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Am J Physiol Heart Circ Physiol 281: H661-H668, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 2, H661-H668, August 2001

Endotoxin stimulated cytokine production in rat vascular smooth muscle cells

Kristina Detmer1, Zhongbiao Wang1, Debra Warejcka2, Sandra K. Leeper-Woodford1, and Walter H. Newman1,2

1 Division of Basic Medical Sciences and 2 Department of Surgery, Mercer University School of Medicine, Macon, Georgia 31207


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Because inflammatory processes may promote the development of atherosclerosis, we examined the activation of cytokine genes in rat vascular smooth muscle cells in vitro after treatment with bacterial lipopolysaccharide (LPS). Interleukin-1 (IL-1), IL-6 and tumor necrosis factor-alpha (TNF-alpha ) mRNA increased in response to LPS. Activation of nuclear factor-kappa B (NF-kappa B) presumably results in NF-kappa B binding to regulatory regions of target genes and activating transcription. We therefore compared the kinetics of NF-kappa B activation, cytokine message production, and TNF-alpha secretion. Maximum active NF-kappa B was found at 30 min after the addition of LPS and decreased thereafter. Increased IL-6 mRNA was detected at 30 min, increased TNF-alpha mRNA at 60 min, and increased IL-1 mRNA at 120 min. Secretion of TNF-alpha was dependent on LPS concentration and was first detected 120 min after LPS addition. Aspirin, which has been shown to inhibit NF-kappa B activation and cytokine secretion in other cell types, did not inhibit NF-kappa B activation or TNF-alpha secretion. However, aspirin reduced the amount of both TNF-alpha and IL-6 mRNA present 30 min after LPS addition by half (P < 0.05).

atherosclerosis; nuclear factor-kappa B; tumor necrosis factor-alpha ; aspirin; cytokine gene transcription


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE DEVELOPMENT OF ATHEROSCLEROTIC lesions resembles an inflammatory process, and bacterial or viral infection has been implicated in the development of atherosclerosis (14). Mediators of the inflammatory process found in atherosclerotic lesions include tumor necrosis factor-alpha (TNF-alpha ), interleukin (IL)-6, and activated nuclear factor-kappa B (NF-kappa B) (5, 17, 25). TNF-alpha and IL-6 are produced by proliferating arterial smooth muscle cells of atherosclerotic plaques but not by smooth muscle cells of normal vessels (2, 21, 25). Activation of the genes for TNF-alpha and IL-6 by lipopolysaccharide (LPS) in vascular smooth muscle has been shown (19, 27, 15) suggesting that secretion of inflammatory cytokines by smooth muscle cells in response to infection contributes to the development of atherosclerosis.

The regulatory regions of the IL-6 and TNF-alpha genes are responsive to NF-kappa B (4). NF-kappa B is a family of Rel-related transcription factors that mediate inflammatory processes. In the cytosol, NF-kappa B proteins form heteromeric complexes with members of a family of inhibitory proteins termed Ikappa B. NF-kappa B is activated after transduction of a proinflammatory signal that results in the phospyhorylation of Ikappa B. The phosphorylated Ikappa B is degraded, liberating NF-kB dimers that translocate to the nucleus and initiate transcription of target genes (3). The predominant NF-kappa B species found in vascular smooth muscle cells are p50 and p65. In normal human arterial blood vessels, staining for p50 and p65 reveals that NF-kappa B is located in the cytoplasm. In atherosclerotic vessels, NF-kappa B is found in the nucleus, consistent with an inflammatory process; in nonatherosclerotic vascular smooth muscle, NF-kappa B is detected in the cytoplasm (5).

Because of similarities in the development of atherosclerosis and inflammatory processes, there is considerable interest in the potential of anti-inflammatory agents to prevent or retard the development of atherosclerotic lesions. Because salicylates, including acetylsalicylic acid (aspirin), prevent the activation of NF-kappa B in some cell types (13, 20), we were interested in whether aspirin would inhibit the activation of NF-kappa B-dependent inflammatory cytokines in vascular smooth cells.

Aspirin and salicylate inhibit the activation of NF-kappa B by blocking the phosphorylation event and hence preventing the degradation of Ikappa B. The mechanism(s) by which aspirin blocks the phosphorylation of Ikappa B is (are) not known. Ikappa B is phosphorylated by a 700 kDa complex, Ikappa B kinase (Ikappa K) (8), which contains two kinases, Ikappa K-1 and Ikappa K-2, capable of phosphorylating Ikappa B (29). The two Ikappa B kinases have been shown to be differentially regulated by upstream kinases activated through different signal transduction pathways (18), consistent with the existence of multiple signal transduction pathways culminating in the activation of NF-kappa B. Salicylate and salicylate derivatives have been shown to interfere with signal transduction to specific mitogen-activated protein kinases (MAPK) in response to cytokines (24). Thus there is the potential that the anti-inflammatory effects of aspirin may be cell-type specific or signal transduction-pathway specific.

The original goal of this study was to characterize the initial steps leading to the production of TNF-alpha by vascular smooth muscle cells in vitro in response to stimulation by LPS. Our findings indicated that both NF-kappa B activation, an increase in TNF-alpha mRNA, and secretion of TNF-alpha protein follow LPS stimulation. To determine whether the activation of NF-kappa B was necessary for activation of the TNF-alpha gene, we measured NF-kappa B activation, TNF-alpha mRNA, and TNF-alpha protein synthesis in rat smooth muscle cells in culture after LPS stimulation in the presence and absence of aspirin. Contrary to expectation, aspirin had no effect on NF-kappa B activation and TNF-alpha protein synthesis. In the presence of aspirin, the amount of TNF-alpha mRNA was ~50% of that found in the absence of aspirin 30 min after LPS stimulation; after 180 min, there was no aspirin effect in the amount of RNA detected. We found a similar decrease of IL-6 mRNA at 30 min but no effect at 180 min after LPS stimulation in the presence of aspirin.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Protocols for animal use were approved by Mercer University's Institutional Animal Care and Use Committee. Thoracic aortas were obtained from pentobarbital- anesthetized male 250-g Sprague-Dawley rats. Under sterile conditions, the aortas were opened and the endothelial cells were removed by rubbing the luminal surface. The aortas were incubated overnight in Dulbecco's modified Eagle's medium (DMEM) plus antibiotics (100 IU/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone). Explants (2 × 2 mm) were then cut, placed in a 10-cm petri dish, and covered with DMEM containing 20% fetal bovine serum (FBS) and antibiotics. The explants were cultured at 37°C in a 5% CO2 incubator. After ~1 wk, the cells that had exited were released with trypsin and passed into 24-well plates and grown to confluence in DMEM containing 5% FBS and antibiotics (complete medium). Each batch of isolated cells was immunostained for smooth muscle alpha -actin with a commercial kit (Sigma). Cell protein was measured by the Bradford method (6). Results reported here were from cells in passages 1-6.

Cell viability assay. To determine whether the concentrations of LPS used in the experiments affected cell viability, vascular smooth muscle cells at confluence in 96-well plates were treated with LPS (20 µg/ml) for 6 h. Cell viability was assessed by the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrozolium bromide (MTT) to formazan. At the end of each experiment, cells were incubated with 100 µl MTT (0.5 mg/ml) dissolved in phenol red-free DMEM plus 0.5% FBS for 4 h. Formazan was extracted with dimethyl sulfoxide and quantitated in a microplate reader at 595 nm. Cell viability in the presence of 20 µg/ml LPS was indistinguishable from viability in the absence of LPS in three individual experiments consisting of five viability determinations.

Cytotoxicity assay for TNF-alpha . Biologically active TNF-alpha in the culture medium was measured by an in vitro cytotoxicity assay using the murine fibroblast L929 cells as described previously (10). L929 cells were seeded into 96-well microtiter plates at an initial density of 5.0 × 104 cells/well in 200 µl of DMEM containing 5% FBS and antibiotics and incubated for 24 h at 37°C in a 5% CO2 incubator. After incubation, the medium was then removed and 50 µl of complete medium containing 20 µg/ml actinomycin D were added to all wells. In each plate, a standard curve (200, 50, 12.5, 5, 1.5, and 0.5 pg/well) of recombinant rat TNF-alpha in complete medium was set up. Test medium (150 µl) was added to the remaining wells. The cells were incubated for 18 h and the medium was decanted. The remaining viable cells were stained with 100 µl/well of 0.1% crystal violet in 20% ethanol, rinsed with phosphate-buffered saline, and air dried. The dye was solubilized by the addition of 100 µl of methanol 5 min before reading the absorbance of each well on an automated microplate reader Elx 800 (Bio-Tek) at 595 nm. To confirm that the cytotoxicity in the L929 assay was due to TNF-alpha , guinea pig anti-murine TNF-alpha antiserum was added to selected samples of medium before assay.

Assay for 6-keto-PGF1alpha . The stable metabolite of prostacyclin 6-keto-PGF1alpha was determined in cell culture medium with a commercially available kit according to the manufacturer instructions (Cayman Chemical).

Electrophoretic mobility shift assay. Whole cell extracts were prepared according to the method of Dent and Latchman (7). To extraction buffer stock (20 mM HEPES, pH 7.8; 450 mM NaCl, 0.4 mM EDTA, and 25% glycerol) was added a complete protease inhibitor cocktail tablet (Boehringer Mannheim) and dithiothreitol (DTT) to 0.5 mM just before use. Extraction buffer was added to frozen vascular smooth muscle cells (150 µl to 100 mm plates, 50 µl to each well of 12-well plates), and the cells were scraped into microfuge tubes. The cells were given two additional freeze/thaw cycles at -70°C and 37°C and centrifuged at 13,000 g for 10 min in a microfuge. If not assayed immediately, supernatants were stored at -70°C. Protein concentrations were determined with the Bio-Rad protein assay kit. Variation in protein concentration among the samples in a given experiment was typically <20% and the samples were assayed without adjusting the protein concentration.

Each electrophoretic mobility shift assay contained 5 µl of whole cell extract, 10 fmol of the P-32 end-labeled, double-stranded oligonucleotide 5'-AGTTGAGGGGACTTTCCCAGGC-3' (Promega), which contains the NF-kappa B consensus-binding sequence, and 0.25 µg of polydeoxyinosinic-deoxycytidylic acid (Boehringer Mannheim) in 10 mM Tris, pH 7.5, 4% glycerol, 1 mM DTT in a total volume of 20 µl. Unlabeled competitor oligonucleotides (1 pmole) were added as indicated. After a 15-min incubation at room temperature, the mixtures were loaded onto a 5% polyacrylamide gel. The gels were electrophoresed at 200 V for 1 h, 20 min in 0.5× Tris-borate-EDTA (0.045 M Tris-borate and 1 mM EDTA). Gels were dried and autoradiographed. Bands corresponding to NF-kappa B were identified by homologous competition experiments. Bands were quantitated by densitometry.

RNAse protection assay. RNA was prepared from cells grown on 100-mm plates and stored at -70°C using the RNeasy mini kit (Qiagen) according to manufacturer directions. Briefly, lysis buffer was applied to the frozen cells; the cell lysate was collected with a cell scraper and applied to a Qiashredder spin column (Qiagen) to shear the genomic DNA. The resulting homogenate was mixed with an equal volume of 70% ethanol and applied to an RNeasy spin column. After washing and elution was completed, the RNA was quantitated by absorbance at 260 nm. The RNA was precipitated by the addition of 0.1 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ethanol and stored at -70°C until use.

The rat cytokine multiprobe set was obtained from PharMingen that consists of DNA templates that can be transcribed to give probes for the following genes: IL-1alpha , IL-1beta , TNF-beta , IL-3, IL-4, IL-5, IL-6, IL-10, TNF-alpha , IL-2, interferon-gamma , L32, and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Probes were made by transcribing 1 h at 20°C 0.5 µg multiprobe DNA with T7 RNA polymerase in a 20-µl reaction volume containing components of the MAXIscript in vitro transcription kit (Ambion) and 100 µCi alpha -[32P]UTP, 800 Ci/mM (Amersham). DNA was degraded by the addition of 4 units of RNase-free DNase and incubation for 15 min at 37°C. Yeast tRNA (2 µg), 0.3 volumes of 7.5 M ammonium acetate, and 2.5 volumes of ethanol were added to precipitate the RNA probes. After 30 min at -70°C, the mixture was centrifuged for 15 min at 13,000 g in a microfuge. The pellet was washed with 90% ethanol, air dried, and dissolved in hybridization buffer (80% deionized formamide/100 mM sodium citrate pH 6.4/300 mM sodium acetate pH 6.4/1 mM EDTA).

Aliquots of the RNA stored as an alcoholic precipitate were centrifuged in a microfuge for 15 min at 13,000 g and the supernatants removed. Hybridization buffer and 300,000-500,000 counts per minute of probe were added to each sample in a total volume of 20 µl. After the mixtures were heated with vortexing for 5 min at 100°C, the mixtures were hybridized overnight at 50°C. RNase digestion and precipitation of the protected fragments was carried out using the RPAII kit (Ambion) according to manufacturer protocols. Protected fragments were resolved by electrophoresis on a 7 M urea/5% polyacrylamide gel in 0.045 M Tris borate/1-mM EDTA buffer. After being dried, the protected fragments were identified by autoradiography or by visualization in a phosphorimager (Molecular Dynamics). The protected fragments were quantitated by densitometry or by analysis with the ImageQuant software accompanying the phosphorimager.

For those experiments where the rat vascular smooth muscle cells were grown in 12-well plates, RNase protection assays were carried out using the Direct Protect RPA kit (Ambion) according the manufacturer protocols. Briefly, hybridization was carried out in the lysis buffer. In these experiments, the probes were redissolved in lysis buffer after precipitation.

Western blot detection of TNF-alpha . After incubation, the cells were washed three times with phosphate-buffered saline and lysed in cell lysis buffer of 50 mM Tris, pH 8.0; 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 100 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1% Nonidet P-40. After being boiled for 5 min, protein concentrations were determined with the Bio-Rad DC protein assay kit (Hercules, CA). Samples (200 µg/lane) were subjected to SDS-polyacrylaminde gel electrophoresis and transferred to a nitrocellulose membrane in 25 mM Tris, 20% methanol, 192 mM glycine, pH 8.3. After membrane transfer, gels were stained for total protein with Coomassie blue to verify transfer efficiency. The membranes were also reversibly stained with ponceau S to assess the equivalence of sample loading and gel transfer. After being blocked in 5% nonfat milk, the membranes were incubated in 2 µg/ml mouse anti-rat-TNF, and followed by horseradish peroxidase-conjugated anti-mouse IgG at 1:3,000 dilution (enhanced chemiluminescence kit, Amersham). The binding of antibody was detected by chemiluminescence and evaluated by densitometry. Prestained protein markers (GIBCO) were used for molecular mass determinations.

Analysis of data. Data were analyzed using Student's t-test, repeated-measures ANOVA, or linear least squares as appropriate.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LPS stimulates the release of TNF-alpha from rat vascular smooth muscle cells in a concentration-dependent manner. In the absence of LPS, TNF-alpha was not detected in the media in which rat vascular muscle cells were growing. Addition of increasing concentrations of LPS resulted in increasing amounts of TNF-alpha production by the cells (Fig. 1A). The amount of TNF-alpha produced became significant (P < 0.05) at concentrations of 2.0 µg/ml LPS.


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  		Fig. 1.   Production of tumor necrosis factor (TNF)-alpha by rat smooth muscle cells in culture in response to lipopolysaccharide (LPS). A: effect of LPS concentration on the amount of TNF-alpha released. Cells were incubated with the indicated concentrations of LPS, and medium was removed at the indicated times and analyzed for TNF-alpha by the L929 cytotoxicity assay. TNF-alpha was undetectable in the absence of LPS. B: time course of TNF-alpha production. Cells were incubated with or without 20 µg/ml LPS added to the culture medium. Medium was removed at the indicated times and analyzed for TNF-alpha by the L929 cytotoxicity assay. Values are means ± SE for cells cultured from 7 rats. *P < 0.05 vs. no LPS.

We used an LPS concentration of 20 µg/ml to determine the time course of TNF-alpha secretion, because the response to LPS was the most robust at that concentration. TNF-alpha was not detected in significant quantities until 120 min after LPS stimulation (Fig. 1B). A similar time course was seen using 2.0 µg/ml LPS (data not shown). Production of TNF-alpha was dependent on new RNA synthesis as treatment of the cells with 20 µg/ml actinomycin D before LPS stimulation abolished TNF-alpha production.

LPS stimulation activates NF-kappa B. Activation of transcription of inflammatory cytokine genes is commonly dependent on NF-kappa B (4). As an initial experiment to determine whether activation of the TNF-alpha gene is directly dependent on NF-kappa B, we examined the time course of NF-kappa B activation by LPS (Fig. 2). We observed a low basal level of NF-kappa B activity. After addition of 20 µg/ml LPS, maximum NF-kappa B activity was reached at 30 min and declined thereafter. Thus, as seen by the comparison of Figs. 1 and 2, there was a substantial lag between the activation of NF-kappa B and the production of TNF-alpha protein.


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  		Fig. 2.   Time course of LPS activation of nuclear factor-kappa B (NF-kappa B) in vascular smooth cells in culture. A: electrophoretic mobility shift assay. Whole cell extracts were made of cells stimulated by LPS for the indicated time and analyzed by the electrophoretic mobility shift assay. Lane 1, unstimulated control; lanes 2-9, after incubation with LPS for the indicated time; lane 10, incubation of the 30 min sample in the presence of 100-fold excess of unlabeled NF-kappa B oligonucleotide; lane 11, incubation of the 30-min sample in the presence of 100-fold excess of unlabeled AP-1 oligonucleotide. B: quantitation of NF-kappa B activation in smooth muscle cells. Time course of NF-kappa B activation by LPS was determined for smooth muscle cells from 3 rats. The amount of NF-kappa B present in each lane was determined by densitometry, normalized to control value, and plotted by means ± SE.

To determine the time course of the effect of LPS on TNF-alpha mRNA, RNase protection assays were carried out. Total RNA was prepared from vascular smooth muscle cells in culture to which 20 µg/ml LPS had been added and allowed to incubate for the indicated time. The probe set used for this experiment contained probes for eleven cytokine genes (IL-1alpha , IL-1beta , TNF-beta , IL-3, IL-4, IL-5, IL-6, IL-10, TNF-alpha , IL-2, and interferon-gamma ) and two housekeeping genes (L32 and GAPDH).

Exposure of rat vascular smooth muscle cells to LPS results in increased IL-6, TNF-alpha , and IL-alpha mRNAs (Fig. 3). TNF-alpha message increases with time starting ~25 min after LPS addition and continuing to 180 min. A linear least squares analysis of the TNF-alpha signal beginning at 20 min after LPS stimulation indicated an x-intercept of 21 min, consistent with NF-kappa B directly activating transcription of the TNF-alpha gene. Thus onset of the increase of TNF-alpha message correlates with NF-kappa B activation. The amount of IL-6 mRNA also has increased 30 min after LPS stimulation, consistent with this gene also being directly activated by NF-kappa B


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  		Fig. 3.   Time course of TNF-alpha mRNA production. Cells were incubated with 20 µg/ml LPS added to the culture medium or without LPS (control). At the indicated times the medium was removed and RNA was prepared. A: RNase protection assay. RNA was analyzed for the presence of cytokine RNAs by RNase protection assay using probes for the following cytokines: interleukin (IL)-1alpha , 432 nucleotides (nt); IL-1beta , 390 nt; TNF-beta , 351 nt; IL-3, 315 nt; IL-4, 285 nt; IL-5, 255 nt; IL-6, 231 nt; IL-10, 210 nt; TNF-alpha , 189 nt; IL-2, 171 nt; interferon-gamma , 158 nt; L32, 141 nt; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 126 nt. Each lane contained 10 µg of total RNA. The sizes of the protected fragments are: IL-1alpha , 403 nt; IL-6, 202 nt; TNFalpha nt; L32, 112 nt; GAPDH, 97 nt. Time course shown is typical of the results obtained with vascular smooth muscle cells isolated from 3 rats. B: TNF-alpha bands visualized after prolonged exposure of the autoradiogram in A. C: uncorrected densitometric tracings of the TNF-alpha bands. Line 1, 15 min; line 2, 30 min; line 3, 60 min; line 4, 120 min; line 5, 180 min. Tracings cover the region from just ~225 nt to 125 nt. Note the anomalously low peak at 60 min and the correspondingly low amount of houskeeping genes at that point. D: densitometric analysis of cytokine production after LPS stimulation. Data were combined from 3 experiments using vascular smooth muscle cells from 3 rats. Area of the peaks from densitometric analysis was corrected for loading errors by normalization to a constant value for L32 and plotted by means ± SE.

By contrast, the appearance of IL-1alpha mRNA was delayed. The delay suggests that activation of the IL-1alpha gene in this system is a downstream event in the amplification cascade initiated by LPS. TNF-alpha has been shown to stimulate production of IL-1alpha in vascular smooth muscle cells (26). Our results are consistent with this observation; the first major increase in IL-1alpha mRNA occurs at 120 min, the time at which secreted TNF-a protein was first detected (Figs. 1B and 3B).

Aspirin does not inhibit NF-kappa B activation and does not affect the secretion of TNF-alpha protein. We attempted to test the hypothesis that activation of the TNF-alpha and IL-6 genes is dependent on NF-kappa B by inhibiting the activation NF-kappa B with 20 mM aspirin. This concentration of aspirin has been shown to inhibit activation of NF-kappa B in several systems (11, 13). However, although aspirin substantially reduced both basal and bradykinin-induced production of 6-keto-PGF1alpha (Table 1), aspirin did not affect the activation of NF-kappa B by 20 µg/ml LPS. Because 2 µg/ml LPS also induced substantial production of TNF-alpha , we repeated the experiment using that concentration of LPS (Fig. 4). Analysis of experiments from cells taken from four different rats showed no significant difference in NF-kappa B activation at either 30 or 180 min, indicating that aspirin did not prevent LPS activation of NF-kappa B.

                              
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  		Table 1.   Effect of aspirin on the production of 6-keto-PGF1alpha from rat vascular smooth muscle cells



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  		Fig. 4.   Aspirin (ASP) fails to block NF-kappa B activation. A: electrophoretic mobility shift assay of whole cell extracts of rat vascular smooth muscle cells. Cells were incubated with or without 20 mM ASP 30 min before addition of 2 µg/ml LPS and incubation for the indicated time. Competition experiments with a 100-fold excess of unlabeled homologous or heterologous oligonucleotide, lanes 7 and 8, were carried out with an extract of cells stimulated with LPS for 30 min without ASP. B: quantitation of NF-kappa B activation in smooth muscle cells. Open bars, minus ASP; solid bars, plus ASP; Con, control. Amount of NF-kappa B present in each lane was determined by densitometry, normalized to the minus ASP control value for each experiment, and plotted by means ± SE. Graph represents the results of smooth muscle cells derived from 4 rats.

Analysis of the amount of IL-6 and TNF-alpha mRNA present at 30 and 180 min after LPS addition showed no difference at 180 min. In the presence of 20 mM aspirin, the amount of IL-6 mRNA found 30 min after LPS addition was 39% of the amount of IL-6 mRNA found 30 min after LPS addition in the absence of aspirin (P < 0.05) (Fig. 5). Similarly, the amount of TNF-alpha mRNA found 30 min after LPS addition in the presence of 20 mM aspirin was 44% of that found in the absence of aspirin (P < 0.05, Fig. 6).


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  		Fig. 5.   Effect of ASP on LPS-induced production of IL-6 mRNA. A: RNase protection assay. Rat smooth muscle cells were incubated with or without 20 mM ASP before addition of 20 µg/ml LPS and incubation for the indicated time. Yeast tRNA, lane 7, was hybridized as a negative control. B: quantitative analysis of IL-6 RNA. Amount of IL-6 present in each lane was determined by densitometric analysis using the ImageQuant software accompanying the phosphorimager and was corrected for loading errors by normalization to a constant value for L32 and plotted by means ± SE. Data represent determinations on smooth muscle cells derived from 4 rats. *P < 0.05 vs. LPS 30 min.



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  		Fig. 6.   Effect of ASP on LPS-induced production of TNF-alpha mRNA. A: RNase protection assay. Rat smooth muscle cells were incubated with or without 20 mM ASP before addition of 20 µg/ml LPS and incubation for the indicated time. Yeast tRNA, lane 7, was hybridized as a negative control. To avoid having the TNF-alpha signal obscured by high background arising from breakdown of the IL-6 band, a probe set was used, in which TNF-alpha was the largest protected band. B: quantitative analysis of TNF-alpha RNA. Amount of each protected band was determined by densitometric analysis using the ImageQuant software accompanying the phosphorimager, corrected for loading errors by normalization to a constant value for L32 and plotted by means ± SE. Data represent determinations on smooth muscle cells derived from 4 rats. *P < 0.05 vs. LPS 30 min. A repeat of the experiment with smooth muscle cells derived from a second set of 4 rats gave similar results.

Western analysis (Fig. 7) showed that aspirin did not affect the translation of TNF-alpha mRNA, because lysates from LPS-stimulated cells showed comparable amounts of TNF-alpha protein whether or not aspirin was present. Dot-blot analysis (not shown) indicated that TNF-alpha protein was secreted into the medium in the presence of aspirin.


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  		Fig. 7.   ASP fails to block TNF-alpha biosynthesis. Western analysis of rat vascular smooth muscle cells. Cells were incubated with 20 mM ASP for 20 min before the addition of 2 µg/ml LPS as indicated. Detection was by chemiluminescence. Results are typical of 3 independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the above experiments showed that LPS induces the activation of NF-kappa B and the dose-dependent production of TNF-alpha by vascular smooth muscle cells in culture. In response to LPS stimulation, increased TNF-alpha mRNA was detected. The initiation of the increase in TNF-alpha mRNA correlated with the activation of NF-kappa B, consistent with the TNF-alpha gene being regulated by NF-kappa B.

Increased IL-6 and IL-1alpha mRNA were also produced in response to LPS; the timing of the onset of the IL-6 increase was also consistent with the gene activation being responsive to NF-kappa B. The increase in IL-1alpha mRNA appeared to be a downstream event. Thus vascular smooth muscle cells were shown capable of producing multiple inflammatory cytokines in response to LPS, indicating the desirability of identifying inflammatory-control agents in this cell type.

The anti-inflammatory effects of aspirin and other salicylates are achieved through multiple mechanisms. In addition to inhibiting prostaglandin-producing cyclooxygenases and preventing NF-kappa B activation, salicylates have also been shown in cardiac fibroblasts to inhibit transcription without inhibiting the activation of NF-kappa B (9). Another mechanism, inhibition of translation, has been shown in the case of IL-1beta induction of nitric oxide synthase in hepatocytes (22). In this system also, the activation of NF-kappa B was unaffected by the presence of salicylates.

The question whether salicylates are capable of inhibiting the activation of NF-kappa B in vascular smooth muscle cells remains unresolved. It has been reported that aspirin inhibits inducible nitric oxide synthase expression and TNF-alpha release from cultured bovine vascular smooth muscle cells stimulated by Il-1beta . In addition, aspirin blocks the activation of NF-kappa B in this system (23). Aspirin also blocks NF-kappa B activation by platelet-derived growth factor in human vascular smooth muscle cells in vitro (16). By contrast, it has been reported that in rat vascular smooth muscle cells neither salicylate nor aspirin affects inducible nitric oxide synthase mRNA expression or NF-kappa B activation in response to IL-1beta and TNF-alpha stimulation (12). Our results are more in agreement with the third report because aspirin did not prevent NF-kappa B activation by LPS and had only transient inhibitory effects on IL-6 and TNF-alpha mRNA levels. The basis for the discrepancies may be a species effect, a result of different aspirin preincubation times, or the result of different signal transduction pathways being activated by different ligands.

Clarification of these points will require the identification of the signal transduction pathways leading to the activation of NF-kappa B. These pathways and the regulation of these pathways will probably vary with ligand and with cell type. In macrophages, salicylates activate phosphorylation of p38 MAPK. The activated p38 MAP kinase inhibits phosphorylation of Ikappa Balpha , thereby preventing its degradation and the release of activated NF-kappa B (1, 24). The p38 MAPK-specific inhibitor SB203580, abolishes salicylate inhibition of NF-kappa B activation. However, in rat vascular smooth muscle cells, LPS activates phosphorylation of p38 MAPK (28). These results suggest that in vascular smooth muscle cells, pathways exist that can override activated p38 MAPK inhibition of NF-kappa B activation. One such potential pathway is known: NF-kappa B inducing kinase activates Ikappa B kinase through a signal transduction pathway that is independent of the MAPK cascade (18).

The failure of aspirin to inhibit the activation of NF-kappa B and subsequent release of inflammatory cytokines in rat vascular smooth muscle cells suggests that cell-type specific anti-inflammatory agents may need to be developed to retard the development of atherosclerosis.


    ACKNOWLEDGEMENTS

This work was supported by grants from the Medcen Foundation of Central Georgia to K. Detmer and W. H. Newman.


    FOOTNOTES

Address for reprint requests and other correspondence: K. Detmer, Division of Basic Medical Sciences, Mercer Univ. School of Medicine, 1550 College St., Macon, GA 31207 (E-mail: detmer_km{at}mercer.edu).

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 26 June 2000; accepted in final form 5 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 281(2):H661-H668
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society


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