| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Baker Medical Research Institute, Melbourne, Victoria 8008, Australia
Address all correspondence and requests for reprints to: Dr. John W. Funder, Baker Medical Research Institute, P.O. Box 6492, St. Kilda Road Central, Melbourne, Victoria 8008, Australia. E-mail: john.funder{at}baker.edu.au
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In contrast with these relatively rapid kinetics, mineralocorticoid effects on other nonepithelial tissues appear much more protracted, in terms of both onset and time to reach plateau levels. Rats administered aldosterone, peripherally or intracerebroventricularly, show consistent elevation of blood pressure some days later, which commonly takes 4–6 wk to reach plateau levels (9). Salt-sensitive rats placed on a 0.9% NaCl diet similarly show aldosterone-dependent increases in blood pressure, with values significantly above control values after 2–3 d and plateau levels at 10–14 d (10).
The effects of mineralocorticoid/salt on cardiac collagen accumulation appear to have an even longer time course than the effects on blood pressure. In the experimental model (uninephrectomized rats receiving 0.75 µg/h aldosterone, sc, and 0.9% NaCl to drink) used by Weber and his associates (11), no differences between control and treated animals were seen for ventricular morphology or cardiac interstitial and perivascular collagen in the first 2 wk of treatment; by 4 wk mean values for interstitial and perivascular collagen had increased, but to a significant level only for right ventricular perivascular collagen (12). In other studies using a similar model Delcayre and his colleagues measured mRNA for collagen types I and III, and again found no difference between controls and rats treated with exogenous aldosterone plus salt for 2 wk (13).
The present studies were designed to explore this lag phase in mineralocorticoid action, and in particular to determine whether a bolus dose of mineralocorticoid might be followed by a more rapid and/or more easily discernible effect. We thus administered deoxycorticosterone acetate (DOCA) im in oil as single 20-mg doses weekly for up to 4 wk, with rats killed 2, 4, 8, 16, and 32 d after the initial injection. In contrast with previous studies, we found relatively prompt elevations in both blood pressure and cardiac fibrosis, with blood pressure 40 mm Hg above control values and levels of collagen type III protein doubling after 4 d of DOCA/salt.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Histology and immunohistochemistry
Six-micron frozen sections from four animals in each group were
cut on a Leica Corp. (Nassloch, Germany) cryostat
transversely through the left and right ventricles, fixed in 4%
formaldehyde for 30 min, and then washed with tap water. Slides were
stained with hematoxylin for 5 min and with eosin for 2 min or with
0.1% Sirius Red in saturated picric acid for 40 min. Slides were
washed for 30 sec with tap water and then rapidly dehydrated with 100%
ethanol and mounted in Depex. All histological and immunohistochemical
scoring was performed blind.
For immunohistochemical analysis the avidin-biotin peroxidase complex technique was used. Transverse sections for both type I and type III collagen were fixed in acetone, treated with 3% hydrogen peroxide, incubated in 2% goat serum, and exposed to avidin and biotin blocking solution (Vector Laboratories, Inc., Burlingame, CA). Sections were then incubated for 16 h with rabbit polyclonal antibodies raised against type I or type III rat collagen (Biodesign, Kennebunkport, ME), followed by biotinylated goat antirabbit antibody (Vector Laboratories, Inc.) for 1 h, avidin-biotin peroxidase complex (Vector Laboratories, Inc.) for 30 min, and 3,3'-diaminobenzidine HCl for 5–10 min. Slides were counterstained with Mayer’s hematoxylin and mounted in Depex.
Terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end
labeling (TUNEL) staining
Frozen sections were fixed with 4% formaldehyde for 30 min,
postfixed in 0.1 M citrate buffer for 1 min with boiling,
and incubated in 0.1 M Tris-HCl buffer with 3% BSA and 3%
hydrogen peroxide. Staining of apoptotic cells was performed with a
terminal deoxynucleotidyltransferase enzyme-based detection kit
(Roche, Mannheim, Germany), followed by
3',3'-diaminobenzidine HCl for 3 min and Mayer’s hematoxylin, and
mounted in Depex.
Other analyses
Sections stained with Sirius Red were analyzed for interstitial,
perivascular, and scar collagen by image analysis as described
previously (14). For interstitial collagen volume
fractions, 20 fields from each heart section were selected at random
for analysis, and the area stained was calculated as a percentage of
the total area within a field. For perivascular collagen, data were
expressed as the area of perivascular collagen per medium area to
correct for differences in vessel size. Total scar collagen area was
calculated as a percentage of the total tissue area. Hydroxyproline in
heart tissue was determined by methods previously described
(17).
Statistics
Results are presented as the mean ± SEM, and
data were analyzed by ANOVA for repeated measures, followed by
post-hoc analysis with Fisher’s test and, where
appropriate, unpaired t test. P < 0.05 was
considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, upper
panel, blood pressure progressively increased with the length of
time of exposure to DOCA/salt. By 4 d the elevation in systolic
blood pressure was of the order of 40 mm Hg, with values rising further
to plateau levels (
200 mm Hg) at 32 d. Body weight (Fig. 1, center panel) showed a different pattern of evolution, with
no significant difference over the first 16 d of exposure, but a
substantial fall by d 32. This decrease should be interpreted with
caution; although treatment was performed in a staggered fashion, so
that changes in weight should reflect intervention rather than age,
rats after 32 d of DOCA/salt treatment were clinically different
from those at earlier time points, a difference underscored by various
other parameters (see below). In contrast with the smooth and
progressive elevation of blood pressure, heart weight remained
essentially unaltered for 8 d of DOCA/salt exposure, with an
approximately 15% increase by d 16 (Fig. 1, lower
panel).
|
Hydroxyproline values rose slowly over the period of study to reach
significance only at 32 d (4.1 ± 0.2 vs. control,
2.8 ± 0.1 mg/g heart weight; P < 0.01). The
pattern of rise was strikingly parallel to that in heart weight (Fig. 1
, lower panel), and the modest overall increase observed
strongly suggests that hydroxyproline assays represent a relatively
insensitive method of gauging early changes in cardiac fibrosis.
That this is indeed the case is clear from the data shown in Figs. 2 and 3.
Figure 2 (upper panel) shows cardiac interstitial collagen
volume fraction as a function of time of exposure to DOCA/salt, and
Fig. 2 (center panel) shows equivalent data for perivascular
collagen in the heart. In both instances there was a progressive rise
in collagen volume fraction; for interstitial collagen this was first
significant at 8 d, and for perivascular collagen this was first
significant at 16 d. Figure 2 (lower panel) shows scar
collagen. Although this also shows a significant increase by d 16, the
pattern of evolution is completely different. This in some part
reflects the absence of scar collagen in all hearts until d 8, when low
levels were found in two of four hearts; in larger part it reflects an
inherently different pattern, which would still be the case even were
increases over control values plotted for perivascular and interstitial
collagen volume fractions. The cellular events underlying these
different patterns are reflected in the temporal differences in tissue
response to injury, detailed below.
|
|
, this is not the case for type I interstitial collagen, which
mirrors both the time course and the extent of the collagen increase
measured with Sirius Red. Immunohistochemical determination of type III
interstitial collagen, however, shows an impressively stepwise increase
with time (Fig. 3, lower panel), with levels at d
4 more than double the control levels, and values at 2 d
statistically significantly higher by unpaired t test than
control values. Type III collagen increased 6-fold over the period of
observation, compared with 3- to 4-fold for type I collagen, or for
total collagen measured with Sirius Red. TGFß1
staining was uniformly negative for cardiac sections in control and
treated animals, although positive staining was seen in renal sections
from treated rats (not shown).
The cellular context of the increased collagen accumulation in Figs. 2 and 3 can be seen in detail in Figs. 4 and 5. When ventricular sections were
stained with hematoxylin and eosin, no morphological changes could be
seen between control (Fig. 4A) and 2 d of treatment (not shown).
By d 4, however, there was clear evidence of focal inflammatory cell
infiltration and cardiac cell necrosis (Fig. 4B), changes seen to a
similar degree in rats exposed to DOCA/salt for 8 d (Fig. 4C);
some scar tissue was seen in two of the four 8 d hearts (Fig. 4D).
In rats given DOCA/salt for 16 or 32 d inflammatory cell
infiltration was lower, and levels of scar tissue were much higher
(Figs. 4, E and F). On TUNEL staining, the progression of apoptosis in
various cell types (Fig. 5) appears to mirror the inflammatory changes;
apoptotic cells were not found in ventricular sections from the control
group (Fig. 5A) or in 2 d rats (data not shown). By 4 d,
however, apoptotic staining was seen throughout the areas of focal
inflammatory cell infiltration (Fig. 5B), persisting at similar levels
in the 8 d group (Fig. 5C). Apoptotic cells appeared to include
cardiomyocytes, fibroblasts, and inflammatory cells on the basis of the
size of the nuclei stained. In scar tissue in 8–32 d DOCA/salt rats,
apoptotic myocytes were also seen, although at the latter time points
the number of apoptotic cells was much lower than that in the areas of
active inflammation on d 4 and 8. Sections from 8, 16, and 32 d
rats are presented as Fig. 5, D, E, and F, respectively.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It is in this context that the studies undertaken in the present work need to be discussed. What they show is distinct time courses for the various parameters measured. Immunohistochemical levels of collagen type III protein 2 d after DOCA administration were uniformly higher than those in control hearts. In the context of previous studies on cardiac fibrosis, this then might be called a "superfast" response. Given that these values are 150% of the control values, it is probable that protein and particularly mRNA levels may be elevated even earlier.
Other fast responses (fast in comparison with previous reports) include blood pressure elevation, interstitial and perivascular collagen volume fractions, collagen type I levels, and both inflammatory cell infiltration and apoptosis. These responses share a similar time course in terms of onset, with substantial increases at 4 and/or 8 d, but differ markedly in terms of offset. Blood pressure rises progressively to what appear to be plateau levels by the end of the study; with more sensitive methods of measuring blood pressure, it is possible that a significant rise may have been seen at the earlier time point, given the extent (40 mm Hg) of the elevation by d 4. In contrast, values for both interstitial and perivascular collagen volume fractions continue to rise steeply over the course of the experiment, as do both collagen type I and type III by immunohistochemistry.
Perivascular collagen values were not significantly higher than control values until d 16, but had doubled, in terms of mean values, by d 4 and are thus included in the category of fast responses. The morphological studies show a third pattern of fast response, with marked inflammatory cell infiltration and evidence of apoptosis at d 4 and 8, essentially clearing by d 16, with only minor cellular indexes present in and around scar tissue. Finally, several parameters do not appear to alter substantially over the first 2 wk of the study, and might be classified as slow responses to mineralocorticoid/salt excess. These include body weight, heart weight, plasma electrolytes, cardiac hydroxyproline content, and scar collagen volume fraction; as noted previously, values at 32 d may need to be treated with caution, given the clinical condition of this group of animals.
Two questions might then be put to these data: how they can be reconciled with previous studies, in which much more protracted time courses have been reported, and the extent to which, together with previous studies, they can serve as a platform on which to base a working hypothesis for the cellular mechanisms involved. In previous time-course studies aldosterone administration had no discernible effect on the indices measured here (blood pressure, interstitial and perivascular collagen, and collagen type I and type III) in the first 2 wk of the study, with angiotensin in parallel and in contrast showing very rapid effects on a similar range of parameters, as a positive control (12). There are two major differences in design between such studies and those detailed in this paper: the choice of mineralocorticoid and the dose and method of administration used.
Both DOCA/salt and aldosterone/salt models of experimental hypertension
are well established. In terms of their effects on cardiac fibrosis,
the two mineralocorticoids have been shown to have similar effects
after 8 wk of treatment, with aldosterone given at 0.75 µg/h by
osmotic minipump and DOCA (20 mg) by weekly injection
(14). Although in the initial study (14) DOCA
appeared to have a greater effect than aldosterone on perivascular
fibrosis, and vice versa for interstitial fibrosis, in
subsequent studies the two steroids had indistinguishable effects
(21). Although DOC and aldosterone have comparable
affinity for MR (22), DOC has only 1/40th the potency in
the in vivo Kagawa bioassay for mineralocorticoid activity
(23), which reflects its very much higher level of plasma
binding (98–99%) than that of aldosterone (50%). The bulk of
plasma binding of DOC is to albumin and thus is essentially
unsaturable, so that the percent free levels will vary only marginally
with plasma concentration.
Although the difference in administered dose between aldosterone (0.75
µg/h) and DOCA (20 mg/wk) is 160-fold, rather than the 40-fold
difference in potency in the Kagawa bioassay, the major difference is
the dosing regimen, constant infusion vs. single bolus dose.
In the heart, MR are operationally unprotected by 11ß-hydroxysteroid
dehydrogenase-2 (24) and thus are presumably normally
overwhelmingly occupied by the physiological glucocorticoids
(cortisol/corticosterone), which circulate at much higher (100-fold)
free concentrations. We have previously shown, for example, that
coinfusion of 30-fold excess corticosterone with 0.75 µg/h
aldosterone lowers the rise in blood pressure and cardiac fibrosis to
half that seen with aldosterone alone (25). The dose of
aldosterone commonly infused is clearly outside the range of normal
aldosterone secretion in the rat, by up to an order of magnitude; at
first approximation, then, perhaps 10% of cardiac MR are aldosterone
occupied during the infusion, clearly sufficient to produce a
relatively gradual effect in terms of fibrosis and hypertrophy.
In contrast, the bolus injection of 20 mg DOCA in oil will produce very high circulating levels of DOC over a period of some time postinjection, with free levels perhaps up to an order of magnitude above those of the circulating glucocorticoids, differences in plasma binding notwithstanding. The levels will obviously fall in line with the half time of clearance of plasma DOC, although release may be attenuated given the depot injection. The two situations are thus very different, with aldosterone predicted to occupy and activate perhaps 10% of cardiac MR over the course of the infusion, and DOC to occupy perhaps 90% of cardiac MR for some time after administration, with occupancy by active mineralocorticoid falling to lower levels thereafter. Whereas over the course of an 8-wk study the outcomes may not be very different, it is plausible that preemptive occupancy of most cardiac MR for some hours may have short-term consequences quite distinct from the constant occupancy and activation of a relatively low percentage of MR, as is the case with aldosterone infusion.
If the initial effects of the very high levels of circulating DOC in the present studies are on the coronary vasculature and/or cardiac myocytes, the effect may be via modulation of transcription or sustained activation of rapid, nongenomic mineralocorticoid effects. In vascular smooth muscle cells aldosterone causes rapid alkalinization of the cell by activation of Na+/H+ exchange; whereas this effect is clearly nongenomic, there is persuasive evidence that it is mediated via classical, 11ß-hydroxysteroid dehydrogenase-2 protected MR (5) In cardiac myocytes the major rapid, nongenomic effect of aldosterone is on Na+/K+/2Cl cotransport, to allow influx of ions and an approximately 40% increase in intracellular Na+ (26). Even very small (4%) increases in ambient Na+ have been shown to have very marked effects in vivo on cardiomyocyte size and rate of protein synthesis (27); the possible effects of an approximately 40% change in intracellular Na+ in response to aldosterone (26) have not been addressed to date.
Cells of the macrophage/monocyte lineage have been shown to express MR (28), as have cardiac myocytes (29); in contrast, in our hands neither T cells nor cardiac fibroblasts have measurable MR levels, and thus may not be direct aldosterone targets. Whatever the mechanism of a presumed action of aldosterone on cardiac myocytes, its effect on fibroblasts may then be paracrine rather than direct; in addition, it is difficult to reconcile a uniquely myocyte effect with the apparent tolerance that characterizes the process of DOC-induced inflammatory cell infiltration and apoptosis. Such an effect may be mediated by activation of MR in cardiac macrophages/monocytes to destroy a particular set of T cells, so that the system is unresponsive to further administration of DOCA. To explore the trigger events in mineralocorticoid/salt cardiac fibrosis additional experiments are clearly required, over shorter time courses and perhaps involving tissue-selective (cardiac myocyte, macrophage/monocyte) deletion of the gene coding for MR.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received February 27, 2001.
Accepted for publication April 26, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  A. J. Rickard, J. Morgan, G. Tesch, J. W. Funder, P. J. Fuller, and M. J. Young Deletion of Mineralocorticoid Receptors From Macrophages Protects Against Deoxycorticosterone/Salt-Induced Cardiac Fibrosis and Increased Blood Pressure Hypertension, September 1, 2009; 54(3): 537 - 543. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J Rickard and M. J Young Corticosteroid receptors, macrophages and cardiovascular disease J. Mol. Endocrinol., June 1, 2009; 42(6): 449 - 459. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Rickard, J. W. Funder, J. Morgan, P. J. Fuller, and M. J. Young Does Glucocorticoid Receptor Blockade Exacerbate Tissue Damage after Mineralocorticoid/Salt Administration? Endocrinology, October 1, 2007; 148(10): 4829 - 4835. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Rickard, J. W. Funder, P. J. Fuller, and M. J. Young The Role of the Glucocorticoid Receptor in Mineralocorticoid/Salt-Mediated Cardiac Fibrosis Endocrinology, December 1, 2006; 147(12): 5901 - 5906. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Michea, A. M. Delpiano, C. Hitschfeld, L. Lobos, S. Lavandero, and E. T. Marusic Eplerenone Blocks Nongenomic Effects of Aldosterone on the Na+/H+ Exchanger, Intracellular Ca2+ Levels, and Vasoconstriction in Mesenteric Resistance Vessels Endocrinology, March 1, 2005; 146(3): 973 - 980. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Barbato, S. Rashid, P. J. Mulrow, J. I. Shapiro, and R. Franco-Saenz Mechanisms for Aldosterone and Spironolactone-Induced Positive Inotropic Actions in the Rat Heart Hypertension, November 1, 2004; 44(5): 751 - 757. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Young and J. W. Funder Eplerenone, But Not Steroid Withdrawal, Reverses Cardiac Fibrosis in Deoxycorticosterone/ Salt-Treated Rats Endocrinology, July 1, 2004; 145(7): 3153 - 3157. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Ling, A. Dai, R. J. Dilley, M. Jones, E. Simpson, P. A. Komesaroff, and K. Sudhir Endogenous Estrogen Deficiency Reduces Proliferation and Enhances Apoptosis-Related Death in Vascular Smooth Muscle Cells: Insights From the Aromatase-Knockout Mouse Circulation, February 3, 2004; 109(4): 537 - 543. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Francis, T. Beltz, A. K. Johnson, and R. B. Felder Mineralocorticoids act centrally to regulate blood-borne tumor necrosis factor-{alpha} in normal rats Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2003; 285(6): R1402 - R1409. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Young and J. Funder Mineralocorticoid Action and Sodium-Hydrogen Exchange: Studies in Experimental Cardiac Fibrosis Endocrinology, September 1, 2003; 144(9): 3848 - 3851. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. J. Brown Eplerenone: Cardiovascular Protection Circulation, May 20, 2003; 107(19): 2512 - 2518. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Casal, J.-S. Silvestre, C. Delcayre, and A. M. Capponi Expression and Modulation of Steroidogenic Acute Regulatory Protein Messenger Ribonucleic Acid in Rat Cardiocytes and after Myocardial Infarction Endocrinology, May 1, 2003; 144(5): 1861 - 1868. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Young, L. Moussa, R. Dilley, and J. W. Funder Early Inflammatory Responses in Experimental Cardiac Hypertrophy and Fibrosis: Effects of 11{beta}-Hydroxysteroid Dehydrogenase Inactivation Endocrinology, March 1, 2003; 144(3): 1121 - 1125. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Rocha, C. L. Martin-Berger, P. Yang, R. Scherrer, J. Delyani, and E. McMahon Selective Aldosterone Blockade Prevents Angiotensin II/Salt-Induced Vascular Inflammation in the Rat Heart Endocrinology, December 1, 2002; 143(12): 4828 - 4836. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
