© 1999 American Heart Association, Inc.
Vascular Biology |
Glucocorticoid Resistance Caused by Reduced Expression of the Glucocorticoid Receptor in Cells From Human Vascular Lesions
From the Divisions of Hematology/Oncology (P.J.B., B.D., V.M.M., C.F., T.A.M.) and Cardiology (S.C.H., E.D.), Department of Medicine, and the Division of Pediatric Endocrinology (R.C.W., H.H.-A.), Department of Pediatrics, Cornell University Medical College, New York, NY.
Correspondence to Timothy A. McCaffrey, PhD, Cornell University Medical College, Division of Hematology/Oncology, Room C606, 1300 York Ave, New York, NY 10021. E-mail tamccaf{at}med.cornell.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—Mechanisms that control the balance between cell proliferation and death are important in the development of vascular lesions. Rat primary smooth muscle cells were 80% inhibited by low microgram doses of hydrocortisone (HC) and 50% inhibited by nanogram concentrations of transforming growth factor-ß1 (TGF-ß1), although some lines acquired resistance in late passage. However, comparable doses of HC, or TGF-ß1, failed to inhibit most human lesion-derived cell (LDC) lines. In sensitive LDC, HC (10 µg/mL) inhibited proliferation by up to 50%, with obvious apoptosis in some lines, and TGF-ß1 inhibited proliferation by more than 90%. Collagen production, as measured by [3H]proline incorporation or RIA for type III pro-collagen, was either unaffected or increased in the LDCs by HC. These divergent responses between LDC lines were partially explained by the absence of the glucocorticoid receptor (GR) and heat shock protein 90 mRNA in 10 of 12 LDC lines, but the presence of the mineralocorticoid receptor and 11ß-hydroxysteroid dehydrogenase type II. Western blot analysis confirmed the absence of the GR protein in cells lacking GR mRNA. Immunohistochemistry of human carotid lesions showed high levels of GR in the tunica media, but large areas lacking GR in the fibrous lesion. Considering the absence of the GR in most lines, the effects of HC may be elicited through the mineralocorticoid receptor. Functional resistance to the antiproliferative and antifibrotic effects of HC may contribute to excessive wound repair in atherosclerosis and restenosis.
Key Words: glucocorticoid receptor • transforming growth factor-ß1 • atherosclerosis • cell proliferation • collagen production • smooth muscle cells • restenosis
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Restenotic narrowing of arteries after angioplasty or endarterectomy is caused principally by fibroproliferative intimal hyperplasia and contractile remodeling by a cell resembling both the vascular smooth muscle cell (SMC) and myofibroblast.1 2 Serial angiography after angioplasty suggests that almost all patients will exhibit a fibroproliferative narrowing after angioplasty, although in most patients the stenosis will spontaneously regress to exhibit a net luminal gain.3 However, in 40% of patients, this lesion fails to regress, but instead progresses to clinical restenosis (reviewed in Reference 44 ). Restenosis appears to be caused by the migration of cells into the intima, subsequent proliferation, and extracellular matrix deposition. In animal models, neointimal regression is caused by apoptosis of the lesion cells.5 In humans, growing evidence suggests that restenosis may be caused by a failure in growth inhibitory and apoptotic systems that would normally mediate lesion regression.6 7 Thus, the persistence and slow proliferation of cells in the injured vessel wall may be caused by a failure in endogenous inhibitory systems.
One defective inhibitory system involves the transforming growth factor-ß1 (TGF-ß1) receptor pathway. Lesion-derived cells (LDCs) from human atherosclerotic plaques are resistant to the inhibitory effects of TGF-ß1 because of a decrease in the TGF-ß type II signaling receptor.7 In some patients, microsatellite instability in the TGF-ß type II receptor is responsible for the resistance to TGF-ß1 in LDCs.8 Another major inhibitory system of SMC proliferation are glucocorticoids (GCs). When the hormone is bound to the glucocorticoid receptor (GR) complex, a fibrosuppressant effect is exerted, which is elicited in several ways: inhibition of SMC proliferation in vitro,9 10 inhibition of collagen production,11 12 13 induction of apoptosis in lymphocytes and fibroblasts (for review see Reference 1414 ), and anti-inflammatory or immunosuppressive actions.15 16 Given these diverse antifibrotic and antiproliferative actions, GCs should be good candidates for preventing restenosis.
Animal studies have generally supported a beneficial effect of GCs on both primary, cholesterol-induced atherosclerosis and postangioplasty restenosis. In cholesterol models of atherosclerosis, treatment with hydrocortisone has consistently been shown to lessen the severity of atherosclerosis in various rabbit models.17 18 19 20 In models of balloon-catheter injury, GCs typically inhibit neointimal proliferation in vivo in both rat21 and rabbit models,22 23 24 although shorter, 1-week treatments with dexamethasone have failed to inhibit intimal hyperplasia in rabbits.25
Despite these in vivo effects of steroids, 2 clinical trials have failed to observe a beneficial role of GCs in preventing restenosis in humans.26 27 Both studies administered high doses of prednisolone, a synthetic GC, after successful coronary angioplasty, and both concluded that high-dose steroid therapy does not influence the overall rate of restenosis after coronary angioplasty when compared with the untreated control groups.
In vitro studies have observed that corticosteroids inhibit proliferation of SMCs derived from atherosclerotic arteries.28 However, to see any significant decrease in cell number (60% to 70%), highly concentrated doses of steroids were needed. In the case of hydrocortisone, 10 000-fold higher than physiological levels were required to see a 70% inhibition of SMC proliferation. Some diseases are known to involve acquired resistance to GCs with time, and this resistance results from the relative inability of GCs to exert their effects on target tissues. Such diseases include asthma,29 rheumatoid arthritis,30 and acute lymphoblastic leukemia.31 The present studies examine the hypothesis that arteriosclerosis may also involve acquired resistance to GCs. The results indicate that the majority of cell lines from human endarterectomy lesions are resistant to the antiproliferative effects of hydrocortisone and this may be caused by the absence of the GR or other members of the GR multiprotein complex.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Vascular Specimens
Vascular lesions were acquired as waste surgical material, under Institutional Review Board approved protocols, from patients undergoing surgical endarterectomy at the New York Hospital/Cornell Medical Center. Portions of the vascular lesions were explanted for cell culture, and the remainder was fixed in 4% buffered formaldehyde and paraffin-embedded for immunohistochemical analysis.
Cell Culture
Vascular lesions from carotid and femoral or iliac
endarterectomy specimens were finely diced, and the
explants placed in 25-cm2 tissue culture flasks
containing medium 199 (M199; Gibco BRL) supplemented with 10% FBS and
gentamicin sulfate (50 µg/mL). LDCs from the explants were
subcultured with trypsin/EDTA (Gibco BRL) at a 1:2 split ratio. Cells
were obtained in approximately 30% of the patient specimens acquired
and could typically be maintained for 5 to 8 passages (p5 through p8),
although most analyses were conducted as soon as sufficient
cell numbers were obtained (p2 through p4). Rat SMCs were explanted
from the aorta of 3-month-old Fisher 344 (Harlan Sprague Dawley,
Indianapolis, IN) rats and also grown in M199 with 10% FBS and
gentamicin sulfate for up to 40 passages.
Cell Proliferation
The effect of the steroids on the rate of DNA synthesis was
examined by semiautomated methods. Cells were plated at
1x104 cells/well of 96-well microtiter plates at
least 24 hours before the assay. Hydrocortisone (0.01 to 10 µg/mL)
was added to M199 plus 1% FBS for 20 hours before the cells were
pulsed with [3H]thymidine (1 µCi/mL; NEN) for
4 hours. Cells were collected with trypsin/EDTA and a cell harvester
(Wallac, Inc), and the DNA-incorporated label was determined by
scintillation counting (Betaplate; Wallac, Inc) (n=3 per point). Cell
proliferation was also determined over a 5-day period with exposure to
hydrocortisone or TGF-ß1, or both, followed by trypsinization and
physical counting of the cells using a particle counter (Coulter
ZBI).
Protein Synthesis
The rate of protein synthesis was measured by
[3H]proline incorporation, which is partially
incorporated into proline-rich proteins such as collagen. Cells were
plated as described for cell proliferation. Hydrocortisone was added to
the cells (M199/1%FBS) at least 4 hours before the cells were pulsed
with [3H]proline (4 µCi/mL;
L-[5-3H]proline; NEN) for 24 hours.
The supernatants were precipitated in 10% trichloroacetic acid (TCA).
The adherent cell monolayer was washed twice with PBS and then
dissolved with 1 mol/L NaOH for 30 minutes at 37°C. The
TCA-precipitated supernatant and cell monolayer were counted using a
scintillation counter. All concentrations were tested in replicates of
3 wells.
Collagen Secretion
Pooled triplicate supernatants from the cell proliferation
assays were analyzed further by radioimmunoassay techniques for
the presence of antigenic material derived from the N-terminal
pro-peptide of type III (PIIINP; Incstar) collagen. A 1-step
equilibrium-type assay using polyclonal rabbit antibodies against
purified human pro-peptide antigen was used. All samples were measured
in duplicate, interpolated to a standard curve, and expressed in
nanogramsx104 cells.
RNA Extraction
Total RNA was extracted from cell cultures by RNAzol B (Tel
Test). Briefly, the supernatant was removed and RNAzol B was added to
the cells and rapidly harvested (0°C to 4°C) with a sterile
scraper. The cell suspension was collected, chloroform was added, and
the mixture was centrifuged at 14 000g for 15
minutes at 4°C. The aqueous phase was collected and mixed with an
equal volume of isopropanol and centrifuged at
14 000g for 15 minutes at 4°C, and the resulting pellet
was washed with ethanol.
Reverse Transcription–Polymerase Chain Reaction (RT-PCR)
The oligonucleotide primers used for RT-PCR
analysis of selected steroid hormone receptors are listed in
the Table
. Total RNA (500 ng) was reverse transcribed into cDNA with
MuLV reverse transcriptase at 42°C with random hexamers (Perkin
Elmer) followed by PCR with Taq polymerase in a 100-µL
reaction containing 0.2 mmol/L of dNTPs and 0.15 µmol/L of
each primer. The PCR profile varied for particular primer pairs but
generally consisted of an initial 1-minute denaturation at 95°C, then
35 cycles of 1-minute denaturation at 95°C, 2-minute annealing at
60°C, 1-minute extension at 72°C, and finally a 10-minute extension
at 72°C. Fifteen microliters of PCR product were separated in
1.5% (wt/vol) agarose gel and stained with ethidium bromide.
|
Western Blot Analysis
The conditioned media of the LDCs was removed and concentrated
(Centricon 10; Amicon), and protein lysates were made from the LDCs.
Lysis buffer (40 mmol/L Tris-base, 1% Triton X-100, 2 mmol/L
MgCl2, 0.1 mmol/L PMSF, 5 µg/mL leupeptin;
pH 8.8) was added to the LDCs, which were then harvested and sonicated
for 5 s. Protein content was determined by the bicinchoninic acid
(BCA) assay (Pierce), and 30 µg was used for analysis.
Samples were boiled for 10 minutes, analyzed by SDS-PAGE, and
electroblotted onto polyvinylidene difluoride (PVDF membranes)
(NEN Life Science) in 160 mmol/L glycine, 25 mmol/L Tris
base, and 20% methanol transfer buffer. The PVDF membrane was then
blocked in Tris-buffered saline (TBS)-milk (20 mmol/L Tris-HCl,
150 mmol/L NaCl, and 4% powdered milk; pH 7.4) for 2 hours. The
membrane was exposed to the anti-GR antibody (diluted 1:100, Novacastra
Laboratories) overnight, then washed in TBS before application of the
peroxidase-conjugated anti-mouse IgG (diluted 1:1000), which was
detected by chemiluminescence (NEN Life Science).
Apoptosis Assays
Terminal Deoxyribonucleotide Transferase-Mediated dUTP
Nick-End Labeling
Fragmented DNA was labeled in situ by use of the terminal
deoxyribonucleotide transferase-mediated dUTP nick-end
labeling (TUNEL) method (Boehringer-Mannheim). Briefly, LDCs
were plated on 8-well chambered glass slides at least 24 hours before
treatment. Hydrocortisone (10 µg/mL) was added to the cells for 24
and 48 hours before the TUNEL assay (M199/1%FBS). The cells were fixed
in 4% buffered formaldehyde and permeabilized (0.1%
Triton X-100, 0.1% sodium citrate). The fragmented DNA was labeled
with fluorescein dUTP at strand breaks identified by
terminal deoxynucleotide transferase (TdT) for 1 hour in
the dark at 37°C. The cells were then analyzed directly under
a fluorescence microscope. Negative controls were performed by
omitting the TdT. For each treatment group 10 fields (approximately 400
cells) were counted and the percentage of TUNEL-positive cells
calculated.
Colorimetric MTT Assay
The MTT reduction by mitochondria is a measure of
metabolism and therefore only detects living
cells.39A Inhibition of MTT reduction has been used as a
quantitative assay for apoptosis. Cells were plated as
described for cell proliferation. Hydrocortisone was added to the cells
(M199/1%FBS) at least 20 hours before the cells were pulsed with MTT
(0.5 mg/mL) for 4 hours. The medium was removed and DMSO was added to
dissolve the dark blue crystals; the mixture was thoroughly shaken at
room temperature for 30 minutes. The optical density of each well was
determined by absorbance at 540 nm.
Immunocytochemistry
Portions of endarterectomy specimens were
fixed in phosphate-buffered 4% formaldehyde, paraffin-embedded, and
immunostained by standard procedures for the avidin-biotin
enhanced immunoperoxidase detection (Vector Laboratories). Primary
antibodies included a monoclonal antibody to detect the GR (NCL-GCR;
Novacastra Laboratories), HAM56 to detect macrophages, a mouse
IgG control antibody (Cappel Cooper Diagnostics), and HHF35
to detect SMC-specific actin (kindly provided by A.
Gown40 ). Positive immunostaining was
detected with DAB, and tissues were counterstained with
hematoxylin.
In Situ Hybridization
LDC lines were plated onto glass multichamber slides (Labtek)
and allowed to adhere for at least 24 hours before fixation with
DEPC-treated 4% buffered formaldehyde for 20 minutes. The fixed
monolayer was prehybridized with hybridization buffer (2 mg/mL
nuclease-free BSA, 20% dextran sulfate, 20 mmol/L EDTA, 1 mg/mL
yeast tRNA, 200 µg/mL poly(A), 4x SSC in Denhardt's solution), and
then the mRNA for the GR was detected in LDC by in situ hybridization
(ISH) of a digoxigenin-labeled probe in hybridization buffer at 45°C
overnight. Free probe was washed with 2x SSC at 37°C. Hybrids were
detected with anti-digoxigenin/alkaline phosphatase visualized with
Fast Red substrate.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Rat SMC Sensitivity to GCs
Primary SMCs isolated from Fisher 344 rat aortas were significantly growth-inhibited (P<0.05) by treatment with increasing concentrations of hydrocortisone for 20 hours (Figure 1A
), as measured by
[3H]thymidine incorporation. In both of the rat
lines tested (R8 and R13), strong inhibition (approximately 60% to
90%) occurred at low doses of hydrocortisone, 0.125 to 1.25 µg/mL.
Higher doses of hydrocortisone further inhibited proliferation, which
was maximal at 1250 µg/mL of hydrocortisone with an 80% to 90%
decrease in DNA synthesis. Later passages of one of the rat SMC lines
(R13-P32) were resistant to the antiproliferative effects of
hydrocortisone, suggesting an acquired resistance to hydrocortisone in
these SMCs. Essentially identical results were obtained with
dexamethasone when corrected for the 30-fold greater
potency of this synthetic steroid (not shown). Further, the
inhibitory effect of GCs on DNA synthesis was corroborated
by marked decreases in cell numbers over a 6-day period.
Dexamethasone, at 0.040 µg/mL (equivalent to 1.2 µg/mL
of hydrocortisone) caused a 62% decrease in cell proliferation of R8
and 40% decrease in R13.
|
The inset in Figure 1
demonstrates the presence of the GR by
RT-PCR (404 bp) in both early (P4) and late passages (P29 or P32) of
these 2 rat SMC lines. Thus, serial subpassage can, but need not, cause
rat SMCs to become resistant to hydrocortisone, and in this
case, the functional resistance is apparently not caused by the loss of
the GR mRNA.
Both rat SMC lines were also sensitive to the antiproliferative effects of TGF-ß1 in early passages, with a 65% decrease in cell proliferation at 10 ng/mL TGF-ß1. Later passages of R13, but not R8, became resistant to the antiproliferative effects of TGF-ß1, thus paralleling the response to hydrocortisone and dexamethasone. TGF-ß resistance with in vitro aging of the R13 line could be attributed to loss of the type II receptor (not shown), in a manner similar to the known loss of the type II receptor in SMCs derived from aged rats.41 TGF-ß resistance may also explain the acquired resistance to hydrocortisone in R13 because hydrocortisone has been reported to inhibit cell proliferation via activation of TGF-ß.42
Effects of Hydrocortisone on DNA Synthesis in LDCs
LDCs were treated with increasing concentrations of hydrocortisone
or TGF-ß1 under conditions similar to the rat studies. After 20 hours
of low-dose hydrocortisone treatment (1 µg/mL), LDC proliferation was
not inhibited (as measured by [3H]thymidine
incorporation) in 5 of 8 lesion-derived SMC lines (Figure 2C; LDC sensitivity is defined as >50%
inhibition of DNA synthesis). This low dose of hydrocortisone is 10
times higher than the physiological levels of
cortisol in the body (average cortisol levels at NY Hospital are
0.12±0.03 µg/mL). Much higher concentrations of hydrocortisone (10
µg/mL; 100-fold higher than physiological), were
needed to produce any detectable inhibition of cell proliferation in 5
of 8 LDC lines. At the highest tested dose, 3 of 8 cell lines remained
resistant to the antiproliferative effects of hydrocortisone
(Figure 2C). Only 2 cells lines, E12sc (a clone of
small cells) and late passage E137, were
inhibited strongly (75%) with high-dose hydrocortisone treatment
(Figure 2A), a result comparable with the sensitive rat SMCs. In
a third line, E47sc (also clonal), hydrocortisone inhibited DNA
synthesis by 50% (Figure 2A).
|
Our laboratory previously has shown that LDCs are resistant to
the inhibitory effects of TGF-ß1.7 Figure 2D confirms that 6 of 8 LDC lines were resistant to the
effects of TGF-ß1. The majority of these resistant cell lines
were also resistant to hydrocortisone (ie, E60, E64, E63, E145,
and E137 early). Two hydrocortisone-sensitive cell lines, E47sc and
E137 late passage, were also extremely sensitive to TGF-ß1,
exhibiting 75% to 90% inhibition of DNA synthesis (Figure 2B).
GC-Induced Apoptosis
GCs have been reported in the literature to induce
apoptosis in fibroblasts and lymphocytes;14
however, their effect on apoptosis of human vascular SMCs has
not been reported. Exposure to hydrocortisone (10 µg/mL) for 48 hours
in a normal human neonatal SMC line (CRL1999) causes a small increase
in the rate of apoptosis compared with untreated control cells
(Figure 3A). A small increase in cell
death was also observed in the E12sc and E47sc LDC lines treated with
hydrocortisone. However, the E85 LDC line, after 48 hours of high-dose
hydrocortisone treatment (10 µg/mL), had the greatest increase of
cell death with a 3-fold increase above basal levels (Figure 3A).
|
The MTT assay was used as a second method of measuring cell death.
After 20 hours of exposure of the normal CRL1999 SMC line to increasing
concentrations of hydrocortisone, a dose of 10 µg/mL hydrocortisone
decreased cell survival by 30% (Figure 3B). Both E12sc and
E47sc LDC lines were resistant to the hydrocortisone-induced
effects on cell death (Figure 3B). E85 was very sensitive to the
apoptotic effects of hydrocortisone, with a 30% increase in
cell death at 10 µg/mL hydrocortisone, a similar pattern as that seen
with the TUNEL assay (Figure 3A). In general, the TUNEL assay
underestimates the number of apoptotic cells because cells
detach and are lost in subsequent washing steps. The MTT assay is more
accurate as only live cells reduce the MTT, but is confounded by the
effect of proliferation in nonapoptotic cells.
Pro-Collagen III Production by LDCs
A major antifibrotic effect of GCs is a decrease in type I and III
collagen synthesis in many cell types, including
fibroblasts12 13 and ureteral SMCs.43 However
the effects of GCs on cells derived from atherosclerotic lesions are
unknown. Given the resistance of most LDC lines to HC, the effect on
protein and collagen synthesis was examined.
After 5 days' treatment of the E47sc LDCs with either hydrocortisone
alone or in the presence of TGF-ß1, the cells were counted and the
supernatants removed for the pro-collagen III RIA (PCNPIII).
Both TGF-ß1 (1 ng/mL) and hydrocortisone (10 µg/mL) inhibited cell
proliferation by up to 50% (Figure 4A).
Analyzing the supernatants from these cells for pro-collagen III
synthesis, hydrocortisone (10 µg/mL) increased pro-collagen
III production by 2-fold (Figure 4B). This effect was
further exaggerated with the addition of TGF-ß1. To confirm this
increase in collagen synthesis, proline incorporation (an indirect
measure of collagen production) was increased markedly in
TGF-ß1- and hydrocortisone-treated cells (Figure 4C). Related
studies observed similar increases in proline incorporation independent
of changes in cell number (not shown). These results suggest that GCs
enhance, not reduce, collagen production in LDCs.
|
Analysis of the GR mRNA in LDCs by RT-PCR
To determine the molecular basis for the resistance to
hydrocortisone, RNA extracted from LDCs was analyzed for the
presence of the human GR by RT-PCR. The primers were directed to exon
2, giving a 428-bp product, and after 35 cycles of PCR, mRNA for
the GR was absent in 10 of 14 LDC lines (Figure 5A; only 6 cell lines shown). LDCs from
patient E137 expressed the GR in early (E137E) but not late (E137L)
passage. Only 3 other lines were observed with detectable GR mRNA
levels, E12sc, E145, which expressed a low level, and normal SMCs
derived from internal mammary arteries (IMA) (Figure 5B).
|
Two GR isoforms have been identified, hGR
and hGRß, which
originate by alternative splicing of exon 9. hGR is fully functional
and hGRß has no hormone-binding activity and is thought to act as a
dominant-negative inhibitor of GR function.44
The cell lines that expressed the GR mRNA in the prior study (IMA,
E12sc, E137E, and E145) were tested for expression of the 2 receptor
isoforms. PCR primers to distinguish between the 2 receptor isoforms
were directed to exon 9 or 9ß, and in all cases the functional
hGR (314-bp PCR product) was present and hGRß absent (452
bp; Figure 5B). As in the prior study, E137E expressed the
highest level of GR mRNA expression.
To confirm the absence of the GR in LDC lines, in situ hybridization
for the GR was performed on a receptor-positive LDC line (E12sc) and a
negative cell line (E47sc). Figure 5C illustrates positive
staining for the GR (anti-sense) in the E12sc LDC line but not E47sc.
The sense controls were negative both in E12sc and E47sc, suggesting
the positive hybridizations were not caused by nonspecific annealing of
probe.
Presence of Heat Shock Protein 70 But Not Heat Shock Protein 90
in LDCs
The unliganded GR is part of a multiprotein complex, comprising 2
molecules of heat shock protein 90 (Hsp 90), 1 molecule of Hsp 70, 1
molecule of Hsp 56, and 1 molecule of Hsp 26. Abnormal Hsp 70 and Hsp
90 have been reported in steroid-resistant acute lymphoblastic
leukemia,31 suggesting that if these proteins are absent
or dysfunctional, the GR multiprotein complex is unable to form and
enter the nucleus, rendering the cells resistant to GCs. RT-PCR
analysis of 6 LDCs and 1 normal SMC line (IMA) indicated that
Hsp 90 (Figure 6, top) was detected only
faintly in 1 line (E85) and was absent in all other lines (182-bp
product). Hsp 70 (Figure 6, middle) was strongly positive in
5 of the cell lines (443 bp) but undetectable in E137 and E47sc. The
presence of the type I receptor for TGF-ß (Alk5) was detected in all
the LDC lines as a positive control for the integrity of the mRNA and
is consistent with prior publications demonstrating loss of the
type II, but not type I, receptor in LDCs.7 8
|
Absence of the GR in LDCs by Western Blot Analysis
Whole-cell lysates were made from LDCs, and 30 µg of protein was
separated on an SDS-PAGE gel and probed with an anti-GR antibody
directed at the immunogenic N-terminal modulating region (Novacastra
Laboratories) for Western blot analysis. The E12sc LDC line was
positive for the GR protein (94 kDa; Figure 7), confirming the positive signal seen
for the GR mRNA (Figure 5). E85 LDCs had a low level of the GR
protein, though the mRNA was not detected by RT-PCR, and 2 different
protein preparations of E47sc confirmed an absence of the GR (Figure 7). As controls, the M199 culture media was negative for the GR
protein and the Jurkat T-lymphocyte cell line was positive.
|
RT-PCR Analysis of the Mineralocorticoid Receptor and
11ß-Hydroxysteroid Dehydrogenase Type II mRNA
The same LDC RNA used to detect the GR mRNA (Figure 5) was
analyzed for both the mineralocorticoid receptor (MR) and
11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2), the enzyme required
for the metabolism of cortisol to cortisone. Because both
mineralocorticoids and GCs are able to signal through the MR with the
same affinity, the specificity of MR is conferred by 11ß-HSD2, which
protects the MR from the large excess of GCs. Figure 8A represents the RT-PCR
analysis for MR mRNA (426 bp). In all LDC lines that have been
tested (n=6), the MR is present. The E85 cell line has an
approximately 150-bp-smaller PCR product than the predicted PCR
product size of 432 bp. The PCR primers anneal to exons 7 and 9,
spanning exon 8 (157 bp), suggesting that the E85 cells lack a key
region of the ligand-binding domain of the MR.45
|
11ß-HSD2 mRNA (Figure 8B; 305 bp) is abundantly present in
E12sc, a cell line that has the GR by RT-PCR (Figure 5) and
Western blot analysis (Figure 7). The E85 cell line,
which has no GR mRNA by RT-PCR (Figure 5) but a low level of the
GR protein (Figure 7), has a low level of 11ß-HSD2 mRNA.
Interestingly, the E47sc LDC line does not express detectable
11ß-HSD2 mRNA by RT-PCR (Figure 8B), nor does it have a GR by
RT-PCR (Figure 5) or Western blot analysis (Figure 7). The 420-bp product corresponds to genomic DNA, confirmed
by its presence in reactions lacking reverse transcription (-RT).
Immunohistochemical Analysis of the GR in
Endarterectomy Lesions
Immunohistochemical analysis of
endarterectomy lesion specimens from 3 patients
demonstrated that the GR was expressed strongly by cells within the
tunica media (Figure 9A). In this
patient, GR staining was also strongly positive around a small
branching blood vessel. However, within the atherosclerotic lesion,
there was only scattered and relatively weak immunoreactivity for the
GR. This confirms the in vitro observation of low levels of GR
expression detected by RT-PCR and Western blot analysis. The
majority of the GR-positive cells were SMC/myofibroblasts as indicated
by the -smooth muscle-actin staining of adjacent sections of the
same patient (Figure 9B). An antibody to detect
macrophages in the lesion (HAM56) showed very little positive
immunoreactivity (Figure 9C). The staining seen in the mouse IgG
control section is only the hematoxylin counterstain (Figure 9D).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Severely narrowed carotid or coronary vessels are commonly revascularized by surgical endarterectomy or balloon angioplasty, respectively. Although initially successful in restoring flow in almost all patients, the major limitation of these procedures is fibroproliferative restenosis of the arteries during the 6-month period after the procedure.4 The findings of this paper illustrate that the majority of cell lines derived from human endarterectomy lesions are resistant to the antiproliferative effects of hydrocortisone, because of decreased expression of the GR, as determined by RT-PCR, Western blot analysis, and immunohistochemistry of the GR in endarterectomy lesions.
Prior studies have shown that cells derived from arteries of aged rats41 and from human atherosclerotic lesions7 are both resistant to the inhibitory effects of TGF-ß1. This resistance could be attributed to the loss of the type II TGF-ß signaling receptor, and in a subset of patients, this loss was caused by microsatellite instability in the TGF-ß type II receptor gene.8 Although TGF-ß1 is one of the key activating and suppressing factors in fibroproliferative disease, the GC system is also recognized as an important fibrosuppressive pathway. Extensive clinical and experimental evidence have suggested that hydrocortisone and its synthetic analogs (ie, dexamethasone and prednisone) suppress fibroproliferative diseases by acting as antiproliferative agents,9 10 antifibrotic agents that reduce collagen production,11 12 13 anti-inflammatory agents,15 16 and apoptotic factors.14
Given these diverse suppressive effects on fibroproliferative pathways, GCs should suppress restenosis, yet published clinical trials indicated that high-dose steroid therapy does not influence the overall rate of restenosis. Stone et al27 randomly assigned 102 patients with documented restenosis after a prior successful coronary angioplasty to receive treatment during repeat coronary angioplasty at the restenotic site. Treated patients received 125 mg methylprednisolone intramuscularly the evening before and the morning of angioplasty and then oral prednisolone (60 mg/d) for 7 days after coronary angioplasty. Angiographic follow-up at 6 months revealed the steroid-treated group had a 59% restenosis rate compared with the no-steroid control group, which had a 56% restenosis rate. A second randomized clinical trial examined 915 patients in total. Treated patients (n=215) received a single dose of methylprednisolone (1 g IV; 2 to 24 hours before coronary angioplasty). Again, angiography at 6 months' follow-up showed a 40% restenotic rate in the steroid-treated group compared with 39% in the control group.26
One possible explanation for the lack of a clinical benefit is insufficient local concentration of the drug at the site of injury. Intramural administration of hydrocortisone incorporated into polymer microspheres caused a significant reduction in post-angioplasty intimal hyperplasia in rabbits.46 Likewise, local delivery of dexamethasone via adventitial cuffs around the rat carotid artery produced a 75% reduction in balloon catheter–induced intimal hyperplasia.21 However, local delivery of dexamethasone by a novel polymer-coated eluting stent in the porcine coronary injury model did not decrease intimal hyperplasia.47
An alternate explanation for the clinical resistance to steroid treatment may be offered by the relative resistance of human LDCs to the antiproliferative effects of hydrocortisone. Analogous to TGF-ß1 resistance caused by loss of the type II receptor, these steroid-resistant cell lines typically lack key elements of the GR complex. Restenotic lesions have a lower rate of apoptotic SMCs and macrophages compared with primary lesions,6 implicating reduced apoptosis as an important mechanism in restenotic lesion formation. The resistance of some LDCs to the apoptotic effects of hydrocortisone is another mechanism by which the lesion might fail to regress. At higher concentrations of hydrocortisone, some LDCs exhibited cell death, possibly explaining the antiproliferative effect of high doses of hydrocortisone. Interestingly, Sato et al.48 reported that human vascular SMCs isolated from IMAs decreased their GR expression after 1 hour's treatment with Lp(a), a risk factor for atherosclerosis.49 These data, coupled with the present studies, suggest lesion cells may be less sensitive to the suppressive effects of steroids in an atherosclerotic environment.
Voisard et al28 cultured cells from human atherosclerotic
lesions and found, in vitro, an antiproliferative effect of
corticosteroids. However, high doses of hydrocortisone
(1250 µg/mL), or its synthetic analogs, prednisolone (750 µg/mL)
and dexamethasone (40 µg/mL), were required to inhibit
SMC proliferation by up to 70% after 5 days in culture. Correcting for
the relative potency of these agents, the effective dose was
approximately 10 000-fold higher than
physiological concentrations. Such high-dose
steroid therapy, in vivo, could only be achieved for short periods by
local administration. Lesion cells that expressed a complete GR
complex, such as E85, required a dose of only 10 µg/mL of
hydrocortisone to significantly inhibit DNA synthesis by up to 80%,
whereas most LDCs lacking the GR complex were dramatically
resistant to steroid therapy. Although it is possible that
cells cultured from these plaques represent a selected subset
of cells with resistance to GCs, 3 facts discount in vitro factors as
the sole explanation for the resistance: (1) although serial subpassage
can be associated with acquired resistance (Figure 1, R13),
other lines (R8) show no decrease in sensitivity, suggesting the
resistance is caused by the expansion of a preexisting subset of
resistant cells; (2) the LDCs exhibited resistance as soon as
it was possible to test, typically within 2 to 4 passages; and (3)
immunostaining suggests the relative absence of the GR
in histological sections of human lesions (Figure 8).
Published evidence has indicated that GCs can act via stimulation of
TGF-ß activity,42 a possibility that would explain the
parallel resistance to these 2 factors in almost all rat (Figure 1) and human (Figure 2) lines examined. A notable
exception was the E12sc LDC line, which is partially
hydrocortisone-sensitive, but TGF-ß1-resistant, suggesting
that hydrocortisone can act via TGF-ß–independent pathways. An
alternate explanation for the parallel resistance to these 2
inhibitory systems is that a common factor is capable of
modulating both the type II TGF-ß receptor and members of the GR
complex. Such a factor would probably exert its effects at the
transcriptional level, because steady-state mRNA levels of both
receptors is decreased.
The loss of the GR in the majority of
endarterectomy cell lines typically correlated with
resistance to the antiproliferative effects of GCs. However, 1 cell
line (E47sc), was extremely sensitive to the inhibitory
effects of hydrocortisone but lacked detectable levels of the GR. This
cell line was positive for the MR but not the enzyme 11ß-HSD2, which
protects the MR from the relative excess of circulating GCs. In the
absence of 11ß-HSD2, GCs bind to the MR with similar affinities as
aldosterone.50 11ß-HSD2 metabolizes cortisol
(hydrocortisone) to cortisone, which is inactive and cannot bind to the
MR.39 Thus, in the absence of 11ß-HSD2, as in E47sc,
hydrocortisone should readily signal through the MR, thus explaining
the functional responses to hydrocortisone in a GR-negative line. The
relationship between hydrocortisone, 11-ß-HSD2, and the MR or GR
complex is shown schematically in Figure 10.
|
Typically, collagen synthesis is inhibited by GCs11 12 13 ; however, in vascular SMCs isolated from either bovine or human aortas, GCs enhance collagen protein synthesis,51 52 although the GR status of these SMCs was not determined. The present data raise the possibility that the relative loss of the GR signaling complex, or reduced expression of 11ß-HSD2, would cause the GCs to interact with the MR and behave as mineralocorticoids. A fibrotic response would result because MR activation typically results in strong induction of type I and type III collagen synthesis.32 53
The human GR gene contains a total of 10 exons and is approximately 80
kb. There are 2 GR isoforms, hGR and hGRß, which originate by
alternative splicing of exon 9.44 hGR is fully
functional and hGRß has no hormone-binding activity.54
These 2 alternatively spliced forms of the GR are able to form
heterodimers but are inactive compared with hGR homodimers, and thus
hGRß is thought to act as an endogenous
inhibitor of GC action.55 The LDC lines that
did express the GR only expressed detectable levels of the active form,
hGR; therefore, the resistance to hydrocortisone in these LDCs is
probably not caused by hGRß inhibiting the actions of hGR.
The unliganded GR is part of a multiprotein complex, comprising 2 molecules of Hsp 90 and 1 each of Hsp 70, Hsp 56, and Hsp 26 (Figure 0). Hsp 90 molecules dissociate from the receptor complex on ligand binding,56 whereas Hsp 56 is required for directing the receptor complex to the nucleus,33 and Hsp 70 is a molecular chaperone for the GR entering the nucleus.34 Abnormal Hsp 70 and Hsp 90 have been reported in steroid-resistant acute lymphoblastic leukemia.31 The absence of the Hsp 90 complex in the LDC lines, and Hsp 70 in E47sc, suggests the resistance to hydrocortisone in these cells is caused not only by an absent GR, but potentially by an absence of other components of the GR multiprotein complex.
To the extent that cells proliferating from human lesions may reflect the properties of cells that respond to vascular injury, such as angioplasty, the results of the present study suggest restenosis may be another disease involving acquired resistance to GCs. The resistance of these LDCs to the antiproliferative and antifibrotic effects of hydrocortisone may contribute to excessive wound repair in atherosclerosis and restenosis. These results may have therapeutic implications if the GR can be replaced in GR-deficient vessels. Steroids are the first line, and in many cases the only line, of therapy for a broad spectrum of inflammatory and fibrotic diseases. Thus, the ability to restore steroid responsiveness might be adapted to treat steroid-resistant fibrosis in other situations such as congestive heart failure, pulmonary fibrosis, keloids, and arthritis.
|
Acknowledgments |
|---|
This work was made possible in part by funds granted to P.J.B. through a fellowship program sponsored by The Charles H. Revson Foundation. The statements made and views expressed, however, are solely the responsibility of the author. The work was further supported by NIH grants to T.A.M. from the National Institutes on Aging (AG12712) and the National Heart, Lung, and Blood Institute (HL56987). H.H.A. was supported, in part, by NIH grant HD00072.
Received September 11, 1998; accepted November 16, 1998.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Austin GE, Ratliff NB, Hollman J, Tabei S, Phillips DF. Intimal proliferation of smooth muscle cells as an explanation for recurrent coronary. J Am Coll Cardiol. 1985;6:369–375.[Abstract]
2. Dartsch PC, Bauridel I, Schinko I, Weiss HD, Hfling B, Betz E. Cell constitution and characteristics of human atherosclerotic plaques selectively removed by percutaneous atherectomy. Atherosclerosis. 1989;80:149–157.[Medline] [Order article via Infotrieve]
3. Mehta VY, Jorgensen MB, Raizner AE, Wolde-Tsadik G, Mahrer PR, Mansukhani P. Spontaneous regression of restenosis: an angiographic study. J Am Coll Cardiol. 1995;26:696–702.[Abstract]
4. Herrman J-PR, Hermans WRM, Vos J, Serruys PW. Pharmacological approaches to the prevention of restenosis following angioplasty: the search for the Holy Grail? (Part I). Drugs. 1993;46:18–52.[Medline] [Order article via Infotrieve]
5. Bochaton-Piallat ML, Gabbiani F, Redard M, Desmouliere A, Gabbiani G. Apoptosis participates in cellularity regulation during rat aortic intimal thickening. Am J Pathol. 1995;146:1059–1064.[Abstract]
6.
Bauriedel G, Schluckebier S, Hutter R, Welsch U,
Kandolf R, Luderitz B, Prescott MF. Apoptosis in
restenosis versus stable-angina
atherosclerosis: implications for the pathogenesis of
restenosis. Arterioscler Thromb Vasc Biol. 1998;18:1132–1139.
7. McCaffrey TA, Consigli S, Du B, Falcone DJ, Sanborn TA, Spokojny AM, Bush H. Decreased type II/type I TGF-ß1 receptor ratio in cells derived from human atherosclerotic lesions: conversion from an antiproliferative to profibrotic response to TGF-ß1. J Clin Invest. 1995;96:2667–2675.
8. McCaffrey TA, Du B, Consigli S, Szabo P, Bray PJ, Hartner L, Weksler BB, Sanborn TA, Bergman G, Bush HL. Genomic instability in the type II TGF-ß1 receptor gene in atherosclerotic and restenotic vascular cells. J Clin Invest. 1997;100:2182–2188.[Medline] [Order article via Infotrieve]
9. Longenecker JP, Kilty LA, Johnson LK. Glucocorticoid influence on growth of vascular wall cells in culture. J Cell Physiol. 1982;113:197–202.[Medline] [Order article via Infotrieve]
10. Berk BC, Vallega G, Griendling KK, Gordon JB, Cragoe E, Canessa M, Alexander RW. Effects of glucocorticoids of Na/H exchange and growth in cultured vascular smooth. J Cell Physiol. 1988;137:391–401.[Medline] [Order article via Infotrieve]
11. Krane SM, Amento EP. Glucocorticoids and collagen disease. Adv Exp Med Biol. 1984;171:61–71.[Medline] [Order article via Infotrieve]
12.
Hamalainen l, Olkarinen J, Kivirikko KI. Synthesis and
degradation of type I procollagen mRNAs in cultured human skin
fibroblasts and the effect of cortisol. J Biol Chem. 1985;260:720–725.
13.
Weiner FR, Cazja MJ, Jefferson DM, Giamvrone MA,
Tur-Kaspa R, Reid LM, Zern MA. The effects of dexamethasone
on in vitro collagen gene expression. J Biol Chem. 1987;262:6955–6958.
14.
Thompson EB. Apoptosis and steroid hormones.
Mol Endocrinol. 1994;8:665–673.
15. Rohhut B, Russo-Marie F. Novel concepts in the mode of action of anti-inflammatory steroids. Agents Actions. 1984;14:171–180.
16. Prescott M, McBride C, Court M. Development of intimal lesions after leukocyte migration into the vascular wall. Am J Pathol. 1989;135:835–846.[Abstract]
17. Bailey JM, Butler J. Pathology: influence of anti-inflammatory agents of experimental atherosclerosis. Nature. 966; 212:731–732.
18. Gordon D, Kobernick SS, McMillan GC, Duff GL. The effect of cortisone on the serum lipids and on the development of experimental cholesterol atherosclerosis in the rabbit. J Exp Med. 1954;99:371–386.[Abstract]
19. Hollander W, Kramsch DM, Franzblau C, Paddock J, Colombo MA. Suppression of atheromatous fibrous plaque formation by antiproliferative and anti-inflammatory drugs. Circ Res. 1974;34:I-131–I-141.
20. Makheja AN, Bloom S, Muesing R, Simon T, Bailey JM. Anti-inflammatory drugs in experimental atherosclerosis, 7: spontaneous atherosclerosis in WHHL rabbits and inhibition by cortisone acetate. Atherosclerosis. 1989;76:155–161.[Medline] [Order article via Infotrieve]
21. Villa AE, Guzman LA, Chen W, Golomb G, Levy RJ, Topol EJ. Local delivery of dexamethasone for prevention of neointimal proliferation in a rat model of balloon angioplasty. J Clin Invest. 1994;93:1243–1249.
22. Oppenheim E, Bruger M. The effect of cortisone and ACTH on experimental cholesterol atherosclerosis in rabbits. Circulation. 1952;6:470–471.
23. Gordon JB, Berk BC, Bettmann MA, Selwyn AP, Rennke H, Alexander RW. Vascular smooth muscle proliferation following balloon injury is synergistically inhibited by low molecular weight heparin and hydrocortisone. Circulation. 1987;76(suppl 4):IV-213. Abstract.
24. Petrik P, Law M, Moore W, Colburn M, Quinones-Baldrich W, Gelabert H. Dexamethasone and enalapril suppress intimal hyperplasia individually but have no synergistic effect. Ann Vasc Surg. 1998;12:216–220.[Medline] [Order article via Infotrieve]
25. Karim MA, Frizzell S, Inman L, Shinn M, Miller DD. In vivo role of glucocorticoid hormone in barotrauma vascular repair and fibrosis. J Mol Cell Cardiol. 1997;29:1111–1122.[Medline] [Order article via Infotrieve]
26.
Pepine CJ, Hirshfeld JW, Macdonald RG, Henderson MA,
Bass TA, Goldberg S, Savage MP, Vetrovec G, Cowley M, Taussig AS,
Whitworth HB, Margolis JR, Hill JA, Bove AA, Jugo RA. A controlled
trial of corticosteroids to prevent restenosis
after coronary angioplasty. Circulation. 1990;81:1753–1761.
27. Stone GW, Rutherford BD, McConahay DR, Johnson WL, Giorgo LV, Ligon RW, Hartzler GO. A randomized trial of corticosteroids for the prevention of restenosis in 102 patients. Cathet Cardiovasc Diagn. 1989;18:227–231.[Medline] [Order article via Infotrieve]
28. Voisard R, Seitzer U, Baur R, Dartsch PC, Osterhues H, Hoher M, Hombach V. Corticosteroid agents inhibit proliferation of smooth muscle cells from human atherosclerotic arteries in vitro. Int J Cardiol. 1994;43:257–267.[Medline] [Order article via Infotrieve]
29. Sher ER, Leung DY, Surs W, Sher ER, Zieg G, Kamada AK, Szefler SJ. Steroid-resistant asthma: cellular mechanisms contributing to inadequate response to glucocorticoid therapy. J Clin Invest. 1994;93:33–39.
30. Schlaghecke R, Kornely E, Wollenhaupt J, Specker C. Glucocorticoid receptors in rheumatoid arthritis. Arthritis Rheum. 1992;35:740–744.[Medline] [Order article via Infotrieve]
31. Kojika S, Sugita K, Inukai T, Saito M, Iijima K, Goi K, Shiraishi K, Mori T, Okazaki T, Kagami K, Ohyama K, Nakazawa S. Mechanisms of glucocorticoid resistance in human leukemic cells: implication of abnormal 90 and 70 kDa heat shock proteins. Leukemia. 1996;10:994–999.[Medline] [Order article via Infotrieve]
32.
Weber KT, Janicki JS, Shroff SG, Pick R, Chen RM,
Bashey RI. Collagen remodeling of the pressure-overloaded hypertrophied
nonhuman primate. Circ Res. 1988;62:757–765.
33.
Czar MJ, Lyons RH, Welsh MJ, Renoir JM, Pratt WB.
Evidence that the FK506-binding immunophilin heat shock protein 56 is
required for trafficking of the glucocorticoid receptor from the
cytoplasm to the nucleus. Mol Endocrinol. 1995;9:1549–1560.
34.
Shi Y, Thomas JO. The transport of proteins into
the nucleus requires the 70-kilodalton heat shock protein or its
cytosolic cognate. Mol Cell Biol. 1992;12:2186–2192.
35. Miesfeld R, Rusconi S, Godowski PJ, Maler BA, Okret S, Wikstroem AC, Gustafsson JA, Yamamoto KR. Genetic complementation to a glucocorticoid receptor deficiency by expression of cloned receptor cDNA. Cell. 1986;46:389–399.[Medline] [Order article via Infotrieve]
36.
Arriza JL, Weinberger C, Cerelli G, Claser TM, Handelin
BL, Housman DE, Evans RM. Cloning of human mineralocorticoid receptor:
structural and functional kinship with the glucocorticoid receptor.
Science. 1987;237:268–275.
37. Agarwal AK, Rogerson FM, Mune T, White PC. Gene structure and chromosomal localization of the human HSD11K gene encoding the kidney (type 2) isozyme of 11ß-hydroxysteroid dehydrogenase. Genomics. 1995;29:195–199.[Medline] [Order article via Infotrieve]
38. Fathallah DM, Cherif D, Dellagi K, Arnaout MA. Molecular cloning of a novel human Hsp70 from a B cell line and its assignment to chromosome 5. J Immunol. 1993;151:810–813.[Abstract]
39.
Funder JW, Pearce PT, Smith R, Smith I.
Mineralocorticoid action: target tissue specificity is enzyme, not
receptor mediated. Science. 1988;242:583–585.
39. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Meth.. 1983;65:55–63.[Medline] [Order article via Infotrieve]
40. Gown AM. Cell type and cell state specific antibodies in the analysis of early lesions of human atherosclerosis. Am J Hypertens. 1992; 5(suppl):114S–117S.
41. McCaffrey TA, Falcone DJ. Evidence for an age-related dysfunction in the antiproliferative response to transforming growth factor-ß in vascular smooth muscle cells. Mol Biol Cell. 1993;4:315–322.[Abstract]
42. Boulanger J, Reyes-Moreno C, Koutsilieris M. Mediation of glucocorticoid receptor function by the activation of latent transforming growth factor-ß in MG-63 human osteosarcoma cells. Int J Cancer. 1995;61:692–697.[Medline] [Order article via Infotrieve]
43. Wolf JS, Soble JJ, Ratliff TL, Clayman RV. Ureteral cell cultures, II: collagen production and response to pharmacologic agents. J Urol. 1996;156:2067–2072.[Medline] [Order article via Infotrieve]
44.
Encio IJ, Detera-Wadleigh SD. The genomic structure of
the human glucocorticoid receptor. J Biol Chem. 1991;266:7182–7188.
45. Geller DS, Rodriguez-Soriano J, Vallo Boado A, Schifter S, Bayer M, Chang SS, Lifton RP. Mutations in the mineralocorticoid receptor gene cause autosomal dominant pseudohypoaldosteronism type I. Nat Genet. 1998;19:279–281.[Medline] [Order article via Infotrieve]
46. Valero F, Hamon M, Fournier C, Meurice T, Flautre B, Van Belle E, Lablanche J, Gosselin B, Bauters C, Bertrand M. Intramural injection of biodegradable microspheres as a local drug-delivery system to inhibit neointimal thickening in a rabbit model of balloon angioplasty. J Cardiovasc Pharmacol. 1998;31:513–519.[Medline] [Order article via Infotrieve]
47. Lincoff AM, Furst JG, Ellis SG, Tuch RJ, Topol EJ. Sustained local delivery of dexamethasone by a novel intravascular eluting stent to prevent restenosis in the porcine coronary injury model. J Am Coll Cardiol. 1997;29:808–816.[Abstract]
48. Sato A, Sheppard KE, Fullerton MJ, Sviridov DD, Funder JW. Glucocorticoid receptor expression is down-regulated by Lp(a) lipoprotein in vascular smooth muscle cells. Endocrinology. 1995;136:3707–3713.[Abstract]
49. Dahlen GH. Lp(a) lipoprotein in cardiovascular disease. Atherosclerosis. 1994;108:111–126.[Medline] [Order article via Infotrieve]
50. Rupprecht R, Reul J, van Steensel B, Spengler D, Soder M, Berning B, Holsboer F, Damm K. Pharmacological and functional characterization of human mineralocorticoid and glucocorticoid receptor ligands. Eur J Pharmacol. 1993;247:145–154.[Medline] [Order article via Infotrieve]
51. Jarvelainen H, Halme T, Ronnemaa T. Effect of cortisol on the proliferation and protein synthesis of human aortic smooth muscle cells in culture. Acta Med Scand Suppl. 1982;660:114–122.[Medline] [Order article via Infotrieve]
52.
Leitman DC, Benson SC, Johnson LK. Glucocorticoids
stimulate collagen and noncollagen protein synthesis in cultured
vascular smooth muscle cells. J Cell Biol. 1984;98:541–549.
53. Medugorac I. Collagen content in different areas of normal and hypertrophied rat myocardium. Cardiovasc Res. 1980;14:551–554.[Medline] [Order article via Infotrieve]
54. Hollenberg SM, Weinberger C, Ong ES, Cerelli G, Oro A, Lebo R, Thompson EB, Rosenfeld MG, Evans RM. Primary structure and expression of a functional human glucocorticoid receptor. Nature. 1985;318:635–641.[Medline] [Order article via Infotrieve]
55. Bamberger CM, Bamberger AM, de Castro M, Chrousos GP. Glucocorticoid receptor ß: a potential endogenous inhibitor of glucocorticoid action in humans. J Clin Invest. 1995;95:2435–2441.
56.
Pratt WB, Sanchez ER, Bresnick EH, Meshinchi S,
Scherrer LC, Dalman FC, Welsh MJ. Interaction of the
glucocorticoid receptor with the Mr 90 000 heat shock
protein: an evolving model of ligand-mediated receptor transformation
and translocation. Cancer Res. 1989;49:2222s–2229s.
57.
Yamazaki M, Akaogi K, Miwa T, Imai T, Soeda E, Yokoyama
K. Nucleotide sequence of a full-length cDNA for 90 kDA
heat-shock protein from human peripheral blood lymphocytes.
Nucleic Acids Res. 1989;17:7108.
58. Franzen P, Dijke P, Ichijo H, Yamashita H, Schulz H, Heldin C-H, Miyazono K. Cloning of a TGFß type I receptor that forms a heteromeric complex with the TGFß type II receptor. Cell. 1993;75:681–692.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  I. Z. Jaffe, Y. Tintut, B. G. Newfell, L. L. Demer, and M. E. Mendelsohn Mineralocorticoid Receptor Activation Promotes Vascular Cell Calcification Arterioscler. Thromb. Vasc. Biol., April 1, 2007; 27(4): 799 - 805. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Matthews, J. Schwartz, S. Cohen, and T. Seeman Diurnal Cortisol Decline is Related to Coronary Calcification: CARDIA Study. Psychosom Med, September 1, 2006; 68(5): 657 - 661. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. Girod and D. J. Brotman Does altered glucocorticoid homeostasis increase cardiovascular risk? Cardiovasc Res, November 1, 2004; 64(2): 217 - 226. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Du, C. Fu, K. C. Kent, H. Bush Jr., A. H. Schulick, K. Kreiger, T. Collins, and T. A. McCaffrey Elevated Egr-1 in Human Atherosclerotic Cells Transcriptionally Represses the Transforming Growth Factor-beta Type II Receptor J. Biol. Chem., December 8, 2000; 275(50): 39039 - 39047. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||