Polycythemia is associated with bone loss and reduced osteoblast activity in mice.

UNLABELLED: Increased fragility has been described in humans with polycythemia vera (PV). Herein, we describe an osteoporotic phenotype associated with decreased osteoblast activity in a mouse model of PV and another mouse of polycythemia and elevated circulating erythropoietin (EPO). Our results are important for patients with PV or those treated with recombinant EPO (rEPO). INTRODUCTION: PV and other myeloproliferative syndromes have been recently associated with an increased risk for fractures. However, the presence of osteoporosis in these patients has not been well documented. EPO, a hormone primarily known to stimulate erythropoiesis, has been shown recently to regulate bone homeostasis in mice. The aim of this study was to examine the bone phenotype of a mouse model of PV and compare it to that of animals with polycythemia caused by elevated circulating EPO. METHODS: Bone mass and remodeling were evaluated by micro-computed tomography and histomorphometry. The JAK2(V617F) knock-in mouse, a model of human PV, manifests polycythemia and low circulating EPO levels. Results from this mouse were compared to wild type (wt) controls and the tg6 transgenic mouse that shows polycythemia caused by increased constitutive expression of EPO. RESULTS: Compared to wt, both JAK2(V617F) and tg6 mice had a decrease in trabecular bone mass. Tg6 mice showed an additional modest decrease in cortical thickness and cortical bone volume per tissue volume (P < 0.01) suggesting a more severe bone phenotype than JAK2(V617F). Decreased osteoblast numbers and bone formation along with normal osteoclast numbers and activity were found in both mice. CONCLUSIONS: This study indicates that PV is associated with low bone mass and decreased osteoblast activity in mice. Our results support future studies of osteoporosis in affected humans. Polycythemia caused by chronically elevated circulating EPO also results in bone loss, and implications on patients treated with rEPO should be evaluated.

Targeted delivery of small interfering RNA to angiogenic endothelial cells with liposome-polycation-DNA particles.

Angiogenesis is an attractive target for cancer therapy, due to its central position in tumor growth and development. Vascular Endothelial Growth Factor (VEGF) and its receptors (VEGFRs) play a key role in the angiogenic process. A promising strategy for targeting VEGF-mediated angiogenesis is RNA interference (RNAi) using short interfering RNA (siRNA). However, for efficacious RNAi a well-designed siRNA delivery system is crucial. Liposome-Polycation-DNA (LPD) particles form a promising system for siRNA delivery to tumors. In order to target angiogenic endothelial cells, LPD particles may be modified with a targeting ligand, such as a cyclic Arg-Gly-Asp (RGD) peptide that specifically binds to integrins expressed on tumor-associated endothelial cells. In the current study, RGD-targeted PEGylated LPD particles containing VEGFR-2 siRNA were prepared and optimized with respect to their size and charge by varying protamine content, carrier DNA content for stronger complexation, and PEGylation density. The size of the optimized particles was around 200 nm and the ζ-potential was approximately +20 mV. The uptake and silencing efficacy of the RGD-targeted PEGylated LPD particles were evaluated in H5V cells (murine endothelial cells) and Human Umbilical Vein Endothelial cells (HUVECs). When compared to non-targeted LPD particles, enhanced uptake and silencing of VEGFR-2 expression was observed for RGD-targeted PEGylated LPD particles. In conclusion, the RGD-targeted PEGylated LPD particles containing VEGFR-2 siRNA presented here may be a promising approach for targeting VEGF-mediated angiogenesis in cancer therapy.

An in vitro assay based on surface plasmon resonance to predict the in vivo circulation kinetics of liposomes.

The adsorption of blood proteins onto liposomes and other colloidal particles is an important process influencing the circulation time. Proteins adsorbed to the surface of liposomes can mediate recognition of the liposomes by macrophages of the reticuloendothelial system (RES) facilitating their clearance from the circulation. Coating liposomes with poly(ethylene glycol) (PEG) decreases the blood clearance considerably, most likely due to reduced protein adsorption and/or liposome aggregation. By using the relation between clearance and protein binding, the present study introduces an in vitro assay measuring interactions of liposomes with proteins to predict their blood clearance in vivo. Such assay is valuable since it limits time and costs, and importantly reduces the number of animals required for pharmacokinetic investigations of new formulations. In the current study, Surface Plasmon Resonance (SPR) and fluorescence Single Particle Tracking (fSPT) were used to study liposome-protein interactions and blood induced liposome aggregation in vitro. By means of SPR the interactions between proteins and liposomes coated with PEG of different molecular weights and at different densities (PEG(2000) in 2.5%, 5% and 7%; PEG(5000) in 0.5%, 1.5% and 2.5%), were measured for several plasma proteins: human serum albumin (HSA), apolipoprotein E (ApoE), α2-macroglobulin (α2-M), ß2-glycoprotein (ß2-G) and fibronectin (Fn). Liposomes coated with PEG interacted less with all proteins, an effect which increased with the PEG surface density. In parallel, fSPT analysis showed that the exposure of liposomes to full blood did not change the liposome size, indicating that aggregation is not a strong attributive factor in the clearance of these liposomes. In addition, the SPR measurements of the interactions between liposomes and proteins were correlated with the blood clearance of the liposomes. For each protein, the degree of protein-liposome interaction as determined by SPR showed a moderate to strong positive correlation with the clearance of the liposome type.

Macrophages and liposomes in inflammatory disease: friends or foes?

Liposome-encapsulated corticosteroids have shown to exert strong beneficial effects in inflammatory diseases, such as arthritis and cancer. To extend the clinical applicability of these potent nanomedicines, the therapeutic effect of dexamethasone phosphate loaded long-circulating liposomes (LCL-DXP) was evaluated in animal models of multiple sclerosis (MS) and Crohn's disease (CD). In mice with experimental autoimmune encephalitis (EAE), a model for MS, treatment with LCL-DXP, but not free DXP, resulted in a decrease in disease activity when compared to PBS treated mice. In contrast, in mice with chronic DSS-induced colitis, a model for CD, treatment with LCL-DXP did not induce an improvement, but in fact worsened the fecal blood loss after treatment, indicating an aggravation of the disease. It is hypothesized that modulation of macrophage polarization towards a M2 phenotype underlies the efficacy of corticosteroid-based drug delivery systems, which is supported by the presented data. On the one hand, M1 polarized macrophages are part of the pathogenesis of MS; the modulation to M2-polarization by LCL-DXP is therefore beneficial. On the other hand, M1-polarized intestinal macrophages fulfill a protective and inflammation-suppressing role in intestinal homeostasis; changing their phenotype to M2 causes reduced protection to invading microorganisms, leading to a more severe intestinal inflammation. These findings therefore indicate that the interplay between the specific phenotype of macrophages and the specific inflammatory context of the inflammatory disease in question may be an important determining factor in the therapeutic applicability of liposomal corticosteroids in inflammatory disease.