Review ArticleTargeting the ACE2–Ang-(1–7) pathway in cardiac fibroblasts to treat cardiac remodeling and heart failure
Research Highlights
► Activation of the renin–angiotensin system (RAS) contributes to cardiac remodeling. ► Cardiac fibroblasts play a key role in this process. ► ACE2 breaks down Ang II and generates the beneficial peptide Ang-(1–7). ► The ACE2–Ang-(1–7) pathway could be a therapeutic target to treat heart failure.
Introduction
Despite important advances in the treatment of heart failure over the past several decades, morbidity and mortality in patients with this condition remain at unacceptably high levels [1], [2]. Remodeling of the heart which occurs in response to injury and/or an increase in wall stress plays a key role in the progressive deterioration of cardiac function that leads to heart failure [3], [4]. Thus, new therapies that can inhibit remodeling would be expected to improve outcomes. Remodeling is characterized by cardiac hypertrophy and dilatation as well as conformational changes in the shape of the heart. Cardiac fibrosis which develops at sites distant from local injury (e.g. in segments of non-infarcted myocardium in patients with ischemic cardiomyopathy) occurs during remodeling [5], [6] and this process adversely affects systolic and diastolic functions of the heart and promotes cardiac arrhythmias. Deposition of extra-cellular matrix (ECM) depends primarily on cardiac fibroblasts which produce many of the interstitial proteins in the heart and release enzymes such as matrix metalloproteinases and tissue inhibitors of metalloproteinases which further regulate the balance between ECM production and breakdown [7], [8], [9], [10], [11]. There is also evidence that cardiac fibroblasts release growth factors that act in a paracrine manner to stimulate cardiomyocyte hypertrophy [12], [13], [14], [15].
In addition to helping maintain cardiovascular homeostasis through its influence on salt and water regulation and vascular tone, the RAS plays an important role in maladaptive cardiac remodeling [5], [16]. The mechanisms through which the RAS contributes to the remodeling process include indirect effects leading to increased load on the heart, interactions with other neurohormonal systems and by direct effects on cardiac cells. Angiotensin II (Ang II), the main effector molecule of the RAS, acts predominantly through its Type 1 receptor (AT1) to activate cardiac fibroblast functions that increase the amount of ECM in the heart [17], [18]. Although Ang II has been shown to stimulate cardiomyocyte hypertrophy, this activity appears to be mainly indirect since application of the peptide to cultured cardiomyocytes has little effect on cell growth or synthesis of proteins [15]. Culture media taken from adult cardiac fibroblasts, however, can stimulate cardiomyocyte hypertrophy and this effect is significantly enhanced by Ang II activation of cardiac fibroblast AT1 receptors [15]. These observations are consistent with evidence that the AT1 receptor density is substantially greater on cardiac fibroblasts than on cardiomyocytes [17]. Moreover, the increase in AT1 receptors that occurs in the heart as it remodels involves predominantly non-myocytes [19], [20]. Thus, the cardiac fibroblasts appear to be the predominant target for the RAS during cardiac remodeling.
The local tissue-based cardiac RAS is regulated independently of the systemic circulatory RAS and its activation is central to the remodeling process [16]. Cells within the heart generate all components of the RAS (or, in the case of renin activity, extract it from the coronary circulation [21]) and both Ang II levels and AT1 receptor density are increased in the myocardium of remodeling hearts [5], [19], [20]. Moreover, results from studies performed in experimental animal models and from clinical trials in human patients provide unequivocal evidence that strategies designed to block RAS activation favorably influence cardiac remodeling [22], [23], [24] and improve outcomes including increased survival [22], [25], [26], [27].
While Ang II through its AT1 receptor is involved in most physiologic and pathophysiologic effects of the RAS, additional pathways within this system that are relevant in health and disease are known to exist. A homologue of angiotensin converting enzyme (ACE) termed angiotensin converting enzyme 2 (ACE2) was reported independently by two groups in 2000 [28], [29]. ACE2 has approximately 40% amino acid sequence similarity with ACE and both enzymes exist as Type I integral membrane-bound glycoproteins. The 805 amino acids of ACE2 include an amino-terminal signal peptide, a catalytic ectodomain, a transmembrane domain and a carboxyl-terminal cytoplasmic domain. The single metalloprotease zinc binding motif is located in the ectodomain and ACE2 functions as a carboxymonopeptidase. As shown in the Fig. 1 it catalyzes the conversion of Ang I to angiotensin-(1–9) which, in turn, can be converted to Ang-(1–7) by other peptidases, and hydrolyzes Ang II to Ang-(1–7) with high catalytic affinity [30]. Thus, whereas ACE generates Ang II, ACE2 reduces Ang II levels while producing Ang-(1–7). Other known substrates of ACE2 include physiologically active peptides such as des-Arg9-bradykinin, apelin-13, dynorphin A (1–13), and β-casomorphin [30].
Cleavage of the carboxyl-terminal amino acid from Ang II results in the formation of Ang-(1–7), a heptapeptide with vasodilatory and cardioprotective properties, that has also been reported to counteract effects of Ang II that promote adverse cardiac remodeling (Fig. 1). To assess the potential role of Ang-(1–7) on cardiac remodeling we measured the effects of this peptide on selected functions of cultured adult rat cardiac fibroblasts (ARCFs) [15]. Pre-treatment of ARCFs with Ang-(1–7) inhibited Ang II stimulated [3H]proline incorporation, suggesting that Ang-(1–7) might reduce fibrous tissue deposition, particularly in situations where Ang II stimulation is involved. Stimulation with Ang-(1–7) by itself also reduced [3H]proline incorporation compared to unstimulated cells. Pretreatment of ARCFs with Ang-(1–7) significantly reduced Ang II stimulated increases in endothelin-1 and leukemia inhibitory factor mRNA, suggesting that the peptide might inhibit synthesis of autocrine/paracrine growth factors by cardiac fibroblasts. To further explore this possibility, cardiomyocytes were exposed to Ang II, Ang-(1–7) or Ang II after Ang-(1–7) pre-treatment. While the angiotensin peptides alone did not induce cardiomyocyte hypertrophy as assessed by [3H]leucine incorporation and atrial natriuretic peptide synthesis, exposure of the cardiomyocytes to ARCF culture media induced a hypertrophic response (compared to ‘mock’ culture media that had not been in contact with the fibroblasts), which increased significantly when the ARCFs had been stimulated with Ang II. This increase, however, was inhibited by pre-treating ARCFs with Ang-(1–7). Overall, these findings suggest that Ang-(1–7) may be playing an important counter-regulatory role during cardiac remodeling by inhibiting cardiac fibroblast ECM synthesis and release of ‘hypertrophic’ growth factors.
The pathways through which Ang-(1–7) initiates potentially favorable anti-remodeling effects have not been fully elucidated. While some investigators report that the peptide can act through well-characterized angiotensin receptors such as AT1 or AT2 [31], [32], [33], there is also evidence that Ang-(1–7) frequently acts via its own dedicated receptor, which is widely regarded to be the Mas receptor [34]. Mas is a G protein-coupled receptor [35] and, although Mas is expressed in cardiac fibroblasts (data not shown), the mechanism by which Ang(1–7) exerts its effects on these cells is currently unknown.
Section snippets
Expression and regulation in animal models
While ACE2 is ubiquitously expressed in multiple organs in both rodents and humans, its expression is particularly high in human heart, kidney and testis [28], [29], [36]. In the heart ACE2 has been found to be localized to endothelial cells, smooth muscle cells, cardiomyocytes, macrophages [29], [37] and myofibroblasts [38]. There is evidence that cardiac ACE2 expression and activity can be regulated at both pre- and post-translational levels. Burell et al. reported increased cardiac ACE2 mRNA
Evidence from gain and loss of function
Distinct substrate specificity of ACE and ACE2 suggests different physiological roles of these enzymes. The possibility that ACE2 could be involved in the regulation of the cardiovascular system has been investigated using genetically engineered mice and gene transfer. Crackower et al. reported impaired cardiac contractility with an increase in local cardiac Ang II level in ACE2 knockout mice and this cardiac phenotype could be rescued by concomitant deletion of the ACE gene [52]. Although
Human data
Increases in cardiac ACE2 expression, protein levels and activity have been reported in patients with heart failure of both ischemic and non-ischemic etiology [37], [71], [72]. Hydrolysis of Ang II by ACE2 is considered to be a key pathway to generate Ang-(1–7) in the failing heart [71]. Soluble ACE2 activity is increased in the blood of heart failure patients and the level correlates with disease severity [44]. An endogenous inhibitor for ACE2 [73] and inhibitory autoantibodies against ACE2
Translation potential, pros and cons
Given that ACE and ACE2 have distinct (even opposing) effects on metabolism of RAS effector peptides that regulate cardiovascular structure and function, drugs which modify the balance of expressions and activities of these enzymes can be viewed as potential therapeutic tools to treat a variety of cardiovascular diseases (Table 1). As already described, ACE inhibitors, ARBs and aldosterone antagonists have been reported to increase ACE2 activity. This mechanism might contribute to their
Most important questions and problems for future research
While evidence is accumulating that targeting ACE2, Ang-(1–7) or the Mas receptor might have therapeutic potential, the effects of enhancing this alternative pathway of the RAS appear to depend on a variety of factors including the approach taken, the specific condition and timing in the progression of the disease state. Strategies that enhance ACE2 activity may prove to be more difficult to develop than those that block its effects. Also, the paucity of information about the pathways involved
Acknowledgments
This work was funded in part by National Institutes of Health grant 1RO1HL091191 to Dr. Greenberg and American Heart Association National Scientist Development Grant 0730126N to Dr. Iwata.
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Drs. Iwata and Cowling contributed equally to the generation of this manuscript.