Imaging Evaluation And Mechanism Of Adipose Tissue Exosomes Regulating Vascular Function In Mice

Background Obesity is an extraordinary prevalent public health problem.Adverse metabolic effects of obesity contribute to the increased risk of coronary heart disease,peripheral vascular disease,stroke and type 2 diabetes.It is estimated that obesity causes 4million deaths worldwide,of which more than two-thirds are associated with cardiovascular diseases（CVDs）.The primary manifestation of obesity is excessive accumulation of adipose tissue.Among them,the increase of visceral adipose tissue is the most critical hazard of obesity,which is closely related to the occurrence and development of cardiovascular disease.The main mechanisms of CVDs induced by obesity are sympathetic nervous system dysfunction,impaired renal and adrenal function,and insulin resistance.Recently,dysregulated adipokines,which are cytokines or growth factors by secreted adipose tissue,are also found to be involved.Adipose tissue produces a variety of adipokines,including leptin and adiponectin,regulating multiple organ functions⁹.Obese adipose tissue had larger volume,stronger inflammatory stimulus,and altered adipokines¹⁰.Decoding the connections between the changed adipokines profile and vascular injury would shed light on how obesity increases the risk of CVDs.Recent studies have shown that exosomes can be internalized in a highly cell type specific manner through the recognition of exosomes surface molecules.In addition to special molecules,the formation of protein crown may be also involved in escaping macrophage clearance and selective delivery into target cells.During circulating in the body,the surface of the nanoparticles（including exosomes）will quickly adsorb protein molecules to form protein crowns,which affect and regulate the behavior and final fate of nanoparticles in vivo.In this study,we found that exosomes from obese visceral adipose tissue is an important causing factor for endothelial dysfunction.Mechanistically,the changed miRNAs encapsulated in the exosomes and the Apo B crown together induce the inflammation in the endothelium,and blocking the process holds great promise for prevention and therapy of obesity associated endothelial dysfunction.Our research explained the mechanism of how obesity causes vascular damage and provide novel insights for the diagnosis,prevention,and treatment of the obesity related vascular diseases.Methods and results1.Declined vascular function in obese miceAfter 12 weeks of high-fat feeding,the mice generally had a 20%higher body weight in comparison with the normal chow diet fed mice,suggesting that high fat dieting successfully induced obesity in mouse model.Ultrasound examination showed that the PWV value in the obese mice was much higher than that of lean mice.Notably,the aortic contraction function did not change significantly.HE staining of aortic artery showed significant increased thickness of the blood vessel in obese mice.DHE staining showed that the ROS generation was also much higher in the blood vessels in obese mice.All the above results indicated that the blood vessel function was damaged in obese mouse model.2.Visceral adipose tissue derived exosomes impair vascular function in obesityIn order to explore whether exosome-mediated intercellular communication participate in this process,we treated the mice with exosome biogenesis inhibitor and examined the vascular function.As expected,GW4869 treatment had no significant effects on PWV and the systolic function of the aorta in the NCD control mice.In contrast,GW4869 treatment significantly reversed the increased PWV in the HFD mice,suggesting that exosomes might be involved in obesity related vascular dysfunction.To further explore whether exosomes derived from visceral adipose tissue participate in this process,we thus isolated exosomes from the visceral adipose tissue as described before.Transmission electron microscopy,particle size analysis and western blotting assay confirmed the identity of exosomes.Both the exosomes from the visceral adipose tissue of lean(Exo^lean)and obese(Exo^obese)mice had similar in vivo distribution profile,mainly in the liver and spleen.In contrast,there were more exosomes distributed in the aorta.In vitro cellular study also revealed that there were more Exo^obeseendocytosed by the HUVECs.We next injected 100μg of Exo^leanor Exo^obeseinto lean mice three times a week,with the mice receiving no treatments as the control.After 4 weeks of treatment,the PWV of the mice in the group injected with Exo^obeseincreased significantly,while no significant change was found in aortic systolic function.HE staining and DHE staining indicated that Exo^obesetreatment increased the vascular wall thickness and increased the content of ROS.Consistent with the in vivo data,Exo^obesetreatment also increased the ROS in the cultured HUVEC cells.Moreover,Western blotting analysis further showed that Exo^obesetreatment increased iNOS expression while decreased e NOS expression in HUVECs.3.Altered miRNAs in the exosomes contribute to the detrimental functionWe next profiled miRNA abundance in the exosomes from the visceral adipose tissue.As expected,there were dozens of miRNAs significantly change in exosomes from obese adipose.For example,miR-206 and miR-146a were found decreased,while miR-23a was found increased,all of which have been found significantly different and closely related to inflammation regulation.To confirm the effects of miR-206,miR-146a,and miR-23a in the exosomes,miR-206 and miR-146a mimics,or miR-23a inhibitors were electroporated into exosomes Consistent with previous findings,exosome based delivery of miR-206 and miR-146a mimics,or miR-23a inhibitors significantly rescued the expression of e NOS while decreased i NOS expression.In the following experiment,we engineered the intervention exosomes by electroporation of miR-206,miR-146a mimics,and miR-23a inhibitors at 1:1:1 ratio.After four weeks of continuous treatments in HFD induced obese mice,Exo ^Interventionsignificantly increased the expression of miR-206 and miR-146a,and decreased miR-23a expression in the aorta.Accordingly,Exo ^Interventiontreatment significantly reduced PWV while didn’t change the aortic systolic function.In addition,Exo ^Interventiontreatment reversed the thickened blood vessel,and reduced the ROS level in the obese mice.4.Apo B crown facilitates the exosomes endocytosed by HUVEC cells and activates inflammationWe also profiled the proteomic differences of exosomes from obese visceral adipose tissue and muscle.Notably,the exosomes from visceral adipose tissue were rich in lipoproteins,such as apo B,suggesting that Apo B containing LDL might interact with exosomes and form a crown like structure.Thus,DiO-labeled exosomes were co-cultured with DiI-labeled highly oxidized low-density lipoprotein.Transmission electron microscopy found that the LDL incorporated on the exosomes,forming a crown like structure.Moreover,the exosome/LDL hybrid could facilitate the exosomes entry into HUVEC cells,as revealed by either fluorescence examination or cel-miR-54 tracking.At the same time,the exosome/LDL hybrid increased ROS generation and induced inflammatory gene expression in HUVEC cells.All of the above data suggest that the ApoB crown like structure,or the LDL/exosome hybrid might be involved in the vascular dysfunction in obesity.Conclusions1.We here have identified that exosomes from obese visceral adipose tissue is an important causing factor for vascular dysfunction,the main ultrasonic manifestation is the acceleration of PWV.2.Mechanistically,the changed miRNAs encapsulated in the exosomes and the ApoB crown together induce the inflammation in the endothelium,and blocking the process holds great promise for prevention and therapy of obesity associated endothelial dysfunction.