Rosuvastatin attenuates hypertension-induced cardiovascular remodeling without affecting blood pressure in DOCA-salt hypertensive rats.

Abstract

The pleiotropic effects of statins represent potential mechanisms for the treatment of end-organ damage in hypertension. This study has investigated the effects of rosuvastatin in a model of cardiovascular remodeling, the DOCA-salt hypertensive rat. Male Wistar rats weighing 300 to 330 g were uninephrectomized (UNX) or UNX and treated with DOCA (25 mg subcutaneously every fourth day) and 1% NaCl in the drinking water. Compared with UNX controls, DOCA-salt rats developed hypertension, cardiovascular hypertrophy, inflammation with perivascular and interstitial cardiac fibrosis, endothelial dysfunction, and prolongation of ventricular action potential duration at 28 days. Rosuvastatin-treated rats received 20 mg/kg/d of the drug in 10% Tween 20 by oral gavage for 32 days commencing 4 days before uninephrectomy. UNX and DOCA-salt controls received vehicle only. Rosuvastatin therapy attenuated the development of cardiovascular hypertrophy, inflammation, fibrosis, and ventricular action potential prolongation, but did not modify hypertension or vascular dysfunction. We conclude that the pleiotropic effects of rosuvastatin include attenuation of aspects of cardiovascular remodeling in the DOCA-salt model of hypertension in rats without altering systolic blood pressure.

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Journal of Cardiovascular Pharmacology

ROSUVASTATIN ATTENUATES HYPERTENSION-INDUCED
CARDIOVASCULAR REMODELLING WITHOUT AFFECTING BLOOD
PRESSURE IN DOCA-SALT HYPERTENSIVE RATS

David Loch1, Scott Levick1, Andrew Hoey2 and Lindsay Brown1


1 Department of Physiology and Pharmacology, School of Biomedical Sciences,
The University of Queensland 4072, AUSTRALIA
2 Centre for Biomedical Research, Faculty of Sciences, University of Southern
Queensland, Toowoomba 4350, AUSTRALIA

Address for Correspondence:
Assoc Prof Lindsay Brown, Department of Physiology and Pharmacology, School of
Biomedical Sciences, The University of Queensland 4072, AUSTRALIA; telephone +61
7 3365 3098; fax +61 7 3365 1766; email l.brown@uq.edu.au

Short Title:
Rosuvastatin and remodelling in DOCA-salt rats

Sources of Support:
This study was supported in part by a grant from AstraZeneca, Macclesfield, Cheshire
U.K.




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ABSTRACT


The pleiotropic effects of statins represent potential mechanisms for the treatment of end-
organ damage in hypertension. This study has investigated the effects of rosuvastatin in a
model of cardiovascular remodelling, the DOCA-salt hypertensive rat. Male Wistar rats
weighing 300-330g were uninephrectomized (UNX) or uninephrectomized and treated
with DOCA (25 mg subcutaneously every fourth day) and 1% NaCl in the drinking
water. Compared with UNX controls, DOCA-salt rats developed hypertension,
cardiovascular hypertrophy, inflammation with perivascular and interstitial cardiac
fibrosis, endothelial dysfunction and prolongation of ventricular action potential duration
at 28 days. Rosuvastatin treated rats received 20mg/kg/day of the drug in 10% Tween 20
by oral gavage for 32 days commencing 4 days before uninephrectomy. UNX and
DOCA-salt controls received vehicle only. Rosuvastatin therapy attenuated the
development of cardiovascular hypertrophy, inflammation, fibrosis and ventricular action
potential prolongation, but did not modify hypertension or vascular dysfunction. We
conclude that the pleiotropic effects of rosuvastatin include attenuation of aspects of
cardiovascular remodelling in the DOCA-salt model of hypertension in rats without
altering systolic blood pressure.

Key Terms: DOCA-salt rat; Rosuvastatin; Hypertension; Fibrosis; Hypertrophy.


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INTRODUCTION
Although many drugs efficiently lower serum cholesterol levels, none have been more
successful and well tolerated than the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-
CoA) reductase inhibitors. The statins, as they are more commonly known, reduce
cardiovascular mortality in primary and secondary prevention trials of coronary heart
disease in patients with high or moderate hypercholesterolaemia [1-5] and even normal
cholesterol levels [1, 5, 6]. This clinical benefit of statins may occur relatively early after
initiation of therapy [3-5] and before any regression in atherosclerotic plaques can be
detected [7]. In addition, their effects differ from those observed after the reduction of
plasma cholesterol levels by surgical therapy [8]. These findings suggest that statins work
by mechanisms additional to a decrement in plasma lipid concentrations and
atherosclerotic plaque prevention or regression. Such pleiotropic effects include
inhibiting the thrombogenic response and reducing oxidative stress and inflammation [9].

More recently, several studies have documented that the pleiotropic effects of statins
extend to the prevention of pathological cardiovascular remodelling in animal models of
human disease, such as rat models of post-infarct heart failure [10] and type II diabetes
mellitus [11]. However, it is unclear whether these cardioprotective effects are shared by
all statins. This is especially important, considering the highly varied chemical structures,
physicochemical and pharmacokinetic/metabolic properties of the many members of the
statin family [12, 13] and the documented differences not only in their lipid lowering
potential, but also in their nonlipid effects, particularly cardiovascular hypertrophy [14-
16].

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Rosuvastatin is a new synthetic and chemically distinct member of the statin family [13].
Clinical evidence has shown that rosuvastatin is the most effective statin with regards to
lipid lowering [17]. However, whether rosuvastatin possesses the ability to exert similar,
or more significant, beneficial effects on cardiovascular remodelling is yet to be
established. There have been a number of publications showing rosuvastatin to be
protective in cardiac ischemia-reperfusion injury models [18-20]. Consequently, we have
investigated the potential pleiotropic effects of rosuvastatin on cardiovascular
remodelling in the DOCA-salt model of hypertension in rats.

METHODS
Ethical Clearance
All experimentation was approved by the Animal Experimentation Ethics Committee of
The University of Queensland under the guidelines of the National Health and Medical
Research Council of Australia.

DOCA-salt hypertensive rats
Male Wistar rats weighing 300-330g (~8 weeks old) were obtained from the Central
Animal Breeding House of The University of Queensland. All rats were
uninephrectomied. This was done under anaesthesia with intraperitoneal tiletamine (25
mg/kg) and zolazepam (25 mg/kg)(Zoletil®) combined with xylazine (10 mg/kg)(Ilium
Xylazil®). Kidneys were visualised by a left lateral abdominal incision. The left kidney
was removed after ligation of adjoining renal vasculature and ureter with sutures. The

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capsule was removed from the left kidney, which was then weighed. Uninephrectomized
rats were given either no further treatment (UNX rats) or 1% NaCl in the drinking water
with subcutaneous injections of deoxycorticosterone acetate (DOCA; 25mg in 0.4ml
dimethylformamide every fourth day) (DOCA-salt rats). Rosuvastatin-treatment groups
received 20mg/kg/day rosuvastatin in 10% Tween 20 by oral gavage for 32 days
commencing 4 days before uninephrectomy. UNX and DOCA-salt controls received
vehicle only. Experiments were performed 28 days after surgery.

Assessment of Physiological Parameters
Systolic blood pressure was measured by tail-cuff plethysmography in rats lightly
anaesthetised with intraperitoneal tiletamine (10 mg/kg) and zolazepam (10 mg/kg). Rats
were euthanased with pentobarbitone (200 mg/kg ip). Blood was taken from the
abdominal vena cava, just caudal to the insertion of renal veins, centrifuged and the
plasma immediately frozen. Plasma sodium and potassium concentrations were measured
by flame photometry. The heart was removed and weighed immediately after death and
expressed as a ratio of the tissue weight (mg) to the total body weight (g). Plasma levels
of total cholesterol were measured by The University of Queensland Veterinary
Pathology Services, Brisbane, Australia.

Isolated Langendorff heart preparation
Rats were anaesthetized with sodium pentobarbitone (100 mg/kg ip) and heparin (200 IU)
was administered via the femoral vein. After allowing two minutes for the heparin to
circulate, the heart was excised and placed in cooled (0°C) crystalloid perfusate (modified

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Krebs-Henseleit solution of the following composition in mM: NaCl 119.1, KCl 4.75,
MgSO4 1.19, KH2PO4 1.19, CaCl2 2.16, NaHCO3 25.0, glucose 11.0). A cannula was
then placed in the heart with its tip immediately above the coronary ostia of the aortic
stump. The cannula was used to perfuse the heart in a non-recirculating Langendorff
fashion at 100cm of hydrostatic pressure. The perfusate temperature was maintained at
37°C and bubbled with 95%O2 / 5%CO2. The apex of the heart was pierced to facilitate
thebesian drainage and paced at 250 bpm.

Left ventricular developed pressure was measured using a balloon catheter inserted into
the left ventricle through the mitral orifice. The catheter was connected via a three-way
tap to a micrometer syringe and to a MLT844 Physiological Pressure Transducer
(ADInstruments) and PowerLab data acquisition unit (ADInstruments). The outer
diameter of the catheter was similar to the mitral annulus to prevent ejection of the
balloon during the systolic phase. After a 5-minute stabilization period, steady-state left
ventricular pressure was recorded from isovolumetrically beating hearts. Increments in
balloon volume were applied to the heart with left ventricular end-diastolic pressure
recorded at approximately 0, 5, 10, 15, 20 and 30mmHg. At the end of the experiment,
the atria and right ventricle were dissected away leaving the left ventricle and septum,
which were blotted dry then weighed.

Myocardial diastolic stiffness was calculated as the diastolic stiffness constant (k,
dimensionless), the slope of the linear relation between tangent elastic modulus (E,
dyne/cm2) and stress (σ, dyne/cm2) [21].

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Isolated thoracic aortic rings
Thoracic aortic rings (approximately 4 mm in length) were suspended with a resting
tension of 10 mN. Cumulative concentration-response curves were performed for
noradrenaline and either acetylcholine or sodium nitroprusside in the presence of a
submaximal (~70%) contraction to noradrenaline.

Quantification of Left Ventricular Collagen
Collagen content was determined by image analysis of picrosirius red-stained sections of
the hearts [22]. In brief, transverse sections were stored initially in Telly’s fixative
(100mL 70% ethanol; 5mL glacial acetic acid; 10mL formaldehyde) for 3 days, then
transferred to modified Bouin’s solution (85 mL saturated picric acid; 5 mL glacial acetic
acid; 10 mL 40% formaldehyde) for two days and then stored in 70% ethanol.

Sections were subsequently embedded in wax and sliced into 10 µm sections. These were
stained with picrosirius red (0.1% Sirius Red F3BA in picric acid). Slides were left in
0.2% phosphomolybdic acid for 5 minutes, washed, left in picrosirius red for 90 minutes,
then in 1 mM HCl for 2 minutes and 70% ethanol for 45 seconds. The stained sections
were mounted with Depex and visualized using a Biorad MRC-1024 confocal laser-
scanning microscope with a Red/Texas Red filter with excitation at 568 nm and green
emission at 609 nm. Images were acquired with an objective lens of 40x magnification
and quantified using NIH-image software (National Institute of Health, USA). At least 4

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areas from each heart were analysed and collagen levels expressed as a percentage of red
area in each image.

Histological collagen results were confirmed by hydroxyproline assay adapted from
Stegemann and Stalder [23]. Approximately 2.5 and 5.0 mg samples of thoracic aorta and
left ventricle respectively were dried for 6 hours at 40oC. Tissues and standards were then
hydrolyzed in 6N HCl at 107oC for 18 hours. The acid was blown off by compressed air
and the hydrolysate reconstituted in distilled water. Chloramine T reagent was added to
each sample for the oxidation step to progress, followed by Ehrlich’s reagent to enable
chromophore development. Absorbance of each sample was read at 550nm in a
spectrophotometer and hydroxyproline content established from a standard curve.

Width of Media in Thoracic Aorta
The width of the media in the thoracic aorta of rats was measured by image analysis of
picrosirius red-stained sections. Section preparation, staining, image acquisition and
analysis were similar to those mentioned above. Three different areas of each aorta were
measured and the results averaged.

Immunofluorescence of ED-1 positive cells in the Left Ventricle
Briefly, 5 µm thick sections were initially incubated with primary antibodies for rat
macrophages (ED1; Serotec mouse anti-rat ED1 diluted 1:15). Omission of primary
antibodies, and staining with an irrelevant mouse immunoglobulin of the same isotype,
served as negative controls. Samples were then incubated with IgG-fluorescein

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conjugated secondary antibody (Chemicon; diluted 1:200). Sections were counterstained
with propidium iodide, mounted and visualized with a confocal laser-scanning
microscope. A zero to four grading scale was used to classify the extent of ED-1 positive
monocyte/macrophage infiltration in the left ventricle. 0 = no inflammatory cells present;
1 = low level of inflammatory cells throughout the left ventricle; 2 = moderate levels of
inflammatory cells throughout the left ventricle and concentrated in mild scarring; 3 =
high levels of inflammatory cells throughout the left ventricle and concentrated in
moderate scarring; 4 = high levels of inflammatory cells throughout the left ventricle and
concentrated in heavy scarring.

Microelectrode studies of isolated left ventricular papillary muscles
Electrophysiological recordings of cardiac action potentials were obtained by
microelectrode single cell impalements of ex vivo left ventricular papillary muscles. Rats
were euthanased by carbon dioxide inhalation with subsequent exsanguination. The heart
was removed and placed in chilled Tyrode’s physiological salt solution (in mM: NaCl
136.9, KCl 5.4, MgCl2.H2O 1.0, NaH2PO4.2H2O 0.4, NaHCO3 22.6, CaCl2.2H2O 1.8,
glucose 5.5, ascorbic acid 0.3, Na2-EDTA 0.05) bubbled with 95%O2 / 5%CO2, where
the left ventricular papillary muscles were promptly dissected out. A stainless steel hook
was placed through the valvular end of the papillary muscle, and a 30G needle was used
to fix the apical end. The needle was subsequently embedded into a rubber base placed in
a 1.0mL experimental chamber continuously perfused with carbogenated, warm
(35±0.5oC) Tyrode’s solution at ~3 mL/min. The hook was attached to a modified sensor
element (SensoNor AE801) connected to an amplifier (World Precision Instruments,

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TBM-4). The muscle was stretched slowly to the required preload (3-5mN). Papillary
contractions were induced by field stimulation (Grass SD-9) via electrodes on each side
of the muscle (stimulation frequency 1 Hz; pulse width 0.5 ms; stimulus strength 20%
above threshold).

The muscle was allowed to equilibrate for 30 minutes then impaled with a microelectrode
(World Precision Instruments, filamented borosilicate glass, outer diameter 1.5 mm) with
a tip resistance of 5-15 MΩ when filled with 3M KCl. Perfusion and recording then
continued for another 30 minutes. Action potential parameters measured were action
potential duration (APD) at 20%, 50% and 90% of repolarization (APD20, ADP50 and
APD90 respectively), action potential amplitude and resting membrane voltage. The
reference electrode was a Ag/AgCl electrode. A Cyto 721 electrometer (World Precision
Instruments) was used to record bioelectrical activity. All signals were recorded via a
PowerLab 4S data acquisition unit (ADInstruments). Data were acquired, derived and
analysed using Chart 4.3 software (ADInstruments).

Data analysis
All results are given as mean ± SEM. The negative log EC50 of the increase in force of
contraction in mN was determined from the concentration giving half-maximal responses
in individual concentration-response curves. These results were analysed by one-way
analysis of variance followed by the Bonferroni post test to determine differences
between treatment groups; p<0.05 was considered significant.


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Drugs
Deoxycorticosterone acetate, acetylcholine, sodium nitroprusside and noradrenaline were
purchased from Sigma Chemical Company, St Louis, MO, USA. Rosuvastatin calcium
was provided by AstraZeneca, U.K.. Noradrenaline, sodium nitroprusside and
acetylcholine were dissolved in distilled water; deoxycorticosterone acetate was
dissolved in dimethylformamide with mild heating.

RESULTS
Control and rosuvastatin-treated UNX rats gained similar weight over the 4 week
protocol. Both DOCA-salt groups, on the other hand, failed to gain body weight in this
period (Table 1). The systolic blood pressure of DOCA-salt rats rose significantly at 2
weeks and even further at 4 weeks (Table 1). Rosuvastatin treatment had no effect on this
developing hypertension in DOCA-salt rats. UNX rats exhibited no alterations in blood
pressure (Table 1). Total plasma cholesterol concentration was significantly augmented
in DOCA-salt rats, while rosuvastatin treatment normalized this parameter in these rats
(Table 1). The concentration of plasma sodium was unaltered amongst the four rat
groups; however, plasma potassium concentration was decreased in both control and
treatment DOCA-salt groups (Table 1).

DOCA-salt animals exhibited an increase in left ventricular mass that was attenuated by
rosuvastatin treatment (Table 1). Fibrosis following an increased infiltration of
inflammatory cells is characteristic of the DOCA-salt rat left ventricle [24]. We have
shown that the left ventricular tissue of DOCA-salt animals exhibited considerable ED1-

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positive inflammatory cell infiltration compared to UNX controls, and this inflammation
was prevented by rosuvastatin (Figure 2). The left ventricular tissue of DOCA-salt
control rats was also found to be substantially fibrosed, as evidenced by a significant
increase in both interstitial and perivascular left ventricular collagen area compared with
UNX controls (Figure 1). Rosuvastatin treatment was successful in attenuating the
increase in interstitial collagen area (Figure 1). The drug also normalized the perivascular
collagen area of DOCA-salt rats (Figure 1). An alternative measure of cardiac fibrosis,
hydroxyproline content, was also elevated in the left ventricle of DOCA-salt control rats
in comparison to UNX animals (Figure 1). Once again, rosuvastatin treatment reduced
this indicator of cardiac fibrosis (Figure 1). The isolated perfused hearts of DOCA-salt
control rats exhibited an increased diastolic stiffness constant compared to UNX animals.
Rosuvastatin treatment of these animals normalized this measure of cardiac stiffness
(Table 1).

Electrophysiological studies of isolated papillary muscles revealed no difference in
resting membrane potential and action potential amplitude between UNX and DOCA-salt
rats (Table 2). DOCA-salt rats showed significant electrical remodelling, manifested as
prolonged action potential durations at 20%, 50% and 90% of repolarization (APD20,
APD50 and APD90) (Table 2, Figure 4). Rosuvastatin treatment significantly attenuated
this prolongation of APD90, but not APD20 or APD50 (Table 2, Figure 4).

The thoracic aorta media width of DOCA-salt rats was found to be significantly increased
compared to UNX rats, with rosuvastatin therapy attenuating this vascular hypertrophy

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(Table 1). In contrast, the hydroxyproline content of these vessels was similar among
groups (Table 1). Vascular smooth muscle dysfunction was evidenced by the reduced
contractile response to noradrenaline, as well as the decreased relaxant response to
sodium nitroprusside in isolated thoracic aortic rings of DOCA-salt rats compared to
UNX controls (Figure 3A and C). There was also an increased potency of noradrenaline-
induced contraction in these rings (Figure 3A). Relaxant responses to acetylcholine were
also reduced in hypertensive rats compared to their normotensive controls (Figure 3B).
Rosuvastatin was without effect on these reduced responses to noradrenaline, sodium
nitroprusside and acetylcholine in DOCA-salt rats (Figure 3A, B and C). Drug therapy
augmented aortic responses to acetylcholine in UNX animals (Figure 3B).

DISCUSSION
Rats represent an excellent model for studying the pleiotropic effects of statins, as their
lipid profile has been demonstrated to be refractory to statin therapy [25] and, in
comparison to humans, their plasma lipid levels are relatively low. In the current study,
we investigated the effects of a new HMG-CoA reductase inhibitor, rosuvastatin, on
cardiovascular remodelling in the DOCA-salt model of hypertension in rats. We show for
the first time that rosuvastatin attenuated left ventricular fibrosis, hypertrophy,
inflammation and action potential prolongation and aortic medial hypertrophy, without
antihypertensive action in this rodent model of end-organ damage.

Statins have been reported to reduce blood pressure in a randomized, double-blind
crossover trial in humans (pravastatin) [26] and hypertensive rodent models (simvastatin

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and lovastatin) [27, 28], including DOCA-salt hypertensive mice (lovastatin) [29]. We,
on the other hand, found that rosuvastatin was without effect on DOCA-salt-induced
hypertension in rats. This difference may be related to model and species differences or
the relative hydrophilicity of rosuvastatin limiting its uptake through their plasma
membrane. However, independent of any anti-hypertensive effect, rosuvastatin attenuated
the increase in left ventricular mass and medial hypertrophy of the thoracic aorta
associated with DOCA-salt treatment. In the genetically hypertensive (GH) rat,
fluvastatin had no effect on blood pressure, but significantly remodelled the mesenteric
resistance and basilar arteries by reducing medial cross-sectional area and increasing
lumen size [30]. Furthermore, long-term administration of simvastatin to rats with aortic
stenosis inhibited left ventricular hypertrophy without effect on the elevated carotid mean
arterial pressure [31]. Taken together, these data support the notion that a reduction of
blood pressure is not the primary factor involved in the inhibitory effect of statins on
cardiovascular hypertrophy.

In the current study, rosuvastatin therapy reduced left ventricular monocyte infiltration.
Such infiltration has been demonstrated to participate in the initiation and progression of
cardiovascular pathology in the DOCA-salt model [24, 32]. HMG-CoA reductase
inhibitors modulate several different components of the inflammatory cascade,
particularly those involving leucocyte-endothelium interactions, such as the down
regulation of cell adhesion molecule expression [9]. Indeed, rosuvastatin inhibited
endothelial cell surface expression of the adhesion molecule P-selectin, and thus

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attenuated leukocyte rolling, adherence and transmigration in normocholesterolaemic rat
mesenteric venules [33].

Inflammation has also been shown to play a role in long-term pathological cardiovascular
changes, such as matrix accumulation [34]. Hence the anti-fibrotic properties of
rosuvastatin in DOCA-salt rats, manifest as reduced LV collagen and hydroxyproline
contents and diastolic stiffness (an indirect measure of total collagen), may in part be due
to a direct inhibitory effect of the drug upon monocyte/macrophage infiltration. This is
supported by Ammarguellat and co-workers [24], who have identified inflammatory
mediators as a major component of cardiac remodelling and critical to the progression of
cardiac fibrosis in this model of mineralocorticoid-induced hypertension. Furthermore,
Kagitani et al. [32] showed that tranilast, an anti-inflammatory drug, suppressed
myocardial fibrosis via inhibition of cytokines, such as monocyte chemotactic protein-1
and interleukin-6, and monocyte/macrophage infiltration in DOCA-salt rats. Taken
together, these results suggest that pharmacological attenuation of the inflammatory
response by rosuvastatin may, in part, be responsible for preventing myocardial fibrosis
in DOCA-salt rats. This effect may also potentially translate to fibrosis-associated
abnormalities, such as diastolic stiffness. Our previous studies with L-arginine, the NO
precursor, and A-127722, a selective ETA-receptor antagonist, have shown attenuation of
inflammatory cell infiltration, myocardial collagen deposition and diastolic stiffness [35,
36].


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