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. Author manuscript; available in PMC: 2011 Jun 1.
Published in final edited form as: Hypertension. 2010 Apr 26;55(6):1476–1483. doi: 10.1161/HYPERTENSIONAHA.109.148783

Elevated MR Activity in Aged Rat VSMCs Promotes a Proinflammatory Phenotype Via ERK1/2 MAPK and EGFR-Dependent Pathways

Alexander W Krug #, Lena Allenhöfer #,§, Robert Monticone #, Gaia Spinetti #, Michael Gekle §§, Mingyi Wang #, Edward G Lakatta #
PMCID: PMC2883813  NIHMSID: NIHMS198685  PMID: 20421514

Abstract

Arterial aging is a predominant risk factor for the onset of cardiovascular diseases such as hypertension, myocardial infarction or stroke. Aging is associated with intravascular renin-angiotensin-system activation, increased vascular stiffness, intimal-media thickening, and a proinflammatory phenotype. Little is known about the influence of aldosterone on arterial aging. Hence, we hypothesized that aldosterone and MR activation might contribute to and possibly accelerate the arterial aging process.

We demonstrate increased mineralocorticoid receptor (MR) expression in whole aortae and early passage aortic vascular smooth muscle cells (VSMCs) from aged (30 months) compared to adult (8 months) F344XBN rats. Sensitivity to aldosterone-induced ERK1/2 MAPK activity is increased in aged cells. MR blockade and ERK1/2 MAPK inhibition prevent age-associated increase of TGF-β, ICAM-1, and pro-collagen-1. Aldosterone increases expression of proinflammatory marker proteins, shifting the phenotype of adult VSMCs towards the proinflammatory phenotype of aged rats. Epidermal growth factor receptor (EGFR) expression is increased with age and by aldosterone, and inhibition of EGFR tyrosine kinase decreases age-associated proinflammatory marker expression.

Our data support the hypothesis that increased constitutive MR signalling may promote and amplify age-associated inflammation that accompanies arterial aging through increased AngII-stimulated expression of MR and enhanced sensitivity to aldosterone-mediated ERK1/2 activation, likely related to increased EGFR expression.

Keywords: aldosterone, arterial aging, vascular smooth muscle cells, mineralocorticoid receptor, inflammation

Introduction

The number of Americans 65 years or older will more than double, from 34.8 million in 2000 to 70.3 million by 2030 according to US Census Bureau data. As a consequence, the USA and other developed societies will be confronted with a substantial rise in aging-associated diseases, such as cardiovascular diseases, the leading cause of death in these societies. While there are well known risk factors for the onset of cardiovascular disease, such as hypertension, diabetes, dyslipidemias, smoking or a sedentary lifestyle, advancing age confers the major risk 1, 2.

Arterial aging is associated with increased expression and activation of the intravascular renin-angiotensin-system (RAS), a proinflammatory phenotype, intimal and medial thickening, vascular stiffening and endothelial dysfunction3, 4. The age-associated arterial inflammatory phenotype includes increased expression of chemokines, such as monocyte chemoattractant protein- (MCP)-1, proinflammatory transcription factors, adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), profibrotic molecules like transforming growth factor (TGF)-β, and increased type-2 matrix metalloproteinase (MMP-2) activity5-7. It has been hypothesized that such age-associated changes render the arteries more susceptible to pathophysiological disease mechanisms, providing the basis for lower threshold, increased severity, and hence, poorer prognosis of the disease8, 9. However, the exact mechanisms and potential stimuli driving the aging-related phenotype are still not completely understood, and no interventional strategy is known to prevent or efficiently retard arterial aging in humans10.

While the classical role of aldosterone is to regulate water and electrolyte balance, and hence blood pressure homeostasis, aldosterone induces structural and functional alterations in the heart, kidneys and vessels, e.g. as myocardial fibrosis, nephrosclerosis, vascular inflammation and remodelling11-13. Accordingly, elevated aldosterone levels are considered an independent cardiovascular risk factor. The Metabolic Syndrome, a cluster of cardiovascular risk factors with increased cardiovascular morbidity and mortality that increases with age14, 15, has been associated with elevated plasma aldosterone levels16-18.

Our purpose was to determine whether age-associated changes of aldosterone or mineralocorticoid receptor (MR) signaling dysregulation occur in VSMCs. Using aortic vascular smooth muscle cells (VSMCs) isolated from Fisher 344 cross-bred Brown Norway (F344XBN) rats, we attempted to elucidate the possible effects of aldosterone and MR signalling on the mechanisms that underlie the arterial inflammation that accompanies arterial aging. The F344XBN rat model has been recommended in the 1990s as the preferred model for aging rat research. For example, the F344XBN rat shows longer maximal life span, normal distribution of age-related pathology and presents less age-related pathologies such as renal dysfunction or cancer than other rat strains. Thus, this model is considered to exhibit age-associated changes as a result of normal aging, rather than merely a reflection of underlying disease. Furthermore, numerous studies over the last decade in this rat model have characterized an age-associated proinflammatory arterial phenotype19, 20.

As a first step, we compared MR expression in whole aortae and early passage VSMCs from adult (8 months) and aged (30 months) animals. We demonstrate that vascular MR is increased in aged animals and sensitivity for aldosterone-induced extracellular signal-regulated kinase (ERK)1/2 activation is enhanced in aged VSMCs. MR blockade by spironolactone or inhibition of the ERK1/2 MAPK pathway by UO126 prevent age-associated upregulation of proinflammatory gene expression, indicating involvement of MR signalling in the development of inflammation that is associated with arterial aging.

Materials and Methods

Animals

All procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee and complied with the guide for the care and use of laboratory animals (National Institutes of Health publication No. 3040-2, revised 1999). Eight- and 30-month-old Fisher344 X Brown Norway rats (F344XBN) were obtained from the National Institute on Aging Contract Colonies (Harlan Sprague Dawley, Indianapolis, IN). Animals were sacrificed by an overdose of sodium pentobarbital, and thoracic aortae were processed immediately.

Vascular smooth muscle cell (VSMC) isolation and cell culture

VSMCs were isolated enzymatically as previously described21, 22 (please see http://hyper.ahajournals.org. for details).

Real-Time PCR Analysis

Total RNA was isolated using the RNeasy kit, according to the manufacturer's instructions (Qiagen). Subsequently, RNA was reverse transcribed using random hexonucleotides for 30 min at 48°C, according to the manufacturer's instructions (Applied Biosystems). Real-time PCR was performed using the SYBR Green protocol in a 384-well plate format, as described previously (Applied Biosystems)5 (please see http://hyper.ahajournals.org. for details).

Western blot analysis

Western blot analysis was performed as described previously23 (please see http://hyper.ahajournals.org. for details).

Quantification of ERK1/2 phosphorylation by in situ cell-based ELISA

For quantification of ERK1/2 phosphorylation we modified an in situ cell-based ELISA, described previously24, 25 (please see http://hyper.ahajournals.org. for details).

Immunohistochemical analysis

Immunohistochemical analysis was performed according to a slightly modified protocol applied by us earlier 26 (please see http://hyper.ahajournals.org. for details).

Statistics

Data are presented as means ± S.E.M. All data were analyzed by one-way or two-way analysis of variance (ANOVA), followed by post hoc comparison procedures (Bonferroni t-test), or t-test as applicable. Differences were considered significant if p was <0.05. In the figure legends n represents the number of animals from which tissue/cells were derived (for a more detailed description of the experimental and statistical approach please see http://hyper.ahajournals.org. for details).

Results

Aldosterone-induced vascular effects are mediated via mineralocorticoid receptor (MR) and mitogen-activated protein kinase kinase (MEK) / extracellular signal-regulated kinase (ERK) 1/2-dependent pathways27 affecting transcription of proinflammatory genes promoting vascular inflammation28-32. As shown in figure 1a MR mRNA is increased in whole aortic tissue from aged animals, as determined by real-time PCR. MR protein expression is enhanced in aortic VSMCs from aged rats. (figure 1 b-c). As shown in figure 1 d-e, Angiotensin II (AngII), but not aldosterone, increases MR protein in adult and aged VSMCs. We compared activation of ERK1/2 MAPK after stimulation with different aldosterone concentrations in VSMCs from adult and aged animals (figure 2a). Aging shifts the aldosterone-induced ERK1/2 phosphorylation dose-response curve to the left and elevates maximal stimulation, while total ERK1/2 expression remains unchanged (figure 2b), indicating increased sensitivity for aldosterone-mediated ERK1/2 activation in aged compared to adult VSMCs.

Figure 1 (a-e). MR mRNA abundance in whole aortae and MR protein is increased in VSMCs from aged animals.

Figure 1 (a-e)

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Expression of MR mRNA in whole aortae of aged (30 months of age) F344XBN rats is higher compared to adult (8 months of age) animals (a). PCR data was normalized to ß-actin. n=4 for all plotted PCR values. Expression of MR is increased in untreated aortic VSMCs from aged compared to adult animals, as demonstrated by Western Blot (b) and immunofluorescence analysis (c). AngII (100 ng/ml), but not aldosterone (100 nmol/l)-treatment stimulates MR expression in adult and aged cells (d-e). Each blot is a representative blot shown above densitometric analysis. Blue indicates nuclear staining, green indicates FITC secondary antibody staining of MR. * p<0.05 vs. adult control. § p<0.05 vs. aged control.

Figure 2 (a-b). Aging increases sensitivity to aldosterone-induced ERK1/2 phosphorylation in aortic VSMCs.

Figure 2 (a-b)

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Phosphorylation of ERK1/2 in VSMCs from aortae of adult and aged F344XBN rats as shown by in situ phospho-ELISA (a). Stimulation time is 10min with various aldosterone concentrations. n=5-6 for all plotted ELISA values. * p<0.05 vs. adult control. Expression of total ERK1/2 is not different in untreated aortic VSMCs from aged compared to adult rats, as demonstrated by Western Blot (b). Each blot is a representative blot shown above densitometric analysis. n.s. non significant.

Cellular expression of TGF-β (2.1±0.3-fold), ICAM-1 (2.1±0.3-fold) and pro-collagen 1 (1.8±0.2-fold) are significantly enhanced in untreated aortic VSMCs from aged compared to adult rats (figure 3 a-c). MR blockade by spironolactone and inhibition of MAPK signaling by UO126 inhibit the age-associated increase of proinflammatory markers (figure 3 a-c). Nanomolar aldosterone concentrations significantly enhanced cellular expression of TGF-ß, ICAM-1 and pro-collagen-1 in cells from adult animals as demonstrated in figure 4 a-c, indicating a shift of marker protein expression in adult cells towards a more aged expression pattern. However, no significant aldosterone-treatment effect could be observed in aged cells (please go to http://hyper.ahajournals.org for details; supplemental figure S2 a-c).

Figure 3 (a-c). MR blockade and inhibition of MAPK signaling reduce age-associated proinflammatory marker expression.

Figure 3 (a-c)

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VSMCs from aortae of adult and aged F344XBN rats. Aged cells were treated with MEK inhibitor UO126 (10 μmol/l) or MR blocker spironolactone (2 μmol/l) for 48h. Each blot is a representative blot shown above densitometric analysis. *p<0.05 vs. adult control; §p<0.05 vs. adult control.

Figure 4 (a-c). Aldosterone shifts the phenotype of adult VSMCs towards the proinflammatory phenotype of aged rats.

Figure 4 (a-c)

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TGF-ß (a), ICAM-1 (b) and pro-collagen-1 (c) expression in VSMCs from aortae of adult and aged F344XBN rats w/wo 48h aldosterone stimulation (1-100 nmol/l). Each blot is a representative blot shown above densitometric analysis. *p<0.05 vs. adult control; no significant differences were observed between control and aldosterone-treated cells in aged animals (p>0.05); see supplementary information for more details.

Both MR and c-src-mediated transactivation of the epidermal growth factor receptor (EGFR) mediate aldosterone-induced ERK1/2 activation in VSMCs, leading to increased proinflammatory marker expression24, 33-34. Figure 5 a-b demonstrates that aging and aldosterone both increase EGFR expression in VSMCs from rat aortae, while total c-src expression remains unchanged in aged compared to adult cells (please go to http://hyper.ahajournals.org for details; supplemental figure S3). Spironolactone as well as UO126 inhibit increased EGFR expression in aged cells (figure 5a), and aldosterone-treatment further increased EGFR expression in aged cells (figure 5c). As shown in figure 6 a-c, inhibition of EGFR tyrosine kinase by AG1478 reduced expression of TGF-β, ICAM-1 and pro-Col-1 in aged cells to a level resembling that of adult cells.

Figure 5 (a-c). Aging and Aldosterone both increase EGFR expression in VSMCs.

Figure 5 (a-c)

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EGFR expression is enhanced in untreated aortic VSMCs from aged compared to adult F344XBN rats, as demonstrated by Western Blot (a) and immunofluorescence analysis (b). Nanomolar aldosterone concentrations (1nmol/l) increases EGFR protein in (a) adult and (c) aged cells. Spironolactone (2 μmol/l) and UO126 (10 μmol/l) inhibit increased EGFR expression in aged cells (a). Blue indicates nuclear staining, green indicates FITC secondary antibody staining of EGFR. Each blot is a representative blot shown above densitometric analysis. *p<0.05 vs. adult control; §p<0.05 vs. aged control; n.s. non significant.

Figure 6. Inhibition of EGFR tyrosine kinase reduces age-associated inflammatory marker expression.

Figure 6

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Aged cells were treated with EGFR tyrosine kinase inhibitor AG1478 (200 nmol/l) for 48h. AG 1478 reduces expression of (a) TGF-ß, (b) ICAM-1 and (c) pro-collagen-1 in aged cells. Each blot is a representative blot shown above densitometric analysis. *p<0.05 vs. adult control; §p<0.05 vs. aged control.

Discussion

Arterial aging is associated with increased arterial RAS expression and activity accompanied by a proinflammatory phenotype which resembles early pathological changes during experimental induction of hypertension and atherosclerosis3, 5, 8, 35. Aldosterone can exert proinflammatory vascular effects via MR- and ERK1/2 MAPK – dependent mechanisms36.

We demonstrate that MR mRNA in the arterial wall and protein levels in isolated VSMCs from F344XBN rats increase with age. Elevated AngII expression and signalling are major characteristics of aged arteries37, 38 and AngII is known to stimulate MR expression11, hereby promoting MR-mediated expression of proinflammatory genes. Accordingly, AngII stimulated MR expression in adult cells to a level resembling that of aged cells, and further increased MR expression in aged VSMCs. However, whether AngII truly is the major factor for increased MR expression in aged rat VSMCs, and whether MR expression is elevated in healthy aged humans as well as potential clinical implications remain to be investigated in future studies. Moreover, aging shifts the aldosterone-induced ERK1/2 phosphorylation dose-response curve to the left and elevates maximal stimulation, indicating increased sensitivity for aldosterone-mediated MAPK activation in aged compared to adult VSMCs. In accordance with our hypothesis that increased MR expression and ERK1/2 MAPK activity are involved in the development of age-associated arterial inflammation, MR blockade by spironolactone and inhibition of MAPK signalling by UO126 reduced expression of proinflammatory markers in aged cells to a level resembling that of adult cells.

Nanomolar aldosterone concentrations increased proinflammatory marker expression in adult cells, indicating a shift towards a more aged phenotype. High (100 nmol/l) aldosterone concentrations may also activate glucocorticoid receptors (GR), possibly opposing MR effects. In contrast, in aged cells aldosterone did not further stimulate proinflammatory marker expression, suggesting that MR is activated by ligand-independent means in aged cells and that no further activation is effected by aldosterone. Jaffe et al have shown that AngII can directly activate the MR in coronary VSMCs39, and local AngII expression is known to be increased in aged arteries38. MR activity can be modulated by a variety of coregulators. For example, rac-1 proved to be a potent activator of MR activity both in the presence and absence of aldosterone40, and results from our previous studies indicate a moderate increase of rac-1 in aged arteries41. Interestingly, in vitro MR overexpression in the absence of steroids also leads to ligand-independent ERK1/2 activation42 supporting the hypothesis that increased proinflammatory marker expression in aged cells, besides possible other mechanisms, might be due to ligand-independent MR and ERK1/2 activation. However, this reflects and untested hypothesis and future research is needed in order to explore in detail the mechanisms that might account for increased MR activity in aged arteries in vivo.

Aldosterone effects involving transcriptional activity of MR are referred to as classical or genomic way of action. Rapid aldosterone effects, also referred to as non-genomic effects, are typically observed within minutes after aldosterone stimulation and include activation of mitogen-activated protein kinases (MAPK) p38, JNK or ERK1/2. Aldosterone-mediated pathologies such as inflammation, remodeling and endothelial dysfunction43 are characterized by activation and crosstalk of genomic effects with rapid signalling pathways11, 27, 43. According to this view, aldosterone-induced activation of MAPKs can modulate the activity and cellular expression pattern of various proteins. For example, in VSMCs aldosterone activates NADPH oxidase in an ERK1/2-dependent way, leading to production of chemokines and cytokines promoting vascular inflammation31, 44. The present study examines the expression of TGF-β, ICAM-1 and pro-Col-1 after 48h aldosterone stimulation, and thus, our results cannot discriminate to which extent genomic, non-genomic as well as secondary compensatory mechanisms eventually contribute to the observed effects. Thus, MR-mediated genomic actions or MR-mediated non-genomic MAPK signalling may both play a significant role in age-associated proinflammatory marker expression in our cell model.

Crosstalk of rapid aldosterone signaling with the EGFR or AngII pathways has been described. Aldosterone has been shown to enhance AngII-mediated ERK1/2 activation in VSMCs27, to increase vascular ACE expression, local AngII concentrations and AT1 receptor expression11. EGFR is a key factor promoting vascular damage and transactivation of EGFR is involved in mediation of vascular AngII, endothelin-1 and catecholamine effects, all of which have been linked to hypertension, vascular inflammation and arteriosclerosis45-48. Age-associated upregulation of EGFR in the arterial wall increases sensitivity for ERK1/2 MAPK activation, eventually enhancing the deleterious effects of AngII, endothelin-1 or aldosterone. Both MR and c-src-mediated transactivation of the EGFR mediate aldosterone-induced ERK1/2 activation in VSMCs24, 34, leading to increased oxidative stress and inflammation28, 33. Our study shows that EGFR expression is stimulated by low aldosterone (1 nmol/l) in adult and aged VSMCs and increases with age, suggesting that aldosterone might indirectly mediate adverse effects in the aging arterial wall by increasing EGFR expression. Accordingly, inhibition of EGFR kinase in aged cells reduced expression of TGF-β, ICAM-1 and pro-Col-1 to levels resembling that of the adult cell. AngII did not elevate EGFR expression (data not shown). Thus, enhanced MR- as well as EGFR expression and signaling may contribute to inflammation in aging arteries.

Vascular aldosterone production and possible age-associated changes might crucially affect arterial aging. However, the evidence for local aldosterone production in the vasculature or the heart is contradictory49. A study by Takeda et al demonstrated aldosterone production from mesenteric arteries of Wistar Kyoto rats50 and human endothelial cells51. In contrast, results from the Gomez-Sanchez group do not support the former results, challenging vascular aldosterone production52. To date it is not known whether aldosterone is produced at a considerable amount in the vessel wall and whether it increases with age.

Our study focused on cell-specific, aging-related vascular alterations in aldosterone/MR signalling. Employing VSMCs from adult and aged F344XBN rats we were able to avoid unknown influences and interactions with other cell types, such as endothelial cells, in animal models. Our results provide in vitro evidence supporting the hypothesis that increased constitutive MR signalling may contribute to age-associated inflammation that accompanies arterial aging through increased AngII-stimulated expression of MR and enhanced sensitivity to aldosterone-mediated ERK1/2 activation, likely related to increased EGFR expression.

Perspectives

Aging is considered the major cardiovascular risk factor. Central arterial aging is characterized by a proinflammatory phenotype leading to arterial remodelling with wall thickening and stiffening. These age-associated changes render the arterial wall a fertile substrate for age-associated diseases such as hypertension and atherosclerosis, indicating that aging and diseases are fundamentally intertwined at the cell and molecular levels. The nature of these age-disease interactions is very complex, including well-defined external risk factors such as NaCl consumption, genetic factors and mechanisms of aging8. This concept calls for a new kind of interventional (preventional) strategies fighting subclinical functional and structural arterial changes in health in order to reduce the incidence and prevalence of cardiovascular diseases. Chronic pharmacological angiotensin converting enzyme (ACE) inhibition or AT1 receptor blockade prevent the onset and progression of age-associated arterial remodelling in animal models53, 54. However, it is thus far unproved if these interventions can slow down age-associated arterial remodelling in healthy individuals who exhibit significant subclinical evidence of “unsuccessful aging”. This report provides evidence for a role of MR and aldosterone signalling in the age-associated inflammation that accompanies arterial aging, expanding the spectrum of factors which might orchestrate arterial aging in vivo. Our in vitro results encourage future studies aiming to further characterize the biological relevance of MR and aldosterone signalling in arterial aging in vivo, and possibly paving the way into a new field of MR blocker application as preventive treatment for cardiovascular diseases.

Supplementary Material

Supp1

Acknowledgments

Sources of funding: This research was supported entirely by the Intramural Research Program of the NIH, National Institute on Aging.

Footnotes

Conflicts of Interest/Disclosures: The authors have nothing to disclose.

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