5-1. The role of ACE2 in the cardiovascular system may be more complex
Angiotensin-converting enzyme II (ACE2) is a critical regulator of the
renin–angiotensin system and the target of a number of highly effective therapeutic agents used to treat cardiovascular diseases. Through its ability to metabolize Ang II to Ang 1-7, it is able to regulate local Ang II levels thereby modulating its effects. Further evidence for a role of ACE2 in maintaining cardiovascular homeostasis via Ang II regulation is provided by studies conducted by Zisman et al. [2003] which detected increased ACE2 and Ang 1-7 forming activity in failing human hearts. The high level of the expression of ACE2 in heart together with its ability to hydrolyze angiotensin peptides have suggested a role for ACE2 in maintaining cardiovascular physiology from the outset. Interestingly, overexpression of ACE2 in cardiac myocytes did cause changes in cardiac conductivity, resulting in arrest and sudden death [Donoghue et al., 2003b]. These contradictory data suggest that the role of ACE2 in the cardiovascular system may be more complex than is immediately apparent.
5-2. Ang II-AT1R modulated ACE2 upregulation was affirmed
In this study, we showed that the cardiac ACE2 was significantly upregulated at transcriptional and translational level in the HCF cells after Ang II treatment. The effect of Ang II-mediated ACE2 upregulation in the cardiac cells was confirmed by the experiments of blocking Ang II−AT1R signaling pathway, including AT1R itself and the downstream
signaling of AT1R, NADPH oxidase and ERK-MAPK cascade (Figure 4-3). The influence of ROS on cardiac ACE2 upregulation was affirmed, suggesting that NADPH oxidase might induce ROS formation after Ang II treatment and then stimulate the ACE2 expression of HCF cells.
5-3. Cardiac ACE2 upregulation is associated with the modulation of Ang II to antagonize the effects of increased Ang II
Several reports showed the experimental results on the regulation of cardiac ACE2
expression associated with Ang II. For example, the studies mentioned that Ang II could reduce ACE2 expression in myocardial infarction or hypertensive rat model, and this effect on ACE2 expression by Ang II could be reversed by the treatment of AT1R blocker [Ishiyama et al., 2004; Ferrario et al., 2005a; Gallagher et al., 2008; Koka et al., 2008]. On the other hand, Yamamuro et al. [2008] reported that aldosterone but not Ang II reduced ACE2 mRNA expression in primary neonatal rat cardiomyocytes. However, above experimental results can not explain the clinical observations that the elevated Ang II paralleled to cardiac ACE2 upregulation was reported in the subjects with cardiovascular diseases, such as myocardial infarction, heart failure and atrial fibrillation [Zisman et al., 2003; Goulter et al., 2004;
Burrell et al., 2005; Epelman et al., 2008]. These clinical data may raise a possibility that cardiac ACE2 upregulation is associated with the modulation of Ang II to antagonize the effects of increased Ang II.
5-4. The upregulated ACE2 might play a compensatory role in maintaining a steady state of RAS
In the present study, the effect of Ang II on the upregulation of ACE2 expression in the HCF cells was found. Our results provide the evidence showing that Ang II could markedly stimulate ACE2 upregulation in the cardiac cells through Ang II−AT1R signaling transduction pathway. We suggested that the different regulation of ACE2 by Ang II may cause by the influence of experimental models of heart physiological condition (health or disease, and progression of disease), species and cell types, and even the aging effect [Shivakumar et al., 2003]. The upregulated ACE2 might play a compensatory role in counteracting the effect from the increased ACE activity and Ang II formation in hearts. This compensatory role, or called protective role, of ACE2 can maintain a steady state of RAS.
5-5. Ang 1-7 provides counter-regulatory effects to Ang II-induced deleterious effects on the cardiac functions
Nowadays, we also demonstrated that the cardiac ACE2 of HCF cells could be significantly upregulated after Ang 1-7 treatment (Figure 4-5). Ang 1-7, another critical angiotensin peptide in RAS, could be converted from Ang II by ACE2 enzyme catalysis, and several studies revealed that Ang 1-7 provides counter-regulatory effects to Ang II-induced
deleterious effects on the cardiac functions [Ferrario et al., 1997; Iwata et al., 2005; Grobe et al., 2007; Pan et al., 2007]. The elevated Ang 1-7 expression in failing heart tissue and ischemia zone of myocardial infarction might cause by the increase of ACE2 expression [Averill et al., 2003; Santos et al., 2005]. Clark et al. [2001] indicated that Ang 1-7 can downregulate AT1R transcription and translation in vascular smooth muscle cells. Recently, it is reported that Mas receptor can interact with the AT1R and antagonize AT1R through the formation of a hetero-oligomeric complex to block the effects of Ang II in cultured
mammalian cells [Kostenis et al., 2005]. Furthermore, Sampaio et al. [2007] showed that Ang 1-7 negatively modulates the activation of AT1R-dependent c-Src and the downstream targets of ERK-MAPK and NADPH oxidase by Mas receptor in endothelial cells.
5-6. Ang 1-7−enhanced ACE2 expression might be independent to the Ang II−AT1R signaling transduction pathway
In this study, the presence of Ang 1-7 significantly increased the ACE2 expression in HCF cells the same as Ang II stimulation. However, the AT1R expression, including at the transcriptional and translational level, in HCF cells did not influence by the Ang 1-7 treatment.
We proposed that the different AT1R regulation in the cardiofibroblasts and endothelial cells is because of cell specificity. To distinguish the signaling pathway in the ACE2 regulation by Ang II and Ang 1-7 becomes more important for the study of ACE2 regulation. HCF cells pretreated with AT1R blocker, Valsartan, could determine the existence of Ang 1-7−AT1R pathway. The results show that HCF cells pretreated with Valsartan have no marked effect on Ang 1-7 induced ACE2 expression. Our result hints that the pathway of Ang 1-7−enhanced ACE2 expression in the cardiac cells might be independent to the Ang II−AT1R signaling transduction pathway.
5-7. ERK-MAPK cascade could be the main pathway to stimulate ACE2 expression
Nie et al. [2009] showed that Ang1-7 stimulated ERK1/2 phosphorylation alone and significantly enhanced Ang II-induced phosphorylation of ERK1/2 in mouse bone
marrow-derived dendritic cells. Studies in mammalian (including human, rat and mouse) cells demonstrated that Ang 1-7 inhibit a lot of processes stimulated by Ang II, such as
vasoconstriction, cell growth and proliferation, proarrhythmia, prothrombogenic actions, and fibrogenic responses as well as activation of the MAPK family [Tallant et al., 1999; Iyer et al., 2000; Ferreira and Santos, 2005; Tallant et al., 2005b; Su et al., 2006]. All of those
processes stimulated by Ang II are mediated by AT1R, and Ang 1-7 takes the inhibitory effects via a specific receptor, the G-protein-coupled receptor Mas, whose relationship with Ang 1-7 was established in mouse kidney and Chinese hamster ovary cells [Santos et al., 2003a]. In this study, we suggest that the induction of phosphorylation of ERK1/2 by Ang II and Ang 1-7 alone are mediated by AT1R and Mas receptor, respectively. The ACE2
expression regulating by NADPH oxidase signaling pathway could even go through ERK-MAPK cascade. It could be explained that ERK-MAPK cascade was the main
pathway to stimulate ACE2 expression. As found in human and rat cells, ACE2 can directly cleave Ang II to form Ang 1-7 [Donoghue et al., 2000a], the coexistence of Mas receptor and ACE2 discovered in the present study suggests that there has an ACE2−Ang 1-7−Mas axis in human cardiofibroblast as well as ACE−Ang II−AT1R axis.
5-8. Distinguishing the signaling pathway in the ACE2 regulation between Ang II and Ang 1-7
To distinguish the signaling transduction pathway in the ACE2 regulation by Ang II and Ang 1-7 and realize the particular signaling cascade become more important for the study of a positive feedback-like loop (i.e., ACE2 catalyzes Ang II to be Ang 1-7 and Ang 1-7 can upregulate ACE2 production) in the cardiac ACE2 expression controlled by angiotensin peptides (Figure 4-16).
Figure 4-16. Schematic representation of interplay of Ang II and Ang 1-7 on the cardiac ACE2 regulation. ACE2, angiotensin-converting enzyme II; Ang II, angiotensin II; Ang 1-7, angiotensin 1-7; AT1R, angiotensin II type I receptor; ERK1/2, extracellular signal-regulated kinases 1/2; MEK1/2, mitogen-activated/ERK kinase 1/2; ROS, reactive oxygen species.
VI. Conclusions
Cardiovascular diseases are predicted to be the most common cause of death worldwide by 2020. In the general population, heart failure is chiefly the end stage of hypertensive, coronary and valvular cardiovascular disease. It is a major and growing problem in most affluent countries because of aging populations of increased size, and the prolongation of the lives of cardiac patients by modern therapy. Whatever the reason for the increasing rate of hospitalization for heart failure, it is clear that this condition is imposing a major burden on the health care system. Heart failure is a multifactorial quantitative trait controlled by both genetic and environmental factors. While much is known about environmental factors that can contribute to high blood pressure, such as diet and physical activity, less is known about the genetic factors that are responsible for predisposition to cardiovascular disease. Thus, the molecular and genetic mechanisms underlying heart failure and other cardiovascular diseases remain obscure.
A major risk factor for heart disease is high blood pressure. One important regulator of blood pressure homeostasis is the renin–angiotensin system (RAS). Recently, a homologue of ACE, termed ACE2, has been identified; it is predominantly expressed in the vascular endothelial cells of the kidney and heart. Unlike ACE, ACE2 functions as a
carboxypeptidase, cleaving a single residue from Ang I, generating Ang 1-9, and a single residue from Ang II to generate Ang 1-7. These in vitro biochemical data suggest that ACE2 may modulate the RAS and thus affect blood pressure regulation. Nevertheless, the in vitro role of ACE2 in the human cardiovascular system and the RAS is not known.
In the present study, human cardiofibroblast (HCF) cells were used to test the regulatory effects of Ang II and Ang 1-7 on the ACE2 expression at transcriptional and translational level.
To summarized, our results demonstrate that cardiac ACE2 was markedly upregulated in the HCF cells stimulated with Ang II, which might explain why the elevated ACE2 expression could be found at the initial stage in failing heart of human. AT1R signaling transduction pathway is involved in the cardiac ACE2 upregulation by Ang II stimulation to confirm the AT1R-dependent effect. Besides, the upregulated ACE2 may increase Ang 1-7 formation from Ang II and then the ACE2 expression was further enhanced by formatted Ang 1-7 via Mas receptor. Cardiac ACE2 upregulation with Ang II and Ang 1-7 stimulation is further confirmed by promoter assay and confocal laser scanning microscopy. The analyzed data showed that human cardiac ACE2 promoter activity was markedly upregulated with
transfection of pGL3 (-516/+20) constructs. This constructs was further incubated with Ang II and Ang 1-7 to verify the role in Ang II− and Ang 1-7−mediated ACE2 promoter activity.
The result showed that human cardiac ACE2 promoter activity was significantly upregulated with Ang II stimulation. This result indicated that ACE2 promoter activity was parallel to the transcriptional and translational expression of ACE2. In addition, the ACE2 florescence imaging also show the consistent results with our previous data.
To sum up, we propose a positive feedback-like loop on the cardiac ACE2 regulation for heart to maintain a steady state of RAS. These effects indicate a protective role of
ACE2−Ang 1-7 axis to counteract Ang II-induced deleterious effects on heart. Our results may present a novel target for developing potential therapeutic strategies in improving serious cardiovascular disease induced by the dysfunction of RAS.
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