Download Ex Vivo Reversal of Heparin-Mediated Cardioprotection by

0022-3565/99/2903-1041$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 290:1041–1047, 1999
Vol. 290, No. 3
Printed in U.S.A.
Ex Vivo Reversal of Heparin-Mediated Cardioprotection by
Heparinase After Ischemia and Reperfusion1
KENNETH S. KILGORE, ELAINE J. TANHEHCO, KEITH B. NAYLOR, and BENEDICT R. LUCCHESI
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan
Accepted for publication April 14, 1999
This paper is available online at http://www.jpet.org
The rapid reversal of heparin’s antithrombotic action may
require the use of protamine sulfate. The protamines are
basic, low-molecular-weight, positively charged proteins having a high affinity for negatively charged molecules, including heparin. However, the use of protamine is associated
with multiple adverse reactions, including acute hypotension, bradycardia, and extension of vascular injury. In light
of these adverse consequences, heparinase, an endoglycosidase that neutralizes heparin by catalytically cleaving the
molecule, has been investigated as a possible alternative to
protamine as an antagonist of heparin-induced systemic anticoagulation (Michelsen et al., 1997; Keller et al., 1998).
Interestingly, in vivo studies have provided evidence that
heparinase III reduces the extent of tissue injury associated
with regional myocardial ischemia and reperfusion (Hayward et al., 1997). The results indicate that heparinase III is
cardioprotective due to its ability to preserve endothelial
function and attenuate neutrophil adherence to the coronary
vascular endothelium. However, it must be emphasized that
the aforementioned study was not conducted in the presence
of previous heparin administration.
Received for publication December 3, 1998.
1
The work in this study was supported by the Cardiovascular Research
Fund at the University of Michigan Medical School.
(p , .05) in the heparin-treated group. Ex vivo administration of
heparinase (5 U/ml) immediately before the onset of global
ischemia was associated with a reversal of the heparin-mediated cardioprotection. The uptake of a radiolabeled antibody to
the intracellular protein myosin and creatine kinase release
were used to determine membrane integrity and discriminate
between viable and nonviable myocardial tissue. The uptake of
radiolabeled antimyosin antibody and release of creatine kinase
after reperfusion were increased in heparin-pretreated hearts
exposed to heparinase, indicating a loss of membrane integrity
and increased myocyte injury. These results demonstrate that
neutralization of heparin by heparinase promotes increased
myocardial injury after reperfusion of the ischemic myocardium.
Heparin and related glycosaminoglycans (GAGs) have
been shown to be of benefit to the ischemic myocardium by
preserving contractile function and reducing tissue injury
(Friedrichs et al., 1994; Black et al., 1995; Gralinski et al.,
1996; Park et al., 1999). It is of interest therefore to determine the effect of heparinase administration in the presence
of tissue-bound heparin. Therefore, the present study was
designed to determine whether the previously observed cardioprotective effects of heparin administration would be negated by the heparin-degrading enzyme heparinase. It was
found that the presence of heparinase during reperfusion
was associated with a loss of heparin-mediated protection as
demonstrated by changes in cardiac functional parameters
and loss of membrane integrity.
Materials and Methods
Guidelines for Animal Research. The procedures used in this
study were in accordance with the guidelines of the University of
Michigan Committee on the Use and Care of Animals. Veterinary
care was provided by the University of Michigan Unit for the Laboratory Animal Medicine. The University of Michigan is accredited by
the American Association of Accreditation of Laboratory Animal
Health Care, and the animal care use program conforms to the
standards in The Guide for the Care and Use of Laboratory Animals,
ABBREVIATIONS: GAGs, glycosaminoglycans; CK, creatine kinase; KH, Krebs-Henseleit.
1041
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 3, 2017
ABSTRACT
Glycosaminoglycans, including heparin, have been demonstrated both in vitro and in vivo to protect the ischemic myocardium against reperfusion injury. In the present study, we
sought to determine whether the cardioprotective effects of
heparin administration could be reversed by the heparin-degrading enzyme heparinase. New Zealand white rabbits were
pretreated with heparin (300 U/kg i.v.) or vehicle (saline). Two
hours after treatment, hearts were removed, perfused on a
Langendorff apparatus, and subjected to 25 min of global
ischemia, followed by 45 min of reperfusion. Hemodynamic
variables were obtained before ischemia (baseline) and every
10 min throughout the reperfusion period. Compared with vehicle-treated rabbits, the left ventricular end-diastolic and left
ventricular developed pressures were improved significantly
1042
Kilgore et al.
ml/min). All hearts were subjected to 25 min of global ischemia
followed by 45 min of reperfusion. Functional parameters were recorded every 10 min during the reperfusion period until termination
of the protocol. A constant temperature of 37°C was maintained
throughout the periods of ischemia and reperfusion.
Preparation of Antimyosin Antibody. Murine monoclonal antimyosin antibody (Mifarmonab F(ab9) was provided by Centocor Inc.
(Malvern, PA). Radioiodination of the antibody was performed by the
chloramine-T method (Sakahara et al., 1987). After incubation with
Na 125I and chloramine-T, the free 125I was removed by Sephadex
G-50 column chromatography. The specific activity of the radiolabeled molecule was between 6 and 12.5 mCi/mg protein.
Determination of 125I-Antimyosin Uptake. Uptake of labeled
antimyosin was determined by perfusing 1.0 mCi of antibody through
the isolated heart for 5 min before terminating the protocol. On
administration of the antimyosin antibody, the hearts were washed
for an additional 5 min with buffer to remove antibody not bound to
myosin. The hearts were dried overnight, weighed, and the uptake of
antimyosin determined by a well-type auto-gamma counter (Minaxi
Auto-Gama; Packard Instrument Co., Downers Grove, IL). The
amount of antimyosin antibody uptake was expressed as a percentage of the perfused dose bound per gram dry weight of the tissue.
Electron Microscopy. On completion of the designated protocol,
hearts were perfused for 3 min with 2.5% glutaraldehyde and 1%
LaCl3 in 0.1 M sodium cacodylate buffer (pH 7.44). The electrondense LaCl3 served as an indicator of arterial capillary endothelium
permeability (Jokelainen et al., 1976). Tissue samples from the left
ventricular myocardium were removed and cut into pieces measuring approximately 1 mm on a side. The samples were fixed for an
additional 2 h at 4°C in the above-mentioned buffer. After washing
with 0.1 M sodium cacodylate buffer, the samples were dehydrated
in an ethanol series and embedded in EM bed-812 (Electron Microscopy Sciences, Ft. Washington, PA). Tissue blocks were sectioned
with a Reichert ultramicrotome and placed on formvar-coated copper
grids and then stained with 4% uranyl acetate. Sections were observed with a Philips CM-10 electron microscope and representative
micrographs from each treatment group obtained.
Release of Creatine Kinase (CK). Effluent from the pulmonary
artery (coronary venous return) was sampled at baseline and at
regular time intervals during the reperfusion period for analysis of
creatine kinase release. CK activity was determined using a kit
purchased from Sigma Chemical Co. (procedure 47-UV; St. Louis,
MO). The assay is based on the modified procedure developed by
Szasz et al. (1976). Briefly, the assay measures the increase in
absorbance at 340 nm produced by the reduction of NAD (nicotinamide adenine dinucleotide) to NADH (nicotinamide adenine dinucleotide, reduced form). The rate of change is proportional to the CK
activity. One unit is defined as the amount of enzyme that produces
one micromole of NADH per minute.
Statistical Analysis. Results are expressed as mean values 6
S.E.M. Comparisons of parameters measured over time were performed using a two-way repeated measures ANOVA to test for group
differences over time followed by Dunnett’s post hoc test. If only two
groups were compared, the Student’s t test for unpaired comparisons
was used. In all cases, a p value of , 0.05 is regarded as significant
and is denoted by an asterisk. Statistical analyses were performed on
a Macintosh computer using Statview SE 1 Graphics (Abacus Concepts, Berkeley, CA).
Results
Cardiac Contractile Parameters. Preliminary studies
examining the effects of heparinase alone indicated concentrations of 1, 5, and 10 U/ml (maximum concentration examined) did not alter cardiac function. Based on these results,
the subsequent studies with heparinase were carried out
using a concentration of 5 U/ml added to the perfusion me-
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 3, 2017
Department of Health, Education, and Welfare Publication Number
(National Institutes of Health) 86-23.
Treatment Groups. The present study consisted of three experimental groups in which each group was treated in vivo 2 h before the
hearts were removed and subjected to perfusion by the Langendorff
method. The treatment regimens were as follows: group I animals
received an i.v. dose of heparin sulfate (300 U/kg) 2 h before removal
of the heart. The dose of heparin was determined based on a previous
study demonstrating the ex vivo cardioprotective effects of the GAG
when administered i.v. in a dose sufficient to increase the activated
partial thromboplastin time, 2- to 3-fold above the control value
(Friedrichs et al., 1994). The hearts were removed 2 h after heparin
pretreatment and subsequently treated with placebo immediately
before the onset of global ischemia. Animals assigned to group II
were treated with heparin as described above, but the isolated hearts
from this group were exposed to heparinase (5 U/ml) immediately
before induction of global ischemia. Group III animals were treated
in vivo with 0.9% sodium chloride solution (drug diluent), 2 h before
removal of the hearts. The concentration of heparinase was obtained
from preliminary studies examining the effect of heparinase alone on
cardiac function. The concentration of heparinase (5.0 U/ml) was
obtained from preliminary studies examining the effect of heparinase alone at concentrations of 1, 5, and 10 U/ml using cardiac
contractile function as a determinant of drug effect. The range of
heparinase concentrations examined in the preliminary studies did
not produce an observable change in left ventricular peak systolic
pressure or left ventricular end-diastolic pressure during 45 min of
exposure to the enzyme in the perfusion medium. Based on the
preliminary examination, a concentration of 5.0 U/ml was selected
for further study. Heparin (porcine intestinal mucosa) and heparinase III were purchased from Sigma Chemical Co. (St. Louis, MO).
Langendorff-Perfused Heart. The Langendorff preparation
used in this study has been described in detail previously (Homeister
et al., 1992). Briefly, male New Zealand White rabbits (1.8 –2.2 kg)
were rendered unconscious by cervical dislocation. The hearts were
excised quickly and the aorta attached to a cannula for perfusion
with a modified Krebs-Henseleit (KH) buffer (pH 7.44, 37°C) at a
constant flow (22–28 ml/min). Buffer was composed of 117 mM NaCl,
4.0 mM KCl, 1.2 mM MgCl2 z 6H2O, 1.1 mM KH2PO4, 25.0 mM
NaHCO3, 2.6 mM CaCl2 z 2H2O, 5.0 mM glucose, 5.0 mM L-glutamate, and 5.0 mM pyruvic acid. The KH buffer passed through a
membrane “lung” composed of 18 feet, 0.058 inches i.d., 0.077 inches
A Silasticô Medical Grade Tubing (Dow Corning, Midland, MI). The
membrane lung was gassed continuously with a mixture of 95%
O2/5% CO2 to achieve an oxygen partial pressure of 500 mm Hg. An
in-line oxygen electrode and digital meter (Instech Laboratories,
Plymouth Meeting, PA) continuously monitored the oxygen tension
in the KH buffer. The hearts were paced through the right atrium
with electrodes attached to a laboratory stimulator (180 impulses/
min, 2 ms duration, 4 V; Grass Instruments SD-5, Quincy, MA).
The pulmonary artery was cannulated to facilitate collection of
fluid from the coronary venous circulation. The pulmonary veins and
the superior and inferior vena cava were ligated. A left ventricular
drain, thermistor probe, and a latex balloon entered via the left
atrium and were secured with a purse string suture at the atrial
appendage. Isovolumetric left ventricular diastolic and systolic pressures were measured with the left ventricular fluid-filled latex balloon. The balloon was filled to achieve a left ventricular end-diastolic
pressure of 5 mm Hg. The monitored physiologic parameters included the left ventricular systolic and diastolic pressures and the
cardiac electrogram, which were recorded continuously on a multichannel polygraph recorder (Grass polygraph model 79D).
Experimental Protocol. Isolated hearts were stabilized under
normoxic conditions for 15 to 20 min before the induction of global
ischemia. The perfusion medium was recirculated throughout the
protocol. Induction of global ischemia was accomplished by stopping
the flow of perfusate to the heart. Reperfusion of the heart was
achieved by turning on the pump to the original flow rate (20 –24
Vol. 290
1999
1043
a cardioprotective effect in the heparin pretreated heart compared with the hearts from vehicle-treated animals.
In contrast, the addition of heparinase (5.0 U/ml) to the
perfusion medium of hearts from heparin-pretreated animals
(group 2) was associated with the reversal of heparin’s cardioprotective effects. The mean left ventricular end-diastolic
pressure at the 45 min time point after reperfusion was
66.0 6 5.1 mm Hg in hearts made ischemic in the presence of
heparinase versus 22.9 6 4.8 mm Hg noted for heparinpretreated hearts administered drug diluent. A similar
change was noted for the left ventricular peak systolic pressure recordings between hearts from groups 1 and 2. The
mean peak systolic pressure in heparinase-perfused hearts
was 84.5 6 3.9 mm Hg at 45 min of reperfusion versus 48.8 6
4 mm Hg in hearts from heparin-pretreated animals. Both
the end-diastolic and peak systolic pressures in heparinpretreated hearts exposed to heparinase were similar to that
noted for vehicle-treated animals. As indicated previously,
significant functional changes were not observed when
hearts were perfused under normoxic conditions for 45 min in
the presence of heparinase (1–10 U/ml).
Determination of 125I-Labeled Antimyosin Uptake as
a Indicator of Tissue Injury. The125I-labeled antimyosin
antibody provides a convenient approach to quantitate the
extent of myocardial tissue injury in hearts subjected to
global ischemia and reperfusion. Compared with hearts from
vehicle-treated animals subjected to global ischemia and
reperfusion, the uptake of the radiolabeled antibody was
decreased significantly in hearts from animals pretreated
with heparin. In contrast, perfusion of the heparin-pretreated hearts with heparinase was accompanied by a
marked accumulation of the antibody in myocardial tissue.
The results are summarized in Fig. 2 and are consistent with
the changes noted in myocardial contractile function, indi-
Fig. 1. Contractile parameters of isolated hearts exposed to 25 min of ischemia and 45 min of reperfusion. A, systolic pressures from hearts of animals
pretreated with vehicle (triangles), heparin (squares), or heparin made ischemic in the presence of heparinase (circles). Values are mean 6 S.E.M. *p ,
.05 versus vehicle pretreatment at the same time point. B, end-diastolic pressures from hearts of animals pretreated with vehicle (triangles), heparin
(squares), or heparin made ischemic in the presence of heparinase (circles). Values are mean 6 S.E.M. *p , .05 versus vehicle at same time point.
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 3, 2017
dium. The decision to use the midpoint concentration was
influenced in part by the high cost of the enzyme.
The observed changes in left ventricular systolic and left
ventricular end-diastolic pressures for each of the three
groups are presented in Fig. 1, A and B, respectively. The
contractile parameters of hearts from vehicle-pretreated animals (group 3), subjected to 25 min of global ischemia followed by 45 min of reperfusion, were characterized by increases in the left ventricular end-diastolic and systolic
pressures occurring within 10 min after restoration of coronary artery perfusion. The observed responses in the group 3
control hearts are characteristic of what is observed when the
heart is subjected to a limited global ischemic insult followed
by reperfusion. Left ventricular pressure development ceases
within minutes after initiation of global ischemia and the left
ventricular end-diastolic pressure increases gradually during
the period of global ischemic arrest. On reperfusion there is a
return of contractile function along with a progressive increase in the left ventricular end-diastolic pressure and a
simultaneous increase in the recorded left ventricular peak
systolic pressure. The difference between the peak systolic
pressure and the end-diastolic pressure represents the left
ventricular developed pressure. Both the end-diastolic and
systolic pressures remained increased throughout the 45 min
of reperfusion as compared with baseline values. The enddiastolic pressure in hearts from animals pretreated (2 h)
with heparin (group 1) was significantly less (p , .05), compared with the corresponding time points in hearts from
vehicle-treated animals (22.9 6 4.8 versus 52.9 6 5.1 mm Hg
at 45 min of reperfusion). The peak systolic pressure also was
significantly less (p , .05) in hearts from animals pretreated
with heparin, compared with vehicle-treated animals (48.8 6
4 versus 89.1 6 6.7 mm Hg at 45 min of reperfusion). Thus,
the combined effects of a reduction in both the left ventricular
peak systolic and end-diastolic pressures are consistent with
Inhibition of Cardioprotection by Heparinase
1044
Kilgore et al.
cating an intensification of tissue injury when the heparinpretreated heart is exposed to heparinase.
Electron Microscopy/Lanthanum Chloride Distribution. Transmission electron microscopy using lanthanum
chloride as an indicator of vessel damage provided morphological data for the analysis of cellular ultrastructure. The
specimen in Fig. 3A was obtained from an animal in group 3.
The perfused heart was exposed to drug vehicle. As observed
in the electron photomicrograph, the effect of ischemia/reperfusion in the presence of vehicle resulted in the failure of
lanthanum to accumulate on the endothelial surface. Extravasation of lanthanum into the perivascular space is apparent.
There is a loss of morphologic structure in both the myocytes
and mitochondria. The observed changes are representative
of the group and suggest irreversible injury as a result of
ischemia/reperfusion.
The morphological appearance of myocardial tissue from a
heparin-pretreated animal is presented in Fig. 3B. Densely
packed mitochondria containing an intact matrix and normal-appearing cristae are apparent. Amorphous densities do
not appear within the mitochondria. The myofibrils are intact with well-aligned Z-bands. The endothelial cells lining
blood vessels in hearts from heparin-pretreated animals
showed a continuous layer of lanthanum on the luminal
surface of the tissue with minimal evidence of extravasation
to the perivascular space. In contrast to the illustration in
Fig. 3A, the heparin-pretreated heart subjected to 25 min of
global ischemia and 45 min of reperfusion retained many
normal morphological features, especially the presence of a
glycocalyx, suggesting that tissue-bound heparin may provide a cardioprotective effect.
The ultrastructural preservation noted above with heparin
pretreatment (Fig. 3B) was absent when hearts from heparin-pretreated animals were exposed to heparinase before
being subjected to reperfusion. The observed morphological
alterations are consistent with those previously associated
with ischemia/reperfusion injury. Endothelial cells exhibited
severe swelling with the appearance of intraluminal protrusions in many of the vessels. The luminal surfaces of the
vessels were disrupted and contained little lanthanum deposition, whereas heavy deposition of lanthanum occurred
within the perivascular space. The mitochondria were swollen with disrupted matrices and contained large, amorphous
densities (arrow), suggestive of irreversible injury. Extensive
myofibrillar damage is apparent, as seen by blurring of the
Z-bands and disruption of the myofibrils. The notable feature
in Fig. 3, A and C is the lack of a glycocalyx, as evidenced by
the failure of lanthanum to deposit on the endothelial surface.
CK Release. The release of the cytosolic enzyme, CK, into
the pulmonary effluent was used as an additional indicator of
an alteration in membrane integrity (Fig. 4). Baseline CK
values were similar in all groups. In control hearts undergoing 25 min of global ischemia and 45 min of reperfusion,
there was an increase in CK activity in the pulmonary artery
effluent after the onset of reperfusion. The CK activity increased within 10 min after initiation of reperfusion and
continued to increase throughout the remainder of the protocol. In hearts from animals pretreated with heparin, CK
activity was significantly less than that noted for control
hearts. In contrast, exposure of heparin-pretreated hearts to
heparinase resulted in an increase in CK activity similar to
that observed in control hearts. CK release from hearts of
heparin-pretreated animals was significantly less at the 20and 30-min time points when compared with heparin-pretreated hearts perfused in the presence of heparinase.
Discussion
The current study derives from an earlier investigation
indicating that the isolated heart of Cynomolgus monkeys
was protected from the damaging effects of global ischemia
and reperfusion if the donor animal had been administered
heparin 2 h or more earlier (our unpublished observations).
The studies were repeated in the rabbit with a similar outcome and with the additional observation that cardioprotection was achieved by pretreating with nonanticoagulant derivatives of heparin, but not if the GAGs were added directly
to the perfusion medium (Friedrichs et al., 1994). The timedependent nature of the cardioprotective effect, and the fact
that protection persisted when the hearts were perfused in
the absence of exogenous GAGs, suggested that the compounds were bound to the cardiac tissue (Hiebert and Jaques,
1976a,b). Subsequent investigation using periodate-Schiff reagent staining of heart tissue indicated that i.v.-administered heparin or heparin analogs were bound to the endothelial cell glycocalyx (Friedrichs et al., 1994; Gralinski et al.,
1996). These observations were extended using in vivo models of myocardial ischemia/reperfusion injury. Heparin
(Black et al., 1995), or its nonanticoagulant derivative Nacetylheparin (Park et al., 1999), protected the myocardium
and resulted in a reduction in infarct size due to ischemia/
reperfusion. Several other laboratories have reported results
in full agreement with our current observations (Hobson et
al., 1988; Kouretas et al., 1998, 1999). There is evidence that
a 2-h in vivo pretreatment regimen with heparin or N-acetyl-
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 3, 2017
Fig. 2. Determination of antimyosin uptake in hearts subjected to global
ischemia and reperfusion. Antimyosin uptake was decreased significantly in hearts from heparin-pretreated animals after 25 min of ischemia and 45 min of reperfusion. In contrast, exposure to heparinase
resulted in an increase in antimyosin uptake. Control hearts from animals administered 0.9% sodium chloride solution exhibited an intermediate degree of antimyosin antibody binding. Values are mean 6 S.E.M.
*p , .05 versus vehicle pretreatment; p , .05 versus heparin pretreatment.
Vol. 290
1999
Inhibition of Cardioprotection by Heparinase
1045
Fig. 3. Representative electron micrographs of hearts from control animals and heparin-pretreated animals after 25 min of ischemia and 45 min of
reperfusion, and hearts made ischemic in the presence of heparinase. A, illustrates the effect of ischemia/reperfusion in the presence of vehicle. There
is a loss of morphologic structure in both the myocytes and mitochondria. Little lanthanum accumulation is noted within the vascular lumen on the
endothelial surface. The endothelial cells lining blood vessels in hearts from heparin-pretreated animals (B) show a uniform layer of lanthanum
(arrows) on the luminal surface (LS), whereas the morphological appearance of the tissue is well preserved. Hearts obtained from heparin-pretreated
animals and perfused in the presence of heparinase (C), show deposition of lanthanum (arrowheads) within the perivascular space (PS) and extensive
morphological alterations consistent with that previously noted for ischemia/reperfusion injury. Original magnification, 85003.
heparin preserves coronary endothelial function after a brief
period of ischemia/reperfusion by a mechanism which is, in
part, due to activation of the nitric oxide-cGMP pathway
(Kouretas et al., 1999).
Despite the extensive clinical use of heparin, the cytoprotective and anti-inflammatory actions of the GAGs have not
been subjected to a critical evaluation. A number of limited
clinical studies have been reported in which heparin is observed to possess an anti-inflammatory and/or cytoprotective
action (Saliba, 1997; Evans et al., 1997; Downing et al.,
1998). The interested reader is referred to an excellent discussion of heparin and its often neglected actions that go
beyond its well known anticoagulant effects (Edens et al.,
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 3, 2017
Fig. 4. Release of cytosolic CK into the pulmonary effluent of isolated
hearts. Vehicle-pretreated hearts show increased release of CK after 20
min of reperfusion as compared with the hearts from heparin-pretreated
animals. Vehicle (triangles, n 5 6), heparin-pretreated (squares, n 5 6),
heparinase (circles, n 5 6). Data are mean 6 S.E.M. *p , .05 versus
vehicle hearts at same time point; ‡p , .05 versus heparinase at same
time point.
1993). Heparin and related GAGs are among the most effective inhibitors of the complement cascade and the anticoagulant property is distinct from the complement inhibitory
activity (Ecker and Pillemer, 1941), thereby providing the
opportunity to develop pharmacologic interventions targeted
to one or more of the serine proteases of the complement
cascade.
Heparin and related GAGs inhibit both the classical and
alternative pathways of complement activation (Ecker and
Gross, 1929; Weiler et al., 1978). Heart tissue is capable of
expressing genes and proteins of the complement system,
although it is not yet known which cell types are responsible.
Myocardial ischemia/reperfusion promotes a rapid expression of mRNA encoding the complement proteins C3 and C9.
These levels exceed those found in normal liver. The observations are consistent with the hypothesis that local production of complement proteins may contribute significantly to
the degree of ischemic injury to the myocardium, and that
complement expression is augmented by reperfusion (Yasojima et al., 1998). The inhibitory action of heparin on the
local tissue complement cascade may provide a partial explanation for the protective effects observed when hearts are
pretreated with heparin before being subjected to ischemia/
reperfusion.
GAGs bind to the glycocalyx of endothelial cells and myocytes, thereby protecting these cell types against injury
(Crarnowska and Karwatowska, 1995). The importance of
heparin and other GAGs is exemplified further by the recognition that neutralization of heparin or heparin sulfate by
protamine or administration of the heparin-degrading enzyme, heparinase, adversely affects the biochemical regulation of endothelial cell-mediated vascular repair (Han et al.,
1997). Many GAG-induced actions have been attributed to
their ability to associate with the glycocalyx (Ruoslahti and
Yamaguchi, 1991), as well as binding to endothelial cell receptors and being internalized (Castellot et al., 1985).
Heparin use may precipitate adverse events (i.e., hemorrhage), or may require that its anticoagulant action be neutralized rapidly. Recently, the endoglycosidase heparinase
has been investigated as an alternative to protamine for the
neutralization of heparin (Michelsen et al., 1997). We sought
to investigate the consequences of heparinase administration
on heparin-mediated cardioprotection. We hypothesized that
1046
Kilgore et al.
lanthanum associated with the luminal surface of the vascular endothelium. Hearts from heparin-pretreated animals
and subjected to ischemia/reperfusion in the presence of heparinase showed little lanthanum retention on the endothelial cells along with vascular injury and loss of myocyte
viability, not noted in hearts pretreated with heparin and not
exposed to heparinase. The latter tolerated the stress of
global ischemia and reperfusion with a lesser degree of injury
assessed by monitoring functional, biochemical, and ultrastructural parameters.
CK is released when myocytes are damaged and membrane integrity is altered (Shell et al., 1971). The increased
release of the enzyme in heparin-pretreated hearts exposed
to heparinase supports the view that heparinase reverses the
protective effects of heparin against ischemia/reperfusion injury. Formation of a heparin– heparinase complex may serve
to amplify the injury associated with the stress imposed by
ischemia/reperfusion.
The data derived from the present study may offer insight
into the cytoprotective actions of heparin. Heparin binds to
the endothelium through charge interactions with glycoproteins and polysaccharides that compose the glycocalyx (Marcum and Rosenberg, 1989). These glycoproteins and polysaccharides are involved in determining tissue structure and
have a role in mediating cell adhesion, migration, and the
activities of membrane-bound chemokines (Hoogenwerf et
al., 1997; Marcum and Rosenberg, 1989). Control of vascular
permeability and membrane integrity is, in part, dependent
on the glycocalyx. Heparinase is known to digest the extracellular matrix, thereby facilitating tissue damage. Perfusion
of isolated lungs with heparinase results in an increase in
radiolabeled albumin uptake and a subsequent increase in
total lung water content (Sunnergren et al., 1987). Based on
the antimyosin and CK data, it is clear that preadministration of heparin acts to preserve endothelial cell and myocyte
membrane integrity of the perfused heart, whereas treatment with heparinase counteracts the protective effect. Electron microscopy using lanthanum chloride indicates that the
glycocalyx of heparin-pretreated hearts exposed to ischemia/
reperfusion in the presence of heparinase undergoes extensive disruption.
The results of this investigation provide support for a cardioprotective action of heparin that is dependent on binding
of the GAG to the endothelial cell. It is not known whether
these findings have a bearing on the potential clinical application of heparinase as a therapeutic intervention for heparin neutralization. Although the study was not designed to
address this question, it does call attention to the fact that
further investigations are needed that focus on the pharmacodynamic events associated with formation of the heparin–
heparinase complex under conditions of altered tissue oxygenation and reperfusion.
References
Black SC, Gralinski MR, Friedrichs GS, Kilgore KS, Driscoll EM and Lucchesi BR
(1995) Cardioprotective effects of heparin or N-acetylheparin in an in vivo model
of myocardial ischaemic and reperfusion injury. Cardiovasc Res 29:629 – 636.
Castellot JJ, Wong K, Herman B, Hoofer RJ, Albertini DF, Wright TC, Caleb BJ and
Karnovsky MJ (1985) Binding and internalization of heparin by vascular smooth
muscle cells. J Cell Physiol 124:13–20.
Crarnowska E and Karwatowska PE (1995) Ultrastructural demonstration of endothelial glycocalyx disruption in the reperfused rat heart: Involvement of oxygen
free radicals. Basic Res Cardiol 90:357–364.
Downing LJ, Strieter RM, Kadell AM, Wilke CA, Greenfield LJ and Wakefield TW.
Low-dose low-molecular-weight heparin is anti-inflammatory during venous
thrombosis (1998) J Vasc Surg 28:848 – 854.
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 3, 2017
the cardioprotective benefits derived from in vivo heparin
pretreatment would be abolished and/or reduced in the presence of the heparinase. The results in the isolated heart
support the concept that the association of heparin with the
cardiac tissue provides a protective action that can be negated by enzymatic cleavage of the GAG from its binding
sites, thereby rendering the heart vulnerable to injury during
ischemia/reperfusion.
A radiolabeled antibody to the cardiac myosin, served as a
specific marker of irreversible injury (Haber et al., 1982).
Membrane damage allows entry of the antibody to the cell
and binding to myosin. At the microscopic level, antimyosin
is bound to myocytes that have undergone extensive damage,
as seen by the loss of cytoplasmic features and nuclear structure (Khaw et al., 1979). Decreased antibody binding in the
hearts of heparin-pretreated animals provides support for
the protective role of heparin. In contrast, when hearts from
heparin-pretreated animals were made ischemic in the presence of heparinase, there was an increase in the uptake of the
antibody. The amount of antimyosin binding in response to
global ischemia and reperfusion was significantly greater in
heparin-pretreated hearts exposed to heparinase compared
with vehicle-treated hearts subjected to the same ischemic
stress, suggesting that the former group incurred a greater
degree of injury. On the other hand, hearts from heparinpretreated animals exhibited a better degree of recovery from
the ischemic insult. The reason for an exaggerated response
to heparinase in the heparin-pretreated heart is unknown.
One may speculate that formation of a heparin– heparinase
complex exacerbates the extent of injury in a manner similar
to untoward reactions that occur when protamine is administered to neutralize heparin (Shastri et al., 1997). Heparinlike molecules may be essential to the biochemical regulation
of vascular repair provided by endothelial cells. The pharmacodynamic actions of heparin neutralization with agents such
as protamine or heparinase should be examined with a focus
on tissue viability as affected by ischemia and reperfusion
(Han et al., 1997). Pretreatment of endothelial cells with
heparinase to alter their glycocalyx composition substantially enhances the formation of reactive oxygen species
(Gorog et al., 1988). It is not known to what extent this latter
mechanism contributed to the heparinase-associated deterioration of cardiac function. The combined cytotoxic effects
resulting from activation of the complement cascade along
with an augmented formation of reactive oxygen species may
provide an explanation for the enhanced antimyosin antibody uptake in the group 3 hearts compared with those in
group 1.
Electron microscopy was performed using lanthanum chloride as a marker of vascular injury and increased permeability to correlate the uptake of the antimyosin antibody with
myocardial damage (Hoffstein et al., 1975; Haack et al.,
1981). Lanthanum chloride binds to a fine filamentous layer
composed of an acidic mucopolysaccharide that lines the luminal surface of intact endothelial cells (Jokelainen et al.,
1976; Weihe et al., 1977). Disruption of the luminal membrane as a result of ischemia/reperfusion results in the loss of
the filamentous layer and a subsequent decrease in lanthanum binding (Haack et al., 1981). Vehicle-pretreated hearts
showed an increase in lanthanum-associated electron opacity
in the perivascular space. Hearts from animals pretreated
with heparin showed the presence of a continuous layer of
Vol. 290
1999
1047
logic, histochemical, autoradiographic and scintigraphic studies. Circulation 60:
1527–1531.
Kouretas PC, Kim YD, Cahill PA, Myers AK, To LN, Wang Y-N, Sitzmann JV and
Hannan RL (1999) Nonanticoagulant heparin prevents coronary endothelial dysfunction after brief ischemia-reperfusion injury in the dog. Circulation 99:1062–
1068.
Kouretas PC, Myers AK, Kim YD, Cahill PA, Myers JL, Wang YN, Sitzmann JV,
Wallace RB and Hannan RL (1998) Heparin and non-anticoagulant heparin preserve regional myocardial contractility after ischemia-reperfusion injury: Role of
nitric oxide. J Thorac Cardiovasc Surg 115:440 – 449.
Marcum JA and Rosenberg RD (1989) Role of endothelial cell surface heparin-like
polysaccharides. Ann NY Acad Sci 556:81–94.
Michelsen LG, Kikura M, Levy JH, Lee MK, Lee KC, Zimmermann JJ and Szlam F
(1997) Heparinase I (neutralase) reversal of systemic anticoagulation. Anesthesiology 85:339 –346.
Park JL, Kilgore KS, Naylor KB, Booth EA, Murphy KL and Lucchesi BR (1999)
N-Acetylheparin pretreatment reduces infarct size in the rabbit. Pharmacology
58:120 –131.
Ruoslahti E and Yamaguchi Y (1991) Proteoglycans as modulators of growth factor
activities. Cell 64:867– 869.
Sakahara H, Endo K, Nakashima T, Koizumi M, Kunimatsu M, Kawamura Y, Ohta
H, Nakamura T, Tanaka H, Kotoura Y, Yamamuro T, Hosoi S, Toyama S and
Torizuka K (1987) Localization of human osteogeneic sarcoma xenographs in nude
mice by a monoclonal antibody labeled with radioiodine and indium-111. J Nucl
Med 28:342–348.
Saliba MJ Jr (1997) The effects and uses of heparin in the care of burns that
improves treatment and enhances the quality of life. Acta Chirurgiae Plasticae
39:13–16.
Shastri KA, Logue GL, Stern MP, Rehman S and Raza S (1997) Complement
activation by heparin-protamine complexes during cardiopulmonary bypass: Effect of C4A null allele. J Thorac Cardiovasc Surg 114:482– 488.
Shell WE, Kjekshus JK and Sobel BE (1971) Quantitative assessment of the extent
of myocardial function in the conscious dog by means of analysis of serial changes
in serum creatine phosphokinase activity. J Clin Invest 50:2614 –2625.
Sunnergren KP, Fairman RP, DeBlois GG and Glauser FL (1987) Effects of protamine, heparinase and hyaluronidase on endothelial permeability and surface
charge. J Appl Physiol 63:1987–1992.
Szasz G, Gruber W and Bernt E (1976) Creatine kinase in serum. I. Determination
of optimum reaction conditions. Clin Chem 22:650 – 656.
Weihe E, Hartschuyh W, Metz J and Bruhl U (1977) The use of ionic lanthanum as
a diffusion tracer and as a marker of calcium binding sites. Cell Tissue Res
178:285–302.
Weiler JM, Yurt RW, Fearon DT and Austen KF (1978) Modulation of the formation
of the amplification convertase of complement, C3b, Bb, by native and commercial
heparin. J Exp Med 147:409 – 421.
Yasojima K, Kilgore KS, Washington RA, Lucchesi BR and McGeer PL (1998)
Complement gene expression by rabbit heart: Upregulation by ischemia and
reperfusion. Circ Res 82:1224 –1230.
Send reprint requests to: Benedict R. Lucchesi, Ph.D., M.D., Department of
Pharmacology, University of Michigan Medical School, 1301C Medical Science
Research Building III, Ann Arbor, MI 48109-0632. E-mail: [email protected]
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 3, 2017
Ecker EE and Gross P (1929) Anticomplementary power of heparin. J Infect Dis
44:250 –253.
Ecker EE and Pillemer L (1941) Anticoagulants and complementary activity. J Immunol 40:73– 80.
Edens RE, Linhardt RJ and Weiler JM (1993) Heparin is not just an anticoagulant
anymore: Six and one-half decades of studies on the ability of heparin to regulate
complement activity, in Complement Today (Cruse JM and Lewis RE Jr eds) vol 1,
pp 96 –130, Karger, Basel.
Evans RC, Wong VS, Morris AI and Rhodes JM (1997) Treatment of corticosteroidresistant ulcerative colitis with heparin–a report of 16 cases. Aliment Pharmacol
Ther 11:1037–1040.
Friedrichs GS, Kilgore KS, Manley PJ, Gralinski MR and Lucchesi BR (1994) Effects
of heparin and N-acetyl heparin on ischemia/reperfusion-induced alterations in
myocardial function in the rabbit isolated heart. Circ Res 75:701–710.
Gorog P, Pearson JD and Kakkar VV (1988) Generation of reactive oxygen metabolites by phagocytosing endothelial cells. Atherosclerosis 72:19 –27.
Gralinski MR, Driscoll EM, Friedrichs GS, DeNardis MR and Lucchesi BR (1996)
Reduction of myocardial necrosis after glycosaminoglycan administration: Effect of
a single intravenous administration of heparin or N-acetylheparin 2 h before
regional ischemia and reperfusion. J Cardiovasc Pharmacol Therapeut 1:219 –228.
Haack DW, Bush LR, Shlafer M and Lucchesi BR (1981) Lanthanum staining of
coronary microvascular endothelium: Effects of ischemic reperfusion, propranolol
and atenolol. Microvasc Res 21:362–376.
Haber E, Katus HA, Hurrell LG, Matsueda GR, Ehrlich P, Zurawski VR and Khaw
BA (1982) Detection and quantification of myocardial cell death: Application of
monoclonal antibodies specific for cardiac myosin. J Mol Cell Cardiol 14(Suppl
3):139 –146.
Han RO, Ettenson DS, Koo EW and Edelman ER (1997) Heparin/heparan sulfate
chelation inhibits control of vascular repair by tissue-engineered endothelial cells.
Am J Physiol 273:H2585–H2595.
Hayward R, Nossuli TO and Lefer AM (1997) Heparinase III exerts endothelial and
cardioprotective effects in feline myocardial ischemia-reperfusion injury. J Pharmacol Exp Ther 283:1032–1038.
Hiebert LM and Jaques LB (1976a) Heparin uptake on endothelium. Artery 2:26 –37.
Hiebert LM and Jaques LB (1976b) The observation of heparin on endothelium after
injection. Thromb Res 8:195–204.
Hobson RW, Wright JG, Fox D and Kerr JC (1988) Heparinization reduces endothelial permeability and hydrogen ion accumulation in a canine skeletal muscle
ischemia-reperfusion model. J Vasc Surg 7:585–591.
Hoffstein ST, Gennaro DE, Fox AC, Hirsch J, Streuli F and Weissmann G (1975)
Colloidal lanthanum as a marker for impaired plasma membrane permeability in
ischemic dog myocardium. Am J Pathol 79:206 –217.
Homeister JW, Satoh PS and Lucchesi BR (1992) Effects of complement activation in
the isolated heart–role of the terminal complement components. Circ Res 71:303–
319.
Hoogenwerf AJ, Kuschert GS, Proudfoot AE, Borlat F, Clark-Lewis I, Power CA and
Wells TN (1997) Glycosaminoglycans mediate cell surface oligomerization of chemokines. Biochemistry 36:13570 –13578.
Jokelainen PT, Haack DW and Rhodin JAG (1976) Permeability of arterial capillary
endothelium, in Microcirculation (Grayson J and Zingg W eds) vol 2, pp 77–78,
Plenum, New York.
Keller FG, DeFazio J, Jencks F, Steiner M, Rogers J and Ritchey AK (1998) The use
of heparinase to neutralize residual heparin in blood samples drawn through
pediatric indwelling central venous catheters. J Pediatrics 132:165–167.
Khaw BA, Fallon JT, Beller GA and Haber E (1979) Specificity of localization of
myosin-specific antibody fragment in experimental myocardial infarction: Histo-
Inhibition of Cardioprotection by Heparinase