S-Adenosylhomocysteine induces apoptosis and phosphatidylserine exposure in endothelial cells independent of homocysteine

J.A. Sipkens, N.E. Hahn, H.J. Blom, S.M. Lougheed, C.D.A. Stehouwer, J.A. Rauwerda, P.A.J. Krijnen, V.W.M. van Hinsbergh, H.W.M. Niessen

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

OBJECTIVE: We have previously shown that homocysteine (Hcy) induces phosphatidylserine (PS) exposure, apoptosis and necrosis in human endothelial cells. Since it has been suggested that S-adenosylhomocysteine (SAH) is the main causative factor in Hcy-induced pathogenesis of cardiovascular disease, we evaluate here whether the cytotoxic Hcy effect in endothelial cells is also SAH dependent.

METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVECs) were exposed to the following conditions: (1) non-treated control (resulting in 2.8 nM intracellular SAH and 3.1 μM extracellular l-Hcy); and incubation with (2) 50 μM adenosine-2,3-dialdehyde (ADA; resulting in 17.7 nM intracellular SAH and 3.1 μM extracellular l-Hcy), (3) 2.5 mM Hcy (resulting in 20.9 nM intracellular SAH and 1.8 mM extracellular l-Hcy), and (4) 1, 10 and 100 μM SAH. We then determined the effect of treatment on annexin V-positivity, caspase-3 activity, cytochrome c release (sub)cellular expression of NOX2, NOX4, p47(phox) and nitrotyrosine, and H(2)O(2). Both Hcy and ADA significantly increased PS exposure (n=5), caspase-3 activity (n=6) and cytochrome c release (n=3). Incubation with extracellular SAH alone did not affect cell viability. Both Hcy and ADA also induced similar increases in nuclear NOX2 and (peri)nuclear NOX4, coinciding with (peri)nuclear p47(phox) expression and local reactive oxygen species (ROS) (n=3). Inhibition of NOX-mediated ROS by the flavoenzyme inhibitor diphenylene iodonium (DPI) significantly decreased apoptosis induction (n=3) and ROS production (n=3).

CONCLUSION: SAH induces PS exposure and apoptosis in endothelial cells independently of Hcy. Our study therefore shows that Hcy-mediated endothelial dysfunction, as determined in the cell model used, is mainly due to SAH accumulation.

Original languageEnglish
Pages (from-to)48-54
Number of pages7
JournalAtherosclerosis
Volume221
Issue number1
DOIs
Publication statusPublished - Mar 2012

Cite this

@article{d0dc1eb07c9f4ca6aa72586d62164dd7,
title = "S-Adenosylhomocysteine induces apoptosis and phosphatidylserine exposure in endothelial cells independent of homocysteine",
abstract = "OBJECTIVE: We have previously shown that homocysteine (Hcy) induces phosphatidylserine (PS) exposure, apoptosis and necrosis in human endothelial cells. Since it has been suggested that S-adenosylhomocysteine (SAH) is the main causative factor in Hcy-induced pathogenesis of cardiovascular disease, we evaluate here whether the cytotoxic Hcy effect in endothelial cells is also SAH dependent.METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVECs) were exposed to the following conditions: (1) non-treated control (resulting in 2.8 nM intracellular SAH and 3.1 μM extracellular l-Hcy); and incubation with (2) 50 μM adenosine-2,3-dialdehyde (ADA; resulting in 17.7 nM intracellular SAH and 3.1 μM extracellular l-Hcy), (3) 2.5 mM Hcy (resulting in 20.9 nM intracellular SAH and 1.8 mM extracellular l-Hcy), and (4) 1, 10 and 100 μM SAH. We then determined the effect of treatment on annexin V-positivity, caspase-3 activity, cytochrome c release (sub)cellular expression of NOX2, NOX4, p47(phox) and nitrotyrosine, and H(2)O(2). Both Hcy and ADA significantly increased PS exposure (n=5), caspase-3 activity (n=6) and cytochrome c release (n=3). Incubation with extracellular SAH alone did not affect cell viability. Both Hcy and ADA also induced similar increases in nuclear NOX2 and (peri)nuclear NOX4, coinciding with (peri)nuclear p47(phox) expression and local reactive oxygen species (ROS) (n=3). Inhibition of NOX-mediated ROS by the flavoenzyme inhibitor diphenylene iodonium (DPI) significantly decreased apoptosis induction (n=3) and ROS production (n=3).CONCLUSION: SAH induces PS exposure and apoptosis in endothelial cells independently of Hcy. Our study therefore shows that Hcy-mediated endothelial dysfunction, as determined in the cell model used, is mainly due to SAH accumulation.",
keywords = "Adenosine, Apoptosis, Caspase 3, Cell Survival, Cells, Cultured, Cytochromes c, Endothelial Cells, Enzyme Inhibitors, Homocysteine, Humans, Hydrogen Peroxide, Membrane Glycoproteins, NADPH Oxidase 2, NADPH Oxidase 4, NADPH Oxidases, Onium Compounds, Phosphatidylserines, S-Adenosylhomocysteine, Tyrosine, Journal Article, Research Support, Non-U.S. Gov't",
author = "J.A. Sipkens and N.E. Hahn and H.J. Blom and S.M. Lougheed and C.D.A. Stehouwer and J.A. Rauwerda and P.A.J. Krijnen and {van Hinsbergh}, V.W.M. and H.W.M. Niessen",
note = "Copyright {\^A}{\circledC} 2011 Elsevier Ireland Ltd. All rights reserved.",
year = "2012",
month = "3",
doi = "10.1016/j.atherosclerosis.2011.11.032",
language = "English",
volume = "221",
pages = "48--54",
journal = "Atherosclerosis",
issn = "0021-9150",
publisher = "Elsevier Ireland Ltd",
number = "1",

}

S-Adenosylhomocysteine induces apoptosis and phosphatidylserine exposure in endothelial cells independent of homocysteine. / Sipkens, J.A.; Hahn, N.E.; Blom, H.J.; Lougheed, S.M.; Stehouwer, C.D.A.; Rauwerda, J.A.; Krijnen, P.A.J.; van Hinsbergh, V.W.M.; Niessen, H.W.M.

In: Atherosclerosis, Vol. 221, No. 1, 03.2012, p. 48-54.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - S-Adenosylhomocysteine induces apoptosis and phosphatidylserine exposure in endothelial cells independent of homocysteine

AU - Sipkens, J.A.

AU - Hahn, N.E.

AU - Blom, H.J.

AU - Lougheed, S.M.

AU - Stehouwer, C.D.A.

AU - Rauwerda, J.A.

AU - Krijnen, P.A.J.

AU - van Hinsbergh, V.W.M.

AU - Niessen, H.W.M.

N1 - Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

PY - 2012/3

Y1 - 2012/3

N2 - OBJECTIVE: We have previously shown that homocysteine (Hcy) induces phosphatidylserine (PS) exposure, apoptosis and necrosis in human endothelial cells. Since it has been suggested that S-adenosylhomocysteine (SAH) is the main causative factor in Hcy-induced pathogenesis of cardiovascular disease, we evaluate here whether the cytotoxic Hcy effect in endothelial cells is also SAH dependent.METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVECs) were exposed to the following conditions: (1) non-treated control (resulting in 2.8 nM intracellular SAH and 3.1 μM extracellular l-Hcy); and incubation with (2) 50 μM adenosine-2,3-dialdehyde (ADA; resulting in 17.7 nM intracellular SAH and 3.1 μM extracellular l-Hcy), (3) 2.5 mM Hcy (resulting in 20.9 nM intracellular SAH and 1.8 mM extracellular l-Hcy), and (4) 1, 10 and 100 μM SAH. We then determined the effect of treatment on annexin V-positivity, caspase-3 activity, cytochrome c release (sub)cellular expression of NOX2, NOX4, p47(phox) and nitrotyrosine, and H(2)O(2). Both Hcy and ADA significantly increased PS exposure (n=5), caspase-3 activity (n=6) and cytochrome c release (n=3). Incubation with extracellular SAH alone did not affect cell viability. Both Hcy and ADA also induced similar increases in nuclear NOX2 and (peri)nuclear NOX4, coinciding with (peri)nuclear p47(phox) expression and local reactive oxygen species (ROS) (n=3). Inhibition of NOX-mediated ROS by the flavoenzyme inhibitor diphenylene iodonium (DPI) significantly decreased apoptosis induction (n=3) and ROS production (n=3).CONCLUSION: SAH induces PS exposure and apoptosis in endothelial cells independently of Hcy. Our study therefore shows that Hcy-mediated endothelial dysfunction, as determined in the cell model used, is mainly due to SAH accumulation.

AB - OBJECTIVE: We have previously shown that homocysteine (Hcy) induces phosphatidylserine (PS) exposure, apoptosis and necrosis in human endothelial cells. Since it has been suggested that S-adenosylhomocysteine (SAH) is the main causative factor in Hcy-induced pathogenesis of cardiovascular disease, we evaluate here whether the cytotoxic Hcy effect in endothelial cells is also SAH dependent.METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVECs) were exposed to the following conditions: (1) non-treated control (resulting in 2.8 nM intracellular SAH and 3.1 μM extracellular l-Hcy); and incubation with (2) 50 μM adenosine-2,3-dialdehyde (ADA; resulting in 17.7 nM intracellular SAH and 3.1 μM extracellular l-Hcy), (3) 2.5 mM Hcy (resulting in 20.9 nM intracellular SAH and 1.8 mM extracellular l-Hcy), and (4) 1, 10 and 100 μM SAH. We then determined the effect of treatment on annexin V-positivity, caspase-3 activity, cytochrome c release (sub)cellular expression of NOX2, NOX4, p47(phox) and nitrotyrosine, and H(2)O(2). Both Hcy and ADA significantly increased PS exposure (n=5), caspase-3 activity (n=6) and cytochrome c release (n=3). Incubation with extracellular SAH alone did not affect cell viability. Both Hcy and ADA also induced similar increases in nuclear NOX2 and (peri)nuclear NOX4, coinciding with (peri)nuclear p47(phox) expression and local reactive oxygen species (ROS) (n=3). Inhibition of NOX-mediated ROS by the flavoenzyme inhibitor diphenylene iodonium (DPI) significantly decreased apoptosis induction (n=3) and ROS production (n=3).CONCLUSION: SAH induces PS exposure and apoptosis in endothelial cells independently of Hcy. Our study therefore shows that Hcy-mediated endothelial dysfunction, as determined in the cell model used, is mainly due to SAH accumulation.

KW - Adenosine

KW - Apoptosis

KW - Caspase 3

KW - Cell Survival

KW - Cells, Cultured

KW - Cytochromes c

KW - Endothelial Cells

KW - Enzyme Inhibitors

KW - Homocysteine

KW - Humans

KW - Hydrogen Peroxide

KW - Membrane Glycoproteins

KW - NADPH Oxidase 2

KW - NADPH Oxidase 4

KW - NADPH Oxidases

KW - Onium Compounds

KW - Phosphatidylserines

KW - S-Adenosylhomocysteine

KW - Tyrosine

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1016/j.atherosclerosis.2011.11.032

DO - 10.1016/j.atherosclerosis.2011.11.032

M3 - Article

VL - 221

SP - 48

EP - 54

JO - Atherosclerosis

JF - Atherosclerosis

SN - 0021-9150

IS - 1

ER -