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Am J Physiol Regul Integr Comp Physiol 292: R373-R381, 2007. First published August 31, 2006; doi:10.1152/ajpregu.00292.2006
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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

Differential expression of neuronal ACE2 in transgenic mice with overexpression of the brain renin-angiotensin system

Marc F. Doobay,1,* Lauren S. Talman,1,* Teresa D. Obr,1,* Xin Tian,1 Robin L. Davisson,1,2 and Eric Lazartigues1,3,4

1Departments of Anatomy and Cell Biology and 2The Cardiovascular Center, Carver College of Medicine, The University of Iowa, Iowa City, Iowa; and 3Pharmacology and Experimental Therapeutics and 4Cardiovascular Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana

Submitted 28 April 2006 ; accepted in final form 26 August 2006


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Angiotensin-converting enzyme 2 (ACE2) is a newly discovered carboxy-peptidase responsible for the formation of vasodilatory peptides such as angiotensin-(1–7). We hypothesized that ACE2 is part of the brain renin-angiotensin system, and its expression is regulated by the other elements of this system. ACE2 immunostaining was performed in transgenic mouse brain sections from neuron-specific enolase-AT1A (overexpressing AT1A receptors), R+A+ (overexpressing angiotensinogen and renin), and control (nontransgenic littermates) mice. Results show that ACE2 staining is widely distributed throughout the brain. Using cell-type-specific antibodies, we observed that ACE2 staining is present in the cytoplasm of neuronal cell bodies but not in glial cells. In the subfornical organ, an area lacking the blood-brain barrier and sensitive to blood-borne angiotensin II, ACE2 was significantly increased in transgenic mice. Interestingly, ACE2 mRNA and protein expression were inversely correlated in the nucleus of tractus solitarius/dorsal motor nucleus of the vagus and the ventrolateral medulla, when comparing transgenic to nontransgenic mice. These results suggest that ACE2 is localized to the cytoplasm of neuronal cells in the brain and that ACE2 levels appear highly regulated by other components of the renin-angiotensin system, confirming its involvement in this system. Moreover, ACE2 expression in brain structures involved in the control of cardiovascular function suggests that the carboxypeptidase may have a role in the central regulation of blood pressure and diseases involving the autonomic nervous system, such as hypertension.

central nervous system; circumventricular organs; volume homeostasis; blood pressure; carboxypeptidase

THE CLASSICAL VIEW OF THE brain renin-angiotensin system (RAS) is an enzymatic cascade by which angiotensinogen (AGT) is successively cleaved by renin and angiotensin-converting enzyme (ACE) and then degraded by angiotensinases to form active and inactive metabolites (3, 35, 40). The effects of central ANG II, i.e., vasoconstriction, vasopressin release, induction of transcription factors, salt appetite, and drinking response, are thought to be mediated primarily by AT1 receptors (32, 41). In rodents, AT1 receptors are subdivided in AT1A and AT1B receptors, and both are expressed in the brain (1). High density of the AT1A receptor is distributed in circumventricular organs, which lack a blood-brain barrier and include the subfornical organ (SFO), organum vasculosum of the lamina terminalis, and area postrema (AP) (28). Other cardiovascular (CV) regulatory areas containing AT1A receptors include the median preoptic area, paraventricular nucleus (PVN), nucleus of the tractus solitarius (NTS), and ventrolateral medulla (VLM) (32).

Recently, a new member of the ACE family was identified and named ACE2 (11, 38). This carboxypeptidase was first sequenced and cloned from human heart failure ventricle (11) and human lymphoma (38) cDNA libraries. These studies reported major expression of ACE2 mRNA in heart and kidneys, but failed to detect it in the brain. Interestingly, these studies also failed to detect ACE in the brain, despite overwhelming reports showing its presence (28). In addition, very recent studies reported ACE2 mRNA in rat medulla oblongata (34) and ACE2 activity in mouse brain (12). ACE2 is believed to degrade ANG II to the vasodilatory peptide angiotensin-(1–7) [ANG-(1–7)] with an affinity 400-fold greater than for ANG I (13, 42). ANG-(1–7) is also present in the brain, where it exerts synergistic or opposite effects to ANG II, but its receptor is still uncertain. One of the possible candidates is the G protein-coupled receptor Mas (36).

Although the presence and therefore the role of ACE2 in the brain is unknown, there is considerable evidence for a role of ANG-(1–7). In addition to its hypotensive effects in hypertensive (5) but not in normotensive (6) rats, studies have shown that ANG-(1–7) may act as an important neuromodulator of cardiac baroreflex mechanisms, following central (6) or peripheral (5) administration, leading to an increased sensitivity of this system (2, 35). On the other hand, ANG-(1–7) antagonists impair baroreflex sensitivity, opposing the effects of losartan on this system (31). As a new component of the RAS, controlling the production of the vasodilatory and antihypertrophic peptide ANG-(1–7), ACE2 provides new possibilities to counterregulate the effects of ANG II and to improve the treatment of hypertension and other CV diseases. However, the relative contribution of ACE2 in the brain of normotensive and hypertensive mouse strains in regulating blood pressure (BP) and CV function is unknown.

Here, we hypothesized that ACE2 is a component of the brain RAS, and its expression is regulated by the other elements of this system. To assess this hypothesis, we used immunohistochemistry and real-time PCR to determine the presence of the ACE2 protein and mRNA in the mouse brain as well as its cellular and regional distribution. In addition, taking advantage of neuron-specific enolase (NSE)-AT1A [with brain-selective overexpression of the rat ANG II type 1A (AT1A) receptor] and R+A+ (with widespread expression of both human renin and human AGT genes) transgenic mouse models exhibiting alterations of the brain RAS components, we investigated the effects of such elements on ACE2 expression in specific brain regions.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transgenic mice and animal husbandry. All transgenic mice used in this study exhibit a C57BL/6J genetic background; therefore, C57BL/6J nontransgenic littermates were used as controls. Transgenic mice (R+A+) harboring both human renin (R+) and human AGT (A+) transgenes were generated by breeding heterozygous R+ transgenic mice with heterozygous A+ transgenic mice, as described previously (29, 37). In this strain, the major phenotype is characterized by a chronic hypertension, concomitant with high ANG II plasma levels. Heterozygous NSE-AT1A transgenic mice were generated as described previously (24). In this strain, the rat AT1A cDNA is driven by a NSE promoter, making its expression specific for neurons. Although not hypertensive, these mice exhibit enhanced pressor and bradycardic responses to ANG II as well as increased baseline water intake and salt appetite. ACE2–/y hemizygotes, nullified for the ACE2 gene and generated through backcrossing for two to four generations onto a C57BL/6J genetic background, were used for our experiments. These transgenic mice were kindly provided by Dr. Curt D. Sigmund from The University of Iowa (R+A+), Dr. Robin L. Davisson from The University of Iowa (NSE-AT1A), and Drs. Susan B. Gurley and Thomas M. Coffman from Duke University (ACE2–/y). All mice were fed standard mouse chow (LM-485; Teklad Premier Laboratory Diets) and water ad libitum. All procedures were approved by the University Animal Care and Use Committee at the University of Iowa.

Immunohistochemistry. Mice (n = 5 per group) were anesthetized and perfused transcardially with 4% paraformaldehyde in phosphate buffer, as described previously (24). Brains were sectioned on a cryostat, and sections were collected in phosphate buffer. Free-floating sections (30 µm, coronal) from transgenic and nontransgenic lines were incubated in 10% normal goat serum (Sigma, St. Louis, MO) for 1 h and then in 0.5% blocking reagent (NEN Life Science Products) for 30 min at room temperature. Sections were then incubated at 4°C with the primary antibody, a rabbit anti-mouse ACE2 antibody (kind gift of Dr. C. M. Ferrario, Wake Forest University, 1:5,000 dilution, 48 h; see Ref. 14 for previous characterization of the antibody). This was followed by tyramide signal amplification using the ABC Vectastain Systems kit (Vector Laboratories, Burlingame, CA), as described by the manufacturer. Briefly, sections were incubated with biotinylated goat anti-rabbit IgG (1:200) and streptavidin-horseradish peroxidse (1:100) for 1 h each. This was followed by a 10-min amplification of biotinyl tyramide (1:100) and 1-h incubation in rhodamine-avidin D (1:100). To assess the specificity of the ACE2 staining, control sections were incubated without primary or secondary antibody. In addition, sections from ACE2–/y mice were similarly processed.

Finally, sections were incubated overnight with a mouse anti-microtubule-associated protein-2 (MAP-2) monoclonal antibody (1:500, Sigma) or a mouse antiglial fibrillary acidic protein (GFAP) monoclonal antibody (1:500, Sigma), followed by 2 h in fluorescein-conjugated goat anti-mouse antibody (1:200, Sigma). At the end of the protocol, sections were incubated for 10 min in DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) staining for double-stranded DNA visualization. Sections were then mounted with buffered glycerol (Molecular Probes, Eugene, OR). Immunostaining was analyzed using fluorescence and UV microscopy (Zeiss LSM 510). Sections were scanned using an argon laser emitting light at 488 nm for visualization of fluorescein and 570 nm for rhodamine.

Quantification was performed by two investigators using NIH Image J software, version 1.33 (http://rsb.info.nih.gov/ij/). The RGB confocal images were loaded into the program and converted to 8-bit gray scale before subtracting background fluorescence equivalently for all images (setting the threshold to 50% maximum intensity). First, the image size was set by entering the appropriate dimensions using a graticule. This was followed by calibration of the optical density (OD) using a Kodak photographic step tablet. The region of interest was then outlined on the gray-scale pictures, and the OD measured in this area. Fluorescence intensity is expressed as OD per square micrometer.

Real-time RT-PCR. Brain tissue punches were collected from transgenic and nontransgenic mice (n = 4 per group). RNA was extracted from the NTS/dorsal motor nucleus of the vagus (NTS/DMNX) and the VLM using an RNeasy mini kit (Qiagen). All samples (0.5 µg) were visualized on a 1% agarose gel to verify their quality. This was followed by first-strand cDNA synthesis, from 1 µg of RNA, using SuperScript II RT, according to the manufacturer's instructions (Invitrogen). The real-time RT-PCR reaction was then performed in quadruplicate using 50 ng of cDNA, mouse ACE2 primers (10 µM) (forward: 5'-ACC CTT CTT ACA TCA GCC CTA CTG-3'; reverse: 5'-TGT CCA AAA CCT ACC CCA CAT AT-3'), mouse beta-actin as internal control (10 µM) (forward: 5'-CCA CCA GTT CGC CAT GGA TGA-3'; reverse: 5'-ACC ATC ACA CCC TGG TGC CTA-3') (Integrated DNA Technologies), and 12.5 µl of SYBRgreen PCR master mix (Applied Biosystems). The reaction mixture was placed into one well of a 96-well plate (Applied Biosystems), and the total reaction volume was brought to 25 µl with diethyl pyrocarbonate-treated water. PCR was performed at 50°C for 2 min and 95°C for 10 min and was run for 40 cycles at 95°C for 15 s and 61°C for 1 min in an ABI Prism 7700 Detection System (Applied Biosystems). The cycle threshold for PCR amplification needed to detect fluorescence was then determined for each unknown cDNA sample. ACE2 mRNA levels in tissues were quantified by comparison to a standard curve previously constructed for each primer set, and message levels were normalized to beta-actin levels in each experiment. The real-time RT-PCR reaction was performed at the University of Iowa DNA Core Facility.

Statistics. Data are expressed as means ± SE. Data were analyzed by one-way ANOVA (following Bartlett's test of homogeneity of variance) followed by Newman-Keuls correction for multiple comparisons between means or Dunnett's multiple-comparison test when appropriate. Statistical comparisons were performed using Prism (version 3.0) software package (GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cellular and regional distribution of ACE2 expression in mouse brain. The first goal of this study was to determine whether ACE2 was expressed in the brain of mice with normal expression of RAS components. Consequently, ACE2 expression was first investigated in coronal brain sections from C57BL\6J mice (nontransgenic littermates). Figure 1 shows typical examples of basal ACE2 expression in the piriform cortex (Fig. 1A), caudate putamen (Fig. 1B), hypoglossal nucleus (Fig. 1C), and primary motor cortex (Fig. 1D) of C57BL\6J mice. Using similar pictures taken throughout the whole brain, assessment of ACE2 expression level was performed using a grading scale, ranging from not detectable to abundant immunostaining (Table 1, Fig. 2). This approach revealed that ACE2 immunostaining is in fact widespread throughout the mouse brain, from the telencephalon to the medulla. To determine the type of brain cells expressing ACE2, double immunohistochemistry was performed with the ACE2 antibody, in combination with cell-specific antibodies such as GFAP (glial cell marker) and MAP-2 (neuronal marker). As illustrated in Fig. 1A, GFAP staining (green) in the piriform cortex does not overlap with ACE2 (red), suggesting that glial cells do not express the carboxypeptidase. Indeed, observation at a higher magnification (Fig. 1B) reveals that the size and morphology of the ACE2 expressing cells are inconsistent with glial cells and are more likely to be neuronal cells. This was confirmed by the clear overlap of MAP-2 (green) and ACE2 (red) immunostainings in the hypoglossal nucleus (Fig. 1C) and primary motor cortex (Fig. 1D). Finally, ACE2 expression appears to be located in the neuron cytoplasm (Fig. 1B).


Figure 1
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  		Fig. 1. Angiotensin-converting enzyme 2 (ACE2) is expressed in the cytoplasm of neuronal cells. A: immunostaining for ACE2 (red), glial fibrillary acidic protein (GFAP; glial cell marker, green), and 4',6-diamidino-2-phenylindole (DAPI; nucleic acid marker, blue) in the piriform cortex shows a lack of colocalization between ACE2 and the astroglia (magnification x40). B: increased magnification (x63) in the caudate putamen shows the absence of GFAP staining in ACE2-positive cells. Immunoreactivity is localized in the cytoplasm and not in the nucleus. Immunostaining for ACE2, microtubule-associated protein-2 (MAP-2; neuronal marker, green), and DAPI in the hypoglossal nucleus (C) and the primary motor cortex (D) shows colocalization (yellow) between ACE2 and MAP-2, suggesting that ACE2 is expressed mostly in neurons. All panels are from C57BL/6J mice.

 

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  		Table 1. Distribution of ACE2 immunofluorescence in nontransgenic and transgenic mouse brains

 

Figure 2
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  		Fig. 2. ACE2 quantification in mouse brain. Levels of ACE2 fluorescence were graded throughout the whole brain using the following: no detectable immunostaining (–), low-level immunostaining (+), abundant immunostaining (++), and highly abundant immunostaining (+++). All panels are from C57BL/6J mice.

 
Specificity of the ACE2 antibody. The ACE2 antibody recognizes the amino acids 198–216 of the mouse ACE2 protein (sequence: DYGDYWRGDYEAEGADGYN). To verify that the ACE2 antibody was indeed targeting the ACE2 amino acid sequence, immunostaining was performed in parallel in C57BL\6J and ACE2 null (ACE2–/y) mice brain sections. Figure 3 shows typical examples of ACE2 immunoreactivity in the SFO and the rostral ventrolateral medulla (RVLM), respectively, of C57BL\6J and ACE2–/y mice. Although the C57BL\6J mouse brain regions exhibit a fair amount of ACE2 immunostaining (Fig. 3, A and B), ACE2–/y mice show no significant staining (Fig. 3, C and D), confirming the specificity of the ACE2 antibody. To confirm this result, several control experiments were also performed. Brain sections incubated with only the primary (ACE2; data not shown) or the secondary (rhodamine-avidin D; Fig. 3, E and F) antibody failed to show any significant level of fluorescence, eliminating the possibility of autofluorescence from the brain tissue as well as other forms of nonspecific immunoreactivity.


Figure 3
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  		Fig. 3. Specificity of the ACE2 antibody. Brain sections of the subfornical organ (SFO; A, C, and E) and rostral ventrolateral medulla (RVLM; B, D, and F) from C57BL/6J (A and B) and ACE2–/y (C–F) mice were incubated with (A–D) and without (E and F) anti-ACE2 antibody. Note that ACE2 immunostaining is restricted to the SFO (A) and absent in the surrounding tissue. The lack of ACE2 immunostaining in ACE2–/y mice (C–F) confirms the specificity of the anti-ACE2 antibody (scale bar: 100 µm).

 
ACE2 protein expression in brain nuclei related to CV function. Observation of the ACE2 immunoreactivity throughout the brain of C57BL\6J mice revealed a widespread distribution, apparently independent of the involvement of any particular nuclei in CV regulation. However, according to Table 1, those CV regulatory areas [e.g., NTS, nucleus ambiguus (NA), AP] are more likely to express a higher level of ACE2 staining than non-CV-related regions (e.g., hippocampus). Figure 4 \. shows that ACE2 immunostaining was positively identified on neurons in the organum vasculosum of the lamina terminalis (Fig. 4A), SFO (Fig. 4B), PVN (Fig. 4C), AP and DMNX (Fig. 4D), NTS (Fig. 4E), and NA and RVLM (Fig. 4F), suggesting that ACE2 could potentially participate in the regulation of CV function and volume homeostasis in those nuclei.


Figure 4
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  		Fig. 4. ACE2 expression in key brain regions involved in the regulation of blood pressure and body fluid homeostasis. A: examples of ACE2 (red) expression in the organum vasculosum of the lamina terminalis (OVLT), an area involved in thirst and salt appetite. Brain nuclei involved in the regulation of cardiovascular function, such as the SFO (B), the magnocellular neurons of the paraventricular nucleus (PVN; C), the area postrema (AP) and the dorsal motor nucleus of the vagus (DMNX; D), the nucleus of tractus solitarii (NTS; E), and the RVLM and the nucleus ambiguus (NA; F), also showed positive staining for ACE2. The neuronal marker MAP-2 is shown in green, and cell nuclei are stained in blue. All panels are from C57BL/6J mice (scale bar: 100 µm).

 
Differential ACE2 expression in transgenic mice. To determine whether ACE2 is an active part of the brain RAS, its expression was studied in brain tissue from mice genetically modified to overexpress components of the RAS in the central nervous system. Table 1 shows the distribution and intensity of ACE2 immunoreactivity in NSE-AT1A and R+A+ mice relative to control C57BL\6J mice. Several areas, at different levels of the brain, such as the bed nucleus of the stria terminalis, the suprachiasmatic nucleus, the lateroanterior hypothalamic nucleus, and the SFO revealed an increase in ACE2 immunostaining in transgenic mice compared with nontransgenic controls. Figure 5, A–C, shows a typical example of the increase in ACE2 immunoreactivity in the SFO. Quantification of ACE2 fluorescence in this area (Fig. 5D) reveals a significant increase in NSE-AT1A (0.95 ± 0.08 OD/µm2, P < 0.01) and even greater in R+A+ (1.27 ± 0.05 OD/µm2, P < 0.001) compared with C57BL\6J (0.54 ± 0.10 OD/µm2) mice. On the other hand, some areas, like the PVN (Table 1) were not significantly affected by the overexpression of brain RAS elements (C57BL\6J: 1.26 ± 0.09, NSE-AT1A: 1.27 ± 0.07, and R+A+: 1.33 ± 0.03 OD/µm2), while others such as the NA (Table 1) and the AP (C57BL\6J: 1.34 ± 0.12, NSE-AT1A: 1.39 ± 0.06, and R+A+: 1.11 ± 0.04 OD/µm2; P < 0.01) exhibited a decrease in ACE2 fluorescence in transgenic mice.


Figure 5
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  		Fig. 5. ACE2 protein expression in the forebrain is genotype dependent. Typical examples in the SFO of the differential expression of ACE2 in C57BL6/J (A), neuron-specific enolase (NSE)-AT1A (B), and R+A+ mice (C). Quantification of ACE2 immunoreactivity (D) shows that expression of the enzyme is modulated by overexpression of central AT1A receptors in NSE-AT1A mice and/or human renin and human AGT in R+A+ mice in the SFO. OD, optical density. **P < 0.01 and ***P < 0.001 vs. nontransgenic mice (scale bar: 100 µm).

 
Because of the importance of brain stem nuclei in the regulation of CV function, ACE2 mRNA was quantified in parallel with the protein expression in the NTS/DMNX and the VLM. Interestingly, while the ACE2-to-beta-actin mRNA ratio was significantly increased in the NTS/DMNX of R+A+ mice (1.8 ± 0.1) compared with controls (1.0 ± 0.1, P < 0.05, Fig. 6A), the protein expression was lowered (1.29 ± 0.09 vs. 1.68 ± 0.05 OD/µm2, respectively, P < 0.05, Fig. 6C). Similarly, in the VLM, the ACE2-to-beta-actin mRNA ratio was significantly decreased in transgenic mice (C57BL\6J: 1.0 ± 0.1; NSE-AT1A: 0.7 ± 0.1; R+A+: 0.5 ± 0.2, P < 0.05, Fig. 6B), while protein expression increased (C57BL\6J: 1.07 ± 0.03, NSE-AT1A: 1.32 ± 0.10, and R+A+: 1.58 ± 0.05 OD/µm2, P < 0.05, Fig. 6D). Taken together, these observations suggest that ACE2 is highly regulated by the other components of the brain RAS.


Figure 6
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  		Fig. 6. ACE2 is differentially expressed in transgenic mouse brain stem. Quantification of ACE2 mRNA expression in the NTS/DMNX (A) and the ventrolateral medulla (VLM; B) and protein immunostaining in the NTS (C) and the RVLM (D). Immunostaining and mRNA expression are inversely correlated in these areas and regulated by the other components of the RAS. *P < 0.05 vs. C57BL6/J mice.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
ACE2, a single amino-acid carboxypeptidase, has recently been identified as a new member of the RAS (11, 38) and is believed to hydrolyze several peptides, including ANG II, but not bradykinin. Early reports suggest that ACE2 may play a crucial role in CV diseases such as hypertension, myocardial infarction, and heart failure by regulating the levels of ANG II (9, 13). Despite the importance of the brain RAS in the pathogenesis of such diseases (10, 46), the presence of ACE2 in the central nervous system is poorly documented. The aim of this study, therefore, was to investigate ACE2 distribution and cellular expression in the brain. The major novel findings of this study are that the ACE2 protein is expressed in the brain, predominantly by neurons in areas involved in the central regulation of CV function as well as in non-CV regions. Additionally, using transgenic mice harboring alterations of the brain RAS components, we determined that ACE2 expression is regulated by the other elements of this system.

Expression of ACE2 was originally identified in the heart, kidney, and testis (11, 38). Later, the carboxypeptidase was also discovered to be a receptor for a coronavirus responsible for the severe acute respiratory syndrome (27), leading to increased interest in the enzyme distribution. Using immunohistochemistry, we extended the list of tissue harboring ACE2 to lungs, nasopharynx, skin, lymph nodes, thymus, bone marrow, spleen, liver, and gastrointestinal tract (17). Despite recent identification of ACE2 activity (12) and mRNA (34) in rodent brain, the presence of the ACE2 protein in the brain remained uncertain.

Our study shows, for the first time, that ACE2 protein and mRNA are expressed in selective nuclei of the mouse brain. Using specific antibodies for neuronal and glial cell markers, in addition to morphological observations, we found that the cells harboring ACE2 are predominantly neurons. This finding contrasts with previous studies relative to ACE2 distribution. Indeed, original reports failed to identify ACE2 in the brain (11, 38). Later, quantitative real-time RT-PCR data reported low levels of ACE2 mRNA in the central nervous system (18), while immunostainings showed that its presence was restricted to endothelial and vascular smooth muscle cells (17). Finally, studies performed in brain primary cell cultures reported that ACE2 was expressed predominantly in glial cells (15), but this observation could be dependent on the culture conditions and the difficulty in maintaining neurons in such cultures. In support of our data, real-time RT-PCR and in situ hybridization studies showed that severe acute respiratory syndrome coronavirus mRNA and virus protein immunoreactivity have been identified in cerebrospinal fluid (20), as well as brain neurons from infected patients (19, 45) and mice (16), suggesting the localization of its receptor, ACE2, in such neurons. In addition, our data show a clear expression of ACE2 in the cytoplasm of neuronal cells. This cytoplasmic expression is in accordance with a previous report using human lung tissue and epithelia samples (17). Moreover, like ACE, ACE2 has been shown to exist as a membrane-bound and a secreted protein (14, 33, 39), consistent with the presence of the enzyme in the cytoplasm.

According to our data, distribution of ACE2 is widespread in the mouse brain, in areas involved or not in the regulation of CV function. More importantly, regional expression of ACE2 in the brain is consistent with the presence of other components of the RAS in the same areas, including AT1 receptors (8, 24), ACE (23), angiotensinases (4), but also ANG-(1–7) (7), confirming ACE2 as a member of the brain RAS. ANG-(1–7), the product of ANG II hydrolysis by ACE2, has been shown, in the brain, to modulate the cardiac baroreflex mechanisms (5, 6) in a similar way than following losartan administration, resulting in increased sensitivity of this system (2, 35). Interestingly, when administered directly in baroreflex areas like the NTS and RVLM, ANG-(1–7) induced depressor and pressor responses, respectively (35). These reverse effects are compelling when paralleled to our data, showing opposite changes in ACE2 levels in those areas, in the chronically hypertensive R+A+ mouse strain, providing additional evidence of the relationship between ACE2 and the regulation of CV function. In addition, previous studies have reported changes in baroreflex sensitivity in R+A+ mice (26, 29), and it might be interesting to determine whether those changes in ACE2 expression levels are the cause or the consequence of the development of hypertension in those animals. Indeed, the elevated BP in this model may have contributed to some of the changes observed in the NTS and the RVLM. For example, the high BP in R+A+ mice could have led to the reduction of ACE2 protein levels in the NTS of this strain. Clearly, more work is needed to identify how ACE2 is regulated, and/or is regulating BP, in these areas.

Another interesting area exhibiting enhanced ACE2 expression in hypertensive mice is the SFO, a circumventricular organ lacking blood-brain barrier and sensitive to peripheral levels of ANG II. This area has previously been shown to play a significant role in the central regulation of BP and volume homeostasis (22, 28). Moreover, the SFO is also connected indirectly to the RVLM via the PVN and is in a position to modulate sympathetic outflow. Our observation of increased ACE2 levels in this region in R+A+ mice could support the idea of a compensatory mechanism promoting the hydrolysis of ANG II and increasing forebrain levels of ANG-(1–7). However, this hypothesis is hard to reconcile with previous studies performed in infarcted rat hearts (21) and in vitro (14), and more work is needed to study our models in similar conditions. Also, noteworthy is the increase in ACE2 levels in the SFO of NSE-AT1A. Indeed, despite a lack of hypertension, the increase in neuronal AT1A receptors in this model has been shown to lead to enhanced water and salt intakes (25). These observations suggest that the exogenous AT1A receptor is able to activate downstream pathways responsible for those changes. Consequently, increased ACE2 protein levels in the SFO, and the lamina terminalis, in general, may suggest the involvement of this carboxypeptidase in the central regulation of body fluid homeostasis.

An interesting finding in our study is that ACE2 mRNA levels are not correlated with the protein levels. Indeed, areas such as the dorsal (NTS/DMNX) and VLM show an inverse correlation between the message and the protein expression. A possible explanation for these discrepancies could be the lack of specificity of the tissue collection for mRNA extraction; the punches collected usually overlap more than one area. In the brain, this mismatch often results from localization of the mRNA in the areas containing the cell bodies and the protein on terminal fibers. Although, in our case, the majority of the staining is associated with the cell bodies. A more exciting hypothesis is that components of the RAS, e.g., the AT1A receptor, might be able to directly or indirectly regulate ACE2 mRNA posttranscriptionally by processes, such as increased stabilization, translation, or degradation of the message. Similar mechanisms have previously been reported for ACE2 and other components of the RAS (30, 43, 44).

It is now well documented that ANG II and ANG-(1–7) produced and, acting locally in the brain, serve a crucial role in CV function. Moreover, there is considerable evidence supporting the sensitivity of the brain RAS to circulating ANG II levels and the role of this system in the pathogenesis of both experimental and genetic hypertension (10). So far, the beneficial effects of the RAS blockade have been attributed to the inhibition of the vasoconstrictor and hypertrophic properties of ANG II. Similarly, to its suspected beneficial effects in the periphery (13), ACE2 in the brain appears to be in a position to buffer the excess ANG II levels in nuclei involved in CV and autonomic regulation.

In summary, this study is the first to show the presence of the ACE2 protein and mRNA in the mouse brain, predominantly in neurons, in regions involved or not in the central regulation of CV function. Our data suggest that ACE2 is part of the brain RAS and highly regulated by the other components of this system. As such, ACE2 could play a major role in the central regulation of autonomic nervous system in buffering the enhanced ANG II levels in diseases such as hypertension and myocardial infarction, where this peptide has been shown to activate pathways leading to an increased sympathetic tone (10, 46). Finally, the evidence of ACE2 as part of the brain RAS introduces a new level of regulation in this system and provides a new tool to fight diseases associated with an imbalance of the autonomic nervous system.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The work described herein was partly funded by The American Physiological Society Postdoctoral Fellowship in Physiological Genomics, a Beginning-Grant-In-Aid (0560007Z) from the American Heart Association Heartland Affiliate, and a R21 grant from the National Institute of Neurological Disorders and Stroke (NS052479) to E. Lazartigues.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Curt D. Sigmund for providing the R+A+ mice, Drs. Thomas M. Coffman and Susan B. Gurley for providing the ACE2–/y mice, and Drs. Martin M. Cassell, Martine Dunnwald, and Xinping Yue for their expertise.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Lazartigues, Louisiana State Univ. Health Sciences Center, Dept. of Pharmacology and Experimental Therapeutics, 1901 Perdido St. P7–1, New Orleans, LA 70112 (e-mail: elazar{at}lsuhsc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* M. F. Doobay, L. S. Talman, and T. D. Obr contributed equally to this work. Back


   REFERENCES
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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