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Endocrinology Vol. 148, No. 9 4371-4384
Copyright © 2007 by The Endocrine Society

Aldose Reductase-Regulated Tumor Necrosis Factor-{alpha} Production Is Essential for High Glucose-Induced Vascular Smooth Muscle Cell Growth

Kota V. Ramana, Ravinder Tammali, Aramati B. M. Reddy, Aruni Bhatnagar and Satish K. Srivastava

Department of Biochemistry and Molecular Biology (K.V.R., R.T., A.B.M.R., S.K.S.), University of Texas Medical Branch, Galveston, Texas 77555-0647; and Institute of Molecular Cardiology (A.B.), University of Louisville, Louisville, Kentucky 40202

Address all correspondence and requests for reprints to: Satish K. Srivastava, Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-0647. E-mail: ssrivast{at}utmb.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Diabetes is associated with increased generation of cytokines and tissue inflammation, but it is unclear how increased cytokine synthesis is causally related to the development of diabetic complications. Here, we report that exposure to high (25 mM) glucose, but not iso-osmotic concentrations of mannitol or 3-methyl glucose, increased TNF-{alpha} secretion by rat and human aortic smooth muscle cells in culture. The increase in TNF-{alpha} production was prevented by actinomycin D and cycloheximide, indicating transcriptional activation of TNF-{alpha} gene. High glucose (HG)-induced TNF-{alpha} release was specifically inhibited by protein kinase C (PKC)-{delta} inhibitor (Rottlerin; EMD Biosciences, San Diego, CA), but not PKC-ß2 inhibitor (CGP53353; Tocris Cookson Inc., Ellisville, MO), indicating the possible involvement of PKC-{delta} in HG signaling. TNF-{alpha} secretion was also prevented by pretreating cells with aldose reductase (AR) inhibitors, sorbinil or tolrestat and in cells treated with antisense AR mRNA. Inhibition of AR also prevented the increase in TNF-{alpha} mRNA. Addition of anti-TNF-{alpha} antibodies or soluble TNF-{alpha} receptors 1 and 2 to the medium or RNA interference ablation of TNF-{alpha} attenuated nuclear factor-{kappa}B activation and prevented HG-stimulated cell growth. These data indicate that AR is required for HG-induced TNF-{alpha} synthesis and release. In vivo, the release of TNF-{alpha} by HG leading to autocrine stimulation of TNF-{alpha} synthesis may be a critical step in the development of the cardiovascular complications of diabetes. Interruption of the autocrine effects of TNF-{alpha} may be a useful strategy for treating diabetic vasculopathies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CARDIOVASCULAR DISEASES ARE the major cause of increased mortality and morbidity associated with insulin resistance and diabetes (1, 2). Biochemical changes associated with metabolic syndrome induce hypertension and dyslipidemia. These changes in turn accelerate atherogenesis and enhance arterial stiffening, which together contribute to the elevated risk and incidence of myocardial infarction and stroke in diabetics (1, 3). Vascular changes contribute also to the development of several other diabetic complications, such as retinopathy, nephropathy, and neuropathy (1, 4). Although the mechanisms by which insulin resistance and diabetes affect cardiovascular diseases remain unclear, recent investigations suggest that inflammation could be a key contributor, and that both systemic and local (tissue-specific) inflammatory responses mediate diabetic changes (5). Even without diabetes, tissue pathology associated with hypertension (5, 6), atherosclerosis (7), and heart failure (8, 9) could in part be attributed to cardiovascular inflammation. However, this inflammation is further exaggerated by diabetes, thereby considerably enhancing the progression and burden of cardiovascular diseases (1, 5, 10).

Inflammatory markers that predict the development of diabetes (11, 12) and the plasma levels of cytokines, such as TNF-{alpha}, are elevated in both type 1 (13, 14) and type 2 diabetics (15, 16). Increased tissue levels of TNF-{alpha} have also been reported in the skeletal muscle of type 2 diabetics (17), in the glomeruli of streptozotocin-treated rats (18), and in the visceral fat and thoracic aorta of obese Zuker rats (19), indicating that diabetes is associated with an increase in systemic as well as local inflammation. This increase in cytokines is thought to contribute to the development of insulin resistance, as well as to the manifestation and severity of diabetes. In agreement with this view, it has been shown that TNF-{alpha} null mice are resistant to obesity-induced insulin resistance (20, 21) and that mice lacking the TNF-{alpha} receptor-2 gene display less severe hyperinsulinemia and weight gain than their wild-type counterparts (22). Moreover, the observations that an increase in systemic cytokine levels by hepatocyte-specific overexpression of I{kappa}B kinase induces local and systemic insulin resistance and hyperglycemia (23), and that TNF-{alpha} blocks the action of insulin by serine phosphorylating insulin receptor substrate 1 (24, 25, 26) further support the notion that inflammation plays a key role in establishing insulin resistance. In addition, TNF-{alpha} and other cytokines also appear to contribute to the development of secondary diabetic complications because TNF-{alpha} levels are elevated in diabetic retinopathy (27), and a progressive increase in inflammatory markers correlates with microangiopathy in type 2 diabetics (28).

Despite overwhelming evidence linking inflammation, insulin resistance, and diabetic complications, the processes that increase cytokine generation in diabetes have not been identified, and it remains unclear whether inflammatory cytokines are markers or mediators of diabetic vasculopathy. Therefore, we studied the direct effects of high glucose (HG) (one of the cardinal features of diabetes) on TNF-{alpha} synthesis and secretion in vascular smooth muscle cells (VSMCs). Our results show that hyperglycemia increases TNF-{alpha} synthesis and that the autocrine effects of TNF-{alpha} secreted from these cells mediate the hyperproliferative effects of HG.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
PBS, penicillin/streptomycin solution, trypsin and fetal bovine serum (FBS), DMEM, and Opti-MEM were purchased from Life Technologies (Gaithersburg, MD). Antibodies against rat and human TNF-{alpha} and soluble TNF receptor (sTNF-R)-1 and sTNF-R2 were obtained from Research Diagnostics Inc. (Flanders, NJ). D609, colchicine C, and nuclear factor (NF)-{kappa}B inhibitors (sc-3060) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rottlerin and CGP53353 were obtained from EMD Biosciences (San Diego, CA) and Tocris Cookson Inc. (Ellisville, MO), respectively. Sorbinil and tolrestat were gifts from Pfizer Inc. (New York, NY) and American Home Products Corp. (Glendale Heights, IL), respectively. Consensus oligonucleotide for NF-{kappa}B transcription factor was obtained from Promega Corp. (Madison, WI). All other reagents were of the highest purity available.

Cell culture
Rat VSMCs were isolated from healthy rat aorta and characterized by smooth muscle cell-specific {alpha}-actin expression. Human VSMCs (T/G HA-VSMC) were obtained from American Type Culture Collection (Manassas, VA). The VSMCs were maintained and grown in endotoxin-free DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 C in a humidified atmosphere of 5% CO2 as described before (29, 30). For glucose stimulation experiments, the cells were serum starved in medium containing 0.1% serum and 5.5 mM glucose. After 24 h, the medium was replaced with fresh medium containing glucose 25 mM for HG and 5.5 mM for normal glucose (NG) with aldose reductase inhibitors (ARIs) or carrier (25% dimethylsulfoxide). The HG medium was prepared freshly before incubation by adding 19.5 mM glucose to DMEM that already contained 5.5 mM glucose. The HG medium was prepared fresh and used immediately to avoid microbial contamination. The cells at the passage number 8–12 were used for this study.

Quantification of TNF-{alpha} in VSMC culture media
Growth-arrested VSMCs in NG (5.5 mM) were treated without or with additional 19.5 mM glucose (HG, 25 mM), 19.5 mM mannitol [19.5 mM mannitol and 5.5 mM glucose (M)], or 19.5 mM 3-O-methyl glucose [19.5 mM 3-O-methyl glucose and 5.5 mM glucose (3-OMG)] for different time intervals (0–24 h) at 37 C in a humidified CO2 incubator. Cells treated with lipopolysaccharide (LPS) (1 µg/ml) in 5.5. mM glucose were used as the positive control. Levels of TNF-{alpha} in the culture media were measured using a rat and human TNF-{alpha} sandwich ELISA kits obtained from BD Biosciences and RayBiotech, Inc. (Norcross, GA), respectively, according to the manufacturer’s instructions. Known concentrations of rat and human TNF-{alpha} were used to generate the standard curves. The assay was linear between 5 and 1000 pg/ml. This range was well within the range of TNF-{alpha} levels measured in the present study. The reaction was initiated by the addition of 0.1 ml of the undiluted media, stopped and read at 450 nm on an ELISA plate reader. The results obtained are expressed as picogram per milliliter of the medium.

RT-PCR analysis of TNF-{alpha}
Total RNA from VSMCs was isolated using the RNeasy kit (QIAGEN, Inc., Valencia, CA) as per supplier’s instructions. Aliquots of RNA (1.5 µg) isolated from each sample were reverse transcribed with Omniscript and Sensiscript reverse transcriptase one-Step RT PCR system with HotStarTaq DNA polymerase (QIAGEN, Inc.) at 55 C for 30 min followed by PCR amplification. The rat TNF-{alpha} oligonucleotide primer sequences were: 5'-ATG AGC ACG GAA AGC ATG ATC-3' (sense) and 5'-AGT AGA CCT GCC CGG ACT CCG-3' (antisense) for TNF-{alpha}; 5'-TGAGACCTTCAACACCCCAG-3' and 5'-TTCATGAGGTAGTCTGTCAGGTCC-3' for ß-actin. The human-specific oligonucleotide primer sequences were: 5'-GAG TGA CAA GCC TGT AGC CCA TGT TGT AGC A-3' (sense) and 5'-GCA ATG ATC CCA AAG TAG ACC TGC CCA GAC T-3' (antisense) for TNF-{alpha}; and 5'-GTT TGA GAC CTT CAA CAC CCC-3' and 5'-GTG GCC ATC TCC TGC TCG AAG TC-3' for ß-actin. The PCR was performed in a GeneAmp 2700 thermocycler (Applied Biosystems, Foster City, CA) under the following conditions: initial denaturation at 95 C for 15 min; 35 cycles of 94 C for 30 sec, 62 C for 30 sec, 72 C for 1 min, and then 72 C for 5 min for final extension. PCR products were electrophoresed with 2% agarose-1x Tris-acetate-EDTA (TAE) gels containing 0.5 µg/ml ethidium bromide. Densitometric analyses were performed to quantify band intensity using the Kodak 1D Image Analysis Software (Eastman Kodak Co., Rochester, NY).

Determination of NF-{kappa}B activation
The cytosolic as well as nuclear extracts were prepared as described before (29). The NF-{kappa}B activity was determined using the calorimetric nonradioactive NF-{kappa}B p65 transcription factor assay kit (CHEMICON International, Inc., Temecula, CA) as per the supplier’s instructions. Briefly, a double-stranded biotinylated oligonucleotide containing the consensus sequence for NF-{kappa}B binding (5'-GGGACTTTCC-3') was mixed with nuclear extract assay buffer provided. After incubation, the mixture (probe + extract + buffer) was transferred to the streptavidin-coated ELISA plate. The color developed read at 450 nm on an ELISA plate reader.

Determination of protein kinase C (PKC) activity
Membrane-bound total PKC activity was measured using the Promega SignaTECT total PKC assay system according to the manufacturer’s instructions and as described earlier (31). Briefly, aliquots of the reaction mixture containing 25 mM Tris-HCl (pH 7.5), 1.6 mg/ml phosphatidylserine, 0.16 mg/ml diacylglycerol (DAG), and 50 mM MgCl2 were mixed with [{gamma}-32P] ATP (3000 Ci/mmol, 10 µCi/µl), and incubated at 30 C for 10 min. The extent of phosphorylation was detected by measuring radioactivity retained on the paper.

Antisense ablation of aldose reductase (AR)
AR was ablated essentially as described earlier (29). Briefly, VSMCs grown in DMEM supplemented with 10% FBS were washed with Opti-MEM and incubated for 60 min with Opti-MEM media before transfection with antisense oligonucleotides. The cells were incubated with 1 µM AR antisense or scrambled control oligonucleotides using LipofectAMINE Plus (15 µg/ml) (Invitrogen, Carlsbad, CA) as the transfection reagent (TR). After 12 h, the medium was replaced with fresh DMEM (containing 10% FBS), and the samples were incubated for another 12 h. AR mRNA levels by RT-PCR and protein levels by Western blot analysis using anti-AR antibodies were measured to determine the extent of AR knockdown.

RNA interference ablation of TNF-{alpha}
The ablation of TNF-{alpha} mRNA was performed essentially as described earlier (31). Briefly, VSMCs were incubated with serum-free medium containing the predesigned rat TNF-{alpha} small interfering RNA (siRNA) (identification no. 189047; Ambion, Inc., Austin, TX) or scrambled siRNA to a final concentration of 100 nM and the RNAiFect TR (QIAGEN, Inc.). After 15 min of incubation at 25 C, the medium was aspirated and replaced with fresh DMEM containing 10% serum. The cells were cultured for 48 h at 37 C, and TNF-{alpha} expression was determined by measuring TNF protein by Western blot analysis using anti-TNF-{alpha} antibodies.

Determination of cell growth
The cells were grown to confluency in DMEM, harvested by trypsinization, and plated at a density of 5000 cells per well in a 96-well plate. When the cells were 60–80% confluent, they were growth arrested in 0.1% FBS. The low-serum levels were maintained during growth arrest to prevent slow apoptosis that accompanies complete serum deprivation. After 24 h, the cells were incubated without or with anti-TNF-{alpha} antibodies (1 µg/ml) or sTNF-R1 and -R2 proteins (0.5 µg/ml each) for 30 min, followed by incubation with HG, mannitol, or 3-O-methyl glucose for 24 h at 37 C in a humidified CO2 incubator. Cell viability was determined by cell counts and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, as described earlier (29). Briefly, for MTT assay, 25 µl of 5 mg/ml MTT was added to each well of the 96-well plate plated with VSMCs. The plate was incubated at 37 C for 2 h. The formazan granules generated by the only live cells were dissolved in 100% dimethylsulfoxide, and absorbance at 562 nm was monitored using an ELISA plate reader. For cell counting, the loss of membrane integrity indicated by the inability of the cells to exclude trypan blue was used to measure cell viability using a hemocytometer under a microscope.

Statistical analysis
Data are presented as mean ± SEM, and the P values were determined using the unpaired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
VSMCs cultured in HG secrete TNF-{alpha}
To examine how HG affects TNF-{alpha} synthesis and secretion, we measured changes in TNF-{alpha} message and protein in VSMCs incubated with HG. Because TNF-{alpha} synthesized by VSMCs is rapidly secreted (32, 33), we also measured changes in TNF-{alpha} levels in the culture medium. As shown in Fig. 1Go, within 3 h of incubation, HG led to an appreciable increase in TNF-{alpha} levels in the medium; thereafter, the levels of TNF-{alpha} in the medium continued to increase progressively for the next 24 h (maximal observation time). When the cells were stimulated with LPS as a positive control, a similar, though more pronounced, increase in TNF-{alpha} level in the medium was observed (Fig. 1Go). TNF-{alpha} was not released by cells treated with equimolar amounts of either mannitol or 3-O-methyl glucose, indicating that the stimulatory effects of HG are not due to an increase in medium osmolarity.


Figure 1
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  		FIG. 1. VSMCs exposed to HG secrete TNF-{alpha}. Rat aortic smooth muscle cells were growth arrested by serum starvation (0.1%) of 24 h in 5.5. mM glucose medium (NG). The cells were then either left untreated or treated with 19.5 mM glucose (HG), mannitol (M), or 3-O-methyl glucose (3-OMG) for indicated times. In separate positive control experiments, VSMCs cultured in NG were treated with LPS (1 µg/ml). The level of TNF-{alpha} in the culture media was determined by ELISA as described in Materials and Methods. The data represent mean ± SEM (n = 4). * and #, P < 0.05; and ** and ##, P < 0.001.

 
To determine whether HG-induced TNF-{alpha} secretion requires de novo protein synthesis or transcriptional activation, we preincubated VSMCs with inhibitors of protein synthesis (cycloheximide or actinomycin D). Cells were incubated with protein synthesis inhibitors before stimulation by glucose, and the levels of TNF-{alpha} in the medium were measured. Incubation with HG for 6–24 h led to a 6- to 12-fold increase in TNF-{alpha} in the medium compared with cells incubated with NG (Fig. 2Go). Treatment with actinomycin D or cycloheximide significantly attenuated HG-induced TNF-{alpha} release. The basal levels of TNF-{alpha} in 5.5 mM glucose were not affected by actinomycin D or cycloheximide. These observations suggest that the increase in TNF-{alpha} by HG is due to de novo synthesis of new protein. To confirm that HG increases transcription of the TNF-{alpha} gene, changes in TNF-{alpha} message were measured by RT-PCR. As shown in Fig. 3Go, exposure to HG for 3 h led to an approximate 3-fold increase in the TNF-{alpha} message. The levels of TNF-{alpha} message increased steadily for the next 24 h; during this period, a maximum of an approximate 6-fold increase in the transcript was observed. Under identical conditions, treatment with equimolar concentrations of mannitol did not increase TNF-{alpha} message, indicating that the effect of HG is independent of changes in medium osmolarity.


Figure 2
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  		FIG. 2. Effect of cycloheximide and actinomycin D on HG-induced TNF-{alpha} secretion. Growth-arrested VSMCs in NG (5.5 mM) were pretreated without or with 2 µg/ml cycloheximide or 10 µg/ml actinomycin D, or vehicle (control), followed by the addition of 19.5 mM glucose (HG) and incubation for the indicated times. The level of TNF-{alpha} in the culture media was determined by ELISA. Data represent mean ± SEM (n = 4). **, P < 0.001 vs. HG-treated cells; and #, P < 0.01 vs. HG and cycloheximide-treated cells.

 

Figure 3
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  		FIG. 3. HG increases TNF-{alpha} mRNA. The growth-arrested VSMCs in NG (5.5 mM) were treated without or with 19.5 mM glucose (HG) or mannitol (M) for the indicated times. Total RNA was isolated from the cells, and 1.5 µg from each sample was reverse transcribed with Omniscript and Sensiscript reverse transcriptase one-Step RT PCR system with HotStarTaq DNA polymerase at 55 C for 30 min, followed by PCR amplification, as described in Materials and Methods. The PCR products were electrophoresed on 2% agarose-1x TAE gels containing 0.5 µg/ml ethidium bromide, and the band intensity was quantified by densitometric analyses. Amplification of the ß-actin gene was used as a sample control. The data represent mean ± SEM (n = 3, where n is the number of independent experimental data sets of RT-PCR analysis). Three agarose gels were run, and only one representative photograph is presented. *, P < 0.001 vs. intensity before exposure to HG (t = 0).

 
Mechanism of increased TNF-{alpha} synthesis
Because transcription of the TNF-{alpha} gene is regulated by NF-{kappa}B (34), we next examined whether up-regulation of TNF-{alpha} by HG was mediated by NF-{kappa}B. For this, serum-starved cells in 5.5 mM glucose were either left untreated or treated with an additional 19.5 mM glucose, and NF-{kappa}B activity in the cells was measured by EMSA. These measurements showed that HG activates NF-{kappa}B (Fig. 4AGo). An increase in NF-{kappa}B activity was evident within 3 h of incubation with HG, and the transcription factor was maintained in a state of approximately 10-fold activation for 24 h. Interestingly, a small but statistically significant increase in NF-{kappa}B was also observed in cells treated with mannitol, and no increase with 3-OMG indicates that an increase in osmolarity could also activate NF-{kappa}B. However, because mannitol and 3-OMG did not induce TNF-{alpha} secretion (Fig. 1Go), we conclude that NF-{kappa}B activation in HG depends upon metabolism and is linked to increased transcription of the TNF-{alpha} gene.


Figure 4
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  		FIG. 4. HG-induced TNF-{alpha} secretion is mediated by PKC and NF-{kappa}B. A, Serum-starved cells were maintained in 5.5 mM glucose or stimulated with HG, mannitol (M), or 3-O-methyl glucose (3-OMG) for the indicated times, and nuclear extracts were prepared. In each sample, NF-{kappa}B was determined calorimetrically using a nonradioactive NF-{kappa}B p65 transcription factor assay kit. Individual EMSA bands are shown in the inset. B, Growth-arrested VSMCs in 5.5 mM glucose (X) were either left untreated or treated with an additional 19.5 mM glucose ({blacksquare}), mannitol (bullet), or 3-O-methyl glucose ({Delta}) for indicated times. PKC activation was determined by the amount of PKC bound to the membrane using the Promega SignaTECT kit. Data represent mean ± SEM (n = 4). *, P < 0.001 vs. NG; and #, P < 0.001 vs. HG.

 
The activation of NF-{kappa}B in VSMCs is known to be mediated by PKC (30, 35); therefore, we next examined whether PKC stimulation precedes NF-{kappa}B activation in cells treated with HG. As shown in Fig. 4BGo, exposure to HG for less than 1 h increased membrane-bound PKC activity 4-fold. In HG, maximal (6- to 7-fold) PKC activation was observed at 3 h, after which PKC activity began to decrease. Even after 24 h of stimulation in HG, the PKC levels in cells remained significantly higher than in cells maintained in 5.5 mM glucose. No increase in membrane-bound PKC was observed in cells treated with mannitol or 3-OMG, indicating that osmotic changes do not lead to PKC activation and that the small increase in NF-{kappa}B in mannitol-treated cells is independent of PKC.

To probe the role of PKC and NF-{kappa}B in increasing TNF-{alpha} synthesis/secretion, we determined whether treatment with PKC or NF-{kappa}B inhibitors would prevent TNF-{alpha} secretion. When cells were treated with a nonspecific total PKC inhibitor, calphostin C or a NF-{kappa}B inhibitor (sc-3060), HG-induced increases in the levels of TNF-{alpha} in the medium were completely prevented (Fig. 5AGo). Treatment with these inhibitors did not affect the basal levels of TNF-{alpha} in the medium. These observations suggest that activation of PKC and NF-{kappa}B is essential for HG-induced increase in the TNF-{alpha} in the culture medium. Previously we have shown that AR inhibition prevents HG-induced activation of PKC-ß2 and PKC-{delta} isozymes in VSMCs (31). To investigate the specific isozymes of PKC involved in the HG-induced production of TNF-{alpha}, we incubated VSMCs with specific inhibitors of PKC-ß2 (CGP 53353) and {delta} (Rottlerin), and measured the TNF-{alpha} release in the culture media. The results shown in Fig. 5BGo suggest that inhibition of PKC-{delta}, but not -ß2, suppressed HG-induced production of TNF-{alpha} in VSMCs, suggesting the role of PKC-{delta} in HG-induced synthesis of TNF-{alpha}.


Figure 5
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  		FIG. 5. Mechanism of HG-induced TNF-{alpha} production. A, Growth-arrested VSMCs in 5.5 mM glucose (NG) were preincubated for 1 h without or with apocyanin (25 µM), D609 (100 µM), calphostin C (0.2 µM), N-acetyl cysteine (NAC) (10 mM), or NF-{kappa}B inhibitor (18 µM), respectively, followed by the addition of 19.5 mM glucose. B, Growth-arrested VSMCs in NG were preincubated with PKC-ß2 inhibitor (CGP 53353; 3 µM) and PKC-{delta} inhibitor [Rottlerin (Rott); 20 µM] for 30 min, followed by the addition of 19.5 mM glucose. At indicated time points, the levels of TNF-{alpha} were measured as described in Materials and Methods. The data represent mean ± SEM (n = 4). *, P < 0.01; and **, P < 0.001 vs. HG-treated cells.

 
Previous studies show that activation of the PKC-NF-{kappa}B pathway in HG depends in part upon the activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (36). In addition, our previous studies show that PKC activation in HG is accompanied by the generation of DAG, which is decreased by inhibiting phosphatidyl choline-phospholipase C (PLC) and AR (31). To examine whether similar signaling pathways regulate TNF-{alpha} generation in HG, we examined the effects of inhibiting PLC, NADPH oxidase, and AR on TNF-{alpha} levels. As shown in Fig. 5AGo, incubation with the phosphatidyl choline-PLC inhibitor markedly decreased TNF-{alpha} levels. A similar decrease in TNF-{alpha} was observed in cells treated with the NADPH oxidase inhibitor, apocyanin, or the antioxidant, N-acetylcysteine. Collectively, these observations support a mechanism in which HG increases TNF-{alpha} levels by stimulating an intracellular signaling pathway that depends upon the activation of PLC and NADPH oxidase. That this mechanism requires AR is suggested by data presented in Fig. 6Go, which show that either pharmacological inhibition of AR by sorbinil or tolrestat or antisense ablation of the AR gene prevents HG-induced TNF-{alpha} levels. Treatment with ARIs did not affect basal levels of TNF-{alpha} in media containing 5.5 mM glucose, mannitol, or 3-OMG. Moreover, HG-induced TNF-{alpha} levels were not prevented in untransfected cells or cells incubated with the transfection medium or transfection medium containing scrambled oligonucleotides. These observations attest to the specific dependence of TNF-{alpha} generation on AR activity. Together, the signaling studies described previously suggest that HG increases TNF-{alpha} secretion by increasing AR and PLC. These processes stimulate PKC and NF-{kappa}B, which in turn increase transcription of the TNF-{alpha} gene.


Figure 6
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  		FIG. 6. AR regulates HG-induced TNF-{alpha} production. A, Growth-arrested VSMCs in 5.5 mM glucose (NG) were preincubated for 1 h without or with sorbinil or tolrestat (10 µM each), followed by the addition of glucose (19.5 mM) and incubation for the indicated times. The inset in B shows AR protein and mRNA levels in the antisense transfected VSMCs. B, AR antisense ablated VSMCs were incubated with HG for the indicated times. The data represent mean ± SEM (n = 4). **, P < 0.001 vs. cells incubated in HG. AN, AR-antisense oligo; C, control; SC, scrambled.

 
TNF-{alpha} regulates hyperglycemic signaling and mitogenesis
Given our observation that HG induces VSMCs to secrete TNF-{alpha}, we tested whether this increase in TNF-{alpha} mediates HG-induced cell signaling and growth. To delineate the potentially autocrine effects of TNF-{alpha}, we interrupted TNF-{alpha} signaling by adding either anti-TNF-{alpha} antibodies or sTNF-R1 and -R2 to the incubation medium. For assessing changes in cell signaling, we examined changes in PKC and NF-{kappa}B, both of which were dramatically activated by HG (Fig. 4Go, A and B). Because our results show that stimulation with HG for 1 h causes a dramatic increase in PKC (Fig. 4BGo), we examined early changes in PKC. As shown in Fig. 7AGo, incubation with HG, but not mannitol or 5.5 mM glucose, caused a 2- to 3-fold increase in the membrane activity of PKC in 1 h. This early increase in PKC was not inhibited by either the anti-TNF-{alpha} antibody or sTNF-R. However, these inhibitors led to a remarkable decrease in PKC activity between 3 and 24 h of incubation in HG, at the time (3 h or more) when the concentration of TNF-{alpha} began to increase significantly in the medium (Fig. 1Go). These observations support the notion that PKC activation is in part due to autocrine stimulation of TNF-{alpha}. That activation of PKC is associated with NF-{kappa}B activation is suggested by the observation that 3-h treatment with anti-TNF-{alpha} antibody or sTNF-R prevented NF-{kappa}B activation and that in the treated cells, NF-{kappa}B was maintained in the repressed state even after 24 h of incubation with HG. These data indicate that initial stimulation of PKC by HG is independent of TNF-{alpha}, but once PKC is stimulated and TNF-{alpha} is secreted, sustained activation of PKC and NF-{kappa}B is due to an autocrine effect of TNF-{alpha} (Fig. 7Go).


Figure 7
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  		FIG. 7. Activation of PKC and NF-{kappa}B by HG is mediated by TNF-{alpha}. A, Growth-arrested VSMCs in 5.5 mM glucose (NG) were either left untreated or stimulated with an additional 19.5 mM glucose (HG) in the absence or presence of 1 µg/ml anti-TNF-{alpha}-antibodies or 0.5 µg/ml each of sTNF-R1 and sTNF-R2 for indicated times. PKC activation was determined by the amount of PKC bound to the membrane using the Promega SignaTECT kit. B, Growth-arrested VSMCs in 5.5 mM glucose (NG) were either left untreated or stimulated with an additional 19.5 mM glucose (HG) in the absence and presence of 1 µg/ml anti-TNF-{alpha}-antibodies or 0.5 µg/ml each of sTNF-R1 and sTNF-R2 for the indicated times. Nuclear extracts were prepared, and NF-{kappa}B was determined by a calorimetric nonradioactive NF-{kappa}B p65 transcription factor assay kit as per the supplier’s instructions. Data represent mean ± SEM (n = 4). ab or AB, Antibodies.

 
Stimulation of PKC and NF-{kappa}B by TNF-{alpha} also led to an autocrine increase in TNF-{alpha} synthesis. Consistent with results shown in Fig. 3Go, exposure of VSMCs to HG led to a progressive increase in TNF-{alpha} mRNA, which was prevented by anti-TNF-{alpha} antibodies in the medium (Fig. 8Go). The increase in TNF-{alpha} was also prevented by pretreating the cells with sorbinil, suggesting that AR is an integral component of this feed-forward autocrine loop in which TNF-{alpha} secreted from smooth muscle cell stimulates its own synthesis.


Figure 8
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  		FIG. 8. Inhibition of AR interrupts autocrine stimulation of TNF-{alpha} synthesis by TNF-{alpha}. Growth-arrested VSMCs in 5.5 mM glucose (NG) were either left untreated or stimulated with an additional 19.5 mM glucose (HG) in the absence or the presence of 1 µg/ml anti-TNF-{alpha}-antibodies or sorbinil (10 µM) for indicated times. Total RNA was isolated from the cells, and 1.5 µg from each sample was reverse transcribed with Omniscript and Sensiscript reverse transcriptase one-Step RT PCR system with HotStarTaq DNA polymerase at 55 C for 30 min, followed by PCR amplification as described in Materials and Methods. The PCR products were electrophoresed with 2% agarose-1x TAE gels containing 0.5 µg/ml ethidium bromide, and the band intensity was quantified by densitometric analyses. Amplification of the ß-actin gene was used as a sample control. The data represent mean ± SEM (n = 3, where n is number of independent RT-PCR analyses). *, P < 0.001 vs. band intensity compared with HG at indicated times. Ab, Antibodies.

 
In addition to alteration in cell signaling, HG also increases VSMC growth (37, 38, 39, 40). Therefore, to test whether the mitogenic effects of glucose are also mediated by TNF-{alpha}, we stimulated serum-starved VSMCs with a medium containing 25 mM glucose, mannitol, or 3-OMG. For control, serum-starved cells were treated with medium containing 5.5 mM glucose. In comparison to cells stimulated with mannitol or 3-OMG or maintained in 5.5 mM glucose, cells incubated with HG displayed significantly higher growth rates as assessed by direct cell count and the MTT assay (Fig. 9Go, A and B). The increase in cell density in HG medium was significantly prevented by treating the cells with either the anti-TNF-{alpha} antibodies or the sTNF-R. Although the cells treated with anti-TNF-{alpha} antibodies and sTNF-R displayed slightly higher growth in HG than the cells in NG, the reduction in cell growth by these treatments was dramatic and consistent with an autocrine growth stimulatory effect of TNF-{alpha}. This was further confirmed by knocking down TNF-{alpha} expression in VSMCs by siRNA. We have ablated TNF-{alpha} protein levels by more than 80% using rat TNF-{alpha}-siRNA (Fig. 10AGo, inset), and were then challenged with HG for 24 h to determine growth and 3 h to determine NF-{kappa}B activation. As observed with TNF-{alpha} antibodies and soluble receptors, the TNF-{alpha} knockdown in VSMCs significantly prevented HG-induced growth (Fig. 10AGo) and NF-{kappa}B activation (Fig. 10BGo). These observations thus indicate that TNF-{alpha} is an essential mediator of VSMC growth in HG and that the mitogenic effects of HG are sustained by autocrine stimulation of TNF-{alpha}.


Figure 9
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  		FIG. 9. TNF-{alpha} mediates HG-induced VSMC growth. Growth-arrested VSMCs in 5.5 mM glucose (NG) were either left untreated or stimulated with an additional 19.5 mM glucose (HG), mannitol (M), or 3-O-methyl glucose (3-OMG) in the absence or the presence of 1 µg/ml anti-TNF-{alpha}-antibodies or 0.5 µg/ml each of sTNF-R1 and sTNF-R2 for 24 h. A, Cell growth was determined by counting the number of cells, and (B) MTT assay (OD562). Bars represent mean ± SEM (n = 4). **, P < 0.001 vs. HG; #, P < 0.001 vs. NG; $, P < 0.01 vs. NG; and @, nonsignificant vs. NG. AB, Antibodies.

 

Figure 10
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  		FIG. 10. Ablation of TNF-{alpha} prevents HG-induced VSMC growth. TNF-{alpha}-siRNA transfected or scrambled siRNA transfected VSMCs in NG with 0.1% serum were either left untreated or stimulated with additional 19.5 mM glucose (HG) for 24 h. A, Cell growth was determined by MTT assay (OD562). B, NF-{kappa}B activation was determined calorimetrically using a nonradioactive NF-{kappa}B p65 transcription factor assay kit. The inset in A represents Western blot analysis for the TNF-{alpha} and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in transfected cells. Bars represent mean ± SEM (n = 4). *, P < 0.001 vs. HG; #, P < 0.001 vs. NG. C, Control; S, scrambled siRNA; T, TNF-{alpha} siRNA.

 
Response of human VSMCs to HG is similar to rat VSMCs
To examine how human VSMCs respond to HG, we measured changes in cell growth and TNF-{alpha} production after exposure of these cells to HG. Treatment of human VSMCs with HG caused a 40% increase in the cell growth, which was significantly (80–85%) attenuated by either AR inhibition or TNF-{alpha} antibodies, or sTNF-R1/sTNF-R2 (Fig 11Go). Furthermore, as shown in Fig. 12AGo, incubation of human VSMCs with HG led to an appreciable increase in TNF-{alpha} levels in the medium within 3 h, which continued to increase for the next 24 h (maximal observation time). The basal amount of TNF-{alpha} released by human VSMCs is significantly (~6-fold) higher than that released by rat VSMCs under similar conditions (Fig. 1Go). Stimulation of human VSMCs with LPS led to a marked (~8-fold) increase in TNF-{alpha} level in the medium (data not shown). Changes in TNF-{alpha} gene transcription were further confirmed directly by measuring changes in TNF-{alpha} message by RT-PCR. As shown in Fig. 12BGo, exposure of human VSMCs to HG for 6 and 12 h led to an approximate 3- and 7-fold increase in the TNF-{alpha} message, respectively. Preincubation of the cells with either sorbinil or TNF-{alpha} antibodies or soluble TNF-{alpha} receptors significantly (~80%) prevented the HG-induced increase in TNF-{alpha} mRNA levels. Together, these data demonstrate that the behavior of human VSMCs is similar to rat VSMCs and that TNF-{alpha} is an essential mediator of HG-induced growth of VSMCs of both the species.


Figure 11
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  		FIG. 11. TNF-{alpha} mediates HG-induced human VSMC growth. Growth-arrested human VSMCs in 5.5 mM glucose (NG) were either left untreated or stimulated with an additional 19.5 mM glucose (HG) in the absence or the presence of 1 µg/ml anti-TNF-{alpha}-antibodies or 0.5 µg/ml each of sTNF-R1 and sTNF-R2 or 10 µM sorbinil for 24 h. Cell growth was determined by MTT assay. Bars represent mean ± SEM (n = 3). *, P < 0.001 vs. HG; #, P < 0.001 vs. NG.

 

Figure 12
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  		FIG. 12. Inhibition of AR interrupts autocrine stimulation of TNF-{alpha} synthesis by TNF-{alpha} in human VSMCs. Growth-arrested human VSMCs in 5.5 mM glucose (NG) were either left untreated or stimulated with an additional 19.5 mM glucose (HG) in the absence or the presence of 1 µg/ml anti-TNF-{alpha} antibodies or 0.5 µg/ml each of sTNF-R1 and sTNF-R2, or 10 µM of sorbinil for indicated times. A, TNF-{alpha} protein levels were determined using an ELISA kit and (B) mRNA levels by RT-PCR as described in Materials and Methods. Amplification of the ß-actin gene was used as a sample control. The data represent mean ± SEM (n = 3). Bars represent mean ± SEM (n = 3; where n is number of independent RT-PCR analyses). *, P < 0.001 vs. HG; #, P < 0.001 vs. NG. AB, Antibodies.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major finding of this study is that HG causes TNF-{alpha} release from VSMCs, which causes autocrine stimulation of its own synthesis and increases cell growth. As reported by us previously (29), the amount of TNF-{alpha} released is sufficient by itself to stimulate VSMC growth. The mechanism of HG-induced increase in TNF-{alpha} production appears to involve activation of PLC, PKC, and NF-{kappa}B, and an increase in the transcription of the TNF-{alpha} gene. The stimulation of this signaling pathway is sustained in part by changes in the cellular redox state and by AR. The significance of HG-induced TNF-{alpha} release relates to the observation that inhibition of the autocrine effects of TNF-{alpha} prevents regenerative stimulation of PKC and NF-{kappa}B, as well as HG-stimulated VSMC growth. Significantly, HG-induced growth of human VSMCs was also prevented by soluble TNF-{alpha} receptors and sorbinil, and inhibition of AR prevented autocrine TNF-{alpha} synthesis in human VSMCs. This suggests that AR-sensitive hyperglycemic growth of VSMCs due to autocrine TNF-{alpha} production and stimulation may be of human relevance as well. Collectively, these results firmly establish a causal relationship between HG and TNF-{alpha} release, and implicate TNF-{alpha} as an obligatory mediator of the mitogenic effects of HG on VSMCs.

Although diabetes is known to be associated with increased levels of cytokines and tissue inflammation (13, 14, 15, 17, 18, 19), it remains unclear whether increased cytokines are a cause or consequence of diabetes (5). In this regard, our results showing that TNF-{alpha} secretion is increased by HG (in the absence of the confounding influence of hyperinsulinemia or dyslipidemia) provide support to the idea that cytokine generation in diabetes is a consequence of hyperglycemia. This view is in agreement with the observation that hyperglycemia acutely increases the levels of circulating cytokines in humans (41). Although hyperglycemia also increases the release of TNF-{alpha} from monocytes (42), most of the circulatory cytokines in diabetic patients are derived from noncirculating cells (43). Thus, an increase in TNF-{alpha} secretion by VSMCs suggests that in diabetic animals and humans, the vessel wall (smooth muscle as well as possibly endothelial cells) might be a significant source of cytokine production. Therefore, increased vascular production of TNF-{alpha} may be one reason why cardiovascular diseases are so profoundly affected by diabetes, and why diabetes and cardiovascular diseases have common risk factors and substrata [the "common soil hypothesis" (44, 45)]. Given that cytokines such as TNF-{alpha} and IL-1 play a critical role in endothelial dysfunction (46, 47, 48) and in promoting atheroma formation and plaque rupture (7, 49), it appears likely that increased cytokine production could contribute to accelerated atherogenesis and lesion instability in diabetics.

Glucose-induced TNF-{alpha} production could also be a significant mechanism underlying many of the biochemical manifestations of hyperglycemic injury. Previous studies report that HG profoundly affects intracellular cell signaling (3, 4, 50, 51). The effects of glucose range from stimulation of MAPKs and protein kinases, to alterations in gene transcription. Most significantly, exposure to HG leads to the activation of the polyol pathway and stimulation of PKC. These changes are accompanied by increased reactive oxygen species (ROS) generation and the accumulation of advanced glycosylation end products. Collectively, these changes alter cell growth and induce inflammation. Within this paradigm, NF-{kappa}B plays a key role in mediating proinflammatory changes. Indeed, the TNF-{alpha} promoter itself contains a NF-{kappa}B binding site, and, therefore, NF-{kappa}B activation and TNF-{alpha} are subject to positive auto-regulation (34). A critical role of NF-{kappa}B in vascular complications is supported by the observations that diabetes is associated with chronic activation of NF-{kappa}B (52), and that the nuclear translocation and transcription of NF-{kappa}B are increased by HG (53) and advanced glycosylation end products (54). Moreover, activation of NF-{kappa}B by HG is prevented by inhibiting PKC or AR (30), or by antioxidant interventions (55), suggesting that HG activates NF-{kappa}B by an AR-PKC-dependent mechanism that is sustained by oxidative stress. Our current results provide further support to this view. The observation that HG-induced TNF-{alpha} secretion was prevented by inhibiting AR, PKC, or NADPH oxidase suggests that increased synthesis of TNF-{alpha} in HG is mediated by the activation of PKC and AR, and ROS generation by NADPH oxidase (Fig. 13Go). Moreover, our results demonstrate that this is not a simple linear signaling cascade culminating in the production of TNF-{alpha} but a regenerative loop in which the secreted TNF-{alpha} causes autocrine stimulation of PKC and NF-{kappa}B via a feed-forward mechanism in which TNF-{alpha} stimulates its own synthesis. The presence of this regenerative loop is supported by the observation that even though the initial increase in membrane translocation of PKC was insensitive to TNF-{alpha}, prolonged activation of PKC and NF-{kappa}B was abolished by anti-TNF-{alpha} antibodies, or sTNF-Rs or TNF-{alpha}-siRNA, thus identifying for the first time an autocrine loop linking HG with inflammation. Our results using Rottlerin, an inhibitor that specifically inhibits PKC-{delta} in vivo (56, 57), suggest involvement of this enzyme in the secretion of HG-induced TNF-{alpha} in VSMCs. However, in vitro kinase assays the specificity for PKC-{delta} inhibition has been questioned (58) and shown to inhibit other kinases, such as PRAK (MAPK-activated protein kinase 5) and MAPKAP-2 (MAPK-activated protein kinase-2).


Figure 13
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  		FIG. 13. Schematic representation that shows HG-induced production of TNF-{alpha} regulates the VSMC growth by autocrine regulatory loop. Inhibition of signaling intermediates that lead to TNF-{alpha} synthesis in VSMCs could prevent autocrine loop and thereby cell growth.

 
The functional significance of the link between HG and inflammation is provided by our observation that quenching TNF-{alpha} prevents HG-induced cell growth of both rat and human VSMCs. Some previous studies show that HG increases VSMC proliferation (37, 38, 39, 40, 59), whereas others report that VSMC growth is unaffected by hyperglycemia (60, 61). Reasons for such discrepancy are not known and could relate to differences in culture conditions, VSMC phenotype, or the presence or absence of serum. Our studies showing that the hyperproliferative response to HG is due to TNF-{alpha} release and autocrine stimulation suggest that culture conditions such as cell density, the level of TNF-{alpha} generated in the medium, and the number of cell passages could significantly affect glucose-stimulated growth, and such procedural differences may explain, at least in part, why HG-induced VSMC hyperproliferation could have been missed in some cell culture studies. Furthermore, given the observation that inhibition of AR prevents HG-induced PKC activation in human coronary artery smooth muscle cells as well (62), it appears that the AR-mediated regulatory axis controlling PKC activity is preserved in humans as well.

Our studies showing that VSMC growth in HG-containing medium is sustained by TNF-{alpha} production and is prevented by sequestering TNF-{alpha} in the medium in both human and rat cells identify TNF-{alpha}, and not HG, as the proximal VSMC mitogen. TNF-{alpha} by itself is a potent VSMC mitogen (29), and, therefore, if generated in high enough concentration, could be sufficient for inducing VSMC growth. That TNF-{alpha} is also a critical VSMC mitogen in diabetic vessels in vivo is suggested by the observation that transfection with adenovirus construct expressing a dominant negative mutant of TNF-{alpha} receptor suppresses neointimal formation in femoral artery of obese Zucker rats (19). However, the contribution of TNF-{alpha} to VSMC growth in diabetic arterial lesions and its ability to sustain vascular inflammation remain to be assessed. In cell culture experiments, stimulation with HG led to a dramatic increase in TNF-{alpha} production, which was similar in extent or least, half as much, as that induced by LPS. However, in vivo, the inflammatory responses to HG are likely to be much smaller than to LPS, and hyperglycemic production of TNF-{alpha} may be balanced either by an increase in TNF-{alpha} degradation or washout from the vessel. The increase in circulatory cytokines (TNF-{alpha}, IL-6, and IL-18) during hyperglycemic clamp in humans is rapidly reversed within a few hours, suggesting that hyperglycemic increases in cytokines in vivo are likely to be transient. However, in cell culture studies, such removal mechanisms may be absent, allowing TNF-{alpha} to accumulate to much higher levels than would be achieved in vivo.

Our current data showing that TNF-{alpha} is an essential mediator of HG-induced cell growth provide an explanation as to why inhibition of AR prevents both TNF-{alpha} and HG-mediated cell growth because the two pathways are identical. Nonetheless, the specific growth-mediating pathways that require AR remain unclear. Our earlier observations indicate that ARIs prevent growth by preventing NF-{kappa}B activation (30, 31). However, it has been recently shown that NF-{kappa}B is not required for VSMC growth (63), therefore, there may be additional AR-sensitive NF-{kappa}B-independent pathways that mediate TNF-{alpha}-induced cell growth. We show that AR inhibition prevents activation of PKC, which could activate TNF-{alpha}-converting enzyme, which in turn can convert inactive TNF-{alpha} in the membranes to active TNF-{alpha} and its release into the medium. This could be one of the possible initial triggers for the release of TNF-{alpha} in hyperglycemia. The TNF-{alpha} thus released could initiate an autocrine loop for the propagation of hyperglycemic signals. Further studies are required to identify these pathways, and to delineate processes involved in the cross talk between inflammatory and growth-promoting pathways in tissues experiencing hyperglycemia.

Hyperglycemia by itself is a significant causative factor in the development of secondary insulin resistance in both type 1 and 2 diabetes (64, 65), but the mechanism of glucose-induced insulin resistance remains unclear. Based on our observation that HG stimulates TNF-{alpha} secretion and the well-described ability of TNF-{alpha} to antagonize insulin signaling (20, 21, 22, 23, 24, 25, 26), we speculate that hyperglycemia causes insulin resistance by inducing cytokine production from peripheral tissues. Cytokine production may represent a physiological response to HG that could potentially prevent intracellular hyperglycemia by antagonizing insulin signaling via serine phosphorylation of insulin receptor substrate-1. Although additional in vivo studies are required to address this issue directly, the observations that PKC inhibition prevents HG-induced release of TNF-{alpha} in VSMCs (this study) as well as insulin resistance in fibroblasts (66) suggest that both insulin resistance and cytokine generation depend upon similar signaling pathways. Significantly, lipid-induced insulin resistance is also associated with the accumulation of DAG, and the activation of PKC and NF-{kappa}B (67), consistent with a common role of the PKC-NF-{kappa}B pathway in regulating both cytokine production and insulin sensitivity.

In summary, the results of this study provide compelling evidence supporting the notion that HG increases transcription of TNF-{alpha} gene, and induces the release of TNF-{alpha} protein from rat and human VSMCs in culture. These findings suggest that HG increases TNF-{alpha} production via an AR-PKC-NADPH oxidase-dependent pathway, which in turn leads to NF-{kappa}B activation. The increase in TNF-{alpha} causes autocrine stimulation of PKC and NF-{kappa}B, and appears to be an essential mediator of HG-induced VSMC growth. Hyperglycemia-stimulated release of TNF-{alpha} and related cytokines from VSMCs could mediate diabetes-induced acceleration of atherogenesis and endothelial dysfunction in humans. Therefore, antiinflammatory interventions in general and anti-TNF-{alpha} therapy in particular may be useful in preventing and treating the cardiovascular complications of diabetes.


   Footnotes
 
This work was supported by National Institutes of Health Grants GM71036 (to K.V.R.), DK36118 (to S.K.S.), and ES 11860 (to A.B.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 21, 2007

Abbreviations: AR, Aldose reductase; ARI, aldose reductase inhibitor; DAG, diacylglycerol; FBS, fetal bovine serum; HG, high glucose; LPS, lipopolysaccharide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NF, nuclear factor; NG, normal glucose; PLC, phospholipase C; PKC, protein kinase C; ROS, reactive oxygen species; siRNA, small interfering RNA; sTNF-R, soluble TNF receptor; TR, transfection reagent; VSMC, vascular smooth muscle cell.

Received April 20, 2007.

Accepted for publication June 8, 2007.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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