PRECLINICAL STUDY
Angiotensin Receptor-1 Blocker Inhibits Atherosclerotic Changes and Endothelial Disruption of the Aortic Valve in Hypercholesterolemic Rabbits
Kumiko Arishiro, MD*,
Masaaki Hoshiga, MD, PhD*,*,
Nobuyuki Negoro, MD, PhD*,
Denan Jin, MD, PhD ,
Shinji Takai, PhD,
Mizuo Miyazaki, MD, PhD,
Tadashi Ishihara, MD, PhD* and
Toshiaki Hanafusa, MD, PhD*
* First Department of Internal Medicine, Osaka Medical College, Takatsuki, Osaka, Japan
Department of Pharmacology, Osaka Medical College, Takatsuki, Osaka, Japan.
Manuscript received August 23, 2006;
revised manuscript received November 1, 2006,
accepted November 23, 2006.
* Reprint requests and correspondence: Dr. Masaaki Hoshiga, First Department of Internal Medicine, Osaka Medical College, 2-7 Daigakumachi, Takatsuki, Osaka 569-8686, Japan. (Email: in1026{at}poh.osaka-med.ac.jp).
This work was partly presented at the 55th Annual Scientific Session of the American College of Cardiology, Atlanta, Georgia, March 11 to 14, 2006, and published in abstract form (J Am Coll Cardiol 2006;47 Suppl A:284A).
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Abstract
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Objectives: We sought to examine the effect of angiotensin receptor blocker (ARB) on the formation of lesions in the aortic valves of hypercholesterolemic rabbits.
Background: Recently, atherosclerosis has been recognized as a mechanism that is responsible for calcific aortic stenosis. The effect of ARBs might help to prevent aortic stenosis because they have multiple antiatherosclerotic effects.
Methods: Male Japanese white rabbits (n = 36) were separated as follows: control with chow diet (C) and vehicle (V) groups, both of which were fed a 1% cholesterol diet for 8 weeks, and an ARB group (A), which was fed a 1% cholesterol diet for 8 weeks with ARB (olmesartan, 1 mg/kg/day) for the last 4 weeks.
Results: This dose of olmesartan did not affect either blood pressure or cholesterol levels. Dietary cholesterol induced fatty deposition with macrophage accumulation and osteopontin coexpression in valve leaflets, whereas ARB decreased macrophage accumulation (% area: V, 9.3 ± 0.34; A, 1.4 ± 0.30; p = 0.003) and osteopontin expression (p = 0.017). Angiotensin-converting enzyme was also up-regulated in V and decreased by olmesartan (p = 0.015). Immunohistochemistry with anti-CD31 antibody revealed that dietary cholesterol disrupted and olmesartan preserved endothelial integrity on the lesion-prone aortic side of the valve (% CD31-positive circumference: V, 30 ± 3.7; A, 62 ± 4.8; p = 0.003). Numbers of alpha-smooth muscle actin-positive myofibroblasts were increased in V and decreased by olmesartan (p = 0.003). Real-time polymerase chain reaction revealed that increased amounts of messenger ribonucleic acid for osteoblast-specific transcription factor core binding factor alpha-1 in V were diminished by olmesartan.
Conclusions: Atherosclerotic changes in the aortic valves of rabbits fed with cholesterol were inhibited by ARB, whereas endo-thelial integrity was preserved and transdifferentiation into myofibroblasts and/or osteoblasts in valve leaflets was inhibited.
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Abbreviations and Acronyms
| | ACE = angiotensin-converting enzyme | | ARB = angiotensin receptor blocker | | AS = aortic stenosis | | Cbfa-1 = core binding factor alpha-1 | | DNA = deoxyribonucleic acid | | eNOS = endothelial nitric oxide synthase | | RNA = ribonucleic acid | | RT-PCR = reverse transcriptional-polymerase chain reaction | | TGF = transforming growth factor |
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Calcific aortic valve stenosis is a common medical condition in the elderly (1) and it is currently the most common indication for valve replacement (2). Accumulating evidence suggests that the calcific aortic stenosis (AS) disease process has many similarities to vascular atherosclerosis (3–8). The prevention or retardation of AS progression is evolving but this has not yet been achieved in the clinical setting. Two classes of drugs, namely hydroxymethylglutaryl coenzyme-A reductase inhibitors (statins) and inhibitors of the renin-angiotensin system, might be useful because they have multiple antiatherosclerotic effects (9,10). Rajammanaan et al. (11) have reported recently that atorvastatin inhibits atherosclerotic changes of aortic valves in cholesterol-fed rabbit models. Clinical trials of statins for patients with AS presently are underway (6,12–15).
In addition, the renin-angiotensin system might be involved in AS progression because angiotensin-converting enzyme (ACE) is up-regulated in human calcific aortic valves (16), which raises the possibility that ACE inhibitors or angiotensin receptor blocker (ARB) might inhibit AS progression, but no animal data and conflicting clinical data have been generated (14,17). The present study tests whether the ARB (olmesartan) prevents atherosclerotic changes in the aortic valves of rabbit models fed dietary cholesterol.
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Methods
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Animal experiments.
Thirty-six male Japanese white rabbits (2.5 to 3.0 kg) were housed at room temperature with a 12-h light-dark cycle and provided with tap water ad libitum throughout the experimental period. All animal studies were performed according to the "Position of the American Heart Association on Research Animal Use," adopted by The American Heart Association on November 11, 1984, and proceeded with the approval of the Institutional Animal Care and Use Committee of the Osaka Medical College.
After 4 weeks of a normal standard diet, the rabbits were separated into 3 groups. The control group (C; n = 12) remained on the normal diet until sacrifice. The olmesartan (A; n = 12) and vehicle (V; n = 12) groups received a diet containing 1% cholesterol (Oriental Yeast Co., Osaka, Japan) for 8 weeks until sacrifice. Olmesartan (a gift from Sankyo Pharmaceutical Co., Tokyo, Japan) was prepared by emulsification with carboxymethyl-cellulose in water. The suspension (1 mg/kg/day of olmesartan) was administered orally via a gastric tube to the A group during the last 4 weeks. The V group received 1% carboxymethyl-cellulose by mouth via a gastric tube during the last 4 weeks.
Blood pressure was directly measured at the central artery of the ear using a SURFLO Flash catheter (Terumo, Tokyo, Japan) and transducer 30 min before sacrifice. Blood samples were collected thereafter, and total plasma cholesterol and triglycerides were measured enzymatically (Sigma-Aldrich Co., St. Louis, Missouri). At the end of the protocol, the rabbits were sacrificed by venous injection with an overdose of sodium pentobarbital.
Tissue preparation.
After sacrifice, the ascending aortic section containing the aortic valve was dissected and rinsed in phosphate-buffered saline. The aortic root was opened longitudinally, and one leaflet from each aortic valve dissected with the adjacent aorta was fixed in 4% paraformaldehyde in phosphate-buffered saline for 1 h. Thereafter, samples for histological and immunohistochemical analyses were taken vertically through the cusp and sinus near the center of the leaflet according to the method of Otto et al. (3), embedded in Tissue-Tek O.C.T. compound (Sakura Finetechnical, Tokyo, Japan), and snap-frozen in liquid nitrogen. The remaining 2 leaflets from each valve were frozen immediately for mRNA detection.
Histological and immunohistochemical staining.
Frozen sections were cut into 3- to 5-µm slices and stained with hematoxylin and eosin as well as Oil red O. Immunohistochemical studies were performed using the following primary antibodies; a mouse monoclonal antibody against rabbit macrophages (RAM-11, 1:1,000 dilution, DAKO, Carpinteria, California), anti- -smooth muscle actin (1A4, 1:1,000 dilution, DAKO), osteopontin (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, California), anti-ACE (1:200, Chemicon International, a Millipore Company, Temecula, California), anti-CD31 (1:100, DAKO), and antiendothelial nitric oxide synthase (anti-eNOS) (1:50, BD Transduction Laboratories, Lexington, Kentucky).
Frozen sections were air-dried, fixed in acetone, and endogenous peroxidase was block by incubation with 3% hydrogen peroxide (H2O2). Tissue sections were incubated overnight with primary antibodies at 4°C, then with biotinylated anti-mouse immunoglobulin (E0354, 1:200 dilution, DAKO) at room temperature for 30 min. The sections were visualized using NiCl2-enhanced 3,3'-diaminobenzidine (LY. 186, Dojin Chemical, Kumamoto, Japan) and counterstained with 0.25% methyl green or stained with AEC (Nichirei, Tokyo, Japan) without counterstaining.
Oil red O staining and immunohistochemistry other than for CD31 and eNOS were quantified by computer-assisted image analysis using NIH-image (1.61). Positive areas are expressed as ratios (%) of whole valve leaflet areas. To quantify endothelial integrity using CD31- and eNOS-immunohistochemistry, each positive length on the valve surface was measured and calculated as a ratio (%) of the total circumference of the valve surface using computer software (MCID 4.0, Imaging Research Inc., St. Catherines, Ontario, Canada).
Ribonucleic acid (RNA) isolation and real-time reverse transcriptional-polymerase chain reaction (RT-PCR).
We assessed valvular gene expression by using RT-PCR. Frozen valve specimens were disrupted in TRIzol reagent (Invitrogen, Carlsbad, California) using a motorized homogenizer (Microtec Nition, Chiba, Japan). Total RNA extracted using the RNeasy Mini Kit (Qiagen, Valencia, California) was purified and determined spectrophotometrically (ratio 260/280 nm) and by gel electrophoresis. For reverse transcription, complementary deoxyribonucleic acid (DNA) was synthesized from 2 µg of total RNA by Avian Myeloblastosis Virus reverse transcriptase using first strand complementary DNA synthesis as described in the protocol supplied with the RT-PCR Kit (Roche Diagnostics, Basel, Switzerland).
The synthesized complementary DNA was quantified by SYBRGreen I dye PCR analysis of each gene using the LightCycler system according to the manufacturer’s protocol (Roche Diagnostics).
The primers were: core binding factor alpha-1 (Cbfa-1) forward, 5'-CCGCACGACAACCGCACCAT-3' and reverse, 5'-CGCTCCGGCCCACAAATCTC-3' (289 base pairs) as described by Komori et al. (18) and GAPDH forward, 5'-TCACCAGGGCTGCTTTTAAC-3' and reverse, 5'-GCGTTGCTGACAATCTTGAG-3' (397 base pairs). The amount of Cbfa-1 complementary DNA was normalized relative to the respective quantity of glyceraldehyde-3-phosphate dehydrogenase complementary DNA.
Statistical analysis.
Quantitative data are expressed as means ± SEM. Data were statistically analyzed using an unpaired t-test for parametric data or Mann-Whitney test for nonparametric data (only for real-time RT-PCR data) using a Macintosh computer (Apple, Cupertino, California) with StatView software (SAS Institute, Cary, North Carolina. Values of p < 0.05 were considered significant.
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Results
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Serum cholesterol increased in rabbits after 8 weeks on a high cholesterol diet from 32.3 ± 9.4 mg/dl in the control group (C) to 1,510 ± 265 mg/dl in the vehicle group (V) and 2,027 ± 239 mg/dl in the olmesartan group (A). The difference in cholesterol levels between A and V groups was not statistically significant. Blood pressures were similar for all 3 groups (C: 100 ± 7.0/72.7 ± 4.6 mm Hg, V: 100 ± 9.7/75.7 ± 6.0 mm Hg and A: 94.7 ± 11.3/72.3 ±5.8 mm Hg; p = NS).
Cholesterol feeding induced lipid accumulation with macrophage infiltration, especially on the aortic side of the valve (Fig. 1A). Olmesartan significantly decreased lipid deposition (% Oil red O area: V, 11.5 ± 2.1; A, 3.4 ± 0.48; p = 0.005) (Fig. 1B) and macrophage accumulation in the valve leaflet (% RAM-11 area: V, 9.3 ± 0.34; A, 1.4 ± 0.30; p = 0.003) (Fig. 1C). Osteopontin, a calcification mediator, was present at very low levels in control valves (% area in C: 0.30 ± 0.34%) but was increased by cholesterol feeding (% area in V: 9.0 ± 0.50%). Olmesartan treatment was associated with a significant decrease in osteopontin (% area in A: 3.6 ± 0.34%, p = 0.017 vs. V) (Fig. 1D).
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Figure 1 Olmesartan Inhibits Lesion Formation of the Aortic Valve in Hypercholesterolemic Rabbits
(A) a, d, g = control; b, e, h = hypercholesterolemic; c, f, i = hypercholesterolemic olmesartan treated. a to c = hematoxylin and eosin (H&E) staining; d to f = macrophage (RAM11) staining; g to i = osteopontin staining. Inset in b shows Oil red O staining. Insets in e and h represent positive staining at greater magnification. Arrowheads indicate aortic side of valve leaflet. Bar = 200 µm. Quantitation of Oil red O (B), macrophage (C), and osteopontin (D)-stained areas corrected for total area. A = olmesartan; Ao = aorta; V = vehicle.
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Cholesterol feeding was associated with a significant increase in leaflet ACE immunostaining (p = 0.001), which was substantially blunted with olmesartan (C: 1.7 ± 1.9%, V: 35 ± 17% and A: 9.9 ± 5.4%, p = 0.015 vs. V) (Fig. 2).
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Figure 2 Olmesartan Inhibits Up-Regulation of ACE in the Aortic Valve Induced by Dietary Cholesterol
Angiotensin-converting enzyme (ACE) immunostaining (black) in sections of aortic valve from control (A), hypercholesterolemic (B), and hypercholesterolemic olmesartan-treated (C) rabbits. Sections are counterstained with methyl green. Inset in B shows the aorta from the same section. Arrowheads indicate aortic side of the valve leaflet. (D) Quantitation of ACE-stained area corrected for total area. Ao = aorta. Bar = 200 µm.
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We next analyzed the endothelial integrity of the aortic valve in this model. CD31 immunostaining (Fig. 3) showed that endothelial integrity was impaired on the aortic surface of the leaflets after cholesterol feeding (p = 0.003), although it was maintained on the left ventricular surface. Olmesartan significantly reversed this disruption on the aortic surface of the valve leaflets (C: 66 ± 6.2%, V: 30 ± 3.7% and A: 62 ± 4.8%, p = 0.003 vs. V). Figure 4
shows immunohistochemical staining for the endothelial marker, eNOS. Because the intensity was similar on both the aortic and ventricular surfaces of the valve leaflets, we analyzed eNOS expression on the whole valve surface. Olmesartan increased eNOS expression as compared with high-cholesterol group (V: 24 ± 5.0 vs. A: 48 ± 5.4%, p = 0.008).
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Figure 3 Olmesartan Preserves Endothelial Integrity of the Aortic Valve
CD31 immunostaining of aortic valve endothelium from control (A), hypercholesterolemic (B), and hypercholesterolemic olmesartan-treated (C) rabbits. Positive staining is shown as red. Arrowheads indicate aortic side of valve leaflet. (D) Quantitation of CD31-positive circumference corrected for valve surface (on each side of valve). Bar = 200 µm. A = olmesartan; Ao = aorta; C = control; V = vehicle.
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Figure 4 Olmesartan Restores eNOS Expression in the Aortic Valve
Immunostaining for endothelial nitric oxide synthase (eNOS) in aortic valve from control (A), hypercholesterolemic (B), and hypercholesterolemic olmesartan-treated (C) rabbits. Positive staining is shown as red. Arrowheads indicate aortic side of valve leaflet. (D) Quantitation of eNOS-positive circumference corrected for total valve surface. Ao = aorta. Bar = 200 µm.
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The transition from valvular interstitial cells to myofibroblasts is a critical change in valvular pathology. We examined myofibroblasts by immunohistochemical staining using alpha-smooth muscle actin antibody (Fig. 5). In the control, chow-fed group, alpha-smooth muscle actin-positive myofibroblasts were sparse and dietary cholesterol obviously increased the alpha-smooth muscle actin-positive area (% area C: 0.34 ± 0.13 vs. V: 7.5 ± 2.2; p = 0.008). Olmesartan decreased the number of myofibroblasts in the valve leaflet (% area: 0.34 ± 0.22%, p = 0.003 vs. V).
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Figure 5 Olmesartan Decreases the Number of Myofibroblasts in the Aortic Valve
Immunostaining for alpha-smooth muscle actin (black) in sections of aortic valve from control (A), hypercholesterolemic (B), and hypercholesterolemic olmesartan-treated (C) rabbits. Sections are counterstained with methyl green. Arrowheads indicate aortic side of valve leaflet. (D) Quantitation of alpha-smooth muscle-stained area corrected for total area. Ao = aorta. Bar = 200 µm.
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Finally, Cbfa-1 messenger RNA expression was examined by real-time RT-PCR (Fig. 6). Dietary cholesterol increased, whereas olmesartan significantly decreased Cbfa-1 mRNA expression (analyzed by Mann-Whitney test).
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Figure 6 Olmesartan Diminishes Cbfa-1 mRNA Expression in the Aortic Valve
Real-time reverse transcriptional polymerase chain reaction of core binding factor alpha-1 (Cbfa-1) message from aortic valves performed as detailed in the Methods section (n =12 rabbits per group; *p < 0.05). GAPDH = glyceraldehyde-3-phosphate dehydrogenase; mRNA = messenger ribonucleic acid.
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Discussion
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The present study demonstrates that the ARB (olmesartan) inhibits lesion formation in aortic valve leaflets of hypercholesterolemic rabbits without significantly altering plasma cholesterol levels or blood pressure. Together with inhibiting the formation of aortic valve lesions, treatment with olmesartan was associated with maintained endothelial integrity and inhibition of the transdifferentiation of valvular fibroblasts into myofibroblasts. To our knowledge, this is the first evidence that a blocker of the renin-angiotensin system can inhibit lesion formation in an animal model of aortic valve disease.
Others have reported that activation of renin-angiotensin system is involved in the pathogenesis of aortic valve lesions (16,19). These reports indicated that ACE, angiotensin II, and the angiotensin II type 1 receptor are upregulated in diseased aortic valves of humans. The present study showed that dietary cholesterol upregulated ACE in the aortic valve, suggesting that in this rabbit model, originally described by Rajamannan et al. (11,20): 1) activation of the renin-angiotensin system is a characteristic of this model, as is the case with human aortic valve lesions (16), and 2) blockade of the renin-angiotensin system inhibits the formation of lesions. The mechanisms by which ARB inhibits lesion formation in the aortic valve are not known but may be similar to those implicated as antiatherogenic effects, such as suppressing inflammation (21), inhibiting macrophage accumulation (22), and decreasing oxidative stress (23,24).
In addition, ACE immunostaining was diffusely positive in the valve leaflets after cholesterol feeding (Fig. 2B), suggesting that ACE is associated with not only macrophages but also low-density lipoprotein particles in extracellular matrix such as biglycan and versican, as already shown in human aortic valves (16). Treatment with ARBs decreased ACE expression in the valve leaflets (Fig. 2C). One proposed mechanism of this effect is that ARBs can decrease extracellular matrix production, which is already shown in rat myocardial infarct models (25), probably through an inhibition of paracrine release of transforming growth factor (TGF)-beta (26). This down-regulation of extracellular matrix may lead to decreased retention of lipoproteins (27), which contain ACE (16), as discussed in recent review by O’Brien et al. (28).
Disruption of endothelial integrity may be an important contributor to the formation of aortic valve lesions as well as to the development of atherosclerosis (29). The present study demonstrates, using CD31 immunohistochemistry, that the endothelial integrity of the aortic valve is disrupted in animals fed dietary cholesterol. Interestingly, this disruption is localized to the aortic, rather than the ventricular, surface of the aortic valve leaflet. Our findings with CD31 antibody are consistent with various reports showing that the aortic side of the aortic valve is more susceptible to lesion formation (30,31). In addition, this study demonstrates olmesartan not only maintains endothelial integrity but also preserves eNOS expression. The mechanisms by which ARBs reduce endothelial damage are not known, but may be similar to those reported for vascular injury (32).
In addition, olmesartan decreased myofibroblast accumulation in the valve leaflets. Myofibroblasts are believed to differentiate from valvular interstitial cells and TGF-beta is critical for this trans-differentiation (33,34). Moreover, angiotensin II can stimulate TGF-beta expression in cardiac myofibroblasts (35). Thus the inhibition of differentiation into myofibroblasts in the valve leaflet might be one mechanism whereby ARB inhibited lesion formation in the present study.
Findings from cell culture (33) and rabbit models (11) indicate that myofibroblasts in valve leaflets transdifferentiate into osteoblast-like cells. This study confirms that dietary cholesterol induces the osteoblast-specific transcription factor, Cbfa-1 (11), but now also demonstrates that ARB treatment decreases Cbfa-1 expression. This result confirms that myofibroblast-osteoblast transition, which is a critical pathway in development of calcific valve lesions, is a component of this model but also demonstrates that this transdifferentiation is inhibited by ARB treatment.
Rajamannan et al. (11) have demonstrated that statin inhibits the formation of lesions in the aortic valve of the rabbit model. However, the clinical efficacy of statin therapy for calcific aortic valves is now controversial. Small, retrospective studies have demonstrated a lower rate of progression of AS with statin therapy (6,12–14). However, more recently, a small, prospective randomized trial showed no benefit of statin therapy in inhibiting AS progression during about a 2-year follow-up (15). These results indicate that, in the clinical setting, effective statin therapy might require longer treatment periods or targeting of the disease at an earlier disease stage, i.e., aortic sclerosis (28). The present study uncovered the first evidence that ARBs are effective against aortic valve lesions in the rabbit model, thus encouraging clinical studies of blockers of the renin-angiotensin system in patients with calcific aortic valve disease. One concern is that the therapy might target an earlier stage of disease because we used the same model as Rajamanaan et al. (11).
Here, we used an ARB to block the renin-angiotensin system. The question now arises as to whether ARBs or ACE-inhibitors are superior in preventing the formation of aortic valve lesions. Because rabbits do not produce chymase, another angiotensin II synthesizing enzyme, this model might not be able to address this issue. However, recent data have shown that the expression of chymase as well as ACE is increased in the diseased aortic valve in humans (19), raising the possibility that ARBs have more potential than ACE inhibitors for preventing AS.
Study limitations.
This study has several limitations. First, the rabbit model in this study is a rapidly progressive model, which is different from degenerative calcific aortic valve in human. Therefore, we must be always cautious when we apply short-time result in animal model to human slowly progressive disease, such as atherosclerosis and aortic valve calcification. Second, the administration of olmesartan caused a small reduction in systolic blood pressure, though the magnitude of reduction was not statistically significant. Although the range of blood pressure was normal and this dose of olmesartan reportedly does not affect blood pressure in normotensive rabbits (36), the possibility remains that this small decrease in blood pressure might affect the formation of valve lesions. Further studies are needed to determine whether lowering blood pressure with agents other than an ARB can inhibit aortic valve lesion formation.
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Acknowledgments
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The authors thank Dr. K. O’Brien (University of Washington, Seattle) for helpful comments and Ms. E. Kohbayashi for excellent technical support.
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Footnotes
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This study was supported in part by the High-Tech Research Program of Osaka Medical College, Osaka, Japan.
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Abstract
Methods