Daniel Zahner

Bone marrow transplantation demonstrates medullar origin of CD34+ fibrocytes and ameliorates hepatic fibrosis in Abcb4−/− mice

Bone marrow (BM)‐derived stem cells and CD34+ fibrocytes are associated with fibrogenesis in several organs. In an Abcb4−/− mouse model for sclerosing cholangitis alpha‐smooth muscle actin‐positive (α‐SMA+) myofibroblasts are thought to play a pivotal role in hepatic fibrogenesis. The aim of this study was 2‐fold: (1) to demonstrate that the origin of an important fibrogenetic cell population is the BM; and (2) to investigate whether transplantation of BM (BM‐Tx) affects liver function, staging, and grading. Surrogate markers for fibrogenesis and regulation of hepatic stellate cells (HSC) as well as progenitor‐cell‐derived fibrocytes in liver tissue were analyzed by quantitative real‐time polymerase chain reaction (PCR) and immunohistology. After lethal irradiation of recipient mice, BM‐Tx was carried out by way of tail vein injection of BM cells from marker protein donors (green fluorescent protein, GFP+) or Abcb4−/− mice as control (syngeneic Tx). Parameters of liver function were assessed serologically and histologically. Activated HSC of α‐SMA+/CRP2+ phenotype were expressed in ≈50% of proliferating bile ducts, whereas fibrotic liver parenchyma showed no expression thereof. Epithelial mesenchymal transfer (EMT) was visualized in the areas of proliferating bile ducts. The hematopoietic origin of CD34+ fibrocytes was demonstrated immunohistologically in livers of BM chimeric mice. These CD34+ cells infiltrated hepatic lobules from portal fields and developed a desmin+ phenotype expressing collagen type I in fibrotic parenchyma as well as in vitro after isolation by magnetic cell separation. Transplantation of GFP+/Abcb4+ BM improved liver function and staging compared with sham transplantation, but no significant differences were noticed among allogeneic and syngeneic Tx. Conclusion: The present study is the first to identify that both BM‐derived fibrocytes and HSC are involved in biliary fibrogenesis in Abcb4−/− mice. Our data suggest that changes in immunity subsequent to BM‐Tx may alter hepatic fibrosis. (HEPATOLOGY 2009.)

A new metaphyseal bone defect model in osteoporotic rats to study biomaterials for the enhancement of bone healing in osteoporotic fractures

The intention of this study was to establish a new critical size animal model that represents clinically relevant situations with osteoporotic bone status and internally fixated metaphyseal defect fractures in which biomaterials for the enhancement of fracture healing in osteoporotic fracture defects can be studied. Twenty-eight rats were ovariectomized (OVX) and treated with a calcium-, phosphorus-, vitamin D3-, soy- and phytoestrogen-free diet. After 3months Dual-energy X-ray absorptiometry measurements showed statistically significant reductions in bone mineral density of the spine of -25.9% and of the femur of -21.3% of the OVX rats compared with controls, confirming osteoporosis in the OVX rats. The OVX rats then underwent either 3 or 5mm wedge-shaped osteotomy of the distal metaphyseal area of the femur that was internally stabilized with a T-shaped mini-plate. After 42days biomechanical testing yielded completely unstable conditions in the 5mm defect femora (bending stiffness 0Nmm(-2)) and a bending stiffness of 12,500Nmm(-2) in the 3mm defects, which showed the beginning of fracture consolidation. Micro-computed tomography showed statistically significant more new bone formation in the 3mm defects (4.83±0.37mm(2)), with bridging of the initial fracture defect area, compared with the 5mm defects (2.68±0.34mm(2)), in which no bridging of the initial defect was found. These results were confirmed by histology. In conclusion, the 5mm defect can be considered as a critical size defect model in which biomaterials can be tested.

Induction of osteoporosis with its influence on osteoporotic determinants and their interrelationships in rats by DEXA

Summary Background As women are the population most affected by multifactorial osteoporosis, research is focused on unraveling the underlying mechanism of osteoporosis induction in rats by combining ovariectomy (OVX) either with calcium, phosphorus, vitamin C and vitamin D2/D3 deficiency, or by administration of glucocorticoid (dexamethasone). Material/Methods Different skeletal sites of sham, OVX-Diet and OVX-Steroid rats were analyzed by Dual Energy X-ray Absorptiometry (DEXA) at varied time points of 0, 4 and 12 weeks to determine and compare the osteoporotic factors such as bone mineral density (BMD), bone mineral content (BMC), area, body weight and percent fat among different groups and time points. Comparative analysis and interrelationships among osteoporotic determinants by regression analysis were also determined. Results T scores were below-2.5 in OVX-Diet rats at 4 and 12 weeks post-OVX. OVX-diet rats revealed pronounced osteoporotic status with reduced BMD and BMC than the steroid counterparts, with the spine and pelvis as the most affected skeletal sites. Increase in percent fat was observed irrespective of the osteoporosis inducers applied. Comparative analysis and interrelationships between osteoporotic determinants that are rarely studied in animals indicate the necessity to analyze BMC and area along with BMD in obtaining meaningful information leading to proper prediction of probability of osteoporotic fractures. Conclusions Enhanced osteoporotic effect observed in OVX-Diet rats indicates that estrogen dysregulation combined with diet treatment induces and enhances osteoporosis with time when compared to the steroid group. Comparative and regression analysis indicates the need to determine BMC along with BMD and area in osteoporotic determination.

Effects of Multi-Deficiencies-Diet on Bone Parameters of Peripheral Bone in Ovariectomized Mature Rat

Many postmenopausal women have vitamin D and calcium deficiency. Therefore, vitamin D and calcium supplementation is recommended for all patients with osteopenia and osteoporosis. We used an experimental rat model to test the hypothesis that induction of osteoporosis is more efficiently achieved in peripheral bone through combining ovariectomy with a unique multi-deficiencies diet (vitamin D depletion and deficient calcium, vitamin K and phosphorus). 14-week-old Sprague-Dawley rats served as controls to examine the initial bone status. 11 rats were bilaterally ovariectomized (OVX) and fed with multi-deficiencies diet. Three months later the treated group and the Sham group (n = 8) were euthanized. Bone biomechanical competence of the diaphyseal bone was examined on both, tibia and femur. Image analysis was performed on tibia via µCT, and on femur via histological analysis. Lower torsional stiffness indicated inferior mechanical competence of the tibia in 3 month OVX+Diet. Proximal metaphyseal region of the tibia showed a diminished bone tissue portion to total tissue in the µCT despite the increased total area as evaluated in both µCT and histology. Cortical bone showed higher porosity and smaller cross sectional thickness of the tibial diaphysis in the OVX+Diet rats. A lower ALP positive area and elevated serum level of RANKL exhibited the unbalanced cellular interaction in bone remodeling in the OVX+Diet rat after 3 month of treatment. Interestingly, more adipose tissue area in bone marrow indicated an effect of bone loss similar to that observed in osteoporotic patients. Nonetheless, the presence of osteoid and elevated serum level of PTH, BGP and Opn suggest the development of osteomalacia rather than an osteoporosis. As the treatment and fracture management of both osteoporotic and osteomalacia patients are clinically overlapping, this study provides a preclinical animal model to be utilized in local supplementation of minerals, drugs and growth factors in future fracture healing studies.

Pathological Impact of Hepatitis B Virus Surface Proteins on the Liver Is Associated with the Host Genetic Background

Background While the immune pathogenesis caused by hepatitis B virus (HBV) infection has been studied extensively, little is known about direct pathogenic effects of HBV surface proteins. Here, we have investigated pathological cellular effects of HBV surface protein expression in the liver of transgenic mice with different genetic background. Methods The impact of HBV surface protein expression on the liver was studied in two mouse strains, BALB/c and C57BL/6. Histology and hydroxyproline assays were performed to investigate liver morphology and fibrosis. Gene expression and signaling were analyzed by microarray, qPCR and Western blotting. Results Expression of HBV surface proteins in the liver of transgenic mice induced activation of protein kinase-like endoplasmic reticulum kinase (PERK) and eukaryotic initiation factor 2α (eIF2α) phosphorylation. Phosphorylation of eIF2α resulted in activation of the ER stress markers glucose regulated protein (GRP) 78 and pro-apoptotic C/EBP homologous protein (CHOP) in transgenic mice on BALB/c genetic background leading to stronger liver injury and fibrosis in comparison with transgenic mice on C57BL/6 background. Hepatic stellate cells represented the main collagen-producing liver cells in HBV transgenic mice. The key regulators of hepatocyte proliferation, transcription factors c-Jun and STAT3 were activated in HBV transgenic mice. Tumour incidence in transgenic mice was strain- and sex-dependent. Conclusions Extent of liver injury, fibrosis, and tumour development induced by hepatic HBV surface protein expression considerably depends on host genetic background.

Implications of combined Ovariectomy/Multi-Deficiency Diet on rat bone with age-related variation in Bone Parameters and Bone Loss at Multiple Skeletal Sites by DEXA

Background Osteoporosis is a multi-factorial, chronic, skeletal disease highly prevalent in post-menopausal women and is influenced by hormonal and dietary factors. Because animal models are imperative for disease diagnostics, the present study establishes and evaluates enhanced osteoporosis obtained through combined ovariectomy and deficient diet by DEXA (dual-energy X-ray absorptiometry) for a prolonged time period. Material/Methods Sprague-Dawley rats were randomly divided into sham (laparotomized) and OVX-diet (ovariectomized and fed with deficient diet) groups. Different skeletal sites were scanned by DEXA at the following time points: M0 (baseline), M12 (12 months post-surgery), and M14 (14 months post-surgery). Parameters analyzed included BMD (bone mineral density), BMC (bone mineral content), bone area, and fat (%). Regression analysis was performed to determine the interrelationships between BMC, BMD, and bone area from M0 to M14. Results BMD and BMC were significantly lower in OVX-diet rats at M12 and M14 compared to sham rats. The Z-scores were below −5 in OVX-diet rats at M12, but still decreased at M14 in OVX-diet rats. Bone area and percent fat were significantly lower in OVX-diet rats at M14 compared to sham rats. The regression coefficients for BMD vs. bone area, BMC vs. bone area, and BMC vs. BMD of OVX-diet rats increased with time. This is explained by differential percent change in BMD, BMC, and bone area with respect to time and disease progression. Conclusions Combined ovariectomy and deficient diet in rats caused significant reduction of BMD, BMC, and bone area, with nearly 40% bone loss after 14 months, indicating the development of severe osteoporosis. An increasing regression coefficient of BMD vs. bone area with disease progression emphasizes bone area as an important parameter, along with BMD and BMC, for prediction of fracture risk.

Bone marrow transplantation improves hepatic fibrosis in Abcb4−/− mice via Th1 response and matrix metalloproteinase activity

Objective Reports on the effects of bone marrow-derived cells on hepatic fibrosis are contradictory. Impaired fibrosis but increased inflammation has recently been demonstrated 10 weeks after bone marrow transplantation (BM-Tx) in Abcb4−/− mice. It is hypothesised that BM-Tx might have long-term therapeutic potential by altering the immunological and matrix remodelling processes leading to hepatic regeneration. Methods After lethal irradiation of recipient mice, BM cells from GFP+ donor mice (allogeneic Tx) or Abcb4−/− mice (syngeneic Tx) were transplanted via tail vein injection. Readouts were performed 2, 10 and 20 weeks after Tx. Liver integrity was assessed serologically and histologically. Surrogate markers for fibrogenesis, T helper (Th) response, inflammation, graft-versus-host disease and fibrolysis were analysed by quantitative real-time PCR, zymography and immunohistology. Results 20 weeks after syngeneic and allogeneic BM-Tx, hepatic grading and staging were significantly improved. In contrast, 2 weeks after BM-Tx inflammatory grading, expression of inflammatory cell markers and associated chemokines and their receptors were increased and subsequently declined. In parallel, CD8+/GFP+ donor-derived T cells infiltrated the liver 2 weeks after BM-Tx. The Th1 cyokine interferon γ was increased 2 and 10 weeks after BM-Tx whereas the Th2 associated interleukin 13 was not altered. The gene expression of matrix metalloproteinases MMP-2, MMP-7, MMP-9 and MMP-13 was transiently upregulated and MMP-9 protein remained elevated 20 weeks after BM-Tx with enhanced gelatinase activity located within the fibrotic areas. Neutrophils were identified as major sources of MMP-9. Conclusion These results show that BM-Tx causes an antifibrotic Th1 response combined with transient inflammatory effects and subsequently upregulated MMP activity. Antifibrotic Th polarisation and prolonged proteolytic activity, especially of MMP-9, might be responsible for long-term amelioration of hepatic fibrosis.

[Ovariectomy and calcium/vitamin D2/D3 deficient diet as a model of osteoporosis in the spine of Sprague-Dawley rats].

BACKGROUND Osteoporosis is a widespread disease characterised by low bone mass and structural deterioration of bone resulting in an increased susceptibility to fractures. Osteoporosis affects women more frequently than men; every second woman older than 50 years suffers from an osteoporotic fracture, frequently a vertebral fracture. The aim of this study was to induce osteoporosis in rats to establish an osteoporotic small-animal model that simulates the human pathology particularly in the spine. Therefore, bone density parameters, which are routinely determined in the spine of osteoporotic patients, were investigated by Dual-Energy X-ray Absorptiometry (DEXA). MATERIALS AND METHODS Fourteen-week-old female Sprague-Dawley rats (n = 50) were either sham-operated (control group: sham) or ovariectomised (experimental group). Ovariectomised rats were further divided into two groups; one received calcium/vitamin D2/D3 deficient diet (OVX + diet), and the other received subcutaneous steroid injections (dexamethasone 0.3 mg/kg body weight) twice a month (OVX + steroid). Rats were scanned by DEXA at three time points (Month = M, 0 M, 1 M and 3 M). DEXA measurement of the spine delivered T-value, Z-value, bone mineral content (BMC), and the scanned area. Fifteen female patients at an age of 57-72 years were scanned in 8-10 regions of the spine (150 measurements). T-values and Z-values were pre-calculated based on patient databases. Statistical analysis was performed using two-way ANOVA followed by Bonferroni correction, with significance considered at p < 0.05. RESULTS T-value and Z-value of both rat groups were compared with the patient data as well as with each others. Both treated rat groups revealed significantly lower T- and Z-values than controls. Despite the significant difference, the reference line (-2.5 for T-value and -1.5 for Z-value) was only reached by the OVX + diet group. On the other hand, the sham group showed an increase in BMC over time, while no change was seen in OVX + diet or OVX + steroid. Bone area demonstrated a significant increase up to M3. However, the increase in bone area within the OVX + diet group was notably higher than in both sham and OVX + steroid groups. Patients showed significantly lower T- and Z-values than sham and OVX + steroid but insignificant ones when compared with OVX + diet. CONCLUSION A reproducible vertebral osteoporosis can be generated in a rat model by combination of ovariectomy with administration of a calcium/vitamin D3 deficient diet. T- and Z-values of this experimental group mimicked values obtained from osteoporotic patients, reflecting a simulation of their pathology. Interestingly, the increase in bone area over time with the steady BMC results in lower mineral density (BMD) of the OVX + diet group. Therefore, this rat model presents a reliable experimental set-up that may serve as a tool to better understand and treat osteoporosis.

Characterization of bone turnover and energy metabolism in a rat model of primary and secondary osteoporosis.

BACKGROUND An experimental rat model served for evaluation of bone- and energy metabolism in early and late stages of osteoporosis. For the early stage, we hypothesized that bilateral ovariectomy (OVX)+multi-deficiency diet (OVXD; depletion of vitamin D, calcium, vitamin K, phosphorus) would induce increased bone turnover while the late stage would be characterized by enhanced bone catabolism. Obesity, insulin resistance and hyperleptinemia would be seen during the whole course of disease. Healthy female Sprague Dawley rats (n=41) aged 10 weeks were randomly assigned to sham and treatment groups and sacrificed at 3, 12, and 14 months after the study began. RESULTS In the early phase, OVXD was associated with an increase in body weight, but not, however, in later stages. There was a decrease in bone mineral density and relative bone volume (BV/TV) as assessed by Dual Energy X-ray Absorptiometry and micro computed tomography that was most severe in the later stages of disease, indicating bone catabolism. Osteocalcin limiting bone formation was increased initially, whereas later stages (14 months) were characterized by elevated osteopontin, suggesting bone remodeling. Severe hyperparathyroidism was present during all stages of disease. Only the early phases of disease were characterized by hyperinsulinemia and increased adrenocorticotrophic stimulating hormone, whereas in the late stage hypoleptinemia rather than hyperleptinemia was seen. CONCLUSION Markers of bone and energy metabolism reflected both an increased bone turn over and ongoing bone remodeling associated with initial hyperinsulinemia. Osteopontin and osteocalcin can be used to differentiate early and late stages of osteoporosis.

Hepatitis B virus surface proteins accelerate cholestatic injury and tumor progression in Abcb4-knockout mice

Understanding of the pathophysiology of cholestasis associated carcinogenesis could challenge the development of new personalized therapeutic approaches and thus improve prognosis. Simultaneous damage might aggravate hepatic injury, induce chronic liver disease and even promote carcinogenesis. We aimed to study the effect of Hepatitis B virus surface protein (HBsAg) on cholestatic liver disease and associated carcinogenesis in a mouse model combining both impairments. Hybrids of Abcb4-/- and HBsAg transgenic mice were bred on fibrosis susceptible background BALB/c. Liver injury, serum bile acid concentration, hepatic fibrosis, and carcinogenesis were enhanced by the combination of simultaneous damage in line with activation of c-Jun N-terminal kinase (JNK), proto-oncogene c-Jun, and Signal transducer and activator of transcription 3 (STAT3). Activation of Protein Kinase RNA-like Endoplasmic Reticulum Kinase (PERK) and Eukaryotic translation initiation factor 2A (eIF2α) indicated unfolded protein response (UPR) in HBsAg-expressing mice and even in Abcb4-/- without HBsAg-expression. CONCLUSION Cholestasis-induced STAT3- and JNK-pathways may predispose HBsAg-associated tumorigenesis. Since STAT3- and JNK-activation are well characterized critical regulators for tumor promotion, the potentiation of their activation in hybrids suggests an additive mechanism enhancing tumor incidence.Understanding of the pathophysiology of cholestasis associated carcinogenesis could challenge the development of new personalized therapeutic approaches and thus improve prognosis. Simultaneous damage might aggravate hepatic injury, induce chronic liver disease and even promote carcinogenesis. We aimed to study the effect of Hepatitis B virus surface protein (HBsAg) on cholestatic liver disease and associated carcinogenesis in a mouse model combining both impairments. Hybrids of Abcb4−/− and HBsAg transgenic mice were bred on fibrosis susceptible background BALB/c. Liver injury, serum bile acid concentration, hepatic fibrosis, and carcinogenesis were enhanced by the combination of simultaneous damage in line with activation of c-Jun N-terminal kinase (JNK), proto-oncogene c-Jun, and Signal transducer and activator of transcription 3 (STAT3). Activation of Protein Kinase RNA-like Endoplasmic Reticulum Kinase (PERK) and Eukaryotic translation initiation factor 2A (eIF2α) indicated unfolded protein response (UPR) in HBsAg-expressing mice and even in Abcb4−/− without HBsAg-expression. CONCLUSION: Cholestasis-induced STAT3- and JNK-pathways may predispose HBsAg-associated tumorigenesis. Since STAT3- and JNK-activation are well characterized critical regulators for tumor promotion, the potentiation of their activation in hybrids suggests an additive mechanism enhancing tumor incidence.