title: Homocysteine induces cerebral endothelial cell death by activating the acid sphingomyelinase ceramide pathway.
abstract: Homocysteine (Hcy) levels may rise after a stroke, but the mechanism of Hcy-induced cerebral endothelial cell (CEC) dysfunction has not been explored. In this study we examined the role of the acid sphingomyelinase (Asm)-ceramide pathway in the molecular mechanism of Hcy-induced CEC dysfunction. Murine CECs were prepared from fresh mouse brains. CECs were treated with 50-500 μM Hcy and 30-100 μM C2-ceramide for 48 h. Sphingomyelinase assays were performed to determine Asm activity. Quantitative assessments of cell survival and death by the MTT reduction and LDH release were conducted. Treatment of murine CECs with Hcy and ceramide caused cell death in a dose-dependent manner as determined by LDH and MTT assays. 250 μM Hcy and 50 μM C2-ceramide caused 50% cell death. Hcy induced murine CEC death also occurred in a time-dependant manner with substantial cell death noted as early as 24h after Hcy exposure. C2-ceramide-induced murine CEC death occurred earlier than Hcy-induced cell death by about 18h. Hcy treatment increased Asm activity and intracellular ceramide accumulation. This study demonstrated that Hcy and C2-ceramide can cause murine CEC death. Hcy induces CEC death possibly by activating the Asm-ceramide pathway.
authors.affiliation: Department of Neurology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC. jiunntay@ndmctsgh.edu.tw
authors.forename: Jiunn-Tay
authors.initials: JT
authors.lastname: Lee
authors.forename: Giia-Sheun
authors.initials: GS
authors.lastname: Peng
authors.forename: Shao-Yuan
authors.initials: SY
authors.lastname: Chen
authors.forename: Chang-Hung
authors.initials: CH
authors.lastname: Hsu
authors.forename: Chun-Chieh
authors.initials: CC
authors.lastname: Lin
authors.forename: Chun-An
authors.initials: CA
authors.lastname: Cheng
authors.forename: Yaw-Don
authors.initials: YD
authors.lastname: Hsu
authors.forename: Jiann-Chyun
authors.initials: JC
authors.lastname: Lin
mesh_terms: D000639:Amitriptyline / Q000494:pharmacology; D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002540:Cerebral Cortex / Q000187:drug effects* / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology; D006710:Homocysteine / Q000037:antagonists & inhibitors / Q000633:toxicity*; D051379:Mice; D061251:Primary Cell Culture; D015398:Signal Transduction / Q000187:drug effects; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity; D013997:Time Factors
chemical_list: C064769:N-acetylsphingosine; D006710:Homocysteine; D000639:Amitriptyline; D013108:Sphingomyelin Phosphodiesterase; D013110:Sphingosine
keywords: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; ANOVA; Ac-LDL; Asm; CECs; Cell death; Ceramide; Cerebral endothelial cells; DMEM; Dulbecco's Modified Eagle Medium; FCS; HBSS; Hank's balanced salt solution; Hcy; Homocysteine; LDH; MTHFR; MTT; Nsm; ROS; Reactive oxygen species; Spearman's correlation coefficient; Stroke; TLC; Thin layer chromatography; acetylated low density lipoprotein; acid sphingomyelinase; analysis of variance; cerebral endothelial cells; fetal calf serum; homocysteine; lactate dehydrogenase; methylene tetrahydrofolate reductase; neutral sphingomyelinase; ρ