Bone Marrow Mesenchymal Stem Cell-derived Exosomes On Calcification Of Vascular Smooth Muscle Cells

Background: Vascular calcification is one of the common pathological phenomena of chronic kidney disease(CKD),which is closely related to the occurrence of cardiovascular events in chronic kidney disease.In patients with chronic kidney disease,there is a disorder of calcium and phosphorus metabolism,uremic toxins are significantly increased,and vascular smooth muscle cells(VSMCs)are induced to transform to an osteogenic phenotype through mechanisms such as inflammation and oxidative stress,which are considered to be calcification of the vascular media The main cellular mechanism.In recent years,bone mesenchymal stem cells(BMSCs)and their derived exosomes have made significant progress in the treatment of cardiovascular diseases.BMSCs are the earliest non-hematopoietic stem cells isolated from bone marrow and are considered to be the precursor cells of bone marrow stromal cells.BMSCs grow adherently under standard culture conditions,have multiple differentiation potentials,and can differentiate into chondrocytes,adipocytes,and osteoblasts.In the past decade,it has become a promising therapeutic tool for the treatment of many diseases.Conditioned medium from BMSCs is a well-known rich source of autologous cytokines and is commonly used in current clinical medicine for tissue regeneration,which can improve myocardium heart function after infarction,increase ALP activity,and specific osteogenic markers such as RUNX2 and Msx2.Exosomes are tiny membrane vesicles that can be secreted by most cells,with a lipid bilayer membrane structure with a diameter of about 40-150 nm.It is formed by the invagination of the cell membrane to form endosomes,and then the endosomal membrane invades again and wraps various signaling molecules in the cell,such as proteins,lipids,DNA,and various RNAs(mRNA、miRNA、lncRNA)to form multiple vesicles Exosomes,the membrane-like structures released by the way of exocytosis by the combination of multivesicular bodies and cell membranes are exosomes.The formed exosomes can either stay around the exosome-producing cells,or they can release the signal molecules wrapped by them to the recipient cells,and then affect the function of the recipient cells through autocrine,paracrine,or hormone-like secretion.In the early days,exosomes were considered to be just a kind of cell waste,but now exosomes are widely studied in the prevention,diagnosis,and treatment of tumors,immunity,and lots of diseases.The number of exosomes produced by BMSCs is higher than other cell types,such as myoblasts,acute monocytic leukemia cells,or embryonic kidney cells.The molecules carried by the exosomes produced by BMSCs in different growth periods and under different induction conditions are different,and they play a vital role in regulating the osteogenic differentiation of VSMCs.When exosomes fuse with the receptor cell membrane,they are considered to be an important carrier for cell-to-cell communication.Under different intervention conditions,the expression profile of the contents of exosomes will change to a certain extent.It is a controllable and non-cell-dependent drug carrier.Compared with young rats,BMSCS-derived exosomes from old rats can induce insulin resistance in normal rats.Therefore,we speculated that BMSCS-derived exosomes of different ages may also have different effects on VSMCs.Based on the above research background,this topic will explore the effect and theory of exosomes derived from bone marrow mesenchymal stem cells(BMSCs-Exos)of rats of different ages on VSMCs calcification.Objective: To investigate the role of exosomes derived from BMSCs in rats of different ages in regulating the calcification of vascular smooth muscle cells by affecting the expression of Sp1.Methods: 1.Use differential adhesion method to isolate and culture primary SD rat BMSCs,collect BMSCs-derived exosomes by ultracentrifugation,observe the morphological characteristics of exosomes by transmission electron microscope,and detect the expression of exosomal marker proteins CD9 and Tsg101 by Western blotting.Nano-particle-tracking Analysis(NTA)analyzes the size and distribution of exosomes.2.Induce calcification of VSMCs by using a complete medium containing and without 10mmol/L β-glycerophosphate(β-GP).The experimental cells are divided into β-GP group and blank control group,and cultured for 6 days and 9days respectively,14 days.Alizarin Red S staining was used to detective the formation of calcium nodules to determine whether the in vitro vascular smooth muscle cell calcification model was successfully established.RT-qPCR and Western Blot were used to detect Sp1,RUNX2,and αSMA mRNA expression;Western blotting was used to detect Sp1,RUNX2,and αSMA protein expression levels.3.Culture the BMSCs in serum-free medium for 72 hours,and collect the BMSCs condition medium(CM)of young rats(3 months old)and old rats(18months old)respectively,denoted as Young-BMSCs-CM and Aged-BMSCs-CM;Exosomes(Exos)in the two groups of BMSCs-CM were collected by ultracentrifugation,denoted as Young-Exos,Aged-Exos.Divide VSMCs into blank control group,β-glycerophosphate sodium(β-GP)group,β-GP + young rat BMSCs-Exos(Young-Exos)group and β-GP + old rat BMSCs-Exos(Aged-Exos)group.Except for the blank control group,the other 3 groups were cultured with a complete medium containing 10 mmol/L β-GP for 14 days to induce calcification of VSMCs.Alizarin red S staining method was used to detect calcification in each group.The RT-qPCR method was used to observe Sp1,RUNX2,and αSMA mRNA;the Western blotting method was used to observe the protein expression of Sp1,RUNX2,and αSMA.4.The male SD rats were randomly divided into a calcification group and a control group,with 10 rats in each group.The calcification group was given highdose vitamin D3(600,000 U/kg/day,for 6 consecutive days)subcutaneously to induce calcification of the thoracic aorta in rats,and an in vivo model of the calcification of the thoracic aorta in rats was established.The negative control group was used the same amount of normal saline.The rats were fed normally during the subcutaneous injection intervention,and they were free to eat and drink.They were sacrificed 3 days after the intervention.The rat thoracic aorta tissue was separated,and part of it was fixed in 4% paraformaldehyde for making pathological sections,and the remaining part was stored in RNA protection solution and then stored in liquid nitrogen for nucleic acid and protein detection.After paraffin embedding,the sections were sliced,and the calcium salt silver nitrate staining method(von kossa staining)and hematoxylin-eosin staining method(HE staining)were used to observe the pathological structure changes and calcium salt deposition of the rat’s thoracic main tissue to verify the size Establishment of a model of thoracic aorta calcification in mice.Immunohistochemistry and RT-qPCR were used to detect the expression of Sp1,RUNX2,and αSMA genes in the calcified thoracic aorta to explore the mechanism of vascular calcification in vivo and in vitro.Results: 1.The BMSCs of primary male SD rats showed adherent growth,large volume,and polygonal shape,and most of the cells had long protrusions and were connected.The positive rate of BMSCs marker protein CD34 and CD45 expression was <5%,and the positive rate of marker protein CD44 and CD90 expression was >95%.Electron microscope observation of BMSCs exosomes showed typical saucer-type characteristics.Western blotting detected the expression of exosomal marker proteins CD9 and Tsg101.Zeta View particle concentration and diameter analysis showed that the original concentration of the tested vesicles was 5.2×108 Particles/mL,and the particle size Located between30～200nm.2.The rat AC model was successfully established.Compared with the aortic tissue of the control group,the normal structure of the arteries in the calcification group was destroyed,a large number of collagen fibers and elastic fibers were arranged disorderly,and there was obvious breakage and degradation.A large amount of black calcium salt deposits can be seen;RT-qPCR detection of Sp1 and RUNX2 expression was significantly increased in the calcification group,whileαSMA gene was significantly decreased in the calcification group,and the difference was statistically significant.Immunohistochemical staining showed that,compared with the control group,the staining of Sp1 and RUNX2 in the calcification group was significantly deeper and the expression level was higher,while the color of αSMA was lighter and the expression level was lower.By adjusting the background and calculating the density analysis per unit area,the differences in expression levels are statistically significant.3.β-GP successfully induced the calcification of VSMCs.Compared with the blank control group,the VSMCsβ-GP group showed obvious orange-red calcium salt nodules,and as β-GP induced VSMCs calcification time,on the 6th,9th day,On the 14 th day,the calcium nodules increased significantly.The more orange-red calcium nodules,the more obvious the color.Rat VSMCs appear osteogenic differentiation.In the in vitro model,the expression of RUNX2 is significantly increased at both the gene level and the protein level,and the gene water and protein expression of Sp1 is also significantly increased,and the difference is statistically significant.4.After intervention with BMSCs-Exos,the number of calcium nodules in the β-GP+Young-Exos group was significantly reduced compared with the β-GP group and β-GP+Aged-Exos group,and the orange-red color was lighter.Compared with the blank control group,the expression of Sp1 and RUNX2 protein in the β-GP group increased,and the expression of αSMA protein decreased.The difference between the two groups was statistically significant;compared with the β-GP group,the Sp1 and RUNX2 protein in the β-GP+YoungExos group The expression decreased,and the expression of αSMA protein increased,and the difference was statistically significant,but there was no significant difference between the β-GP+Aged-Exos group and the β-GP group(P>0.05).Conclusion: The results of this experiment suggest that high-dose vitamin D3 and β-GP can establish in vitro and in vitro calcification models in rats and promote osteogenic differentiation of VSMCs.Sp1 is up-regulated in calcified VSMCs and tissues.The effects of BMSCs-Exos on arterial calcification in rats of different ages are different.Among them,Young-Exos intervention can reduce VSMCs calcification.The mechanism may be related to the down-regulation of Sp1 and RUNX2 expression and up-regulation of αSMA expression.