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Am J Physiol Heart Circ Physiol 294: H2879-H2888, 2008. First published April 25, 2008; doi:10.1152/ajpheart.91406.2007
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Angiotensin II and tumor necrosis factor-{alpha} synergistically promote monocyte chemoattractant protein-1 expression: roles of NF-{kappa}B, p38, and reactive oxygen species

Masao Takahashi,1 Etsu Suzuki,2 Ryo Takeda,1 Shigeyoshi Oba,1 Hiroaki Nishimatsu,3 Kenjiro Kimura,2 Tetsuo Nagano,4 Ryozo Nagai,1 and Yasunobu Hirata1

1Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo; 2Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki; and 3Department of Urology, Faculty of Medicine, and 4Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan

Submitted 6 December 2007 ; accepted in final form 23 April 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We examined whether ANG II and TNF-{alpha} cooperatively induce vascular inflammation using the expression of monocyte chemoattractant protein (MCP)-1 as a marker of vascular inflammation. ANG II and TNF-{alpha} stimulated MCP-1 expression in a synergistic manner in vascular smooth muscle cells. ANG II-induced MCP-1 expression was potently inhibited to a nonstimulated basal level by blockade of the p38-dependent pathway but only partially inhibited by blockade of the NF-{kappa}B-dependent pathway. In contrast, TNF-{alpha}-induced MCP-1 expression was potently suppressed by blockade of NF-{kappa}B activation but only modestly suppressed by blockade of p38 activation. ANG II- and TNF-{alpha}-induced activation of NF-{kappa}B- and p38-dependent pathways was partially inhibited by pharmacological inhibitors of ROS production. Furthermore, ANG II- and TNF-{alpha}-stimulated MCP-1 expression was partially suppressed by ROS inhibitors. We also examined whether endogenous ANG II and TNF-{alpha} cooperatively promote vascular inflammation in vivo using a wire injury model of the rat femoral artery. Blockade of both ANG II and TNF-{alpha} further suppressed neointimal formation, macrophage infiltration, and MCP-1 expression in an additive manner compared with blockade of ANG II or TNF-{alpha} alone. These results suggested that ANG II and TNF-{alpha} synergistically stimulate MCP-1 expression via the utilization of distinct intracellular signaling pathways (p38- and NF{kappa}B-dependent pathways) and that these pathways are activated in ROS-dependent and -independent manners. These results also suggest that ANG II and TNF-{alpha} cooperatively stimulate vascular inflammation in vivo as well as in vitro.

blood vessels; signaling pathway; atherosclerosis

IT IS WELL KNOWN that the renin-angiotensin system (RAS) is implicated in the pathogenesis of atherosclerosis and restenosis after angioplasty (4, 8, 23, 24). However, several reports have demonstrated that blockade of the RAS is insufficient to inhibit vascular inflammation in animal models, including humans (9, 15, 16). Although the reason for this discrepancy remains unclear, the role of the RAS in vascular inflammation may differ depending on the animal species and experimental model used. It is also possible that apart from the RAS, some other factors such as proinflammatory cytokines are also implicated in vascular inflammation and that blockade of the RAS alone is not always sufficient to inhibit vascular inflammation. To reduce morbidity and mortality caused by cardiovascular diseases, it will be necessary to find molecules that cooperate with the RAS to stimulate vascular inflammation.

TNF-{alpha} is a proinflammatory cytokine that is known to be involved in the development of vascular inflammation such as atherosclerosis. It has been demonstrated that the progression of atherosclerosis is retarded in mice deficient in both apolipoprotein E and TNF-{alpha} compared with apolipoprotein E-null mice (2). Furthermore, we have demonstrated that blockade of endogenous TNF-{alpha} using an adenovirus expressing a dominant negative mutant of the receptor for TNF-{alpha} inhibited neointimal formation in obese Zucker rats, an animal model of Type 2 diabetes (34). Thus, it is possible that TNF-{alpha}, as well as the RAS, plays a pivotal role in vascular inflammation.

Monocyte chemoattractant protein (MCP)-1 is a peptide that induces the migration of monocytes/macrophages in the vessel wall and is reportedly implicated in the formation of vascular diseases such as atherosclerosis and restenosis after angioplasty (1, 11, 20, 35). It has been reported that ANG II as well as TNF-{alpha} stimulates MCP-1 production in blood vessels (3, 10, 40). Several intracellular signaling pathways have been identified that mediate MCP-1 expression. Among them, NF-{kappa}B is a well-known transcription factor that mediates TNF-{alpha}-induced MCP-1 expression, especially in vascular endothelial cells (7). In contrast to the established role of NF-{kappa}B in TNF-{alpha}-induced MCP-1 expression, its role in ANG II-induced MCP-1 expression remains to be fully determined. Although ANG II reportedly activated the NF-{kappa}B-dependent pathway in vascular smooth muscle cells (VSMCs) and vascular endothelial cells, few studies have examined the functional role of the NF-{kappa}B-dependent pathway in ANG II-induced MCP-1 expression (22, 27, 29, 38). MAPK p38 is also known to be implicated in TNF-{alpha}-induced MCP-1 expression in vascular endothelial cells, although its effect on TNF-{alpha}-induced MCP-1 expression is reportedly modest (12). We have shown that ANG II induces MCP-1 expression via p38-mediated activation of the transcription factor MEF2 in VSMCs and that ANG II-induced MCP-1 expression is potently inhibited by blockade of the p38-dependent pathway (32). Thus, ANG II and TNF-{alpha} may utilize distinct sets of intracellular signaling pathways to stimulate MCP-1 expression. Interestingly, both NF-{kappa}B- and p38-dependent pathways can be, at least partly, activated by ROS (13, 21).

We hypothesized that ANG II and TNF-{alpha} would cooperatively stimulate vascular inflammation. To test this hypothesis, we examined whether ANG II and TNF-{alpha} would cooperatively stimulate MCP-1 expression. We examined the role of NF-{kappa}B- and p38-dependent pathways in the induction of MCP-1 expression. We also examined the role of ROS in the induction of MCP-1 expression. Finally, we examined the cooperative effect of endogenous ANG II and TNF-{alpha} in vivo using a wire injury model of the rat femoral artery.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. N-acetylcysteine (NAC) was purchased from Sigma-Aldrich (St. Louis, MO). Apocynin, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), rotenone, and human TNF-{alpha} were obtained from Wako Pure Chemical (Osaka, Japan). Valsartan (Val) was kindly supplied by Novartis Pharma (Basel, Switzerland). ANG II was purchased from the Peptide Institute (Osaka, Japan). Antibodies against the type 1 receptor for ANG II (AT1R), the type 1 receptor for TNF-{alpha} (TNFR1), p38, and the inhibitor of NF-{kappa}B ({alpha}-isoform; I{kappa}B-{alpha}) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphospecific anti-p38 and anti-I{kappa}B-{alpha} antibodies, which recognize catalytically active p38 and I{kappa}B-{alpha}, respectively, were obtained from New England BioLabs (Beverly, MA).

Cell culture. VSMCs were cultured from rat thoracic aortas following the explant method, as previously described (26). NIH3T3 cells were cultured using DMEM supplemented with 10% FBS.

Western blot analysis. Western blot analysis was performed as previously described (30).

RNA extraction and real-time PCR. Total RNA was extracted using TRIzol Reagent (GIBCO-BRL, Rockville, MD) according to the instructions provided by the manufacturer. To extract total RNA from the rat femoral artery, the femoral artery was homogenized in TRIzol Reagent. After phenol-chloroform extraction, a small amount of total RNA was coprecipitated with tRNA (Sigma-Aldrich). Total RNA was subjected to reverse transcription using an Omniscript RT kit (QIAGEN, Tokyo, Japan). The expression of MCP-1 and GAPDH was examined by real-time PCR using SYBR green dye as previously described (28). The primers used were as follows: rat MCP-1 sense 5'-CTCAGCCAGATGCAGTTAATGC-3', rat MCP-1 antisense 5'-TCTCCAGCCGACTCATTGG-3', rat GAPDH sense 5'-GTATGACTCTACCCACGGCAAGT-3', and rat GAPDH antisense 5'-TTCCCGTTGATGACCAGCTT-3'. Real-time PCR was performed using an ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA). To confirm that no significant amounts of primer dimers were formed, dissociation curves were analyzed.

Detection of ROS. Intracellular ROS production was detected using the fluorescent probe DCFH-DA. Cultured rat VSMCs were loaded with DCFH-DA (10 µmol/l) for 30 min at 37°C and washed twice with PBS. Fluorescence images were obtained with a CSU21 laser scanning confocal microscope system (Yokogawa Electronic, Tokyo, Japan) coupled to an ORCA-ER high-resolution digital charge-coupled device camera (Hamamatsu Photonics, Shizuoka, Japan). Fluorescence was detected at an excitation wavelength of 488 nm and an emission wavelength of 510 nm. The relative fluorescence intensity was measured using an AQUACOSMOS image-analysis system (Hamamatsu Photonics).

Plasmids. Cloning of hemagglutinin epitope-tagged mouse I{kappa}B-{alpha} was performed by RT-PCR using total RNA extracted from NIH3T3 cells. The primers used were as follows: sense 5'-TTTCAGCCAGCTGGGCACGGCCA-3' and antisense 5'-TTATAATGTCAGACGCTGGCCTCCA-3'. The construction of I{kappa}B-{alpha} S32A/S36A (I{kappa}B{alpha}AA), in which Ser32 and Ser36 were substituted with alanine, was performed by PCR. The primer used to introduce the mutation was as follows: 5'-GACGATCGCCACGACgcCGGCCTGGACgCCATGAAGGACGAGGA-3' (where the lowercase bold letters indicate the nucleotide substitutions to induce the mutation). The entire DNA sequence was determined by a cycle sequence reaction using a CEQ8000 DNA sequencer (Beckman Coulter, Fullerton, CA). The construction of wild-type MEK6 and MEK6 S207A/T211A (MEK6AA), in which Ser207 and Thr211 were replaced with alanine, were as previously described (31). Isolation of the promoter region of the human MCP-1 gene (2,644 bp, pGL2/–2644 human MCP-1 promoter) was performed by PCR as previously described (28).

Adenovirus. Replication-defective adenovirus that expressed I{kappa}B{alpha}AA (AdI{kappa}B{alpha}AA) was constructed according to the method previously described (31) using an AdMax kit (Microbix Biosystems, Toronto, ON, Canada). The construction of adenoviruses that expressed MEK6AA (AdMEK6AA) and a carboxyl terminal-truncated form of rat TNFR1 (AdTNFR{Delta}C) have been previously described (32, 34). A recombinant adenovirus that expressed green fluorescence protein (AdGFP) was obtained from Quantum Biotechnologies (Montreal, QC, Canada).

Transient transfection. pRL-TK, which encodes the SeaPansy luciferase gene, was purchased from Toyo Ink (Tokyo, Japan) and used as the internal control for luciferase assays. To examine the activity of the MCP-1 promoter, rat VSMCs were transiently transfected with the reporter plasmid pGL2/–2644 human MCP-1 promoter along with pRL-TK using LIPOFECTAMINE (Life Technologies, Rockville, MD). Rat VSMCs were also cotransfected with expression plasmids encoding I{kappa}B{alpha}AA (pcDNA3HA/I{kappa}B{alpha}AA) and MEK6AA (pcDNA3HA/MEK6AA) in some experiments to examine the effects of these mutants on the activities of the promoter. The total amounts of plasmid DNA transfected in VSMCs were adjusted using the expression vector pcDNA3. A dual-luciferase assay was performed using a luminometer (Lumat LB 9507, Berthold, Bad Wildbad, Germany). SeaPansy luciferase activity was used as the internal control to normalize the promoter activity.

Animal experiments. All procedures involving experimental animals were approved by the institutional committee for animal research of Tokyo University. Male Wistar rats (7 wk old) were purchased from Charles River Laboratories (Wilmington, MA). They were fed a standard chow and had free access to water. Wistar rats were divided into five groups (10 rats/group) as follows: the control group, a group with AdGFP infection, a group treated with Val, a group with AdTNFR{Delta}C infection, and a group treated with Val plus AdTNFR{Delta}C infection. Rats in the Val (3 mg·kg–1·day–1)-treated groups received an oral dose of Val dissolved in distilled water starting 2 days before and until 2 wk after a wire injury. Rats in the other groups received the same amount of distilled water.

Rat femoral artery injury. Transluminal mechanical injury to the rat femoral artery was performed as previously described (28). Rats were anesthetized with pentobarbital injected intraperitoneally, and a groin incision was made under a surgical microscope. A guidewire (0.46 mm diameter) was introduced through a small muscular branch of the femoral artery proximally to the aortic bifurcation and withdrawn. Adenoviruses (1 x 108 plaque-forming units) or saline were injected at this time, as previously described (32). Femoral arteries were harvested 2 wk after the wire injury.

Histochemical analysis. Femoral arteries were fixed by perfusion with 4% paraformaldehyde and processed for paraffin embedding. Cross sections (2 µm) were cut, deparaffinized, rehydrated, and stained with hematoxylin and eosin. For immunohistochemistry, sections were incubated with mouse anti-rat ED1 antibody (Serotec, Oxford, UK) diluted at 1:800. Sections were then incubated with biotinylated anti-mouse secondary antibody and finally horseradish peroxidase-labeled streptavidin according to the instructions provided by the manufacturer (DAKO, Copenhagen, Denmark). Sections were counterstained with hematoxylin.

Statistical analysis. Values are means ± SE. Statistical analyses were performed using ANOVA followed by the Student-Neumann-Keul test. Differences with a P value of <0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ANG II and TNF-{alpha} stimulate MCP-1 expression in a synergistic manner. We first examined whether ANG II and TNF-{alpha} cooperatively stimulated MCP-1 expression in cultured VSMCs using real-time PCR analysis (Fig. 1). ANG II significantly promoted MCP-1 expression in a dose-dependent manner, and ANG II (10–7 mol/l)-induced MCP-1 expression was significantly suppressed by pretreatment with Val, suggesting that the ANG II induction of MCP-1 expression was mediated by the AT1R. TNF-{alpha} also significantly stimulated MCP-1 expression in a dose-dependent manner, and TNF-{alpha} (10 ng/ml)-induced MCP-1 expression was significantly inhibited when VSMCs were infected with AdTNFR{Delta}C. When VSMCs were stimulated with ANG II and TNF-{alpha} simultaneously, MCP-1 expression further increased. Interestingly, although a low dose of ANG II (10–9 mol/l) only slightly stimulated MCP-1 expression, the combined treatment of ANG II (10–9 mol/l) and TNF-{alpha} (10 ng/ml) significantly enhanced MCP-1 expression compared with TNF-{alpha} (10 ng/ml) treatment alone. Furthermore, although a low dose of TNF-{alpha} (0.1 ng/ml) only slightly promoted MCP-1 expression, it significantly enhanced ANG II (10–7 mol/l)-induced MCP-1 expression when VSMCs were stimulated with both TNF-{alpha} (0.1 ng/ml) and ANG II (10–7 mol/l). These results suggested that ANG II and TNF-{alpha} stimulated MCP-1 expression in a synergistic manner.


Figure 1
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  		Fig. 1. ANG II and TNF-{alpha} stimulate monocyte chemoattractant protein (MCP)-1 expression in a synergistic manner. Cultured rat vascular smooth muscle cells (VSMCs) were serum starved for 3 days and stimulated with ANG II and/or TNF-{alpha} for 4 h. Some cells were pretreated with valsartan (Val; 200 nmol/l) for 30 min. AdTNFR{Delta}C infection [at a multiplicity of infection (MOI) of 20] was performed when VSMCs were serum starved. Total RNA was extracted and used for real-time PCR analysis. GAPDH expression was used as an internal control. *P < 0.05, {dagger}P < 0.01, and {ddagger}P < 0.001, respectively, vs. the nonstimulated control (Cont) (n = 6 each); §P < 0.01 vs. ANG II (10–7 mol/l) stimulation (n = 6); **P < 0.01 vs. TNF-{alpha} (10 ng/ml) stimulation (n = 6).

 
We therefore examined whether ANG II and TNF-{alpha} stimulated the expression of TNFR1 and AT1R, respectively. TNF-{alpha} did not significantly stimulate AT1R expression until 8 h poststimulation, nor did ANG II significantly stimulate TNFR1 expression (Fig. 2), suggesting that ANG II and TNF-{alpha} stimulated MCP-1 expression in a synergistic manner at levels located downstream of their receptor expressions.


Figure 2
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  		Fig. 2. Expression of the ANG II type 1 receptor (AT1R) and TNF-{alpha} type 1 receptor (TNFR1) in VSMCs. A: effect of TNF-{alpha} on AT1R expression. Cultured rat VSMCs were serum starved for 3 days and stimulated with 10 ng/ml TNF-{alpha} for up to 8 h. Protein extracts were subjected to Western blot analysis (top) using anti-AT1R antibody. The bottom histogram shows the relative intensity of each band (n = 6). B: effect of ANG II on TNFR1 expression. VSMCs were serum starved for 3 days and stimulated with 10–7 mol/l ANG II for up to 8 h. Western blot analysis (top) was performed using anti-TNFR1 antibody. The bottom histogram shows the relative intensity of each band (n = 6).

 
Effect of blockade of the NF-{kappa}B-dependent pathway on ANG II- and TNF-{alpha}-induced MCP-1 expression. To clarify the intracellular signaling pathways that mediate ANG II- and TNF-{alpha}-induced MCP-1 expression, we first examined the role of the NF-{kappa}B-dependent pathway. To study the activation of the NF-{kappa}B-dependent pathway, we examined the phosphorylation of I{kappa}B-{alpha}. TNF-{alpha} stimulated phosphorylation of I{kappa}B-{alpha} in a time-dependent manner, and its phosphorylation peaked at ~2–5 min poststimulation (Fig. 3A). ANG II also stimulated phosphorylation of I{kappa}B-{alpha} in a time-dependent manner. Interestingly, its phosphorylation peaked at ~20–30 min after stimulation, which was apparently later than I{kappa}B-{alpha} phosphorylation was observed in TNF-{alpha} stimulation. We also examined the effects of several ROS inhibitors on I{kappa}B-{alpha} phosphorylation. Apocynin (an inhibitor of NADPH oxidase), rotenone (an inhibitor of the mitochondrial respiratory chain), and NAC (an antioxidant) partially inhibited TNF-{alpha}- and ANG II-induced I{kappa}B-{alpha} phosphorylation (Fig. 3B). TNF-{alpha}-induced MCP-1 expression was significantly suppressed to a nonstimulated basal level when VSMCs were infected with AdI{kappa}B{alpha}AA. In contrast, although AdI{kappa}B{alpha}AA infection significantly inhibited ANG II-induced MCP-1 expression, its effect was modest (Fig. 3C). We also used the I{kappa}B-{alpha} inhibitor IMD-0354 and obtained basically the same results (data not shown). These results suggested that, although the NF{kappa}B-dependent pathway is implicated in both TNF-{alpha}- and ANG II-induced MCP-1 expression, its contribution is larger in TNF-{alpha}-induced MCP-1 expression than in ANG II-induced MCP-1 expression.


Figure 3
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  		Fig. 3. Role of the NF-{kappa}B-dependent pathway in ANG II- and TNF-{alpha}-induced MCP-1 expression. A: time course of I{kappa}B-{alpha} phosphorylation in response to ANG II and TNF-{alpha} stimulation. Cultured rat VSMCs were serum starved for 3 days and stimulated with ANG II (10–7 mol/l) or TNF-{alpha} (10 ng/ml) for the indicated periods. Western blot analysis was performed using anti-phosphospecific I{kappa}B-{alpha} antibody (p-I{kappa}B-{alpha}) and anti-I{kappa}B-{alpha} antibody (I{kappa}B-{alpha}), which recognizes total I{kappa}B-{alpha}. Shown are representative results of 3 independent experiments in which the same results were obtained. B: effect of ROS inhibitors on ANG II- and TNF-{alpha}-induced I{kappa}B-{alpha} phosphorylation. Cultured rat VSMCs were serum starved for 3 days. Some cells were pretreated with apocynin (Apo; 30 µmol/l), rotenone (Rote; 10 µmol/l), or N-acetylcysteine (NAC; 500 µmol/l) for 30 min and stimulated with ANG II (10–7 mol/l) and TNF-{alpha} (10 ng/ml) for 20 and 5 min, respectively. Western blot analysis was performed as described in A. Shown are representative results of 3 independent experiments in which the same results were obtained. C: effect of blockade of the NF-{kappa}B-dependent pathway on ANG II- and TNF-{alpha}-induced MCP-1 expression. Cultured rat VSMCs were serum starved for 3 days and stimulated with ANG II (10–7 mol/l) or TNF-{alpha} (10 ng/ml) for 4 h. AdI{kappa}B{alpha}AA infection (20 MOI) was performed when VSMCs were serum starved. Total RNA was extracted and used for real-time PCR analysis. GAPDH expression was used as an internal control. *P < 0.01 vs. ANG II stimulation; {dagger}P < 0.01 vs. TNF-{alpha} stimulation (n = 6).

 
Effect of blockade of the p38-dependent pathway on ANG II- and TNF-{alpha}-induced MCP-1 expression. We next examined the role of the p38-dependent pathway, because we have shown that ANG II-induced MCP-1 expression is mediated by p38-induced activation of MEF2 (32). To study the activation of the p38-dependent pathway, we examined the phosphorylation of p38. Both TNF-{alpha} and ANG II stimulated p38 phosphorylation in a time-dependent manner, and its phosphorylation peaked at ~10–15 min poststimulation (Fig. 4A). ROS inhibitors partially inhibited TNF-{alpha}- and ANG II-induced p38 phosphorylation (Fig. 4B). TNF-{alpha}-induced MCP-1 expression was modestly but significantly suppressed when VSMCs were infected with AdMEK6AA (Fig. 4C). In contrast, AdMEK6AA infection potently and significantly inhibited ANG II-induced MCP-1 expression to a nonstimulated basal level. These results suggested that the p38-dependent pathway is implicated in both TNF-{alpha}- and ANG II-induced MCP-1 expression, even though its contribution is larger in ANG II-induced MCP-1 expression than in TNF-{alpha}-induced MCP-1 expression.


Figure 4
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  		Fig. 4. Role of the p38-dependent pathway in ANG II- and TNF-{alpha}-induced MCP-1 expression. A: time course of p38 phosphorylation in response to ANG II and TNF-{alpha} stimulation. Experiments were performed as described in Fig. 3. Western blot analysis was performed using anti-phosphospecific p38 antibody (p-p38) and anti-p38 antibody (p38), which recognizes total p38. Shown are representative results of 3 independent experiments in which the same results were obtained. B: effect of ROS inhibitors on ANG II- and TNF-{alpha}-induced p38 phosphorylation. Experiments were performed as described in Fig. 3. Cultured rat VSMCs were stimulated with ANG II (10–7 mol/l) and TNF-{alpha} (10 ng/ml) for 15 min. Shown are representative results of 3 independent experiments in which the same results were obtained. C: effect of blockade of the p38-dependent pathway on ANG II- and TNF-{alpha}-induced MCP-1 expression. Experiments were performed as described in Fig. 3. AdMEK6AA infection (20 MOI) was performed when VSMCs were serum starved. *P < 0.05 vs. ANG II stimulation; {dagger}P < 0.01 vs. TNF-{alpha} stimulation (n = 6).

 
Blockade of both NF-{kappa}B- and p38-dependent pathways is sufficient to inhibit ANG II- and TNF-{alpha}-induced MCP-1 expression. We therefore examined whether blockade of both the NF-{kappa}B- and p38-dependent pathways would inhibit ANG II- and TNF-{alpha}-induced MCP-1 expression. VSMCs were infected with both AdI{kappa}B{alpha}AA and AdMEK6AA and stimulated with ANG II and TNF-{alpha} simultaneously. Infection with AdI{kappa}B{alpha}AA and AdMEK6AA potently and significantly suppressed ANG II- and TNF-{alpha}-induced MCP-1 expression to a nonstimulated basal level (Fig. 5), suggesting that NF-{kappa}B- and p38-dependent pathways are major pathways that mediate ANG II- and TNF-{alpha}-induced MCP-1 expression.


Figure 5
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  		Fig. 5. Blockade of both NF-{kappa}B- and p38-dependent pathways is sufficient to inhibit ANG II- and TNF-{alpha}-induced MCP-1 expression. Cultured rat VSMCs were serum starved for 3 days and stimulated with ANG II (10–7 mol/l) and TNF-{alpha} (10 ng/ml) simultaneously for 4 h. Infection with AdGFP (30 MOI) or with both AdI{kappa}B{alpha}AA and AdMEK6AA (15 MOI each) was performed when VSMCs were serum starved. Total RNA was extracted and used for real-time PCR analysis. GAPDH expression was used as an internal control. *P < 0.05 vs. ANG II and TNF-{alpha} stimulation (n = 4 each).

 
ANG II and TNF-{alpha} additively stimulate MCP-1 promoter activity. To further examine the mechanism by which ANG II and TNF-{alpha} synergistically stimulate MCP-1 expression, we examined the effect of ANG II and TNF-{alpha} stimulation on MCP-1 promoter activity. When VSMCs were stimulated with ANG II, MCP-1 promoter activity significantly increased 8–12 h after stimulation (Fig. 6A). MCP-1 promoter activity also significantly increased 4–12 h after stimulation with TNF-{alpha}. We therefore stimulated VSMCs with ANG II and TNF-{alpha} for 12 h. Stimulation with both ANG II and TNF-{alpha} additively induced MCP-1 promoter activity compared with either stimulation alone (Fig. 6B). This increased activation was significantly suppressed almost to basal, nonstimulated levels by cotransfection with expression plasmids encoding I{kappa}B{alpha}AA and MEK6AA. These results suggested that ANG II and TNF-{alpha} synergistically stimulate MCP-1 expression, at least partly, at the level of transcription and that both NF-{kappa}B- and p38-dependent pathways are implicated in this transcriptional activation.


Figure 6
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  		Fig. 6. ANG II and TNF-{alpha} additively stimulate MCP-1 promoter activity. A: time course of MCP-1 promoter activity. Rat VSMCs were transfected with 2.0 µg pGL2/–2644 human MCP-1 promoter and 0.25 µg pRL-TK. Rat VSMCs were serum starved for 48 h and stimulated with ANG II or TNF-{alpha} for the indicated periods. SeaPansy luciferase activity was used as the internal control. The relative luciferase activity observed in nonstimulated control cells (0 h) was calculated as 1.0, and the fold induction at each time point is indicated. *P < 0.05 vs. 0 h; {dagger}P < 0.01 vs. 0 h (n = 6). B: ANG II and TNF-{alpha} additively enhance MCP-1 promoter activity. Rat VSMCs were transfected with 1.5 µg pGL2/–2644 human MCP-1 promoter and 0.25 µg pRL-TK along with 1.0 µg pcDNA3HA/I{kappa}B{alpha}AA and pcDNA3HA/MEK6AA in some experiments. The total amount of plasmid DNA transfected in VSMCs was adjusted using the expression vector pcDNA3. Rat VSMCs were serum starved for 48 h and stimulated with both ANG II and TNF-{alpha} for 12 h. SeaPansy luciferase activity was used as the internal control. The relative luciferase activity observed in nonstimulated control cells was calculated as 1.0, and the fold induction in each group is indicated. *P < 0.05 vs. control (n = 6); {dagger}P < 0.01 vs. control; {ddagger}P < 0.01 vs. ANG II stimulation; §P < 0.01 vs. TNF-{alpha} stimulation; **P < 0.01 vs. ANG II plus TNF-{alpha} stimulation.

 
Effect of ROS inhibitors on ANG II- and TNF-{alpha}-induced ROS production. Because ROS inhibitors inhibited ANG II- and TNF-{alpha}-stimulated phosphorylation of I{kappa}B-{alpha} and p38, we measured ROS production in response to ANG II and TNF-{alpha} stimulation. We first examined the time course of ROS production in response to ANG II or TNF-{alpha} stimulation. Intracellular ROS levels started to increase 1 h after stimulation with ANG II or TNF-{alpha} and peaked at ~4 h poststimulation (data not shown). We therefore detected ROS production 4 h after ANG II and TNF-{alpha} stimulation. ANG II and TNF-{alpha} significantly stimulated ROS production; however, the extent of ROS production in response to ANG II was modest provided that DCFH-DA was used as the probe (Fig. 7, A and B). When VSMCs were stimulated with ANG II and TNF-{alpha} simultaneously, ROS production further increased. We basically examined the effects of ROS inhibitors on ROS production in VSMCs stimulated with ANG II and TNF-{alpha} simultaneously, because it was easier to detect the effects of ROS inhibitors (Fig. 7, C and D). Apocynin and rotenone partially but significantly suppressed ROS production induced by ANG II and TNF-{alpha}. NAC significantly inhibited ROS production to a similar level as observed in nonstimulated control cells. NAC also significantly suppressed ROS production to the nonstimulated basal level when VSMCs were stimulated with ANG II alone or TNF-{alpha} alone (data not shown). When VSMCs were pretreated with both apocynin and rotenone, ROS production was significantly inhibited to the same level as observed in NAC-pretreated cells. These results suggested that NADPH oxidase and the mitochondrial respiratory chain were the major sources of ROS produced in response to ANG II and TNF-{alpha} in VSMCs.


Figure 7
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  		Fig. 7. Detection of ROS production in VSMCs. A: effect of ANG II and TNF-{alpha} on ROS production. Cultured rat VSMCs were serum starved for 3 days and stimulated with ANG II (10–7 mol/l) and/or TNF-{alpha} (10 ng/ml) for 4 h. Cells were incubated with the fluorescence dye 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) during the last 30 min. B: statistical analysis of ROS production. The histogram shows relative fluorescence intensity (n = 6 each). *P < 0.01 vs. nonstimulated control; {dagger}P < 0.01 vs. TNF-{alpha} stimulation; {ddagger}P < 0.001 vs. ANG II stimulation. C: effect of ROS inhibitors on ROS production. Cultured rat VSMCs were serum starved for 3 days. Some cells were pretreated with apocynin (30 µmol/l), rotenone (10 µmol/l), or NAC (500 µmol/l) for 30 min and stimulated with ANG II (A; 10–7 mol/l) and TNF-{alpha} (T; 10 ng/ml) for 4 h. Cells were incubated with DCFH-DA during the last 30 min. D: statistical analysis of ROS production. The histogram shows relative fluorescence intensity (n = 8 each). *P < 0.0001 vs. nonstimulated control; {dagger}P < 0.001 vs. ANG II and TNF-{alpha} stimulation.

 
ANG II- and TNF-{alpha}-induced MCP-1 expression is partially inhibited by ROS inhibitors. We also examined the role of ROS in ANG II- and TNF-{alpha}-induced MCP-1 expression (Fig. 8). VSMCs were stimulated with ANG II and TNF-{alpha} simultaneously, and the effects of ROS inhibitors were examined. Although NAC potently inhibited ROS production to a nonstimulated basal level, as shown in Fig. 7, MCP-1 expression was only partially suppressed by treatment with NAC. Apocynin and rotenone also partially but significantly suppressed ANG II- and TNF-{alpha}-induced MCP-1 expression. When VSMCs were pretreated with both apocynin and rotenone, ANG II- and TNF-{alpha}-induced MCP-1 expression was significantly suppressed to a similar level as observed with NAC pretreatment. ROS inhibitors also partially but significantly suppressed MCP-1 expression induced by ANG II alone (percent inhibition: apocynin, 35.9 ± 15; rotenone, 55 ± 1.6; and NAC, 57.6 ± 7.5; P < 0.05 vs. 10–7 mol/l ANG II stimulation, n = 4) or TNF-{alpha} alone (percent inhibition: apocynin, 46.9 ± 6.3; rotenone, 43.5 ± 12.4; and NAC, 54.4 ± 13.6; P < 0.05 vs. 10 ng/ml TNF-{alpha} stimulation, n = 4). In contrast, these ROS inhibitors did not affect MCP-1 expression in nonstimulated control cells, suggesting that these ROS inhibitors did not cause cytotoxic effects at the doses used in this study. Collectively, these results suggested that ROS played a role in ANG II- and TNF-{alpha}-induced MCP-1 expression, although ROS-independent pathways might also be implicated in ANG II- and TNF-{alpha}-induced MCP-1 expression.


Figure 8
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  		Fig. 8. Effects of ROS inhibitors on MCP-1 expression. VSMCs were serum starved for 3 days and pretreated with NAC (500 µmol/l), apocynin (30 µmol/l), or rotenone (10 µmol/l) for 30 min. VSMCs were then stimulated with 10–7 mol/l ANG II and 10 ng/ml TNF-{alpha} for 4 h. MCP-1 expression was analyzed by real-time PCR. Expression of GAPDH was used as an internal control. *P < 0.0001 vs. nonstimulated control (n = 6); {dagger}P < 0.01 vs. ANG II and TNF-{alpha} stimulation (n = 6); {ddagger}P < 0.05 vs. ANG II and TNF-{alpha} stimulation (n = 6).

 
Effect of blockade of endogenous ANG II and TNF-{alpha} on neointimal formation, macrophage infiltration, and MCP-1 expression. It has been reported that MCP-1 plays pivotal roles in neointimal formation via stimulation of macrophage infiltration in blood vessels (1, 11, 20). It has also been shown that macrophages are critically implicated in neointimal formation (6, 14). Since we found that ANG II and TNF-{alpha} synergistically stimulated MCP-1 expression in cultured VSMCs, we examined whether endogenous ANG II and TNF-{alpha} cooperatively stimulated neointimal formation and macrophage infiltration using a wire injury model of the rat femoral artery. Val (3 mg·kg–1·day–1) did not significantly alter blood pressure, which was measured by the tail cuff method (data not shown). Neointimal formation (the ratio of the intimal to medial area) was significantly inhibited by Val (Fig. 9) compared with that in control rats. AdTNFR{Delta}C infection also significantly suppressed neointimal formation, whereas AdGFP infection did not significantly suppress neointimal formation, compared with control rats. When rats were subjected to both Val administration and AdTNFR{Delta}C infection to block both RAS and TNF-{alpha} signaling, neointimal formation was more significantly inhibited in an additive manner. In accordance with these results, infiltration of macrophages in the neointima was significantly suppressed by Val administration or AdTNFR{Delta}C infection (Fig. 10). When rats were treated with Val and exposed to AdTNFR{Delta}C infection, the number of macrophages that infiltrated the neointima further decreased. Furthermore, MCP-1 expression in the wire-injured femoral artery was significantly suppressed by Val administration or AdTNFR{Delta}C infection (Fig. 11). When rats were subjected to both Val administration and AdTNFR{Delta}C infection, MCP-1 expression was further suppressed. These results suggested that endogenous ANG II and TNF-{alpha} additively stimulated neointimal formation via, at least partly, stimulation of MCP-1 expression and macrophage infiltration.


Figure 9
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  		Fig. 9. A: effects of blockade of endogenous ANG II and TNF-{alpha} on neointimal formation. Val was administered to Wistar rats starting 2 days prior to wire injury. The left femoral artery was wire injured, and AdGFP or AdTNFR{Delta}C was injected at this time. Neointimal formation was analyzed histologically 2 wk after the injury. B: the ratio of the intimal to medial area (I/M ratio) was compared among the groups (n = 10 each). *P < 0.001 vs. control; {dagger}P < 0.01 vs. AdGFP infection; {ddagger}P < 0.05 vs. Val administration; §P < 0.05 vs. AdTNFR{Delta}C infection.

 

Figure 10
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  		Fig. 10. A: effects of blockade of endogenous ANG II and TNF-{alpha} on macrophage infiltration. Macrophages in the neointima were positively stained for ED1. B: numbers of macrophages infiltrated in the neointima were compared among the groups (n = 10 each). *P < 0.001 vs. control; {dagger}P < 0.05 vs. AdGFP infection; {ddagger}P < 0.05 vs. Val administration; §P < 0.05 vs. AdTNFR{Delta}C infection.

 

Figure 11
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  		Fig. 11. Effects of blockade of endogenous ANG II and TNF-{alpha} on MCP-1 expression. RNA was extracted from the wire-injured femoral artery, and MCP-1 expression was analyzed by real-time PCR. Expression of GAPDH was used as an internal control (n = 10 each). *P < 0.05 vs. control; {dagger}P < 0.05 vs. AdGFP infection, {ddagger}P < 0.05 vs. Val administration; §P < 0.05 vs. AdTNFR{Delta}C infection.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Although it is well known that the RAS plays pivotal roles in the pathogenesis of atherosclerosis and vascular restenosis, blockade of the RAS alone did not always result in sufficient inhibition of vascular restenosis in some reports (9, 15, 16). We therefore hypothesized that some other proinflammatory factors might stimulate vascular inflammation in concert with the RAS and that blockade of these factors as well as the RAS would be necessary to sufficiently inhibit vascular inflammation. Since both ANG II and TNF-{alpha} reportedly stimulate MCP-1 expression in blood vessels (3, 10, 40), we tested our hypothesis that ANG II and TNF-{alpha} cooperatively stimulate vascular inflammation using MCP-1 expression as a marker of vascular inflammation. We showed in this study that ANG II and TNF-{alpha} synergistically promoted MCP-1 expression.

To examine the mechanisms by which ANG II and TNF-{alpha} synergistically stimulate MCP-1 expression, we first examined the role of the NF-{kappa}B-dependent pathway. TNF-{alpha} stimulated NF-{kappa}B activation, as assessed by I{kappa}B-{alpha} phosphorylation, and TNF-{alpha}-induced MCP-1 expression was potently suppressed to a level similar to the nonstimulated basal level by blockade of the NF-{kappa}B-dependent pathway using AdI{kappa}B{alpha}AA. This result was compatible with that of a previous report (7) that showed that the NF-{kappa}B-dependent pathway was critically implicated in MCP-1 expression in vascular endothelial cells. In contrast, although ANG II stimulated I{kappa}B-{alpha} phosphorylation, its time course was apparently different from that observed in TNF-{alpha}-induced I{kappa}B-{alpha} phosphorylation. Furthermore, ANG II-induced MCP-1 expression was only partially suppressed by AdI{kappa}B{alpha}AA infection. Although ANG II reportedly activates the NF-{kappa}B-dependent pathway in VSMCs and vascular endothelial cells, few studies have examined the role of the NF-{kappa}B-dependent pathway in ANG II-induced MCP-1 expression by blocking, specifically, the activation of the NF-{kappa}B-dependent pathway (22, 27, 29, 38). Our results suggested that the NF-{kappa}B-dependent pathway plays a major role in TNF-{alpha}-induced MCP-1 expression but not in ANG II-induced MCP-1 expression in VSMCs.

We, therefore, examined the role of the p38-dependent pathway in ANG II- and TNF-{alpha}-induced MCP-1 expression. ANG II activated the p38-dependent pathway, and ANG II-induced MCP-1 expression was potently inhibited by AdMEK6AA infection to a level similar to the nonstimulated basal level. In contrast, although TNF-{alpha} stimulated the p38-dependent pathway, TNF-{alpha}-induced MCP-1 expression was modestly inhibited by AdMEK6AA infection. These results were compatible with those of a previous report (12) that demonstrated that blockade of the p38-dependent pathway partially inhibited TNF-{alpha}-induced MCP-1 expression in vascular endothelial cells. Thus, it appears that ANG II and TNF-{alpha} utilize distinct intracellular signaling pathways to induce MCP-1 expression and that this can be a mechanism by which ANG II and TNF-{alpha} synergistically stimulate MCP-1 expression.

Because NF-{kappa}B- and p38-dependent pathways are well-known redox-sensitive pathways (13, 21), and ANG II- and TNF-{alpha}-induced phosphorylation of I{kappa}B-{alpha} and p38 was inhibited by ROS inhibitors, we examined the role of ROS in MCP-1 expression. We found that NAC, apocynin, and rotenone partially but significantly inhibited ANG II- and TNF-{alpha}-induced MCP-1 expression, suggesting that ROS are implicated in ANG II- and TNF-{alpha}-induced MCP-1 expression. A growing body of evidence suggests that ROS play pivotal roles in physiological and pathophysiological functions of vascular tissues via interactions with a variety of intracellular signaling pathways (37). Among several pathways that produce ROS in blood vessels, NADPH oxidase and the mitochondrial respiratory chain are well-known pathways that mediate ANG II- and TNF-{alpha}-induced ROS production, especially in vascular endothelial cells (3, 5, 18, 19, 25, 36). It has also been reported that ROS are implicated in MCP-1 expression (17, 33, 39). In fact, it has been reported that blockade of ROS production was sufficient to potently inhibit TNF-{alpha}-induced MCP-1 expression (3). However, we did not find such a potent effect of ROS inhibitors on ANG II- and TNF-{alpha}-induced MCP-1 expression in rat VSMCs. Although the precise reasons for this discrepancy are not clear, the effect of ROS inhibitors may differ depending on the species and cell types. Our results suggested that ROS-independent activation of NF-{kappa}B- and p38-dependent pathways occurred and that these ROS-independent pathways were also implicated in ANG II- and TNF-{alpha}-induced MCP-1 expression.

Although ROS inhibitors suppressed the phosphorylation of I{kappa}B-{alpha} or p38 that peaked within ~2–20 min after stimulation with ANG II or TNF-{alpha}, an apparent increase of ROS production was detected 1 h or later after stimulation with ANG II or TNF-{alpha}. This result may look curious because ROS production should have increased before the phosphorylation of I{kappa}B-{alpha} or p38 occurs, if ROS is really implicated in the phosphorylation of I{kappa}B-{alpha} and p38. This is probably due to the detection limit of DCFH-DA that we used in this study.

To study whether endogenous ANG II and TNF-{alpha} cooperatively enhance vascular inflammation in vivo, we used a wire injury model of the rat femoral artery and examined the effects of the blockade of ANG II and TNF-{alpha} on neointimal formation. Neointimal formation, macrophage infiltration, and MCP-1 expression in the rat femoral artery after wire injury were further inhibited by blockade of both ANG II and TNF-{alpha} using Val and AdTNFR{Delta}C compared with blockade of ANG II alone or TNF-{alpha} alone. Our results suggested that endogenous ANG II and TNF-{alpha} cooperatively enhanced vascular inflammation in vivo via, at least in part, stimulation of MCP-1 expression and macrophage infiltration. However, because this model is not always suitable for studying the mechanisms of atherosclerosis, utilization of other models such as apolipoprotein E-null mice will be necessary to examine the role of endogenous ANG II and TNF-{alpha} in the progression of atherosclerosis.

Taken together, our results suggest the following scenario (Fig. 12). Both ANG II and TNF-{alpha} stimulate ROS production via activation of NADPH oxidase and mitochondrial respiratory chains, which, in turn, stimulates redox-sensitive pathways such as NF-{kappa}B and p38. However, both ANG II and TNF-{alpha} also activate NF-{kappa}B and p38 in a ROS-independent manner, and these ROS-independent pathways are also critical for full activation of NF-{kappa}B and p38. ANG II mainly utilizes the p38-dependent pathway to promote MCP-1 expression, whereas TNF-{alpha} mainly utilizes the NF-{kappa}B-dependent pathway to stimulate MCP-1 expression. When cells are stimulated with both ANG II and TNF-{alpha}, ANG II-induced activation of the p38-dependent pathway and TNF-{alpha} stimulation of the NF-{kappa}B-dependent pathway appear to synergistically stimulate MCP-1 expression. Given that both ANG II and TNF-{alpha} stimulate NF-{kappa}B and p38, it is unclear how ANG II and TNF-{alpha} selectively utilize p38 and NF-{kappa}B, respectively, to stimulate MCP-1 expression. ANG II and TNF-{alpha} may activate distinct sets of nuclear cofactors that modulate the activity of NF-{kappa}B or p38-dependent transcription factors, such as MEF2. Future studies are required to clarify this point.


Figure 12
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  		Fig. 12. Schematic representation of the proposed intracellular signaling pathways that mediate ANG II- and TNF-{alpha}-induced MCP-1 expression. NADPH, NADPH oxidase; Mit: mitochondrial respiratory chain.

 
In summary, ANG II and TNF-{alpha} synergistically stimulated MCP-1 expression via utilization of distinct intracellular signaling pathways, i.e., p38- and NF-{kappa}B-dependent pathways. These pathways were activated in ROS-dependent and -independent manners. This cooperative action of ANG II and TNF-{alpha} appears to promote vascular inflammation in vivo. Although blockade of the RAS is a useful and established strategy to reduce cardiovascular events, anti-proinflammatory cytokine therapy may be necessary in the future to further reduce the morbidity and mortality caused by cardiovascular diseases.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Core Research for Evolutional Science and Technology) (to T. Nagano and Y. Hirata). This work was also supported in part by Ministry of Education, Culture, Sports, Science and Technology of Japan Grants-In-Aid 17590709 (to E. Suzuki) and 19590966 (to K. Kimura).


   ACKNOWLEDGMENTS
 
The authors thank Novartis Pharma for supplying valsartan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Suzuki, Institute of Medical Science, St. Marianna Univ. School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki 216-8512, Japan (e-mail: esuzuki-tky{at}umin.ac.jp)

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.


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 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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