| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Review Articles |
1 Division of Nephrology, Department of Medicine, Duke University and Durham VA Medical Centers, Durham, NC, USA
 |
Abstract |
|---|
 Top Abstract References |
|---|
(Received 10 February 2008;
accepted after revision 27 February 2008; first published online 30 March 2008)
Corresponding author T. M. Coffman: Division of Nephrology, Department of Medicine, Duke University and Durham VA Medical Centers, Nephrology Research, DUMC 103015, Durham, NC 27710, USA. Email: tcoffman{at}duke.edu
Cardiac phenotypes in ACE2 knockout mice
Angiotensin-converting enzyme 2 was originally cloned from a cDNA library prepared from the left ventricle of a failing human heart (Donoghue et al. 2000) and was found to have substantial (>40%) homology with angiotensin-converting enzyme (ACE). Accordingly, there was speculation that this gene might have an important cardiovascular role. Several groups in the field sought to characterize the physiological functions of ACE2 in vivo by generating ACE2 knockout mice (Table 1).
|
Subsequently, Yamamoto and associates published their experiments using a distinct line of mice deficient in ACE2 (Yamamoto et al. 2006). This line was generated by deleting exon 3 of the Ace2 gene, including the splice donor and acceptor sites, resulting in the absence of detectible ACE2 mRNA and protein. In contrast to the findings of Crackower and colleagues, baseline cardiac function and morphology appeared normal in this ACE2-deficient mouse line. For example, heart-to-body weight ratio, echocardiographic parameters, changes in left ventricular pressure over time (dP/dt), systolic left ventricular pressures and cardiac morphology were virtually identical in ACE2-knockout mice and the wild-type control animals.
Our group developed a third line of ACE2-deficient mice, also generated by replacing nucleotides +1069 to +1299 of the Ace2 gene with a neomycin cassette (Gurley et al. 2006). Angiotensin-converting enzyme 2 mRNA and protein were undetectable in these animals. The ACE2-null mice appeared normal, had a normal lifespan and were fertile. Moreover, their cardiac function, assessed by echocardiography in the conscious state, was completely normal and not different from their wild-type littermates. Although the study of Crackower and associates had suggested that cardiac abnormalities were more pronounced in male mice and became worse with age, we found normal fractional shortening and left ventricular dimensions in a cohort of male ACE2-deficient mice more than 17 months old.
The dramatic difference in phenotypes between our ACE2-deficient line and that reported by Crackower and associates cannot be easily explained by differences in the gene disruption methodology, since the exon encoding the active site of the enzyme was deleted in both lines. One common cause of variable phenotypes in transgenic mouse models is a difference in genetic backgrounds. Moreover, our initial experiments, as well as the previous two reports from the other groups, were carried out in mice with randomly mixed genetic backgrounds derived from C57BL/6 and 129.
To assess the potential for background gene effects as an explanation for the variability of the cardiac phenotype, we back-crossed the Ace2 null mutation for more than six generations onto each of the parental lines (C57BL/6 and 129/SvEv) and assessed cardiac function by echocardiography in conscious, inbred C57BL/6- and 129/SvEv-Ace2–/y male mice at 3 and 6 months of age. Similar to our findings in the older mice on a mixed genetic background, inbred C57BL/6- and 129/SvEv-Ace2–/y male mice also had normal cardiac systolic function compared with their wild-type littermates at 3 and 6 months of age. Thus, a modifying influence of genetic background does not seem to explain the different cardiac phenotypes.
Another factor that might have impacted the assessments of cardiac function was the use of anaesthesia for the echocardiographic measurements. General anaesthesia has well-recognized effects to depress cardiac function (Vatner & Braunwald, 1975). Compared with our studies performed in conscious, unanaesthetized animals, it is possible that the use of anaesthesia in the previous studies of Crackower and colleagues might have uncovered apparent abnormalities in cardiac function (Crackower et al. 2002) Alternatively, other unrecognized factors in the environment might affect the cardiac response to ACE2 deficiency. It is worth noting that a subsequent publication using the same line originally generated by Crackower and associates reported normal cardiac function in the ACE2-deficient mice, suggesting that the overt cardiac phenotype may have been lost in subsequent generations (Wong et al. 2007).
Additional insight into this issue is perhaps provided by the studies of Yamamoto and associates. These authors tested whether responses to pressure-overload caused by transverse aortic constriction (TAC) was affected by ACE2 deficiency. After TAC, they found that cardiac hypertrophy, assessed as heart-to-body weight ratio, was more severe in the ACE2 knockout mice than wild-type control animals. The cardiac hypertrophy was characterized by worse LV dilatation, myofibrillar disarray and fibrotic changes in the ACE2 knockout mice. By echocardiography, left ventricular diastolic and systolic dimensions after TAC were significantly larger, and systolic function, assessed by left ventricular fractional shortening, was impaired in ACE2-deficient mice compared with wild-type control animals; fractional shortening was reduced by 14% in the ACE2 knockout mice. The authors measured lung-to-body weight ratios following TAC as an assessment of congestive heart failure and found them to be higher in the ACE2 knockout mice than in control animals. Finally, cardiac death with pulmonary congestion after TAC occurred with a greater frequency in ACE2-deficient mice compared with wild-type control animals. Taken together, these studies would suggest that ACE2 does not necessarily play a critical role in regulating normal cardiac function, but may be important in modulating responses to pressure overload and perhaps other stresses and injuries.
Angiotensin-converting enzyme 2 and blood pressure regulation
Owing to its homology with ACE and its potent in vitro actions to metabolize angiotensin peptides, it was anticipated that ACE2 might also play a role in regulation of blood pressure. Some of the studies by Crackower and associates appeared to support this hypothesis. In studies of three rat models of genetic hypertension, namely the Sabra-salt-sensitive rat, the stroke-prone spontaneously hypertensive rat (SHR) and hypertensive BB.X rats, the Ace2 gene was mapped to quantitative trait loci (QTLs) associated with elevated blood pressure. Furthermore, levels of Ace2 mRNA and ACE2 protein were reduced in hypertensive rats compared with their respective normotensive controls. However, in their knockout studies, the authors reported no effect of the Ace2-null mutation on blood pressures in younger animals, and found frank reductions in blood pressure in older male mice that were ACE2-deficient. Since these animals had a cardiac contractility defect that became more severe with age, a separate role for ACE2 in the regulation of blood pressure would be difficult to discern in this setting.
In contrast, since there was no abnormal cardiac phenotype in our ACE2-deficient mice (Gurley et al. 2006), the line could be used to study the role of ACE2 in regulation of blood pressure without concern for potentially confounding effects of impaired cardiac function. Thus, we carefully assessed the consequences of ACE2 deficiency on blood pressure in separate comparisons of cohorts of mice with mixed or inbred backgrounds. In animals with genetic backgrounds that were a random mix of C57BL/6 and 129, the effect of ACE2 on blood pressure was inconsistent. In one early cohort of mixed-background mice, blood pressures were higher in ACE2-deficient mice than in their wild-type littermates. However, in a second cohort, back-crossed two generations onto the 129/SvEv background, there was no difference in blood pressure between ACE2-deficient and control mice. This variability in blood pressure suggested that there might be genetic modifiers in the parental backgrounds affecting this trait.
To test for this possibility, we examined blood pressures in the lines of inbred ACE2-deficient mice described above. Blood pressures of inbred 129/SvEv ACE2-deficient mice were virtually identical to those of 129/SvEv control animals. By contrast, on the C57BL/6 background, ACE2 deficiency was associated with a modest, but statistically significant, increase in blood pressure of 
7 mmHg. This level of increase was very similar to that observed in the initial cohort of mixed-background animals and in the more recent report by Wong et al. (2007). Taken together, these findings suggest that background genes can significantly modify the relatively modest impact of ACE2 on normal blood pressure homeostasis. Therefore, in a permissive genetic milieu, ACE2 may contribute to determining baseline level of blood pressure.
Angiotensin-converting enzyme 2 and angiotensin peptide metabolism
Analogous to the actions of ACE, in vitro studies have demonstrated the capacity of ACE2 to metabolize angiotensin peptides, including angiotensins I and II (Donoghue et al. 2000; Vickers et al. 2002). While ACE is a dicarboxypeptidase, ACE2 is a monocarboxypeptidase. Hydrolysis of angiotensin II by ACE2 generates a peptide with putative biological actions: angiotensin(1–7) (Vickers et al. 2002). Accumulating evidence suggests that angiotensin(1–7) causes vasodilatation and natriuresis and may promote reduced blood pressures (Ferrario et al. 1991). It is therefore conceivable that ACE2 might influence blood pressure and cardiovascular functions by at least two mechanisms: (1) by metabolizing and degrading pools of active angiotensin II; and (2) by production of angiotensin(1–7). Angiotensin-converting enzyme 2-null mouse lines have been used to examine the actions of ACE2 in metabolism of angiotensin peptides in vivo.
Crackower and associates found increased levels of angiotensin I and II in kidneys and hearts of ACE2-deficient mice, along with increased circulating levels of angiotensin II in plasma. However, the interpretation of these findings is confounded to some extent by the cardiomyopathy that was present in these animals. Reduced cardiac output is a potent stimulus for activation of the renin–angiotensin system, and elevated levels of angiotensin I and II are typically seen in heart failure. To explore the mechanism of the cardiac abnormality and to examine the potential role of the RAS, the authors intercrossed their ACE2-deficient line with mice lacking conventional ACE (Krege et al. 1995; Esther et al. 1996). The combination of ACE deficiency with the ACE2 knockout restored normal cardiac systolic function, suggesting that the abnormal cardiac phenotype associated with ACE2 deficiency depends on normal activity of the RAS, including a normal capacity to generate angiotensin II.
Yamamoto and associates also evaluated the effects of the ACE2 mutation on other components of the RAS. Unlike the Crackower study, they found that angiotensin II levels in plasma and in the heart were similar in ACE2 knockout and wild-type mice at baseline. After TAC, cardiac angiotensin II levels increased significantly in both groups, but the extent of increase was greater in the ACE2-deficient animals (8-fold) compared with the wild-type control animals (5-fold). There was also a trend towards higher plasma levels of angiotensin II in the knockout mice after TAC. Finally, they measured downstream targets of angiotensin II in the heart, including activation of mitogen-activated protein kinases (MAP kinases). After TAC, levels of activated extracellular signal related-kinases (ERKs) and c-jun N-terminal kinases (JNKs) were significantly enhanced in hearts of ACE2-deficient mice compared with wild-type control animals. Administration of the angiotensin receptor blocker, candesartan, attenuated the hypertrophic response and fibrosis seen in the ACE2 knockout mice after TAC, suggesting that the cardiac injury was mediated by angiotensin II. Furthermore, candesartan treatment prevented the impairment of cardiac contractility in ACE2-deficient mice after TAC.
Similar to Yamamoto and associates, we found no significant differences in plasma angiotensin II levels between wild-type and ACE2-deficient mice. However, 10 min after an acute intravenous infusion of angiotensin II, we found that plasma levels of angiotensin II were almost threefold higher in ACE2-deficient mice than in control animals, suggesting that ACE2 is a key pathway for the metabolism of angiotensin II in vivo. In angiotensin II-dependent hypertension, intrarenal actions of angiotensin II contribute to antinatriuresis, and elevation of blood pressure is associated with accumulation of angiotensin II peptide in the kidney (Wang et al. 2000; Zhuo et al. 2002). Owing to the high levels of ACE2 expression in the kidney (Donoghue et al. 2000) and our demonstration of the key contribution of ACE2 to angiotensin II metabolism, we posited that ACE2 might function to attenuate the hypertensive actions of angiotensin II. To test this hypothesis, we compared the responses of ACE2-deficient and wild-type mice to chronic infusion of angiotensin II, a widely used model of angiotensin-dependent hypertension (Mitchell & Navar, 1995). Chronic infusion of 40 pmol min–1 of angiotensin II over 14 days caused a significant increase in blood pressure in wild-type mice, as expected (Kawada et al. 2002; Francois et al. 2004). Administration of an equivalent concentration of angiotensin II to ACE2-deficient mice caused a significantly larger increase in blood pressure that was almost twofold greater than in control animals. Thus, the absence of ACE2 enhances the severity of angiotensin II-dependent hypertension.
To determine whether the exaggerated severity of hypertension in ACE2-deficient mice was associated with altered metabolism of angiotensin II in the kidney, we compared the levels of renal angiotensin peptides in the two groups using a novel application of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Compared with other available techniques, this method affords advantages in specificity and sensitivity for angiotensin peptide detection (Desiderio et al. 2000; Kokko & Dix, 2002). After chronic angiotensin II infusion, we found that renal angiotensin II levels were more than fivefold higher in ACE2-deficient mice than in control animals, indicating that the more severe hypertension in ACE2-deficient mice is due to impaired metabolism of angiotensin II in the kidney. Thus, ACE2 appears to have a critical role in regulating local concentrations of angiotensin II in the kidney. Owing to the powerful effects of angiotensin II to stimulate renal sodium reabsorption, this represents a potent pathway for controlling blood pressure.
Our findings suggest that ACE2 regulates cardiovascular responses by degrading angiotensin II and attenuating its actions at the angiotensin II type 1 receptor. However, hydrolysis of angiotensin II by ACE2 may generate angiotensin(1–7). It is therefore conceivable that production of angiotensin(1–7) by ACE2 may have physiological consequences and that reduced synthesis of angiotensin(1–7) might also contribute to the proclivity of ACE2-deficient mice to develop hypertension. However, the robust, fivefold increase in renal angiotensin II levels observed in the ACE2-deficient mice is probably sufficient to explain the difference in severity of hypertension, independent of any potential effect of angiotensin(1–7).
Conclusion
Taken together, studies using mouse lines with targeted deletion of the Ace2 gene have provided a number of contributions to understanding the role of ACE2 in cardiovascular functions. Despite a lack of uniformity in certain phenotypes, some common themes have emerged from these studies carried out by several investigative groups. First, ACE2 appears to have only modest effects on baseline cardiovascular functions and blood pressure control; these effects can be substantially modulated by genetic and, perhaps, environmental factors. In contrast, the activity of ACE2 may have more profound effects on susceptibility to pathological states, such as hypertension and cardiac hypertrophy. These actions require an active RAS, including capacity for generation of angiotensin II and activation of angiotensin II type 1 receptors. The contribution of ACE2 to cardiovascular physiology and disease is probably due to its role in metabolizing and degrading pools of angiotensin II, along with its contribution to synthetic pathways for angiotensin(1–7).
|
References |
|---|
|
TopAbstractReferences |
|---|
Desiderio DM, Wirth U, Lovelace JL, Fridland G, Umstot ES, Nguyen TM, Schiller PW, Szeto HS & Clapp JF (2000). Matrix-assisted laser desorption/ionization mass spectrometric quantification of the mu opioid receptor agonist DAMGO in ovine plasma. J Mass Spectrom 35, 725–733.[CrossRef][Medline]
Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N, Donovan M, Woolf B, Robison K, Jeyaseelan R, Breitbart R & Acton S (2000). A novel ACE-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1–9. Circ Res 87, 1–9.
Esther C, Howard T, Marino E, Goddard J, Capecchi M & Bernstein K (1996). Mice lacking angiotensin converting enzyme have low blood pressure, renal pathology, and reduced male fertility. Lab Invest 74, 953–965.[Medline]
Ferrario C, Brosnihan K, Diz D, Jaiswal N, Khosla M, Milsted A & Tallant E (1991). Angiotensin-(1–7): a new hormone of the angiotensin system. Hypertension 18, III126–III133.[Medline]
Francois H, Athirakul K, Mao L, Rockman H & Coffman TM (2004). Role for thromboxane receptors in angiotensin-II-induced hypertension. Hypertension 43, 364–369.
Gurley SB, Allred A, Le TH, Griffiths R, Mao L, Philip N, Haystead TA, Donoghue M, Breitbart RE, Acton SL, Rockman HA & Coffman TM (2006). Altered blood pressure responses and normal cardiac phenotype in ACE2-null mice. J Clin Invest 116, 2218–2225.[CrossRef][Medline]
Kawada N, Imai E, Karber A, Welch WJ & Wilcox CS (2002). A mouse model of angiotensin II slow pressor response: role of oxidative stress. J Am Soc Nephrol 13, 2860–2868.
Kokko KP & Dix TA (2002). Monitoring neurotensin[8–13] degradation in human and rat serum utilizing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal Biochem 308, 34–41.[CrossRef][Medline]
Krege J, John S, Langenbach L, Hodgin J, Hagaman J, Bachman E, Jennette J, O'Brien D & Smithies O (1995). Male-female differences in fertility and blood pressure in ACE-deficient mice. Nature 375, 146–149.[CrossRef][Medline]
Mitchell K & Navar L (1995). Intrarenal actions of angiotensin II in the pathogenesis of experimental hypertension. In Hypertension: Pathophysiology, Diagnosis and Management, ed. Laragh J & Brenner B, pp. 1437–1450. Raven Press, New York.
Tipnis S, Hooper N, Hyde R, Karran E, Christie G & Turner A (2000). A human homolog of angiotensin-converting enzyme. J Biol Chem 275, 33238–33243.
Vatner S & Braunwald E (1975). Cardiovascular control mechanisms in the conscious state. N Engl J Med 293, 970–976.[Medline]
Vickers C, Hales P, Kaushik V, Dick L, Gavin J, Tang J, Godbout K, Parsons T, Baronas E, Hsieh F, Acton S, Patane M, Nichols A & Tummino P (2002). Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase. J Biol Chem 277, 14838–14843.
Wang C-T, Chin S & Navar L (2000). Impairment of pressure-natriuresis and renal autoregulation in ANG II-infused hypertensive rats. Am J Physiol Renal Physiol 279, F319–F325.
Wong DW, Oudit GY, Reich H, Kassiri Z, Zhou J, Liu QC, Backx PH, Penninger JM, Herzenberg AM & Scholey JW (2007). Loss of angiotensin-converting enzyme-2 (Ace2) accelerates diabetic kidney injury. Am J Pathol 171, 438–451.
Yamamoto K, Ohishi M, Katsuya T, Ito N, Ikushima M, Kaibe M, Tatara Y, Shiota A, Sugano S, Takeda S, Rakugi H & Ogihara T (2006). Deletion of angiotensin-converting enzyme 2 accelerates pressure overload-induced cardiac dysfunction by increasing local angiotensin II. Hypertension 47, 718–726.
Zhuo JL, Imig JD, Hammond TG, Orengo S, Benes E & Navar LG (2002). Ang II accumulation in rat renal endosomes during Ang II-induced hypertension: role of AT1 receptor. Hypertension 39, 116–121.
|
Acknowledgements |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. K. Raizada and J. F. R. Paton Recent advances in the renin-angiotensin system: angiotensin-converting enzyme 2 and (pro)renin receptor Exp Physiol, May 1, 2008; 93(5): 517 - 518. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
