Molecular Mechanism Of Endothelial Cell Metabolic Memory Leading To Cardiovascular Dysfunction In Diabetes

Diabetes mellitus(DM)is a chronic metabolic disease related to the continuous increase of blood glucose level,and the total number of diabetes patients in China has reached 130 million,ranking first in the world.A series of complications such as retinopathy and cardiovascular disease caused by long-term hyperglycemia have high disability and mortality rates.Studies have shown that more than 50% of diabetic patients die of cardiovascular disease,indicating that cardiovascular complications have become the main cause of high mortality in patients with DM.More importantly,increasing clinical studies have found that the long-term damage left by high-glucose “metabolic memory” cannot be completely reversed after the blood glucose level is controlled.This has gradually become one of the main reasons why the cardiovascular complications of diabetes are difficult to be prevented and effectively reduced.However,the molecular mechanism behind “metabolic memory” is still not very clear and needs further study.This study conducted a series of studies on the relationship between high glucose metabolism memory of vascular endothelial cells and cardiovascular dysfunction in diabetes and its mechanism.First of all,this study shows that endothelial-mesenchymal transition(EndoMT)has a “metabolic memory” phenomenon.High glucose(HG,35 m M D-glucose)could induce EndoMT in endothelial cells(ECs).However,when endothelial cells were treated with high glucose for 4 days,they were changed to normal medium(5.6m M D-glucose + 29.4 m M L-glucose)and cultured for another 4 days,EndoMT phenotype of ECs still existed.In addition,the effect of the latter in inducing EndoMT is even stronger than simply using a high glucose medium for 8 days,suggesting that there is a “metabolic memory” phenomenon of EndoMT induction by HG in ECs.The results further showed that treatment with HG significantly downregulated the protein expression of NRF2,activated transforming growth factor-?(TGF-?)signal,enhanced nuclear factor kappa-B(NF-?B)signal,increased reactive oxygen species(ROS)level,reduced the content of nitric oxide(NO)and downregulated miR-29 expression in ECs.And these changes could be remembered by ECs and cannot be quickly cleared with the improvement of glucose concentration in the culture environment,resulting in long-term continuous damage to ECs.More importantly,this study found that using NRF2 agonist tert-Butylhydroquinone(tBHQ)could significantly reverse the “metabolic memory” effect of EndoMT,suggesting that the down-regulation of NRF2 protein is a necessary condition for high glucose induced metabolic memory in ECs.We further found that NF-?B(p65)could significantly up-regulate the expression of miR-27a-3p under high glucose condition,and miR-27a-3p could directly target the 3'UTR(untranslated region)of NRF2 m RNA and down-regulate its protein expression in ECs.Therefore,this study indicates that NF-?B(p65)-miR-27a-3p-NRF2-ROS-TGF-?-EndoMT is a potential molecular mechanism of endothelial cell “metabolic memory” induced by high glucose.And the activation of this signaling pathway could further amplify the inflammatory signal in ECs by enhancing intracellular ROS,thereby forming a NF-?B-miR-27a-3p-NRF2-ROSNF-?B feedback loop,which persists the induction of EndoMT induction during endothelial cell “metabolic memory”.Furthermore,we found that using miR-27a-3p inhibitor could also partialy reverse the “metabolic memory” effect of EndoMT.In addition,treatment with NRF2 activator tBHQ or miR-27a-3p inhibitor in animal experiments significantly increased the left ventricle ejection fraction(LVEF)and and significantly reduced peri-cardiovascular fibrosis in diabetic mice.Through the above research:(1)It is the first time to find that down-regulation of NRF2 protein induced by high glucose is a necessary condition for EndoMT induction and is a potential cause of endothelial cell metabolic memory;(2)We reveals NF-?B(p65)-miR-27a-3p-NRF2-ROS-TGF-?-EndoMT pathway is a new molecular mechanism of endothelial cell metabolic memory;(3)It is the first time to propose that using tBHQ or miR-27a-3p inhibitor to destroy endothelial cell metabolic memory could effectively improve the defect that the use of insulin alone can not delay cardiac insufficiency in the process of treating diabetes.The above studies on the molecular mechanism of endothelial cell metabolic memory and its association with cardiovascular dysfunction provide a new strategy and direction for the treatment of cardiovascular complications of diabetes.It is proposed for the first time that targeting NRF2 or miR-27a-3p may be an effective strategy for blocking endothelial cell metabolism memory and inhibiting diabetes cardiovascular dysfunction and other diabetes complications.