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Stroke. 2000;31:1744-1751

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(Stroke. 2000;31:1744.)
© 2000 American Heart Association, Inc.

Original Contributions

Oxygen-Glucose Deprivation Induces Inducible Nitric Oxide Synthase and Nitrotyrosine Expression in Cerebral Endothelial Cells

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

From the Department of Neurology, Washington University School of Medicine, St. Louis, Mo, and the Department of Anesthesiology and Biochemistry (J.S.B.), School of Medicine, University of Alabama, Birmingham.

Correspondence to Chung Y Hsu, MD, PhD, Department of Neurology, Box 8111, Washington University School of Medicine, 660 S Euclid Ave, St. Louis, MO 63110. E-mail hsuc{at}neuro.wustl.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
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 transferase–mediated 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-NG-(1-iminoethyl)ornithine 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.


Key Words: apoptosis • blood-brain barrier • cerebral ischemia • free radicals • nitric oxide


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
The brain requires a continuous supply of oxygen and glucose to maintain normal function. Loss of this supply, even for a short duration, leads to irreversible brain injury, including degeneration of neurons and other cell elements. Cerebral endothelial cells (ECs) have been shown to be more resistant than neurons or glia to ischemic insult.1 2 However, cerebral endothelial injury or death after ischemia may exacerbate brain damage and contribute to postischemic secondary injury characterized by the breakdown of the blood-brain barrier, leading to increased vascular permeability and vasogenic brain edema.3 Endothelial injury enhances leukocyte adhesion4 and also serves as a source of oxygen-derived free radicals.5 6

Various therapeutic strategies aimed at reducing EC activation and injury in the postischemic inflammatory reaction have been explored.4 However, the molecular mechanism of cerebral EC death after ischemia has not been systematically studied. NO is a major signaling molecule generated in various cell types in the brain, including cerebral ECs.7 8 9 NO has also been implicated in tissue damage under a number of experimental paradigms, including brain injury after cerebral ischemia.10 11 12 NO synthesis is catalyzed by NO synthase (NOS). Of the 3 known isoforms of NOS, NO generated by neuronal NOS contributes to ischemic neuronal death,13 14 whereas constitutively expressed endothelial NOS may serve to protect cerebral ECs from ischemic insult.15 In disease states, including infection and ischemia, a third isoform of NOS, namely, inducible NOS (iNOS) may be expressed and contributes to inflammatory processes and ischemic brain damage.16 17 18 In the brain, iNOS expression has been noted in the inflammatory cells, such as the polymorphonuclear cells that infiltrate the ischemic brain and glial elements, including microglia and astrocytes.12 19 20 Cerebral ECs are also capable of expressing iNOS and producing NO under inflammatory conditions.21 22 iNOS immunoreactivity has also been noted in cerebral ECs in the ischemic brain.17 In general, massive NO production by iNOS as occurs in various pathological states is considered cytotoxic.23 However, NO derived from iNOS has recently been shown to be cytoprotective in a number of cell types.24 25 26 Whether iNOS expression plays any role in the ischemic death of cerebral ECs has not been studied previously. In the present study, we sought to explore morphological, biochemical, and pharmacological features and the molecular mechanism of ischemic cerebral EC death by use of an in vitro model based on oxygen-glucose deprivation (OGD) in primary cultures of bovine cerebral ECs. The in vitro system allowed the characterization of molecular events relevant to iNOS expression.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
Bovine Cerebral EC Culture
Bovine cerebral ECs were prepared and characterized as previously described.27 28 Briefly, fresh bovine brains in ice-cold Hanks’ balanced salt solution (HBSS, GIBCO-BRL) with antibiotics were freed of meninges and superficial blood vessels. The gray matter was homogenized and filtered, and the resulting microvessel fraction was then sequentially digested with collagenase B (4 mg/mL) for 2 hours and collagenase/dispase (1 mg/mL, Boehringer-Mannheim) for 8 hours, followed by centrifugation in 40% Percoll solution. The second band containing microvessels was collected and washed before plating onto collagen-coated dishes. Bovine cerebral ECs migrating from vessels were pooled to form a culture of proliferating endothelial cells that were maintained in medium containing 10% FCS, heparin (0.5 mg/mL), and endothelial growth supplements (75 µg/mL, Sigma Chemical Co). Bovine cerebral ECs of passages 4 to 15, which were uniformly positive for factor VIII and vimentin (>95% EC purity) and exhibited the characteristic bradykinin receptors,29 were grown to 70% to 80% confluence before the experiments.

Combined OGD
Confluent bovine cerebral ECs were transferred into a temperature-controlled (37±1°C) anaerobic chamber (Forma Scientific) containing a gas mixture composed of 5% CO2, 10% H2, 85% N2, and 0.02% to 0.2% O2.30 The culture medium was replaced and washed with 0.5 mL of preheated deoxygenated glucose-free HBSS 4 times and then kept in the same medium in the hypoxia chamber for 1 to 8 hours. In some experiments, bovine cerebral ECs that underwent OGD were returned to a normoxic incubator under 5% CO2/95% air for 16 hours (reoxygenation). Bovine cerebral ECs without OGD or with anoxia without glucose deprivation (normoglycemic anoxia) served as controls.

Assessment of Bovine Cerebral EC Death
Trypan Blue Test
At the end of OGD, bovine cerebral ECs were incubated in the medium containing 0.4% trypan blue for 1 hour. To dissociate the cells, 0.05% trypsin and 0.53 mmol/L EDTA were added. Cell viability was determined by light microscopy. Cells that excluded trypan blue were considered viable.28

MTT Assay
At the end of OGD, DMEM and MTT (Sigma) reagent (0.5 mg/mL) were added for 4 hours, followed by lysis solution (10% SDS in 0.01N HCl) for 14 hours. Absorbance was read at 540 nm in a multiple reader.28

LDH Release
Bovine cerebral EC death was also quantitatively assessed by measuring the extent of LDH release into the medium after OGD for 1 to 8 hours. The amount of LDH released after bovine cerebral EC lysis by 0.5% Triton 100 constitutes 100% cell death or "full kill." The extent of cell death was expressed as percentage of full kill.28 31

Quantification of Cytoplasmic Histone-Associated DNA Fragments by ELISA
A prominent feature of apoptosis is DNA fragmentation. A Cell Death Detection ELISA kit (Boehringer-Mannheim) was used to quantitatively determine the levels of histone-associated DNA fragments, including mononucleosomes and oligonucleosomes after OGD. The assay was based on the sandwich-enzyme immunoassay principle with the use of mouse monoclonal antibodies directed at DNA and histone. This assay allows the determination of mononucleosome and oligonucleosome levels in the cell lysates.28 32 Increases in DNA fragmentation over control values were quantitatively determined by an enrichment factor based on the following formula: enrichment factor=milliunits of the treated sample/milliunits of the vehicle-treated sample, where milliunits=absorbance (10-3).

Assessment of DNA Fragmentation by Agarose Gel Electrophoresis
A DNA isolation kit from Promega (catalog No. A1120) was used for the extraction of DNA after OGD. The cells in 100-mm dishes were lysed by the addition of 1.5 mL cell lysis solution and treated with RNase A solution. Proteins were precipitated by a solution provided with the DNA isolation kit, and DNA was hydrated. The DNA samples (10 µg per lane) were electrophoresed at 75 V for 2 hours in 1.5% agarose gel containing 0.4 µg/mL ethidium bromide in a Tris-acetate buffer (0.4 mol/L Tris, 0.25 mol/L sodium acetate, and 0.22 mmol/L EDTA, pH 7.8). DNA was visualized through ultraviolet transillumination and photographed. The ladder consists of DNA fragments, which differ in multiples of 180 to 200 bp.28

DAPI Staining
Bovine cerebral ECs on coverslips were incubated in 1 µg/mL of 4',6-diamidino-2-phenylindole dihydrochloride (DAPI, Molecular Probes), a fluorescent probe for DNA, for 10 minutes after fixation with 4% paraformaldehyde. After they were washed with PBS, the slides were examined under a fluorescence microscope.

TUNEL Staining
Confluent bovine cerebral ECs grown on coverslips were subjected to OGD for 8 hours, followed by fixation with 4% paraformaldehyde. Terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) staining was performed according to the ONCOR kit protocol (catalog No. S7100-kit) as has been previously described.28

RNA Isolation
RNA isolation has been previously described.21 In brief, total RNA from bovine cerebral ECs was prepared with the use of TRI reagent from Molecular Research Center, Inc. Cells were lysed and extracted by adding 1.0 mL of TRI reagent. The lysate was added to 100 µL of chloroform, and the solution was mixed and centrifuged. The supernatant was removed, mixed with an equal volume of isopropanol, and kept at 4°C for at least 90 minutes. After centrifugation at 14 000g for 30 minutes at 4°C, the pellet was washed with 75% ethanol and then centrifuged again for 10 minutes at 4°C. The RNA fraction was then resuspended in water. Total RNA was quantified by spectrophotometry.

RT-PCR
Reverse transcription (RT)–polymerase chain reaction (PCR) for iNOS has also been reported.21 Briefly, equal amounts of RNA (2 µg) were reverse-transcribed with oligo(dT) and 500 µmol/L dNTPs (BRL Life Technologies, Inc), 20 U Rasin (Promega), 200 µmol/L dithiothreitol, and 200 U reverse transcriptase (BRL) for 50 minutes at 42°C. After incubation, the sample was heated for 5 minutes at 95°C, diluted, and divided into aliquot portions. The PCR was performed with a reaction mixture containing cDNA transcribed from 50 ng RNA, 100 µmol/L dNTPs, 1 µmol/L of each primer, [{alpha}-32P]dCTP, 1.5 mmol/L MgCl2, and 2.5 U Taq polymerase (BRL). PCR reaction conditions were as follows: 25 cycles (each cycle consisted of 1 minute at 94°C, 1 minute at 55°C, and 2 minutes at 72°C) and a delay time of 10 minutes. The conditions were determined in preliminary studies to be within the linear range in terms of RT input and PCR cycles. The PCR products were electrophoresed through a 12% PAGE gel and visualized by PhosphorImager (Molecular Dynamics) quantification. The relative mRNA levels of each gene were determined after normalization on the base of endogenous cyclophilin mRNA.21 The PCR experiments were repeated 3 times each by using separate sets of cultures.

Western Blot Analysis
Detection of iNOS and nitrotyrosine expression in bovine cerebral ECs by Western blot analysis has been previously reported.21 Briefly, bovine cerebral ECs were homogenized by sonification in a Western blot buffer (10 mmol/L Tris-HCl containing 2 mmol/L EDTA, 1 mmol/L benzamidine, 1 mmol/L phenylmethylsulfonyl fluoride, 2 mmol/L iodoacetate, 5 mmol/L N-ethylmaleimide, and 0.1 U/mL aprotinin, pH 7.2) and centrifuged at 10 000g for 15 minutes. Twenty micrograms of protein from the supernatant of each sample was loaded onto an 8% polyacrylamide gel, resolved by SDS/PAGE, and transferred to nitrocellulose membranes by electrophoresis. For iNOS assay, the membranes were blocked in TBST buffer containing 20 mmol/L Tris-HCl, 5% nonfat milk, 150 mmol/L NaCl, and 0.05% Tween 20 (pH 7.5) for 1 hour at room temperature. For detection of nucleotides, the membrane was quickly washed 3 times with 100 mmol/L sodium dithionite, which was dissolved in 100 mmol/L nitrogen-bubbled sodium bicarbonate (pH 10) to eliminate nonspecific protein binding to nucleotide antibody, and then the membrane was washed with TBST as described above. Primary polyclonal antibody against a mouse macrophage iNOS (Transduction Laboratory) at a 1:500 dilution or monoclonal anti-nitrotyrosine antibody33 at a 1:2000 dilution was added to the membrane and incubated at 4°C overnight or for 2 hours at room temperature. The membranes were washed with TBST 3 times at 10-minute intervals, incubated with the second antibody (goat anti-rabbit IgG conjugated with alkaline phosphatase [1:5000 for iNOS] and sheep anti-mouse IgG conjugated with horseradish peroxidase [1:7000 for nitrotyrosine]) at 37°C for 1 hour, and then washed 3 times each at 10-minute intervals with TBST and 2 times each for 2 minutes with TBS (TBST without Tween 20). The color reaction based on the Blot AP System was as described in the technical manual provided by Promega for iNOS expression or the chemiluminescence method as described in Amersham Life Science protocol RPN2106 for nitrotyrosine.

Immunocytochemical Staining for Cytochrome c and Nitrotyrosine
Bovine cerebral ECs were fixed with 4% paraformaldehyde and washed 3 times with PBS. For indirect immunofluorescence, the primary antibodies were monoclonal antibodies against cytochrome c (1:100, Pharmingen) and nitrotyrosine (1:100, Transduction Labs). The secondary antibody was goat anti-mouse IgG conjugate/rhodamine34 (1:100, Promega). Negative controls were prepared with omission of the primary antibody. Additional negative control studies were conducted by incubation with the anti-nitrotyrosine antibody in the presence of exogenous nitrotyrosine (10 mmol/L, Transduction Labs).

Treatment With Caspase and NOS Inhibitors
In some experiments, the bovine cerebral ECs in 24-well plates were treated with 50 µmol/L of zVAD-fmk (Enzyme Systems Products) before undergoing OGD for 8 hours. In a separate set of experiments, cells in 24-well plates were treated with 1 µmol/L of NOS inhibitors, L-NG-(1-iminoethyl)ornithine (NIL) or nitro-L-arginine (L-NA), or an NO scavenger, the Fe2+-N-methyl-D-glucamine dithiocarbamate complex [(MGD)2-Fe2+], before undergoing OGD for 8 hours. After the OGD treatment, the extent of cell death was assessed by the MTT assay as explained above.

Statistical Analyses
Quantitative data are expressed as mean±SD on the basis of at least 2 separate experiments of triplicate samples. Difference among groups was statistically analyzed by 1-way ANOVA followed by the Bonferroni post hoc test. Comparison between 2 experimental groups was based on the 2-tailed t test. A value of P<0.05 was considered significant.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
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 down arrowIntroduction 
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Time Course of OGD-Induced Bovine Cerebral EC Death
Bovine cerebral ECs that underwent OGD for 1 hour showed little morphological change and sustained virtually no cell death. Changes in cell morphology occurred if OGD duration was >=2 hours. The cell bodies became thin, elongated, and shrunken. The spaces between cells were greater in cells with OGD than in control cells (Figure 1Down). The extent of OGD-induced cell death was assessed by counting the number of living cells that exclude trypan blue (Figure 2ADown). The extent of cell death was {approx}20%, {approx}35%, and {approx}40%after 4, 6, and 8 hours, respectively, of OGD. Similar time-dependent OGD was also noted on the basis of the MTT assay (Figure 2BDown) and LDH release (Figure 2CDown).



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  		Figure 1. Morphological changes in bovine cerebral ECs after OGD. Bovine cerebral ECs grown in 24-well plates were washed with nitrogen-saturated HBSS 3 times and incubated with the same medium in an oxygen-free chamber for 2 to 8 hours. Phase-contrast light microscopy shows normal bovine cerebral ECs (A) and bovine cerebral ECs with OGD for 2 hours (B) and 8 hours (C). Note subtle changes in cell shape after 2-hour OGD. With 8-hour exposure, cell shrinkage became evident. Magnification x100.



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  		Figure 2. OGD-induced bovine cerebral EC death determined by trypan blue exclusion (A), MTT assay (B), and the extent of LDH release (C). Data are expressed as mean±SD from 3 separate experiments in quadruplicate. *P<0.05 vs control cells (without OGD).

OGD-Induced DNA Fragmentation and Cytochrome c Release
We determined the extent of DNA fragmentation by quantitative assessment of cytoplasmic histone-associated DNA fragments. OGD gradually increased DNA fragmentation in a time-dependent manner (Figure 3ADown). DNA laddering was also evident after OGD exposure for >=4 hours (Figure 3BDown). An increase in nuclear DNA strand breaks after OGD was confirmed morphologically by TUNEL staining (Figure 3CDown). Furthermore, DAPI staining showed cell nuclear condensation after OGD (Figure 4Down). Cytochrome c release into the cytosol was also noted after OGD (Figure 4Down). Together, these findings are compatible with the contention that OGD-induced bovine cerebral EC death may involve an apoptotic process.



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  		Figure 3. OGD-induced DNA fragmentation. A, OGD-induced generation of cytoplasmic histone-associated DNA fragments on the basis of ELISA. Data are expressed as mean±SD from 3 experiments in quadruplicate. *P<0.05 vs control cells (without OGD). B, OGD-induced DNA laddering revealed by gel electrophoresis. Lanes are as follows: 1, DNA marker; 2, control; 3, 2-hour OGD; 4, 4-hour OGD; 5, 6-hour OGD; and 6, 8-hour OGD. C, OGD-induced DNA fragmentation reflected by TUNEL staining. TUNEL-positive cells containing immunoreactivity of DNA strand breaks were seen after OGD (8 hours), but no staining was detected in the control cells without OGD.



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  		Figure 4. Nuclear condensation and cytochrome c release visualized by fluorescence microscopy. Immunolocalization of cytochrome c (anti–cytochrome c, red) and of nuclear morphology (DAPI, blue) is shown in bovine cerebral ECs without (control) and with OGD for 8 hours (OGD). After treatment, cells were fixed, stained, and observed under fluorescence microscope with use of a CCD camera. Note normal nuclear morphology in control cells compared with nuclear condensation in OGD cells. A diffuse redistribution of cytochrome c into the cytosol after OGD compared with the punctate pattern in the absence of OGD is also noted. Bar=25 µm for both panels..

OGD-Induced iNOS Expression
iNOS mRNA expression in bovine cerebral ECs was increased after OGD on the basis of RT-PCR. An increase in iNOS mRNA expression was detectable after OGD for >=2 hours. Peak expression occurred after OGD for 6 hours (Figure 5ADown). iNOS expression at the protein level was confirmed by Western blot analysis (Figure 5BDown).



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  		Figure 5. A, OGD-induced iNOS mRNA expression in bovine cerebral ECs by RT-PCR. Bovine cerebral ECs grown in 100-mm dishes underwent OGD for 2 to 6 hours. Total RNA was extracted for RT-PCR. Lanes are as follows: 1, control; 2, OGD for 2 hours; 3, OGD for 4 hours; and 4, OGD for 6 hours. B, OGD-induced iNOS protein expression in bovine cerebral ECs as shown by Western blot analyses. Bovine cerebral ECs grown in 100-mm dishes underwent OGD for 2 to 8 hours. Proteins were extracted and subjected to Western blot analysis with use of polyclonal antibodies against iNOS. Lanes are as follows: 1, control; 2, OGD for 2 hours; 3, OGD for 4 hours; 4, OGD for 6 hours; and 5, OGD for 8 hours.

Expression of Nitrotyrosine in Bovine Cerebral ECs During OGD
NO and superoxide (O2-) interact to form peroxynitrite (ONOO-).35 ONOO- is a highly toxic reactive oxygen species, which in turn reacts with tyrosine in proteins to form nitrotyrosine, a stable oxidation product.33 Western blot analysis detected an increase in nitrotyrosine formation in bovine cerebral ECs after OGD. Nitrotyrosine expression increased with the duration of OGD. A single protein band showing nitrotyrosine immunoreactivity was {approx}68 kDa (Figure 6Down). Nitrotyrosine immunoreactivity was also detected in bovine cerebral ECs after OGD by immunocytochemistry (Figure 7Down).



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  		Figure 6. OGD-induced nitrotyrosine expression in bovine cerebral ECs as shown by Western blot analysis. Bovine cerebral ECs grown in 100-mm dishes underwent OGD for 2 to 8 hours. Proteins were extracted and subjected to Western blot analysis with use of a monoclonal antibody against nitrotyrosine. Lanes are as follows: 1, control; 2, OGD for 2 hours; 3, OGD for 4 hours; 4, OGD for 6 hours; and 5, OGD for 8 hours.



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  		Figure 7. Immunocytochemical studies of OGD-induced nitrotyrosine expression in bovine cerebral ECs. Bovine cerebral ECs grown in 24-well plates underwent OGD as described above for 4 hours, followed by exposure to normoxia for 16 hours. Bovine cerebral ECs were fixed and examined with indirect immunofluorescence microscopy with use of a monoclonal antibody against nitrotyrosine. Left panels, Phase-contrast morphology of bovine cerebral ECs. Right panels, Fluorescence micrographs of the same field (x40 objective, silicon-intensified target camera with constant setting for all fluorescence images). A, Control. B, OGD for 4 hours followed by reoxygenation for 16 hours. C, Same conditions as in panel B but in the presence of nitrotyrosine. D, Same conditions as in panel B but with omission of the primary antibody.

Effects of Caspase Inhibitor, NOS Inhibitors, and NO Scavenger on OGD-Induced Bovine Cerebral EC Death
zVAD-fmk, a broad-spectrum caspase inhibitor, was effective in reducing OGD-induced bovine cerebral EC death (Figure 8ADown). NOS inhibitors, including NIL, a selective iNOS inhibitor, and L-NA, a nonspecific NOS inhibitor, partially reduced bovine cerebral EC death. (MGD)2-Fe2+, an NO scavenger,36 was also effective (Figure 8BDown).



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  		Figure 8. Effect of caspase inhibitor zVAD-fmk, NOS inhibitors, and NO scavenger on OGD-induced bovine cerebral EC death. A, Cell survival in bovine cerebral ECs grown in 24-well plates that underwent OGD for 8 hours in an OGD chamber. zVAD-fmk (50 µmol/L) was added before OGD. B, Cell survival in bovine cerebral ECs after similar OGD treatment with or without NIL (1 µmol/L), L-NA (1 µmol/L), and (MGD)2-Fe2+ (1 µmol/L). Data are expressed as mean±SD from 3 experiments in quadruplicate. *P<0.05 vs control cells (without OGD). #P<0.05 vs cells with 8-hour OGD.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
Ischemic or hypoxic insults can cause cell death by both necrosis and apoptosis.37 38 We found that OGD-induced bovine cerebral EC death exhibited biochemical, morphological, and pharmacological features suggestive of apoptosis. DNA fragmentation in bovine cerebral ECs after OGD was noted on the basis of ELISA, TUNEL stain, and gel electrophoresis. Immunostaining showed cytochrome c release. Cytochrome c is a mitochondrial respiratory component that translocates to the cytosol and activates DEVD (Asp-Glu-Val-Asp)-specific caspases in cells dying of apoptosis.39 Although none of the findings described above is fully specific for apoptosis, the observation that zVAD-fmk, a broad-spectrum caspase inhibitor, could substantially increase the cell viability from {approx}25% to {approx}75% strengthens the role of apoptosis in OGD-induced bovine cerebral EC death.

Cerebral ECs have been shown to express iNOS and produce NO under inflammatory conditions.21 22 iNOS immunoreactivity has also been identified in cerebral ECs in the ischemic brain.17 40 Similarly, hypoxia enhances iNOS expression in cytokine-treated murine macrophages,41 in rat mesangial cells,42 and in the rat lung.43 In the present study, we noted that OGD induced iNOS mRNA and protein expression in bovine cerebral ECs on the basis of RT-PCR and Western blot. Expression of iNOS is associated with prolonged production of high levels of NO.23 Overproduction of NO may exacerbate ischemic brain injury.10 11 NO, when produced in excess, reacts with O2- to form ONOO-, which has been proposed to play an important role in the cellular damage associated with the overproduction of NO.35 NO and O2- may act synergistically to enhance ONOO--mediated toxicity in cerebral endothelial cells.44 ONOO- reacts with proteins and results in the oxidation of tryptophan and cysteine residues. This process also leads to the nitration of tyrosine, formation of dityrosine, and 2,4-dinitrophenylhydrazine–reactive carbonyls, leading to protein fragmentation.45 The formation of 3-nitrotyrosine represents a likely ONOO--mediated protein modification that may be different from modifications mediated by other reactive oxygen species.46 We found that OGD also enhanced nitrotyrosine expression at 4 hours and peaked at 8 hours. Nitrotyrosine was identified in a band with a molecular size of {approx}68 kDa. The particular protein species that is vulnerable to nitration remains to be identified. The expression of nitrotyrosine in bovine cerebral ECs after OGD was further confirmed by immunohistochemical studies. The observation that nitrotyrosine immunoreactivity could be blocked by exogenous nitrotyrosine suggests specificity of nitrotyrosine moiety in bovine cerebral ECs after OGD.

The role of NO in the regulation of apoptosis is complex. Although NO has been shown to induce apoptosis in several cell types, it is cytoprotective in others, depending on the particular biological conditions, the concentration and rate of NO production, and its redox state.23 47 48 NO-mediated apoptosis has been reported in macrophages,49 astrocytes,50 and PC12 cells.51 On the other hand, NO protects against apoptosis in neurons,52 hepatocytes,53 human umbilical venous cells,54 and B lymphocytes.55 The protective effect of NO could be either via cGMP-mediated interruption of apoptotic signaling, Bcl 2 upregulation, or a direct inhibition of caspase activity.25 52 56 57 58 In contrast, the proapoptotic effects may be due to a mechanism involving excitotoxic mediators, Ca2+ overload and the subsequent activation of caspases,59 proteosome inhibition leading to p53 accumulation,60 or increased Bax production.61 NO production after increased iNOS activity has also shown conflicting effects on apoptosis.24 25 26 62 Results derived from the present study revealed that the extent of bovine cerebral EC death with features suggestive of apoptosis was dependent on the duration of OGD and was correlated with the expression of iNOS. It is interesting to note that cerebral ECs were more resistant to OGD than were cortical neurons in culture. Neurons exposed to OGD for 1 hour showed extensive neuronal death over a period of 24 hours.30 In contrast, bovine cerebral ECs exposed to OGD for up to 2 hours showed virtually no delayed cell death during the same period. The in vitro findings are consistent with the in vivo observation showing that ECs are more resistant to focal cerebral ischemia than are neurons.2

To further explore whether iNOS expression contributes to OGD-induced bovine cerebral EC death, we tested NOS inhibitors that are either nonspecific (L-NA) or iNOS selective (NIL). L-NA and NIL were effective in partially reducing OGD-induced death in bovine cerebral ECs. The notion that bovine cerebral EC death after OGD is mediated by NO is further suggested by the cytoprotective role of an NO scavenger, (MGD)2-Fe2+. Because iNOS was induced and because it produces substantially more NO than that catalyzed by endothelial NOS, the findings are consistent with a causal role of iNOS in OGD-induced cell death. However, to what extent iNOS-mediated cell death is apoptotic in nature cannot be ascertained. Also, the protection of bovine cerebral EC death by these agents was not complete, suggesting that mechanisms other than the iNOS pathway may contribute to OGD-induced bovine cerebral EC death.

In summary, results from the present study show that apoptosis may be involved in OGD-induced bovine cerebral EC death. The OGD death paradigm probably involves multiple mechanisms, with iNOS expression contributing partially to bovine cerebral EC death. Understanding the mechanism of cerebral EC death after OGD may aid in the development of therapeutic strategies to reduce secondary ischemic brain injury caused by postischemic hypoperfusion and blood-brain barrier dysfunction.


*    Acknowledgments
 
This work was supported in part by NIH grants NS25545, NS28995, and NS37230 and an Office of Naval Research grant. We thank Y. Kim, Andrew Tsung, and Dr Jinming Xu for technical assistance and Dr Monte Lai of Medinox Pharmaceuticals (San Diego, Calif) for the generous supply of Fe2+-N-methyl-D-glucamine dithiocarbamate complex.

Received February 10, 2000; revision received March 30, 2000; accepted April 4, 2000.


*    References
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 up arrowMaterials and Methods
 up arrowResults
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 *References
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Editorial Comment

Costantino Iadecola, MD, Guest Editor

Center for Clinical and Molecular Neurobiology, Departments of Neurology and Neuroscience, University of Minnesota, Minneapolis, Minnesota


*    Introduction 
up arrowTop
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 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 up arrowReferences
 *Introduction 
down arrowReferences 
 
Most of the effort in elucidating the cellular biological mechanisms of ischemic brain injury has appropriately focused on neurons. However, there is increasing evidence that ischemia-induced vascular damage is an integral step in the cascade of the cellular and molecular events initiated by cerebral ischemia.R1 While endothelial cells express critical inflammatory mediators in response to ischemia, alterations in integrin expression and endothelial function may lead to increased vascular permeability, edema formation, and hemorrhage.R2 However, little is known about the effects of hypoxia-ischemia on cerebral endothelial cells. In the accompanying article, Xu and colleagues investigated the mechanisms of endothelial injury in bovine cerebral endothelial cell cultures subjected to oxygen-glucose deprivation. They found that oxygen-glucose deprivation induces expression of inducible nitric oxide synthase (iNOS) in endothelial cells and leads to cell death by a mechanism resembling apoptosis. Pharmacological inhibition of iNOS attenuates endothelial cell death, which suggests that NO produced by iNOS, probably through peroxynitrite, its reaction product with superoxide, is involved in the killing.

This is an important contribution that sheds light on a poorly understood, yet highly relevant aspect of the cellular biology of cerebral ischemia. The observation that endothelial cells undergo apoptosis is an exciting new finding that provides a previously unrecognized mechanism by which antiapoptotic agents, such as caspase inhibitors, may protect the ischemic brain.R3 Furthermore, the observation that iNOS may be linked to endothelial apoptosis provides an insight into the pathogenic significance of the endothelial iNOS expression observed in models of cerebral ischemia and in human stroke as well.R4 R5 R6 While large amounts of NO generated by iNOS could kill endothelial cells by an apoptotic mechanism, they could also produce endothelial cell dysfunction, resulting in vascular dysregulation and exacerbation of ischemia. The latter possibility is supported by recent findings that iNOS gene transfer to cerebral arteries blocks endothelium-dependent relaxation of the transfected vessel.R7 Therefore, the findings of the present study suggest that vascular iNOS expression could contribute to brain injury by multiple mechanisms.

Received February 10, 2000; revision received March 30, 2000; accepted April 4, 2000.


*    References 
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 up arrowReferences
 up arrowIntroduction 
 *References 
 

  1. del Zoppo GJ, von Kummer R, Hamann GF. Ischaemic damage of brain microvessels: inherent risks for thrombolytic treatment in stroke. J Neurol Neurosurg Psychiatry.. 1998;65:1–9.[Free Full Text]
  2. Barone F, Feuerstein GZ. Inflammatory mediators and stroke: new opportunities for novel therapeutics. J Cereb Blood Flow Metab.. 1999;19:819–834.
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  4. Forster C, Clark HB, Ross ME, Iadecola C. Inducible nitric oxide synthase expression in human cerebral infarcts. Acta Neuropathol (Berl).. 1999;97:215–220.
  5. Iadecola C, Zhang F, Xu X, Casey R, Ross ME. Inducible nitric oxide synthase gene expression in brain following cerebral ischemia. J Cereb Blood Flow Metab.. 1995;15:378–384.[Medline] [Order article via Infotrieve]
  6. Galea E, Golanov EV, Feinstein DL, Kobylarz KA, Glickstein SB, Reis DJ. Cerebellar stimulation reduces inducible nitric oxide synthase expression and protects brain from ischemia. Am J Physiol.. 1998;274:H2035–H2045.
  7. Gunnett CA, Faraci FM, Chu Y, Brooks RM, Heistad DD. Impaired endothelium-dependent relaxation following gene transfer of iNOS: role of superoxide and L-arginine. FASEB J.. 2000;14:A118. Abstract.



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