Abstract
Vascular calcification, especially medial artery calcification, is associated with cardiovascular death in patients with diabetes mellitus and chronic kidney disease (CKD). To determine the underlying mechanism of vascular calcification, we have demonstrated in our previous report that advanced glycation end-products (AGEs) stimulated calcium deposition in vascular smooth muscle cells (VSMCs) through excessive oxidative stress and phenotypic transition into osteoblastic cells. Since AGEs can induce apoptosis, in this study we investigated its role on VSMC apoptosis, focusing mainly on the underlying mechanisms. A rat VSMC line (A7r5) was cultured, and treated with glycolaldehyde-derived AGE-bovine serum albumin (AGE3-BSA). Apoptotic cells were identified by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. To quantify apoptosis, an enzyme-linked immunosorbent assay (ELISA) for histone-complexed DNA fragments was employed. Real-time PCR was performed to determine the mRNA levels. Treatment of A7r5 cells with AGE3-BSA from 100 µg/mL concentration markedly increased apoptosis, which was suppressed by Nox inhibitors. AGE3-BSA significantly increased the mRNA expression of NAD(P)H oxidase components including Nox4 and p22phox, and these findings were confirmed by protein levels using immunofluorescence. Dihydroethidisum assay showed that compared with cBSA, AGE3-BSA increased reactive oxygen species level in A7r5 cells. Furthermore, AGE3-induced apoptosis was significantly inhibited by siRNA-mediated knockdown of Nox4 or p22phox. Double knockdown of Nox4 and p22phox showed a similar inhibitory effect on apoptosis as single gene silencing. Thus, our results demonstrated that NAD(P)H oxidase-derived oxidative stress are involved in AGEs-induced apoptosis of VSMCs. These findings might be important to understand the pathogenesis of vascular calcification in diabetes and CKD.
Highlights
Vascular complication is an important aspect of the pathological course of diabetes mellitus, and affects the disease-related morbidity and mortality
We found that advanced glycation end-products (AGEs)-induced reactive oxygen species (ROS) generation via NAD(P)H oxidase plays an important role in vascular smooth muscle cells (VSMCs) apoptosis, as it does in calcium deposition [19]
The cells were treated with control BSA or AGE3-BSA in calcification medium
Summary
Vascular complication is an important aspect of the pathological course of diabetes mellitus, and affects the disease-related morbidity and mortality. United Kingdom Prospective Diabetes Study (UKPDS) demonstrated that intensive glycemic control started at the time of diagnosis is associated with a significantly reduced risk of microvascular disease, myocardial infarction and death from any cause in type 2 diabetes [2]. Such findings suggest a salutary effect of intensive therapy on the risk of CV events. AGE-RAGE signaling can induce oxidative stress and tumor necrosis factor–α, both of them activate NF-κB, form a positive feed-back loop leading to the development of CV diseases [8] Such findings are supported by study with diabetes model mouse, showing that the defect of RAGE prevented the development of AGEs-induced atherosclerosis [9]. We found that AGE-induced ROS generation via NAD(P)H oxidase plays an important role in VSMC apoptosis, as it does in calcium deposition [19]
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