© 1999 American Heart Association, Inc.
Vascular Biology |
Endothelial Nuclear Factor-
B Translocation and Vascular Cell Adhesion Molecule-1 Induction by Complement
Inhibition With Anti-Human C5 Therapy or cGMP Analogues
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Abstract |
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Abstract—We have previously shown that reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) leads to the activation and deposition of complement. In the present study, we investigated whether the terminal complement complex (C5b-9) influences HUVEC nuclear factor-
B (NF-B) translocation and vascular cell adhesion
molecule-1 (VCAM-1) protein expression after
hypoxia/reoxygenation by decreasing
endothelial cGMP. Additionally, we investigated the
action of anti-human C5 therapy on endothelial cGMP,
NF-B translocation, and VCAM-1 protein expression.
Reoxygenation (0.5 to 3 hours, 21% O2) of
hypoxic (12 hours, 1% O2) HUVECs in human serum (HS)
significantly increased C5b-9 deposition, VCAM-1 expression, and
NF-B translocation compared with
hypoxic/reoxygenated HUVECs treated with the recombinant
human C5 inhibitor h5G1.1-scFv. Acetylcholine (ACh)-induced
cGMP synthesis was significantly higher in normoxic HUVECs compared
with hypoxic HUVECs reoxygenated in HS but did not differ
from hypoxic HUVECs reoxygenated in buffer or HS treated
with h5G1.1-scFv. Treatment of hypoxic/reoxygenated HUVECs
with h5G1.1-scFv or cGMP analogues significantly attenuated NF-B
translocation and VCAM-1 protein expression. Treatment with NO
analogues, but not a cAMP analogue, cGMP antagonists, or an
NO antagonist, also significantly attenuated VCAM-1
expression. We conclude that (1) C5b-9 deposition, NF-B
translocation, and VCAM-1 protein expression are increased in hypoxic
HUVECs reoxygenated in HS; (2)
reoxygenation of hypoxic HUVECs in HS, but not buffer
alone, attenuates ACh-induced cGMP synthesis; and (3) treatment of
hypoxic/reoxygenated HUVECs with h5G1.1-scFv attenuates
C5b-9 deposition, NF-B translocation, and VCAM-1 expression while
preserving ACh-induced cGMP synthesis. C5b-9–induced VCAM-1 expression
may thus involve an NO/cGMP-regulated NF-B translocation
mechanism.
Key Words: adhesion molecules • hypoxia • inflammation • nitric oxide • immunotherapy
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Increasing evidence suggests that the terminal complement complex (C5b-9) plays an integral role in the pathogenesis of atherosclerosis1 2 3 4 5 and vascular injury after ischemia-reperfusion, cardiopulmonary bypass, and acute myocardial infarction.6 7 8 9 10 11 In addition to amplifying the local inflammatory response by inducing endothelial interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) secretion,12 C5b-9 also influences endothelial vascular tone13 14 15 and expression of leukocyte adhesion molecules (eg, vascular cell adhesion molecule-1 [VCAM-1],16 intercellular adhesion molecule-1 [ICAM-1],17 and endothelial leukocyte adhesion molecule-1 [E-selectin]16 ). Yet the mechanisms by which C5b-9 influences endothelium-dependent relaxation and leukocyte adhesion molecule expression have not been fully elucidated.
Vascular tone and leukocyte adhesion molecule expression can be
regulated by NO.18 19 NO synthesized by the vascular
endothelium induces vasodilation by activation of
soluble guanylate cyclase and the subsequent increase of
cGMP.20 In addition to being a potent smooth muscle
relaxant, NO inhibits platelet aggregation21 and
suppresses endothelial neutrophil adhesion molecule
expression.19 Adhesion molecules regulated by NO include
P-selectin,22 VCAM-1,23 and
ICAM-1.24 Specifically, increased levels of NO are
associated with decreased leukocyte adhesion molecule
expression.23 24 25 26 The mechanisms by which NO modulates
expression of these adherence molecules is unclear but have been
speculated to include NO-dependent regulation of oxidant-responsive
transcription via nuclear factor-B (NF-B)23 26
and/or endothelial cGMP
concentrations.25 26 27 28 Indeed, the addition of
8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), but not
8-Br-cAMP, after inhibition of endothelial NO synthesis
has been shown to reduce both endothelial adhesion
molecule expression and leukocyte adherence.25 27 28 Thus,
decreased levels of intracellular cGMP could potentially compromise
vascular blood flow owing to a loss of
endothelium-dependent relaxation and increased adhesion
of neutrophils to the endothelium.
We recently demonstrated that reoxygenation of hypoxic
human endothelial cells increases NF-B translocation
and activates complement.29 30 In the present
study, we investigated whether the terminal complement components
influence endothelial NF-B translocation and VCAM-1
expression after hypoxia/reoxygenation by a
cGMP-dependent mechanism. We demonstrate that C5b-9–induced VCAM-1
expression may involve an NO/cGMP-regulated NF-B translocation
mechanism.
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Methods |
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Cell Culture
Human umbilical vein endothelial cells (HUVECs) were obtained and cultured as previously described.29 In brief, HUVECs were harvested with 0.1% collagenase (Worthington Biochemical Corp) and suspended in medium 199 containing 20% heat-inactivated bovine calf serum (Gibco Life Technologies Inc). The cells were initially seeded in 75-cm2 flasks (Corning Costar) and incubated at 37°C in 95% air and 5% CO2. When confluent, the endothelial cells were passaged with 0.5% trypsin-EDTA. Endothelial cell purity was assessed by their phase-microscopic "cobblestone appearance," uptake of fluorescent acetylated LDL, and the presence of von Willebrand factor. All experiments were conducted on HUVECs during passages 1 to 3.
C5b-9 ELISA
A C5b-9–specific cell-surface ELISA was developed using a
monoclonal antibody (mAb) that recognizes a neoepitope on human C9 in
the terminal complement complex (C5b-9) (clone B7; a gift from Dr B.P.
Morgan, University of Wales College of Medicine, Cardiff, UK).
Inhibition of C5b-9 formation was achieved by using the recombinant
human C5 inhibitor, h5G1.1-scFv, to block the cleavage of
C5 into C5a and C5b.31
HUVECs were grown to confluence on 0.1% gelatinized 96-well plastic plates (Corning Costar). The plates were then subjected to 0 (normoxia) or 12 hours of hypoxia in a humidified, sealed chamber (Coy Laboratory Products, Inc) at 37°C that was gassed with 1% O2, 5% CO2, and the balance N2 as described previously.29 After the specified period of normoxia or hypoxia, the cell medium was aspirated, and 100 µL of either 30% human serum (HS; pooled donor sera) diluted in Hanks’ balanced salt solution (HBSS) or 30% HS treated with h5G1.1-scFv (20 µg/mL) was added to each well. The cells were then reoxygenated for 30 minutes at 37°C in 95% air and 5% CO2. The cells were washed and then lightly fixed with 1% paraformaldehyde (Sigma Chemical Co) for 30 minutes. The cells were washed again and incubated at 4°C for 1.5 hours with the anti-C9 neoepitope mAb (20 µg/mL) or an inappropriate isotype control antibody (mAb GS1 to porcine C5a).32 The cells were then washed and incubated at 4°C for 1 hour with 50 µL of peroxidase-conjugated polyclonal goat anti-mouse secondary antibody (1:1000 dilution, Cappel). After the cells were washed, the plates were developed with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid and read (Molecular Devices) at 405 nm. Background optical density of the isotype control antibody was subtracted from all wells. This experiment was performed 4 times with 6 wells per experimental group (n=4).
HUVEC Immunohistochemistry and Confocal Microscopy
C5b-9 deposition and VCAM-1 expression were studied by
immunofluorescent confocal microscopy on HUVECs grown on Labtek
tissue-culture slides (Nunc). HUVECs were subjected to 0 or 12 hours of
hypoxia followed by 3 hours of reoxygenation in
the presence of 30% HS or 30% HS treated with h5G1.1-scFv (20
µg/mL), 8-Br-cGMP (10 or 100 µmol/L), or
N-2,2'-O-dibutyrylguanosine 3',5'-cyclic
monophosphate (dibutyryl-cGMP; 10 or 100 µmol/L). The cells were
then washed, fixed in 4% paraformaldehyde, washed
again, and blocked with 5% goat serum to prevent nonspecific secondary
antibody staining. The cells were incubated (overnight at 4°C) with
the primary antibody (20 µg/mL of the anti-C9 neoepitope mAb or a 1:1
dilution of mAb 6G10, an anti–VCAM-1 mAb obtained from the
Developmental Studies Hybridoma Bank, University of Iowa, Iowa City,
IA33 ). The cells were then washed and incubated (2
hours at room temperature) with an FITC-conjugated goat anti-mouse IgG
(1:1000 dilution, Jackson Immunoresearch). The cells were also
incubated with propidium iodide (20 µg/mL) for 10 minutes to stain
the nuclei. After being washed, the slides were coated with antifade
mounting medium (Molecular Probes), covered, and analyzed with
a Zeiss laser scanning confocal microscope. Controls with secondary
antibody only were processed as above, omitting the primary antibody to
determine nonspecific binding. All analyses were conducted at
the same pinhole, voltage, and laser settings. This experiment was
performed 3 times.
cGMP ELISA
HUVEC cGMP concentrations were determined by ELISA. HUVECs
(100-mm Petri dishes) were subjected to 0 or 12 hours of
hypoxia. The cell medium was then aspirated, and 1 of the
following was added to each plate: (1) 30% HS, (2) vehicle (HBSS), or
(3) 30% HS treated with 10 µg/mL h5G1.1-scFv. The cells were then
reoxygenated for 30 minutes. After the plates were washed,
the cells were stimulated with 10 µmol/L acetylcholine (ACh) and
100 µmol/L isobutylmethylxanthine for 30
minutes. The cells were then processed for measurement of cGMP by using
a commercially available ELISA kit (Cayman Chemical). This experiment
was performed 3 times with 3 or 4 plates per experimental group (n=3).
VCAM-1 ELISA
VCAM-1 protein expression was measured by ELISA with an mAb to
human VCAM-1 (mAb 6G10).33 HUVECs were subjected to 0 or
12 hours of hypoxia. One hour before
reoxygenation, select wells were treated with 1 of the
following compounds: (1) 10 or 100 µmol/L of the cGMP analogues
8-Br-cGMP or dibutyryl-cGMP; (2) 10 or 100 µmol/L of the cAMP
analogue dibutyryl-cAMP; (3) 100 µmol/L of the cGMP
antagonists
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or
LY8358334 35 ; (4) 1 or 10 µmol/L of the NO
analogues S-nitrosoglutathione (SNOG) or
S-nitroso-N-acetylpenicillamine
(SNAP)36 37 ; or (5) 10 µmol/L of the NO
antagonist
NG-nitro-L-arginine
methyl ester (L-NAME).38 The cell medium was then
aspirated, and vehicle (HBSS), 30% HS, or 30% HS treated with
h5G1.1-scFv (20 µg/mL) was added. The cells were then
reoxygenated for 3 hours (VCAM-1 protein expression was
measured at 3 hours based on pilot studies). The cells were washed,
fixed (1% paraformaldehyde), washed again, and
incubated at 4°C for 1.5 hours with the anti-human VCAM-1 mAb. After
being washed, the cells were incubated for 1 hour at 4°C with a
peroxidase-conjugated goat anti-mouse secondary antibody (Cappel). An
inappropriate isotype control antibody (mAb GS1 to porcine
C5a)32 was used in all experiments to assess background
optical density. The background optical density was subtracted from all
wells. This experiment (6 wells per experimental group) was performed 3
times (n=3).
Western Analysis of HUVEC NF-B p65 Subunit
HUVECs were subjected to 0 or 12 hours of hypoxia and
then reoxygenated for 30 minutes in the presence of (1)
30% HS, (2) 30% HS plus h5G1.1-scFv (20 µg/mL), (3) 30% HS plus
8-Br-cGMP (100 µmol/L), or (4) 30% HS plus dibutyryl-cGMP
(100 µmol/L). After reoxygenation, nuclear
protein lysates (20 µg) were resolved by SDS–polyacrylamide
gel electrophoresis and subjected to Western blot analysis with
a polyclonal antibody to the p65 subunit of NF-B (Santa Cruz
Biotechnology) as described previously.30 39
Statistical Analysis
Data analyses were performed using Sigma Stat (Jandel
Scientific). C5b-9 deposition, VCAM-1 protein expression, and
intracellular cGMP concentrations in normoxic and hypoxic HUVECs
(ELISA) were analyzed by 2-way ANOVA. NF-B translocation
(densitometry) was analyzed by 1-way ANOVA. All pairwise
multiple comparisons were made using the Student-Newman-Keuls test with
significant differences between groups being defined at
P<0.05. C5b-9 deposition or VCAM-1 expression (ELISA;
Figures 2
, 5, and 6) were normalized to hypoxic
HUVECs reoxygenated in 30% HS. All data are expressed as
mean±SEM.
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Results |
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Effect of Anti-Human C5 Therapy on Endothelial C5b-9 Deposition
We have previously shown that reoxygenation of hypoxic HUVECs leads to the activation and deposition of complement.29 To evaluate the effectiveness of anti-C5 therapy in inhibiting terminal complement complex formation in this model, C5b-9 deposition was studied by immunofluorescent confocal microscopy (Figure 1
) on
normoxic HUVECs and hypoxic (12 hours) HUVECs reoxygenated
(30 minutes) in 30% HS treated with or without h5G1.1-scFv (20
µg/mL). Staining for C5b-9 (green) on hypoxic HUVECs
reoxygenated in 30% HS (Figure 1B) was
significantly greater than in normoxic HUVECs bathed in 30% HS (Figure 1A). In contrast, no staining for C5b-9 was observed on hypoxic
HUVECs reoxygenated in HS treated with h5G1.1-scFv (Figure 1C). Thus, reoxygenation of hypoxic HUVECs leads
to increased C5b-9 deposition that is inhibited by h5G1.1-scFv.
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To further confirm and quantitate these findings,
endothelial C5b-9 deposition after
hypoxia/reoxygenation was measured by ELISA.
C5b-9 deposition on hypoxic/reoxygenated HUVECs was
significantly greater (2-fold) than in normoxic HUVECs
(OD405 0.29±0.03 versus 0.14±0.01,
respectively, P<0.05). In contrast, C5b-9 deposition on
hypoxic HUVECs reoxygenated in HS treated with 20 µg/mL
h5G1.1-scFv was significantly (P<0.05) less than in hypoxic
HUVECs reoxygenated in vehicle-treated HS (Figure 2
). Thus, anti-human C5 therapy inhibits
endothelial C5b-9 formation after
hypoxia/reoxygenation.
Effect of Complement on Endothelial cGMP
We have previously shown that deposition of C5b-9 on the vascular
endothelium results in a functional loss of
NO-dependent relaxation.13 14 15 We thus investigated
whether C5b-9 attenuates ACh-induced increases in
endothelial cGMP. Intracellular concentrations of cGMP
were measured by ELISA in normoxic and hypoxic (12 hours) HUVECs
reoxygenated (30 minutes) in the presence of buffer or 30%
HS treated with 0 or 10 µg/mL h5G1.1-scFv (Figure 3). Reoxygenation of
hypoxic HUVECs in buffer alone did not attenuate ACh-induced increases
in cGMP compared with normoxic cells. Thus,
hypoxia/reoxygenation alone does not attenuate
ACh-induced increases in cGMP. In contrast, ACh-induced cGMP synthesis
was significantly decreased in hypoxic HUVECs reoxygenated
in HS compared with normoxic HUVECs. Inhibition of C5b-9 formation with
h5G1.1-scFv significantly attenuated cGMP loss in hypoxic HUVECs
reoxygenated in HS. Thus, anti-C5 therapy preserves
ACh-induced increases in cGMP after
hypoxia/reoxygenation.
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Effect of Anti-Human C5 Therapy or cGMP Analogues on
Endothelial NF-B Translocation
C5b-9 is known to induce NF-B translocation.40 Yet
the mechanism by which C5b-9 induces NF-B activation is unknown. To
demonstrate that C5b-9 induces NF-B translocation by decreasing
endothelial cGMP, Western blot analysis of the
nuclear p65 subunit of NF-B was performed in normoxic HUVECs and
hypoxic (12 hours) HUVECs reoxygenated (30 minutes) in 30%
HS, 30% HS treated with 20 µg/mL h5G1.1-scFv, or 30% HS treated
with 100 µmol/L 8-Br-cGMP or dibutyryl-cGMP (Figure 4A). NF-B p65 band density was
significantly increased (densitometry, P<0.05) in hypoxic
HUVECs reoxygenated in 30% HS (lane 2) compared with
normoxic cells (lane 1). In contrast, the NF-B p65 band density was
similar to that of normoxic cells when hypoxic HUVECs were
reoxygenated in 30% HS treated with h5G1.1-scFv (lane 3),
8-Br-cGMP (lane 4), or dibutyryl-cGMP (lane 5). The mean integrated
optical density of the 3 separate experiments is presented in
Figure 4B. These data demonstrate that anti-C5 or cGMP analogue
therapy attenuates complement-induced NF-B translocation in human
endothelial cells.
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Effect of Anti-C5 Therapy or NO/cGMP Analogues on
Endothelial VCAM-1 Expression
Restoration of cGMP loss after inhibition of
endothelial NO synthesis has been shown to reduce both
leukocyte adhesion molecule expression and leukocyte
adherence.25 27 28 Since endothelial
VCAM-1 expression is induced in part by NF-B26 41 and
C5b-9,16 we hypothesized that anti-C5 or NO/cGMP analogue
therapy would significantly attenuate VCAM-1 expression after
endothelial
hypoxia/reoxygenation. VCAM-1 protein
expression was observed by immunofluorescent confocal
microscopy (Figure 5) on normoxic HUVECs
and hypoxic (12 hours) HUVECs reoxygenated (3 hours) in
30% HS or 30% HS treated with 20 µg/mL h5G1.1-scFv or with 10 or
100 µmol/L 8-Br-cGMP or dibutyryl-cGMP. Staining for VCAM-1
(green) on hypoxic HUVECs reoxygenated in 30% HS (Figure 5B) was significantly greater than in normoxic HUVECs (Figure 5A). In contrast, VCAM-1 staining was significantly decreased on
hypoxic HUVECs reoxygenated in 30% HS treated with
h5G1.1-scFv (Figure 5C), 8-Br-cGMP (Figure 5D), or
dibutyryl-cGMP (Figure 5E). Furthermore, addition of 8-Br-cGMP
or dibutyryl-cGMP to HS decreased VCAM-1 staining in a dose-dependent
manner (data not shown).
Endothelial VCAM-1 expression after
hypoxia/reoxygenation was also measured by
ELISA to further confirm and quantitate the immunofluorescent
confocal microscopy findings. VCAM-1 expression on hypoxic (12 hours)
HUVECs reoxygenated (30 minutes) in 30% HS was
significantly greater (3-fold) than on normoxic HUVECs
(OD405 0.06±0.01 versus 0.02±0.01,
respectively, P<0.05). In contrast, VCAM-1 expression on
hypoxic HUVECs reoxygenated in buffer or in HS treated with
20 µg/mL h5G1.1-scFv was significantly (P<0.05) less than
on hypoxic HUVECs reoxygenated in untreated HS (Figure 6A). Similarly, treatment with cGMP
(8-Br-cGMP or dibutyryl-cGMP, 10 or 100 µmol/L) or NO analogues
(SNOG or SNAP, 1 or 10 µmol/L; Figures 6A and 6B,
respectively) significantly attenuated VCAM-1 expression
(P<0.05). In contrast, treatment with the NO
antagonist L-NAME (10 µmol/L) significantly
augmented VCAM-1 expression compared with untreated control
(P<0.05). VCAM-1 expression was unchanged after treatment
with the cAMP analogue dibutyryl-cAMP (10 µmol/L) or the cGMP
antagonists ODQ or LY83583 (100 µmol/L). Thus,
anti-C5 or NO/cGMP analogue therapy decreases
endothelial VCAM-1 expression after
hypoxia/reoxygenation.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Increased attention in recent years has focused on the role of complement in the pathogenesis of human atherosclerosis and cardiovascular disease.1 42 43 44 In addition to causing direct tissue injury, complement may also indirectly mediate vascular injury by stimulating leukocyte activation and chemotaxis and by increasing endothelial leukocyte adhesion molecule expression.16 17 45 However, the intracellular mechanisms by which the terminal complement components induce endothelial leukocyte adhesion molecule expression are not completely characterized. We have previously shown that reoxygenation of hypoxic human endothelial cells activates complement.29 As C5b-9 is known to increase endothelial VCAM-1 expression16 and to inhibit NO-dependent relaxation,13 14 15 we investigated whether C5b-9 influences endothelial NF-
B
translocation and VCAM-1 expression after
hypoxia/reoxygenation by decreasing
intracellular cGMP. Additionally, we investigated the action of
anti-human C5 therapy on endothelial cGMP, NF-B
translocation, and VCAM-1 expression. In the first set of experiments, cell-surface deposition of C5b-9 was studied by immunofluorescent confocal microscopy and ELISA to demonstrate activation of the terminal complement components after reoxygenation of hypoxic human endothelial cells. Reoxygenation of hypoxic human endothelial cells resulted in complement activation and endothelial C5b-9 deposition. Furthermore, the anti-C5 inhibitor h5G1.1-scFv significantly attenuated C5b-9 deposition after hypoxia/reoxygenation. Thus, these data demonstrate that complement activation in this model proceeds through activation of the terminal complement components.
Endothelial VCAM-1 protein expression is regulated in
part by NF-B26 and can be induced by
C5b-9.16 As C5b-9 is known to inhibit NO-dependent
relaxation,13 14 15 activate NF-B,40
and increase VCAM-1 expression,16 we investigated whether
C5b-9 influences endothelial NF-B translocation and
VCAM-1 protein expression by decreasing endothelial
cGMP. Stimulation of HUVECs with ACh in the present study
significantly increased cGMP to levels consistent with previous
reports.46 Reoxygenation of hypoxic
HUVECs in human sera significantly decreased ACh-induced increases
in cGMP. Inhibition of C5b-9 formation with h5G1.1-scFv significantly
attenuated cGMP loss. Although reoxygenation of hypoxic
HUVECs is known to generate reactive oxygen species,30
reoxygenation of hypoxic HUVECs in buffer did not
result in a loss of ACh-induced cGMP generation. Thus, the observed
decrease in cGMP in hypoxic HUVECs reoxygenated in human
sera is unlikely due to oxygen-derived free-radical production
but is instead complement dependent. Furthermore, this observation is
consistent with our previous report that superoxide dismutase
does not prevent C5b-9–induced loss of
endothelium-dependent relaxation.15
Although the mechanism by which C5b-9 decreases
endothelial cGMP is unknown at present, future
studies examining the effect of C5b-9 on intracellular ion transport
and kinases known to be involved in
endothelium-dependent relaxation, such as protein
kinase C, are warranted.47
Having demonstrated that C5b-9 attenuated ACh-induced increases in
endothelial cGMP, we investigated the action of C5b-9
on endothelial NF-B translocation and VCAM-1 protein
expression after hypoxia/reoxygenation.
Reoxygenation of hypoxic HUVECs in the presence of
human sera significantly increased NF-B translocation and VCAM-1
expression. Addition of h5G1.1-scFv or cGMP analogues (ie, 8-Br-cGMP or
dibutyryl-cGMP) to human sera significantly attenuated
endothelial NF-B translocation and VCAM-1
expression. As endothelial VCAM-1 expression is
regulated in part through NF-B,26 these data suggest
that C5b-9 may regulate NF-B translocation and VCAM-1 expression by
decreasing endothelial cGMP. Along these lines, the NO
analogues SNOG or SNAP, but not the cAMP analogue dibutyryl-cAMP,
significantly attenuated VCAM-1 expression. In contrast, treatment with
the NO antagonist L-NAME significantly increased VCAM-1
expression. These data are in agreement with previous studies in which
NO or cGMP analogues, but not cAMP analogues, decreased
endothelial VCAM-1.26 Furthermore, our
finding that C5b-9 increased NF-B translocation and VCAM-1
expression is also consistent with recent reports in which
C5b-9 has been shown to activate endothelial
NF-B12 and to increase endothelial
VCAM-1 expression in a dose- and time-dependent manner.16
Our findings extend these past studies to suggest that C5b-9–induced
increases in VCAM-1 expression may involve an NO/cGMP-regulated NF-B
mechanism. Finally, these findings also suggest that anti-C5 therapy
may represent a novel therapeutic strategy for regulating C5a-
and C5b-9–induced proinflammatory events.
In summary, reoxygenation of hypoxic human
endothelial cells activates complement,
resulting in the formation and endothelial deposition
of C5b-9. Additionally, C5b-9 decreases intracellular cGMP,
activates NF-B, and increases endothelial
VCAM-1 protein expression. Endothelial NF-B
translocation and VCAM-1 protein expression after
hypoxia/reoxygenation are significantly
attenuated by anti-C5 or cGMP analogue therapy. C5b-9–induced
increases in VCAM-1 expression may thus involve an NO/cGMP-regulated
NF-B mechanism. Furthermore, these data suggest that the decreased
ability of the endothelium to generate cGMP after C5b-9
deposition may be important in the regulation of blood flow and
leukocyte adherence in human disease states in which complement
activation and endothelial dysfunction are known to
occur.
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Acknowledgments |
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Sources of support for these studies included HL-03854 (to C.D.C.), GM-07592 (to C.D.C.), the Foundation for Anesthesia Education and Research (to C.D.C.), HL-56086 (to G.L.S.), and an American Heart Association Established Investigator Award (to G.L.S.). We wish to thank Margaret M. Morrissey for assistance with HUVEC culture.
Received December 22, 1998; accepted April 5, 1999.
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