| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of 1 Anesthesiology, 2 Pharmacology, and 3 Hematology, Lille University Hospital, 59037 Lille, France; and 4 Department of Anesthesiology, University of Rochester, Rochester, New York 14642
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
To investigate whether impaired endothelial function was related to alteration of nitric oxide (NO) formation during endotoxic shock, we studied the effects of supplementation of L-arginine (L-Arg), D-arginine (D-Arg), and NG-nitro-L-arginine methyl ester (L-NAME), on endothelial function and structure in a rabbit model. Endotoxic shock was induced by a single lipopolysaccharide bolus (0.5 mg/kg iv, Escherichia coli endotoxin). Coagulation factors and expression of monocyte tissue factor were determined by functional assays. Endothelium-dependent vascular relaxation was assessed by in vitro vascular reactivity. Immunohistochemical staining (CD31) was performed to assess damaged endothelial cell surface of the abdominal aorta. These parameters were studied 5 days after the onset of endotoxic shock and were compared under three conditions: in absence of treatment, with L-Arg or D-Arg supplementation, or with L-NAME. Both L-Arg and D-Arg significantly improved endothelium-dependent relaxation and endothelial morphological injury. L-NAME did not alter endothelial histological injury induced by lipopolysaccharide. These data indicate that arginine supplementation nonspecifically prevents endothelial dysfunction and histological injury in rabbit endotoxic shock. Moreover, L-Arg has no effect on coagulation activation and expression of monocyte tissue factor induced by endotoxic shock.
endothelium; endotoxic shock; lipopolysaccharide; NG-nitro-L-arginine methyl ester; tissue factor; monocyte
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
ENDOTHELIAL DYSFUNCTION PLAYS a crucial role in the pathophysiology of septic shock due to gram-negative bacteria (8). Alteration in endothelial function, such as impaired release of endothelium-derived nitric oxide (NO), could alter physiological regulation of blood flow distribution by interfering with metabolic vasodilation in states of limited O2 supply (25). Indeed, endothelium-derived NO production has been reported to be reduced during endotoxemia (17, 28). This might be associated with the risk of multiple organ dysfunction syndrome (3). In contrast, enhanced inducible NO synthase (iNOS) production has been proposed as a mechanism to explain loss of vascular responsiveness to vasoconstrictor agents in endotoxemia (12, 22). In agreement with this, it has been suggested that inhibition of NO synthase is beneficial to restoring contractility (18, 23), and the use of NO synthase inhibitors has been proposed as a treatment. However, the inhibition of NO synthase was demonstrated to be detrimental during endotoxemia or sepsis (20, 21, 27). To block or to enhance NO production during sepsis or septic shock remains, therefore, a matter of controversy.
Our laboratory previously investigated endothelial function in a rabbit endotoxic shock model (14). In that study, it was reported that impaired endothelium-dependent relaxation of aorta in response to acetylcholine (ACh) was still present and almost maximal 5 days after lipopolysaccharide (LPS) injection. This endothelial dysfunction was associated with endothelial histology injury. Our results, along with those of others (13, 17, 19), suggest that an alteration in ACh receptor-to-NO synthase coupling and/or a reduced production of endothelium-derived NO causes this attenuated endothelium-mediated vasorelaxation. A similar type of endothelial dysfunction has been described in hypercholesterolemia and in angioplasty-associated vascular injury in rabbits (6, 11). Altered NO-mediated relaxation was prevented by administration of L-arginine (L-Arg), the precursor of NO synthase (6, 11). Interestingly, L-Arg has been shown to improve survival in septic animals (10, 15).
To explore further whether impaired endothelium-dependent relaxation of the aorta in a rabbit endotoxic shock model was related to alteration of NO formation, we examined the effect of L-Arg, its stereoisomer D-arginine (D-Arg), and of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, on endothelial function and structure in a rabbit endotoxic shock model.
| |
MATERIALS AND METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The animal experiments were approved by the French Agricultural Office for the care of animal subjects, and the care and handling of the animals were in agreement with the European legislation for animal research.
Animal Model
Experiments were carried out on male New Zealand White rabbits weighing 2.5-3.0 kg. All rabbits were obtained from the Charles River laboratory (St. Aubin-lès-Elbeuf, France). Animals were maintained throughout on a standard rabbit chow diet with food (200 g/day) containing 1.25% of arginine and water ad libitum. Rabbits were assigned to one of seven groups. For the endotoxin animals (4 groups), conscious animals were rapidly injected intravenously via a marginal ear vein with 0.5 mg/kg body wt of purified LPS endotoxin (Escherichia coli serotype O55:B5 from a single batch, Sigma Chemical, St. Louis, MO).One group received LPS alone (LPS control group, n = 8), and the other three groups received LPS and were supplemented with either L-Arg (LPS+L-Arg group, n = 8), D-Arg (LPS+D-Arg group, n = 5), or L-NAME (LPS+L-NAME group, n = 6). For the sham animals (3 groups), animals were injected intravenously with saline and supplemented with either L-Arg (L-Arg group, n = 6) or L-NAME (L-NAME group, n = 6) or were not supplemented (control group, n = 8).
L-Arg or D-Arg (1,500 mg · kg
1 · day1) or
L-NAME (15 mg · kg1 · day1)
supplementation in the drinking water was initiated 3 days before the
onset of endotoxic shock and was maintained for 5 days afterward. Before the initiation of supplementation, 3 days after supplementation, and 5 days after LPS injection, blood samples were obtained from animals in each group for measurement of plasma arginine levels. Plasma
was deproteinized with 10% sulfosalicylic acid and analyzed for free
arginine with an automated amino acid analyzer (model LC 300, Biotronic
Instruments, Berlin, Germany).
Four hours after LPS or saline injection, arterial blood gases were measured in the seven groups. Coagulation and hematological parameters were measured in all groups 5 days after LPS or saline injection.
All animals were killed on day 5 (D5). They were anesthetized with pentobarbital sodium (30 mg/kg iv; Specia, Paris, France) and injected with heparin (500 IU/kg iv; Panpharma, Fougères, France) to prevent coagulation. Abdominal aortas were quickly removed, placed into cold (4°C) Krebs-Henseleit (KH) buffer (pH 7.40 at 37°), and gassed with 5% CO2 in O2. Abdominal aortic rings were obtained as previously described (11). Briefly, the abdominal aorta was carefully trimmed of extravascular tissues and cut into rings of ~3.0 mm in length in the KH buffer. From each rabbit aorta, four rings, chosen randomly, were studied.
Vascular Responses
Each ring was mounted on two stainless steel wire supports inserted through the lumen. To determine the possible existence of iNOS, vascular endothelium of one ring of each abdominal aorta was previously mechanically removed with a small wooden applicator. Rings were suspended in organ chambers (Radnoti Glass Technology, Monrovia, CA) filled with 40 ml KH solution, warmed (37°C), and continuously oxygenated (95% O2-5% CO2). One support was fixed, and the other was connected to an isometric force-displacement transducer. Incoming signals from the transducers were amplified by signal conditioners and sent to an Intel 486-based computer, after analog-to-digital conversion. This system allowed the continuous screen visualization and computer storage of changes in isometric force.Over a 60-min "step-tension" period, the vascular rings were
stretched to a force of 8.0 g. In preliminary experiments,
8.0 g was determined to be the optimal point of ring
length-tension relation. After a stabilization period of 60 min at the
resting wall tension, all rings were preconstricted with 70 mM KCl to ensure their viability. The rings were contracted with
L-phenylephrine hydrochloride (PE; concentration range from
109 to 105 M; Sigma Chemical).
Endothelium-dependent or -independent relaxant effects were established
when the constrictor response to PE was stable. The relaxant response
to ACh (concentration range from 109 to
3.105 M; Sigma) and to calcium ionophore A23187
(concentration ranging from 109 to 3.106 M;
Sigma Chemical) was also measured. Similarly, the response to sodium
nitroprusside (SNP, concentration range from 109 to
3.105 M; Sigma Chemical) was obtained. The same range of
SNP concentrations was also applied to rings from which the vascular
endothelium was removed.
Removal of the endothelium was verified by the failure of these rings
to relax in response to submaximal concentrations of ACh
(3.107 M). A final series of experiments was performed in
L-NAME-treated rabbits that either received or did not
receive endotoxin. Rings were incubated with L-Arg
(102 M) 30 min before the protocol of ACh-induced
relaxation to avoid remnant effects of L-NAME and to
separate LPS-induced endothelial dysfunction and
L-NAME-induced NO synthase blockade.
Immunohistochemical Staining of Vascular Endothelium
Aorta segments were fixed with paraformaldehyde (4%), then cryoprotected by immersion in sucrose 30%. Tissues were embedded in optimal cutting temperature (methyl methacrylate) compound, frozen in isopentane, and stored at
70°C. Tissue sections were cut 6 µm thick. The endothelial cell layer was stained by using an antibody
against the endothelium-specific intercellular adhesion molecule
CD31-PECAM1. Briefly, frozen sections were air-dried for 1 h,
incubated with peroxidase-blocking reagent, then rinsed in
phosphate-buffered saline (PBS) for 10 min and blocked with 10% horse
serum in PBS for 10 min. The sections were then incubated at 37°C
overnight with a mouse-prepared monoclonal primary antibody to CD31
(Dako, Carpinteria, CA) diluted 1:20 in PBS. After extensive washing in
PBS (3 × 10 min), an anti-mouse biotinylated secondary antibody
was applied for 1 h. After three washings with PBS, the sections
were incubated for 1 h in a solution of avidin-biotin-peroxidase preformed complex (ABC Vector, Vectastain kit, Vector Laboratories, Burlingame, CA). The peroxidase activity was revealed by using hydrogen
peroxide and diaminobenzidine as a chromogen. Finally, sections were
counterstained with hematoxylin and mounted with Permount (Fisher
Scientific, Elancourt, France). In each experiment, negative controls
without the primary antibody were included to check for nonspecific staining.
For quantification of endothelial injury, three nonconsecutive cross sections per aortic segment were microscopically photomicrographed (model Axioskop 20, Zeiss, Le Pecq, France). After photographic reconstruction of each tissue section, each picture was digitalized for computerized analysis (Color Image 1.32 Software). The surface area of endothelial cell injury (including three types: subendothelial vacuolization, detachment of endothelial cells, and endothelial denudation) was measured and expressed as percentage of total circumference of each section.
Coagulation
Blood was sampled at day 1 (D1) and at D5 under sterile conditions from the ear artery. Samples collected on EDTA were used for blood cell counts (Coulter MAXM, Beckman, Fullerton, CA). The total white blood cell count was verified manually. Peripheral blood smears for differential white cell counts were stained with May Grünwald Giemsa. Each count was performed by three investigators, who were blinded to the treatment allocation. Factor II and V levels were determined by an automated clotting assay (STA, Stago, Asnières, France) using calcified rabbit brain thromboplastin and human factor deficient plasma (Stago). Prothrombin index was measured by an automated clotting assay using calcified rabbit brain thromboplastin (Stago). Fibrinogen levels were measured by the Clauss technique (Biomérieux, Lyon, France).Measurement of Monocyte Tissue Factor Procoagulant Activity
The possible activation of monocyte tissue factor (TF) procoagulant activity after a single endotoxin injection was investigated in the LPS group, the LPS+L-Arg group, and the control group, respectively. Determination of TF activity was made by a method adapted from Carson (5, 7). After gradient centrifugation isolation, the mononuclear cells were resuspended in RPMI 1640 (GIBCO). Aliquots of cell preparations were cultured for 16 h at 37°C in a humidified 5% CO2 atmosphere, without or with stimulation by 1 µg/ml of endotoxin (LPS; 055:B5; Sigma Chemical), which corresponds to 5,000 endotoxin U/ml. These are referred to as unstimulated and stimulated cells, respectively. At the end of the incubation period, cells were resuspended and frozen at80°C. The lysed cell suspensions (50 µl) were incubated at 37°C
in a microtiter plate (2 min) and mixed with CaCl2 (0.25 M;
50µl) (3 min of incubation) and prothrombin concentrate complex
(Laboratoire de Fractionnement et des Biotechnologies, Les Ulis,
France) as a source of factor VII (50 µl, 3 U/ml). After
addition of 50 µl of the chromogenic substrate S2765 (Biogenic,
Maurin, France), the change in optical density at 405 nm was quantified
with a microplate reader and converted into units of TF activity from
log-log plots of serial dilutions of a rabbit brain thromboplastin
(CI+, Stago). Arbitrarily, 1 ml of thromboplastin was assigned a value
of 1,000 U/ml of TF. Results were expressed as mU/103 monocytes.
Solutions
Drugs used for vasomotor experiments as well as KH components were dissolved in deionized water. The KH solution had the following composition (in mM): 118 NaCl, 4.6 KCl, 27.2 NaHCO3, 1.2 MgSO4, 1.2 KH2PO4, 1.75 CaCl2, 0.03 Na2EDTA, and 11.1 dextrose. Gases were purchased from Air
Liquide Santé (Paris, France).
Statistical Analysis
Results are expressed as means ± SE; n represents the number of rabbits. Hematology and coagulation results were compared by using unpaired Student's t-test. Plasma arginine levels were compared via Wilcoxon's test for paired groups and Mann-Whitney's U-test for unpaired groups. Determination of the PE concentration eliciting 50% of the maximal constriction response (EC50) was calculated using a semilogistic regression. EC50 values were compared using the Mann-Whitney test. Relaxation responses were expressed as percent reduction of maximal vascular PEinduced tension. Mean intergroup differences were tested by repeated-measures ANOVA. If a significant value was found, Scheffé's test for multiple comparisons was used. Significance was accepted at P < 0.05.| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Clinical Parameters
There was a significant body weight loss of ~5-9% in the endotoxic animals compared with the control group at D1 and D5, respectively. The overall mortality for the nonsupplemented septic group was 40%, whereas it was 21.4% in the LPS+L-Arg group (P < 0.05 vs. LPS group), 20% in the LPS+L-NAME group (P < 0.05 vs. LPS group), and 0% in the LPS+D-Arg group, (P < 0.05 vs. LPS group), respectively. Five-day survivors were considered permanent survivors because all animals were killed at 5 days.Ex Vivo Measurements
Arterial blood gas analysis. Arterial blood-gas analysis was performed 4 h after LPS injection. Metabolic acidosis confirmed endotoxic shock (bicarbonate = 11.40 ± 0.92 in LPS group vs. 23.90 ± 0.81 in control group, P < 0.05). Acidosis was not significantly different between the endotoxic animals with or without treatment (bicarbonate = 11.33 ± 0.63 in LPS+L-Arg group, 8.33 ± 0.39 in LPS+D-Arg group, 12.30 ± 3.29 in LPS+L-NAME group, not significant vs. LPS group).
Plasma arginine levels and hematological modifications.
L-Arg and D-Arg supplementation increased
plasma arginine levels 3 days (D0) after initiation of supplementation
and remained elevated after 8 days (D5) in the L-Arg group,
the LPS+L-Arg group, and the LPS+D-Arg group
compared with at baseline. In contrast, 5 days after LPS injection,
plasma arginine levels decreased significantly in the nonsupplemented
LPS group but not significantly in the supplemented LPS group (Fig.
1).
|
Immunohistochemical staining.
In the control group, endothelial cells were stained by
immunohistochemical label (PECAM1/CD31) and appeared intact. LPS
injection induced three types of endothelial cell injury at D5:
subendothelial vacuolization, detachment of endothelial cells, and
endothelial denudation. In endotoxic rabbits, the surface area of
endothelial cell injury was 23.3 ± 0.8% of the total endothelial
surface area of the abdominal aorta (Fig.
2). Both L-Arg and
D-Arg supplementation decreased LPS-induced endothelial
cell injury (7.0 ± 0.8% and 10.1 ± 1.5%, respectively,
P < 0.05 vs. LPS group), whereas L-NAME did not alter LPS-induced endothelial cell injury (22.1 ± 1.9%).
|
In Vitro Vasoreactivity
Vascular contraction.
In control animals, the response to PE was larger in
endothelium-denuded rings than in intact rings; the PE EC50
was significantly increased in the presence of the endothelium. The
same results were observed in the L-Arg, the
LPS+L-arg, and the LPS+D-Arg groups, respectively. In contrast, there was no longer any
endothelium-dependent contraction modulation in the LPS, the
L-NAME, and the LPS+L-NAME groups,
respectively, because EC50 was similar in aortic rings in
the presence and absence of endothelium (Fig.
3A).
|
Endothelium-dependent and endothelium-independent relaxation.
The in vitro relaxation response of aortic rings was tested after PE
precontraction with ACh, A23187, and SNP. In the control group, the
maximum relaxation induced by ACh was 80.6 ± 3.1%. The response
to calcium ionophore A23187 was similar (79.0 ± 2.5%). SNP, an
endothelium-independent relaxing agent, caused a relaxation response of
92.9 ± 2.4%. In contrast, LPS injection was associated with a
loss of ACh-induced relaxation. At D5, ACh-induced relaxation was
decreased (P < 0.05 vs. control group), with a maximum
relaxation of 50.0 ± 6.1%. Both L-Arg and
D-Arg prevented this altered ACh-induced relaxation
(P < 0.05 vs. LPS group) (Fig. 4A). L-NAME did
not aggravate LPS-altered ACh-induced relaxation. Altered ACh-induced
relaxation in the L-NAME group was completely reversed by
an excess of L-Arg but was only partially reversed in the
LPS+L-NAME group (Fig.
5A).
|
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
In the present study, we confirmed that a single injection of endotoxin (E. coli, 0.5 mg/kg) was rapidly associated with metabolic acidosis and with prolonged activation of monocyte TF expression and endothelial cell injury and dysfunction (14). Decreased endothelium-dependent relaxation in response to ACh was still present 5 days after LPS injection, whereas SNP-induced endothelium-independent relaxation was not altered. These results suggest that the impaired endothelium-dependent relaxation is not due to a decreased ability of vascular smooth muscle to respond to NO. Our study data also demonstrate that L-Arg supplementation was able, as in other types of endothelial injury (6, 11), to prevent this altered endothelium-dependent relaxation to ACh. This improved function was associated with restoration of endothelial morphology. However, this effect of L-Arg is nonspecific because D-Arg gave, in this model, the same results on endothelium function and morphology.
These data suggest that the NO pathway is not centrally involved in the functional and structural improvements observed with L-Arg in large conduit vessels from endotoxic shock rabbits. These results are not in agreement with studies in which hypercholesterolemia or balloon vascular injury in rabbits was associated with impaired endothelium-dependent relaxation and improved by supplemented L-Arg diet (6, 11). It is important to emphasize, however, that neither Cooke et al. (6) nor Hamon et al. (11) used D-Arg as a control treatment.
Endothelial dysfunction is a common feature of sepsis and septic shock induced by endotoxin-containing gram-negative bacteria, and it may be induced by a single injection of bacterial endotoxin used to induce septic shock in animals (26). Impairment of endothelial function and structure was restored by both L-Arg and D-Arg supplementation. Therefore, an effect of L-Arg on the endothelial synthesis and release of NO through L-Arg availability and/or metabolism is unlikely.
In the present study, L-Arg supplementation did not prevent activation of coagulation and induction of monocyte TF expression. This result is different from that of a recent study in which L-Arg supplementation inhibited increased monocyte TF expression observed in the hypercholesterolemic rabbit after angioplasty (7). This may be due to differences in the type of vascular injury. It must be underlined also that, in this previous study, D-Arg was not used as control.
Our results also showed that plasma arginine levels were decreased in endotoxic animals. L-Arg or D-Arg supplementation increased plasma arginine levels 3 days after initiation of treatment. Arginine levels remained elevated for days after the onset of endotoxic shock, while supplementation in drinking water was maintained. The decrease of plasma arginine levels after endotoxic shock induction may be explained by an increased utilization of arginine for NO synthesis. This, however, does not seem to be the case because the results were identical whether L-Arg or D-Arg was used for supplementation. Another proposed explanation for decreased arginine levels in septic animals may be impaired intestinal arginine absorption. Madden et al. (16) demonstrated a 400% increase in plasma arginine level 30 min after gavage with 100 mg arginine in nonseptic animals, whereas this increase was not found in septic animals. Gardiner et al. (9) investigated intestinal arginine absorption in two different models of sepsis (cecal ligation and puncture, or intraperitoneal injection of LPS). They concluded that, in both models, sepsis resulted in impaired intestinal amino acid uptake. In our model, this mechanism may play a role and explain the decrease in arginine level observed at D5 in supplemented animals. This effect is, again, nonspecific because the results are comparable whether L-Arg or D-Arg was given for supplementation.
L-Arg restored the relaxation response to ACh after LPS
injection without affecting endothelium-dependent relaxation in control animals. Because the same effect was observed with D-Arg
supplementation, we propose that a nonspecific effect of
L-Arg leads to improved endothelial function. Consistent
with a non-NO-dependent mechanism, A-23187 induced similar
endothelium-dependent relaxation in both control and endotoxic rabbit
aortic rings. The calcium ionophore A23187 directly stimulates
conversion of L-Arg to NO in contrast to ACh, which
requires muscarinic receptors to allow calcium entrance and calmodulin
activation. Aoki et al. (2) and Kessler et al. (13) demonstrated that endotoxin or cytokines, such as
tumor necrosis factor-
(TNF-) or interleukin-1
(IL-1), were
able to impair the receptor-mediated release of NO in cat or rabbit carotid arteries. Cycloheximide, an inhibitor of protein synthesis, blocked this TNF--induced impaired relaxation, suggesting
that TNF- or IL-1 induces synthesis of one or several key
messenger proteins interfering with the ACh-receptor-NO synthase
pathway (2, 13). Whether L-Arg or
D-Arg can interfere with this protein synthesis was not
clarified by our study.
It has also been proposed that cytokines induce generation of iNOS in the vascular wall, thus affecting the signal transduction cascade in endothelial cells by inhibiting receptor mechanisms (13). Because iNOS is L-Arg blood concentration dependent for NO synthesis, increased extracellular L-Arg should have resulted in iNOS-derived overproduction of NO (23) and increased endothelium-dependent relaxation dysfunction (13). We can speculate that, in our model, this is unlikely because L-Arg did not cause major endothelial dysfunction.
Our data show that ACh-mediated relaxation is inhibited by in vivo pretreatment with L-NAME in endotoxic, as well as control, rabbits. This effect of L-NAME is completely reversible by incubation of rings from nonendotoxic rabbits with in vitro excess L-Arg, demonstrating remnant effects of L-NAME in ex vivo rings from these animals. In contrast, L-Arg only partially reversed L-NAME blockade in rings from the endotoxic rabbits. It is important to emphasize, however, that in vivo L-NAME pretreatment did not aggravate either endothelial denudation or ACh-induced altered relaxation. This suggests, again, that altered NO production by NO synthase in LPS-induced septic shock is not responsible, by itself, for endothelial dysfunction and morphological injury.
In the present study, the sensitivity to PE was decreased in aortic rings after LPS injection. In various animal models, induction of iNOS after an endotoxin challenge is usually observed within 3-12 h (12, 22). In a rabbit model (14), our laboratory previously demonstrated impaired smooth muscle contractility in response to PE 24 h after 0.5 mg/kg LPS injection. Contractility to PE was restored in the presence of L-NAME, suggesting the presence of iNOS in smooth muscle cells. This effect was still present, although less consistent, 5 days after LPS injection. Whether endothelial cell dysfunction triggers iNOS expression, or the reverse, remains controversial (1, 13). Adeagbo and Triggle (1) demonstrated that removal or damage of endothelial cells triggers the induction of iNOS in vascular smooth muscle cells of rat aorta. However, Kessler et al. (13) showed that decreased endothelial NO synthase activity due to cytokine-induced sustained generation of iNOS affects the signal transduction cascade in endothelial cells. Our results show that supplying L-Arg or D-Arg restores the sensitivity of smooth muscle cells to PE. This might be related to restoration of endothelial structure and function, which might allow limited iNOS expression.
Actually, our study demonstrates that recovery of endothelial function is associated with morphological restoration. This preservation of endothelial cell structure is obtained by L-Arg as well as D-Arg. It is well known that arginine increases protein synthesis in sepsis. León et al. (15) showed that arginine supplementation improved fibrinogen and acute-phase protein synthesis during sepsis in the rat. This may be an explanation for the protective effect of L-Arg on endothelial morphology in our model. However, we did not notice a significant increase in fibrinogen synthesis in our supplemented animals.
We observed endothelial cell abnormalities, even in surviving animals at D5 after LPS injection, suggesting that mortality was not directly related to endothelial dysfunction in endotoxic shock animals. We also observed that L-Arg or D-Arg supplementation decreased the mortality rate of endotoxic animals. This is consistent with a study by Madden et al. (16). They showed that arginine supplementation, given before sepsis induction, significantly increased animal survival. The mechanism by which arginine improves survival in our model remains unknown. Arginine has been shown to have multiple beneficial effects on T cell-mediated immunity (4) and protein synthesis in sepsis (15). On the other hand, it has been suggested that administration of NO synthase inhibitor increases (18, 24) or decreases (19, 21, 27) survival in animal sepsis. Mortality and tissue damage were well linked in these previous reports (19, 21, 27). Our present study showed that administration of L-NAME aggravated neither mortality nor endothelial morphologic injury in endotoxic rabbits.
It should be noted that the present study focused on the mechanisms involved in vascular endothelial cell dysfunction during endotoxic shock. This study was limited to arteries that are not involved in the microcirculatory regulation of blood flow. Results might be different at the microvascular level. The severity of septic shock might have been lower in surviving animals, and the absence of resuscitation may have modified our results.
In summary, our results indicate that, in endotoxin shock rabbits, 1) L-Arg or D-Arg supplementation prevents endothelial dysfunction by restoration of endothelial histological injury, which probably improves coupling between endothelial ACh receptor and NO synthase in a nonspecific fashion (i.e., in a manner independent of NO synthase substrate availability); 2) L-Arg or D-Arg supplementation decreases mortality; 3) L-Arg has no effect on coagulation activation and expression of monocyte TF; and 4) L-NAME does not aggravate endothelial histological injury and mortality rate.
| |
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: Benoît Vallet, Département d'Anesthésie-Réanimation Chirurgicale II, CH & U Lille-Hôpital Claude Huriez, Rue Michel Polonovski, 59037 Lille Cédex, France (E-mail: bvallet{at}chru-lille.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 20 March 2000; accepted in final form 12 June 2000.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Adeagbo, ASO,
and
Triggle CR.
Interactions of nitric oxide synthase inhibitors and dexamethasone with -adrenoceptor-mediated responses in rat.
Br J Pharmacol
109:
495-501,
1993[ISI][Medline].
2.
Aoki, N,
Siegfried M,
and
Lefer AM.
Anti-EDRF effect of tumor necrosis factor in isolated, perfused cat carotid arteries.
Am J Physiol Heart Circ Physiol
256:
H1509-H1512,
1989
3.
Astiz, ME,
and
Rackow EC.
Septic shock.
Lancet
351:
1501-1505,
1998[ISI][Medline].
4.
Barbul, A,
Wasserkrug HL,
Seifter G,
Rettura E,
Levenson SM,
and
Efron G.
Immunostimulatory effect of arginine in normal and injured rats.
J Surg Res
29:
228,
1980[ISI][Medline].
5.
Carson, S.
Continuous chromogenic tissue factor assay: comparison to clot-based assays and sensitivity established using pure tissue factor.
Thromb Res
47:
379-387,
1987[ISI][Medline].
6.
Cooke, JP,
Singer AH,
Tsao P,
Zera P,
Rowan RA,
and
Billingham ME.
Antiatherogenic effect of L-arginine in the hypercholesterolemic rabbit.
J Clin Invest
90:
1168-1172,
1992.
7.
Corseaux, D,
Le Tourneau T,
Six I,
Ezekowitz MD,
McFadden EP,
Meurice T,
Asseman P,
Bauters C,
and
Jude B.
Enhanced monocyte tissue factor response after experimental balloon angioplasty in hypercholesterolemic rabbit: inhibition with dietary L-arginine.
Circulation
98:
1776-1782,
1998
8.
Cotran, RS,
and
Pober JS.
Cytokine-endothelial interactions in inflammation, immunity, and vascular injury.
J Am Soc Nephrol
1:
225-235,
1990[Abstract].
9.
Gardiner, KR,
Gardiner RE,
and
Barbul A.
Reduced intestinal absorption of arginine during sepsis.
Crit Care Med
23:
1227-1232,
1995[ISI][Medline].
10.
Gianotti, L,
Alexander JW,
Pyles T,
and
Fukushima R.
Arginine-supplemented diets improve survival in gut-derived sepsis and peritonitis by modulating bacterial clearance: the role of nitric oxide.
Ann Surg
217:
644-654,
1993[ISI][Medline].
11.
Hamon, M,
Vallet B,
Bauters C,
Wernert N,
McFadden EP,
Lablanche JM,
Dupuis B,
and
Bertrand M.
Long-term oral administration of L-arginine reduces intimal thickening and enhances neoendothelium-dependent acetylcholine-induced relaxation after arterial injury.
Circulation
90:
1357-1362,
1994
12.
Julou-Schaeffer, G,
Gray GA,
Fleming I,
Schott C,
Parratt JR,
and
Stoclet JC.
Loss of vascular responsiveness induced by endotoxin involves L-arginine pathway.
Am J Physiol Heart Circ Physiol
259:
H1038-H1043,
1990
13.
Kessler, P,
Bauersachs J,
Busse R,
and
Schini-Kerth VB.
Inhibition of inducible NO synthase restores the impaired endothelium-dependent relaxations in isolated arteries exposed to proinflammatory mediators.
Arterioscler Thromb Vasc Biol
17:
1746-1755,
1997
14.
Leclerc J, Pu Q, Corseaux D, Haddad E, Decoene C, Bordet R, Six I, Jude
B, and Vallet B. A single endotoxin injection in rabbit causes
prolonged blood vessel dysfunction and procoagulant effect. Crit
Care Med In press.
15.
León, P,
Redmond HP,
Stein TP,
Shou J,
Schluter MD,
Kelly C,
Lanza-Jacoby S,
and
Daly JM.
Arginine supplementation improves histone and acute-phase protein synthesis during gram-negative sepsis in the rat.
J Parenter Enteral Nutr
15:
503-508,
1991[Abstract].
16.
Madden, HP,
Breslin RJ,
Wasserkrug HL,
Efron G,
and
Barbul A.
Stimulation of T cell immunity by arginine enhances survival in peritonitis.
J Surg Res
44:
658-663,
1988[ISI][Medline].
17.
Myers, PR,
Zhong Q,
Jones JJ,
Tanner MA,
Adams HR,
and
Parker JL.
Release of EDRF and NO in ex vivo perfused aorta: inhibition by in vivo E. coli endotoxemia.
Am J Physiol Heart Circ Physiol
268:
H955-H961,
1995
18.
Nava, E,
Palmer RMJ,
and
Moncada S.
The role of nitric oxide in endotoxic shock: effects of NG-monomethyl-L-arginine.
J Cardiovasc Pharmacol
20, Suppl 12:
132-134,
1992.
19.
Park, JL,
Chang KM,
Lee SH,
and
Shin SH.
Protective effect of nitric oxide in an endotoxin-induced septic shock.
Am J Surg
171:
340-345,
1996[ISI][Medline].
20.
Parker, JL,
and
Adams HR.
Selective inhibition of endothelium-dependent vasodilatator capacity by Escherichia coli endotoxemia.
Circ Res
72:
539-551,
1993
21.
Pastor, CB,
Teissere E,
Vicaut E,
and
Payen D.
Effects of L-arginine and L-nitro-arginine treatment on blood pressure and cardiac output in a rabbit endotoxin shock model.
Crit Care Med
22:
465-469,
1994[ISI][Medline].
22.
Rees, DD,
Monkhouse JE,
Cambridge D,
and
Moncada S.
Nitric oxide and the haemodynamic profile of endotoxin shock in the conscious mouse.
Br J Pharmacol
124:
540-546,
1998[ISI][Medline].
23.
Schott, CA,
Gray GA,
and
Scoclet JC.
Dependence of endotoxin-induced vascular hyporeactivity on extracellular L-arginine.
Br J Pharmacol
108:
38-43,
1993[ISI][Medline].
24.
Strand, ØA,
Leone AM,
Giercksky KE,
Skovlund E,
and
Kirkebøen KA.
NG-monomethyl-L-arginine improves survival in a pig model of abdominal sepsis.
Crit Care Med
26:
1490-1499,
1998[ISI][Medline].
25.
Vallet, B.
Vascular reactivity and tissue oxygenation.
Intensive Care Med
24:
3-11,
1998[ISI][Medline].
26.
Vallet, B,
and
Leclerc J.
Endothelial cell dysfunction in septic shock.
In: Yearbook of Intensive Care and Emergency Medicine, edited by Vincent JL.. Berlin: Springer, 1997, p. 133-141.
27.
Wright, CE,
Rees DD,
and
Moncada S.
Protective and pathological roles of nitric oxide in endotoxin shock.
Cardiovasc Res
26:
48-57,
1992
28.
Young, JS,
Headrick JP,
and
Berne RM.
Endothelial-dependent and -independent responses in the thoracic aorta during endotoxic shock.
Circ Shock
35:
25-30,
1991[ISI][Medline].
This article has been cited by other articles:
|
|
 |
|
  N. Matsuda, Y. Takano, S.-i. Kageyama, N. Hatakeyama, K. Shakunaga, I. Kitajima, M. Yamazaki, and Y. Hattori Silencing of caspase-8 and caspase-3 by RNA interference prevents vascular endothelial cell injury in mice with endotoxic shock Cardiovasc Res, October 1, 2007; 76(1): 132 - 140. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Matsuda, Y. Hattori, Y. Takahashi, J. Nishihira, S. Jesmin, M. Kobayashi, and S. Gando Therapeutic effect of in vivo transfection of transcription factor decoy to NF-{kappa}B on septic lung in mice Am J Physiol Lung Cell Mol Physiol, December 1, 2004; 287(6): L1248 - L1255. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Matsuda, Y. Hattori, X.-H. Zhang, H. Fukui, O. Kemmotsu, and S. Gando Contractions to Histamine in Pulmonary and Mesenteric Arteries from Endotoxemic Rabbits: Modulation by Vascular Expressions of Inducible Nitric-Oxide Synthase and Histamine H1-Receptors J. Pharmacol. Exp. Ther., October 1, 2003; 307(1): 175 - 181. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. C. Aird The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome Blood, May 15, 2003; 101(10): 3765 - 3777. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
