5-1. Regulation of angiotensin converting enzyme II by angiotensin peptides We reported that ACE2 expression could be detected in human cardiac myocytes and fibroblasts. This result is confirmed by a previous report (Guy et al., 2008), although other studies show a limited amount of ACE2 mRNA and no ACE2 enzyme activity in cardiac fibroblasts of neonatal rats (Grobe et al., 2007; Gallagher et al., 2008). In this study, we selected cardiac fibroblasts over cardiac myocytes to test the hypothesis of angiotenin peptides-regulated ACE2 expression based on the following three reasons. First, cardiac fibroblasts are the most abundant cell type present in the myocardium (estimated 2/3 of myocardial cells) (Grove et al., 1969). Second, AT1R are much more abundant on cardiac fibroblasts than on cardiac myocytes (Greenberg et al., 2005). Third, cardiac fibroblasts are mainly responsible for the deposition of extracellular matrix (ECM) in heart. Cardiac remodeling is characterized by the proliferation of cardiac fibroblasts and abnormal ECM metabolism leading to cardiac fibrosis (Cohn et al., 2000; Porter and Turner, 2009).
In the present study, we found that Ang II-induced ACE2 expression in HCF cells. This result contrasts with other studies indicating that Ang II reduces ACE2 expression (Gallagher et al., 2008; Koka et al., 2008) and that RAS blockade through ACE inhibitors (to inhibit Ang II synthesis) or AT1R antagonists (to reduce Ang II activity) induces ACE2 expression
(Ishiyama et al., 2004; Ferrario et al., 2005; Kaiqiang et al., 2009). We do not challenge the concept that inhibition of Ang II synthesis or Ang II activity could induce ACE2 mRNA and protein expression in vivo or in vitro as reported previously. Instead, we emphasize that ACE2 regulation by angiotensin peptides may be largely dependent on the pathological or
physiological process present in the study model, including disease state and species-specific variations.
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Additionally, we do not exclude the possibility that the stage of cell differentiation could affect ACE2 gene regulation by Ang II. The primary HCF cells provided by ScienCell
Research Laboratories may be isolated from fetal hearts. Gene regulation is altered in cardiac tissues at different stages of differentiation and in different diseased conditions (Razeghi et al., 2001). This is supported by the reported differential response of cardiac fibroblasts from young adult and senescent rats to Ang II (Shivakumar et al., 2003). Varied cell sources could explain some of the contradiction between our data and previous reports. However, cardiac fibroblasts are the most prevalent cell type in heart and play a key role in regulating normal myocardial function, this study provides valuable data on the effect of angiotensin peptides on cardiac fibroblast ACE2 expression.
We currently show increased ACE2 expression by Ang II stimulation could be abolished by both Val and PD98059 pretreatment. These data suggest that Ang II could stimulate ACE2 expression in HCF cells through the Ang II-AT1R signaling pathway. Our results suggest that up-regulated ACE2 may play a compensatory role in counteracting the effects of increased cardiac Ang II formation. This compensatory or protective role may serve as a means to maintain a steady state within RAS. We also demonstrated that ACE2 expression in HCF cells could be up-regulated by Ang 1-7. Ang 1-7, an angiotensin peptide of RAS, can be converted from Ang II by ACE2 enzyme catalysis. Several studies reveal that Ang 1-7 provides
counter-regulatory effects to the deleterious effects of Ang II on cardiac function (Grobe et al., 2007; Pan et al., 2007; Mercure et al., 2008). The reported elevation of Ang 1-7 expression in failing heart tissue and in the ischemic zone following MI might result from increased ACE2 expression (Averill et al., 2003; Santos et al., 2005). Loss of ACE2 severely impairs cardiac function (Crackower et al., 2002), and Kassiri et al. hypothesize that loss of ACE2 would accelerate maladaptive left ventricular remodeling in response to MI (Kassiri et al., 2009).
ACE2 may modify AT1R expression by altering the balance between the Ang II and Ang
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1-7 (Zucker et al., 2009). ACE2 regulation by angiotensin peptides may be influenced by the experimental model, age, species, cell type, state of health or type of cardiac disease
(Shivakumar et al., 2003). In vascular smooth muscle cells, Clark et al. (Clark et al., 2003) indicated that Ang 1-7 down-regulates AT1R transcription and translation. In endothelial cells, Ang 1-7 reduces activation of AT1R-dependent c-Src protein and the downstream targets of ERK-MAPK via the Mas receptor (Sampaio et al., 2007). In the present study, both Ang 1-7 and Ang II comparably increased ACE2 expression in HCF cells. Additionally, Ang 1-7 increased the activation of p-ERK1/2 proteins (Fig. 4-6A). Furthermore, the increased ACE2 expression induced by Ang 1-7 could not be reversed by pretreatment with the AT1R inhibitor Val (Fig. 4-6B). These results suggest that the up-regulation of ACE2 expression by Ang 1-7 in cardiac fibroblasts may be independent of the Ang II-AT1R signaling pathway. This result is inconsistent with findings in vascular smooth muscle cells (Clark et al., 2001) and
endothelial cells (Sampaio et al., 2007). The reasons for these differences are unclear, but we propose the existence of cell-type-specific differences in AT1R regulation among
cardiofibroblasts, vascular smooth muscle cells and endothelial cells.
We report, for the first time, that Ang 1-7 increases ACE2 expression in HCF cells in vitro. Increased ACE2 expression could promote the conversion of Ang II to Ang 1-7 and
thereby increase cardiac Ang 1-7. Furthermore, increased Ang 1-7 may up-regulate ACE2 expression in certain physiological conditions. This positive feedback loop may promote the Ang II conversion into Ang 1-7 to maintain a static state of cardiac Ang II concentration (Fig.
5-1). This proposed regulation of cardiac ACE2 expression maybe important for cardiac response to physiological stresses that would abnormally increase Ang II concentration.
Furthermore, we propose that abnormal regulation on cardiac ACE2 expression may be related to cardiac pathophysiological processes such as hypertension (Koka et al., 2008), ischemia (Ishiyama et al., 2004) and atrial fibrillation (Pan et al., 2007).
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Fig. 5-1. Schematic diagram representing the interplay of Ang II and Ang 1-7 on cardiac ACE2 regulation. This scheme indicates that Ang II-up-regulated ACE2 may increase Ang 1-7 formation from Ang II, which can further increase ACE2 expression through a positive feedback loop. ACE2, angiotensin converting enzyme II; Ang II, angiotensin II; Ang 1-7, angiotensin 1–7; AT1R, angiotensin II type 1 receptor; ERK1/2, extracellular signal-regulated kinases 1/2; MEK1/2, mitogen-activated/ERK kinase 1/2.
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5-2. Identifying the regulatory element for human angiotensin-converting enzyme 2 (ACE2) expression
To investigate the molecular mechanism by which Ang II regulates the expression of ACE2, we examined the promoter activity its gene, ace2. Using sequence deletion and
site-directed mutation analyses, we identified a region upstream of ace2, at –516/–481 domain, that is required for Ang II-activated transcription. We also demonstrated that the sequence ATTTGGA is the Ang II responsive element.
From this study, the results of the promoter activity assay are consistent with those that show cardiac ACE2 was significantly up-regulated at both transcriptional and translational levels in HCFs after Ang II stimulation-presumably via the Ang II-AT1R signaling pathway.
Several reports have shown that elevated Ang II levels were observed in conjunction with cardiac ACE2 up-regulation in subjects with cardiovascular disease, (e.g., MI, heart failure and atrial fibrillation) both in the clinic and in animal experiments (Zisman et al., 2003;
Goulter et al., 2004; Burrell et al., 2005; Pan et al., 2007; Epelman et al., 2008). This raises the possibility that cardiac ACE2 up-regulation is associated with the modulation of the effect of Ang II, by an antagonist for example, which diminishes the effect of increased Ang II.
Based on the results of Ang II-stimulated ACE2 up-regulation in HCFs, we suggest that the regulation of ACE2 by Ang II may be largely dependent on pathological and/or physiological conditions, and that up-regulated ACE2 may play a compensatory role in counteracting the effects from the increased ACE activity and Ang II production in the heart. This compensatory or protective role of ACE2 may serve to maintain homeostasis within the RAS.
In addition to the angiotensin peptides in the RAS, inflammation plays a key role in the initiation, progression, and clinical outcome of cardiovascular diseases. Substantial evidence suggests the involvement of the inflammatory and immune systems in adverse remodeling of cardiac failure and hypertrophy (Torre-Amione, 2005; Yndestad et al., 2007; Wynn, 2008). In
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this study, we attempted to evaluate whether the expression of ACE2 could be modulated by pro-inflammatory factors in HCFs. We examined the effects of two pro-inflammatory cytokines, TGF-β1 and TNF-α on the expression ACE2. Increased doses of TGF-β1 and TNF-α did not cause significant change in ACE2 expression, however, nor in the promoter activity of ace2. This result confirms a previous report that ACE2 expression was not affected by TNF-α, IL-1 and chronic hypoxia in human cardiac myofibroblasts (Guy et al., 2008). It has been shown that Ang II can induce TGF-β1 and TNF-α expression in cardiac cells via the Ang II/AT1R signaling pathway (Kalra et al., 2002; Schultz Jel et al., 2002; Rosenkranz, 2004). We therefore suggest that Ang II-stimulated ACE2 up-regulation may occur via a TGF-β1/TNF-α independent pathway-although the results of ACE2 modulation by
angiotensin and the cytokines reported here may be dependent on the specific experimental models used.
The sequence ATTTGGA is a potential binding domain for the transcriptional factor Ikaros. Ikaros was originally found to function as a key regulator of lymphocyte
differentiation (Lo et al., 1991; Georgopoulos et al., 1992). Subsequent studies demonstrated the role of Ikaros in normal hematopoiesis (Lopez et al., 2002), and in the migration and invasion of extravillous trophoblasts in early placentation (Yamamoto et al., 2005). In a recent study, it was reported that Ikaros primes the lymphoid transcriptional program in
hematopoietic stem cells, and that loss of Ikaros may confer aberrant self-renewing properties on myeloid progenitors (Yoshida et al., 2010); yet despite the clearly important biological role of Ikaros, its mechanism of action remains elusive. Consensus DNA recognition sequences for Ikaros have been unusually difficult to define because of several encoded Ikaros isoforms (Molnar and Georgopoulos, 1994) and because multiprotein complexes containing Ikaros family members have not been purified to homogeneity (Sridharan and Smale, 2007). From sequence analysis (using TFSEARCH) the potential binding domain of Ikaros was found in
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the regulatory region of ace2, but was not found in ace gene. This may explain why some factors have been shown to regulate ace and ace2 differently (Hamming et al., 2008; Koka et al., 2008; Zhang et al., 2009).
We report here for the first time the characterization of the regulatory element of human gene, ace2, and provide insight into the molecular mechanism controlling cardiac ACE2 expression in HCFs. We have identified the –516/–481 sequence domain within the upstream region of ace2 as a putative protein binding domain for modulation of ACE2 expression, which is associated with the Ang II signaling pathway. Furthermore, a potential regulatory element, ATTTGGA, within the –516/–481 promoter region of ace2 is responsible for Ang II stimulation, and this is unaffected by the pro-inflammatory cytokines, TGF-β1 and TNF-α.
Our results suggest that the –516/–481 domain of ace2 is involved in modulating ACE2 expression, and may be a binding domain for Ikaros, or other unidentified regulatory factor(s).
Investigating the regulatory role of Ikaros on ace2 and other potential regulatory factor(s) would lead to a greater understanding of the molecular mechanisms that regulate ACE2 expression.
5-3. The association between ACE2, MMP-2 and angiotensin peptides
In recent study, the relationship between Ang II and MMP-2 had been investigated in cell level. Human umbilical vein endothelial cells (HUVECs) treated with Ang II were induced TNF-α and MMP-2 release, and reduced the secretion of TIMP-2 via AT1R (Arenas et al., 2004). However, the association between ACE2 and MMP-2 is unclear. In this study, ACE2 lentivirus, TLC-ACE2 was used to infected HCFs to obtain HCFs/ACE2 and investigated the associated with ACE2 overexpression and MMP-2 activity. The results showed ACE2
overexpression enhanced MMP-2 activity and the Ang II and Ang 1-7 suppressed the
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activation of ERK1/2 via AT1R and Mas receptor in HCFs/ACE2, respectively. Ang II was through AT1R to induce ERK1/2 activation and MMP-2 activity. Additionally, Ang
II-AT1R-ERK1/2 and Ang 1-7-Mas-ERK1/2 axes also affected the ACE2 shedding from the cell membrane of HCFs/ACE2.
Cardiac fibroblasts and myocytes are the major cell type in heart tissue. The role of Ang II in modulating cardiac fibroblast activity is well accepted (Campbell et al., 1997; Kawano et al., 2000), but the actions of ACE2 in cardiac cells is unclear. Grobe and his group examined the effects of ACE2 gene delivery to cultured cardiac fibroblasts after acute hypoxic exposure.
They utilized neonatal rat cardiac myocytes and cardiac fibroblasts to indicate that endogenous ACE2 activity is observed in cardiac myocytes, but not in cardiac fibroblasts (Grobe et al., 2007). Because of cardiac fibroblasts are the major cell type found in an infarct zone following a MI, lenti-ACE2 was been used to induce ACE2 expression of cardiac fibroblasts and revealed that ACE2 overexpression significant attenuated TGF-β and
hypoxia/re-oxygenation-induced collagen production by the cardiac fibroblasts. In addition, the expression of ACE, ACE2 and AT1R were been detected in human cardiac myofibroblasts isolated from patients undergoing coronary artery bypass surgery (Guy et al., 2008). It also revealed that ACE2 can been released into extracellular medium and evidenced that ACE2 expression was not been regulated by TNF-α, IL-1β (Turner et al., 2007) or chronic hypoxia in human cardiac myofibroblasts. In addition, cardiac myocytes were isolated from diabetes rat and evidenced that ACE2 overexpression in vitro decreased high glucose (HG)-induced Ang II production, collagen accumulation and TGF-β expression in cardiac fibroblasts, and attenuated myocyte hypertrophy, myocardial fibrosis, and left ventricular (LV) remodeling (Dong et al., 2012). All results supported our results in HCFs and appeared that ACE2 plays a protect role in cardiac fibroblasts, myofibroblasts and myocytes to opposite to Ang II induced TGF-β expression and collagen accumulation, some difference of results maybe from the
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different species and cell types (Guy et al., 2008).
Besides the discussion of cardiac cell, ACE2 also been investigated in animal model, especially the study of ACE2 overexpression and knockout. Huentelman (2005) and his coworker inserted a cDNA of mACE2 into the pTY.EF1.IRES.EGFP lentivirus cloning vector to construct the lenti-mACE2 and beginning the study of ACE2 overexpression. They injected the lenti-mACE2 into the left cardiac ventricular cavity to established ACE2 overexpression mice and infused Ang II to estimate the body weight and myocardial fibrosis. The results showed ACE2 overexpression were significant attenuation of the increased heart weight to body weight ratio and myocardial fibrosis induced by Ang II infusion (Huentelman et al., 2005). This lenti-mACE2 also used in spontaneously hypertensive rat (SHR) and coronary artery ligation (CAL) rat, the results demonstrated that ACE2 overexpression improved high blood pressure, left ventricular (LV) wall thickness and perivascular fibrosis in SHR mice (Díez-Freire et al., 2006; Der et al., 2008). Furthermore, the cardiac fibroblasts and cardiac myocytes of ACE2 overexpression mice were isolated and indicated that cardiac fibroblasts were not expressing ACE2, but ACE2 overexpression suppressed the
hypoxia/re-oxygenation-induced collagen and TGF-β production in cardiac fibroblasts (Grobe et al., 2007).
In RAS and ECM, the further investigation indicated that ACE2 overexpression reduces Ang II levels and enhances Ang 1-7 to improve heart dysfunction (Dong et al., 2008; Guo et al., 2008). ACE2 overexpression inhibited cell growth, MMP-2 and MMP-9 expression, VEGFa production, and ACE and AT1R expression in human lung cancer xenografts and A549 cells in vitro (Feng et al., 2011). In MI and SHR rat, ACE2 overexpression inhibiting ACE, Ang II and collagen expression, up-regulating Ang 1-7 and MMP-2 expression to attenuate LV fibrosis, and to improve LV remodeling and systolic function (Rentzsch et al., 2008; Zhao et al., 2010; Dong et al., 2012). These evidences reveal that Ang II mediated
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AT1R to induce NADPH oxidase and MMP activation, AT1R blocker and Ang 1-7
supplementation inhibited NADPH oxidase and MMP activation (Kassiri et al., 2009; Bodiga et al., 2011). These results demonstrated that ACE2 overexpression regulates ACE-Ang II-AT1R axis and ACE2-Ang 1-7-Mas axis to affect the expression of pro-inflammatory cytokines, collagen and MMPs. These results also suggest that ACE2 serves as a protective mechanism and associated with MMPs expression, especially MMP-2 and MMP-9, to improve the hypertension, heart dysfunction and cardiac fibrosis. The significant references were listed in Appendix 8-3.
ACE2 knockout (KO) mice were also established in 2006 by Gurley and his group. They evidenced that ACE2-deficient mice are not any structural abnormalities and no effect on baseline blood pressures, moreover, acute Ang II infusion increased 3-fold higher Ang II concentration to cause hypertension in ACE2-deficient mice than in controls (Gurley et al., 2006). The further studies were using the different heart diseases model such as transverse aortic constriction (TAC) and left anterior descending artery ligation in ACE2 KO mice to investigate the effect of ACE2 deficient (Yamamoto et al., 2006; Kassiri et al., 2009). In MI which induced by left anterior descending artery ligation, ACE2 deficiency leads to increase phosphorylation of ERK1/2 and JNK1/2 signaling pathways, up-regulate MMP-2, MMP-9 and inflammatory cytokines and the chemokine such as interferon-gamma, interleukin-6, monocyte chemoattractant protein-1. Moreover, loss of ACE2 also associated with the
increased expression and phosphorylation of p47phox, Ang II levels, NADPH oxidase activity, and superoxide generation, which could lead to enhanced MMP-mediated degradation of the extracellular matrix in ACE2-deficient myocardium and eccentric remodeling, increased pathological hypertrophy, and worsening of systolic performance (Bodiga et al., 2011; Patel et al., 2012). These results support our data and appeared that loss of ACE2 facilitates adverse post-MI ventricular remodeling associated with MMP-2, MMP-9 and inflammatory factor
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(Kassiri et al., 2009).
In addition, ACE2 KO mice received TAC was significant increased the Ang II concentration in heart and plasma compared to WT mice. The enhanced Ang II was induce mitogen-activated protein (MAP) kinases, MMPs and activation of NADPH oxidase, decrease cardiac contractility to develop cardiac hypertrophy and dilatation, revealed that Ang II is a major factor in development of cardiac hypertrophy and dilatation (Yamamoto et al., 2006;
Patel et al., 2012). Therefore, some studies utilized Ang II infusion in WT and ACE2 KO mice to worse cardiac fibrosis and pathological hypertrophy. The WT and ACE2 KO mice infused Ang II and recombinant human ACE2 (rhACE2) revealed that Ang II induced collagen and MMP-2 expression, NADPH oxidase activity, hypertension, myocardial hypertrophy, fibrosis, and diastolic dysfunction were been attenuated by rhACE2 (Zhong et al., 2010; Alghamri et al., 2012). The significant references were listed in Appendix 8-4.
In this study, ACE2 shedding also been investigated in HCFs/ACE2 treated with angiotensin peptides to discuss the role that ACE2 plays in the cardiac fibrosis. In 2005, ADAM17 had been demostrated that involved with the regulated ectodomain shedding of ACE2. ADAM17 stimulated ACE2 shedding mediated phorbol ester-inducible ectodomain shedding in HEK293 cells and Huh7 cells, the ADAM17 overexpression and knockout were used to identify with this observation (Lambert et al., 2005). ADAM17 recognized in the site Arg(708) and Arg(710) within ACE2 peptides sequence and cleaved ACE2 peptide sequence between Arg(708) and Ser(709) (Lai et al., 2011). In addition, these results also had been affirmed in 3T3-L1 adipocytes and HeLa cells (Gupte et al., 2008; Jia et al., 2009). In RAS, ACE2 is homologous to one of the active sites of ACE and has 40% overall identity to ACE.
However, as like captopril or other ‘classical’ ACE-inhibitors are not inhibiting the activity of ACE2, ADAM17 is not suitable to ACE shed (Zisman, 2005). Allinson and his group used antisense oligonucleotide of ADAM17 to reveal ADAM17 supressed was not effect ACE
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shedding with the mercurial compound 4-aminophenylmercuric acetate (APMA) stimulated the shedding of ACE in human SH-SY5Y cells (Allinson et al., 2004).
The further study indicated that ACE2 plays a protector in severe acute respiratory
The further study indicated that ACE2 plays a protector in severe acute respiratory