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Discrepancy Between Plasma and LungAngiotensin-Converting Enzyme Activity inExperimental Congestive Heart FailureA Novel Aspect of Endothelium Dysfunction

Huaming Huang, Jean-Frangois Arnal, Catherine Llorens-Cortes, Mireille Challah,Francois Alhenc-Gelas, Pierre Corvol, Jean-Baptiste Michel

Abstract The renin-angiotensin and cardiac natriuretic sys-tems play an important role in the pathophysiology of conges-tive heart failure (CHF). The status of the membrane-boundpulmonary and renal activities of three ectoenzymes involvedin the regulation of these systems - angiotensin-convertingenzyme (ACE), neutral endopeptidase (NEP), and aminopep-tidase A (APA) -was investigated in Wistar rats 3 monthsafter induction of myocardial infarction (MI) and in sham-operated (control) rats. Plasma renin activity and ACE activ-ity, plasma angiotensin II (Ang II) levels, and atrial natriureticfactor levels were simultaneously determined. The lung ACEactivity was decreased in MI rats compared with control rats(P<.0001), and this decrease depended on the severity of theheart failure. In contrast, plasma ACE activity was increasedin MI rats (P<.01), and this increase was also proportional tothe severity of MI. Northern blot analysis showed that the lungACE mRNA level in severe MI rats was half that of the controlrats. Renal ACE activity of the MI rats was not affected, and

ongestive heart failure (CHF) is a syndrome inC which the heart is unable to deliver nutrient

blood to peripheral tissues at normal levels ofventricular filling pressure. The syndrome is most oftencaused by impaired left ventricular performance and,particularly in severe or decompensated CHF, is accom-panied by complex abnormalities of the peripheralcirculation.' Activation of the renin-angiotensin systemis claimed to occur in CHF.23 Plasma renin activity andrenal renin content are known to increase in clinical andexperimental myocardial infarction (MI).2,3 Plasma an-giotensinogen is decreased in patients with severe heartfailure.4 Despite the importance of the renin-angioten-sin system in the pathogenesis of CHF, relatively little isknown about the status of the enzymes that can modifythe activity of the effector hormone, angiotensin II (AngIL), in this setting.

Angiotensin-converting enzyme (ACE, EC 3.4.15.1)plays an important role in the regulation of the renin-angiotensin system by converting angiotensin I (Ang I)into Ang 11.5,6 This enzyme is a membrane-bound zincmetalloprotease predominantly present at the luminal

Received October 22, 1993; accepted June 2, 1994.From the Institut National de la Sante et de la Recherche

Medicale, Paris, France.Correspondence to Dr Pierre Corvol, Co1llge de France, 3 rue

d'Ulm, 75005 Paris, France.© 1994 American Heart Association, Inc.

neither renal or pulmonary NEP nor pulmonary APA activi-ties were altered. Thus, lung ACE gene expression appears tobe both organ- and enzyme-specifically regulated during CHF.Whereas plasma renin was increased in heart failure rats,plasma Ang II levels were not different from those of controlrats. Thus, decreased lung ACE activity could possibly con-tribute to keeping plasma Ang II levels in the normal range.The decrease in lung ACE activity and mRNA levels, com-bined with increased plasma ACE activity, represents a novelaspect of endothelial dysfunction in CHF. This dissociationbetween the membrane-bound endothelial enzyme and itscirculating counterpart emphasizes the importance of simulta-neously assessing the circulating and tissue components of therenin-angiotensin system in heart failure. (Circ Res. 1994;75:454-461.)Key Words * neutral endopeptidase * aminopeptidase A

* myocardial infarction * renin-angiotensin system *atrial natriuretic factor

surface of the vascular endothelium.5,7 ACE is alsopresent at the surface of some epithelia, such as that ofthe renal proximal tubule,8 and is expressed by inflam-matory cells and fibroblasts.6 In plasma, ACE is foundin soluble form. Aminopeptidase A (APA, EC 3.4.11.7)is another endothelium-associated zinc metallopro-tease.9 This enzyme metabolizes Ang II to angiotensinIII (Ang IIJ).9 Thus, one key function of the endothelialcell is to metabolize vasoactive peptides through theaction of peptidases such as ACE and APA. Althoughthere is recent evidence that endothelial cell functionmay be altered in both experimental animals10,11 andpatients2'13 with CHF, little is known about the impactof this syndrome on endothelial cell ACE and APA.Neutral endopeptidase (NEP, EC 3.4.24.11), a mem-brane-bound zinc metalloprotease, is widely distributedin peripheral organs14 and is particularly abundant inthe microvilli of renal proximal tubule epithelial cells15but low in concentration in endothelial cells.16 NEPplays an important role in the renal degradation ofvasoactive peptides such as atrial natriuretic factor(ANF), angiotensins, or bradykinin.'7

Therefore, ACE, APA, and NEP are important regu-latory enzymes of the vasoactive peptide systems. Theaim of the present study was to investigate whether theirenzymatic activity varied in a rat model of chronic heartfailure. In this study, enzymatic activity was assessed inthe lung, which because of its large vascular bed contains

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Huang et al Decrease in Lung Angiotensin-Converting Enzyme in Heart Failure 455

the highest quantity of endothelial ACE in the organismand is believed to be the major site of Ang II formation,and in the kidney, since altered NEP activity may be ofparticular physiological relevance in this organ.

Materials and MethodsExperimental Design

Normotensive male Wistar rats (Iffa Credo) weighing 300 to320 g were used. Left ventricular infarction was produced byligation of the left coronary artery under ether anesthesia asdescribed by Fishbein et al'8 and Pfeffer et al.19 The leftdescending anterior coronary artery was ligated proximally toobtain a large infarct leading to decompensated heart failure.Control rats were sham-operated by using a similar procedure(thoracotomy) without coronary ligation.The experimental period was 3 months. Systolic blood

pressure and heart rate were measured weekly by the tail-cuffmethod (W+W electronic recorder 8005, Apelab), and theanimals were examined twice weekly. At the end of the firstmonth, each rat was placed in a metabolic cage and urine wascollected for 15 hours.A first set of experiments was designed to examine possible

modifications in vascular endothelium-bound ectoenzymes.Although disturbances of the peripheral circulation are mostlikely to occur in severe and decompensated heart failure, thisstage is rapidly lethal, making it difficult to obtain decompen-sated heart failure animals. In order not to lose the decom-pensated heart failure animals, rats showing overt signs ofheart failure (the presence of two of the following three signs:no weight gain, breathlessness, and respiratory frequency>30/min) were killed between the end of the first month andthe end of the third month. A control animal was killed on thesame day. At the end of the experimental protocol, unanes-thetized rats were killed by decapitation. Blood samples werecollected into prechilled 10-mL tubes containing 2 mg/mLsodium EDTA, 4.25 mg/mL phenylmethylsulfonyl fluoride,and 200 000 protease inhibitor units/mL aprotinin to preventenzymatic degradation of ANF. These samples were centri-fuged for 10 minutes at 3000g, and the plasma was removedand frozen at -70°C. At the same time, lung and kidney wererapidly excised, rinsed in cold phosphate-buffered saline,frozen in liquid nitrogen, and stored at -70°C. The heart wasexcised and weighed. The left ventricle was then separatedfrom the two atria and weighed.A second set of experiments was conducted to evaluate the

physiological consequences of ACE changes on the othercomponents of the plasma renin-angiotensin system, includingplasma Ang II levels. A total of 31 rats were treated aspreviously described, but blood samples were collected at thetime of decapitation into prechilled 10-mL tubes containing 25mmol/L orthophenanthroline, 0.125 mol/L sodium EDTA, 1mmol/L MK422 (Merck Sharp & Dohme), 10 gmol/L pepsta-tin A, and 2% ethanol (final concentration) for the Ang II leveldetermination and also into heparinized 10-mL tubes for themeasurement of angiotensinogen levels, plasma renin activity(PRA), plasma renin concentration (PRC), and ACE activity.

Infarct Size DeterminationThe left ventricle was opened with an incision along the

septum from base to apex. Both ventricles were rinsed, blotteddry, and weighed immediately. Myocardial infarction size wasmeasured by use of techniques previously described by Chienet al.20 Incisions were made in the left ventricle so that thetissue could be pressed flat. The circumferences of the leftventricle and the region of infarction were outlined on a clearplastic sheet for both the endocardial and epicardial surfaces.Infarct size was calculated and expressed as a percentage ofleft ventricular surface area, based on the weight of the areasmarked on the plastic sheet. The average of endocardial andepicardial surface areas was reported.

The degree of heart failure in the MI group was determinedaccording to one major criterion, lung water content indicatingthe presence of edema, and three minor criteria as follows:heart-left ventricle weight index > 1.5 x control, urinarycGMP >3 x control, and infarct size .35%. Lung watercontent was the difference between wet and dry weights afterdehydration in acetone diethyl ether and drying under aircurrent for 24 hours. Lung water content was expressed as apercentage of lung wet weight. When edema and/or threeminor criteria were present, the rat was assigned to the severeMI subgroup. When only one or two minor criteria werepresent, the rat was assigned to the moderate MI subgroup.

Tissue Membrane PreparationLung or kidney was homogenized in 10 vol cold Tris-HCl

buffer (0.05 mol/L, pH 7.4) with a Teflon-glass homogenizer.The homogenate was centrifuged for 15 minutes at lOOOg, andthe resulting supernatant was collected and submitted toanother centrifugation at 16 00g for 30 minutes in a Spincoultracentrifuge (Beckman Instruments). The supernatant fluidwas discarded, and the pellet was superficially washed threetimes with 10 mL cold buffer. It was then resuspended in 10mL fresh buffer using a Dounce homogenizer and aliquoted.Aliquots were sonicated for 10 seconds in position 3 using amodel W-10 sonicator (Electro-mech Instrument Co) beforeuse as an enzyme source.

Preparation of Plasma Samples for ACEActivity Measurement

Plasma samples were dialyzed against 20 mmol/L potassiumphosphate buffer in a 10-mm-diameter dialysis bag (Pro-science, 1:500 [vol/vol]) two times for 24 hours each at 4°C toeliminate the EDTA present in the plasma samples. Thesesamples were then used for measurement of ACE activity.

Determination of Plasma ACE, Tissue ACE, NEP,and APA Enzymatic ActivitiesACE EnzEymatic ActivityACE activity was estimated by measuring the hydrolysis of

100 gmol/L [glycine-1-14C]hippuryl-L-histidyl-L-leucine (3mCi/mmol, Amersham) in the presence or absence of 10`mol/L captopril. Incubations (final volume, 100 ,L) wereperformed at 37°C under conditions of initial velocity mea-surement in 0.1 mol/L phosphate buffer, pH 8.0, containing 0.3mol/L NaCl and 10 gmol/L ZnCl2 and were stopped after 30minutes by adding 50 gL of 0.3N HCl, as described byCushman and Cheung.21 `4C metabolites were separated fromthe substrate by adding 0.5 mL ethyl acetate to the acidifiedmedium of the enzymatic reaction and shaking for 1 minute.After centrifugation at 18 OOOg for 7 minutes, a 0.3-mL aliquotof the organic layer containing `4C metabolites was counted byscintillation spectrometry (Pico-Fluor 15, Packard InstrumentCo). Contamination by the substrate was <3%, and recoveryof the metabolites was >90%.

NEP Enzymatic ActivityNEP activity was determined according to the method of

Llorens-Cortes et a122 by measuring the hydrolysis of 20nmol/L [3HID-Ala2,D-Leu'-enkephalin (50 Ci/mmol, Dositek)in the presence or absence of 10-' mol/L DL-thiorphan.Puromycin (10-4 mol/L, Sigma) and captopril (10` mol/L,Bristol-Myers Squibb Institute for Medical Research) wereadded to prevent aminopeptidase and ACE activities fromdegrading the substrate. Incubations were performed at 37°Cunder conditions of initial velocity measurement and were

stopped after 30 minutes by the addition of 25 gL 0.3N HCl.'H metabolites were isolated from the substrate by chroma-tography on polystyrene bead columns (Porapak Q, 100-120mesh, Waters Assoc) and counted by liquid scintillation spec-trometry (Aquasol-2, NEN Research Products). Addition of



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10` mol/L DL-thiorphan diminished by 99% the hydrolysis ofthe substrate, which was equal in these conditions to thereaction blank obtained by adding HCI before incubation.

Aminopeptidase A Enzymatic ActivityAPA activity was determined by measuring the hydrolysis of

0.2 mmol/L (final concentration) a-glutamyl-,-naphthylamidein the presence or absence of CaCl2 (4 mmol/L, final concen-tration) in 50 mmol/L Tris-HCl buffer at 37°C for 1 hour, asdescribed by Lojda and Gossrau.9

Protein DeterminationProtein content was assayed by the method of Lowry et al,23

with bovine serum albumin used as a standard.

Analysis of Lung ACE mRNA by Northern BlotTotal lung RNA was prepared by the method of Chomczin-

ski and Sacchi.24 Briefly, tissue was homogenized in a mixtureof 4 mol/L guanidinium thiocyanate, 25 mmol/L sodium citrate(pH 7.0), 0.5% sarkosyl, and 0.1 mol/L ,8-mercaptoethanol; 50,L of 2 mol/L sodium acetate (pH 4.0), 500 ,L water-saturated phenol, and 100 ,L chloroform:isoamyl alcohol(49:1 [vol/voll) were sequentially added to 500 ,uL of theguanidinium solution, mixed, and cooled on ice for 15 minutes.After centrifugation, the aqueous phase containing the RNAwas transferred to fresh tubes, and the phenol extraction stepwas repeated once. The final aqueous phase was mixed with500 ,uL isopropanol and chilled with dry ice for 1 hour toprecipitate RNA. The precipitate was recovered by centrifu-gation, washed once with 70% ethanol, dried in vacuum, andfinally dissolved in water treated with 0.1% diethylpyrocarbon-ate. In all experiments, the quality of isolated mRNA wasverified by gel electrophoresis. Twenty micrograms of totalRNA was denatured in a mixture containing 50% formamideand 20% formaldehyde for 12 minutes at 65°C. The denaturedRNA was fractionated by gel electrophoresis in 1% agarosegels containing 20 mmol/L MOPS, 5 mmol/L sodium acetate,1 mmol/L EDTA (pH 8.0), and 10% formaldehyde. Afterelectrophoresis at 90 V for 3 hours, the gel was washed twicefor 10 minutes in 10x saline sodium citrate buffer (SSC) andsoaked in 10x SSC, and the RNA was transferred to a HybondN nylon membrane (Amersham) by capillary action. Themembrane was cross-linked by exposure to UV light and bakedat 80°C for 1 hour. A 1922-bp insert of human ACE cDNAfragment25 was isolated from a plasmid vector (Bluescript)containing 3334 bp of human endothelial ACE cDNA26 byendonuclease digestion followed by agarose gel electrophore-sis. The fragment was labeled with [a-32P]dCTP (3000 Ci!mmol, Amersham) by random priming and was then used as aprobe for ACE mRNA detection. The blot was prehybridizedfor 2 hours at 42°C and then hybridized at 42°C for 20 hours in40% formamide, 10x Denhardt's solution, 50 mmol/L Tris-HCI (pH 7.4), 4x SSC, 0.1% sodium pyrophosphate, 1%sodium dodecyl sulfate (SDS), 50 bg/mL salmon sperm DNA,50 gg/mL yeast tRNA, and the 32P-labeled cDNA fragment ofthe human ACE at 5 x 106 dpm/mL. Filters were subsequentlywashed for 25 minutes at room temperature, 20 minutes at42°C in lx SSC and 0.1% SDS, and then 20 minutes at 42°Cin 0.2x SSC and 0.1% SDS. The blots were exposed to ahyperfilm 3-Max (Amersham) at -80°C for 72 hours in thepresence of intensifying screens. Densitometric analysis of theautoradiograms was performed with a model 620 video densi-tometer (Bio-Rad Laboratories).To determine the amount of ACE mRNA relative to total

RNA present in a given tissue sample, two separate densito-metric scans were carried out. First, to provide a relativequantification of the total amount of RNA applied to eachlane of the gel, a photographic negative (Polaroid 665) of thegel taken under UV light was scanned across the 28S band.Then, after hybridization with the cDNA probe, the ACEmRNA bands were quantified in the same way. In a second

Northern blot using seven lung mRNA samples from thesham-operated group and seven from the severe MI group, thelung ACE mRNA was normalized to glyceraldehyde-3-phos-phate dehydrogenase (GAPDH) mRNA. The human GAPDHcontrol probe (Clontech), labeled with [32P]dCTP (DuPont-New England Nuclear) to a specific activity of 109 cpm/rtgusing a random-primer labeling system (Multiprime DNAlabeling system, Amersham), was used to rehybridize the RNAblots as described above. Data concerning mRNA content arepresented in arbitrary units, defined by the area under thepeak of the ACE-specific band from the autoradiogram,divided by the area under the peak of the 28S RNA applied tothe gel or by the area under the peak of GAPDH mRNA.

Determination of Plasma ANF, PRA, PCR, PlasmaAngiotensinogen, and Urinary cGMP Levels

Plasma ANF was extracted on a C18 Sepak cartridge column(Waters Assoc) and determined by nonequilibrium radioim-munoassay using a double antibody to separate the free andthe bound fractions.27 Urinary cGMP levels were measured byradioimmunoassay using an Amersham kit (TRK 500).PRA was determined according to the method of Menard

and Catt.28 PRC was measured by radioimmunoassay of Ang Igenerated by incubation with an excess of rat renin substrate.28Plasma angiotensinogen was estimated after addition of sub-maxillary gland renin (20x10` Goldblatt units) to obtainexhaustion of the angiotensinogen, as described by M6nardand Catt.28 Plasma samples were diluted to be 1:400 in theassay mixture and incubated for 1 hour at 37°C. The amount ofAng I was measured by radioimmunoassay. The plasma angio-tensinogen concentration is expressed in nanograms Ang I permilliliter.

Measurement of Plasma Ang IIPlasma Ang II was measured by use of high-performance

liquid chromatography (to separate Ang II from Ang I and itsmetabolites) combined with a sensitive radioimmunoassaydescribed previously.29

Statistical MethodsResults are expressed as mean±SEM. Differences in blood

pressure and body weight were evaluated by two-way ANOVAwith repeated measures (comparison of the two groups). Thedifference in each biological parameter (in lung, kidney, orplasma) was evaluated by a one-factor ANOVA of the increaseor decrease of each variable measured. One-way ANOVA wasfollowed by Scheffe's F test to compare the effect of differentpathophysiological conditions on these parameters. Linearregression curves and correlation coefficients were obtained bythe least-squares method.

ResultsBlood Pressure and Body WeightThe overall spontaneous mortality in MI was 48%.

These rats were not included and analyzed in thepresent study. Of the 31 MI rats, 7 were killed after theend of the first month because of overt signs of heartfailure. A group of 7 control rats was killed at the sametime. The remaining 24 MI and 18 sham-operated ratssurvived until the end of the experimental period. Ofthe MI rats that survived until the end of the study, 12were considered to have severe CHF. Thus, a total of 19rats with severe CHF and 12 with moderate CHF werestudied. MI caused a significant fall in systolic bloodpressure that persisted throughout the study (P<.0001)(Fig 1). There was no significant difference in bodyweight between rats with severe MI and those withmoderate MI. As shown in Table 1, body weights for the



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FIG 1. Line graph shows weekly blood pressure recordingsduring the experimental period of myocardial infarction (Ml). oindicates sham-operated rats (n=25); o, moderate Ml rats(n=12); and A, severe Ml rats (n=19). Ml caused asignificantfallin systolic blood pressure that persisted throughout the study(P< .0001).

severe CHF group were less than those for the controlgroup at the end of the experimental period.

Heart Parameters and EdemaBoth the total heart weight and the ratio of total heart

weight to body weight (heart weight index) were signifi-cantly increased in the MI rats compared to sham-operated rats (P<.0001) (Table 1). The absolute weightof the myocardium hemodynamically upstream from theinfarcted left ventricle (heart-left ventricle weight) andits ratio to body weight (heart-left ventricle weightindex) were also significantly increased in the MI rats(P<.0001) (Table 1). Infarction size averaged 29±+1% ofthe left ventricle in rats with moderate MI and 37+1% inrats with severe MI (P<.0001). Lung water contentincreased proportionally to the severity of MI (P<.0001,Table 1). Pleural effusion was present in 85% of thesevere MI rats, with 15% of the severe MI rats having a

peritoneal effusion (Table 1).

Biochemical ParametersPlasma ANF levels were significantly increased in MI

rats (P<.0001), and the increase was related to theseverity of MI (Table 2). They were highly correlated to

TABLE 2. Plasma Renin Activity, Plasma AtrialNatriuretic Factor, and Urinary cGMP Levels in RatsWith Myocardial Infarction and Sham-Operated Rats

Plasma Urinary PlasmaANF Levels, cGMP, Renin Activity,

pg/mL nmol/h ng/mL per hour

Control(n=25) 238±28 1.13±0.07 5.62±0.44Moderate Ml(n=12) 694±83* 2.53±1.20* 6.04±0.68Severe Ml(n=19) 1011±61 *t 4.93±1 .20*t 8.92±3.89*tP value(ANOVA) <.0001 <.0001 <.001

ANF indicates atrial natriuretic factor; Ml, myocardial infarction.Values are mean±SEM from 12 to 25 determinations.*P<.05 vs sham-operated (control) value; tP<.05 vs moder-

ate Ml value.

the heart weight index (R2=.67, P<.0001) and to theheart-left ventricle weight index (R2=.65, P<.0001).

Urinary excretion of cGMP was significantly increasedin MI rats (P<.0001), and this increase was also propor-tional to the severity of MI (Table 2). The changes inurinary cGMP excretion were closely correlated with thechange in plasma ANF levels (R2=.52, P<.0001).PRA was significantly higher in the severe than in the

moderate MI rats (P<.002) (Table 2).Plasma ACE activity was significantly increased in MI

rats compared with control rats (19.4+0.9 versus 14.7±0.5nmol min-m mL-1, P<.01) (Table 3, Fig 2A). PlasmaACE activity was positively correlated to markers ofseverity: plasma ANF (r=.50, P<.0001), cGMP (r=.51,P<.001), and right ventricular weight (r=.58, P<.001).

Measurement of Tissue ACE, NEP,and APA ActivitiesLung ACE activity in the MI group was decreased

to 30% below that of the control group (481+35 ver-sus 718±41 pmol- min-1 * mg-1, P<.0001) (Table 3).The decrease in lung ACE activity was more pro-nounced in severe MI rats (400+ 36 pmol * min-m * mg-1

44% decrease) than in moderate MI rats (613+52pmol* min-l . mg-1, 15% decrease) (P<.0001) (Table 3,

TABLE 1. Rat Body Weight, Heart Weight, and Edema at End of Myocardial Infarction Protocol

Control Moderate Ml Severe Ml P Value(n=25) (n=12) (n=19) (ANOVA)

Body weight (at death), g 497±10 488±28 444+15* <.05

Heart weight, mg 1330±27 1545+39 2050+53*t <.0001

Heart- LV weight, mg 389+7 543±26* 1032±45*t <.0001

Heart weight index, mg/g 2.69±0.06 3.26±0.17 4.68±0.17*t <.0001

Heart-LV weight index, mg/g 0.81±0.02 1.17±0.11 2.38±0.14*t <.0001

Infarction size, % 0±0 29+1 37±1 <.0001

Lung water content, % 77.8±0.2 79.1 ±0.1* 81.1 ±0.3*t <.0001

Pleural effusion, No. of rats 0 0 14

Pleural plus peritoneal effusion, No. of rats 0 0 3

Ml indicates myocardial infarction; LV, left ventricular; and heart-LV weight index, heart weight minus LV weight indexed to bodyweight. Values are mean±SEM from 1 1 to 25 determinations.*P<.05 vs control value; tP<.05 vs moderate Ml value.



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TABLE 3. Tissue Angiotensin-Converting Enzyme, Aminopeptidase A, and Neutral Endopeptidase Activities and PlasmaAngiotensin-Converting Enzyme Activity in Rats With Myocardial Infarction and Sham-Operated Rats

ACE Activity NEP Activity APA Activity

Plasma, Lung, Kidney, Lung, Kidney, Lung,nmol-min-1nmL pmol*min1mg-1 pmol*min-1*mg-1 pmol*min-'mg-1 pmol*min-1.mg-1 nmomin 1 *mg1

Control(n=25) 14.7+0.5 717±41 12.3+0.8 1.86+0.07 13.38+0.74 63.4+3.0

Moderate Ml(n=12) 17.5+#1.2 613-+52 13.3-+1.6 1.59+.~0.12 12.05+~-0.91 52.5+4.3

Severe Ml(n=19) 20.5+0.5* 400-+-36*t 13.2+1.1 1.71+0.09 12.35+0.76 58.2+4.7

All Ml (n=31) 19.4+0.9* 481 +35* 13.2+0.9 1.67+0.07 12.25±0.58 56.0±3.3

P value(ANOVA) <.0001 <.0001 NS NS NS NS

ACE indicates angiotensin-converting enzyme; NEP, neutral endopeptidase; APA, aminopeptidase A; and Ml, myocardial infarction.Values are mean±SEM from 12 to 25 determinations.*Pc 05 vs sham-operated (control) value; tPc.05 vs moderate Ml value.

Fig 2B). Renal ACE activity remained unchanged. Heartfailure did not influence renal or pulmonary NEP activityor pulmonary APA activity (Table 3).

Lung ACE mRNA ExpressionACE mRNA expression in lungs from heart failure and

sham-operated rats was assessed by Northern blot analy-sis. A rat ACE mRNA hybridizable to the human ACEprobe was detected by Northern blot, with a size of =n4 kb.A representative Northern blot in Fig 3 clearly shows arelative decrease in lung ACE mRNA levels in severe MIrats compared with sham-operated rats. The densitomet-ric absorbance of the ACE mRNA-specific band, revealedby densitometric quantification, was 2.47±0.44 units(mean-+-SEM) in the severe MI group as compared with7.96±0.44 units in the sham-operated group. The 28SrRNA band was 20.74+3.06 units in the severe MI ratsand 32.93+2.38 units in the sham-operated rats. Theoptical density (OD) ratio of ODACE mRNA to OD2ssrRNA was 0.13+0.02 in the severe MI rats and was0.25±0.02 in the sham-operated rats (P<.01) (Fig 3). Asecond Northern blot was performed using seven lungmRNA samples from the severe MI group and seven fromsham-operated controls. The lung ACE mRNA was nor-malized to GAPDH mRNA. The OD ratio of ODACF toODGAPDH mRNA was significantly decreased from 4.7 ±0.9in the sham-operated group to 2.0±0.3 in the severe MI

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FIG 2. Bar graphs show effects of heart failure induced by myo-cardial infarction (Ml) on plasma angiotensin-converting enzyme(ACE) activity (A), lung ACE activity (B), and lung ACE mRNA levels(C). Each bar in panels A and B represents the mean±SEM from12 to 25 determinations. ACE-specific mRNA levels were halved inlung from severe Ml rats compared with lung from control rats (fivedeterminations in each group) (C). C indicates control; M, moder-ate Ml; and S, severe Ml. *P<.05 or greater vs sham-operatedvalue; tP-.05 or greater vs moderate Ml value.

group (P<c02) (Fig 4). Therefore, the ACE mRNA levelwas halved in the lung from the severe MI group com-pared with the control group. There was a close correla-tion between lung ACE mRNA levels and ACE activity inthese two sets of experiments (R2=.77, P<.001).

Consequences of Severe MI on the CirculatingRenin-Angiotensin SystemAs shown in Table 4, this second series of experi-

ments provided results identical to those of the previ-ous experiments concerning blood pressure, myocar-dial hypertrophy, and urinary cGMP excretion. Thedecrease in pulmonary ACE activity and the increasein plasma ACE activity closely confirmed the results of

Lung ACE activity(pmol/min/mg protein)

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FIG 3. Bar graph (A) and gels (B and C) show expression of lungangiotensin-converting enzyme (ACE) mRNA in severe myocardialinfarction rats versus sham-operated (control) rats. Total RNAs (20gg per lane) prepared from lungs of myocardial infarction or controlrats were fractionated in a 1 % agarose formaldehyde gel, trans-ferred onto Hybond N membrane, and hybridized with d_32p-labeled human endothelial ACE cDNA (B). The corresponding ACEactivities of these animals are shown (A). 18S and 28S ribosomalbands from the same Northern blot analysis shown in panel C werescanned to define the amount of RNA in each lane.



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FIG 4. Gels (top) and bar graph (bottom) show expression oflung angiotensin-converting enzyme (ACE) mRNA in rats withsevere myocardial infarction (infarct, n=7) versus sham-oper-ated control rats (control, n =7). mRNAs were analyzed byNorthern blotting followed by hybridization to 32P-labeled humanendothelial ACE cDNA and glyceraldehyde-3-phosphate-dehy-drogenase (GAPDH). OD indicates optical density. The level ofACE mRNA was significantly increased in severe myocardialinfarction (F=7.7, P<.02).

the first experiment. Plasma angiotensinogen was de-creased in severe MI rats. PRA and PRC were slightlyincreased in severe MI rats, whereas plasma Ang II

levels were unchanged. The ratios of Ang II to PRAand to PRC were decreased in severe MI rats com-

pared with control rats.

DiscussionIn this model of experimental heart failure, the

activities of enzymes regulating vasoactive peptideswere evaluated. The most striking finding was that lungACE activity was decreased in MI rats compared withcontrol rats and that this decrease was dependent on theseverity of the heart failure. In contrast, plasma ACEactivity was increased in MI rats, and this increase was

also proportional to the severity of CHF. Lung ACEmRNA level in severe MI rats was half that of thecontrol rats. Renal ACE activity, however, was not

affected in the MI rats, and neither renal or pulmonaryNEP nor pulmonary APA activities were altered.

In the present study, MI induced cardiac hypertrophyand a decrease in systolic blood pressure. PRA, plasmaANF level, and urinary eGMP excretion were increased inrelation to the severity of MI. a finding consistent withprevious studies.'922030 ACE is responsible for the conver-sion of Ang I to Ang II; only the latter has importantphysiological effects on vasoconstriction and on stimula-tion of aldosterone release. In rat lung, ACE is predomi-nantly expressed in vascular endothelial cells and consti-tutes an important endothelial cell marker.6h7 In thepresent study, the change in endothelial lung ACE activitywas organ specific, as demonstrated by the finding thatACE activity in the kidney, in contrast to that in the lung,was not affected. This finding suggests a cell-specificregulation of ACE because lung ACE activity reflectsmainly endothelial ACE activity whereas renal ACE ac-tivity reflects mainly epithelial ACE activity. Furthermore,there was no alteration in the activity of lung APA,indicating that the change in lung ACE activity was

enzyme specific rather than a generalized effect on mem-brane protein expression in pulmonary cells.The decrease in lung ACE activity was paralleled by

a decrease in ACE mRNA levels as determined byNorthern blot, suggesting that the decreased lung ACEactivity could be attributed to diminished ACE biosyn-thesis as a result of the decreased ACE mRNA levels.This fall in mRNA levels may be due to alterations inthe transcription rate of the ACE gene or in the stabilityof its mRNA, or both.

Alteration in ACE gene expression could result sche-matically from either humoral neuroendocrine changesor local hemodynamic factors. Thyroid hormone andcorticosteroids have strong effects on ACE expression invivo and/or in vitro.31-33 However, such mechanisms seemnot to be involved, since changes in these two hormoneshave not been reported in heart failure. Lung edema, inaddition to the chronic decrease in blood flow, contrib-utes to blood desaturation and hypoxia in lung tissue.Studies by Oparil et aD34 indicated a progressive reduc-

TABLE 4. Characteristics of Rats Showing Effects of Experimental Heart Failure on Components of theRenin-Angiotensin System

Control Moderate Ml Severe Ml P Value(n=10) (n=11) (n-10) (ANOVA)

Systolicblood pressure, mmHg 129+3 120+4 111+3* <.01

Heart- LV weight index, mg/g 0.80-+0.04 1.07±0.05* 2.04+ 0.1 0* <.0001Urinary cGMP, nmol/h 1 .4+ 0.1 2.5 + 0.3* 4.6+0.3* <.0001

Plasma ACE activity, nmol mL-1 min-1 13.1+0.6 13.7+0.7 18.2+0.6* <.0001Pulmonary ACE activity, pmol mg-1 min-1 786+35 803±42 405±50* <.0001

PRA, ng Ang mL- h-1 3.64+0.51 5.26±0.60 5.82+0.77 <.07

PRC, ng Ang mL-1 h1 17.1+1.2 25.7+3.1 28.0+3.4* <.05

Plasma angiotensinogen, ng Ang l/mL 989±49 903±34 780 ±57* <.05

Plasma Ang II, pg/mL 5.3±0.7 6.8+0.8 6.0+1.0 NS

Ang Il/PRA 1.61+0.14 1.37+0.17 1.10±0.09* <.05

Ang Il/PRC 0.33+-0.03 0.28+0.03 0.22±0.02* <.05

Ml indicates myocardial infarction; LV, left ventricle; ACE, angiotensin-converting enzyme; PRA, plasma renin activity; Ang 1,angiotensin l; PRC, plasma renin concentration; and Ang II, angiotensin 11. Values are mean+SEM.*P<.05 vs control group.

4



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tion in lung ACE activity and an increase in renal ACEactivity in rats exposed chronically (2 and 4 weeks) tohypoxia and a reversion to basal values after return to anormoxic environment. No significant alteration in serumACE content was observed. In the present study, areduction in lung ACE activity was observed, but plasmaand kidney ACE did not follow the evolutionary patterndescribed in hypoxia. Therefore, the changes observed inlung and plasma ACE in heart failure do not seem to beexplained by hypoxia alone. Concomitant elevation ofpulmonary artery pressure and lumen area and decreasein cardiac output leading to a decrease in shear stress isanother putative hemodynamic cause of the alteredexpression of the ACE gene in pulmonary endothelialcells. This could be due to a specific alteration in cellfunction in response to the mechanical stimulus. Anassociation between decrease in shear stress and alteredendothelial cell function has already been found in CHFby investigators who studied endothelium-derived relax-ing factor activity in that setting.1"The vascular endothelium plays an important role in

the control of vasomotor tone by releasing vasoactivesubstances and by metabolizing vasoactive peptidesthrough ectoenzymes such as ACE, APA, and alsoNEP.16 Endothelial functional impairments related tothe release of vasoactive mediators have been demon-strated in experimental and human CHF.10-13 The find-ing of the present study may indicate a novel form ofendothelial dysfunction in CHF consisting of an alter-ation in ACE gene expression.

In heart failure rats, a paradoxical increase in plasma-soluble ACE activity was found, in contrast to thedecrease in lung ACE. This increase in plasma ACEactivity was proportional to the severity of MI. Severalpossible mechanisms could be responsible, individuallyor in combination, for the increase in plasma ACEactivity: (1) Endothelial alteration in heart failure mayalter the ACE solubilization process at the plasma mem-brane, resulting in increased ACE release.35 Althoughthe lung seems to be one of the most important sites ofACE synthesis, ACE is synthesized by all the otherendothelial cells throughout the body, a process that maybe altered in heart failure. (2) Extraendothelial sourcessuch as mononuclear inflammatory cells and fibroblastsmay contribute to the increased plasma ACE activity.26,36The observed increase in cardiac ACE gene expressionin heart failure rats37 and the ACE overexpression in thescar tissue of the infarcted area38 could also contribute toelevated plasma ACE activity. ACE could be releasedfrom the hemodynamically stressed scar tissue of theinfarcted area into the circulation; this remains to bedemonstrated. (3) Finally, increased plasma ACE activ-ity might be the consequence of a decreased circulatingACE turnover rate due to the altered liver function inheart failure, as found for other proteins.4 Indeed, aspreviously reported in patients with severe heart failure,4plasma angiotensinogen was decreased in severe MI rats,whereas cardiac angiotensinogen mRNA has been foundto be increased in this model.39We investigated the physiological consequences of

decreased lung ACE activity and increased plasma ACEactivity by quantifying Ang II in the circulation.Whereas PRC was significantly increased in heart fail-ure rats, plasma Ang II levels were not different from

to PRA and to PRC were decreased in severe MI ratscompared with control rats. This suggests that de-creased pulmonary ACE may prevent excess Ang II

formation. Furthermore, this observation is in agree-ment with the concept that pulmonary ACE is more

important than its plasma counterpart for the conver-sion of Ang I into Ang l1.6 Given the observations thatpharmacological ACE blockade improves left ventricu-lar function in heart failure40 and that a high level ofACE expression may be deleterious for the coronary

circulation in healthy individuals,41 the present down-regulation of pulmonary ACE may have importantpathophysiological consequences.Most of the MI rats in the present study had high

plasma ANF levels, indicating the activation of thecardiac natriuretic system in response to cardiac over-

load. NEP is one of the main enzymes involved in the invivo degradation of this peptide, especially in the kid-ney. Our results showed that renal and pulmonaryepithelial NEP activities were essentially unchanged inthis model of heart failure. These findings suggest thatalteration of NEP synthesis in the kidney is not involvedin the blunted response to ANF in heart failure.42,43Furthermore, the renal NEP activity may be highenough to degrade ANF in heart failure.44 Anotherpossibility derives from the fact that although NEP inrodent kidney is predominantly located at the tubularbrush border,15 it is also present, in much loweramounts, at the entire glomerular epithelial cellularsurface.15 Consequently, a local increase in glomerularNEP activity in the heart failure rat cannot be ruled outsince it would not be detectable because of the predom-inance of tubular NEP activity.

In summary, we have demonstrated that lung ACEactivity is decreased and plasma ACE activity is in-creased in rats with chronic heart failure. The decreasein lung ACE activity was correlated to the decrease inACE mRNA levels, suggesting that decreased lungACE activity can be attributed to diminished ACEbiosynthesis. Whereas PRA and PRC were significantlyincreased in heart failure rats, plasma Ang II levels werenot different from those of control rats. Thus, decreasedlung ACE activity possibly contributing to limitation ofAng LI formation represents another functional alter-ation of the renin-angiotensin system in congestiveheart failure. Complex alterations of the renin-angio-tensin system occur in congestive heart failure, includ-ing opposite changes in plasma and tissue angiotensin-ogen45 and ACE. Quantification of only the circulatingcomponents of the renin-angiotensin system in heartfailure is probably not sufficient to assess the status ofthis system in this pathophysiological state and may atleast in part explain the absence of correlation withhemodynamic parameters.46

AcknowledgmentsThis study was partly supported by the Institut National de

la Sante et de la Recherche Medicale and by a grant from theBristol-Myers Squibb Institute for Medical Research. Wethank Dr B. Greenberg for critical review of this manuscript.

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H Huang, J F Arnal, C Llorens-Cortes, M Challah, F Alhenc-Gelas, P Corvol and J B Michelexperimental congestive heart failure. A novel aspect of endothelium dysfunction.Discrepancy between plasma and lung angiotensin-converting enzyme activity in

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