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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:2854.)
© 1999 American Heart Association, Inc.

Vascular Biology

Augmented Expression of Inducible NO Synthase in Vascular Smooth Muscle Cells During Aging Is Associated With Enhanced NF-B Activation

Zhong-qun Yan; Allan Sirsjö; Marie-Luce Bochaton-Piallat; Giulio Gabbiani; Göran K. Hansson

From the Cardiovascular Research Laboratory (Z.-q.Y., A.S., G.K.H.), Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden, and the Department of Pathology (M.-L.B.-P., G.G.), University of Geneva, Geneva, Switzerland.

Correspondence to Dr Zhong-qun Yan, Center for Molecular Medicine (L8:03). Karolinska Hospital, S-17176 Stockholm, Sweden. E-mail zhong-qun.yan{at}cmm.ki.se

	Abstract

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Abstract—Vascular smooth muscle cells (SMCs) are important targets for endothelium-derived nitric oxide (NO), but this production is attenuated in injured and diseased arteries and during aging. However, SMCs can produce NO themselves by expressing an inducible form of NO synthase (iNOS) under inflammatory conditions and in the repair process after arterial injury. We examined iNOS expression in SMCs derived from the aortic media of newborn, young adult, and old rats. Our results show that SMCs from newborn rats cannot produce significant amounts of NO on stimulation with interferon- plus lipopolysaccharide or interleukin-1ß. In contrast, SMCs from old rats exhibit markedly enhanced iNOS activity. The difference in iNOS activity between the newborn and the old SMCs was closely correlated with levels of iNOS protein, mRNA, and gene promoter activity. Similarly, intercellular adhesion molecule-1 (ICAM-1) was also expressed more abundantly in the old than in the newborn SMCs in response to cytokines. Both iNOS and ICAM-1 are transcriptionally regulated by nuclear factor B (NF-B). Our data demonstrate an intense transactivation of NF-B in old SMCs on tumor necrosis factor- stimulation but only a weak one in newborn SMCs. The difference in the NF-B activation could be explained by a much faster and more extensive IB degradation in old than in newborn SMCs. These data indicate that the capability to respond to proinflammatory stimuli by activating NF-B differs between SMCs at different stages of development. This results in differential capability to express NF-B–dependent genes such as iNOS and ICAM-1, which could have implications for host defense and the pathogenesis of vascular diseases.

Key Words: age • vascular smooth muscle cells • nitric oxide • inducible nitric oxide synthase • nuclear factor-B

	Introduction

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Nitric oxide (NO) is an important regulator of vascular tone, smooth muscle growth, and platelet adhesion.¹ It is normally produced by vascular endothelial cells, which constitutively express an NO synthase (endothelial NO synthase, eNOS, or NOS3). One of the main targets of NO is vascular smooth muscle cells (SMCs); nitrosylation of guanylate cyclase triggers cyclic GMP production, which leads to vascular relaxation by interfering with myosin phosphorylation. During inflammation, proinflammatory cytokines and endotoxins induce expression of another NO synthase isoform, inducible NO synthase (iNOS, or NOS2), in several cell types including SMCs. This is followed by the production of large amounts of NO and results in nitrosylation of key proteins in the producing SMCs. Thus, cytokine treatment of SMCs is associated with relaxation, growth inhibition, and inhibition of mitochondrial respiration. This is probably due to nitrosylation of guanylate cyclase, Ras proteins, and enzymes of the mitochondrial respiratory chain.²

In vivo, vascular injury leads to iNOS-dependent, NO-mediated SMC relaxation and inhibition of platelet adhesion.³ Thus, the loss of eNOS-expressing endothelium is compensated for by the expression of iNOS in the SMC population. iNOS expression is particularly prominent in the type of SMCs that populate the arterial intimal lesion at the site of injury, whereas SMCs derived from the normal arterial media express much lower levels of iNOS and produce much less NO on cytokine stimulation.⁴ The induction of iNOS may therefore be of critical importance in the response-to-injury program of blood vessels.

Whereas eNOS is expressed constitutively and regulated by Ca²⁺/calmodulin at the level of enzyme activity, iNOS is regulated mainly at the transcriptional level and is expressed only in the activated cell. Activating stimuli include proinflammatory factors interleukin-1ß (IL-1ß), tumor necrosis factor- (TNF-), lipopolysaccharide (LPS), and interferon- (IFN-). These external factors have been shown to act on specific elements of the iNOS promoter. In SMCs and macrophages, B elements are critical for iNOS expression.^{5 6 7} The nuclear factor B (NF-B) binding to the promoter element is a heterodimer, composed of the subunits p65 (Rel-A) and p50 or p52.^{8 9} In quiescent cells, NF-B is sequestered in the cytosol owing to its association with its inhibitor IB.^{10 11 12} During activation by a multitude of stimuli such as TNF-, LPS, and IL-1ß, IBs are rapidly degraded via the ubiquitin-proteasome pathway. This permits the translocation of NF-B to the nucleus, where the activated NF-B interacts with the regulatory B element in promoters and enhancers,^{13 14} thereby controlling expression of iNOS and the genes encoding adhesion molecules, growth factors, cytokines, and their receptors in the vessel wall.^{15 16}

Ample evidence suggests that expression of vascular genes is not only subject to the influence of pathological conditions but also to developmental regulation during embryonal and postnatal life. Aging is considered an independent risk factor for atheromatous lesions. However, it is not well established whether SMCs undergo intrinsic changes during aging. Previous work has shown that SMCs cultured from the same arterial segment during different situations may yield whole populations with distinct phenotypic features.^{17 18 19} Two main SMC populations have been described: 1 spindle-shaped, classically growing in hills and valleys, and 1 epithelioid-shaped, growing in a monolayer.^{17 18 19} Spindle SMCs do not completely stop growing after reaching confluence, and this characteristic leads to their classic appearance in hills and valleys, whereas epithelioid cells show contact inhibition but, when sparse, can grow in the absence of serum factors. Aortic SMCs cultured from newborn rats have the features of a spindle population. Moreover, they exhibit differentiation markers in vitro, including contractile protein expression, whereas SMCs originating from old animals are epithelioid, do not need serum to grow, and dedifferentiate rapidly in vitro.²⁰ SMCs originating from adult animals have intermediate features of the 2 aforementioned populations, although they exhibit more spindle- than epithelioid-type SMC features.²⁰ When clones are derived from the aortic media of an adult animal, the majority of them exhibit spindle features and a minority exhibit epithelioid features, suggesting that the whole population of an artery is heterogeneous.²¹ Thus, it is tempting to speculate that with age, the epithelioid SMC population overcomes the spindle population in the rat aorta. The mechanisms of this modulation are not clear, but it is conceivable that this change in SMC features corresponds to biological and functional changes of the arterial wall. For example, it has been reported that newborn and adult SMCs express little or no cellular retinol binding protein-1 in vivo and in vitro and are relatively insensitive to retinoid action. However, SMCs from old rats as well as from the experimental intimal thickening after a balloon-induced endothelial lesion express high amounts of this protein and are sensitive to the action of retinoic acid or retinol.^{22 23} Thus far, little is known regarding the regulation of iNOS in SMCs with aging.

The present study was designed to characterize the developmental regulation of iNOS expression in VSMCs and the molecular mechanisms involved. The results of this study indicate that transcription of the iNOS gene is deficient in newborn SMCs but dramatically enhanced in SMCs from old rats. This postnatal regulation of iNOS expression is governed by differential activation of the NF-B pathway.

	Methods

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Materials
Murine recombinant IFN-, TNF-, and IL-1ß were purchased from Genzyme. Pyrrolidinedithiocarbamate (PDTC) and bacterial LPS (from Escherichia coli serotype O55:B5) were from Sigma. MG132 (carbobenzoxoyl-Leu-Leu-Leu-H) was purchased from Biomol. All reagents except the LPS itself contained <12 pg/mL endotoxin by the Limulus amebocyte lysate assay.

Cell Culture
The thoracic aortic media of 4-day-, 6-week-, and 18-month-old Wistar rats was carefully dissected and digested enzymatically, as previously described.²⁴ The handling of experimental animals used to obtain cells was in line with institutional guidelines and approved by the regional ethical committee. SMCs were plated on 100-mm plastic Petri dishes at a density of 2x10⁴ cells/cm² in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal calf serum (FCS, Seromed Biochem), 100 U/mL penicillin, and 100 U/mL streptomycin. Cell populations were brought to passage 10 in the same medium and on dishes of the same size. They were characterized as previously described.^{20 24}

NO₂^- Assay
The accumulation of nitrite (NO₂^-), a stable end product of NO formation, in conditioned medium was measured as an indicator of NO production.²⁵ Cell-free conditioned medium (100 µL) was incubated for 10 minutes with 100 µL of Griess reagent at room temperature, and the absorbance at 540 nm was measured in a VERSAmax microplate reader. NO₂^- in the samples was calculated from a standard curve of sodium nitrite. For comparison of NO production by different types of SMCs, NO₂^- values were normalized by cell number.

Flow Cytometry
SMCs were detached from dishes by trypsinization and rinsed with DMEM/10% FCS. An indirect immunofluorescence staining was performed to detect RT1B (a major histocompatibility complex [MHC] class II gene product) and intercellular adhesion molecule-1 (ICAM-1). In brief, cells were incubated for 15 minutes on ice with monoclonal mouse anti-rat RT1B (OX6, Serotec) or monoclonal mouse anti-rat ICAM-1 (CD54, Serotec). After being rinsed, the cells were stained with phycoerythrin-labeled rabbit anti-mouse IgG and fixed with 1% p-formaldehyde in PBS. Controls were incubated with a nonspecific hybridoma protein, MOPC21. Approximately 5000 cells from each culture were analyzed by an FACSCalibur (Becton Dickinson).

RNA Isolation and Northern Blotting
Total RNA was extracted from cells with an RNA isolation kit (Promega). RNA was size-fractionated on 1% agarose gels containing 660 mmol/L formaldehyde and transferred to Hybond-N nylon membranes (Amersham Corp). A 4100-bp, full-length iNOS cDNA was labeled with [-³²P]dCTP by using a random-priming DNA labeling kit (Amersham). Filters were prehybridized for 2 to 5 hours at 42°C with a solution containing 5x SSPE (1x SSPE is 150 mmol/L NaCl, 10 mmol/L sodium phosphate, and 1 mmol/L EDTA), 5x Denhardt’s reagent, 50% deionized formamide, 100 mg/mL salmon sperm DNA, and 0.1% SDS and hybridized overnight at 42°C in the same buffer containing 10⁶ counts per minute/mL of denatured probe. After hybridization, the filters were washed twice for 10 minutes at room temperature with 2x SSPE and 0.1% SDS, for 20 minutes at 65°C with 1x SSPE and 0.1% SDS, and for 15 minutes at 65°C with 0.1x SSPE and 1% SDS before autoradiography. To normalize hybridization signals for variations in loading and/or transfer, filters were initially visualized for 18S rRNA by methylene blue staining. The density of bands was scanned and then evaluated by the program NIH Image 1.61.

Transfection and CAT Assay
Cells (50% confluent in 6-well plates) were preincubated in OptiMEM medium (Life Technologies, Inc) for 2 hours at 37°C and transfected with 2 µg of iNOS promoter–chloramphenicol acetyltransferase (CAT) reporter plasmid (Oxford Biomedical Research, Inc) by using lipofectin (8 µg/well, Life Technologies, Int). This construct contains the 5' flanking region (1749 bp, from nucleotides -1588 to +161 as the mRNA initiation site) of the iNOS gene, which harbors at least 24 elements homologous to consensus sequences for inducibility by LPS, TNF-, IL-1ß, and IFN-.²⁶ Two micrograms of the ß-galactosidase expression vector plasmid, pSV–ß-galactosidase vector (control, Promega), was cotransfected as an internal control for transfection efficiency. Twenty hours after transfection, cells were treated for 20 hours with a combination of 100 U/mL IFN- and 10 µg/mL LPS. The cells were harvested and assayed for CAT activity by thin-layer chromatography. ß-Galactosidase activity was measured spectrophotometrically at 420 nm by the generation of o-nitrophenol from o-nitrophenyl-ß-D-galactopyranoside.

Electrophoretic Mobility Shift Assay (EMSA)
Nuclear extracts were prepared from cells cultured in 100-mm dishes as described²⁷ and nuclear protein concentrations determined by using the bicinchoninic acid method (Pierce). The nuclear extract (2 µg of protein) was preincubated for 10 minutes in the reaction buffer (10 mmol/L HEPES, pH 7.9, 10% glycerol, 60 mmol/L KCl, 5 mmol/L MgCl₂, 0.5 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L PMSF, and 2 mg of poly[dI-dC), followed by incubation for 30 minutes at room temperature with 50 000 cpm of ³²P-labeled NF-B probe (double-stranded oligonucleotides containing an NF-B consensus binding site: 5'-AGT TGA GGG GAC TTT CCC AGG C-3', Promega). DNA-protein complexes were electrophoresed on 7% native polyacrylamide gels in low-ionic-strength buffer (22.3 mmol/L Tris-borate, 0.5 mmol/L EDTA, pH 8). Dried gels were analyzed by autoradiography. In some cases, the incubation of nuclear extracts with ³²P-labeled NF-B probe was performed in the presence of excess unlabeled NF-B probe or the irrelevant oligonucleotide activator protein (AP)-1 (Promega). For supershift analysis, rabbit anti-p65 polyclonal antibodies (Santa Cruz Biotechnology) were incubated with the nuclear extracts for 15 minutes before the addition of radiolabeled probe.

Immunolocalization of NF-B
SMCs were plated on glass coverslips and either treated with 200 U/mL TNF- or left untreated. After treatment, the cells were fixed with cold methanol and acetone. Intracellular p65 was visualized by indirect immunofluorescence with the use of polyclonal rabbit anti-p65 antibodies (Santa Cruz Biotechnology) followed by FITC-labeled goat anti-rabbit IgG (DAKOPATTS).

Immunoblotting Analysis of IB
SMCs were washed with PBS and then resuspended in the sample buffer containing 10 mmol/L HEPES (pH 7.8), 10 mmol/L KCl, 2 mmol/L MgCl₂, 1 mmol/LDTT, 0.1 mmol/L EDTA, 0.1 mmol/L PMSF, and 10 mmol/L NaF. After 10 minutes on ice, cells were lysed with 1% Nonidet P-40, and the cells were centrifuged for 1 minute at 10 000g. The supernatant, containing the cytoplasmic fraction, was recovered. One volume of 2x Laemmli buffer containing 20% ß-mercaptoethanol was added, and the samples were boiled. The cytoplasmic protein was fractionated by 10% SDS–polyacrylamide gel electrophoresis and transferred to Hybond-C-extra nitrocellulose membranes (Amersham Pharmacia Biotech). Membranes were then preincubated with 5% dry milk powder in PBS and incubated for 1 hour with a 1:1000-diluted anti-IB antibody (C-21, Santa Cruz Biotechnology) that was subsequently visualized using an Amersham Pharmacia enhanced chemiluminescence system.

Statistics
Unless otherwise indicated, data are presented as mean±SEM. Comparison of means was performed using Student’s t test.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Expression of iNOS by VSMCs During Postnatal Development
The capability of expressing iNOS was studied in VSMCs derived from rats of different ages. To induce iNOS expression, VSMCs isolated from the media of newborn (4 days), young adult (6 weeks), and old (18 months) rats were stimulated with 10 µg/mL LPS plus 100 U/mL IFN-, which synergistically activate iNOS transcription. Activity of iNOS was determined by measuring NO₂^- concentrations in culture media. As shown in Figure 1A, cells derived from old rats showed an iNOS activity that was nearly 3-fold higher than that of young adult SMCs and 5-fold that of newborn SMCs. These differential responses in iNOS induction to LPS and INF- were further examined by using a panel of proinflammatory factors including IL-1ß, TNF-, and IFN-. As shown in Figure 1B, SMCs from old rats exhibited a dose-dependent response to IL-1ß, which induced maximal iNOS activity at a dose of 3.3 ng/mL. In contrast, the same dose of IL-1ß (3.3 ng/mL) only induced 1/6 of that iNOS activity when given to newborn SMCs. Other cytokines used alone exerted a weak or no effect on iNOS induction in the SMCs (Figure 1C). The IL-1ß–induced NO production was prevented by PDTC and MG132, which are inhibitors of NF-B activation.^{28 29} Taken together, these results indicate that iNOS activity in SMCs is closely related to the age of the donor and that activation of NF-B is involved.

View larger version (15K): [in this window] [in a new window]   Figure 1. Activity of iNOS in SMCs from donors of different age. A, NO production by SMCs derived from the media of newborn, young adult, and old rats. SMCs were exposed to IFN- (100 U/mL) plus LPS (10 µg/mL). iNOS activity was assessed at the indicated times by determining NO2- concentration in conditioned medium. B, Dose-response induction of iNOS in newborn and old SMCs treated with IL-1ß. NO2- levels were assessed 24 hours after exposure to IL-1ß. C, Differential induction of iNOS in response to IL-1ß (1 ng/mL), LPS (20 µg/mL), TNF- (250 U/mL), and INF- (200 U/mL) in the absence or presence of PDTC (5 µmol/L) or MG132 (0.5 µmol/L). NO2- levels were determined 24 hours after exposure to the cytokines. Data are presented as mean±SEM of 3 experiments. Asterisks indicate significant differences compared with newborn SMCs. *P<0.05 compared with newborn SMCs; #P<0.05 compared with IL-1ß alone.

iNOS Expression and Promoter Activity in Newborn and Old SMCs
Because iNOS is regulated mainly at the transcriptional level, we next examined iNOS transcripts by Northern blotting. As shown in Figure 2, large amounts of iNOS mRNA were found in old SMCs after 6 and 24 hours of exposure to LPS and IFN-, but little was detected in the newborn SMCs. To assess the transcriptional activity of iNOS, newborn and old SMCs were transfected with a plasmid construct containing a mouse iNOS promoter/enhancer, which drives a CAT reporter gene.⁷ CAT activity in the transfected cells was determined 20 hours after stimulation with IFN- plus LPS. Neither newborn nor old SMCs showed CAT activity under unstimulated conditions. On stimulation with IFN- and LPS, old SMCs expressed high CAT activity, but an identical treatment could not trigger the newborn SMCs to produce CAT activity (Figure 3).

View larger version (40K): [in this window] [in a new window]   Figure 2. Expression of iNOS mRNA. Newborn and old SMCs were maintained in quiescent medium (DMEM+0.4% FCS) and stimulated with IFN- (100 U/mL) plus LPS (10 µg/mL) for the indicated time. A, iNOS mRNA was analyzed by Northern blotting. 18S rRNA, stained with methylene blue, was used as a loading reference. O indicates old SMCs; N, newborn SMCs. B, The abundance of iNOS mRNA was quantified and presented as arbitrary units after subtraction of background. Data are presented as mean±SEM of 3 experiments.

View larger version (14K): [in this window] [in a new window]   Figure 3. Transcriptional activation of the iNOS gene promoter in newborn and old SMCs. The cells were transiently cotransfected with iNOS-CAT and ß-galactosidase plasmids. Thereafter, the cells were exposed for 20 hours to IFN- (100 U/mL) plus LPS (10 µg/mL). CAT activity was assessed by thin-layer chromatography and normalized by ß-galactosidase activity. Data shown are mean±SEM of 3 experiments. *P<0.05 compared with newborn SMCs.

Induction of MHC Class II Protein and ICAM 1 in SMCs
Although activation of the NF-B signal transduction pathway is crucial for expression of the iNOS gene, its full transcriptional activation requires additional activation of the Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway induced by IFN-.³⁰ The lack of iNOS gene expression in newborn SMCs led us to investigate whether other genes regulated by NF-B and STAT-1 could be expressed in SMCs. To this end, induction of MHC class II and ICAM-1 in SMCs was examined, because expression of MHC class II is known to be regulated substantially by STAT-1, and ICAM-1 to a great extent by NF-B–mediated signaling.^{31 32} Figure 4 demonstrates that newborn SMCs responded to IFN- by expressing the MHC class II gene product RT1B at a similar level as did old SMCs. Neither TNF- nor LPS was able to induce RT1B expression in newborn or old SMCs (Figure 4A). These results suggest that there was no significant difference between newborn and old SMCs in IFN-–induced, STAT-1–mediated gene expression.

View larger version (40K): [in this window] [in a new window]   Figure 4. Flow-cytometric analysis of surface molecule expression on newborn and old SMCs. A, Histograms showing expression of RT1B (an MHC class II gene product). Newborn and old SMCs were unstimulated (control) or treated with TNF- (200 U/mL) or IFN- (100 U/mL) for 48 hours, stained with the mouse anti-rat RT1B monoclonal antibody OX6 followed by phycoerythrin-labeled anti-mouse-IgG, and then analyzed by flow cytometry. B, Histograms showing induction of ICAM-1 expression. Newborn and old SMCs were exposed to medium alone (control), TNF- (200 U/mL), or LPS (10 µg/mL). After 48 hours, cells were stained with mouse anti-rat ICAM-1 monoclonal antibody followed by phycoerythrin-labeled anti-mouse-IgG and analyzed by flow cytometry. Background fluorescence was established by incubation of the cells with the phycoerythrin-conjugated monoclonal antibody alone. X and y axes represent the mean of fluorescence intensity and cell counts, respectively. Data from 1 of 3 similar experiments are presented. C, Dose dependence of TNF-–induced ICAM-1 expression on new- born and old SMCs. The cells were incubated with medium alone (control) or with the indicated dose of TNF- for 48 hours. Surface expression of ICAM-1 was measured by flow cytometry. Expression of ICAM-1 is presented as the mean fluorescence intensity. Values are the mean±SD of 3 independent determinations.

Unlike RT1B, expression of ICAM-1 was markedly different between the old and newborn SMCs. In old SMCs, treatment with TNF- (200 U/mL) or LPS (10 µg/mL) strongly induced ICAM-1 expression, resulting in 3-fold and 2.5-fold increases in the mean fluorescence intensity, respectively. In comparison, the identical treatments exerted much weaker effects on newborn SMCs (Figure 4B).

Figure 4C demonstrates that a low dose of TNF- (20 U/mL) was enough to induce ICAM-1 expression in old SMCs but insufficient for newborn SMCs. Effective induction of ICAM-1 in the newborn SMCs required at least 200 U/mL TNF-, a concentration 10 times higher than that needed for the old SMCs. This result supports the notion that newborn SMCs exhibit a reduced NF-B–dependent gene expression.

Activation of NF-B in Old and Newborn SMCs
Given the observation that there was a marked difference in the expression of NF-B–regulated genes in SMCs, depending on the age of the donor, the transactivation of NF-B in newborn and old SMCs was determined by EMSA. After treatment with TNF- for 30 minutes, a strong NF-B binding complex was observed by EMSA in the nuclear extract of old SMCs but not in newborn SMCs (Figure 5). Formation of this protein-DNA complex could be prevented by pretreating the cells with PDTC, an antioxidant that blocks the release of the NF-B protein from its cytoplasmic inhibitor, IB.²⁸ The composition of these protein-DNA complexes was analyzed by using specific antibodies to members of the NF-B/Rel family. In the SMC extracts, antibodies to the p65 subunit of NF-B produced a supershifted band in addition to decreasing the intensity of the NF-B consensus-binding protein (Figure 5).

View larger version (63K): [in this window] [in a new window]   Figure 5. EMSA of NF-B complexes in newborn and old SMCs. Nuclear extracts were prepared from cells treated with 100 U/mL TNF- for 30 minutes with or without PDTC (100 µmol/L, pretreated for 20 minutes). The 32P-labeled oligonucleotide corresponding to a consensus B site was incubated with 2 µg of nuclear protein and an antibody to the p65 subunit of NF-B. Data are representative of 3 experiments.

Activation of NF-B in SMCs was also determined in terms of the translocation of p65. In unstimulated SMCs, p65 was sequestered in the cytoplasm, and no significant difference in the distribution of p65 protein could be noticed between old and newborn SMCs (Figures 6A and 6D). Treatment with TNF- (200 U/mL) resulted in translocation of p65 into the nuclei of all old SMCs. This was completely prevented by pretreatment of the cells with PDTC. However, very few of the newborn SMCs exhibited such p65 translocation when stimulated with TNF- (Figure 6E). Together with the EMSA data, this result suggests that newborn SMCs cannot mount an appropriate NF-B activation on stimulation with proinflammatory agents.

View larger version (61K): [in this window] [in a new window]   Figure 6. Transactivation of NF-B in newborn and old SMCs in response to TNF-. Old SMCs (A through C) and newborn SMCs (D through F) were plated on glass coverslips and left untreated (A, D), treated with 200 U/mL TNF- for 30 minutes (B, E), or pretreated with 100 µmol/L PDTC for 15 minutes followed by TNF- for 30 minutes (C, F). The intracellular location of p65 was detected by indirect immunofluorescence with an anti-p65 antibody. Original magnification x200. Data are representative of 3 experiments.

To clarify whether the low NF-B activity in newborn SMCs on cytokine stimulation was due to a deficiency at the receptor level, receptor-independent activation of NF-B was examined in SMCs by using H₂O₂. As illustrated in Figure 7A, a prolonged-exposed EMSA gel revealed that both newborn and old SMCs bore constitutive NF-B activity (refereed to as C2) under resting conditions. Stimulating SMCs with H₂O₂ rapidly initiated NF-B activation in old SMCs. This was characterized by the appearance of an intense B binding complex referred to as C1 (lanes 5 and 6 in Figure 7A). However, this B binding complex was not detected in the newborn SMCs receiving an identical treatment. The specificity of B binding proteins was further verified by competition analysis of the nuclear extracts derived from H₂O₂-activated SMCs as used in Figure 7A, lanes 2 and 5. The unlabeled NF-B probe could effectively remove C1 and C2 but to a much less extent affected the formation of C3 (Figures 7B and 7C). Together with the results of the supershift assay, these observation suggest that C1 and C2 represent the specific NF-B signals. The nature of C3 remains unclear.

View larger version (41K): [in this window] [in a new window]   Figure 7. Activation of NF-B in newborn and old SMCs in response to H2O2. A, Lack of NF-B activation in newborn SMCs treated with H2O2. Nuclear proteins were derived from SMCs treated for the indicated periods with 250 µmol/L H2O2. The NF-B binding activity of 2 µg of nuclear protein was displayed by EMSA by using a 32P-labeled oligonucleotide corresponding to the B site. The resultant nuclear extract–DNA complexes are referred to as C1, C2, and C3. B and C, Specificity of NF-B complex. Nuclear extracts derived from SMCs treated for 5 minutes with H2O2 were incubated with a 32P-labeled NF-B probe plus a 25- to 100-fold excess of unlabeled NF-B probe or AP-1 probe. Formation of C1 and C2 has been substantially prevented by the unlabeled NF-B but not by the AP-1 probe. C, Samples without the unlabeled probes. The exposure of these autoradiograms was prolonged to visualize the NF-B complex in newborn SMCs.

Cytokine-Induced Degradation of IB in Old and Newborn SMCs
NF-B translocation depends on the dissociation of NF-B dimers from their cytosolic inhibitors, the IB proteins. This step is preceded by phosphorylation of IB by IB kinases, which are activated on ligation of TNF and IL-1 receptors,^{33 34 35} and is followed by ubiquitination that targets IB for proteasomal degradation.^{11 36 37} To investigate whether the difference in NF-B activation between old and newborn SMCs was due to differences in the handling of its inhibitor, we analyzed cellular levels of the main IB protein IB by Western blotting. As shown in Figure 8, IB was present in roughly equal amounts in old and newborn SMCs. TNF- stimulation led to a rapid disappearance of IB in old SMCs, whereas degradation of IB was minimal in newborn cells (Figure 8). A similar pattern of IB degradation was also observed in cells stimulated with IL-1ß (Figure 8). Noticeably, the inducible degradation of IB was enhanced in SMCs with aging and was closely correlated with the responsiveness of NF-B activation. Thus, these data have disclosed that differential degradation of IB determines the cytokine response in SMCs.

View larger version (39K): [in this window] [in a new window]   Figure 8. Cytokine-induced degradation of IB in newborn and old SMCs. Western blot analysis of IB in newborn and old SMCs after exposure to 100 U/mL TNF- or 0.5 ng/mL IL-1ß for the indicated time periods. Data are representative of 2 experiments.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
VSMCs are primary targets of endothelium-derived NO in the regulation of vascular tone. This capability is reduced with age³⁸ and in arterial disease.³⁹ However, SMCs can also be induced to produce large amount of NO themselves after deendothelializing arterial injury in vivo and on stimulation with proinflammatory cytokines in culture. The data reported here indicate (1) that SMCs of old rats have a much higher capability to produce NO than do SMCs from the media of young adult or newborn rats; (2) that this is due to differences in their capability to transcribe the iNOS gene; and (3) that this depends on the responsiveness of the NF-B transduction pathway.

iNOS is largely regulated at the transcriptional level. Its promoter is complex in rodents and even more so in humans, in whom it covers 16 kb of an upstream sequence.⁴⁰ The rodent iNOS promoter contains NF-B sites, interferon-–activated and IFN-RE sites, and AP-1 and hypoxia response elements.^{6 26 41} This promoter is known to be activated by NF-B–activating cytokines, ie, TNF- or IL-1ß, and by LPS. Full activation in SMCs requires additional stimulation with IFN-,^{30 42} suggesting synergistic action between NF-B and STAT-1 on the promoter.

The promoters of iNOS derived from the mouse and rat are highly homologous.^{6 26} Both promoters show similar features, especially the essential role of NF-B in the cytokine-induced transcriptional activation of the iNOS gene. Transfecting SMCs with a mouse iNOS promoter-reporter construct, we observed dramatic differences in the activation of the iNOS gene between SMCs from rats of different ages, which were also correlated with the levels of NO production and amounts of iNOS transcription when stimulated with a combination of LPS and IFN-. This difference in iNOS induction was even more pronounced when IL-1ß was applied alone. In fact, almost no NO production could be detected in cultures of newborn SMCs when treated with IL-1ß. Because IL-1ß is a potent NF-B activator, this suggests that SMCs derived from rats of different ages might differ in NF-B responsiveness. This notion received further support from the finding that 2 different inhibitors of the NF-B pathway, PDTC and MG132, blunted IL-1ß–induced iNOS expression. This interpretation was also indicated by the observation that another NF-B–dependent gene, ICAM-1, was insufficiently expressed in newborn SMCs but fully expressed in old SMCs after TNF- stimulation. In contrast, IFN-–induced, Jak/STAT-dependent expression of the MHC class II gene RT1B was equal in newborn and old SMCs.

EMSA analysis of NF-B activation revealed a prominent B binding complex in nuclear extracts of TNF-–treated old but not newborn SMCs. This complex was identified as NF-B by supershifting with specific antibodies against p65 and by its sensitivity to an inhibitor of NF-B activation, PDTC. Immunofluorescence microscopy with anti-p65 also clarified that the protein was present in both old and newborn SMCs but was translocated to the nucleus only in the former but not the latter on TNF- stimulation. Our present data demonstrate that the responsiveness of NF-B transactivation is markedly enhanced in VSMCs with aging and also show that NF-B is a key mechanism governing the expression of iNOS and ICAM-1, 2 examples of inflammatory genes.

The degradation of IB, as a crucial regulatory mechanism of NF-B activation, behaves differentially between the newborn and the old SMCs. The old SMCs were associated with rapid IB degradation after cytokine stimulation; this should release NF-B to permit nuclear translocation and transcriptional activation of iNOS. In contrast, IB degradation was minimal in newborn SMCs, which therefore may account for the weak activation of NF-B in response to stimuli. However, the mechanisms governing IB are equivocal. Characterization of IB kinase may assist in a further understanding of the altered responsiveness of NF-B in SMCs with aging.

It is unlikely that the lack of iNOS response was due to specific cytokine receptor defects because TNF-, IL-1ß, nor LPS could induce significant iNOS expression and NF-B activation in newborn SMCs, although these stimuli act on different receptors. Furthermore, H₂O₂, which is a receptor-independent inducer, was incapable of activating NF-B in the newborn SMCs, while it was effective in old SMCs. This argues against the possibility of a receptor deficiency. Instead, the defect was associated with insufficient IB degradation, resulting in a subdued NF-B signal transduction pathway.

Moreover, it is unlikely that the differences between the different cell types would be due to other properties of the cultures than the age of the donor, because the phenotype of each different SMC type is characteristic for the site and age of the donor and is stable in culture.²⁰ Several cell populations derived from different isolations were used, which reduced the likelihood that our findings could be due to random differences between cultures.

Cernadas et al⁴³ have recently demonstrated that the expression of iNOS is upregulated in rat arteries with aging. Accordingly, it has been shown that young mice produce much less NO and TNF- than do old mice when challenged with LPS.⁴⁴ Together with our observations, these data conceivably indicate that expression of the iNOS gene is subjected to developmental regulation. It should be noted, however, that the regulation of iNOS expression is likely to be more complex in humans than in rodents,^{1 6} and it will be necessary to evaluate the NF-B/iNOS pathway in human SMCs before definitive conclusions can be drawn regarding human disease.

iNOS is a high-output NOS that produces sufficient NO to induce apoptosis in target cells. However, it may also be beneficial under certain circumstances. Thus, we have previously found that iNOS is rapidly induced in arterial SMCs after angioplastic injury.⁴⁵ This response serves to inhibit platelet adhesion and relax vascular tone.³ In this way, intimal SMCs may substitute for the loss of NO-producing endothelial cells by synthesizing NO via the iNOS pathway. Similarly, an enhanced iNOS response of SMCs may compensate for the reduction in endothelium-derived NO production in the aged organism³⁸ and in atherosclerotic lesions.⁴⁶ The amounts of NO produced and, hence, the concentration of the inducing stimuli are likely to determine whether this NO synthesis is beneficial or detrimental. The results of the present study have identified the capability of the SMC to mount NF-B activation on stimulation as an important level of regulation of the iNOS/NO response. Further studies will be needed to clarify the role of such differences in the signal transduction machinery in host defense and vascular disease.

	Acknowledgments

 
This study was supported by the Swedish Medical Research Council (project No. 6816 and 2042), the Swedish Heart-Lung Foundation, the Swedish Cancer Society, the Axel and Margaret Ax:son Johnson Foundation, the Nanna Svartz’ Fund, King Gustaf V 80th Anniversary Fund, the Swedish Society for Medical Research, and the Swiss National Science Foundation (grant No. 3100.50568–97). We thank Dr W. Erl for advice on flow cytometry and Ingrid Thörnberg for technical assistance.

Received March 29, 1999; accepted June 10, 1999.

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