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Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Endothelial cell ICAM-1 upregulation in
response to TNF-
is mediated in part by reactive oxygen species
(ROS) generated by the endothelial membrane-associated NADPH oxidase
and occurs maximally after 4 h as the synthesis of new protein is
required. However, thrombin-stimulated P-selectin upregulation is
bimodal, the first peak occurring within minutes. We hypothesize that
this early peak, which results from the release of preformed P-selectin
from within Weibel-Palade bodies, is mediated in part by ROS generated from the endothelial membrane-associated xanthine oxidase. We found
that this rapid expression of P-selectin on the surface of endothelial
cells was accompanied by qualitatively parallel increases in ROS
generation. Both P-selectin expression and ROS generation were
inhibited, dose dependently, by the exogenous administration of
disparate cell-permeable antioxidants and also by the inhibition of
either of the known membrane-associated ROS-generating enzymes NADPH
oxidase or xanthine oxidase. This rapid, posttranslational cell
signaling response, mediated by ROS generated not only by the classical
NADPH oxidase but also by xanthine oxidase, may well represent an
important physiological trigger of the microvascular inflammatory response.
signal transduction; NADPH oxidase; xanthine oxidase; oxidant signaling
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ENDOTHELIAL CELLS
interact with circulating leukocytes via the expression of cell
surface-associated molecules and thereby effect a variety of
physiological and pathophysiological functions that comprise the
microvascular inflammatory response. These include the rolling,
accumulation, diapedesis, and activation of circulating polymorphonuclear leukocytes, facilitating host defense against circulating microorganisms (27, 32), as well as the
initiation of leukocyte-mediated tissue injury following
ischemia-reperfusion (15). The upregulation of
these cell surface molecules in response to circulating cytokines is
mediated, at least in part, by reactive oxygen species (ROS) generated
locally at sites of tissue injury or inflammation (2, 42).
For example, the upregulation of endothelial ICAM-1 expression after
TNF- stimulation appears to require the generation of ROS by the
endothelial membrane-associated NADPH oxidase (2).
Moreover, these ROS signaling mechanisms have been found to interact
with other, more classical signal transduction pathways, including the
upregulation of the nuclear transcription factors NF-
B and activator
protein-1 (AP-1) (29, 34, 35, 45, 50), and thereby appear
to play important signaling roles in the expression of a variety of
cell surface molecules (2, 45), in the intracellular
expression of heat shock proteins (30) and nitric oxide
(14), as well as in the regulation of TNF- production
(13), Fas ligand expression (48), and
apoptosis (11).
Conceptually, cellular signaling can be divided into rapid, posttranslational signaling (protein modification, which occurs within minutes) and the slower responses, which require the synthesis of new protein, including those cited above. Conceptually discrete from control by protein synthesis, there are a number of examples of rapid posttranslational protein modification as a means for the control of cellular signaling, including the complement cascade, the clotting cascade, and the renin-angiotensin axis (37). ROS, which are rapidly generated, highly reactive, and short-lived (nanoseconds to seconds) molecules, with short diffusion radii (often ångstroms to micrometers), are well suited to serve as second messengers for such rapid signaling systems. ROS-generating enzymes such as xanthine oxidase, whose activity is upregulated not only by protein synthesis, but also posttranslationally by limited proteolysis (4-6), could be well suited for this role as well.
P-selectin is one of the first cell surface molecules to be expressed on the endothelial cell in response to inflammatory stimuli and triggers initial neutrophil rolling along the microvascular endothelium (1, 10, 23, 25, 31, 38, 46). Its expression on the endothelial cell luminal surface in response to circulating mediators such as thrombin is bimodal, with an early peak occurring within minutes and a late peak occurring after 4-6 h (1). Whereas the late peak is transcriptionally regulated via protein synthesis, the early peak reflects the release of preformed P-selectin from Weibel-Palade (WP) bodies located inside the cell membrane (8, 10, 19, 20, 33, 51). However, the mechanisms by which this rapid P-selectin expression is triggered remain to be fully elucidated. We evaluated the hypothesis that this early, posttranslational response to stimulation by a prototypical agonist, thrombin, in human umbilical vein endothelial cells (HUVEC) is mediated, at least in part, by ROS generated from endothelial xanthine oxidase as well as from endothelial membrane-associated NAPDH oxidase.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Human Umbilical Vein Endothelial Cells
HUVEC were harvested from freshly and anonymously acquired human umbilical cords by a previously described and validated collagenase treatment protocol (22, 41), which had been approved by The Johns Hopkins University Joint Committee for Clinical Investigation. Briefly, fresh human umbilical cord segments were washed and flushed with Ca2+/Mg+-free PBS to remove clotted blood. Sixty milliliters of a 0.1% solution of collagenase type II (Worthington Biochemical; Lakewood, NJ) dissolved in Ca2+/Mg+-free PBS were gently infused into the umbilical vein and incubated at 37°C for 20 min. The collagenase-endothelial cell suspension was then allowed to drain slowly into a 50-ml tube. This tube was centrifuged for 10 min at 1,000 revolutions/min, and the pellet was then resuspended in a 10-ml stock of endothelial cell growth medium (EGM-2) supplemented with 2% heat-inactivated fetal calf serum, thymidine (10 µmol/l), glutamine (1.57 mmol/l), heparin sodium (10 IU/ml), penicillin (100 IU/ml), streptomycin (68.6 mmol/l), amphotericin B (135 mmol/l), and endothelial cell growth factor (588 pg/l) (all from Clonetics; Walkersville, MD).These primarily harvested HUVEC were plated onto 25-cm2 tissue culture flasks (Becton Dickinson; Franklin Lakes, NJ) coated with 57 ng/l of fibronectin (GIBCO-BRL; Gaithersburg, MD) and confirmed to be endothelial cells by their characteristic cobblestone appearance and positive labeling with acetylated low-density lipoprotein (Ac-LDL) (Biomedical Technologies; Stoughton, MA) and rabbit anti-human factor VIII antibody (Dako; Carpinteria, CA). After these cells had grown to confluence at 37°C, 5% CO2, they were expanded by brief trypsinization with 0.05% trypsin (GIBCO-BRL) in PBS containing 0.53 mmol/l EDTA (GIBCO-BRL) and then grown on 96-well microplates (Becton Dickinson Labware) in EGM-2 at 37°C for 48 h before use. For the purposes of this study, the first passage of primary HUVEC cultures expressed P-selectin, manifested WP bodies, and had significant activities of ROS-generating enzymes, including membrane-associated NADPH oxidase and xanthine oxidase (21, 22, 24, 47). All HUVEC were used at passage 1, because pilot experiments had shown the rapid loss of P-selectin expression, both constitutive and responsive, as well as WP body morphology with subsequent propagation (data not shown).
Measurement of Endothelial Cell Surface P-selectin Expression by ELISA
Because the focus of this study was the rapid, postsynthetic translocation of functional P-selectin to the endothelial cell surface, P-selectin expression on the surface of HUVEC was measured discriminately by modified ELISA with nonpermeabilized monolayers. At the completion of an experiment entailing the application of the appropriate agonist and/or antagonist, the media were removed and the HUVEC monolayers were fixed with 2% paraformaldehyde (J. T. Baker, Phillipsburg, NJ) in a sodium phosphate buffer (pH 7.4) with 0.1 mol/l L-lysine monohydrochloride (Sigma; St. Louis, MO) and 10 mmol/l sodium m-periodate (Sigma) for 30 min at 4°C and then incubated with 1% BSA (Sigma) in PBS containing 0.1 mol/l glycine (Baker) overnight at 4°C. After being washed twice with 0.1% BSA in PBS, the fixed monolayers were probed with a monoclonal rabbit anti-human P-selectin antibody (PharMingen; San Diego, CA) for 1 h at 37°C. After being washed again four times with 0.1% BSA in PBS, the HUVEC were incubated with a peroxidase-conjugated mouse anti-rabbit IgG secondary antibody (Sigma) for 1 h at 37°C. After a final wash with 0.1% BSA in PBS, the developing substrate, 0.2% H2O2 and 3.7 mmol/l O-phenylenediamine (Sigma), was added for 4 min, and the reaction was then stopped with 1 mol/l H2SO4. The plates were read on a spectrophotometric microplate reader (Molecular Devices; Sunnyvale, CA) at 520 nm. This assay thereby discriminates the surface expression of P-selectin from intracellular levels.Localization of WP Body Morphology by Electron Microscopy
WP bodies have a characteristic appearance under the electron microscope, both in their resting state beneath the cell membrane and when actively degranulating (9, 24, 51). Although this morphological approach is far from definitive and does not lend itself to repetitive nor quantitative assay, we did want to document the visualization of WP degranulation in parallel with the quantitative estimation of the upregulation of endothelial surface P-selectin expression. Confluent HUVEC monolayers, before and after treatment with thrombin, were washed three times with 0.1 mol/l sodium cacodylate buffer (Ted Pella; Redding, CA), fixed for 1 h with 3% glutaraldehyde (EM Science; Gibbstown, NJ), postfixed in osmium tetrachloride (EM Science), dehydrated through graded ethanol, and embedded in epoxy resin (Epox 812; Ernest F. Fullam, Latham, NY). Sections were examined with a Philips 201 electron microscope (FEI; Hillsboro, OR) to visualize the WP bodies.Measurement of ROS Generation
Having noted a marked, rapid increase in P-selectin expression by HUVEC after thrombin stimulation (see RESULTS), we estimated ROS generation in association with this response. Four traditionally used assays for the measurement of ROS generation [cytochrome c reduction (17), nitroblue tetrazolium reduction (3), lucigenin-enhanced chemiluminescence (18), and 2,7-dichlorofluorescin diacetate fluorescence (12)] were found to be insufficiently sensitive to measure the low, physiological levels of ROS released into the supernatant in this system. A newer assay using N-acetyl-3,7-dihydroxyphenoxazine (Amplex red) as an indicator reagent (36, 52) proved to be relatively more sensitive, specific, and reproducible when compared with the more traditional assays: Amplex red reacts with intracellular hydrogen peroxide, and perhaps with other ROS, to form an adduct that can be detected at excitation 530 nm/emission 590 nm (36, 52). Although quantitatively somewhat indiscriminant in that net intracellular and extracellular ROS generation is detected, and not the more physiologically relevant, localized subcellular change in ROS concentration, it is nonetheless the most usable and reproducible method of assessing trends in total ROS generation available at this time. However, it should be appreciated that the thresholds for the detection of ROS using this assay are apparently much higher than the corresponding thresholds for P-selectin detection by ELISA, making definitive quantitative correlations between P-selectin expression and ROS generation at the lower doses of stimulation/inhibition not possible. Therefore, HUVEC monolayers in 96-well plates were incubated with or without antioxidants or inhibitors of ROS-generating enzymes for 30 min at 37°C. These cells were then incubated with thrombin (0.3-1.0 U/ml; Sigma) or menadione (as a positive control, 3-100 µmol/l; Sigma) and Amplex red (50 µmol/l, Molecular Probes; Eugene, OR) for 3 min and then read on the microplate reader at 530 nm. Blank wells without HUVEC were loaded with Amplex red alone, and this background fluorescence was subtracted from experimental wells (standard curve not shown).Experimental Protocols
Agonists. HUVEC were treated with varying doses of thrombin (0.3-10 U/ml, Sigma) based on previously published reports (47), and our own preliminary dose- and time-response curves, and cell surface P-selectin expression and ROS generation were assayed; dose- and time-response curves were thereby generated.
Antagonists. To determine whether the correlation among thrombin stimulation, ROS generation, and P-selectin expression was causal in nature, we assayed P-selectin expression in response to thrombin stimulation in the presence or absence of various lipid soluble (cell permeable) antioxidants, pyrrolidine dithiocarbamate (PDTC, 1-30 µmol/l, Sigma), 1,3-dimethyl-2-thiourea (DMTU, 1-30 mmol/l, Aldrich Chemical; Milwaukee, WI), N-acetylcysteine (NAC, 1-30 mmol/l, Sigma), and DMSO (0.3-10 mmol/l, Sigma), as well as the lipid-insoluble (cell impermeable) antioxidants human erythrocyte catalase (30-300 U/ml, Calbiochem; San Diego, CA) and bovine erythrocyte superoxide dismutase (30-300 U/ml, Calbiochem). HUVEC were pretreated independently with one of the above antioxidants for 30 min at 37°C before thrombin stimulation. Antioxidant concentrations were based on previously reported doses in other experimental systems (2, 49) and on our own preliminary dose- and time-response curves. In addition to P-selectin expression, we wanted to assess whether ROS generation might also be inhibited by the use of antioxidants. Although this, in fact, represents circular reasoning because antioxidants by definition scavenge the very substance one is trying to measure, for the sake of completeness we selected the antioxidant that was least effective at inhibiting P-selectin expression (PDTC) and assessed ROS generation in its presence.
Noting a marked inhibition of both P-selectin expression and ROS generation by the above-mentioned antioxidants, we evaluated the role of each of our previously defined candidate ROS-generating enzyme systems. HUVEC were pretreated with one of the following enzyme inhibitors for 30 min at 37°C before thrombin stimulation and assay of P-selectin expression and/or ROS generation: NADPH oxidase was inhibited with diphenylene iodonium chloride (DPI, 3-100 µmol/l; Sigma), xanthine oxidase was inhibited with allopurinol (3-300 µmol/l, Aldrich Chemical) or oxypurinol (3-300 µmol/l, Sigma), mitochondrial NADH oxidase was inhibited with rotenone (3-100 µmol/l, Sigma), mitochondrial cytochrome bc1 complex was inhibited with antimycin A (3-100 µmol/l, Sigma), cyclooxygenases were globally inhibited with indomethacin (3-100 µmol/l, Sigma), the cytochrome P-450 system was inhibited with metyrapone (3-100 µmol/l, Sigma), cytochrome oxidase was inhibited with sodium azide (3-100 µmol/l, Sigma), and nitric oxide synthases were inhibited with NG-monomethyl-L-arginine (L-NMMA, 3-100 µmol/l, Sigma). All inhibitor concentrations were based on previously published reports (2, 49) and on our own preliminary dose- and time-response curves (data not shown).Statistical Analysis
All values were expressed as means ± SD. Apparent differences in dose- and time-response curves were evaluated by one-way ANOVA. Values of P
0.05 were considered to be
statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Thrombin Stimulated Marked, Dose-Dependent Upregulation of HUVEC Surface P-Selectin Expression
P-selectin was constitutively expressed at low levels on the surface of passage 1 HUVEC. Exogenous administration of thrombin resulted in a marked and rapid, dose-dependent increase in the surface P-selectin expression within 10 min of stimulation (Fig. 1, A and B). Electron microscopic visualization of HUVEC before and 10 min after stimulation with thrombin (1 U/ml) revealed a mobilization of the morphologically characteristic WP bodies from their "resting" intracytoplasmic location to the cell surface in response to thrombin stimulation (Fig. 2, A and B).
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Thrombin Stimulation Upregulated HUVEC ROS Generation in Parallel With Rapid P-Selectin Expression
The Amplex red assay detected significant increases in ROS generation in response to menadione (positive control) or thrombin stimulation (Fig. 3, A and B). This dose response to thrombin stimulation paralleled qualitatively the P-selectin response (Fig. 1).
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Inhibition of Thrombin-Stimulated P-selectin Expression and ROS Generation by Antioxidants
A series of conventionally employed, lipid-soluble (cell membrane permeable) antioxidants (PDTC, DMTU, NAC, and DMSO) each inhibited the thrombin-induced P-selectin expression on HUVEC to varying degrees (Fig. 4, A-D). Interestingly, catalase, an antioxidant generally thought to be cell impermeable, also inhibited this response, to a small but statistically significant degree (Fig. 4E). Not surprisingly, however, exogenously administered (cell impermeable) SOD had little effect (Fig. 4F). Similarly, PDTC inhibited thrombin-induced ROS generation by HUVEC in a dose-dependent manner (Fig. 5).
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Inhibition of Thrombin-Induced P-selectin Expression and ROS Generation by Specific Inhibitors of Various ROS Generating Enzymes
Selective inhibition of NADPH oxidase with DPI or xanthine oxidase with allopurinol or oxypurinol each resulted in a dose-dependent inhibition of thrombin-stimulated P-selectin expression (Fig. 6, A-C) and ROS generation (Fig. 7). Reportedly effective concentrations of inhibitors of other ROS-generating enzymes, including mitochondrial NADH dehydrogenase (rotenone), cytochrome bc1 complex (antimycin A), cyclooxygenases (indomethacin), cythochrome P-450 (metyrapone), cytochrome oxidase (sodium azide), and nitric oxide synthase (L-NMMA), failed to inhibit this response to any degree (data not shown). The dose-dependent inhibition by allopurinol of the thrombin-induced P-selectin expression paralleled qualitatively its inhibition of ROS generation (Fig. 8).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The choice of primarily harvested HUVEC for these studies was selective. Although these cells are derived from large vessels and not the venular microvasculature where this characteristic vascular inflammatory response takes place in vivo, work in our laboratory and others (21, 22, 24, 47) has indicated that these cells maintain many important properties of the microvascular endothelium, including P-selectin expression, WP body formation, and xanthine oxidase activity. Although we attempted to repeat these experiments by using a small vessel-derived, simian virus-transformed, murine microvascular endothelial cell line (39), we found that these cell lines no longer retained the capability to express P-selectin and were therefore not usable for this study. These HUVEC were the only cell cultures, primary or propagated, that in our hands simulated the P-selectin expression of the venules in vivo, where we and others (1) have noted a parallel responsiveness to stimulation and oxidant inhibition.
Some ELISA protocols for the quantification of P-selectin expression do not discriminate intracellular from cell surface protein expression. We sought to discriminately measure the surface expression of P-selectin, because this is where the functionally consequential response is manifested. Therefore, we employed a modified ELISA to include the fixation of HUVEC with paraformaldehyde without subsequent permeabilization, thereby rendering the cell membrane impermeable to the antibody and thus allowing the discriminant measurement of cell surface P-selectin.
ROS detection remains technically challenging, related primarily to the low amplitude of local oxidant fluxes and the relatively short half-lives of these species. Using four conventional assays for ROS generation, we (2) and others (40) have been unable to reproducibly detect the low levels of ROS generation associated with physiological signaling (versus those generated under injurious conditions) (7, 16) in a variety of settings. We repeated these assays in our current system and were again unable to detect these physiological levels of ROS generation. However, the Amplex red assay proved sensitive enough to reproducibly detect measurable physiological increases in net cellular oxidant generation, which were inhibited by antioxidants and ROS-generating enzyme inhibitors. Although insufficiently quantitatively sensitive to provide more than qualitative indications of directional ROS fluxes and indiscriminant in that net intracellular and extracellular ROS generation are both measured and not the more physiologically relevant, localized subcellular (plasma membrane associated) change in ROS concentration, it is nonetheless the most useful and reproducible method of assessing directional trends in ROS generation available to us at this time. Careful review of the data presented reveals that the lower doses of thrombin stimulation (0.3 U/ml) clearly have an effect on P-selectin expression (Fig. 1) but no measurable effect on ROS generation (Fig. 3). This disparity is probably due to a higher threshold for detection of a response by the less sensitive ROS generation assay. When one normalizes this sensitivity baseline to account for these apparent differences in threshold (as we have done in Fig. 8), it becomes clear that the responses of these two end points to thrombin stimulation appear to be qualitatively parallel.
Traditionally, we and others have used menadione as an internal
standard for ROS generation assays (2), which raises the question of
whether menadione might also trigger P-selectin expression as a result
of these ROS. However, we have found that with primarily harvested HUVEC, many noxious stimuli, such as menadione, trigger the
rapid expression of surface P-selectin directly. This results in the
maximal expression of preformed P-selectin such that any additional
component that might be mediated by ROS cannot be detected. For
example, our repeated (and successful) attempts to utilize an
adenoviral vector to effect stable, intracellular overexpression of SOD
have resulted in a rapid, pronounced increase in surface P-selectin
expression, even when a control (Ad-
-gal) virus was used (data not
shown). Therefore, we are unable to definitively assess the role of
menadione-generated ROS on this response.
We found that the previously well-characterized, thrombin-induced,
rapid upregulation of endothelial surface P-selectin expression (47) was dependent on ROS generated by the endothelial
cells, because it was inhibited by a variety of lipid-soluble
antioxidants as well as specific inhibitors of the membrane-associated
enzymes NADPH oxidase and xanthine oxidase, which generate these ROS. Although such ROS are traditionally known for their role in
oxidant-mediated tissue injury following ischemia and
reperfusion via the upregulation of cellular adhesion molecules
(44), we postulate that these same ROS can participate
actively in cell signaling under these more physiological conditions.
Their localized generation (by selective localization of ROS-generating
enzymes on the cell membrane), high reactivity, and short half-lives
with correspondingly short diffusion radii make them remarkably well
suited for such highly localized, rapid signal transduction. We
(45) have recently reported that endothelial cell ICAM-1
upregulation in response to TNF- is mediated in part by ROS
generated by this same NAPDH oxidase but not by xanthine oxidase. This
response occurs maximally at 4 h, consistent with reports that it
is dependent on new protein synthesis (28). Other studies
have indicated that ROS signal transduction processes may interact with
more traditional signaling mechanisms to effect either upstream or
downstream processes. For example, it has been found that ROS can
upregulate NF-kB and AP-1 expression and that the endogenous
overexpression of intracellular antioxidants inhibits these process in
vitro (29, 34, 35, 45, 50).
Endothelial cell P-selectin expression in response to stimulation with
thrombin or histamine occurs in a bimodal distribution, with an early
peak appearing within minutes and a later peak after about 4 h
(1). The early peak involves the degranulation of preformed WP bodies, which contain already synthesized P-selectin (8, 10, 19, 20, 33, 51). We found that this early expression was associated with a parallel rise in ROS generation and
that it was inhibited by various cell-permeable antioxidants, as well
as specific inhibitors of ROS generation by either NADPH oxidase or
xanthine oxidase. Whereas it is not surprising to find that NADPH
oxidase was involved in this response because it parallels the slower
transcriptional regulation of ICAM-1 protein synthesis in response to
TNF- (2), the finding that xanthine oxidase can mediate
this particular response is novel. Whereas xanthine oxidase, known in
this context primarily for the generation of the oxidant flux that
triggers microvascular inflammation and tissue injury after
ischemia-reperfusion (44), is also important for
ROS-mediated microbial killing by periotneal macrophages (41, 49) and Kupffer cells (43), it does not appear to
play an important role in ICAM-1 expression in response to TNF-
(2). However, the posttranslational control of rapid
P-selectin surface expression from preformed intracellular stores does
appear to be controlled, at least in large part, by ROS generated from
xanthine oxidase.
A number of important questions regarding the mechanism of this rapid,
thrombin-induced P-selectin expression remain unanswered. Although we
postulate that the ROS generated from xanthine oxidase in this system
are controlled by the limited proteolysis of xanthine dehydrogenase to
xanthine oxidase (4-6), we have yet to formally address this issue in this system. Moreover, the mechanisms by which
this ROS-mediated signaling pathway interacts with more classical
signaling pathways, such as the recently described role of NF-B in
the thrombin-stimulated, transcriptionally mediated upregulation of
P-selectin expression and consequent leukocyte recruitment
(26), remain to be defined. Finally, confirmation of the
function of this mechanism in vivo and determination of its
physiological (e.g., microbicidal) or pathophysiological (e.g., microvascular inflammatory tissue injury) roles remain to be determined.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institutes of Health Grant DK-31764.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. B. Bulkley, Blalock 685, The Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD 21287-4685 (E-mail: gbulkley{at}jhmi.edu).
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.
July 26, 2002;10.1152/ajpheart.01001.2001
Received 15 November 2001; accepted in final form 23 July 2002.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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