FGF-23 Is a Negative Regulator of Prenatal and Postnatal Erythropoiesis

Abnormal blood cell production is associated with chronic kidney disease (CKD) and cardiovascular disease (CVD). Bone-derived FGF-23 (fibroblast growth factor-23) regulates phosphate homeostasis and bone mineralization. Genetic deletion of Fgf-23 in mice (Fgf-23^−/−) results in hypervitaminosis D, abnormal mineral metabolism, and reduced lymphatic organ size. Elevated FGF-23 levels are linked to CKD and greater risk of CVD, left ventricular hypertrophy, and mortality in dialysis patients. However, whether FGF-23 is involved in the regulation of erythropoiesis is unknown. Here we report that loss of FGF-23 results in increased hematopoietic stem cell frequency associated with increased erythropoiesis in peripheral blood and bone marrow in young adult mice. In particular, these hematopoietic changes are also detected in fetal livers, suggesting that they are not the result of altered bone marrow niche alone. Most importantly, administration of FGF-23 in wild-type mice results in a rapid decrease in erythropoiesis. Finally, we show that the effect of FGF-23 on erythropoiesis is independent of the high vitamin D levels in these mice. Our studies suggest a novel role for FGF-23 in erythrocyte production and differentiation and suggest that elevated FGF-23 levels contribute to the pathogenesis of anemia in patients with CKD and CVD.     

Homozygous ablation of fibroblast growth factor-23 results in hyperphosphatemia and impaired skeletogenesis, and reverses hypophosphatemia in Phex-deficient mice.

Fibroblast growth factor-23 (FGF-23), a recently identified molecule that is mutated in patients with autosomal dominant hypophosphatemic rickets (ADHR), appears to be involved in the regulation of phosphate homeostasis. Although increased levels of circulating FGF-23 were detected in patients with different phosphate-wasting disorders such as oncogenic osteomalacia (OOM) and X-linked hypophosphatemia (XLH), it is not yet clear whether FGF-23 is directly responsible for the abnormal regulation of mineral ion homeostasis and consequently bone development. To address some of these unresolved questions, we generated a mouse model, in which the entire Fgf-23 gene was replaced with the lacZ gene. Fgf-23 null (Fgf-23-/-) mice showed signs of growth retardation by day 17, developed severe hyperphosphatemia with elevated serum 1,25(OH)2D3 levels, and died by 13 weeks of age. Hyperphosphatemia in Fgf-23-/- mice was accompanied by skeletal abnormalities, as demonstrated by histological, molecular, and various other morphometric analyses. Fgf-23-/-) mice had increased total-body bone mineral content (BMC) but decreased bone mineral density (BMD) of the limbs. Overall, Fgf-23-/- mice exhibited increased mineralization, but also accumulation of unmineralized osteoid leading to marked limb deformities. Moreover, Fgf-23-/- mice showed excessive mineralization in soft tissues, including heart and kidney. To further expand our understanding regarding the role of Fgf-23 in phosphate homeostasis and skeletal mineralization, we crossed Fgf-23-/- animals with Hyp mice, the murine equivalent of XLH. Interestingly, Hyp males lacking both Fgf-23 alleles were indistinguishable from Fgf-23/-/ mice, both in terms of serum phosphate levels and skeletal changes, suggesting that Fgf-23 is upstream of the phosphate regulating gene with homologies to endopeptidases on the X chromosome (Phex) and that the increased plasma Fgf-23 levels in Hyp mice (and in XLH patients) may be at least partially responsible for the phosphate imbalance in this disorder.     

Ancient split of major genetic lineages of European Black Pine: evidence from chloroplast <Emphasis>DNA</Emphasis>

The European Black Pine (Pinus nigra Arn.) has a long and complex history. Genetic distance and frequency analyses identified three differentiated genetic groups, which corresponded to three wide geographical areas: Westerns Mediterranean, Balkan Peninsula and Asia Minor. These groups shared common ancestors (14.75 and 10.72 Ma). The most recent splits occurred after the Messinian Salinity Crisis (4.37 Ma) and the Early–Middle Pleistocene Transitions (0.93 Ma). The posterior ancestral population size (Na) is 260,000–265,000 individuals. Each pool is further fragmented, with evidence of a phylogeographic structure (N _st  > G _st ) typically observed in some natural populations from the Western Mediterranean region and the Balkan Peninsula. The laboratory analysis was performed by fragment analysis—i.e. electrophoretic sizing of polymerase chain reaction fragments, combined with the sequencing analysis of 33 % of all individuals as a control. Intense sampling of chloroplast DNA polymorphisms (3154 individuals and 13 markers: SNPs and SSRs) over the full area of the species’ natural distribution indicated moderate among-population variability (G _st(nc) ≤ 0.177) in various parts of its range. These results indicate that the natural populations have long migration histories that differ from one another and that they have been strongly phylogeographically affected by complex patterns of isolation, speciation and fragmentation. Long and varying climatic fluctuations in the region of the principal genetic group have been the probable cause of different forest community associations with different successional patterns resulting in interglacial refugia vs. macro long-term refugia.     

Biochemical properties of encapsulated high-density 3-D HepG2 aggregates formed in an ultrasound trap for application in hepatotoxicity studies

This paper describes the alginate encapsulation of preformed high-density 3-D HepG2 cell aggregates that guarantees good maintenance of liver-specific biomarker expression. The process involves forming a high-density (≥7 × 10⁴ cells/aggregate) discoid 3-D cell aggregate in an ultrasound trap, which is subsequently recovered and encapsulated in alginate/CaCl₂ hydrogel. Glucose secretion/consumption, lactate release, detoxifying enzyme capacity, cytokeratin-18 expression as well as hypoxia were characterized in encapsulated 3-D HepG2 aggregates over 10 days in culture. Encapsulated 3-D HepG2 aggregates released glucose into the media, although this ability was exhibited only after 1 day in culture and was subsequently lost over the ensuing 9 days. In contrast, lactate was constantly released into the media. Significantly more lactate was secreted after 3 days in culture indicating a more hypoxic environment and hence a higher rate of anaerobic glycolysis. Aggregates consistently expressed cytokeratin-18. Cytochrome P450-1A1 activity reached a maximum on day 1 of culture followed by a progressive reduction to basal levels, while P450-3A4 activity was up-regulated in a time-dependent manner reaching a peak on day 7 in culture. Glutathione-S-transferase activity, on the other hand, was at more physiological levels and remained constant over the 10-day culture period. The ultrasound trap allowed the rapid (within 5 min) generation of uniformly shaped and sized aggregates. The results reported here suggest that ultrasound-formed 3-D HepG2 aggregates can serve as alternative in vitro models providing a quick outlook on toxicity, in a tissue-mimetic manner, thus offering the future option of a cost-effective screening platform for pharmaceutical development.     

The genetic background of gallstone formation: An update

Gallstone disease is one of the most prevalent gastrointestinal diseases with a substantial burden to health care systems that is expected to increase in ageing populations at risk. This review summarizes recent data on the genetic background of cholesterol gallstones and the role of biliary lipid composition. Three previously unknown non-synonymous mutations in the ABCB4 gene encoding the hepatobiliary phospholipid-flippase MDR3 are presented.     

Morphometric analysis of AgNORs in thin-layer, liquid-based liver specimens

OBJECTIVE: To detect argyrophilic nucleolar organizer regions (AgNORs) in ThinPrep (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) liver fine needle aspiration (FNA) specimens and to define the diagnostic value of their quantitative analysis in the evaluation of hepatic lesions. STUDY DESIGN: ThinPrep liquid-based FNA biopsy specimens from 49 malignant and benign liver lesions were resampled, fixed in 95% ethanol and stained with the AgNOR technique in accordance with the 1-step colloid method. The specimens included 11 benign and 38 malignant lesions (23 poorly differentiated hepatocellular carcinomas [HCCs] and 15 poorly differentiated metastatic adenocarcinomas [MCs]). Morphometric analysis was performed using a Zeiss Axiolab microscope (Carl Zeiss GmbH, Jena, Germany) with a mechanical stage fitted with a Sony-iris CCD videocamera (Tokyo, Japan). The videocamera was connected to a Pentium III P/C (Intel Corp., Santa Clara, California, U.S.A.) loaded with the appropriate image analysis software. The measurements were performed with ImageScan software (Jandel Scientific, Erkrath, Germany). The number of AgNORs per nucleus (NN) and the total area per nucleus occupied by AgNORs (AR) were calculated semiautomatically. Statistical analysis was performed using the SPSS software package (Chicago, Illinois, U.S.A.). RESULTS: The least significant deviance test for multiple comparisons revealed that NN differed significantly between the 3 groups of samples examined (P < .0001). The mean NN values in HCCs and MCs were significantly different (P < .0001). Logistic regression model demonstrated that as NN increased, the probability of a MC diagnosis decreased (<4%). AR values were different at a statistically significant level only between benign and malignant specimens (P = .00006), not between HCCs and MCs (P = .933). CONCLUSION: Quantitative analysis of AgNORs in ThinPrep specimens could be a diagnostically useful method in liver disease.     

Transfection of liver in vivo by biolistic particle delivery

We describe the delivery of reporter gene constructs to rat liver through the use of the Helios Gene Gun system. The effectiveness of this transfection method is illustrated by describing its use for determining in vivo the role of a DNA element that regulates cytochrome P450 2B1 (CYP2B1) gene expression in response to xenobiotics. DNA was delivered to the liver of an anesthetized animal via DNA-coated gold microcarriers. The highest level of reporter gene expression was obtained about six hours posttransfection; however, at this time endogenous CYP2B1 mRNA is transiently induced by the anesthetic treatment. The optimal time for investigating expression of a reporter gene under the control of CYP2B1 regulatory elements was 24 h after transfection, by which time the inductive effect of the anesthetic had ceased. Reporter gene expression subsequently declined rapidly to a low level by 48 h. In the transfected liver the heterologous SV40 promoter was about eight-fold stronger than the minimal CYP2B1 promoter. However, when attached to the phenobarbital response element both promoters give the same fold-induction of reporter activity in response to phenobarbital.