Vascular Endothelial-Specific Dimethylarginine Dimethylaminohydrolase-1–Deficient Mice Reveal That Vascular Endothelium Plays an Important Role in Removing Asymmetric Dimethylarginine
Background— Asymmetrical methylarginines inhibit NO synthase activity and thereby decrease NO production. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) degrades asymmetrical methylarginines. We previously demonstrated that in the heart DDAH1 is predominantly expressed in vascular endothelial cells. Because an earlier study showed that mice with global DDAH1 deficiency experienced embryonic lethality, we speculated that a mouse strain with selective vascular endothelial DDAH1 deficiency (endo-DDAH1−/−) would largely abolish tissue DDAH1 expression in many tissues but possibly avoid embryonic lethality.
Methods and Results— By using the LoxP/Cre approach, we generated the endo-DDAH1−/− mice. The endo-DDAH1−/− mice had no apparent defect in growth or development compared with wild-type littermates. DDAH1 expression was greatly reduced in kidney, lung, brain, and liver, indicating that in these organs DDAH1 is distributed mainly in vascular endothelial cells. The endo-DDAH1−/− mice showed a significant increase of asymmetric dimethylarginine concentration in plasma (1.41 μmol/L in the endo-DDAH1−/− versus 0.69 μmol/L in the control mice), kidney, lung, and liver, which was associated with significantly increased systolic blood pressure (132 mm Hg versus 113 mm Hg in wild-type). The endo-DDAH1−/− mice also exhibited significantly attenuated acetylcholine-induced NO production and vessel relaxation in isolated aortic rings.
Conclusions— Our study demonstrates that DDAH1 is highly expressed in vascular endothelium and that endothelial DDAH1 plays an important role in regulating blood pressure. In the context that asymmetric methylarginines are broadly produced by many type of cells, the strong DDAH1 expression in vascular endothelium demonstrates for the first time that vascular endothelium can be an important site to actively dispose of toxic biochemical molecules produced by other types of cells.
Received September 9, 2008; accepted September 1, 2009.
Nitric oxide (NO) produced by NO synthases (NOS) exerts many important biological functions. Asymmetric dimethylarginine (ADMA) and NG-monomethyl-l-arginine (L-NMMA) are endogenous NOS inhibitors that competitively inhibit NO production. Recent reports have demonstrated that accumulation of asymmetrical methylarginines is a major risk factor for cardiovascular diseases including hypertension,1,2 congestive heart failure,3,4 stroke,5 coronary heart disease,6 atherosclerosis,7 and diabetes mellitus.8
Clinical Perspective on p 2229
ADMA and L-NMMA are eliminated principally by metabolism to l-citrulline by the enzyme dimethylarginine dimethylaminohydrolase (DDAH),9,10 with a small contribution from renal excretion.11 Two isoforms of DDAH have been identified, DDAH112 and DDAH2.13 Overexpression of either DDAH1 or DDAH2 in transgenic mice led to decreased plasma ADMA levels, increased NO bioavailability, and decreased blood pressure.14,15 Conversely, global heterozygous DDAH1 gene deficiency or treatment of wild-type mice with selective DDAH inhibitors resulted in decreased acetylcholine-induced NO production and vasodilation in aortic rings.16 This study also showed that homozygous global DDAH1 gene deletion in mice was lethal in utero and that the total DDAH activity of lung, liver, and kidney was significantly decreased in heterozygous DDAH1-deficient mice.16 It was reported that the distribution of DDAH1 is similar to neuronal NOS, whereas the distribution of DDAH2 is similar to endothelial NOS, suggesting that DDAH1and DDAH2 are expressed mainly in neuronal tissue and vascular endothelium, respectively.17 However, we recently found that DDAH1 is highly expressed in vascular endothelial cells in hearts,18 which is consistent with another early report showing strong DDAH1 expression in renal vascular endothelial cells.19
In the present study, using LoxP/Cre strategy, we have generated homologous vascular endothelial-specific DDAH1 gene–deficient (endo-DDAH1−/−) mice. The endo-DDAH1−/− mice had significantly increased plasma ADMA levels, decreased acetylcholine-induced NO production and vessel relaxation in isolated vessel rings, and elevation of systolic blood pressure, indicating that vascular endothelial DDAH1 regulates NO bioavailability and vessel tone. Most interestingly, we found that DDAH1 expression was greatly reduced in tissues obtained from liver, lung, aorta, brain, skeletal muscle, and kidney in some of the endo-DDAH1−/− mice, indicating that in these organs DDAH1 is expressed predominantly in vascular endothelial cells. In the context that asymmetric methylarginines are produced by all kinds of cells, the strong expression of DDAH1 in endothelium indicates that vascular endothelial cells represent the important site for removal of asymmetric methylarginines. Because vascular endothelial cells actively take up asymmetric methylarginines, this distribution of DDAH1 in the endothelial cells suggests that the vascular endothelium not only produces biological molecules that regulate functions of other cells but also contributes to disposing of metabolic by-products generated by other cells, a biological function previously unknown for the endothelium.
Generation of Endo-DDAH1−/− Mice
The genomic DNA of DDAH 1 was amplified by polymerase chain reaction (PCR), subcloned into pGEM-Teasy vector (Promega), and confirmed by DNA sequencing. The targeting vector was constructed as shown in Figure 1A and introduced into CJ7 embryonic stem cells by electroporation. Homologous recombinant clones were identified by Southern blot (Figure 1B), and 2 independent clones were used to generate chimeras with C57BL/6J blastocysts. The heterozygous DDAHflox/+ were obtained by inbreeding the male chimeras with female C57BL/6J and identified by Southern blot and PCR with the use of primer pairs 5′-AAT CTG CAC AGA AGG CCC TCA A-3′/5′-TTC TGA ATC CCA GCC GTC TGA A and 5′-AGG ATG ATC TGG ACG AAG AGC A-3′/5′-TTC TGA ATC CCA GCC GTC TGA A-3′ to identify wild-type allele and floxed allele, respectively. The endothelial-specific DDAH1 knockout mice were generated by crossing DDAH1flox/+ with Tie2-Cre transgenic mice (Jackson Laboratory, B6.Cg-Tg [Tek-cre]12Flv/J). The endo-DDAH1−/− (DDAH1flox/flox/Tie2-Cre) was genotyped by PCR with the use of the primer pairs listed above to identify DDAH1flox/flox and primer pair 5′-GCG GTC TGG CAG TAA AAA CTA TC-3′/5′-GTG AAA CAG CAT TGC TGT CAC TT-3′ to identify mice with Tie2-Cre. The deletion of DDAH1 exon 4 was confirmed by PCR with the use of primer pair 5′-TGT CCA CAA GGG ATG CAA ACA-3′/5′-AAG CCC ATT GTG AAG TAG-3′. The disruption of DDAH1 expression was confirmed by Western blot.
Tissue samples were frozen immediately in liquid nitrogen after collection. Proteins from tissue homogenates were separated on sodium dodecyl sulfate–polyacrylamide gels and transferred to polyvinylidene fluoride nitrocellulose membranes. The primary antibody against DDAH1 was developed in our laboratories.18 DDAH2 antibody was from Santa Cruz Biotechnology, Santa Cruz, Calif. The horseradish peroxidase–conjugated secondary antibodies (Bio-Rad, Hercules, Calif) were detected with enhanced chemiluminescent substrate (Amersham, GE Healthcare, Piscataway, NJ).
Determination of Plasma and Tissue ADMA Levels
Plasma ADMA levels were determined by a validated enzyme-linked immunosorbent assay method20 (DLD Diagnostika GmbH, Hamburg, Germany). Tissue ADMA and symmetric dimethylarginine (SDMA) concentrations were determined as described previously.21
Mice were anesthetized with 1.5% isoflurane. Echocardiographic images were obtained with a Visualsonics Veve 770 system as described previously.22
Evaluation of Blood Pressure and Left Ventricular Hemodynamics
Invasive hemodynamic studies were performed as described previously.23 Briefly, mice were anesthetized with 3% isoflurane, incubated with the use of a 24-gauge catheter, and ventilated with a mouse ventilator (HSE Harvard Apparatus). The ventilation rate was set at 120 breaths per minute with a tidal volume of 250 to 300 μL. The anesthesia was maintained with 2% isoflurane. For the hemodynamic measurement, a 1.2F pressure catheter (Scisense Inc, Ontario, Canada) was introduced into the right common carotid artery for aortic pressure measurement and then advanced into the left ventricle (LV) for measurement of LV pressure, its first derivative (LV dP/dt), and heart rate. Data represent the mean of at least 10 beats of recording during stable hemodynamic conditions. Tail blood pressure was determined in conscious mice with the XBP 1000 system (Kent Scientific, Torrington, Conn).
Measurement of NO Production and Vessel Relaxation
Cross sections of aorta (≈2 mm) were incubated with 10 μmol/L NO-specific fluorescent probe 4,5-diaminofluoresceine diacetate (DAF-2 DA) (Sigma, St Louis, Mo) dye and 100 μmol/L l-arginine (Sigma) at 37°C for 30 minutes and then washed with Dulbecco’s phosphate-buffered saline to remove free dye. For those sections treated with NG-nitro-l-arginine methyl ester (L-NAME), 100 μmol/L L-NAME was added during the last 20 minutes of DAF-2 DA incubation, and no l-arginine was added. For measurement of NO production, DAF-2 was excited at a wavelength of 490 nm with an emitted fluorescence at 515 nm. The fluorescence was captured by an Olympus FluoView 1000 confocal microscope. The signal intensity was recorded every 10 seconds for 5 minutes for each study.
For measuring vessel relaxation, mouse thoracic aortic rings were mounted in myographs (in PSS buffer bubbled with 95% O2 and 5% CO2 at 37°C) at 0.25g resting tension. Vessels were then contracted with 10−7 mol/L phenylephrine as described previously.24 After the vessel contraction was stabilized, acetylcholine or sodium nitroprusside was added at indicated doses.
Values are expressed as mean±SEM. At least 4 animals from each group were used for each study. Unpaired Student t tests were performed to compare endo-DDAH1−/− mice with their littermate controls. For the study of acetylcholine-induced NO production in isolated aortic rings, 3-way repeated-measures ANOVA was used. Two-way repeated-measures ANOVA was used for the study of acetylcholine-induced vessel dilation in aortic rings. If the ANOVA demonstrated a significant effect, post hoc comparisons were made pairwise with the Fisher least significant difference test. P<0.05 was considered statistically significant.
Endothelium-Specific Deletion of DDAH1 Gene
The targeting vector was generated by inserting DDAH1 genomic DNA into pflox-FRT-Neo vector (Figure 1A).25 Two LoxP sites were introduced into the third and fourth introns of DDAH1 gene. The targeting vector was linearized with Not I before transfection into CJ7 embryonic stem cells by electroporation. The homologous recombinant clones were identified by Southern blot (Figure 1B) and injected into blastocysts from C57BL/6J mice to generate chimeras. The male chimeras were inbred with female C56BL/6J, and germ-line transmitted heterozygous mice DDAH1flox/+ were identified by Southern blot (Figure 1B). The DDAH1flox/+ mice were crossed with Tie2-Cre transgenic mice (Jackson Laboratory, B6.Cg-Tg [Tek-cre]12Flv/J) that have endothelial-specific expression of Cre. The double heterozygous DDAH1flox/+/Tie2-Cre were identified by PCR and further inbred with DDAH1flox/+/Tie2-Cre or crossed with DDAH1flox/+ to generate DDAH1flox/flox/Tie2-Cre. Three types of control mice were generated from this breeding: DDAH1flox/flox, DDAH1+/+/Tie2-Cre, and DDAH1+/+. There were no significant differences in terms of DDAH1 protein level, LV function, blood pressure, heart rate, and ratio of heart weight to body weight (data not shown) among these 3 control mice. Therefore, in the following studies, DDAH1flox/flox/Tie2-Cre were crossed with DDAH1flox/flox to generate DDAH1flox/flox/Tie2-Cre (endo-DDAH1−/−), and their DDAH1flox/flox littermates were used as controls.
The endo-DDAH1−/− mice were viable and grew and developed normally. PCR with the use of primers surrounding exon 4 showed that exon 4 was deleted in heart, lung, and kidney of endo-DDAH1−/− mice (Figure 1C). Because the amplification of the exon 4 floxed allele (≈1.8-kb PCR product) is less efficient than the amplification of exon 4 deleted allele (≈1.3-kb PCR product), only the exon 4 deleted allele could be detected by PCR of endo-DDAH1−/− genomic DNA (Figure I in the online-only Data Supplement).
Endothelium-Specific Deletion of the DDAH1 Gene Greatly Reduces the Expression of DDAH1 in Several Organs
Although the expression levels of DDAH1 among different organs were dramatically different, Western blot with DDAH1-specific antibody detected almost no DDAH1 protein expressed in lung, kidney, brain, liver, and skeletal muscle (Figure 2A) of the endo-DDAH1−/− mice. After prolonged exposure, a small amount of DDAH1 could be detected in the endo-DDAH1−/− heart (Figure 2A) and aorta (Figure II in the online-only Data Supplement). The amount of DDAH1 in the heterozygous DDAHflox/+/Tie2-Cre mice in these organs was ≈50% that of control (Figure 2A). This result suggested that DDAH1 is highly expressed in endothelium of these organs.
Endothelium-Specific Deletion of DDAH1 Gene Has No Effect on DDAH2 Expression
To determine whether endothelial DDAH1 gene deletion would cause compensatory upregulation of DDAH2 expression, the protein content of DDAH2 was determined in several organs of endo-DDAH1−/− mice. As shown in Figure 2B, the protein levels of DDAH2 in the endo-DDAH1−/− heart, lung, and kidney was not significantly different from control, indicating that depletion of DDAH1 in endothelium did not affect the expression of DDAH2 in these organs.
Endo-DDAH1−/− Increases ADMA Levels in Plasma, Kidney, Lung, and Liver and Reduces Acetylcholine-Induced NO Generation and Vasodilation in Aortic Rings
With the depletion of DDAH1 in the endothelium, plasma ADMA levels in the endo-DDAH1−/− mice were >75% higher than in the control mice (1.41 μmol/L in endo-DDAH1−/− mice versus 0.69 μmol/L in the control mice; P<0.05; Figure 3A). In addition, the ADMA content in lung, liver, and kidney was significantly higher in the endo-DDAH1−/− mice compared with control (Figure 3B), suggesting that endothelial DDAH1 plays a pivotal role not only in clearing circulating ADMA but also in degrading ADMA in these organs. The SDMA content was not different between the endo-DDAH1−/− and control mice in lung and kidney. In liver, however, SDMA was significantly increased by ≈60% in the endo-DDAH1−/− mice compared with control (Figure 3C).
To determine whether the increased ADMA level influences NO availability, we measured NO generation in response to acetylcholine stimulation in isolated aortic rings using DAF-2 DA. Endothelial-specific deletion of DDAH1 gene attenuated acetylcholine-induced NO production in the aortic rings derived from the endo-DDAH1−/− mice, whereas 100 μmol/L of L-NAME almost completely blocked the acetylcholine-induced NO generation from both control and endo-DDAH1−/− aortic rings (Figure 3D). These data demonstrate that disruption of endothelial DDAH1 impairs acetylcholine-induced NO generation. In addition, the endo-DDAH1−/− significantly reduced aortic ring relaxation in response to acetylcholine but had no effect on their relaxation in response to the NO donor sodium nitroprusside (Figure 3E and 3F).
Endo-DDAH1−/− Mice Have Increased Blood Pressure
To determine whether vascular endothelial DDAH1 is involved in regulating vascular tone, blood pressure was evaluated in both conscious mice and mice during anesthesia. Measured in the conscious state by the tail-cuff method, the endo-DDAH1−/− mice exhibited significantly increased systemic blood pressure (132±2 mm Hg in the endo-DDAH1−/− mice versus 113±3 mm Hg in the control mice) (Figure 4A). Invasive hemodynamic studies showed that both the systolic blood pressure (endo-DDAH1−/−, 110±2 mm Hg versus control, 99±3 mm Hg; Figure 4B and 4D) and the mean aortic pressure (endo-DDAH1−/−, 89±2 mm Hg versus control, 75±3 mm Hg; Figure 4C and 4D) were significantly higher in endo-DDAH1−/− mice than in their control littermates. Vascular resistances were calculated with the use of blood pressure (tail-cuff method) and cardiac output. The result showed that vascular resistance (endo-DDAH1−/−, 5.06±0.36 mm Hg/mL per minute versus control, 3.67±0.45 mm Hg/mL per minute) was significantly increased in endo-DDAH1−/− mice.
Endo-DDAH1−/− Mice Have Normal Ventricular Size and Function at 3 Months of Age
Aortic and LV systolic pressures were significantly increased in the endo-DDAH1−/− mice (Figure 4B and 4C). Despite this significant increase of blood pressure, cardiac structure and function of the young endo-DDAH1−/− mice remained normal compared with control littermates. There was a trend toward an increase in the ratios of heart weight, LV weight, right ventricular weight, and lung weight to tibial length in the endo-DDAH1−/− mice. However, none of these changes were statistically significant. LV ejection fraction and dimensions were not significantly different between control and endo-DDAH1−/− mice. Taken together, at 3 months of age, the endo-DDAH1−/− mice had not developed LV hypertrophy or dysfunction. However, DDAH1 depletion may cause subtle changes in the cardiac structure and functions.
The significant findings of this study are as follows: (1) endo-DDAH1−/− greatly reduced DDAH1 expression in organs such as lung, liver, kidney and brain; (2) endo-DDAH1−/− causes an increase of the endogenous NOS inhibitor ADMA in plasma, kidney, liver, and lung; (3) endo-DDAH1−/− mice have increased systemic blood pressure; and (4) endo-DDAH1−/− has no significant impact on growth and development or on cardiac function in young mice.
Because asymmetric methylarginines are produced by almost all type of cells and broadly inhibit all 3 NOS isoforms, the endo-DDAH1−/− and the DDAH1flox/flox mice are anticipated to be useful tools for the study of DDAH1 and asymmetrical methylarginines in the development of clinical complications such as hypertension, diabetes mellitus, and congestive heart failure. These strains may be particularly useful for the study of tissue-specific NO bioavailability in various organs under physiological or pathological conditions.
It is well known that the endothelium regulates many biological and physiological functions, including the control of vascular tone, leukocyte and biomolecule trafficking, cell growth and proliferation/angiogenesis, and inflammatory responses. However, it was not previously known that vascular endothelium can play an important role in removing toxic biochemical molecules. Our findings that endothelial-specific DDAH1 gene deletion reduced DDAH1 expression in all organs tested and increased plasma and tissue ADMA levels demonstrate that the vascular endothelium has a unique function to dispose of asymmetrical methylarginines produced in other tissues. In addition, it is perceivable that the strategic distribution of DDAH1 in vascular endothelium would benefit endothelial cells by degrading the endogenous NOS inhibitors and promoting local endothelial NOS activity and NO production.
The elevated ADMA level in the endo-DDAH1−/− mice is associated with a moderate increase of systemic blood pressure in these mice. This moderate increase of blood pressure in the endo-DDAH1−/− mice essentially duplicates the phenotype observed in young endothelial NOS knockout mice.26 Reports from several clinical studies showed that ADMA levels increase in patients with hypertension.27–29 Thus, decreased DDAH activity and accumulation of ADMA may have a causal relationship with hypertension. Furthermore, endo-DDAH1−/− impairs acetylcholine-induced NO production and vasodilation in isolated aortic rings. Studies using selective DDAH inhibitors or in the heterozygous global DDAH1-null mice showed similar results.16 These data suggest that DDAH1 is critical in ADMA clearance, endothelium-dependent vasodilation, and maintenance of normal blood pressure.
In some of the endo-DDAH1−/− mice, DDAH1 expression was almost undetectable in many organs. In the context that a previous study showed that global homozygous DDAH1 is lethal at the embryonic stage,16 the finding that depletion of DDAH1 has no significant effects on growth and development is unanticipated. Because the expression of Tek/Tie2 is detectable at around embryonic day 8.0 in the mouse,30,31 whereas the expression of DDAH1 seems to be important in trophoblast invasion and embryo implantation32 (which takes place during embryonic day 4 to 6 in most laboratory rodents33), a potential explanation for the discrepancy might be that DDAH1 expression is required by some biological processes before the Tie2 promoter becomes active during embryogenesis. Therefore, DDAH1 expression is not indispensable for embryonic development after Tie2 expression occurs. Alternatively, the construct from the previous global DDAH1−/− may exert some unwanted effects in addition to the deletion of the DDAH1 gene. Another possible explanation might be that DDAH1 expression is not totally abolished in our tissue-specific DDAH1−/− strain. Thus, the residual DDAH1, particular the DDAH1 expressed in nonendothelial cells, may be critical for the survival and early development of embryos. Further studies are needed to address this question.
DDAH1 protein is highly expressed in kidney, liver, brain, and lung. Kidney and liver are reported to be the 2 organs that have the highest capacity for ADMA uptake and clearance.34 We found that kidney had the highest free ADMA of all organs tested and that endothelial-specific DDAH1 deletion caused significant ADMA accumulation in both kidney and liver. We also observed a significant 45% increase of pulmonary ADMA in the endo-DDAH1−/− mice. Overall, these results suggest that DDAH1 is essential in regulating ADMA levels in these organs. The long-term physiological impact of increased ADMA levels, and consequently decreased NO bioavailability, in the endo-DDAH1−/− mice is yet to be determined. Although SDMA cannot be metabolized by DDAH1, the SDMA content in liver was also significantly increased in the endo-DDAH1−/− mice. Ogawa et al35 identified a common pathway for degradation of both ADMA and SDMA through the formation of α-ketoacid analogues and the oxidatively decarboxylated products of the α-ketoacids. When the major metabolic route to eliminate ADMA by DDAH1 is not available, increased amounts of ADMA may compete with SDMA for this alternative route for degradation. This could contribute to the elevation of liver SDMA in endo-DDAH1−/− mice.
The cellular distribution of DDAH1 has been a subject of controversy for years. Thus, with the use of an antibody detecting a protein at ≈34 kDa, an early study from Tojo et al19 demonstrated that DDAH1 was strongly expressed in vascular endothelial cells. We found recently that DDAH1 was expressed primarily in vascular endothelial cells in hearts,18 and we confirmed the specificity of the antibody used in our study by using selective DDAH1 gene silencing in cultured human endothelial cells. In the present study, we found that endothelial-specific DDAH1 gene deletion resulted in greatly reduced DDAH1 protein (at ≈34 kDa) in kidney, liver, lung, skeletal muscle, and brain, and this was accompanied by increased tissue and plasma ADMA levels. Moreover, the Tie2-Cre strain of mouse used in this study has been well defined,36 and studies demonstrated that the regulatory cassette that controls Cre expression in this strain has uniform and specific endothelial-specific promoter/enhancer activity at both embryonic and adult stages.36,37 Thus, our data indicated that DDAH1 is highly expressed in vascular endothelial cells.
However, Western blot analysis in several recent studies identified a protein at 38 to 40 kDa as DDAH1.38 With the use of the same antibody as for the Western blot in their immunohistochemical staining, these studies demonstrated that DDAH1 was also expressed in cell types other than endothelial cells, such as bronchial smooth muscle cells,38 proximal tubules of the nephron,39 and vascular smooth muscle cells.40 Because the antibody used in the aforementioned studies is not available to us, we are not sure whether the discrepancy among different groups is due to the inconsistent labeling of protein size of DDAH1 or is an issue of antibody specificity. Therefore, the exact cause of the discrepancy regarding DDAH1 distribution is not clear at the present time. It is hoped that the tissue samples from our DDAH1-deficient mice can help to address this question. Moreover, because Western blots may not be able to detect the residual amount of DDAH1 in the whole organ extracts, the data from our study cannot exclude small amounts of DDAH1 expressed in cells other than endothelial cells. Indeed, our data demonstrated that DDAH1 was detectable in heart and aorta obtained from endo-DDAH1−/− mice, indicating that DDAH1 is at least also expressed in cardiac myocytes18 and vascular smooth muscle cells.40
There are several limitations in the present study. To confirm the tissue-specific DDAH1 deletion in endo-DDAH1−/− mice, we have made a significant effort to develop antibodies suitable for immunohistochemical staining of DDAH1 in mouse tissues. Unfortunately, we are still currently unable to detect DDAH1 in mouse tissue samples by immunohistochemistry. Therefore, we cannot exclude the possibility that DDAH1 expression might also be disrupted in other types of cells in this endo-DDAH1−/− strain. In addition, although we found that there is no upregulation of DDAH2 in the heart, lung, and kidney in the endo-DDAH1−/− mice, we did not determine endothelial DDAH2 expression and the relative contribution of DDAH1 and DDAH2 on endothelial DDAH activity in these mice. These are the limitations of this study. Furthermore, Tie2-expressing endothelial progenitor cells may theoretically differentiate into non–endothelial cell types that have little or no Tie2 expression at later developmental or adult stages. Thus, even though a differentiated cell may have no Tie2 promoter activity (or Cre expression), the DDAH1 gene may have been deleted in their precursor cells before differentiation. Therefore, although Tie2-Cre mice have uniform and specific endothelial-specific promoter/enhancer activity; we could not totally exclude the possibility of DDAH1 gene deletion in cell types other than vascular endothelial cells. This is the limitation of using the Tie2-Cre strain to generate endothelial-specific gene-deficient strains.
In summary, our present study demonstrates that DDAH1 is greatly reduced in several organs in the endo-DDAH1−/− strain, indicating that vascular endothelium is an important site for removal of toxic asymmetric methylarginines. In addition, we found that endo-DDAH1−/− mice have increased plasma and tissue ADMA levels and blood pressure, indicating that vascular DDAH1 plays an important role in regulating NO bioavailability and blood pressure. Our study also suggests that DDAH1 expression may not be indispensable for embryonic development after Tie2 expression occurs. Finally, the DDAH1flox/flox and endo-DDAH1−/− mouse strains could be of considerable utility for studies of NO bioavailability in many physiological and pathological conditions, possibly in a tissue-specific fashion.
The endo-DDAH1−/− mouse strain was developed with help from the Mouse Genetics Laboratory of the University of Minnesota. We thank Dr Haoyu Yu for her assistance with statistical data analysis.
Sources of Funding
This study was supported by National Heart, Lung, and Blood Institute grant HL71790 (Dr Chen) from the National Institutes of Health. Dr Xu is a recipient of an American Heart Association Postdoctoral Fellowship. Drs Hu and Zhang are recipients of the Scientist Development Award from the American Heart Association National Center.
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Asymmetrical methylarginines asymmetric dimethylarginine (ADMA) and NG-monomethyl-l-arginine are endogenous NO synthase inhibitors. Recent clinical studies demonstrated that the elevation of ADMA levels is associated with many pathological conditions. Thus, ADMA becomes an independent risk indicator for cardiovascular diseases. Asymmetrical methylarginines are eliminated from the body mainly through degradation by dimethylarginine dimethylaminohydrolase (DDAH). Thus, impaired DDAH activity due to either loss-of-function polymorphisms of the DDAH gene or posttranslational modification of DDAH protein may cause accumulation of asymmetrical methylarginines. In this study, we found that endothelial-specific depletion of DDAH1 in endo-DDAH1−/− mice leads to increases of plasma and tissue ADMA concentration and systemic blood pressure, as well as decrease of acetylcholine-induced NO generation and vasodilation in the isolated vessel rings. These results demonstrated that endothelial DDAH1 plays an important role in metabolizing ADMA and regulating blood pressure and vessel tone. In addition, the endo-DDAH1−/− and the DDAH1flox/flox mouse strains will become useful tools for studies of the NO signaling pathway in various tissues under physiological or pathological conditions.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.819912/DC1.