Role Of Perivascular Adipose Tissue-derived Let-7b In Vascular Inflammation Via ADRB3

Background and Objective: Vascular injury caused by hypertension is a chronic low-grade inflammation process,especially a growing number of studies have shown that inflammatory response in vascular adventitial not only involved in hypertension,but also involved in atherosclerosis and vascular restenosis.As the important composition of outer membrane,perivascular adipose tissue secretes a variety of biological active substances involved in regulation of vascular pathophysiology function.Our previous study found that perivascular adipose tissue secretes inflammatory factors involved in vascular remodeling in DOCA-salt hypertension model.And the expression of inflammatory factor is regulated by many factors,including at the transcription and post-transcription level.MicroRNAs are newly discovered regulatory factors.MicroRNAs in peripheral adipose tissue,however,whether to participate in the regulation of vascular inflammation is still not clear.In this study,we try to seek microRNAs in perivascular adipose tissue associated eith inflammation in the DOCA-salt mice.Methods: 1.Establish animal model.DOCA-salt hypertensive mice with uninephrectomy were buried with DOCA pump(50mg)subcutaneously.Blood pressure were measured before and after DOCA-salt treatment by tail cuff method.The structure and inflammation phenotype were stained by H&E or immunohistochemical staining method.2.The miRNA expression profile were measured by chip and then we validated miRNAs of interest by LNA-qRT-PCR.3.The interaction between miRNA and its targeted genes were examined by dual luciferase report gene experiment and verified the relationship between miRNA and the target gene by gain or loss of miRNA by PCR,western blot.4.The expression of target genes detected by western blot and immunofluorescence staining and study its effect on inflammatory factor at a cellular level.Results: 1)DOCA-salt caused a significant increase in blood pressure and mice in this group had thickened thoracic aorta vascular wall,smaller cell size in PVAT,increased proinflammatory cytokines OPN protein and macrophage marker F4/80.These suggests that inflammation occured in perivascular adipose tissue which structural change.2)Compared with control group,microRNAs expression profile of DOCA-salt mice among PVAT,mesenteric adipose tissue(MAT)and aorta were different.After Venn diagram and signal value analysis,we selected let-7b as candidates and validated by LNA-qRT-PCR.let-7b decreased significantly up to 1.7 fold in DOCA-salt PVAT.3)Through target genes predicting for let-7b and related analysis,ADRB3 may be involved in vascular inflammation process.4)ADRB3 was a target of let-7b which was validated by dual luciferase reporter gene system and by gain or loss of let-7b expression.Furthermore,compared with control group,let-7b in DOCA-salt PVAT was significantly reduced while ADRB3 protein level increased significantly.5)Angiotensin II caused increasing of ADRB3 protein and OPN in adipocytes while let-7b decreased slightly..The ADRB3 selective agonist CL316243 had similar effect.However,let-7b antagonists significantly increased ADRB3 protein levels,but the expression of OPN was not increased obviously.These suggested that activating ADRB3 can increase OPN level.Conclusion: Let-7b in perivascular adipose tissue can regulate ADRB3 protein expression,and activating ADRB3 caused the expression of proinflammatory factor OPN.These suggested that let-7b may indirectly involved in vascular adventitia inflammation process by adjusting ADRB3,providing a theoretical basis for new therapeutic targets in cardiovascular disease research.