Role of ecNOS-derived NO in mediating TNF-induced endothelial barrier dysfunction

Kathleen Bove1, Paul Neumann2, Nancy Gertzberg2, and Arnold Johnson1,2

1 Research Service, Stratton Veterans Affairs Medical Center, and 2 Center for Cardiovascular Science, Albany Medical College, Albany, New York 12208


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We tested the hypothesis that endothelial cell nitric oxide synthase (ecNOS) mediates the tumor necrosis factor (TNF)-alpha -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<UP><SUB>2</SUB><SUP>−</SUP></UP>, the oxidation product of NO), and barrier function (albumin clearance rate). PEM were incubated with TNF (50 ng/ml) for 0.5, 2, 4, and 24 h. TNF induced a decrease in ecNOS mRNA at 2, 4, and 24 h. TNF induced an acute (0.5 h) increase followed by a protracted decrease (4-24 h) in ecNOS protein levels. The other NOS isotypes, inducible and brain NOS, could not be detected in the PEM using RT-PCR and Western blot assay. ecNOS antisense oligonucleotide decreased ecNOS protein, which prevented the increase in NO and albumin permeability at TNF-4 h. Spermine-NONOATE, the NO agonist, ablated the protective effect of ecNOS antisense oligonucleotide on albumin permeability in response to TNF-4 h. However, ecNOS antisense oligonucleotide had no effect on the TNF-induced increase in albumin permeability at 24 h despite prevention of the increase in NO. The data indicate that the isotype ecNOS mediates generation of NO and the acute (i.e., 4 h) barrier dysfunction; however, the prolonged (i.e., 24 h) increase in the TNF-induced increase in endothelial permeability is independent of NO.

antisense; edema; messenger ribonucleic acid; permeability; transcription


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TUMOR NECROSIS FACTOR (TNF)-alpha 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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.

RT-PCR. First-stand cDNA was generated from 1.0 mg of total RNA using SuperScript II reverse transcriptase (GIBCO BRL, Gaithersburg, MD). Quantitative PCR was performed according to the GeneAmp DNA Amplification Reagent Kit (Perkin Elmer, Foster City, CA) using 32P-labeled dCTP. Oligonucleotide paired primers for bovine ecNOS and iNOS were purchased from GIBCO BRL. Quantum RNA 18S Internal Standard Kit (Ambion, Austin, TX) was used as an internal control. PCR samples were fractionated by electrophoresis on an 8% PAGE and quantified by phosphorImage analysis (STORM 860; Molecular Dynamics, Sunnyvale, CA). The linear range of amplification was determined for each primer pair. Results are expressed as the ratio of the gene of interest to 18S ribosomal RNA.

OLIGONUCLEOTIDE PRIMERS. The sequences of the bovine oligonucleotide primer pairs for ecNOS and iNOS are ecNOS forward, 1100-CTGGTACATGAGCACGGAGA-1119; ecNOS reverse, 1434-AGTTGACCATCTCCTGGTGG-1415; iNOS forward, 32-CAGGAACCTACCAGCTGACC-51, and iNOS reverse, 252-CTGGGGGAACACAGTGATG-234. The expected PCR product size for each pair of oligonucleotide primers were as follows: 1) ecNOS, 335 bp; 2) iNOS, 211 bp; and 3) 18S, 488 bp.

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 alpha 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.

TOTAL PROTEIN. Total protein concentrations were determined in diluted samples with Coomassie Plus protein assay reagent (Pierce, Rockford, IL) against protein standards consisting of 50% BSA-50% gamma -globulin.

SDS-PAGE. The samples were diluted (0.5:1) in Laemmli buffer (187.5 mM Tris · HCl, 30% glycerol, 15% 2-mercaptoethanol, 6% SDS, and 0.006% bromphenol blue, pH 6.8) and boiled for 5 min. The lysate proteins were separated by SDS-PAGE on 7.5% gels using standard procedures (Mini-Protean II Electrophoresis Cell Manual; Bio-Rad, Hercules, CA). All lanes were loaded so that each lane contained 8-12 mg of total protein.

WESTERN BLOT. The polyacrylamide gels were transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, MA) with use of standard procedures (Mini-Trans Blot Electrophoretic Transfer Cell Manual, Bio-Rad). The PVDF membranes were stained with Ponceau red and digitized by scanning (Scan Jet 4C; Hewlett Packard Canberra, Downers Grove, IL) for measurement of total protein per lane. Then, the PVDF membranes were treated with Blotto (5% nonfat dry milk) in Tween 20-Tris-buffered saline solution (0.1% Tween 20, 10 mM Tris, and 100 mM NaCl, pH 7.5) to prevent nonspecific binding of antibody to the PVDF membrane.

PRIMARY AND SECONDARY ANTIBODIES. A rabbit polyclonal antibody to bovine ecNOS which recognizes the amino acid residues 596-609 was used (1:10,000 dilution in Blotto). In addition, both mono- and polyclonal antibodies to mouse iNOS, amino acid residues 961-1144 and mono- and polyclonal antibodies to human bNOS, amino acid residues 1095-1289, were used (Transduction Laboratories, Lexington KY). Goat alkaline phosphatase-conjugated anti-rabbit or -mouse was used as the secondary antibody. All incubations of the membranes were done for 30 min at 37°C. The unbound antibodies were removed by washing five times for 5 min each with Tris-buffered saline solution at room temperature. The PVDF membranes were developed with the chemiluminescent substrate CDP-Star (Tropix, Bedford, MA) and exposed to X-OMAT AR, Ortho-G, or BioMax ML film for 0.5-15 min.

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

NO<UP><SUB>2</SUB><SUP>−</SUP></UP> levels, an index of ·NO, were measured with an colorimetric assay using Griess reagent as previously reported (9, 14, 21, 38). In brief, after treatments, the cell medium (100 ml) was added to Griess reagent (100 ml) and read spectrophotometrically.

Assay 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<UP><SUB>2</SUB><SUP>−</SUP></UP> (14), and permeability (14) during a 4-h incubation with TNF using pulmonary arterial (14) or microvessel endothelial cells (8).

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<UP><SUB>2</SUB><SUP>−</SUP></UP> data were converted to the experimental-to-control ratio before statistical analysis. All data are reported as means ± SE. Significance was at P < 0.05. There are 5-10 samples per group in all studies.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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Fig. 1.   Tumor necrosis factor (TNF) induces a decrease in endothelial cell nitric oxide synthase (ecNOS) mRNA. Pulmonary microvessel endothelial monolayers (PEM) were incubated with TNF for 0.5, 2.0, 4.0, and 24 h. A: representative PAGE of DNA generated from primers for ecNOS mRNA and 18S ribosome using RT-PCR in the Con and TNF-4 h groups. iNOS, inducible nitric oxide synthase. B: graphic quantification of the mean levels of the ecNOS mRNA after correction by the 18S ribosome after TNF. #Different from the respective Con group.

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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Fig. 2.   TNF induces a biphasic change in ecNOS protein. PEM were incubated with Con or TNF for 0.5, 2.0, 4.0, and 24 h. In separate studies, PEM were preincubated for 4 h with antisense (Anti) or nonsense (Non) oligonucleotide followed by the TNF. A: representative Western blot of total PEM lysate in the Con, Anti, Non, and TNF-24 h groups. B: the time course of the mean levels of the ecNOS 135-kDa protein expressed as experimental-to-control ratio (Exp/Con) only in the Con and TNF-treated groups. #Different from the respective Con group.

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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Fig. 3.   ecNOS antisense depletes ecNOS protein. PEM were incubated with TNF for 0.5 (A), 4 (B), or 24 h (C). In separate studies, PEM were preincubated for 4.0 h with Con, Anti, or Non followed by the TNF. The mean levels of the ecNOS 135-kDa protein are expressed as experimental-to-baseline ratio (Exp/Bas). #Different from the respective untreated group. *Different from the Con group. $Different from the TNF group.

ecNOS Mediates TNF-Induced Acute Increase in NO<UP><SUB>2</SUB><SUP>−</SUP></UP>

Figure 4A shows the levels of NO<UP><SUB>2</SUB><SUP>−</SUP></UP> in extracellular medium after treatment with control vehicle or TNF-4 h (50 ng/ml) in the oligonucleotide ecNOS (i.e., Anti and Non)-treated groups. In the TNF-4 h and Non + TNF-4 h groups, there was an increase in NO<UP><SUB>2</SUB><SUP>−</SUP></UP> compared with the Con group. In the Non group and Anti-treated groups, there was no increase in NO<UP><SUB>2</SUB><SUP>−</SUP></UP>. We previously showed that the increase in NO<UP><SUB>2</SUB><SUP>−</SUP></UP> is prevented by the NOS inhibitor aminoguanidine (9, 14) and is L-arginine dependent (9, 14). Figure 4B shows the levels of NO<UP><SUB>2</SUB><SUP>−</SUP></UP> in extracellular medium after treatment with control vehicle or TNF-24 h (50 ng/ml) in the oligonucleotide ecNOS (i.e., Anti and Non)-treated groups. There was no significant difference in the NO levels in any of the TNF-treated groups compared with the Con group. However, there was less NO<UP><SUB>2</SUB><SUP>−</SUP></UP> in the Anti + TNF-24 h group compared with the TNF-24 h group. Thus the data derived from Figs. 3 and 4 show ecNOS antisense treatment decreases the level of NO<UP><SUB>2</SUB><SUP>−</SUP></UP>, indicating that ecNOS mediates the generation of NO.


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Fig. 4.   TNF-induces an ecNOS dependent increase in nitric oxide (NO). PEM were incubated with TNF for 4 (A) or 24 h (B). In separate studies, PEM were preincubated for 4 h with Con, Anti, or Non followed by the TNF. The mean levels of NO<UP><SUB>2</SUB><SUP>−</SUP></UP> in medium were measured with Griess reagent. *Different from the Con group. #Different from the TNF group

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<UP><SUB>2</SUB><SUP>−</SUP></UP> levels in the Anti + Sper-NO + TNF-4 h group (Con-4 h group, 860.0 ± 59.0 pmol/well < Anti + Sper-NO + TNF-4 h, 3,720.0 ± 90.2 pmol/well, P < 0.05). Spermine did not affect the antisense-mediated decrease in NO<UP><SUB>2</SUB><SUP>−</SUP></UP> levels (Anti + Sper + TNF-4 h, 1,075.0 ± 124.7 pmol/well). The albumin clearance rate in the aminoguanidine (Amino) and Amino + TNF-4 h groups was similar to the value in the control group. Therefore, the data from the experiments using ecNOS antisense, spermine-NO, and aminoguanidine support the concept that ecNOS derived NO mediates the TNF-induced acute increase in albumin permeability of PEM.


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Fig. 5.   Depletion of ecNOS prevents acute barrier dysfunction induced by TNF. PEM were incubated with Con, TNF, spermine (Sper), spermine-NONOATE (Sper-NO), and aminoguanidine (Amino) for 4.0 h. In separate studies, PEM were preincubated for 4 h with Con, Anti, or Non oligonucleotide to ecNOS followed by the above treatments. PEM were assayed for endothelial albumin clearance rate. *Different from Con group.

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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Fig. 6.   Depletion of ecNOS does not prevent prolonged barrier dysfunction induced by TNF. PEM were incubated with TNF and Amino for 24 h. In separate studies, PEM were preincubated for 4 h with Con, Anti, or Non oligonucleotide to ecNOS, followed by Con or TNF treatments. PEM were assayed for endothelial albumin clearance rate. *Different from Con group.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha (8). Our data in progress using pulmonary microvessel endothelium indicate that inhibition of PKC-alpha activity prevents the TNF-induced increase in NO (28). In response to TNF, the PKC-alpha -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-alpha /epsilon 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-alpha (8) and NO (9), which points to the cascade TNF-alpha right-arrow PKC-alpha right-arrow ecNOS right-arrow RNS right-arrow barrier dysfunction in acute lung injury. The role of PKC-alpha 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 right-arrow 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-alpha (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-kappa 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.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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