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2812
Basic Science Reports
Soluble Complement Receptor Type 1 Inhibits
the Complement Pathway and Prevents
Contractile Failure in the Postischemic Heart
Evidence That Complement Activation Is Required for
Neutrophil-Mediated Reperfusion Injury
Suresh M.L. Shandelya, MD; Periannan Kuppusamy, PhD; Ahvie Herskowitz, MD;
Myron L. Weisfeldt, MD; Jay L. Zweier, MD
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Background. Complement-mediated neutrophil activation has been hypothesized to be an important
mechanism of reperfusion injury. It has been proposed that soluble complement receptor 1 (sCR1), a
potent inhibitor of both classical and alternative complement pathways, may prevent the complementdependent activation of polymorphonuclear leukocytes (PMNs) that occurs within postischemic myocardium and thereby inhibit PMN-derived free radical generation and prevent postischemic contractile
failure. Therefore, we performed studies to determine the effects of sCRi on contractile function, PMN
adhesion, complement deposition, and PMN-derived free radical generation in the postischemic heart.
Methods and Results. Studies were performed in an isolated rat heart model in which the isolated effects
of given cellular or humoral factors could be determined. Plasma and PMNs were present to study the
effects of sCRi on contractile function, coronary flow, leukocyte adhesion, complement deposition, and
PMN-derived free radical generation. Isolated rat hearts were perfused by the method of Langendorff
(n=10 in each group) and subjected to 20 minutes of global ischemia and reperfusion with PMNs and
plasma in the presence or absence of sCR1. Left ventricular developed pressure (LVDP), coronary flow
(CF), left ventricular end-diastolic pressure (LVEDP), and rate-pressure product (RPP) were measured
during the preischemic period, during 1-minute control infusion of PMNs and plasma, and on reflow
following 20 minutes of global ischemia. During the preischemic control infusion, no significant
alterations in the physiologic parameters were observed, and there was no measurable free radical
generation. Reperfusion with sCRI markedly improved the recovery of postischemic contractile function.
LVDP after 45 minutes of reperfusion was 76+9.8% compared with 32±6.2% (P<.001). In addition,
significant improvements in LVEDP, RPP, and CF were observed in hearts treated with sCRi. Additional
experiments were also performed to determine the effect of sCRI on complement-mediated PMN
activation. Measurements of PMN-derived free radical generation were performed in both isolated PMNs
and the coronary effluent of hearts using electron paramagnetic resonance spectroscopy (EPR) with the
spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). EPR measurements in both isolated PMNs and
coronary effluent demonstrated that sCRl blocked complement-mediated free radical generation from the
PMNs. Increased accumulation of PMNs was observed both in hearts treated with sCRl and in those not
treated with sCRl. Immunohistochemical staining of the postischemic myocardial tissue demonstrated
marked complement deposition on the endothelial surface of small arterioles and capillaries, which was
prevented by sCRI treatment. Thus, sCRI did not prevent PMN adhesion but did prevent complement
deposition with activation of the PMN oxidative burst.
Conclusions. The potent complement inhibitor sCRl was found to be effective at preventing postischemic
myocardial contractile dysfunction and enhancing the recovery of coronary flow. This study demonstrated
that complement activation occurs in postischemic myocardium and is necessary for activation of the
neutrophil oxidative burst with the generation of reactive oxygen free radicals. The process of neutrophil
adhesion, however, was not affected by sCRl and was independent of complement factors. These findings
demonstrate the sCRi is a highly potent agent at preventing complement-mediated PMN activation and
secondary free radical generation in the postischemic heart. This genetically engineered protein appears
to be a promising therapeutic agent in the prevention of myocardial reperfusion injury. (Circulation.
1993;88:2812-2826.)
KEY WORDS * free radicals * reperfusion * myocardium * ischemia * complement
Shandelya et al Complement Receptor 1 Prevents Postischemic Injury
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It has been suggested that the activation of the complement pathway within ischemic myocardium can
promote the increased recruitment of intracardiac
inflammatory cells, which then results in further myocardial injury.1-3 There has been increasing evidence from
both animal and clinical studies that complement split
products are formed in ischemic myocardium. With the
activation of the complement cascade, the split products
C3a and C5a are generated, resulting in increased activation of polymorphonuclear leukocytes (PMNs), which
then adhere to the vascular endothelium and further
occlude the coronary microcirculation, potentially resulting in a no-reflow phenomenon.4-6 Further experiments
have demonstrated that the anaphylatoxins C3a and C5a
constrict vascular smooth muscle and thereby cause a
significant decrease in coronary flow by increasing the
arterial vascular resistance.7,8 Longhurst et a19 have shown
that intracoronary administration of purified porcine C5a
in swine produces reductions in both coronary blood flow
and left ventricular function. In addition, several studies
have shown that the complement system is an important
mediator in acute myocardial ischemia. However, it is not
clear whether the complement activation that occurs
causes myocardial injury with impaired cardiac contractile
function.10-13 Therefore, it has been suggested that activation of the complement system of proteins may be an
important mechanism in the pathogenesis of postischemic
injury, but questions remain regarding the significance of
this complement activation.
The activation of the complement system in response
to ischemia could result in either direct complementmediated injury or injury secondary to complementmediated neutrophil infiltration and activation with the
generation of toxic oxygen free radicals.14-17
We developed an isolated rat heart model in which the
effects of isolated cellular or humoral factors on contractile function, coronary flow, cellular adhesion, and free
radical generation can be readily studied. In this isolated
rat heart model, we performed studies using a recombinant form of complement receptor 1, sCR1, which was
genetically engineered to lack both the transmembrane
as well as the cytoplasmic domains, resulting in a soluble
molecule. This sCR1 has been previously shown to be
highly effective at suppressing both the classical and the
alternative complement pathways.18
In the present study, we performed the first experiments to determine the efficacy of sCR1 in preventing
postischemic contractile dysfunction. Further experiments were performed to determine the effect of sCR1
on PMN adhesion, complement deposition, and free
radical generation. These experiments demonstrate that
complement activation is required for PMN-mediated
reperfusion injury and that sCR1 is highly effective at
preventing this injury. It was observed that the process
of PMN adhesion was independent of complement,
whereas complement was required for activation of the
PMN mediated oxidative burst. sCR1 was shown to
Received April 16, 1993; revision accepted July 7, 1993.
From the Department of Medicine, Division of Cardiology,
Electron Paramagnetic Resonance Laboratories, The Johns Hopkins Medical Institutions, Francis Scott Key Medical Center,
Baltimore, Md.
Correspondence to Dr Jay L. Zweier, Division of Cardiology,
Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Johns
Hopkins Medical Institutions, Baltimore, Md 21224.
2813
effectively block complement-mediated PMN free radical generation.
Methods
Isolated Heart Perfusion
Female Sprague-Dawley rats (weight, 250 to 400 g)
were heparinized and anesthetized with intraperitoneal
pentobarbital. The hearts were excised, the ascending
aorta was cannulated, and retrograde perfusion was initiated. The hearts were then perfused with Krebs bicarbonate perfusate (17 mmol/L glucose, 120 mmol/L sodium
chloride, 25 mmol/L sodium bicarbonate, 2.5 mmol/L
calcium chloride, 0.5 mmol/L EDTA, 5.9 mmol/L potassium chloride, and 1.2 mmol/L magnesium chloride) at
37°C with a constant pressure of 80 mm Hg. The perfusate
was bubbled with 2 /min of 95% 02-5% CO2 gas. A
sidearm in the perfusion line allowed infusion of leukocytes and plasma directly into the heart (Fig 1, top). To
measure contractile function, a latex balloon was inserted
through an opening in the left atrium across the mitral
valve into the left ventricular cavity and connected to a
pressure transducer as described previously.14 The balloon
was initially inflated with a volume of distilled water
sufficient to produce an end-diastolic pressure in the range
of 8 to 14 mm Hg. Subsequent measurements of developed pressures were calculated as the difference between
the peak systolic and end-diastolic pressure. Left ventricular pressure was recorded with a Gould RS3400 fourchannel recorder. Coronary flow and heart rates were
measured periodically every 5 minutes before ischemia
and after 20 minutes of global ischemia for 45 minutes of
reperfusion.
Leukocyte (PMN) Preparation
Human neutrophils were prepared by the method of
Kensler and Trush,19 which yields PMNs with a purity of
>95%. Freshly sampled blood (50 mL) was drawn from
volunteer donors in heparinized 10-mL vacutainers and
centrifuged at 500g in a Beckman TJ-6 for 10 minutes. The
plasma and buffy coat were aspirated, leaving the red cell
layer. The cells were then mixed with an equal volume of
6% dextran (1 g 500 K, 5 g 80 K, and 100 mL 0.9% normal
saline), with the tubes rinsed thoroughly to secure all the
cells. The mixture was then transferred to 30-mL plastic
syringes that were inverted and incubated at 37°C for 1
hour until a clear separation was obtained. Next, the
upper layer was ejected through a 16-gauge needle with a
90' bend into 50-mL plastic centrifuge tubes and spun for
10 minutes at 500g. The supernatant was discarded, and
the pellet was resuspended in ice-cold ACK lysing buffer
(0.155 mol/L NH4Cl, 0.01 mol/L KHCO3, and 0.1 mmol/L
EDTA at pH 7.4) and respun for 10 minutes at 500g.
Finally, the pellet was washed, resuspended in Dulbecco's
phosphate-buffered saline plus 1% glucose, and spun for 5
minutes at 340g. A small volume of the cells resuspended
in Dulbecco's phosphate-buffered saline was counted using a hemocytometer.
Rat Plasma
Rat blood was obtained by performing a closed-chest
intracardiac puncture with a 10-mL heparinized syringe
and immediately centrifuging it at 500g for 15 minutes;
Circulation Vol 88, No 6 December 1993
2814
_
95 % 02
5% CO,
1
INFUSION:
PMNa +Ra (Rat A Hmw
PMNa +Pm (Rat & Hur) +sgm
Gould RS3400
Chart Recorder
80 mm Hg
Constant Pressure
F ___1
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FIG 1. Top, Diagram of the isolated
heart preparation used. Bottom, Diagram of the experimental protocol.
EXPERIMENTAL PROTOCOL
WSEONPERIOD
PREMTIhC PrOD
T
10.15
N
srUBILIZAMON
,
1 MSN
PREWosQ
r
10.1SMEN
r
30SEC
IRON*
POST
NHONA 1NMMON
r
20MIN
SCUENA
BERE
ISHMA
*INFLSIONW7TH& W7TTHOVfSCR)
RA
SMIN
4oMIN
REOW
EINFSION
U1M
TPL4SMA ORI MMUMJASORBEDHUMANPL4SMA & PMMN
the plasma was aspirated into
and kept on ice.
a
50-mL centrifuge tube
Preparation of Human Plasma
Human plasma was obtained from healthy donors
after whole blood was centrifuged for the isolation of
neutrophils. The human plasma was treated with homogenized rat heart tissue to absorb any cross-reactive
antibodies to rat heart antigens. The rat heart tissue was
homogenized in a tissue grinder (Omni 5000) in the
presence of 25 mL Dulbecco's phosphate-buffered saline. Subsequently, this tissue was centrifuged in a
high-speed centrifuge at 5000g for 30 minutes at 40C.
This step was repeated three times until the supernatant
was clear. The pellet was then incubated in the presence
of 10 mL of human plasma at 40C for 20 minutes. After
this incubation, the immunoabsorbed plasma was separated from the heart tissue by centrifugation at 40C. The
plasma was then aspirated into a 50-mL centrifuge tube
and stored on ice for use, and the homogenized rat
heart tissue pellet was discarded.
Electron Paramagnetic Resonance Measurements
Hearts were isolated and perfused as described above
except that no EDTA was included in the perfusate in
each of the groups. The experimental protocol remained the same except that the spin trap 5,5 -dimethyl1-pyroline-N-oxide (DMPO) was infused through a second sidearm located at the level of the heart with a final
concentration of 40 mmol/L, as described previously.'4
Periodic collections of the effluent were made in 20second aliquots during the control period and also for
the first 2, 5, 7.5, and 10 minutes during reperfusion. In
addition, experiments were performed to detect the
generation of oxygen free radicals from PMNs plus
plasma in the presence or absence of zymosan, an
activator of the complement cascade.20 Care was taken
to keep the DMPO-containing solutions covered to
prevent any light-induced degradation. The DMPO
purchased from Aldrich was further purified by double
distillation. Electron paramagnetic resonance (EPR)
spectra were recorded in flat cells at room temperature
with a Bruker-IBM ER 300 spectrometer operating at
X-band with a TM 110 cavity using a modulation
Shandelya et al Complement Receptor 1 Prevents Postischemic Injury
frequency of 100 kHz, modulation amplitude of 0.5 G,
microwave power of 20 mW, microwave frequency of
9.77 GHz, and acquisitions of 10 1-minute scans. The
digitized Bruker spectral data files were transferred to
an AST 386 personal computer for analysis. Software
capable of isotropic spectral simulation, developed in
this laboratory, was used for component analysis of
experimental spectra as described previously.21 Spectral
simulations consisting of linear combinations of the
component signals were performed to match the observed spectra. From the weighted intensities of each
component in these simulations, the relative amount of
each component signal was determined. The total radical concentration was then determined from the ratio
of the double integral of the observed spectra to the
known concentration of 2,2,6,6-tetramethylpiperidinoxy
(TEMPO) free radical in aqueous solution.
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Immunohistochemistry
Immunocytochemical staining was performed on
hearts removed from female Sprague-Dawley rats
weighing 250 to 400 g that had been perfused in the
presence or absence of sCR1. Hearts were quickly
removed from the cannula and embedded in OCT
compound (Miles Scientific, Naperville), immediately
quick-frozen in liquid nitrogen, and stored at -700C
until ready for staining. Cryostat sections (6 gm) were
placed onto pretreated slides (Histostik, Accurate
Chemical, Westbury, NY) and dried in a desiccator at
40C overnight. The tissue sections were warmed to room
temperature, fixed at room temperature (alum filtered)
for 10 minutes, and then washed twice in Tris-buffered
saline, pH 7.6, for 5 minutes. To eliminate endogenous
peroxidase activity, the sections were incubated for 30
minutes in a TBS-milk solution (TBS with 0.55 Carnation nonfat dry milk containing 0.5% hydrogen peroxide
and 1% normal goat serum). This and all subsequent
incubations were performed at room temperature in a
humidified chamber. The sections were washed with the
TBS-milk solution for 5 minutes and then incubated for
1 hour with CSb-9 antibody (1:200). The sections were
washed in TBS and then in TBS-milk solution for 5
minutes each and then incubated for 1 hour with
biotinylated goat anti-mouse IgG (Sigma Chemical Co,
St Louis, Mo) diluted in TBS-milk solution with 1%
normal goat serum. After washing, the sections were
incubated for 5 minutes with avidin-peroxidase-diluted
TBS-milk solution, washed in TBS-milk solution for 5
minutes followed by TBS for 5 minutes, and then
incubated with filtered DAB substrate solution (0.05
mol/L Tris buffer [pH 7.6] containing 0.06% 3,3'diamino-benzidine tetrahydrochloride and 0.003% hydrogen peroxide) for 8 minutes. The sections were
rinsed in TBS and then in running tap water followed by
a 2-minute immersion in a copper sulfate solution
(0.5% copper sulfate in 0.15 NaCI) and again in running
tap water for 2 minutes, and counter stained in Mayersmodified hematoxylin (Poly-Scientific, Bay Shore, NY).
Following immersion in running tap water for 5 minutes, the sections were dehydrated in a graded ethanol
series, cleared in toluene, and mounted using Diatex
(American Scientific Products; McGaw Park, Ill).22
2815
Histology
After completion of the experiments, hearts were
quickly removed from the cannula, and the ventricles
were sliced into sections 3 to 5 mm thick. The hearts
were immediately immersed and stored in 10% Formalin at 40C. Histologic processing was done by conventional methods. Sections were stained with hematoxylin
and eosin. The histologic sections were examined for
the extent of polymorphonuclear binding to the vascular
endothelium as well as for infiltration into myocardial
interstitium.
Measurement of Complement Activation
Measurement of complement activation by red cell
lysis assay was performed to determine if the mixture of
human PMNs and rat plasma caused any nonspecific
activation of complement. In these assays, 0.4 mL of the
PMN-containing buffer and 0.1 mL plasma were added
to 0.5 mL packed human blood cells and incubated at
37°C for 30 minutes. The red blood cells were then
pelleted by centrifugation at 50g for 5 minutes. Spectrophotometric measurements of hemoglobin in the
supernatant solution were then performed. No hemolysis was observed on mixing the PMNs and plasma,
which demonstrated that there was no nonspecific complement activation. On addition of the complement
activator zymosan (1 mg/mL), however, marked hemolysis was observed.
Chemicals
Zymosan and other chemicals used were purchased
from Sigma Chemical Co. Double-distilled deionized
water was used to prepare the perfusate and other
solutions. DMPO, 97% pure, was purchased from Aldrich Chemical Co and further purified by double distillation. Recombinant sCR1 was provided by T Cell
Sciences, Boston, Mass.
Experimental Protocol
After a 10- to 15-minute equilibration period, baseline left ventricular developed pressure, left ventricular
end-diastolic pressure, and coronary flow were measured (Fig 1, bottom). The hearts were subjected to a
1-minute preischemic control infusion with PMNs and
plasma and then were allowed to equilibrate again with
Krebs bicarbonate buffer for a period of 10 minutes,
during which measurements of coronary flow and developed pressure were noted. Hearts subsequently received a 30-second infusion of PMNs and plasma with
or without sCR1 before the onset of the 20-minute
period of 37°C global ischemia. At the onset of ischemia, the balloon was deflated. The intraventricular
balloon volume was then reinflated with the same
volume as previously used to set the baseline end-diastolic pressure immediately after the onset of reflow. At
the onset of reperfusion, the hearts were reperfused for
the first 5 minutes with PMNs and plasma in the
presence or absence of sCR1, after which perfusion was
continued with Krebs buffer alone for a total of 45
minutes of reflow, during which time serial measurements of coronary flow and developed pressure were
performed every 5 minutes. One or more sidearm ports
were placed just above the aortic cannula, allowing
administration of cells, plasma, or both (Fig 1, top). To
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Circulation Vol 88, No 6 December 1993
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determine free radical generation, hearts were similarly
perfused in each of the groups in the presence of 40
mmol/L DMPO, as described above.
Two sets of experiments were performed-one in
which rat plasma was used and the other in which
immunoabsorbed human plasma was used-to access
the efficacy of sCR1 at inhibiting both human complement and rat complement. Therefore, there were four
experimental groups, with 10 hearts in each group.
Group 1. Hearts were subjected to ischemia and
reperfusion in the presence of PMNs and rat plasma,
without sCR1. The PMNs were suspended in 5 mL of
Dulbecco's phosphate-buffered saline with 0.1% glucose and then infused during a 1-minute preischemic
control infusion, a 30-second period prior to ischemia,
and then again during the first 5 minutes of reperfusion.
The cells and plasma were infused through a sidearm at
1:20 dilution to achieve a final concentration of 300 000
PMNs/mL.
Group 2. Hearts were subjected to ischemia and
reperfusion in the presence of PMNs and rat plasma, in
the presence of sCR1 suspended in plasma. The PMNs
and plasma were infused as described for group 1. The
final concentration of sCR1 administered to hearts was
10 pug/mL.
Group 3. Hearts were subjected to ischemia and
reperfusion in the presence of PMNs and immunoabsorbed human plasma, in the absence of sCR1. The
PMNs and plasma were infused as described in group 1.
Group 4. Hearts were subjected to ischemia and
reperfusion in the presence of PMNs and immunoabsorbed human plasma, in the presence of sCR1. PMNs
and plasma were infused as described for group 1. The
final concentration of sCR1 administered to hearts was
10 gg/mL.
Validation of the Model
The present model was developed to enable the study
of the effect of isolated cellular and humoral factors on
myocardial contractile function and cellular injury in
the postischemic heart, as described previously.14 In the
present study, we sought to determine the efficacy of
sCR1 at inhibiting either rat or human complement
thereby preventing neutrophil-mediated myocardial
reperfusion injury.
Human PMNs can be isolated in large numbers, 30 to
60 million, and can be readily purified from other blood
elements by procedures that do not result in activation
of the cells. In practice, 50 mL blood is required to
isolate sufficient cells for one or two experiments.
Therefore, human PMNs are used in this model. To
similarly isolate this number of PMNs from the rat,
complete phlebotomy of 10 animals would be required,
and it is difficult to purify the cells to >70% purity.
On infusion of rat plasma in rat hearts, no toxicity was
seen and no alterations in left ventricular developed
pressure occurred. To verify that the human PMNs did
not activate rat complement or cause other adverse
reactions, all hearts studied were first subjected to
control preischemic infusion of both the PMNs and
plasma, and it was observed in all hearts studied that
after infusion the left ventricular developed pressure
and coronary flow were not significantly altered. In
contrast, if complement was activated with 1 mg/mL
zymosan or PMNs activated with 200 ng/mL TPA,
phorbol 12-myristate 13-acetate, marked sustained injury was observed with a >60% decrease in contractile
function. To further validate the observations that
mixture of the human PMNs and rat plasma did not
cause PMN activation, EPR measurements were performed on solutions of PMNs and plasma. When
300000 PMNs/mL were mixed with 5% plasma in
phosphate-buffered saline in the presence of 50 mmol/L
DMPO, no signal was observed. Repeated measurements with up to 1 million PMNs/mL and up to 50%
plasma also showed no significant signal, confirming
that the rat plasma did not cause activation of an
oxidative burst from the human PMNs. To further
confirm that the PMNs did not induce any activation of
complement, red cell lysis assays were performed as
described above. These assays demonstrated that mixture of human PMNs and rat plasma did not result in
any measurable complement activation. If complement
was intentionally activated with 1 mg/mL zymosan,
however, marked red cell lysis and marked free radical
generation were observed. Thus, these validation experiments demonstrated that there was no baseline complement activation, neutrophil activation, or alterations
in preischemic function in this rat heart model perfused
with human PMNs and rat plasma.
To determine if there were any adverse effects of
human plasma on rat hearts, pilot studies were performed infusing human plasma into control rat hearts.
Even at a 1:20 dilution, human plasma resulted in
irreversible asystole. This appeared to be due to crossreactive human antibodies reacting with the rat antigens. We observed that this toxicity could be prevented
by immunoabsorption of the human plasma with rat
heart homogenate using the procedure described above.
To verify that immunoabsorption of human plasma did
not deplete the complement proteins, we performed
experiments to determine if these complement proteins
remained. EPR experiments were conducted with
PMNs and immunoabsorbed plasma with 1 mg/mL
zymosan added to activate the complement system of
proteins. In the presence of zymosan, free radical
generation was observed identical to that of native
human plasma. In the presence of sCR1 (10 ,ug/mL),
suppression of the complement pathway was observed,
and free radical generation was abolished. These experiments demonstrated that the process of immunoabsorption of human plasma with rat heart homogenate at
4°C did not inactivate the complement system of proteins. Further validation of this was done with the
measurement of complement activation by red cell lysis
in the presence of PMNs and plasma. In the presence of
zymosan (1 mg/mL), marked hemolysis was observed.
To verify that remixing of human PMNs and human
plasma did not activate complement or cause other
adverse reactions, all hearts studied were first subjected
to control preischemic infusion of both the PMNs and
human plasma, and it was observed in all hearts studied
that after infusion left ventricular developed pressure
and coronary flow were not significantly altered. In
contrast, if complement was activated with 1 mg/mL of
zymosan or PMNs activated with 200 ng/mL TPA,
phorbol 12-myristate 13-acetate, marked sustained injury was again observed
contractile function.
with
a
>60%
decrease in
Shandelya et al Complement Receptor 1 Prevents Postischemic Injury
2817
I
0
20
5
10
FIG 2. Plots demonstrating the recovery of left
ventricular developed pressure (LVDP). Top, Data
obtained using the model with rat plasma. Closed
circles, Hearts without soluble complement receptor 1 (sCR1) treatment; open circles, hearts that
were treated with sCR1 (10 jig/mL). Bottom, Data
obtained using the model with immunoabsorbed
human plasma. Closed squares, Hearts infused in
the absence of sCR1; open squares, hearts infused with sCR1 (10 ,gg/mL).
15
Time Course ofReperfusion
ml
I4
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5
10
15
0
5
20
10
15
Time Course ofReperfusion
20
25
Thus, these validation experiments demonstrated
that there was no baseline complement activation, neutrophil activation, or alteration in preischemic function
with the use of either rat plasma or immunoabsorbed
human plasma in this model.
Statistical Analysis
Data are presented as the mean±1 SEM. Comparisons between the groups during preischemic control
infusion as well as postischemic groups were made by a
one-way ANOVA designed for repeated measures.
Since multiple comparisons were made, a conventional
value of P<.05 was accepted as statistically significant.
Results
Data
Physiologic
Experiments were conducted to evaluate the efficacy
of sCR1 at preventing the alterations in hemodynamic
parameters that occur secondary to complement-mediated neutrophil activation. Two sets of experiments
were performed - one in which rat plasma was used and
the other in which immunoabsorbed human plasma was
used -to assess the efficacy of sCR1 at inhibiting both
human complement and rat complement.
Left ventricular developed pressure. GROUPS 1 AND 2
(RAT PLAsMA). Preischemic baseline pressures for hearts
in the absence or presence of sCR1 were 135±7.2 and
132±8.5 mm Hg, respectively. On reperfusion following
20 minutes of ischemia, the sCR1-treated hearts exhib-
30
45
ited higher recovery of left ventricular developed pressure. By 45 minutes of reflow marked differences were
seen with developed pressures of only 43.3 ±9.5 mm Hg
for hearts not treated with sCR1 and 97.2±11.9 mm Hg
for those hearts treated with sCR1 (Fig 2, top). This
corresponds to a final recovery of developed pressure of
31.9±6.2% for hearts not treated with sCR1 and
76±9.8% for sCR1-treated hearts. With the administration of sCR1, a more than twofold increase in left
ventricular developed pressure was observed that was
highly significantly different with P<.0001.
GROUPS 3 AND 4 (HUMAN PLASMA). Preischemic baseline pressures were 118±3.0 mm Hg for the untreated
hearts and 123±4.7 mm Hg for the sCR1-treated group.
Immediately following reperfusion, the sCR1-treated
hearts exhibited higher recovery of left ventricular
developed pressure, and by 45 minutes of reflow marked
differences were seen with developed pressures of only
53±2 mm Hg for hearts not treated with sCR1 and
121.8±4.8 mm Hg for those hearts treated with sCR1
(Fig 2, bottom). This corresponds to a final recovery of
87.1±15.2% for hearts treated with sCR1 and
36.2±1.4% for untreated hearts. Both groups were
significantly different with P<.0001. Thus, in these
experiments with either rat plasma or human plasma,
similar marked decreases in developed pressures were
observed after 45 minutes of reflow. Reperfusion in the
presence of sCR1 improved the recovery of left ventric-
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Circulation Vol 88, No 6 December 1993
Time Course ofReperfsion
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FIG 3. Plots showing the recovery of ratepressure product (RPP). Top, Data obtained
using the model with rat plasma. Closed circles, Hearts infused in the absence of soluble
complement receptor 1 (sCR1); open circles,
hearts that were treated with sCR1 (10 jig/mL).
Bottom, Data obtained using the model with
immunoabsorbed human plasma. Closed
squares, Hearts infused in the absence of
sCR1; open squares, hearts that were treated
with sCR1 (10 jig/mL).
I
5
10
15
20
Time Course of Reperfusion
ular contractile function with a more than twofold
increase in developed pressure.
Rate-pressure product. The product of heart rate and
left ventricular developed pressure, the rate-pressure
product was determined as a measure of cardiac contractile work.
GROUPS 1 AND 2 (RAT PLASMA). Preischemic baseline
rate-pressure product was 39.4±2.3 min' mm Hg for
hearts not treated with sCR1 and 34.0±2.3 min'
mm Hg for those hearts treated with sCR1. On reflow
following 20 minutes of global ischemia, the hearts
treated with sCR1 were observed to have a much higher
recovery of rate-pressure product. After 45 minutes of
reperfusion, the rate-pressure product for hearts
treated with sCR1 was 24.7±3.3 min' mm Hg but only
12.1±3.1 min' mm Hg for those hearts not treated with
sCR1 (Fig 3, top). Thus, hearts treated with sCR1
exhibited a 2.5-fold increase in the recovery of ratepressure-product hearts with sCR1 treatment, with
recovery of 76±10.3% of basal function while only
30±7.0% recovery was observed in untreated hearts.
The two groups were significantly different with a
P<.001.
GROUPS 3 AND 4 (HUMAN PLAsMA). Experiments were
also conducted with immunoabsorbed human plasma in
the presence and absence of sCR1. Preischemic baseline rate-pressure product in the absence of sCR1 was
38.0+2.8 min' mm Hg and 32.1+1.9 min' mm Hg in
the presence of sCR1. Again, on reflow following 20
minutes of global ischemia, the hearts treated with
sCR1 were observed to have a much higher recovery of
rate-pressure product. After 45 minutes of reperfusion,
the rate-pressure product for hearts treated with sCR1
was 27.2±2.1 min' mm Hg compared with a 14.3±1.4
min' mm Hg in untreated hearts (Fig 3, bottom). The
hearts treated with sCR1 demonstrated a more than
twofold greater final recovery of rate-pressure product
compared with untreated hearts with an 83.8±6.4%
recovery versus a 25.0±5.3% recovery in the absence of
sCR1. The two groups were significantly different at
P<.001.
These data demonstrate that sCR1 treatment resulted in much higher recovery of cardiac contractile
work in hearts subjected to ischemia and reflow in the
presence of PMNs and either rat or human plasma. This
confirms that sCR1 has efficacy in blocking both human
and rat complement pathways.
End-diastolic pressure. GROUPS 1 AND 2 (RAT PLASMA).
Preischemic baseline end-diastolic pressure was measured for hearts in the presence and absence of sCR1
Shandelya et al Complement Receptor 1 Prevents Postischemic Injury
00
Time Course
ofReperfusion
2819
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FIG 4. Plots showing the recovery of left
ventricular end-diastolic pressure (LVEDP).
Top, Data obtained using the model with rat
plasma. Closed circles, Hearts infused in
the absence of soluble complement receptor 1 (sCR1); open circles, hearts that were
treated with sCR1 (10 gg/mL). Bottom, Data
obtained using the model with immunoabsorbed human plasma. Closed squares,
Hearts infused in the absence of sCR1;
open squares, hearts that were treated with
sCR1 (10 gg/mL).
to
Time Course of Reperfusion
and was not significantly different with values of 11±1
and 10±1 mm Hg, respectively. After reperfusion, much
higher values of left ventricular developed pressure
were observed in the untreated hearts. Marked differences were observed as early as the first 5 minutes of
reflow and persisted out to 45 minutes of reflow with a
final recovery of end-diastolic pressure in hearts treated
with sCR1 of 37.2± 9.5 mm Hg compared with a value of
75.8±7.2 mm Hg in untreated hearts (Fig 4, top). These
groups were significantly different at P<.001.
GROUPS 3 AND 4 (HUMAN PLASMA). Preischemic baseline end-diastolic pressure was again fixed at similar
values of approximately 10 mm Hg in both groups. After
reperfusion, much higher values of end-diastolic pressure were again observed in hearts not treated with
sCR1. Following 20 minutes of global ischemia and 45
minutes of reperfusion, the left ventricular end-diastolic
pressure of hearts treated with sCR1 was 21.7±2.3 and
56±2.3 mm Hg for hearts that did not receive sCR1 (Fig
4, bottom). Both groups were significantly different at
P<.001. Thus, in hearts subjected to ischemia and
reflow in the presence of either rat or human plasma,
sCR1 resulted in markedly lowered values of left ventricular end-diastolic pressure after reperfusion.
Coronary flow. GROUPS 1 AND 2 (RAT PLASMA). Coronary flow was measured before the onset of global
ischemia and was identical in both groups with values of
16+1 mL/min for those hearts that did not receive sCR1
and 17±1 mL/min for the hearts treated with sCR1.
Throughout the entire 45 minutes of reperfusion, however, marked differences were seen with higher recovery
in the sCR1-treated hearts (Fig 8). The final recovery of
coronary flow was 12.8±1.6 mL/min in hearts treated
with sCR1 versus 7.5±2.2 mL/min in untreated hearts
(Fig 5, top), which corresponds to 73.7±8.6% and
43.7±9.9% recoveries, respectively. The two groups
were significantly different at P<.001.
GROUPS 3 AND 4 (HUMAN PLASMA). Experiments conducted in the presence of human plasma demonstrated
similar cardioprotective effects with baseline coronary
flows of 15.3±1.3 mL/min in the absence of sCR1 and
15.5±1 mL/min in the presence of sCR1. Following 45
minutes of reflow, hearts treated with sCR1 had much
higher coronary flow, with a recovery of 14.25±1.3
mL/min, a 91.8±4.2% recovery compared with hearts
not receiving sCR1, which recovered to 5.13±1.3 mL/
minm a 33.3+7.8% recovery (Fig 5, bottom). Both groups
were significantly different at P<.0001.
Therefore, it was observed that sCR1 treatment
significantly increased the recovery of coronary flow in
either model.
Polymorphonuclear Leukocyte Adherence
Leukocyte adherence was measured by performing a
differential count on the perfusate solution entering the
2820
Circulation Vol 88, No 6 December 1993
.t
3
FIG 5. Plots showing the recovery of coronary flow (CF). Top, Data obtained using
the model with rat plasma. Closed circles,
Hearts infused in the absence of soluble
complement receptor 1 (sCR1); open circles, hearts that were treated with sCR1 (10
jig/mL). Bottom, Data obtained using the
model with immunoabsorbed human
plasma. Closed squares, Hearts infused in
the absence of sCR1; open squares, hearts
that were treated with sCR1 (10 ug/mL).
0
Time Course ofReperfusion
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10U
.1
5
10
15
0
20
5
10
is
20
25
30
45
Time Course ofReperfision
heart cannula and the coronary effluent collected during control infusion prior to ischemia and the first 5
minutes of reflow. Prior to ischemia during control
infusion, only approximately 25% to 30% of the PMNs
remained adherent with more than 70% of the infused
PMNs appearing in the effluent. After ischemia, as
reported previously, there was a marked increase in
PMN adherence within the heart. Adherence of PMNs
were quite similar in all four groups studied, suggesting
that the mechanism of neutrophil adherence is largely
independent of the complement system of proteins
(Table 2). In the model with rat plasma in the absence
of sCR1 treatment, 90% of the PMNs remained within
the heart, while with sCR1 treatment, 70% of the PMNs
remained. In the model with human plasma, in either
the presence or absence of sCR1, approximately 70% of
the PMNs remained in the heart. Thus, in postischemic
hearts treated with sCR1 approximately 70% to 90% of
the PMNs infused remain within the heart. These data
suggested that the process of PMN adherence was
largely independent of complement.
Histology
Histologic sections were examined from hearts that
were treated with and without sCR1. In hearts that
received sCR1, the myofibrillar structure of the myocytes
as well as the interstitial spaces appeared normal,
whereas in hearts that were not treated with sCR1, areas
of myocyte necrosis were seen as reported previously in
this model.14 In both sCR1-treated and untreated hearts,
PMNs were seen adherent to the endothelial surfaces of
arterioles and capillaries; however, only in untreated
hearts were PMNs noted to have migrated across the
endothelium. As noted previously, in the absence of
treatment, degranulating PMNs were observed adjacent
to cardiac myocytes. Therefore, sections from sCR1treated hearts appeared different from those of untreated hearts with PMNs only observed adherent to the
endothelium without endothelial transmigration and
myocardial structure appeared normal (Fig 6).
Immunohistochemistry
Hearts were subjected to the protocol described above
in the presence or absence of sCR1. These hearts were
stained with monoclonal antibody to the C5b-9 attack
complex using a standard immunoperoxidase method.
Normal rat heart tissue failed to reveal any staining.
Control, nonischemic rat hearts subjected to perfusion
with neutrophils and plasma failed to demonstrate any
staining. Standard controls using either no primary or no
Shandelya et al Complement Receptor 1 Prevents Postischemic Injury
2821
TABLE 1. Final Recovery Physiologic Parameters
RPP
LVDP
Groups: n=10 In
each group
CF
LVEDP,
mm Hg
75.8±7.2
37±9.5
56±2.3
% Rec.
% Rec.
% Rec.
min-1 mm Hg
mL/min
7.5+2.2
43.7+9.9
30+7.0
12.1+3.1
32±6.2
76+9.8
24.7±3.3
76±10.0
12.8±1.6
73.7+8.6
36.2±1.4
53±2.0
14.3+1.4
25.0+5.3
5.13+1.3
33.3±7.8
121.8±4.8
87.1±15.2
27.2+2.1
83.8±6.4
21.7+2.3
14.3+1.3
91.8+4.2
74.6±11.9X 64.5+8.8
XX
22.7±3.0
66.9±7.0
45.7±5.7
10.8±1.7
71.2±8.8
85.4+10.5
60.6±9.9
22.4±3.2
59.8±9.8
42.7+7.4
12.0±1.7
68.3+8.7
75.8+7.0
63.6±7.2
20.0±1.8
62.8±5.5
46.0±5.9
10.1±0.9
68.0±5.5
LVDP indicates left ventricular developed pressure; RPP, rate-pressure product; LVEDP, left ventricular end-diastolic pressure; and
CF, coronary flow.
*Experiments conducted with rat plasma.
tExperiments conducted with immunoabsorbed human plasma.
*Control data with neutrophils only, plasma, or perfusate alone, shown for comparison as previously described.14
No sCR1 (r)*
sCR1 (r)*
No sCR1 (h)t
sCR1 (h)t
PMNs onlyt
Plasma onlyt
Perfusate alone
mm Hg
43.3+9.5
97.2+12.0
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
primary and secondary antibodies failed to show any
staining. In rat hearts subjected to ischemia and reperfusion, marked positive staining with anti C5b-9 was observed, while no staining with a control isotype-matched
irrelevant antibody was observed. The distribution of
positive staining was seen throughout both ventricles but
did not appear random; rather, discrete patches staining
arterioles and microcirculatory capillaries were present.
Even within these stained patches, not all capillaries
appeared positive. No interstitial or myocyte sarcolemmal
staining was present. sCR1-treated rat hearts subjected to
ischemia and reperfusion revealed only very faint staining
within rare patches of microcirculatory capillaries. No
dense staining of capillaries or staining of arterioles,
interstitial spaces, or myocyte surfaces was seen (Fig 7).
Therefore, in sCR1-treated hearts, there was marked
diminution in complement deposition compared with untreated hearts.
Measurement of Free Radical Generation
It has been suggested that oxygen free radicals are
important mediators of the myocardial injury that occurs on postischemic reperfusion23-25 and that PMNs are
important sources of this radical generation. To assess if
the complement blocker sCR1 could prevent this neutrophil-mediated free radical generation, experiments
were performed to determine if sCR1 could prevent
complement-mediated activation of the neutrophil oxiTABLE 2. Polymorphonuclear Leukocyte Adherence
Postischemic Infusion
Groups
No sCR1 (r)*
sCR1(r)*
No sCR1 (h)t
sCR1 (h)t
PMNs In
PMNs
PMNs Infused,
nxl16
Effluent,
nxlO65
Adherent,
nxlO6l
26.4+3.5
30.1±2.0
27.3±3.1
28.2±2.8
2.7±0.92
9.1±2.30
23.7±3.4
21.0±3.5
18.9±2.6
19.9±1.8
8.4±-f..41.3
8.3±0.9
PMN indicates polymorphonuclear leukocyte.
*Experiments conducted with rat plasma.
tExperiments conducted with immunoabsorbed human
plasma.
dative burst and prevent the generation of reactive
oxygen free radicals in vitro. Complement was activated
with 1 mg/mL zymosan, a potent activator of the
complement pathway, and added to PMNs in the presence or absence of sCR1. The overall kinetics of free
radical generation was measured for a 35-minute period
by EPR spectroscopy in the presence of the spin-trap
DMPO, at a 50 mmol/L concentration. In the presence
of zymosan, marked free radical generation was observed with prominent DMPO-OOH and DMPO-OH
adducts signals. These signals were totally quenched in
the presence of 200 U/mL SOD, demonstrating that the
observed signals were derived from superoxide. The
intensity of these radical signals peaked after 20 minutes. In the presence of sCR1, however, complete
inhibition of free radical generation was observed (Fig
8). Further experiments were performed in the isolated
rat heart model to determine if sCR1 also prevented the
generation of PMN-derived free radicals in the heart.
Hearts were subjected to 20 minutes of global ischemia
and reperfused with the presence or absence of sCR1.
These hearts were also perfused with 40 mmol/L concentration of the spin-trap DMPO, and the coronary
effluent was sampled every 20 seconds for the first 2, 5,
7.5, and, finally, 10 minutes of reflow. We have previously demonstrated in the presence of both PMNs and
plasma that the total magnitude of free radical generation is increased with a prolongation in the duration of
free radical generation. In isolated hearts treated with
sCR1, the magnitude and duration of radical generation
were observed to be less than that of hearts reperfused
with PMNs and plasma in the absence of the drug.
Radical generation in sCR1-treated hearts was similar
to that observed in the absence of PMNs and plasma,
consisting of a short burst peaking at 30 seconds of
reflow with no radical generation observable after 2
minutes of reflow. In the absence of sCR1 treatment,
radical generation was prolonged and persisted for
more than 10 minutes as reported previously.14 Thus,
reperfusion with sCR1 prevented the increased magnitude and duration of free radical generation that occurs
in hearts secondary to PMN-mediated free radical
generation. This suggests that complement is required
for PMN-mediated free radical generation in the postischemic heart (Fig 8).
Wid:gf4X,0$-2'&;?KA.tvS<iEQs_jV?:'7d,g-4.
Circulation Vol 88, No 6 December 1993
2822
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Discussion
There is considerable evidence that myocardial ischemia is associated with the activation of the complement
system and that this process promotes further cardiac
injury with the enhancement of a series of inflammatory
events, including neutrophil chemotaxis and activation.26,27 This cellular inflammatory response with PMN
activation has been hypothesized to result in the increased generation of reactive oxygen free radicals28'29
and coronary endothelial dysfunction,30'31 which in turn
Shandelya et al Complement Receptor 1 Prevents Postischemic Injury
FIG 6. Facing page. A, Photomicrograph showing the
histology of postischemic rat myocardium that was subjected to ischemia and reperfusion in the presence of
soluble complement receptor 1 (sCR1), 10 gg/mL. Original magnification x100. Note the normal myocardial
structure of the myocytes, sinusoids, and the small arterioles. This is in contrast to hearts not treated with sCR1
as shown previously.14 B, Photomicrograph of an intramural artery demonstrating the numerous neutrophils
adherent to the endothelium, with no infiltration of neutrophils beyond the endothelial lining of the vessel.
2823
cause further myocardial injury.32-34 In the presence of
the nonspecific complement inhibitor cobra venom factor, it has been demonstrated that myocardial injury can
be significantly reduced. With the administration of
cobra venom factor in primates before ligation of the
coronary artery, a reduction in infarct size was observed
24 hours later.3
Therefore, it has been hypothesized that new therapeutic agents with the ability to inhibit and block the
complement cascade could be effective in preventing
the myocardial damage that occurs after ischemia. One
such novel and highly potent inhibitor is a genetically
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
FIG 7. A, 4mmunoperoxidase
staining with a monoclonal
antibody to C5b-9 of a frozen
section of left ventricular myocardium from a soluble complement receptor 1 (sCR1i)treated rat subjected to 20
minutes of ischemia followed
by 45 minutes of reperfusion
with neutrophils and rat
plasma. Only very faint staining of rare capillaries (large
arrowhead) can barely be apprecated and most capilares
are devoid of any staining
(small arrowheads). No staining is seen within a small arteriole as well (arrow). (x 40O.)
B, Immunoperoxidase staining with a monoclonal anti-
body to C5b-9 of a frozen section of left ventricular
myocardium from a rat subjected to 20 minutes ischemia
followed by 45 minutes of
reperfusion with neutrophl
and rat plasma. A representative patch of myocardium with
POsitive staining is shown.
Note the dense staining within
the lumen of a small, branchand
arteriole (arrows)
ingollwdby4
intso
~5 ~
j$>v;
25t~~(
*2s60~
~
within
a microcirculatory capN
~~~~illary (arrowhead). Other
*
neighboring capillaries appear devoid of staining. No
appreciable staining is noted
within the myocardial interstitium or on myocyte surfaces.
2824
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
3440
Circulation Vol 88, No 6 December 1993
3465
3490
3515
Magnetic Field (Gauss)
FIG 8. Electron paramagnetic resonance spectra of free
radical generation by isolated neutrophils in the presence
of activated complement. Spin-trapping experiments
were performed on 1 0 neutrophils/mL in the presence of
50 mmol/L DMPO. A, Neutrophils+ rat plasma activated in
the presence of zymosan 200 ,g/mL. B, PMNs+rat
plasma activated in the presence of zymosan (200 Lg/
mL) +soluble complement receptor 1 (sCR1) (10 ,g/mL).
C, Neutrophils+ human plasma activated in the presence
of zymosan (200 ,ug/mL). D, PMNs+human immunoabsorbed plasma activated in the presence of zymosan
(200,g/mL)+sCR1 (10 ,ug/mL).
engineered soluble form of complement receptor 1,
sCR1. It has been hypothesized that a more specific and
potent complement inhibitor such as sCR1 might be an
effective therapeutic agent at preventing reperfusion
injury in the clinical setting of acute myocardial infarction. We have previously shown that sCR1 inhibits both
the classical and the alternative complement pathways
and have demonstrated that sCR1 can decrease infarct
size in rats.18 It was not known, however, whether a
specific complement inhibitor such as sCR1 could enhance the recovery of cardiac contractile function and
coronary flow in postischemic myocardium. There are
also a number of important unanswered mechanistic
questions regarding the effect of complement inhibition
on neutrophil adhesion, chemotaxis, and activation as
well as the generation of highly reactive oxygen free
radicals. An understanding of the effect of complement
inhibition with respect to these mechanisms of injury is
essential to enable a rational comparison of the potential efficacy of this approach in preventing reperfusion
injury compared with that of other new agents such as
those directed at scavenging free radicals or preventing
neutrophil adhesion. Therefore, we performed the present study in an isolated heart model where these
mechanistic and functional questions could be readily
addressed.
We designed this study to evaluate the specific role of
complement activation on neutrophil-mediated contractile dysfunction, neutrophil adhesion and activation,
and oxygen free radical generation. The efficacy of
sCR1 at preventing contractile dysfunction, alterations
in coronary flow, complement deposition, as well as
neutrophil adhesion, chemotaxis, and activation with
the generation of reactive free radicals was determined.
The complement system of proteins includes five
regulatory proteins that inhibit the proteolytic enzymes
that activate C3 and C5, namely, the C3 and C5
convertases of the alternative and classical pathways.
These regulatory components consist of two plasma
proteins, factor H35,36 and C4-binding protein (C4bp),37,38 and three membrane proteins complement receptor 1,39-41 decay accelerating factor,42 and membrane
cofactor protein.43 This set of proteins have commonly
referred to as the regulator of complement activation
(RCA) proteins because of their structural and functional similarities. Based on this molecular and functional similarity the RCA family has been regarded as a
potential group of proteins that could selectively inhibit
complement activation. Homeister et al44 have shown
that purposeful heterologous activation of the complement system in rabbit hearts perfused with human
plasma generates Bb, C3a, C5a, and C5b-9. Their
observation of increased amounts of Bb indicates that
the alternative pathway was activated in their rabbit
heart model. It has been hypothesized that in ischemic
myocardium, either the alternative or classical complement pathway could be activated. Therefore, sCR1,
which uniquely blocks both pathways, may have a
greater potential for benefit in the prevention of myocardial reperfusion injury than other complement-directed therapeutic agents.
sCR1 has a molecular weight of 200 kd consistent
with a deletion of 67 residues of the full-length receptor.35 The mechanism of sCR1 inhibition of the complement pathway has been extensively investigated. Fearon
et a118 suggested that sCR1 has the greatest potential of
the RCA family because it has the specificity for binding
C3b and C4b, with distinct sites for both proteins,
thereby possessing the capacity for displacement of the
catalytic subunits from the C3 or C5 convertases of both
the pathways and also activating as cofactor for the
degradation of C3b and C4b by factor 1.
In the present study, experiments were performed in
an isolated rat heart model in the presence of either rat
or human complement factors and human PMNs. sCR1
blocks formation of the membrane attack complex
C5b-9 in both human plasma and rat plasma by interaction with the C3 and C5 convertase of both complement pathways.16'45 We have demonstrated that sCR1
has marked cardioprotective effects in preventing the
neutrophil-mediated reperfusion injury that occurs secondary to the activation of complement. Efficacy was
observed against either rat complement or human com-
Shandelya et al Complement Receptor 1 Prevents Postischemic Injury
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
plement. Greater than twofold increased coronary flow
was observed in the sCR1-treated hearts than in similar
untreated hearts. In the hearts treated with sCR1, much
lower end-diastolic pressures were observed demonstrating improved diastolic relaxation of the left ventricle, most likely secondary to decreased myocyte calcium
loading. sCR1 treatment resulted in a more than twofold reduction in left ventricular end-diastolic pressure.
Both postischemic recovery of left ventricular developed pressure and the rate-pressure product were
markedly increased in the sCR1-treated hearts compared with untreated controls with a more than twofold
increase seen. Thus, sCR1 treatment greatly improved
the recovery of both contractile function and coronary
flow in postischemic hearts. The recovery in left ventricular developed pressure, rate-pressure product, enddiastolic pressure, and coronary flow observed in the
presence of sCR1 was identical to that of hearts subjected to ischemia in the absence of PMNs, plasma, or
PMNs and plasma, demonstrating that sCR1 totally
prevented complement-mediated and PMN-mediated
injury (Table 1).
Immunohistochemical staining of the tissue of hearts
subjected to ischemia and reperfusion in the presence of
PMNs and plasma demonstrated the presence of complement deposition throughout both ventricles with the
C5b-9 attack complex localized primarily on the endothelium of the arterioles and capillaries. In normally
perfused hearts in the presence of PMNs and plasma or
in postischemic hearts in the absence of PMNs and
plasma, complement deposition was not seen. In hearts
subjected to ischemia and reperfusion in the presence of
PMNs and plasma, sCR1 prevented this complement
deposition. These studies demonstrate that complement
is activated in ischemic myocardium with the deposition
of the membrane attack complex on the coronary
endothelium and that sCR1 effectively prevents this
complement deposition.
In postischemic hearts, increased PMN adhesion was
noted; however, sCR1 did not prevent this increase. No
significant alternations in PMN accumulation or adherence were observed in the presence or absence of sCR1
in measurements of the arterial venous gradient in PMN
concentration or the histologic visualization of PMNs
adherent to the vascular endothelium. This suggests in
this model that PMN adhesion is not complement
dependent. The process of PMN adhesion is probably
primarily controlled by the expression of PMN and
endothelial adhesion molecules. In postischemic myocardium upregulation of adhesion molecule surface
expression has been hypothesized to occur, and this
process alone may be responsible for the increased
PMN adhesion that was observed in the postischemic
myocardium. In both in vivo canine and feline models of
myocardial ischemia and reperfusion it has been demonstrated that specific antibodies to the CD18 neutrophil adhesion molecule can prevent neutrophil accumulation and decrease infarct size.46
Electron paramagnetic resonance studies were performed that demonstrated that sCR1 could prevent the
neutrophil-derived oxidative burst triggered by complement activation. sCR1 totally prevented the generation
of superoxide and superoxide-derived free radicals that
occurs in neutrophils incubated in the presence of
activated complement. In the postischemic hearts, sCR1
2825
prevented the amplification in the magnitude and duration of free radical generation that is observed in the
presence of PMNs and plasma. Thus, sCR1 did not
prevent PMN adhesion within postischemic myocardium, but it did prevent the complement-dependent
process of free radical generation from these adherent
PMNs. These data suggest that while the process of
PMN adhesion within ischemic myocardium is complement independent, complement is required for PMN
activation with the generation of reactive superoxide
and superoxide-derived free radicals, which in turn
cause tissue injury.
In summary, these studies demonstrate that complement-mediated neutrophil activation occurs in the postischemic heart. This process causes marked reperfusion
injury with impaired contractile function and decreased
coronary flow. Neutrophil adhesion was increased in
postischemic myocardium, and this process was observed to be independent of the presence of complement proteins. Activation of the neutrophil oxidative
burst, however, was observed to be complement dependent. sCR1 was observed to be a particularly potent
inhibitor of this neutrophil-derived complement dependent postischemic injury. It totally prevented the neutrophil-derived alterations in contractile function and
coronary flow and prevented neutrophil-derived free
radical generation. Thus, sCR1 appears to be a promising therapeutic agent in the prevention of myocardial
reperfusion injury. Since it is a recombinant human
molecule, which is highly effective in inhibiting human
complement, it should be suitable for extension to
clinical use in humans.
Acknowledgments
Supported by National Institutes of Health grants HL17655-18 and HL-383224. J.L.Z. is also supported by an
Established Investigator Award from the American Heart
Association. We gratefully acknowledge the valuable advice
and comments of Dr Douglas Fearon, Johns Hopkins University School of Medicine. We would like to thank Dr Robert L.
Engler, University of California San Diego School of Medicine, and Dr Lewis Becker, Johns Hopkins University School
of Medicine, for helpful comments and advice.
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S M Shandelya, P Kuppusamy, A Herskowitz, M L Weisfeldt and J L Zweier
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Circulation. 1993;88:2812-2826
doi: 10.1161/01.CIR.88.6.2812
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