Effects Of Exosomes Secreted By Adventitial Fibroblasts Induced By High Phosphorus On Vascular Calcification

Aims:Vascular calcification often occurs in patients with chronic renal failure,which significantly increases the incidence of cardiovascular events and seriously affects the prognosis of patients.Due to the impairment of renal excretion of phosphate,the patients with chronic renal failure are in a state of severe hyperphosphatemia.High phosphorus is closely related to vascular calcification,which is mainly manifested by promoting the differentiation of vascular smooth muscle cells（VSMCs）into osteoblast-like cells through different mechanisms.Previous studies mainly focused on the direct calcification promoting effect of high phosphorus on VSMCs,recent studies have shown that exosomes secreted by adventitial fibroblasts（AFs）can directly affect the function of VSMCs.This study aims to investigate the effects of exosomes secreted by high phosphorus-induced AFs on the calcification of VSMCs and vascular calcification and its mechanism involved.Methods:In vitro,3.5 m M inorganic phosphorus（HPi）was used to simulate the high phosphorus state in vivo,and 0.9 m M inorganic phosphorus（NPi）was used as control.Exosomes（Exos）in the supernatant of AFs were extracted by the differential centrifugation method.Exosomes were identified by transmission electron microscopy,nanoparticle size analysis,and western blot.Western blot was used to detect the expressions of Runx2 and BMP2 in VSMCs,Alkaline phosphatase（ALP）activity assay kit and ALP staining kit were used to detect the activity of ALP,Alizarin red staining was used to analyze the formation of mineralized nodules in VSMCs.Through literature review,we selected eight miRNAs that are highly expressed in AFs and related to osteogenic differentiation,and then used quantitative real-time polymerase chain reaction（q RT-PCR）technology was used to detect the expression of miRNAs in exosomes and cells.The effects of exosomes secreted by AFs overexpressing or downexpressing miR-21-5p on the calcification of VSMCs were investigated by transfecting miR-21-5p mimics,miR-21-5p inhibitor and their controls into AFs.The potential target genes of miR-21-5p were predicted by bioinformatics techniques,and the targeting relationship between miR-21-5p and cysteine-rich motor neuron1 protein（Crim1）was verified by luciferase reporter assay.The expression of Crim1 in VSMCs was silenced by si RNA transfection technology to study the effects of Crim1 on the calcification of VSMCs.We used spectral imaging instrument to detect vascular uptake of exosomes in mice.The 5/6 nephrectomy with high phosphorus diet were used to establish the mouse model of chronic renal failure with vascular calcification,exosomes secreted by AFs were intravenously injected into the mice to observe the effects of AFs secreted exosomes on vascular calcification in mice with chronic renal failure.Results:The exosomes isolated from the supernatant of AFs were typical cup-shaped or ellipsoidal structures with a diameter of 50-150 nm,expressed exosomal surface markers TSG101,CD9 and CD81,and could be taken up by VSMCs.The supernatant of HPi treated AFs(AFs ^HPi-CM)promoted the calcification of VSMCs,which was partially blocked by the exosomes biogenesis/release inhibitor GW4869.Exosomes secreted by HPi-induced AFs(AFs ^HPi-Exos)can significantly promote the expression of Runx2,BMP2,increase ALP activity and the formation of mineralized nodules in VSMCs.miR-21-5p was enriched in AFs ^HPi-Exos,and exosomes secreted by AFs with overexpression of miR-21-5p significantly enhanced osteoblast-like differentiation of VSMCs,while exosomes secreted by AFs with downexpression of miR-21-5p antagonized high phosphorus-induced osteoblast-like differentiation of VSMCs.There is a direct targeting relationship between miR-21-5p and Crim1 gene,and silencing the expression of Crim1 can promote the calcification of VSMCs.Exosomes secreted by AFs can be taken up by mouse thoracic aorta,and AFs ^HPi-Exos can significantly promote vascular calcification in chronic renal failure mice.Conclusions:miR-21-5p is enriched in exosomes secreted by high phosphorus-induced AFs,and miR-21-5p promoted the calcification of VSMCs and vascular calcification by inhibiting the expression of the downstream target gene Crim1.