia-inducible factor 1-α (HIF1-α) signalling seemed also relevant. CONCLUSION: MSC secreted proteins, which differed depending on cell source and degree of differentiation. The factors might address inflammatory and growth factor pathways as well as chemo-attraction and innate immunity. Since these are prone to dysregulation in most liver diseases, MSC release hepatotropic factors, potentially supporting liver regeneration.

Pathological implications of cadherin zonation in mouse liver.

Both acute and chronic liver diseases are associated with ample re-modeling of the liver parenchyma leading to functional impairment, which is thus obviously the cause or the consequence of the disruption of the epithelial integrity. It was, therefore, the aim of this study to investigate the distribution of the adherens junction components E- and N-cadherin, which are important determinants of tissue cohesion. E-cadherin was expressed in periportal but not in perivenous hepatocytes. In contrast, N-cadherin was more enriched towards the perivenous hepatocytes. In agreement, ß-catenin, which links both cadherins via α-catenin to the actin cytoskeleton, was expressed ubiquitously. This zonal expression of cadherins was preserved in acute liver injury after treatment with acetaminophen or partial hepatectomy, but disrupted in chronic liver damage like in non-alcoholic steatohepatitis (NASH) or α1-antitrypsin deficiency. Hepatocyte proliferation during acetaminophen-induced liver damage was predominant at the boundary between the damaged perivenous and the intact periportal parenchyma indicating a minor contribution of periportal hepatocytes to liver regeneration. In NASH livers, an oval cell reaction was observed pointing to massive tissue damage coinciding with the gross impairment of hepatocyte proliferation. In the liver parenchyma, metabolic functions are distributed heterogeneously. For example, the expression of phosphoenolpyruvate carboxykinase and E-cadherin overlapped in periportal hepatocytes. Thus, during liver regeneration after acute damage, the intact periportal parenchyma might sustain essential metabolic support like glucose supply or ammonia detoxification. However, disruption of epithelial integrity during chronic challenges may increase susceptibility to metabolic liver diseases such as NASH or vice versa. This might suggest the regulatory integration of tissue cohesion and metabolic functions in the liver.

Induction of ketosis in rats fed low-carbohydrate, high-fat diets depends on the relative abundance of dietary fat and protein.

Low-carbohydrate/high-fat diets (LC-HFDs) in rodent models have been implicated with both weight loss and as a therapeutic approach to treat neurological diseases. LC-HFDs are known to induce ketosis; however, systematic studies analyzing the impact of the macronutrient composition on ketosis induction and weight loss success are lacking. Male Wistar rats were pair-fed for 4 wk either a standard chow diet or one of three different LC-HFDs, which only differed in the relative abundance of fat and protein (percentages of fat/protein in dry matter: LC-75/10; LC-65/20; LC-55/30). We subsequently measured body composition by nuclear magnetic resonance (NMR), analyzed blood chemistry and urine acetone content, evaluated gene expression changes of key ketogenic and gluconeogenic genes, and measured energy expenditure (EE) and locomotor activity (LA) during the first 4 days and after 3 wk on the respective diets. Compared with chow, rats fed with LC-75/10, LC-65/20, and LC-55/30 gained significantly less body weight. Reductions in body weight were mainly due to lower lean body mass and paralleled by significantly increased fat mass. Levels of ß-hydroxybutyate were significantly elevated feeding LC-75/10 and LC-65/20 but decreased in parallel to reductions in dietary fat. Acetone was about 16-fold higher with LC-75/10 only (P < 0.001). In contrast, rats fed with LC-55/30 were not ketotic. Serum fibroblast growth factor-21, hepatic mRNA expression of hydroxymethylglutaryl-CoA-lyase, peroxisome proliferator-activated receptor-γ coactivator-1α, and peroxisome proliferator-activated receptor-γ coactivator-1ß were increased with LC-75/10 only. Expression of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase was downregulated by 50-70% in LC-HF groups. Furthermore, EE and LA were significantly decreased in all groups fed with LC-HFDs after 3 wk on the diets. In rats, the absence of dietary carbohydrates per se does not induce ketosis. LC-HFDs must be high in fat, but also low in protein contents to be clearly ketogenic. Independent of the macronutrient composition, LC-HFD-induced weight loss is not due to increased EE and LA.

The generation of hepatocytes from mesenchymal stem cells and engraftment into murine liver.

Donor organ shortage is still the major obstacle for the clinical application of hepatocyte transplantation in the treatment of liver diseases. However, generation of hepatocyte-like cells from mesenchymal stem cells (MSCs) has become a real alternative to the isolation of primary hepatocytes. MSCs are extracted from the tissue by collagenase digestion and enriched by their capacity to grow on plastic surfaces. Enriched cells display distinct mesenchymal surface markers and are capable of multiple lineage differentiation. In the presence of specific growth conditions, the cells adopt functional features of differentiated hepatocytes. After orthotopic transplantation, differentiated human stem cells engraft in the host liver parenchyma of immunocompromised mice. This protocol describes the in vitro differentiation of stem cells from human bone marrow and their transplantation into livers of immunodeficient mice. The cell culture procedures take about 4-5 weeks, and cells engrafted in the mouse liver may be detected 2-3 months after transplantation.

Propranolol impairs liver regeneration after partial hepatectomy in C57Bl/6-mice by transient attenuation of hepatic lipid accumulation and increased apoptosis.

OBJECTIVE: Acute hepatic fat accumulation appears to be crucial for liver regeneration after partial hepatectomy. Since fatty acids in the liver are provided by catecholamine-induced lipolysis in the adipose tissue, we investigated whether beta-adrenergic blockade of lipolysis might affect liver regeneration. MATERIAL AND METHODS: Mice were treated with propranolol prior to partial hepatectomy. Subsequently, liver regeneration was evaluated histologically, by determination of the relative liver weight and the mitotic index at different time points after surgery. RESULTS: Liver mass restoration was delayed by propranolol, which was associated with a lower hepatic triglyceride content. Ki-67 labelling indicated that liver regeneration was attenuated by propranolol through inhibition of mitosis. Hepatocytes were arrested in the G1 phase of the cell cycle, as shown by the expression of G1-related proteins such as proliferating cell nuclear antigen, cyclin D1 and cyclin-dependent kinase-2, and underwent apoptosis as indicated by detection of poly(adenosine diphosphate-ribose) polymerase fragments. beta-adrenergic blockade of the host animal did not provide transplanted hepatocytes with a growth advantage over host cells. CONCLUSION: Impairment of liver regeneration by propranolol is related to the inhibition of acute hepatic fat accumulation and to a predisposition of hepatocytes to apoptosis.