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Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:2623-2629

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:2623.)
© 1999 American Heart Association, Inc.

Vascular Biology

Endothelial Nuclear Factor-{kappa}B Translocation and Vascular Cell Adhesion Molecule-1 Induction by Complement

Inhibition With Anti-Human C5 Therapy or cGMP Analogues

Charles D. Collard; Azin Agah; Wende Reenstra; Jon Buras; Gregory L. Stahl

*    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-{kappa}B (NF-{kappa}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-{kappa}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-{kappa}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-{kappa}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-{kappa}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-{kappa}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-{kappa}B translocation mechanism.


Key Words: adhesion molecules • hypoxia • inflammation • nitric oxide • immunotherapy


*    Introduction
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 *Introduction
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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-{kappa}B (NF-{kappa}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-{kappa}B translocation and activates complement.29 30 In the present study, we investigated whether the terminal complement components influence endothelial NF-{kappa}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-{kappa}B translocation mechanism.


*    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-{kappa}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-{kappa}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-{kappa}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 2Down, 5Down, and 6Down) were normalized to hypoxic HUVECs reoxygenated in 30% HS. All data are expressed as mean±SEM.



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  		Figure 2. h5G1.1-scFv–mediated inhibition of endothelial C5b-9 deposition (ELISA). C5b-9 deposition was measured by ELISA on hypoxic (12 hours) HUVECs reoxygenated (30 minutes) in the presence of 30% HS treated with or without h5G1.1-scFv (20 µg/mL). h5G1.1-scFv significantly inhibited endothelial C5b-9 deposition. Data are normalized to hypoxic HUVECs reoxygenated in 30% HS (vehicle). n=4, *P<0.05 compared with vehicle.



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  		Figure 5. Immunohistochemical analysis of endothelial VCAM-1 expression. Immunofluorescent confocal microscopy demonstration of VCAM-1 expression (green) on normoxic and hypoxic (12 hours) HUVECs reoxygenated (3 hours) in the presence of 30% HS or 30% HS treated with 8-Br-cGMP (10 or 100 µmol/L), dibutyryl-cGMP (10 or 100 µmol/L), or h5G1.1-scFv (20 µg/mL). VCAM-1 expression on hypoxic HUVECs reoxygenated in 30% HS (B) was significantly greater than in normoxic HUVECs (A). In contrast, VCAM-1 staining was significantly decreased on hypoxic HUVECs reoxygenated in 30% HS treated with h5G1.1-scFv (C), 8-Br-cGMP (D), or dibutyryl-cGMP (E). Panels D and E depict the 100 µmol/L data only. Propidium iodide (red) was used to stain nuclei. Magnification 40x water objective.



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  		Figure 6. VCAM-1 protein expression after endothelial hypoxia/reoxygenation (ELISA). A, VCAM-1 protein expression was measured by ELISA on hypoxic (12 hours) HUVECs reoxygenated (3 hours) in buffer, 30% HS, or 30% HS treated with ODQ (100 µmol/L), LY83583 (100 µmol/L), dibutyryl-cAMP (10 µmol/L), 8-Br-cGMP (10 µmol/L), dibutyryl-cGMP (10 µmol/L), or h5G1.1-scFv (20 µg/mL). Data are normalized to hypoxic HUVECs reoxygenated in 30% HS (vehicle). VCAM-1 expression on hypoxic HUVECs reoxygenated in HS was significantly greater than on hypoxic HUVECs reoxygenated in buffer. Treatment with h5G1.1-scFv or the cGMP analogues 8-Br-cGMP or dibutyryl-cGMP significantly attenuated VCAM-1 expression. VCAM-1 expression was not attenuated by the cAMP analogue dibutyryl-cAMP or the cGMP antagonists ODQ or LY83583. B, VCAM-1 protein expression was measured by ELISA on hypoxic HUVECs reoxygenated in 30% HS or 30% HS treated with L-NAME, SNOG, or SNAP. Data are normalized to hypoxic HUVECs reoxygenated in 30% HS (vehicle). The NO analogues SNOG and SNAP significantly decreased HUVEC VCAM-1 expression in a dose-dependent manner. L-NAME significantly augmented VCAM-1 expression. n=3, *P<0.05 compared with vehicle.


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
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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 1Down) 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 1BDown) was significantly greater than in normoxic HUVECs bathed in 30% HS (Figure 1ADown). In contrast, no staining for C5b-9 was observed on hypoxic HUVECs reoxygenated in HS treated with h5G1.1-scFv (Figure 1CDown). Thus, reoxygenation of hypoxic HUVECs leads to increased C5b-9 deposition that is inhibited by h5G1.1-scFv.



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  		Figure 1. Immunohistochemical analysis of endothelial C5b-9 deposition. Immunofluorescent confocal microscopy demonstration of C5b-9 deposition (green) on normoxic and hypoxic (12 hours) HUVECs reoxygenated (30 minutes) in the presence of 30% HS treated with or without h5G1.1-scFv (20 µg/mL). C5b-9 deposition on hypoxic HUVECs reoxygenated in 30% HS (B) was significantly greater than normoxic HUVECs bathed in 30% HS (A). In contrast, no staining for C5b-9 was observed on hypoxic HUVECs reoxygenated in 30% HS treated with h5G1.1-scFv. Propidium iodide was used to stain nuclei (red). Magnification 40x water objective.

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 2Up). 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 3Down). 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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  		Figure 3. Endothelial cGMP after hypoxia/reoxygenation in the presence and absence of human serum. Intracellular cGMP concentrations were measured by ELISA in normoxic and hypoxic (12 hours) HUVECs reoxygenated (30 minutes) in buffer or 30% HS treated with or without h5G1.1-scFv (10 µg/mL). ACh (10 µmol/L)-induced cGMP synthesis in normoxic or hypoxic HUVECs reoxygenated in buffer was not significantly different. In contrast, ACh-induced cGMP synthesis was significantly higher in normoxic HUVECs incubated in HS compared with hypoxic HUVECs reoxygenated in untreated HS (*P<0.05). Inhibition of C5b-9 formation with h5G1.1-scFv significantly attenuated cGMP loss in hypoxic HUVECs reoxygenated in HS. +P<0.05, n=3.

Effect of Anti-Human C5 Therapy or cGMP Analogues on Endothelial NF-{kappa}B Translocation
C5b-9 is known to induce NF-{kappa}B translocation.40 Yet the mechanism by which C5b-9 induces NF-{kappa}B activation is unknown. To demonstrate that C5b-9 induces NF-{kappa}B translocation by decreasing endothelial cGMP, Western blot analysis of the nuclear p65 subunit of NF-{kappa}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 4ADown). NF-{kappa}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-{kappa}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 4BDown. These data demonstrate that anti-C5 or cGMP analogue therapy attenuates complement-induced NF-{kappa}B translocation in human endothelial cells.



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  		Figure 4. Western blot analysis of the HUVEC NF-{kappa}B p65 subunit. Nuclear protein lysates were resolved by SDS–polyacrylamide gel electrophoresis and subjected to Western blot analysis with a polyclonal antibody to the p65 subunit of NF-{kappa}B. NF-{kappa}B p65 band density (A) was increased in hypoxic (12 hours) HUVECs reoxygenated (30 minutes) in 30% HS (lane 2) compared with normoxic cells bathed in 30% HS (lane 1). In contrast, NF-{kappa}B p65 band density was significantly attenuated when hypoxic HUVECs were reoxygenated in 30% HS treated with h5G1.1-scFv (20 µg/mL) (lane 3), 100 µmol/L 8-Br-cGMP (lane 4), or 100 µmol/L dibutyryl-cGMP (lane 5). This panel is representative of 3 separate experiments. The mean integrated optical band density for the 3 separate experiments is depicted in B. +P<0.05 compared with normoxic cells; *P<0.05 compared with untreated hypoxic cells.

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-{kappa}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 5Up) 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 5BUp) was significantly greater than in normoxic HUVECs (Figure 5AUp). In contrast, VCAM-1 staining was significantly decreased on hypoxic HUVECs reoxygenated in 30% HS treated with h5G1.1-scFv (Figure 5CUp), 8-Br-cGMP (Figure 5DUp), or dibutyryl-cGMP (Figure 5EUp). 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 6AUp). 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 6AUp and 6BUp, 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.


*    Discussion
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 up arrowMethods
 up arrowResults
 *Discussion
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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-{kappa}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-{kappa}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-{kappa}B26 and can be induced by C5b-9.16 As C5b-9 is known to inhibit NO-dependent relaxation,13 14 15 activate NF-{kappa}B,40 and increase VCAM-1 expression,16 we investigated whether C5b-9 influences endothelial NF-{kappa}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-{kappa}B translocation and VCAM-1 protein expression after hypoxia/reoxygenation. Reoxygenation of hypoxic HUVECs in the presence of human sera significantly increased NF-{kappa}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-{kappa}B translocation and VCAM-1 expression. As endothelial VCAM-1 expression is regulated in part through NF-{kappa}B,26 these data suggest that C5b-9 may regulate NF-{kappa}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-{kappa}B translocation and VCAM-1 expression is also consistent with recent reports in which C5b-9 has been shown to activate endothelial NF-{kappa}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-{kappa}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-{kappa}B, and increases endothelial VCAM-1 protein expression. Endothelial NF-{kappa}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-{kappa}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.


*    Acknowledgments
 
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.


*    References
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