Homocysteine-induced apoptosis in endothelial cells coincides with nuclear NOX2 and peri-nuclear NOX4 activity.
Apoptosis of endothelial cells related to homocysteine (Hcy) has been reported in several studies. In this study, we evaluated whether reactive oxygen species (ROS)-producing signaling pathways contribute to Hcy-induced apoptosis induction, with specific emphasis on NADPH oxidases. Human umbilical vein endothelial cells were incubated with 0.01-2.5 mM Hcy. We determined the effect of Hcy on caspase-3 activity, annexin V positivity, intracellular NOX1, NOX2, NOX4, and p47(phox) expression and localization, nuclear nitrotyrosine accumulation, and mitochondrial membrane potential (ΔΨ m). Hcy induced caspase-3 activity and apoptosis; this effect was concentration dependent and maximal after 6-h exposure to 2.5 mM Hcy. It was accompanied by a significant increase in ΔΨ m. Cysteine was inactive on these parameters excluding a reactive thiol group effect. Hcy induced an increase in cellular NOX2, p47(phox), and NOX4, but not that of NOX1. 3D digital imaging microscopy followed by image deconvolution analysis showed nuclear accumulation of NOX2 and p47(phox) in endothelial cells exposed to Hcy, but not in control cells, which coincided with accumulation of nuclear nitrotyrosine residues. Furthermore, Hcy enhanced peri-nuclear localization of NOX4 coinciding with accumulation of peri-nuclear nitrotyrosine residues, a reflection of local ROS production. p47(phox) was also increased in the peri-nuclear region. The Hcy-induced increase in caspase-3 activity was prevented by DPI and apocynin, suggesting involvement of NOX activity. The data presented in this article reveal accumulation of nuclear NOX2 and peri-nuclear NOX4 accumulation as potential source of ROS production in Hcy-induced apoptosis in endothelial cells.
Department of Pathology, VU University Medical Centre, Room 0E46, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.
Jessica A
JA
Sipkens
Nynke
N
Hahn
Carlien S
CS
van den Brand
Christof
C
Meischl
Saskia A G M
SA
Cillessen
Desirée E C
DE
Smith
Lynda J M
LJ
Juffermans
René J P
RJ
Musters
Dirk
D
Roos
Cornelis
C
Jakobs
Henk J
HJ
Blom
Yvo M
YM
Smulders
Paul A J
PA
Krijnen
Coen D A
CD
Stehouwer
Jan A
JA
Rauwerda
Victor W M
VW
van Hinsbergh
Hans W M
HW
Niessen
D017209:Apoptosis / Q000187:drug effects*; D002467:Cell Nucleus / Q000187:drug effects* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006710:Homocysteine / Q000494:pharmacology*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects
D008562:Membrane Glycoproteins; D006710:Homocysteine; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; C060505:neutrophil cytosolic factor 1