Mice with Cardiac-Restricted Angiotensin-Converting Enzyme (ACE) Have Atrial Enlargement, Cardiac Arrhythmia, and Sudden Death
2004; Elsevier BV; Volume: 165; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)63363-9
ISSN1525-2191
AutoresHong Xiao, Sébastien Fuchs, Duncan J. Campbell, William Lewis, Samuel C. Dudley, Vijaykumar S. Kasi, Brian D. Hoit, George Keshelava, Hui Zhao, Mario R. Capecchi, Kenneth E. Bernstein,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoTo investigate the local effects of angiotensin II on the heart, we created a mouse model with 100-fold normal cardiac angiotensin-converting enzyme (ACE), but no ACE expression in kidney or vascular endothelium. This was achieved by placing the endogenous ACE gene under the control of the α-myosin heavy chain promoter using targeted homologous recombination. These mice, called ACE 8/8, have cardiac angiotensin II levels that are 4.3-fold those of wild-type mice. Despite near normal blood pressure and a normal renal function, ACE 8/8 mice have a high incidence of sudden death. Both histological analysis and in vivo catheterization of the heart showed normal ventricular size and function. In contrast, both the left and right atria were three times normal size. ECG analysis showed atrial fibrillation and cardiac block. In conclusion, increased local production of angiotensin II in the heart is not sufficient to induce ventricular hypertrophy or fibrosis. Instead, it leads to atrial morphological changes, cardiac arrhythmia, and sudden death. To investigate the local effects of angiotensin II on the heart, we created a mouse model with 100-fold normal cardiac angiotensin-converting enzyme (ACE), but no ACE expression in kidney or vascular endothelium. This was achieved by placing the endogenous ACE gene under the control of the α-myosin heavy chain promoter using targeted homologous recombination. These mice, called ACE 8/8, have cardiac angiotensin II levels that are 4.3-fold those of wild-type mice. Despite near normal blood pressure and a normal renal function, ACE 8/8 mice have a high incidence of sudden death. Both histological analysis and in vivo catheterization of the heart showed normal ventricular size and function. In contrast, both the left and right atria were three times normal size. ECG analysis showed atrial fibrillation and cardiac block. In conclusion, increased local production of angiotensin II in the heart is not sufficient to induce ventricular hypertrophy or fibrosis. Instead, it leads to atrial morphological changes, cardiac arrhythmia, and sudden death. The renin-angiotensin system (RAS) is a key regulator of blood pressure and electrolyte homeostasis. A critical component of this system is angiotensin-converting enzyme (ACE), which produces the eight amino acid peptide angiotensin II, the effector molecule of the RAS.1Corvol P Williams TA Soubrier F Peptidyl dipeptidase A: angiotensin I-converting enzyme.Methods Enzymol. 1995; 248: 283-305Crossref PubMed Scopus (229) Google Scholar ACE is a zinc metallopeptidase located on the cell surface of endothelium. In this location, ACE produces angiotensin II adjacent to vascular smooth muscle, a critical target organ for this vasoconstrictor. ACE is also produced by a variety of other tissues including renal tubular epithelium, activated macrophages, proximal gut epithelium, and areas of the brain. Endothelium and these other tissues make the isozyme of ACE, termed somatic ACE, which consists of two catalytic domains that are independently capable of producing angiotensin II. Studies of knockout mice established that somatic ACE influences blood pressure and other cardiovascular functions.2Krege JH John SW Langenbach LL Hodgin JB Hagaman JR Bachman ES Jennette JC O'Brien DA Smithies O Male-female differences in fertility and blood pressure in ACE-deficient mice.Nature. 1995; 375: 146-148Crossref PubMed Scopus (607) Google Scholar, 3Esther Jr, CR Howard TE Marino EM Goddard JM Capecchi MR Bernstein KE Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility.Lab Invest. 1996; 74: 953-965PubMed Google Scholar In contrast, within the testis, developing male germ cells produce a different ACE isozyme called testis ACE, which plays an important role in normal male reproduction.4Hagaman JR Moyer JS Bachman ES Sibony M Magyar PL Welch JE Smithies O Krege JH O'Brien DA Angiotensin-converting enzyme and male fertility.Proc Natl Acad Sci USA. 1998; 95: 2552-2557Crossref PubMed Scopus (304) Google Scholar In addition to regulating normal physiology, substantial evidence suggests that the RAS plays an important role in disease, including heart disease.5Dzau VJ Cell biology and genetics of angiotensin in cardiovascular disease.J Hypertens Suppl. 1994; 12: S3-S10PubMed Google Scholar Genetic studies reported a link between somatic ACE polymorphisms and the incidence of cardiac hypertrophy, sudden cardiac death, and acute coronary events.6Malik FS Lavie CJ Mehra MR Milani RV Re RN Renin-angiotensin system: genes to bedside.Am Heart J. 1997; 134: 514-526Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar This is consistent with the clinical effectiveness of ACE inhibitors in treating heart failure.7Garg R Yusuf S Overview of randomized trials of angiotensin-converting enzyme inhibitors on mortality and morbidity in patients with heart failure: Collaborative Group on ACE Inhibitor Trials.JAMA. 1995; 273: 1450-1456Crossref PubMed Scopus (1792) Google Scholar The beneficial effects of ACE inhibitors may not be solely the result of blood pressure reduction since other antihypertensive drugs do not produce the same effect. Rather, ACE may directly influence heart function through the local production of angiotensin II. Studies have found that angiotensinogen, renin, and ACE exist in the heart, implying that local generation of angiotensin II may affect cardiac functions including pathological formation of cardiac hypertrophy and fibrosis.8Dostal DE Baker KM The cardiac renin-angiotensin system: conceptual, or a regulator of cardiac function?.Circ Res. 1999; 85: 643-650Crossref PubMed Scopus (305) Google Scholar, 9Neri Serneri GG Boddi M Coppo M Chechi T Zarone N Moira M Poggesi L Margheri M Simonetti I Evidence for the existence of a functional cardiac renin-angiotensin system in humans.Circulation. 1996; 94: 1886-1893Crossref PubMed Google Scholar, 10Bader M Role of the local renin-angiotensin system in cardiac damage: a mini-review focusing on transgenic animal models.J Mol Cell Cardiol. 2002; 34: 1455-1462Abstract Full Text PDF PubMed Scopus (80) Google Scholar To investigate the local, cardiac effects of angiotensin II, several investigators created transgenic models with overexpression of angiotensinogen,11Mazzolai L Nussberger J Aubert JF Brunner DB Gabbiani G Brunner HR Pedrazzini T Blood pressure-independent cardiac hypertrophy induced by locally activated renin-angiotensin system.Hypertension. 1998; 31: 1324-1330Crossref PubMed Scopus (183) Google Scholar ACE,12Tian XL Costerousse O Urata H Franz WM Paul M A new transgenic rat model overexpressing human angiotensin-converting enzyme in the heart.Hypertension. 1996; 28 (Abstract): 520Google Scholar, 13Higaki J Aoki M Morishita R Kida I Taniyama Y Tomita N Yamamoto K Moriguchi A Kaneda Y Ogihara T In vivo evidence of the importance of cardiac angiotensin-converting enzyme in the pathogenesis of cardiac hypertrophy.Arterioscler Thromb Vasc Biol. 2000; 20: 428-434Crossref PubMed Scopus (30) Google Scholar angiotensin II receptors,14Hein L Stevens ME Barsh GS Pratt RE Kobilka BK Dzau VJ Overexpression of angiotensin AT1 receptor transgene in the mouse myocardium produces a lethal phenotype associated with myocyte hyperplasia and heart block.Proc Natl Acad Sci USA. 1997; 94: 6391-6396Crossref PubMed Scopus (194) Google Scholar, 15Paradis P Dali-Youcef N Paradis FW Thibault G Nemer M Overexpression of angiotensin II type I receptor in cardiomyocytes induces cardiac hypertrophy and remodeling.Proc Natl Acad Sci USA. 2000; 97: 931-936Crossref PubMed Scopus (315) Google Scholar, 16Hoffmann S Krause T van Geel PP Willenbrock R Pagel I Pinto YM Buikema H van Gilst WH Lindschau C Paul M Inagami T Ganten D Urata H Overexpression of the human angiotensin II type 1 receptor in the rat heart augments load induced cardiac hypertrophy.J Mol Med. 2001; 79: 601-608Crossref PubMed Scopus (52) Google Scholar or even angiotensin II peptide17van Kats JP Methot D Paradis P Silversides DW Reudelhuber TL Use of a biological peptide pump to study chronic peptide hormone action in transgenic mice: direct and indirect effects of angiotensin II on the heart.J Biol Chem. 2001; 276: 44012-44017Crossref PubMed Scopus (74) Google Scholar in the heart. These studies generated controversy in that some models presented with cardiac hypertrophy and fibrosis, while other animal models lacked a cardiac phenotype in the absence of external stimuli. Here we report a new mouse model, called ACE 8/8, created using targeted homologous recombination in mouse ES cells. These mice overexpress ACE in the heart, but differ from transgenic models in that they lack ACE expression in such traditional ACE expressing tissues as vascular endothelium, kidney, gut, and brain. Thus, rather than adding cardiac ACE expression to endogenous ACE, our model substitutes cardiac ACE expression for the disseminated presence of ACE in a wild-type mouse. As a result, angiotensin II levels in cardiac tissue are greater than four times that of control mice. Surprisingly, ACE 8/8 mice have normal ventricular size and function. The blood pressure of the mice is near normal. However, these mice have very marked enlargement of the left and right atria. This is associated with cardiac arrhythmia and a marked incidence of sudden death. We conclude that increased angiotensin II within the heart is not associated, a priori, with ventricular fibrosis, enlargement, or dysfunction. In contrast, atrial enlargement develops as a result of abnormal amounts of cardiac ACE and angiotensin II, and this appears independent of blood pressure elevation. A 10.7-kb fragment of mouse genomic DNA was cloned from a mouse CC1.2 ES cell library. This contained 2.4 kb of the somatic ACE promoter, the somatic ACE transcription start site, and 8.3 kb of genomic sequence encompassing somatic ACE exons 1 through 12. A neomycin cassette (called KT3NP4) was inserted into a unique BssH II restriction site located within the 5′ untranslated region of somatic ACE.18Cole J Quach du L Sundaram K Corvol P Capecchi MR Bernstein KE Mice lacking endothelial angiotensin-converting enzyme have a normal blood pressure.Circ Res. 2002; 90: 87-92Crossref PubMed Scopus (66) Google Scholar A 4.4-kb α-myosin heavy chain (α-MHC) promoter was cloned by PCR amplification from an α-MHC plasmid construct sent to us by Dr. Jim Gulick, Cincinnati Children's Hospital Medical Center. The α-MHC promoter was placed immediately 3′ to the neomycin cassette. The ACE.8 targeting construct was linearized and electroporated into R1 ES cells derived from a 129/SVx129/SvJ F1 embryo. Individual ES cell clones were screened for targeted homologous recombination using a combination of PCR and genomic Southern blot analysis. The generation of chimeric mutant mice was performed as previously described.3Esther Jr, CR Howard TE Marino EM Goddard JM Capecchi MR Bernstein KE Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility.Lab Invest. 1996; 74: 953-965PubMed Google Scholar Chimeric mice were mated to C57BL/6 mice to generate F1 mice. Heterozygous F1 mice were bred to create F2 offspring of wild-type (WT), heterozygous (HZ), and homozygous ACE.8 (8/8) mice. All studies were performed on F2 or F3 generation litters generated from the breeding of heterozygous animals. Age and gender matched littermate controls were used in all studies. Animal procedures were approved by Institutional Animal Care and Use Committee and were supervised by the Emory University Division of Animal Research. Genomic DNA was obtained through tail clipping. Three primers were used for PCR genotyping: a reverse primer located in the first exon of the ACE gene (5′-CCACCTCGGCACTCGAGTTATAGCTTCAG-3′); a forward wild-type primer located in the 5′ untranslated region of the ACE gene, (5′-TCTAGCTTCCTCTGAGAGAGCCCGATCTAG-3′); and a forward mutant primer located on the 3′ end of the α-MHC promoter (5′-CCACCTCGGCACTCGAGTTATAGCTTCAG-3′). A 450-bp fragment was amplified for the wild-type allele and a 742-bp fragment was amplified for the mutant allele. Cardiac puncture was performed on anesthetized mice to collect blood in heparinized tubes. Plasma was obtained by centrifugation of blood samples at 4°C for 10 minutes at 2000 × g. Animals were then sacrificed and tissue samples were collected. Individual tissues were briefly homogenized at low speed in ACE homogenization buffer (50 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 25 mol/L ZnCl2, and 1 mmol/L PMSF). These homogenates were centrifuged at 10,000 × g and the supernatant discarded. The pellets were then resuspended in ACE homogenization buffer containing 0.5% Triton X-100 and vigorously re-homogenized. The tissue homogenates were again spun at 10,000 × g and supernatants were used for ACE activity measurement. Due to the small size of atrial tissues, a small hand-held motorized glass-pestle homogenizer was used following the same procedure. ACE activity was measured using the ACE-REA kit from American Laboratory Products Company, Ltd. (Alpco, Windham, NH). ACE activity assay was performed following the kit instructions and activity was defined as that inhibited by captopril. Protein concentration was measured using BCA Protein Assay Reagent kit (Pierce, Rockford, IL). Tissue ACE activity was calculated as ACE units per μg protein. For Western blot, tissue homogenates were prepared as described for the ACE activity assay. Protein samples (20 μg per lane) were separated on an 8% SDS gel and transferred to a nitrocellulose membrane. The membrane was blotted using a rabbit polyclonal anti-mouse ACE antibody19Langford KG Shai SY Howard TE Kovac MJ Overbeek PA Bernstein KE Transgenic mice demonstrate a testis-specific promoter for angiotensin-converting enzyme.J Biol Chem. 1991; 266: 15559-15562Abstract Full Text PDF PubMed Google Scholar and exposed to X-ray film using the enhanced chemiluminescence method. For histological analysis, tissue samples were taken at euthanasia and preserved in 10% neutral-buffered formalin. Lung tissues were infused with formalin through the trachea. Tissues were then embedded in paraffin using standard procedures. Sections were stained for hematoxylin and eosin, or picro-sirius red using standard techniques. For immunohistochemistry, both ACE 8/8 and wild-type tissues were placed on a single slide. Immunohistochemical detection of ACE was performed as previously described.19Langford KG Shai SY Howard TE Kovac MJ Overbeek PA Bernstein KE Transgenic mice demonstrate a testis-specific promoter for angiotensin-converting enzyme.J Biol Chem. 1991; 266: 15559-15562Abstract Full Text PDF PubMed Google Scholar ACE 8/8 and wild-type mice were anesthetized with a mixture of ketamine (125 mg/kg) and xylazine (12.5 mg/kg) administered by IP injection. Blood was collected from the inferior vena cava directly into a syringe containing 5 ml 4 mol/L guanidine thiocyanate (GTC) using a 25-gauge needle. Tissues were then rapidly removed and immediately rinsed briefly in cold isotonic saline, weighed, and homogenized in 5 ml GTC. The GTC blood and tissue homogenates were then frozen at −80°C and shipped on dry ice to St. Vincent's Institute of Medical Research where peptide measurements were performed. Angiotensin I, angiotensin II, and bradykinin peptides were measured using HPLC-based radioimmunoasays as previously described.20Campbell D Lawrence A Kladis A Duncan A-M Strategies for measurement of angiotensin and bradykinin peptides and their metabolites in central nervous system and other tissues Edited by Smith AI Methods.Neurosci. 1995; 23: 328-343Crossref Scopus (25) Google Scholar The method allows analysis of both angiotensin I and angiotensin II peptides in the same sample during a single HPLC run thus reducing the variance of the peptide ratio. Data from one outlier ACE 8/8 mouse was eliminated from both the angiotensin and bradykinin calculations because the data were greater than 3 standard deviations removed from the means. Systolic blood pressure was measured in conscious mice using a Visitech Systems BP2000 automated tail cuff system (Apex, NC) as previously described.3Esther Jr, CR Howard TE Marino EM Goddard JM Capecchi MR Bernstein KE Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility.Lab Invest. 1996; 74: 953-965PubMed Google Scholar Mice were trained in the apparatus for 5 days before data were collected. The blood pressure of an animal was the average of 80 measurements over an additional 4 days. Spot urine samples were collected before and after 24 hours of water deprivation. Urine samples were spun at 5500 × g to precipitate particulates. Urine osmolality was determined using a Wescat 5500 Vapor Pressure Osmometer (Wescor Inc., Logan, UT). Mice were euthanized and the hearts were isolated. The whole hearts were briefly rinsed in 0.9% saline to remove blood. Both atria were carefully removed from the ventricles at the atrial-ventricular septum. The atria and ventricles were then blotted dry and weighed separately. Mice ages 7 to 11 weeks were anesthetized with tribromoethanol (0.25 mg/g body weight). M-mode echocardiogram studies were performed as described.21Sutliff RL Haase C Russ R Hoit BD Morris R Norman AB Lewis W Cocaine increases mortality and cardiac mass in a murine transgenic model of acquired immune deficiency syndrome.Lab Invest. 2003; 83: 983-989Crossref PubMed Scopus (8) Google Scholar M-mode measurements of end-diastolic dimension (EDD), end-systolic dimension (ESD), intraventricular wall septum thickness (IVS) and end-diastolic posterior wall thickness (PW) were made from original tracings. Calculated variables included the following: left ventricular fractional shortening (FS = (EDD − ESD)/EDD), relative wall thickness (RWT = (IVS+PW)/EDD), LV mass = 1.06 × ((EDD + PW + IVS)3 − (EDD)3, and LV mass normalized by body weight (LV/BW). Echocardiography was analyzed by B.D.H. who was blinded to genotype of the mice. Mice ages 8 to 12 weeks were anesthetized with a mixture of ketamine (125 mg/kg) and xylazine (12.5 mg/kg) administered by IP injection. The mice were placed on a heated pad during the surgery. A 1.4 French Millar high fidelity pressure catheter (SPR-671, AD Instruments, CO) was inserted into the right carotid artery and then advanced into the left ventricle. The catheter was calibrated using an external analog manometer. Data were recorded using a Powerlab system and Chart 5 software (AD Instruments, CO) with a sample speed of 1 k/s. Heart rate, left ventricular (LV) systolic pressure, and LV end-diastolic pressure were calculated directly from LV pressure wave forms. LV dP/dt max and LV dP/dt min were obtained as the first-degree differential of the LV pressure. The time constant of isovolumic LV relaxation, t, was estimated by an unweighted non-linear least squares method from 42 individual wave forms. ECG recordings of awake, free-moving mice were obtained using a telemetry method. After mice were sedated with an IP injection of ketamine and xylazine mix (125 mg/kg and 12.5 mg/kg), an EA-F20 ECG transmitter (Data Sciences, MN) was implanted in the intraperitoneal cavity. The positive lead of the transmitter was tunneled subcutaneously to the left anterior chest wall above the apex of the heart and the negative lead to the right shoulder. This configuration approximates lead II on the surface ECG. After 24 hours for recovery from the surgery, the ECG was recorded digitally for 2 minutes at the beginning of each hour using a 500 Hz A/D converter. ECG data were analyzed using Dataquest ART Software, version 2.3 (Data Sciences, MN). For data presentation, recordings were filtered with 100 Hz low-pass filter to reduce noise levels. Signal averaging was used before interval and waveform analysis (ECG Analysis Software 4.0, Data Sciences, MN). For this analysis, a 2-minute stretch of ECG recording was used and complexes were identified by the T-end fit method with a filter cut-off of 100 Hz and a T-end threshold of 30%. Interval correction for heart rate was calculated using Bazett's formula.22Bazett HC An analysis of the time relations of electrocardiograms.Heart. 1920; 7: 353-370Google Scholar All data were expressed as means ± SE. The significance of the difference between two groups was obtained by an unpaired Student's t-test. The significance of the difference among multiple groups was obtained using analysis of variance and the Tukey HSD test. Homologous recombination was used to modify the ACE gene so that ACE was produced specifically by cardiac tissue. For this purpose, a targeting vector was made in which a neomycin resistance cassette and a 4.4-kb portion of the α-MHC promoter were inserted into a BssH II restriction site, positioned between the start of somatic ACE transcription and translation (Figure 1). This strategy positions the neomycin resistance cassette to block any influence of the endogenous somatic ACE promoter on ACE gene transcription. It also positions the mouse α-MHC promoter, a well-known cardiac-specific promoter, to control the transcription of somatic ACE. This strategy does not alter the testis ACE promoter and the resulting mice were predicted to be fully fertile. We refer to this new line of mice as ACE.8 since it was the eighth modification of the ACE gene prepared in our laboratory. Targeted homologous recombination in embryonic stem cells was performed as previously described.18Cole J Quach du L Sundaram K Corvol P Capecchi MR Bernstein KE Mice lacking endothelial angiotensin-converting enzyme have a normal blood pressure.Circ Res. 2002; 90: 87-92Crossref PubMed Scopus (66) Google Scholar Proper homologous targeting was verified by both genomic Southern blot analysis and by PCR. Chimeric mice were bred to produce agouti F1 offspring; male and female heterozygous F1 mice were used to produce F2 mice. Of 368 F2 mice genotyped, 96 (26%) were wild-type, 177 (48%) were heterozygous, and 95 (26%) were ACE 8/8 mice. This Mendelian ratio indicates that the ACE 8/8 mutation does not significantly increase mortality before weaning at 3 weeks of age. ACE 8/8 mice appeared grossly normal. Body weight, as determined weekly between 3 and 8 weeks of age, showed no difference between ACE 8/8 mice and wild-type littermates (data not shown). To evaluate the tissue distribution of ACE, wild-type and ACE 8/8 mice were sacrificed and tissue extracts of individual organs were tested for ACE activity (Figure 2A). ACE 8/8 mice had a marked increase in cardiac ACE activity. Specifically, wild-type mice had ACE activity levels in atria and ventricles of 1.2 ± 0.2 U/μg protein and 0.8 ± 0.1 U/μg protein, while ACE activity in ACE 8/8 mice increased about 100-fold to 106.4 ± 7.3 U/μg protein in the atria and 104.5 ± 4.5 U/μg protein in ventricles. Significant ACE activity was also detected in lung and plasma where ACE 8/8 mice had 43% and 56%, respectively, of the activity found in wild-type mice. ACE levels in the testis were similar to those of wild-type. In contrast, kidney (Figure 2A), intestine, spleen, brain, muscle, fat, and liver had virtually undetectable ACE activity in ACE 8/8 mice. In particular, the kidney represents a major change from wild-type mice as this organ normally expresses a substantial amount of ACE activity in both vascular endothelium and proximal tubular epithelium. ACE activity in the heart, plasma, and kidney of heterozygous mice was intermediate that of wild-type and ACE 8/8 mice (data not shown). To confirm the results obtained with ACE activity assays, we also performed Western blot analysis of tissue extracts from wild-type and ACE 8/8 mice (Figure 2B). This analysis confirmed the ACE activity data in that a large amount of ACE protein was detected in the whole heart of ACE 8/8 mice. Also, significant ACE protein was observed in lung and plasma. In contrast, no ACE protein was detected in tissue extracts of the kidney in ACE 8/8 mice. Testis ACE protein expression was not altered. In agreement with the ACE activity assay results, ACE protein was also undetectable in the intestine, brain, muscle, liver, and fat of the ACE 8/8 mice by Western blot (data not shown). To understand ACE expression patterns in more detail, we performed immunohistochemistry using an anti-ACE antibody (Figure 3). These data were consistent with both the enzyme activity assay and the Western blot analysis. In wild-type mice, cardiac myocytes produced little amount of ACE. In the ACE 8/8 heart, high levels of ACE were found in both ventricles and atria. The ACE was identified on the cell surface of cardiac myocytes. This tissue pattern of distribution is expected as ACE is a membrane-anchored protein normally localized on the surface of cells. Interestingly, there was one tissue within the heart that underexpressed ACE. This tissue was vascular endothelium, which produced ACE in wild-type animals but not in ACE 8/8 mice (Figure 3D). A similar situation was observed in the kidney where wild-type mice expressed ACE in proximal tubular epithelium and vascular endothelium (Figure 4). The kidneys from ACE 8/8 mice had no such expression. In mice that are null for all ACE expression (ACE knockout mice), the kidney shows a phenotype characterized by under-development of the renal medulla and papilla, expansion of the renal calyx and vascular smooth muscle hyperplasia resulting in vascular wall thickening.3Esther Jr, CR Howard TE Marino EM Goddard JM Capecchi MR Bernstein KE Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility.Lab Invest. 1996; 74: 953-965PubMed Google Scholar Such a phenotype was not observed in the ACE 8/8 mice where renal medullary development was normal, and vascular wall thickness was equivalent to that of wild-type mice. In contrast to the heart and the kidney, the pattern of ACE expression in the lung was complex (Figure 4). In wild-type animals, lung endothelium produced abundant ACE, which was distributed homogeneously throughout the lung parenchyma. In the ACE 8/8 mice, however, lung parenchymal expression of ACE was patchy, with microscopic areas of tissue producing the enzyme immediately juxtaposed to similar-sized areas lacking discernable immunoreactive ACE (Figure 4D). It was difficult to determine which cell types in the lung parenchyma were responsible for the ACE expression (smooth muscle, type 1 epithelial cells, or endothelium). A more consistent pattern of ACE expression was observed in the pulmonary artery branches accompanying the bronchial tree. Here, high level ACE expression was observed in vascular smooth muscle (Figure 4H). In contrast, blood vessel endothelium appeared to produce little, if any, ACE. The expression of ACE in pulmonary tissues of ACE 8/8 mice was not a complete surprise. The original characterization of the α-MHC promoter in transgenic mice documented promoter activity in pulmonary vascular smooth muscle.23Subramaniam A Jones WK Gulick J Wert S Neumann J Robbins J Tissue-specific regulation of the alpha-myosin heavy chain gene promoter in transgenic mice.J Biol Chem. 1991; 266: 24613-24620Abstract Full Text PDF PubMed Google Scholar As to why lung parenchyma expressed ACE in a patchy pattern, this must reflect heterogeneity of the lung tissue and differential recognition of the α-MHC promoter. Our hypothesis was that mice with ACE expression shifted to cardiac tissue would generate high levels of cardiac angiotensin II. To measure this, wild-type and ACE 8/8 mice were sacrificed, and ventricles, kidney, and plasma were prepared for HPLC determination of angiotensin peptide levels (Figure 5, a to c). In the ventricles, there was a marked difference in the tissue content of angiotensin II, with ACE 8/8 mice having 4.3-fold the angiotensin II concentration of ventricles from wild-type mice (8/8: 179.7 ± 24.6; WT: 41.5 ± 12.1 fmol/g, P < 0.001). In contrast, the angiotensin I levels were not significantly different between ACE 8/8 and wild-type mice. A useful measure is the angiotensin II/angiotensin I ratio, which was significantly elevated in the ACE 8/8 mice, reflecting the increased ACE activity in the heart. In blood of ACE 8/8 mice, the angiotensin II levels were not significantly different from wild-type. Angiotensin I levels were elevated compared to wild-type values (P < 0.06) resulting in an angiotensin II/angiotensin I ratio that was less than wild-type mice (P < 0.05). In the kidneys of the ACE 8/8 mice, the angiotensin II level was decreased while the angiotensin I level was elevated compared to those of wild-type mice (P < 0.05), resulting in a reduced angiotensin II/angiotensin I ratio (P < 0.01). This is consistent with decreased renal ACE activity in ACE 8/8 mice. In summary, the concentration of angiotensin II was markedly increased in the heart, in agreement with what we predicted from the increased cardiac ACE expression. A decreased ratio of angiotensin II/angiotensin I in the kidney and plasma was consistent with the decreased ACE activity in these tissues. In addition to converting angiotensin I to angiotensin II, ACE also inactivates bradykinin peptide by converting bradykinin-(1–9) into bradykinin-(1–7) (Figure 5, d to f). We measured the tissue and blood bradykinin peptide levels using an HPLC-based method. In the ventricles of ACE 8/8 mice, bradykinin-(1–9) levels were slightly increased and bradykinin-(1–7) was slightly decreased, but neither measure reached significance. These data were surprising in that we predicted a significant decrease of bradykinin-(1–9) due to increased degradation by ACE. Both blood and kidney levels of bradykinin peptides were also no different from those of wild-type mice. In total, these data suggest that, in vivo, ACE may play a l
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