| Objective: Arterial calcification is an actively regulated process similar to bone formation,and its cytological basis is the differentiation of vascular smooth muscle cells（VSMCs）to osteoblast-like cells,which is manifested by an increased expression of osteogenesis-related indicators.Recent studies have revealed that osteoblast exosomes（OC-Exos）can be involved in regulating osteoblast function.In this study,we isolated mouse mesenteric VSMCs and established an in vitro cell model of arterial calcification,intervened in calcified VSMCs with OC-Exos,detected the expression of calcification-related molecules,observed the effect of OC-Exos on osteoblastic differentiation of VSMCs,and sought new strategies to prevent and treat arterial calcification.Methods: The experiment had three stages.Phase Ⅰ: Isolation and identification of VSMCs from mice and establishment of arterial calcification cell modelThe VSMCs were isolated,and the expression of α-SMA was detected by cellular immunofluorescence（IF）to identify VSMCs.10 m M sodium β-glycerophosphate（β-GP）was used to interfere with VSMCs to establish the arterial calcification cell model,and the expression of related calcification molecules Runx2 and BMP-2 was detected by Western blot,and calcified nodules were observed by alizarin red staining.The formation of calcified nodules was observed by western blot.Phase Ⅱ: Induction of OCs differentiation and extraction of ExosRAW264.7（mouse mononuclear macrophages）were purchased and induced to differentiate into osteoclasts using macrophage colony-stimulating factor（M-CSF）and nuclear factor-κB receptor-activating factor ligand（RANKL）,and OCs were identified by TRAP staining and ghost pen cyclic peptide staining.OCs supernatant cultures were collected and Exos extraction was performed using ultracentrifugation,the diameter size was observed by electron microscopy,and the expression of Exos-specific transmembrane protein molecules CD63 was detected by immunoblotting（WB）.Phase Ⅲ: Observation of the effect of OC-Exos on osteoblast-like differentiation of VSMCsFirst,four subgroups were set:（1）blank group,（2）calcification group,（3）calcification + OC-Exos group,（4）calcification +RAW264.7-Exos group: to observe the effect of Exos secreted by RAW264.7,which was not differentiated into osteoclasts,on osteogenic differentiation of VSMCs.Groups（1）（2）（3）were induced with β-GP,and VSMCs were collected after 7 days.Group（1）VSMCs were cultured in complete medium without β-GP for 7 days,and cells were collected to extract total cellular proteins,and the expression of calcification-related signaling molecules Runx2 and BMP-2 were detected using Western blot.Results:1.Primary VSMCs from mice were successfully obtained,and IF results showed positive expression of α-SMA;WB and alizarin red staining results showed that β-GP significantly increased the expression of Runx2,BMP-2 and the formation of mineralized nodules.2.The results of TRAP staining and ghost pen cyclic peptide staining showed that intervention on mice mononuclear macrophages RAW264.7 cells with M-CSF and RANKL appeared more than3-nucleated cells,mature osteoclasts,after 2 days,and the number of osteoclasts gradually increased with the extension of induction time,and the highest number of mature osteoclasts was observed at day 4.3.Cell supernatant was collected and osteoclast exosomes were extracted.40-50 nm vesicular structure of exosomes could be observed under electron microscope,and WB results showed positive expression of exosome-specific marker molecule CD63.4.Osteoclast exosomes significantly reduced the expression of Runx2 and BMP-2 in VSMCs.Conclusion: Osteoclast exosomes exerted an inhibitory effect on osteoblast-like differentiation of VSMCs,which provides a new therapeutic direction for exogenous factors to improve arterial calcification. |
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