Contrasting effects of aliskiren versus losartan on hypertensive vascular remodeling

(1)

Contrasting effects of aliskiren

versus

losartan on hypertensive vascular remodeling

Alisson Martins-Oliveira

a

, Michele M. Castro

a

, Diogo M.M. Oliveira

a

, Elen Rizzi

a

, Carla S. Ceron

a

,

Danielle Guimaraes

a

, Rosana I. Reis

b

, Claudio M. Costa-Neto

b

, Dulce E. Casarini

c

, Amanda A. Ribeiro

c

,

Raquel F. Gerlach

d

, Jose E. Tanus-Santos

a,

⁎

a_{Department of Pharmacology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Brazil}

b_{Department of Biochemistry and Immunology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Brazil} c_{Department of Medicine, Nephrology Division, Federal University of Sao Paulo, Brazil}

d_{Department of Morphology, Estomatology and Physiology, Dental School of Ribeirao Preto, University of Sao Paulo, Brazil}

a b s t r a c t

a r t i c l e

i n f o

Article history:

Received 9 February 2012 Accepted 14 March 2012 Available online 6 April 2012

Keywords: Aliskiren

Angiotensin type II receptor antagonist Hypertension

Losartan

Matrix metalloproteinases Vascular remodeling.

Background:Hyperactivation of the renin–angiotensin system contributes to hypertension-induced upregu-lation of vascular matrix metalloproteinases (MMPs) and remodeling, especially in the two kidney, one clip (2K1C) hypertension model. We hypothesized that the AT1R antagonist losartan or the renin inhibitor aliskiren, given at doses allowing similar antihypertensive effects, could preventin vivovascular MMPs upre-gulation and remodeling, and collagen/elastin deposition found in 2K1C hypertension by preventing the ac-tivation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and transforming growth factor-β1(TGF-β1). We also hypothesized that aliskiren could enhance the effects of losartan.

Methods:2K1C rats were treated with aliskiren (50 mg.kg−1_.day−1_{), or losartan (10 mg.kg}−1_.day−1_{), or} both by gavage during 4 weeks.

Results:Aliskiren, losartan, or both drugs exerted similar antihypertensive effects when compared with 2K-1C rats treated with water. Aliskiren reduced plasma renin activity in both sham and 2K-2K-1C rats. Losartan alone or combined with aliskiren, but not aliskiren alone, abolished 2K1C-induced aortic hypertrophy and hyperplasia, and prevented the increases in aortic collagen/elastin content, MMP-2 levels, gelatinolytic activity, and expression of phospho-ERK 1/2 and TGF-β1. No signiﬁcant differences were found in the aortic expression of the (pro)renin receptor.

Conclusions:Thesefindings show that although losartan and aliskiren exerted similar antihypertensive effects, only losartan prevented the activation of vascular profibrotic mechanisms and MMP upregulation associated with vascular remodeling in 2K1C hypertension. Ourfindings also suggest that aliskiren does not enhance the protective effects exerted by losartan.

1. Introduction

Hyperactivation of the renin

–

angiotensin system (RAS) contributes

to hypertension and promotes cardiovascular remodeling

[1]

. Mounting

evidence indicates that a proteolytic imbalance involving upregulated

matrix metalloproteinases (MMPs), especially MMP-2

[2]

, plays a

major role in these alterations, especially in the two kidney, one clip

(2K1C) hypertension model

[3,4]

. Indeed, angiotensin II increases

MMP-2 expression and activity

[5,6]

via activation of angiotensin II

type 1 receptors (AT

1

R)

[1,7,8]

and therefore promotes cardiovascular

remodeling

[2]

. Moreover, increased renin and prorenin formation

acti-vates the (pro)renin receptor (PRR)

[9]

, which is widely expressed in

renal mesangial and in vascular smooth muscle cells (VSMCs)

[10,11]

.

Importantly, PRR activation exerts dual molecular functions by

stimu-lating signaling pathways that are both dependent and independent

of angiotensin II (ANG II) generation

[12]

. The last one elicits

intracellu-lar signaling that upregulates pro

ﬁ

brotic pathways including

extracel-lular regulated kinase 1/2 (ERK 1/2) and transforming growth

factor-β

1

(TGF-

β

1

)

[13,14]

, which contribute to hypertensive cardiovascular

remodeling.

While antihypertensive drugs interfering with the RAS exert bene

ﬁ

-cial effects against the cardiovascular remodeling associated with

hy-pertension

[1]

, it is not known whether AT

1

R antagonists affect

pro

ﬁ

brotic pathways and vascular MMP upregulation in hypertensive

animals or humans. Moreover, although aliskiren (a direct renin

inhib-itor) decreases angiotensin II levels and improves cardiac remodeling

[15]

, its effects on hypertension-induced vascular MMP upregulation

and remodeling are not known. While compelling results suggest that

aliskiren enhances the protective effects of AT

1

R antagonists

[16,17]

,

the effects of this drug combination on the increases in vascular

MMPs and remodeling associated with hypertension are not known.

–

⁎Corresponding author at: Department of Pharmacology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Av. Bandeirantes, 3900, 14049-900 Ribeirao Preto, SP, Brazil. Tel.: +55 16 3602 3163; fax: +55 16 3602 0220.

E-mail addresses:tanus@fmrp.usp.br,tanussantos@yahoo.com(J.E. Tanus-Santos).

Contents lists available at

SciVerse ScienceDirect

International Journal of Cardiology

(2)

In this study, we hypothesized that the AT

1

R antagonist losartan

or the renin inhibitor aliskiren prevents

in vivo

vascular MMPs

upre-gulation and remodeling, and elastin/collagen deposition found in

2K1C hypertension

[4,18]

by preventing the activation of pro

ﬁ

brotic

pathways involving ERK 1/2 and TGF-

β

1

. Because increased

trans-mural pressure is sensed by integrins

[19]

, which transform signals

into adaptive vascular remodeling, thus affecting vascular MMP

activ-ities

[20]

, we compared the effects of losartan and aliskiren given at

doses that allowed very similar antihypertensive effects in pilot

stud-ies. Moreover, we hypothesized that aliskiren could enhance the

ef-fects of losartan, as previously suggested

[16]

.

2. Methods

2.1. Animals and treatments

This study was approved by the Institutional Animal Care and Use Committee of the Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, and the animals were handled according to the guiding principles published by the National Institutes of Health. Male Wistar rats (180–200 g) were maintained on 12-h light/dark cycle at 25 °C with free access to rat chow and water.

2K1C hypertension was induced by clipping the left renal artery with a silver clip (0.2 mm). Sham-operated rats underwent the same surgical procedure (under general anesthesia with ketamine 100 mg/kg and xylazine 10 mg/kg i.p.), except for the place-ment of the renal artery clip. The systolic blood pressure (SBP) was measured by tail-cuff plethysmography weekly, and the rats were considered hypertensive when SBP >160 mm Hg two weeks after the surgery.

Animals were randomly assigned to one of eight groups: 2K1C and Sham groups that received tap water; 2K1C and Sham groups that received losartan (Cozaar®; Merck Sharp & Dohme) at 10 mg/kg per day[8]; 2K1C and Sham groups that received aliskiren at 50 mg/kg[21](Rasilez®; Novartis) per day; and 2K1C and Sham groups that received the combination of aliskiren 50 mg/kg + losartan 10 mg/kg per day. The treatments were started two weeks after 2K1C hypertension was induced and main-tained for additional four weeks. All treatments were given daily by oral gavage and distilled water was used as vehicle.

2.2. Plasma renin activity assay

Blood samples were collected in tubes containing 5.0 mmol/L of EDTA. Blood sam-ples were centrifuged at 2000 ×gfor 30 min at 4 °C. Plasma renin activity was deter-mined by assessing the generation of angiotensin I by reversed phase HPLC after incubation of plasma with a synthetic tetradecapeptide substrate at 37 °C, as previous-ly described[22].

2.3. Morphometric analysis of the vascular wall

At the end of the experiments, animals were anesthetized and killed by decapita-tion. The thoracic aorta was harvested, cleaned of connective tissue, and immediately fixed in 4% phosphate-buffered paraformaldehyde, pH 7.4, and embedded in paraffin blocks. Four micrometer thick slices were stained with hematoxylin and eosin. Media cross-sectional area (CSA), media to lumen diameter (M/L) and the number of VSMC were quantified as previously described[23]. Sirus red and Orceine staining were used to assess aortic collagen and elastin contents using ImageJ Program (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA,http:// imagej.nih.gov/ij/, 1997–2011)[4].

2.4. Measurement of aortic MMP-2 levels

Aortic tissues were homogenized in buffer containing 20 mmol/L Tris–HCl, pH 7.4, 1 mmol/L 1,10-phenanthroline, 1 mmol/L phenylmethylsulfonyl ﬂuoride (PMSF), 1 mmol/L N-ethylmaleimide (NEM), and 10 mmol/L CaCl2. Tissue extracts were normalized for protein concentration and gelatin zymography was performed as previously described[24]. The forms of MMP-2 were identiﬁed as bands at 75, 72 and 64 kDa[24].

2.5. In-situ zymography and immunoﬂuorescence for MMP-2

MMPs activity was measured in the media and intima of frozen thoracic aorta using DQ Gelatin as thefluorogenic substrate (E12055, Molecular Probes, Oregon, USA). Proteolytic activity was detected as bright greenfluorescence, indicating sub-strate breakdown and MMP enzymatic activity. Aortic MMPs activity was also co-localized with aortic MMP-2 levels by immunofluorescence. After DQ gelatin, tissue sections were incubated with MMP-2 primary mouse human monoclonal anti-body (1:1000; Chemicon) for 1 h in dark humidified chambers. Redfluorescence was visualized by adding the rhodamine conjugated as secondary antibody (1:200; Chemi-con) for 1 h. Sections were examined withfluorescent microscopy (Leica Imaging Sys-tems Ltd., Cambridge, England) and the image was captured at X400. MMP activity and MMP-2 levels were evaluated by using ImageJ Program.

2.6. Western blotting

Aortas from 2K1C and sham rats were homogenized on ice-cold RIPA buffer. Forty mi-crograms of protein extracts were subject to SDS-PAGE using a 12% polyacrylamide gel. The proteins were then transferred onto nitrocellulose membranes and blocked with TBST (NaCl 100 mmol/L; Tris–Cl 100 mmol/L; Tween 0.1%) containing 5% bovine serum albumin. The membranes were incubated overnight at 4 °C with anti-MMP-2 (1:1000, Chemicon), anti-TGF-β1 (1:1000, Chemicon), anti-phosphorylated-ERK 1/2 (anti-p-ERK 1/2; 1:2000, Santa Cruz Biotechnology) or anti-prorenin receptor (anti-PRR; 1:250, Abcam) antibodies. Anti-β-actin (1:10,000, Millipore) was used as a loading control. The antibodies were washed in TBST and incubated with horseradish peroxidase (HRP)-secondary goat mouse antibody (1:2000, Millipore) or HRP-(HRP)-secondary goat anti-rabbit antibody (1:1000, Millipore) for 1 h. Immunolabeled proteins were visualized using chemiluminescence ECL (Millipore) and registered by ImageQuant 350 detection system (GE Healthcare). The signal intensities were quantiﬁed using ImageJ Program (NIH— National Institute of Health).

2.7. Statistical analysis

Results are expressed as means ± S.E.M. Comparisons between groups were assessed by two-way or one way ANOVA followed by the Bonferroni test. A probability valueb0.05 was considered signiﬁcant.

3. Results

3.1. Effects of aliskiren and losartan on SBP levels and plasma renin

activity

We found similar baseline SBP in all experimental groups and no

effects on SBP in the Sham groups (

Fig. 1

). While SBP increased

pro-gressively in 2K1C rats treated with water (208 ± 2 mm Hg),

treat-ment of 2K1C rats with aliskiren, losartan, or both drugs exerted

similar antihypertensive effects, especially after 4 weeks of treatment

(SBP = 157 ± 2 mm Hg, 158 ± 2 mm Hg, 146 ± 2 mm Hg,

respective-ly;

Fig. 1

, P

b

0.05). Similar body weight gain was found in the eight

0 1 2

90 120 150 180 210 240 270

3 4 5 6

Sham

Sham LOS Sham ALK

Sham ALK/LOS 2K1C

2K1C LOS 2K1C ALK

2K1C ALK/LOS

*

* *

*

aliskiren and/or losartan

or vehicle _# _# #

Time (Weeks)

Systolic Blood Pressure

(mmHg)

A

B

Vehicle ALK LOS ALK/LOSVehicle

ALK LOS ALK/LOS 0.0

0.1 0.2 0.3 0.4 0.5

Sham 2K-1C

**

Plasma renin activity (ng Ang I.ml

-1.

h

-1)

(3)

experimental groups (P > 0.05; data not shown). The plasma renin

ac-tivity was signi

ﬁ

cantly reduced in sham rats treated with aliskiren

when compared with Sham + vehicle group (

Fig. 1

B, P

b

0.05).

Treat-ment with aliskiren signi

ﬁ

cantly attenuated plasma renin activity in

2K-1C rats when compared with 2K1C rats treated with vehicle

(

Fig. 1

B, P

b

0.05).

3.2. Aliskiren treatment did not prevent the vascular remodeling

2K1C hypertension induced vascular remodeling as co1mpared to

sham-operated rats, and the antihypertensive drugs had no effects in

the sham groups (

Fig. 2

A

–

D). Whereas losartan alone or combined

with aliskiren abolished the increases in VSMCs number, CSA, and

Sham

2K1C

ALK LOS

Vehicle

ALK LOS ALK/LOS

ALK/LOS

Vehicle

B

A

200µm

Vehicle ALK LOS_ALK/LOSVehicle ALK LOS_ALK/LOS 0.0

200000.0 400000.0 600000.0 800000.0

*

Sham 2K1C

Elastin surface (

µ

m

2)

Collagen

ALK LOS

Vehicle ALK/LOS

C

Vehicle ALK LOS_ALK/LOSVehicle ALK LOS_ALK/LOS

0.0 500000.0 1000000.0 1500000.0

Sham 2K1C

*

Collagen surface (

µ

m

2)

Vehicle

ALK LOS

Vehicle ALK/LOS

Elastin

D

Fig. 3.Effects of aliskiren and/or losartan on collagen and elastin content in the aortic media. Panels A and C show representative photomicrographs of aortic samples (400×) stained by Sirus red and Orceine, respectively. Panel B and Panel D show the values for surface area of collagen and elastin in the aortic media. Data are shown as mean ± S.E.M. (n = 4–6 per group). *Pb0.05versusthe Sham groups andversusthe 2K1C + LOS and the 2K1C + ALK/LOS groups.

A

ALK LOS ALK/LOS Vehicle

2K1C

Sham

LOS

ALK ALK/LOS

Vehicle

C

B

VehicleALK LOS_ALK/LOSVehicle ALK LOS_ALK/LOS

0 50000 100000 150000 200000 250000

Sham 2K1C

*

CSA (

µ

m

2)

Vehicle ALK LOS_ALK/LOSVehicle ALK LOS_ALK/LOS

0 5 10 15 20

Sham 2K1C

*

M/L (%)

D

0 20 40 60

Sham 2K1C

*

Cells/

µ

m aorta

200µm

Fig. 2.Effects of aliskiren and/or losartan on the structural modiﬁcations induced by 2K1C hypertension in the aortas from rats. Panel A shows representative photomicrographs of aortic samples (400×) stained by hematoxylin and eosin (H&E). Panel B shows the values for vascular smooth muscle cell number per length of aorta. Panel C shows the values for thoracic aorta medial cross-sectional area (CSA). Panel D shows the values for media to lumen ratio (M/L). Data are shown as mean ± S.E.M. (n = 5–6 per group). *Pb0.05versusthe Sham groups andversusthe 2K1C + LOS and the 2K1C + ALK/LOS groups.

(4)

M/L ratio found in hypertensive rats (P

b

0.05;

Fig. 2

A

–

D), aliskiren

alone exerted no signi

ﬁ

cant effects, even though all treatments had

similar antihypertensive effects. In parallel with these

ﬁ

ndings, we

found increased total collagen

ﬁ

ber and elastin content per

cross-sectional area in 2K1C rats compared with the sham groups

(P

b

0.05;

Fig. 3

A

–

D). Treatment with losartan or aliskiren + losartan,

but not with aliskiren alone, attenuated the increases in these

param-eters (P

b

0.05;

Fig. 3

).

3.3. Assessment of aortic MMP-2 levels by zymography

Gelatin zymography was used to assess MMP-2 levels in aortic

ex-tracts because this protease has been implicated in hypertensive

vas-cular remodeling

[4,18]

. 2K1C hypertension increased the aortic

levels of the 75 kDa, 72 kDa, and 64 kDa MMP-2 forms (P

b

0.05;

Fig. 4

). While all treatments reduced 75-kDa MMP-2 levels, only

losartan or aliskiren + losartan treatments abolished

hypertension-induced increases in the 64-kDa MMP-2 form (P

b

0.05;

Fig. 4

).

3.4. Assessment of aortic gelatinolytic activity and MMP-2 levels

Hypertension signi

ﬁ

cantly increased aortic gelatinolytic activity, as

revealed by increased green

ﬂ

uorescence in

Fig. 5

A. While treatment

with losartan or with aliskiren+losartan abolished the increases in

aor-tic gelatinolyaor-tic activity found in 2K1C rats (P

b

0.05;

Fig. 5

A

–

B),

treat-ment with aliskiren alone exerted no such effect (P>0.05;

Fig. 5

A

–

B).

Interestingly, aortic MMP-2 levels assessed by immuno

ﬂ

uorescence

(red

ﬂ

uorescence in

Fig. 5

A) paralleled and co-localized with aortic

gelatinolytic activity (

Fig. 5

A

–

C).

3.5. Aortic expression of MMP-2, PRR, p-ERK 1/2 and TGF-

β

1

protein

levels

Hypertension signi

ﬁ

cantly increased the vascular expression of

MMP-2, p-ERK 1/2, and TGF-

β

1

, as shown in

Fig. 6

(all P

b

0.05).

While treatment of 2K1C rats with losartan or with aliskiren +

losar-tan abolished the increases in aortic expression of MMP-2, ERK 1/2,

and TGF-

β

1

(all P

b

0.05;

Fig. 6

A, C, and D, respectively), treatment

with aliskiren alone exerted no such effect (P > 0.05;

Fig. 6

A, C, and

D).

We found no signi

ﬁ

cant changes in PRR expression in

hyperten-sive rats compared with the Sham group, and no signi

ﬁ

cant effects

for drug treatments (P > 0.05;

Fig. 6

B).

4. Discussion

This is the

ﬁ

rst study to show that the antihypertensive effects of

losartan are associated with prevention against activation of vascular

pro

ﬁ

brotic mechanisms and MMP-2 upregulation found in 2K1C

hy-pertension. Another important

ﬁ

nding is that aliskiren, given at a

dose exerting similar antihypertensive effects to those found with

losartan, was not effective in preventing pro

ﬁ

brotic mechanisms

and vascular remodeling. Moreover, aliskiren did not improve the

protective effects exerted by losartan.

Clinical studies suggested that both AT

1

R antagonists or aliskiren

exert comparable antihypertensive effects

[17,25,26]

. While losartan

and aliskiren exerted similar effects on blood pressure in the present

study, the combination of both drugs did not lower blood pressure

signi

ﬁ

cantly more than either monotherapy, and this

ﬁ

nding

appar-ently contrasts with clinical

ﬁ

ndings

[17]

. However, it is reasonable

to believe that the differences between treatments with respect to

vascular remodeling, MMP-2, or other pro

ﬁ

brotic factors are not

due to differences in antihypertensive effects and therefore

differ-ences in transmural pressure, which affects vascular MMPs

[20]

.

As expected from previous studies

[23,24,27]

, 2K1C hypertension

in-duced vascular hypertrophy, increased vascular collagen and elastin

contents, and enhanced MMP-2 levels, probably as a result of increased

angiotensin II levels

[6,28]

. These

ﬁ

ndings were associated with

in-creased expression of pro

ﬁ

brotic factors including p-ERK 1/2 and

TGF-β

1,

which clearly contribute to VSMC growth and rearrangement of

ex-tracellular matrix. The increased vascular MMP-2 levels in 2K1C rats

may activate TGF-

β

1

and enhance TGF-

β

1

intracellular signaling, thus

leading to

ﬁ

brosis with increased deposition of collagen and elastin

[29,30]

. The antigrowth effects exerted by losartan in the present

study are supported by previous results indicating similar effects on

small resistance arteries from rats treated with angiontensin II

[8]

.

Con-sistent with the notion that the AT

1

R enhances collagen gene

expres-sion via ERK 1/2 signaling

[31]

, we found increased levels of p-ERK 1/

2 and collagen deposition in the aorta from 2K1C rats, and these

changes were prevented with losartan, thus strongly suggesting a

major role for the AT

1

R in the vascular alterations of 2K1C hypertension.

75 kDa 72 kDa

64 kDa

Std

Vehicle ALK LOS ALK/LOS Vehicle ALK LOS ALK/LOS

Sham

2K1C

0.0 0.2 0.4 0.6 0.8

Sham 2K1C

*

75 kDa MMP-2 _{(Arbitrary units)}

B

A

VehicleALK LOS_ALK/LOSVehicleALK LOS_ALK/LOS 0.0

0.5 1.0 1.5 2.0

Sham 2K1C

**

0.1 0.2 0.3 0.4

Sham 2K1C

#

C

D

(5)

In contrast with losartan, aliskiren monotherapy was not effective

in preventing the structural and biochemical alterations discussed

above, even though similar antihypertensive effects were found

with both drugs. In fact, the lack of signi

ﬁ

cant effects for aliskiren

on p-ERK 1/2 and on TGF-

β

1

levels aligns with the lack of signi

ﬁ

cant

effects on MMP-2 levels and on the other biochemical and

morpho-logical parameters studied here. Moreover, it is intriguing that

aliski-ren did not improve the protective effects exerted by losartan, thus

contrasting with previous experimental studies

[16]

. These

ﬁ

ndings

suggest that renin inhibition may not have signi

ﬁ

cant effects on

fun-damental mechanisms promoting hypertensive vascular remodeling,

even though antihypertensive effects are found with aliskiren.

In line with this suggestion above, previous studies have shown

that the concentrations of aliskiren necessary to block rat renin are

approximately 100 times higher than those required to block

human renin

[32]

. However, in the present study, aliskiren clearly

suppressed plasma renin activity in 2K-1C, thus con

ﬁ

rming previous

ﬁ

ndings from a study using aliskiren at the same dose used here

[21]

. In addition, the decreased plasma renin activity that we found

in 2K-1C, untreated rats is in agreement with previous studies

show-ing that plasma renin activity is reduced after four weeks, durshow-ing the

more chronic phase of 2 K-1 C hypertension

[33,34]

.

The lack of signi

ﬁ

cant antigrowth effects for aliskiren may be

at-tributed to a compensatory elevation in plasma renin concentration

in 2K1C hypertension

[35]

and augmented PRR activation. While

overexposure of the PRR to renin/prorenin could downregulate PRR

[13,36,37]

, we found that 2K1C hypertension or the antihypertensive

treatments exerted no effects on the expression of PRR in the aorta.

Therefore, it is possible that treatment with aliskiren enhanced PRR

signaling and activated mitogen-activated protein kinases

(MAPK)-dependent pathways. This results in VSMCs proliferation

[14]

and

overexpression of matrix proteins and pro

ﬁ

brotic proteins including

ERK 1/2 and TGF-

β

1

[38

–

40]

, especially in a context of unblocked

AT

1

R, which contributes to MAPK activation

[41]

. In contrast, while

the treatment with losartan may also increase renin concentrations,

this AT

1

R blocker directly prevents the activation of AT

1

R and MAPK

signaling pathways

[41]

, probably exerting more effective antigrowth

effects as consistently suggested by our results.

In conclusion, our

ﬁ

ndings support the idea that the

antihyperten-sive effects exerted by losartan and aliskiren are associated with

con-trasting antigrowth effects. Only losartan prevented

in vivo

vascular

MMP upregulation and remodeling, and the pro

ﬁ

brotic alterations

found in 2K1C hypertension. Thus, our results suggest that blockade

of AT

1

R promotes better protection against the vascular remodeling

of 2K1C hypertension than renin inhibition, and that aliskiren does

not improve the effects exerted by losartan.

Acknowledgments

This study was funded by: Fundação de Aparo a Pesquisa do

Estado de São Paulo (FAPESP), Conselho Nacional de

Desenvolvi-mento Cientí

ﬁ

co e Tecnológico (CNPq) and Coordenadoria de

Fig. 5.Effects of aliskiren, losartan, or aliskiren + losartan on aorticin situgelatinolytic activity and MMP-2 levels in the aortas from Sham and 2K1C rats. Panel A shows represen-tative photomicrographs ofin situgelatinolytic activity (× 400), MMP-2 detected by immunofluorescence (M = media; L = lumen), and their co-localization (merge) in vessel sur-face. Panel B shows the quantification of percentage of vessel surface area covered by bright greenfluorescence, which reflects gelatinolytic activity. Panel C shows the quantification of percentage of vessel surface area covered by bright redfluorescence, which reflects MMP-2 levels. Data are shown as mean ± S.E.M. (n = 5–6 per group). *Pb0.05versusthe Sham groups andversusthe 2K1C + LOS and the 2K1C + ALK/LOS groups.

(6)

Aperfeiçoamento de Pessoal de Nível Superior (CAPES). This work

was supported by FAPESP, CAPES, and CNPq. The authors of this

man-uscript have certi

ﬁ

ed that they comply with the principles of Ethical

Publishing in the International Journal of Cardiology.

References

[1] Bader M. Tissue renin–angiotensin–aldosterone systems: targets for pharmaco-logical therapy. Annu Rev Pharmacol Toxicol 2010;50:439–65.

[2] Martinez-Lemus LA, Zhao G, Galinanes EL, Boone M. Inward remodeling of resis-tance arteries requires reactive oxygen species-dependent activation of matrix metalloproteinases. Am J Physiol Heart Circ Physiol 2011;300:H2005–15. [3] Guimaraes DA, Rizzi E, Ceron CS, et al. Doxycycline dose-dependently inhibits

MMP-2-mediated vascular changes in 2K1C hypertension. Basic Clin Pharmacol Toxicol 2011;108:318–25.

[4] Castro MM, Rizzi E, Figueiredo-Lopes L, et al. Metalloproteinase inhibition amelio-rates hypertension and prevents vascular dysfunction and remodeling in renovas-cular hypertensive rats. Atherosclerosis 2008;198:320–31.

[5] Jimenez E, Perez de la Blanca E, Urso L, Gonzalez I, Salas J, Montiel M. Angiotensin II induces MMP 2 activity via FAK/JNK pathway in human endothelial cells. Bio-chem Biophys Res Commun 2009;380:769–74.

[6] Luchtefeld M, Grote K, Grothusen C, et al. Angiotensin II induces MMP-2 in a p47phox-dependent manner. Biochem Biophys Res Commun 2005;328:183–8. [7] Walter A, Etienne-Selloum N, Sarr M, Kane MO, Beretz A, Schini-Kerth VB.

Angio-tensin II induces the vascular expression of VEGF and MMP-2in vivo: preventive effect of red wine polyphenols. J Vasc Res 2008;45:386–94.

[8] Brassard P, Amiri F, Schiffrin EL. Combined angiotensin II type 1 and type 2 recep-tor blockade on vascular remodeling and matrix metalloproteinases in resistance arteries. Hypertension 2005;46:598–606.

[9] Nguyen G, Delarue F, Burckle C, Bouzhir L, Giller T, Sraer JD. Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin. J Clin Invest 2002;109:1417–27.

[10] Sakoda M, Ichihara A, Kurauchi-Mito A, et al. Aliskiren inhibits intracellular angio-tensin II levels without affecting (pro)renin receptor signals in human podocytes. Am J Hypertens 2010;23:575–80.

[11] Sakoda M, Ichihara A, Kaneshiro Y, et al. (Pro)renin receptor-mediated activation of mitogen-activated protein kinases in human vascular smooth muscle cells. Hypertens Res 2007;30:1139–46.

[12] Nguyen G. Renin/prorenin receptors. Kidney Int 2006;69:1503–6.

[13] Feldman DL, Jin L, Xuan H, et al. Effects of aliskiren on blood pressure, albumin-uria, and (pro)renin receptor expression in diabetic TG(mRen-2)27 rats. Hyper-tension 2008;52:130–6.

[14] Liu G, Hitomi H, Hosomi N, et al. Prorenin induces vascular smooth muscle cell pro-liferation and hypertrophy via epidermal growth factor receptor-mediated extracellu-lar signal-regulated kinase and Akt activation pathway. J Hypertens 2011;29:696–705. [15] Westermann D, Riad A, Lettau O, et al. Renin inhibition improves cardiac function and remodeling after myocardial infarction independent of blood pressure. Hy-pertension 2008;52:1068–75.

[16] Yamamoto E, Kataoka K, Dong YF, et al. Aliskiren enhances the protective effects of valsartan against cardiovascular and renal injury in endothelial nitric oxide synthase-deﬁcient mice. Hypertension 2009;54:633–8.

[17] Oparil S, Yarows SA, Patel S, Fang H, Zhang J, Satlin A. Efﬁcacy and safety of com-bined use of aliskiren and valsartan in patients with hypertension: a randomised, double-blind trial. Lancet 2007;370:221–9.

VehicleALK LOS ALK/LOSVehicle

ALK LOS ALK/LOS 0.0

0.2 0.4 0.6 0.8 1.0

Sham 2K1C

*

PhosphoERK 1/2/

β

-actin

(Arbitrary units)

44 kDa

42 kDa

p-ERK1/2

-actin

43 kDa

B

VehicleALK LOS_ALK/LOSVehicleALK LOS_ALK/LOS

0.0 0.5 1.0 1.5

Sham 2K1C

*

MMP-2/

β

-actin

MMP-2

-actin

A

72 kDa

43 kDa

0.5 1.0 1.5 2.0 2.5

Sham 2K1C

*

TGF-β

1/

β

-actin

VehicleALK LOSALK/LOSVehicle ALK LOSALK/LOS

0.0 0.2 0.4 0.6 0.8

Sham 2K1C

PRR/

β

-actin

TGF- 1

-actin

PRR

-actin

25 kDa

43 kDa

39 kDa

C

D

(7)

[18] Castro MM, Rizzi E, Prado CM, Rossi MA, Tanus-Santos JE, Gerlach RF. Imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases in hypertensive vascular remodeling. Matrix Biol 2010;29:194–201.

[19] Martinez-Lemus LA, Wu X, Wilson E, et al. Integrins as unique receptors for vas-cular control. J Vasc Res 2003;40:211–33.

[20] Chesler NC, Ku DN, Galis ZS. Transmural pressure induces matrix-degrading activ-ity in porcine arteriesex vivo. Am J Physiol 1999;277:H2002–9.

[21] Nussberger J, Aubert JF, Bouzourene K, Pellegrin M, Hayoz D, Mazzolai L. Renin in-hibition by aliskiren prevents atherosclerosis progression: comparison with irbe-sartan, atenolol, and amlodipine. Hypertension 2008;51:1306–11.

[22] Prieto MC, Gonzalez-Villalobos RA, Botros FT, et al. Reciprocal changes in renal ACE/ANG II and ACE2/ANG 1–7 are associated with enhanced collecting duct renin in Goldblatt hypertensive rats. Am J Physiol Renal Physiol 2011;300:F749–55. [23] Castro MM, Rizzi E, Rodrigues GJ, et al. Antioxidant treatment reduces matrix

metalloproteinase-2-induced vascular changes in renovascular hypertension. Free Radic Biol Med 2009;46:1298–307.

[24] Ceron C, Castro M, Rizzi E, et al. Spironolactone and hydrochlorothiazide exert anti-oxidant effects and reduce vascular matrix metalloproteinase-2 activity and expres-sion in a model of renovascular hypertenexpres-sion. Br J Pharmacol 2010;160:77–87. [25] Pool JL, Schmieder RE, Azizi M, et al. Aliskiren, an orally effective renin inhibitor,

provides antihypertensive efﬁcacy alone and in combination with valsartan. Am J Hypertens 2007;20:11–20.

[26] Stanton A, Jensen C, Nussberger J, O'Brien E. Blood pressure lowering in essential hy-pertension with an oral renin inhibitor, aliskiren. Hyhy-pertension 2003;42:1137–43. [27] Cau SBA, Guimaraes DA, Rizzi E, et al. Pyrrolidine dithiocarbamate downregulates

vascular matrix metalloproteinases and ameliorates vascular dysfunction and remodeling in renovascular hypertension. Br J Pharmacol 2011;164:372–81. [28] Wang M, Zhang J, Spinetti G, et al. Angiotensin II activates matrix

metalloprotei-nase type II and mimics age-associated carotid arterial remodeling in young rats. Am J Pathol 2005;167:1429–42.

[29] Wang M, Zhao D, Spinetti G, et al. Matrix metalloproteinase 2 activation of trans-forming growth factor-beta1 (TGF-beta1) and TGF-beta1-type II receptor signal-ing within the aged arterial wall. Arterioscler Thromb Vasc Biol 2006;26:1503–9.

[30] Sauvage M, Hinglais N, Mandet C, et al. Localization of elastin mRNA and TGF-beta1 in rat aorta and caudal artery as a function of age. Cell Tissue Res 1998;291:305–14.

[31] Tharaux PL, Chatziantoniou C, Fakhouri F, Dussaule JC. Angiotensin II activates col-lagen I gene through a mechanism involving the MAP/ER kinase pathway. Hyper-tension 2000;36:330–6.

[32] Wood JM, Maibaum J, Rahuel J, et al. Structure-based design of aliskiren, a novel orally effective renin inhibitor. Biochem Biophys Res Commun 2003;308:698–705. [33] Okamura T, Miyazaki M, Inagami T, Toda N. Vascular renin–angiotensin system in

two-kidney, one clip hypertensive rats. Hypertension 1986;8:560–5.

[34] Nakada T, Iijima Y, Kubota Y, Watanabe M, Ishigooka M, Suzuki H. Increased vas-cular collagen and noncollagenous protein synthesis contributes to sustain chronic phase of two-kidney, one-clip renovascular hypertension. J Urol 1996;156:1180–5. [35] Krebs C, Hamming I, Sadaghiani S, et al. Antihypertensive therapy upregulates renin and (pro)renin receptor in the clipped kidney of Goldblatt hypertensive rats. Kidney Int 2007;72:725–30.

[36] Schefe JH, Menk M, Reinemund J, et al. A novel signal transduction cascade involv-ing direct physical interaction of the renin/prorenin receptor with the transcrip-tion factor promyelocytic zincﬁnger protein. Circ Res 2006;99:1355–66. [37] Schefe JH, Neumann C, Goebel M, et al. Prorenin engages the (pro)renin receptor

like renin and both ligand activities are unopposed by aliskiren. J Hypertens 2008;26:1787–94.

[38] Danser AH. The increase in renin during renin inhibition: does it result in harmful effects by the (pro)renin receptor? Hypertens Res 2010;33:4–10.

[39] Nguyen G, Danser AH. Prorenin and (pro)renin receptor: a review of available data from in vitro studies and experimental models in rodents. Exp Physiol 2008;93:557–63. [40] Huang Y, Wongamorntham S, Kasting J, et al. Renin increases mesangial cell trans-forming growth factor-beta1 and matrix proteins through receptor-mediated, an-giotensin II-independent mechanisms. Kidney Int 2006;69:105–13.