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causes reversible in vivo systemic vascular barrier
dysfunction via NO-dependent and -independent
mechanisms
Departments of 1 Surgery and
2 Pathology, Tumor necrosis factor (TNF-
blood-brain barrier; capillary permeability; cytokines; endothelium; lung; nitric oxide
SEPSIS IS CHARACTERIZED by refractory hypotension,
capillary leakage, and multiple organ dysfunction. Recent evidence
suggests that many of the effects of the septic mediators endotoxin,
tumor necrosis factor- An important role for NO in septic shock has been suggested by
observations that iNOS expression and increased NO production occurs
during septic shock (44) and inhibition of NOS reverses TNF- The barrier function of vascular endothelium regulates movement of
macromolecules and cells across vessel walls into tissues. Impaired
vascular barrier function results in increased permeation of
macromolecules and may result in edema formation, leukocyte migration
into the tissues, and frank organ damage. Systemic administration of
high doses of TNF- NO contributes to increased vascular permeation by macromolecules
during cardiac allograft rejection (50), in diabetic animals (46), in
response to increased blood flow (54), and in response to various
inflammatory and noninflammatory agonists, including endotoxin (3, 22,
24, 25, 45, 55). Conversely, inhibition of basal endogenous NO
synthesis has been demonstrated to increase vascular permeation and
leukocyte adherence (19, 20). Thus it is unclear whether TNF- The present study hypothesized that
1) TNF- Experimental Groups
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
) and nitric
oxide (NO) are important vasoactive mediators of septic shock. This
study used a well-characterized quantitative permeation method to
examine the effect of TNF- and NO on systemic vascular barrier
function in vivo, without confounding endotoxemia, hypotension, or
organ damage. Our results showed 1)
TNF- reversibly increased albumin permeation in the systemic
vasculature (e.g., lung, liver, brain, etc.); 2) TNF- did not affect
hemodynamics or blood flow or cause significant tissue injury;
3) pulmonary vascular barrier
dysfunction was associated with increased lung water content and
impaired oxygenation; 4) TNF-
caused inducible nitric oxide synthase (iNOS) mRNA expression in the
lung and increased in vivo NO production;
5) selective inhibition of iNOS with
aminoguanidine prevented TNF--induced lung and liver vascular
barrier dysfunction; 6)
aminoguanidine prevented increased tissue water content in
TNF--treated lungs and improved oxygenation; and
7) nonselective inhibition of NOS with NG-monomethly-L-arginine
increased vascular permeation in control lungs and caused severe lung
injury in TNF--treated animals. We conclude that
1) TNF- reversibly impairs
vascular barrier integrity through NO-dependent and -independent
mechanisms; 2) nonselective NOS
inhibition increased vascular barrier dysfunction and caused severe
lung injury, whereas selective inhibition of iNOS prevented impaired
endothelial barrier integrity and pulmonary dysfunction; and
3) selective inhibition of iNOS may
be beneficial in treating increased vascular permeability that
complicates endotoxemia and cytokine immunotherapy.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(TNF-), interleukin-1 (IL-1), and
interferon-
are mediated by increased production of nitric oxide
(NO) (30). NO is synthesized from
L-arginine by the NO synthases
(NOS). Endothelial cell NOS is constitutively expressed (cNOS) and
generates small amounts of NO in response to physical and receptor
stimuli. Conversely, inducible NOS (iNOS) produces much
larger amounts of NO for sustained time periods, is expressed in
diverse cell types, and is principally implicated in the
pathophysiological actions of NO.
- and
endotoxin-induced hypotension in some models (15, 43). However, the
role of NOS inhibition in the treatment of septic shock is
controversial, because nonselective inhibition of cNOS and iNOS
increased mortality from cytokine- or endotoxin-induced septic shock
(7, 33, 51), whereas selective inhibition of iNOS (52) or nonselective
inhibition of cNOS and iNOS together with replacement of basal NO
production with an NO donor (51) ameliorated the hypotension and
mortality of endotoxemic shock. The deleterious effects of nonselective
inhibition of cNOS and iNOS may reflect inhibition of the
vasoregulatory functions of basal endothelial NO production, such as
regulation of vascular tone, platelet aggregation, and neutrophil
adhesion.
produces hypotension and organ damage
characteristic of septic shock (23, 36, 47). At lower doses, TNF-
modulates the barrier function of endothelial cell monolayers (4, 38, 42) and increases local vascular permeation after intradermal or
intramuscular injection (1, 53). However, there are discordant reports
as to whether TNF- increases systemic vascular permeability in vivo
(2, 6, 13, 18, 26, 34, 41).
modulates systemic vascular barrier function in vivo, whether this
contributes to vascular barrier dysfunction during endotoxemia, and
whether the effects of TNF- are mediated through NO-dependent
mechanisms.
would reversibly increase
pulmonary and systemic vascular permeation independent of changes in
blood flow or hemodynamics, 2)
TNF- would induce iNOS expression and increased NO production in the
affected tissues, and 3) selective
inhibition of iNOS would ameliorate TNF--induced systemic vascular
barrier dysfunction. This study used a well-characterized quantitative
double-tracer permeation and microsphere method (45, 46, 50) to examine
systemic vascular permeation of macromolecules (albumin), tissue blood
flow, and systemic hemodynamics after systemic administration of a low
dose of recombinant TNF-, avoiding the confounding effects of
endotoxemia, hypotension, and organ damage. Specifically, this study
examined 1) whether TNF-
increased systemic vascular permeation and whether this was independent of changes in blood flow or hemodynamics or associated tissue injury,
2) whether TNF--induced pulmonary
vascular barrier dysfunction was associated with increased lung water
content and/or impaired gas exchange,
3) whether increased vascular
permeation was associated with iNOS expression and increased NO
production, and 4) whether treatment
with the selective iNOS inhibitor aminoguanidine (28, 49) or the
nonselective cNOS and iNOS inhibitor
NG-monomethyl-L-arginine
(L-NMMA) ameliorated pulmonary
and systemic vascular barrier dysfunction.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
[20
µg/kg in 2.5 ml of phosphate-buffered saline (PBS)-0.5% bovine serum
albumin (BSA) in these and subsequent groups receiving TNF- (2.7 × 105 U/µg per
manufacturer's L929 cytotoxicity assay; Genzyme, Cambridge, MA)]; 2) controls at 18 h
after intraperitoneal injection of 2.5 ml PBS-BSA (maximal effect of
TNF- seen at 18 h); 3) at 18 h after intraperitoneal injection of TNF- that was boiled for 30 min
before injection; 4) at 18 h after
intraperitoneal injection of TNF- in animals treated with the iNOS
inhibitor aminoguanidine (subcutaneous injection of 200 mg/kg of
aminoguanidine in 1 ml of PBS at 3 and 15 h after TNF- injection)
(28, 49); 5) at 18 h after
intraperitoneal injection of TNF- in animals treated with the cNOS
and iNOS inhibitor L-NMMA (60 mg/kg ip in 1 ml PBS at 3 h after TNF- injection); and
6) controls treated with
aminoguanidine, L-NMMA, or
D-NMMA (60 mg/kg ip in PBS) in a
similar fashion. All chemicals were from Sigma Chemical (St. Louis, MO)
unless otherwise noted.
Vascular Albumin Permeation, Regional Blood Flow, and Hemodynamics
Vascular albumin permeation was determined by a well-described (45, 46, 50) quantitative isotope dilution technique based on the sequential injection of 125I- and 131I-labeled BSA. 125I-BSA was used to quantify vascular albumin filtration after 10 min of tracer circulation. 131I-BSA circulated for 2 min and served as a plasma volume marker to allow for correction of the 125I-BSA activity contained within the tissue vasculature, with the assumption that little or no 131I-BSA was filtered across the endothelium during this short time period (11). 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. 125I, 131I, and 46Sc were from DuPont NEN (Boston, MA). Purified monomer BSA (1 mg) was iodinated with 1 mCi of 125I or 131I by the iodogen method as described previously (46).Experimental procedure. Rats were anesthetized with thiopental sodium (65 mg/kg ip), and core body temperature was maintained at 37 ± 0.5°C. The animals were breathing room air spontaneously, and a tracheotomy was performed to prevent upper airway obstruction. The left femoral vein, right carotid artery, and both iliac arteries were cannulated with heparinized polyethylene tubing (0.58-mm internal diameter; 400 U heparin/ml). The right iliac artery cannula was connected to a pressure transducer for blood pressure monitoring, and the left iliac artery cannula was connected to a Harvard Bioscience (South Natick, MA) model 940 constant-withdrawal pump set at 0.055 ml/min. The experimental procedure was as follows: 1) at time 0, 0.2 ml of 125I-BSA was injected intravenously via the left femoral vein catheter and the withdrawal pump was started; 2) at 8 min, 0.2 ml of 131I-BSA was injected intravenously to serve as a plasma volume marker to allow for correction of the 125I-BSA activity contained within the tissue vasculature; 3) at 9 min, 46Sc-labeled 11.4-µm microspheres were injected into the left ventricle via the right carotid artery catheter to measure tissue blood flow and cardiac output; and 4) at 10 min, the heart was excised to stop all blood flow, the withdrawal pump was stopped, the tissues were removed and weighed, and tissue tracer content was determined by gamma spectrometry. Tissue dry weights were determined after drying in an 80°C oven. Systemic arterial pH, arterial PO2 (PaO2), and arterial PCO2 (PaCO2) were determined in a 0.2-ml blood sample removed from the left iliac artery cannula before tracer injection using a calibrated blood gas analyzer (model 170; Corning).
Data analysis. A quantitative index of 125I-BSA clearance in each tissue was calculated as previously described (45, 46, 50). Briefly, 125I-BSA activity in each tissue was corrected for 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. Vascular-corrected 125I-BSA tissue activity was divided by the time-averaged 125I-BSA plasma activity in the withdrawal syringe and the tracer circulation time (10 min) and then normalized per gram of tissue wet weight. Tissue blood flow was calculated by dividing tissue 46Sc activity by total 46Sc activity in the withdrawal syringe and multiplying by the pump withdrawal rate. Cardiac output was calculated by dividing 46Sc activity injected by the 46Sc activity in the withdrawal pump sample and then multiplying by the pump withdrawal rate. Peripheral resistance was derived by dividing mean arterial blood pressure by cardiac output.
iNOS Ribonuclease Protection Assay
Lungs were rapidly excised and flash-frozen in liquid nitrogen, and total RNA was extracted using guanidinium thiocyanate as described (50). mRNA expression was analyzed by ribonuclease (RNase) protection assay using an Ambion RPA II kit (Austin, TX) as described (50). Triplicate 5-µg samples of RNA were hybridized to 1 × 105 counts/min of 32P-labeled rat iNOS antisense RNA probe. RNase digestion after probe hybridization to tissue iNOS mRNA leaves a protected fragment of 227 bases in length (bases 1,189-1,415 of the iNOS coding region). Rat cyclophilin probe 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.Serum Nitrite/Nitrate Levels
Systemic arterial serum nitrite/nitrate levels were measured in blood samples taken from the left iliac artery cannula as described (29, 50). Red blood cells were removed by centrifugation, and the serum was filtered through an Ultrafree-MC microcentrifuge filter (Millipore, Bedford, MA). After conversion of nitrate to nitrite with nitrate reductase, total nitrite was measured by reaction with 2,3-diaminonaphthalene to form 1H-naphthotriazole, a fluorescent product, as described (29).Histology
Tissues were harvested; fixed in 10% neutral, buffered formaldehyde; embedded in paraffin; sectioned; stained with hematoxylin and eosin; and examined by a pathologist blinded to group identity.Statistical Analysis
Data were compared between groups using one-way analysis of variance with the Tukey honestly significant difference post hoc test because of multiple comparisons using SYSTAT 5.0 (SYSTAT, Evanston, IL). Data are reported as means ± SD of n animals per experimental group with P < 0.05 considered statistically significant.| |
RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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TNF- Caused Reversible Systemic Vascular Barrier
Dysfunction
-treated animals was increased
above controls at 12-18 h after TNF- injection (20 µg/kg ip)
and returned to baseline control levels by 72 h after TNF- injection
(Fig. 1). Vascular permeation
in the heart was not affected by TNF-
(P > 0.6 vs. controls). Boiled
TNF- had no effect on vascular permeation in any tissue
(n = 3;
P > 0.5 vs. controls). Treatment
with a lower dose of TNF- (4 µg/kg ip) caused a small increase in
permeation in the lung, liver, and brain which did not achieve
statistical significance (n = 3;
P > 0.2 vs. controls).
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Effect of NOS Inhibitors on Vascular Permeation
TNF- treatment induced iNOS mRNA expression in the lung in vivo
(Fig. 2). iNOS mRNA was detected in lung
tissue harvested at 4 h after TNF- injection but was not present at
8 or 12 h after TNF- injection (Fig. 2). iNOS mRNA was not detected
in control lungs. iNOS expression was not assessed in other tissues. Increased endogenous NO production was demonstrated by elevated systemic serum levels of nitrite/nitrate (end products of NO
metabolism) at 12 and 18 h after TNF- injection which returned to
baseline control values by 36 h after TNF- injection (Fig.
3). The increased NO production in
TNF--treated animals was reduced by treatment with the selective
iNOS inhibitor aminoguanidine (Fig. 3). The role of iNOS and NO in
increased vascular permeation after injection of TNF- was then
examined (determined at 18 h after TNF- injection).
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Vascular permeation in the lung and liver of animals that were injected
with TNF- and then treated with the selective iNOS inhibitor
aminoguanidine was significantly reduced compared with that in animals
treated with TNF- alone (Fig. 4).
Aminoguanidine did not detectably affect vascular permeation in the
rest of the TNF--treated tissues or in control tissues (Fig. 4).
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Treatment with the nonselective NOS inhibitor
L-NMMA significantly increased permeation in
the control lung (Fig. 4).
L-NMMA did not detectably affect
vascular permeation in other control tissues (Fig. 4). Treatment with
the inactive enantiomer D-NMMA did not affect permeation in control tissues [e.g., permeation of
1,184 ± 132 vs. 1,128 ± 192 µg plasma · g
tissue
wt
1 · min1
for lung and 1,247 ± 211 vs. 1,223 ± 68 µg
plasma · g tissue wt1 · min1
for liver in D-NMMA-treated
controls (n = 4) and untreated
controls (n = 12), respectively;
P > 0.9].
Vascular permeation could not be assessed in animals injected with
TNF- and then treated with L-NMMA because
all four animals died during measurement of vascular permeation as a
result of lung injury (see Pulmonary Tissue Water
Content, Gas Exchange, and Histology).
In comparison, the mortality was 0 of 12 in controls, 1 of 16 in
TNF--treated animals, and 1 of 13 in TNF- + aminoguanidine-treated animals.
Pulmonary Tissue Water Content, Gas Exchange, and Histology
TNF- did not cause histologically detectable injury in the liver,
brain, or muscle (data not shown). The lungs from TNF--treated animals showed minimal injury, characterized by minimal disruption of
the alveolar architecture and rare areas of a mild inflammatory infiltrate and red blood cell extravasation into the interstitium (Fig.
5). Conversely, lungs from the four
L-NMMA- and TNF--treated animals that died during measurement of vascular permeation and from
four additional L-NMMA- and
TNF--treated animals (n = 8) showed
severe injury, characterized by widespread obliteration of the alveoli,
interstitial thickening, inflammatory infiltrate, and red blood cell
extravasation. L-NMMA did not
cause injury in control lungs (Fig. 5).
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TNF--treated animals had significantly higher lung water content
than controls, which was prevented with aminoguanidine (Table 1). TNF--treated animals had reduced
systemic PaO2 on room air ventilation
compared with untreated controls which was significantly improved with
aminoguanidine treatment (Table 1). TNF- and aminoguanidine did not
affect systemic pH or PaCO2.
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Tissue Blood Flow and Hemodynamics
TNF- increased blood flow in the liver at 18 h after injection but
did not affect the rest of the tissues at 18 h after injection or any
tissue at 6, 12, 36, or 72 h after injection (Table
2; data not shown for other time points;
P > 0.5). Aminoguanidine reduced
blood flow in the TNF--treated liver but did not affect blood flow
in the rest of the TNF--treated tissues or affect blood flow in any
control tissue (Table 2). L-NMMA
increased blood flow in the control cecum but did not affect blood flow in any other control tissue (Table 2).
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Body weight, mean systemic arterial blood pressure, cardiac output, and
total peripheral resistance were not significantly different from
controls at 6, 12, 18, 36, or 72 h after TNF- injection (Table 3;
data only shown for 18 h; P > 0.4).
Treatment with L-NMMA or
aminoguanidine did not affect these parameters in controls
and/or TNF--treated animals (Table
3).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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This study examined the effect of the vasoactive cytokine TNF- on
systemic vascular endothelial barrier function in rats, without the
confounding variables of endotoxemia, hypotension, or organ damage.
This study demonstrated that 1)
TNF- reversibly increased albumin permeation in the systemic
vasculature (e.g., lung, liver, brain, etc.),
2) TNF- did not detectably affect hemodynamics or tissue blood flow or cause significant lung injury at
the doses tested, 3) pulmonary
vascular barrier dysfunction was associated with increased lung water
content and impaired oxygenation, 4)
TNF- induced iNOS mRNA expression in the lung and increased systemic
serum nitrite/nitrate levels indicative of increased in vivo NO
production, 5) selective inhibition
of iNOS with aminoguanidine prevented TNF--induced lung and liver vascular barrier dysfunction, 6)
aminoguanidine prevented increased tissue water content in
TNF--treated lungs and improved oxygenation, and
7) nonselective inhibition of NOS
with L-NMMA increased vascular permeation in control lungs and caused severe lung injury in
TNF--treated animals.
Modulation of Systemic Vascular Barrier Function by
TNF-
reversibly increased systemic vascular
albumin permeation without significantly affecting tissue blood flow or
hemodynamics suggests that the increased permeation resulted from
impaired endothelial barrier integrity and increased endothelial
permeability to macromolecules. Vascular barrier dysfunction appeared
to be a specific effect of TNF-, because the carrier solution had no
effect in controls and boiling abolished the effect of TNF- (boiling
would not be expected to alter the effect of any endotoxin
contamination of the TNF-). The 12- to 18-h lag time after injection
of TNF- before development of systemic vascular barrier dysfunction
is unlikely to reflect the route of administration, because TNF-
would be expected to be rapidly absorbed after intraperitoneal administration. Rather, this lag time is most likely required for
protein or mRNA synthesis or for cytoskeletal rearrangement. This is
consistent with observations that TNF- required 4-12 h to
increase permeability of endothelial cell monolayers through cytoskeletal rearrangement and formation of intercellular gaps (4, 38,
42). The transient nature of increased vascular albumin permeation in
the present study, together with these previous observations that
TNF- increased endothelial cell monolayer permeability without
altering cell viability (4, 38), suggests that TNF- caused specific
disruption of endothelial barrier function without endothelial cell
cytotoxicity in our model.
Others have demonstrated that 1)
intradermal or intramuscular injection of TNF- increased local
vascular permeability (1, 53) and 2)
systemic administration of high doses of TNF- caused septic shock
and multiple organ damage (23, 36, 47). However, there are discordant
reports as to whether TNF- increases lung and/or systemic
vascular permeability in vivo (2, 6, 13, 18, 26, 34, 41). To our
knowledge, the present study is the first to demonstrate that low doses
of TNF- caused reversible systemic vascular barrier dysfunction in
the absence of hemodynamic alterations or systemic organ damage.
TNF--induced vascular barrier dysfunction may contribute to
capillary leakage and organ dysfunction during sepsis.
An intriguing finding of this report is that systemic administration of
TNF- increased vascular permeability in the brain by fourfold versus
controls. Brain microvascular endothelial cells and surrounding
astrocytes form a very tight barrier to permeation by macromolecules.
Permeability of this blood-brain barrier (BBB) is increased during
experimental meningitis produced by the intracisternal injection of
IL-1 or TNF- (17, 35). 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 increased during
cardiac allograft rejection (50) but is not affected during diabetes
(46) or by systemic administration of histamine, leukotriene
B4, prostaglandin
E2, or atrial natriuretic factor
(16, 40, 48). Others have demonstrated that, whereas intracisternal
injection of TNF- increased BBB permeability (17, 35), intravenously
administered TNF- did not affect BBB permeability (10, 17). The
significance and mechanism of systemic TNF- modulating BBB function
requires further investigation.
Role of NO in TNF--Induced Vascular Barrier
Dysfunction
-induced systemic vascular barrier
dysfunction because 1) TNF-
induced transient iNOS expression in the lung that preceded the
increased vascular permeability, 2)
TNF- caused increased endogenous NO production that paralleled the
increased vascular permeation, and
3) inhibition of NO production with
aminoguanidine prevented vascular barrier dysfunction in the lung and
liver. Aminoguanidine is 10- to 100-fold selective for iNOS versus cNOS
(28, 49) and does not significantly inhibit cNOS in vivo at the doses
used in this study (50). Aminoguanidine has other effects in addition
to inhibiting iNOS, including reducing advanced glycation end-product
formation and inhibiting diamine oxidase and aldose reductase (5, 14,
21). However, we are not aware of evidence to suggest that advanced
glycation end products or polyol pathway products are elevated after
TNF- administration. Thus the beneficial effect of aminoguanidine in
ameliorating TNF--induced lung and liver vascular barrier
dysfunction is most likely mediated through inhibition of NO production
by iNOS.
The present observations are consistent with previous reports that NOS
inhibitors prevented or reduced increased vascular permeation by
macromolecules during cardiac allograft rejection (50), in diabetic
animals (46), in response to increased blood flow (54), and in response
to various inflammatory and noninflammatory agonists (3, 22, 24, 25,
45, 55). Because aminoguanidine did not affect vascular permeation in
several systemic tissues in the present study, our results indicate
that TNF- caused systemic vascular barrier dysfunction through both
NO-dependent and -independent mechanisms. NO-dependent vascular barrier
dysfunction in the lung and liver may partially reflect the high number
of resident mononuclear inflammatory cells in these tissues which can
be induced to express iNOS. Cellular localization of iNOS expression in
the lung and liver during TNF--induced vascular barrier dysfunction
requires further investigation.
Disparate Effects of NOS Inhibitors on Lung Vasculature
Although some previous studies indicate that TNF- increases
endothelial cell monolayer permeability (4, 38, 42) and increases lung
vascular permeation in vivo (13, 41), others demonstrate that TNF-
does not increase vascular permeability in vivo (2, 6, 18, 26, 34). In
the present study, a low dose of TNF- increased pulmonary vascular
permeation and tissue water content in the absence of changes in
pulmonary blood flow or systemic hemodynamics. The reduced systemic
PaO2 in TNF--treated animals, which
was improved with aminoguanidine, suggests that increased pulmonary
vascular permeation and water content resulted in impaired oxygenation
and that NO contributed to this impaired gas exchange. NO may increase
vascular permeation directly by increasing venular leaky sites (24, 25)
through a guanosine 3',5'-cyclic monophosphate mechanism
(25, 54) or indirectly by increasing production of prostaglandins (39)
or other vasoactive mediators. Alternatively, increased vascular
permeability may result from NO-mediated endothelial cell cytotoxicity
(32), although this is unlikely given the reversibility of vascular barrier dysfunction and the minimal histological evidence of
TNF--induced lung injury. These observations suggest that TNF--
and NO-mediated vascular barrier dysfunction may contribute to
increased pulmonary and systemic vascular permeability during sepsis.
Perhaps the most important observations in this study are that
aminoguanidine prevented TNF--induced vascular barrier
dysfunction, whereas L-NMMA
increased vascular permeation in control lungs and caused severe lung
injury in TNF--treated animals. These results suggest that, although
NO generated by iNOS may contribute to increased vascular permeability,
nonselective inhibition of iNOS and cNOS results in increased pulmonary
vascular barrier dysfunction and potentiation of TNF--induced lung
injury. Increased pulmonary vascular barrier dysfunction after
inhibition of cNOS is consistent with observations that inhibition of
basal NO production increased vascular permeation and leukocyte
adherence and emigration, which were prevented with anti-adhesion
molecule antibodies (19, 20, 31). Lung injury may reflect inhibition of
basal endothelial NO production, resulting in platelet aggregation,
increased neutrophil adhesion, and altered regulation of pulmonary
vascular tone. Potentiation of cytokine-induced pulmonary vascular
barrier dysfunction and lung injury may contribute to the increased
mortality reported with nonselective inhibition of iNOS and cNOS during
cytokine- or endotoxin-induced septic shock (7, 33, 51). Furthermore, prevention of pulmonary vascular barrier dysfunction with selective inhibition of iNOS may contribute to the reduced mortality of endotoxemic animals treated with aminoguanidine (52).
Alternatively, L-NMMA-induced
lung injury in TNF--treated animals may reflect toxicity of
L-NMMA that is unrelated to
inhibition of cNOS or iNOS. Hepatic toxicity has been previously
reported for L-NMMA (7). Thus
our data suggest that further investigation of
L-NMMA-induced pulmonary injury
in both normal and septic conditions is warranted.
Our results also suggest that the systemic vascular leak syndrome and
resulting pulmonary edema that complicate IL-2 immunotherapy (37),
which is associated with elevated circulating levels of TNF- (27),
ameliorated by neutralization of TNF- (8, 9) and associated with
increased NO production in humans (12), may at least partially reflect
TNF-- and NO-mediated pulmonary and systemic vascular barrier
dysfunction. Thus the role of NO and iNOS and the effect of iNOS
inhibition in the IL-2-induced systemic vascular leak syndrome warrant
further investigation.
In conclusion, systemic administration of low-dose TNF- reversibly
increased systemic vascular permeation and tissue water content without
associated tissue injury or alterations in blood flow or hemodynamics.
TNF- induced iNOS mRNA expression in the lung and increased in vivo
NO production. Inhibition of iNOS with aminoguanidine prevented
TNF--induced vascular barrier dysfunction in the lung and liver but
not in other affected tissues. Vascular barrier dysfunction in the lung
was associated with impaired oxygenation, which was prevented with
aminoguanidine. L-NMMA increased vascular permeation in the control lung and produced severe lung injury in
TNF--treated animals. These observations suggest that TNF- impairs endothelial barrier integrity without associated cytotoxicity through NO-dependent and -independent mechanisms. Whereas nonselective inhibition of iNOS and cNOS increased vascular barrier dysfunction and
caused severe lung injury, selective inhibition of iNOS prevented impaired endothelial barrier integrity and pulmonary dysfunction. Thus
selective inhibition of iNOS may be beneficial in treating the
increased vascular permeability that complicates endotoxemia and
cytokine immunotherapy.
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ACKNOWLEDGEMENTS |
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This study was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant F-32-HL-09021 (N. K. Worrall), National Eye Institute Grant EY0660 and NHLBI Grant HL-39934 (J. R. Williamson), and NHLBI Grant R-29-HL-46387 (T. B. Ferguson, Jr.); the Kilo Diabetes and Vascular Research Foundation (J. R. Williamson); and the Monsanto-Searle/Washington University Biomedical Program (J. R. Williamson and T. B. Ferguson, Jr.).
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FOOTNOTES |
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Address for reprint requests: J. R. Williamson, Dept. of Pathology, Box 8118, 660 S. Euclid Ave., St. Louis, MO 63110.
Received 3 March 1997; accepted in final form 6 August 1997.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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P < 0.005 vs. control;
P < 0.0002 vs. control, P < 0.05 vs. TNF-
