Oxygen-glucose deprivation induces inducible nitric oxide synthase and nitrotyrosine expression in cerebral endothelial cells

Jan Xu, Luming He, S. Hinan Ahmed, Sha Wei Chen, Mark P. Goldberg, Joseph S. Beckman, Chung Y. Hsu

Research output: Contribution to journalArticle

81 Citations (Scopus)

Abstract

Background and Purpose: The cerebral endothelial cells (ECs) are a primary target of hypoxic or ischemic brain insults. EC damage may contribute to postischemic secondary injury. Massive production of NO after inducible NO synthase (iNOS) expression has been implicated in cell death. This study aimed to characterize bovine cerebral EC death in relation to iNOS expression after oxygen-glucose deprivation (OGD) in vitro. Methods: OGD in bovine cerebral ECs in culture was induced by deleting glucose in the medium and by incubating the cells in a temperature-controlled anaerobic chamber. The extent of cell death was assessed by trypan blue exclusion, MTT assay, and LDH release. ELISA, gel electrophoresis, and staining by terminal deoxynucleotidyl transferasemediated dUTP nick end-labeling were used to examine DNA fragmentation. The expression of iNOS mRNA and protein was detected by reverse transcription-polymerase chain reaction and Western blotting, respectively. Nitrotyrosine expression was confirmed with Western blot analysis and immunostaining. Results: Bovine cerebral EC death was dependent on the duration of OGD and showed selected biochemical, morphological, and pharmacological features suggestive of apoptosis. OGD also induced the expression of iNOS mRNA and protein in bovine cerebral ECs. Increased expression of nitrotyrosine, the product formed by peroxynitrite reaction with proteins, was also detected after OGD. The involvement of iNOS in EC death was suggested by partial reduction of cell death by NO synthase inhibitors, including L-N(G)-(1-iminoethyl)omithine and nitro-L-arginine, and an NO scavenger, the Fe2+-N-methyl-D-glucamine dithiocarbamate complex. Conclusions: OGD-induced bovine cerebral EC death involves an apoptotic process. Induction of iNOS with subsequent peroxynitrite formation may contribute to bovine cerebral EC death caused by OGD.

Original languageEnglish (US)
Pages (from-to)1744-1751
Number of pages8
JournalStroke
Volume31
Issue number7
StatePublished - Jul 2000

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Nitric Oxide Synthase Type II
Endothelial Cells
Nitric Oxide Synthase
Cell Death
Oxygen
Glucose
Peroxynitrous Acid
Western Blotting
3-nitrotyrosine
Messenger RNA
Proteins
Trypan Blue
DNA Fragmentation
Reverse Transcription
Arginine
Electrophoresis
Cell Culture Techniques
Gels
Enzyme-Linked Immunosorbent Assay
Pharmacology

Keywords

  • Apoptosis
  • Blood-brain barrier
  • Cerebral ischemia
  • Free radicals
  • Nitric oxide

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine
  • Neuroscience(all)

Cite this

Xu, J., He, L., Ahmed, S. H., Chen, S. W., Goldberg, M. P., Beckman, J. S., & Hsu, C. Y. (2000). Oxygen-glucose deprivation induces inducible nitric oxide synthase and nitrotyrosine expression in cerebral endothelial cells. Stroke, 31(7), 1744-1751.

Oxygen-glucose deprivation induces inducible nitric oxide synthase and nitrotyrosine expression in cerebral endothelial cells. / Xu, Jan; He, Luming; Ahmed, S. Hinan; Chen, Sha Wei; Goldberg, Mark P.; Beckman, Joseph S.; Hsu, Chung Y.

In: Stroke, Vol. 31, No. 7, 07.2000, p. 1744-1751.

Research output: Contribution to journalArticle

Xu, J, He, L, Ahmed, SH, Chen, SW, Goldberg, MP, Beckman, JS & Hsu, CY 2000, 'Oxygen-glucose deprivation induces inducible nitric oxide synthase and nitrotyrosine expression in cerebral endothelial cells', Stroke, vol. 31, no. 7, pp. 1744-1751.
Xu, Jan ; He, Luming ; Ahmed, S. Hinan ; Chen, Sha Wei ; Goldberg, Mark P. ; Beckman, Joseph S. ; Hsu, Chung Y. / Oxygen-glucose deprivation induces inducible nitric oxide synthase and nitrotyrosine expression in cerebral endothelial cells. In: Stroke. 2000 ; Vol. 31, No. 7. pp. 1744-1751.
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AU - Xu, Jan

AU - He, Luming

AU - Ahmed, S. Hinan

AU - Chen, Sha Wei

AU - Goldberg, Mark P.

AU - Beckman, Joseph S.

AU - Hsu, Chung Y.

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N2 - Background and Purpose: The cerebral endothelial cells (ECs) are a primary target of hypoxic or ischemic brain insults. EC damage may contribute to postischemic secondary injury. Massive production of NO after inducible NO synthase (iNOS) expression has been implicated in cell death. This study aimed to characterize bovine cerebral EC death in relation to iNOS expression after oxygen-glucose deprivation (OGD) in vitro. Methods: OGD in bovine cerebral ECs in culture was induced by deleting glucose in the medium and by incubating the cells in a temperature-controlled anaerobic chamber. The extent of cell death was assessed by trypan blue exclusion, MTT assay, and LDH release. ELISA, gel electrophoresis, and staining by terminal deoxynucleotidyl transferasemediated dUTP nick end-labeling were used to examine DNA fragmentation. The expression of iNOS mRNA and protein was detected by reverse transcription-polymerase chain reaction and Western blotting, respectively. Nitrotyrosine expression was confirmed with Western blot analysis and immunostaining. Results: Bovine cerebral EC death was dependent on the duration of OGD and showed selected biochemical, morphological, and pharmacological features suggestive of apoptosis. OGD also induced the expression of iNOS mRNA and protein in bovine cerebral ECs. Increased expression of nitrotyrosine, the product formed by peroxynitrite reaction with proteins, was also detected after OGD. The involvement of iNOS in EC death was suggested by partial reduction of cell death by NO synthase inhibitors, including L-N(G)-(1-iminoethyl)omithine and nitro-L-arginine, and an NO scavenger, the Fe2+-N-methyl-D-glucamine dithiocarbamate complex. Conclusions: OGD-induced bovine cerebral EC death involves an apoptotic process. Induction of iNOS with subsequent peroxynitrite formation may contribute to bovine cerebral EC death caused by OGD.

AB - Background and Purpose: The cerebral endothelial cells (ECs) are a primary target of hypoxic or ischemic brain insults. EC damage may contribute to postischemic secondary injury. Massive production of NO after inducible NO synthase (iNOS) expression has been implicated in cell death. This study aimed to characterize bovine cerebral EC death in relation to iNOS expression after oxygen-glucose deprivation (OGD) in vitro. Methods: OGD in bovine cerebral ECs in culture was induced by deleting glucose in the medium and by incubating the cells in a temperature-controlled anaerobic chamber. The extent of cell death was assessed by trypan blue exclusion, MTT assay, and LDH release. ELISA, gel electrophoresis, and staining by terminal deoxynucleotidyl transferasemediated dUTP nick end-labeling were used to examine DNA fragmentation. The expression of iNOS mRNA and protein was detected by reverse transcription-polymerase chain reaction and Western blotting, respectively. Nitrotyrosine expression was confirmed with Western blot analysis and immunostaining. Results: Bovine cerebral EC death was dependent on the duration of OGD and showed selected biochemical, morphological, and pharmacological features suggestive of apoptosis. OGD also induced the expression of iNOS mRNA and protein in bovine cerebral ECs. Increased expression of nitrotyrosine, the product formed by peroxynitrite reaction with proteins, was also detected after OGD. The involvement of iNOS in EC death was suggested by partial reduction of cell death by NO synthase inhibitors, including L-N(G)-(1-iminoethyl)omithine and nitro-L-arginine, and an NO scavenger, the Fe2+-N-methyl-D-glucamine dithiocarbamate complex. Conclusions: OGD-induced bovine cerebral EC death involves an apoptotic process. Induction of iNOS with subsequent peroxynitrite formation may contribute to bovine cerebral EC death caused by OGD.

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KW - Blood-brain barrier

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KW - Free radicals

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