Effects Of Endothelial Exosomes Stimulated By Oxidized Low Density Lipoprotein On Osteoblastic Differentiation Of Smooth Muscle Cells

Objective:Arterial calcification refers to the deposition of calcium,phosphorus and other minerals in the medial side of the vascular wall,which is an active regulatory process similar to osteogenesis.Osteoblast-like cells of vascular smooth muscle cells(VSMCs)differentiate into their characteristic lesions.Previous studies have shown that endothelial cells(ECs),which can directly contact the risk factors of arterial calcification such as high glucose,high fat and high phosphorus in the blood,can participate in the osteogenic differentiation of VSMCs through paracrine action.In this study,ECs were treated with oxidized low density lipoprotein(ox-LDL)of different concentrations to detect the functional changes of ECs,and then 100 μg/m L ox-LDL interferes with ECs,extracts the supernatant and exosomes(Exos)to culture VSMCs,and observes the calcification of VSMCs.Finally,the ECs are sequenced to observe the differential expression of m RNA.The purpose of this study is to study the effect and mechanism of exos secreted by ox-LDL interfering ECs on the osteogenic differentiation of VSMCs,so as to further clarify the mechanism of coronary calcification.Methods: The experiment had three stages.Phase I: Culture of mouse aorta ECs and VSMCsExtracting aortic ECs using tissue block attachment method failed to pass through after extraction.Purchase ECs and VCMCs cell lines for culture and passage for subsequent experiments.Phase II: ox-LDL interferes with ECs and extracts supernatant and secretionIntervention of ECs with different concentrations of ox-LDL and detection of cell proliferation,migration,and tubular function were conducted.Finally,100mg/L ox-LDL was determined to complete the subsequent experiment.ECs were cultured to 70% and divided into two groups.The blank group was further cultured with high sugar DMEM,while the high fat group was further cultured with high sugar DMEM containing 100mg/L ox-LDL.After 48 hours,the supernatant and Exos were extracted and identified using electron microscopy,particle size,and Western blot(WB).Phase III: To observe the role of EC-supernatant and EC-Exos in VSMC.EC-supernatant intervention was divided into 4 groups:(1)control group: high glucose DMEM;(2)Blank supernatant group: blank ECs-supernatant;(3)High fat group: high sugar DMEM containing100mg/L ox-LDL;(4)High-fat supernatant group: high-fat group ECs-supernatant;Each group was added with the same volume of complete medium as the intervention,and VSMCs were cultured after mixing.EC-Exos intervention was divided into three groups:(1)control group cultured VSMCs in complete medium;(2)Blank Exos group contains 100μg/m L blank EC-Exos complete medium culture VSMCs;(3)Fat Exos group contains 100μg/m L fat EC-Exos complete medium culture VSMCs.24 hours later,VSMCs were collected,total RNA was extracted,and the expression levels of Runt-related transcription factor 2(Runx2)and Bone morphogenetic protein2(BMP2)in VSMCs of calcification groups were detected by real-time fluorescence quantitative polymerase chain reaction(RT-q PCR);After 96 hours,VSMCs were dissociated to obtain protein.The expression of Runx2 and BMP2 in VSMCs was detected by WB method.Phase IV: Detect the differential expression of blank ECs and high-fat ECs m RNACollect blank ECs and high-fat ECs in the second stage for m RNA sequencing,screen out differentially expressed m RNA,and conduct bioinformatics analysis.Results:1.The primary ECs of mice were extracted,but the cells died after passage.Purchase ECs and VSMCs cell lines to complete the follow-up experiment.2.Ox-LDL has a concentration dependent promoting effect on the proliferation,migration,and tubular ability of ECs.100mg/L ox-LDL was selected for subsequent experiments.3.Collect the blank ECs-supernatant and high-fat ECs-supernatant,extract the exocrine body by ultracentrifugation,and observe the double-layer membrane structure under the electron microscope,which is round and cup-shaped;The particle size test results showed that the average particle size of blank ECs-Exo was 122.4 nm,and the average particle size of high-fat ECs-Exo was 126.6 nm;WB results showed that CD63 and CD9 in the two groups were positive.4.The RNA and protein of blank ECs-supernatant and high-fat ECs-supernatant intervened VSMCs were extracted.Using RT-q PCR technique,we confirmed that the expression of BMP2 and Runx2 in VSMCs was significantly increased with EC-supernatant intervention in high-fat and 100μg/m L ox-LDLgroup than in VSMCs with EC-supernatant intervention in blank group;WB found that compared with the normal control group,the relative expressions of BMP2 and Runx2 in the hyperlipidemic group and 100 μ g/m Lox-LDL treated VSMCs were significantly higher than those in the normal control group.5.RNA and protein of blank ECs-Exos and high-fat ECs-Exos intervened VSMCs were extracted.Using RT-q PCR technique,we confirmed that the expression of BMP2 and Runx2 in EC-Exos intervened VSMCs in high-fat group were significantly higher than those in blank group;WB results showed that the relative expression of BMP2 and Runx2 in EC-Exos intervened VSMCs in high-fat group was significantly higher than that in EC-Exos intervened VSMCs in blank group.6.The m RNA sequencing results of blank ECs and high fat ECs suggest that compared with the control group ECs,14522 genes were selected from the high fat group ECs,of these genes,424 were significantly different,while 191 of these genes were elevated and 233 were decreased.GO expression profile data show that the change of the gene expression is closely related to the body’s defense response,cholesterol synthesis and innate immune response.KEGG mainly enriched signal pathways such as tumor necrosis factor and adhesion.Conclusion: The Exos of endothelial cells stimulated by ox-LDL promotes the osteogenic differentiation of smooth muscle cells.