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INTRODUCTION
METHODS
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DISCUSSION
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Recent studies demonstrated that activation of the nuclear enzyme
poly(ADP-ribose) polymerase-1 (PARP-1) by oxidant-mediated DNA
damage is an important pathway of tissue injury in conditions associated with oxidative stress. Using a dual approach of PARP-1 suppression, by genetic deletion or pharmacological inhibition with
the phenanthridinone PARP inhibitor PJ-34, we now demonstrate an essential role of PARP-1 in the development of pulmonary inflammation induced by lipopolysaccharide (LPS). PARP-1+/+ and
PARP-1
/ mice received an intratracheal instillation of LPS (50 µg),
followed after 24 h by bronchoalveolar lavage to measure the cytokines TNF-
, IL-1
, and IL-6, the chemokines MIP-1 and MIP-2,
leukocyte counts and myeloperoxidase activity (neutrophil accumulation), protein content (high permeability edema), and nitrite/
nitrate (nitric oxide production). Malondialdehyde (an index of lipid peroxidation) was measured in lung tissue. Similar experiments were conducted in BALB/c mice treated with PJ-34 or vehicle. The absence of functional PARP-1 reduced LPS-induced increases of cytokines and chemokines, alveolar neutrophil accumulation, lung hyperpermeability, NO production, and lipid peroxidation. Histological analysis revealed attenuated lung damage after PARP inhibition. Our findings support a mechanistic role of PARP-1 in the
regulation of LPS-induced lung inflammation. Pharmacological inhibition of PARP may be useful in clinical conditions associated
with overwhelming lung inflammation.
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Keywords: lung; ARDS; lipopolysaccharide; poly(ADP-ribose) polymerase; chemokines
Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear DNA-binding enzyme present throughout the phylogenetic spectrum that participates in DNA repair in response to genotoxic
stress (1, 2). DNA single-strand breakage is the obligatory
trigger of PARP-1 activation, which, in turn, cleaves its substrate nicotinamide adenine dinucleotide (NAD+) into nicotinamide and ADP-ribose, and covalently attaches ADP-ribose units to various nuclear proteins (3). This process results in a
reduced cellular content in NAD+, slowing the rate of glycolysis, mitochondrial respiration, and high-energy phosphate generation, ultimately leading to cell death via the necrotic pathway (4). In addition, PARP-1 has been recently shown to be
involved in the regulation of inflammatory processes, being
functionally associated with important transcription factors,
notably nuclear factor (NF)-
B (5, 6).
Acute respiratory distress syndrome (ARDS) is a severe form of lung inflammation most commonly complicating gram-negative septic shock. Endotoxin released from the bacterial cell wall is considered to be an important eliciting factor in the development of ARDS in this setting, which triggers the intrapulmonary production of various proinflammatory mediators (7). We hypothesized that PARP-1 activation resulting from this overwhelming oxidative stress might participate in the development of acute lung injury induced by intratracheally administered lipopolysaccharide (LPS). Using a dual approach of both genetic suppression of PARP-1 and pharmacological PARP inhibition with the novel, phenanthridinone compound PJ-34 (10, 11), we demonstrate that activation of PARP-1 is a pivotal mechanism of LPS-induced pulmonary inflammation.
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In vivo studies were performed in accordance with National Institutes of Health guidelines and with the approval of the local institutional animal care and use committee.
Intratracheal LPS Administration
The experiments were conducted both in male BALB/c mice, 8-10 wk
old, and in PARP-1-deficient (PARP-1/) and wild-type (PARP-1+/+)
littermates (genetic background: 129/Sv × C57BL6), as previously described (12). The animals were anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (30 mg/kg), given intraperitoneally. A
1-cm midline cervical incision was made to expose the trachea. Intratracheal administration of LPS (Escherichia coli, O127:B8, 50 µg/
mouse) or vehicle (phosphate-buffered saline [PBS] pH 7.4), in a volume of 100 µl, was performed with a bent 27-gauge tuberculin syringe
(13, 14). The cervical incision was closed with 5.0 silk suture and
the mice returned to their cage. The animals recovered rapidly after
surgery. In preliminary studies, a time course of the pulmonary inflammatory response was investigated. We found that 24 h after LPS
injection there was a marked degree of pulmonary inflammatory response, which largely resolved by 3 d. Therefore, in all subsequent
studies, measurements were taken at 1-d post-LPS, which was considered the peak time of the pulmonary inflammatory alterations.
Bronchoalveolar Lavage
Twenty-four hours after the surgery, the mice were reanesthetized with
ketamine/xylazine intraperitoneally. A laparotomy was performed and
the animals were bled by transection of the inferior vena cava to reduce hemorrhage in the lungs. Bronchoalveolar lavage was performed
by the intratracheal instillation of 1 ml PBS (pH 7.4) into the exposed
lungs (maintained within the thoracic cavity). The lavage fluid was reinfused three times into the lungs before final collection. The bronchoalveolar lavage fluid (BALF) was then centrifuged at 5,000 rpm
for 10 min and the cell-free supernatant was frozen at 
70° C until
further analysis. The volume of cell-free supernatants was measured
for each animal. In addition, a cell count was performed in the BALF
taken from PARP-1+/+ and PARP-1/ animals. The cells were resuspended in a volume of 0.5 ml (0.4 ml PBS and 0.1 ml 0.4% Trypan
blue) and total leukocytes were counted in a hemocytometer. Differential cell counts were also performed and were found, in all experimental groups, to be approximately 90% neutrophilic and approximately 5% lymphocytic; there was no significant difference between
experimental groups. Lungs were then removed and either processed
for wet-to-dry ratio determinations or immediately frozen in liquid nitrogen and stored at 70° C.
Treatment Groups
BALB/c mice challenged with intratracheal LPS were treated with PJ-34
(20 mg/kg, n = 12) or vehicle (isotonic saline, 0.5 ml, n = 12), administered intraperitoneally, 1 h after surgery. The treatment was repeated
after 6 and 12 h. A group of mice (n = 8) was challenged with intratracheal PBS instead of LPS (sham mice). In the experiments using genetically engineered mice, seven PARP-1+/+ and seven PARP-1/
mice were challenged with LPS, whereas five PARP-1+/+ and five
PARP-1/ mice received PBS (sham animals).
Measurements
Cytokines and chemokinesThe production of the proinflammatory cytokines tumor necrosis factor (TNF)-, interleukin (IL)-1, and IL-6,
as well as of the chemokines macrophage inflammatory protein
(MIP)-1 (a CC chemokine) and MIP-2 (a CXC chemokine), was determined in the BALF using commercially available enzyme-linked immunosorbent assay (ELISA), following the protocol provided by the manufacturer (R&D Systems, Minneapolis, MN). BALFs were diluted (1:2 to 1:5) in animals challenged with LPS, whereas they were assayed undiluted in sham animals. Results are expressed as picogram (or nanogram) per milliliter BALF.
Myeloperoxidase activityThe activity of myeloperoxidase (MPO), an indicator of neutrophil accumulation, was determined directly in the BALF or in whole lung homogenates. Pieces of lung tissue or an aliquot of the BALF (20 µl) were mixed with a solution of 1.6 mM tetramethylbenzidine and 1 mM hydrogen peroxide. Activity was measured spectrophotometrically as the change in absorbance at 650 nm at 37° C, using a Spectramax microplate reader (Molecular Devices, Sunnyvale, CA). Results are expressed as milliunits of MPO activity per milliliter BALF or per milligram tissue protein.
Nitrate/nitrite concentrationThe pulmonary production of nitric oxide (NO) was determined by the measurement of nitrate and nitrite, the stable end products of NO metabolism, in the BALF. First, nitrate was reduced to nitrite by incubation with nitrate reductase (610 mU/ml) and NADPH (170 mM) at room temperature for 3 h. After 3 h, nitrite concentration in the samples was measured by the Griess reaction, by adding 100 µl of Griess reagent (0.1% naphthalethylenediamine dihydrochloride in H2O and 1% sulfanilamide in 5% concentrated H3PO4; vol 1:1). The optical density at 550 nm (OD 550, corrected for absorbance at 650 nm) was measured in a Spectramax microplate reader. Nitrite concentrations were calculated by comparison of OD 550 of standard solutions of sodium nitrite prepared in PBS. Nitrate/nitrite concentrations are expressed in nanomoles per milliliter BALF.
Protein assayThe amount of proteins in the BALF was assayed using the Bradford assay and is expressed in milligrams protein per milliliter BALF.
Malondialdehyde formation in lung homogenatesMalondialdehyde formation was utilized to quantify the lipid peroxidation in the lung and measured as thiobarbituric acid-reactive material. Lung samples were homogenized (50 mg/ml) in 1.15% KCl buffer. Homogenates (200 µl) were then added to a reaction mixture consisting of 1.5 ml 0.8% thiobarbituric acid, 200 µl 8.1% sodium dodecyl sulfate, 1.5 ml 20% acetic acid (pH 3.5), and 600 µl distilled H2O and heated at 90° C for 45 min. After cooling to room temperature, the samples were cleared by centrifugation (10,000 × g, 10 min) and their absorbance measured at 532 nm, using 1,1,3,3-tetramethoxypropane as an external standard. The level of lipid peroxides was expressed as nanomoles MDA per milligram protein, measured by the Bradford assay.
Lung Histology and Immunohistochemistry
Histopathological changes induced by LPS were evaluated in BALB/c mice treated or not with PJ-34 (n = 4 mice/group). Twenty-four hours after surgery, the animals were anesthetized and killed by exsanguination and the lungs were inflated-fixed with 4% paraformaldehyde. Paraffin-embedded lungs were sectioned at 3 µm and stained with hematoxylin and eosin for morphological analysis.
To confirm the PARP inhibitory effect of PJ-34, PARP activity in
tissues was measured by using an immunohistochemical method of
PARP activity utilizing biotinylated NAD (11, 15). Briefly, cryosections (10 µm) were fixed for 10 min in 95% ethanol at 20° C and
then rinsed in PBS. Sections were permeabilized by 1% Triton X-100
in 100 mM Tris pH 8.0, for 15 min. Reaction mixture (10 mM MgCl2,
1 mM dithiothreitol, 30 µM biotinylated NAD+, in 100 mM Tris,
pH 8.0) was then applied to the sections for 30 min at 37° C. Reaction
mixes containing 5 mM 3-aminobenzamide or biotinyl-NAD+ free reaction mix were used as controls. After three washes in PBS, incorporated biotin was detected by peroxidase-conjugated streptavidine
(1:100, 30 min, room temperature). After 3 × 10 min washes in PBS,
color was developed with cobalt-enhanced nickel-DAB substrate.
Sections were counterstained in Nuclear Fast Red, dehydrated, and
mounted in Vectamount.
Data Analysis
All the data were statistically evaluated using analysis of variance, followed by post-hoc analysis with Tukey's test when appropriate. Significance was assigned to p < 0.05.
Chemicals
Unless otherwise specified, all chemicals were purchased from Sigma Chemicals (St. Louis MO). PJ-34 was synthesized as previously described (11).
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PARP-1 Suppression Reduces the Production of Proinflammatory Cytokines
Figure 1 shows the results of the measurements of the proinflammatory cytokines TNF- (Figure 1A) and IL-1 (Figure 1B)
in PARP-1+/+ and PARP-1/ mice. Baseline cytokines were
undetectable in the BALF from sham animals. A massive production of cytokines was measured in PARP-1+/+ mice instilled with LPS, which was largely reduced in PARP-1/ animals. In BALB/c mice, treatment with PJ-34 also resulted in a
significant downregulation of the production of TNF- (Figure 1D). However, contrasting with the beneficial effect of the
genetic suppression of PARP-1 on IL-1, the effect of PJ-34
was only partial and did not reach significance (Figure 1E).
PARP deficiency and pharmacological inhibition of PARP
with PJ-34 also tended to reduce LPS-induced IL-6 production (see Figure E1A and E1C in the online data supplement).
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Reduction of Chemokine Expression in the Absence of Functional PARP-1
In the absence of LPS exposure, there were no detectable levels of the chemokines MIP-1 and MIP-2 in the BALF. In
PARP-1+/+ mice challenged with LPS, both chemokines increased to 442 ± 37 pg/ml BALF (MIP-1, Figure 1C) and
1203 ± 152 pg/ml BALF (MIP-2; see Figure E1B in the online
data supplement). These levels were significantly lower in
PARP-1/ mice, reaching 244 ± 26 pg/ml BALF for MIP-1
and 310 ± 36 pg/ml BALF for MIP-2 (p < 0.05 for both chemokines). In BALB/c mice, although PJ-34 significantly reduced the expression of MIP-1 (Figure 1F), it did not significantly attenuate that of MIP-2 (see Figure E1D in the online
data supplement).
Effect of PARP-1 Suppression on Neutrophil Accumulation
As illustrated in Figure 2, a large increase in MPO activity,
indicative of the accumulation of polymorphonuclear cells, was detected in the BALF of PARP-1+/+ mice (Figure 2A) and
BALB/c mice (Figure 2B) after LPS administration. Genetic
deletion of PARP-1 was associated with a significant reduction in MPO activity in the BALF, an observation that was reproduced in BALB/c mice treated with PJ-34. Similarly, a significant increase in whole lung MPO activity was noted after
LPS injection, which was markedly reduced in the absence of
functional PARP (not shown). In addition, the massive leukocyte
infiltration (mainly neutrophilic) of the alveolar spaces noted
in the PARP-1+/+ mice (from 5 ± 1 × 104 to 149 ± 15 × 104 cells/
BALF, p < 0.01) was significantly prevented in PARP-1/
animals (80 ± 15 × 104 cells/BALF, p < 0.01).
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Reduction of High Permeability Pulmonary Edema by Suppression of PARP-1 Activation
We have measured the protein content of the BALF as an indicator of high permeability pulmonary edema. In sham mice
from the PARP-1 colony, a small amount of proteins was recovered in the BALF. Following LPS, the protein content increased largely in the PARP-1+/+ mice, but less so in the
PARP-1/ mice. Similarly, in BALB/c mice, PJ-34 provided a
significant protection against the increase in BAL protein content (Figure 3A and 3B). Similar findings were obtained when
measuring lung wet-to-dry ratios. For instance, LPS increased
wet-to-dry ratios from 4.4 ± 0.1 to 5.4 ± 0.2 (p < 0.01) in the
BALB/c mice, and treatment with PJ-34 reduced this value to
4.8 ± 0.1 (p < 0.05, n = 5). Similarly, in PARP+/+ animals LPS
induced a significant increase in wet-to-dry ratio (from 4.7 ± 0.1 to 6.3 ± 0.6, n = 5, p < 0.01), but no significant increase
was seen after LPS in the PARP-deficient mice (4.6 ± 0.3 to
4.7 ± 0.3, n = 5).
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Decreased Production of NO in the Absence of Functional PARP-1
Increased NO production due to the induced expression of
type II NO synthase is a typical consequence of LPS administration (17). LPS induced a significant increase of NOx in the
BALF of PARP-1+/+ animals and BALB/c mice, which was
significantly (p < 0.01) attenuated in PARP-1/ mice as well
as in BALB/c mice treated with PJ-34 (Figure 3C and 3D).
Genetic Deletion or Pharmacological Inhibition of PARP-1 Decreases Lipid Peroxidation
An increased formation of MDA, attesting of enhanced lipid
peroxidation, was observed in PARP-1+/+ and BALB/c mice
after LPS administration. In contrast, MDA increased significantly less in lungs from PARP-1/ mice and BALB/c mice
receiving PJ-34 (data not shown).
Confirmation of the PARP Inhibitory Effect of PJ-34 in Vivo
PJ-34 treatment abolished the activity of poly(ADP-ribose) polymerase, as measured by an immunohistochemical functional assay (see Figure E2 in the online data supplement), confirming that the current pharmacological dosing regimen with PJ-34 was sufficient to block PARP activation in vivo.
PARP Inhibition with PJ-34 Reduces the Morphological Alterations Induced by LPS in the Lung
In animals treated with vehicle, the morphological alterations were characterized by an increased thickness of the alveolar septa and diffuse alveolar flooding with both leukocytes and erythrocytes (see Figure E3A in the online data supplement). Treatment with PJ-34 reduced the damage induced by LPS, notably by decreasing the extent of alveolar hemorrhages and the leukocyte extravasation into the alveolar spaces (see Figure E3B in the online data supplement).
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DISCUSSION |
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The data presented in this study support the proposal that PARP-1 activation plays a central role in the development of lung inflammation following the intratracheal administration of LPS. Suppression of PARP-1 either by genetic deletion or pharmacological inhibition markedly downregulated the expression of proinflammatory mediators and reduced the amount of extravasated leukocytes (notably polymorphonuclear [PMN] cells) in the alveolar spaces. These effects were associated with a significant suppression of high permeability pulmonary edema and lipid peroxidation and an improvement of lung morphology.
Background and Previous Work
The obligatory trigger of PARP-1 activation is the formation of
DNA breaks induced by free radicals and oxidants, most notably peroxynitrite, formed from the rapid reaction of NO with
the superoxide radical (O2) (18). The enhanced production
of both NO and O2 during acute lung inflammation also favors the formation of peroxynitrite, as attested by the formation
of 3-nitrotyrosine, an indirect marker of peroxynitrite generation, in the lungs of endotoxin-treated rats (8) and in autopsy
specimens of sepsis-induced diffuse alveolar damage (9). Several
earlier reports have implicated a role of PARP activation in
experimental models of pulmonary inflammation. Pulido and coworkers reported that pharmacological inhibition of PARP suppressed the decrease in lung ATP levels and attenuated the dysfunction of pulmonary vasorelaxation induced by the systemic
administration of LPS (19). In a similar model, we found that
3-aminobenzamide, a prototypical PARP inhibitor, reduced
pulmonary microvascular leakage (20). In a nonseptic model
of lung inflammation, PARP inhibition prevented edema formation in isolated rat lungs exposed to the excitatory amino acid
N-methyl-D-aspartate (21). Finally, we have demonstrated that
peroxynitrite-dependent activation of PARP caused energy depletion and increased permeability in human pulmonary epithelial cells in vitro (22). Our present data further extend these
findings, by demonstrating a pivotal role of PARP-1 in the
regulation of the inflammatory response in a clinically relevant model of acute lung injury.
Suppression of Proinflammatory Cytokine Expression and NO Overproduction by Genetic Deletion of PARP-1
The upregulation of proinflammatory cytokine expression and of nitric oxide production induced by LPS in PARP-1+/+ mice was largely abolished in mice genetically deficient in PARP-1, an effect that may be ascribed to at least two distinct mechanisms. First, overactivation of PARP-1, by depleting the cellular stores in NAD+ and ATP, has been shown to mediate necrotic cell death in conditions of overwhelming oxidative stress (4, 23). In such conditions, the absence of functional PARP-1 shifts the necrotic cell population toward the normal and apoptotic cell populations, which is considered beneficial, as necrotic but not apoptotic cells release their content into the extracellular space, resulting in a progressive amplification of the initial inflammatory process (4, 23, 24).
The second mechanism linking PARP-1 to the regulation
of the inflammatory response relies in the direct interaction of
PARP-1 with several distinct proinflammatory signaling pathways. Two recent studies have demonstrated a functional
association between PARP-1 and the transcription factor nuclear factor-kappa B (NF-B), which is upstream of the synthesis
of acute phase proinflammatory mediators (5, 6). The defective NF-B-dependent transcription activation resulting from
PARP-1 suppression has been associated in vivo with a reduced production of TNF- and interferon (IFN)-
, a decreased
expression of iNOS, and an improved survival rate in response to endotoxin (6). Furthermore, the absence of PARP-1 or its pharmacological inhibition has been shown to suppress
LPS-mediated induction of mitogen-activated protein (MAP)
kinase activity (25) and oxidant-induced activation of the transcription factor AP-1 (26), which both play a major role in the
pleiotropic transduction of intracellular inflammatory cascades. In addition to NF-B, MAP kinase, and AP-1, a number of other signaling pathways may be functionally regulated
by PARP-1, in view of the growing list of transcription factors
or transcription coactivators shown to be associated with
PARP-1, including AP-2 (27), TEF-1 (28), oct-1 (29), and
YY1 (30). Because PARP inhibition and PARP deficiency resulted in a general downregulation of the inflammatory response, it is not possible to define the relative contribution of
the various affected mediators to the pathogenesis of LPS- induced lung injury. For instance, the fact that NO production was downregulated does not necessarily mean that the NO production by iNOS has significantly contributed to the pathogenesis of the disease in the current model.
Regulation of Leukocyte Trafficking by PARP-1 during Acute Lung Inflammation
It is widely acknowledged that polymorphonuclear neutrophils are important cellular effectors of the diffuse alveolar damage characterizing ARDS (31, 32). Here, we report that PARP-1-deficient mice are protected against the flooding of alveolar spaces by leukocytes, notably PMN (as attested by decreased MPO activity in the BALF) induced by LPS. These observations are consistent with previous findings supporting a role of PARP-1 in the regulation of leukocyte trafficking in conditions such as inflammation, shock, and reperfusion injury (12, 13, 33). Both in vitro and in vivo data support the proposal that this effect could be ascribed, at least in part, to a reduced expression of the adhesion molecules ICAM-1 and P-selectin by suppression of PARP-1 activity (34). Here, we propose an additional mechanism related to the downregulation of chemokine production in the absence of functional PARP-1.
Chemokines (and other chemoattractant molecules), play a
central role in the pathophysiology of ARDS by directing the
migration of inflammatory cells through chemotactic gradients (35). In rodents, the CXC chemokines MIP-2 and KC,
and the CC chemokine MIP-1 represent powerful granulocyte-activating chemokines (36, 37). The extensive release of
both MIP-1 and MIP-2 into the BALF of PARP-1+/+ mice
exposed to LPS was largely reduced in PARP-1/ animals.
This is consistent with recent work showing a reduced production of MIP-1 in PJ-34-treated or in PARP-1-deficient macrophages and fibroblasts stimulated with LPS in vitro (38).
The functional association between PARP-1 and the transcription factors NF-B and AP-1 is a likely mechanism underlying the effect of PARP-1 suppression on chemokine expression. These observations may be clinically relevant, as a
direct correlation has been established between the BALF
levels of the CXC chemokine IL-8, the severity of lung inflammation, and mortality in patients with ARDS (39). PARP-1
appears to play a critical role in regulating leukocyte trafficking, acting both at the level of adhesion molecules and at the
level of chemokine expression.
Suppression of PARP-1 Reduces High Permeability Pulmonary Edema and Lipid Peroxidation
The typical lesion in ARDS is characterized by widespread
destruction of the alveolar epithelium and flooding of the alveolar spaces with proteinaceous exudates containing large
amounts of neutrophils (42). The reduced production of proinflammatory mediators and leukocyte infiltration in the lungs
of PARP-1/ mice translated into a significant reduction of
high permeability pulmonary edema, evidenced by a decreased
protein content in the BALF. This finding may have clinical
implications, as the formation of high permeability edema eventually results in alveolar collapse, reduced lung compliance,
and severe gas exchange abnormalities (43), alterations that
are further exacerbated by the initiation of mechanical ventilation in the clinical setting (44). An additional effect of PARP-1
suppression was a reduced formation of malondialdehyde, indicative of decreased oxidant stress and lipid peroxidation. Because the formation of oxidants is a prerequisite for the activation of PARP-1, these data indicate that PARP-1 may enhance its own activation by favoring the formation of oxidants.
The beneficial effects were obtained while PJ-34 administration was delayed after the instillation of LPS, which is relevant to the clinical scenario. Thus, although part of the effects attributed to PARP-1 suppression are related to an interference with early phenomena, such as the transcriptional activation of proinflammatory genes, our data indicate that pharmacological inhibition of PARP remains a valid therapeutic option in the posttreatment paradigm. PARP-1 inhibition may represent a novel, useful adjunct for the therapy of clinical conditions associated with severe pulmonary inflammation, most notably ARDS.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Csaba Szabo, M.D., Ph.D., Inotek Corporation, Suite 419 E, 100 Cummings Center, Beverly, MA 01915. E-mail: szabocsaba{at}aol.com
(Received in original form June 13, 2001 and accepted in revised form October 22, 2001).
Supported, in part, by National Institutes of Health Grant ROIGM60915 to C. Szabó. L. Liaudet is on leave from the Critical Care Division, Department of Internal Medicine, University Hospital, Lausanne, Switzerland, and is supported by a Grant from the ADUMED foundation (Switzerland). F.G. Soriano is on leave from Department of Critical Care Medicine, Hospital das Clinicas da Universidade de Sao Paulo, Brazil, and is supported by a fellowship from FAPESP (Brazil). P. Pacher is on leave from the Department of Pharmacology and Pharmacotherapy, Semmelweis University Medical School, Budapest, Hungary.This article has on online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org
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References |
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