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(Hypertension. 1995;25:343-349.)
© 1995 American Heart Association, Inc.

Articles

Glucocorticoids Induce Angiotensin-Converting Enzyme Expression in Vascular Smooth Muscle

Robert S. Fishel; Steven Eisenberg; Shaw-Yung Shai; Robert A. Redden; Kenneth E. Bernstein; Bradford C. Berk

From the Cardiology Division, Department of Medicine (R.S.F., S.E., R.A.R.) and Department of Pathology (S.-Y.S., K.E.B.), Emory University, Atlanta, Ga; and the Cardiology Division, Department of Medicine (B.C.B.), University of Washington, Seattle.

	Abstract

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Abstract Angiotensin-converting enzyme (ACE) activity plays a central role in vessel growth and remodeling as shown by the fact that ACE inhibitors reduce neointimal proliferation after rat carotid injury. To investigate the mechanisms that regulate smooth muscle cell ACE expression, we studied the effects of steroids on ACE activity and mRNA in cultured rat aortic smooth muscle cells. ACE activity was present at low levels independent of growth state. In response to the glucocorticoid dexamethasone (100 nmol/L for 72 hours), ACE activity (hydrolysis of [³H]benzoyl-Phe-Ala-Pro) increased 10.1±3.1-fold. The increase in activity occurred within 12 hours and peaked after 72 hours of treatment. The increase in ACE activity was specific for glucocorticoids and paralleled their potency (dexamethasone>hydrocortisone=prednisolone). Dexamethasone increased the steady-state level of ACE mRNA in a concentration-dependent manner (21.4±0.4-fold at 100 nmol/L for 72 hours). Dexamethasone stimulation of ACE expression appeared to be due to both increased transcription and stabilization of ACE enzyme mRNA. This was suggested by the finding that dexamethasone stimulated nuclear run-on expression of ACE mRNA by only threefold, in contrast to the 21-fold increase in steady-state mRNA. These findings establish that ACE is a dynamically regulated enzyme in rat aortic smooth muscle cells. In addition, the present findings suggest an important role for stress steroids in the vascular response to injury in vivo.

Key Words: steroids • dexamethasone • endothelium • angiotensin converting enzyme • muscle, smooth, vascular

	Introduction

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Angiotensin-converting enzyme (ACE), a zinc-containing dipeptidase, catalyzes the proteolytic cleavage of angiotensin I (Ang I) to Ang II.¹ The generation of this physiological pressor and vascular growth factor by ACE is believed to be of major importance in the maintenance of normal vascular tone and is also thought to play a role in angiogenesis,² vascular remodeling,³ the vascular response to injury,⁴ and atherogenesis.⁵ Recent demonstration of the importance of Ang II as a smooth muscle cell growth factor in vivo⁶ and in vitro⁷ further indicates the importance of ACE activity.

Studies of ACE activity have revealed that this enzyme is expressed in a variety of tissues, including pituitary, adrenal,⁸ kidney, intestine,⁹ heart,¹⁰ macrophages,¹¹ and testis.¹² In the vasculature, ACE is thought to be expressed predominantly on the luminal surface of the endothelium.¹³ Because of this, the endothelium is believed to be the major source for Ang II generation from Ang I.

Powell et al⁴ and others¹⁴ have demonstrated that ACE inhibitors significantly decrease myointimal proliferation after balloon injury in the rat carotid and aorta. Although inhibition of circulating ACE may mediate this phenomenon, a large body of evidence suggests that the renin-angiotensin system, present locally in the vasculature, may be more important.¹⁵ For example, vascular smooth muscle cells express angiotensinogen mRNA¹⁶ and bind the ACE inhibitor ramiprilat.¹⁷ In addition, Ang I has been shown to increase intracellular calcium in cultured vascular smooth muscle cells. Because this increase was inhibitable by the Ang II receptor blocker saralasin, Andre et al¹⁸ concluded that ACE present in the smooth muscle cells had converted Ang I to Ang II. ACE may also be important in the vessel wall by virtue of its effects on local bradykinin metabolism. A recent study found that most ACE inhibitor suppression of rat carotid neointimal proliferation is prevented by administration of the B₂ bradykinin antagonist Hoe 140.¹⁹ Thus, ACE appears to regulate both the renin-angiotensin and kinin-kininogen systems of the vessel wall.

ACE expression is not static but may be dynamically regulated in a tissue-specific manner. For example, rat cardiac ACE expression can be induced by pressure-overload ventricular hypertrophy.²⁰ In the endothelial cell, high-level ACE expression can be induced by density-dependent growth arrest,^{21 22} hypoxia,²³ glucocorticoids,^{24 25 26} and endothelin²⁷ and by raising cyclic AMP.²⁸ Of greatest relevance, a recent report by Rakugi et al²⁹ demonstrated an increase in ACE in the neointima of rat aorta after balloon injury, suggesting that ACE is dynamically regulated during the vessel response to injury. ACE induction by glucocorticoids appears to be ubiquitous, as it is shown in macrophages³⁰ and endothelial cells.^{24 25 26 31 32}

Because elevations of stress steroids may be important in the vessel response to injury, we investigated ACE regulation in rat aortic smooth muscle cells (RASMCs). Cultured RASMCs express ACE activity comparable to that seen in cultured bovine aortic endothelial cells (BAECs). In response to physiological levels of the glucocorticoid dexamethasone (based on equipotent levels of cortisol), ACE mRNA and protein levels increased. ACE induction by steroids was proportional to the relative glucocorticoid potency of the steroids and was mediated in part by increased ACE gene transcription as demonstrated by nuclear run-on analysis.

	Methods

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Tissue Culture
Endothelial cells were isolated from fetal and adult bovine aortas by endothelial abrasion and collagenase digestion as previously described.³³ Clones were established by dilutional plating and used between passages 2 and 8. Rat heart endothelial cells were isolated from 12-week-old Sprague-Dawley rats with the use of the Langendorff technique and collagenase digest procedure described by Diglio et al.³⁴ Cells were characterized as endothelial cells based on their cobblestone morphology, ability to take up fluorescently labeled di-Ac-low-density lipoprotein, and staining with anti–factor VIII antibodies. RASMCs were isolated from adult Sprague-Dawley rats as previously described³⁵ and used between passages 6 and 15. Three different RASMC preparations were used for the experiments described. These cells were characterized as smooth muscle based on their growth in hills and valleys and expression of smooth muscle–specific -actin mRNA.³⁶ Cells were grown at 37°C in 5% CO₂ in Dulbecco's modified Eagle's medium supplemented with 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% (vol/vol) heat-inactivated calf serum (RASMCs) or 10% (vol/vol) fetal calf serum (BAECs). Dexamethasone (Sigma Chemical Co) was prepared as a stock solution of 10 mg/mL in absolute ethanol and diluted to yield a final concentration of 0.01% ethanol. Cells were plated at 1x10⁴ cells/cm², grown for 24 to 48 hours until 70% confluent in 10% serum, and then, before treatment with dexamethasone, growth arrested for 24 hours in medium containing 0.4% serum as previously described.³⁷ The use of 10% calf serum was avoided because serum frequently contains significant levels of glucocorticoid activity. No significant difference in glucocorticoid responsiveness was observed when serum-free medium was used instead of 0.4% serum (not shown). However, use of 0.4% serum significantly reduced the variability present in triplicate determinations.

ACE Assays
Cells were harvested into 4 mL ACE buffer (0.05 mol/L HEPES [pH 7.5], 0.1 mol/L NaCl, 0.05% [vol/vol] Triton X-100) and sonicated for 1 minute. The homogenate was then frozen, stored for up to 2 weeks, and thawed before ACE assay. ACE activity was assayed based on the hydrolysis of [³H]benzoyl-Phe-Ala-Pro (Ventrex Laboratories) using the manufacturer's protocol. Samples were assayed for total cellular protein by the Bradford protein assay (Biorad), and identical protein concentrations were added to all assays done. Each experimental sample was assayed in triplicate. Activity calculations were based on Michaelis-Menton first-order kinetics. ACE activity was expressed as ramiprilat-inhibitable (1 µmol/L ramiprilat, Upjohn Co) hydrolytic activity during 30 minutes of incubation per microgram of protein using arbitrary enzymatic units. These units represent a percent utilization of substrate, and according to Ventrex Laboratories, a value of 10.0 U/µg protein is equivalent to 0.6 pmol substrate hydrolyzed per microgram protein. Because this calculation is logarithmic in its formula, a value of 3.0 U/µg is equivalent to 0.3 pmol/µg protein.

mRNA Preparation and Analysis
Total cellular RNA was isolated by the guanidinium isothiocyanate–cesium chloride protocol.³⁸ When required, poly(A⁺) mRNA was selected from total RNA using an oligo(dT)-cellulose spin column (one pass) according to the manufacturer's protocol (Pharmacia). Twenty-five micrograms total RNA or 8 µg poly(A⁺) RNA was size fractionated by electrophoresis on a 1% agarose/2% formaldehyde denaturing gel. After transfer to nylon,³⁹ the RNA was cross-linked to the membrane with the use of UV irradiation (Stratalinker, Stratagene). After 4 hours of prehybridization in 50% (vol/vol) formamide, 5x SSC (1x SSC=0.15 mol/L NaCl and 0.015 mol/L sodium citrate), 5x Denhardt's (1x Denhardt's=0.02% [wt/vol] each of Ficoll, polyvinylpyrrolidone, and bovine serum albumin), 50 mmol/L sodium phosphate (pH 6.5), and 250 µg/mL sheared salmon sperm DNA at 42°C, the Nytran membrane was hybridized in the above solution containing 10% (wt/vol) dextran sulfate (except Fig 4) and 1x10⁶ cpm cDNA probe for 16 hours at 42°C. For hybridization, the 1033-bp EcoRI fragment of the mouse ACE cDNA clone ACE.31⁴⁰ or ACE.315 (Fig 4 only) was used. Northern blots were reprobed with the rat GAPDH cDNA (a full-length rat cDNA, clone pRGAPD 13⁴¹ ) where indicated. Appropriate cDNAs were radiolabeled with the use of a GIBCO/BRL random primer labeling kit as per the manufacturer's protocol with [-³²P]dCTP (specific activity, 3000 Ci/mmol; DuPont–New England Nuclear) and purified with the use of polyacrylamide chromatographic spin columns (Bio-Rad Laboratories). After hybridization, the nylon membrane was washed three times in 2x SSC and 0.2% sodium dodecyl sulfate (SDS) (15 minutes, 25°C) and twice in 0.1x SSC and 0.1% SDS (15 minutes, 60°C). The membrane was then exposed to Kodak X-Omat AR x-ray film with an intensifying screen at -70°C for 16 hours.

View larger version (54K): [in this window] [in a new window]   Figure 4. Northern blots show concentration response for induction of angiotensin-converting enzyme (ACE) mRNA in rat aortic smooth muscle cells by dexamethasone (Dex). Cells were treated with the indicated dexamethasone concentrations for 72 hours. Total RNA was prepared and poly(A+) mRNA purified by one pass through an oligo(dT) spin column. Equal amounts (8 µg per lane) were then loaded, and Northern blot analysis was performed with a mouse ACE cDNA. Ethidium bromide staining of the gel is shown in B. Results are typical of three experiments, each performed with a different cell preparation.

Nuclear Run-on Assay
RASMCs were growth arrested for 24 hours in medium supplemented with 0.4% serum, and nuclei from 2x10⁸ cells were prepared by the technique of Groudine et al.⁴² Run-on transcription was carried out at 30°C for 30 minutes as previously described.⁴³ Ten micrograms of the mouse ACE cDNA EcoRI restriction fragment, 2 µg of the rat GAPDH cDNA Xba I/Pst I restriction fragment, and 10 µg of pBluescript were denatured, neutralized, and applied to a Nytran membrane with the use of a slot-blot apparatus. The cDNA was fixed with UV irradiation and prehybridized for 3 hours in 100 mmol/L TES-HCl buffer (pH 7.4), 0.2% SDS, 10 mmol/L EDTA, 0.3 mol/L NaCl, 1x Denhardt's, and 250 µg/mL yeast tRNA. The membrane was hybridized in the above solution containing 1x10⁷ cpm/mL ³²P-labeled nuclear RNA transcripts for 48 hours at 65°C. Membranes were washed twice in 2x SSC and 0.1% SDS (15 minutes, 25°C) and twice in 0.1x SSC and 0.1% SDS (60 minutes, 60°C).

Data Analysis and Densitometry
The intensity of hybridization signals on autoradiograms was measured by transmitive densitometry and a Silverscanner (LaCie, Inc) using PHOTOSHOP software (Adobe Systems, Inc) and a Macintosh IIcx computer (Apple Computers Inc). Measurement of autoradiograms was performed with IMAGE software (W. Rasband, National Institutes of Health, Bethesda, Md). For some experiments, autoradiographic data were obtained with a phosphoimager (Molecular Dynamics) and quantification performed with phosphoimager software. For ACE mRNA, the 4.2-kb band was quantified, and for GAPDH mRNA, the single 2.3-kb band was quantified. ACE activity data are presented as mean±SEM, with each assay done in triplicate. Student's t tests, unpaired and two-tailed, were used to compare autoradiogram quantification and changes in ACE activity. A value of P.05 was considered significant.

	Results

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ACE Activity Is Inducible by Dexamethasone in RASMCs
Measurement of ACE activity in subconfluent RASMCs and BAECs that had been growth arrested for 24 hours (0.4% calf serum) showed that the two cell types had comparable ramiprilat-inhibitable ACE activity (0.34±0.01 U/µg per minute for RASMCs versus 0.27±0.01 for BAECs) (Fig 1). Baseline ACE activity was similar in cells maintained continuously in 10% calf serum (not shown). Exposure of subconfluent BAECs to increasing concentrations of dexamethasone for 72 hours resulted in a 3.6±0.1-fold induction in BAEC ACE activity. This increase in ACE activity in BAECs was maximal at 3 nmol/L dexamethasone, and the EC₅₀ was approximately 1 nmol/L. The concentration response for ACE induction by dexamethasone in RASMCs was quite different (Fig 1). There was no induction of ACE at dexamethasone concentrations less than 3 nmol/L. At 100 nmol/L dexamethasone, RASMCs exhibited a 14.1±0.5-fold induction in ACE activity over baseline in the cell preparations used for these experiments. Among the three RASMC preparations used for this work, the maximal induction in ACE activity was 10.1±2.2-fold (range, 4.1- to 14.1-fold). Additional experiments showed no further increase in ACE activity at 500 nmol/L dexamethasone, which may be related to a decrease in protein synthesis (see below). The EC₅₀ for stimulation of RASMC ACE activity by dexamethasone was at least 30 nmol/L. It was important to normalize for protein because dexamethasone caused a concentration-dependent decrease in RASMC protein content as previously described³⁷ (Fig 2).

View larger version (22K): [in this window] [in a new window]   Figure 1. Line graph shows dexamethasone dose response for induction of angiotensin-converting enzyme (ACE) activity in rat aortic smooth muscle cells (RASMCs) and bovine aortic endothelial cells (BAECs). Growth-arrested subconfluent RASMCs and BAECs were treated for 72 hours with the indicated dexamethasone concentrations. BAECs and RASMCs demonstrated comparable baseline ACE activity that increased a maximum of 3.6±0.1-fold and 14.1±0.5-fold, respectively, in the experiment shown. Similar results were obtained for BAECs obtained from both fetal (shown) and adult aortas. Inset, Linear representation of the same data illustrates the increased sensitivity of BAECs to low dexamethasone concentrations compared with RASMCs. Rat heart endothelial cells also showed significant increases in ACE activity at low dexamethasone concentrations (3.2±0.9-fold at 3 nmol/L dexamethasone). Results are mean±SEM of a single experiment performed in triplicate and are representative of three similar experiments performed with different RASMC preparations.

View larger version (13K): [in this window] [in a new window]   Figure 2. Line graph shows that dexamethasone decreases total cellular protein in rat aortic smooth muscle cells. Dexamethasone treatment of cells (0 to 500 nmol/L for 72 hours) resulted in a concentration-dependent decrease in total cellular protein. The maximal decrease was 51% of control values. Data are the mean of two experiments, each performed in triplicate. Bovine aortic endothelial cell protein content did not decrease when these cells were exposed to 500 nmol/L dexamethasone (data not shown).

Dexamethasone increased RASMC ACE activity within 12 hours, and the effect was maximal after 72 hours of treatment (Fig 3). In separate experiments, cell growth was continued to achieve density-dependent growth arrest as assessed by confluence. As previously reported by our laboratory²² and others,²¹ after complete growth arrest of BAECs (3 days after confluence), a large increase in ACE activity occurred in BAECs (5.6±0.9 U/µg per minute), which did not occur with growth-arrested RASMCs (0.26±0.09 U/µg per minute).

View larger version (14K): [in this window] [in a new window]   Figure 3. Line graph shows the time course for induction of angiotensin-converting enzyme (ACE) activity by dexamethasone. Cells were exposed to 100 nmol/L dexamethasone for the indicated times, and ACE activity was measured as described in "Methods." Rat aortic smooth muscle cell ACE activity increased within 12 hours of treatment. Control cells were maintained in 0.4% calf serum/Dulbecco's modified Eagle's medium. Results are triplicate determinations of a single experiment and are representative of two experiments. Absolute ACE values in this experiment were significantly lower than those obtained in Fig 1, partly because of the use of a different cell preparation.

ACE mRNA Is Induced by Dexamethasone in RASMCs
Treatment of RASMCs with increasing concentrations of dexamethasone for 72 hours resulted in a concentration-dependent increase in ACE mRNA levels (Fig 4). In response to the highest dexamethasone concentration studied (500 nmol/L for 72 hours), there was a 33.8±1.3-fold absolute increase in ACE mRNA (Fig 5). Dexamethasone increased ACE mRNA levels within 6 hours (Fig 6). Both 4.2- and 5.1-kb mRNA transcripts were observed, as previously described,⁴⁰ although only the 4.2-kb transcript was quantified. In the experiment shown, GAPDH mRNA levels decreased significantly after exposure to dexamethasone, consistent with previous studies that showed dexamethasone-mediated decreases in RNA levels in RASMCs.^{37 44}

View larger version (16K): [in this window] [in a new window]   Figure 5. Bar graph shows densitometric quantitation of rat aortic smooth muscle cell angiotensin-converting enzyme (ACE) mRNA induction by dexamethasone. The autoradiographic signals for ACE hybridization on the Northern blots from two experiments (using cell preparations shown in Figs 1 and 4) were measured with a phosphoimager. The 4.2-kb band was used for analysis. Absorbance in control cells (no dexamethasone added) was normalized to 1.0 for each experiment.

View larger version (56K): [in this window] [in a new window]   Figure 6. Northern blots show time course for induction of rat aortic smooth muscle cell angiotensin-converting enzyme (ACE). Cells were exposed to 500 nmol/L dexamethasone (Dex) for the indicated times, and total RNA was prepared. Poly(A+) mRNA was prepared as in Fig 4 and analyzed on Northern blots (8 µg per lane). The blot was sequentially hybridized with ACE and GAPDH cDNA probes. Results are typical of two experiments.

Dexamethasone stimulation of ACE mRNA expression appeared to require new protein synthesis. Preincubation of RASMCs for 30 minutes with 10 µmol/L cycloheximide completely inhibited subsequent induction of ACE activity by 500 nmol/L dexamethasone (48 hours). This treatment caused a greater than 90% decrease in ACE mRNA induction (not shown).

Induction of ACE in RASMCs Is Specific for Glucocorticoids
To determine the specificity of dexamethasone action, we exposed RASMCs to steroids with varying glucocorticoid activity. After treatment of RASMCs for 72 hours with 100 nmol/L dexamethasone, prednisilone, hydrocortisone, deoxycorticosterone, or testosterone, ACE activity increased in proportion to relative glucocorticoid potency (11.7±0.3-, 3.8±0.1-, 3.6±0.2-, 2.0±0.8-, and 1.3±0.5-fold, respectively) (Fig 7). Thus, ACE induction by steroids in RASMCs appears to be specific for glucocorticoids.

View larger version (13K): [in this window] [in a new window]   Figure 7. Bar graph shows that angiotensin-converting enzyme (ACE) induction in rat aortic smooth muscle cells is specific for glucocorticoids. Cells were treated for 72 hours with 100 nmol/L dexamethasone, prednisilone, hydrocortisone, deoxycorticosterone, or testosterone. ACE activity was measured as described. Protein concentrations per dish were 0.58, 0.75, 0.94, 1.06, and 1.03 mg, respectively; the control was 1.08 mg. Results are the mean of two experiments performed in triplicate.

Dexamethasone Increases ACE mRNA Expression in Part by Stimulating Gene Transcription
To study the effect of dexamethasone on ACE transcription, we treated RASMCs with 500 nmol/L dexamethasone for 72 hours and measured ACE mRNA synthesis by nuclear run-on. Dexamethasone treatment stimulated transcription of the RASMC ACE gene by 3.2-fold (Fig 8). GAPDH hybridization was equivalent in dexamethasone-treated cells and control cells, indicating a similar transcription rate of this gene.

View larger version (58K): [in this window] [in a new window]   Figure 8. Blots show that dexamethasone increases angiotensin-converting enzyme (ACE) transcriptional rate. Rat aortic smooth muscle cells were treated for 72 hours with 100 nmol/L dexamethasone or vehicle (0.01% ethanol), and nuclei were isolated for nuclear run-on analysis. RNA transcripts were labeled with 32P and hybridized to filter-immobilized mouse ACE cDNA (10 µg), pBluescript (10 µg), and GAPDH cDNA (2 µg). Results are representative of two experiments using cell preparations shown in Figs 1 and 4.

	Discussion

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The major finding of this study is that expression of ACE protein and mRNA is highly regulated by dexamethasone in RASMCs. This regulation is specific for glucocorticoids and occurs at concentrations that may be present during physiological stress in vivo. Dexamethasone-stimulated ACE expression in RASMCs appeared to be due to both increased transcription of the RASMC ACE gene and stabilization of ACE mRNA.

The stimulation of ACE expression by dexamethasone differed significantly when the nuclear run-on study (Fig 8) was compared with the steady-state mRNA studies (Figs 4 through 6). The nuclear run-on study showed an approximately threefold increase in ACE gene transcription in response to 500 nmol/L dexamethasone, whereas Northern blot analysis showed an approximately 34-fold increase. Although there are consensus sequences for the glucocorticoid response element (TGTTGT) in the mouse ACE promoter at -810 bp^{40 45} and in the human promoter at -935, -794, and -685 bp from the transcription start site,⁴⁶ these regulatory elements do not appear to be functional when transfected into endothelial cells.⁴⁷ Thus, an important mechanism for glucocorticoid-mediated increases in ACE mRNA expression may be stabilization of ACE mRNA. This effect of glucocorticoids has been previously demonstrated for the human growth hormone and phosphoenolpyruvate carboxykinase genes.^{48 49} Of interest, glucocorticoids may also destabilize mRNA, as shown for JE mRNA in RASMCs.⁴⁴ The stimulation of gene expression observed by nuclear run-on may be due to activation of glucocorticoid regulatory elements. However, based on the study of Testut and colleagues,⁴⁷ the regulatory element must be located more than 3.6 kb upstream from the transcription start site or within the gene itself. Future studies of the ACE promoter region and its interactions with nuclear transcription factors will be required to resolve this issue.

Although BAECs and RASMCs expressed essentially identical amounts of ACE when measured in subconfluent cells (growth arrested for only 24 hours in 0.4% calf serum), there were two important differences in ACE expression between RASMCs and BAECs: (1) When BAECs were growth arrested in a density-dependent manner (96 hours in 0.4% serum), they demonstrated a very large increase in ACE activity, whereas RASMC ACE activity did not change significantly during growth arrest; (2) BAECs were very sensitive to low concentrations of glucocorticoids as shown by an EC₅₀ of 3 nmol/L for dexamethasone; in contrast, the EC₅₀ in RASMCs was at least 30 nmol/L. Thus, BAECs were at least 10 times as sensitive to glucocorticoids as RASMCs. Because the species studied were different, we cannot be certain whether this is a tissue- or species-related difference. However, rat heart endothelial cells also exhibited similar increases in ACE expression in response to dexamethasone (not shown), suggesting that these differences are related to cell type. Although BAECs were more sensitive than RASMCs to low dexamethasone concentrations, RASMCs exhibited much greater maximal ACE activity in response to dexamethasone (in growing cells). For example, 100 nmol/L dexamethasone increased BAEC ACE activity by approximately fourfold, whereas RASMC ACE activity increased approximately 10-fold. By comparison, growth-arrested BAECs increased ACE activity by approximately 20-fold.²² Thus, under certain conditions, RASMCs may express ACE activity comparable to that of BAECs.

The present findings suggest that glucocorticoids may be important regulators of smooth muscle cell ACE expression in vivo. Glucocorticoid levels (predominantly cortisol) in human plasma may increase 50-fold during stressful conditions such as surgery and extreme athletic competition and be equivalent in glucocorticoid potency to 100 nmol/L dexamethasone.^{50 51} Thus, particularly under stress, endothelial and vascular smooth muscle cells may be exposed to glucocorticoid concentrations that approximate those used in these experiments. Sustained elevation of glucocorticoids, as occurs in disease states such as Cushing's disease, are associated with hypertension and vascular damage. Although the major cause of these pathological processes is renal salt and water retention, the present data suggest that local responses of the renin-angiotensin system in the blood vessel may also be important. Recent findings from Rakugi et al²⁹ and Fishel et al⁵² demonstrate that balloon injury of the rat aorta is associated with increased ACE expression. This expression is greatest in the neointimal vascular smooth muscle cells, supporting results of the present study that ACE is dynamically regulated in vascular smooth muscle cells.

In summary, our findings further support the concept of a highly regulated renin-angiotensin system present locally within the vessel wall. Future study of the interactions between steroids and other local and systemic mediators may provide insight into the mechanisms by which ACE inhibitors prevent myointimal proliferation after angioplasty and the role of the renin-angiotensin system in vascular growth and remodeling.

	Acknowledgments

 
Drs Bernstein and Berk are Established Investigators of the American Heart Association and the George M. O'Brien Kidney Research Center at Emory University. This work was supported by grants DK 39777, DK 44280, DK 45215, and HL-44721 from the National Institutes of Health, Bethesda, Md, as well as Grants-In-Aid from the American Heart Association and the Georgia Affiliate of the American Heart Association. We wish to thank Garnett S. Huguley of the Upjohn Co for generously providing the ramiprilat used in our ACE assays and Marshall A. Corson for helpful discussions.

	Footnotes

 
Reprint requests to Bradford C. Berk, MD, PhD, Cardiology Division, RG-22, University of Washington School of Medicine, Seattle, WA 98195.

Received June 6, 1994; first decision August 9, 1994; accepted August 31, 1994.

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