Prolonged Effects of Short-Term Fosinoprilon Blood Pressure and Vascular Morphologyand Function in RatsDamiano Rizzoni, Maurizio Castellano, Enzo Porteri, Giorgio Bettoni, Maria Lorenza Muiesan,Angelo Cinelli, Roberto Zulli, and Enrico Agabiti Rosei

The aim of this study was to evaluate the delayedeffects of an angiotensin converting enzyme (ACE)inhibitor on blood pressure and on structural andfunctional alterations in mesenteric smallresistance arteries of spontaneously hypertensiverats (SHR). The ACE inhibitor fosinopril (25 mg/kg/day) was administered according to threedifferent schedules: in one group of SHR from 4 to8 weeks of age (n 5 12), in a second group from 8to 12 weeks of age (n 5 15), and in a third groupfrom 4 to 12 weeks of age (n 5 12). Eighteenuntreated SHR and 18 untreated Wistar-Kyoto ratsserved as controls. About half the animals in eachgroup were killed at 13 weeks of age, and theremaining were killed at 38 weeks of age. Afterdeath, relative left ventricular mass (left ventricularweight/body weight) was calculated. Vascularmorphology (media : lumen ratio) and function(responses to norepinephrine and acetylcholine) inmesenteric small resistance arteries were thenassessed using a micromyographic technique.

Short-term fosinopril, given either before or after

the development of hypertension, persistentlyreduced (but did not normalize) systolic bloodpressure, vascular structural alterations, andreactivity to norepinephrine in mesentericresistance arteries in SHR. These favorable effectswere maintained at least for 26 to 30 weeks aftertreatment withdrawal. The endothelium-dependentvasodilator response to acetylcholine was improvedat 13 but not at 38 weeks of age, in treated SHR.

Therefore, the vascular response tonorepinephrine seems to be dependent mainly onthe structure of the vessels, whereas endothelialfunction is probably more linked to thehemodynamic load. Am J Hypertens 1997;10:1034–1043 © 1997 American Journal ofHypertension, Ltd.

KEY WORDS: Inbred spontaneously hypertensive rats,angiotensin converting enzyme inhibitors, vascularresistance, hypertrophy, fosinopril, acetylcholine,endothelium, endothelium-derived relaxing factor(EDRF).

Received May 31, 1996. Accepted February 27, 1997.From the Cattedra di Semeiotica e Metodologia Medica, U.O.P.

Scienze Mediche, University of Brescia, Brescia, Italy.This work has been performed under the auspices of the Euro-

pean Working Party on Resistance Artery Disease (EURAD), sup-ported by the European Community under the BIOMED 1 program.

Address correspondence and reprint requests to Prof. EnricoAgabiti Rosei, Cattedra di Semeiotica e Metodologia Medica, Sci-enze Mediche, University of Brescia, c/o 1a Medicina, Spedali Civili.25100 Brescia, Italy.

The development of hypertension in the spon-taneously hypertensive rat (SHR) is usuallyassociated with the presence of vascularstructural alteration1,2 and impairment of

endothelial function.3–6 Both these factors may con-tribute to an imbalance between vasoconstriction andvasodilatation.3,7 The prevention or regression ofstructural alterations in small arteries is associated

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© 1997 by the American Journal of Hypertension, Ltd. 0895-7061/97/$17.00Published by Elsevier Science, Inc. PII S0895-7061(97)00166-0

with a significant attenuation of the development ofhigh blood pressure values.8–18

Several angiotensin converting enzyme (ACE) in-hibitors (captopril, enalapril, cilazapril, and perindo-pril) have been shown to possess beneficial effects onvascular structure and were able to determine a moreor less pronounced delay in the development or rede-velopment of hypertension,8–17 especially if treatmentwas started very early, before the onset of overt hy-pertension. In fact, these studies suggest the possibleexistence of a “critical phase” for the pharmacologicalintervention during the development of hypertensionand for the possible interference with the long-termcourse of the disease.

It has also been observed that chronic therapy withACE inhibitors is able to induce an improvement ofthe vasodilator response to acetylcholine (an index ofthe endothelial function) in SHR.13,19–21 However, it isnot known if this favorable response is still presentafter a prolonged treatment withdrawal.

The ACE inhibitor fosinopril is a relatively newcompound showing interesting pharmacokinetic andpharmacodynamic properties. In addition, fosinoprilshares with zofenopril the highest lipid solubilityamong the ACE inhibitors currently available,22 andcould therefore be more effective in penetrating vas-cular tissue sites and inhibiting actions of the renin-angiotensin system within the walls of arteries and ofthe heart.

Therefore, the aim of our study was to evaluate thedelayed effects of the ACE inhibitor fosinopril, givenbefore or after the development of hypertension, onblood pressure and cardiovascular structural alter-ation, as well as on functional responses to norepi-nephrine and acetylcholine of mesenteric resistancearteries of SHR.

METHODS

Seventy-five male rats [57 SHR and 18 Wistar-Kyoto(WKY)] were included in the study. Animals wereobtained from Charles River Laboratory (Calco, Italy).The rats were housed two to a cage in a room in whichtemperature was controlled between 23°C and 25°C,and a 12-h light/dark cycle was maintained. Food andwater were supplied ad libitum. Fosinopril was ad-ministered in drinking water, at the dosage of 25mg/kg per day, according to three different schedules:in one group of SHR from the fourth to the eighthweek of age (FOS 4 to 8, n 5 12), in a second groupfrom the eighth to the twelfth week of age (FOS 8 to12, n 5 15), and in a third group from the fourth to thetwelfth week of age (FOS 4 to 12, n 5 12). EighteenSHR and 18 WKY were kept untreated as controls.About half the animals of each group were killed atthe age of 13 weeks, whereas the remaining were

killed at 38 weeks. The protocol of the study is similar,in part, to that proposed by Harrap and colleagues.9

Systolic blood pressure values and heart rate weremeasured noninvasively (tail cuff method, IITC LifeScience Instruments, Woodland Hills, CA) in con-scious rats every week. Animals were warmed prior toblood pressure measurement. More than 12 measure-ments per session were performed, and then the val-ues were averaged to obtain one weekly pressure,after discarding the first 3 measurements.

On the day of death, the animals were weighed andthen killed by decapitation. The heart was promptlydissected, dried, and weighed. The weight of the leftventricle plus septum was measured and the relativeleft ventricular mass (RLVM: left ventricular plus sep-tum weight/body weight) was calculated. At the sametime, mesenteric vessels corresponding to the secondbranch (about 140 to 200 mm of average diameter inrelaxed conditions, 2 mm long) were dissected fromthe surrounding fat from each rat. Vessels weremounted as a ring preparation on an isometric myo-graph (410 A, JP Trading, Aarhus, Denmark), bythreading onto two stainless steel wires (40 mm diam-eter). The wires were attached to a force transducerand micrometer, respectively, as previously describedby Mulvany and coworkers.1,2 Vessels were warmedto 37°C and allowed to equilibrate for at least 30 minin physiological saline solution (PSS) with the follow-ing composition (in mmol/L): NaCl 119, NaHCO3 24,KCl 4.7, KH2PO4 1.18, MgSO4 1.17, CaCl2 2.5 andglucose 5.5, kept constantly at 37°C and bubbled with5% CO2 in O2. The vessel internal circumference wasset to give a wall tension of 0.1 mN/mm.

Vessel wall and media thicknesses were measuredat 12 sites that were then averaged, using a lightmicroscope with immersion lens (Lab 20, Carl ZeissS.p.A., Milan, Italy) at 6003 magnification, which pro-vides a resolution of 0.2 mm. Lower magnification wasused for measurement of the distance between thewires and length of the blood vessel. The resting ten-sion-internal circumference relation was determined,and vessels were set to the normalized circumferenceL1, where L1 5 0.9 L100 and L100 is the internal circum-ference the vessels would have had in vivo, whenrelaxed and under a transmural of 100 mm Hg asdescribed previously by Mulvany et al.1,2 From L1 thenormalized internal diameter L1 was calculated. As-suming that the cross-sectional area remains constantwhen the vessel is extended to L1, the wall and mediathickness were automatically calculated also in nor-malized condition. Wall and media thickness of bloodvessels in normalized condition (vessels extended toL1) were obtained assuming a constant wall and me-dia volume, from wall and media cross-sectional areacalculated from wall and media thickness measured inunstretched vessels, as previously described.1,2,23

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A “remodeling index” was calculated in untreatedSHR and WKY, according to Heagerty and col-leagues,24 expanding a previous observation of Baum-bach and Heistad.25 It quantifies how much of thevascular structural alteration may be explained by arearrangement of the same material around a nar-rowed lumen, without cell growth.

The formula for calculation of remodeling index isas follows:

ID remodeled 5 Î~EDh!2 2 4 3 CSAn/p

RI 5 100 3~IDn! 2 ~ID remodeled!

~IDn! 2 ~IDh!

where CSA, media cross-sectional area; ID, media in-ternal diameter (media 1 intima); ED, media externaldiameter; n, normals; h, hypertensives; and RI, remod-eling index.

Each vessel was then stimulated as follows:

• Three stimulations (2 min for each) with PSS inwhich NaCl was substituted with KCl on anequimolar basis (K-PSS), and two stimulations withK-PSS containing 10 mmol/L norepinephrine;

• A cumulative dose-response curve to norepineph-rine at the following concentrations: 0.01, 0.03, 0.1,0.3, 1, 3, and 10 mmol/L, 3 min per concentration;and

• A cumulative dose-response curve to acetylcholineat the following concentrations: 0.001, 0.003, 0.01,0.03, 0.1, 0.3, 1, 3, and 10 mmol/L, 3 min per con-centration, after precontraction with 5 mmol/L nor-epinephrine. The response to acetylcholine was ex-pressed as the percent decrease of the wall tensionobtained with norepinephrine precontraction.

If the vessels produced rhythmic activity, the re-sponse was measured from the mean active force forthe last 20 sec of each period. The responses of bloodvessels are expressed as wall tension (active forcedivided by two times the segment length). Activemedia stress is the active wall tension divided by themedia thickness. Morphological and functional resultsfrom two different blood vessels in each rat wereaveraged to provide one mean observation per sub-ject. For further details about the methods used, seereferences 18 and 26. To be sure of the absence ofendothelial damage, possibly occurring during themounting procedure, in a small subgroup of vessels(two in each group of rats) we have tested the pres-ence or absence of endothelium, by standard electronmicroscopy techniques, in the biological specimens,fixed after morphological and functional evaluations.In each case, the endothelium was intact.

Statistical Analysis All data are expressed asmean 1 SEM. One-way analysis of variance (ANOVA)

and Bonferroni’s correction for multiple comparisonswere used to evaluate differences among groups.Two-way ANOVA for repeated measures was usedfor blood pressure and heart rate (group 3 time) aswell as for dose-response curves to norepinephrineand acetylcholine (group 3 dose) (BMDP StatisticalSoftware programs 7D, 1V, and 2V, BMDP StatisticalSoftware Inc., Los Angeles, CA).

RESULTS

No significant difference in the body weight amongthe five groups was detected at the beginning of thestudy (4 weeks of age, data not shown).

Blood Pressure Systolic arterial pressure (SAP) val-ues in WKY and in untreated SHR from the fourth tothe thirty-eighth week are reported in Figure 1. At 4weeks of age untreated SHR still had SAP in thenormal range (129 6 7.35 v 133 6 4.31), whereas at 8weeks SAP was significantly increased (180 6 6.33 v151 6 4.01 P , .01) compared to WKY. At 13 weeks ofage, the increase of SAP values in untreated SHRcompared to WKY was almost fully developed, with asmall further increase at 38 weeks (Figure 1, Table 1).SAP was significantly reduced in all treated groupswhile the drug was administered (on average, 25 to 45mm Hg less than agematched, untreated SHR;ANOVA P , .01 at least in any case). After treatmentwithdrawal, SAP remained significantly lower in alltreated groups, compared to untreated SHR (ANOVAP , .001 in all cases) (Figure 1), without significantdifferences among them. The prevention of hyperten-sion was only partial (ANOVA P , .01 v untreatedWKY); however, the effect was still present at the endof the period of observation (ANOVA P , .01 at leastv untreated SHR in the last 4 weeks) (Figure 1).

Cardiac Morphology Values for heart weight, leftventricular weight, body weight, and RLVM of SHRand WKY at different ages are reported in Table 1. At13 and at 38 weeks of age, untreated SHR showed anincreased RLVM compared to untreated WKY. A sig-nificant reduction of RLVM was observed in allgroups of treated SHR at 13 weeks of age, whereas at38 weeks the reduction was no longer statisticallysignificant.

Vascular Morphology Values for media thickness,wall thickness, media cross-sectional area, internal di-ameter, and media/lumen ratio (M/L) of SHR andWKY at different ages are reported in Table 2. Un-treated SHR clearly showed the presence of vascularstructural alterations at 13 and at 38 weeks of age.Treatment with fosinopril induced a reduction ofM/L, which was detectable at the thirteenth week ofage (1 or 5 weeks after treatment withdrawal). At 38weeks of age, M/L, media thickness, and wall thick-

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ness were significantly lower in all treated groupscompared to untreated SHR. The normalized internaldiameter was smaller in the untreated SHR comparedto untreated WKY at 13 weeks of age; treatment withfosinopril was associated with an increase in the normal-ized internal diameter at 13 but not at 38 weeks of age.

The remodeling index in untreated SHR was 77% at13 weeks and 72% at 38 weeks. Therefore, more than70% of the increase in media/lumen ratio could beexplained by a remodeling process, whereas cellgrowth played a minor role.

Vascular Function No significant difference amongthe groups (WKY, untreated and treated SHR) in theresponse to KPSS was observed either at 13 or at 38weeks (data not shown).

Endothelial Function Both at 13 and at 38 weeks, aright-shifted dose-response curve to acetylcholine(ANOVA between curves: P 5 .001) was observed inuntreated SHR compared with WKY. An evident im-provement of endothelial function, as suggested by a

left-shifted dose-response curve to acetylcholine wasobserved in all treated groups (ANOVA betweencurves: P , .01 at least compared with untreated SHR)at 13 weeks, whereas at 38 weeks there was no differ-ence between previously treated and untreated SHR(Figure 2, Table 3). In FOS 4 to 8 and FOS 8 to 12 at 13weeks, the shift to the left of the dose-response curveto acetylcholine was evident only at the higher doses.No significant difference among the groups in thesensitivity to acetylcholine, as expressed by log EC50(ie, the logarithm of the median effective concentration)was observed either at 13 or at 38 weeks (Table 3).

At 13 weeks of age, a statistically significant cor-relation between SAP at the moment of death andmaximal vasodilatation induced by acetylcholine(1025 mol/L) (r 5 0.66, P , .001) was observed,when all the SHR (treated and untreated) weretaken into account (n 5 29) (Figure 3). The correla-tion between EC50 and SAP was not statisticallysignificant (r 5 0.24, P 5 NS). No correlations havebeen observed at 38 weeks of age (n 5 35) between

FIGURE 1. Line graphs show time course of systolic arterial pressure from 4 to 38 weeks in rat groups killed at 38 weeks.Wistar-Kyoto (WKY, n 5 7) rats (top left) and rats treated with fosinopril from 4 to 8 weeks of age (FOS 4–8, n 5 6) (top right),from 8 to 12 weeks of age (FOS 8–12, n 5 8) (bottom left), and from 4 to 12 weeks of age (FOS 4–12, n 5 6) (bottom right) arecompared with untreated spontaneously hypertensive rats (SHR, n 5 8). See text for significance of differences among groups.

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SAP (thirty-eighth week) and EC50 r 5 0.27 or SAPthirty-eighth week—maximal acetylcholine-inducedvasodilatation: r 5 20.03, P 5 NS).

At 13 weeks of age, the correlation with maximalacetylcholine-induced vasodilatation was slightlycloser if the average SAP of the previous 8 weekswas considered (r 5 0.74, P , .001), whereas no

difference was observed at 38 weeks of age (r 520.17, P 5 NS). The latter correlation became sta-tistically significant if the WKY were included (r 50.61, P , .001) (Figure 3).

Response to Norepinephrine No significant differenceamong the groups (WKY, untreated and treated SHR)

TABLE 1. SYSTOLIC ARTERIAL PRESSURE AND HEART RATE AT TIME OF DEATH, BODY WEIGHT,AND CARDIAC MASS INDICES OF THE EXPERIMENTAL GROUPS

SystolicArterialPressure(mm Hg)

Heart Rate(beats/min)

BodyWeight (g)

HeartWeight (g)

LVW(g)

RLVM(g/kg)

SHR 13 weeks untreated,n 5 10 225 6 10.2 464 6 15.4 255 6 9.05 1.01 6 0.02 0.77 6 0.02 3.01 6 0.05

WKY 13 weeks untreated,n 5 11 158 6 8.81* 385 6 9.29* 302 6 5.49* 1.17 6 0.08 0.77 6 0.03 2.53 6 0.10**

SHR 13 weeks FOS 4–8,n 5 6 190 6 10.9*† 453 6 6.98 305 6 13.5** 1.09 6 0.03 0.79 6 0.04 2.60 6 0.06***

SHR 13 weeks FOS 8–12,n 5 7 188 6 9.51*† 444 6 12.0 263 6 9.40† 1.01 6 0.01 0.73 6 0.01 2.79 6 0.08*

SHR 13 weeks FOS 4–12,n 5 6 187 6 8.65*† 451 6 11.2 276 6 8.95† 1.03 6 0.02 0.74 6 0.02 2.68 6 0.06**

SHR 38 weeks untreated,n 5 8 263 6 13.1 424 6 17.6 402 6 17.1 1.60 6 0.03 1.20 6 0.03 3.03 6 0.18

WKY 38 weeks untreated,n 5 7 164 6 3.28*** 415 6 3.88 358 6 13.75 1.23 6 0.02*** 0.75 6 0.04*** 2.13 6 0.14**

SHR 38 weeks FOS 4–8,n 5 6 220 6 9.75*† 423 6 5.26 409 6 14.8 1.36 6 0.06** 0.98 6 0.04** 2.41 6 0.10

SHR 38 weeks FOS 8–12,n 5 8 227 6 9.31*† 408 6 19.3 388 6 11.2 1.37 6 0.04** 1.01 6 0.04** 2.60 6 0.11†

SHR 38 weeks FOS 4–12,n 5 6 228 6 9.22*† 446 6 15.6 357 6 15.43 1.30 6 0.05** 0.98 6 0.05** 2.75 6 0.13†

* P , .05; ** P , .01; *** P , .001 v untreated SHR; † P , .05 at least v untreated WKY.

SHR, spontaneously hypertensive rats; WKY, Wistar-Kyoto rats; FOS 4–8, rats receiving fosinopril from 4 to 8 weeks of age; FOS 8–12, rats receivingfosinopril from 8 to 12 weeks of age; FOS 4–12, rats receiving fosinopril from 4 to 12 weeks of age; LVW, left ventricular weight; RLVM, relative leftventricular mass (LVW/body weight).

TABLE 2. MORPHOLOGICAL CHARACTERISTICS OF THE MESENTERIC RESISTANCE VESSELS

MT (mm) WT (mm) MCSA (mm2) ID (mm) M/L

SHR 13 weeks untreated, n 5 10 22.4 6 1.21 36.1 6 1.27 14493 6 1250 182 6 6.14 0.123 6 0.003WKY 13 weeks untreated, n 5 11 16.2 6 0.66‡ 29.8 6 0.93‡ 12068 6 771 216 6 9.44* 0.076 6 0.005‡SHR 13 weeks FOS 4–8, n 5 6 21.3 6 1.38# 35.1 6 1.85# 15138 6 1439 217 6 15.7* 0.099 6 0.006#SHR 13 weeks FOS 8–12, n 5 7 22.0 6 0.70# 36.2 6 1.38# 15981 6 844 212 6 5.71* 0.104 6 0.04*#SHR 13 weeks FOS 4–12, n 5 6 21.0 6 1.50# 35.2 6 2.21# 15056 6 1180 212 6 12.6* 0.100 6 0.07*#SHR 38 weeks untreated, n 5 8 30.2 6 1.30 47.0 6 1.89 24566 6 2.740 223 6 20.6 0.141 6 0.009WKY 38 weeks untreated, n 5 7 23.2 6 0.68‡ 37.3 6 1.27† 17532 6 1392 258 6 16.4 0.091 6 0.006‡SHR 38 weeks FOS 4–8, n 5 6 22.4 6 1.52† 37.2 6 2.21† 17225 6 1586 218 6 9.92 0.103 6 0.007*SHR 38 weeks FOS 8–12, n 5 8 23.8 6 0.76† 39.6 6 0.95† 18400 6 795 220 6 7.34 0.109 6 0.006*SHR 38 weeks FOS 4–12, n 5 6 25.9 6 1.35* 41.4 6 2.17 20883 6 1127 229 6 15.4 0.106 6 0.011*

* P , .05; † P , .01, ‡ P , .001 v untreated SHR, # P , .05 at least v untreated WKY.

MT, media thickness; WT, wall thickness; MCSA, media cross-sectional area, ID; normalized internal diameter; M/L; media/lumen ratio; for otherdefinitions, see Table 1.

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in the sensitivity to norepinephrine as expressed byEC50 was observed either at 13 or at 38 weeks (data notshown).

Both at 13 and at 38 weeks an increased reactivity tonorepinephrine in terms of wall tension was observedin untreated SHR compared with WKY (dose-re-sponse curve to norepinephrine: ANOVA betweencurves: P , .05). In all treated groups, both at 13 andat 38 weeks, the reactivity to norepinephrine was re-duced compared with untreated SHR (ANOVA be-tween curves: P , .05 at least compared with un-treated SHR) (Figure 4). There were no statisticallysignificant differences in the maximal wall tensionamong groups (Table 4). When the vascular responsewas normalized for the media thickness (active mediastress), no difference was observed between untreatedSHR and WKY at any age, as well as between treatedand untreated SHR (ANOVA between curves: P . .10,data not shown) (Table 3).

DISCUSSION

This study shows for the first time that a short-termtreatment with the ACE inhibitor fosinopril is associ-ated with a prolonged delay in the development orredevelopment of hypertension. This finding is similarto what has been observed in other studies on SHRtreated with a few ACE inhibitors,8–17 which showeda persistent reduction of blood pressure 12 to 28 weeksafter treatment withdrawal. However, different ACEinhibitors could have different effects on blood vesselsand on the time course of blood pressure after treat-ment withdrawal, and therefore it is not completelyestablished whether this beneficial effect could be at-tributed to the whole pharmacological class. Themechanisms involved in this intriguing phenomenonare still a matter of speculation.

FIGURE 2. Line graphs show dose-response curve to acetyl-choline in mesenteric resistance vessels of Wistar-Kyoto rats(hollow circles) and spontaneously hypertensive rats (SHR)(untreated: filled circles; receiving fosinopril from 8 to 12 weeksof age: filled triangles up, from 4 to 8 weeks of age: filled tri-angles down, from 4 to 12 weeks of age: filled squares) at 13 (top)and 38 (bottom) weeks of age; see text for significance of differ-ences among groups. See Table 4 for the number of rats in eachgroup.

TABLE 3. MAXIMAL WALL TENSION AND ACTIVE MEDIA STRESS IN RESPONSE TO NOREPINEPHRINE,EC50, AND MAXIMAL PERCENT REDUCTION IN WALL TENSION IN RESPONSE TO ACETYLCHOLINE

Wall TensionNorepinephrine

1025 mol/L(mN/mm)

Active Media StressNorepinephrine

1025 mol/L(KPa)

Percent Reductionin Wall Tension

Acetylcholine1025 mol/L

Sensitivity toAcetylcholine

(log EC50)

SHR 13 weeks untreated, n 5 10 3.98 6 0.76 170 6 21.5 240.4 6 4.58 28.06 6 0.59WKY 13 weeks untreated, n 5 6 2.67 6 0.37 184 6 32.4 273.9 6 5.60‡ 28.26 6 0.23SHR 13 weeks FOS 4–8, n 5 6 3.05 6 0.54 148 6 27.6 265.4 6 5.91† 27.81 6 0.49SHR 13 weeks FOS 8–12, n 5 7 3.62 6 0.45 164 6 19.9 271.5 6 3.15‡ 26.63 6 0.37SHR 13 weeks FOS 4–12, n 5 6 2.87 6 0.46 137 6 18.5 273.2 6 8.11† 25.89 6 0.85SHR 38 weeks untreated, n 5 8 3.72 6 0.45 118 6 18.2 237.3 6 2.31 24.91 6 0.70WKY 38 weeks untreated, n 5 7 1.76 6 0.33* 82.2 6 14.8 286.4 6 2.12‡ 26.72 6 0.17SHR 38 weeks FOS 4–8, n 5 6 2.96 6 0.65 136 6 30.1 231.3 6 4.98 26.38 6 0.61SHR 38 weeks FOS 8–12, n 5 8 2.10 6 0.40* 88.6 6 16.9 231.7 6 3.45 25.06 6 0.57SHR 38 weeks FOS 4–12, n 5 6 2.20 6 0.55 146 6 21.7 237.3 6 4.11 25.29 6 0.67

* P , .05; † P , .01; ‡ P , .001 v untreated SHR. Definitions are as in Table 1.

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Two of the previously mentioned studies havepointed out the importance of an early onset of thetreatment, in fact no delay in the redevelopment ofhypertension was observed if treatment was started at14 to 20 weeks of age.9,10 In the present study, theeffect on systolic blood pressure of the antihyperten-sive treatment started before or during the develop-ment of hypertension was superimposable. However,it is possible that if the onset of treatment is furtherdelayed, its effect on the redevelopment of hyperten-sion could be less evident, or absent. These data are in

contrast to what we have previously observed usingthe calcium entry blocker nitrendipine, which did notinduce any delay in the development of hypertensionif treatment was started at 8 weeks of age.18 Moreover,the delay in the development of hypertension ob-served in rats treated earlier with nitrendipine (from 4week of age) ceased at about 30 to 32 weeks of age,even if a reduction of vascular structural alterationcould still be observed at 38 weeks of age.18

In the present study, the administration of fosino-pril was associated with the regression of vascularhypertrophy, which was evident in all groups oftreated SHR regardless of the time of onset of treat-ment, and it was maintained over the whole period ofobservation. Our data are in keeping with those ob-tained by Harrap and colleagues,9 who observed that

FIGURE 3. Correlation between maximal acetylcholine-in-duced (10 mmol/L) percent reduction in wall tension and systolicarterial pressure (average of the previous 8 weeks) both at 13 weeksof age (top) and 38 weeks of age (bottom) in treated and un-treated spontaneously hypertensive rats (SHR). SHR: untreated:filled circles, receiving fosinopril from 8 to 12 weeks of age: filledtriangles up, from 4 to 8 weeks of age: filled triangles down, from4 to 12 weeks of age: filled squares, Wistar-Kyoto rats (WKY):hollow circles. See Table 4 for the number of rats in each group. Ifthe WKY are also included, the correlation coefficients are 0.75(P , .001) at 13 weeks of age, and 0.61 (P , .001) at 38 weeks ofage.

FIGURE 4. Line graphs show dose-response curve to norepi-nephrine in mesenteric resistance vessels of Wistar-Kyoto rats(hollow circles) and spontaneously hypertensive rats (SHR)(untreated: filled circles; receiving fosinopril from 8 to 12 weeksof age: filled triangles up, from 4 to 8 weeks of age: filledtriangles down, from 4 to 12 weeks of age: filled squares) at 13(top) and 38 (bottom) weeks of age; see text for significance ofdifferences among groups. See Table 4 for the number of rats ineach group.

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in perindopril-treated SHR there was a persistent reduc-tion of M/L in mesenteric small resistance arteries after12 weeks of therapy withdrawal. However, in thisstudy,9 morphological data about mesenteric resistancearteries have been provided for only one therapeuticscheme (SHR treated from 6 to 10 weeks of age).

Fosinopril treatment was also associated to a reduc-tion in RLVM in all groups of treated SHR, 1 to 5weeks after treatment withdrawal. This reduction wasno longer statistically significant 26 to 30 weeks aftertreatment was stopped. This finding may suggest thatthe antiproliferative action of the ACE inhibitor ismore effective or prolonged in vascular than in cardiactissue, or that cardiac mass is more directly influencedby an albeit partial increase of the hemodynamic loadthan resistance arteries.

It is well known that vascular structural changesin hypertension may act as vascular amplifier, ca-pable of enhancing any hypertensive stimulus.7,27

We found an increased response to norepinephrinein SHR at both 13 and 38 weeks of age, in terms ofwall tension but not in terms of active media stress.Similar results are reported in other studies,28 –30

and clearly suggest that the enhanced response ob-served in SHR is mainly related to the structuralchanges present in this strain (ie media remodeling).When a significant reduction of vascular structuralalterations was obtained by fosinopril treatment (at13 and at 38 weeks of age), a trend toward normal-ization of the dose-response curve to norepineph-rine was also observed. It is therefore possible thatthe beneficial effect of antihypertensive therapywith ACE inhibitors on vascular structure couldplay a role in the persistent reduction in bloodpressure observed after treatment withdrawal.31

An impairment of endothelial functions is a com-mon feature in SHR3– 6 and it may contribute to themaintenance of hypertension in this strain. Acetyl-choline is able to induce the release of endothelium-derived relaxing factor (EDRF), and therefore thevasodilator response to acetylcholine may be anuseful tool to evaluate the endothelial function.3 Inour study, a clear alteration of the vascular responseto acetylcholine (as expressed by differences in thedose-response curves) was observed in 13 and 38week old SHR, compared with WKY. The biochem-ical mechanisms underlying endothelial dysfunc-tion are still unclear even if a major role could beplayed by reduced synthesis of EDRF/nitric oxide(NO) or an enhanced release of endothelium-de-rived contracting factors. In previous studies, ACEinhibitors, either added to the organ bath32,33 orgiven as chronic therapy13,19 –21 have been shown toimprove the vasodilator response to acetylcholine insmall resistance arteries of SHR. It has been specu-lated that these drugs may improve endothelial

function by increasing the release of EDRF/NO. Inone of these studies,13 the increase in the vasodilatorresponse to acetylcholine observed after therapywith ramipril was associated with an increased aor-tic cyclic GMP content, suggesting that all theEDRF/NO cascade is activated, even if it is not clearif this is due to an increased synthesis/release ofNO or to an increased activity of guanlylate cyclase.However, no data are presently available about theendothelial function after a prolonged treatmentwithdrawal. In our study, a short-term treatmentwith fosinopril determined an improvement of theendothelial function, in those rats in which a satis-factory control of blood pressure was obtained up to1 to 5 weeks before death. On the contrary, in thoserats in which therapy was withdrawn 26 to 30 weeksbefore death (and in which systolic blood pressureremained constantly above the values observed inWKY controls) no improvement of endothelial alter-ations could be observed, despite persistent signif-icant reduction of vascular structural changes. Sim-ilar findings have been obtained using the calciumantagonist nitrendipine.18

Therefore, a clear dissociation between vascularstructural alterations and endothelial function wasclearly observed; in a previous study we observed theabsence of any endothelial dysfunction in young SHRwho showed the presence of vascular hypertrophy.23

The observation of improvements of endothelial func-tion during therapy with other drugs, such as calciumchannel blockers18,20,21 gives more strength to the hy-pothesis that the effect is not specific, and a hemody-namic rather than a structural factor is involved bothin the genesis and in the prevention or regression ofendothelial dysfunction in SHR.

The lack of an improvement of endothelial func-tion at 38 weeks of age in the groups of rats previ-ously treated with fosinopril, and in which the he-modynamic load is reduced, but not normalized,would suggest the necessity of an almost completenormalization of blood pressure in order to obtain asignificant prevention of the onset of endothelialdysfunction. On the other hand, an alternative ex-planation is that fosinopril may possess a direct,beneficial effect on endothelial function, which isstill present after 5 but not after 26 weeks of treat-ment withdrawal. In this case the extent of the over-all reduction of blood pressure would be less im-portant. However, we have previously demon-strated that when fosinopril is administered at low,nonhypotensive doses, no effect on endothelialfunction may be observed.34

In the present study, the observation of a closecorrelation between SAP values and maximal ace-tylcholine-induced reduction in wall tension at 13weeks of age supports the hypothesis of a relevant

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role of the hemodynamic load in the genesis or inthe maintenance of endothelial dysfunction. Thelack of a correlation between SAP values and endo-thelial function at 38 weeks could be explained, atleast in part, by a lingering effect of the ACE inhib-itor, present at 13 but not at 38 weeks, or by otherendothelial changes that occur over time (ie due toaging) that disrupt the pressure:endothelial dys-function relationship.

In conclusion, in SHR fosinopril at any therapeuticregimen may prevent or regress, at least in part, car-diovascular hypertrophy. The beneficial effect of fosi-nopril on vascular structural alterations and on sys-tolic arterial pressure was present even at 38 weeks ofage, 26 to 30 weeks after treatment withdrawal. More-over, the vascular response to norepinephrine seemsto be dependent mainly on the structure of the vessels,whereas endothelial function is probably more linkedto the hemodynamic pattern.

ACKNOWLEDGMENTS

We thank Stefano Collatina, MD (Bristol Myers SquibbS.p.A., Rome, Italy), for having provided fosinopril, andMiss Alessandra Panarotto for her technical assistance.

REFERENCES

1. Mulvany MJ, Halpern W: Contractile properties ofsmall resistance vessels in spontaneously hypertensiveand normotensive rats. Circ Res 1977;41(1):19–26.

2. Mulvany MJ, Hansen PK, Aalkjaer C: Direct evidencethat the greater contractility of resistance vessels inspontaneously hypertensive rats is associated with anarrowed lumen, a thickened media, and an increasednumber of smooth muscle cell layers. Circ Res 1978;43(6):854–864.

3. Luscher TF, Tschudi MR, Dohi Y: Resistance arteries inhypertension: role of the endothelium, in Mulvany MJ,Aalkjaer C, Haegerty AM, et al (eds): Resistance Arter-ies, Structure and Function. Amsterdam, Elsevier Sci-ence Publishers B.V., 1991, pp 312–315.

4. Tesfamariam B, Halpern W: Endothelium-dependentand endothelium independent vasodilatation in resis-tance arteries from hypertensive rats. Hypertension1988;11(5):440–444.

5. Yang ST, Mayhan WG, Faraci FM, Heistad DD: Endo-thelium-dependent response of cerebral blood vesselsduring chronic hypertension. Hypertension 1991;17(5):612–618.

6. Andriantsitohaina R, Stoclet JC, Bukoski RD: Role ofendothelium on the effects of neuropeptide Y in mes-enteric resistance arteries of spontaneously hyperten-sive and Wistar-Kyoto normotensive rats. J PharmacolExp Ther 1991;257(1):276–278.

7. Lever AF: Slow pressor mechanisms in hypertension: arole for hypertrophy of resistance vessels? J Hypertens1986;4:515–524.

8. Christensen KL, Jespersen LT, Mulvany MJ: Develop-ment of blood pressure in spontaneously hypertensiverats after withdrawal of long-term treatment related tovascular structure. J Hypertens 1989;7:83–90.

9. Harrap SB, Van der Merwe WM, Griffin SA, et al: Briefangiotensin converting enzyme inhibitor treatment inyoung spontaneously hypertensive rats reduces bloodpressure long term. Hypertension 1990;16:603–614.

10. Adams MA, Bobik A, Korner PI: Enalapril can preventvascular amplifier development in spontaneously hy-pertensive rats. Hypertension 1990;16:252–260.

11. Lee RMKW, Berecek KH, Tsoporis J, et al: Prevention ofhypertension and vascular changes by captopril treat-ment. Hypertension 1991;17:141–150.

12. Wu JN, Berecek KH: Prevention of genetic hyperten-sion by early treatment of spontaneous hypertensiverats with the angiotensin converting enzyme inhibitorcaptopril. Hypertension 1993;22:139–146.

13. Gohlke P, Lamberty V, Kuwer I, et al: Long term lowdose angiotensin converting enzyme inhibitor treat-ment increases vascular cyclic guanosine 39,59 mono-phosphate. Hypertension 1993;22:682–687.

14. Kost CK, Jr., LI P, Jackson EK: Blood pressure aftercaptopril withdrawal from spontaneously hypertensiverats. Hypertension 1995;25:82–87.

15. Giudicelli JF, Freslon JL, Glasson S, Richer C: Captopriland hypertension development in SHR. Clin Exp Hy-pertens 1980;2:1083–1096.

16. Freslon JL, Giudicelli JF: Compared myocardial andvascular effects of captopril and dihydralazine duringhypertension development in spontaneously hyperten-sive rats. Hypertension 1983;80:533–543.

17. Thybo NK, Korsgaard N, Eriksen S, et al: Dose-depen-dent effects of perindopril on blood pressure and smallartery structure. Hypertension 1994;23:659–666.

18. Rizzoni D, Castellano M, Porteri E, et al: Delayed de-velopment of hypertension after short-term nitrendip-ine treatment. Hypertension 1994;24:131–139.

19. Clozel M, Kuhn H, Hefti F, Baumgartner HR: Endothe-lial dysfunction and subendothelial monocyte macro-phages in hypertension. Effect of angiotensin convert-ing enzyme inhibition. Hypertension 1991;18(2):132–141.

20. Tschudi MR, Criscione L, Novosel D, et al: Antihyper-tensive therapy augments endothelium-dependent re-laxation in coronary arteries of spontaneously hyper-tensive rats. Circulation 1994;89:2212–2218.

21. Dohi Y, Criscione L, Pfeiffer K, Luscher TF: Angioten-sin blockade or calcium antagonists improve endothe-lial dysfunction in hypertension: studies in perfusedmesenteric resistance arteries. J Cardiovasc Pharmacol1994;24:372–379.

22. Opie LH: ACE-inhibitors: agents and pharmacokinet-ics, in Opie LH (ed), Angiotensin converting enzymeinhibitors. Scientific basis for clinical use. New York,Wiley-Liss, 1992.

23. Schiffrin EL, Deng LY, Larochelle P: Effects of ab-blocker or a converting enzyme inhibitor on resis-tance arteries in essential hypertension. Hypertension1994;23:83–91.

24. Heagerty AM, Aalkjaer C, Bund SJ, et al: Small arterystructure in hypertension. Dual process of remodelingand growth. Hypertension 1993;21:391–397.

25. Baumbach GL, Heistad DD: Remodeling of cerebral

AJH–SEPTEMBER 1997–VOL. 10, NO. 9, PART 11042 RIZZONI ET AL

arterioles in chronic hypertension. Hypertension 1989;13:968–972.

26. Rizzoni D, Castellano M, Porteri E, et al: Vascular struc-tural and functional alterations before and after thedevelopment of hypertension in SHR. Am J Hypertens1994;7:193–200.

27. Korner PI, Bobik A, Jennings GL, et al: Significance ofcardiovascular hypertrophy in the development andmaintenance of hypertension. J Cardiovasc Pharmacol1991;17(suppl 2):S25–S32.

28. Mulvany MJ, Nyborg N: An increased calcium sensi-tivity of mesenteric resistance vessels in young andadult spontaneously hypertensive rats. Br J Pharmacol1980;71:585–596.

29. Mulvany MJ, Aalkjaer C, Christensen J: Changes innoradrenaline sensitivity and morphology of arterialresistance vessels during development of high bloodpressure in spontaneously hypertensive rats. Hyper-tension 1980;2:664–671.

30. Kong JQ, Taylor DA, Fleming WW. Mesenteric vascu-lar response of young spontaneously hypertensive rats.J Pharmacol Exper Ther 1991;258(1):13–17.

31. Chen DG, Jin XQ, Wang HJ, Chen SC: Mechanismsresponsible for sustained hypotension after captopriltreatment. J Hypertens 1995;13:1113–1121.

32. Kerth PA, Vanhoutte PM: Effects of perindoprilat onendothelium-dependent relaxation and contractions inisolated blood vessels. Am J Hypertens 1991;4:226S–234S.

33. Mombouli JV, Nephtali M, Vanhoutte PM: Effect of theconverting enzyme inhibitor cilazaprilat on endotheli-um-dependent responses. Hypertension 1991;18(supplII):II-22–II-29.

34. Rizzoni D, Castellano M, Porteri E, et al: Effects of lowand high doses of fosinopril on the structure and func-tion of resistance arteries. Hypertension 1995;26:118–123.

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