| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
GROWTH, DIFFERENTIATION, AND APOPTOSIS
in cerebral vascular endothelial cells
1Laboratory for Research in Neonatal Physiology, Department of Physiology, Vascular Biology Center, University of Tennessee Health Science Center, Memphis, Tennessee; and 2Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania
Submitted 25 January 2006 ; accepted in final form 30 June 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
(TNF-) causes oxidative stress and apoptosis in a variety of cell types. Heme oxygenase (HO) degrades heme to bilirubin, an antioxidant, and carbon monoxide (CO), a cell cycle modulator, and a vasodilator. Newborn pig cerebral microvascular endothelial cells (CMVEC) highly express constitutive HO-2. We investigated the role of HO-2 in protection against TNF--induced apoptosis in cerebral vascular endothelium. In CMVEC from mice and newborn pigs, 15 ng/ml TNF- alone, or with 10 µg/ml cycloheximide (CHX) caused apoptosis detected by nuclear translocation of p65 NF-
B, caspase-3 activation, DNA fragmentation, cell-cell contact destabilization, and cell detachment. TNF- did not induce HO-1 expression in CMVEC. CMVEC from HO-2 knockout mice showed greater sensitivity to apoptosis caused by serum deprivation and TNF- than did wild-type mice. TNF- increased reactive oxygen species generation, including hydrogen peroxide and superoxide radicals, as detected by dihydrorhodamine-123 and dihydroethidium. The TNF- response was inhibited by superoxide dismutase and catalase suggesting apoptosis is oxidative stress related. Inhibition of endogenous HO-2 in newborn pig CMVEC increased oxidative stress and exaggerated apoptosis caused by serum deprivation and TNF-. In HO-1-overexpressing CMVEC (HO-1 selective induction by cobalt portophyrin), TNF- did not cause apoptosis. A CO-releasing compound, CORM-A1, and bilirubin blocked TNF--induced reactive oxygen species accumulation and apoptosis consistent with the antioxidant and antiapoptotic roles of the end products of HO activity. We conclude that HO-2 is critical for protection of cerebrovascular endothelium against apoptotic changes induced by oxidative stress and cytokine-mediated inflammation. carbon monoxide; bilirubin; vascular injury; reactive oxygen species; heme oxygenase; cycloheximide
(TNF-) plays a major role in inflammatory responses causing apoptosis by signaling through death receptors. The receptor of TNF- (TNFR1) recruits cytoplasmic signaling molecules and forms the death-induced signaling complex, which activates serine proteases, known as caspases, that execute the terminal phases of apoptosis (1, 8, 62). In the brain, TNF- produced by astrocytes, microglia, and endothelial cells contribute to inflammation, sepsis, stroke, and seizures (3, 21, 23, 37, 42). Seizure-induced neuronal degeneration has been linked to TNFR1-mediated apoptosis (4, 57).
Endothelial cells from cerebral microvessels release vasoactive factors and messenger molecules that play key roles in the regulation of cerebral blood flow. Inflammation and oxidative stress following ischemia, seizures, and brain trauma can cause severe endothelial cell injury (6, 29, 31, 64). Endothelial cells are also involved in inflammatory and immune reactions by producing and responding to cytokines (19, 29, 31, 64, 68). Exposure of endothelial cells to TNF- may result in endothelial dysfunction (1, 8, 14, 24, 62). However, very little is known about apoptotic effects of TNF- in cerebral endothelial cells comprising the blood-brain barrier.
Generation of reactive oxygen species (ROS) plays a crucial role in endothelial death induced by TNF- (12, 15, 52). Endogenous antioxidant stress defense mechanisms are essential for protection from TNF--induced apoptosis. Heme oxygenase (HO) metabolizes pro-oxidant heme to carbon monoxide (CO), a gaseous messenger molecule, and biliverdin/bilirubin, potent antioxidants (35, 36). Induction of the stress-responsive HO-1 protected vascular endothelial cells from TNF--mediated apoptosis (7, 8).
Cerebral vascular endothelial cells are characterized by high expression and activity of the constitutive HO-2 isoform (48). In cerebral vasculature, HO-1 is not expressed under baseline conditions in vivo and is not induced by epileptic seizures (49), hydrogen peroxide-induced oxidative stress or the excitatory neurotransmitter, glutamate, in vitro (50). The significance of constitutive HO-2 in oxidative stress-induced injury and vascular cell death is poorly understood. HO-2 plays an important role in the regulation of cerebral blood flow in newborn pigs and rats (9, 32, 39, 48). HO-2 activity is critical in preventing seizure-related cerebral vascular injury in vivo (9, 49). Evidence in vitro suggests that HO-2 also contributes to protection against oxidative stress. HO-2 gene deletion increased hemin-induced injury in astrocytes (11) and sensitized cerebral vascular endothelial cells to glutamate-induced apoptosis (50). However, HO-2 did not reduce oxidative stress-mediated damage caused by arachidonic acid, hydrogen peroxide, and peroxynitrite in cerebral endothelial cells (50). Moreover, in astrocytes, HO-2 deletion sensitized the cells to oxidative stress-related hemin injury (11). Therefore, it appears HO-2 provides a cell- and stimuli-specific cytoprotection, rather than general antioxidant protection.
The present study, conducted in primary cultures of CMVEC from newborn pigs and wild-type (WT) and HO-2 knockout (KO) mice, addresses the hypotheses that 1) TNF- causes cerebral vascular endothelial death by apoptosis, 2) HO-2 provides endogenous protection against TNF--induced endothelial apoptosis, and 3) CO and bilirubin, the products of HO-2 pathway, protect cerebral vascular endothelium from oxidative stress and apoptosis caused by TNF-.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
HO-2 KO mice. The HO-2 KO mice used in this study were descendents of those produced by Poss et al. (50) by replacing a region of mouse HO-2 sequence that corresponds to rat HO-2 exons 4 and 5 with a neomycin resistance cassette containing a pgk promoter. The HO-2 KO mice were cross-bred with WT C57B1/6 x 129/Sv mice (50). All mice were the product of heterozygous matings, so the HO2-KO and HO2-WT animals are of the same genetic background. The genotype was identified by PCR as described previously (50). The mice were bred and then euthanized at 3–4 mo of age at Thomas Jefferson University for isolation of cerebral vascular endothelial cells.
Isolation of cerebral microvessels and cell cultures. Cerebral cortex was obtained from anesthetized newborn pigs and HO-2 KO or WT mice (3 to 4 mo old) (48). CMVEC were isolated from cerebral vessels (60–300 µm) by collagenase-dispase digestion, and separated by centrifugation in a Percoll gradient (48). CMVEC were plated on Matrigel-coated plates or glass coverslips, and cultured in DMEM with 20% FBS, 30 µg/ml endothelial cell growth supplement, 1 U/ml heparin, and antibiotic/antimycotic mixture for 5–6 days until confluence was reached. All experiments were performed on confluent quiescent cells in primary or first passage cultures. To achieve quiescence, CMVEC were serum starved overnight in DMEM with 0.1% FBS.
Experimental treatments.
Confluent quiescent CMVEC were incubated with 15 ng/ml of TNF- and/or 10 µg/ml CHX for 3–6 h at 37°C, in the presence or absence of tin protoporphyrin (SnPP, 20µM), chromium mesoporphyrin (CrMP, 20 µM), N-tosyl-L-phenylalanine chloromethyl ketone (TPCK, 20–40 µM), parthenolide (40–60 µM), superoxide dismutase (SOD) or its permeable analogue (PEG-SOD, 1,000 U), PEG-catalase (500 U), bilirubin (0.1–1 µM) or CORM-A1 (1–50 µM). All protoporphyrin solutions were freshly prepared by dissolving in Dulbecco’s phosphate-buffered saline (DPBS) containing 0.5% ethanolamine. Bilirubin was dissolved in distilled water with the addition of 1–2 drops of NaOH, and pH was adjusted to 7.4 with Tris·HCl. To stabilize bilirubin, a bilirubin/albumin mixture (molar ratio, 1:5) was prepared (18). A CO donor, sodium borocarbonate (CORM-A1), a boron-based carbonylating agent that slowly releases CO in neutral and acidified aqueous solutions (41), was diluted in DPBS immediately before the experiment.
Quantitative detection of DNA fragmentation by ELISA.
DNA fragmentation was detected with an apoptosis detection kit (Roche Applied Science, Indianapolis, IN), which allows quantitative detection of cytoplasmic histone-associated-DNA fragments (mono- and oligonucleosomes). Quiescent CMVEC were treated with TNF- (15 ng/ml) and/or CHX (10 µg/ml) for 3 h. The detached cells were collected for counting. The attached cells were washed with DPBS and lysed with the solution provided. Aliquots of the nuclei-free supernatant were placed in streptavidin-coated wells and incubated with anti-histone-biotin antibody and anti-DNA peroxidase-conjugated antibody for 2 h at room temperature. Immunobilized DNA fragments were visualized with 2,2'-azino-di(3-ethylbenzthiazolin-sulfonate), and the absorbance at 405 nm was detected (5).
Protein detection. CMVEC were lysed with the mammalian protein extraction reagent buffer (M-PER; Pierce, Rockford, IL). The protein contents were measured by the bicinchoninic acid method using a kit from Pierce, or the dot-blot method which allows determination of proteins dissolved in Laemmli sample buffer by their ability to quantitatively bind amidoblack 10B (25).
HO-1 overexpression in porcine CMVEC. Cobalt protoporphyrin (CoPP) is a potent and selective HO-1 inducer in vitro and in vivo (49). Quiescent CMVEC were treated with 20µM of CoPP in serum-depleted DMEM (0.1% FBS) for 3 h. After treatment, CoPP was removed from the media, and the incubation continued overnight in fresh DMEM with 0.1% FBS. Under these conditions, HO-1 expression was upregulated 10-fold.
Western blot analysis. CMVEC were solubilized in Laemmli sample buffer (100°C for 10 min). Proteins (20–50 µg/lane) were separated by 15% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% BSA-0.1% Tween 20. Caspase-3 is expressed as an inactive 32-kDa precursor from which a variety of cleaved active subunits (11–19 kDa) are proteolytically generated during apoptosis. Caspase-3 activation was detected by formation of the 17- and 19-kDa proteolytic fragments using highly selective polyclonal antibodies (caspase-3-Asp 175, Cell Signaling, Beverly, MA). To detect HO-1, membranes were probed with polyclonal antibody against human HO-1 (1:5,000) (SPA 895, Stressgen, Victoria, Canada), followed by peroxidase-conjugated anti-rabbit IgG (Sigma, St. Louis, MO). To normalize caspase-3 and HO-1 expression to actin, the membranes were reprobed with monoclonal anti-actin (Roche Molecular Biochemicals, Indianapolis, IN) (1:10,000), followed by peroxidase-conjugated anti-mouse IgG (1:10,000). The immunocomplexes were visualized with the Western lightning chemiluminescence kit (Perkin Elmer Life Sciences, Boston, MA) and quantified by digital densitometry using NIH Image J 1.33 software.
Immunostaining.
Control or treated cells were fixed with 3.7% paraformaldehyde (pH 7.4, 20 min), permeabilized with 0.1% Triton X-100 (20 min), and blocked with 5% BSA/PBS (1 h), at room temperature. The cells were incubated with monoclonal antibodies against NF-B p65 (1:30) (BD Transduction, San Diego, CA), or 
-catenin (1:20) (Cell Signaling) for 1 h at 37°C, followed by FITC-conjugated secondary antibody (Vector Laboratories, Burlingame, CA) for 1 h at 37°C. For F-actin staining, cells were incubated with rhodamine-phalloidin (1:100, Molecular Probes, Eugene, OR). Coverslips were mounted using antifade mounting medium (Vector Laboratories). Slides were viewed using an Image Deconvolution system consisting of Nikon Diaphot microscope with fluorescein filter coupled to MacQuadra 950 computer with a Mac processor 601 in conjuntion with a cooled charge-coupled device camera (model 250 CH; Nikon). Images were deconvolved using IPLab Spectrum software for image collection. Digital processing of the images was done using Photoshop (Adobe Systems).
Cytotoxicity assay. Cytotoxicity was estimated by release of a cytosolic adenylate kinase (AK) activity into the medium (cytolysis) detected using the ToxiLight bioluminescent cytotoxicity kit (Cambrex, Rockland, ME).
Detection of oxidant generation.
To assess intracellular ROS, we used two independent oxidant-sensitive probes, dihydrorhodamine-123 (DHR123) and dihydroethidium (DHE) (50). DHR123, a cell-permeable nonfluorescent compound, is oxidized to fluorescent rhodamine 123 (Rh123) by a variety of ROS, especially H2O2 and peroxynitrite, and accumulates in mitochondria. DHE, a superoxide-sensitive probe, is oxidized by O2–· to ethidium and oxyethidium, intercalates with DNA in the nucleus and emits red fluorescence. To visualize ROS formation in live cells by fluorescence microscopy, CMVEC were preloaded with 50 µM DHR123 (20 min at 37°C) and incubated with 15 ng/ml TNF- for an additional 20 min. Cells were rapidly rinsed 3 times with DPBS and observed under a Nikon Diaphot microscope with a fluorescein filter. For spectroscopic measurements of ROS generation, CMVEC were incubated with 15 ng/ml TNF- (alone or with antioxidants as indicated) for 3 h. DHR123 (50 µM) or DHE (20 µM) were added to the incubation media for additional 20 min. To detect overall cytokine-induced oxidative stress, both attached and floating cells were collected by scraping and aspiration. Collected cells were diluted 10 times with ice-cold PBS and pelleted at 3,000 g for 10 min at 4°C. The process was repeated twice to remove free probes. The cell pellet was disrupted by sonication in 100 µl of ice-cold DPBS, and cell debris was pelleted. Supernatant was transferred to Falcon 96-well black microplates in duplicates, and fluorescence was measured with a Fusion HT Universal Microplate Analyzer (Packard Instrumental) using excitation and emission wavelengths 470/525 nm for Rh123, or 490/605 nm for ethidium.
Statistical analysis. Data are presented as means ± SE of absolute values or percent of control. Data were analyzed by analysis of variance, followed by the Tukey-Kramer multiple-comparison test to isolate differences between groups. P < 0.05 was considered significant in all statistical tests.
Materials.
Cell culture reagents were purchased from Life Technologies (Gaithersburg, MD), Hyclone (South Logan, UT) and Amersham Pharmacia Biotech (Piscataway, NJ). Human and rat TNF- and Matrigel were from BD Biosciences (Bedford, MA). CORM A-1 was a generous gift from Tyco Healthcare/Mallincrodt (Petten, The Netherlands). SnPP and CoPP were purchased from Frontier Scientific (Logan, UT). DHR123 and DHE were from Molecular Probes. All other reagents were from Sigma.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
in mouse CMVEC.
Primary cultures of CMVEC isolated from HO2-KO and HO2-WT mice brains were established (50). Genotypes of HO2-KO and WT mice were confirmed by PCR, immunoblotting, and immunostaining for HO-2 (50). HO-1 expression was not upregulated in HO-2 gene-deleted CMVEC (50). We compared the apoptotic effects of rat TNF- and CHX, alone or in combination, in WT and HO2-KO mouse CMVEC. Following overnight serum withdrawal, HO2-KO CMVEC had increased apoptosis as indicated by DNA fragmentation (Fig. 1A), cell detachment (Fig. 1B), and caspase-3 activity (Fig. 1C). TNF- (15 ng/ml) combined with CHX (10 µg/ml) increased apoptosis in mouse CMVEC, whereas TNF- or CHX were without effects (Figs. 1, A–C). HO2-KO CMVEC showed much greater sensitivity to TNF/CHX induced apoptosis evidenced by greater DNA fragmentation (Fig. 1A), cell detachment (Fig. 1B), and caspase-3 activity (Fig. 1C) compared with WT CMVEC. These data indicate that HO-2 gene deletion increases the sensitivity of CMVEC to TNF/CHX induced apoptosis.
|
-induced NF-B translocation in mouse CMVEC.
Transcription factor NF-B, a critical mediator of inflammatory apoptotic responses, resides in the cytoplasm of unstimulated cells. Upon initiation of apoptosis, NF-B translocates to the nucleus (40, 52). In WT CMVEC, NF-B is in the cytoplasm, whereas in HO2-KO CMVEC nuclear localization is already detectable under basal condition (Fig. 2, A and D). In HO2-KO CMVEC, nuclear translocation of NF-B was observed after a 15-min treatment with TNF-/CHX in 40 ± 10% cells (5 microscopic fields in 3 independent preparations), whereas in WT CMVEC, NF-B remained in the cytoplasm (no cells in 5 microscopic fields in 3 independent preparations) (Fig. 2, B and E). After a 1-h treatment with TNF-/CHX, complete cytosolic-nuclear translocation of NF-B was observed in both WT and HO2-KO cells (90 ± 5% and 95 ± 5% of the total cells, respectively, 5 microscopic fields in 3 independent preparations; Fig. 2, C and F). These data indicate the HO2 gene deletion accelerates the cytokine-induced NF-B nuclear translocation, a key indicator of apoptosis.
|
-induced cytotoxicity in HO2-KO CMVEC.
Cytolysis, a major indicator of cytotoxicity, was measured by release of cytosolic adenylate kinase (AK) into the media. In WT CMVEC, no cytotoxic effects were observed following a 3-h treatment with rat TNF- (15 ng/ml) and/or CHX (10 µg/ml). In contrast, in HO2-KO CMVEC, AK activity was increased to 230 ± 50% following a 3-h incubation with TNF-/CHX (P < 0.05 compared with baseline values, N = 4 independent cultures). Neither TNF- nor CHX alone had cytotoxic effects in HO2-KO CMVEC.
TNF- induces apoptosis in CMVEC from newborn piglets.
We investigated whether TNF-, alone or in combination with CHX, causes apoptosis in CMVEC from newborn pigs that constitutively express high HO-2 activity. In porcine CMVEC, human TNF- alone (15 ng/ml, 3 h) caused significant DNA fragmentation and cell detachment (threefold above baseline; see Fig. 3, A and B). CHX (10 µg/ml) alone increased DNA fragmentation and cell detachment and greatly potentiated TNF--induced apoptosis. These data suggest that in contrast to mouse WT cells, TNF- alone is a potent inducer of apoptosis in piglet CMVEC.
|
does not upregulate HO-1 in mouse or piglet CMVEC.
In piglet cerebral microvessels, HO-2 is highly expressed (Fig. 4A), while HO-1 is not detectable (49). In cultured mouse and piglet CMVEC, baseline HO-1 protein level is detectable (Fig. 4, B and C). Prolonged (6–12 h) treatment with TNF- or TNF-/CHX did not upregulate HO-1 expression in HO2-KO (mouse) or HO2-WT (mouse and piglet) cells (Fig. 4, B and C).
|
-induced apoptosis.
To investigate whether constitutive HO-2 plays a role in CMVEC survival, we used SnPP (20 µM) that effectively inhibits HO activity in CMVEC (33–35, 38). SnPP increased the basal level of apoptosis caused by serum deprivation as evidenced by DNA fragmentation (Fig. 5A) and caspase-3 activation (Fig. 5B). Furthermore, SnPP increased DNA fragmentation and caspase-3 activation caused by TNF- alone or in combination with CHX (Fig. 5, A and B). In contrast, SnPP did not enhance apoptotic changes induced by CHX alone (Fig. 5, A and B).
|
B in TNF--induced apoptosis.
The translocation of NF-B from cytoplasmic to nuclear sites is the key event of apoptosis (40, 52). Human TNF- (15 ng/ml, 3 h) and CHX (10 µg/ml, 3 h), alone or in combination, caused NF-B nuclear translocation in piglet CMVEC (Fig. 6, A–D), consistent with apoptosis. To determine the role of NF-B, we used TPCK, a serine/threonine protease inhibitor that selectively inhibits NF-B signaling (65) and parthenolide, a sesqiterpene lactone, that inhibits NF-B activation by directly interacting with p65 subunit of NF-B and, to a minor degree, by inhibition of IB- degradation (22). TPCK (20–40 µM) and parthenolide (40–60 µM) blocked or greatly reduced DNA fragmentation response to TNF-, CHX, and TNF/CHX (Fig. 7A). Both inhibitors dose-dependently inhibited caspase-3 activation: 1) in low concentrations, they prevented complete proteolysis of the zymogen to a 17-kDa fragment, resulting in accumulation of a 19-kDa band was accumulated; 2) in higher concentrations, both completely blocked caspase-3 activation evidenced by the absence of both the 17- and 19-kDa bands (Fig. 7B). These data indicate that NF-B has pro-apoptotic role in TNF--evoked apoptosis signaling in CMVEC.
|
|
-induced apoptosis.
CoPP is a potent HO-1 inducer in CMVEC (49). We investigated whether HO-1 is protective against TNF--mediated cell death. HO-1 expression in piglet CMVEC was upregulated 
10-fold by a 3-h treatment with 20 µM CoPP, followed by overnight incubation in the starvation medium (Fig. 8B). HO-1 overexpressing CMVEC did not respond to TNF- or TNF-/CHX by apoptosis-related DNA fragmentation and caspase-3 activation (Fig. 8, A and B). Surprisingly, inhibition of HO-1 activity by SnPP did not restore apoptotic responses to TNF- and/or CHX (Fig. 8, A and B).
|
-induced apoptosis in piglet CMVEC.
We investigated whether bilirubin or CO can protect piglet CMVEC from TNF--induced apoptosis. CMVEC were treated with TNF- and/or CHX for 3 h in the absence or presence of bilirubin and CORM-A1, a spontaneous CO donor. Bilirubin (1 µM) inhibited DNA fragmentation (Fig. 9A) and caspase-3 activation (Fig. 9B) caused by TNF-. Bilirubin was less effective in inhibiting TNF-/CHX-induced apoptosis, although reduction of DNA fragmentation and caspase-3 activation were also observed (Fig. 9, A and B). CORM-A1 (50 µM) completely prevented DNA fragmentation and caspase-3 activation in response to TNF- alone and greatly reduced apoptotic changes caused by a TNF-/CHX treatment (Fig. 10, A and B). These data indicate that both bilirubin and CO are anti-apoptotic factors that attenuate TNF--induced endothelial death.
|
|
-induced oxidative stress in piglet CMVEC.
To determine whether HO-2 inhibition alters ROS formation, CMVEC were treated with HO inhibitors, SnPP or CrMP, for 3 h. ROS production was measured with two independent probes, DHR123 (total ROS formation) and DHE (a superoxide-sensitive probe). SnPP (20 µM) and CrMP (20 µM) greatly increased ROS formation. Rh123 fluorescence, 32 ± 3, 168 ± 12, and 234 ± 25 optical density units in control, SnPP-treated, and CrMP-treated cells, respectively (P < 0.05 compared with control values, n = 3 independent cultures in duplicates); ethidium fluorescence, 10 ± 2, 35 ± 6, and 31 ± 7 in control, SnPP-treated, and CrMP-treated cells, respectively (P < 0.05 compared with the control values, n = 3 independent cultures in duplicate). These data demonstrate antioxidant potential of endogenous HO-2 activity.
TNF- (15 ng/ml, 3 h) greatly increased ROS formation detected by Rh123 and ethidium fluorescence microscopy (Fig. 11, A and B) and spectroscopy (Fig. 12, A and B). TNF- also disrupted the junctional integrity of cerebral vascular endothelium as detected by -catenin immunostaining. In control CMVEC, -catenin, an integral component of cell-cell adherence junctions responsible for the maintenance of endothelial barrier functions, is localized to the CMVEC periphery (Fig. 11C). In TNF--treated cells, a notable disruption of the cell-cell contacts was observed as evidenced by punctate staining, membrane blebbing, and decreased appearance of -catenin at the endothelial adherence junction areas (Figs. 11D).
|
|
(Fig. 12, A and B). SOD and its permeable derivative PEG-SOD (1,000 U/ml) blocked TNF--induced Rh123 and ethidium fluorescence signals (Fig. 12, A and B). The cell-permeable derivative of catalase, 500 U/ml PEG-catalase, also reduced Rh123 fluorescence (Fig. 12A). Overall, these data indicate that endogenous HO-2 metabolites exert antioxidant effects in serum-starved and TNF--treated CMVEC. |
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
, alone (in piglet cells) or in combination with the protein synthesis inhibitor CHX (in piglet and mouse cells), causes oxidative stress-mediated apoptosis detected by caspase-3 activation, NF-B cytoplasmic-nuclear translocation, cell-cell contact disruption, DNA fragmentation and cell detachment, 2) HO2 gene deletion and inhibition of HO-2 activity greatly potentiate TNF- and/or TNF-/CHX-induced apoptosis, 3) products of endogenous HO-2 activity, CO and bilirubin, protect against oxidant-mediated apoptotic responses to TNF-, 4) TNF- does not induce HO-1 in pig or mouse HO2-KO or WT cells. Overall, these data strongly suggest that endogenous HO-2 is a physiologically important component of antioxidant protection from inflammation-induced cell damage and death in the cerebrovascular endothelium.
TNF- is a pleiotropic proinflammatory cytokine that may cause cell death by apoptosis (5, 10, 52). In the brain, endogenous TNF- produced by astrocytes, infiltrating macrophages, and cerebrovascular endothelium, contributes to neurological disease including inflammation, stroke, and seizures (3, 21, 23, 37, 42). Accumulating evidence demonstrates that TNF- contributes to detrimental effects of epileptic seizures in the brain. In pediatric patients with histories of febrile convulsions, TNF- levels in blood were steadily elevated (63). During sustained epileptic seizures in animal models, TNF- production by the brain increases (53, 63). Seizure-evoked neuronal degeneration has been linked to TNFR1-mediated apoptosis (4, 57). Epileptic seizures cause sustained cerebrovascular endothelial injury in the newborns (49). Our data indicate that TNF- may be involved in the pathophysiology of cerebrovascular injury by initiating endothelial apoptosis that includes cytoplasmic-nuclear NF-B translocation, caspase-3 activation, DNA fragmentation, disruption of cell-cell contacts, and cell detachment.
Many cell types respond to TNF- only when intracellular protein synthesis is inhibited at the transcriptional or translational levels, implying that apoptosis involves de novo production of intracellular inducers, including short-lived transcriptional regulators and anti-apoptotic factors (5, 27, 51). We found species- and/or age-specific differences in the responses of cerebral vascular endothelium to TNF-. In adult mouse CMVEC, TNF- initiated apoptosis only when the cells were sensitized by cycloheximide, a protein synthesis inhibitor. In newborn pig CMVEC, TNF- alone was sufficient to induce apoptosis, although cycloheximide also greatly potentiated cell death. We therefore conclude that inhibition of de novo protein synthesis is required for the induction of cell death in mouse and, to a lesser extent, in piglet CMVEC.
We provide evidence that in cerebral vascular endothelium, apoptotic responses to TNF- are mediated by oxidative stress. Oxidative stress is the consequence of an imbalance between the formation of ROS and the antioxidant capacity of the cell (43). ROS include superoxide anion radical, hydrogen peroxide, peroxynitrite, and hydroxyl radical that readily react with various biological molecules including membrane lipids, cytoskeletal proteins, and DNA. TNF- rapidly and steadily increased ROS formation in CMVEC as detected using DHR123 (overall ROS production, preferentially hydrogen peroxide and peroxynitrite) and a superoxide-selective probe, DHE. SOD and its permeable analogue, PEG-SOD, as well as PEG-catalase blocked both the ROS and apoptosis responses to TNF-, indicating that superoxide and hydrogen peroxide are among the leading factors in the cytokine-induced endothelial damage.
HO-1 gene belongs to a group of antioxidant stress response elements, which can be induced by a variety of oxidative stress factors and is important in cell survival against oxidative stress (7, 13, 44, 45, 66). HO-1 is not expressed in cerebral vessels under physiological conditions, whereas in cultured mouse and pig CMVEC, basal HO-1 protein level is detectable due to the presence of growth-stimulating factors in the culture media. In contrast to other cell types, in cerebral vascular endothelium in vitro HO-1 is induced only by severe oxidative stress induced by peroxynitrite, but not hydrogen peroxide or excitotoxic glutamate (50). Our current study in pig and mouse CMVEC showed that HO-1 is not induced during oxidative stress conditions caused by apoptotic concentrations of TNF- alone or even combined with CHX. CoPP is the most potent and selective HO-1 inducer in cerebral vascular endothelium in vivo and in vitro (49). Antiapoptotic effects of HO-1 are clearly indicated by complete resistance of HO-1 overexpressing CMVEC to TNF--induced apoptosis. Surprisingly, we found that in HO-1-overexpressing CMVEC, the HO inhibitor, SnPP, did not reverse apoptosis protection to the cytokine. In contrast, SnPP greatly potentiated apoptotic responses in control CMVEC. It is possible that HO-1 and HO-2 isoforms have distinct sensitivity and/or selectivity to pharmacological inhibitors. Still it is also conceivable that HO-1 has anti-apoptotic endothelial protective functions that are not related to heme metabolizing activity. Indeed, in cerebral vascular endothelium HO-1 is localized to the nuclear envelope and the nucleus (47, 50), indicating possible nuclear functions of the isoform. However, because HO-1 is not inducible by TNF-, HO-1 cannot be considered a physiologically significant anti-apoptotic endogenous factor that may protect from cerebrovascular endothelial damage induced by TNF--mediated inflammation.
The cerebral microcirculation is characterized by a high expression and activity of constitutive HO-2 (35, 38). HO-2-produced CO is a paracrine and autocrine relaxing factor in the cerebral circulation that contributes to vasodilator responses to glutamate, hypoxia, and seizures in newborn pigs (9, 33–35, 49) and adult rats (39). Recently, we demonstrated that inhibition of HO-2 activity or HO-2 gene deletion exaggerated oxidative stress and apoptosis induced by glutamate in cerebral vascular endothelium (50). Our present data in newborn pigs and WT and HO-2 gene-deleted mice show that pharmacological inhibition or complete elimination of HO-2 activity causes excessive ROS formation during basal conditions and in response to TNF- stimulation, indicating that HO-2 is an antioxidant and antiapoptotic factor in cerebral circulation.
Bilirubin, a product of HO/biverdin reductase reactions, was long considered a toxic metabolite of heme catabolism responsible for the clinical manifestation of jaundice, particularly in neonates. Recently, the potent biological properties of bilirubin as a major physiological antioxidant have been recognized (61). Bilirubin is an effective scavenger of superoxide and peroxyl radicals (38, 61). Antioxidant properties of bilirubin have been also suggested based on the ability of biliverdin/bilirubin to undergo the redox cycling (2, 18, 36, 38, 61). We confirm the antioxidant potency of bilirubin in our experimental system by measuring ROS formation in TNF--treated cells. Increasing evidence suggests that bilirubin acts as a powerful chain-breaking antioxidant that may exert cytoprotective effects (17, 18, 61). In nervous tissue, bilirubin serves as an important cytoprotective factor (2, 17, 18). Our previous (50) and present data demonstrate that bilirubin is an extremely efficient antiapoptotic factor that protects cerebrovascular endothelium from oxidative stress-related apoptosis induced by glutamate and TNF-. Therefore, bilirubin can provide physiological cytoprotection against oxidant-mediated cell and tissue injury.
While accumulating evidence indicates that CO may also act as anti-apoptotic cytoprotective factor (28, 35, 50, 67), the possibility of antioxidant properties of CO needs to be addressed. Recently, we demonstrated that CO is a potent antioxidant and antiapoptotic factor that reduces glutamate-induced apoptosis in cerebral vascular endothelium (50). Our present data provide direct evidence that CO administered in the form of a CO-releasing molecule, CORM-A1, blunts severe oxidative stress caused by TNF- by reducing hydrogen peroxide and superoxide anion formation. Astoundingly, CORM-A1 was even more effective than SOD, catalase, or bilirubin, in preventing oxidative stress-related apoptosis in response to TNF- in cerebral vascular endothelium. On the basis of CO chemistry, it is unlikely that CO acts as a ROS scavenger. However, due to its heme-binding capacity, CO may inhibit oxidant-producing enzymes, including heme-containing mitochondrial cytochromes and the electron transport chain components, thus decreasing ROS formation. Recent papers present experimental evidence that CO and CO-releasing molecules do inhibit mitochondrial function and ROS generation (16, 30, 54, 55, 58, 59). Anti-oxidant properties of CO may also be due to interaction with the heme proteins of the NF-B signaling pathway (7, 8).
Nuclear transcription factor NF-B plays a key role in controlling apoptosis (26). The cellular consequences of the NF-B pathway, that include translocation to the nuclear sites upon activation, are complex and may produce both pro- and anti-apoptotic effects in a stimuli-dependent manner (20, 26, 40, 60). Cytoplasmic-nuclear NF-B translocation was observed in CMVEC in response to TNF-, CHX, or TNF-/CHX. HO-2 KO CMVEC showed accelerated NF-B translocation consistent with exaggerated apoptotic responses, including DNA fragmentation. NF-B signaling inhibitors, TPCK and parthenolide, blocked DNA fragmentation and caspase-3 activation initiated by TNF-, CHX, or TNF-/CHX. These findings suggest that NF-B plays a proapoptotic role in TNF-- and TNF-/CHX-induced apoptosis in cerebral vascular endothelium.
We present evidence that TNF- may cause disruption of the blood-brain barrier. -Catenin, a component of adherence junctions, is responsible for the maintenance of endothelial barrier functions. Junctional disassembly and modifications of the cadherin/catenin complex may lead to increased paracellular permeability of the endothelial barrier. -Catenin integrity is important for the survival from TNF--induced apoptosis (46, 56). TNF--mediated apoptosis in CMVEC was accompanied by loss of cell-cell contacts and disruption of -catenin at the cell periphery. These results suggest that progression of apoptosis in cerebral vascular endothelium is concomitant with the loss of integrity of adherence junctions resulting in cell detachment from the substratum.
Overall, the pleiotropic cytokine TNF- causes oxidative stress-mediated apoptosis in adult mouse and newborn piglet cerebral vascular endothelial cells. CO and bilirubin, products of heme degradation by the HO pathway, are potent antioxidants and antiapoptotic factors against endothelial injury caused by TNF-. HO-1 is not induced in cerebral vascular endothelium by TNF-, and, therefore, is not physiologically relevant to endogenous protection against the cytokine-induced cerebrovascular injury. On the other hand, the products of HO-2 activity appear to be essential components in endogenous defense against TNF--induced apoptosis in the cerebral vascular endothelium.
|
GRANTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
2. Baranano DE and Snyder SH. Neural roles for heme oxygenase: contrasts to nitric oxide synthase. Proc Natl Acad Sci USA 98: 10996–11002, 2001.
3. Benveniste EN. Inflammatory cytokines within the central nervous system: sources, function, and mechanism of action. Am J Physiol Cell Physiol 263: C1–C16, 1992.
4. Bengzon J, Mohapel P, Ekdahl CT, and Lindvall O. Neuronal apoptosis after brief and prolonged seizures. Prog Brain Res 135: 111–119, 2002.[Medline]
5. Bhattacharya S, Ray RM, Viar MJ, and Johnson LR. Polyamines are required for activation of c-Jun NH2-terminal kinase and apoptosis in response to TNF- in IEC-6 cells. Am J Physiol Gastrointest Liver Physiol 285: G980–G991, 2003.
6. Bidmon HJ, Gorg B, Palomero-Gallagher N, Schliess F, Gorji A, Speckmann EJ, and Zilles K. Bilateral, vascular and perivascular glial upregulation of heat shock protein-27 after repeated epileptic seizures. J Chem Neuroanat 30: 1–16, 2005.[CrossRef][ISI][Medline]
7. Brouard S, Otterbein LE, Anrather J, Tobiasch E, Bach FH, Choi AM, and Soares MP. Carbon monoxide generated by heme oxygenase-1 suppresses endothelial cell apoptosis. J Exp Med 192: 1015–1026, 2000.
8. Brouard S, Berberat PO, Tobiasch E, Seldon MP, Bach FH, and Soares MP. Heme oxygenase-1-derived carbon monoxide requires the activation of transcription factor NF-kappa B to protect endothelial cells from tumor necrosis factor-alpha-mediated apoptosis. J Biol Chem 277: 17950–17961, 2002.
9. Carratu P, Pourcyrous M, Fedinec A, Leffler CW, and Parfenova H. Endogenous heme oxygenase prevents impairment of cerebral vascular functions caused by seizures. Am J Physiol Heart Circ Physiol 285: H1148–H1157, 2003.
10. Chen G and Goeddel DV. TNF-R1 signaling: a beautiful pathway. Science 296: 1634–1635, 2002.
11. Chen J and Regan RF. Heme oxygenase-2 gene deletion increases astrocyte vulnerability to hemin. Biochem Biophys Res Commun 318: 88–94, 2004.[CrossRef][ISI][Medline]
12. Chiu JJ, Wung BS, Shyy JY, Hsieh HJ, and Wang DL. Reactive oxygen species are involved in shear stress-induced intercellular adhesion molecule-1 expression in endothelial cells. Arterioscler Thromb Vasc Biol 17: 3570–3577, 1997.
13. Choi AM and Alam J. Heme-oxygense-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury. Am J Respir Cell Mol Biol 15: 9–19, 1996.[Abstract]
14. Cohen GM. Caspases: the executioners of apoptosis. Biochem J 326: 1–16, 1997.[ISI][Medline]
15. Corda S, Laplace C, Vicaut E, and Duranteau J. Rapid reactive oxygen species production by mitochondria in endothelial cells exposed to tumor necrosis factor-alpha is mediated by ceramide. Am J Respir Cell Mol Biol 24: 762–768, 2001.
16. Di Noia MA, Van Driesche S, Palmieri F, Yang LM, Quan S, Goodman AI, and Abraham NG. Heme oxygenase-1 enhances renal mitochondrial transport carriers and cytochrome C oxidase activity in experimental diabetes. J Biol Chem 281: 15687–15693, 2006.
17. Dore S, Goto S, Sampei K, Blackshaw S, Hester LD, Ingi T, Sawa A, Traystman RJ, Koehler RC, and Snyder SH. Heme oxygenase-2 acts to prevent neuronal death in brain cultures and following transient cerebral ischemia. Neuroscience 99: 587–592, 2000.[CrossRef][ISI][Medline]
18. Dore S, Takahashi M, Ferris CD, Zakhary R, Hester LD, Guastella D, and Snyder SH. Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury. Proc Natl Acad Sci USA 96: 2445–2450, 1999.
19. Ergonul Z, Hughes AK, and Kohan DE. Induction of apoptosis of human microvascular endothelial cells by Shiga toxin 1. J Infect Dis 187: 154–158, 2003.[CrossRef][ISI][Medline]
20. Fan C, Yang J, and Engelhardt JF. Temporal pattern of NFkappaB activation influences apoptotic cell fate in a stimuli-dependent fashion. J Cell Sci 115: 4843–4853, 2002.[Medline]
21. Freyer D, Manz R, Ziegenhorn A, Weih M, Angstwurm K, Docke WD, Meisel A, Schumann RR, Schonfelder G, Dirnagl U, and Weber JR. Cerebral endothelial cells release TNF-alpha after stimulation with cell walls of Streptococcus pneumoniae and regulate inducible nitric oxide synthase and ICAM-1 expression via autocrine loops. J Immunol 163: 4308–4314, 1999.
22. Garcia-Pineres AJ, Castro V, Mora G, Schmidt TJ, Strunck E, Pahl HL, and Merfort I. Cysteine 38 in p65/NF-kappaB plays a crucial role in DNA binding inhibition by sesquiterpene lactones. J Biol Chem 276: 39713–39720, 2001.
23. Gourin CG and Shackford SR. Production of tumor necrosis factor-alpha and interleukin-1beta by human cerebral microvascular endothelium after percussive trauma. J Trauma 42: 1101–1107, 1997.[ISI][Medline]
24. Green DR and Evan GI. A matter of life and death. Cancer Cell 1: 19–30, 2002.[CrossRef][ISI][Medline]
25. Henkel AW and Bierger SC. Quantification of proteins dissolved in an electrophoresis sample buffer. Anal Biochem 223: 329–331, 1994.[CrossRef][ISI][Medline]
26. Karin M, Cao Y, Greten FR, and Li ZW. NF-kappaB in cancer: from innocent bystander to major culprit. Nat Rev Cancer 2: 301–310, 2002.[CrossRef][ISI][Medline]
27. Karsan A, Yee E, and Harlan JM. Endothelial cell death induced by tumor necrosis factor is inhibited by the Bcl-2 family member, A.1. J Biol Chem 271: 27201–21204, 1996.
28. Kim HP, Wang X, Zhang J, Suh GY, Benjamin IJ, Ryter SW, and Choi AM. Heat shock protein-70 mediates the cytoprotective effect of carbon monoxide: involvement of p38 beta MAPK and heat shock factor-1. J Immunol 175: 2622–2629, 2005.
29. Kimura H, Gules I, Meguro T, and Zhang JH. Cytotoxity of cytokines in cerebral microvascular endothelial cell. Brain Res 990: 148–156, 2003.[CrossRef][ISI][Medline]
30. Li MH, Cha YN, and Surh YJ. Carbon monoxide protects PC12 cells from peroxynitrite-induced apoptotic death by preventing the depolarization of mitochondrial transmembrane potential. Biochem Biophys Res Commun 3: 984–990, 2006.
31. Lin R, Liu J, Peng N, Gan W, Wang W, Han C, and Ding C. Lovastatin reduces apoptosis and downregulates the CD40 expression induced by TNF-alpha in cerebral vascular endothelial cells. Curr Neurovasc Res 3: 41–47, 2006.[CrossRef][ISI][Medline]
32. Leffler CW, Nasjletti A, Yu C, Johnson RA, Fedinec A, and Walker N. Carbon monoxide and cerebral microvascular tone in newborn pigs. Am J Physiol Heart Circ Physiol 276: H1641–H1646, 1999.
33. Leffler CW, Balabanova L, Fedinec AL, Waters CM, and Parfenova H. Mechanism of glutamate stimulation of CO production in cerebral microvessels. Am J Physiol Heart Circ Physiol 285: H74–H80, 2003.
34. Leffler CW, Balabanova L, Sullivan CD, Wand X, Fedinec AL, and Parfenova H. Regulation of CO production in cerebral microvessels of newborn pigs. Am J Physiol Heart Circ Physiol 285: H292–H297, 2003.
35. Leffler CW, Parfenova H, Jaggar JH, and Wang R. Carbon monoxide and hydrogen sulfide: gaseous messengers in cerebrovascular circulation. J Appl Physiol 100: 1065–1076, 2006.
36. Maines MD. The heme oxygenase system and its functions in the brain. Cell Mol Biol (Oxf) 46: 573–585, 2000.
37. Medana IM, Hunt NH, and Chaudhri G. Tumor necrosis factor-alpha expression in the brain during fatal mouse cerebral malaria: evidence for production by microglia and astrocytes. Am J Pathol 150: 1473–1486, 1997.[Abstract]
38. Minetti M, Mallozzi C, Di Stasi AM, and Pietraforte D. Blirubin is an effective antioxidant of peroxynitrite-mediated protein oxidation in human blood plasma. Arch Biochem Biophys 352: 165–174, 1998.[CrossRef][ISI][Medline]
39. Montecot C, Seylaz J, and Pinard E. Carbon monoxide regulates cerebral blood flow in epileptic seizures but not in hypercapnia. Neuroreport 9: 2341–2346, 1998.[ISI][Medline]
40. Mercurio F and Manning AM. NF-kappaB as a primary regulator of the stress response. Oncogene 18: 6163–6171, 1999.[CrossRef][ISI][Medline]
41. Motterlini R, Sawle P, Hammad J, Baines S, Alberto R, Foresti R, and Green CJ. CORM-A1: a new pharmacologically active carbon monoxide-releasing molecule. FASEB J 19: 284–286, 2005.
42. Nadeau S and Rivest S. Role of microglial-derived tumor necrosis factor in mediating CD14 transcription and nuclear factor kappa B activity in the brain during endotoxemia. J Neurosci 20: 3456–3468, 2000.
43. Nakao N and Brundin P. Neurodegeneration and glutamate induced oxidative stress. Prog Brain Res 116: 246–263, 1998.
44. Otterbein LE, Mantell LL, and Choi AM. Carbon monoxide provides protection against hyperoxic lung injury. Am J Physiol Lung Cell Mol Physiol 276: L688–L694, 1999.
45. Otterbein LE and Choi AM. Heme oxygenase: colors of defense against cellular stress. Am J Physiol Lung Cell Mol Physiol 279: L1029–L1037, 2000.
46. Pallares J, Martinez-Guitarte JL, Dolcet X, Llobet D, Rue M, Palacios J, Prat J, and Matias-Guiu X. Abnormalities in the NF-kappaB family and related proteins in endometrial carcinoma. J Pathol 204: 569–577, 2004.[CrossRef][ISI][Medline]
47. Porter AG and Janicke RU. Emerging roles of caspase-3 in apoptosis. Cell Death Differ 6: 99–104, 1999.[CrossRef][ISI][Medline]
48. Parfenova H, Neff RA III, Alonso JS, Shlopov BV, Jamal CN, Sarkisova SA, and Leffler CW. Cerebral vascular endothelial heme oxygenase: expression, localization, and activation by glutamate. Am J Physiol Cell Physiol 281: C1954–C1963, 2001.
49. Parfenova H, Carratu P, Tcheranova D, Fedinec A, Pourcyrous M, and Leffler CW. Epileptic seizures cause extended postictal cerebral vascular dysfunction that is prevented by HO-1 overexpression. Am J Physiol Heart Circ Physiol 288: H2843–H2850, 2005.
50. Parfenova H, Basuroy S, Bhattacharya S, Tcheranova D, Qu Y, Regan RF, and Leffler CW. Glutamate induces oxidative stress and apoptosis in cerebral vascular endothelial cells: contributions of HO-1 and HO-2 to cytoprotection. Am J Physiol Cell Physiol 290: C1399–C1410, 2006.
51. Polunovsky VA, Wendt CH, Ingbar DH, Peterson MS, and Bitterman PB. Induction of endothelial cell apoptosis by TNF : modulation by inhibitors of protein synthesis. Exp Cell Res 214: 584–594, 1994.[CrossRef][ISI][Medline]
52. Rahman A, Kefer J, Bando M, Niles WD, and Malik AB. E-selectin expression in human endothelial cells by TNF--induced oxidant generation and NF-B activation. Am J Physiol Lung Cell Mol Physiol 275: L533–544, 1998.
53. Rizzi M, Perego C, Aliprandi M, Richichi C, Ravizza T, Colella D, Veliskova J, Moshe SL, De Simoni MG, and Vezzani A. Glia activation and cytokine increase in rat
P < 0.05, compared with corresponding control values.
