© 1996 American Heart Association, Inc.
Articles |
Inhibition of Inducible Nitric Oxide Synthase Prevents Myocardial and Systemic Vascular Barrier Dysfunction During Early Cardiac Allograft Rejection
Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994.
From the Division of Cardiothoracic Surgery, Department of Surgery (N.K.W., G.M.S., T.B.F.), and the Department of Pathology (K.C., W.S.A., R.G.T., J.R.W.), Washington University School of Medicine, and the Departments of Molecular Pharmacology (T.P.M.) and Cellular and Molecular Biology (P.M.S.), Searle Research and Development, Monsanto Co, St Louis, Mo.
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Abstract |
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Abstract NO is produced during cardiac allograft rejection by expression of inducible NO synthase (iNOS) in the rejecting heart. Recent evidence indicates that NO modulates vascular permeability under both physiological and pathophysiological conditions. The present study explored the effects of early acute cardiac allograft rejection, and specifically the effects of NO, on myocardial and systemic vascular barrier function using a quantitative double-tracer permeation method in a rat cardiac transplant model. Early allograft rejection increased albumin permeation twofold to fivefold in the allograft heart and systemic vasculature (brain, lung, sciatic nerve, diaphragm, retina, muscle, kidney, and uvea) compared with isografts and controls. There were no detectable differences in regional blood flow or hemodynamics, suggesting that increased albumin permeation resulted from increased vascular permeability. iNOS mRNA was expressed in the allograft heart and native lung and was associated with increased serum nitrite/nitrate levels. iNOS inhibition with aminoguanidine prevented or attenuated allograft heart and systemic vascular barrier dysfunction and reduced allograft serum nitrite/nitrate levels to isograft values. Aminoguanidine did not affect the mild histological changes of rejection present in allografts. These data demonstrate the novel observations that (1) endothelial barrier function is compromised in the systemic vasculature, particularly in the brain, remote from the site of allograft rejection; (2) allograft vascular barrier dysfunction is associated with increased NO production and iNOS mRNA expression in the affected tissues (eg, native lung and grafted heart); and (3) inhibition of NO production by iNOS prevents vascular barrier dysfunction in the allograft heart and systemic vasculature.
Key Words: blood-brain barrier • capillary permeability • endothelium • nitric oxide • cardiac transplantation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although cardiac transplantation has evolved into an effective therapeutic option in the management of end-stage heart disease, its overall clinical success is limited by acute and chronic allograft rejection. The host immune response to allogeneic tissue is characterized by the accumulation of an intense inflammatory cell infiltrate in the graft that ultimately results in destruction of the transplanted organ. The microvascular endothelium is recognized to be an active participant in inflammatory processes, including acute allograft rejection.1 2 3 The barrier function of vascular endothelium regulates movement of macromolecules, solutes, and blood cells across vessel walls into tissues and is modulated by various mechanisms. Endothelial activation during allograft rejection is characterized by (1) expression of adhesion molecules in the rejecting organ,1 2 3 (2) migration of leukocytes into the allograft,4 (3) increased graft microvascular permeation by plasma proteins and fluid,5 and (4) edema formation in the allograft.4 The free radical NO has recently been implicated in mediating increased vascular permeation by macromolecules during endotoxin-induced uveitis6 and meningitis,7 in solid neoplasms,8 in diabetic animals,9 10 in response to increased blood flow,11 and in response to agonists such as endotoxin,12 endothelin-1,13 substance P,14 carageenin,15 leukotriene C4,16 histamine,17 18 and ADP and bradykinin.19 NO is synthesized from the amino acid L-arginine by the NOS family of enzymes and is involved in diverse physiological and pathophysiological processes, including host immune defense, allograft rejection, vasoregulation, neurotransmission, and diabetes.20 21 22 23
NO is produced during cardiac allograft rejection by expression of the
inducible isoform of NOS, iNOS, in the rejecting
heart.22 23 Treatment of cardiac allograft recipients with
aminoguanidine, a selective iNOS
inhibitor,9 24 25 26 significantly attenuated
acute rejection, as demonstrated by prolonged graft survival and
improved graft contractile function.23 The role of NO in
mediating endothelial barrier dysfunction in the
transplanted organ has not been described. The systemic sequelae of
clinical transplant rejection suggested that allograft rejection may
also cause systemic vascular barrier dysfunction. The possibility that
increased NO production may at least partially mediate systemic
vascular dysfunction during allograft rejection was suggested by
observations that (1) vasoactive cytokines such as TNF-
,
IL-1, and 
-IFN, which are expressed during allograft
rejection27 and can induce iNOS expression in
endothelial cells,20 21 have been detected
in the systemic circulation during allograft
rejection28 29 and (2) NO can react with sulfhydryl groups
of plasma proteins to form biologically active NO
adducts.30 31 32 33 The present study explored the effects
of early cardiac allograft rejection, and specifically the effects of
increased NO production, on myocardial and systemic vascular
barrier function using a quantitative double-tracer permeation
method in a rat model of cardiac transplantation.
The present study determined that early allograft rejection increased vascular albumin permeation in the grafted heart and the systemic vasculature (particularly in the brain) before significant necrosis of the grafted organ and in the absence of alterations of tissue blood flow or hemodynamic parameters. Allograft vascular barrier dysfunction was associated with increased NO production and with iNOS mRNA expression in the affected tissues (eg, native lung and grafted heart). Inhibition of iNOS with aminoguanidine prevented or attenuated endothelial barrier dysfunction in both the grafted heart and the systemic vasculature.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Heterotopic Cardiac Transplant Model
Male 220- to 270-g Lewis (RT-1l major histocompatability antigen haplotype) and ACI (RT-1a) rats were purchased from Harlan-Sprague-Dawley (Indianapolis, Ind). Animals received standard rat chow and water ad libitum and were housed and cared for in accordance with guidelines set forth by the Washington University Committee for the Humane Care of Laboratory Animals and the National Institutes of Health regarding laboratory animal welfare. Allogeneic (Lewis donor to ACI recipient) and syngeneic (ACI to ACI) heterotopic intra-abdominal cardiac transplantation was performed as described previously.23 34 Briefly, after aseptic preparation of the skin of the donor and recipient animals, anesthesia was induced with subcutaneous ketamine (100 mg/kg) and inhaled methoxyflurane. After preparation of the recipient's infrarenal abdominal aorta and inferior vena cava, the donor heart was rapidly excised after hypothermic arrest and ligation of the venae cavae and pulmonary veins. Iced saline was used for topical cooling, and the donor aorta and pulmonary artery were anastomosed to the recipient aorta and inferior vena cava, respectively. A sham operation, consisting of laparotomy, cross-clamping of the abdominal aorta and inferior vena cava for 30 minutes, phlebotomy of 0.5 mL of blood, and immediate replacement with 2 mL of 0.9% NaCl, was also performed.
Aminoguanidine Administration
Immediately after transplantation or sham operation, the right
external jugular vein was cannulated with a silicone catheter connected
to an ALZET osmotic infusion pump (model 2ML1, ALZA Corp) implanted in
the intrascapular subcutaneous space. Isografts and allografts received
continuous intravenous infusion of aminoguanidine
hemisulfate in 0.9% NaCl (375
mg·kg-1·d-1)
from the time of transplantation until determination of vascular
albumin permeation on POD 4. Sham-operated animals received
continuous intravenous infusion of 0.9% NaCl in a similar
manner. Normal animals received aminoguanidine in a similar manner for
72 hours before determination of vascular albumin
permeation.
Assessment of Vascular Albumin Permeation, Regional
Blood Flow, and Hemodynamic
Parameters
Eight experimental groups of rats were studied (all on POD 4,
except as listed): normal ACI group (n=16), aminoguanidine-treated
normal ACI group (n=3), sham-operated ACI group (n=7), ACI to ACI
isograft group (n=11), aminoguanidine-treated isograft group (n=6),
Lewis to ACI allograft group (n=11), aminoguanidine-treated
allograft group (n=10), and allograft group on POD 6 (n=2). Ten
additional animals were excluded from analysis because of
perioperative death, death during cannulation for the
vascular albumin permeation measurements, or the presence of a
unilateral kidney or infarct in the grafted heart.
Regional vascular albumin permeation was determined by a well-described9 10 35 36 quantitative isotope dilution technique based on the sequential injection of albumin labeled with two different iodine isotopes. [125I]BSA was used to quantify vascular albumin filtration after 10 minutes of tracer circulation. [131I]BSA circulated for 2 minutes and served as a plasma volume marker to allow for correction of the [125I]BSA activity contained within the tissue vasculature, assuming that little or no [131I]BSA was filtered across the endothelium during this short time period.37 If [131I]BSA did permeate across the endothelium during this short circulation time, then the correction would overestimate the intravascular content of [125I]BSA and, consequently, underestimate the degree of permeation across the endothelium. 46Sc-labeled microspheres were injected for simultaneous measurement of regional blood flow and hemodynamic parameters.10
Animal and Radiolabeled Tracer Preparation
Rats were anesthetized with thiopental (65 mg/kg body
wt, injected intraperitoneally), and core body
temperature was maintained at 37±0.5°C using heat lamps and a 37°C
surgical tray. The left femoral vein, right carotid artery, and both
iliac arteries were cannulated with polyethylene tubing (0.58-mm
internal diameter) filled with heparinized saline (400 U heparin/mL).
The left femoral vein cannula was used for tracer injection, and the
right iliac artery cannula was connected to a pressure transducer for
BP monitoring. The left iliac artery cannula was connected to a 1-mL
syringe attached to a Harvard Bioscience model 940
constant-withdrawal pump set at 0.055 mL/min. The tip of the right
carotid artery cannula was advanced into the left ventricle of the
native heart and was used for injection of radiolabeled
microspheres. The trachea was intubated and connected to a
small-rodent respirator for continuous ventilatory support.
125I, 131I, and 46Sc were from
DuPont-NEN. Purified monomer BSA (1 mg) was iodinated with
1 mCi of 125I or 131I by the iodogen method as
described previously.10
Experimental Procedure
Vascular albumin permeation and blood flow were assessed
simultaneously. At time 0, [125I]BSA (in 0.2
mL of saline) was injected intravenously, and the
withdrawal pump was started. Eight minutes later, 0.2 mL of
[131]BSA was injected, and 1 minute later,
46Sc-labeled 11.4-µm microspheres were
injected slowly over 
30 seconds. At the 10-minute mark, the native
heart was excised to stop all blood flow, the withdrawal pump was
stopped, and various organs/tissues were removed for analysis
of tracer content by -spectrometry. The left cerebral and
cerebellar hemispheres were removed en bloc, and the left eye was
dissected as described previously.35 36 Tissue samples and
arterial plasma samples were weighed before determination
of 125I, 131I, and 46Sc activities
in a -spectrometer interfaced with a DEC-5000 computer (Digital
Equipment Corp), in which data were corrected for background and stored
for subsequent analysis. Tissue dry weights were determined
after drying in an 80°C oven for 1 week.
Data Analysis
A quantitative index of [125I]BSA tissue clearance
was calculated as previously described9 10 35 36 and
expressed as micrograms plasma per gram tissue wet weight per minute.
Briefly, [125I]BSA activity in each tissue was corrected
for tissue intravascular content of this tracer by subtracting the
product of [131I]BSA tissue activity multiplied by
the ratio of [125I]BSA to [131I]BSA
activities in the arterial plasma sample obtained at the
end of the experiment. Vascular-corrected [125I]BSA
tissue activity was divided by the time-averaged
[125I]BSA plasma activity (obtained from a well-mixed
sample of plasma taken from the withdrawal syringe) and the tracer
circulation time (10 minutes) and then normalized per gram tissue wet
weight. To calculate regional blood flows, total 46Sc
activity in each tissue was divided by total 46Sc activity
in the reference blood sample obtained from the withdrawal syringe,
then multiplied by the pump withdrawal rate, and expressed as
milliliters per gram tissue wet weight per minute.10 36
Local vascular resistance in each tissue was determined by dividing the
animal's mean arterial BP by blood flow to that tissue.
Cardiac output was calculated by dividing total counts per minute of
46Sc injected by the 46Sc activity in the
withdrawal pump sample and then multiplied by the pump withdrawal
rate.38 Peripheral resistance was derived by
dividing mean arterial BP by cardiac output. Tissue wet
weight–to–dry weight ratios were calculated.
Spectrofluorometric Determination of Serum
Nitrite/Nitrate
Systemic arterial serum nitrite/nitrate levels were
measured in 0.2-mL blood samples taken from the left iliac artery
cannula before tracer injection. Red blood cells were removed by
centrifugation, and the resulting serum was filtered
through an Ultrafree-MC microcentrifuge filter (Millipore
Corp) to remove hemoglobin resulting from cell lysis. After conversion
of nitrate to nitrite with nitrate reductase, total nitrite was
measured by reaction with 2,3-diaminonaphthalene (Aldrich Chemical
Co; all other chemicals were from Sigma Chemical Co) under acidic
conditions to form 1-(H)-naphthotriazole, a
fluorescent product, as described
previously.39 Formation of
1-(H)-naphthotriazole was measured using a Pandex
fluorescent plate reader (IDEXX Laboratories) with excitation
at 365 nm and emission read at 450 nm.
iNOS mRNA Detection
Tissue was harvested by rapid excision and flash-frozen in
liquid nitrogen, and total RNA was then extracted using guanidinium
thiocyanate as described previously.40 mRNA expression was
analyzed by ribonuclease protection assay using an Ambion RPA
II kit. Duplicate 5-µg samples of total RNA were hybridized to
1x105 cpm of 32P-labeled rat iNOS antisense
RNA probe. The iNOS probe was generated from
lipopolysaccharide-stimulated rat white blood cell RNA
by reverse transcriptase–polymerase chain reaction amplification
of a 907-base iNOS fragment (corresponding to bases 509 to 1415 of the
rat iNOS coding region), as described previously,23 and
cloned into the Invitrogen pCRII vector. The 295-base iNOS probe was
then generated by linearization with Bsa I and transcription
with T7 polymerase. RNase digestion after probe
hybridization to rat tissue iNOS mRNA leaves a protected fragment of
227 bases in length, corresponding to bases 1189 to 1415 of the coding
region of rat iNOS. Rat GAPDH riboprobe was purchased from Ambion and
used as an internal control. Fragments were separated by
electrophoresis on an 8% polyacrylamide/8 mol/L urea gel and
visualized by autoradiography.
In Vivo ecNOS Activity
In vivo ecNOS activity was assessed by measuring the peak
increase in mean arterial BP following
intravenous injection of the NOS inhibitor
L-NMMA. Allografts and aminoguanidine-treated allografts (375
mg·kg-1·d-1
IV beginning at time of transplantation as in permeation experiments)
were anesthetized on POD 4 with thiopental (65 mg/kg body wt,
injected intraperitoneally), core body temperature
was maintained at 37±0.5°C using heat lamps and a 37°C surgical
tray, and the left femoral vein and artery were cannulated as described
above. After stabilization of arterial BP, increasing
amounts of L-NMMA in a constant volume (0.5 mL/kg) were injected
intravenously, and the peak percentage increase in mean
arterial BP from baseline was recorded for each dose of
L-NMMA.
Histology
At the time of measurement of vascular albumin
permeation, a portion of the grafted heart was excised, fixed in 10%
neutral buffered formaldehyde, embedded in paraffin, and sectioned.
After staining with hematoxylin and eosin, grafted hearts were graded
for acute rejection using a modification of Billingham's
criteria.4 In a masked fashion, four separate sections
from each specimen were graded for both interstitial
infiltrate and myocyte necrosis as follows: 0, no infiltrate or
necrosis; 1, mild scattered mononuclear infiltrate or rare necrosis; 2,
moderate infiltrate or patchy necrosis; 3, moderately severe infiltrate
or necrosis; 4, severe infiltrate or necrosis; and 5, complete
rejection.
Statistical Analysis
Tissue vascular albumin permeation, regional blood flow,
tissue vascular resistance, and tissue wet–to–dry weight
ratios were compared between groups using one-way ANOVA with
Tukey's HSD post hoc test because of multiple comparisons. Body
weight, hematocrit, hemodynamic parameters,
and serum nitrite/nitrate levels were also compared using one-way
ANOVA with Tukey's HSD post hoc test. Histological
scores were compared using Student's t test. BP response to
L-NMMA was compared using repeated-measures ANOVA with
one factor. Data were analyzed using SYSTAT 5.0 (SYSTAT Inc)
and are reported as mean±SD, with P<.05 considered to
indicate statistical significance.
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Results |
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Effect of Early Allograft Rejection on Graft Vascular Albumin Permeation
Vascular albumin permeation in the native heart did not differ between groups (Fig 1
). Vascular albumin
permeation in the isograft transplanted heart (subsequently referred to
as the isograft heart) was increased twofold versus the isograft native
heart. Vascular albumin permeation in the allograft
transplanted heart (subsequently referred to as the allograft heart)
was increased fourfold versus the allograft native heart and twofold
versus the isograft heart. Similar endothelial
dysfunction was observed in the allograft heart on POD 6 (n=2; 3301 and
3327 µg plasma·g tissue
wt-1·min-1,
respectively).
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P=.0001 for
grafted heart vs animal's native heart; 
P=.05 for
grafted heart vs animal's native heart.
Effect of Early Allograft Rejection on Systemic Vascular
Albumin Permeation
Vascular albumin permeation was significantly increased in
the brain, sciatic nerve, forelimb muscle, retina, uvea, diaphragm,
lung, and kidney of the allograft group compared with the control,
sham-operated, and isograft groups (Fig 2).
Similar increases in systemic vascular permeation were
present in the allograft group on POD 6 (n=2; eg, brain, 276 and
288 µg plasma·g tissue
wt-1·min-1;
lung, 2643 and 3014 µg plasma·g tissue
wt-1·min-1;
and muscle, 151 and 149 µg plasma·g tissue
wt-1·min-1).
Vascular albumin permeation in the isograft group was increased
above the levels found in the control and/or sham group in the brain,
sciatic nerve, retina, and diaphragm. There were no detectable
differences in vascular albumin permeation in any of the
tissues examined in sham-operated animals compared with control
animals (P
.5 versus control animals for each tissue).
There was no detectable difference between groups for vascular
albumin permeation in the thoracic aorta, skin, jejunum, or
spleen (data not shown).
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NO Production and iNOS mRNA Expression
Arterial serum nitrite/nitrate levels, which are
stable breakdown products of NO and thus reflect NO
production, were determined at the time of measurement of
vascular albumin permeation on POD 4 (Table 1).
Similar to our previous findings,23 allograft serum
nitrite/nitrate levels were significantly elevated above control, sham,
and isograft levels. Aminoguanidine treatment reduced allograft serum
nitrite/nitrate levels to isograft values. Aminoguanidine treatment had
no effect on isograft serum nitrite/nitrate levels
(P=.99).
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Ribonuclease protection assay analysis demonstrated that
elevated allograft serum nitrite/nitrate levels were associated with
iNOS mRNA expression in the allograft heart (Fig 3).
iNOS mRNA was not detected in isograft or control hearts. Lung tissue
was also analyzed to determine if allograft systemic vascular
barrier function was associated with iNOS expression in systemic
tissues. iNOS mRNA was present in left lung tissue from all three
cardiac allografts examined but was not present in isograft or
control lungs (Fig 3; iNOS mRNA expression was not examined in other
systemic tissues affected by the rejection process).
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Effect of Aminoguanidine on Graft and Systemic Vascular
Albumin Permeation
Given that allograft vascular barrier dysfunction was associated
with increased NO production and iNOS mRNA expression in the
allograft native lung and grafted heart, we examined whether increased
graft and systemic vascular albumin permeation was mediated by
NO. Aminoguanidine treatment reduced vascular albumin
permeation in the allograft heart to baseline isograft levels (Fig 1
,
P=.0001 versus untreated allograft hearts and
P=.3 versus isograft hearts). Similarly, aminoguanidine
treatment reduced systemic vascular albumin permeation to
isograft and/or control levels in all affected allograft tissues (Fig 2, P.6 versus isograft group). To determine if the
aminoguanidine-mediated reduction in allograft heart and systemic
vascular albumin permeation was an effect of aminoguanidine on
the endothelium independent of the rejection process,
we assessed the effect of aminoguanidine on albumin permeation
in control and isograft animals. Aminoguanidine did not affect
albumin permeation in normal animal tissues or in the isograft
heart or systemic tissues (Figs 1 and 2; P.9 versus
untreated controls and isografts, respectively).
Effect of Rejection and Aminoguanidine on Tissue Water
Content
Significantly higher tissue wet weight–to–dry weight
ratios were demonstrated in the brain, lung, and grafted heart in the
allograft group compared with the control and isograft groups (Table 2; not measured in other organs with increased vascular
albumin permeation). Aminoguanidine treatment significantly
reduced or prevented the increased tissue water content in allograft
tissues.
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Regional Blood Flow, Tissue Vascular Resistance, and
Hemodynamics
Tissue blood flow and vascular resistance were determined using
46Sc-labeled microspheres (Table 3). Regional blood flow and/or tissue vascular
resistance in the uvea and sciatic nerve were different in the sham,
isograft, and/or allograft groups versus the control group. However,
there were no differences in blood flow or vascular resistance in the
uvea and sciatic nerve between the isograft and allograft groups. There
were no detectable intergroup differences in regional blood flow or
vascular resistance in the rest of the tissues. Blood flow and vascular
resistance in the grafted heart were not different between groups but
were significantly less than in the native heart in each of the groups
(P<.01). Aminoguanidine treatment had no effect on tissue
blood flow or vascular resistance in any tissue in the allograft,
isograft, or control groups (Table 3; data not shown for
aminoguanidine-treated isograft and control groups).
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Hematocrit, cardiac output, and total peripheral resistance
were different from control values in some experimental groups (Table 4). However, there were no detectable differences in
hematocrit, mean systemic arterial BP, cardiac output,
total peripheral resistance, and body weight between the
sham, isograft, and allograft groups. Aminoguanidine treatment had no
effect on hemodynamic parameters in the
control, isograft, or allograft groups (data not shown for
aminoguanidine-treated control or isograft groups).
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Effect of Aminoguanidine on ecNOS Activity
To determine if aminoguanidine treatment selectively inhibited
iNOS in this model, in vivo ecNOS activity was assessed on POD 4 in the
allograft group and aminoguanidine-treated allograft group (same
dose as permeation experiments). The peak increase in mean
arterial BP following intravenous injections of
increasing amounts of L-NMMA was measured. There was no detectable
effect of aminoguanidine treatment on the BP response to L-NMMA
infusion (Fig 4, P>.5).
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Effect of Aminoguanidine on Graft Histology
The untreated allograft group demonstrated mild
histological changes consistent with an early
time point in the rejection process. There was a patchy mononuclear
infiltrate and scattered areas of myocyte necrosis. There was no
detectable effect of aminoguanidine treatment on the degree of
mononuclear infiltrate (histological score of 1.4±0.4
versus 1.3±0.4 [0 to 5 scale]) or myocyte necrosis (1.6±0.3 versus
1.5±0.3 [0 to 5 scale], n=6, P>.7 for infiltrate and
necrosis) at this early time point in the rejection process.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We and others have recently demonstrated that iNOS is expressed in cardiac allografts during acute rejection.22 23 Furthermore, we have demonstrated that inhibition of iNOS with aminoguanidine attenuated acute rejection, as manifested by prolonged graft survival, improved graft contractile function, and reduced myocyte necrosis and leukocyte migration into the graft.23 These observations prompted the present investigation examining the effect of early allograft rejection, and specifically the effect of increased NO production, on myocardial and systemic vascular barrier function. The data in the present study indicate that during early cardiac allograft rejection (1) vascular permeation by macromolecules is increased in the allograft heart and systemic vasculature and is associated with increased tissue water content, (2) allograft vascular barrier dysfunction is associated with increased NO production and iNOS mRNA expression in the affected tissues (eg, native lung and grafted heart), and (3) inhibition of iNOS with aminoguanidine prevented or significantly attenuated endothelial barrier dysfunction and the increased tissue water content in the allograft heart and systemic vasculature. Increased vascular permeation in the rejecting heart is consistent with previous observations.5 To our knowledge, this is the first report demonstrating that (1) systemic vascular barrier function is compromised in allograft recipients and (2) inhibition of NO production by iNOS prevents or attenuates endothelial barrier dysfunction in both the grafted heart and the systemic vasculature.
Mechanisms of Endothelial Dysfunction
Role of NO
The likelihood that NO contributed to increased graft and systemic
vascular permeation in allograft recipients is supported by (1) iNOS
mRNA expression in allograft tissues that demonstrated vascular barrier
dysfunction (eg, native lung and grafted heart), (2) elevated serum
nitrite/nitrate levels in allografts that were reduced to isograft
values with aminoguanidine, (3) reduction of increased albumin
permeation and increased tissue water content in the allograft heart
and all affected systemic tissues to isograft and/or control levels
with aminoguanidine, (4) observations that selective iNOS inhibition
with aminoguanidine attenuated increased vascular permeation by
macromolecules in diabetic animals9 10 and during
endotoxin-induced uveitis6 and
meningitis,7 and (5) reports that NOS
inhibitors attenuated increased vascular permeation caused
by various agonists.12 13 14 15 16 17 18 19
We and others have demonstrated that aminoguanidine is 10-fold to 100-fold selective for iNOS versus ecNOS.9 24 25 26 The aminoguanidine dose used in the present study is similar to that used in our previous in vivo studies,6 23 41 ie, 400 to 600 mg·kg-1·d-1. The selectivity of aminoguanidine for iNOS rather than ecNOS in the present study is demonstrated by (1) no difference in mean arterial BP between aminoguanidine-treated and untreated animals and (2) no detectable effect of aminoguanidine treatment on in vivo BP response to L-NMMA infusion. Aminoguanidine has effects other than inhibiting iNOS, including reducing advanced glycation end-product formation42 and inhibiting diamine oxidase43 and aldose reductase.44 However, these effects are unlikely to explain the beneficial effect of aminoguanidine in ameliorating vascular barrier dysfunction during allograft rejection for the following reasons: (1) there is no evidence to suggest that advanced glycation end products are elevated in the allografts; (2) since diamine oxidase deaminates histamine, aminoguanidine would reduce histamine metabolism and thus possibly increase vascular leakiness by potentiating the effects of histamine, as we have reported previously45 ; and (3) we have demonstrated that aminoguanidine is a very poor inhibitor of purified recombinant aldose reductase.10 Thus, the beneficial effect of aminoguanidine in ameliorating vascular barrier dysfunction during allograft rejection is most likely mediated through inhibition of iNOS.
The reason for the organ-selective nature of vascular barrier dysfunction during allograft rejection is not clear from our data and may be related to the early time point in the rejection process and/or the specific methodology used to assess endothelial barrier function. Vascular dysfunction in the allograft group is likely to be multifactorial, because aminoguanidine reduced albumin permeation to isograft levels but not to control levels in the grafted heart and in some systemic tissues. Endothelial dysfunction in isograft tissues, which was not affected by aminoguanidine, and residual endothelial dysfunction in some aminoguanidine-treated allograft tissues are most likely caused by the transplantation procedure itself. Thus, our results indicate that organ harvest, preservation, and/or reimplantation increase vascular permeation in the isograft heart and some systemic tissues. Endothelial barrier dysfunction was further increased in the allograft heart and systemic vasculature by the rejection process through what appeared to be a NO-mediated mechanism. Increased vascular albumin permeation in the allograft heart and systemic vasculature and prevention of this endothelial dysfunction with aminoguanidine appeared to be independent of changes in systemic hemodynamic parameters and of changes in blood flow and vascular resistance in the affected tissues. This suggested that increased albumin permeation resulted from increased endothelial permeability to macromolecules.
Allograft Heart
Endothelial barrier dysfunction in the allograft
heart was associated with increased tissue water content and with only
mild histological changes of acute rejection. This is
consistent with previous observations that early allograft
rejection is associated with edema formation and compromised vascular
barrier function, before significant myocyte necrosis.4 5
The early time point in the rejection process, together with the
observation that aminoguanidine prevented endothelial
dysfunction without altering the mild histological
changes of acute rejection present on POD 4 (in contrast to
attenuation of severe histological changes present
on POD 823 ), suggested that endothelial
barrier dysfunction was a specific effect of acute rejection and of NO.
Increased vascular permeability in the allograft heart may result from
several mechanisms, including (1)
pathophysiological regulation of
endothelial cell barrier integrity by
NO,16 17 18 19 (2) NO-mediated endothelial cell
cytotoxicity,46 which at this early point in the rejection
process is not manifested histologically, and (3)
increased synthesis of inflammatory
prostaglandins47 48 or other mediators caused
by NO. These results suggest that attenuation of allograft rejection
previously demonstrated with inhibition of iNOS23 may be
at least partially mediated through prevention of vascular barrier
dysfunction in the allograft heart.
Expression of iNOS has recently been demonstrated in the allograft heart during chronic rejection.49 Modulation of the inflammatory response (with a diet deficient in essential fatty acids) resulted in a significant decrease in the degree of transplant arteriosclerosis and was correlated with decreased iNOS expression. Whether pharmacological inhibition of iNOS will attenuate transplant arteriosclerosis and whether NO-mediated vascular barrier dysfunction contributes to transplant arteriosclerosis during chronic rejection remain to be explored.
Allograft Systemic Vasculature
Since systemic endothelial dysfunction remote from
localized inflammatory processes has not been previously reported to
our knowledge, the observation that endothelial barrier
function is compromised in the systemic vasculature during early
allograft rejection is of particular interest. Also of note is that the
increase in vascular permeation and tissue water content in the
systemic tissues was of approximately the same magnitude as that in the
rejecting heart. Our results suggest that allograft systemic vascular
barrier dysfunction is mediated by iNOS mRNA expression and increased
NO production in the affected systemic tissues. This is
supported by our observations that (1) iNOS mRNA is present in the
allograft native lung, associated with increased lung vascular
permeability and water content, and (2) iNOS inhibition with
aminoguanidine prevented allograft systemic vascular barrier
dysfunction. Systemic iNOS expression may be induced by
cytokines such as TNF-, IL-1, and -IFN, which are
present during allograft rejection,27 have been
detected in the systemic circulation during allograft
rejection,28 29 and can induce iNOS expression in the
endothelium.20 21 This mechanism is also
supported by evidence that injection of low doses of TNF- increased
systemic albumin permeation without affecting tissue blood flow
or hemodynamic parameters.50
Cytokine-induced systemic endothelial
dysfunction is analogous to the systemic vascular leak syndrome
accompanying IL-2 treatment for tumors,51 although the
role of NO in this syndrome has not been evaluated.
Two other potential actions of NO may contribute to systemic vascular barrier dysfunction and cannot be discounted by our data. First, NO produced in the graft may circulate and act systemically. This seems unlikely given the short half-life of NO. However, several groups have postulated that NO-like activity may be stabilized in vivo.30 31 32 33 52 53 Recent reports demonstrate that NO can react with sulfhydryl groups of plasma proteins to form biologically active NO adducts with half-lives significantly longer than that of NO itself.30 31 32 33 Large amounts of NO are produced during allograft rejection as manifested by elevated serum nitrite/nitrate levels and by electron paramagnetic resonance spectroscopic analysis of the grafted heart23 54 and of allograft erythrocytes in the systemic circulation.54 Thus, NO produced in the allograft heart may form NO adducts, which then circulate and act systemically. Second, inhibition of NO production in the graft may prevent elaboration of other vasoactive mediators that act systemically. Production and release of these other mediators could be specifically dependent on NO production (eg, leukotrienes or prostaglandins47 48 ) or may be nonspecifically blocked by the iNOS inhibition attenuating myocyte damage (eg, release of ANF). This seems unlikely for the following reasons: (1) injection of leukotriene-B4, prostaglandin-E2, or ANF did not affect albumin permeation in brain or sciatic nerve,55 56 which were both profoundly affected by the rejection process in the present report; (2) in contrast to acute rejection, ANF produced changes in tissue blood flow and vascular resistance56 ; and (3) aminoguanidine did not alter the histological changes of acute rejection in the present study.
Allograft Blood-Brain Barrier
Perhaps the most significant finding of the present study is
that allograft rejection increased vascular permeability in the brain
by fourfold compared with control values. Brain microvascular
endothelial cells and surrounding astrocytes form a
very tight barrier to permeation by macromolecules. Permeability of
this BBB is increased by intrinsic central nervous system processes
such as meningitis7 57 58 and focal cerebral
ischemia.59 However, the effect of systemic
processes on BBB function is not well described. Our previous studies
have demonstrated that albumin permeation in the brain is not
affected during diabetes9 10 36 37 38 or by systemic
administration of histamine,45
leukotriene-B4,55
prostaglandin-E2,55 or
ANF.56 Others have demonstrated that bilateral
adrenalectomy increases BBB permeability to albumin by up to
1.4-fold,60 suggesting that
corticosteroids help maintain BBB integrity and adding
significance to the present study reporting a fourfold elevation in
BBB permeability to albumin during acute allograft rejection.
To our knowledge, the present report is the first to demonstrate
that (1) allograft rejection is associated with compromised BBB
integrity and (2) a selective inhibitor of iNOS attenuates
this BBB dysfunction.
Our observation that iNOS inhibition significantly attenuated BBB dysfunction during allograft rejection is perhaps not surprising given that (1) NO or NO donors activate soluble guanylate cyclase in brain endothelial cells and thus increase the formation of cGMP,61 (2) cGMP increases BBB permeability to macromolecules,62 (3) NOS inhibitors attenuate increased BBB permeability during focal cerebral ischemia,63 and (4) aminoguanidine attenuates BBB dysfunction in experimental endotoxin-induced meningitis.7 The exact mechanism causing impaired BBB function during allograft rejection has implications beyond the field of transplantation, including intrinsic neuropathophysiology, brain function during systemic pathophysiological processes, and drug delivery into the central nervous system.
Modulation of Vascular Permeability by NO
NO has diverse effects on the endothelium. Our
results demonstrate that during acute allograft rejection, inhibition
of iNOS prevents increased vascular permeability. NOS
inhibitors also prevented or attenuated increased vascular
permeation by macromolecules during endotoxin-induced
uveitis6 and meningitis,7 in solid
neoplasms,8 in diabetic animals,9 10 in
response to increased blood flow,11 and in response to
various agonists.12 13 14 15 16 17 18 19 These studies are similar in that
they examined increased vascular permeation caused by agonists or
during pathological conditions. Conversely, inhibition of basal NO
production increased vascular permeation in
endothelial monolayers,64 feline small
intestine,65 rat mesenteric venules,66 and
rat coronary circulation.67 Increased vascular
permeation seen with inhibition of basal NO production may
reflect increased leukocyte adherence and endothelial
activation, given that (1) impairment of constitutive NO
production promoted leukocyte adherence and
emigration68 69 and (2) antibodies to adhesion molecules
prevented increased vascular permeation caused by NOS
inhibitors.65 Conversely, inhibition of iNOS
reduced cellular infiltrate in experimental autoimmune
encephalomyelitis,41 in endotoxin-induced
uveitis,6 and in the latter stages of allograft
rejection.23 These diverse effects of NO on the
endothelium may be partially explained by NO serving to
maintain endothelial integrity under
physiological conditions, whereas
pathophysiological production of larger
amounts of NO increases endothelial permeability.
Conclusions
Early allograft rejection increased vascular albumin
permeation and tissue water content in both the grafted heart and the
systemic vasculature (particularly in the brain). In the absence of
changes in regional blood flow or hemodynamic
parameters, the increased albumin permeation is
indicative of increased endothelial permeability.
Allograft vascular barrier dysfunction was associated with iNOS mRNA
expression in the affected tissues and with elevated serum
nitrite/nitrate levels. Inhibition of iNOS with aminoguanidine
prevented or significantly attenuated endothelial
barrier dysfunction in the allograft heart and systemic vasculature and
reduced allograft serum nitrite/nitrate levels to isograft values.
These data indicate that NO contributes to compromised vascular barrier
function in both the allograft heart and the systemic vasculature
during early allograft rejection. Inhibition of NO production
may provide a novel therapeutic modality in the management of acute
allograft rejection and other immune-mediated processes and may
attenuate systemic sequelae of these processes.
|
Selected Abbreviations and Acronyms |
|---|
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|
Acknowledgments |
|---|
This study was supported by National Institutes of Health grants F32 HL-09021 (Dr Worrall), EY0660 and HL-39934 (Dr Williamson), and R29 HL-46387 (Dr Ferguson); the Kilo Diabetes and Vascular Research Foundation (Dr Williamson); and the Monsanto-Searle/Washington University Biomedical Program (Drs Williamson and Ferguson). We would like to thank Dr Richard B. Schuessler for help with the statistical analysis.
|
Footnotes |
|---|
Reprint requests to T. Bruce Ferguson, Jr, MD, Division of Cardiothoracic Surgery, Suite 3108, Queeny Tower, One Barnes Hospital Plaza, St Louis, MO 63110.
Received June 23, 1995; accepted January 16, 1996.
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References |
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