Aldosterone and Angiotensin II Synergistically Stimulate Migration in Vascular Smooth Muscle Cells Through c-Src-Regulated Redox-Sensitive RhoA Pathways
Objective— Synergistic interactions between aldosterone (Aldo) and angiotensin II (Ang II) have been implicated in vascular inflammation, fibrosis, and remodeling. Molecular mechanisms underlying this are unclear. We tested the hypothesis that c-Src activation, through receptor tyrosine kinase transactivation, is critically involved in synergistic interactions between Aldo and Ang II and that it is upstream of promigratory signaling pathways in vascular smooth muscle cells (VSMCs).
Methods and Results— VSMCs from WKY rats were studied. At low concentrations (10−10 mol/L) Aldo and Ang II alone did not influence c-Src activation, whereas in combination they rapidly increased phosphorylation (P<0.01), an effect blocked by eplerenone (Aldo receptor antagonist) and irbesartan (AT1R blocker). This synergism was attenuated by AG1478 and AG1296 (inhibitors of EGFR and PDGFR, respectively), but not by AG1024 (IGFR inhibitor). Aldo and Ang II costimulation induced c-Src-dependent activation of NAD(P)H oxidase and c-Src-independent activation of ERK1/2 (P<0.05), without effect on ERK5, p38MAPK, or JNK. Aldo/Ang II synergistically activated RhoA/Rho kinase and VSMC migration, effects blocked by PP2, apocynin, and fasudil, inhibitors of c-Src, NADPH oxidase, and Rho kinase, respectively.
Conclusions— Aldo/Ang II synergistically activate c-Src, an immediate signaling response, through EGFR and PDGFR, but not IGFR transactivation. This is associated with activation of redox-regulated RhoA/Rho kinase, which controls VSMC migration. Although Aldo and Ang II interact to stimulate ERK1/2, such effects are c-Src-independent. These findings indicate differential signaling in Aldo-Ang II crosstalk and highlight the importance of c-Src in redox-sensitive RhoA, but not ERK1/2 signaling. Blockade of Aldo/Ang II may be therapeutically useful in vascular remodeling associated with abnormal VSMC migration.
Compelling data supports the concept that combination of aldosterone and angiotesin II (Ang II) receptor blockade may be therapeutically beneficial in ameliorating vascular injury in cardiovascular disease.1–4⇓⇓⇓ Ang II is the principle vasoactive mediator of the renin-angiotensin-aldosterone system (RAAS), which has various actions including vasoconstriction, tissue remodeling, inflammation, oxidative stress, and aldosterone release.5 Originally described as an important regulator of blood pressure and electrolytic balance, aldosterone is now considered a key player in cellular processes underlying cardiovascular hypertrophy and fibrosis.6,7⇓ Several lines of evidence have suggested complex interactions between the effects of Ang II and aldosterone in vivo.1,8,9⇓⇓ Mineralocorticoid receptor antagonism reduces blood pressure, partially restores endothelium dependent relaxation, and improves cardiovascular remodeling induced by Ang II, whereas hypertensive and profibrotic effects of aldosterone infusion are ameliorated by Ang II receptor blockade, implicating cross-talk between aldosterone and Ang II.10,11⇓ More than 20 years ago it was shown that aldosterone stimulates expression of AT1 receptors (AT1R).12 Oxidative stress mediated by nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] oxidase activation appears to be implicated in the reciprocal interaction between Ang II and aldosterone.1,8,9⇓⇓
Cellular mechanisms and signaling pathways implicated in the potential synergistic effect of Ang II and aldosterone are currently subjects of investigation. Min et al demonstrated the involvement of mitogen-activated protein kinases (MAPKs) in the cross-talk of growth-promoting signaling between Ang II and aldosterone.13 These authors also reported interactions between aldosterone and Ang II in vascular smooth muscle cell (VSMC) senescence involving oxidative stress and Ki-ras2A.14 Vascular activation of MAPKs has been reported to be dependent on the nonreceptor tyrosine kinase c-Src,15 although c-Src-independent mechanisms have also been demonstrated.16 To date, at least 14 Src-related kinases have been identified, of which the 60-kDa c-Src is the most abundantly expressed isoform in VSMCs.17 c-Src is an early response signaling molecule for Ang II and aldosterone.15,18⇓ In addition, c-Src, as a critical proximal regulator of NAD(P)H oxidase-driven superoxide anion generation, contributes to amplification of oxidative stress-induced vascular redox-sensitive MAPK activation and to upregulation of proinflammatory and profibrotic genes.19 c-Src also plays an important role in Ca2+ mobilization and sensitization mechanisms including phospholipase C phosphorylation, inositol 1,4,5-trisphosphate formation, and RhoA/Rho kinase activation.20 Rho-dependent pathways are activated by several stimuli and mediate cellular functions other than VSMC contraction including actin cytoskeleton organization; cell adhesion, proliferation, inflammation, and migration, all of which are associated with vascular remodeling.21,22⇓ Recent evidence indicates that RhoA/Rho kinase is involved in the progression of organ target damage induced by both Ang II and aldosterone.23,24⇓
c-Src holds a key position in both Ang II and aldosterone signaling.15,25⇓ We recently identified a novel nongenomic signaling pathway for aldosterone, involving c-Src-dependent activation of p38 MAPK and NAD(P)H oxidase-mediated generation of superoxide anion in VSMCs.25 We also highlighted the importance of c-Src in the molecular and cellular processes underlying vascular activation of MAPK-dependent growth signaling and oxidative stress by Ang II.15 On the other hand, ET-1-mediated activation of ERK1/2 seems to be independent of c-Src.16 Except for a few studies, most previous investigations examined Ang II and aldosterone interactions at high pharmacological concentrations.
Based on the premise that costimulation of aldosterone and Ang II receptors may amplify vascular injury, we sought to understand in greater detail molecular mechanisms underlying crosstalk between aldosterone and Ang II focusing on c-Src and its signal transduction through MAP kinases, NADPH oxidase, and RhoA/Rho kinase. We also questioned the role of receptor tryrosine kinase transactivation in c-Src signaling and evaluated whether aldosterone and AT1 receptor costimulation augments VSMC migration important in vascular remodeling.
This study was approved by the Animal Ethics Committee of the University of Ottawa and performed according to the recommendations of the Canadian Council for Animal Care. VSMCs derived from adult male WKY (16 weeks old) were studied. Mesenteric arteries were isolated and characterized as described in detail previously.26 Briefly, mesenteric beds were cleaned of adipose and connective tissue; VSMCs were dissociated by enzymatic digestion of vascular arcades for 60 minutes at 37°C. Cell suspension was centrifuged and resuspended in Dulbecco modified Eagle medium containing 10% fetal calf serum, 2 mmol/L glutamine, 20 mmol/L HEPES (pH 7.4), and antibiotics. At subconfluence, culture medium was replaced with serum-free medium for 24 hours to render the cells quiescent. Low-passage cells (passages 4 to 7) were studied. To characterize the synergistic effect on c-Src phosphorylation, cells were stimulated with Ang II and aldosterone individually or in association at high (10 nmol/L) and low concentrations (0.1 nmol/L) for 5 and 30 minutes previously determined from full concentration-response curves (100 to 0.01 nmol/L). High concentrations were those that individually induced nearly maximal response of c-Src phosphorylation. Low concentrations were those that individually did not display any effect on the kinase phosphorylation. Cells were preexposed for 30 minutes to antagonists and inhibitors at concentrations previously determined as follows: 10 μmol/L irbesartan (selective AT1R antagonist), 10 μmol/L eplerenone (selective aldosterone receptors antagonist), 10 μmol/L PP2 (selective Src inhibitor), 10 μmol/L apocynin (NAD(P)H oxidase inhibitor) and 10 μmol/L fasudil (Rho kinase inhibitor), 1 μmol/L tyrphostin A23 (tyrosine kinase inhibitor), 1 μmol/L AG1478 (EGFR kinase inhibitor), 1 μmol/L AG1296 (PDGFR kinase inhibitor), 1 μmol/L AG1024 (IGFR kinase inhibitor).27–31⇓⇓⇓⇓
Proteins were extracted from VSMCs, separated by electrophoresis on a 10% polyacrylamide gel, and transferred onto a nitrocellulose membrane as previously described.20 Nonspecific binding sites were blocked with 5% skim milk in Tris-buffered saline solution with Tween for 1 hour at 24°C. Membranes were then incubated with phospho-specific antibodies (1:1000) overnight at 4°C. Antibodies were as follows: anti-c-Src (Tyr418) (Biosource), anti-p38MAPK (Thr180/Tyr182), anti-extracellular signal regulated kinase (ERK) 5 (Thr218/Tyr220), anti-SAPK/JNK (Thr183/Tyr185), and anti-ERK 1/2 (Thr202/Tyr204) (Cell Signaling). The respective nonphospho-antibodies (1:2000) were also used in the present study: c-Src (Biosource); p38 MAPK, ERK 5, SAPK/JNK and ERK 1/2 (Cell Signaling). After incubation with secondary antibodies, signals were revealed with chemiluminescence, visualized by autoradiography, and quantified densitometrically. Results were normalized by the total protein and expressed as percentage of vehicle used in the experimental protocols.
Measurement of NAD(P)H Oxidase Activity
The lucigenin-derived chemiluminescence assay was used to determine NAD(P)H oxidase activity in total protein cell homogenates as previously described.25 Activity was expressed as arbitrary units/mg protein.
Assessment of RhoA and Rho Kinase Activity
Activation of RhoA and Rho kinase was assessed by evaluating their translocation from the cytosol to the membrane. Cells were homogenized in lysis buffer (50 mmol/L Tris/HCl [pH 7.4], 5 mmol/L EGTA and 2 mmol/L EDTA, 0.1 mmol/L PMSF, 1 μmol/L pepstatin A, 1 μmol/L leupeptin, and 1 μmol/L aprotinin) and fractionated to obtain cytosol- and membrane-rich fractions. Homogenates were centrifuged at 50 000g for 1 hour at 4°C, thereby isolating cytosolic fraction in the supernatant. The particulate fraction was resuspended in lysis buffer containing 1% Triton X-100. Protein analysis was performed by Western blotting as described above using anti-RhoA (1:1000) and anti-Rho kinase (1:1500) antibodies from Santa Cruz Biotechnology. RhoA activity was also evaluated by using the absorbance based G-LISA RhoA activation assay kit (Cytoskeleton; Cat. # BK124). The method detects RhoA-GTP bound in cell lysates.
After stimulation, cell suspensions (2.5×104 cells) were seeded into 24-well inserts with 8-μm pore matrigel-coated membranes (BD Biocoat Growth Factor Reduced Matrigel Invasion Chamber, BD Biosciences). After 4 hours, cells were removed from the upper side of the membrane with a cotton swab, leaving those that migrated through the membrane to the lower side. Cells at the lower side were fixed using paraformaldehyde (4%) for 20 minutes and incubated with hematoxylin for 2 minutes. Membranes were washed 5 times and prepared for light microscopy. Microphotographs of 5 different fields were taken and cells were counted. The average number of migrating cells was determined for each experimental condition.
Effects of aldosterone, Ang II, and aldosterone combined with Ang II were determined as the percent increase over control, with the control normalized to 100%. Results are presented as mean±SEM and compared by ANOVA or by the Student t test when appropriate. Values of P<0.05 were considered to be significant.
Synergistic Effect of Ang II and Aldosterone on c-Src Phosphorylation
At high concentrations Ang II and aldosterone individually induced c-Src phosphorylation after 5 and 30 minutes. High dose Ang II and aldosterone costimulation did not elicit additional c-Src phosphorylation compared with individual stimulations (Figure 1A). At low concentrations, combined stimulation with Ang II and aldosterone induced c-Src phosphorylation, whereas no effects were observed when cells were individually stimulated with agonists (Figure 1B). The synergism between Ang II and aldosterone on c-Src phosphorylation observed at low concentrations was abolished by irbesartan and eplerenone, demonstrating the role of AT1 and mineralocorticoid receptors in this effect (Figure 1C). The synergism on c-Src phosphorylation was also inhibited by the tyrosine kinase inhibitor, tyrphostin A23 (Figure 1C). EGFR and PDGFR, but not IGFR inhibitors, blocked the c-Src response to Ang II/aldosterone costimulation, demonstrating that EGFR and PDGFR are upstream of the synergism between Aldo and Ang II on c-Src activation (Figure 1C).
Synergistic Effect of Ang II and Aldosterone on NAD(P)H Oxidase-Induced Superoxide Anion Generation
Neither Ang II nor aldosterone alone induced activation of NAD(P)H oxidase at low concentrations (supplemental Figure IA, available online at http://atvb.ahajournals.org). Combined low concentrations of Ang II and aldosterone significantly increased NAD(P)H oxidase-derived superoxide anion generation in VSMCs (supplemental Figure IA). To establish the functional importance of c-Src synergistically induced by Ang II and aldosterone, generation of intracellular superoxide anion was evaluated in the presence of PP2. Inhibition of c-Src with PP2 abolished the synergistic effect of Ang II/aldosterone on superoxide anion generation (supplemental Figure IB). This effect was also inhibited by irbesartan and eplerenone (supplemental Figure IB). The pharmacological inhibitors did not influence the basal state of NAD(P)H oxidase activity.
Effect of Ang II and Aldosterone on MAPK Phosphorylation
We examined whether the synergistic interaction between Ang II and aldosterone is implicated in the activation of MAPKs. Supplemental Figure II demonstrates the effect of low concentrations of Ang II and aldosterone in p38 MAPK, ERK 5, and SAPK/JNK phosphorylation. p38 MAPK phosphorylation was unaffected by individual or costimulation with Ang II and aldosterone (supplemental Figure IIA). Individually, aldosterone, but not Ang II, induced ERK 5 phosphorylation. No synergism was observed regarding ERK 5 activation (supplemental Figure IIB). Both aldosterone and Ang II alone induced SAPK/JNK phosphorylation within 5 minutes of stimulation. However, no additional effect was observed in costimulated cells (supplemental Figure IIC). At high concentrations Ang II and aldosterone individually induced phosphorylation of p38 MAPK, ERK 5, and SAPK/JNK after 5 and 30 minutes (supplemental Figures III through V). Ang II and aldosterone costimulation did not induce additional MAPK phosphorylation as compared with individual stimulations.
VSMCs stimulation with low concentrations of Ang II and aldosterone individually did not increase ERK 1/2 phosphorylation (supplemental Figure VIA). However, Ang II and aldosterone synergistically induced ERK 1/2 phosphorylation (supplemental Figure VIA). To further assess whether c-Src is involved in this response, ERK 1/2 activation was evaluated in the presence of PP2. Synergism on ERK 1/2 phosphorylation was not abolished by PP2 treatment (supplemental Figure VIB).
Synergistic Effect of Ang II and Aldosterone on the RhoA/Rho Kinase Pathway
In VSMCs, RhoA and Rho kinase translocation from the cytosol to the membrane was observed with Ang II and aldosterone in combination, without any individual effect of the agonists at low concentrations (Figure 2A and 2B). This synergistic effect was inhibited by AT1 and mineralocorticoid receptors antagonists irbesartan and eplerenone, respectively (Figure 2A and 2B). In parallel with these findings, RhoA activation was also synergistically increased by Ang II and aldosterone (Figure 2C), and this effect was abolished by c-Src and NAD(P)H oxidase inhibitors, PP2 and apocynin, respectively (Figure 2D).
Synergism Between Aldosterone and Angiotensin II on VSMC Migration
Ang II and aldosterone alone, at low concentrations, were unable to induce VSMC migration. Costimulation with aldosterone and Ang II increased VSMCs migration (supplemental Figure VII), and this effect was c-Src and Rho kinase-dependent because it was abolished by PP2 and fasudil, their respective inhibitors (Figure 3).
An expanded results section is available in the supplemental materials (http://atvb.ahajournals.org).
Cross-talk between aldosterone and Ang II in vascular cells may lead to amplified cellular responses contributing to vascular remodeling in cardiovascular disease. Min et al demonstrated that aldosterone exerts a synergistic mitogenic effect with Ang II through ERK1/2-mediated pathways in VSMCs.13 Whether other vascular responses are similarly influenced remains unclear. Here we show that VSMC migration is stimulated by aldosterone/Ang II interactions through c-Src pathways that regulate redox-sensitive RhoA/Rho kinase (Figure 4). These effects depend on transactivation of receptor tyrosine kinases, specifically EGFR and PDGFR. Although ERK1/2 is activated by aldosterone/Ang II, as previously reported,13 this does not seem to involve c-Src. Taken together we demonstrate for the first time that synergistic interactions between aldosterone and Ang II at (patho)physiological concentrations involve c-Src-dependent and c-Src-independent pathways. Such differential signaling may underlie distinct vascular responses. Whereas c-Src-insensitive ERK1/2 may be involved in VSMC growth, receptor tyrosine kinase-activated c-Src-regulated NADPH oxidase/RhoA may be important in VSMC migration.
Increasing evidence indicates that aldosterone and Ang II function interdependently to regulate vascular function. Ang II stimulates both systemic and local aldosterone production, while aldosterone can amplify Ang II effects by increasing the expresssion of AT1 receptors and angiotensin converting enzyme.12,32–34⇓⇓⇓ Additionally, actions that are usually attributed to direct effects of Ang II may be mediated, at least in part, by aldosterone. Animal studies using Ang II-infused rats showed that the mineralocorticoid receptor antagonist spironolactone reduces blood pressure and partially improves endothelium dependent relaxation.10 Spironolactone has been shown to ameliorate cardiac hypertrophy, inflammation, and extracellular matrix production in Ang II-induced hypertension35 and in human heart.36 Conversely hypertensive and profibrotic effects induced by aldosterone infusion were reduced by AT1R blockade.9
The molecular basis underlying cross-talk between aldosterone and Ang II is complex, involving multiple receptors and many signaling pathways. Similar to our findings here others have demonstrated rapid signaling by aldosterone through classical mineralocorticoid receptors (eplerenone-inhibitable), which potentiates Ang II/AT1R-mediated actions.11,37⇓ The nonclassical aldosterone receptor has also been implicated in aldosterone/Ang II signaling.11 Aldosterone synergistically augments ERK1/2 activation, JNK phosphorylation, Ki-ras2A induction, and oxidative stress, processes involved in VSMC growth, senescence, fibrosis, and inflammation.11,14⇓ Here we expand and develop these findings by demonstrating that aldosterone and Ang II synergistically induce migration through c-Src-dependent signaling pathways.
c-Src plays an important role in Ang II signaling in VSMCs. It regulates many molecular processes including MAPK phosphorylation and RhoA activation.18,19⇓ It is also an upstream regulator of NADPH oxidase as well as a downstream target of the oxidase.19,38,39⇓⇓ Of significance c-Src is involved in nongenomic redox signaling by aldosterone in vascular cells.25 Here, we show that c-Src plays a role in redox-regulated RhoA/Rho kinase but not in ERK1/2 signaling in response to aldoterone/Ang II stimulation. Moreover we demonstrate that this interaction is mediated through receptor tyrosine kinases, specifically PDGFR and EGFR, because selective EGFR and PDGFR inhibitors blocked aldosterone/Ang II-mediated c-Src phosphorylation. This is not a generalized phenomenon, because AG1024, an IGFR inhibitor, did not influence synergistic effects on c-Src. Such selectivity may relate to subcellular localization of EGFR, PDGFR, AT1, and mineralocorticoid receptors in caveolae/lipid rafts. This may facilitate synergistic interactions between receptors to amplify downstream signaling events. In support of this, mineralocorticoid receptors may colocalize with AT1R in cholesterol-rich domains, which are endowed with receptor tyrosine kinases and c-Src.40
Rho-kinase, a downstream target of small GTP-binding protein RhoA, plays a crucial role in numerous vascular functions, including contraction, cytoskeleton organization, cell adhesion, proliferation, and migration.41 Different upstream signals can converge toward RhoA activation.42,43⇓ Activation of the RhoA/Rho kinase pathway has been implicated in vascular remodeling in Ang II-induced and salt-sensitive hypertension as well as in aldosterone/salt-induced hypertension.22–24,44⇓⇓⇓ Here we provide molecular and cellular evidence that RhoA/Rho kinase is involved crosstalk between aldosterone and Ang II. Combination Ang II and aldosterone induced translocation of RhoA and Rho kinase from the cytosol to the membrane, key steps in the activation of this pathway. Further confirmation was obtained by the evaluation of RhoA activity, which was increased by the combined stimulation. PP2 and apocynin inhibited these effects, indicating the importance of c-Src and NAD(P)H oxidase-drived ROS generation in the synergistic activation of RhoA/Rho kinase pathway by aldosterone and Ang II. From a functional viewpoint we show that aldosterone and Ang II synergistically stimulate VSMC migration, a response that is inhibited by fasudil and PP2. Together these data underlie a significant role of c-Src in mediating RhoA/Rho kinase-dependent cell migration in Ang II and aldosterone synergism. Our data also suggest that NAD(P)H oxidase influences the synergism, because inhibition of superoxide anion production by the oxidase abolished RhoA activation. Previous studies identified JNK and RhoA in Ang II-regulated VSMC migration.23 However those experiments assessed effects of high concentrations of Ang II and did not examine the paradigm of aldosterone/Ang II cross-talk.
Similar to other studies we found amplified ERK1/2 phosphorylation in response to combination low dose aldosterone and Ang II.11 This effect was not impaired by PP2, indicating that c-Src is not involved in this synergism. These findings differ to those where higher concentrations of Ang II and aldosterone (10−8 to 10−5 mol/L) are known to activate ERK1/2 in a c-Src-dependent manner,15–18⇓⇓⇓ highlighting the fact that common signaling pathways can be activated through different mechanisms when cells are exposed to agonists alone or in combination, at low or at high concentrations. Not all MAP kinases are influenced by combination aldosterone/Ang II. Despite the fact that ERK 5, p38MAPK, and SAPK/JNK are activated by Ang II, we failed to show a synergistic response of these MAPKs in the presence of aldosterone.
In summary our data elucidate some novel molecular mechanisms whereby interactions between aldosterone and Ang II, at physiologically relevant concentrations, modulate VSMC migration. We show that aldosterone and Ang II via mineralocorticoid and AT1 receptors synergistically trigger c-Src signaling through EGFR and PDGFR, but not IGFR transactivation. This is associated with activation of redox-regulated RhoA/Rho kinase, which regulates VSMC migration. Although aldosterone and Ang II interact to stimulate ERK1/2, such effects are not reliant on c-Src. Hence we identify 2 different signaling pathways in aldosterone/Ang II synergistic crosstalk, namely c-Src-dependent redox-sensitive RhoA/Rho kinase, important in VSMC migration and c-Src-independent ERK1/2 signaling, shown to be important in VSMC growth. Targeting some of these signaling events using combination AT1 and mineralocorticoid receptor antagonists may provide important vascular protection in cardiovascular disease.
Original received July 19, 2007; final version accepted April 30, 2008.
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