A Role for Angiotensin-Converting Enzyme in the Characterization, Enrichment, an - 《stemcells》干细胞学杂志干细胞之家



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作者：Diana A. Leporea, Vanta J. Jokubaitisb, Paul J. Simmonsb, Kelly N. Roeszlera, Ralph Rossic, Karl Bauerd, Paul Q. Thomase作者单位：aPituitary Research Unit, Murdoch Childrens Research Institute, Royal Childrens Hospital, Parkville, Victoria, Australia;bStem Cell Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia;cFlow Cytometry Unit, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia;dMa




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      【摘要】( W* F/ d! Y5 @) r% P
   Recently, we described a rare cell type within the adult murine pituitary gland with progenitor cell hallmarks (PCFCs). PCFCs are contained exclusively within a subpopulation of cells that import fluorescent &szlig;-Ala-Lys-N-AMCA (7-amino-4-methylcoumarin-3-acetic acid). Herein, we investigate the utility of cell surface molecules angiotensin-converting enzyme (ACE) and stem cell antigen-1 (Sca-1) to further enrich for PCFCs. ACE and Sca-1 were expressed on 61% and 55% of AMCA CD45¨CCD31¨C cells, respectively, and coexpressed on 38%. ACE Sca-1 AMCA  cells enriched for PCFCs by 195-fold over unselected cells. ACE AMCA  cells enriched for PCFCs by 170-fold, and colonies were twofold larger than for AMCA  selection alone. Conversely, ACE¨C-selected cells reduced both colony-forming activity and size. Notably, colonies generated from AMCA  cells obtained from ACEnull mice were 2.7-fold smaller than for wild-type mice. These data identify ACE as a previously unrecognized marker of PCFCs and suggest that ACE is functionally important for PCFC proliferation. Anatomically, the cells that imported AMCA and expressed ACE were situated in the marginal epithelial cell layer of the pituitary cleft and in the adjacent subluminal zone, thus supporting previous proposals that the luminal zone is a source of precursor cells in the adult pituitary. " N- N% ?* f8 @: t9 |2 e
      【关键词】 Angiotensin-converting enzyme Pituitary Tissue stem cells -Amino--methylcoumarin--acetic acid% D) J3 S7 {8 P* s$ K+ H1 e
   INTRODUCTION

Growth hormone (GH) deficiency is a significant clinical problem . However, many of the key features of PCFCs are unclear, including their precise location within the pituitary gland and their repertoire of expression of cell surface molecules. We reasoned that identifying cell surface molecules whose expression was restricted to PCFC would provide an improved means to prospectively isolate these progenitors and thereby facilitate the study of their properties in vitro and their anatomical location in vivo in the adult pituitary gland.
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To this end, we examined the expression of two cell surface glycoproteins, stem cell-associated antigen-1 (Sca-1) and angiotensin-converting enzyme (ACE), on PCFCs. Initially defined as a marker of murine hemopoietic stem cells .
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In recent years, it has become clear that ACE is also a regulator of hemopoietic stem cell proliferation. This was first indicated by the observation that occasional hypertensive patients treated with specific inhibitors of ACE activity developed granulocytopenia, aplastic anemia, and pancytopenia , ACE may also control proliferation of other stem/progenitor cells. To date, this possibility has not been explored./ @  C- M- C8 E2 ?; o

In the current study, we investigated the potential of ACE and of Sca-1 to enrich for PCFCs in the pituitary gland. Here, we show that 60% of AMCA  cells express ACE and that this marker, in combination with positive selection for AMCA uptake, allows PCFCs to be enriched 170-fold. Further, we show that PCFC proliferation is increased in ACE-selected cultures and markedly reduced in cells isolated from ACEnull mice. Dual fluorescence analysis of adult pituitaries revealed that ACE AMCA  cells are located in the marginal epithelial cell (MEC) layer and in the subluminal region. Together, the data indicate that ACE may be an important mediator of PCFC proliferation and provide novel insight into the topographical location of PCFCs within the pituitary gland.; {9 q0 B" [! u7 @) u2 N
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MATERIALS AND METHODS9 h( q+ G' S' @! g" P5 @
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Isolation of Pituitary Single-Cell Suspensions and Uptake of ß-Ala-Lys-N-AMCA" ^7 x0 e5 e! S9 g5 ]

The pituitary gland was removed from HS DOLA female mice aged 5¨C6 weeks, digested into a single-cell suspension, and incubated in the presence of the dipeptide-fluorophore ß-Ala-Lys-N-AMCA .

Antibody Labeling and Fluorescence-Activated Cell Sorting

Single-cell pituitary suspensions were prepared and loaded with AMCA as described above. Cells were then incubated with a panel of rat anti-mouse antibodies to cell membrane-bound Sca-1, ACE, CD45 (leukocytes), and CD31 (endothelium). Briefly, cells were incubated with rat anti-mouse ACE (10 µg/ml, clone 230214; R&D Systems Inc., Minneapolis, http://www.rndsystems.com) or immunoglobulin (Ig)G2a nonbinding isotype control antibody (10 µg/ml; BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen) for 30 minutes on ice, followed by two washes in wash buffer (phosphate-buffered saline ) was added to cells for 5 minutes. To this were added rat-anti-mouse CD45-PE-cyanine (Cy7) (0.4 µg/ml), Sca-1-fluorescein isothiocyanate (FITC) (2.5 µg/ml), and CD31-allophycocyanin (APC) (0.5 µg/ml) or relevant nonbinding isotype control antibodies (all from BD Pharmingen) for a further 20 minutes on ice. Flow cytometric analysis and sorting were performed on a FACS Vantage SE with DIVA option (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) using Fluoro Gold (1:10,000 methanosulfonate; Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) to exclude nonviable cells.' S9 B! I- c, J! S) \

Live, single-nucleated pituitary cells were sorted by size and their exclusion of Fluoro Gold. Cell doublets were excluded by a combination of high forward scatter height and area characteristics . AMCA  cells were excited using an I90 laser (Coherent Scientific Pty. Ltd., Inc., Hilton, South Australia, Australia, http://www.coherent.com.au) tuned to 350 nm and run at 50 mW; the emission was then collected with a 450/30-nM filter. Relevant nonbinding isotype control-labeled cells were used to set gates for Sca-1-FITC  and ACE-PE  cells. Individual, viable AMCA  CD45¨C CD31¨C cells were sorted on the basis of Sca-1 and ACE positivity into 96-well culture plates (NUNC A/S, Roskilde, Denmark, http://www.nuncbrand.com). Cells were cultured in medium containing Ham's F-12 medium/Dulbecco's modified Eagle's medium (50%:50% vol/vol) supplemented with 10% fetal calf serum (FCS) at 36¡ãC.
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Single-Cell Cloning, Clonogenic Titration

Single cells selected by fluorescence-activated cell sorting (FACS) and seeded into 96-well plates were scored for CFA after 10¨C15 days in culture. The criterion used for defining a colony was one cluster of closely packed cells per culture of greater than or equal to approximately 25 cells in number. Single-cell colonies were stained with nuclear stain 4',6-diamidino-2-phenylindole, dilactate (DAPI) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) at a final concentration of 2.5 µg/ml. Colonies were photographed using a Leica DM IRB inverted microscope and Leica DFC 480 camera (Leica, Heerbrugg, Switzerland, http://www.leica.com). The size of the colonies was determined by counting the number of DAPI  nuclei using the semiautomated cell-counting program Image J 1.36B (NIH, Bethesda, MD, http://www.rsb.info.nih.gov/ij).

Clonogenic titer curves were performed by seeding cells (1, 3, 5, 10, or 20 cells per well, n = 24 cultures per cell concentration) into 96-well plates. The clonogenicity was determined as previously described  or wild-type mice (n = 5 each), a generous gift from Dr. Michael McKinley (Howard Florey Institute, Melbourne, Australia), were used to assess the clonogenic activity of cells isolated from pituitaries in an ACEnull background.- ?# a2 J& D% Y& n7 _9 K
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Statistical Analysis( j3 w7 |% I$ ?: Z, T

Multiple group comparisons of the single-cell cloning data or clonogenic activity were performed using the one-way analysis of variance (ANOVA) and Tukey's post hoc test. Comparison of two groups such as the colony size was carried out using the Student's t-test for unequal variances.

Immunofluorescence Staining of Pituitary Sections

For in situ analysis of the ß-Ala-Lys-N-AMCA  cell population, the whole pituitary was placed in HEPES-buffered saline (HBS) containing 20 µM AMCA at 36¡ãC for 3 hours. The AMCA-labeled pituitary was rinsed in HBS and post-fixed in 4% wt/vol paraformaldehyde in PBS for 15 minutes in the dark, embedded in Optimal Cutting Temperature medium (Tissue Tek, Torrance, CA, http://www.sakura-americas.com), and then cryosectioned into 5-µM slices. Sections were analyzed using ultraviolet fluorescence microscopy.
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To detect ACE  cells in pituitary sections, we used an ACE rabbit polyclonal antibody raised against the cell membrane-bound amino terminus of ACE (amino acids 1¨C170; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com) at a final concentration of 4 µg/ml. Briefly, pituitary sections were treated with 0.1% vol/vol Triton X in PBS for 10 minutes. Sections were then blocked for 15 minutes using 1% wt/vol bovine serum albumin and 10% vol/vol FCS in PBS and incubated in the presence of the primary antibody diluted in the blocking agent overnight at 4¡ãC. Excess primary antibody was rinsed away with PBS, and sections were blocked again as above followed by incubation with secondary antibody (goat anti-rabbit IgGs conjugated to Alexa Fluor 488 at a final concentration of 1.3 µg/ml) for 2 hours at room temperature. Images were captured using an Olympus FV1000 laser scanning confocal microscope or on an inverted IX81 microscope (Olympus, Tokyo, http://www.olympus-global.com). AMCA was excited with a 405-nm laser and Alexa-488 with a 488-nm argon ion laser.6 b7 ~: x/ _; V. Q; E! g4 z: x
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RESULTS

ACE and Sca-1 Are Expressed by a Subpopulation of AMCA  Cells

We have previously shown that approximately 3.7% of viable adult pituitary cells specifically import the fluorescent dipeptide ß-Ala-Lys-N-AMCA and that PCFCs are contained exclusively within this population . ACE and Sca-1 staining was detected on 61% and 55% of AMCA CD45¨CCD31¨C cells, respectively (Table 1; Fig. 1). Colabeling with ACE and Sca-1 showed that the AMCA  population contains ACE Sca-1  (38.2% ¡À 6.5%), ACE Sca-1¨C (22.9% ¡À 3.9%), ACE¨CSca-1  (16.6% ¡À 4.9%), and ACE¨CSca-1¨C (16.4% ¡À 4.2%) cells (Fig. 1).

Figure 1. Flow cytometric analysis of Sca-1 and ACE expression in AMCA-loaded pituitary cell preparations. Pituitary single-cell suspensions incubated in the presence of AMCA were labeled with antibodies to Sca-1 and ACE or their respective nonbinding isotype control antibodies. (A): Single cells were selected by excluding doublets and debris (gate 1). (B): Live cells were selected by their exclusion of the viability dye (gate 2). (C): Leukocytes and endothelial cells were excluded by selecting cells that were CD45¨C and CD31¨C (gate 3). (D): AMCA  cells were selected (gate 4). (E): Isotype control antibodies IgG2a-FITC and IgG2a-PE. (F): Flow cytometric analysis showing Sca-1 and ACE expression on AMCA-loaded CD45¨CCD31¨C pituitary cell preparations. AMCA CD45¨CCD31¨C cells that were ACE Sca-1¨C, ACE Sca-1 , Sca-1¨CACE¨C, or Sca-1 ACE¨C were selected for single-cell cloning and for limiting dilution assays. Abbreviations: ACE, angiotensin-converting enzyme; AMCA, 7-amino-4-methylcoumarin-3-acetic acid; FITC, fluorescein isothiocyanate; Sca-1, stem cell antigen-1.: H  b, I, u( g( B
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Table 1. Summary of CFA, recovery, and relative percentages of subpopulations of AMCA  cells

Significant Enrichment of PCFCs by ACE but Not Sca-1 Selection
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To determine whether PCFCs express ACE and/or Sca-1, we performed clonogenicity assays on AMCA  subpopulations coselected for these antigens. Labeled cells were sorted using the selection parameters shown in Figure 1F and seeded individually into 96-well plates. Approximately 18% (one in six cells) of AMCA  cells (n = 671) exhibited CFA (Fig. 2), but this increased significantly when sorted for the ACE  population (n = 480). One-third of AMCA ACE  cells exhibited CFA, a significant 1.8-fold increase compared with selection based on AMCA  alone (p

Figure 2. CFA of AMCA  pituitary cells. Single cells were cultured following selection as shown in Figure 1F. The number of colonies derived from single-cell clones is expressed as a percentage of the total number of cells seeded for each cell phenotype. Total numbers of cells plated: AMCA  (n = 671), ACE  (n = 480), AMCA ACE¨C (n = 480), AMCA ACE Sca-1  (n = 672), AMCA ACE¨CSca-1¨C (n = 575), AMCA Sca-1  (n = 192), and AMCA Sca-1¨C (n = 192). Data are derived from three independent experiments. CFA for AMCA ACE  cells is significantly greater than for AMCA  cells (p ! Q+ H1 s9 S; l0 l1 d
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To confirm these results, limiting dilution growth analysis  was used to compare the CFA of ACE , ACE¨C, ACE Sca-1 , ACE¨CSca-1 , and AMCA  populations. Consistent with single-cell assays, ACE selection of AMCA  cells increased the frequency of colony-forming cells from approximately one in six to one in three cells, whereas ACE¨C selection exhibited a marked reduction in CFA (Fig. 3A). Sca-1 coexpression further enriched the CFA of AMCA ACE  cells to approximately one in two cells (Fig. 3B). Together, these data indicate that the major determinant of clonogenicity is ACE expression and that maximum enrichment (195-fold) of CFA with high recovery (79%) can be obtained based on selection of ACE Sca  cells (Table 1).

Figure 3. Limiting dilution analysis. The clonogenic activity of AMCA  cells that expressed ACE and/or Sca-1 was compared with control AMCA  cultures using limiting dilution analysis. Cells were sorted into 96-well plates (n = 24 per cell concentration), and the clonogenicity was determined using the Poisson distribution analysis method . The data are derived from three independent experiments. (A): One in six AMCA  gives rise to colonies, versus one in three AMCA ACE . There was no detectable clonogenic activity in the ACE¨C fraction of AMCA  cells. (B): ACE  cultures subfractionated according to Sca-1 expression compared with ACE¨C. Clonogenic activity for ACE SCA -selected cells was one in two cells. (C): Phase-contrast microscopy view of a single-cell colony derived from AMCA ACE -selected pituitary colony-forming cells, composed mainly of cells that were stellate-like with long cytoplasmic processes and scattered with round refractile cells. Magnification: x200. (D): Single-cell colonies derived from AMCA ACE  or ACE¨C cultures were fixed on day 10 of culture. Nuclei were detected by staining with 4',6-diamidino-2-phenylindole, and the nuclei were counted to determine the total cell number per colony. The scatter plots show the individual sizes of each colony. The total numbers of colonies counted were n = 124 and n = 59, respectively. The mean colony size ¡À SEM is shown beside each scatter plot. ACE¨C cultures were significantly smaller that ACE  cultures (p " R' L) ^/ G' C' F+ u6 g2 W9 K

A comparison of the morphology of colonies derived from single-cell cultures of AMCA ACE  and AMCA ACE¨C cells revealed marked differences. Colonies grown from individually sorted AMCA ACE  cells resembled our previously described AMCA -derived colonies . These colonies were comprised of cells that were mainly stellate in shape with long cytoplasmic processes (Fig. 3C). Also scattered throughout the colonies were small, round refractile cells. Rare GH /prolactin (PRL)  stellate or round cells were detected in some colonies, demonstrating that single AMCA ACE  cells are capable of differentiating into multiple cell types. In comparison, ACE¨C-derived colonies were poorly arranged and were less compact. These colonies lacked the small round cells and rare GH /PRL  cells that were present in the ACE -derived colonies.+ w0 D# z; H6 [& h5 r2 b

ACE Expression in PCFCs Correlates with Greater Colony Expansion1 R* w- X) K; m5 @- E# _& p
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To investigate the effect of ACE expression on the proliferative potential of PCFCs, the size of single-cell colonies derived from AMCA ACE  cells versus AMCA ACE¨C cells was compared. The average size of ACE  or ACE¨C colonies at day 10 was 102 ¡À 10 cells (n = 124) and 59 ¡À 9 cells (n = 30), respectively (p ; d: U/ j2 |2 x: s  ^0 B; V

Reduction in PCFC Colony Size in the Absence of ACE

To further investigate the importance of ACE activity in PCFC colony expansion, we compared the CFA and the sizes of AMCA  single cell-derived colonies generated from ACEnull and wild-type littermates. ACEnull mice carry an insertional mutation that inactivates the protein .4 ?( w4 z3 H% L# G7 X* P1 w
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No difference was detected in the percentage frequency of CFA between ACEnull and wild-type mice. However, the size of single cell-derived colonies arising from AMCA  cells isolated from ACEnull mice was considerably smaller than those from wild-type mice: mean colony size on day 15 of culture 301 ¡À 70 (n = 25) compared with 832 ¡À 244 (n = 26) in wild-type mice (p

Figure 4. Size of single-cell colonies derived from ACEnull mice. Pituitary cell suspensions derived from ACEnull mice (n = 5) and wild-type controls (n = 5) were loaded with 7-amino-4-methylcoumarin-3-acetic acid. The size of colonies arising from single-cell cultures was determined by counting 4',6-diamidino-2-phenylindole nuclei on day 15 of culture. Twenty-four and 25 single cell-derived colonies, respectively, were counted, and the individual sizes are shown as a scatter plot. The average size is shown as the mean ¡À SEM. The mean size of ACEnull colonies was significantly less than that of wild-type mice (p + _; ]7 V2 N1 N: ]' u2 O' N
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Like that of their AMCA ACE -derived counterparts, the morphology of colonies arising from the pituitaries of wild-type mice was heterogeneous in nature, comprising mainly stellate cells scattered with round cells and rare GH /PRL  cells. The smaller, diffuse colonies from ACEnull mice did not contain the small, round nor the GH /PRL  cells.8 ]4 n# I8 d" L

AMCA ACE  Cells in the Adult Pituitary Gland Are Located in the MEC Zone0 V  Y. r1 a$ u" e6 h+ C7 D9 Y! g
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To determine the location of ACE AMCA  cells in the pituitary, AMCA-loaded pituitary glands were immunostained with antibody to cell surface ACE. As reported in our previous study , the MECs that line the pituitary cleft import AMCA (Fig. 5A). AMCA  cells that coexpressed ACE were detected on the luminal surfaces that lined both the anterior and intermediate lobes (Fig. 5B, arrow a). Dual-positive cells were also found in the lateral MEC region of the lumen (Fig. 5C) and in the subluminal zone (Fig. 5C, arrow b). In addition, ACE  cells could be found scattered throughout the anterior lobe in a coronal arrangement around the lumen; some were stellate in shape. However, we were unable to detect any overlap of AMCA and ACE expression in this area (data not shown).

Figure 5. Immunofluorescence of AMCA-loaded pituitary showing the location of ACE AMCA  cells. (A): AMCA-loaded pituitary showing AMCA  cells in blue. These can be found both in the marginal epithelial lining of the gland (arrow a), including the lateral region (arrow L), and in the anterior lobe (arrow b). (B): MECs of the pituitary luminal lining that import AMCA and express ACE: (bi) AMCA  cells (blue), (bii) ACE  (green), and (biii) merged image showing dual positive cells detected both in the epithelial layer (arrow a) and subluminal zone (arrow b). (biv) Confocal image of MEC showing cell surface ACE (green) and intracellular AMCA (blue). (C): Cells located in the lateral regions of the pituitary gland that are (ci) AMCA  and (cii) ACE . (ciii) Merged image showing dual positive cells. Arrow b indicates dual positive cells that are located in the subluminal zone. Magnifications: x100 (A), x400 (Bi¨Ciii), x600 (Biv), x200 (C). Abbreviations: ACE, angiotensin-converting enzyme; AMCA, 7-amino-4-methylcoumarin-3-acetic acid; MEC, marginal epithelial cell.4 e  x# E, k5 q2 Q$ {+ R

DISCUSSION+ y* d  }3 V5 h% ~) o3 D" _
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We have previously described a rare pituitary cell type, termed PCFCs, that has progenitor cell hallmarks, including CFA and apparent potential to differentiate into cells that are immunopositive for pituitary hormones. In the present study, we investigated whether PCFCs express cell surface antigens that could be used to enrich for PCFCs and to study their anatomical location within the pituitary gland. We found that PCFCs express the stem cell markers ACE and Sca-1 and can be enriched 195-fold through selection of cells that coexpress these markers and possess AMCA-dipeptide importation activity. Further, we demonstrated that in the absence of ACE activity PCFC colony size is significantly reduced, suggesting that ACE is functionally important for PCFC proliferation activity. Finally, we identified a subpopulation of AMCA  cells in the anterior pituitary that express ACEs that are predominantly located in the epithelial lining of the pituitary cleft and in subluminal areas.3 G3 @: I2 P, |9 ~

The expression of stem cell-associated antigens ACE and/or Sca-1 on subpopulations of AMCA  cells further supports our previous study that the AMCA  population is heterogenous, containing not only typical folliculo-stellate cells but also a cell subtype with progenitor cell properties (PCFCs) . The expression of ACE on AMCA  cells correlated with increased clonogenicity in both single-cell cloning and titration curve assays, indicating the importance of ACE as a marker for the PCFC subpopulation. Sca-1, although found not to be critical for the colony-forming potential of PCFCs, was often coexpressed with ACE, consistent with the progenitor-like nature of PCFCs. Because the majority of PCFCs were recovered in the ACE  fraction, the expression of cell surface ACE can be used to distinguish the clonogenic subpopulation from the remainder of AMCA  cells, allowing highly enriched preparations of PCFCs to be generated. This will facilitate the preparation of purified PCFC populations for additional functional and expression profiling studies., w+ u( v- v8 t; ]6 }6 p; j

The size of single-cell colonies derived from ACE AMCA  cells was significantly larger than ACE¨CAMCA  selected colonies, indicating that ACE is an important regulator of PCFC proliferation. This conclusion is further supported by the significantly reduced colony size observed for ACEnull versus wild-type mice. ACE, however, does not appear to be critical for PCFC detection or attachment per se, given that colonies are still able to be generated from ACEnull mice. How does ACE regulate PCFC colony size? Two possible pathways by which ACE could act in controlling PCFC proliferation are through its role in the RAS and/or through the degradation of circulating cell cycle inhibitor, AcSDKP. Firstly, via the RAS system, ACE may exert its effect through the ACE product angiotensin II to stimulate proliferation of PCFCs. Angiotensin II may alone stimulate PCFC proliferation or synergize with putative PCFC expansion factors in a way similar to which Angiotensin II synergizes with erythropoietin to expand the erythroid progenitor compartment . Given that PCFCs are cultured in serum-supplemented medium, cell surface ACE may promote PCFC proliferation in vitro by degrading AcSDKP." N% a" Q* ^# o4 n8 B

Taken together, the increased clonogenicity observed in ACE AMCA  cultures and decreased colony size for those derived from ACEnull mice observed in the current study suggest that ACE activity may play a role in controlling PCFCpro-liferation. Hence, it is possible that the ACE-related control mechanisms of stem/progenitor cell proliferation previously described in the hemopoietic system may also operate in other organ systems, including the pituitary gland.* w1 Q. E4 ~' U
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Although the exact role of PCFCs in the pituitary is not known, our recent study suggests that PCFCs may act as progenitors for hormone-secreting cells . The differentiation potential of ACE AMCA  cells found within the subluminal zone and MEC region of the pituitary gland remains to be explored.
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CONCLUSION

Our data indicate that PCFCs reside within the MEC zone of the adult pituitary and express cell surface markers typical of stem/progenitor cells, Sca-1 and ACE, the latter of which appears to be functionally important for PCFC proliferation. These findings will facilitate further investigations into the niche of PCFCs, the control of their proliferation, gene expression analysis, and the preparation of enriched putative progenitor cell populations for in vitro and in vivo differentiation studies.6 n8 s1 W6 ]* \8 }! h
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DISCLOSURES7 R5 P, T) e: F  x6 o" n' w. U+ H! ~

The authors indicate no potential conflicts of interest.3 ?. R) K- l! \& f, A& Z' D) E0 h

ACKNOWLEDGMENTS* b1 m4 b6 l+ |

We thank Dr. Michael McKinley (Howard Florey Institute, Melbourne, Australia) for providing the ACEnull mice. This work was funded by the Australian National Health and Medical Research Council. P.Q.T. is an RD Wright Fellow of the Australian National Health and Medical Research Council. P.J.S. is currently affiliated with the Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, Texas 77030, USA.
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