RAS and connective tissue in the heart

The International Journal of Biochemistry & Cell Biology 35 (2003) 919–931

Review

RAS and connective tissue in the heart Yao Sun, Karl T. Weber∗ Department of Medicine, Division of Cardiovascular Diseases, University of Tennessee Health Science Center, Rm. 353 Dobbs Research Institute, 951 Court Avenue, Memphis, TN 38163, USA Received 15 July 2002; received in revised form 2 October 2002; accepted 2 October 2002

Abstract Circulating angiotensin (Ang) II has well-known endocrine properties in the cardiovasculature. AngII, produced de novo within the heart, has various autocrine and paracrine properties on resident cells expressed via AT1 receptor–ligand binding. Herein, we review the heart’s renin-angiotensin system and its role in connective tissue turnover involving heart valve leaflets and fibrous tissue that appears at sites of injury, such as following myocardial infarction. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Angiotensin receptors; Renin; Angiotensin converting enzyme; Infarct scar; Myocardial infarction

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Tissue RAS and the heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Normal heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Infarcted heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cells and RAS in the heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Normal heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Infarcted heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Function of RAS in the heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Connective tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Normal heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Infarcted heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. ACE inhibition and AT1 receptor antagonism in the injured heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction ∗ Corresponding author. Tel.: +1-901-448-5759; fax: +1-901-448-8084. E-mail address: [email protected] (K.T. Weber).

Circulating angiotensin (Ang) II, a derivative of angiotensinogen and product of angiotensin convert-

1357-2725/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 2 ) 0 0 2 7 6 - 5

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ing enzyme (ACE)-based conversion of biologically inactive AngI, has multiple well-known endocrine properties in the cardiovasculature. AngII, produced de novo within the heart, independent of plasma angiotensinogen, plasma renin activity, and endothelial cell-bound ACE, has various autocrine and paracrine properties on resident cells that include: cardiomyocytes, representing but one-third of all cells found in the myocardium; and the remaining two-thirds which consist of fibroblasts, endothelial and vascular smooth muscle cells, and macrophages. Based on current evidence AT1 receptor–ligand binding accounts for the majority of these respective endocrine and auto-/paracrine actions of AngII on blood vessels and cardiac tissue. Herein, we review the heart’s renin-angiotensin system (RAS) and its involvement in high turnover connective tissue formation normally found in heart valve leaflets and that which appears at sites of repair (e.g. following myocardial infarction, MI). A role for AngII in regulating connective tissue formation in other tissues has been reviewed elsewhere (Weber, 1999; Weber, Swamynathan, Guntaka, & Sun, 1999).

2. Tissue RAS and the heart 2.1. Normal heart As shown in Fig. 1, AngII can be generated directly from angiotensinogen or from its immediate precursor, the decapeptide AngI. Enzymes involved in the generation of this octapeptide from AngI include ACE, chymase, chymostatin-sensitive Ang generating enzyme and carboxypeptidase (Dell’Italia & Husain, 2000; Dell’Italia, Perry, & Oparil, 2001). A review of each of these enzymes is beyond the scope of this report. Moreover, the contribution of these enzymes in AngII generation would appear to be species dependent. Our work has focused entirely on rodents, where ACE is dominant, and therefore we will confine our attention to it in addressing tissue RAS. Many tissues have the capacity to generate Ang peptides via tissue RAS. By in situ hybridization and immunohistochemistry, Sun et al. (Sun, Zhang, Zhang, & Weber, 2001) found renin expression (mRNA and protein) to be undetectable in the normal rat myocardium, but high within heart valve leaflets (see Fig. 2, panels A and B). Quantitative in vitro autoradiography

Fig. 1. The generation of angiotensin II and bradykinin and their stimulatory (+) or inhibitory (−) influence on fibrous tissue formation is depicted herein. Multiple pathways are involved in the generation of Ang peptides. These include angiotensin converting enzyme (ACE), chymase, chymostatin-sensitive Ang generating enzyme (CAGE), and carboxypeptidase (CPase). Reproduced with permission from Weber (1995).

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Fig. 2. Renin, ACE and AngII receptor expression in the rat cardiac valves. Detected by in situ hybridization, renin mRNA is expressed in all heart valve leaflets. Shown here is renin expression in tricuspid valve leaflets (V) (panel A). Normal myocardium does not express renin mRNA (panel A). The in situ hybridization image shown in panel A was obtained from the tissue section shown in panel B stained with hematoxylin and eosin. By in vitro autoradiography, high-density ACE and AngII receptor binding are found in heart valve leaflets. Shown here are aortic valve leaflets (panels C and D, respectively), while ACE and AngII receptor binding densities in atria are low.

identifies a similar heterogenous distribution to ACE and AngII receptor binding densities within the heart. Low ACE binding density, for example, is present throughout the myocardium of the right and left atria and ventricles of the adult rat heart, as is the case for AngII receptor binding. High-density binding for ACE and AngII receptors, on the other hand, is found within all four heart valve leaflets (see Fig. 2, panels C and D) (Pinto, Viglione, & Saavedra, 1991; Sun, Diaz-Arias, & Weber, 1994; Yamada, Fabris, Allen, & Johnston, 1991). Autoradiography further identifies heart valve leaflets as sites of high-density receptor binding for TGF-␤1 , a fibrogenic cytokine (Sun, Diaz-Arias, & Weber, 1994; Sun, Zhang, Zhang, & Ramires, 1998). By in situ hybridization marked mRNA expression of type I collagen is seen in valve leaflets and where, unlike normal myocardium, collagen turnover is high (Laurent, 1987). Valve leaflets and their chordae tendineae represent exteriorized portions of the heart’s extracellular

matrix (Robinson, Geraci, Sonnenblick, & Factor, 1988). Residing within leaflet connective tissue and responsible for matrix formation are valvular interstitial cells, also termed myofibroblasts because of their expression of ␣-smooth muscle actin microfilaments (Filip, Radu, & Simionescu, 1986). The anatomic concordance between renin, ACE and receptors for AngII and TGF-␤, together with marked type I collagen mRNA expression, in heart valve leaflet tissue implicates the heart’s RAS and de novo generation of AngII in governing leaflet myofibroblast collagen turnover via autocrine-based regulation of TGF-␤1 . Such has been demonstrated for cultured valve leaflet myofibroblasts (Campbell & Katwa, 1997; Katwa et al., 1996) and will prove the case for the infarct scar (vida infra). Elevations in plasma AngII found in renovascular hypertension raise collagen synthesis of tricuspid, mitral and aortic valve leaflets (Willems, Havenith, Smits, & Daemen, 1994a).

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Fig. 3. Renin, ACE, AngII receptor expression in the infarcted rat heart. Following MI, high-density renin mRNA expression is observed at the site of MI, as well as inflamed pericardium and endocardium at week 1 (panel A) and which remains elevated over the course of 4 weeks (not shown). ACE and AngII receptor binding density was also markedly increased at the same sites. Panels B and C, respectively, demonstrate ACE and AngII receptor binding densities in the infarcted heart at week 4 post MI. Detected by immunohistochemical ␣-SMA labeling, abundant myofibroblasts (MF) are seen at the site of MI (panel D) and account for the expression of these components of the heart’s renin-angiotensin system.

2.2. Infarcted heart The mRNA expression for renin is upregulated at the site of MI (see Fig. 3, panel A) and other sites of injury in the infarcted rat heart model. As observations in heart valve leaflets would predict high-density ACE binding is found in the infarct scar and is related to fibrous tissue formation. Temporal and spatial responses in autoradiographic ACE binding have been assessed in a rat heart model of MI. Other forms of injury involving cardiac and noncardiac tissues that appear in this model were also examined. Each served as positive controls in the analysis of ACE and its relationship to tissue repair. As shown in Table 1, they included: the foreign-body fibrosis that surrounds a silk ligature placed around the left coronary artery and fibrosis of visceral pericardium that accompanies manual handling of the heart in sham-operated, non-

Table 1 Sites of high-density autoradiographic ACE binding in injured rat tissuesa Cardiac myocyte necrosis due to: Acute anterior myocardial infarction secondary to left coronary artery ligation Endogenous catecholamine release induced by AngII Exogenous catecholamine (isoproterenol) administration Potassium depletion with chronic aldosterone treatment Endocardial fibrosis that follows mural thrombus formation in infarcted ventricle Fibrosis of visceral pericardium following pericardiotomy and manual handling of the heart Foreign-body fibrosis surrounding silk ligature placed around left coronary artery or used to suture incised skin Fibrosis at sites of thromboembolic renal infarction a High-density ACE binding is anatomically coincident with high-density AngII and TGF-␤ receptor binding densities and mRNA expression of TGF-␤1 and type I collagen.

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infarcted controls; renal thromboembolic infarction; and incised/sutured skin. Serial sections involving injured and noninjured tissues were examined together with picrosirius red histochemistry and videodensitometry, in vitro quantitative autoradiography and quantitative in situ hybridization to address the temporal and spatial appearance of RAS components that included localization of ACE, AngII and TGF-␤ binding densities and mRNA expression of TGF-␤1 and type I collagen, together with fibrillar collagen accumulation. Following left coronary artery ligation and the appearance of an anterior transmural MI, high-density renin mRNA expression and ACE binding density first appear at the infarct site on day 7 coincident with the initial accumulation of fibrillar collagen. As a fibrillar collagen network forms scar tissue over the course of 8 weeks, the density of renin expression and ACE binding at this site increases progressively (Sun, Cleutjens, Diaz-Arias, & Weber, 1994). Renin mRNA expression and ACE binding density in the infarcted rat heart 4 weeks after MI is shown in panels A and B of Fig. 3. The appearance of fibrosis at sites remote to the infarct, including the noninfarcted left ventricle, interventricular septum and right ventricle, are also sites of renin expression and high-density ACE binding. The appearance of fibrosis at these remote sites is directly related to the extent of infarction (Smits, van Krimpen, Schoemaker, Cleutjens, & Daemen, 1992; van Krimpen et al., 1991a; Volders et al., 1993). When the transmural infarct is extensive, the entire myocardium (including infarcted and noninfarcted ventricular tissue) is involved in tissue repair and subsequent structural remodeling by fibrous tissue. Noninfarct-related sites of injury and repair (see Table 1) serve to further address the relationship between the appearance of renin, ACE and fibrosis. Sham operation includes manual handling of the heart. This alone leads to inflammation and subsequent fibrosis of the visceral pericardium. Silk ligature placement around the left coronary artery or within skin to close surgical incision are each associated with a foreign-body fibrosis. The appearance of a mural thrombus in the infarcted left ventricle can be associated with subsequent endocardial fibrosis and on occasion thromboembolic renal infarction. At these sites of repair, high-density renin mRNA expression and autoradiographic ACE binding are temporally

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and spatially concordant with fibrous tissue formation (Sun et al., 1994, 2001). Nonischemic models of cardiac myocyte necrosis and repair have also been examined relative to ACE expression. They included: endogenous release of catecholamines that accompanies AngII infusion from implanted mini-pump (Ratajska, Campbell, Sun, & Weber, 1994) or administration of isoproterenol, a synthetic catecholamine (Benjamin et al., 1989); and chronic (>3 weeks) administration of aldosterone by mini-pump in uninephrectomized rats on a high salt diet and which is accompanied by enhanced urinary potassium excretion and subsequent cardiac myocyte potassium depletion with necrosis (Campbell, Janicki, Matsubara, & Weber, 1993; Darrow & Miller, 1942). At each site of nonischemic cardiac myocyte necrosis, and irrespective of its etiologic basis, the temporal and spatial appearance of high-density ACE binding is coincident with tissue repair and the deposition of scar tissue (Sun, Ratajska, Zhou, & Weber, 1993; Sun & Weber, 1996a) to resemble reparative responses observed with ischemic necrosis following MI. Thus, irrespective of the etiologic basis of injury, the tissue involved, or the presence of repair in the setting of ischemic or nonischemic cardiomyocyte necrosis, a recruitable source of renin and ACE appear at sites of fibrous tissue formation. Examination of serial heart sections of the infarcted rat heart further demonstrates high-density renin expression and ACE binding to be spatially and temporally concordant with marked autoradiographic AngII (see Fig. 3, panel C) and TGF-␤ receptor binding densities and expression of TGF-␤1 and type I collagen mRNAs (by in situ hybridization) at these sites of repair. Collectively, these findings in various injured tissues implicate both tissue AngII and TGF-␤1 as a common signaling pathway involved in promoting repair (Weber, 1997a). What is the cellular source of this recruitable RAS? 3. Cells and RAS in the heart 3.1. Normal heart Using monoclonal antibodies and immunolabeling cells expressing renin and ACE in normal rat heart, valve leaflets were identified as valvular interstitial cells residing within leaflet matrix. These

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myofibroblasts contain ␣-smooth muscle actin (␣-SMA) microfilaments and are contractile (Filip et al., 1986; Katwa et al., 1995; Messier et al., 1994). Cultured myofibroblasts, obtained from intact rat heart valve leaflets and maintained under serum-deprived conditions, demonstrate autoradiographic ACE binding and converting enzyme (and kininase II) activities to substrates that include AngI, bradykinin, substance P and enkephalin (Katwa et al., 1995). 3.2. Infarcted heart Renin is expressed by macrophages and myofibroblasts found at the infarct site. ACE-positive cells are seen at and remote to the infarct site and involve endothelial cells of the neovasculature (constitutive ACE), macrophages and myofibroblasts (recruitable ACE) (Falkenhahn et al., 1995; Sun & Weber, 1996b). Within 24 h of MI, macrophages appear at the interface between viable and necrotic myocardium; by day 3, fibroblasts co-aggregate with macrophage clusters bordering on the infarct. Thereafter, fibroblast differentiation follows resulting in the ␣-SMA positive myofibroblast phenotype which proliferates and migrates into the site of necrosis during the remainder of week 1. A combination of cell growth with spatial control of growth and fibrillar collagen assembly governs rebuilding of infarcted tissue. Myofibroblasts at sites of repair are aligned parallel to epi- and endocardium following transmural MI and parallel to the long axis of viable myocytes in nontransmural MI and suggest spatial alignment of myofibroblasts and their actin filaments are important to their function which serve to prevent tissue deformation. Recent evidence implicates transmission of polarity signals and homologues of Drosophila tissue polarity genes frizzled 2 in the expression and alignment of ␣-SMA positive myofibroblasts at the infarct site (Blankesteijn, Essers-Janssen, Verluyten, Daemen, & Smits, 1997). By immunolabeling activated macrophages and myofibroblasts at the infarct site are each renin- and ACE-positive. Beyond day 14, the gradual disappearance of macrophages from this site leaves only myofibroblasts as renin and ACE-expressing cells. Persistent high-density ACE binding and renin mRNA expression are present at the infarct site long after MI. This is based on ␣-SMA positive myofibroblasts, which remain in infarct scar tissue for prolonged

periods of time. In the infarcted human heart these myofibroblasts persist at the site of MI for years (Willems, Havenith, De Mey, & Daemen, 1994b). Myofibroblasts and their cell–cell and cell–matrix connections confer contractile behavior to scar tissue and this accounts for thinning of the infarct scar. Mediators of scar tissue contraction include AngII and endothelins (Gabbiani, Ryan, & Majno, 1971). These contractile fibroblast-like cells are a common feature of repair seen in diverse injured tissues of rat and man (Desmouliére & Gabbiani, 1996). Myofibroblasts and macrophages are not normal residents of the heart. Following MI, myofibroblasts appear at the site of MI, proliferate and express ACE. Macrophages infiltrate into the site of MI and also contribute to ACE expression. However, macrophages do not proliferate. By autoradiography, ACE at the site of infarction is primarily located on cell membranes, but not in interstitial space. It suggests that high ACE activity at the site of MI is not a result of circulating soluble ACE recruited into the infarct site. Myofibroblasts have considerable phenotypic and functional diversity (Skalli et al., 1989). Immunolabeling of ␣-SMA, vimentin and desmin defines myofibroblast phenotype at the infarct site. Fibroblast-like cells express vimentin (V). ACE-labeled fibroblasts present in the infarct scar and involved in the expression of fibrillar collagen mRNA are also positive for ␣-SMA (A). These VA-positive myofibroblasts are instrumental to tissue repair, including wound contraction. They are likewise found in connective tissue that comprises endocardial fibrosis, pericardial fibrosis, renal scarring and foreign-body fibrosis (Sun & Weber, 2000). Unlike incised skin, where myofibroblasts contribute to tissue repair and then progressively disappear through programmed cell death (apoptosis) coincident with wound closure and scar tissue formation at week 4 (Desmouliére, Redard, Darby, & Gabbiani, 1995), VA phenotype remains at the infarct site for prolonged periods (Sun & Weber 1996b; Willems et al., 1994b). An abnormal persistence of VA positive cells in healed skin is associated with hypertrophic scarring. Whether progressive fibrosis that appears at and remote to MI found in the infarcted heart is related to its persistent myofibroblasts is presently uncertain. Nonetheless, it is clear that the infarct scar is not inert tissue. Indeed it is a dynamic

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tissue containing persistent, metabolically active cells nourished by a neovasculature (Sun & Weber, 2000). In vitro emulsion autoradiography identifies VApositive myofibroblasts as expressing AngII receptors (Sun & Weber, 1996c). Based on displacement studies using either an AT1 receptor antagonist (losartan) or AT2 receptor antagonist (PD123,177) the great majority of AngII receptors in the infarcted rat heart are of the AT1 subtype. Myofibroblasts found at sites of microscopic scarring involving both infarcted and noninfarcted tissue express TGF-␤1 and its receptors (Sun et al., 1998). 4. Function of RAS in the heart 4.1. Connective tissue Renin, ACE and AngII receptors impart connective tissue cells with metabolic activity (Weber, Sun, Katwa, & Cleutjens, 1995). Scar tissue renin activity is independent of plasma renin activity (Sun et al., 2001). ACE substrate utilization involves substances contributing to cell behavior, including cell growth, apoptosis and fibrillar collagen turnover (Weber, Sun, Katwa, & Cleutjens, 1995). ACE and in its dual capacity as kininase II catabolizes AngI, bradykinin, AcSDKP, substance P and enkephalins, each of which are involved in inflammation and repair. AcSDKP is a recently described tetrapeptide found in a variety of tissues (Pradelles, Frobert, Créminon, Ivonine, & Frindel, 1991). It suppresses cell growth by preventing the recruitment of pluripotent cells into the S-phase of the cell cycle; instead, they remain quiescent in the Go-phase (Azizi et al., 1996). Matrix homeostasis depends on the turnover of fibrillar type I and III collagens, as well as fibroblast replication and survival. Following injury, matrix and the interstitial space become a dynamic microenvironment consisting of: macrophages, fibroblasts and endothelial cells; and various soluble, matrix- and cell membrane-bound molecules that operate in an orchestrated balance of reciprocal regulation between competitive stimulators and suppressors of cell behavior and matrix chemistry. AngII is both a growth stimulator and inhibitor based on its respective binding with AT1 and AT2 receptors (Nakajima et al., 1995; Stoll et al., 1995). Expression of angiotensino-

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gen, an aspartyl protease (e.g. renin, cathepsin D), and ACE are a differentiated function of myofibroblasts (Katwa et al., 1996, 1997, 1998). Local AngII also regulates multiple stimulatory and inhibitory factors involved in collagen formation and cell growth and survival. These include: endothelins, aldosterone, catecholamines, and TGF-␤ family of polypeptides as stimulators; and bradykinin, nitric oxide, prostaglandins and natriuretic peptides as inhibitors. 4.2. Normal heart ACE bound to myofibroblasts residing in valve leaflet matrix demonstrates both ACE and kininase II activities. Cultured leaflet myofibroblasts contain all components requisite to Ang peptide generation and AT1 receptor–ligand binding regulates their synthesis of collagen and expression of TGF-␤1 (Campbell & Katwa, 1997; Katwa et al., 1995, 1996). Suprarenal aortic banding is associated with increased expression of type I and III collagens in tricuspid and mitral valve leaflets and suggests a role for circulating AngII and leaflet AT1 receptor binding in response to renal ischemia (Willems et al., 1994a). Loose and dense connective tissue formation is a dynamic process during early growth and development of newborn rats. Treatment of 4-week-old rats with enalapril in nondepressor dosage attenuates cardiac and vascular collagen accumulation in right and left ventricles, aorta and systemic arteries compared to untreated, age-matched control rats (Keeley, Elmoselhi, & Leenen, 1992). No such study has yet been conducted with an AngII receptor antagonist. In rats with a genetic predisposition to hypertension, treatment with either quinapril or hydralazine in depressor dosage during early growth and development prevented the appearance of hypertension in adulthood, however, only quinapril attenuated expected development of vascular connective tissue seen in age-matched, untreated hypertensive controls (Albaladejo et al., 1994). 4.3. Infarcted heart The number of ACE transcripts found in homogenates prepared from explanted failing human heart tissue is increased, compared to nonfailing donor heart tissue (Studer et al., 1994). ACE activity

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has been examined in tissue obtained from the failing, infarcted and noninfarcted human heart tissue (Hokimoto, Yasue, Fujimoto, Sakata, & Miyamoto, 1995). Homogenates of transmural tissue blocks obtained from tissue adjacent to visible scar tissue at the time of aneurysmectomy, revealed ACE activity. Infarct tissue ACE activity exceeded that of control tissue several fold and the extent of activity was related to the severity of tissue damage. In rat heart tissue homogenates prepared from sites remote to a large transmural anterior MI demonstrate ACE activity the extent to which correlates with infarct size (Hirsch, Talsness, Schunkert, Paul, & Dzau, 1991). As a corollary, the presence of fibrosis at sites remote to a transmural MI is dependent on the size of the infarct (van Krimpen et al., 1991a). ACE activity of fibrous tissue has been demonstrated in the rat heart with fibrosis of the visceral pericardium that appears 4 weeks after pericardiotomy (Ou, Sun, Ganjam, & Weber, 1996) and in subcutaneous pouch tissue, which appeared 2 weeks after instillation of chemical irritant (Sun, Ramires, Zhou, Ganjam, & Weber, 1997). AngII generation (from AngI substrate) found in each preparation was abrogated by lisinopril. Cultured myofibroblasts obtained from 4-week-old infarct scar tissue possess all components requisite to generation of Ang peptides including angiotensinogen, cathepsin D, and ACE and expression of AT1 receptors (Katwa et al., 1997). Thus, fibrous tissue with its myofibroblast composition is capable of de novo AngII generation and whose biologic actions include autocrine regulation of collagen turnover. A paradigm of tissue repair has been proposed in which ACE and local AngII are integral to the orderly and sequential nature of repair that eventuates in fibrosis (Weber, 1997b). ACE is involved in a two-part de novo generation of AngII within granulation tissue that forms at sites of injury. The first component to local AngII generation is provided by activated macrophages. In an autocrine manner, macrophage-derived AngII regulates expression of TGF-␤1 that induces phenotype conversion of co-aggregating fibroblasts. VA positive myofibroblasts next generate AngII, whose autocrine induction of TGF-␤1 regulates collagen turnover at sites of fibrous tissue formation, including infarcted and noninfarcted myocardium. AngII generation at the infarct site is

related to the extent of the myofibroblast response which, in turn, is governed by the degree of myocyte necrosis. A large transmural MI, with extensive macrophage and myofibroblast responses, generates a large amount of AngII. Soluble AngII reaches remote sites via its diffusion through tissue fluid to promote fibrosis. Activation of fibrogenesis would therefore be expected to be greatest at sites closest to the anterior MI (e.g. interventricular septum) and less so at more remote sites (e.g. right ventricle). Expression of type I and III collagens is greater and persists longer in the septum compared to right ventricle in rat hearts following left coronary artery ligation (Cleutjens, Verluyten, Smits, & Daemen, 1995). A persistence of metabolically active myofibroblasts at the infarct site perpetuates such remodeling. Elevations in circulating AngII are due to RAAS activation accompanies impaired renal perfusion in heart failure or unilateral renal artery stenosis. It represents a signal that gains entry to the heart’s tissue fluid where it promotes further postinfarct fibrosis by AT1 receptor binding. An ongoing perivascular/interstitial fibrosis of noninfarcted myocardium is therefore not only related to local AngII elaborated by active myofibroblasts in the infarct scar, but also to elevations in circulating effector hormones of the RAAS (Weber & Brilla, 1991). AngII and aldosterone each induce a proinflammatory phenotype of vasoactive segments of the arterial circulation. Inflammatory cells and myofibroblasts surround small intramyocardial coronary arteries and arterioles prior to vascular remodeling by fibrous tissue (Nicoletti & Michel, 1999). Like those found in infarct scar, these myofibroblasts express ACE independent of circulating AngII (Sun et al., 1993). AT1 receptor antagonism prevents this perivascular/interstitial fibrosis even when circulating levels of this peptide are suppressed by long-term aldosterone administration (Robert et al., 1999).

5. ACE inhibition and AT1 receptor antagonism in the injured heart Salutary clinical responses to ACE inhibition are likely to include a prevention of adverse structural remodeling of infarcted and noninfarcted myocardium by fibrous tissue. Evidence supporting a contribution of locally produced AngII in regulating myofibroblast

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collagen synthesis is obtained using pharmacologic probes that interfere with local AngII generation (i.e. ACE inhibition) or occupancy of its AT1 receptor prior to circulating RAAS activation. Captopril or enalapril attenuate infarct size and expansion and attenuate the rise in hydroxyproline concentration at the infarct site in dogs following coronary artery occlusion (Jugdutt, 1995; Jugdutt, Schwarz-Michorowski, & Khan, 1992; Jugdutt, Khan, Jugdutt, & Blinston, 1995). The potential additional contribution of bradykinin, an inhibitor of growth, to tissue repair and which would accompany ACE inhibition has been investigated in rats. A BK2 receptor antagonist (Hoe140 or icatibant) accentuates collagen accumulation remote to the MI site (Wollert et al., 1997). Losartan attenuates, but does not prevent infarct scar formation (Frimm, Sun, & Weber, 1997). Moreover, the expected rise in tissue AngII concentration found at the infarct site 3 weeks post coronary artery ligation is markedly attenuated by either delapril or TCV-116, an AT1 receptor antagonist (Yamagishi, Kim, Nishikimi, Takeuchi, & Takeda, 1993). These findings raise the prospect that the number of myoFb and/or their AngII-generating activity per cell at sites of repair may be influenced by AngII. Fibrous tissue formation at sites remote to MI is also influenced by these pharmacologic interventions. Perindopril, given 1 week after MI, attenuates the endocardial fibrosis that appears in the nonnecrotic segment of the rat left ventricle (Michel et al., 1988). Captopril, commenced at the time of coronary artery ligation, attenuates the expected fibrosis of noninfarcted rat left and right ventricles (van Krimpen et al., 1991b; Bélichard et al., 1994) and proliferation of fibroblasts and endothelial cells that appears at remote sites 1 and 2 weeks following MI (van Krimpen et al., 1991b). Under these circumstances, captopril prevents the rise in LV end diastolic pressure that appears in untreated rats and which is not the case in propranolol-treated rats. Captopril also reduces inducibility of ventricular arrhythmias in this model (Bélichard et al., 1994). When initiated 3 weeks post MI, well after the tissue repair process has commenced and progressed, captopril does not prevent fibrosis remote to the infarct site or the rise in ventricular stiffness (Litwin, Litwin, Raya, Warner, & Goldman, 1991). Losartan prevents fibrosis at remote sites (Frimm et al., 1997; Smits et al., 1992; Schieffer et al., 1994), but not the cellular prolifera-

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tion that appears (Smits et al., 1992). Others did not find an inhibition of type I and III collagens mRNA expression at remote sites (Dixon, Ju, Jassal, & Peterson, 1996; Hanatani et al., 1995) and have suggested posttranslational modification in collagen turnover to explain why fibrosis fails to appear at remote sites (Dixon et al., 1996). In vitro and in vivo studies have demonstrated that AngII-induced cardiac fibrosis involves its stimulation of TGF-␤1 synthesis (Agarwal, Siva, Dunn, & Sharma, 2002; Campbell & Katwa, 1997; Sun et al., 1998), mediated by AT1 receptors. Enhanced TGF-␤1 production in the infarcted heart is significantly attenuated by an AT1 receptor antagonist (Sun et al., 1998). These favorable tissue protective effects of ACE inhibition or AT1 receptor antagonism are not confined to the infarcted heart. These interventions prevent the appearance of fibrosis in diverse organs with experimentally induced or naturally occurring tissue injury and where circulating RAAS is not activated. These include: pericardial fibrosis postpericardiotomy (Ou et al., 1996); tubulointerstitial fibrosis associated with unilateral urethral obstruction (Ishidoya, Morrissey, McCracken, Reyes, & Klahr, 1995; Ishidoya, Morrissey, McCracken, & Klahr, 1996; Kaneto, Morrissey, McCracken, Reyes, & Klahr, 1994; Morrissey, Ishidoya, McCracken, & Klahr, 1996; Pimentel, Martinez-Maldonado, Wilcox, Wang, & Luo, 1993; Pimentel et al., 1995; Yanagisawa, Morrissey, Morrison, & Klahr, 1990), toxic nephropathy (Cohen, Moulder, Fish, & Hill, 1994; Diamond & Anderson, 1990; Lafayette, Mayer, & Meyer, 1993), cyclosporine (Burdmann et al., 1995), remnant kidney (Anderson, Rennke, & Brenner, 1986; Ikoma, Kawamura, Kakinuma, Fogo, & Ichikawa, 1991; Shibouta et al., 1996; Tanaka, Sugihara, Tatematsu, & Fogo, 1995) or renal injury following irradiation (Juncos, Carrasco Dueñas, Cornejo, Broglia, & Cejas, 1993); cardiovascular and glomerulosclerosis that appear in stroke-prone spontaneously hypertensive rats (Kim et al., 1994, 1995; Nakamura et al., 1994, 1996); interstitial pulmonary fibrosis that follows irradiation (Ward, Molteni, & Ts’ao, 1989; Ward, Molteni, Ts’ao, & Hinz, 1990; Ward, Molteni, Ts’ao, Kim, & Hinz, 1992) or monocrotaline administration (Molteni, Ward, Ts’ao, Soliday, & Dunnes, 1985); and subcutaneous pouch tissue in response to croton oil (Sun et al., 1997). Attenuation of fibrous tissue

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formation by these interventions in diverse organs with various forms of injury supports the importance of local AngII in promoting fibrosis. A more detailed review of AngII and tissue repair involving systemic organs can be found elsewhere (Weber, 1997a; Luft, 2001). Our recent study has shown that 4 weeks of chronic ALDO/salt treatment is accompanied by activation of NF␬B and expression of inflammatory and fibrogenic mediators of repair at vascular and nonvascular sites of cardiac injury. These responses are spatially concordant with the expression of ACE and AT1 receptors by activated macrophages and myofibroblasts. Our findings further demonstrate that an AT1 receptor antagonist attenuates the expression of these mediators. Hence, we would implicate tissue AngII, produced de novo at sites of repair in the injured rate heart, in contributing to inflammatory responses and healing in this rat model where circulating AngII is suppressed by chronic ALDO and salt treatment.

6. Summary Macrophage and myofibroblast renin and ACE (recruitable), respectively, regulate local concentrations of AngI and AngII involved in tissue repair. De novo generation of AngII modulates expression of TGF-␤1 whose autocrine/paracrine properties regulate collagen turnover in heart valve leaflets, an exteriorized portion of the normal extracellular matrix, and at sites of fibrous tissue formation that appear in response to various forms of injury involving diverse tissues. Persistent myofibroblasts and their RAS activity at the infarct site contribute to the progressive fibrosis found at and remote to sites of MI. Activation of the circulating RAAS with sustained elevations in plasma AngII and aldosterone further induce the recruitable form of ACE bound to macrophages and myofibroblasts. Locally produced AngII from this source promotes perivascular fibrosis of intramural vessels of noninfarcted myocardium. At these remote sites, such adverse structural remodeling by fibrous tissue eventuates in ischemic cardiomyopathy, a major etiologic factor involved in the appearance of chronic cardiac failure and which contributes to its progressive nature.

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