regulation of myocardial fibrillar collagen by angiotensin ii. a role in hypertensive heart disease?

J Mol Cell Cardiol 34, 1585ÿ1593 (2002)

doi:10.1006/jmcc.2002.2081, available online at http://www.idealibrary.com on1

Cardiac Renin±Angiotensin Aldosterone System

Regulation of Myocardial FibrillarCollagen by Angiotensin II. A Role inHypertensive Heart Disease?Arantxa GonzaÂlez, BegonÄ a LoÂpez, RamoÂn Querejeta and Javier DõÂez

Division of Cardiovascular Pathophysiology, School of Medicine, and Department of Cardiology andCardiovascular Surgery, University Clinic, University of Navarra, Pamplona (AG, BL, JD),and Division of Cardiology, Donostia Hospital, San SebastiaÂn (RQ), Spain

(Received 30 May 2002, accepted for publication 3 June 2002)

A. GONZAÂ LEZ, B. LOÂ PEZ, R. QUEREJETA AND J. DIÂEZ. Regulation of Myocardial Fibrillar Collagen by Angiotensin II. ARole in Hypertensive Heart Disease? Journal of Molecular and Cellular Cardiology (2002) 34, 1585±1593. Collagentypes I and III (Col I and Col III) are the major ®brillar collagens produced by ®broblasts and myo®broblasts in theadult heart. Fibrillar collagen of the heart provides the structural scaffolding for cardiomyocytes and coronaryvessels and imparts cardiac tissue with physical properties that include stiffness and resistance to deformation. Inaddition, ®brillar collagen may also act as a link between contractile element of adjacent cardiomyocytes and as aconduit of information that is necessary for cell function. As in other organs, collagen turnover of normal adultheart results from the equilibrium between the synthesis and degradation of Col I and Col III. A number of factorshave been described that may alter the balance in favor of either the synthesis (e.g., angiotensin II±ANG II-) or thedegradation. Predominance of synthesis over degradation leads to increased Col I and Col III deposition or ®brosisthat accompanies cardiac diseases such as hypertensive heart disease. Fibrosis alters myocardial structure andfunction and adversely afects the clinical outcome of hypertensive patients. Various lines of evidence suggest thatbesides hypertension, systemically and/or locally produced ANG II may participate in the development of hyper-tensive myocardial ®brosis via activation of ANG II type 1 receptors (AT1R). The potential clinical relevance of thispossibility is linked to the ability of antihypertensive drugs such as angiotensin converting enzyme inhibitors(ACEIs) and AT1R antagonists (ARAs) to reverse myocardial ®brosis beyond their antihypertensive ef®cacy.

# 2002 Elsevier Science Ltd. All rights reserved.

Key Words: Angiotensin II; Arterial hypertension; Collagen; Fibrosis; Myocardium.

Fibrillar Collagen

Every organ is composed of highly differentiated,very specialized parenchymal cells surrounded bystroma consisting of extracellular matrix (ECM),tissue ¯uid and undifferentiated, pluripotent mesen-chymal cells. The maintenance of matrix integrityinvolves the synthesis and degradation of its majorcomponents, including collagens, glycoproteins,glycosaminoglycans and proteoglycans.

Please address all correspondence to: Dr Javier DõÂez, DivisioÂn de Fisiop31080 Pamplona, Spain. Tel: 34-948-425600; Fax: 34-948-425649

0022±2828/02/121585�09 $35.00/0

Biochemical aspects

Collagens comprise a family of structural macro-molecules of the extracellular matrix that exhibitdiversity at the molecular and supramolecularlevels. By de®nition, a collagen is a structural pro-tein of the ECM that contains at least one domainin the characteristic triple helical conformation.1

The triple helix is formed by three polypeptidechains (a chains). All collagen molecules are

atologõÂa Cardiovascular, Facultad de Medicina, C/ Irunlarrea s/n,; E-mail: [email protected]

# 2002 Elsevier Science Ltd. All rights reserved.

1586 A. GonzaÂlez et al.

characterized by a central collagen domain com-posed of repeating Gly-Xaa-Yaa triplets, a highcontain of proline, alanine, and lysine residues andnoncollagenous domains at their terminal ends.2,3

To date, 20 different collagen types have beenidenti®ed.4

Vertebrate collagens can be divided into twogroups based on their primary structure and supra-molecular organization: ®bril-forming (or ®brillar)collagens and non®brillar collagens.1,2 The ®brillargroup is composed of collagen types I, II, III, V, andXI. These molecules contain triple helical domainsof about 1000 amino acids, highly conservedcarboxy-terminal noncollagenous domains of about250 amino acids, and variable amino-terminal non-collagenous domains of 50±520 amino acids.2 Col Iis present in most tissues, and is the most abundantprotein in the human body. Col III is the secondmost common of the collagens, found in associationwith Col I, except in bones, which almost solelycontain Col I.

Synthesis and degradation

Several cells are capable of synthesising collagens(osteoblasts, endothelial cells, smooth muscle cells)but most derive from ®broblasts (Fig. 1). The struc-tural genes for the various collagen proteins arescattered throughout the genome, without appar-ent organization. For instance the genes coding forthe a1(I) and a2(I) chains of Col I are located onchromosomes 17 and 7, respectively, while thegene for the a1(III) chain of Col III is situated onchromosome 2.5 Despite being localized widely apart

Preprocollagen ty

Procollagen typ

Procollagen typ

Collagen types I an

Collagen types I

Telopep

Degradation

α1 (I), α2(I), and FibroblastMyofibroblast

Other cell types

Interstitium

DNA

Collagen types I a

Figure 1 Diagramatic depiction of the different steps of ®bril

from each other, the expressions of the two Col Igenes are usually extremely thightly co-regulated.In ®broblasts also the expressions of the Col I andCol III usually change hand in hand.

A variety of growth factors, cytokines andhormones modulate expression of collagen genes.These include activators and inhibitors, whichprobably act by different signalling mechanisms.2

Multiple signal transduction pathways involv-ing receptor or non-receptor tyrosine kinases,G-protein-coupled transmembrane receptors,cytokine-receptors and integrins, and a battery ofkinases (protein kinase C, MAP kinases, JAK/STATkinases) have been shown to be involved in theregulation of collagen gene expression in ®bro-blasts,6 but to date the entire pathway for any ofthese agents has not been elucidated.

The biosynthesis of collagens follows the normalpattern of protein synthesis, but it differs from thebiosynthesis of many proteins in that the newlyformed a chains undergo a number of post-transla-tional modi®cations.7 In fact, during the translationof messenger RNA into polypeptide, prolyl and lysylresidues of the nascent chains are hydroxylated byprolyl and lysyl hydroxylases, respectively. This isfollowed by glycosylation of certain hydroxylysylresidues to form galactosylhydroxylysines or gluco-sylgalactosylhydroxylysines. Following these modi-®cations the a chains assembly into triple-helicalmolecules, after which they are secreted to theextracellular space.

The secreted triple-helical molecules are extracel-lularly modi®ed in the interstitial space prior to theirassembly into supramolecular structures.7 Forinstance ®brillar collagens are processed from a

pes I and III

es I and III

es I and III

d III molecules

and III fibers

tides

products

α1(III) chains

nd III fibrils

Synthesis

Assembly

Degradation

lar collagens types I and III turnover.

1587Angiotensin II and Cardiac Collagen

proform to the actual collagen forms by speci®cproteinases.3,7 Thus amino- and carboxy-terminalextension propeptides of secreted procollagen types Iand III are cleaved off before ®brils are formed. Thefunction of propeptides in the ECM is unknown, butthey may serve as modulators of ®bril growth, orconstitute feedback regulation.8 The propeptideswith a molecular weight of 42±100 kDa, aredrained from the interstitial space by lymphaticsand across the capillary wall.9 Once the circulationis reached, the liver and kidneys are the mainorgans for excretion.10

During their spontaneous assembly into ®brils,collagen molecules are cross-linked by pyridiniumand deoxy-pyridinium containing bonds. Althoughthe resulting collagen ®bril is very robust, it canbe degraded by collagenase, a member of thematrix metalloproteinases (MMPs) family of zinc-containing endoproteinases.11 This enzyme, ®rstsynthesized as inactive zymogen precursor, requiresto be activated by proteolytic cleavage. Fully acti-vated collagenase can be inhibited by interactionwith naturally occurring, four speci®c tissue inhib-itors of MMPs (TIMP).12 Thus, the actual activity ofcollagenase depends on the rate of synthesis, acti-vation, and the balance between active enzyme andinhibitors.

There is evidence that procollagen moleculesundergo also intracellular degradation, within min-utes of synthesis.13 This process, that occurs withinthe lysosome or within the cisternae of the endo-plasmic reticulum or Golgi aparatus, may allow theadaptive advantages of rapid collagen turnoverwithout compromising the supportive role of ®bri-llar collagen.

Normal collagen homeostasis involves a balancedequilibrium between synthesis and degradation(Fig. 1).14 There normally exists a reciprocal regu-lation between stimulators and inhibitors whosebiological properties neutralize one another tomaintain a zero-sum balance on collagen turnover(e.g., synthesis equals degradation). An imbalancein the stimulator-inhibitor ratio in favor of stimula-tors accounts for collagen accumulation. Incontrast, the overabundance of inhibitors on stimu-lators may lead to collagen network dysruption.

Fibrillar Collagen in the Heart

General aspects

In addition to ®broblasts, myo®broblasts govern®brogenesis in the heart.15 Myo®broblasts arisefrom interstitial ®broblasts and/or pericytes, express

a-smooth muscle actin and are contractile.16,17

Although signals that determine the appearance ofthe myo®broblast phenotype are not entirely cer-tain, recent data suggest that transforming growthfactor-b1 (TGF-b1) induces the differentiation of®broblasts to myo®broblasts.18 Myo®broblastshave a higher activity for collagen production that®broblasts.18 Interestingly, it has been shown thatcultured cardiac myo®broblasts express requi-site components of the renin±angiotensin system,including angiotensinogen, renin and angiotensinconverting enzyme (ACE), and are able to generateANG II de novo.19

Although several collagen types are transientlyexpressed during embrionic development and mayplay important roles in migration, differentiationand organization of the heart, Col I and Col III arethe major ®brillar collagens of the adult heart.20

The Col I/Col III ratio for human heart, usingcyanogen bromide extraction, has been reported at1.3±1.9:1.21 Studies using monospeci®c antibodiesto each type of collagen and cDNA probes for theidenti®cation of mRNA transcripts for collagentypes in isolated cardiac cells have led to the con-clusion that, in the ventricular myocardium, ®bro-blasts and myo®broblasts are responsible for thebiosynthesis of Col I and Col III.22,23 In the heart,more than half of the collagen synthesized isdegraded intracellularly, with the remaining eitherdeposited or degraded extracellularly.24 From thepioneer work by Montfort and Perez-Tamayo25 itis well known that collagenase is present in thenormal myocardium. Collagenase is produced by®broblasts, in¯ammatory cells, as well cardiomyo-cytes,26,27 and it is predominantly present in itslatent form.28 As in other organs, collagen turnoverof normal adult heart results from the equilibriumbetween the synthesis and degradation of Col I andCol III. A number of factors have been described thatmay alter the balance in favor of either the synthesisor the degradation (Table 1).29,30

Fibrillar collagen of the heart provides the struc-tural scaffolding for cardiomyocytes and coronaryvessels and imparts cardiac tissue with physicalproperties that include stiffness and resistance todeformation.30,31 In addition, ®brillar collagenmay also act as a link between contractile elementof adjacent cardiomyocytes and as a conduit ofinformation that is necessary for cell function. Thisis supported by the observation that the interactionof collagen ®bers in the heart with cardiomyo-cytes occurs at regions near the Z band, and thecell attachment is mediated by speci®c moleculesbelonging to the family of integrins.32 Thus, cardiac®brillar collagen not only determines tensile

Table 1 Factors governing ®brillar collagen turnover inthe myocardium

Factors that mainly facilitate collagen synthesisAngiotensin IITransforming growth factor bOther growth factors (PDGF, bFGF, IGF-1)AldosteroneDeoxycorticosteroneEndothelin-1CatecholaminesInterleukin-1Adhesion moleculesOstepontin

Factors that mainly facilitate collagen degradationBradykininProstaglandinsNitric oxideNatriuretic peptidesTumoral necrosis factor-aInterferon-gGlucocorticoidsN-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP)

ANG II

AT1R

STIMULATION INHIBITION

TGF-β1

MAPK/ERK /Smad CTGF

Synthetic pathways Degradative pathways

> Procollagen type I < Collagenase > Procollagen type III

Figure 2 Summary of angiotensin II effects on ®brillarcollagen metabolism acting via the AT1 receptor in car-diac ®broblasts and myo®broblasts.


strength and compliance of the left ventricularchamber, but also integrates individual cardiomyo-cyte contractions into a coordinated biologic pumpfor expulsion of blood into the systemic and pulmon-ary circulations.

Regulation by ANG II

Increasing evidence strongly supports the notionthat ANG II in¯uences both ®brillar collagen syn-thesis and degradation (Fig. 2). In vitro studies of ratand human cardiac ®broblasts and myo®broblastshave shown that angiotensin II stimulates cellproliferation and ®brillar collagen synthesis viaAT1R.33±38 Results in the literature indicate thatthe proliferative response of ®broblasts to ANG IImight well be mediated by stimulation of the syn-thesis of growth or in¯ammatory substances likeplatelet-derived growth factor (PDGF) and cyto-kines, by integrin activation due to secreted extra-cellular matrix proteins (e.g., osteopontin), or by acombination of these mechanisms.39,40

Different signalling pathways of the AT1R maysubserve collagen synthesis.41 Irrespective of sig-nalling mechanisms, the end result is activation oftranscription factors which bind to various `̀ cis-acting'' elements in the regulatory sequences ofa1(I), a2(I) and a1(III) collagen genes.42,43

A number of studies provide strong evidencethat ANG II regulates collagen synthesis by cardiac®broblasts via speci®c growth factors.44 For inst-

ance, angiotensin II has been shown to induce Col Igene expression via activation of both MAP/ERkinase pathway and TGF-b1 signaling pathways(e.g., connective tissue growth factorÐCTGF- andSmad proteins).42,45 In addition, Pathak et al.46

have provided evidence that a cardiomyocyte cofac-tor is an important mediator of ANG II-induced Col Iand Col III mRNA synthesis in a rat cell coculturemodel.

There is some in vivo evidence that ANG II alsoin¯uences post-translational processing of cardiac®brillar collagen. It has been shown that ANG IIinfusion is associated with stimulation of prolyl4-hydroxylase (an enzyme that mediates hydroxy-lation of procollagen a-chains in the endoplasmicreticulum of cardiac ®broblasts) in the rat leftventricle.47 In addition, it has been reported thatimmunoreactive prolyl 4-hydroxylase concentra-tion decreases signi®cantly in the ventricle of post-myocardial infarction rats treated with the ARAlosartan.48

In addition to collagen synthesis, ANG II stimula-tion of the AT1R has been shown to regulatecollagen degradation by attenuating interstitialcollagenase activity in adult rat33 and human49

cardiac ®broblasts and by enhancing TIMP-1 pro-duction in rat heart endothelial cells.50

The role of angiotensin II type 2 receptor (AT2R)signaling on cardiac collagen metabolism is stilldebatable because of con¯ictive data. Some studiessuggest that AT1R and AT2R induce phenotypicallyopposite responses. In experiments performed in®broblasts isolated from myopathic hamster hearts,Ohkubo et al.51 provide evidence that the AT2R is anegative regulator of cell growth and collagen syn-thesis. These ®ndings are consonant with in vivodata reported by Liu et al.52 demonstrating thatthe anti®brotic effect of ARAs is mediated in


part by activation of AT2R in a model of heartfailure induced myocardial infarction in rats. Con-versely, other evidence suggest that simultaneousactivation of both AT1R and AT2R may stimulate asignaling pathway that results in upregulation ofcollagen in parallel rather than in opposite direc-tions. Brilla et al.53 reported that ANG II stimulatedcollagen synthesis by both AT1R and AT2R in cul-tured adult rat cardiac ®broblasts and that ANG II-induced inhibition of collagenase activity wasspeci®cally mediated by AT2R. In support of these®ndings are recent observations by Ichihara et al.54

showing the abolition of ANG II-induced cardiac®brosis in mice lacking the AT2R gene.

Upregulation of Fibrillar Collagen inHypertensive Heart Disease

General aspects

It is now recognized that the remodeling of thecollagen matrix of the heart occurs during patho-logic conditions and aging.55 The remodelingincludes quantitative and qualitative changes inthe ®brillar collagen matrix of the heart. To datemost reported cases of pathologic remodeling areassociated with increased Col I and Col III depositionor ®brosis that accompanies cardiac diseases suchas hypertensive heart disease (Fig. 3), ischemiccardiomyopathy, diabetic cardiomyopathy, andhypertrophic cardiomyopathy.56 Such an adverseaccumulation of collagen ®bers initially raises myo-cardial stiffness, thus compromising diastolic ®lling;its continued accumulation further increases stiff-ness and impairs contractile behavior, coronary

Figure 3 Histological section of myocardial specimenbiopsy from a hypertensive patient with severe myocar-dial ®brosis (Picrosirius red stain �20).

reserve and electrical activity, thus leading to det-rimental outcomes.57,58

As shown by in vivo experiments chronic pressureoverload of the heart stimulates both procollagengene expression and collagen protein synthesisleading to excessive collagen deposition and ®bro-sis.59 In addition, in vitro studies have shown thatprocollagen type I synthesis is stimulated in cardiac®broblasts submitted to cyclic mechanical load.59

Thus, hemodynamic overload of the left ventricledue to systemic hypertension may play a role inmyocardial ®brosis.

The observation that myocardial ®brosis is pre-sent not only in the left ventricle but also in theright ventricle and the interventricular septumof animals60±63 and patients with arterial hyper-tension64±66 suggests that besides hypertension,some systemically and/or locally produced humoralfactor may also contribute to hypertensive myocar-dial ®brosis. Various lines of evidence support a rolefor ANG II as one potential candidate factor.67

A role for ANG II in hypertensive myocardial ®brosis?

Endogenous elevations in circulating ANG II thataccompany unilateral renal artery stenosis60,61 orthe infusion of exogenous ANG II68 are associatedwith increased blood pressure and ®brosis. Theappearance of such ®brous tissue formation is pre-ceded by increased expression of AT1R, TGF-b1, andmRNA for Col I and Col III.69±71 In addition, devel-opment of ®brosis involves proliferating ®broblastsand cell differentiation into myo®broblasts.72,73

Two observations suggest that the ability of ANG IIto induce cardiac ®brosis in these models is inde-pendent of its hypertensive action. First, ®brosis inthe renal artery stenosis model develops in bothlow-pressure right and left atria and right ventricleand high-pressure left ventricle.60 Second, cardiac®brosis in the ANG II infusion model can be pre-vented by either ACEIs or ARAs, but not hydrala-zine or prazosin, despite a similar antihypertensiveef®cacy of these compounds.74,75

Pharmacological interventions with ACEs andARAs have underscored the potential importanceof ANG II in the mediation of cardiac ®brosis inprimary hypertension. In spontaneously hyperten-sive rats (SHR) with left ventricular hypertrophy(LVH) myocardial ®brosis has been shown to regressby treatment with the ACEI lisinopril.76 This effectocurred independently of the drug's antihyper-tensive effect.77 On the other hand, it has beenfound that chronic AT1R blockade with losartanresulted in reversal of ®brosis, inhibition of the

Table 2 Effects of antihypertensive treatment on blood pressure and cardiac ®brosis in patientswith hypertensive heart disease

Parameter Brilla's study LoÂpez's study

Effect oflisinopril

Effect ofhydrochlothiazide

Effect oflosartan

Effect ofamlodipine

Systolic blood pressure* ÿ1 ÿ5 ÿ36 ÿ25Diastolic blood pressure* ÿ2 ÿ1 ÿ14 ÿ26Mean blood pressure* ÿ2 ÿ3 ÿ18 ÿ22Collagen volume fraction** ÿ0.6 �0.1 ÿ1.7 ÿ0.62

Data represent absolute variations induced by treatment in mean values of the different parameters tested.Values are given as mmHg (*) and % (**). (Adapted from ref. 79 and 80.)


post-transcriptional synthesis of procollagen type I,inhibition of TIMP-1 expression and stimulation ofcollagenase activity in the left ventricle of adultSHR.63,78 Analysis of the individual data showedthat the intensity of these changes was independentof the antihypertensive ef®cacy of the drug.63,78

The ®brogenic role of ANG II in essential hyper-tension has been investigated in 2 recent pros-pective trials of limited size using biopsy-provenmyocardial ®brosis in patients with LVH (Table 2).Brilla et al.79 randomized 35 previously treatedpatients with controlled blood pressure to receiveeither the ACEI lisinopril or the diuretic hydrochlor-othiazide for 6 months. Only patients randomized tolisinopril had a signi®cant reduction in myocardial®brosis. Blood pressure reduction was similar inpatients treated with either lisinopril or hydrochloro-thiazide. On the other hand, LoÂpez et al.80 studied37 treated patients with uncontrolled blood pres-sure. After randomization, 21 patients wereassigned to the ARA losartan and 16 to the calciumchannel blocker amlodipine for 12 months. Where-as myocardial ®brosis decreased signi®cantly inlosartan-treated patients, this parameter remainedunchanged in amlodipine-treated patients. A simi-lar reduction of blood pressure in losartan-treatedpatients than in amlodipine-treated patients wasreported in this study. Collectively, these obser-vations support the concept that in addition to pres-sure overload, ANG II participates in myocardial®brosis in primary hypertension.

Perspectives

Structural homogeneity of cardiac tissue is gov-erned by mechanical and humoral factors thatregulate cell growth, apoptosis, phenotype, andECM turnover. ANG II has endocrine, autocrineand paracrine properties that in¯uence the behav-ior of cardiac cells and matrix via AT1R binding.

Thus, various paradigms have been suggested,including ANG II-mediated upregulation of Col Iand Col III formation and deposition in cardiacconditions such as hypertensive heart disease. Toreduce the risk of heart failure that accompanieshypertensive heart disease, its adverse structuralremodeling (e.g., myocardial hypertrophy and ®bro-sis) must be targeted for pharmacological interven-tion. Thus, cardioprotective agents must reverse notonly the exaggerated growth of cardiac cells, butalso regress existing abnormalities in ®brillar colla-gen. Available experimental and clinical data sug-gest that agents interfering with either ACE or AT1Rmay provide such a cardioprotective effect. Albeitpreliminary, these data set the stage for large-scaleclinical studies aimed to proof the usefulness of theconcept.

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regulation of myocardial fibrillar collagen by angiotensin ii. a role in hypertensive heart disease?

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