Evaluation of the long-term skeletal effect induced by teratogen 5-aza-2′deoxycytidine on offspring of high (C3H/HeJ) and low (C57BL/6J) bone mass phenotype mice

The long term skeletal effects of antenatal exposure to teratogen 5-deoxy-2′-cytidine (5-AZA) were studied using two inbred strains, C3H/HeJ (C3H, with inherently stronger bones) and C57Bl/6J (C57, with weaker bones). We previously reported that in-utero exposure to 5-AZA resulted in loss of bone quality in 3- and 6-mo-old C3H offspring. In this study, we further examined whether the long-term effects of an acute teratogenic exposure are still evident in older mice. Bone phenotypes of 12 mo-old mice exposed to a single injection of 5-AZA on day 10 of their mother's pregnancy were evaluated by micro-computed tomography and compared to the untreated controls.The main observation of this study is that 5-AZA-induced loss of bone length was registered in 12-mo-old C57 and C3H males. As expected, we did not find differences in the 3rd lumbar vertebra since in-utero exposure to 5-AZA was shown to affect the limb buds but not the axial skeleton. Trajectory of changes in bone phenotypes from ages 3 mo through 6 mo to 12 mo was also compared; 5-AZA-exposed C57 males had consistently lower femoral length and trabecular BMD than age-matched controls. In summary, by characterizing teratogen-exposed C57 and C3H mice, we further confirmed that the adaptive response to antenatal insults continue into mid-life of the mice as well as there is a sex-specificity of these responses. Keywords: Bone loss, Genetic heterogeneity, Adult mice, Developmental origin of disease

Erythropoietin enhances Kupffer cell number and activity in the challenged liver

Erythropoietin (EPO) is the main hormone driving mammalian erythropoiesis, with activity mediated via the surface receptor, EPO-R, on erythroid progenitor cells. Recombinant human EPO is currently used clinically for the treatment of anemia in patients with end-stage renal disease, and in certain cancer patients suffering from anemia induced either by the tumor itself or by chemotherapy. EPO-R expression is also detected in non-erythroid cells, including macrophages present in the peritoneum, spleen, and bone marrow (BM). Here we demonstrate that Kupffer cells (KCs) - the liver-resident macrophages - are EPO targets. We show that, in vitro, EPO initiated intracellular signalling and enhanced phagocytosis in a rat KC line (RKC-2) and in sorted KCs. Moreover, continuous EPO administration in mice, resulted in an increased number of KCs, up-regulation of liver EPO-R expression and elevated production of the monocyte chemoattractant CCL2, with corresponding egress of Ly6C(hi) monocytes from the BM. In a model of acute acetaminophen-induced liver injury, EPO administration increased the recruitment of Ly6C(hi) monocytes and neutrophils to the liver. Taken together, our results reveal a new role for EPO in stimulating KC proliferation and phagocytosis, and in recruiting Ly6C(hi) monocytes in response to liver injury

Beta Palmitate Improves Bone Length and Quality during Catch-Up Growth in Young Rats

Palmitic acid (PA) is the most abundant saturated fatty acid in human milk, where it is heavily concentrated in the sn-2-position (termed beta palmitate, BPA) and as such is conserved in all women, regardless of their diet or ethnicity, indicating its physiological and metabolic importance. We hypothesized that BPA improves the efficiency of nutrition-induced catch up growth as compared to sn-1,3 PA, which is present in vegetable oil. Pre-pubertal male rats were subjected to a 17 days food restriction followed by re-feeding for nine days with 1,3 PA or BPA-containing diets. We measured bone length, epiphyseal growth plate height (EGP, histology), bone quality (micro-CT and 3-point bending assay), and gene expression (Affymetrix). The BPA-containing diet improved most growth parameters: humeri length and EGP height were greater in the BPA-fed animals. Further analysis of the EGP revealed that the hypertrophic zone was significantly higher in the BPA group. In addition, Affymetrix analysis revealed that the diet affected the expression of several genes in the liver and EGP. Despite the very subtle difference between the diets and the short re-feeding period, we found a small but significant improvement in most growth parameters in the BPA-fed rats. This pre-clinical study may have important implications, especially for children with growth disorders and children with special nutritional needs

Erythropoietin directly stimulates osteoclast precursors and induces bone loss

Erythropoietin (EPO) primarily regulates red blood cell formation, and EPO serum levels are increased on hypoxic stress (e.g., anemia and altitude). In addition to anemia, recent discoveries suggest new therapeutic indications for EPO, unrelated to erythropoiesis. We investigated the skeletal role of EPO using several models of overexpression (Tg6 mice) and EPO administration (intermittent/continuous, high/low doses) in adult C57Bl6 female mice. Using microcomputed tomography, histology, and serum markers, we found that EPO induced a 32%-61% trabecular bone loss caused by increased bone resorption (+60%-88% osteoclast number) and reduced bone formation rate (-19 to -74%; P < 0.05 throughout). EPO targeted the monocytic lineage by increasing the number of bone monocytes/macrophages, preosteoclasts, and mature osteoclasts. In contrast to the attenuated bone formation in vivo, EPO treatment in vitro did not inhibit osteoblast differentiation and activity, suggesting an indirect effect of EPO on osteoblasts. However, EPO had a direct effect on preosteoclasts by stimulating osteoclastogenesis in isolated cultures (+60%) via the Jak2 and PI3K pathways. In summary, our findings demonstrate that EPO negatively regulates bone mass and thus bears significant clinical implications for the potential management of patients with endogenously or therapeutically elevated EPO levels.-Hiram-Bab, S., Liron, T., Deshet-Unger, N., Mittelman, M., Gassmann, M., Rauner, M., Franke, K., Wielockx, B., Neumann, D., Gabet, Y. Erythropoietin directly stimulates osteoclast precursors and induces bone loss

Erythropoietin Mediated Bone Loss in Mice Is Dose-Dependent and Mostly Irreversible

Recent studies have demonstrated that erythropoietin (EPO) treatment in mice results in trabecular bone loss. Here, we investigated the dose-response relationship between EPO, hemoglobin (Hgb) and bone loss and examined the reversibility of EPO-induced damage. Increasing doses of EPO over two weeks led to a dose-dependent increase in Hgb in young female mice, accompanied by a disproportionate decrease in trabecular bone mass measured by micro-CT (µCT). Namely, increasing EPO from 24 to 540 IU/week produced a modest 12% rise in Hgb (20.2 ± 1.3 mg/dL vs 22.7 ± 1.3 mg/dL), while trabecular bone volume fraction (BV/TV) in the distal femur decreased dramatically (27 ± 8.5% vs 53 ± 10.2% bone loss). To explore the long-term skeletal effects of EPO, we treated mice for two weeks (540 IU/week) and monitored bone mass changes after treatment cessation. Six weeks post-treatment, there was only a partial recovery of the trabecular microarchitecture in the femur and vertebra. EPO-induced bone loss is therefore dose-dependent and mostly irreversible at doses that offer only a minor advantage in the treatment of anemia. Because patients requiring EPO therapy are often prone to osteoporosis, our data advocate for using the lowest effective EPO dose for the shortest period of time to decrease thromboembolic complications and minimize the adverse skeletal outcome

Perturbed bone composition and integrity with disorganized osteoblast function in zinc receptor/Gpr39-deficient mice

Changes in bone matrix composition are frequently found with bone diseases and may be associated with increased fracture risk. Bone is rich in the trace element zinc. Zinc was established to play a significant role in the growth, development, and maintenance of healthy bones; however, the mechanisms underlying zinc effects on the integrity of the skeleton are poorly understood. Here, we show that the zinc receptor (ZnR)/Gpr39 is required for normal bone matrix deposition by osteoblasts. Initial analysis showed that Gpr39-deficient (Gpr39-/-) mice had weaker bones as a result of altered bone composition. Fourier transform infrared spectroscopy analysis showed high mineral-to-matrix ratios in the bones of Gpr39-/- mice. Histologic analysis showed abnormally high numbers of active osteoblasts but normal osteoclast numbers on the surfaces of bones from Gpr39-/- mice. Furthermore, Gpr39-/- osteoblasts had disorganized matrix deposition in vitro with cultures exhibiting abnormally low collagen and high mineral contents, findings that demonstrateacell-intrinsic role for ZnR/Gpr39 in these cells. We show that both collagen synthesis and deposition by Gpr39-/- osteoblasts are perturbed. Finally, the expression of the zinc transporter Zip13 and a disintegrin and metalloproteinase with thrombospondin motifs family of zinc-dependent metalloproteases that regulate collagen processing was downregulated in Gpr39-/- osteoblasts. Altogether, our results suggest that zinc sensing by ZnR/Gpr39 affects the expression levels of zinc-dependent enzymes in osteo-blasts and regulates collagen processing and deposition

Increased epo levels are associated with bone loss in mice lacking phd2 in epo-producing cells

The main oxygen sensor HIF prolyl hydroxylase 2 (PHD2) is a critical regulator of tissue homeostasis during erythropoiesis, hematopoietic stem cell maintenance and wound healing. Recent studies point towards a role for the PHD2-erythropoietin (EPO) axis in the modulation of bone remodeling, even though the studies produced conflicting results. Here, we used a number of mouse strains deficient of PHD2 in different cell types to address the role of PHD2 and its downstream targets HIF-1α and HIF-2α in bone remodeling. Mice deficient for PHD2 in several cell lineages, including EPO-producing cells, osteoblasts and hematopoietic cells (CD68:cre-PHD2f/f ) displayed a severe reduction of bone density at the distal femur as well as the vertebral body due to impaired bone formation but not bone resorption. Importantly, using osteoblast- (Osx:cre-PHD2f/f ) and osteoclast-specific PHD2 knock-out mice (Vav:cre- PHD2f/f ), we show that this effect is independent of the loss of PHD2 in osteoblast and osteoclasts. Using different in vivo and in vitro approaches, we here demonstrate that this bone phenotype, including the suppression of bone formation, is directly linked to the stabilization of the α-subunit of HIF-2, and possibly to the subsequent moderate induction of serum EPO, which directly influenced the differentiation and mineralization of osteoblast progenitors resulting in lower bone density. Taken together, our data identify the PHD2:HIF-2α:EPO axis as a so far unknown regulator of osteohematology by controlling bone homeostasis. Further, these data suggest that patients treated with PHD inhibitors or EPO should be monitored with respect to their bone status

The Non-Erythropoietic EPO Analogue Cibinetide Inhibits Osteoclastogenesis In Vitro and Increases Bone Mineral Density in Mice

The two erythropoietin (EPO) receptor forms mediate different cellular responses to erythropoietin. While hematopoiesis is mediated via the homodimeric EPO receptor (EPOR), tissue protection is conferred via a heteromer composed of EPOR and CD131. In the skeletal system, EPO stimulates osteoclast precursors and induces bone loss. However, the underlying molecular mechanisms are still elusive. Here, we evaluated the role of the heteromeric complex in bone metabolism in vivo and in vitro by using Cibinetide (CIB), a non-erythropoietic EPO analogue that exclusively binds the heteromeric receptor. CIB is administered either alone or in combination with EPO. One month of CIB treatment significantly increased the cortical (~5.8%) and trabecular (~5.2%) bone mineral density in C57BL/6J WT female mice. Similarly, administration of CIB for five consecutive days to female mice that concurrently received EPO on days one and four, reduced the number of osteoclast progenitors, defined by flow cytometry as Lin&minus;CD11b&minus;Ly6Chi CD115+, by 42.8% compared to treatment with EPO alone. In addition, CIB alone or in combination with EPO inhibited osteoclastogenesis in vitro. Our findings introduce CIB either as a stand-alone treatment, or in combination with EPO, as an appealing candidate for the treatment of the bone loss that accompanies EPO treatment