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(The FASEB Journal. 2007;21:156-166.)
© 2007 FASEB

Vascular expression of germinal ACE fails to maintain normal blood pressure in ACE–/– mice

Sean P. Kessler*, Preenie deS. Senanayake{dagger}, Christina Gaughan* and Ganes C. Sen*,1

* Department of Molecular Genetics, Lerner Research Institute, and

{dagger} Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, USA

1Correspondence: Department of Molecular Genetics- NE20, Lerner Research Institute, 9500 Euclid Ave., Cleveland, Ohio 44195, USA. E-mail: seng{at}ccf.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Maintenance of normal blood pressure is critical for preserving the integrity of the cardiovascular system. Angiotensin 1-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-aldosterone system (RAAS) and the renal tubuloglomerular feedback response. Although the two structurally related isozymic forms of ACE both generate the vasoactive octapeptide angiotensin II (Ang II) with equal efficiency, both are expressed in a nonoverlapping tissue-restricted fashion. To discriminate the precise physiological role of each ACE in its requisite tissue in vivo, we expressed one ACE isoform exclusively in a single cell type of an Ace null mouse. Previously, we demonstrated that vascular endothelial cell-specific expression of transgenic somatic ACE (sACE) could restore normal blood pressure of Ace-null mice. In this current study, we expressed germinal ACE (gACE) in the vascular endothelial cells of the Ace null mouse. These mice exhibited correct renal structure, renal function, and normal growth rates. Although the mice had elevated levels of gACE bound to vascular endothelial cells and high levels of gACE and Ang II in the circulating serum, blood pressure was restored only partially. This study demonstrated that gACE, even when expressed in the vasculature, could not functionally substitute for sACE.—Kessler, S. P., Senanayake, P. deS., Gaughan, C., Sen, G. C. Vascular expression of germinal ACE fails to maintain normal blood pressure in ACE-/- mice.


Key Words: ANG-converting enzyme • transgenic mice • renin-angiotensin system


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM (RAAS) (1) has been well recognized for its critical role in modulating a wide variety of physiological functions, most notably the regulation of renal and cardiovascular mechanisms aimed at maintaining fluid and electrolyte homeostasis (1) . The pivotal physiological role of angiotensin 1-converting enzyme (ACE) within the RAAS cannot be overstated. ACE is chiefly responsible for controlling vascular resistance and electrolyte balance by generating the vasoactive peptide angiotensin II, (Ang II) and by inactivating bradykinin, a well-known vascular endothelium dilator (2) . Historically, successful reduction of pathophysiological effects of hypertension due to an overactive RAAS, has relied heavily on blockade of ACE enzyme activity with a plethora of ACE-specific inhibitors and Ang II receptor blockers. Novel inhibitors with even greater specificity should be forthcoming since the crystal structure of two domains of ACE have recently been solved (3 , 4) .

ACE is expressed in two tissue-restricted isozymic forms. The somatic form (sACE) is expressed in vascular endothelial cells, renal proximal tubule epithelial cells, circumventricular organs of the brain, intestinal brush border cells, macrophages, monocytes, T-cells, and Leydig cells of the testis (5 6 7 8 9 10 11 12) . The germinal isozyme (gACE) is expressed exclusively in maturing sperm (11) . Both isoforms are structurally related in that they are encoded by a single gene and arise from alternate transcription initiation and post-transcriptional splicing (13 , 14) . Both share a completely homologous C-domain, including the His-Glu-X-X-His catalytic active site center and intracellular membrane anchoring tail that contains a cleavage target site for ACE secretion from the cell surface (15 16 17 18 19) . However, both isoforms possess a unique N-domain, which, in the case of the somatic isoform, broadens its substrate specificity in somatic tissue sites. The somatic N-domain, highly homologous with the C-domain inclusive of an additional His-Glu-X-X-His active site, cleaves LHRH and the hematopoeitic peptide AcSDKP 30 and 40 times faster than the C-domain of sACE, respectively (15 , 20) . An alternate substrate preference for the gACE N-domain remains to be identified, although the recent discovery that gACE cleaves glycosylphosphatidylinositol-anchored (GPI) proteins from the surface of sperm has generated new insight into the function of gACE with respect to male fertility (21) . However, recently this observation has been seriously questioned by two other groups (22 , 23) .

Although ACE inhibitors have been widely prescribed for many years to control hypertension, it was unclear what side effects may result from global, prolonged, or permanent inhibition of ACE activity. Though the Ace –/– mouse models exhibit reduced blood pressure, they also display a debilitating phenotype, most notably renal atrophy, hydronephrosis, renal vascular hyperplasia, anemia, runting, and male sterility (24 25 26) . This phenotype made it very clear that ACE plays additional roles beyond maintaining blood pressure, especially with regard to renal function and development as well as in male reproductive functions. This concept, that ACE is required for additional functions, was further strengthened by the recent identification of ACE N-domain-like and C-domain-like proteins in lower invertebrates (Drosophila) and their developmental-specific expression patterns (27) . To discern the tissue-specific, isozyme-specific, physiological role of each ACE isoform, we have utilized a transgenic approach to develop unique mouse strains that express a single ACE protein in a target cell-type of an Ace –/– mouse. Our studies have revealed that expression of the correct ACE isoform in its natural location restores a limited functional deficiency observed in the ACE null mouse. For example, expression of gACE exclusively in sperm (Pg strain) restores Ace –/– male fertility and expression of sACE exclusively in vascular endothelial cells (Ts strain) restores blood pressure. However, blood pressure and renal structure/function are not corrected in the Pg strain and male mice remain sterile in the Ts strain (26 , 28 , 29) .

One question still remained unanswered. Why are two ACE isoforms expressed in distinct tissue sites instead of one isoform expressed in every ACE-expressing tissue? Could one ACE isoform reciprocate the function of the other if expressed in the alternate cell-type? To address this question, we have developed mouse strains that express the isoforms in the noncognate tissue. We have previously demonstrated that sACE expressed in sperm fails to restore the fertility of an Ace –/– male mouse (Ps strain) (30) . In this current study, we report the results from a reciprocal experimental strain. We have generated two independent strains of mice that express gACE in vascular endothelium of an Ace –/– mouse (Tg strain). Despite expressing gACE at levels equivalent to or substantially higher than either wild-type (Wt) or Ts strains in vascular endothelial cells or in the serum, blood pressure was only partially restored. However, all renal defects and defective growth rates were cured. Taken together, our mouse strains reveal that neither gACE nor sACE can fully reciprocate the other isozyme’s function in the alternate cell-type.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transgene construction
The rabbit somatic ACE cDNA was replaced by the rabbit gACE cDNA in the tie-sACE construct (plasmid AP008) previously used to generate the Ts mouse strain (29) . The resulting plasmid, tie-rACET/pcDNA3, was verified by transient transfection into HT1080 cells. Expression of full-length rabbit gACE by this construct was verified by Western blotting transfected cell extracts with polyclonal anti-rabbit ACE antisera #447 as described previously (30) . The 4100 bp Tie-gACE-BGHpA (Tg) transgene was subsequently released from this plasmid by HindIII and AseI digestion and sent to the University of Cincinnati Transgenic Mouse Core Facility for the generation of transgenic mice utilizing standard techniques.

Southern blot hybridization
Southern blot genotyping was performed as described previously utilizing SacI digestion of genomic tail-snip DNA (26) . Heterozygosity or homozygosity of the transgene was determined by normalizing the transgene Imagequant value to the endogenous mouse Ace gene value in the same genomic DNA sample. The endogenous Ace genotype is determined by the presence of a WT 6.4 kB SacI genomic fragment or the disrupted 8.4 kB SacI (Ace null) genomic fragment (26) . The presence of the tie-gACE transgene is indicated by hybridization of the probe with a 3.4 kB fragment.

Establishment of transgenic lines and male fertility tests
The tie-gACE transgene was microinjected into the pronuclei of FVB strain zygotes at the University of Cincinnati transgenic core utilizing standard techniques. Adult FVB tie-gACE-BGHpA (Tg) transgenic founder mice (Ace +/+, Tg ± Line 4200) and (Ace +/+, Tg ± Line 4000) were independently mated with Ace ± FVB mice (30) to generate Ace ±, Tg ± mice. Interbreeding between male and female Ace ±, Tg ± mice within the same line was performed to generate the Ace –/–, Tg ± (Tg Line 4200) and (Tg Line 4000) experimental mice. Genotyping of all mice was performed by Southern blotting as described above. Expression of gACE in each transgenic line was first confirmed by observing the presence of gACE in the serum by Western blot with anti-angiotensin 1-converting enzyme #447 antisera. Wt, Ace–/– (KO) and transgenic Ts strain mice, which exhibit all normal WT phenotypes [(blood pressure, renal structure/function, growth rates) except male fertility], were all used as the control groups (29) .

For fertility comparison, adult Tg males were mated with six WT adult FVB strain females (Jackson Labs, West Grove, PA, USA) for 15 d; the equivalent to three complete estrous cycles as described previously (30) . Each mating consisted of two females per male. Females were observed for plugs. If no pups were produced within 22 d from experimental male removal, the same females were mated with adult Ace +/+ (Wt) males for 10 d. The number of pups per litter was noted.

ACE enzyme assay
The standard ACE assay, which measures ACE cleavage of the AngI analog Hip-His-Leu, was performed by incubating 25 µg total protein extract from Wt, Ts, Tg (Line 4200), and Tg (Line 4000) adult mouse lung and kidney or 1 µl of serum from retro-orbital eye bleed (16 , 30) . All tissues were rinsed extensively in PBS prior to homogenization in ACE lysis buffer as described previously (29) . All tissues and serum originated from five age-matched FVB strain adult mice. Activity values are reported as micromoles His-Leu per micrograms protein or per microliter serum liberated from Hip-His-Leu after a 1-h incubation in a Falcon microtiter plate with shaking at 37C. Samples were read on a SpectraMax Gemini XPS spectrofluorimeter plate reader. Triplicate activity values from each mouse sample were averaged to determine an activity average for each mouse lung, kidney, and serum sample. Data points represent the average activity values from 5 age-matched adult mice ± 95% confidence interval (CI) for the mean.

Ang II measurements
For each genotype, the blood from four adult mice of the same sex and age were pooled to achieve a 1 ml plasma sample. Duplicate samples from each plasma pool were extracted for the measurement of Ang II levels as described previously (29 , 31) . The results represent the arithmetic mean of assaying a minimum of 5 pools from each genotype ±95% CI for the mean.

Histology and immunohistochemistry
Age-matched, adult organs were paraffin-embedded, cross-sectioned at 2.5 µm thickness, and hematoxylin and eosin stained by the Histology Core (Lerner Research Institute, Cleveland, OH, USA). Immunohistochemistry was performed following deparaffinization as described previously (29) . Slides were incubated in 10 mM sodium citrate, pH 6.0 for 30 min at 25°C, then returned to PBS. The slides were blocked for 2 h at 25°C in PBS + 10% horse serum + 0.3% Triton X-100 (blocking buffer). The polyclonal goat anti-angiotensin 1-converting enzyme antibody (Ab) #447 (32) , diluted 1:1000 in blocking buffer or the monoclonal anti-rabbit ACE Ab 3C5 (a generous gift from Sergei Danilov at the University of Chicago) (33) diluted 1:25, was applied to a slide in a humid chamber for 16 h at 4°C. Following washes in PBS + 0.3% Triton X-100 (PBST), anti-goat-FITC (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was applied to polyclonal ACE stained slides at 1:3000 dilution in blocking buffer or a anti-mouse AlexaFlour 594 (red) (Molecular Probes, Eugene, OR, USA) diluted 1:1500 in blocking buffer to each section for 2 h in the dark at 25°C. Following washes in PBST, Vectashield ± Dapi (Vector Laboratories, Burlingame, CA, USA) diluted 1:1 in PBS was applied. All stained slides were visualized with a Leica digital fluorescent microscope (Bannockburn, IL, USA) and Adobe Photoshop software (San Jose, CA, USA).

Water uptake and urine output measurement
Age-matched, adult mice of the following genotypes (Wt, KO, Ts, Tg line 4200, and Tg line 4000) were individually placed in a Nalgene metabolic cage supplied with powdered standard chow and water ad libitum. Reported data reflect the average daily (24 h) water consumption and urine vol produced for five consecutive days for each of five mice of the same genotype ±95% CI.

Blood pressure measurement
The noninvasive computerized RTBP007 tail cuff blood pressure system (Harvard Apparatus, Holliston, MA, USA) was used to obtain systolic blood pressure on conscious mice as described previously (26 , 29 , 34) . All mice were housed separately, fed autoclaved chow with water ad libitum, and were maintained on a 12 h light/dark cycle. Each adult mouse was trained for 4 d to acclimate them to the apparatus and restraint. All measurements and training were performed on consecutive days between 12 and 3 pm each day. Computer recorded measurements were then taken for five consecutive days following training. A minimum of 10 blood pressure readings per mouse per day were used to calculate the average daily blood pressure for each FVB mouse. The average blood pressure for each mouse was then calculated by averaging the daily blood pressure of each mouse over the five consecutive days of readings. The data were presented as the arithmetic mean systolic blood pressure for each genotype ±95% CI.

Statistics
Data are presented as arithmetic means and variations as 95% confidence interval of the mean. Significance values were obtained by unpaired t test, comparing experimental Tg strains to the Wt control strain or the ACE null (KO) strain as indicated in the figure legend. The probability value for significance was defined as P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experimental mice
All mice, including Wt, KO, and Tie-sACE (Ts) control groups, were of the FVB strain (Table 1 ). The Tie-gACE (Tg) experimental mice were generated by crossing Ace ± mice with mice carrying the Tg transgene. The transgene consisted of rabbit gACE cDNA, a polyadenylation and splicing cassette from the bovine growth hormone gene (26) , and the 808 bp murine Tie-1 promoter, which was previously shown to direct expression to the vascular endothelial cells (29) . The Tie-gACE-BGHpA transgene (Tg) was assembled as shown in Fig. 1 A and tested in vitro by transient transfection into HT1080 cells (data not shown). An ACE activity assay and Western blot of extracts prepared from cells transfected with the plasmid containing the Tg transgene confirmed synthesis of the full-length, enzymatically active gACE protein (data not shown). The HindIII-AsnI fragment containing the Tg transgene (Fig. 1A ) was microinjected into pronuclei of FVB zygotes and then implanted in the uteri of pseudopregnant mothers. Tail DNA of resulting pups was digested with SacI and analyzed by Southern blotting using a 405 bp rabbit ACE cDNA fragment as the probe (shown as Southern Probe in Fig. 1A ). Two independent founders carrying the Tg transgene were separately crossed with Ace ± FVB mice, and the progenies were genotyped by Southern blot analysis. Both of the lines transmitted the transgene to their progenies. The two transgenic lines (4200 and 4000) were mated (Ace±Tg± males with Ace±Tg± females of the same line) to generate the Ace– /–Tg ± (Tg Line 4200) and Ace–/–Tg± (Tg Line 4000) experimental mouse genotypes used for further physiological characterization (Fig. 1B ). Ace–/–Ts+/+ (Ts strain) control mice were generated by mating Ace±Ts+/+ males with Ace– /–Ts+/+ females as described previously (29) . Ace null control mice were generated by mating Ace± male mice with Ace–/– female mice.


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  		Table 1. Transgenic strains


Figure 1
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  		Figure 1. Generation of experimental mice. The Tie-1-gACE-BGHpA transgene (Tg) and rabbit ACE cDNA Southern probe (black bar) were designed as shown in (A). A representative Southern blot genotype of the Tg experimental mice is shown in (B). The genomic DNA cut with SacI yields a 3.4 kB transgenic band, a 6.6 kB native Ace allelic band, and an 8.4 kB disrupted Ace allelelic band. A Western blot (C) of serum from experimental Ace ±, Tg ± mice (line 4000 and line 4200) was probed with anti-angiotensin 1-converting enzyme Ab #447 and enhanced chemiluminescence (ECL) detection reagent.

Previous experiments have demonstrated that the natural cleavage-secretion process catalyzed by a membrane secretase efficiently cleaves gACE peptides from the surface of somatic cells in culture (16) . The Tg transgene was designed to express gACE in vascular endothelial cells. Since both sACE and gACE contain a homologous C-domain anchoring tail, we expected to find soluble gACE circulating in the serum in addition to the naturally produced soluble sACE in mice of Ace±Tg± genotype following this natural cleavage-secretions process. Serum obtained from retro-orbital eye bleed from Tg Line 4200 and Tg Line 4000 mice both contained the 100 kDa gACE protein in addition to the 170 kDa endogenously produced sACE (Fig. 1C ). Although both serum samples contained equivalent levels of sACE, detected by Western blotting with #447 anti-angiotensin 1-converting enzyme antisera, Line 4000 mice had substantially higher levels of gACE in the serum compared to Line 4200 (Fig. 1C ).

An in vitro ACE enzyme assay demonstrated that both experimental transgenic Tg lines, in the Ace-null background, produced significantly higher levels of gACE in serum as compared to Wt and Ts strain mice (Fig. 2 A). Although the Tg Line 4200 and Ts strain mice displayed significantly lower ACE activity in lung extracts compared to Wt mice, the ACE activity in the lungs of Line 4000 mice was not significantly different from that produced by Wt mice (Fig. 2B ). In kidney, the three transgenic lines produced similar levels of ACE, which were much lower than those in Wt mice (Fig. 2C ). This difference was possibly due to the lack of expression of the transgenic protein in proximal tubules of the kidney. Both Tg lines displayed normal health and growth rates similar to the Ts strain, which were previously shown to have normal physiology and blood pressure and were comparable to Wt FVB mice (29) .


Figure 2
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  		Figure 2. ACE activity assay. Age-matched, adult serum (A), lung extract (B), and kidney extract (C) were prepared from Wt and strain Ts, Tg (Line 4200) and Tg (Line 4000) mice as described in the Experimental Procedures. Lung or kidney protein extract (25 µg) or serum (1 µl) was assayed for ACE activity, measured as µmoles of His-Leu generated by cleavage of Ang I analog Hip-His-Leu in 1 h at 37°C. Each bar represents the average value from triplicate samples from five mice of the same genotype. Error bars indicate the 95% CI for each data sets. *P ≤ 0.001**P ≤ 0.0005, ***P ≤ 0.00005 vs. Wt control strain mice.

We also determined the ability of the gACE produced in the Tg lines to cleave Ang I in vivo by measuring the level of Ang II in the plasma of each strain. Plasma, pooled from 4–5 adult mice of the same genotype, was assayed for Ang II. In Fig. 3 , we report that the levels of Ang II produced in the Tg Line 4200 and Tg Line 4000 strains did not significantly differ from the Ang II levels observed in the Wt and Ts strains. As expected, the Ang II present in the plasma of KO mice, most likely generated by the chymase pathway, was significantly lower than all other strains in this study.


Figure 3
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  		Figure 3. Plasma Ang II measurements. Plasma (1 ml) pooled from 4 adult mice of the same age, sex, and genotype was assayed for Ang II levels as described in Experimental Procedures. Each data point is the average of duplicate measurements (pg/ml plasma) from five independent pools (±95% CI). *P ≤ 0.005 vs. Wt control strain mice.

Expression profile of the transgene
For identifying the transgene-expressing cell type in a tissue, immunohistochemistry (IHC) was performed on thin sections of tissues from Tg strain mice using a polyclonal anti-rabbit-angiotensin 1-converting enzyme Ab. As shown in Fig. 4 , the gACE produced by the Tg transgene was expressed in brain, heart, liver, lung, kidney, and spleen of Tg Line 4000. The same was true for Line 4200 (not shown). In all tissues, expression was restricted to the vascular endothelial cells lining the lumen of blood vessels. We did not observe staining in any other cell type in these tissues in either Tg strain. Therefore, the Tie-1 promoter that previously demonstrated strict tissue-specificity in our Ts line (29) also has demonstrated strict vascular endothelial cell-specific gene expression in these two new Tg transgenic lines. However, in the kidneys of both Ts and Tg mice, ACE was expressed in the microcapillaries of the glomeruli, whereas this was not the case for ACE expression in the Wt mouse. In contrast, as expected, transgenic sACE or gACE was not expressed in the proximal tubules (Fig. 5 ).


Figure 4
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  		Figure 4. Immunohistochemistry of ACE expression. All organs were removed from Tg (Line 4000) adult mice, paraffin embedded, and sectioned at 3 µm thickness. Following paraffin removal, slides were probed with goat anti-angiotensin 1-converting enzyme antiserum #447 and anti-goat FITC. Slides were mounted with VectaShield + 4',6'-diam idino-2-phenylidole (DAPI) and viewed with a Leica fluorescent microscope at 40x magnification. The vessel luminal walls in all organs appear brightly stained green, while nuclei appear blue.


Figure 5
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  		Figure 5. Immunohistochemistry of renal vessels. Age-matched, adult kidneys from Wt and transgenic (Ts and Tg) mice were prepared and stained for ACE expression with goat anti-angiotensin 1-converting enzyme antiserum #447 and anti-goat FITC as described in Materials and Methods. Slides were mounted with VectaShield + DAPI and viewed with a Leica fluorescent microscope at 40x magnification. ACE expressed by Wt mouse proximal tubule epithelial cells (PT) and transgenic mouse gomerular microcapillaries (G) and vascular endothelium in luminal vessel walls (V) is indicated by arrows.

To correlate the level of ACE activity observed in the lung samples obtained from the Ts and Tg strain mice (Fig. 2) with the level of ACE produced in the vascular endothelial cells of these same strains, we repeated the IHC staining on lung sections with a monoclonal antibody (mAb) that specifically recognizes a unique epitope of the rabbit ACE C-domain (33) . This epitope, common to both gACE and sACE, is not present in the murine ACE protein. As shown in Fig. 6 , we observed greater levels of gACE produced by vascular endothelial cells in the lung of Tg Line 4200 mice as compared to the level of sACE produced by the same cell type in the Ts strain. Significantly increased staining was observed in lung sections from Tg Line 4000 mice as compared to both Line 4200 mice as well as mice of the Ts strain. No detectable expression of gACE was found in other vascular cell types in the lung, nor in any other cells in either Tg experimental mouse strain with this monoclonal anti-rabbit ACE Ab. In addition, this Ab failed to bind murine sACE in Wt mice, which confirmed the specificity of this Ab.


Figure 6
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  		Figure 6. Immunohistochemistry of lung vessels. Age-matched, adult lungs from Ts, Tg (Line 4200), Tg (Line 4000), and Wt mice were prepared and stained for transgenic rabbit ACE expression with anti-rabbit ACE mAb 3C5 and AlexaFlour (red) as described in Materials and Methods. Slides were mounted with VectaShield + DAPI (blue) and viewed with a Leica fluorescent microscope at 40x magnification. Rabbit ACE expressed by vascular endothelium in luminal vessel walls of transgenic lungs appears red while absence of murine ACE staining in Wt lungs indicates the specificity of this mAb.

Physiological properties of the experimental mice
Ace –/– FVB mice are runty and fail to thrive (26) . However, we have previously demonstrated that transgenic expression of sACE, in either the vascular endothelial cells or renal proximal tubule epithelial cells, restores Ace–/– mice to normal health and survival (29) . We have also reported that mice, serendipitously expressing gACE in the serum, also exhibit correct renal structure and function in addition to overall restored health (26) . In the current study, we observed a similar health profile for Tg mice of both Line 4200 and Line 4000. Both groups exhibit normal growth rates and fecundity; they are indistinguishable in size from Wt or Ts strain mice.

A major phenotype of Ace –/– mice is male sterility. Only Ace–/– mice expressing gACE in sperm have restored fertility (30) . All of our other Ace–/– mice expressing sACE in sperm (Ps strain), or sACE in other somatic tissues (i.e., vasculature, kidneys or serum), remained sterile (29 , 30) . Similarly, male fertility was not corrected in the Tg experimental male mice, despite gACE expression in the vasculature of the testis (data not shown). In addition, the presence of high levels of gACE in the serum and vasculature of both Tg lines did not adversely affect or reduce the fertility of Ace+/+ or Ace± male mice that were used to transmit the transgene in our experimental mouse breeding scheme.

Kidneys of Ace –/– FVB mice have very striking structural defects: the remnant renal structure of the older mice expands to the point of rupture as large volumes of fluid accumulate in a membranous cortical sac. This abnormality is visible from the outside of the mouse as the kidneys protrude from the posterior of Ace–/– mice. In younger adults, the cortex becomes thinner and a large cavity appears in the center (Fig. 7 B vs. A). The endothelial cells and smooth muscle cells of the arterial vessel wall become hyperplastic (marked V in Fig. 7B vs. A ). These structural defects were completely cured in both of the Tg experimental mouse strains (Fig. 7D, E ). Their kidneys, including the arterial walls, returned to the same phenotype as observed in the Ts mouse strain (Fig. 7C ).


Figure 7
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  		Figure 7. Renal histology. Hematoxylin- and eosin-stained slides from 2-mo-old kidneys of age-matched mice reveal abnormal renal perforation and thickened renal blood vessel (arrow+V) in Ace null (KO) mice (B) compared to Wt control mice (A). The absence of the kidney perforation and the restoration of normal renal vessel thickness are shown in the Ts strain (C), the Tg (Line 4200) (D), and Tg (Line 4000) (E). All kidneys were photographed at 0.6x (whole cross section) or 40x (blood vessel) with a digital Leica light microscope.

Since the kidneys of both Tg strain mice exhibit normal structure, we expected a corresponding return to normal renal function in these mice. To assess the overall fluid homeostasis in the Tg experimental lines, we measured the amount of urine output and water uptake over a 5 day period. The average water uptake and urine output in the Tg strain mice was indistinguishable from that observed in Wt and Ts strain mice (Fig. 8 ). Therefore, gACE can reciprocate the function of sACE with respect to the restoration of renal structure and functions, since transgenic expression of gACE in the vasculature, but absent in the proximal tubule, was sufficient for maintaining normal renal structure and functions.


Figure 8
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  		Figure 8. Renal function analysis. Mean urine output (±95% CI) produced in a 24-h period from Wt, KO, Ts, Tg (Line 4200), and Tg (Line 4000) mice reported in milliters (A). Mean water vol (in milliliters), consumed during the same 24-h period is noted in (B). A minimum of 5, age-matched adult mice was studied over a period of five consecutive days.

The primary aim of this study was to determine whether gACE could substitute for sACE and restore Ace-null mouse blood pressure to normal levels. To examine this parameter, we used the computerized tail-cuff plethysmography method for measuring systolic blood pressure. Our results demonstrated that both Tg Line 4200 and Tg Line 4000 failed to maintain normal blood pressure as compared to the Ts strain (Fig. 9 ). The mean systolic blood pressure of Wt mice was 118 mm Hg, whereas the corresponding numbers were: Ace–/– mice, 86 mm Hg; Ts mice, 119 mm Hg; Line 4200 mice, 96 mm Hg; and Line 4000 mice, 100 mm Hg. In Line 4200, despite expression of equivalent levels of gACE in the serum and lung and Ang II in the plasma, as compared to the Ts strain, the blood pressure restoration was only 32% of what was restored in the Ts strain. In Line 4000, the corresponding figure was 45%, although the transgene was expressed at a much higher level. These results demonstrate a major difference in the abilities of sACE and gACE to regulate blood pressure, since blood pressure was fully restored in the mice that expressed sACE in vascular endothelial cells (Ts strain) but not in mice expressing gACE in the same cell type, even when gACE was vastly over-expressed.


Figure 9
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  		Figure 9. Systolic blood pressure measurements. The noninvasive, computerized tail cuff plethysmography method was used to determine the systolic blood pressure of adult male and female KO, Wt, Ts, Tg (Line 4200), and Tg (Line 4000) FVB strain mice. The mean blood pressure (in mmHg±95%CI) for each genotype, separated by sex, was calculated on the mean daily blood pressure over a 5-day reading period for the number of mice indicated as (n). A minimum of 10 readings per day was used to calculate the daily blood pressure. *P < 0.0001 vs. Ts control strain mice. •P < 0.01 vs. KO control strain mice. {blacksquare}P < 0.0001 vs. KO control strain.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The tissue-restricted, isozyme-specific expression pattern of both ACE isoforms would suggest that these two, structurally related enzymes cannot physiologically reciprocate each other’s function in the noncognate cell type. If this were not the case, just one ACE isoform would exist that could perform all of the jobs that both gACE and sACE accomplish individually. In support of this concept was the discovery of alternative substrates for the N-domain of sACE and the most recent discovery that gACE cleaves GPI-anchored peptides located on the surface of sperm (15 , 20 , 21) .

To test our hypothesis that discrete, independent, physiological functions are attributed to each isoform and that these functions are dictated in part by the local environment in which each is expressed, we have utilized a transgenic approach to express one ACE isoform in the non-native cell type of an ACE null mouse. We have previously demonstrated that sACE failed to reciprocate the fertility function of gACE. ACE null mice expressing gACE on sperm (Pg strain) were fertile, whereas ACE null mice expressing sACE on sperm (Ps strain) remained sterile (30) . In this current study, we have demonstrated that gACE expression in vascular endothelial cells (Tg strain) failed to maintain normal blood pressure. This result is in contrast to our Ts strain that maintained normal blood pressure by expressing sACE in vascular endothelial cells (29) . In all four strains (Pg, Ps, Ts, and now Tg), more than adequate levels of the selected ACE isoform were expressed in the target cell-type as compared to control groups. Taken together, these animal models prove that ACE-regulated blood pressure and male fertility are strictly dependent on the entire structure of an ACE isoform since the common C-domain, inclusive of the Ang II generating active site center, was present in each of the transgene-expressing targeted cell types.

It is widely accepted that a major role of ACE within the RAAS is to regulate blood pressure and fluid homeostasis by generating sufficient levels of the vasopressor peptide Ang II. In support of this concept, numerous studies have demonstrated that in vivo pharmacological blockade of ACE activity is vitally important for managing renal failure, cardiovascular disease, hypertension, and stroke (35) . However, the significance of the precise site of Ang II generation has not been fully appreciated. Our results demonstrate that the local generation of Ang II at the cell surface matters greatly in the final effect of ACE-mediated blood pressure regulatory events. In two of our animal models (Gs and Tg), we have achieved more-than-sufficient levels of circulating ACE and Ang II, yet blood pressure was not restored to normal levels (29) . This result suggests a clear distinction between the function of circulating ACE and membrane-bound ACE. In support of this concept, a study utilizing domain-specific ACE inhibitors revealed that membrane-bound ACE-mediated vascular pressor responses can be blocked by lower ACE inhibitor doses than that which is required to inhibit AngII generation by circulating ACE (36) .

Why was gACE, even when expressed in vascular endothelial cells, unable to restore blood pressure of Ace–/– mice as competently as sACE? It appears that gACE, though capable of a partial restoration of blood pressure, somehow falls short of maintaining completely normal blood pressure when compared to sACE function. This difference cannot be attributed to differences in the levels of expression of the two isozymes: gACE was expressed at a comparable level to Ts mice in Line 4200 and at a much higher level in Line 4000, as observed in lungs stained with the 3C5 Ab and by lung ACE activity. Hence, the difference must lie in the intrinsic properties of the two isozymes. The N-terminal domain of sACE, absent in gACE, may exert functional constraints on the C-terminal domain. Alternatively, one might envision that gACE is inefficient in cleaving Ang I on the surface of the vascular bed, which may affect negatively the autocrine and paracrine functions of Ang II in the vasculature. Germinal ACE may reside on the cell surface in a conformation more favorable to GPI-anchored protein cleavage of sperm proteins rather than AngI cleavage, or the size of gACE may be functionally limiting. Somatic ACE, which is twice the size of gACE, may generate Ang II and then directly hand it off to the neighboring ANG receptors, thereby triggering local smooth muscle cell vasoconstriction and blood pressure regulation. Germinal ACE may be too short to hand off the Ang II that it generates to nearby receptors. Alternatively, secretase-specific cleavage may release gACE faster than sACE. If gACE is secreted more efficiently from the cell-surface, it may not remain membrane-bound long enough to generate sufficient local Ang II before being released into the serum. This, however, seems unlikely due to the intense staining of membrane-bound gACE in the lung vasculature in both Tg strains (Fig. 6) and the calculated ratios between the lung-bound and the serum ACE activities in the different strains of mice (Fig. 2A, B ). It is also possible that cell-bound sACE has another enzymatic property, not shared by gACE, which is required for full blood pressure maintenance (37) . Irrespective of the nature of the exact mechanism, our results indicate that the low level of Ang II in the blood of Ace –/– mice may not be the sole, or even the primary, reason for their low blood pressure.

Of interest is the observation that in both Ts and Tg strains, renal structure and function were maintained in the absence of proximal tubule-expressed ACE. This result suggests that ACE may serve yet another physiological purpose in proximal tubules since its absence has no detrimental effect on renal function and maintenance of fluid homeostasis in these mice. A second interesting observation is that in the Line 4000 mice, the serum AngII level was slightly, but not significantly, lower than that in the Wt, Ts and Tg Line 4200 mice, although the Tg Line 4000 mice expressed substantially more ACE in both lung and serum (Figs. 2 , 3) . Perhaps overcrowding of gACE at the cell surface may present a situation whereby ACE is sterically impeded from efficiently cleaving Ang I at the vascular endothelial cell surface.

In this study, we have tested the functional reciprocity of gACE for sACE in somatic cells. Previously, we studied mice, which expressed soluble gACE in the serum (Pg400 strain); their blood pressure was not restored (26) . However, we believed the inability of gACE to restore blood pressure in the Pg400 strain was due to the fact that it was not produced in vascular endothelial cells and because the gACE produced was not retained on the surface of vascular endothelial cells. In contrast, the Tg strains in this report exhibited more than adequate levels of correctly targeted, membrane-bound gACE in the vasculature; however, blood pressure was not fully restored. Our results point to a structural distinction between sACE and gACE, because both proteins failed to fully reciprocate the function of the other isozyme in the noncognate tissue site. As more structural details are revealed in future modeling studies, we will be able to devise a clearer model of the physiological action of ACE on the vascular wall. Nevertheless, we now possess two animal models demonstrating that high serum ACE levels, elevated plasma Ang II levels, or correct renal structures do not correlate with blood pressure. This may call into question the use of these parameters as predictive markers for hypertension.


   ACKNOWLEDGMENTS
 
We thank the following individuals for expert technical assistance: Theresa Rowe, David Young, Paulette Zavacky, Bryan Costin, and Thomas Scheidemantel. We thank Sergei Danilov at the University of Chicago for providing the 3C5 monoclonal antibody and for technical advice. This study was supported by the National Institutes of Health grant HL-48258.

Received for publication June 16, 2006. Accepted for publication August 21, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Soffer, R. L. (1981) Angiotensin-converting enzyme. Soffer, R. L. eds. Biochemical Regulation of Blood Pressure ,123-164 Wiley-Interscience New York, NY, USA.
  2. Corvol, P., Williams, T. A., Soubrier, F. (1995) Peptidyl dipeptidase A: angiotensin I-converting enzyme. Methods Enzymol. 248,283-305[Medline]
  3. Natesh, R., Schwager, S. L., Evans, H. R., Sturrock, E. D., Acharya, K. R. (2004) Structural details on the binding of antihypertensive drugs captopril and enalaprilat to human testicular angiotensin I-converting enzyme. Biochemistry 43,8718-8724[CrossRef][Medline]
  4. Corradi, H. R., Schwager, S. L., Nchinda, A. T., Sturrock, E. D., Acharya, K. R. (2006) Crystal structure of the N domain of human somatic angiotensin I-converting enzyme provides a structural basis for domain-specific inhibitor design. J. Mol. Biol. 357,964-974[CrossRef][Medline]
  5. Caldwell, P. R., Seegal, B. C., Hsu, K. C., Das, M., Soffer, R. L. (1976) Angiotensin-converting enzyme: vascular endothelial localization. Science 191,1050-1051[Abstract/Free Full Text]
  6. Sibony, M., Gasc, J. M., Soubrier, F., Alhenc-Gelas, F., Corvol, P. (1993) Gene expression and tissue localization of the two isoforms of angiotensin I converting enzyme. Hypertension 21,827-835[Abstract]
  7. Saavedra, J. M., Chevillard, C. (1982) Angiotensin-converting enzyme is present in the subfornical organ and other circumventricular organs of the rat. Neurosci. Lett. 29,123-127[CrossRef][Medline]
  8. Berg, T., Sulner, J., Lai, C. Y., Soffer, R. L. (1986) Immunohistochemical localization of two angiotensin I-converting isoenzymes in the reproductive tract of the male rabbit. J. Histochem. Cytochem. 34,753-760[Abstract]
  9. Bruneval, P., Hinglais, N., Alhenc-Gelas, F., Tricottet, V., Corvol, P., Menard, J., Camilleri, J. P., Bariety, J. (1986) Angiotensin I converting enzyme in human intestine and kidney. Ultrastructural immunohistochemical localization. Histochemistry 85,73-80[CrossRef][Medline]
  10. Friedland, J., Setton, C., Silverstein, E. (1978) Induction of angiotensin converting enzyme in human monocytes in culture. Biochem. Biophys. Res. Commun. 83,843-849[CrossRef][Medline]
  11. Langford, K. G., Zhou, Y., Russell, L. D., Wilcox, J. N., Bernstein, K. E. (1993) Regulated expression of testis angiotensin-converting enzyme during spermatogenesis in mice. Biol. Reprod. 48,1210-1218[Abstract]
  12. Murphy, T. J., Alexander, R. W., Griendling, K. K., Runge, M. S., Bernstein, K. E. (1991) Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature 351,233-236[CrossRef][Medline]
  13. Howard, T. E., Shai, S. Y., Langford, K. G., Martin, B. M., Bernstein, K. E. (1990) Transcription of testicular angiotensin-converting enzyme (ACE) is initiated within the 12th intron of the somatic ACE gene. Mol. Cell. Biol. 10,4294-4302[Abstract/Free Full Text]
  14. Kumar, R. S., Thekkumkara, T. J., Sen, G. C. (1991) The mRNAs encoding the two angiotensin-converting isozymes are transcribed from the same gene by a tissue-specific choice of alternative transcription initiation sites. J. Biol. Chem. 266,3854-3862[Abstract/Free Full Text]
  15. Ehlers, M. R., Riordan, J. F. (1991) Angiotensin-converting enzyme: zinc- and inhibitor-binding stoichiometries of the somatic and testis isozymes. Biochemistry 30,7118-7126[CrossRef][Medline]
  16. Sen, I., Samanta, H., Livingston, W. D., Sen, G. C. (1991) Establishment of transfected cell lines producing testicular angiotensin-converting enzyme. Structural relationship between its secreted and cellular forms. J. Biol. Chem. 266,21985-21990[Abstract/Free Full Text]
  17. Isaac, R. E., Williams, T. A., Sajid, M., Corvol, P., Coates, D. (1997) Cleavage of arginyl-arginine and lysyl-arginine from the C-terminus of pro-hormone peptides by human germinal angiotensin I-converting enzyme (ACE) and the C-domain of human somatic ACE. Biochem. J. 328,587-591[Medline]
  18. Williams, T. A., Barnes, K., Kenny, A. J., Turner, A. J., Hooper, N. M. (1992) A comparison of the zinc contents and substrate specificities of the endothelial and testicular forms of porcine angiotensin converting enzyme and the preparation of isoenzyme-specific antisera. Biochem. J. 288,875-881[Medline]
  19. Santhamma, K. R., Sadhukhan, R., Kinter, M., Chattopadhyay, S., McCue, B., Sen, I. (2004) Role of tyrosine phosphorylation in the regulation of cleavage secretion of angiotensin-converting enzyme. J. Biol. Chem. 279,40227-40236[Abstract/Free Full Text]
  20. Rousseau, A., Michaud, A., Chauvet, M. T., Lenfant, M., Corvol, P. (1995) The hemoregulatory peptide N-acetyl-Ser-Asp-Lys-Pro is a natural and specific substrate of the N-terminal active site of human angiotensin-converting enzyme. J. Biol. Chem. 270,3656-3661[Abstract/Free Full Text]
  21. Kondoh, G., Tojo, H., Nakatani, Y., Komazawa, N., Murata, C., Yamagata, K., Maeda, Y., Kinoshita, T., Okabe, M., Taguchi, R., Takeda, J. (2005) Angiotensin-converting enzyme is a GPI-anchored protein releasing factor crucial for fertilization. Nat. Med. 11,160-166[CrossRef][Medline]
  22. Leisle, L., Parkin, E. T., Turner, A. J., Hooper, N. M. (2005) Angiotensin-converting enzyme as a GPIase: a critical reevaluation. Nat. Med. 11,1139-1140[CrossRef][Medline]
  23. Fuchs, S., Frenzel, K., Hubert, C., Lyng, R., Muller, L., Michaud, A., Xiao, H. D., Adams, J. W., Capecchi, M. R., Corvol, P., Shur, B. D., Bernstein, K. E. (2005) Male fertility is dependent on dipeptidase activity of testis ACE. Nat. Med. 11,1140-1142; author reply 1142–1143[CrossRef][Medline]
  24. Krege, J. H., John, S. W., Langenbach, L. L., Hodgin, J. B., Hagaman, J. R., Bachman, E. S., Jennette, J. C., O’Brien, D. A., Smithies, O. (1995) Male-female differences in fertility and blood pressure in ACE-deficient mice. Nature 375,146-148[CrossRef][Medline]
  25. Esther, C. R., Jr, Howard, T. E., Marino, E. M., Goddard, J. M., Capecchi, M. R., Bernstein, K. E. (1996) Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility. Lab. Invest. 74,953-965[Medline]
  26. Kessler, S. P., Gomos, J. B., Scheidemantel, T. S., Rowe, T. M., Smith, H. L., Sen, G. C. (2002) The germinal isozyme of angiotensin-converting enzyme can substitute for the somatic isozyme in maintaining normal renal structure and functions. J. Biol. Chem. 277,4271-4276[Abstract/Free Full Text]
  27. Houard, X., Williams, T. A., Michaud, A., Dani, P., Isaac, R. E., Shirras, A. D., Coates, D., Corvol, P. (1998) The Drosophila melanogaster-related angiotensin-I-converting enzymes Acer and Ance–distinct enzymic characteristics and alternative expression during pupal development. Eur. J. Biochem. 257,599-606[Medline]
  28. Ramaraj, P., Kessler, S. P., Colmenares, C., Sen, G. C. (1998) Selective restoration of male fertility in mice lacking angiotensin-converting enzymes by sperm-specific expression of the testicular isozyme. J. Clin. Invest. 102,371-378[Medline]
  29. Kessler, S. P., Senanayake, P. S., Scheidemantel, T. S., Gomos, J. B., Rowe, T. M., Sen, G. C. (2003) Maintenance of normal blood pressure and renal functions are independent effects of angiotensin-converting enzyme. J. Biol. Chem. 278,21105-21112[Abstract/Free Full Text]
  30. Kessler, S. P., Rowe, T. M., Gomos, J. B., Kessler, P. M., Sen, G. C. (2000) Physiological non-equivalence of the two isoforms of angiotensin- converting enzyme. J. Biol. Chem. 275,26259-26264[Abstract/Free Full Text]
  31. Senanayake, P. S., Smeby, R. R., Martins, A. S., Moriguchi, A., Kumagai, H., Ganten, D., Brosnihan, K. B. (1998) Adrenal, kidney, and heart angiotensins in female murine Ren-2 transfected hypertensive rats. Peptides 19,1685-1694[CrossRef][Medline]
  32. Sadhukhan, R., Sen, G. C., Ramchandran, R., Sen, I. (1998) The distal ectodomain of angiotensin-converting enzyme regulates its cleavage-secretion from the cell surface. Proc. Natl. Acad. Sci. U. S. A. 95,138-143[Abstract/Free Full Text]
  33. Balyasnikova, I. V., Metzger, R., Franke, F. E., Danilov, S. M. (2003) Monoclonal antibodies to denatured human ACE (CD 143), broad species specificity, reactivity on paraffin sections, and detection of subtle conformational changes in the C-terminal domain of ACE. Tissue Antig. 61,49-62[CrossRef]
  34. Krege, J. H., Hodgin, J. B., Hagaman, J. R., Smithies, O. (1995) A noninvasive computerized tail-cuff system for measuring blood pressure in mice. Hypertension 25,1111-1115[Abstract/Free Full Text]
  35. Meier, P., Maillard, M., Burnier, M. (2005) The future of angiotensin II inhibition in cardiovascular medicine. Curr. Drug Targets Cardiovasc. Haematol. Disord. 5,15-30[CrossRef][Medline]
  36. Van Esch, J. H., Tom, B., Dive, V., Batenburg, W. W., Georgiadis, D., Yiotakis, A., van Gool, J. M., de Bruijn, R. J., de Vries, R., Danser, A. H. (2005) Selective angiotensin-converting enzyme C-domain inhibition is sufficient to prevent angiotensin I-induced vasoconstriction. Hypertension 45,120-125[Abstract/Free Full Text]
  37. Jaspard, E., Alhenc-Gelas, F. (1995) Catalytic properties of the two active sites of angiotensin I-converting enzyme on the cell surface. Biochem. Biophys. Res. Commun. 211,528-534[CrossRef][Medline]




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