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Original Studies |
Departments of Medicine (N.J.B., K.-S.K., L.S.B., J.H.N., D.E.V.), Pharmacology (N.J.B., K.-S.K., Y.-Q.C., D.E.V.), and Radiology (S.G.M.), Vanderbilt University Medical Center, Nashville, Tennessee 37232; and Veterans Administration Medical Center, Nashville, Tennessee 37212
Address all correspondence and requests for reprints to: Nancy J. Brown, M.D., 560 MRB-I, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6602. E-mail: nancy.brown{at}mcmail.vanderbilt.edu
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Abstract |
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Introduction |
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In salt-deplete normotensive subjects, we observed that plasma PAI-1 concentrations correlate significantly with aldosterone concentrations (16). One possible explanation for this correlation is that Ang II increases the synthesis of both aldosterone (19) and PAI-1 (5, 6). However, the identification of a glucocorticoid-responsive element (GRE) in the promoter of the PAI-1 gene (20) and the observation that glucocorticoids increase PAI-1 expression in cultured cells (20, 21, 22, 23, 24, 25, 26, 27) suggest an alternative hypothesis that the mineralocorticoid aldosterone also regulates PAI-1 expression. The present study tests this hypothesis both in vitro and in vivo. To test this hypothesis in vitro, we examined the effect of the glucocorticoid dexamethasone and the mineralocorticoid aldosterone on PAI-1 expression in rat aortic smooth muscle (RASM) cells transfected with a series of luciferase reporter constructs containing variable lengths of the human PAI-1 promoter. To test the hypothesis in humans, we measured local plasma PAI-1 antigen concentrations in patients with primary hyperaldosteronism who were undergoing selective adrenal vein sampling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FBS was obtained from HyClone Laboratories, Inc. (Logan, UT). Tissue culture medium, transferrin, and ascorbic acid were obtained from Life Technologies, Inc. (Gaithersburg, MD). Ang II was obtained from Bachem (Bubendorf, Switzerland); aldosterone was purchased from Sigma (St. Louis, MO), and dexamethasone was obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI). Restriction enzymes were obtained from Promega Corp. (Madison, WI), and the Klenow fragment of DNA polymerase I was purchased from New England Biolabs, Inc. (Beverly, MA).
Cell culture
RASM cells were isolated as previously described (28). Cells
were maintained in DMEM with 10% FBS, penicillin (100 U/mL),
streptomycin (100 µg/mL), and 25 mmol/L HEPES. For these studies,
RASM cells were subcultured 10–16 times. The cells were identified as
smooth muscle cells by both appearance and positive fibrillar staining
with a smooth muscle-specific 
-actin antibody (Roche Molecular Biochemicals, Mannheim, Germany). Human umbilical vein
endothelial cells (HUVECs) were propagated from pooled primary cultures
of human umbilical veins as previously described (29). HUVECs were
grown on gelatin-coated plates in DMEM containing 25 mmol/L HEPES
buffer and 2.0 mmol/L glutamine supplemented with 20% FBS, 50 µg/mL
endothelial mitogen, 50 U/mL penicillin, and 50 µg/mL streptomycin.
Cells subcultured fewer than three times were used in these
experiments. HUVECs were grown to confluence in 100-mm tissue culture
dishes, washed twice with sterile PBS, and then incubated overnight
with serum-free DMEM.
Reporter constructs
PAI-1-luciferase reporter vectors were constructed by cloning various portions of the PAI-1 promoter into the pGL2-Basic luciferase vector (Promega Corp.) as previously described (30). The original promoter construct (-6.4 kbp) of the PAI-1 promoter was provided by Dr. David J. Loskutoff (Scripps Research Institute, La Jolla, CA).
Site-directed mutagenesis
Base substitutions were made by oligonucleotide-mediated
mutagenesis in the region identified P-box, corresponding to
nucleotides -71 to -47 (Fig. 1
). The
mutant oligonucleotides used as primers are illustrated in Fig. 1
together with the corresponding wild-type PAI-1 sequence. The mutations
were made with Altered Sites in vitro mutagenesis kit
(Promega Corp.). Mutation accuracy was checked by
restriction enzyme mapping and direct sequencing.
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For transient transfection experiments, RASM cells were cultured in 35-mm six-well plates at density of 1 x 105 cells/well. After cells reached 70–80% confluence, they were transfected using the diethylaminoethyl-dextran method (31) with 1 µg of the PAI-1 pGL-2 construct and 0.8 µg of the plasmid pMVS-ß-Gal, which contains the lacZ gene under control of the Moloney sarcoma virus long terminal repeat. The control plasmid has been examined on several occasions and has never demonstrated any response to Ang II (data not shown). After overnight incubation, the medium was replaced with serum starvation medium (5.5 mmol/L glucose, DMEM containing 5 x 10-7 mol/L insulin, 5 µg/mL transferrin, and 0.2 mmol/L ascorbic acid) supplemented with aldosterone or dexamethasone for 24 h. The following morning, the cells were then kept in glucose-deficient DMEM supplemented with Ang II and or/dexamethasone or aldosterone. After 6 h, the cells were harvested with reporter lysis buffer. The luciferase assays were performed according to the protocol of Promega Corp. A 50-µL sample of the extract was used for determination of ß-galactosidase activity by colorimetric assay (32). The luciferase activity was corrected for ß-galactosidase activity and is presented as corrected light units.
Northern blot analysis
For the Northern blot analysis, cells were grown in 100-mm tissue culture dishes. When the cells reached 80% confluence, the cells were then kept in the DMEM supplemented with different concentrations of Ang II, aldosterone, dexamethasone, and spironolactone. In all studies in RASM, the duration of agonist exposure was 6 h. In HUVECs the effect of time on aldosterone-induced PAI-1 expression was determined by varying the duration of aldosterone exposure: 0.5, 1, 2, 4, 6, 8, and 24 h. In addition, studies were performed in the presence of 5 µmol/L actinomycin D. Total cellular ribonucleic acid (RNA) isolation and Northern blot analysis were carried out as previously described (6). For PAI-1 antigen measurements, conditioned media were collected, centrifuged to remove cellular debris, and stored at -70 C until assays were performed.
Human subjects
Six consecutive patients with biochemical evidence of primary hyperaldosteronism who had been referred for adrenal vein sampling were asked to participate in the study. Biochemical evidence was defined as failure of serum aldosterone to suppress to less than 10 ng/dL after iv infusion of 1.5 L normal saline or 24-h urinary aldosterone excretion greater than 17 µg/24 h. Baseline aldosterone concentration and PRA were 41.4 ± 10.7 (range, 20.4–86) ng/dL and 0.41 ± 0.08 (range, 0.2–0.66) ng Ang I/mL·h, respectively. Three of the six patients had a single adrenal nodule by computed tomography scan, and two others had generalized enlargement of one adrenal gland. All patients had been off spironolactone for at least 3 weeks before adrenal vein sampling and were taking potassium supplementation to maintain normokalemia. All patients gave informed consent for the collection of blood for measurement of PAI-1 and tissue plasminogen activator (t-PA) antigen, in addition to aldosterone and cortisol concentrations, at each site. The consent form was approved by the Vanderbilt University institutional review board.
Adrenal vein sampling procedure
Adrenal vein sampling was performed in the morning in all but one patient. The right groin was prepped and draped in a sterile fashion. Lidocaine was administered as a local anesthetic. Using a Seldinger technique the right common femoral vein was punctured, and a wire was advanced into the inferior vena cava (IVC). A catheter was advanced into the left renal vein and pulled back to engage the orifice of the left adrenal vein, and blood samples were obtained. A second catheter was then used to select the right adrenal vein and obtain samples. In some of the cases, a small amount of contrast was injected to confirm catheter placement. After adrenal vein sampling, blood samples were obtained from the IVC. The total duration of each study was less than 1 h.
Laboratory analysis
Blood samples were collected on ice and centrifuged at 0 C for 20 min. All plasma or serum was separated and stored at -70 C until the time of assay. Blood for measurement of PAI-1 and t-PA was collected in standard Vacutainer tubes containing 0.105 mol/L sodium citrate (Becton Dickinson and Co., Rutherford, NJ) to prevent release of platelet PAI-1. PAI-1 and t-PA antigen levels were determined using a two-site enzyme-linked immunosorbent assay (Biopool AB, Umea, Sweden) as previously described (15). In our laboratory, the coefficients of variation for repeated measures of t-PA antigen and PAI-1 antigen are 5.9% and 8.1%, respectively. Blood for PRA was collected in tubes containing ethylenediamine tetraacetate. PRA was measured by RIA for Ang I formation at pH 7.4 and 37 C (33). Serum aldosterone and cortisol were assayed using a commercially available RIA kits. For aldosterone (Diagnostic Products, Los Angeles, CA) the intra- and interassay coefficients of variation were 6% and 10%, respectively. For cortisol (INCSTAR Corp., Stillwater, MN), the intra- and interassay coefficients of variation were 4.5% and 6.7%, respectively.
Statistical analysis
The effects of hormones on PAI-1 expression, as measured by luciferase activity, were compared using unpaired t test or Mann-Whitney test, as appropriate. The dose effect of dexamethasone or aldosterone was determined by one-way ANOVA followed by Bonferroni correction for multiple comparisons. In patients with hyperaldosteronism, comparison between IVC and adrenal vein was made using paired t test or Wilcoxon signed ranks test, as appropriate. All P values are two-sided. P < 0.05 was judged to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether dexamethasone and aldosterone regulate PAI-1
gene expression, we measured the effects of these steroids on
luciferase activity in RASM transiently transfected with constructs
containing a 3-kb portion of the human PAI-1 promoter and 72
nucleotides of the PAI-1 5'-untranslated region fused to a luciferase
reporter gene (Figs. 2 and 3). Neither 1 µmol/L dexamethasone
alone nor 1 µmol/L aldosterone alone increased luciferase activity
compared to the control value (Fig. 2). Ang II (100 nmol/L, a
concentration previously shown to maximally stimulate PAI-1 expression)
increased luciferase activity 3-fold compared to the control value.
However, in the presence of 100 nmol/L Ang II, both dexamethasone
(effect of dose: F = 7.8; P = 0.022) and
aldosterone (effect of dose: F = 40.4; P < 0.001)
caused a concentration-dependent increase in PAI-1 expression (Fig. 3).
To determine whether the effect of aldosterone on PAI-1 gene expression
was cell or species specific, the relative expression of PAI-1
messenger RNA (mRNA) in HUVECs was measured by Northern blotting and
phosphorimaging (Fig. 4). In these
experiments, aldosterone enhanced the effect of Ang II on PAI-1
expression in a concentration-dependent manner. Enhancement of PAI-1
expression was seen after 6–8 h of exposure to aldosterone but not
after shorter exposure. Treatment with 5 µmol/L actinomycin D blocked
the effect of aldosterone on PAI-1 mRNA expression (data not shown).
The effect of aldosterone on PAI-1 expression (not shown) and protein
synthesis (Fig. 4) was attenuated by an excess concentration (10
µmol/L) of the mineralocorticoid receptor antagonist
spironolactone.
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To localize the regulatory elements required for transcriptional
activation of the PAI-1 gene by dexamethasone and aldosterone,
progressive 5'-promoter deletion constructs containing different
portions of the PAI-1 promoter and 72 nucleotides of the PAI-1
5'-untranslated region were fused to a luciferase reporter gene
construct. In addition, site-directed mutagenesis was performed on the
sequence from -71 to -47 to introduce an EcoRI restriction
site in a P-box, localized close to the glucocorticoid-responsive
element (Fig. 1
). In the longer constructs, aldosterone and
dexamethasone acted synergistically with Ang II to increase PAI-1
expression (Fig. 2). Thus, relative luciferase expression during
combined steroid/Ang II administration was significantly greater than
that during either steroid alone plus that during Ang II alone
(e.g. P = 0.04 for relative luciferase
activity measured during combined treatment with aldosterone and Ang II
vs. that measured during aldosterone alone plus that
measured during Ang II alone for the 1.3-kb construct). In the 884- and
107-bp constructs, the effects of aldosterone and dexamethasone were
additive to the effect of Ang II. However, mutation of the P-box
abolished the effect of either steroid on PAI-1 expression. This result
supports the involvement of the nearby glucocorticoid-responsive
element in dexamethasone and aldosterone activation of the PAI-1
gene.
Adrenal vein sampling
Table 1 lists local venous
aldosterone and cortisol concentrations in the six patients who
underwent adrenal vein sampling. In several patients there was
difficulty cannulating the right adrenal vein. Samples were judged to
have been successfully obtained from the right adrenal vein if there
was a step-up in cortisol concentration compared to the concentration
in the IVC (Table 1). However, comparison of cortisol concentrations in
the right and left adrenal veins suggests that incomplete cannulation
of the right adrenal vein was common. In two patients, aldosterone
hypersecretion lateralized to one adrenal gland (cases 3 and 5); thus,
the aldosterone/cortisol ratio was significantly higher in blood
obtained from the right adrenal vein compared to that obtained from the
left adrenal vein or the IVC in both patients. The concentrations of
both cortisol (by definition) and aldosterone were significantly higher
in either adrenal vein compared to those in the IVC (Fig. 5).
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shows local venous PAI-1 antigen
and t-PA antigen concentrations in the six patients. No sample was
obtained from the right adrenal vein in two of the patients (cases 1
and 4). PAI-1 antigen concentrations measured in either adrenal vein
were significantly higher compared to concentrations in the IVC
(P = 0.007; Fig. 6). In
contrast, there was no difference in t-PA antigen concentrations
between the IVC and the adrenal veins. To assess specifically the
effect of aldosterone production on PAI-1 antigen, we examined the
relationship between local aldosterone concentrations and PAI-1
concentrations within each patient. In the two patients in whom
aldosterone hypersecretion lateralized to one side, and thus in whom
there was a range of aldosterone concentrations, local PAI-1 antigen
concentrations correlated significantly with aldosterone concentrations
in case 3 (P = 0.013), but not in case 5
(P = 0.17; Fig. 7). The
relationship between PAI-1 antigen concentrations and cortisol
concentrations was not significant in either of these patients.
Interindividual variation in PAI-1 antigen concentrations made it
difficult to measure the relationship between aldosterone and PAI-1
across patients. To control for this, local venous PAI-1 antigen
concentrations were normalized to IVC PAI-1 antigen concentrations
within the same individual. There was a significant correlation between
normalized PAI-1 antigen and the aldosterone/cortisol ratio
(r2 = 0.5; P = 7 x
10-4) in venous samples from all six patients
combined.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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50 nmol/L) is approximately 10-fold higher than
that measured in peripheral venous blood, but is comparable to
concentrations measured in the adrenal vein. On the other hand,
aldosterone induced PAI-1 expression in HUVECs at physiological
concentrations. The mechanism for the interactive effect of Ang II and steroids on PAI-1 expression is not addressed in the present study. One possible mechanism involves an effect of the steroids on Ang II receptor expression. Previous studies in VSMCs and rat aortic rings suggest that aldosterone and corticosterone increase Ang II-stimulated contractions through up-regulation of AT1 receptor binding (34, 35). However, although in VSMCs the effect of Ang II on PAI-1 expression appears to be mediated through the AT1 receptor (36), in endothelial cells the effect of Ang II on PAI-1 expression is mediated through the AT4 receptor (6). Thus, studies are needed to determine the effects of dexamethasone and aldosterone on Ang II binding and on the Ang II concentration-PAI-1 response curves in specific cell types.
Corticosteroids such as dexamethasone and aldosterone bind to intracellular type I receptors (37). Although aldosterone binds to the mineralocorticoid receptor with relative specificity, glucocorticoids bind to both the mineralocorticoid and glucocorticoid receptors (38). In vivo, mineralocorticoid specificity is maintained by conversion of cortisol to its inactive metabolite cortisone by 11ß-hydroxysteroid dehydrogenase (11ßHSD). In culture, VSMCs express mRNA of the type 1 isoform, whereas endothelial cells express both type 1 and type 2 isoforms of 11ß-hydroxysteroid dehydrogenase (39) Because dexamethasone is a poor substrate for the type 1 isoform (40), it is possible that both dexamethasone and aldosterone were acting through a mineralocorticoid receptor in the present experiments.
After translocation, ligand-activated mineralocorticoid or glucocorticoid receptors bind to hormone response elements in the regulatory region of target gene promoters and mediate transcriptional activity (38). In the present study, inhibition of transcription with actinomycin D blocked the effect of aldosterone on PAI-1 expression. In addition to binding to a nuclear receptor, aldosterone appears to alter electrolyte fluxes in a number of cell types through rapid interaction with a nongenomic receptor (41). In the present study, the time course of the effect of aldosterone on PAI-1 expression and the finding that this effect is attenuated by spironolactone suggest that aldosterone induces PAI-1 expression and synthesis through a classical mineralocorticoid receptor pathway.
Based on functional studies, van Zonneveld et al. have identified two GREs within the human PAI-1 promoter, one located between positions -100 and +75 and a second region in the promoter located between nucleotides -800 and -549 (20). These investigators identified a sequence at position -64 to -59 whose complementary strand (5'-TGTTCC-3') resembles one half of a GRE (20). Data from the present study suggest that the effect of either dexamethasone or aldosterone on PAI-1 expression requires interaction with the GRE located between -100 and +75, as mutations in the region (107 mPbox) abolished the effect of either steroid on PAI-1 expression. However, we cannot exclude the possibility that the mutations introduced at -57 and -59 to -61 could have altered the function of a putative Ang-responsive element at -48 to -38 (42), particularly as the mutations reduced the response to Ang II alone. The loss of synergy between Ang II and steroids with truncation of the PAI-1 promoter from 1.3 kb to 884 bp is compatible with the existence of a second GRE upstream.
This study is the first to examine the effects of adrenal steroids on PAI-1 synthesis in humans. To do this we adopted a strategy of measuring local plasma PAI-1 concentrations in patients undergoing selective adrenal vein sampling. Because each subject served as his own control and because samples were obtained within a narrow period of time, confounding factors, such as salt intake (16), medication use (16), and glucose and insulin concentrations (43, 44), were held constant within subject. The results of the study suggest that in humans, as we observed in vitro, adrenal steroids influence PAI-1 production. Thus, PAI-1 antigen concentrations were 25% higher in samples obtained from the adrenal veins compared to concentrations in the IVC. In contrast, t-PA antigen concentrations were similar in the adrenal veins and IVC. In vivo, the effect of adrenal steroids on PAI-1, although significant, appears to be modest; this may reflect the importance of a number of other neurohumoral factors, such as Ang II (5, 6), renin (45), and glucose and insulin (30, 43, 44) on the regulation of PAI-1 synthesis. In particular, if as suggested in vitro, aldosterone interacts with Ang II to increase PAI-1 expression, suppressed renin activity and Ang II in patients with primary hyperaldosteronism may attenuate the effect of aldosterone.
One of the limitations of the adrenal vein sampling study is that the results do not allow us to distinguish with certainty between the effects of cortisol and aldosterone on PAI-1 synthesis. In only two patients did aldosterone hypersecretion lateralize to one adrenal gland. This relates in part to difficulty sampling from the right adrenal vein. Nevertheless, the relationship between local aldosterone and PAI-1 concentrations in one of these patients together with the correlation between normalized PAI-1 and the aldosterone/cortisol ratio in all patients combined suggest that, as we observed in vitro, aldosterone regulates PAI-1 synthesis in humans. The present study also does not allow us to exclude the hypothesis that adrenal products other than aldosterone or cortisol increase PAI-1 expression. Catecholamines do not appear to alter PAI-1 concentrations in humans (46, 47); however, other factors present in adrenal venous blood could conceivably alter PAI-1 expression. In addition, the present study does not address the cell source of PAI-1 measured in the adrenal vein.
The findings that the mineralocorticoid aldosterone increases PAI-1 expression in cultured cells and that PAI-1 antigen concentrations are increased in adrenal venous blood in humans have significant clinical implications. Emerging data indicate that aldosterone causes myocardial (48), vascular (49), and renal fibrosis (50) in animal models and that hyperaldosteronism is associated with vascular dysfunction in humans (51). Because PAI-1 plays a role in the regulation of fibrosis (14), increased PAI-1 expression could contribute to the fibrotic effects of aldosterone. Given data suggesting that aldosterone is produced locally in the vasculature (52), aldosterone may exert an autocrine or paracrine effect on fibrinolysis. High concentrations of aldosterone observed in obese, hypertensive, insulin-resistant patients (53) could contribute to the increased PAI-1 concentrations observed in this population (54). Perhaps most importantly, although ACE inhibitors have been shown to lower mortality due to myocardial ischemia in patients with left ventricular dysfunction (3, 4), aldosterone concentrations have been demonstrated to return toward baseline or escape during chronic ACE inhibition (55). Recently, coadministration of an aldosterone antagonist (56) has been shown to reduce mortality 26% in heart failure patients treated with ACE inhibitor. The results of the present study suggest a possible mechanism for this favorable effect of aldosterone receptor antagonism; however, studies of the effect of aldosterone antagonists on fibrinolytic balance are needed to test this hypothesis.
In summary, this study demonstrates that both glucocorticoids and mineralocorticoids interact synergistically and in a concentration-dependent fashion with Ang II to enhance PAI-1 expression in both transfected RASMs and HUVECs. The effect of the steroids on PAI-1 expression in RASM is abolished by mutation of the human PAI-1 promoter in the region of a putative GRE. Studies in patients undergoing selective adrenal vein sampling confirm the relevance of these findings in humans and suggest a potential mechanism for recently reported favorable effects of aldosterone receptor antagonism on cardiovascular mortality.
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Acknowledgments |
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Footnotes |
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Received April 9, 1999.
Revised August 30, 1999.
Accepted September 21, 1999.
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