Clinical Science (2014) 127, 123–134 (Printed in Great Britain) doi: 10.1042/CS20130382

Aliskiren limits abdominal aortic aneurysm, ventricular hypertrophy and atherosclerosis in an apolipoprotein-E-deficient mouse model

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Sai-Wang SETO∗ , Smriti M. KRISHNA∗ , Corey S. MORAN∗ , David LIU∗ and Jonathan GOLLEDGE∗ † ∗ Vascular Biology Unit, Queensland Research Centre for Peripheral Vascular Disease, School of Medicine and Dentistry, James Cook University, Townsville, Queensland 4811, Australia †Department of Vascular and Endovascular Surgery, Townsville Hospital, Townsville, Queensland 4814, Australia

Abstract Aliskiren is a direct renin inhibitor developed to treat hypertension. Several clinical studies have suggested that aliskiren has beneficial effects on cardiovascular diseases beyond its antihypertensive effect. In the present study, we examined whether aliskiren limits the progression of AAA (abdominal aortic aneurysm), VH (ventricular hypertrophy) and atherosclerosis in an AngII (angiotensin II)-infused mouse model. ApoE − / − (apolipoprotein-Edeficient) mice were infused subcutaneously with AngII (1000 ng/kg of body weight per day; 4 weeks) to induce AAA and VH. At the completion of the AngII infusion, mice were randomly allocated to three groups to receive vehicle control, low-dose aliskiren (10 mg/kg of body weight per day) or high-dose aliskiren (50 mg/kg of body weight per day) for 4 weeks. Suprarenal aortic diameter assessed by ultrasound was significantly smaller in mice administered aliskiren at days 42 and 56. Aliskiren also significantly reduced the normalized heart weight, ventricular myocyte cell width and aortic arch atherosclerosis. Aliskiren lowered PRR (pro-renin receptor) expression and MAPK (mitogen-activated protein kinase) activity in the suprarenal aorta and heart. Aortic infiltration of T-lymphocytes and macrophages was reduced by aliskiren. In conclusion, aliskiren limits the progression of AAA, VH and atherosclerosis in an AngII-infused mouse model.

Clinical Science

Key words: abdominal aortic aneurysm, angiotensin II, atherosclerosis, pro-renin receptor, ventricular hypertrophy

INTRODUCTION Animal studies suggest that AngII (angiotensin II) contributes to numerous cardiovascular diseases, such as hypertension, atherosclerosis, AAA (abdominal aortic aneurysm) and VH (ventricular hypertrophy) [1–4]. Trials are underway investigating the efficacy of ARBs (angiotensin receptor blockers) and ACEIs (angiotensinconverting enzyme inhibitors) as therapies to limit aortic aneurysm expansion; however, currently there is no clear evidence of their efficacy in this regard [5]. Aliskiren is the first renin inhibitor approved for the treatment of primary hypertension by the US Food and Drug Administration [6]. It suppresses the activity of the RAS (renin–angiotensin system) by direct inhibition of renin, preventing the conversion of angiotensinogen into AngI (angiotensin I), a rate-limiting step

of AngII generation. The antihypertensive effect of aliskiren has been demonstrated in various animal models and clinical trials [7–10]. Studies have suggested that aliskiren can provide cardiovascular protection beyond its anti-hypertensive effect [9,10]. The RAS functions to generate AngII at both the systemic and local levels. Intracellular generation of AngII has been suggested to mediate local physiological activities in, for example, cardiac myocytes, VSMCs (vascular smooth muscle cells) and renal mesangial cells [7,8]. In a diabetic rat model, aliskiren has been shown to reduce oxidative stress and cardiac fibrosis via the suppression of intracellular, but not plasma, AngII levels [3]. The more pronounced cardiac-protective effect of aliskiren when compared with an ARB and an ACEI in that study suggested the possibility that aliskiren could also act via BP (blood pressure)and AngII-independent mechanisms. Indeed, Lu et al. [9] have

Abbreviations: AAA, abdominal aortic aneurysm; ACEI, angiotensin-converting enzyme inhibitor; AngII, angiotensin II; ApoE, apolipoprotein E; ARB, angiotensin receptor blocker; AT1 R, AngII type 1 receptor; AT2 R, AngII type 2 receptor; BP, blood pressure; CCD, charge-coupled-device; CI, confidence interval; CV, coefficient of variation; ERK, extracellular-signal-related kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H&E, haematoxylin/eosin; HDL, high-density lipoprotein; HRP, horseradish peroxidase; IHC, immunohistochemistry; IQR, interquartile range; LDL, low-density lipoprotein; Ldlr, LDL-receptor; MAPK, mitogen-activated protein kinase; MMD, mean maximum diameter; MMP, matrix metalloproteinase; PRR, pro-renin receptor; RAS, renin–angiotensin system; SBP, systolic BP; SRA, suprarenal aorta; TC, total cholesterol; VH, ventricular hypertrophy; VSMC, vascular smooth muscle cell. Correspondence: Professor Jonathan Golledge (email [email protected]).

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demonstrated that renin inhibition by aliskiren at 2.5 mg/kg of body weight markedly reduced atherosclerotic lesion size and formation within the aortic root and arch in high-fat-fed Ldlr − / − [LDL (low-density lipoprotein)-receptor-deficient] mice without lowering BP [9]. Similarly, a study has shown that aliskiren reduced atherogenesis in ApoE − / − (apolipoprotein-E-deficient) mice independent of BP by reducing vascular NADPH oxidase activity and up-regulating pro-angiogenic cells [10]. The MAPK (mitogen-activated protein kinase) signalling pathway plays an important role in numerous cardiovascular diseases [11–14]. ERK (extracellular-signal-related kinase) is one of the key enzymes in the MAPK signalling pathway [15]. ERK regulates macrophage and T-cell activation and MMP (matrix metalloproteinase) activity, contributing to the development and progression of AAA [13,16,17]. A recent study demonstrated that ERK-deficient mice failed to develop AAA upon elastase perfusion [13], suggesting an important role of MAPK signalling in AAA. Although the antihypertensive and atheroprotective effects of aliskiren have been demonstrated in different animal and clinical studies [9,18,19], the effect of aliskiren in limiting AAA progression has not been assessed to our knowledge. We hypothesized that aliskiren would limit AAA and VH by altering local RAS receptor expression, including down-regulating the PRR (pro-renin receptor), the AT1 R (AngII type 1 receptor) and MAPK. The aim of the present study was to examine whether aliskiren limited the progression of AAA, VH and atherosclerosis within an ApoE − / − mouse model.

MATERIALS AND METHODS Animals Male ApoE − / − mice (C57BL/6J background; obtained from Animal Resources Centre, Canning Vale, Western Australia) at 6 months of age were housed under a 12-h light/dark cycle (re◦ late humidity, 55–60 %; temperature, 22 + − 1 C) and were given standard chow and water ad libitum. AAA was induced by infusion of AngII via osmotic mini-pump (ALZET model 2004, BioScientific) as described previously [20]. In brief, mice were anaesthetized by intraperitoneal injection of ketamine (150 mg/kg of body weight) and xylazine (10 mg/kg of body weight). Osmotic mini-pumps (ALZET model 1004) were placed into the subcutaneous space along the dorsal midline to deliver AngII at 1000 ng/kg of body weight per min (Sigma–Aldrich) or vehicle over 28 days. All animals were maintained on a normal laboratory diet throughout the infusion period. Mice were monitored fortnightly by ultrasound sonography by two trained independent observers, as described previously [20]. All animal protocols conformed to the Guide for the Care and use of Laboratory Animals by the United States National Institutes of Health and the Australian Code of Practice for the Care and Use of Animals for Scientific Purpose (7th Edition, 2004). Institutional ethics approval was obtained from James Cook University (Ethics number A1580) before commencement of the study.

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Experimental design In experiment 1, mice receiving AngII (n = 6) or vehicle (saline) control (n = 6) were killed by CO2 asphyxiation at the end of week 4. Aortas were isolated under a dissection microscope (Leica) for Western blot analysis. In experiment 2, mice (n = 27) received AngII for 4 weeks to induce AAA and VH. After 4 weeks of AngII infusion, mice were randomly allocated to receive vehicle (water; n = 9), low-dose aliskiren (10 mg/kg of body weight per day; n = 9) or high dose aliskiren (50 mg/kg of body weight per day; n = 9) subcutaneously delivered via osmotic mini-pumps for 4 weeks. No AngII was administered to the mice between weeks 5 and 8. Mice infused with vehicle (saline) or AngII for 4 weeks were referred to as saline control and baseline control respectively. Mini-pump implantation was performed under anaesthesia using intraperitoneal injection of ketamine (150 mg/kg of body weight) and xylazine (10 mg/kg of body weight). All mice were maintained in a barrier caging system and fed on a normal mouse laboratory diet during the experiment. Mice were killed at the end of week 8. Hearts (weight recorded) and aortas were isolated under a dissection microscope (Leica) and stored in OCT at − 80 ◦ C for future analysis.

Non-invasive tail-cuff BP and heart rate measurement BP and heart rate were measured before AngII infusion (baseline) and every 2 weeks throughout the experiment (days 14, 28, 42 and 56) using a computerized non-invasive tail-cuff system (Kent Scientific). Animals were habituated to the device before assessment. Good reproducibility of this technique has been established previously {mean difference for heart rate (beats/min), 21.1 [95 % CI (confidence interval), 14.8–27.5], and mean difference for SBP (systolic BP) (mmHg), 12.3 (95 % CI, 8.4–16.1)}.

Ultrasound measurement of the abdominal aortic diameter The maximum diameter of the abdominal aorta within the SRA (suprarenal aorta) region was estimated using ultrasound immediately before AngII infusion (baseline) and every 2 weeks throughout the experiment (days 14, 28, 42 and 56). Mice were immobilized by intraperitoneal injection of ketamine (40 mg/kg of body weight) and xylazine (4 mg/kg of body weight). Ultrasound was performed in B-mode using a MyLa 70 VETXY platform (Esaote) with an LA435 linear transducer (Esaote) at an operating frequency of 12 MHz. Maximum outer SRA diameter was measured at peak systole using the calliper measurement feature as described previously [20,21]. A good inter-observer reproducibility of this method has been established previously [mean difference (mm), 0.92 (95 % CI, 0.883–0.946); average CV (coefficient of variation), 9.5%]. All measurements were obtained by two observers blinded to the treatment groups.

Aortic morphometry Aortas were isolated free from fat and connective tissue from their origin at the left ventricle to the iliac bifurcation and placed on a black background before being digitally photographed. Diameters of the aortic arch, thoracic aorta, SRA and infrarenal aorta were measured using computer-aided analysis (Adobe

Aliskiren limits aortic aneurysm

Photoshop). MMD (mean maximum diameter) was then calculated by averaging the values collected from these four segments. Good intra-observer reproducibility of this measurement method has been previously confirmed by our group [20,21].

Quantification of atherosclerosis Sudan IV staining was performed on aortic arch samples (region from aortic valve to the left subclavian artery) to identify atherosclerotic plaque as described previously [20]. In brief, aortic arches were opened longitudinally and fixed in 75 % ethanol for 15 min. Sections were then stained with Sudan IV solution (0.1 % Sudan IV dissolved in equal parts of acetone and 70 % ethanol) for 10 min, followed by a 15 min 80 % ethanol wash. Stained aortic arches were digitally photographed and the atherosclerotic plaque areas were measured using computer-aided analysis (Adobe Photoshop). Good intra-observer reproducibility of this technique has been previously confirmed by our group [20,21].

Plasma high-density lipoprotein-, low-density lipoprotein- and total cholesterol measurements The plasma samples were snap-frozen in liquid nitrogen and stored at − 80 ◦ C for subsequent assessments. Measurements of HDL (high-density lipoprotein)-cholesterol, LDL-cholesterol and TC (total cholesterol) were performed using a commercially available fluorescence assay (Abcam) in accordance with the manufacturer’s instructions.

Western blot analysis Sample from the left ventricle and SRA were homogenized in the presence of protease inhibitors to obtain protein extracts. Protein concentrations were determined using a Bradford protein assay kit (Bio-Rad Laboratories). Samples (25 μg of protein per lane) were separated by SDS/PAGE (10 % gel). After electrophoresis (110 V, 90 min), the separated proteins were transferred (15 mA, 60 min) on to a PVDF membrane (BioRad Laboratories). Non-specific sites were blocked with 5 % non-fat dry milk for 60 min, and the blots were then incubated overnight at 4 ◦ C with an anti-ATP6IP2 (PRR) antibody (1:1000 dilution; ab40790, Abcam) (Supplementary Figure S1 at http://www.clinsci.org/cs/127/cs1270123add.htm), anti-AT1 R antibody (1:1000 dilution; SAB3500209, Sigma–Aldrich), antiAT2 R (AngII type 2 receptor) antibody (1:1000 dilution; sc-7421, Santa Cruz Biotechnology), anti-MMP-9 antibody (1:1000 dilution; ab38898, Abcam), anti-p44/42 MAPK (ERK1/2) antibody (1:1000 dilution; 4695S, Cell Signaling Technology) or anti-[phospho-p44/42 MAPK (ERK1/2) (Thr202 /Tyr204 )] antibody (1:1000 dilution; 4370S, Cell Signaling Technology). HRP (horseradish peroxidase)-conjugated anti-(rabbit IgG) (1:1000 dilution; P0448, DakoCytomation) or HRP-conjugated anti-(goat IgG) (1:1000 dilution; P0449, DakoCytomation) was used to detect the binding of its corresponding antibody. An anti-(β-actin) antibody (1:10000 dilution; ab8227, Abcam) or anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody (1:5000 dilution; 2118S, Cell Signaling Technology) was used to verify the equal loading of protein in each lane. The protein expression was estimated with Western Lightning Chemiluminescence

Reagent Plus (PerkinElmer Life Sciences) and quantified by Quantity One (version 4.6.7) software (Bio-Rad Laboratories).

Histology At study completion, mice hearts were isolated and weighed. Heart weight was divided by total mouse body weight and expressed as the normalized heart weight (mg/g). A total of five hearts and five SRA segments from each group were selected randomly and processed for H&E (haematoxylin/eosin) staining. Frozen ventricular sections were cut (5-μm-thick) and stained with H&E. Digital images were captured by a Zeiss microscope fitted with a CCD (charge-coupled-device) camera and collected on to a computer supported with Zeiss AxioVision (version 4.8.2) software. Average myocyte width was measured within ventricular sections by examining 30 randomly chosen myocytes in the cross-sections using computer-aided analysis (Adobe Photoshop), as described previously [22]. The reproducibility of this technique was assessed in 13 mice [mean difference (μm), 0.65 (95 % CI, 0.06–4.07); average CV, 24.97%].

Immunohistochemistry SRA segments from five mice from each group were selected using a random number generator. Serial cryostat sections 5-μmthick were cut from each SRA segment and processed for IHC (immunohistochemistry) as described previously [20]. Briefly, serial frozen sections were air-dried, fixed in acetone for 10 min at − 20 ◦ C, air-dried again and rehydrated with PBS before being incubated in PBS containing 3 % H2 O2 and 0.1 % sodium azide to block endogenous peroxidase activity. Slides were then incubated in 2 % normal goat serum (S1000, Vector) in PBS and endogenous avidin and biotin blocked using a commercial kit (SP2001, Vector), and incubated overnight at 4 ◦ C with the primary antibodies anti-CD68 (1:200 dilution; MEC13.3, BD Pharmingen) and antiCD3 (1:200 dilution; AB5690, Abcam). Sections were rinsed twice in PBS and then incubated with the appropriate secondary antibody [biotinylated anti-(rabbit IgG) (BA-1000, Vector) or biotinylated anti-(rat IgG) (BA-9400, Vector)] diluted 1:100 to detect the binding of its corresponding primary antibody. Sections were washed twice in PBS as above and incubated in peroxidise– avidin–biotin for 30 min (PK6100, Vector). Slides were incubated in the peroxidase substrate diaminobenzidine (SK4105, Vector), counterstained in Mayer’s haematoxylin, dehydrated, cleared in xylene and mounted in Entellan mounting medium (Electron Microscopy Sciences). Sections were stained simultaneously using identical reagents and incubation times and negative controls were included. Stained sections were photographed using a Nikon microscope fitted with a CCD camera (Diagnostic Instruments) and Nikon software (Bio-Rad Laboratories). Identical exposure times and settings were used for all sections. Image analysis was performed on digital tiff images using Adobe Photoshop CS3 extended software. For each section, the total tissue area and area of staining were measured using the ‘Selection Tool’ and ‘Record Measurements’ functions. Staining was expressed as a percentage of total tissue area (i.e. area macrophage staining/total tissue area×100). We have previously shown this technique to be reproducible [20].

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Figure 1 Effect of AngII on aortic PRR levels in ApoE − / − mice Upper panel, Western blot analysis of PRR protein expression in the SRA from ApoE − / − mice receiving vehicle or AngII infusion for 4 weeks. Lower panel, densitometric analysis of PRR expression relative to β-actin. Results are medians (IQR), n = 6 per group. A.U., arbitrary units.

Statistical analyses A D’Agostino–Pearson test was used to test the normality of the data. Results are medians [IQR (interquartile range)] for nonnormally distributed data, and means + − S.E.M. for normally distributed data. For non-normally distributed data, comparisons were made using Mann–Whitney U test or Kruskal–Wallis test, followed by Dunn’s multiple comparisons test, where appropriate. Normally distributed data were compared using ANOVA, followed by Bonferroni’s multiple comparisons test. Differences were considered to be statistically significant at P < 0.05. All statistical analysis was performed using GraphPad Prism 5 software.

RESULTS Effect of AngII on aortic PRR levels in ApoE − / − mice AngII infusion for 4 weeks caused an up-regulation of PRR levels in the SRA in ApoE − / − mice when compared with saline-infused controls (P = 0.0022) (Figure 1).

Effects of AngII infusion and aliskiren on BP and heart rate AngII infusion caused a time-dependent increase in SBP in ApoE − / − mice throughout the 28-day infusion period (Figure 2A). BP was similar in mice randomized to different groups at day 28. SBP returned to the baseline level after day 28 in a time-dependent manner in all groups. SBP was significantly lower in mice that received high-dose aliskiren (50 mg/kg of body weight per day) at days 42 and 56 when compared with the vehicle control group (Figure 2A). Mice that received high-dose aliskiren (50 mg/kg of body weight per day), but not the other groups, had significantly lower SBPs at day 56 when compared with baseline (day 0) (P = 0.03, n = 9 per group) (Figure 2B).

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Figure 2 Effect of AngII infusion and aliskiren on BP (A) SBP in ApoE − / − mice that received vehicle control, low-dose aliskiren (10 mg/kg of body weight per day) or high-dose aliskiren (50 mg/kg of body weight per day) for 4 weeks following after 4 weeks + S.E.M., n = 9 per group. (B) Comof AngII infusion. Results are means − parison of SBP at day 0 and 56 in ApoE − / − mice receiving vehicle, low– dose aliskiren (10 mg/kg of body weight per day) or high-dose aliskiren (50 mg/kg of body weight per day) after 4 weeks of AngII infusion. Results are means + − S.E.M., n = 9 per group.

Heart rate was not altered throughout the whole period of study (results not shown).

Effect of aliskiren administration on plasma HDL-cholesterol, LDL-cholesterol and TC in AngII-infused ApoE − / − mice Plasma HDL-cholesterol, LDL-cholesterol and TC were measured in a subset of mice at days 28 and 56 to examine whether aliskiren administration altered plasma cholesterol concentrations. Plasma HDL-cholesterol, LDL-cholesterol and TC were not significantly different between days 28 and 56 in all groups. Moreover, no significant difference was observed between mice receiving vehicle control, low-dose aliskiren or high-dose aliskiren (n = 5 per group) (Table 1).

Effects of AngII and aliskiren administration on aortic aneurysm As expected from the design of the study, median maximum SRA diameter at day 28 was similar in mice allocated to vehicle control and aliskiren [control, 1.17 + − 0.04 mm; low-dose aliskiren (10 mg/kg of body weight per day), 1.16 + − 0.07 mm; and high-dose aliskiren (50 mg/kg of body weight per day), 1.17 + − 0.04 mm]. Aliskiren administrated at either dose significantly suppressed the progression of SRA dilation as assessed by ultrasound (Figure 3A and Supplementary Figure S2 at http://www.clinsci.org/cs/127/cs1270123add.htm). Aortas collected at the end point (day 56) are shown in Supplementary

Aliskiren limits aortic aneurysm

Table 1

Plasma HDL-cholesterol, LDL-cholesterol and TC levels in ApoE − / − mice before (day 28) and after (day 56) receiving vehicle control, or a low or high dose of aliskiren Values are means + − S.E.M., n = 5 per group. Aliskiren (mg/kg of body weight per day) Parameter

Vehicle

10

50

HDL-cholesterol (mg/dl) Day 28 Day 56

66.1 + − 10.33 68.2 + − 13.5

55.2 + − 15.6 34.8 + − 9.9

64.7 + − 8.1 47.8 + − 16.0

LDL-cholesterol (mg/dl) Day 28 Day 56

379.0 + − 79.9 282.0 + − 72.1

305.9 + − 59.1 156.1 + − 38.4

257.4 + − 143.2 273.2 + − 98.4

817.6 + − 121.9 519 + − 36.5

776.0 + − 170.8 695.4 + − 124.5

666.8 + − 109.2 818.1 + − 237.4

TC (mg/dl) Day 28 Day 56

Figure S3 at http://www.clinsci.org/cs/127/cs1270123add.htm. The median aortic MMD was significantly smaller in mice receiving high-dose aliskiren (50 mg/kg of body weight per day) as measured by morphometry (P = 0.041) (Figure 3B). However, the aortic MMD was not different in the groups receiving vehicle control or low-dose aliskiren (10 mg/kg of body weight per day) (P > 0.05) (Figure 3B).

Aliskiren reduces the progression of aortic arch Sudan IV staining area Aortic arch Sudan IV staining area in mice that received AngII infusion for 4 weeks was significantly greater than in mice that received saline infusion for 4 weeks (P = 0.041) (Figure 3C). Aortic arch Sudan IV staining area in mice that receiving 4 weeks of AngII infusion followed by 4 weeks of vehicle control was significantly greater than that in mice that were killed directly after 4 weeks of AngII infusion (P = 0.035) (Figure 3B). However, aortic arch Sudan IV staining area in mice receiving either lowdose aliskiren (10 mg/kg of body weight per day) or high-dose aliskiren (50 mg/kg of body weight per day) was not significantly different compared with the baseline control (AngII infused for 28 days) group (Figure 3C).

Aliskiren reduces normalized heart weight and cardiac myocyte cell width in AngII-infused ApoE − / − mice Normalized heart weight in mice receiving vehicle was significantly greater than that in the baseline control group (P = 0.049) (Table 2). However, normalized heart weight in mice that received either low-dose aliskiren (10 mg/kg of body weight per day) or high-dose aliskiren (50 mg/kg of body weight per day) was not significantly different compared with the baseline control group (Figure 4A). In addition, cardiac myocyte cell width in mice that received aliskiren was significantly smaller when compared with the vehicle control group (Figure 4B).

Effects of aliskiren on aortic PRR, AT1 R, AT2 R and MMP-9 levels in AngII-infused ApoE − / − mice Western blotting was used to estimate PRR, AT1 R, AT2 R and MMP-9 levels in the SRA of mice completing the study. Aortic

MMP-9 and PRR levels were significantly reduced in mice receiving high-dose aliskiren (50 mg/kg of body weight per day) when compared with the baseline control group (Figures 5A, 5B and 5E). Low-dose aliskiren administration appeared to suppress SRA PRR and MMP-9 levels; however, these trends did not reach statistical significance. Aortic AT1 R and AT2 R levels were not altered by aliskiren administration (Figures 5A, 5C and 5D).

Effects of aliskiren on cardiac PRR levels and Akt activity in AngII-infused ApoE − / − mice Aliskiren administration caused a reduction in cardiac PRR level (Figure 6). Akt activity was estimated as the ratio of phosphorylated to total protein expression. However, no significant effect of aliskiren administration on the phosho-Akt/total Akt ratio in the heart was found (P = 0.70, n = 3 per group) (results not shown).

Effects of aliskiren on aortic and cardiac MAPK activity in AngII-infused ApoE − / − mice MAPK activity was estimated as the ratio of phosphorylated ERK1/2 to total ERK1/2. Aliskiren administration caused a significant reduction in both the aortic (Figure 7A) and cardiac (Figure 7B) phospho-ERK1/2/total-ERK1/2 ratio when compared with the baseline control group.

Aliskiren reduced inflammatory cell infiltration in AngII-infused ApoE − / − mice We assessed the histology of SRA samples obtained at the completion of the studies using H&E staining. SRA sections demonstrated marked inflammatory cell infiltration within the media and adventitia of mice receiving vehicle control, which appeared to be substantially reduced in animals receiving high dose aliskiren (Figure 8A). IHC suggested that aliskiren inhibited aortic infiltration of T-lymphocytes and macrophages. There was reduced CD3 + staining in SRA sections from mice that received both lowdose aliskiren (10 mg/kg of body weight per day; P = 0.063) or high-dose aliskiren (50 mg/kg of body weight per day; P = 0.031) when compared with the vehicle control group (Figure 8B and

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Figure 3 Effect of aliskiren on aortic diameter, aortic morphometry and aortic arch Sudan IV staining area in AngII-infused ApoE − / − mice (A) In vivo suprarenal aortic diameter in ApoE − / − mice that received vehicle control, low-dose aliskiren (10 mg/kg of body weight per day) or high-dose aliskiren (50 mg/kg of body weight per day) for 4 weeks after 4 weeks of AngII infusion. Results are means + − S.E.M., n = 9 per group. (B and C) Comparison of the aortic morphometry (B) and aortic arch Sudan IV staining area (C) in ApoE − / − mice receiving saline control (day 28) (n = 6) or at day 56 that received vehicle (n = 9), lowdose aliskiren (10 mg/kg of body weight per day) (n = 9) or high-dose aliskiren (50 mg/kg of body weight per day) (n = 9) after 4 weeks of AngII infusion. Results are medians (IQR). Results are medians (IQR).

8Dii). The SRA macrophage staining area was lower in mice that received high-dose aliskiren (P = 0.031) when compared with the vehicle control group (Figure 8C and 8Di). Table 2

Figure 4 Aliskiren reduces normalized heart weight and cardiac myocyte cell width in AngII-infused ApoE − / − mice (A) Comparison of normalized heart weight (heart weight/body weight) of ApoE − / − mice at baseline (day 28 control) or at day 56 that received vehicle, low-dose aliskiren (10 mg/kg of body weight per day) or high-dose aliskiren (50 mg/kg of body weight per day) after 4 weeks of AngII infusion. Results are means + − S.E.M., n = 9 per group. (B) Representative images and quantification of the cardiac myocyte cell width in ApoE − / − mice receiving vehicle, low-dose aliskiren (10 mg/kg of body weight per day) or high-dose aliskiren (50 mg/kg of body weight per day). Results as means + − S.E.M., n = 5 per group.

DISCUSSION In the present study, we have demonstrated that PRR levels are up-regulated in the aorta of ApoE − / − mice in response to AngII infusion. Most importantly, our results have shown that the direct renin inhibitor aliskiren reduces the progression of

Body weight, heart weight and normalized heart weight in ApoE − / − mice that received vehicle control, or the low or high dose of aliskiren + S.E.M. ∗ P = 0.015 and †P = 0.049 compared with the baseline control. Values are means − Aliskiren (mg/kg of body weight per day) Parameter

Baseline control

Vehicle

10

50

Body weight (g)

30.01 + − 0.78 170 + − 5.35 5.68 + − 0.16

28.18 + − 1.08 182.2 + − 7.41 6.50 + − 0.25†

27.85 + − 0.80 155.6 + − 6.26 5.60 + − 0.22

∗ 26.18 + − 0.78 150.0 + − 3.73 5.76 + − 0.16

Heart weight (mg) Normalized heart weight (mg/g)

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Aliskiren limits aortic aneurysm

Figure 6 Effect of aliskiren on cardiac PRR levels in AngII-infused ApoE − / − mice (A) Representative immunoblots of PRR protein levels in the left ventricle of ApoE − / − mice at baseline (day 28) control or at day 56 that received vehicle, low-dose aliskiren (10 mg/kg of body weight per day) or high-dose aliskiren (50 mg/kg of body weight per day) following 4 weeks of AngII infusion. (B) Densitometric analysis of PRP levels relative to GAPDH is shown in the box plots. Results are medians (IQR), n = 5 per group. A.U., arbitrary units.

Figure 5 Effect of aliskiren on aortic PRR, AT1 R, AT2 R and MMP-9 levels in AngII-infused ApoE − / − mice (A) Representative immunoblots of PRR, AT1 R, AT2 R and MMP-9 protein levels in the SRA of ApoE − / − mice at baseline (day 28) control or at day 56 that received vehicle, low-dose aliskiren (10 mg/kg of body weight per day) or high-dose aliskiren (50 mg/kg of body weight per day) following 4 weeks of AngII infusion. (B–E) Densitometric analysis of the (B) PRR, (C) AT1 R, (D) AT2 R and (E) MMP-9 protein levels relative to GAPDH. Results are medians (IQR), n = 5 per group. A.U., arbitrary units.

VH and atherosclerosis in AngII-infused ApoE − / − mice at both sub-antihypertensive (10 mg/kg of body weight per day) and antihypertensive (50 mg/kg of body weight per day) doses. In addition, an antihypertensive dose of aliskiren suppressed the progression of AAA in AngII-infused ApoE − / − mice. In a relatively unique way, we focused on the efficacy of aliskiren in limiting complications of AngII infusion after the organ damage was established, rather than during this process, since this is likely to be most clinically relevant. The PRR has been shown to be involved in the pathogenesis of numerous cardiovascular diseases, such as cardiomyopathy and hypertension [23,24]. In the present study, we have shown that the PRR level was markedly increased in the aorta of ApoE − / − mice after 28 days of AngII infusion when compared with the vehicle control group. The possibility of direct PRR activation by renin or pro-renin in organs other than the kidney is controversial [25,26]. Recent work suggests that enhanced expression or activation of the PPR could have detrimental effects via AngII-independent mechanisms in both in vitro and in vivo studies [27,28]. Indeed, infusion of HRP, a PRR blocker, decreased cardiac AngII level and attenuated cardiac fibrosis without effects on plasma RAS

activity in SHRs (spontaneously hypertensive rats). That study suggested the local effect of the PRR in cardiovascular complications [29]. Moreover, HRP suppressed nephropathy and reduced glomerulosclerosis in both a diabetic mouse model deficient in AT1a R and a PRR transgenic rat, suggesting that the effects of activation of the PRR can be independent of AngII generation [30,31]. Putting our present findings and other previous reports together, the PRR could play a significant role in the pathogenesis of cardiovascular diseases, including AAA. Hence drugs or therapies that reduce the expression or activity of this receptor could be potentially beneficial. However, the exact role of the PRR in AAA has not been directly addressed in the present study and requires further investigation using techniques which more specifically up- or down-regulate this receptor. It remains uncertain whether the up-regulation of the PRR demonstrated in AAA samples in the present study results from direct changes in vascular cell expression or are due to changes in cellular composition within the aorta, such as influx of inflammatory cells. The antihypertensive property of aliskiren has been extensively studied in both animal models and clinical studies [6,32]. Large numbers of reports have suggested that this antihypertensive effect of aliskiren is caused by inhibiting AngII generation by regulating the rate-limiting step of the RAS. To our knowledge, the present study is the only one that has investigated the effect of aliskiren in the AngII-infused ApoE − / − mouse, therefore two doses of aliskiren were chosen based on previous mouse studies, which suggested that 50 mg/kg of body weight per day was required to achieve BP reduction [33,34]. In keeping with these previous reports, we found that only mice receiving aliskiren at 50 mg/kg of body weight per day and not at 10 mg/kg of body weight per day had a significant reduction in BP in comparison with the control group. This result suggests that the BP-dependent

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Figure 7 Effect of aliskiren on aortic and cardiac MAPK activity in AngII-infused ApoE − / − mice (A and B) Representative immunoblots of phospho-ERK1/2 and total-ERK1/2 protein expression in the SRA (A) and left ventricle (B) of ApoE − / − mice at baseline (day 28) control or day 56 that received vehicle, low-dose aliskiren (10 mg/kg of body weight per day) or highdose aliskiren (50 mg/kg of body weight per day) following 4 weeks of AngII infusion. Densitometric analysis of phospho-ERK1/2 protein relative to total ERK1/2 is shown in the box plots. Results are medians (IQR), n = 5 per group. A.U., arbitrary units.

and -independent effects of aliskiren can be studied by using the sub-hypotensive dose (10 mg/kg of body weight per day) and hypotensive dose (50 mg/kg of body weight per day) of aliskiren in the AngII-infused ApoE − / − mouse model. AngII-infusion induces atherosclerosis and AAA in ApoE − / − mice [20]. Consistent with previous reports [20,35], marked aortic arch intimal atherosclerosis and SRA dilation were observed in AngII-infused mice. Previous studies have suggested that atherosclerosis and AAA induced by AngII infusion in ApoE − / − mice is not a direct consequence of elevated BP [35,36]. In the present study, aortic arch intimal atherosclerosis continued to progress in mice receiving vehicle control, whereas mice receiving aliskiren at 50 mg/kg of body weight had similar levels of atherosclerosis to baseline controls. Administration of aliskiren at 10 mg/kg of body weight did not affect BP, but significantly suppressed the progression of aortic arch

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intimal atherosclerosis. A similar finding was also observed by Lu et al. [9], who reported that aliskiren reduced atherosclerosis with no significant correlation between BP and atherosclerotic lesion size in a hypercholesterolaemia-induced mouse model of atherosclerosis. More recent studies support the theory that BP reduction is not responsible for all the anti-atherosclerotic effects of aliskiren [10,19]. In addition, a recent study has shown that aliskiren inhibits aortic root atherosclerosis in Ldlr − / − mice without changing plasma cholesterol concentrations [37]. In line with these studies, we found that aliskiren inhibited atherosclerosis progression in AngII infused ApoE − / − mice without altering plasma HDL-cholesterol, LDL-cholesterol or TC levels, indicating that aliskiren inhibits atherosclerosis via a mechanism independent of cholesterol-lowering. In contrast with our study, Rateri et al. [55] demonstrated a non-significant increase in atherosclerosis in mice after cessation of AngII infusion. Other investigators have, however, shown that atherosclerosis progresses with aging in ApoE − / − mice fed on regular chow [38,39]. The disparate findings may relate to the different ages of mice used in the present investigation and those of mice used by Rateri et al. [55]. The most novel finding from the present study is that aliskiren limits AAA progression within a mouse model. Expression of the PRR has been demonstrated in human VSMCs, and PRRmediated MAPK signalling has been shown to be involved in vascular remodelling [40]. Deficiency of MAPK has previously been shown to protect against AAA development in a mouse model [13]. Given the importance of MAPK signalling in AAA, we hypothesized that aliskiren could reduce PRR and MAPK activity in the aorta of AngII-infused ApoE − / − mice. Previous in vitro studies have demonstrated that aliskiren reduces PRR expression in cultured human aortic smooth muscle cells [41]. In the present study, aliskiren significantly reduced SRA PRR levels and MAPK activation. MAPK activation has been shown previously to promote MMP secretion [12], VSMC apoptosis [42] and fibrosis [14]. Although a study has demonstrated that AngII-mediated aneurysm progression involves regulation of the activity of the MAPK signalling pathway via stimulating both the AT1 R and the AT2 R [14], our present study found no difference in AT1 R and AT2 R levels in mice receiving aliskiren. Our findings suggest that aliskiren suppressed aortic dilatation independent of the down-regulation of AT1 R and AT2 R. Interestingly, we demonstrated a suppression of aortic PRR levels and MAPK activity in mice receiving aliskiren, indicating the possible role of PPR and MAPK activity in AAA. Indeed, a recent in vitro study [28] has demonstrated that overexpression of the human PRR induces oxidative stress via activation of MAPK, which was not reversed by losartan, an AT1 R blocker, suggesting that the PRR can mediate MAPK signalling via an AngII-independent mechanism. Aortic infiltration by macrophages and T-lymphocytes is one of the characteristics of AAA [20]. Numerous studies have reported that activation of MAPK plays an important role in macrophage and T-lymphocyte activation [16,17,43] and aortic wall breakdown [13]. In our present study, aliskiren reduced aortic wall macrophage and T-lymphocyte infiltration within the media and adventitia. These results suggest that the down-regulation of the PRR could reduce MAPK activation, which subsequently suppresses aortic inflammation via inhibition of macrophage and

Aliskiren limits aortic aneurysm

Figure 8

Aliskiren reduces inflammatory cell infiltration in AngII-infused ApoE − / − mice (A) H&E micrographs showing varying degrees of inflammation in mice receiving (i and iv) vehicle, (ii and v) low-dose aliskiren (10 mg/kg of body weight per day) or (iii and vi) high-dose aliskiren (50 mg/kg of body weight per day) for 4 weeks after 4 weeks of AngII infusion. Scale bar, 500 μm in (Ai)–(Aiii) and 50 μm in (Aiv)–(Avi); ∗ indicates the lumen. (B and C) Representative IHC micrographs showing CD3 staining (for T-lymphocytes) (B) and CD68 staining (for macrophages) (C) in mice receiving (i) vehicle, (ii) low-dose aliskiren (10 mg/kg of body weight per day) or (iii) high-dose aliskiren (50 mg/kg of body weight per day) after 4 weeks of AngII infusion; ∗ indicates the lumen. Scale bar, 50 μm. (D) Quantification of (n = 5 SRA per group) (Di) T-lymphocyte and (Dii) macrophage infiltration. Five harvested SRA segments were randomly selected from each group for IHC for T-lymphocytes (CD3) and macrophages (CD68). Staining area (four to five high-power fields from each section) was estimated in each section from each group by computer-aided analysis. Results are medians (IQR), n = 5 per group.

T-lymphocyte infiltration; however, a more direct examination of this hypothesis is needed. Numerous studies have demonstrated that AngII infusion induces VH in mice [44,45]. The increase in BP caused by AngII infusion can elevate cardiac afterload, leading to VH, although BPindependent mechanisms are also involved [44–46]. In the present study, aliskiren administered at 10 mg/kg of body weight per day reduced the normalized heart weight and myocyte cell width without modifying BP, suggesting that aliskiren can effectively inhibit heart damage independent of its antihypertensive effects. Similar to our study, Pilz et al. [47] have reported that aliskiren attenuated heart and renal damage in a double-transgenic rat model. Moreover, a recent study has demonstrated that aliskiren protects against doxorubicin-induced cardiomyopathy in rats [48]. Aliskiren has been reported to reduce cardiac PRR expression and improve cardiac function [49]. Similar to previous reports, we observed that aliskiren significantly reduced PRR levels in the hearts of AngII-infused ApoE − / − mice at both of the doses tested. We investigated the impact of PRR down-

regulation by aliskiren on the downstream signalling pathways. Our study showed that aliskiren markedly reduced ERK1/2 phosphorylation in the hearts of the AngII-infused ApoE − / − mice, suggesting the suppression of MAPK activity. Previous studies have demonstrated that activation of the ERK1/2 signalling pathway causes VH via multiple mechanisms, such as oxidative stress or up-regulation of pro-inflammatory cytokines [50–52]. Given the importance of MAPK signalling in the development and progression of VH [50,51], our results suggest that aliskiren could provide cardioprotection by reducing PRR levels and subsequently decreasing MAPK signalling. Indeed, activation of the PRR has been shown to trigger intracellular signalling by activating the MAPK pathway [53]. Although a previous study has suggested that aliskiren could attenuate VH via inhibition of Akt activity in an experimental myocardial infarction mouse model [54], we found no evidence that aliskiren altered Akt activity in the heart. More direct examination of the role of the PRR in VH is needed to establish the suggested role of this receptor in VH.

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Rateri et al. [55] reported that continuous infusion of AngII is required for AAA progression and maintenance of hypertension in the ApoE − / − mouse model; however, some studies report different findings [56,57]. Indeed, a previous study has shown that aortic diameter continues to expand for a further 20 weeks after infusion was stopped. In the same study, it was reported that aortic ACE and chymase activities were markedly enhanced after 4 weeks of AngII infusion. Chymase, but not ACE, activity remained high 20 weeks after AngII infusion was stopped [56]. Moreover, it was reported that MMP-9 levels continued to increase after AngII infusion was ceased, suggesting that aortic inflammation remained activated for a prolonged period. These results suggest that aortic remodelling continues to progress after AngII infusion has stopped. In the present study, aortic diameter continued to increase, whereas BP immediately reduced, when AngII infusion was ceased at day 28. These results suggest that increased BP requires continuous infusion of AngII, whereas AAA progression continues once initiated by AngII. In addition, and in line with our present results, Ayabe et al. [58] reported that atherosclerosis continued to progress in ApoE − / − mice after 2 weeks of AngII infusion, suggesting that a transient increase in AngII can have a prolonged effect on atherosclerosis progression. The AngII-infused ApoE − / − mouse mimics many major characteristics of human AAA, including activation of the inflammatory response, thrombus formation, medial degradation and male preponderance [20,21,59]. We designed our present study in a unique way to look at the effect of aliskiren in limiting the progression of AAA, VH and atherosclerosis after the organ damage was established. Our results suggest that aliskiren significantly suppressed the progression of these diseases in the mouse model, highlighting the potential of aliskiren as a therapeutic agent for these conditions. There are, however, several differences between human AAA and the AngII-infused mouse model, such as the acute onset of pathology [60,61]. Thus the translation of findings from this and other mice models remains uncertain. Ultimately, clinical trials are required to examine the therapeutic value of agents such as aliskiren for AAA. The results of the present study should be interpreted taking into account a number of limitations. Sample sizes were relatively small, although typical of studies of this type. Only one model of AAA was investigated. The findings of ultrasound and aortic morphometry were not in complete agreement since ultrasound suggested that low-dose aliskiren reduced AAA progression, although this finding was not confirmed by morphometric analysis. Although the present study found no difference in aortic AT1 R and AT2 R levels in mice receiving aliskiren, analysis of other local RAS components, such as AngII, ACE, ACE2 and renin, should also be considered in future studies. Finally, although we examined the PRR levels within the aorta, we did not directly examine the effect of up- or down-regulation of this receptor on AAA. PRR-deficiency models should be used to identify whether aliskiren has any PRR-independent effect on MAPK activation in future studies. In conclusion, the findings of the present study suggest that aliskiren suppresses progression of AAA, VH and atherosclerosis within a mouse model.

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CLINICAL PERSPECTIVES

r r

r

Aliskiren has been suggested to have beneficial effects on cardiovascular diseases beyond its antihypertensive effect, but the effect of aliskiren on AAA has not been investigated. In the present study, we show that aortic PRR levels are increased in a mouse model of AAA. Aliskiren suppressed progression of AAA, VH and atherosclerosis associated with down-regulation of the PRR and reduction in the downstream signalling of this receptor. This study suggests that aliskiren may provide a number of cardiovascular-protective benefits.

AUTHOR CONTRIBUTION

Sai-Wang Seto conducted the experiments, analysed and interpreted the data, performed the statistical analysis and drafted the paper. Smriti Krishna conducted the IHC experiments and analysed the data. David Liu assisted with assessment of VH. Corey Moran and Jonathan Golledge conceived and designed the research, obtained funding and made critical revisions of the paper. All authors revised and approved the final paper.

ACKNOWLEDGEMENTS

We thank Novartis for providing aliskiren for the present study. We also thank Dr Carla Ewels for advice on statistical analysis.

FUNDING

This work was supported by Novartis, the National Health and Medical Research Council and the Office of Health and Medical Research, Australia. S.-W.S. is a recipient of fellowships from the Australia Government National Health and Medical Research Council [grant number 1016349] and from the National Heart Foundation, Australia [grant number PF12B6825]. J.G. is supported by fellowships from the Queensland Government and the National Health and Medical Research Council, Australia.

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Received 15 July 2013/23 December 2013; accepted 30 January 2014 Published as Immediate Publication 30 January 2014, doi: 10.1042/CS20130382

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 C The Authors Journal compilation  C 2014 Biochemical Society

Clinical Science (2014) 127, 123–134 (Printed in Great Britain) doi: 10.1042/CS20130382

SUPPLEMENTARY ONLINE DATA

Aliskiren limits abdominal aortic aneurysm, ventricular hypertrophy and atherosclerosis in an apolipoprotein-E-deficient mouse model Sai-Wang SETO∗ , Smriti M. KRISHNA∗ , Corey S. MORAN∗ , David LIU∗ and Jonathan GOLLEDGE∗ † ∗ Vascular Biology Unit, Queensland Research Centre for Peripheral Vascular Disease, School of Medicine and Dentistry, James Cook University, Townsville, Queensland 4811, Australia †Department of Vascular and Endovascular Surgery, Townsville Hospital, Townsville, Queensland 4814, Australia

Figure S1 Western blot analysis of PRR levels in the left ventricle of ApoE − / − mice at baseline (day 28) control or at day 56 that received vehicle, aliskiren (10 mg/kg of body weight per day) or aliskiren (50 mg/kg of body weight per day) following 4 weeks of AngII infusion

Correspondence: Professor Jonathan Golledge (email [email protected]).

 C The Authors Journal compilation  C 2014 Biochemical Society

S.-W. Seto and others

Figure S2

Representative ultrasound images of suprarenal aortas of ApoE − / − mice at baseline (day 28) control or at day 56 that received vehicle, aliskiren (10 mg/kg of body weight per day) or aliskiren (50 mg/kg of body weight per day) following 4 weeks of AngII infusion

 C The Authors Journal compilation  C 2014 Biochemical Society

Aliskiren limits aortic aneurysm

Figure S3

Images of harvested aortas of ApoE − / − mice that received vehicle, aliskiren (10 mg/kg of body weight per day) or aliskiren (50 mg/kg of body weight per day) following 4 weeks of AngII infusion

Received 15 July 2013/23 December 2013; accepted 30 January 2014 Published as Immediate Publication 30 January 2014, doi: 10.1042/CS20130382

 C The Authors Journal compilation  C 2014 Biochemical Society

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Aliskiren limits abdominal aortic aneurysm, ventricular hypertrophy and atherosclerosis in an apolipoprotein-E-deficient mouse model.

Aliskiren is a direct renin inhibitor developed to treat hypertension. Several clinical studies have suggested that aliskiren has beneficial effects o...
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