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Review Articles |
1 Max-Delbrück-Center for Molecular Medicine (MDC), D-13092 Berlin-Buch, Germany 2 Institute for Experimental Medicine, Russian Academy of Medical Sciences, St Petersburg, Russia
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(Received 15 November 2007;
accepted after revision 17 December 2007; first published online 21 December 2007)
Corresponding author M. Bader: Max-Delbrück-Center for Molecular Medicine (MDC), Robert-Rössle-Strasse 10, D-13092 Berlin-Buch, Germany. Email: mbader{at}mdc-berlin.de
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Introduction |
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Discovery of the Mas gene
The MAS gene codes for a G protein-coupled cell surface receptor (GPCR). It was discovered in 1986 and was originally described as a protooncogene owing to its ability to transform NIH 3T3 cells in a tumorigenicity assay in nude mice with DNA from a human epidermoid carcinoma cell line (Young et al. 1986). By in situ hybridization, Rabin et al. (1987) mapped the human MAS gene to the distal half of chromosome 6q (6q24–q27), within a region frequently rearranged in malignant cells. Subsequently, it was shown that activation of MAS additionally appears after transfection of human acute myelocytic leukaemia DNA (Janssen et al. 1988) and human ovarian carcinoma DNA (van 't Veer et al. 1988). Most probably, the rearrangement of the 5' non-coding sequences that appeared during transfection was responsible for the activation of MAS, which in turn led to the tumorigenicity in nude mice. Nevertheless, in all three primary tumours the MAS gene was not rearranged and had a weak focus-inducing activity in transfected NIH 3T3 cells. Further studies showed that Mas could promote the growth of rodent fibroblasts in serum-free medium (Andrawis et al. 1992). In contrast however, overexpression of Mas in cones of retina induces cell death without tumour formation (Xu et al. 2000). Furthermore, experiments with transgenic mice overexpressing Mas ubiquitously and specifically in the brain did not reveal any tumour formation (N.A. and M.B., unpublished data), confirming that the Mas gene per se has no oncogenic activity. Therefore, most of the follow-up studies were focused on the elucidation of the physiological role of Mas and of the signalling pathways employed by this receptor.
Structure of the Mas gene
The analysis of the human MAS cDNA sequence revealed an open reading frame that codes for a 325-amino-acid protein (Young et al. 1986). The sequence of the Mas protein indicates that it belongs to the class of GPCRs, which share a conserved structure, consisting of seven membrane-spanning 
-helices and hydrophilic N- and C-terminal ends. More specifically, Mas belongs to the Class A Orphan GPCRs (http://www.gpcr.org/7tm/multali/multali.html). In fact, Mas was the first member of a GPCR subfamily, which includes proteins sharing around 35% homology with Mas at the protein level. Besides the Mas-related gene (mrg; Monnot et al. 1991) and the rat thoracic aorta gene (RTA; Ross et al. 1990), the subfamily includes a group of approximately 50 GPCRs related to Mas, called Mrg (Mas-related genes), a subset of which is expressed in specific subpopulations of sensory neurones that detect painful stimuli (Dong et al. 2001; Burstein et al. 2006).
In addition to the human gene, the Mas gene was isolated and characterized in rats and in mice (Young et al. 1988; Metzger et al. 1995). The mouse and rat homologues of the protein are 324 amino acids long and show 97% identity between each other, compared with 91% between the mouse and human forms, indicating that Mas is highly conserved, except in its hydrophilic amino-terminal domain. The recent advance in the sequencing of genomes of model organisms has revealed that the Mas gene is also conserved in other species, including rhesus monkeys, opossums, dogs and chickens, whereas in frogs, zebrafish and Fugu there is no Mas homologue (http://ecrbrowser.dcode.org).
Mas gene structure, as well as the regulation of Mas expression at the transcriptional level, is relatively poorly studied. As in 90% of mammalian GPCRs, the open reading frame of Mas is not interrupted by introns (Jackson et al. 1988; Gentles & Karlin, 1999), whereas at least in the mouse several 5' untranslated exons exist (Schweifer et al. 1997; and N.A. and M.B., unpublished data).
Expression of the Mas gene
Brain. Brain was the first organ in which Mas was found to be highly expressed (Young et al. 1988). Detailed examination of Mas expression in rat brain by these investigators using RNase protection assay (RPA) revealed that high levels of Mas transcripts are present in the hippocampus and cerebral cortex. The cellular localization and the distribution of Mas mRNA in the rat brain have been studied using in situ hybridization (Bunnemann et al. 1990). Strong, specific signals were demonstrated in the dentate gyrus, the CA3 and CA4 areas of the hippocampus, the olfactory tubercle, the piriform cortex and the olfactory bulb, while a weak to moderate labelling was present all over the neocortex, especially in the frontal lobe. In the mouse, the distribution of Mas mRNA in the brain is comparable to that in the rat, being highest in the hippocampus and piriform cortex, as also detected by in situ hybridization (Metzger et al. 1995).
Martin et al. (1992) showed by in situ hybridization and RPA that Mas mRNA is expressed in a subpopulation of neurones in both the adult and developing rat central nervous system (CNS). In the adult CNS, Mas mRNA was most abundant in hippocampal pyramidal neurones and dentate granule cells but was also present at low levels in the cortex and thalamus. Recently, Mas expression was also discovered in cardiovascular regions of the brain by Western blot and immunofluorescence (Becker et al. 2007).
The study of Mas expression during rat ontogenesis showed that Mas is first expressed in the developing rat CNS at postnatal day 1. Even at this early stage in CNS development, the pattern of Mas expression was similar to that seen in the adult (Martin et al. 1992). Furthermore, brief seizure episodes led to a significant and transient increase in Mas mRNA in the rat hippocampus, which may contribute to anatomical and physiological plasticity associated with intense activation of hippocampal pathways (Martin & Hockfield, 1993).
Kitaoka et al. (1994) investigated the distribution of MAS expression in the rhesus macaque retina by in situ hybridization. They demonstrated a weak positive signal above the neurones of the retina, suggesting that MAS can be used as a possible marker for the retinal pigment epithelium.
Testis. We examined the ontogenetic profile of Mas expression and could demonstrate high levels of Mas mRNA in mouse and rat testis (Metzger et al. 1995; Alenina et al. 2002a). Testicular Mas mRNA from rats was shown to be markedly increased during development, beginning 5 weeks after birth and reaching the highest concentrations at 25 weeks of age (Metzger et al. 1995). A similar profile of Mas expression in testis was also observed in mice: Mas mRNA is not detectable in testis of newborn animals, but its expression is dramatically upregulated, starting 2 weeks after birth and continuously increasing during puberty until 6 months of age when it becomes maximal (Alenina et al. 2002a). Additional experiments using in situ hybridization in 3-month-old mouse testis demonstrated a distinct expression of the Mas gene in Leydig and Sertoli cells, being much more pronounced in Leydig cells (Alenina et al. 2002a). Interestingly, the well-controlled regulation of Mas expression in testis coincides temporally with the transformation of immature adult Leydig cells to mature cells, indicating that Mas is a marker for adult Leydig cells and may be involved in the function of this cell type.
Cardiovascular and other tissues. Reverse transcriptase-polymerase chain reaction (RT-PCR) and RPA have demostrated low levels of Mas expression also in other tissues of mice, such as heart, kidney, lung, liver, spleen, tongue and skeletal muscle (Villar & Pedersen, 1994; Metzger et al. 1995). Using fluorescent in situ hybridization, we could confirm the presence of Mas mRNA in kidney, mostly in the renal cortex (Fig. 1A). In the spleen, Mas was detected in the pulp near the blood vessels (Fig. 1B), in the trabeculae and in the connective tissue of the capsule (data not shown). In the heart, low levels of Mas transcripts were detected in cardiomyocytes (Fig. 1C) and much higher concentrations in the endothelium of coronary arteries (Fig. 1D). Ferrario et al. (2005) also confirmed cardiac Mas mRNA expression in rats and showed its downregulation by AT1 receptor blockade.
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We also found Mas expression in the smooth muscle of uterus and in bone marrow, whereas lung, liver and ovary were free of Mas transcripts (N.A. and E.L.P., unpublished data).
Signalling of Mas
Considerable efforts have been made to find out which signal transduction pathways are employed by Mas. Zohn et al. (1998) suggested that Mas mediates the activation of GTP-binding protein Rac-dependent signalling pathway. Rac belongs to the Ras superfamily of GTPases. Mas and Rac 1 activate c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase. Moreover, expression of Mas in cells leads to activation of phospholipase C, indicating that Mas couples to the Gq/11 family of heterotrimeric G proteins (Canals et al. 2006). One of the major pathways of Mas signalling in the cardiovascular system is the phosphorylation of Akt. Giani et al. (2007) showed that this pathway is activated by Ang-(1–7) in the heart and can be blocked by the specific Ang-(1–7) antagonist, A-779, indicating that the effect is mediated by Mas. Another study using cardiomyocytes showed an inhibition of MAP kinase activation by Ang-(1–7) which could be blocked by antisense oligonucleotides against Mas (Tallant et al. 2005).
Interaction of Mas with the renin–angiotensin system (RAS)
The first attempts to clarify the function of Mas protein in Xenopus oocytes characterized it as an Ang II receptor (Jackson et al. 1988). However, the activation of inward currents by Ang II in Mas-injected Xenopus oocytes was not inhibited by Ang II antagonists. In addition, the major Ang II receptors, AT1 and AT2, were discovered afterwards and exhibited only 8 and 19% amino-acid homology, respectively, to the Mas protein (Sasaki et al. 1991; Mukoyama et al. 1993). In contrast, Ambroz et al. (1991) showed that the intracellular Ca2+ increase in Mas-transfected cells after Ang II treatment was only observed in cells endogenously expressing the AT1 receptor. Thus, Mas seems to be a modulator of AT1 signalling. Wolf & Neilson (1992) demonstrated that permanent transfection of a murine renal tubular cell line with Mas changed the hypertrophic actions of Ang II into a proliferative response. Recently, it was shown that Mas can hetero-oligomerize with the AT1 receptor and inhibit the actions of Ang II, thus being a physiological antagonist of the AT1 receptor (Kostenis et al. 2005). Possibly by this direct interaction, Mas is able to rescue binding and functionality of an AT1 receptor double mutant in CHO or COS7 cells (Santos et al. 2007). Furthermore, Sampaio et al. (2007) recently showed in endothelial cells that Ang-(1–7), via Mas, counteracts Ang II- and AT1-dependent c-Src activation and thereby ERK1/2 phosphorylation and the generation of reactive oxygen species by NAD(P)H oxidase. Comparable results have been obtained in proximal tubular cells of the kidney (Su et al. 2006). Interestingly, none of these studies found a direct inhibitory activity of Ang-(1–7) on ERK1/2 phosphorylation, in contrast to the studies in cardiomyocytes mentioned above (Tallant et al. 2005).
Although Mas negatively influences the signalling of the AT1 receptor, it induces an upregulation of receptor binding (Kostenis et al. 2005). This could be explained by a constitutive capacity of Mas activating the Gq/11 and stimulating protein kinase C-dependent phosphorylation of the AT1 receptor at its C-terminus (Canals et al. 2006). Mas-knock-out mice were used to clarify the interaction between Mas and AT1. The distribution of cells expressing AT1 receptors in different limbic and thalamic brain structures in Mas-knock-out and in wild-type mice was the same when assessed using immunohistochemistry (Von Bohlen und Halbach et al. 2000). Within the amygdala, however, Ang II induced an increase in the field potentials in wild-type mice but reduced them in Mas-knock-out animals. Moreover, mice lacking the Mas gene show enhanced Ang II-mediated vasoconstriction in mesenteric microvessels (Peiro et al. 2007).
There is additional evidence that Mas might interact functionally not only with AT1 but also with AT2 receptors (Castro et al. 2005).
Mas knock-out mice
Mas is an Ang-(1–7) receptor. After its discovery in 1986, the direct function of Mas was unclear for more than a decade. Only after the generation of Mas knock-out mice in 1998 (Walther et al. 1998) was it possible to uncover the ligand binding to Mas. In these mice, the binding of Ang-(1–7) to kidney sections was abolished (Santos et al. 2003). Accordingly, Mas-deficient mice completely lack the antidiuretic action of Ang-(1–7) after an acute water load. Furthermore, Mas-deficient aortas lose their Ang-(1–7)-induced relaxation response. Coincident with this finding, AVE0991, a non-peptide mimetic of Ang-(1–7), was found to induce an equipotent vasodilator effect to Ang-(1–7) in aortic rings in wild-type mice. This effect could be blocked by two specific Ang-(1–7) receptor antagonists, A-779 and D-Pro7-Ang-(1–7). As expected, AVE0991 failed to induce vasodilatation in Mas-deficient aortic rings (Lemos et al. 2005). As observed previously for Ang-(1–7), the antidiuretic effect of AVE0991 after water load was also blunted in Mas-knock-out mice.
In-vitro studies confirmed that Ang-(1–7) is a Mas ligand, since it bound to Mas-transfected cells and elicited arachidonic acid release (Santos et al. 2003).
Collectively, these findings identify Mas as a functional receptor for Ang-(1–7) and provide a clear molecular basis for the physiological actions of this peptide. Nevertheless, it cannot be excluded that Mas may have additional ligands and that Ang-(1–7) may have additional receptors.
Cardiovascular phenotype. Targeted deletion of the genomic region coding for the amino-terminal 253 amino acids of Mas, including six transmembrane domains, led to a loss of Mas expression (Walther et al. 1998). The homozygous Mas-deficient mice on the mixed 129xC57BL/6 genetic background were healthy, grew normally, had normal Ang II plasma levels and showed no obvious developmental abnormalities.
Central autonomic control. Blood pressure variability (BPV) and heart rate variability (HRV) are relevant predictors of arterial hypertension and cardiovascular diseases in humans. Reduced HRV is correlated with an increased mortality in heart failure patients. Both male and female anaesthetized Mas-knock-out mice (on the mixed genetic background) were normotensive and showed no alteration in baseline heart rate (Walther et al. 1998). However, Mas-deficient females in particular showed a strong reduction of HRV (Walther et al. 1999). Despite the finding that the influence of Mas deficiency on BPV was much lower than on HRV in females, a significant increase in BPV was observed in Mas-knock-out males. Moreover, there were sex-specific differences in the sensitivity of the baroreflex between wild-type and Mas-knock-out animals, which, however, did not reach statistical significance (Walther et al. 2000b). Taken together, the autonomic balance was shifted in favour of the sympathetic tone in both sexes. These alterations are probably related to the described baroreflex-modulating actions of Ang-(1–7) in the rostral ventral lateral medulla (RVLM) of the brainstem (Wang et al. 2003; Silva-Barcellos et al. 2004; Alzamora et al. 2006) and suggest a protective function of the Ang-(1–7)–Mas axis in the central regulation of cardiovascular parameters.
Cardiac function. Isolated hearts of Mas knock-out and wild-type mice treated with the Ang-(1–7) antagonist A-779 presented a decreased contractility and heart rate (Castro et al. 2006). Upon global ischaemia, hearts from wild-type mice showed a significant decrease in systolic tension and an increase in diastolic tension. During reperfusion, both parameters were increased. Depletion or blockade of Mas markedly attenuated these changes in isolated hearts. These results indicate that Mas plays an important protective role in cardiac function during ischaemia–reperfusion, which is in keeping with the beneficial cardiac and coronary effects previously described for Ang-(1–7).
Endothelial function. Endothelial dysfunction is an initial step in the pathogenesis of cardiovascular diseases. Angiotensin-(1–7)-mediated relaxation of isolated mesenteric arteries was equally impaired in both wild-type mice pretreated with A-779 and Mas-deficient mice (Peiro et al. 2007). Importantly, the response to the endothelium-dependent vasorelaxants, bradykinin (BK) and acetylcholine (ACh), was comparably inhibited, while endothelium-independent vessel relaxation by sodium nitroprusside (SNP) was unaltered in these vessels. The A-779-induced impairment of endothelial function was confirmed in vitro, since BK-mediated nitric oxide (NO) release was increased by Ang-(1–7) and blunted by A-779 pretreatment in primary human endothelial cell cultures.
We have recently studied Mas-deficient mice on different genetic backgrounds because it is known that different mouse strains show completely different responses to cardiovascular interventions such as renal ablation and deoxy corticosterone acetate (DOCA) salt treatment (Pillebout et al. 2001; Hartner et al. 2003). Indeed, Mas-deficient mice on the FVB/N genetic background exhibited higher blood pressures compared to control animals when measured by telemetry (Xu et al. 2008). Confirming the data from isolated vessels (Peiro et al. 2007), these Mas-knock-out mice also exhibited impaired endothelial function, decreased NO production and lower endothelial NO synthase expression. The NAD(P)H oxidase catalytic subunit gp91phox protein content was higher in Mas-knock-out mice than in control animals, while superoxide dismutase and catalase activities were reduced. The superoxide dismutase mimetic, tempol, decreased blood pressure in Mas-knock-out mice, but had a minimal effect in control mice.
Brain phenotype.
Morphology. Despite the high expression of Mas in the brain, no obvious alterations could be detected in the morphology of the hippocampus and its subregions, indicating that the cytoarchitectural distribution patterns and the fine wiring of neuronal subtypes are not affected by Mas ablation.
Long-term potentiation and memory. In addition to Ang II and Ang IV (Wright & Harding, 2004), Ang-(1–7) may also be involved in learning and memory, since Ang-(1–7) enhances long-term potentiation (LTP) in the hippocampus (Hellner et al. 2005). The Ang-(1–7) antagonist A-779 blocked this LTP-enhancing effect of Ang-(1–7) in wild-type mice, and low concentrations of Ang-(1–7) did not change the magnitude of CA1 LTP in Mas-deficient mice. The dramatic improvement of LTP in the CA1 region of the hippocampus after theta-burst stimulation observed in Mas-knock-out mice is in agreement with these data (Walther et al. 1998). Furthermore, Mas ablation leads to an improved maintenance of LTP in the dentate gyrus. Thus, Mas mediates the LTP-promoting effect of Ang-(1–7) in the brain.
In contrast to its significant effects on LTP, which would classify Mas as a memory suppressor gene (Abel et al. 1998), its ablation and overexpression did not result in clear changes of spatial learning in both Morris Water Maze and Shuttle box experiments (Walther et al. 1998; and N.A., unpublished data).
Anxiety. To check on changes in anxiety, Mas-deficient animals (Walther et al. 1998) were examined in the Elevated-Plus Maze, which is based upon the natural aversion of rodents for open spaces. Thus, mice exposed to the maze usually prefer the closed arms over the open arms. The Mas-deficient male mice entered the open arms of the maze significantly less often and spent less time on this section than did the control animals (Walther et al. 1998), whereas Mas-deficient females showed no differences (Walther et al. 2000a). Thus, the behavioural data showed a significantly higher level of anxiety in Mas-deficient male mice, implying that the lack of Mas protein influences anxiety in a sex-specific manner.
Testis phenotype. Although Mas is highly expressed in testis, Mas-deficient mice were fertile and had an equal number of male and female offspring (Walther et al. 1998). In spite of increased relative weights of testes in Mas-knock-out males, the number and motility of spermatocytes, as well as testis morphology, were unchanged.
Depletion of Mas affects the expression of important determinants of steroidogenesis in the testis, such as decreased steroidogenic acute regulatory protein and 3β-hydroxysteroid-dehydrogenases (3β-HSD), which are key enzymes for the biosynthesis of testosterone in Leydig cells (Xu et al. 2007). Thus, Mas may be relevant for the regulation of androgen metabolism in the testis.
Imprinting of the Mas gene. Mas-deficient mice were also instrumental in clarifying a scientific dispute about Mas as an imprinted gene. In 1992, two independent groups mapped the mouse Mas gene to the proximal portion of mouse chromosome 17 (Cebra-Thomas et al. 1992; al Ubaidi et al. 1992). The human gene and the rat gene were also localized to chromosomes 6q25.3–q26 and 1, respectively (Riesewijk et al. 1996; and M.B., unpublished data). In all three species, Mas is located in a cluster of genes, which was suggested to be imprinted in close proximity to the paternally imprinted Igf2r gene (Barlow et al. 1991). Villar & Pedersen (1994) investigated the allele-specific expression pattern of the Mas gene by RT-PCR. They demonstrated a maternal imprinting of Mas, at least during embryonic development between days 11 and 12.5 post coitum, and for some organs, such as tongue and heart, also at later time points. However, Schweifer et al. (1997) showed that Mas is biallelically expressed in organs of adult mice, whereas imprinting of the human gene was discussed controversially (Riesewijk et al. 1996; Miller et al. 1997). Riesewijk et al. (1996) demonstrated that the MAS gene, like the IGF2R gene, is not imprinted in humans, whereas Miller et al. (1997) showed monoallelic MAS expression in human breast tissue by RT-PCR. The situation with Mas imprinting was finally clarified after discovery of a maternally imprinted Antisense RNA (AS RNA), which starts from a promoter in intron 2 of the Igf2r gene and extends into the Mas gene up to a few hundred base pairs upstream of the Mas translation start (Lyle et al. 2000). Using RNase protection assay and mice with one genetically deleted Mas allele, we and others showed that in mice not Mas but the AS RNA is maternally imprinted in both embryos and adult organs (Wutz et al. 1997; Hu et al. 1999; Lyle et al. 2000; Alenina et al. 2002b). Thus, owing to the lack of strand selectivity in the RT-PCR used by Villar & Pedersen (1994), the maternally imprinted RNA detected by them was most probably not the coding mRNA but non-coding AS RNA whose transcript partly overlaps the Mas gene in an antisense orientation.
Transgenic rat models with increased Ang-(1–7)
TGR(A1–7)3292.
In our attempts to functionally characterize the Ang-(1–7)–Mas axis, we also generated the transgenic rat model TGR(A1–7)3292 (Santos et al. 2004). These animals express an engineered fusion protein which drives a chronic elevation of Ang-(1–7). The peptide is directly released during secretion from this protein by proteolytic action of the enzyme furin (Methot et al. 1997). The Cytomegalovirus promoter/enhancer was used, which directed transgene expression exclusively to the testes. Measurements of Ang-(1–7) peptide level via radioimmunoassay revealed a 4.5-fold higher concentration in the testis, as well as 
2.5-fold increase in venous and arterial blood in the transgenic animals compared with control rats. This indicates that in this model the testes are functioning as biological Ang-(1–7) infusion pumps.
Radiotelemetry measurements revealed no differences in arterial blood pressure between transgenic rats and control animals but they uncovered an increased heart rate and cardiac contractility. This may be a compensatory mechanism or a direct cardiac or central effect of chronic Ang-(1–7) augmentation. Furthermore, an attenuation of the development of heart hypertrophy by isoprenaline and an improvement of postischaemic systolic function were observed in TGR(A1–7)3292 (Santos et al. 2004). The latter was measured in isolated hearts perfused according to the Langendorff technique, showing reduced duration of reperfusion arrhythmias in the transgenic rat line.
Furthermore, we used the same model to investigate the effects of chronic Ang-(1–7) elevation on renal function (Ferreira et al. 2006). The TGR(A1–7)3292 rats show a reduction in basal urinary flow, leading to increased urinary osmolality and osmolal clearance. This antidiuretic effect was related neither to changes in the glomerular filtration rate nor to the excretion of sodium and potassium. Furthermore, we could not find changes in vasopressin release or V2 receptor expression. These data reinforce the theory about the antidiuretic effect of Ang-(1–7) and confirm the results of previous studies in which acute and chronic administration of the selective Mas antagonists, A-779 or D-Pro7-Ang-(1–7), were used to produce diuresis (Pinheiro et al. 2004).
A more recent study with TGR(A1–7)3292 rats investigated systemic and regional haemodynamics using fluorescent microspheres (Botelho-Santos et al. 2007). The chronic overproduction of Ang-(1–7) led to changes in the regional blood flow, resulting in an increase of vascular conductance in the kidneys, lungs, adrenals, spleen, brain, testis and brown fat tissue. Furthermore, the transgenic rats showed a significant increase in stroke volume and cardiac index, as well as a decreased total peripheral resistance.
In conclusion, TGR(A1–7)3292 rats provided further evidence for a crucial role of Ang-(1–7) in cardiac and renal function, as well as in the tonic control of blood distribution.
TGR(SM22ACE2). Another factor of rising importance in the RAS is the carboxypeptidase angiotensin-converting enzyme 2 (ACE2), which serves as a negative regulator. It has emerged as a crucial enzyme to counterbalance the actions of ACE in determining the levels of Ang II in tissues, as well as to produce Ang-(1–7). Crackower et al. (2002) have shown that loss of ACE2 leads to age-dependent progressive ventricular dilatation and reduced systolic performance in the heart. Furthermore, age-dependent cardiomyopathy in ACE2-null mice has been related to increased Ang II. In contrast, Donoghue et al. (2003) reported sudden death due to cardiac arrhythmias in transgenic mice overexpressing ACE2 in the heart. Consequently, there are still missing, as well as conflicting, data about the importance of this enzyme.
Therefore, we generated a transgenic rat model expressing the human ACE2 gene in vascular smooth muscle cells on the genetic background of spontaneously hypertensive stroke-prone rats (SHR-SP) which are genetically deficient in this enzyme (Crackower et al. 2002). Initial results show increased Ang-(1–7) and slightly decreased Ang II levels in the circulation and a reduction in mean arterial blood pressure in these transgenic rats, TGR(SM22ACE2) (Rentzsch et al. 2007). Furthermore, intra-arterial administration of Ang II led to a reduced vasoconstriction response in the transgenic rats compared with SHR-SP control animals. This effect was abolished by prior administration of an ACE2 inhibitor. Endothelial function was tested in vitro as well as in vivo. For this purpose, endothelium-dependent and -independent agents such as ACh and SNP, respectively, were applied to the descending thoracic aorta, and blood pressure was monitored. Endothelial function turned out to be significantly improved in TGR(SM22ACE2) rats compared with SHR-SP control animals. These data demonstrate that vascular ACE2 overexpression in SHR-SP rats reduces hypertension, probably by locally degrading Ang II and improving endothelial function. Thus, ACE2 seems to play a crucial role particularly in pathological situations. This is in accordance with recently published data from Yamazato et al. (2007) and Feng et al. (2007). Yamazato et al. (2007) showed that lentivirally expressed ACE2 in the RVLM in the brainstem was associated with a decrease in mean arterial pressure exclusively in the spontaneously hypertensive rat. Feng et al. (2007) generated a transgenic mouse model overexpressing human ACE2 exclusively in neurones. These mice were normal in baseline conditions but showed a blunted pressor response to Ang II. Taken together, these data indicate that ACE2 is not essential for baseline blood pressure regulation but becomes important in pathophysiological situations, when it protects tissue from the actions of Ang II by locally degrading this peptide and generating Ang-(1–7), which has opposing actions.
Conclusions
In conclusion, Mas is expressed in the vascular wall, and animals lacking the Mas gene develop endothelial dysfunction and become hypertensive. Accordingly, animals overproducing the ligand for Mas, Ang-(1–7), exhibit improved endothelial function and protection from pathophysiological challenges to the cardiovascular system. These data characterize the Ang-(1–7)–Mas axis as an important system in vascular control, which is to some extent involved in basic cardiovascular regulation but has even greater implications in hypertension and other diseases affecting vascular homeostasis.
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