title: 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 p22(phox), 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 p22(phox). Double knockdown of Nox4 and p22(phox) 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.
authors.affiliation: Department of Internal Medicine 1, Shimane University Faculty of Medicine, Shimane 693-8501, Japan. syoko@med.shimane-u.ac.jp.
authors.forename: Sayo
authors.initials: S
authors.lastname: Koike
authors.affiliation: Department of Laboratory Medicine, Shimane University Faculty of Medicine, Shimane 693-8501, Japan. syano@med.shimane-u.ac.jp.
authors.forename: Shozo
authors.initials: S
authors.lastname: Yano
authors.affiliation: Department of Internal Medicine 1, Shimane University Faculty of Medicine, Shimane 693-8501, Japan. s-tanaka@med.shimane-u.ac.jp.
authors.forename: Sayuri
authors.initials: S
authors.lastname: Tanaka
authors.affiliation: Department of Laboratory Medicine, Shimane University Faculty of Medicine, Shimane 693-8501, Japan. abdullah@med.shimane-u.ac.jp.
authors.forename: Abdullah M
authors.initials: AM
authors.lastname: Sheikh
authors.affiliation: Department of Laboratory Medicine, Shimane University Faculty of Medicine, Shimane 693-8501, Japan. anagai@med.shimane-u.ac.jp.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nagai
authors.affiliation: Department of Internal Medicine 1, Shimane University Faculty of Medicine, Shimane 693-8501, Japan. sugimoto@med.shimane-u.ac.jp.
authors.forename: Toshitsugu
authors.initials: T
authors.lastname: Sugimoto
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D017127:Glycation End Products, Advanced / Q000737:chemistry / Q000633:toxicity*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012710:Serum Albumin, Bovine / Q000737:chemistry
chemical_list: D017127:Glycation End Products, Advanced; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D012710:Serum Albumin, Bovine; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498911:Nox4 protein, rat; C515768:Cyba protein, rat; D002118:Calcium
keywords: NAD(P)H oxidase; advanced glycation end-products; apoptosis; chronic kidney disease; diabetes; oxidative stress; vascular calcification; vascular smooth muscle cell