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Journal of the American College of Cardiology Vol. 47, No. 11, 2006© 2006 by the American College of Cardiology Foundation ISSN 0735-1097/06/$32.00P

RECLINICAL STUDY

evelopment of Mild Aortic Valvetenosis in a Rabbit Model of Hypertension

uis A. Cuniberti, PHD,* Pablo G. Stutzbach, FACC, MD,† Eduardo Guevara, FACC, MD,†ustavo G. Yannarelli, MSC,* Rubén P. Laguens, MD, PHD,† Roberto R. Favaloro, MD, PHD†uenos Aires, Argentina

OBJECTIVES This study was designed to investigate the association between hypertension and aortic valvestenosis (AVS) in a rabbit model.

BACKGROUND Degenerative AVS is a prevalent disease in elderly persons. Its molecular mechanisms remainunclear, in part because of the absence of experimental models. Epidemiologic data suggesta link between hypertension and AVS. However, there has been no evidence of a cause-effectrelationship.

METHODS New Zealand White rabbits were divided into two groups: 1) animals (n � 20) instrumentedaccording to one-kidney/one-clip hypertensive model; and 2) control animals (n � 10) shamoperated. Echocardiography (S12 MHz) was used to assess aortic valve (AV) morphology andfunction as well as left ventricular mass at baseline and after two and four months ofhypertension.

RESULTS Blood pressure and left ventricular mass increase were highly significant in the animal modelbut not in controls at two months, without noticeable AV function abnormalities. After 4months, however, 14 hypertensive survived animals showed a 14.6% reduction of AV area(0.240 � 0.063 cm2 vs. 0.205 � 0.060 cm2, p � 0.05), a 19.6% increase of AV thickness(0.056 � 0.011 cm vs. 0.067 � 0.010 cm, p � 0.001), a 40.4% increase of transvalvular meangradient (5.35 � 2.26 mm Hg vs. 7.51 � 3.73 mm Hg, p � 0.05) and a 63.6% increase oftransvalvular maximal gradient (10.56 � 3.68 mm Hg vs. 17.28 � 10.95 mm Hg, p � 0.05).Control animals did not show significant changes.

CONCLUSIONS We report a novel experimental model of AVS in rabbits that may prove useful in studyingthe progression of the disease and the efficacy of new treatments. The present findings supportthe hypothesis of a causal link between hypertension and AVS. (J Am Coll Cardiol 2006;

ublished by Elsevier Inc. doi:10.1016/j.jacc.2005.12.070

47:2303–9) © 2006 by the American College of Cardiology Foundation

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ortic valve stenosis (AVS) is a chronic progressive condi-ion and is the most common form of acquired valve heartisease in developed countries (1). Despite the incidence ofegenerative AVS and its increasing associated morbiditynd mortality, the molecular and cellular mechanisms of thisisorder remain unclear, in part because of the absence ofppropriate experimental models (2–4). Epidemiologictudies have identified several atherosclerotic risk factors forhe progression and outcome of AVS, including age, smok-ng, high cholesterol levels, low high-density lipoproteinholesterol levels, hypertension (HTA), lipoprotein(a) lev-ls, and diabetes (5–12). Although recent observations haveeinforced the suggested association between HTA and theevelopment of AVS (13,14), comorbidities associated withigh blood pressure (high blood cholesterol, hyperinsulin-mia, and so on) might be relevant confounding factorsathologically related to AVS progression, and so far noertain evidence of a causal link has been found. In fact,merging animal models of AVS have focused on the

From the *Lipid and Atherosclerosis Research Laboratory, Department of Pathol-gy, Favaloro University, Buenos Aires, Argentina; and the †Institute of Cardiologynd Cardiovascular Surgery of the Favaloro Foundation, Buenos Aires, Argentina.his work was supported by grant CID 107 from Favaloro University.

mManuscript received August 22, 2005; revised manuscript received October 6,

005, revised December 13, 2005.

evelopment of AVS essentially using hypercholesterolemiconditions as valvular insult (15–17).

Histologic studies show that an early step in therogression of AVS is oxidized lipid and lipoproteinepositions, macrophage and T-lymphocyte infiltration,nd microscopic calcification, particularly in the aorticide of the valve (18,19). This prominent feature fits withhe notion that arterial segments subjected to turbulentlood flow, such as those at branch points or arterialurfaces of aortic valve cups, show a predisposition toesion development that is intensified by hypertension20 –22). A possible mechanism that could explain theeleterious effects of HTA is, at least in part, the synergyetween elevated blood pressure and other atherogenictimuli to induce oxidative stress (23). The strikingorrelation between disturbed blood flow patterns andtherosclerosis has led to various recent studies (24,25) inn attempt to define a mechanistic role for hemodynam-cs forces in the pathogenesis of AVS.

We hypothesized that sustained hypertension per seould lead to valve damage with abnormalities in the valve’sorphology and function. Therefore, the aim of the present

tudy was to investigate the development of AVS in a rabbit

odel of hypertension.

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2304 Cuniberti et al. JACC Vol. 47, No. 11, 2006Emerging Model of AVS in Rabbits June 6, 2006:2303–9

ETHODS

nimals. Thirty adult New Zealand White rabbits (weight.7 to 3.2 kg) were randomly divided into two groups: 1)ypertensive animals (n � 20) induced by one-kidney/one-lip Goldblatt model (26), and 2) sham-operated normo-ensive controls (n � 10) with unilateral nephrectomy andontralateral renal artery non-clipping. Animals wereoused in individual cages, quarantined for seven daysefore surgery, fed normal chow and water ad libitum, andreated according to the Guide for the Care and Use ofaboratory Animals published by the U.S. National Insti-

utes of Health.Direct blood pressure measurements and blood samples

ere obtained from all animals before the surgical procedurend two and four months thereafter through catheterizationf the central ear artery. Serum samples were immediatelyrozen and kept at �80°C until laboratory determinationsere carried out.chocardiography. Transthoracic echocardiography waserformed by applying standard practice guidelines at base-

ine and at two and four months of follow-up. Animals wereedated with an intramuscular injection of ketamine (20g/kg) and xilazine (1 mg/kg). Ultrasound images were

btained with a 12-MHz phased-array probe connected toSonos 5500 echograph (Philips Medical Imaging, An-

over, Massachusetts). A parasternal short-axis view at theid left ventricular (LV) level was used to measure the

ollowing parameters: LV end-systolic and end-diastolicimensions and ventricular septum and LV posterior wallhickness. The LV mass was measured by M-mode echo-ardiography with the use of the Devereux formula (27).elative wall thickness (RWT) was measured as RWT �� (PWT/LVIDd). The peak and mean aortic valve flow

elocities were determined by continuous-wave Dopplerchocardiography by systematically sampling the flow fromifferent windows and averaging the values for four to fiveeats. The maximal instantaneous gradient across the aorticalve and the mean gradient were derived from aorticoppler velocities by the modified Bernoulli equation. The

ortic valve area was measured by the standard continuityquation in the same manner as in humans with suspectedVS (28). To minimize the intra- and inter-assay variabil-

ty, all echocardiographic imaging and analyses were carriedut by the same investigator, who was kept blind regarding

Abbreviations and AcronymsAV � aortic valveAVS � aortic valve stenosisHDL � high-density lipoproteinHTA � hypertensionLV � left ventricularPWT � posterior wall thicknessRWT � relative wall thickness

roup allocation of individual animals throughout the study.*

istology. After four months’ follow-up, rabbits received aethal anesthetic dose; the hearts were excised; and the aorticalves were immediately dissected free of surrounding tissue,insed in phosphate-buffered saline, and prepared for mac-oscopic evaluation. One suitable leaflet from each aorticalve was fixed in 4% neutral-buffered formalin and pro-essed for paraffin embedding. Serial cross-sections (5-�mhick) were prepared with hematoxylin and eosin and

asson’s trichromic staining for histologic analysis.tatistical analysis. Continuous variables are expressed asean � SD. Repeated-measurement one-way analysis of

ariance with the post hoc Newman-Kleus test was used forntragroup comparisons to assess changes in blood pressureevels and echocardiographic parameters over time. Thenpaired t test was used for intergroup comparisons. Arobability value �0.05 was considered statisticallyignificant.

ESULTS

linical and laboratory characteristics. All rabbits in theontrol group remained alive to the end of the study,hereas six animals in the hypertensive group died prema-

urely (five from histologically verified hemorrhagic strokend one from nonverified cause). Rabbits in group 1niformly developed hypertension within two to threeeeks from surgery. As shown in Table 1, at two months

ystolic and diastolic blood pressure levels significantlyncreased by 36.3% and 47%, respectively. Systolic, but notiastolic, blood pressure levels additionally increased to

able 1. Clinical and Laboratory Characteristics Over Studyeriod

Group IHTA

Group IIControl p Value

umber of rabbits(M/F)

Baseline 20 (9/11) 10 (5/5) —2 months 17 (8/9) 10 (5/5) —4 months 14 (6/8) 10 (5/5) —

ody weight, kgBaseline 3.1 � 0.4 2.9 � 0.4 NS2 months 3.7 � 0.5* 3.6 � 0.4* NS4 months 3.7 � 0.6* 3.7 � 0.5* NS

ystolic blood presure,mm Hg

Baseline 102.9 � 6.4 99.0 � 7.4 NS2 months 140.3 � 15.9* 99.0 � 5.2 �0.00014 months 161.5 � 23.0*† 101.2 � 7.5 �0.0001iastolic blood presure,

mm HgBaseline 81.8 � 3.5 80.0 � 4.1 NS2 months 120.2 � 16.1* 80.8 � 4.4 �0.00014 months 119.8 � 16.3* 80.0 � 6.2 �0.0001

otal cholesterol,mg/dl

Baseline 84.2 � 30.1 86.2 � 34.6 NS2 months 93.6 � 32.6 88.2 � 20.2 NS4 months 93.2 � 19.8 92.8 � 32.7 NS

p � 0.001 compared with baseline; †p � 0.001 compared with 2 months.HTA � hypertension; NS � not significant.

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2305JACC Vol. 47, No. 11, 2006 Cuniberti et al.June 6, 2006:2303–9 Emerging Model of AVS in Rabbits

6.9% over baseline at four months. Blood pressure levelsid not change in control animals throughout the studyeriod. Body weight increased similarly in animals from theontrol and the hypertensive groups (3.7 � 0.5 kg and 3.7 �.6 kg, respectively, p � NS). Total plasma cholesterol levelsere transiently and slightly higher after surgery to a similar

xtent in the control and the hypertensive group, but theyemained within the normal range throughout the study92.8 � 32.7 mg/dl and 93.2 � 19.8 mg/dl, respectively,� NS).chocardiographic evaluation of AVS progression. As

hown in Table 2, the thickness of ventricular septum andhe LV posterior wall, as well as RWT and LV mass,howed a highly significant increase after two and fouronths in the hypertensive group. At this time, aortic valve

AV) area significantly decreased by 14.6% (0.240 � 0.063m2 vs. 0.205 � 0.060 cm2, p � 0.05), whereas AVhickness, transvalvular mean gradient, and transvalvularaximal gradient significantly increased 19.6% (0.056 �

.011 cm vs. 0.067 � 0.010 cm, p � 0.001), 40.4% (5.35 �

.26 mm Hg vs. 7.51 � 3.73 mm Hg, p � 0.05), and 63.6%

able 2. Changes in Echocardiographic Characteristics Overtudy Period

Group IHTA

Group IIControl p Value

osterior wallthickness, mm

Baseline 3.10 � 0.46 3.11 � 0.36 NS2 months 3.57 � 0.53† 3.10 � 0.47 �0.054 months 3.87 � 0.61†‡ 3.13 � 0.37 �0.01

eptum thickness, mmBaseline 3.00 � 0.41 3.06 � 0.38 NS2 months 3.49 � 0.47† 3.01 � 0.36 �0.014 months 3.64 � 0.54†‡ 2.79 � 0.28 �0.001

V mass, gBaseline 4.48 � 0.95 4.18 � 0.77 NS2 months 5.22 � 1.07† 4.12 � 0.71 �0.014 months 6.40 � 2.26†� 3.93 � 0.81 �0.01

alve thickness, cmBaseline 0.056 � 0.011 0.055 � 0.007 NS2 months 0.058 � 0.010 0.055 � 0.007 NS4 months 0.067 � 0.010†� 0.053 � 0.007 �0.001

ortic valve area, cm2

Baseline 0.240 � 0.063 0.228 � 0.062 NS2 months 0.256 � 0.077 0.222 � 0.048 NS4 months 0.205 � 0.060*§ 0.236 � 0.041 NSaximal gradient,

mm HgBaseline 10.56 � 3.68 10.98 � 3.88 NS2 months 7.88 � 3.29 10.73 � 3.57 NS4 months 17.28 � 10.95*§ 9.15 � 3.64 �0.05ean gradient,

mm HgBaseline 5.35 � 2.26 5.07 � 1.93 NS2 months 4.43 � 2.47 4.84 � 1.58 NS4 months 7.51 � 3.73*‡ 4.14 � 1.47 �0.05

p � 0.05; †p � 0.001 compared with baseline; ‡p � 0.05; §p � 0.01; �p � 0.001ompared with 2 months.

HTA � hypertension; LV � left ventricular; NS � not significant.

10.56 � 3.68 mm Hg vs. 17.28 � 10.95 mm Hg, p � c

.05), respectively, in the hypertensive group. None of thesechocardiographic changes was evident in the control groupFig. 1). An intergroup comparison at four months revealedhat the hypertensive condition was associated with valvehickening (26.4%, p � 0.001), increased transvalvularean gradient (81.4%, p � 0.05), increased transvalvularaximal gradient (88.9%, p � 0.05), and a nonsignificant

eduction of AV area (13.1%).ssociation between severity of hypertension and AVS.s shown in Table 3, at four months of follow-up thereere positive associations between systolic blood pressure

nd echocardiographic measures of LV remodeling such aseptum thickness (p � 0.0001), posterior wall thicknessp � 0.05), and LV mass (p � 0.0005). Similarly, signifi-ant associations were observed between systolic bloodressure and measures of AVS, including valve thicknessp � 0.005) and maximal and mean gradients (p � 0.01 and

� 0.05, respectively). Finally, AV area changes did notorrelate with a hypertensive state (p � 0.10).

acroscopic and histologic evaluation. Visual examina-ion of excised aortic valves from hypertensive animalsevealed a progressive degeneration of the leaflet morphol-gy during follow-up. Whereas valves from control animalsad a clear glistening appearance (Fig. 2A), those fromypertensive animals presented signs of tissue hypertrophyFigs. 2B and 2C). Microscopic analysis showed a promi-ent lesion development on the aortic side of the valveharacterized by inflammation nodules extending to theortic base and to the tip region of the leaflets (Fig. 3).

ISCUSSION

he progression of calcified AVS has long been considered“wear and tear” phenomenon—a consequence of aging

oupled with increased hemodynamic/biomechanicaltresses on the aortic valve. For most of the patientsrogressing to severe stenosis, surgical intervention is re-uired (29,30). More recently, classical risk factors fortherosclerotic disease were implicated as accelerating con-itions of AVS. Although an association between hyperten-ion and AVS has been reported almost uniformly inultiple clinical studies (5,6,12–14,31), most of these ob-

ervations have been limited by small sample size; possibleelection bias; nonrandomization; cross-sectional nature;etrospective study design; and comorbidities associatedith hypertension, such as hypercholesterolemia, insulin

esistance, obesity, and so on.To our knowledge, this study is the first to demonstrate a

irect association between experimental hypertension andevelopment of aortic stenosis without the potential influ-nce of common clinically relevant confounding factors.he comparable results obtained by morphologic valve

ssessment, combined with functional echocardiographicnalysis and histopathology examination, support our

onclusions.

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2306 Cuniberti et al. JACC Vol. 47, No. 11, 2006Emerging Model of AVS in Rabbits June 6, 2006:2303–9

igure 1. Effect of hypertension (HTA) on aortic valve by echocardiographic assessment. Transthoracic echocardiography was used to measure theollowing morphologic and functional parameters at baseline and at two and four months of follow-up: valve thickness (A), aortic valve area (AVA) �

ontinuity (B), maximal gradient (C), and mean gradient (D) in hypertensive (left) and control rabbits (right).

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2307JACC Vol. 47, No. 11, 2006 Cuniberti et al.June 6, 2006:2303–9 Emerging Model of AVS in Rabbits

The association between hypertension and aortic valveisease may find several explanations. Pathogenically, hy-ertension may intensify the recognized hemodynamic dis-urbances that affect the aortic side of the valve (25,32), andhis degenerative process could be accelerated in our studyy the coupled high cardiac frequency of the rabbit.A prominent characteristic of degenerative aortic valvular

tenosis in humans is the presence of inflammatory infil-rates composed of macrophages, T-cells, and lipids, par-icularly in the fibrosa, the anatomic layer of the valveocated immediately below the endothelium on the aorticide of the valve (18). It is noteworthy that these character-stics are strikingly similar to the histologic findings in our

odel of inflammatory nodules in the aortic side of thealves, which suggest that the valvular lesions induced byypertension in the rabbit well reproduce one of the majorarly features of human valvular disease. Yet the preciseubcellular/molecular mechanisms responsible for the in-reased site-specific inflammatory activity of the valvesemain to be clarified.

In this study, we cannot exclude that various humoral/etabolic modifications inherent to the experimental modelight have contributed to the development of valvular

isease, including electrolyte imbalances, hyperreninemia,yperaldosteronemia, other neurohormonal changes, or a

igure 2. Aortic valve photographs from rabbits over study period. The a

Table 3. Relationship Between EchocardiograpFour Months of Follow-Up

85–119(n � 10)

(A)

PWT, mm 3.13 � 0.40Septum thickness, mm 2.79 � 0.28RWT 0.448 � 0.074LV mass, g 3.94 � 0.81Valve thickness, cm 0.053 � 0.007Aortic valve area, cm2 0.236 � 0.041Maximal gradient, mm Hg 9.15 � 3.64Mean gradient, mm Hg 4.14 � 1.47

p values derived from one-way ANOVA after Newman-Kleu0.05 compared with B.

LV � left ventricular; NS � not significant; PWT � posteblood pressure.

B) hypertension (HTA) after two months; (C) HTA after four months. The coypertensive animals show marked signs of tissue hypertrophy after four month

ild loss of renal function. Yet, one or more of thesearameters are also commonly altered in patients withypertension, which makes the model even more evocativef the human condition.Another advantage of this study as compared to other

revious experimental models of AVS is its use of two-imensional Doppler echocardiography as the test of choiceo assess valve morphology and to quantify the extent ofhanges in valvular function. This is pertinent in that thisiagnostic tool is considered the “noninvasive gold stan-ard” for the diagnosis of aortic valve stenosis (33). Thispproach was recently proposed by Drolet et al. (16) andas successfully reproduced in the present study.Although the ultrasonographic cardiac assessment of

mall animals may raise practical difficulties and inaccura-ies, the method may be useful and show fair reproducibilityn middle-size animals such as the rabbits weighing around.7 kg used in this protocol.Unlike other previously published models of hypertension

nduced by acute pressure overload (34,35), the model used inhis study more suitably reproduces a chronic hypertensive state36). Some of our notable findings can be summarized asollows: 1) a drop in transvalvular gradients at two months,hich indicates that intraventricular pressure is conserved inypertensive early state; and 2) a heterogeneous response to

valve is shown from the ascending aorta. (A) Control after four months;

arameters and Systolic Blood Pressure at

BP (mm Hg)

p Value

120–149(n � 6)

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150–180(n � 8)

(C)

78 � 0.65* 3.92 � 0.75* �0.0543 � 0.41† 3.87 � 0.58‡ �0.000161 � 0.054† 0.584 � 0.078‡ �0.00116 � 1.68† 7.11 � 1.92‡ �0.000568 � 0.010* 0.067 � 0.010† �0.00529 � 0.040 0.184 � 0.063 NS59 � 3.65 21.30 � 12.37†§ �0.0136 � 3.18 8.01 � 4.29* �0.05

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2308 Cuniberti et al. JACC Vol. 47, No. 11, 2006Emerging Model of AVS in Rabbits June 6, 2006:2303–9

ustained hypertension in terms of mean gradient and valveeaflet thickening, which indicates that valvular susceptibil-ty to hemodynamic damage has a significant interindividualariability. This heterogeneous response likely mimics thelinical condition epidemiologically associated with progres-ion of valvular aortic stenosis.

Although the present findings support a role for highlood pressure in the pathogenesis of mild AVS, furtherxperiments using other hypertensive models of experimen-al chronic hypertension in different animal species shoulde performed to confirm this association. Moreover, longererm studies (6 to 12 months) are required to evaluatehether the early lesions induced by hypertension in ourodel will ultimately result in advanced disease with pro-

ressive leaflet thickening and eventually calcification andevere aortic valve stenosis.

Finally, a role of atherogenic lipoproteins as risk factors,ot only for classical atherosclerosis but also for aortic valvetenosis, has recently been emphasized, and this notion isupported by several sources of data. First, at least threexperimental models of hypercholesterolemia-induced valvealcification and stenosis were recently reported (15–17);econd, high blood lipids are epidemiologically associatedith progression of AVS; and third, there is some contro-

igure 3. Light microscopy of rabbit aortic valves after four months withA) Low-magnification view (�25); (B) high-magnification view (�400)hickness and development of inflammation nodules.

ersial evidence that progression of AVS can be reduced by v

tatins owing to their lipid-lowering activity and/or some ofheir pleiotropic effects.

Although in the studies reported herein the animals wereormolipidemic, we cannot exclude a pathogenic role oflood lipids. Indeed, hypertension augments infiltration ofacromolecules, proteins, and lipoproteins through the

ndothelial barrier and promotes vascular oxidative stress23), potentially augmenting the susceptibility of the vesselven to physiologic plasma levels of atherogenic lipopro-eins. Thus, we speculate that lipids beneath a hypertensivetate, even within the normal range, may represent a continu-us risk factor for AVS. Furthermore, it could be interesting tossess the ability of statins to prevent the progression of mildVS in the hypertensive-normolipidemic rabbit model pre-

ented in this study.In summary, we report here an experimental animalodel of acquired AVS induced by high blood pressure,hich may prove useful to investigate the precise mecha-isms underlying the association between hypertension andVS and the efficacy of different medical treatments toelay, or even hinder, the advancement of the disease.

cknowledgmentshe authors thank Dr. María I. Besanson and Pedro Iguain for

son trichrome stain. A1 and B1 � control; A2 and B2 � hypertension.tic valves from hypertensive animals are characterized by increased valve

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2309JACC Vol. 47, No. 11, 2006 Cuniberti et al.June 6, 2006:2303–9 Emerging Model of AVS in Rabbits

auna for assistance during surgical procedures, and Dr. Pabloerba, MD, for his review of the manuscript.

eprint requests and correspondence: Dr. Luis A. Cuniberti,aboratorio de Investigación en Lípidos y Aterosclerosis, Univer-

idad Favaloro, Solis 453, 4to piso, (C1078AAI), Buenos Aires,rgentina. E-mail: cuniberti@favaloro.edu.ar.

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