The Primary Research On Mechanism Of Epicardial Adipose Tissue-Derived Exosomes Regulating Medial Arterial Calcification In Diabetes Mellitus

Aim:the medial arterial calcification is a severe cardiovascular complications for diabetic mellitus patients which can cause vascular dysfunction.Fundamental studies have demonstrated that osteogenic trans-differentiation of vascular smooth muscle cell(VSMC)in arterial wall micro-enviroment is the key process in medial arterial calcification.Epicardial adipose tissue(EAT),the adipose tissue located between the myocardium and the visceral pericardium,is a unique and multifaceted fat depot with local and systemic effects.The functional and anatomic proximity of EAT to the myocardium and coronary arteries enables endocrine,paracrine,and vasocrine effects on the heart and coronary arteries in physiological or pathophysiological condition.So EAT displays a large secretosome,which regulates physiological and pathological processes in the heart.Previous studies have indicated that epicardial adipose tissue regulate the atherosclerosis and arterial calcification via adipokine or exosome.And a clinical sectional-cross study indicated that EAT volume was independently associated with CAC in diabetes mellitus patients.These sparks us that epicardial adipose tissue may also exert effects on the medial arterial calcification mediated by exosomes which are important messengers to deliver information between cells and tissues.Considered the complicated origin of epicardial adipose tissue-derived exosomes and potential multiple mechanism participating in the medial arterial calcification,we firstly explored whether the adipose-derived mesenchymal stem cell(ADSC)plays a role in medial arterial calcification.By isolation of exosomes secreted by high glucose induced-/normal glucose induced-ADSC,we demonstrated the mechanism of high glucose induced-ADSC-derived exosomes(HG-ADSC Exo)regulating the osteogenic trans-differentiation of VSMC in vitro.Moreover,we harvested the EAT from diabetic/non-diabetic patients and EAT were cultured in vitro to isolate the exosomes from their supernatants.We also primarily investigated the EAT-derived exosomes whose regulation of osteogenic trans-differentiation of VSMC.Finally,we observed the transportation and location of ADSC Exo in vivo via the living imaging technology to identify the mechanism of ADSC Exo regulating the medial arterial calcification.Methods:(1)We used the 25m M high concentration of glucose(HG)complete medium and 5m M normal concentration of glucose(NG)complete medium to mimic the hyperglycemia in diabetes mellitus patients and normal blood glucose respectively.And we isolated the diabetic/non-diabetic patients’EAT into complete medium in vitro under the permission of the Ethnics Committee of the Second Xiang-Ya hospital,Central South University.By the differential centrifugation method,we successfully isolated the four types of cell/tissue-derived exosomes:(1)high glucose-stimulated exosomes(HG-ADSC Exo)from adipose-derived mesenchymal stem cells(ADSC),(2)normal glucose-stimulated exosomes(NG-ADSC Exo)from adipose-derived mesenchymal stem cells(ADSC),(3)DM~+-EAT-derived Exosomes,and(4)DM~--EAT-derived Exosomes.To authenticate whether the isolated extracellular vesicles were exosomes,we used the transmission electron microscopy,and nanoparticle size analysis.The Runx2 and BMP2protein expression were determined by Western blot to evaluate the pro-calcific effects of exosomes on VSMC in vitro.(2)Real-time fluorescent quantitative PCR was used to evaluate the relative expression level of miRNAs which were selected by previous studies:hsa-miR-214-3p;miR-153-3p;miR-100-5p;miR-133a-3p;miR-125b-3p;miR-204-5p.The potential target gene of miR-133a-3p was predicated by bio-informatics analysis.(3)Through the in vivo imaging technology for mice,we identified the the entering process and location of Di R-labeled Exo through the vein injection.Results:(1)Exosomes isolated from the supernatant of adipose tissue-derived mesenchymal cells culture media has a cup-shaped and three-dimensional structure with a diameter of 50-100nm.(2)Treatment with HG-ADSC Exo in VSMC resulted in decreased Runx2 and BMP2 expression.Exosomes isolated from the supernatant of DM~+/DM~-patients’EAT culture media through the exactly same method as the harvest of ADSC Exo,treatment with DM~+/DM~--EAT Exo in VSMC both resulted in increased Runx2 expression compared to the blank group.(3)Moreover,the q RT-PCR shows that miR-133a-3p is up-regulated in HG-ADSC Exo.Based on the bio-information database:Targetscan,Starbase and database for target gene associated with vascular calcification:Gene portals,Genecard,we selected the Rho A gene as the potential target gene of miR-133a-3p.(4)In vivo imaging for mice showed that transportation of Di R-labeled ADSC Exo into mice thoracic aorta.Conclusions:(1)High glucose-stimulated exosomes(HG-ADSC Exo)from adipose-derived mesenchymal stem cells inhibits the osteogenic trans-differentiation of VSMC.(2)HG-ADSC Exosomal miR-133a-3p is up-regulated and the Rho A gene as its predicted potential target gene.(3)There is no obvious difference of pro-calcific effects on VSMC between DM~+-EAT-derived exosomes and DM~--EAT-derived exosomes.The mechanism of epicardial adipose tissue-derived exosomes regulating medial arterial calcification in diabetes mellitus is still not elucidated,and more studies on EAT-derived exosomes in medial arterial calcification will be needed.