Extracellular vesicles from endothelial progenitor cells prevent steroid-induced osteoporosis by suppressing the ferroptotic pathway in mouse osteoblasts based on bioinformatics evidence

Abstract

Abnormal antioxidative capabilities were observed in the pathogenesis of steroid-induced osteoporosis (SIOP). Ferroptosis is a recently discovered type of cell death that is characterized by the overproduction of ROS in response to GPX4 and system Xc− downregulation, which is mediated by an Fe2+ fenton reaction. However, investigations focusing on the relationship between ferroptosis and steroid-induced bone disease remain limited. In the present study, high-dose dexamethasone was used to establish a mouse SIOP model, and extracellular vesicles extracted from bone marrow-derived endothelial progenitor cells (EPC-EVs) alleviated the pathological changes in SIOP via microtomography (micro-CT), with elevations in bone volume (BV), bone surface (BS), trabecular thickness (Tb.Th), and trabecular connectivity density (Conn-D) and decreases in trabecular separation (Tb.sp) and the structure model index (SMI). Histopathological analysis, such as haematoxylin and eosin (HE) and Masson staining, showed that EPC-EVs treatment increased the volume and density of the trabecular bone and bone marrow. RNA sequencing (RNA-seq) and bioinformatics analysis revealed subcellular biological alterations upon steroid and EPC-EVs treatment. Compared with the control, high-dose dexamethasone downregulated GPX4 and system XC−, and the Kyoto Encyclopedia of Genes and Genomes (KEGG)-based gene set enrichment analysis suggested that the ferroptotic pathway was activated. In contrast, combination treatment with EPC-EVs partly reversed the KEGG-mapped changes in the ferroptotic pathway at both the gene and mRNA expression levels. In addition, alterations in ferroptotic marker expression, such as SLC3A2, SLC7A11, and GPX4, were further confirmed by RNA-seq. EPC-EVs were able to reverse dexamethasone treatment-induced alterations in cysteine and several oxidative injury markers, such as malondialdehyde (MDA), glutathione (GSH), and glutathione disulphide (GSSG) (as detected by ELISA). In conclusion, EPC-EVs prevented mouse glucocorticoid-induced osteoporosis by suppressing the ferroptotic pathway in osteoblasts, which may provide a basis for novel therapies for SIOP in humans.

miR‑483‑3p promotes the osteogenesis of human osteoblasts by�targeting Dikkopf 2 (DKK2) and the Wnt signaling pathway

“…Three different fields of vision of each section were observed using an optical microscope (cKX53, Olympus corporation) selected to calculate the mean length and thickness of the trabecula in each sample. during the decalcification process, the flexibility of all samples was evaluated daily and the hardness was assessed by needling until all samples were flexible and easily pierced by the needle (21).…”

“…All experiments were conducted in accordance with the approved guidelines. Histopathological analysis was performed on bone tissue samples using hematoxylin & eosin (H&E) staining and Masson's staining following previously described methods ( 21 ).…”

“…All bone samples were decalcified using ethylenediamine tetraacetic acid (EDTA) for 3-5 days following 4% formalin fixation and were then subjected to paraffin embedding, sectioning H&E staining, and Masson's staining (Beyotime Institute of Biotechnology, Inc.) according to previously described methods ( 21 ). Three different fields of vision of each section were observed using an optical microscope (CKX53, Olympus Corporation) selected to calculate the mean length and thickness of the trabecula in each sample.…”

The Role of Extracellular Vesicles (EVs) in the Epigenetic Regulation of Bone Metabolism and Osteoporosis

“…Finally, peripheral blood mononuclear cells transfer via EVs the chemokine receptor CCR5 to endothelial cells [112]. Endothelial precursors have been described to secrete EVs able to attenuate steroid-induced osteoblast apoptosis and autophagy, being able to upregulate glutathione peroxidase 4, system Xc − , and cysteine levels while reducing malondialdehyde and reactive oxygen species production [113].…”

Extracellular Vesicles in Cell Biology and Medicine

“…For the treatment of immune-related diseases, the application of an inflammatory stimulus is shown to improve the anti-inflammatory and/or immunosuppressive potential of EVs secreted by adipose mesenchymal stem cells 5 . Cytoprotection of stressed cardiomyocytes through the use of EVs derived from mesenchymal stromal cells 6 , and the prevention of glucocorticoid-induced osteoporosis -through the suppression of osteoblasts' ferroptic pathway -by EVs extracted from bone marrow-derived endothelial progenitor 7 , were also demonstrated. As well as applying naturally occurring EVs, one of the published original Articles has demonstrated engineered EVs as a feasible therapeutic tool.…”