Advanced Glycation End-Products Induce Apoptosis of Vascular Smooth Muscle Cells: A Mechanism for Vascular Calcification

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.

TDB protects vascular endothelial cells against oxygen-glucose deprivation/reperfusion-induced injury by targeting miR-34a to increase Bcl-2 expression

“…Moreover, TUNEL assay was used to detect apoptotic cells that undergo extensive DNA degradation during the late stages of apoptosis. The assay principle is based on the presence of nicks in the damaged DNA which can be recognized by terminal deoxynucleotidyl transferase or TdT44. The apoptotic cells were stained in brown in TUNEL assay.…”

“…3 mm coronal brain sections were cut and stained with 0.1% TTC solution44. TTC was reduced by succinate dehydrogenase (mitochondria) into red formazan in the viable region, and it remained unstained or colourless in the nonviable infarct region.…”

The link between advanced glycation end products and apoptosis in delayed wound healing

“…Vascular smooth muscle cells also display apoptosis through oxidative stress in the presence of AGEs . Two proteins, Nox4 and p22 phox , were elevated due to increased levels of AGEs.…”

“…Two proteins, Nox4 and p22 phox , were elevated due to increased levels of AGEs. These proteins increase NAD(P)H oxidase activity, which has been shown to induce apoptosis in vascular cells by increasing superoxide levels . Apoptosis of bone‐forming cells, or osteoblasts, through AGE/RAGE interaction was also reported, suggesting that AGEs contribute to impaired bone healing as is seen in chronic diabetics …”

Association of the roles of advanced glycation end products and osteocalcin between bone metabolism and vascular failure

“…AGEs induce the osteoblastic differentiation and mineralization of pericytes 16) , which have the plasticity to differentiate into smooth muscle cells. Furthermore, AGEs increase oxidative stress by stimulating NADPH oxidase (Nox) expression and induce vascular smooth muscle cell apoptosis and osteoblastic transdifferentiation 17,18) (Figure 1). Several types of AGEbinding proteins have been identified 19) .…”