ENPP1 Was Involved In Ages-induced Vascular Calcification Through Autophagy And Oxidative Stress

Background and objectiveVascular calcification is highly prevalent in patients with chronic kidney disease(CKD)and diabetes mellitus(DM),which is associated with increased morbidity and mortality.Ectonucleotide pyrophosphatase/phosphodiesterase 1(ENPP1),a type ? transmembrane glycoprotein,exerts an important role in regulating soft tissue mineralization through the generation of pyrophosphate.However,whether it is involved in vascular calcification caused by chronic kidney disease or diabetes remains unknown.Advanced glycation end products,as toxin widely existed in both diseases,may play an important role in related vascular calcification.This study was aimed to understand the interaction between AGEs and ENPP1during AGEs-induced vascular calcification.and to further explore the pathogenesis of vascular calcification.Methods(1)AGEs were incubated with high glucose and bovine serum albumin.The effect of AGEs on cell viability of HASMCs culture in vitro were examined with CCK-8 assay.(2)HASMCs were incubated with or without AGEs in the presence of?-GP,RUNX2 and ALP were evaluated by western blot,and alizarin red S staining and calcium content assay were used to evaluate the calcium deposition.(3)HASMCs were incubated with different dose of AGEs for 12,24,48 and 72 hours,RT-qPCR and western blot were used to evaluated the expression of ENPP1,meanwhile,we used immunohistochemical staining to detect the expression of ENPP1 in diabetic rats'aorta.(4)Western blot was used to evaluate the change of autophagy level during AGEs-induced vascular calcification,then we used chloroquine and rapamycin were used to regulate the level of autophagy to detect whether autophagy was involved in AGEs-mediated ENPP1 expression and vascular calcification.(5)DCFH-DA probe and MitoSOX~TMM assay were used to detect the intracellular ROS level during AGEs-induced calcification;then we used N-acetyl-cysteine to reduce the intracellular ROS level to detect whether oxidative stress was involved in AGEs-mediated ENPP1 expression and vascular calcification.Results(1)AGEs ranging from 50-800?g/ml did not affect the cell viability of HASMCs after being treated for 24-72 hours.(2)Compared with control group,?-GP could significantly increase the expression of RUNX2 and ALP,and calcium deposition also increased measured by alizarin red S staining and calcium content assay,meanwhile,AGEs significantly aggravated these processes.(3)After being treated with AGEs in different concentration,the expression of ENPP1 increased in a dose-dependent manner;meanwhile,we incubated HASMCs with 200?g/ml AGEs in the presence of10?M?-GP for 12,24,48 and 72 hours respectively,a time-dependent increase for ENPP1 was observed.(4)AGEs increased the autophagy level during HASMCs calcification,and inhibition of autophagy with chloroquine could decrease the expression of ENPP1 and aggravate the pro-calcification effect of AGEs;meanwhile,rapamycin could partly alleviate HASMCs calcification and upregulate the ENPP1 expression.(5)Intracellular ROS was significantly upregulated during AGEs-induced calcification and reduction of ROS level with NAC could partly alleviate the pro-calcification effect of AGEs.Conclusion1.AGEs could aggravate the process of HASMCs calcification.2.ENPP1 was upregulated during AGEs-induced calcification.3.Autophagy and oxidative stress were involved in vascular calcification and the expression of ENPP1.