Lipocalin 2 Influences Bone and Muscle Phenotype in the MDX Mouse Model of Duchenne Muscular Dystrophy.

Lipocalin 2 (Lcn2) is an adipokine involved in bone and energy metabolism. Its serum levels correlate with bone mechanical unloading and inflammation, two conditions representing hallmarks of Duchenne Muscular Dystrophy (DMD). Therefore, we investigated the role of Lcn2 in bone loss induced by muscle failure in the MDX mouse model of DMD. We found increased Lcn2 serum levels in MDX mice at 1, 3, 6, and 12 months of age. Consistently, Lcn2 mRNA was higher in MDX versus WT muscles. Immunohistochemistry showed Lcn2 expression in mononuclear cells between muscle fibres and in muscle fibres, thus confirming the gene expression results. We then ablated Lcn2 in MDX mice, breeding them with Lcn2-/- mice (MDXxLcn2-/-), resulting in a higher percentage of trabecular volume/total tissue volume compared to MDX mice, likely due to reduced bone resorption. Moreover, MDXxLcn2-/- mice presented with higher grip strength, increased intact muscle fibres, and reduced serum creatine kinase levels compared to MDX. Consistently, blocking Lcn2 by treating 2-month-old MDX mice with an anti-Lcn2 monoclonal antibody (Lcn2Ab) increased trabecular volume, while reducing osteoclast surface/bone surface compared to MDX mice treated with irrelevant IgG. Grip force was also increased, and diaphragm fibrosis was reduced by the Lcn2Ab. These results suggest that Lcn2 could be a possible therapeutic target to treat DMD-induced bone loss.

Effects of osteoblast-derived extracellular vesicles on aggressiveness, redox status and mitochondrial bioenergetics of MNNG/HOS osteosarcoma cells.

Osteosarcoma is the most common primary bone malignancy. The crosstalk between osteosarcoma and the surrounding tumour microenvironment (TME) drives key events that lead to metastasization, one of the main obstacles for definitive cure of most malignancies. Extracellular vesicles (EVs), lipid bilayer nanoparticles used by cells for intercellular communication, are emerging as critical biological mediators that permit the interplay between neoplasms and the tumour microenvironment, modulating re-wiring of energy metabolism and redox homeostatic processes. We previously showed that EVs derived from the human osteosarcoma cells influence bone cells, including osteoblasts. We here investigated whether the opposite could also be true, studying how osteoblast-derived EVs (OB-EVs) could alter tumour phenotype, mitochondrial energy metabolism, redox status and oxidative damage in MNNG/HOS osteosarcoma cells.These were treated with EVs obtained from mouse primary osteoblasts, and the following endpoints were investigated: i) cell viability and proliferation; ii) apoptosis; iii) migration and invasive capacity; iv) stemness features; v) mitochondrial function and energy metabolism; vi) redox status, antioxidant capacity and oxidative molecular damage. OB-EVs decreased MNNG/HOS metabolic activity and viability, which however was not accompanied by impaired proliferation nor by increased apoptosis, with respect to control. In addition, OB-EV-treated cells exhibited a significant reduction of motility and in vitro invasion as compared to untreated cells. Although the antioxidant N-acetyl-L-cysteine reverted the cytotoxic effect of OB-EVs, no evidence of oxidative stress was observed in treated cells. However, the redox balance of glutathione was significantly shifted towards a pro-oxidant state, even though the major antioxidant enzymatic protection did not respond to the pro-oxidant challenge. We did not find strong evidence of mitochondrial involvement or major energy metabolic switches induced by OB-EVs, but a trend of reduction in seahorse assay basal respiration was observed, suggesting that OB-EVs could represent a mild metabolic challenge for osteosarcoma cells. In summary, our findings suggest that OB-EVs could serve as important means through which TME and osteosarcoma core cross-communicate. For the first time, we proved that OB-EVs reduced osteosarcoma cells' aggressiveness and viability through redox-dependent signalling pathways, even though mitochondrial dynamics and energy metabolism did not appear as processes critically needed to respond to OB-EVs.