Human Mesenchymal Stromal Cells Resolve Lipid Load in High Fat Diet-Induced Non-Alcoholic Steatohepatitis in Mice by Mitochondria Donation.

Mesenchymal stromal cells (MSC) increasingly emerge as an option to ameliorate non-alcoholic steatohepatitis (NASH), a serious disease, which untreated may progress to liver cirrhosis and cancer. Before clinical translation, the mode of action of MSC needs to be established. Here, we established NASH in an immune-deficient mouse model by feeding a high fat diet. Human bone-marrow-derived MSC were delivered to the liver via intrasplenic transplantation. As verified by biochemical and image analyses, human mesenchymal stromal cells improved high-fat-diet-induced NASH in the mouse liver by decreasing hepatic lipid content and inflammation, as well as by restoring tissue homeostasis. MSC-mediated changes in gene expression indicated the switch from lipid storage to lipid utilization. It was obvious that host mouse hepatocytes harbored human mitochondria. Thus, it is feasible that resolution of NASH in mouse livers involved the donation of human mitochondria to the mouse hepatocytes. Therefore, human MSC might provide oxidative capacity for lipid breakdown followed by restoration of metabolic and tissue homeostasis.

Mesenchymal stromal cells mitigate liver damage after extended resection in the pig by modulating thrombospondin-1/TGF-ß.

Post-surgery liver failure is a serious complication for patients after extended partial hepatectomies (ePHx). Previously, we demonstrated in the pig model that transplantation of mesenchymal stromal cells (MSC) improved circulatory maintenance and supported multi-organ functions after 70% liver resection. Mechanisms behind the beneficial MSC effects remained unknown. Here we performed 70% liver resection in pigs with and without MSC treatment, and animals were monitored for 24 h post surgery. Gene expression profiles were determined in the lung and liver. Bioinformatics analysis predicted organ-independent MSC targets, importantly a role for thrombospondin-1 linked to transforming growth factor-ß (TGF-ß) and downstream signaling towards providing epithelial plasticity and epithelial-mesenchymal transition (EMT). This prediction was supported histologically and mechanistically, the latter with primary hepatocyte cell cultures. MSC attenuated the surgery-induced increase of tissue damage, of thrombospondin-1 and TGF-ß, as well as of epithelial plasticity in both the liver and lung. This suggests that MSC ameliorated surgery-induced hepatocellular stress and EMT, thus supporting epithelial integrity and facilitating regeneration. MSC-derived soluble factor(s) did not directly interfere with intracellular TGF-ß signaling, but inhibited thrombospondin-1 secretion from thrombocytes and non-parenchymal liver cells, therewith obviously reducing the availability of active TGF-ß.

Mitochondrial Transfer by Human Mesenchymal Stromal Cells Ameliorates Hepatocyte Lipid Load in a Mouse Model of NASH.

Mesenchymal stromal cell (MSC) transplantation ameliorated hepatic lipid load; tissue inflammation; and fibrosis in rodent animal models of non-alcoholic steatohepatitis (NASH) by as yet largely unknown mechanism(s). In a mouse model of NASH; we transplanted bone marrow-derived MSCs into the livers; which were analyzed one week thereafter. Combined metabolomic and proteomic data were applied to weighted gene correlation network analysis (WGCNA) and subsequent identification of key drivers. Livers were analyzed histologically and biochemically. The mechanisms of MSC action on hepatocyte lipid accumulation were studied in co-cultures of hepatocytes and MSCs by quantitative image analysis and immunocytochemistry. WGCNA and key driver analysis revealed that NASH caused the impairment of central carbon; amino acid; and lipid metabolism associated with mitochondrial and peroxisomal dysfunction; which was reversed by MSC treatment. MSC improved hepatic lipid metabolism and tissue homeostasis. In co-cultures of hepatocytes and MSCs; the decrease of lipid load was associated with the transfer of mitochondria from the MSCs to the hepatocytes via tunneling nanotubes (TNTs). Hence; MSCs may ameliorate lipid load and tissue perturbance by the donation of mitochondria to the hepatocytes. Thereby; they may provide oxidative capacity for lipid breakdown and thus promote recovery from NASH-induced metabolic impairment and tissue injury.

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.

The High Calcium, High Phosphorus Rescue Diet Is Not Suitable to Prevent Secondary Hyperparathyroidism in Vitamin D Receptor Deficient Mice.

The vitamin D receptor (VDR) knockout (KO) mouse is a common model to unravel novel metabolic functions of vitamin D. It is recommended to feed these mice a high calcium (2%), high phosphorus (1.25%) diet, termed rescue diet (RD) to prevent hypocalcaemia and secondary hyperparathyroidism. First, we characterized the individual response of VDR KO mice to feeding a RD and found that the RD was not capable of normalizing the parathyroid hormone (PTH) concentrations in each VDR KO mouse. In a second study, we aimed to study whether RD with additional 1 and 2% calcium (in total 3 and 4% of the diet) is able to prevent secondary hyperparathyroidism in the VDR KO mice. Wild type (WT) mice and VDR KO mice that received a normal calcium and phosphorus diet (ND) served as controls. Data demonstrated that the RD was no more efficient than the ND in normalizing PTH levels. An excessive dietary calcium concentration of 4% was required to reduce serum PTH concentrations in the VDR KO mice to PTH levels measured in WT mice. This diet, however, resulted in higher concentrations of circulating intact fibroblast growth factor 23 (iFGF23). To conclude, the commonly used RD is not suitable to normalize the serum PTH in VDR KO mice. Extremely high dietary calcium concentrations are necessary to prevent secondary hyperthyroidism in these mice, with the consequence that iFGF23 concentrations are being raised. Considering that PTH and iFGF23 exert numerous VDR independent effects, data obtained from VDR KO mice cannot be attributed solely to vitamin D.

Vitamin D receptor knockout mice exhibit elongated intestinal microvilli and increased ezrin expression.

In addition to its principle function as a calcium regulator, vitamin D can affect cell and tissue morphology. The intestine is an important target tissue of vitamin D, as it must ensure the efficient transport of nutrients across the epithelium while excluding the passage of harmful molecules and bacteria into the organism. These functions require a highly organized morphology, which may be modified by vitamin D deficiency. To elucidate the role of vitamin D in gut morphology and barrier function, we compared the enterocyte microstructures, gut permeability, and cytoskeletal and cell junction protein expression in vitamin D receptor (VDR) knockout (KO) and wild-type (WT) mice. We found that the duodenal epithelial cells in the VDR-KO mice had longer microvilli (+19%) than those of the WT mice (P < .05). Interestingly, microvilli elongation in the VDR-KO mice was associated with higher messenger RNA and protein expression of ezrin, which is involved in the regulation of microvillus morphogenesis. Intestinal tight junction width and permeability were assessed by measuring the fluorescein isothiocyanate dextran concentrations in plasma; the concentrations were comparable between the 2 groups of mice. We further observed a decrease in the messenger RNA and protein expression of the calcium-transporting tight junction protein claudin-2 in the VDR-KO mice compared with the WT mice (P < .05). In conclusion, the mice lacking VDR had longer enterocyte microvilli, likely as a result of increased ezrin expression. However, the morphology of the tight junctions and the intestinal permeability for large molecules were not affected.

Vitamin D receptor regulates intestinal proteins involved in cell proliferation, migration and stress response.

BACKGROUND: Genome-wide association studies found low plasma levels of 25-hydroxyvitamin D and vitamin D receptor (VDR) polymorphisms associated with a higher prevalence of pathological changes in the intestine such as chronic inflammatory bowel diseases. METHODS: In this study, a proteomic approach was applied to understand the overall physiological importance of vitamin D in the small intestine, beyond its function in calcium and phosphate absorption. RESULTS: In total, 569 protein spots could be detected by two-dimensional-difference in-gel electrophoresis (2D-DIGE), and 82 proteins were considered as differentially regulated in the intestinal mucosa of VDR-deficient mice compared to that of wildtype (WT) mice. Fourteen clearly detectable proteins were identified by MS/MS and further analyzed by western blot and/or real-time RT-PCR. The differentially expressed proteins are functionally involved in cell proliferation, cell adhesion and cell migration, stress response and lipid transport. Mice lacking VDR revealed higher levels of intestinal proteins associated with proliferation and migration such as the 37/67 kDa laminin receptor, collagen type VI (alpha 1 chain), keratin-19, tropomyosin-3, adseverin and higher levels of proteins involved in protein trafficking and stress response than WT mice. In contrast, proteins that are involved in transport of bile and fatty acids were down-regulated in small intestine of mice lacking VDR compared to WT mice. However, plasma and liver concentrations of cholesterol and triglycerides were not different between the two groups of mice. CONCLUSION: Collectively, these data imply VDR as an important factor for controlling cell proliferation, migration and stress response in the small intestine.

Maternal vitamin D deficiency causes smaller muscle fibers and altered transcript levels of genes involved in protein degradation, myogenesis, and cytoskeleton organization in the newborn rat.

SCOPE: Epidemiologic data reveal associations between low serum concentrations of 25-hydroxyvitamin D (25(OH)D) and higher risk of falls and muscle weakness. Fetal stage is critical for the development of skeletal muscle, but little information is available on the impact of maternal vitamin D deficiency on muscles of offspring. METHODS AND RESULTS: To investigate the morphology and transcriptome of gastrocnemius muscle in newborns in response to maternal vitamin D deficiency, 14 female rats were fed either a vitamin D3 deficient (0 IU/kg) or a vitamin D3 adequate diet (1000 IU/kg) 8 weeks prior to conception, during pregnancy, and lactation. Analysis of cholecalciferol, 25(OH)D3 and 1,25-dihydroxyvitamin D3 show that dams fed the vitamin D deficient diet and their newborns suffered from a relevant vitamin D deficiency. Muscle cells of vitamin D deficient newborns were smaller than those of vitamin D adequate newborns (p < 0.05). Muscle transcriptome of the newborns revealed 426 probe sets as differentially expressed (259 upregulated, 167 downregulated) in response to vitamin D deficiency (fold change ≥1.5, p < 0.05). The effected genes are involved in protein catabolism, cell differentiation and proliferation, muscle cell development, and cytoskeleton organization. CONCLUSION: Maternal vitamin D deficiency has a major impact on morphology and gene expression profile of skeletal muscle in newborns.

Vitamin D receptor deficiency and low vitamin D diet stimulate aortic calcification and osteogenic key factor expression in mice.

Low levels of 25-hydroxy vitamin D (25(OH)D) are associated with cardiovascular diseases. Herein, we tested the hypothesis that vitamin D deficiency could be a causal factor in atherosclerotic vascular changes and vascular calcification. Aortic root sections of vitamin D receptor knockout (VDR(-/-)) mice that were stained for vascular calcification and immunostained for osteoblastic differentiation factors showed more calcified areas and a higher expression of the osteogenic key factors Msx2, Bmp2, and Runx2 than the wild-type mice (P<0.01). Data from LDL receptor knockout (LDLR(-/-)) mice that were fed western diet with either low (50 IU/kg), recommended (1,000 IU/kg), or high (10,000 IU/kg) amounts of vitamin D(3) over 16 weeks revealed increasing plasma concentrations of 25(OH)D (P<0.001) with increasing intake of vitamin D, whereas levels of calcium and phosphorus in plasma and femur were not influenced by the dietary treatment. Mice treated with the low vitamin D diet had more calcified lesions and a higher expression of Msx2, Bmp2, and Runx2 in aortic roots than mice fed recommended or high amounts of vitamin D (P<0.001). Taken together, these findings indicate vitamin D deficiency as a risk factor for aortic valve and aortic vessel calcification and a stimulator of osteogenic key factor expression in these vascular areas.

Mouse γ-butyrobetaine dioxygenase is regulated by peroxisome proliferator-activated receptor α through a PPRE located in the proximal promoter.

Convincing evidence from studies with peroxisome proliferator-activated receptor (PPAR)α-deficient mice suggested that the carnitine biosynthetic enzyme γ-butyrobetaine dioxygenase (BBD) is regulated by PPARα. However, the identification of BBD as a direct PPARα target gene as well as its exact regulation remained to be demonstrated. In silico-analysis of the mouse BBD promoter revealed seven putative peroxisome proliferator response elements (PPRE) with high similarity to the consensus PPRE. Luciferase reporter gene assays using mutated and non-mutated serial 5'-truncation BBD promoter reporter constructs revealed that one PPRE located at -75 to -87 relative to the transcription start site in the proximal BBD promoter is probably functional. Using gel shift assays we observed in vitro-binding of PPARα/RXRα heterodimer to this PPRE confirming that it is functional. In conclusion, the present study clearly shows that mouse BBD is a direct PPARα target gene and that transcriptional up-regulation of mouse BBD by PPARα is likely mediated by binding of the PPARα/RXR heterodimer to one PPRE located in its proximal promoter region. The results confirm emerging evidence from recent studies that PPARα plays a key role in the regulation of carnitine homeostasis by controlling genes involved in both, carnitine synthesis and carnitine uptake.