diverse factors ace molecularbiology and

Br Heart J (Supplement) 1994; 72: 3-10

BASIC SCIENCE

Diverse factors influencing angiotensinmetabolism during ACE inhibition: insights frommolecular biology and genetic studies

Kevin Morgan

Departments ofCardiology andHistochemistry, RoyalPostgraduate MedicalSchool, HammersmithHospital, LondonK MorganCorrespondence to:Dr K Morgan,Department ofHistochemistry,Royal PostgraduateMedical School,Hammersmith Hospital,London W12 ONN.

Reduction of angiotensin converting enzyme(ACE) activity can profoundly benefit aproportion of patients receiving cardiac careby improving cardiovascular function,reducing the risk of myocardial infarction, andextending life expectancy.' 2 The long termresponse to ACE inhibition, however, remainsunpredictable in patients, and issues currentlyrequiring attention are whether and how ACEinhibition might be improved such that alarger proportion of patients can be effectivelytreated.Although plasma ACE activity can be

essentially abolished pharmacologically, en-zyme activity is not permanently reduced tonegligible values in all human tissues.3 Theexistence of pathophysiologically important,patient specific differences in angiotensin II orbradykinin production during ACE inhibitionmight account, in part, for the outcome indifferent subjects.New insights into the relevance of residual

or altered angiotensin metabolism duringtreatment with ACE inhibitors might be closeat hand following advances in the molecularbiology of ACE, cardiac chymase, and othercomponents of the renin-angiotensin andbradykinin systems.

Current evidence indicates that activation ofthe plasma renin-angiotensin system in heartfailure is a secondary line of endocrineresponse enlisted after increases in plasmaatrial natriuretic peptide and arginine vaso-pressin have already occurred.4 The activationis usually transient, largely reversible, andassociated with phases of acute decompen-sation followed by stabilisation, unless endstage disease develops or diuretics areintroduced. It is suspected, however, thatlocalised tissue specific systems such as theintracardiac renin-angiotensin system mightbe significantly activated from an early stageduring the progression of heart failure,although it is not known precisely what factorsacting within the heart or other organs mightinfluence the sequence of events leading to thischange.

Progress towards the identification ofimportant modifications in ACE productionand function of the renin-angiotensin systemin heart failure has been made in animalmodels of cardiovascular disease. Determiningwhich of the molecular changes seen inanimals are most relevant to humans must

now be evaluatedcomplicating humanaccount.

after taking severalspecific factors into

Response of renin-angiotensin system toACE inhibitorsSeveral predisposing factors in humans in-fluence the response of the renin-angiotensinsystem to ACE inhibitors.

GENETIC INFLUENCES

So far, a tentative molecular-genetic linkbetween ACE and the occurrence of humancardiovascular disease has been established. Acommon, simple genetic variation existingwithin intron 16 of the human ACE gene islinked to determination of plasma, andperhaps tissue, activities of ACE.5 6 PlasmaACE activity in subjects with the ACE DDgenotype can be twofold higher than that inthose with the ACE II genotype. The ACE Dallele is apparently associated with increasedrisk of myocardial infarction and perhaps inthe development of end stage disease throughdifferent aetiologies in white people.'-"Around 30% of white patients with heartfailure attending hospital clinics are homo-zygous for the ACE D allele, the proportionincreasing according to the severity of disease.Raised baseline plasma ACE activity in someof these patients may have contributed to thegeneration of damaged or inappropriatelyrepaired cardiovascular tissue through longterm increased production of angiotensin IIaround the primary lesion, though acontribution from closely linked and unrelatedgenes cannot be ruled out."The precise molecular mechanism through

which possession of the D allele contributes toraised plasma ACE activity is not yet under-stood; a structural change located elsewherewithin the ACE gene might provide theanswer. Theoretically, the expression of theD allele may be raised in some tissues,the release of the protein it encodes fromthe plasma membrane may be increased, orthe degradation of excess enzyme may bereduced. Studies aimed at understanding thebiochemical-genetic regulation of ACEproduction and its relation to local haemo-dynamic, endocrine, and histological re-organisation during ACE inhibition will be offundamental importance for drug targeting in

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the future, but progress may be delayed by thecurrent absence of similar ACE geneticvariants in animal models.

It has been difficult to establish a clear rolefor human plasma or tissue concentrations ofrenin'2-14 or aldosterone'5'7 in the generationof predisposition to cardiovascular disease.The existing biochemical data suggest thatACE activity is not the only determinantgoverning the cascade but that interaction ofvarious protein and peptide componentsdetermines net angiotensin production. Thediscovery of a common allelic variant ofhuman angiotensinogen is thought to be sig-nificant."8 This common molecular variationspecifies the substitution of a methionine(Met) residue by a threonine (Thr) residue at

position 235 of the mature protein. Individualshomozygous for the Thr235 residue usuallyhave high baseline plasma concentrations ofangiotensinogen, and there may be largeethnic differences in allele frequencies."

Uncertainties about the importance ofraised plasma angiotensinogen concentrationand its consequences for the response to ACEinhibition are raised by the discovery of thisangiotensinogen variant which parallel thoseassociated with the ACE gene dimorphism.Does a combination of alleles specifying raisedangiotensinogen and ACE result in a high riskgroup of patients? About 5-10% of patientswith heart failure in the United Kingdom are

homozygous for both the angiotensinogen andACE alleles (K Morgan et al, unpublished dataon 300 patients). Are plasma or tissue angio-tensinogen, renin, and angiotensin I concen-

trations raised during treatment with ACEinhibitors to an extent that might outweighany genetically influenced interindividualdifferences in baseline angiotensin IIproduction? Further clinical studies are

required to resolve these questions.

ENDOTHELIAL FUNCTION

Although further investigation is required todetermine how subjects respond to ACEinhibition according to their genetic make up,molecular interactions within different organs

at the blood-endothelial interface are likely tobe important in all patients.20 Whereascoronary endothelial angiotensin receptorscolocalise with ACE binding, they are alsofound at lower concentrations in the mediallayer of the vessel wall.2' In addition, thoughresidual angiotensin-forming activity occurs inthe vascular beds of a range of different organsduring ACE inhibition, it is currently unclearwhich locations within the coronary tree are

most important with respect to occurrence ofdeleterious vascular events mediated by angio-tensins. The fine details of the molecularorganisation of the renin-angiotensin systemalong the coronary vascular tree, where ACEis found associated predominantly with

coronary endothelium and perivascular nerves,require further molecular investigation.22

Perturbation of endothelial cell ACEturnover is presumably an early event in theresponse to ACE inhibition. Enzyme-inhibitorcomplexes may be internalised and degraded

or ACE secretase activity, which releasesmembrane bound ACE, may be altered.23Theoretically, endothelial cells might respondto reduced circulating angiotensin II concen-trations by upregulating cell surface angio-tensin receptor concentrations or down-regulating angiotensin degradation.

In response to raised circulating concen-trations of angiotensinogen, prorenin, andactivated renin secreted from the liver andkidneys, respectively, ACE inhibited endo-thelial cells might begin sequestering andmetabolising these extra circulatingcomponents. Although specific cell surfacereceptors mediating uptake of angiotensinogenand renin have not been identified, alterationsin their post-translational glycosylation statesknown to occur during ACE inhibition mightaffect their interaction with the endothelial cellsurface and their metabolism in someorgans.24 25A localised tissue specific contribution to

vascular angiotensin production by initiationof de novo synthesis of ACE, angiotensinogen,and renin within certain endothelial cells as aresult of activation of gene transcriptionremains a distinct possibility26-28 (despiteconsiderable difficulties associated with its invivo and in vitro analysis29 30), provided thatthere is efficient translation of the messengerRNA (mRNA) and secretion of the correctlyprocessed mature proteins.3' Does inductionof endothelial gene expression result inresistance to ACE inhibition in certainpatients?

EXTENT OF VASCULAR DISEASE

Dysfunctional endothelial cells surroundingatherosclerotic plaques may well responddifferently to ACE inhibition than do normalcells. Activated endothelial cells and vascularsmooth muscle cells in injured arteries expressvarious trophic factors capable of modulatingcell function. Paracrine or autocrine inductionof angiotensin production may already exist inthe diseased coronary arteries of some patientsbefore treatment with ACE inhibitors. Parallelchanges in the generation of bradykinin withinthe damaged vessel wall by tissue kallikreinactivity 2-15 might also occur.The regulation of vascular wall angiotensin

and bradykinin peptide concentrations mightbe modulated at several levels.36-38 The localbalance of angiotensin and bradykinin peptidemay be influenced by the leucocyte chemo-attractant and proadhesive properties of angio-tensin and the subsequent production ofangiotensinogen and ACE by infiltratingcells.3942The high ligand binding density of ACE

localised in perivascular nerves suggests thatoperation of extrinsically controlled neuralmechanisms might also be important indetermining the vascular response to ACEinhibition. How vascular innervation relates tocardiac angiotensin metabolism requiresfurther research. Within the myocardium,nerve fibres, blood vessels, fibroblasts, andmast cells43 are juxtaposed within the cardiacinterstitium, which consists of an extracellular

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glycoprotein network supporting myofibrilbundles anchored in parallel array. Nervescontaining neuropeptide synthase and nitricoxide synthase are distributed throughout theatria and ventricles.44 45 The nerves areassociated with blood vessels and myocytes,with the atria having more nerve fibres thanthe ventricles. Around coronary blood vesselsthere is an inverse relation between externalvessel diameter and the extent of innervation.The number of nerve fibres containingsubstance P, a potential ACE substrate, is low.Ganglion cells containing nitric oxide synthasein the subepicardium and interatrial regionsare surrounded by terminals positive forsubstance P. Connective tissue mast cellsoccur in close proximity to interstitial nervefibres in some tissues, and it has beenhypothesised that they metabolise peptidesreleased into the interstitium from nerves.Whether the cardiac innervation contributes tothe release of components of the renin-angio-tensin system into the interstitium andmodulation of peptide and vasodilator actionis uncertain.

REGIONAL DIFFERENCES IN INTRACARDIAC

DISTRIBUTION OF COMPONENTS OF RENIN-

ANGIOTENSIN SYSTEM

AtriaHuman atrial cardiac myocytes manufactureangiotensinogen, renin, ACE, and angiotensinreceptors and show a positive inotropiccontractile response to angiotensin JI.46-48Neural reflex mediated activation of atrialrenin-angiotensin activity might occur in re-sponse to failing cardiac output in an attemptto improve the dynamics of ventricular filling.Raised atrial pressure leading to increasedventricular filling pressure has been hypoth-esised to have deleterious effects on damagedventricles by contributing to end diastolicstretch of ventricular sarcomeres. Atrial ACEinhibition is therefore thought to be beneficial,at least mechanically. There remains, however,a poor understanding of potential interactionsbetween atrial renin-angiotensin activity andthe dysregulated production of atrial natriureticpeptide seen in heart failure.

Impulse conduction systemConcentrations of angiotensinogen, renin, andangiotensin receptors in cardiac conductiontissue differ significantly from the distributionof ACE in rat hearts. The concentration ofACE in the sinoatrial and atrioventricularnodes is relatively low compared with theconcentrations of angiotensinogen and angio-tensin receptors. This observation has raisedspeculation concerning the mode of angio-tensin II production and function inconduction tissue.The function of the sinoatrial node is often

disturbed in patients with heart failure, withabout 300/o of patients showing atrial fibril-lation, and particularly high concentrations ofangiotensin receptors and nerve fibres havebeen noticed in the atrioventricular node inrats. Conduction cells, neural tissue, and thenodal artery might be involved in angio-

tensin II metabolism in these histologicallyspecialised locations.49 Although atrioventricu-lar node dysfunction is not a commonpersistent problem in patients with heartfailure, the conduction system runningthrough the ventricular endocardium may,nevertheless, represent an important site ofresidual intracardiac angiotensin generationaffecting intercellular communication duringACE inhibition.50 51

VentriclesExperimental evidence suggests that myo-cardial ACE gene expression is increased inrats with augmented ventricular wall stressand heart failure produced as a result ofoverload.52 A chamber specific fourfoldincrease in the levels of ACE mRNA, atwofold elevation of ACE protein, and afourfold increase in ACE activity occurs in thefree wall, septum, and ventricular apex.53

After induction of left ventricular infarctionin rats there is a 1*8-fold increase in thedensity of myocyte angiotensin II receptorsassociated with myocyte hypertrophy54 whileleft ventricular angiotensinogen expression isonly transiently increased, about 1-7-fold,subsiding to control levels at some pointbetween five days and 25 days afterinfarction.55 56 Increased ACE expression canremain detectable one month after in-farction.57 These observations are consistent,perhaps, with a role for tissue angiotensinogenproduction in the acute phase separate fromthe function of chronically raised ACE activityand angiotensin receptor density. Induction ofscar formation and peri-infarct inflammationand angiogenesis may be important processesaffecting activation of the cardiac renin-angio-tensin system soon after infarction, whereaslocalised release of growth factors in mechan-ically stressed tissues might account forinduction and maintenance of ACE and angio-tensin receptor expression in hypertrophy andscar expansion over the long term. Raisedcardiac ACE and angiotensinogen concentra-tions can be induced in the absence ofischaemia or increases in ventricular mass58 byheart failure induced by increased heart rate.

It is not clear whether changes in geneexpression of the ventricular renin-angiotensinsystem at different times after infarction orhypertrophy or in response to ACE inhibitorsare beneficial, detrimental, or ineffectual.Most experimental evidence indicates thatACE inhibitors can limit infarct size andventricular dilatation and slow hypertrophy,but it is not certain whether these effects aremediated by indirect haemodynamic changesor by direct cardiac structural-functionalchanges mediated by gene expression.59

Analysing the human cardiac renin-angiotensin systemDetection of raised ACE gene expression inthe human left ventricle (up to twofoldincreases) has so far been described only in aproportion of patients with end stage heartfailure60; distinct mechanisms might still be

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involved in the induction of raised veiACE expression in human heartaccording to disease aetiology.

It is now possible to analyse ACexpression in small cardiovasculaisamples derived from different regiorheart using reverse transcription-polchain reaction amplification (fig 1). Aqualitative studies are straightforwarc'titative studies are more time consun

require application of rigorous control,competitive polymerase chain reaction(fig 2).61

Localised cardiac chymase producHuman cardiac chymase is fairly abuiventricular tissue.62 It is synthesised a

Figure 1 Polyacrylamide gel autoradiograph showing qualitative analysis of ti,expression of genes coding for angiotensinogen (1), renin (2, negative), angiote,

converting enzyme (3), cardiac chymase (4), glyceraldehyde 3-phosphate dehyi(5), and transferrin receptor (6) in a single human tissue sample taken from t),

wall of the right ventricle of an explanted heart; reverse transcriptase-polymeras,reaction was used to amplify mRNA (M, markers).

M 1 2 3 4 5 6 78

:. -_~~~~~~~~~~~~~~~~~~~~~~~~~~~.. .....

..................~~~~~ ~ ~ ~ ~~~~~~~~~~~~~~~~~~~~~~;:,.:: ...

Figure 2 Polyacrylamide gel autoradiograph showing a competitive polymerasreaction method for measuring ACE gene expression in human tissues by titratiendogenous message (lower band) against a competitor template (upper band).Quantification is possible after titration when the two cDNA molecules are am,the same extent (lane 4) following the addition of a predetermined amount of cmolecule. (M, markers.)

ntricular protein63 and secreted into the interstitium.43failure The purified enzyme has a restricted substrate

specificity for angiotensin I and neurotensin.64_E gene It generates angiotensin II more efficientlyr tissue than does ACE, whereas it cleaves neurotensinis of the somewhat less efficiently in the same micro-lymerase molar range. Chymase mediated generation ofilthough interstitial angiotensin II within the ventriclesi, quan- is potentially of great importance, provided.ling and that local angiotensin I production is sufficients using a in view of its potential inotropic, hypertrophic,strategy and tissue remodelling effects.

Chymase activity has been analysed indifferent chambers of the heart. Likewise, wehave analysed chymase gene expression in

ction different regions of the heart. Using reversendant in transcriptase-polymerase chain reaction ampli-is a pre- fication we confirmed that the highest human

cardiac chymase gene expression is restrictedto ventricular tissue, although spliced mRNAcan be detected in atrial tissue at lower levelsin a proportion of patients.6" The analysis ofmechanisms modulating cardiac chymase geneexpression in different patients and its relationto disease progression should have importantimplications for understanding the role ofangiotensins in heart failure in the future.

Interpreting the importance of alteredcardiac ACE gene expressionCardiac ACE might have distinct roles in thepeptide hormone response to ischaemia,damage repair, wall stress, and maintenace ofhypertrophy according to its intracardiacdistribution and activity. Determining whichintracardiac sites respond favourably tointervention with ACE inhibitors may require

he a detailed understanding of regional intra-wnsin cardiac ACE production and intracardiac sig-drogenase nal transduction pathways.e cfhaein The activated angiotensin II receptor sub-

type 1 (AT1) and its associated heterotrimericGTP binding protein (Gq subtype) are linkedto increased intracellular production ofinositol triphosphate, raised intracellularcalcium ion concentrations, generation of dia-cylglycerol (DAG),65 and inhibition ofadenylate cyclase activity.66 Angiotensindependent increases in intracellular calciumare associated with stimulation of tyrosinekinase activity,67 whereas release of DAGactivates members of the protein kinase Cfamily of enzymes capable of phosphorylatingserine and threonine residues within targetproteins, including intracellular structuralproteins, contractile proteins, components ofthe protein biosynthesis machinery, and genetranscription factors relevant to cardiacpathology.68-72Exogenous angiotensin II upregulates net

cardiomyocyte gene expression via a proteinkinase C-fos/jun dependent pathway,73 butwhether this pathway normally stimulatesfeedback inhibition of ACE production by

;e chain activation of repressors which recognise theion of ACE gene remains to be determined.

plified to Ventricular hypertrophy in spontaneously-ompetitor hypertensive rats is associated with a threefold

increase in ATIA gene expression and a

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corresponding twofold increase in receptordensity.74 The relative concentrations of AT,and AT2 subtypes does not seem to bechanged and upregulation of expression isreversible. Although the human ATIA receptorgene has been characterised,75 no alterations inthe levels of its expression have been describedso far in failing hearts.76The distribution and modulation of cardiac

bradykinin receptors is less well characterised.Blunted intracellular responsiveness to raisedplasma concentrations of atrial natriureticpeptide secreted from atria and ventriclescorrelates with altered cyclic GMP (cGMP)metabolism. Cardiac responsiveness to otheragents which activate increases in intracellularcGMP, such as the bradykinin-nitric oxidepathway, might also be compromised in heartfailure. ACE inhibition does, however, seem tohave cardioprotective effects through thebradykinin and nitric oxide pathways in some

experimental systems.77-79

Disease related regional disturbances ofcardiac phospholipid, intracellular calcium,and cyclic nucleotide metabolism in failinghearts may exert an important influence on

ACE gene expression. Uncoordinated hyper-stimulation of cardiac intracellular signaltransduction probably occurs, in part, as a

result of compromised energy metabolism.Changes in cardiac energy metabolism in heartfailure are thought to be largest in the sub-endocardium, where increased wall stress andventricular filling pressure contribute todecreased perfusion.80 An infarction covering40%/ of the left ventricle in rats is associatedwith a prolonged 15% decrease in ATPcontent in the remaining viable tissue.Changes in the septum and right ventricle are

also prolonged but are less pronounced.8'Hypoxic cardiomyocytes release high concen-

trations of AMP, which can be metabolised toadenosine and inosine at the extracellularsurface of endothelial cells.82 Sustainedincreased extracellular concentrations ofadenosine might modify myocardial andendothelial cell function by activation ofadenosine receptors and inhibition of theeffects of adrenergic stimulation either at thelevel of adenylate cyclase or adrenergicreceptor phosphorylation. Although negativefeedback by extracellular adenosine mayprimarily be a protective mechanism operatingagainst excessive adrenergic stimulationin the endocardium, it might seriouslycompromise other processes dependent on

signal transduction through the cyclic AMP(cAMP) pathway. Unravelling the relationbetween altered cAMP status, ACE pro-duction, and altered cardiac angiotensin IIresponsiveness83 may identify cardiovascularregulatory pathways important during ACEinhibition.

The proximal 5' promoter regions of thehuman and mouse ACE genes have beendescribed,84 but precise molecular mechan-isms regulating cardiac ACE gene expressionare currently undefined. Molecular control ofrenin gene expression has been characterisedmore extensively than the other components

of the renin-angiotensin system.8594 Inductionof renal renin expression is intimatelyassociated with the intracellular level ofcAMP, and expression of the gene in extra-renal tissues is normally repressed by a nuclearprotein which blocks the cAMP responsiveDNA sequence element located in the 5'promoter region of the gene. Regionalmyocardial renin expression might beexpected to remain repressed in heart failure,in which stimulation through the adenylatecyclase cascade may be defective. However,increases in atrial renin gene expression havebeen shown by dietary salt deprivation.95 Thismay represent an atrial autocrine or paracrineresponse aimed at reinforcing angiotensinmediated downregulation of atrial natriureticpeptide production in the salt depleted state.It is unknown whether this mechanism isdefective in heart failure.

Intracardiac levels of angiotensinogen geneexpression are higher than those of ACE orrenin and they increase in aging rat hearts.96Molecular mechanisms governing its ex-pression in the liver have been defined interms of DNA sequence elements involved incytokine mediated acute phase transcriptionactivation and the response to glucocorticoidsand angiotensin II.90-92Mechanisms controlling the activity of the

cardiac kallikrein-kinin system have not beenstudied extensively, although the existence ofkallikrein and kininogen in rat heart and bloodvessels has been proved.97-99 Concentrations ofbradykinin peptides and their response toACE inhibition have been measured in a rangeof rat tissues, including the heart, where thereis a significant increase in their concentrationafter drug treatment.'00 A correspondingactivation of bradykinin receptors presumablyoccurs, but whether there is parallelmodulation of local kininogen and kallikreinactivation is uncertain. Expression of thekininogen gene displays differential 3' terminalexon usage for the production of highmolecular weight and low molecular weightkininogens.'0' In the liver expression of thelow molecular weight transcript, designedprimarily for bradykinin production, pre-dominates. 102

Further studies of factors affectingangiotensin metabolism during ACEinhibitionHUMAN GENETIC STUDIES

The long term response to treatment withACE inhibitors in patients classified accordingto angiotensinogen and ACE genotype shouldshow the existence of any adverse contributionmade by the possession of these alleles. Ifthere is an adverse effect then studies of ethnicvariations in allele frequencies and the effectsof dose modifications or use of alternativeACE inhibitors may be required to improvethe targeting of existing treatment.

PATHOLOGICAL TISSUE SAMPLES

The demonstration of abnormal concen-trations or unusual isoforms of components of

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the renin-angiotensin system and theirlocalisation within atherosclerotic arteries orparticular regions of hypertrophied heartswould be of mechanistic importance. In asurvey of 30 samples from the right atrialappendage we found an unusual cDNAspecies encoding angiotensinogen in onesample.6' Interpreting the relevance of suchfindings will require more investigation at themolecular level.

NEW COMPONENTS OF THE RENIN-

ANGIOTENSIN SYSTEM

Further study of several recently characterisedcomponents of the renin-angiotensin systemshould provide new insights into the func-tional organisation of the system in differentorgans. More details about the molecularbiology of prorenin converting enzyme,'03renin binding protein,'04 ACE secretase, theangiotensin II type 2 receptor,'05 106 and angio-tensinases'07 108 and their responses to ACEinhibition should be forthcoming in the nearfuture.

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