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Cardiovascular Research 2007 76(3):506-516; doi:10.1016/j.cardiores.2007.07.008
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Copyright © 2007, European Society of Cardiology

Cross-talk between aldosterone and angiotensin II in vascular smooth muscle cell senescence

Li-Juan Min, Masaki Mogi, Jun Iwanami, Jian-Mei Li, Akiko Sakata, Teppei Fujita, Kana Tsukuda, Masaru Iwai and Masatsugu Horiuchi*

Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University, Graduate School of Medicine, Tohon, Ehime 791-0295, Japan

*Corresponding author. Tel.: +81 89 960 5248; fax: +81 89 960 5251. horiuchi{at}m.ehime-u.ac.jp

Received 1 February 2007; revised 17 June 2007; accepted 11 July 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 Appendix A
 Acknowledgments
 References
 
Objective Our aim was to examine the possible cross-talk of angiotensin II (Ang II) and aldosterone (Aldo) in the regulation of vascular cell senescence in cultured vascular smooth muscle cells (VSMC).

Methods VSMC were prepared from thoracic aorta of adult male Sprague–Dawley rats. Cellular senescence was evaluated by senescence-associated β-galactosidase (SA-β-gal) staining and expression of p21, p53, p16, and p27. Oxidative stress was determined by measuring NADPH oxidase activity and superoxide production. Signal transduction was examined by immunoblot analysis with or without RNA interference methods.

Results Persistent Ang II (100 nM) stimulation increased SA-β-gal-stained VSMC and enhanced expression of p21, p53, p16, p27 and Ki-ras2A. These effects of Ang II were markedly inhibited by treatment with a selective AT1 receptor blocker, valsartan, but partially attenuated by a mineralocorticoid receptor antagonist, spironolactone. The culture medium of VSMC treated with Ang II (100 nM) showed a time-dependent increase in Aldo concentration, which increased senescent VSMC. Antioxidant, N-acetyl-L-cysteine or superoxide dismutase attenuated Ang II- or Aldo-induced VSMC senescence and Ki-ras2A expression. A lower dose combination of Ang II (100 pM) and Aldo (1 pM) significantly enhanced SA-β-gal-stained VSMC with increases in expression of p21, p53, p16, p27 and Ki-ras2A, oxidative stress, and activity of transcription factors such as NF-{kappa}B, AP-1, whereas Ang II or Aldo alone at these doses did not affect these parameters. Ki-ras2A-siRNA treatment attenuated senescent VSMC, expression of p21, p53, p16 and p27, oxidative stress induced by Ang II or a lower dose combination of Ang II and Aldo.

Conclusion These results suggest that Ang II and Aldo exert cross-talk in VSMC senescence with involvement of oxidative stress and Ki-ras2A, and could provide a therapeutic benefit for age-related vascular disorders by blockade of both Ang II and Aldo.

KEYWORDS Renin–angiotensin system; Aging; Receptor; Signal transduction; Smooth muscle


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 Appendix A
 Acknowledgments
 References
 
The renin–angiotensin–aldosterone system (RAAS) regulates cardiovascular homeostasis and tissue responses [1]. Increasing evidence, including our recent study, suggests that aldosterone (Aldo) could play a role in vascular remodeling independent of Ang II, and act synergistically with Ang II, involving mineralocorticoid receptor (MR) activation [2–5]. Recently, histological examination of human atherosclerotic lesions has revealed that endothelial cells and vascular smooth muscle cells (VSMC) exhibit morphological features of cellular senescence [6,7]. Moreover, senescence-associated β-galactosidase (SA-β-gal)-positive vascular cells have been demonstrated in human atherosclerotic plaques of coronary arteries obtained from patients with ischemic heart disease [8]. Thus, these data support the notion that vascular cell senescence plays a critical role in age-related vascular disorders. Recently, it has been reported that Ang II induced p21-dependent premature senescence of VSMC in vitro [9]. Moreover, in animal models, the increase in local RAAS activity with aging and the efficacy of inhibitors of RAAS in blocking age-related hypertension suggest that Ang II and Aldo are closely associated with aging and age-related vascular disorders [10–12]. However, the functions of Ang II and Aldo and their possible cross-talk in regulating vascular cell senescence remain largely unknown.

Numerous studies suggest that Ang II is a potent mediator of oxidative stress [13,14] that can trigger activation of multiple signaling pathways contributing to vascular damage, such as atherosclerosis [15]. In addition, Aldo is also suggested to have a direct effect on oxidative stress in mediating cardiovascular fibrosis, which involves an interaction with Ang II [16]. An increase in the concentration of reactive oxygen species (ROS), either by increasing the oxygen partial pressure or by treating cells with H2O2, can induce premature senescence in human diploid fibroblasts [17,18], indicating that oxidative stress is one of the major determinants of a senescent cell phenotype. Despite these accumulating evidences, it is not clear whether oxidative stress is involved in Ang II and Aldo-mediated vascular cell senescence. Ras can act as an important signaling molecule of Ang II and Aldo [5,19], and closely links to oxidative stress [20]. There is a growing body of evidence that Ras plays a critical role in premature senescence, in contrast to its mitogenic activity [21–23]. And our previous study has reported the possible cross-talk of growth-promoting signaling between Ang II and Aldo in VSMC involving upregulation of Ki-ras2A, which is a member of the Ras family [5,19]. Thus, we speculated that there might also be cross-talk of Ang II and Aldo in vascular cell senescence-related Ki-ras2A signaling.

Therefore, to explore the roles of Ang II and Aldo in regulating vascular cell senescence, we examined the possible cross-talk between Ang II and Aldo in cellular senescence using cultured VSMC. To elucidate the cellular and molecular mechanisms, we especially focused on a key molecule, Ki-ras2A and examined the possible involvement of oxidative stress.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 Appendix A
 Acknowledgments
 References
 
2.1 Cell culture
Vascular smooth muscle cells (VSMC) were isolated from male adult Sprague–Dawley rat thoracic aorta (Clea Japan Inc., Tokyo, Japan) as previously described; these cells exclusively express the AT1 receptor but not the AT2 receptor [5]. The experimental protocol was approved by the Animal Studies Committee of Ehime University which is based on the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The cells were cultured on 100-mm dishes in Dulbecco's Modified Eagle Medium (DMEM) containing low-glucose (1 g/l) (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cells at passages 3 to 8 were used for the experiments. Subconfluent cells were serum-starved for 48 h to induce a quiescent state before the experiments. In all experiments, to expose to Ang II and/or Aldo or other reagents for the indicated time, the conditioned medium was aspirated and we added quickly the fresh culture medium without FBS in the presence or absence of Ang II and/or Aldo or other reagents every 24 h. We counted suspended cells in conditioned medium and did not observe significant differences in cell numbers.

2.2 Senescence-associated β-galactosidase (SA-β-gal) staining
Senescence-associated β-galactosidase (SA-β-gal) activity was detected as a biomarker for cellular senescence [22,23]. Cells that were 50–60% confluent and quiescent cultured on 6-well plates were exposed to different experimental conditions. SA-β-gal staining was performed using a Senescence Detection Kit (Bio Vision, Mountain View, CA, USA). The cells were photographed at x20 magnification, and counterstained with 4'6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) for 10 min to count the numbers of total and SA-β-gal-positive cells in five randomly chosen fields per group at x10 magnification.

2.3 Ang II receptor binding assay
The change of expression of AT1 receptor by Aldo was measured by radioligand binding assay as described previously [24].

2.4 Aldo receptor binding assay in VSMC fractions
The cytosolic fractions of VSMC were isolated as described previously [25,26]. For radioligand binding assay, each fraction (50 µg) was incubated for 4 h at room temperature in binding buffer [20 mM Tris/HCL (PH 7.5), 1 mM PMSF, 10 µg/ml aprotinin] containing [3H]-Aldo (100 nM) in the absence (for total count) or presence of spironolactone (10 µM), which biological blocking effect of Aldo on mineralocorticoid receptor (MR) is stronger than that on glucocorticoid receptor (GR). Bound and unbound [3H]-Aldo were separated by ice-cold 0. 5% dextran-coated charcoal (Sigma-Aldrich Corp., in binding buffer) treatment and centrifuged at 12,000 xg at 4 °C for 3 min. [3H]-Aldo binding to each fraction was determined by counting the radioactivity of the supernatant with a liquid scintillation β-counter. The difference between the total count and the count from samples incubated with spironolactone could be considered as the expression of intracellular receptor MR [27,28].

2.5 Immunoblot analysis
Subconfluent and quiescent VSMC cultured in 100-mm dishes were exposed to different experimental conditions. The proteins were subjected to SDS-PAGE and immunoblotted with anti-Ki-ras2A antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-p21, p53, p16 or p27 antibody (Cell Signaling Technology Inc., Beverly, MA), or anti-{alpha} smooth muscle actin antibody (Sigma-Aldrich, Inc., St. Louis, MO). The bands of proteins were visualized with an ECL system (Amersham Biosciences). Densitometric analysis was performed using NIH image software [5,29].

2.6 Determination of NADPH oxidase activity
Activity of NADPH oxidase in total protein of cell homogenates was measured with a luminescence assay as previously described [13], using 500 µM lucigenin as the electron acceptor and 100 µM NADPH as the substrate. The reaction was started by the addition of NADPH to the protein. Chemiluminescence was monitored with a luminometer (AB-2200, ATTO Corp., Tokyo, Japan).

2.7 Detection of superoxide anion in VSMC
Histological detection of superoxide anion in VSMC was carried out as described previously [30]. Cells were fixed with paraformaldehyde for 10 min and immediately incubated with dihydroethidium (DHE; 10 µM) in PBS for 30 min at 37 °C in a humidified chamber protected from light. DHE is oxidized on reaction with superoxide to ethidium, which binds to DNA in the nucleus and fluoresces red. For detection of ethidium, samples were examined with a Leica DMI6000B (Leica Microsystems, Wetzlar, Germany) equipped with a computer-based imaging system, FW4000 (Leica). The intensity of the fluorescence was analyzed and quantified using computer-imaging software (Densitograph, ATTO Corporation, Tokyo, Japan).

2.8 Luciferase activity assay
NF-{kappa}B and AP-1 activities were analyzed by luciferase activity assay. VSMC seeded in 6-well plates were transfected with 1 µg of NF-{kappa}B-luciferase Reporter Vector or AP-1-luciferase Reporter Vector (Panomics, Dumbarton Circle, Fremont, CA) using Lipofectamine PLUS (Invitrogen Corp. Carlsbad, CA) according to the instructions of manufacturer. To ensure the efficiency of equivalent transfection, we cotransfected pRL-null Vector expressing Renilla luciferase reporter gene as a control (Promega, Madison, WI). Forty-eight hours post-transfection, stimulated cells were lysed and subjected to luciferase activity assay, using Dual-Glo Luciferase Assay System (Promega, Madison, WI) on a luminometer (AB-2200, ATTO Corp., Tokyo, Japan). Data were expressed as fold change after normalization to the activity of Renilla luciferase.

2.9 RNA interference (RNAi) of Ki-ras2A
For small interfering RNA (siRNA) assay, VSMC were transiently transfected with a scrambled version of siRNA of Ki-ras2A as a control or Ki-ras2A-specific siRNA, a cocktail of three siRNAs designed by B-Bridge (Sunnyvale, CA), using Lipofectamine PLUS (Invitrogen Corp. Carlsbad, CA). Thirty-six hours after transfection, cells were subjected or not to various stimuli [5,31] and subjected to experiments. The sequences of siRNAs targeting Ki-ras2A and the scrambled version as a control are shown in Supplementary Tables 1 and 2.

2.10 Materials
Reagents not listed above were as follows: Aldo, spironolactone, N-acetyl-L-cysteine and superoxide dismutase were obtained from Sigma-Aldrich. An AT1 receptor blocker, valsartan, was donated by Novartis Pharma AG (Basel, Switzerland). All other reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan).

2.11 Statistical analysis
All values are expressed as mean±SEM in the text and figures. Data were evaluated by ANOVA followed by post-hoc analysis for multiple comparisons. Differences with P<0.05 were considered to be significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
Appendix A
 Acknowledgments
 References
 
3.1 Ang II induced VSMC senescence
VSMC treated with Ang II (100 nM) in serum-free DMEM medium for 7 days exhibited an enlarged size and flattened morphology, which are characteristics of the senescent phenotype of VSMC, whereas VSMC cultured in serum-free DMEM without Ang II showed similar morphology to those before treatment with Ang II (data not shown). With Ang II (100 nM) treatment, total cell number per field at x10 magnification increased time-dependently detected by 4'6-diamidino-2-phenylindole (DAPI) staining and reached a plateau at around 5 days (data not shown). Supplementary Fig. 1 is the photographs of increased SA-β-gal- and DAPI-stained VSMC by Ang II (100 nM) stimulation for 14 days. SA-β-gal activity is shown as the percentage of SA-β-gal-positive cell number to total cell number. The percentage of SA-β-gal-positive VSMC was significantly increased by Ang II (100 nM) stimulation in a time-dependent manner, starting 5 days after Ang II stimulation (Fig. 1A). Ang II (100 nM) treatment also increased expressions of p21, p53, p16, and p27 time-dependently (Fig. 1B and C). Treatment with a selective AT1 receptor blocker, valsartan (10 µM), markedly inhibited Ang II-induced SA-β-gal activity and expressions of p21, p53, p16, and p27. Interestingly, treatment with a mineralocorticoid receptor (MR) antagonist, spironolactone (10 µM), partially attenuated these effects of Ang II (Fig. 1D, E and F).


Figure 1
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  		Fig. 1 Ang II induces VSMC senescence. (A) Time course of SA-β-gal activity after stimulation with Ang II (100 nM). SA-β-gal activity was performed by SA-β-gal staining, the ratio of SA-β-gal-positive cells to total cells is shown. (B) Time course of expressions of p21, p53, p16, and p27 after stimulation with Ang II (100 nM). Expression of p21, p53, p16 or p27 was determined by Western blot analysis as described in "Materials and Methods", densitometric measurements are shown in (C). Fifty–60% confluent and quiescent VSMC were treated with new serum-free DMEM in the presence or absence of Ang II (100 nM), changing the old medium every 24 h for the indicated time. (D) Effects of valsartan (10 µM) and spironolactone (10 µM) on Ang II (100 nM)-induced SA-β-gal activity after 5 days of treatment. (E), (F) Effects of valsartan (10 µM) and spironolactone (10 µM) on Ang II (100 nM)-induced expressions of p21, p53, p16, and p27 after 5 days of treatment. Fifty–60% confluent and quiescent VSMC were treated with new serum-free DMEM in the presence or absence of Ang II (100 nM) with or without valsartan (10 µM) or spironolactone (10 µM) every 24 h until day 5. Similar results were obtained in three different culture lines. Values are expressed as mean±SEM (n=3). Ang II, angiotensin II; Val, valsartan; Spiro, spironolactone. *P<0.05 vs. control. #P<0.05 vs. Ang II (+).

 
3.2 Aldo secreted by Ang II stimulation promoted VSMC senescence
Therefore, we assessed the association between Ang II and Aldo in VSMC senescence. To investigate the interaction between the expression of Ang II receptors and cytosolic Aldo receptors, we performed radioligand receptor binding assay. AT1 receptor binding density in VSMC showed no significant change with Aldo stimulation (1 nM) for 5 days (basal level; 60.1±4.5 fmol/106 cells vs. after 5 days of Aldo treatment; 62.4±5.7 fmol/106 cells). Similarly, cytosolic Aldo receptor binding density was not significantly altered by Ang II (100 nM) stimulation for 5 days (basal level; 78.4±8.0 cpm/µg protein vs. after 5 days of Ang II treatment; 88.7±7.9 cpm/µg protein). Next, we measured Aldo concentration in the culture medium and observed that Ang II (100 nM) enhanced Aldo secretion in a time-dependent manner with Aldo concentration 0.59±0.026 nM after 5 days of stimulation (Supplementary Fig. 2A). Treatment with Aldo for 5 days at the doses in these conditioned media resulted in dose-dependent increases in SA-β-gal-stained VSMC and expressions of p21, p53, p16, and p27 (Supplementary Fig. 2B and C). These effects of Aldo were effectively blocked by spironolactone (10 µM), but not significantly changed by valsartan (10 µM) (Fig. 2). These results suggest that Aldo secreted by Ang II contributes at least partly to Ang II-induced VSMC senescence.


Figure 2
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  		Fig. 2 Aldo secreted by Ang II induces VSMC senescence. (A) Effect of Aldo (0.8 nM) on SA-β-gal activity after 5 days of stimulation. (B) Effect of Aldo (0.8 nM) on expressions of p21, p53, p16, and p27 after 5 days of stimulation. Expression of p21, p53, p16 or p27 was determined by Western blot analysis, densitometric measurements are shown in (C). Fifty–60% confluent and quiescent VSMC were treated with new serum-free DMEM in the presence or absence of Aldo (0.8 nM) with or without spironolactone (10 µM) and/or valsartan (10 µM), changing the old medium every 24 h until day 5. Similar results were obtained in three different culture lines. Values are expressed as mean±SEM (n=3). Aldo, aldosterone; Spiro, spironolactone; Val, valsartan. *P<0.05 vs. control. #P<0.05 vs. Aldo (+).

 
3.3 Ki-ras2A was involved in Ang II- or Aldo-mediated VSMC senescence
Ras is an important signaling molecule that has been demonstrated to promote vascular cell senescence [23]. Ras expression is regulated by Ang II and Aldo stimulation [5,19]. We also previously reported that Aldo increased Ki-ras2A expression through MR, thereby potentiating Ang II-mediated VSMC proliferation [5]. Here, we examined the possible role of Ki-ras2A in VSMC senescence. Ki-ras2A expression was increased after 1–7 days of stimulation with Ang II (100 nM) or Aldo (0.8 nM) (data not shown). Valsartan markedly inhibited Ang II-mediated Ki-ras2A expression after 3 days of stimulation (Fig. 3A). Spironolactone partially blocked Ang II induced-Ki-ras2A expression and markedly inhibited Aldo-induced Ki-ras2A expression (Fig. 3A and B). We examined the effect of inhibition of Ki-ras2A expression on VSMC senescence. Knockdown of the Ki-ras2A gene with RNA interference methods was performed, and the effectiveness of Ki-ras2A-siRNA was evaluated by determining Ki-ras2A expression by immunoblot analysis. Ki-ras2A expression was significantly attenuated in Ki-ras2A-siRNA-treated VSMC, but not in scrambled-siRNA-treated cells (Fig. 3C). Treatment with Ki-ras2A-siRNA markedly attenuated Ang II-induced SA-β-gal activity and expressions of p21, p53, p16, and p27 (Fig. 3D and E).


Figure 3
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  		Fig. 3 Role of Ki-ras2A in Ang II- or Aldo-induced VSMC senescence. Effect of Ang II (100 nM) (A) or Aldo (0.8 nM) (B) on Ki-ras2A expression after 5 days of stimulation. Administration of Ang II or Aldo with or without valsartan and/or spironolactone to VSMC was similar to those described in Figs. 1 and 2Go. Expression of Ki-ras2A was determined by Western blot analysis. (C) Effect of Ki-ras2A RNA interference on Ki-ras2A expression in VSMC performed by Western blot analysis. Inhibitory effects of Ki-ras2A-siRNA on Ang II (100 nM)-induced SA-β-gal activity (D) and expressions of p21, p53, p16, and p27 (E) after 5 days of stimulation are shown. Thirty-six hours after transfection with scrambled-siRNA or Ki-ras2A-siRNA, old cell culture medium were replaced with new serum-free DMEM in the presence or absence of Ang II (100 nM) every 24 h until day 5. Similar results were obtained in three different culture lines. Values are expressed as mean±SEM (n=3). Ang II, angiotensin II; Aldo, aldosterone; Val, valsartan; Spiro, spironolactone. *P<0.05 vs. control. #P<0.05 vs. Ang II (+) or Aldo (+).

 
3.4 Oxidative stress was important for Ang II- or Aldo-mediated VSMC senescence
Oxidative stress has been reported to induce cellular senescence, and associate with Ras signaling [22]. Moreover, it is well known that Ang II is a mediator of oxidative stress by activating mainly NADPH oxidase, resulting in superoxide production [13,14,16]. Thus, we examined the production of superoxide and NADPH oxidase activity by Ang II or Aldo stimulation in senescent VSMC. Ang II (100 nM) or Aldo (0.8 nM) treatment significantly increased fluorescence intensity of ethidium after 5 days of treatment. VSMC treated with Ang II (100 nM) or Aldo (0.8 nM) also exhibited a time-dependent increase in NADPH oxidase activity (Fig. 4A). Next, we examined the effects of inhibition of oxidative stress, using an antioxidant, N-acetyl-L-cysteine (NAC) or superoxide dismutase (SOD) as a free radical scavenger, on Ang II- or Aldo-induced VSMC senescence after 5 days of treatment. NAC (1 mM) or SOD (250 U/ml) attenuated Ang II or Aldo-induced SA-β-gal activity and Ki-ras2A expression (Fig. 4B and C). Ki-ras2A expression induced by Ang II was markedly inhibited by spironolactone with NAC (1 mM) or SOD (250 U/ml) (Fig. 4B). These results indicate that oxidative stress is involved in Ang II- or Aldo-induced VSMC senescence, and that Ki-ras2A could act one of down-stream targets of oxidative stress. ROS generated by NADPH oxidase can function as messenger molecules during pathophysiologic processes and have been shown to interact with down-stream signaling systems including activation of nuclear factor kappa B (NF-{kappa}B) and activator protein 1(AP-1) transcription factors in terms of cell proliferation, cell cycle and vascular remodeling [32]. Ang II has been shown to activate NF-{kappa}B and AP-1 in VSMC [33]. Therefore, we examined the possibility that Ang II or Aldo induced VSMC senescence via oxidative stress-regulated NF-{kappa}B or AP-1 activation. We observed that Ang II (100 nM) or Aldo (0.8 nM) administration for 5 days increased both NF-{kappa}B and AP-1 activities determined by luciferase activity assay (Fig. 4D).


Figure 4
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  		Fig. 4 Role of oxidative stress in Ang II- or Aldo-induced VSMC senescence. (A) Effect of Ang II (100 nM) or Aldo (0.8 nM) on superoxide production (a) and NADPH oxidase activity (b) after 5 days of stimulation. The intensity analysis of superoxide production was performed by using computer-imaging software as described in "Materials and Methods". Inhibitory effects of antioxidants, NAC (1 mM) and SOD (250 U/ml) with or without spironolactone (10 µM), on Ang II (100 nM)- (B) or Aldo (0.8 nM)-induced (C) SA-β-gal activity (a) and Ki-ras2A expression (b). (D) Effect of Ang II (100 nM) or Aldo (0.8 nM) on NF-{kappa}B (a) and AP-1 (b) activities. The activities of NF-{kappa}B and AP-1 were assayed by luciferase activity assay as described in "Materials and Methods". Administration of all reagents to VSMC was performed by changing the medium every 24 h with new medium containing freshly prepared reagents until day 5. Similar results were obtained in three different culture lines. Values are expressed as mean±SEM (n=3). Ang II, angiotensin II; Aldo, aldosterone; NAC, N-acetyl-L-cysteine; SOD, superoxide dismutase; Spiro, spironolactone. *P<0.05 vs. control. #P<0.05 vs. Ang II (+) or Aldo (+).

 
3.5 Treatment with combination of lower doses of Aldo and Ang II induced VSMC senescence
To explore the possible synergistic effect of Ang II and Aldo on VSMC senescence, in the following experiments, we used lower doses of Ang II (100 pM) and Aldo (1 pM). Treatment with Ang II or Aldo at these doses alone for 5 days did not significantly affect the percentage of SA-β-gal-positive VSMC and expressions of p21, p53, p16, and p27; however, combination treatment with Ang II and Aldo at these doses for 5 days markedly increased the percentage of SA-β-gal-positive cells and expressions of p21, p53, p16, and p27 (Fig. 5A). These effects of Ang II with Aldo were markedly inhibited by valsartan or spironolactone (Fig. 5A), suggesting that Aldo and Ang II could synergistically promote VSMC senescence. AT1 receptor and cytosolic Aldo receptor binding density in VSMC showed no significant change after lower doses of Ang II or Aldo stimulation (data not shown). Stimulation with Ang II at a dose of 100 pM did not increase Ki-ras2A expression, whereas Aldo treatment at a dose of 1 pM increased Ki-ras2A expression slightly in a time-dependent manner (data not shown). Interestingly, addition of Ang II (100 pM) to Aldo (1 pM) further increased Ki-ras2A expression time-dependently compared to that in Aldo (1 pM)-treated VSMC (Supplementary Fig. 3). The increase in Ki-ras2A expression induced by co-treatment with Ang II (100 pM) and Aldo (1 pM) was significantly blocked by both valsartan and spironolactone (Fig. 5B). We previously reported that this dose of Ang II (100 pM) did not increase Aldo secretion [5]. Therefore, the synergistic effects of Ang II and Aldo at these doses on VSMC senescence could be due to the synergistic effect on Ki-ras2A induction. Lower dose Ang II (100 pM) or Aldo (1 pM) treatment did not affect superoxide production, NADPH oxidase activity, NF-{kappa}B and AP-1 transcriptional activities; however, combination treatment with Ang II and Aldo at these doses strongly enhanced superoxide production, NADPH oxidase activity, NF-{kappa}B and AP-1 transcriptional activities determined after 5 days of treatment compared to Ang II or Aldo alone (Fig. 5C and D). Moreover, the combination of lower doses of Ang II (100 pM) and Aldo (1 pM) synergistically induced-VSMC senescence and oxidative stress at day 5 were attenuated in Ki-ras2A-siRNA-treated cells but not in scrambled-siRNA-treated cells (Fig. 5E and F).


Figure 5
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  		Fig. 5 Combination of lower doses of Aldo and Ang II synergistically induces VSMC senescence. (A) Effects of lower dose of Ang II (100 pM) with or without Aldo (1 pM) on SA-β-gal activity (a) and expressions of p21, p53, p16, and p27 (b, c) after 5 days of treatment. (B) Effect of lower dose combination of Ang II (100 pM) and Aldo (1 pM) on Ki-ras2A expression. (C) Effects of lower dose of Ang II (100 pM) with or without Aldo (1 pM) on superoxide production (a) and NADPH oxidase activity (b) after 5 days of stimulation. (D) Effects of lower dose combination of Ang II (100 pM) and Aldo (1 pM) on NF-{kappa}B (a) and AP-1 (b) activities. (E) Effect of Ki-ras2A RNA interference on SA-β-gal activity (a) and expressions of p21, p53, p16, and p27 (b, c) by combination treatment of lower doses of Ang II (100 pM) and Aldo (1 pM) for 5 days. (F) Effect of Ki-ras2A RNA interference on superoxide production (a) and NADPH oxidase activity (b) after 5 days of stimulation with lower dose combination of Ang II (100 pM) and Aldo (1 pM). Administration of all reagents to VSMC was performed by medium change every 24 h with new medium containing freshly prepared reagents until day 5. Similar results were obtained in three different culture lines. Values are expressed as mean±SEM (n=3). Ang II, angiotensin II; Aldo, aldosterone; Val, valsartan; Spiro, spironolactone. *P<0.05 vs. control. #P<0.01 vs. Ang II (+) Aldo (+).

 
4. Discussion
The present study examined the effects of the interaction between Ang II and Aldo on VSMC senescence. We demonstrated that persistent stimulation with Ang II (100 nM) caused sustained increases in the ratio of SA-β-gal-positive cell number to total cell number of cultured VSMC and expressions of p21, p53, p16, and p27 starting 5 days after stimulation, whereas the Ang II-induced increase in VSMC growth reached an apparent plateau after 5 days of stimulation, suggesting that Ang II could promote VSMC senescence. Ang II-stimulated senescence was markedly inhibited by the AT1 receptor blocker, valsartan, but partially attenuated by the mineralocorticoid receptor (MR) antagonist, spironolactone. Moreover, SA-β-gal activity and expressions of p21, p53, p16, and p27 in VSMC were also increased by treatment with Aldo at concentrations of 0.4 nM and higher, which could be obtained by stimulation with Ang II (100 nM) in the conditioned medium, indicating that the possible Aldo secretion by AT1 receptor activation and that Ang II-induced VSMC senescence is, at least partly mediated by Aldo secreted by Ang II. Thus, our study consists with previous observation that AT1 receptor activation enhances Aldo secretion [34]. Furthermore, we observed that a combination of lower doses of Ang II (100 pM) and Aldo (1 pM) significantly induced senescent VSMC, whereas senescent cells was not stimulated by Ang II or Aldo alone at these doses. Therefore, these results indicate that the possible cross-talk between Ang II and Aldo could induce VSMC senescence through at least two distinct mechanisms: i) Aldo secreted by Ang II contributes at least partly to Ang II-mediated VSMC senescence, and ii) Ang II and Aldo at even non-effective doses synergistically exaggerates the senescence of VSMC. Moreover, we demonstrated that the senescent interaction between Ang II and Aldo was not involving the change of receptor expression of each other. Therefore, this senescent response of VSMC could be mediated via direct signaling cross-talk between the Aldo receptor and AT1 receptor.

Ras is an oncoprotein that has been extensively studied for its key roles in proliferation and differentiation of many cell types, in addition to promoting tumoriogenesis [35]. It has been recently suggested that Ras induces premature senescence in vascular cells, thereby promoting human atherosclerosis [23]. It is well recognized that Ang II mediates cell growth via a Ras-dependent mechanism [36]. It has also been reported that Aldo rapidly induces Ras protein level through a genomic mechanism, thereby promoting cell proliferation [5]. In the present study, we demonstrated that Ang II or Aldo alone, and their combination even at lower doses increased Ki-ras2A expression. Valsartan markedly inhibited Ang II-induced Ki-ras2A expression, whereas spironolactone partially attenuated this Ang II-mediated increase and strongly blocked Aldo-stimulated Ki-ras2A expression. Not only valsartan but also spironolactone inhibited Ki-ras2A expression induced by a combination of lower doses of Ang II and Aldo. Moreover, in Ki-ras2A-siRNA-transfected VSMC, Ang II-mediated VSMC senescence was attenuated. These results suggest that Ki-ras2A plays a critical role in Ang II- and/or Aldo-mediated VSMC senescence. Accordingly, we postulated that there are three different possible pathways in VSMC senescence induced by Ang II and/or Aldo, namely Ang II-mediated, Aldo-mediated, and a synergistic interaction of Ang II- and Aldo-induced Ki-ras2A expression. Our recent study also demonstrated that the cross-talk between Ang II and Aldo in VSMC proliferation closely associates with Ki-ras2A [5]. Therefore, we can assume that Ang II and Aldo initially stimulate mitogenic signaling in VSMC, resulting in proliferation, and consequently, persistent mitogenic stimulation with Ang II and Aldo would turn their signaling to induce cellular senescence, and Ki-ras2A might play an important role in determining the fate of VSMC. However, the switching point between proliferation and senescence is an enigma. More detailed analysis of the roles of Ki-ras2A signaling in VSMC proliferation, senescence and phenotypic changes could contribute to further understanding of the pathogenesis of vascular remodeling.

It has been suggested that oxidative stress plays a role in the onset and pathogenesis of cardiovascular diseases [37], involving cellular senescence [17,18] and activation of Ras [38]. Moreover, accumulating evidence suggests that Ang II and Aldo mediate oxidative stress by activating NADPH oxidase, which results in superoxide production [13,14,16]. Accordingly, we observed that persistent Ang II and/or Aldo stimulation results in increases in superoxide production and NADPH oxidase activity. Using antioxidant, NAC or SOD as free radical scavenger, we further demonstrated that Ang II- and/or Aldo-induced senescence could be mediated, at least in part, by oxidative stress. Increasing evidence suggests that oxidative stress attributable to excessive production of ROS such as superoxide anion modulates various biologic functions by stimulating transduction signals. Superoxide anion is mainly produced by a major enzymatic source, NADPH oxidase in vascular cells [13,39]. NADPH oxidase consists of membrane components, nox1, nox4, and p22phox, and cytoplasmic components, p47phox and rac-1 [40]. It has been reported that NADPH oxidase activity requires the synthesis and activity of the NADPH oxidase subunit components, which mediates superoxide release. Therefore, data from present study support the possibility that in senescent VSMC, Ang II and/or Aldo stimulation produces superoxide anion could be due to the increase in translocation of rac-1, phosphorylation of p47phox, or enhanced binding to p22phox, thereby contributing to accumulation of oxidative stress. In the present study, we also observed that NAC or SOD effectively suppressed Ang II- or Aldo-stimulated Ki-ras2A expression. Thus, it is suggested that oxidative stress could trigger VSMC senescence in response to Ang II and/or Aldo by upregulation of Ki-ras2A in addition to its direct effects on VSMC senescence. Indeed, Ang II- or Aldo-mediated oxidative stress and Ki-ras2A expression concomitantly occurred. Moreover, we speculate that the secreted Aldo by Ang II plays a role in possible Ang II-mediated oxidative stress/Ki-ras2A cascade in regulating VSMC senescence, since the Ang II-induced increase in Ki-ras2A expression was further inhibited by NAC or SOD with spironolactone. Our finding was also consistent with the previous observation of a tight link between oxidative stress and Ras signaling [20,38]. The superoxide anion may function as a signaling molecule, mediating increased activity of transcription factor such as NF-{kappa}B, AP-1 [32]. Consistent with this result, we observed that persistent Ang II and/or Aldo stimulation increased activity of NF-{kappa}B, AP-1 in senescent VSMC. It has been shown that superoxide anion produced by NADPH oxidase can activate several receptor and nonreceptor protein tyrosine kinases possibly via inhibition of a protein tyrosine phosphatase, these tyrosine kinases include EGF receptor [41]. Our previous study suggests that EGF receptor transactivation plays a critical role in the synergistic mitogenic response of Ang II and Aldo [5]. Thus, our present results lead to the speculation that the senescent signaling cross-talk between the AT1 receptor and Aldo receptor might involve EGF receptor transactivation, which links oxidative stress and Ki-ras2A pathway and causes the activation of transcription factors such as NF-{kappa}B and AP-1. It seems that oxidative stress-induced DNA damage is another cellular senescence pathway independent of a Ras-mediated mechanism. Our results also implicate that Ang II- and/or Aldo-induced oxidative stress could trigger VSMC senescence independent of Ki-ras2A expression. Moreover, in this study, Ki-ras2A-siRNA treatment attenuated Ang II and Aldo-induced superoxide production and NADPH oxidase activity. Therefore, it is indicated that oxidative stress could also act as a down-stream target of Ki-ras2A to mediate senescence-inducing effects of Ang II and/or Aldo. However, in the present study, it is not clear whether oxidative stress and telomere shortening contributed independently or dependently of each other to Ang II- and/or Aldo-mediated VSMC senescence.

In conclusion, our present study suggests that a cross-talk of Ang II and Aldo plays a role in the regulation of VSMC senescence, thereby contributing to cardiovascular disease development. This study highlights the importance of RAAS in vascular cell senescence, which influences vascular aging and age-related vascular diseases. Moreover, our findings suggest potential tools for anti-senescence therapy for age-related vascular disorders by blockade of both Ang II and Aldo.

Time for primary review 25 days


   Appendix A
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 Appendix A
Acknowledgments
 References
 
Supplementary data
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2007.07.008.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 Appendix A
 Acknowledgments
References
 
This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan to M.H. and the Novartis Foundation for Gerontological Research to M.H. and the Suzuken Memorial Foundation to M.M.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 Appendix A
 Acknowledgments
 References
 

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