1 Research Service, Stratton Veterans Affairs Medical Center, and 2 Center for Cardiovascular Science, Albany Medical College, Albany, New York 12208
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ABSTRACT |
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We tested the hypothesis that endothelial cell
nitric oxide synthase (ecNOS) mediates the tumor necrosis factor
(TNF)--induced increase in nitric oxide (NO) and albumin
permeability in pulmonary microvessel endothelial monolayers (PEM). PEM
lysates were analyzed for ecNOS mRNA (RT-PCR), ecNOS protein (Western
immunoblot), NO levels (NO
antisense; edema; messenger ribonucleic acid; permeability; transcription
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INTRODUCTION |
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TUMOR NECROSIS
FACTOR (TNF)- is a mediator of sepsis syndrome and the adult
respiratory distress syndrome (1, 40). TNF induces an
increase in pulmonary vascular permeability in vivo (22),
in the isolated lung (19), and in pulmonary arterial and
microvessel endothelial monolayers (8, 9, 16). We previously demonstrated in bovine pulmonary artery or bovine pulmonary microvessel endothelium that TNF induces a nitric oxide (NO)-dependent 1) lipid peroxidation (21), 2)
glutathione depletion and oxidation (33), 3)
protein oxidation and nitration (9, 14, 33), 4)
activation of activating protein-1 (14), and 5)
an increase in albumin permeability (9).
NO is made by the enzyme NO synthase (NOS) that exists as three different NOS isotypes characterized by gene location, primary nucleotide and amino acid sequences, and mechanism of activation (11, 17, 27, 35, 41). The constitutive NOS group includes type I neuronal (brain NOS, bNOS) and type III endothelial cell (ecNOS) NOS (11, 17). The ecNOS mediated generation of NO (e.g., in response to acetylcholine and bradykinin) is classically induced by increases in intracellular Ca2+ levels (11, 17). The inducible group includes type II inducible NOS (iNOS). iNOS is constitutively activated because of the increased affinity of the cofactor Ca2+/calmodulin for iNOS (11, 17, 27); therefore, iNOS-mediated generation of NO is dependent on increases in iNOS protein (e.g., in response to TNF in macrophages) rather than increased intracellular Ca2+. In pulmonary artery endothelium, the isotype ecNOS is the most prevalent, whereas bNOS and iNOS are not usually detected (11, 17).
The consensus of the literature indicates that TNF decreases ecNOS mRNA and ecNOS protein in pulmonary and systemic arterial endothelium (11, 17, 43, 44); however, the effect of TNF on ecNOS in pulmonary microvessel endothelial cells is not known. The elucidation of the role of a specific isotype of NOS during the inflammatory response in pulmonary microvessel endothelial cells is underscored by our conclusion that TNF induces NO-dependent pulmonary endothelial barrier dysfunction (9, 14). It is not known which isotype of NOS is responsible for the increase in NO and microvessel endothelial barrier dysfunction. The investigation of the pathobiology of pulmonary microvessel endothelial cells is necessary because the probable primary fluid exchange site in high-permeability pulmonary edema is in the microcirculation (25). Thus the purpose of this study was to document a role for ecNOS in the TNF-induced generation of NO and increased permeability in pulmonary microvessel endothelial cells.
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MATERIALS AND METHODS |
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Reagents
All reagents were obtained from Sigma (St. Louis, MO) unless otherwise noted.Pulmonary Microvessel Endothelial Cell Culture
Bovine pulmonary microvessel endothelial cells derived from fresh calf lungs were obtained at 4th passage (Vec Technologies, Rensselaer, NY) and serially cultured from 4 to 12 passages (8, 14). The culture medium contained Dulbecco's modified Eagle's medium (DMEM; GIBCO BRL, Grand Island, NY) supplemented with 20% fetal bovine serum (HyClone Laboratories, Logan, UT), 15 mg/ml endothelial cell growth supplement (Upstate Biotechnology, Lake Placid, NY), and 1% nonessential amino acids (GIBCO BRL).Pulmonary microvessel endothelial monolayers (PEM) were maintained in 5% CO2 plus humidified air at 37°C and reached confluence within two to three population doublings, which took 4-5 days. The preparations were identified as endothelial monolayers by 1) the characteristic "cobblestone" appearance using contrast microscopy showing 2) the presence of factor VIII-related antigen (indirect immunofluorescence), 3) the uptake of acylated low-density lipoproteins, and 4) the absence of smooth muscle actin (indirect immunofluorescence).
Detection of ecNOS
RT-PCR.
rna isolation.
RNA was extracted using Tri Reagent (Molecular Research Center,
Cincinnati, OH). The phenolic layer of the extraction preparation containing cellular proteins was stored at 4°C for later
purification. The total RNA was resuspended in Tris-EDTA buffer and
stored at 20°C before RT-PCR.
Immunoblot for protein.
preparation of pem lysate fractions.
PEM were seeded (2 × 105 cells) into six-well plastic
culture plates and incubated for 3-4 days until confluent
(1.2 × 106 cells). After interventions, the PEM were
washed on ice two times with ice-cold PBS containing 15 mM EDTA and 6 mM EGTA. Then 200 ml of ice-cold extraction buffer [50 mM Tris, 2 mM
EGTA, 5 mM EDTA, 150 mM 2-mercaptoethanol, 5 µg/ml
1-antitrypsin, 30 µg/ml antipain, 1.5 KIU/ml
aprotinin, 100 mM benzamidine, 150 nM calpeptin, 15 mg/ml leupeptin, 5 mg/ml pepstatin A, 3 mM 4-(2-aminoethyl)benezenesulfonylfluoride, HCl (AEBSF; Calbiochem, San Diego, CA), 0.2% SDS, and 0.1% Triton X-100] were added to the cells. The wells were scraped with a plastic cell lifter, and the viscous contents sheared in a
microcentrifuge tube by drawing 10 times through a tuberculin syringe
with a 22-gauge needle. The cell lysates were then centrifuged at
16,000 g, 4°C for 20 min, and the supernatants were stored
at
70°C.
Quantification of autoradiographs. The optical density of the autoradiographs was digitized, and the bands were quantified with Sigma Scan Pro as in our previous studies (SPSS Scientific Software, San Rafael, CA) (8, 14). Each band was analyzed by fill area (manual threshold 100) multiplied by intensity to give total intensity units. All Western blots were corrected for protein loading, transfer efficiency, and interblot variability by dividing the density of each band by the total protein density of its respective lane initially obtained by Ponceau red staining and then normalizing each band to its control value (experimental/control). There were 5-10 separate experiments for each group.
Determination of NO Level
NOAssay of Endothelial Permeability
Polycarbonate micropore membranes (13-mm diameter, 0.8-µm pore size; Corning Costar, Cambridge, MA) were gelatinized (type II calf skin gelatin) as previously described (8, 9), mounted on plastic cylinders (9-mm ID; Adaps, Dedham, MA), and sterilized by ultraviolet light for 12-24 h. Endothelial cells (1 × 105 in 0.50 ml of DMEM) were then seeded to the gelatinized membranes and cultured for 3-5 days (37°C, 5% CO2) to allow the cells to be confluent.The experimental apparatus for the study of transendothelial transport in the absence of hydrostatic and oncotic pressure gradients has been described (8, 9). In brief, the system consists of two compartments separated by a microporous polycarbonate membrane lined with the endothelial cell monolayer as previously described. The luminal (upper) compartment (0.7 ml) was suspended in the abluminal (lower) compartment (25 ml). The lower compartment was stirred continuously for complete mixing. The entire system was kept in a water bath at a constant temperature of 37°C. The fluid height in both compartments was the same to eliminate convective flux.
Endothelial permeability was characterized by the clearance rate of albumin labeled with Evans blue dye using our previously published technique (8) adapted from Patterson et al. (32). Hanks' balanced salt solution (HBSS) containing 0.5% BSA and 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer was used on both sides of the monolayer. The albumin was labeled with Evans blue dye by addition (20 µl) of stock Evans blue (20 mg/ml in HBSS) to the upper compartment (680 µl). The absorbance of free Evans blue in the luminal and abluminal compartments was always less than 1% of the total absorbance of Evans blue. At the beginning of each study, an upper compartment sample (700 µl) was diluted 1:100 to determine the initial absorbance of the luminal compartment. Samples (300 µl) were taken from the lower compartment (abluminal) every 5 min for 60 min. The absorbance (620 nm) of the samples was measured in a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). The clearance rate of Evans blue albumin was determined by least squares linear regression between 10 and 60 min for the control and experimental groups.
Treatments
General. PEM cultured in 35- or 60-mm dishes were used for the isolation of mRNA and proteins. PEM cultured in 35-mm wells were used for the NO assay. The lysis or assay buffer was added to the PEM after aspiration of the treated culture medium.
TNF. Highly purified recombinant human TNF from Escherichia coli (Calbiochem) in a stock solution of 10 µg/ml was used. The endotoxin level was less than 0.1 ng/mg of TNF as determined by standard Limulus assay. We previously showed that the boiling of TNF for 45 min prevents the effect of TNF in our system (8, 9, 14), indicating no effect of endotoxin contamination. TNF is used at a dose of 50 ng/ml. Our preliminary studies using TNF in a dose range of 10-50 ng/ml final concentration indicated that 50 ng/ml induced an increase in permeability similar to our previous studies that used TNF (1,000 U/ml) from a different and defunct TNF manufacturer (8, 9; Genzyme, Cambridge, MA).
Antisense oligonucleotides. ecnos antisense. Translation of ecNOS mRNA was inhibited using ecNOS antisense oligonucleotides complementary to bovine ecNOS nucleotides 1165-1189 (Oligos Etc., Wilsonville, OR). The chimeric antisense oligonucleotide (5'-CCG-CGT-GTC-GAG-GTC-CAT-GCA-GAC-C-3') and scrambled nonsense oligonucleotide (5'-GAC-GAC-GTG-ACT GAC-CTG-GTG-CCG-C-3') were phosphorothioated DNA (9 nucleotides) and 2'-O-methylated RNA (16 nucleotides) to achieve improved specificity of binding to the mRNA and nuclease resistance. Specificity of the antisense sequence to bovine ecNOS and randomness of the scrambled nonsense was verified by using the Entrez GenBank data base of the National Institutes of Health.
TRANSFECTION OF OLIGONUCLEOTIDES IN PEM. Transfection of oligonucleotides was accomplished by using the cationic liposome carrier Lipofectin (GIBCO BRL). The Lipofectin was prepared in serum-free DMEM (6.6 ml/ml) and added to the PEM for a final concentration of 5.0 mM. Oligonucleotides from a 40 mM stock solution were added to the PEM for a final concentration of 200 nM, swirled five times, and incubated at 37°C for 4 h. The Lipofectin-oligonucleotide-containing medium was removed before experimental treatment.NO-related reagents.
The exogenous NO donor spermine-NONOATE (spermine-NO, 1 µM; LC
Laboratories, Woburn, MA) was used to test the role of NO generation in
the endothelial response to ecNOS antisense and TNF
(26). Spermine (1 mM; LC Laboratories) was used as
the inactive control for spermine-NO (26). NOS activity
was inhibited by coincubation with the NOS substrate antagonist
aminoguanidine (100 mM; LC Laboratories; 8, 14). We previously showed
that a similar dose of spermine-NO reversed the aminoguanidine-induced
prevention of the increase in activating protein-1 activity
(8), NO
Assay of cell viability. trypan blue exclusion. PEM were seeded (2 × 106 cells/well) and grown until confluent in 35-mm well dishes. After respective drug procedures, PEM were washed with PBS (2 ml) followed by treatment with 0.05% trypsin (0.2 ml) for 1.0 min at 37°C. The cells were resuspended in PBS (1 ml). An aliquot of cell suspension (50 µl) was combined with 0.08% trypan blue (50 µl) for 3 min. Then 10 µl of the mixture were counted for cells using a hemocytometer. Cell viability was defined by the following formula: cell viability = (cells excluding trypan blue/total cells) × 100.
CELL NUMBER. Total cell number of PEM was determined as above in conjunction with the trypan exclusion studies.Statistics
A one-way analysis of variance was used to compare values among the treatments. If significance among treatments was noted, a Bonferroni multiple comparison test was used to determine significant differences among the groups (39). A t-test was used when appropriate. Each PEM well and flask represent a single experiment. The NO ![]() |
RESULTS |
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Cell Viability of PEM: Trypan Blue Exclusion and Cell Counts
We previously showed that TNF does not affect cell viability (8, 14). In the present study, treatment with ecNOS oligonucleotides with and without TNF (50 ng/ml) did not affect PEM viability. The level of cell counts [e.g., control (Con)-4 h, 692,250 ± 60,305 vs. TNF-4 h, 582,750 ± 23,443 cells/35 mm; Con-24 h, 728,250 ± 47,526 vs. TNF-24 h, 667,500 ± 87,184 cells/35 mm] and trypan blue exclusion [e.g., Con-4 h, 93.0 ± 1.5 vs. antisense (Anti) + TNF-4 h, 92.8 ± 0.9%; Con-24 h, 93 ± 2 vs. Anti+TNF, 94 ± 1%] were similar among the groups.TNF Induces a Decrease in ecNOS mRNA
Figure 1A shows a representative PAGE for ecNOS mRNA after vehicle or TNF (50 ng/ml) treatment for 4.0 h. There were two demonstrable bands corresponding to 335 and 488 bp that represent the ecNOS mRNA and the mRNA for 18S ribosome, respectively. In addition, the other NOS isoform (iNOS) was not detected despite detection of its positive control (i.e., bovine spleen lysate for iNOS; data not shown).
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Figure 1B shows the graphic quantification ecNOS mRNA corrected by the 18S ribosomal RNA after TNF (50 ng/ml) treatment for 0.5, 2.0, 4.0, and 24 h. There was a trend for the ecNOS mRNA to decrease from 0.5 to 4.0 h in the control groups. There was a decrease in the ecNOS mRNA from 2.0 to 24.0 h in the TNF groups compared with the levels in the respective control groups. There was no consistent effect of TNF on the expression of 18S RNA. Thus the data from Fig. 1 indicates that the pool of ecNOS mRNA decreases in response to TNF.
TNF Induces a Biphasic Change in ecNOS Protein
Figure 2A shows a representative Western blot for ecNOS protein in total lysate after vehicle or TNF (50 ng/ml) treatment for 0.0 and 24 h. There was one major band in each lane corresponding to a molecular mass of 135 kDa, consistent with the ecNOS native enzyme (11, 17). In addition, the other two NOS isoforms, iNOS and bNOS, were not detected despite detection of their positive controls (i.e., bovine spleen lysate for iNOS and bovine brain lysate for bNOS; data not shown).
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Figure 2B shows the time course for the change in relative density units for the ecNOS protein at 0.5, 2.0, 4.0, and 24 h after TNF (50 ng/ml). Interestingly, in the TNF-0.5 h group, there was an increase in ecNOS protein compared with the corresponding control group. In the TNF-2 h group, there was no significant change in ecNOS protein compared with the respective control group. However, in the TNF-4 h and TNF-24 h groups, there was a decrease in the ecNOS protein compared with the level in the respective control groups. There was no effect of incubation in the control groups because there was no difference in relative density units between the Con and 0.0 group (i.e., no incubation with vehicle nor TNF, 0.054 ± 0.008 relative density units), and the Con-0.5 h (0.042 ± 0.005 relative density units), Con-2 h (0.063 ± 0.009 relative density units), Con-4 h (0.059 ± 0.005 relative density units), and Con-24-h (0.068 ± 0.014 relative density units) groups. The data from Fig. 2 indicate that TNF treatment induces a biphasic alteration in ecNOS protein characterized by an acute increase followed by a protracted decrease of ecNOS protein from 4 to 24 h.
ecNOS Antisense Specifically Depletes ecNOS Protein
Figure 2A shows representative Western blots for ecNOS protein in total lysate in control and after TNF (50 ng/ml) for 24 h with and without the 4-h pretreatment with ecNOS oligonucleotide. The Western blots indicate that ecNOS antisense effectively depleted the ecNOS protein.Figure 3 shows the change in relative
density units for ecNOS protein after treatment with control vehicle,
TNF-0.5 h (Fig. 3A), TNF-4 h (Fig. 3B), and
TNF-24 h (Fig. 3C) in the oligonucleotide ecNOS [i.e., Anti
and nonsense (Non)]-treated groups. In TNF-0.5 h group (Fig.
3A), there was a decrease in the ecNOS protein in the
Anti + TNF-0.5 h group compared with the level in the TNF-0.5 h
groups. In TNF-4 h group (Fig. 3B), there was a decrease in the ecNOS protein in the Anti + TNF-4 h group compared with the level in the Anti and TNF-4 h groups. The ecNOS antisense substantially decreased the ecNOS protein in the antisense group compared with the
level in the control group. In TNF-24 h group (Fig. 3C),
there was a decrease in the ecNOS protein in the Anti + TNF-24 h
group compared with the level in the Anti and TNF-24 h groups. The
ecNOS antisense substantially decreased the ecNOS protein in the Anti group compared with the level in the Con group. The ecNOS nonsense had
no effect in the Non- and TNF-treated groups (Fig. 3, A-C). The data in Fig. 3 indicate that ecNOS antisense treatment is an
effective tool to induce the specific depletion of ecNOS protein during
the TNF treatments.
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ecNOS Mediates TNF-Induced Acute
Increase in NO
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ecNOS Antisense Prevents TNF-Induced Acute Increase in Endothelial Permeability
Figure 5 shows the albumin clearance rates of PEM that were treated with TNF-4 h (50 ng/ml), spermine-NO (1 µM), or spermine (1 µM) for 4.0 h in the vehicle and oligonucleotide-treated PEM. We used spermine-NO because we wanted to verify that the protective effect of ecNOS antisense was due to depletion of NO during the response to TNF. Figure 5 demonstrates that the albumin clearance rates were greater in the TNF-4 h, Non + TNF-4 h, spermine (Sper)-NO + TNF-4 h and Anti + Sper-NO + TNF-4 h groups compared with the Con group. The albumin clearance rates in the Anti, Anti + TNF-4 h, and Anti + Sper + TNF-4 h groups were similar to the control group. Spermine-NO did not induce a further increase in the Sper-NO + TNF-4 h group compared with the TNF-4 h group. Spermine-NO prevented the ecNOS antisense-induced decrease in NO
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ecNOS Antisense Does Not Affect TNF-Induced Prolonged Increase in Endothelial Permeability
Figure 6 shows the albumin clearance rates of PEM that were treated with TNF (50 ng/ml) for 24 h in the vehicle- and oligonucleotide-treated PEM. The albumin clearance rates were greater in all the TNF-treated groups compared with the Con group. The albumin clearance rates in the Amino, Anti, and Non alone groups were similar to the Con group. Therefore, the data indicate that ecNOS derived NO does not mediate the TNF-induced prolonged (i.e., 24 h) increase in albumin permeability of PEM.
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DISCUSSION |
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The present study indicates in pulmonary microvessel endothelium
that ecNOS mediates the acute (i.e., 4 h) increase in ·NO and
albumin permeability in response to TNF. ecNOS mediated the response to
TNF because 1) TNF induced the release of NO, 2)
iNOS and bNOS were not detected, and 3) antisense to ecNOS
prevented the effects of TNF. The data indicate that the cellular
effects of the antisense on ecNOS were specific for ecNOS because the scrambled nonsense oligonucleotide and the appropriate vehicle had no
effect on the response to TNF. The effect of ecNOS antisense on
permeability was due to inhibition of NO generation because repletion
of NO using spermine-NO ablated the protective effect ecNOS antisense,
whereas the spermine control had no effect in the Anti + TNF-4 h
group. Finally, there was similar cell viability among all the groups.
We previously showed in vitro that TNF induces NO-dependent
ONOO generation (33), protein oxidation
(9), glutathione oxidation (33), and barrier
dysfunction (9) in pulmonary arterial endothelial cells.
The present data agree with the results of others demonstrating that NO
mediates increases in pulmonary arterial endothelial permeability in
response to inflammatory mediators such as TNF (9),
vascular endothelial growth factor (VEGF) (42), phorbol
12-myristate 13-acetate (PMA) (20), and bradykinin
(5). The role of ecNOS and NO on increases in pulmonary
microvessel endothelial permeability in response to TNF has not been
investigated previously. This study is the first to demonstrate that
the isotype ecNOS is essential for TNF-induced acute pulmonary
microvessel barrier dysfunction.
The present study indicates a TNF-induced biphasic alteration in the ecNOS protein pool characterized by an acute increase (i.e., 0.5 h post-TNF) followed by a protracted decrease of the ecNOS protein level by 4.0 h after TNF. To our knowledge, the novel observation of an acute increase in ecNOS protein in response to TNF has not been reported in pulmonary microvascular endothelial cells. Our evidence indicates that translation of ecNOS mRNA is the probable mechanism for the acute increase in ecNOS protein because the ecNOS antisense oligonucleotide prevented the TNF-induced increase in the ecNOS protein pool. Recently, TNF (10 U/ml for 0.5 h) has been shown to activate the regulator of translation enzyme p70/p85 S6 kinase in human umbilical vein endothelial cells (23), consistent with the currently observed acute increase in ecNOS protein. In contrast, the latter decrease in the ecNOS protein is probably mediated in part by the decrease in the pool of ecNOS mRNA that began 2 h after the TNF. In agreement with our finding, it has been shown previously in systemic and pulmonary arterial endothelium that TNF decreases the expression of ecNOS protein (11, 17, 43, 44). Moreover, previous studies indicate that the decrease in expression is caused in part by the degradation of ecNOS mRNA (43, 44). The 3'-untranslated region of the ecNOS mRNA has an element that binds to a protein, possibly induced by TNF, that enhances ecNOS mRNA binding to RNase and subsequent degradation of the RNA (11, 17, 43, 44). Thus similar to other endothelial cell types (11, 17, 43, 44), the results of our study using pulmonary microvascular endothelium indicate that the likely mechanism for the TNF-induced decrease in the ecNOS protein pool is depletion of ecNOS mRNA. Also, a TNF-induced increase in the degradation of the ecNOS protein cannot be discounted because TNF induced a further decrease in the ecNOS protein pool despite the effectiveness of the ecNOS antisense (i.e., decrease in ecNOS protein before TNF).
Interestingly, despite the gradual decrease in the ecNOS protein pool,
there was an increase in the level of NO, which suggests activation of
ecNOS. The direct mechanism for the activation of ecNOS in response to
TNF was not investigated in the present study. We previously indicated
that protein kinase C (PKC) activation mediates TNF-induced alterations
in NO (21, 38), peroxynitrite (33), and
glutathione oxidation (33). In addition, we recently demonstrated that the TNF-induced increase in pulmonary microvessel permeability is dependent on the activation of the isotype PKC- (8). Our data in progress using pulmonary microvessel
endothelium indicate that inhibition of PKC-
activity prevents the
TNF-induced increase in NO (28). In response to TNF, the
PKC-
-mediated phosphorylation of myristolated arginine rich C kinase
substrate may induce the release of calmodulin, resulting in increased
activity of ecNOS (18). In addition, it has been shown
that ecNOS is located in the calveolae membrane microdomain and is
associated with the protein calveolin (6, 7). Calveolin is
hypothesized to inhibit ecNOS, and the effect of calveolin on ecNOS is
prevented following the phosphorylation of calveolin by PKC (6,
7). Huang and Yuan (20) have shown that increases
in microvascular permeability in response to PMA are mediated by NO. Wu
et al. (42) have demonstrated VEGF-induced activation of
PKC mediates NO-dependent venular hyperpermeability. PKC-
/
has
been shown to induce transcription of ecNOS in human vein endothelial
cells (24). Thus a probable downstream target for PKC
activation are pathways leading to generation of reactive nitrogen
species (RNS) via ecNOS. Our previous work indicates that TNF-induced
barrier dysfunction is mediated by both PKC-
(8) and NO
(9), which points to the cascade TNF-
PKC-
ecNOS
RNS
barrier dysfunction in acute lung injury. The role of
PKC-
activation in the activation of ecNOS is now under intense
investigation in our laboratory (28). Conversely, in
bovine aortic endothelial cells, PKC inhibition increased the
expression of ecNOS mRNA, ecNOS protein, and release of nitrogen oxides
(29). Thus the cell type and other experimental differences can influence the role of the PKC in the ecNOS
RNS pathway. Finally, other mechanisms for ecNOS activation in response to
TNF can include activation of the novel kinase PKB [i.e., Akt (4)], and increases in intracellular calcium
(42), tetrahydrobiopterin (3), and
L-arginine (30).
The literature indicates that NO has myriad effects on the homeostasis of the endothelium (12, 27) that can affect endothelial barrier function (8, 9). We previously showed that TNF induces the NO-dependent oxidation of a protein with the molecular mass of actin (9), and our preliminary studies indicate that treatment of PEM with peroxynitrite-treated actin increases protein permeability of PEM (10, 37). It has been shown clearly by other investigators that an alteration in the actin cytoskeleton and extracellular matrix can mediate the increase in permeability of PEM in response to TNF (16, 19, 31). Thus a potential mechanism for the NO-mediated increase in permeability is alteration of cytoskeletal targets (e.g., actin) and extracellular matrix (e.g., fibronectin), with subsequent alterations in cell shape and the dynamics of cell-cell and cell-extracellular matrix interaction (13, 31, 34).
An intriguing finding in the present study is that antisense-induced
depletion of ecNOS protein and prevention of the increase in NO did not
alter the prolonged TNF (i.e., 24 h)-induced barrier dysfunction.
The NO-independent effect at 24 h is evidenced by the TNF-induced
increase in albumin clearance in the Anti + TNF-24 h group despite
the similar NO levels between the Con-24 h and TNF-24 h groups.
Moreover, there was no effect of aminoguanidine on barrier function in
the Amino + TNF-24 h group. Thus the pulmonary microvessel
endothelial cell recruits other permeability-enhancing pathways
that alter endothelial barrier function after the prolonged exposure to
TNF-24 h. TNF has been shown to activate a host of other pathways
that include proteases (31), mediators of
apoptosis (36), PKC- (8, 13), and
mitogen-activated protein kinase (15) that can potentially
induce endothelial barrier dysfunction. In addition, we
previously showed the NO-independent activation of the transcription
factor nuclear factor-
B after 2-4 h of TNF treatment in the
pulmonary microvascular endothelium (14). Thus the
prolonged increase in permeability may be mediated by transcription factor-dependent expression of critical signals and subsequent downstream effects on the cytoskeleton, extracellular matrix, and
barrier function.
In summary, our study indicates for the first time that TNF induces the acute dysfunction of the endothelial barrier, which is dependent on ecNOS mediated generation of NO. However, the prolonged TNF-induced increases in permeability is independent of NO. The development of strategies that target on-going ecNOS activity may provide therapy for the early proinflammatory response during states of TNF-mediated acute lung injury.
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ACKNOWLEDGEMENTS |
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This work was supported by the Department of Veterans Affairs (VA) Medical Research Service Merit Review (A. Johnson), National Heart, Lung, and Blood Institute Grants RO1-HL-48406-07 and RO1-HL-59901-02 (A. Johnson), and VA Merit Review Entry Program Award (K. Bove).
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. Johnson, 151, 113 Holland Ave., Dept. of Veterans Affairs Medical Center, Albany, NY 12208 (E-mail: jmurd{at}msn.com).
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 25 September 2000; accepted in final form 13 November 2000.
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