© 1995 American Heart Association, Inc.
Articles |
Complement-Mediated Loss of Endothelium-Dependent Relaxation of Porcine Coronary Arteries
Role of the Terminal Membrane Attack Complex
From Brigham and Women's Hospital (G.L.S., G.F.), Department of Anesthesia, Anesthesia Research Laboratories, Ischemia Reperfusion Center, Boston, Mass; the Department of Internal Medicine (G.L.S.), Division of Cardiovascular Medicine, University of California, Davis; and the Department of Dermatology (W.R.R.), Boston (Mass) University, School of Medicine.
Correspondence to Gregory L. Stahl, PhD, Brigham and Women's Hospital, Department of Anesthesia, Ischemia Reperfusion Center, 75 Francis St, Boston, MA 02115. E-mail gstahl@zeus.bwh.harvard.edu.
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Abstract Reperfusion of the ischemic myocardium results in the loss of endothelium-dependent relaxation. We have shown recently that the alternate complement pathway is activated immediately on reperfusion of the ischemic porcine myocardium. We hypothesized that complement activation directly attenuates endothelium-dependent relaxation of porcine coronary arteries. Bradykinin (BK) or substance P concentration-dependently relaxed precontracted (U46619, 50 nmol/L) left anterior descending coronary artery (LAD) rings in vitro. Addition of zymosan to human (10%) or porcine (10%) serum for 30 minutes significantly (P<0.05) increased the EC50 of BK-induced LAD relaxation from 4±1 to 418±159 nmol/L (n=8) and from 9±3 to 281±132 nmol/L (n=7), respectively. Similarly, addition of zymosan to 10% human serum (HS) for 30 minutes increased the EC50 of substance P–induced LAD relaxation from 0.4±0.1 to 30±14 nmol/L (n=9, P<.05). Basal release of nitric oxide was reduced significantly in LAD rings exposed to zymosan-activated HS compared with HS alone. Addition of soluble CR1 (sCR1, 10 nmol/L) to zymosan-activated HS preserved BK-induced relaxation (EC50) of the LAD rings (control, 4±1 nmol/L; sCR1+zymosan+serum, 2±1 nmol/L; n=6). Zymosan-activated C8-depleted HS (10%) did not attenuate the EC50 of BK-induced coronary artery relaxation (3±1 to 3±1 nmol/L, n=7, P=NS). Zymosan-activated C8-depleted HS plus C8 (6 µg/mL) increased the EC50 of BK-induced coronary artery relaxation from 4±1 to 423±141 nmol/L (n=12, P<.05). We have further demonstrated that C5b-9 complexes can be found on the luminal surface of LAD endothelial cells after 5 minutes of exposure to zymosan-activated HS by using C5b-9 reactive monoclonal antibody fluorescent immunohistochemistry and confocal microscopy. We conclude that complement activation directly attenuates endothelium-dependent relaxation through the formation of the terminal membrane attack complex (C5b-9).
Key Words: C5b-9 • soluble complement receptor type 1 • bradykinin • substance P • nitric oxide
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Introduction |
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The obligatory role of endothelium in the regulation of vascular tone was first described by Furchgott and Zawadzki.1 This endothelium-dependent relaxing factor (EDRF) has been characterized to be at least partly composed of nitric oxide or a closely related compound.2 Since this discovery, many substances and physiological and pathophysiological conditions have been shown to augment, attenuate, or modulate the synthesis, production, or effects of EDRFs.
Studies of myocardial ischemia and reperfusion have shown marked alterations in endothelium-dependent relaxation of the coronary vasculature.3 4 5 Even brief periods of myocardial ischemia and reperfusion in swine alter endothelium-dependent, but not endothelium-independent, relaxation of coronary arteries.6 Although the mechanisms by which ischemia and reperfusion induce endothelial dysfunction remain unclear, there is reason to suspect that neutrophils (PMNs) and oxygen-derived free radicals play a role. Activated PMNs and oxygen-derived free radicals are toxic to endothelial cells.7 Further, superoxide ions inactivate EDRF and initiate lipid peroxidation, thus altering membrane permeability and leading to endothelial cell dysfunction.8 Additional experimental data demonstrate that monoclonal antibodies against neutrophil or endothelial cell adherence proteins provide not only myocardial protection but also preserve endothelium-dependent relaxation.9 10 11 12
A significant amount of evidence suggests that the complement cascade plays an important role in myocardial ischemia and reperfusion injury. First, the alternate complement pathway is activated immediately on reperfusion of the ischemic porcine myocardium.13 Second, subcellular membrane fragments released after myocardial ischemia have been shown to activate the complement cascade.14 15 Third, depletion of complement with cobra venom factor or inhibition of complement activation with soluble complement receptor type 1 (sCR1) provides protection against experimental models of myocardial ischemia and reperfusion injury.16 17 Fourth, C5b-9 directly induces cardiac dysfunction in the isolated human plasma–perfused rabbit heart.18 19 Fifth, intracoronary infusion of porcine C5a induces myocardial ischemia.20 21 Sixth, recent evidence from our laboratory demonstrates that a monoclonal antibody against porcine C5a protects the ischemic porcine myocardium against reperfusion injury.13 Thus, the complement system plays an important role in myocardial ischemia and reperfusion injury. However, the mechanisms of complement-induced myocardial injury and the importance of the various complement components have not been established fully.
There is evidence to suggest that complement may attenuate endothelial cell function. The terminal membrane attack complex (MAC, C5b-9) is observed along endothelial cells soon after reperfusion and may elicit endothelial cell injury by forming transmembrane channels.16 22 Additionally, complement activation releases heparan sulfate from cultured porcine endothelial cells.23 24 Heparan sulfate tethers superoxide dismutase to endothelial cells and attaches the endothelium to the extracellular matrix.23 Further, complement activation and C5b-9 formation have been shown to upregulate the PMN adherence protein, P-selectin, on endothelial cells.25 26 However, the direct role of the complement system on endothelial cell dysfunction and the loss of endothelium-dependent relaxation is unknown.
Recently, we have observed that the alternate complement cascade is activated immediately on reperfusion of the ischemic porcine myocardium.13 This time course of complement activation parallels the maximal loss of endothelium-dependent relaxation observed in another model of myocardial ischemia and reperfusion.3 In the present study, we investigated the potential direct effects of complement activation on endothelium-dependent relaxation of the porcine coronary vasculature. We hypothesized that complement activation would directly attenuate endothelium-dependent relaxation of porcine coronary arterial rings in vitro in the absence of blood-borne inflammatory cells (ie, PMNs). Our data demonstrate that complement activation directly attenuates endothelium-dependent relaxation of porcine coronary vessels. The presence of C5b-9 on the vascular endothelium, normal endothelium-dependent relaxation of left anterior descending coronary artery (LAD) rings after activation of C8-depleted serum, and abnormal endothelium-dependent relaxation of LAD rings after activation of C8-supplemented C8-depleted serum suggest that the loss of endothelium-dependent relaxation is mediated by the formation of C5b-9.
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Animal and Human Research Guidelines
All procedures in the present study were approved by the Human Subjects Review Committee and the Animal Use and Care Administrative Advisory Committee of the University of California, Davis, and the Harvard Medical Area Standing Committee on Animals.
Materials
Bradykinin, substance P,
N
-nitro-L-arginine methyl ester
(L-NAME), and zymosan were purchased from Sigma Chemical Co. U46619 was
a gift from Upjohn. Complement compounds, including C8 and C8-depleted
human serum, were purchased from Quidel. sCR1 was a gift from
SmithKline and Beecham. Mouse anti-human C5b-9 antibodies were
purchased from Dako and Quidel.
Coronary Vessel Preparation
Domestic swine (35 to 45 kg) were premedicated and anesthetized
as described previously.21 The hearts were removed, 2 to 3
cm of the proximal portion of the LAD was dissected free, and
connective tissue was removed. The vessels were cut into 2- to 3-mm
rings and mounted between a pair of stainless steel hooks. One hook was
attached to a force transducer (Grass model FT03). The other hook was
attached to the bottom of a warmed (37°C) organ bath (5 mL). The
organ bath was filled and oxygenated (95% O2/5%
CO2) with Krebs-Henseleit (KH) buffer as we have described
previously.27 28 Preliminary length-tension studies in our
laboratory demonstrated that a preload of 4 to 6 g was optimal for
contraction and relaxation studies with this size porcine coronary
artery. The KH buffer was replaced every 20 to 30 minutes. The analog
signals were digitized, analyzed, and stored by a commercially
available software system (PO-NE-MAH) on a 486-based
computer.
After an equilibration period of 1 hour, the rings were contracted with
100 mmol/L K+ KH buffer and washed. This procedure was
repeated three times. If necessary, the preload was adjusted back to 4
to 6 g after each 100 mmol/L K+ KH buffer–induced
contraction. The LAD rings then were contracted with U46619 (50
nmol/L), which resulted in a stable contraction for 15 to 20 minutes.
Use of other vasoconstricting agents (ie, norepinephrine,
phenylephrine, prostaglandin F2
, and
acetylcholine) in preliminary studies resulted in poor or unstable
contractions. At the peak of contraction, a dose-response curve to
bradykinin (BK, 10-9 to 10-6 mol/L) or
substance P (10-10 to 10-8 mol/L) was
performed by adding cumulative amounts of these
endothelium-dependent relaxing
agents.29 30 31 The LAD rings then were washed with KH buffer
and allowed to return to baseline.
After 1 hour, each of the rings was then bathed in one of the following supplemented KH buffers: (1) 10% porcine serum, (2) 10% human serum, (3) zymosan (1 mg/mL), (4) 1%, 3%, or 10% porcine serum plus zymosan (1 mg/mL), (5) 1%, 3%, or 10% human serum plus zymosan (1 mg/mL), (6) 10% porcine serum plus zymosan (1 mg/mL) plus sCR1 (10 nmol/L), (7) 10% human serum plus zymosan (1 mg/mL) plus sCR1 (1 nmol/L), (8) C8-depleted human serum (10%) plus zymosan (1 mg/mL), (9) C8-depleted human serum (10%) plus zymosan (1 mg/mL) plus C8 (6 µg/mL), (10) zymosan-activated human serum (10%), or (11) human recombinant C5a (0.1 µmol/L). After 30 minutes, each bath was then washed with normal KH buffer and contracted with U46619 (50 nmol/L), and a second dose-response curve to BK or substance P was generated.
Inhibition of Basal Release of Nitric Oxide
Additional LAD rings were exposed to human serum (10%) or human
serum (10%) plus zymosan (1 mg/mL) for 30 minutes. These vessels then
were washed and exposed to L-NAME (100 µmol/L) to inhibit the basal
release of nitric oxide in these vessels.26
Immunohistochemistry
Purified mouse anti-human C5b-9 (MAC) IgG2 monoclonal antibody
(anti-human MAC, aE11, Dakopats A/S) was used for immunohistochemical
detection of human C5b-9 in the tissues. Fluorescein isothiocyanate
(FITC)–labeled goat anti-mouse IgG F(ab')2 (Jackson Immuno Research
Laboratories) secondary antibody was used for visualization.
Tissue samples were fixed in 2% paraformaldehyde for 2 to 3 hours, then placed in 30% sucrose for 14 to 16 hours at 4°C, and frozen in 2-methyl-butane at -70°C. Frozen tissue sections (10 to 12 µm thin) were prepared after imbedding into Tissue-Tek OCT compound (Miles Diagnostics) on a Zeiss Microm HM 505E cryostat. Multiple sections were mounted on precleaned and coated Superfrost/Plus (Fisher Scientific) microscopic slides, fixed with acetone, air-dried, and processed for immunohistochemical staining.
Tissue samples were blocked with 1% goat serum for 20 minutes at room temperature, washed, and then incubated with anti-human C5b-9 monoclonal antibody in phosphate-buffered saline (PBS) containing 1% bovine serum albumin for 1 hour. After the slides were washed, they were incubated with the FITC-labeled goat anti-mouse secondary antibody for 30 minutes, washed with PBS several times, and covered with antifade solution (SlowFade, Molecular Probes Inc) and coverslips for confocal microscopic analysis.
Confocal Microscopic Analysis
The specimens were analyzed with the Leica confocal laser
scanning microscope equipped with an argon ion laser with an output
power of 2 to 50 mW, two photomultiplier tubes, and narrow band
filters. The argon laser has two excitation wavelengths, 488 and 514
nm. The apertures were set at the minimum size for optimal signal.
Smaller apertures (pinhole) allow a narrower optical cross section and
less background.32 33 The images are en face optical
sections through the vertical axis of the tissue. Each image of the
series was taken at 1.0-µm intervals in the 512x512 pixel format.
The tissue sections for immunohistochemistry were viewed with a x100
(numerical aperture, 1.0) water immersion lens. Optical sections were
taken through 3 to 10 layers, depending on the intensity of the stain.
The images were analyzed, enhanced, and stored on an optical disc.
Pseudocolor images were computer-generated with a twofold computer
enhancement. All samples were collected, analyzed, and enhanced under
the same conditions.
Statistical Analysis
The data are presented as mean±SEM. Relaxation of the
preconstricted LAD rings is presented as a percentage of the
U46619-induced contraction. The median effective concentration
(EC50) of BK or substance P inducing a 50% relaxation of
the preconstricted LAD ring was calculated for each vessel.
Distribution of data was assessed with a Shapiro-Wilk
test.34 A paired t test or the Wilcoxon signed
rank test (analogous nonparametric t test) was used to
establish significance of the EC50 data.35
Differences in relaxation between the control and experimental group at
the various BK or substance P concentrations were analyzed with
Friedman's repeated-measures ANOVA followed by the
Student-Newman-Keuls post hoc test. Statistical significance was taken
at P
.05. Analyses were performed using
CRUNCH 4 (Crunch Software Corp) and
SIGMASTAT (Jandel Scientific) for the IBM computer.
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Results |
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A total of 38 pigs were used in the present study. High K+ (100 mmol/L) KH buffer contracted the LAD rings by 10.5±0.6 g. The first U46619-induced contraction was 73±4% of the 100 mmol/L K+ KH buffer response.
Time Control Experiments
Zymosan, porcine (10%), or human (10%) serum did not
significantly (P>.05) alter U46619-induced contraction of
the LAD rings compared with the first U46619 contraction (7.7±2.4
versus 9.0±3.1 g, 3.9±0.4 versus 5.7±0.7 g, and 4.5±1.1 versus
5.4±1.2 g, respectively.
Fig 1
summarizes the effect of zymosan (Fig 1A), porcine
(Fig 1B), or human (Fig 1C) serum on BK-induced relaxation. BK
concentration-dependently relaxed the LAD rings during control states
in the presence of KH buffer (Fig 1A through 1C). Repeating the BK
dose-response curve after incubation of the LAD rings with zymosan (Fig 1A), 10% porcine serum (Fig 1B), or 10% human serum (Fig 1C) for 30
minutes did not attenuate BK-induced relaxation. The EC50
for BK-induced relaxation of the LAD rings was not significantly
different from the control value when compared with zymosan, porcine,
or human serum (3±1 versus 3±1 nmol/L, 3±1 versus 2±1 nmol/L, and
3±1 versus 4±1 nmol/L, respectively).
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Effect of Zymosan-Activated Human Serum on LAD Ring Contraction and
Relaxation
Zymosan-activated human serum (10%) did not significantly alter
U46619-induced contraction of the LAD rings (n=8) compared with the
first U46619-induced contraction (7.1±1.3 versus 7.8±1.2 g,
P=NS).
Zymosan-activated human serum concentration-dependently attenuated
BK-induced relaxation of the LAD rings (Fig 2). No
significant loss of BK-induced relaxation was observed after a
30-minute incubation with zymosan-activated 1% human serum in six LAD
rings (Fig 2A). The EC50 for BK-induced relaxation of the
LAD rings in the KH buffer (ie, control) was 3±1 nmol/L and increased
to 11±3 nmol/L (P<.05) after incubation of the LAD rings
in 3% human serum activated with zymosan for 30 minutes. A greater
loss of endothelium-dependent relaxation was observed
after incubation (ie, 30 minutes) of eight LAD rings in 10% human
serum activated with zymosan, with the EC50 increasing from
4±1 to 418±159 nmol/L (P<.05). However, nitroglycerin
(10-5 mol/L) completely relaxed all the LAD rings to the
baseline value.
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Effect of Zymosan-Activated Porcine Serum on LAD Ring Contraction
and Relaxation
Zymosan-activated porcine serum (10%) did not significantly alter
U46619-induced contraction of the LAD rings (n=7) compared with the
first U46619-induced contraction (6.1±1.4 versus 7.2±1.7 g,
P=NS).
Zymosan-activated porcine serum attenuated BK-induced relaxation
of the LAD rings (Fig 3). No significant loss of
BK-induced relaxation was observed after a 30-minute incubation with
zymosan-activated 1% or 3% porcine serum in three and seven LAD
rings, respectively (Fig 3A and Fig 3B, respectively). The
EC50 for BK-induced relaxation of the LAD rings was 4±1
nmol/L before and after incubation of the LAD rings in 3%
zymosan-induced activation of porcine serum. The EC50 for
BK-induced relaxation of the LAD rings in the KH buffer (ie, control)
was 2±1 nmol/L and increased to 10±1 nmol/L (P<.05) after
incubation of seven additional LAD rings in 7% zymosan-activated
porcine serum for 30 minutes. A greater loss of
endothelium-dependent relaxation was observed after
incubation of seven LAD rings in 10% zymosan-activated porcine serum,
with the EC50 increasing from 9±3 to 281±132 nmol/L (Fig 3C, P<.05). However, nitroglycerin (10-5
mol/L) completely relaxed all the LAD rings to the baseline value.
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Effect of Zymosan-Activated Human Serum on Substance P–Induced
Relaxation
LAD rings incubated with 10% human serum did not attenuate
the EC50 for substance P–induced relaxation (Fig 4, top). The EC50 for substance P–induced
relaxation of the LAD rings was 0.3±0.1 and 0.5±0.1 nmol/L (n=7,
P=NS) after incubation of the LAD rings in KH buffer (ie,
control) and human serum (10%), respectively. Substance P–induced
relaxation was significantly attenuated when the LAD rings were
incubated with zymosan-activated human serum (Fig 4, bottom). The
EC50 for substance P–induced relaxation for the LAD rings
in the KH buffer was 0.4±0.1 nmol/L and increased to 30±14 nmol/L
(n=9, P<.05) after incubation of nine LAD rings in
zymosan-activated human serum for 30 minutes.
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Effect of Zymosan-Induced Activation of Human Serum on Basal
Release of Nitric Oxide
Fig 5A shows a representative tracing of a
porcine LAD ring receiving L-NAME (100 µmol/L) after exposure to 10%
human serum for 30 minutes. Exposure of six LAD rings to L-NAME after
incubation in 10% human serum increased baseline tension by
8±2%.
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Fig 5B
shows a representative tracing of a porcine LAD ring
receiving L-NAME (100 µmol/L) after exposure to 10% human serum and
zymosan for 30 minutes. Exposure of nine LAD rings to L-NAME after
incubation in 10% human serum and zymosan increased baseline tension
by 2±1%. We observed a significantly greater increase in tension
above baseline after application of L-NAME to LAD rings exposed to 10%
human serum compared with 10% human serum plus zymosan (Fig 5C).
Role of Complement in Loss of Endothelium-Dependent Relaxation
Incubation of four LAD rings with 10% heat-inactivated (56°C
for 30 minutes) human serum plus zymosan (1 mg/mL) for 30 minutes did
not attenuate the EC50 for BK-induced relaxation compared
with the control value (33±7 versus 39±19 nmol/L, respectively;
P=NS).
Administration of sCR1 (1 nmol/L) to zymosan-activated human serum (10%) did not significantly alter U46619-induced contraction of the LAD rings (n=6) compared with the first U46619-induced contraction (7.0±1.9 versus 10.0±2.6 g, respectively; P=NS). Similarly, sCR1 (10 nmol/L) in the presence of zymosan-activated porcine serum (10%) did not significantly alter U46619-induced contraction of the LAD rings (n=6) compared with the first U46619-induced contraction (4.8±0.5 versus 5.9±0.4 g, respectively; P=NS).
sCR1 preserved BK-induced relaxation of the LAD rings incubated with
zymosan-activated human serum (Fig 6A) or
zymosan-activated porcine serum (Fig 6B). The EC50 of
BK-induced relaxation of LAD rings in the KH buffer (ie, control) was
4±1 and 2±1 nmol/L before and after sCR1 treatment of
zymosan-activated human serum (Fig 6A, P=NS). Similarly, 10
nmol/L sCR1 preserved BK-induced relaxation of LAD rings (n=6)
incubated with zymosan-activated porcine serum. The EC50
for BK-induced relaxation was 9±4 and 10±3 nmol/L (P=NS)
before and after sCR1 treatment of zymosan-activated porcine serum,
respectively.
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Effect of C5a on the Loss of Endothelium-Dependent Relaxation
Recombinant human C5a (0.1 µmol/L, Sigma) failed to attenuate
BK-induced relaxation in six LAD rings. The EC50 (3±1
nmol/L) for BK-induced relaxation remained unchanged after incubation
of the rings with C5a for 30 minutes.
Zymosan-activated human serum was made in a test tube, as we described previously,21 and then added to the LAD rings. In this protocol, addition of zymosan-activated human serum (10%) failed to attenuate BK-induced relaxation in four additional LAD rings (EC50, 4±1 versus 2±1 nmol/L).
Role of C8 in the Loss of Endothelium- Dependent Relaxation
Zymosan-activated C8-depleted human serum (10%) did not
significantly alter U46619-induced contraction of the LAD rings (n=7)
compared with the first U46619-induced contraction (5.8±0.7 versus
4.1±0.9 g, respectively; P=NS). Similarly, addition of C8
(6 µg/mL) to zymosan-activated C8-depleted human serum (10%) did not
significantly alter U46619-induced contraction of the LAD rings (n=12)
compared with the first U46619-induced contraction (3.1±0.7 versus
5.1±0.8 g, respectively; P=NS).
LAD rings incubated with zymosan-activated C8-depleted human serum
demonstrated no significant loss of BK-induced relaxation (Fig 7A). The EC50 for BK-induced relaxation of
the LAD rings was 3±1 nmol/L before and after incubation of the LAD
rings with zymosan-activated C8-depleted human serum. In contrast,
addition of C8 (6 µg/mL) to zymosan-activated C8-depleted human serum
significantly attenuated endothelium-dependent
relaxation (Fig 7B). The EC50 for BK-induced relaxation of
the LAD rings in KH buffer was 4±1 nmol/L and increased significantly
(P<.05) to 423±141 nmol/L after the addition of C8 (6
µg/mL) to zymosan-activated C8-depleted human serum. The
EC50 for BK-induced relaxation of LAD rings after
incubation with 10% C8-depleted serum plus C8 plus zymosan was not
significantly different from that found with 10% human serum plus
zymosan (423±141 versus 418±159 nmol/L, respectively).
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Immunoreactive C5b-9 Is Present on Coronary Vascular Endothelium
After Complement Activation
Representative confocal microscopic images of porcine LAD
coronary arteries are presented in Fig 8. The
luminal side of the LAD ring was exposed to zymosan (1 mg/mL) and 10%
HS for 5 (Fig 8A and 8D) or 30 (Fig 8B and 8E) minutes and then
processed for frozen sectioning and immunofluorescent histochemical
evaluation. A mouse anti-human C5b-9 monoclonal antibody (aE11, Dako)
combined with an FITC-labeled goat anti-mouse secondary antibody
(Jackson Immunochemicals) was used for the detection of C5b-9 protein
complexes. Confocal microscopic analysis was used for
visualization. C5b-9 immunoreactivity (intense fluorescence) can be
detected after a 5-minute exposure of the samples (Fig 8A) to
zymosan-activated 10% human serum on the luminal surface (arrows
denote surface) of endothelial cells, with a further increase after 30
minutes of incubation (Fig 8B). Identical tissue samples stained with
the secondary antibody only (Fig 8D through 8F; control staining for
Fig 8A through 8C, respectively) showed no staining in this region.
Furthermore, LAD rings incubated with 10% human serum for 30 minutes
did not demonstrate the presence of C5b-9 (Fig 8C). However, we noted a
high level of autofluorescence in the region of the elastic lamina in
all tissue samples regardless of treatment. The autofluorescence made
it impossible to use conventional fluorescence microscopy for the
evaluation of the samples and necessitated the use of confocal
microscopy. It should be noted that conventional immunohistochemical
techniques (ie, ABC kit with color reaction for visualization) were
only able to demonstrate the presence of C5b-9 on endothelial cells,
when extremely high concentrations of complement (zymosan-induced
activation of 100% human serum) were used. However, those studies have
also demonstrated that the microscopic structure of the blood vessels
and endothelial cells was intact (data not shown here) when zymosan
activation of 10% human serum was used (ie, same concentration of
activated serum that demonstrated functional loss of
endothelium-dependent relaxation).
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Several important findings were observed in the present study. Activation of 10% human or porcine serum with zymosan attenuated endothelium-dependent relaxation of porcine LAD rings. Basal release of nitric oxide was significantly inhibited by exposure of LAD rings to zymosan-activated human serum. sCR1 prevented zymosan-activated porcine serum– or human serum–induced loss of endothelium-dependent relaxation. Addition of recombinant human C5a or previously generated complement products (ie, zymosan-activated human serum) failed to attenuate endothelium-dependent relaxation. Removal of the complement component C8 in human serum also prevented the loss of endothelium-dependent relaxation induced by zymosan-activated human serum. The addition of C8 to C8-depleted human serum followed by zymosan activation significantly attenuated endothelium-dependent relaxation. Immunohistochemical staining of the coronary vessels demonstrated the presence of immunoreactive C5b-9 on the luminal surface of the vascular endothelium. Thus, we have demonstrated that complement activation directly attenuates endothelium-dependent relaxation of the porcine coronary vasculature, with the concomitant presence of C5b-9 on the endothelial cell membrane. These results suggest that the terminal MAC (ie, C5b-9) initiates the loss of endothelium-dependent relaxation in this model.
Role of Complement in the Loss of Endothelium-Dependent
Relaxation of Coronary Vessels
The role of complement in the loss of
endothelium-dependent relaxation of the coronary
vasculature has not been studied. Complement activation may attenuate
endothelium-dependent relaxation directly or
indirectly. Complement activation results in the production of C5a, a
potent chemotaxin and PMN-aggregating and -activating
anaphylatoxin.36 37 Thus, C5a production following
reperfusion could attenuate endothelium-dependent
relaxation of coronary vessels indirectly by activating PMNs.
Additionally, C5a has been shown to release heparan sulfate from
porcine endothelial cells. Heparan sulfate tethers superoxide dismutase
to the endothelial cell and secures the endothelial cell to the
extracellular matrix.23 24 In the present study, the
addition of a high concentration of human C5a (0.1 µmol/L) did not
attenuate BK-induced relaxation of the porcine LAD rings. Additionally,
the addition of zymosan-activated human serum, which contains C3a/C3a
des-Arg, C5a/C5a des-Arg, and soluble C5b-9, did not attenuate
endothelium-dependent relaxation. These data are
similar to a preliminary study by Rendig et al,38 which
demonstrated that C5a does not attenuate
endothelium-dependent relaxation of the porcine
microvasculature. Further, zymosan-activated C8-depleted human serum
did not attenuate BK-induced relaxation in the present study. One
would expect C5a and C3a to be produced after zymosan activation of
C8-depleted human serum, although C5a and C3a were not measured in
these experiments. Thus, it appears that the anaphylatoxins, C5a and
C3a, do not directly attenuate endothelium-dependent
relaxation of porcine coronary arteries in this model.
The formation of membrane-bound C5b-9 could directly attenuate endothelium-dependent relaxation of the porcine coronary artery rings. It is well known that C5b-9 forms transmembrane pores; in nonnucleated cells, this activity results in cellular lysis. It is less appreciated that the formation of C5b-9 may directly cause cellular activation in the absence of cellular lysis. Lysis of nucleated cells is less likely because of the presence of various membrane-bound complement regulatory proteins or homologous restriction factors, including CD59 (protectin), CD46 (membrane cofactor protein, MCP), CD55 (decay-accelerating factor), and C8 binding protein.39 40 41 42 Endothelial cells have two well-characterized membrane-bound proteins, CD59 and CD55, that restrict complement activation at the plasma membrane.43 The regulation of C5b-9 formation by CD59 and CD55 is generally considered to be species restricted. However, recent observations by Van den Berg and Morgan44 suggest that porcine CD59 may not be homologously restricted. We observed concentration-dependent inhibition of endothelium-dependent relaxation after activation of 3% to 10% human serum. However, we observed a significant loss of endothelium-dependent relaxation of porcine coronary arteries only after activation of 7% to 10% porcine serum. These data suggest that porcine endothelial cell complement-regulatory mechanisms do not efficiently recognize human complement but effectively attenuate low levels of porcine complement activation.
It is unlikely that zymosan-activated serum resulted in the direct lysis of porcine endothelial cells in the present study for several reasons. First, in a series of histochemical studies attempting to demonstrate the presence of C5b-9 on endothelial cells, we observed no damage to the microscopic appearance of the endothelial cells, except when extremely high concentrations of activated complement (100% activated serum) were used. Second, the membrane regulators of complement activation (ie, CD59 and CD55) would have been able to effectively inhibit lytic concentrations of C5b-9 in only 10% serum. Third, endothelial cells exposed to other species of complement are not easily lysed even after 48 hours of exposure to complement.45 Fourth, one would expect to observe a significant attenuation or augmentation of the contractile effect of U46619 in the present study, a finding that was not observed. Therefore, we postulate that it is more likely that C5b-9 formed sublytic concentrations of transmembrane pores in the present study. We speculate that C5b-9 leads to the activation of the endothelium and the loss of endothelium-dependent relaxation. However, this assumption is speculative and warrants further investigation.
Endothelium-Dependent Relaxation and Nitric Oxide Formation
It is well known that porcine coronary arteries and endothelial
cells release multiple EDRFs when exposed to BK.30 31
Thus, the loss of BK-induced porcine coronary artery relaxation
resulted from a loss of endothelium-"dependent"
relaxation in the present study. However, substance P releases only
nitric oxide from porcine endothelial cells.29 Thus, we
conclude that the attenuated substance P–induced relaxation of LAD
rings, exposed to zymosan-activated human serum, was a result of
altered nitric oxide–induced relaxation.
Vascular ring preparations, like those performed in the present study, release basal amounts of nitric oxide from normal functioning endothelial cells.26 We observed that the addition of L-NAME to LAD rings exposed to human serum resulted in contraction of the vascular smooth muscle. Further, LAD rings exposed to 10% human serum relaxed in the presence of BK or substance P. These data demonstrate that both basal nitric oxide and mediator-induced release of nitric oxide are not inhibited by 10% human serum in this model. In contrast, LAD rings exposed to human serum and zymosan contracted significantly less to L-NAME and relaxed significantly less to substance P or BK. Thus, complement attenuates not only the pharmacologically induced release of nitric oxide but the basal release as well. Future studies will investigate the mechanism(s) by which C5b-9 attenuates nitric oxide–induced relaxation.
Possible Role of C5b-9 in Myocardial Ischemia and
Reperfusion
There is reason to suspect that the terminal MAC (C5b-9) is an
important mediator of ischemia and reperfusion injury. First, C5b-9
directly induces myocardial dysfunction in the isolated perfused rabbit
heart.18 19 46 Second, C5b-9 directly modifies myocardial
contractility and intracellular calcium in isolated
myocytes.47 Third, deposition of C5b-9 has been
demonstrated on myocardial cells in infarcted areas obtained from
autopsies, in plasma, and along endothelial cells soon after
reperfusion of ischemic human myocardium.16 22 48 49
However, the importance of the presence of C5b-9 on endothelial cells
is unknown.
In the present study, we demonstrate that either zymosan-activated porcine or human serum results in a loss of endothelium-dependent relaxation of porcine coronary arteries and is associated with deposition of C5b-9 on the endothelial cells. Further, we have shown that the basal release of nitric oxide was also attenuated by zymosan-activated serum. The loss of endothelium-dependent relaxation appears to be mediated by the formation of C5b-9, since depletion and subsequent addition of C8 resulted in normal and abnormal endothelium-dependent relaxation of the LAD rings, respectively. The loss of a functional endothelial cell lining in the myocardium could be potentially harmful to an already ischemic vascular bed. Loss of endothelium-dependent relaxation may predispose the coronary vasculature to vasoconstrictive metabolites formed during ischemia and reperfusion and lead to vasospasm. Along these lines, complement activation has been shown to play a significant role in cerebral vasospasm following subarachnoid hemorrhage in humans.50 These data suggest that complement activation during myocardial ischemia and reperfusion may result in the loss or attenuation of endothelium-dependent relaxation.
Complement activation and C5b-9 formation have been shown to upregulate P-selectin on endothelial cells.25 26 Recent data suggest that inhibition of nitric oxide synthase with L-NAME induces the upregulation of P-selectin.51 P-selectin is necessary for PMN–endothelial cell adherence and the resulting transendothelial migration.9 Furthermore, inhibition of P-selectin function protects the feline myocardium from ischemia and reperfusion injury.9 52 Thus, complement activation may be an early and possibly a necessary factor in the upregulation of P-selectin and transcellular migration of PMNs after ischemia and reperfusion.
Limitations of the Present Model
We have shown recently that complement activation is observed
immediately on reperfusion of the ischemic porcine
myocardium.13 However, the present LAD ring
preparation is not a model of ischemia and reperfusion. Whether
complement-mediated loss of endothelium-dependent
relaxation takes place in vivo remains unknown. Further, regulation of
coronary blood flow is at the arteriolar level. It is unknown at
present whether complement attenuates
endothelium-dependent relaxation of the
microvasculature. Future studies in our laboratory will examine the
effects of complement activation on the microvasculature in vitro and
after ischemia and reperfusion in vivo.
Summary and Conclusion
We have shown that activation of the complement cascade directly
attenuates the endothelium-dependent relaxation of
porcine coronary arteries. The formation of C5b-9 is the contributing
factor in the loss of endothelium-dependent relaxation
after complement activation. These studies demonstrate that in addition
to oxygen-derived free radicals and activated PMNs, complement can
directly attenuate the endothelium-dependent relaxation
of coronary arteries and possibly lead to coronary vasospasm.
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Acknowledgments |
|---|
This study was supported by the American Heart Association, California Affiliate, Inc (91-32A), the Golden Empire Chapter, and the Brigham and Women's Hospital Anesthesia Foundation. Dr Stahl was a research fellow of the American Heart Association, California Affiliate, Inc, during a portion of these studies. We acknowledge the technical assistance of Chris Longhurst and Margaret Morrissey.
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Footnotes |
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Previously presented in part at the Experimental Biology meetings, Anaheim, Calif, April 23-28, 1994, and the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994.
This manuscript was sent to Harold C. Strauss, MD, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Received May 11, 1994; accepted December 16, 1994.
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References |
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