Potent and selective inhibitors of the enzyme dimethylarginine dimethylaminohydrolase (DDAH) are useful as molecular probes to better understand cellular regulation of nitric oxide. Inhibitors are also potential therapeutic agents for treatment of pathological states associated with the inappropriate overproduction of nitric oxide, such as septic shock, selected types of cancer, and other conditions. Inhibitors with structures dissimilar to substrate may overcome limitations inherent to substrate analogs. Therefore, to identify structurally-diverse inhibitor scaffolds, high-throughput screening (HTS) of a 4000-member library of fragment-sized molecules was completed using the Pseudomonas aeruginosa DDAH and human DDAH-1 isoforms. Use of a substrate concentration equal to its KM value during the primary screen allowed for the detection of inhibitors with different modes of inhibition. A series of validation tests were designed and implemented in the identification of four inhibitors of human DDAH-1 that were unknown prior to the screen. Two inhibitors share a 4-halopyridine scaffold and act as quiescent affinity labels that selectively and covalently modify the active-site Cys residue. Two inhibitors are benzimidazole-like compounds that reversibly and competitively inhibit human DDAH-1 with Ligand Efficiency values ≥ 0.3 kcal / mol / heavy (non-hydrogen) atom, indicating their suitability for further development. Both inhibitor scaffolds have available sites to derivatize for further optimization. Therefore, use of this fragment-based HTS approach is demonstrated to successfully identify two novel scaffolds for development of DDAH-1 inhibitors.
Fragment library; High-throughput screen; Inhibitor discovery; Dimethylarginine dimethylaminohydrolase; Nitric oxide
Ischemia-induced angiogenesis is critical for tissue repair, but aberrant neovascularization in the retina causes severe sight impairment. Nitric oxide (NO) has been implicated in neovascular eye disease because of its pro-angiogenic properties in the retina. Nitric oxide production is inhibited endogenously by asymmetric dimethylarginines (ADMA and L-NMMA) which are metabolized by dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2. The aim of this study was to determine the roles of DDAH1, DDAH2, ADMA and L-NMMA in retinal ischemia-induced angiogenesis. First, DDAH1, DDAH2, ADMA and L-NMMA levels were determined in adult C57BL/6J mice. The results obtained revealed that DDAH1 was twofold increased in the retina compared to the brain and the choroid. DDAH2 expression was approximately 150 fold greater in retinal and 70 fold greater in choroidal tissue compared to brain tissue suggesting an important tissue-specific role for DDAH2 in the retina and choroid. ADMA and L-NMMA levels were similar in the retina and choroid under physiological conditions. Next, characterization of DDAH1+/− and DDAH2−/− deficient mice by in vivo fluorescein angiography, immunohistochemistry and electroretinography revealed normal neurovascular function compared with wildtype control mice. Finally, DDAH1+/− and DDAH2−/− deficient mice were studied in the oxygen-induced retinopathy (OIR) model, a model used to emulate retinal ischemia and neovascularization, and VEGF and ADMA levels were quantified by ELISA and liquid chromatography tandem mass spectrometry. In the OIR model, DDAH1+/− exhibited a similar phenotype compared to wildtype controls. DDAH2 deficiency, in contrast, resulted in elevated retinal ADMA which was associated with attenuated aberrant angiogenesis and improved vascular regeneration in a VEGF independent manner. Taken together this study suggests, that in retinal ischemia, DDAH2 deficiency elevates ADMA, promotes vascular regeneration and protects against aberrant angiogenesis. Therapeutic inhibition of DDAH2 may therefore offer a potential therapeutic strategy to protect sight by promoting retinal vascular regeneration and preventing pathological angiogenesis.
•Nitric oxide has been implicated in neovascular eye disease.•Key inhibitor of NO production is ADMA, which is metabolized by DDAH.•DDAH2 deficiency results in elevated ADMA and reduced neovascularization in mice.•Therapeutic inhibition of ADMA or DDAH2 may offer a potential therapeutic strategy.
DDAH1; DDAH2; ADMA; Nitric oxide; Angiogenesis; Retinal neovascularization
Dimethylarginine dimethylaminohydrolase (DDAH) is an endogenous regulator of nitric oxide production and represents a potential therapeutic target. However, only a small number of biologically useful inhibitors have been reported, and many of these are substrate analogues. To seek more diverse scaffolds, we developed a high-throughput screening (HTS) assay and queried two small libraries totaling 2446 compounds. The HTS assay proved to be robust, reproducible, and scalable, with Z′ factors ≥ 0.78. One inhibitor, ebselen, is structurally divergent from substrate and was characterized in detail. This selenazole covalently inactivates DDAH in vitro and in cultured cells. The rate constant for inactivation of DDAH (44000 ± 2400 M–1 s–1) is greater than those reported for any other target, suggesting that this pathway is an important aspect of ebselen's total pharmacological effects.
Dimethylarginine dimethylaminohydrolase; high-throughput screening; ebselen; covalent inhibitors; nitric oxide
Dimethylarginine dimethylaminohydrolase (DDAH) is an endogenous regulator of nitric oxide production and represents a potential therapeutic target. However, only a small number of biologically useful inhibitors have been reported, and many of these are substrate analogs. To seek more diverse scaffolds, we developed a high-throughput screening (HTS) assay and queried two small libraries totaling 2446 compounds. The HTS assay proved to be robust, reproducible and scalable, with Z' factors ≥ 0.78. One inhibitor, ebselen, is structurally divergent from substrate and was characterized in detail. This selenazole covalently inactivates DDAH in vitro and in cultured cells. The rate constant for inactivation of DDAH (44,000 ± 2,400 M−1s−1) is greater than those reported for any other target, suggesting this pathway is an important aspect of ebselen's total pharmacological effects.
Dimethylarginine dimethylaminohydrolase; high-throughput screening; ebselen; covalent inhibitors; nitric oxide
Asymmetric NG,NG-dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is regulated by the enzymatic participants of synthetic and metabolic processes, i.e., type I protein N-arginine methyltransferase (PRMT) and dimethylarginine dimethylaminohydrolase (DDAH). Previous reports have demonstrated that circulating ADMA levels can vary in patients with type 1 and type 2 diabetes mellitus (T2DM). White adipose tissue expresses the full enzymatic machinery necessary for ADMA production and metabolism; however, modulation of the activities of adipose ADMA-related enzymes in T2DM remains to be determined.
A rodent model of T2DM using 11- and 20-week old Goto-Kakizaki (GK) rats was used. The expression and catalytic activity of PRMT1 and DDAH1 and 2 in the white adipose tissues (periepididymal, visceral and subcutaneous fats) and femur skeletal muscle tissue were determined by immunoblotting, in vitro methyltransferase and in vitro citrulline assays.
Non-obese diabetic GK rats showed low expression and activity of adipose PRMT1 compared to age-matched Wistar controls. Adipose tissues from the periepididymal, visceral and subcutaneous fats of GK rats had high DDAH1 expression and total DDAH activity, whereas the DDAH2 expression was lowered below the control value. This dynamic of ADMA-related enzymes in white adipose tissues was distinct from that of skeletal muscle tissue. GK rats had lower levels of serum non-esterified fatty acids (NEFA) and triglycerides (TG) than the control rats. In all subjects the adipose PRMT1 and DDAH activities were statistically correlated with the levels of serum NEFA and TG.
Activities of PRMT1 and DDAH in white adipose tissues were altered in diabetic GK rats in an organ-specific manner, which was reflected in the serum levels of NEFA and TG. Changes in adipose ADMA-related enzymes might play a part in the function of white adipose tissue.
Protein N-arginine methyltransferase 1; Dimethylarginine dimethylaminohydrolase 1 and 2; Non-esterified fatty acids; Triglycerides; Type 2 diabetes mellitus
Nitric oxide (NO) is a potent signaling molecule that needs to be tightly regulated to maintain metabolic and cardiovascular homeostasis. The nitric oxide synthase (NOS)/Dimethylarginine dimethylaminohydrolase (DDAH)/Asymmetric Dimethylarginine (ADMA) pathway is central to this regulation. Specifically, the small molecule ADMA competitively inhibits NOS, thus lowering NO levels. The majority of ADMA is physiologically metabolized by DDAH, thus maintaining NO levels at physiological concentration. However, under pathophysiological conditions, DDAH activity is impaired, in part as a result of its sensitivity to oxidative stress. Therefore, the application of high throughput chemical screening for the discovery of small molecules that could restore or enhance DDAH activity might have significant potential in treating metabolic and vascular diseases characterized by reduced NO levels, including atherosclerosis, hypertension, and insulin resistance. By contrast, excessive generation of NO (primarily driven by iNOS) could play a role in idiopathic pulmonary fibrosis (IPF), sepsis, migraine headaches, and some types of cancer. In these conditions, small molecules that inhibit DDAH activity might be therapeutically useful. Here, we describe optimization and validation of a highly reproducible and robust assay successfully used in a high throughput screen for DDAH modulators.
nitric oxide; asymmetric dimethylarginine; diabetes; hypertension; idiopathic pulmonary fibrosis
Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH-1) can raise endogenous levels of asymmetric dimethylarginine (ADMA) and lead to a subsequent inhibition of nitric oxide synthesis. Herein, N5-(1-imino-2-chloroethyl)-L-ornithine (Cl-NIO) is shown to be a potent time- and concentration-dependent inhibitor of purified human DDAH-1 (KI = 1.3 ± 0.6 μM; kinact = 0.34 ± 0.07 min−1), with > 500-fold selectivity against two arginine-handling enzymes in the same pathway. An activity probe is used to measure the “in cell” IC50 value (6.6 ± 0.2 μM) for Cl-NIO inhibition of DDAH-1 artificially expressed within cultured HEK293T cells. A screen of diverse melanoma cell lines reveals that a striking 50 / 64 (78 %) of melanoma lines tested showed increased levels of DDAH-1 in comparison to normal melanocyte control lines. Treatment of the melanoma A375 cell line with Cl-NIO shows a subsequent decrease in cellular nitric oxide production. Cl-NIO represents a promising tool for the study of methylarginine-mediated nitric oxide control and a potential therapeutic lead compound for other indications with elevated nitric oxide production such as septic shock and idiopathic pulmonary fibrosis.
dimethylarginine; dimethylaminohydrolase; covalent inhibitor; nitric oxide; melanoma
Does inhibition of dimethylarginine dimethylaminohydrolase (DDAH) increase the sensitivity of trophoblasts to TRAIL-induced apoptosis?
Inhibition of DDAH1, but not DDAH2, increases the sensitivity of trophoblasts to TRAIL-induced apoptosis.
WHAT IS KNOWN ALREADY
Successful human pregnancy is dependent on adequate trophoblast invasion and remodelling of the maternal spiral arteries. Increased trophoblast apoptosis is seen in pregnancies complicated by pre-eclampsia. The mechanism underlying this increase is unknown. We have previously shown that nitric oxide (NO) is involved in regulating trophoblast motility and invasion, and have also demonstrated an important role for NO in regulating trophoblast sensitivity to apoptotic stimuli. DDAH is an enzyme that metabolizes asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthesis, previously shown to be elevated in the plasma of pre-eclamptic mothers.
STUDY DESIGN, SIZE, DURATION
This study used the human extravillous trophoblast-derived cell line SGHPL-4 cells. All experiments were performed at least three times.
PARTICIPANTS/MATERIALS, SETTING, METHODS
The effect of DDAH on trophoblast apoptosis was examined using siRNA and time-lapse microscopy. Changes in the expression of DDAH were followed by PCR and western blot analysis. Receptor expression was followed by flow cytometry.
MAIN RESULTS AND THE ROLE OF CHANCE
Inhibiting the expression of DDAH1, but not DDAH2, resulted in a significant increase in the sensitivity of the EVT cell line SGHPL-4 to tumour necrosis factor related apoptosis inducing ligand (TRAIL) induced apoptosis (P < 0.01). This response could be mimicked by the addition of Asymmetric Dimethylarginine (ADMA), an endogenous inhibitor of NO synthesis and the substrate for both isoforms of DDAH. We further showed that this increased sensitivity to apoptosis is accompanied by a significant increase in the expression of TRAIL receptor 2 (TR2; P < 0.05) but not TRAIL receptor 1 (TR1).
LIMITATIONS, REASONS FOR CAUTION
This study was performed only in vitro using a well characterized trophoblast cell line, SGHPL-4, derived from first trimester extravillous trophoblasts.
WIDER IMPLICATIONS OF THE FINDINGS
This study provides new insight into the role of the DDAH/ADMA pathway in the regulation of trophoblast function. Both dysregulation of DDAH and the accumulation of ADMA have been associated with the development of pre-eclampsia. This is the first study to implicate the DDAH/ADMA pathway as a mechanism that might underlie the poor trophoblast invasion seen in this common pregnancy disorder.
STUDY FUNDING/COMPETING INTEREST(S)
B.A.L. was supported by a grant from Action Medical Research UK (SP4577). A.E.W. was supported by a grant from the Wellcome Trust (091550). There are no competing interests and the authors have no conflict interest to declare.
extravillous trophoblast; apoptosis; TRAIL; DDAH; ADMA
Asymmetric dimethylarginine (ADMA), present in human serum, is an endogenous inhibitor of nitric oxide synthase and contributes to vascular disease. Dimethylarginine dimethylaminohydrolase (DDAH) is an ADMA degrading enzyme that has two isoforms: DDAHI and DDAHII. We sought to determine whether serum ADMA levels in type 2 diabetes are influenced by common polymorphisms in the DDAH1 and DDAH2 genes.
Relevant clinical parameters were measured and peripheral whole blood obtained for serum and genetic analysis on 343 participants with type 2 diabetes. Serum ADMA concentrations were determined by mass spectroscopy. Twenty six tag SNPs in the DDAH1 and 10 in the DDAH2 gene were genotyped in all subjects and tested for association with serum ADMA levels. Several SNPs and haplotypes in the DDAH genes were strongly associated with ADMA levels. Most significantly in the DDAH1 gene, rs669173 (p = 2.96×10−7), rs7521189 (p = 6.40×10−7), rs2474123 (p = 0.00082) and rs13373844 (p = 0.00027), and in the DDAH2 gene, rs3131383 (p = 0.0029) and the TGCCCAGGAG haplotype (p = 0.0012) were significantly associated with ADMA levels. Sub-analysis by diabetic retinopathy (DR) status revealed these variants were associated with ADMA levels predominantly in participants without DR. Combined analysis of the most strongly associated SNPs in DDAH1 (rs669173) and DDAH2 (rs3131383) revealed an additive effect (p = 1.37×10−8) on ADMA levels.
Genetic variation in the DDAH1 and 2 genes is significantly associated with serum ADMA levels. Further studies are required to determine the pathophysiological significance of elevated serum ADMA in type 2 diabetes and to better understand how DDAH gene variation influences ADMA levels.
The endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is an independent predictor of cardiovascular and overall mortality. Moreover, elevated ADMA plasma concentrations are associated with the extent of hypertension. However, data from small-sized clinical trials and experimental approaches using murine transgenic models have revealed conflicting results regarding the impact of ADMA and its metabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) in the pathogenesis of hypertension.
Therefore, we investigated the role of ADMA and DDAH1 in hypertension-induced end organ damage using the uninephrectomized, deoxycorticosterone actetate salt, and angiotensin II-induced hypertension model in human DDAH1 (hDDAH1) overexpressing and wild-type (WT) mice. ADMA plasma concentrations differed significantly between hDDAH1 and WT mice at baseline, but did not significantly change during the induction of hypertension. hDDAH1 overexpression did not protect against hypertension-induced cardiac fibrosis and hypertrophy. In addition, the hypertension-induced impairment of the endothelium-dependent vasorelaxation of aortic segments ex vivo was not significantly attenuated by hDDAH1 overexpression. However, hDDAH1 mice displayed an attenuated hypertensive inflammatory response in renal tissue, resulting in less hypertensive renal injury.
Our data reveal that hDDAH1 organ-specifically modulates the inflammatory response in this murine model of hypertension. The lack of protection in cardiac and aortic tissues may be due to DDAH1 tissue selectivity and/or the extent of hypertension by the used combined model. However, our study underlines the potency of hDDAH1 overexpression in modulating inflammatory processes as a crucial step in the pathogenesis of hypertension, which needs further experimental and clinical investigation.
Molecules that block nitric oxide's (NO) biosynthesis are of significant interest. For example, nitric oxide synthase (NOS) inhibitors have been suggested as anti-tumor therapeutics, as have inhibitors of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that catabolizes endogenous NOS inhibitors. Dual-targeted inhibitors hold promise as more effective reagents to block NO biosynthesis than single-targeted compounds. In this study, a small set of known NOS inhibitors are surveyed as inhibitors of recombinant human DDAH-1. From these, an alkylamidine scaffold is selected for homologation. Stepwise lengthening of one substituent converts an NOS-selective inhibitor into a dual-targeted NOS/DDAH-1 inhibitor and then into a DDAH-1 selective inhibitor, as seen in the inhibition constants of N5-(1-iminoethyl)-, N5-(1-iminopropyl)-, N5-(1-iminopentyl)- and N5-(1-iminohexyl)-l-ornithine for neuronal NOS (1.7, 3, 20, >1,900 μM, respectively) and DDAH-1 (990, 52, 7.5, 110 μM, respectively). A 1.9Å X-ray crystal structure of the N5-(1-iminopropyl)-l-ornithine : DDAH-1 complex indicates covalent bond formation between the inhibitor's amidino carbon and the active-site Cys274, and solution studies show reversible competitive inhibition, consistent with a reversible covalent mode of DDAH inhibition by alkylamidine inhibitors. These represent a versatile scaffold for the development of a targeted polypharmacological approach to control NO biosynthesis.
Levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are increased in lung, sputum, exhaled breath condensate and plasma samples from asthma patients. ADMA is metabolized primarily by dimethylarginine dimethylaminohydrolase 1 (DDAH1) and DDAH2. We determined the effect of DDAH1 overexpression on development of allergic inflammation in a mouse model of asthma. The expression of DDAH1 and DDAH2 in mouse lungs was determined by RT-quantitative PCR (qPCR). ADMA levels in bronchoalveolar lavage fluid (BALF) and serum samples were determined by mass spectrometry. Wild type and DDAH1-transgenic mice were intratracheally challenged with PBS or house dust mite (HDM). Airway inflammation was assessed by bronchoalveolar lavage (BAL) total and differential cell counts. The levels of IgE and IgG1 in BALF and serum samples were determined by ELISA. Gene expression in lungs was determined by RNA-Seq and RT-qPCR. Our data showed that the expression of DDAH1 and DDAH2 was decreased in the lungs of mice following HDM exposure, which correlated with increased ADMA levels in BALF and serum. Transgenic overexpression of DDAH1 resulted in decreased BAL total cell and eosinophil numbers following HDM exposure. Total IgE levels in BALF and serum were decreased in HDM-exposed DDAH1-transgenic mice compared to HDM-exposed wild type mice. RNA-Seq results showed downregulation of genes in the inducible nitric oxide synthase (iNOS) signaling pathway in PBS-treated DDAH1-transgenic mice versus PBS-treated wild type mice and downregulation of genes in IL-13/FOXA2 signaling pathway in HDM-treated DDAH1-transgenic mice versus HDM-treated wild type mice. Our findings suggest that decreased expression of DDAH1 and DDAH2 in the lungs may contribute to allergic asthma and overexpression of DDAH1 attenuates allergen-induced airway inflammation through modulation of Th2 responses.
Involvement of nitric oxide (NO) in the pathophysiology of human African trypanosomiasis (HAT) was analyzed in a HAT animal model (rat infected with Trypanosoma brucei brucei). With this model, it was previously reported that trypanosomes were capable of limiting trypanocidal properties carried by NO by decreasing its blood concentration. It was also observed that brain NO concentration, contrary to blood, increases throughout the infection process. The present approach analyses the brain impairments occurring in the regulations exerted by arginase and NG, NG–dimethylarginine dimethylaminohydrolase (DDAH) on NO Synthases (NOS). In this respect: (i) cerebral enzymatic activities, mRNA and protein expression of arginase and DDAH were determined; (ii) immunohistochemical distribution and morphometric parameters of cells expressing DDAH-1 and DDAH-2 isoforms were examined within the diencephalon; (iii) amino acid profiles relating to NOS/arginase/DDAH pathways were established.
Arginase and DDAH activities together with mRNA (RT-PCR) and protein (western-blot) expressions were determined in diencephalic brain structures of healthy or infected rats at various days post-infection (D5, D10, D16, D22). While arginase activity remained constant, that of DDAH increased at D10 (+65%) and D16 (+51%) in agreement with western-blot and amino acids data (liquid chromatography tandem-mass spectrometry). Only DDAH-2 isoform appeared to be up-regulated at the transcriptional level throughout the infection process. Immunohistochemical staining further revealed that DDAH-1 and DDAH-2 are contained within interneurons and neurons, respectively.
In the brain of infected animals, the lack of change observed in arginase activity indicates that polyamine production is not enhanced. Increases in DDAH-2 isoform may contribute to the overproduction of NO. These changes are at variance with those reported in the periphery. As a whole, the above processes may ensure additive protection against trypanosome entry into the brain, i.e., maintenance of NO trypanocidal pressure and limitation of polyamine production, necessary for trypanosome growth.
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase (NOS). ADMA is eliminated largely by the action of dimethylarginine dimethylaminohydrolase1 (DDAH1). Decreased DDAH activity is found in several pathological conditions and is associated with increased risk of vascular disease. Overexpression of DDAH1 has been shown to augment endothelial proliferation and angiogenesis. To better understand the mechanism by which DDAH1 influences endothelial proliferation, this study examined the effect of DDAH1 deficiency on cell cycle progression and the expression of some cell cycle master regulatory proteins. DDAH1 KO decreased in vivo Matrigel angiogenesis and depressed endothelial repair in a mouse model of carotid artery wire injury. DDAH1 deficiency decreased VEGF expression in HUVEC and increased NF1 expression in both HUVEC and DDAH1 KO mice. The expression of active Ras could overcome the decreased VEGF expression caused by the DDAH1 depletion. The addition of VEGF and knockdown NF1 could both restore proliferation in cells with DDAH1 depletion. Flow cytometry analysis revealed that DDAH1 sRNAi knockdown in HUVEC caused G1 and G2/M arrest that was associated with decreased expression of CDC2, CDC25C, cyclin D1 and cyclin E. MEF cells from DDAH1 KO mice also demonstrated G2/M arrest that was associated with decreased cyclin D1 expression and Akt activity. Our findings indicate that DDAH1 exerts effects on cyclin D1 and cyclin E expression through multiple mechanisms, including VEGF, the NO/cGMP/PKG pathway, the Ras/PI3K/Akt pathway, and NF1 expression. Loss of DDAH1 effects on these pathways results in impaired endothelial cell proliferation and decreased angiogenesis. The findings provide background information that may be useful in the development of therapeutic strategies to manipulate DDAH1 expression in cardiovascular diseases or tumor angiogenesis.
Asymmetric methylarginines inhibit nitric oxide (NO) synthase (NOS) activity and thereby decrease NO production. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) degrades asymmetric methylarginines. We previously demonstrated that in the heart DDAH1 is predominantly expressed in vascular endothelial cells. Since an earlier study showed that mice with global DDAH1 deficiency was embryonic lethal, we speculate 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 as compared with wild type littermates. DDAH1 expression was greatly reduced in kidney, lung, brain, and liver, indicating that in these organs DDAH1 is mainly distributed in vascular endothelial cells. The endo-DDAH1−/− mice showed a significant increase of asymmetric methylarginine concentration in plasma (1.41μM in the endo-DDAH1−/− vs. 0.69μM in the control mice), kidney, lung and liver, which was associated with significantly increased systolic blood pressure (132 mmHg vs. 113 mmHg in wild type). The endo-DDAH1−/− mice also exhibited significantly attenuated acetylcholine-induced NO production and vessel relaxation in isolated aortic rings.
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.
Asymmetric dimethylarginine; dimethylarginine dimethylaminohydrolase 1; nitric oxide; gene knockout mice
Hyperhomocysteinemia is a cardiovascular risk factor that is associated with the nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA). Using mice transgenic for overexpression of the ADMA-hydrolyzing enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH1), we tested the hypothesis that overexpression of DDAH1 protects from adverse structural and functional changes in cerebral arterioles in hyperhomocysteinemia.
Methods and Results
Hyperhomocysteinemia was induced in DDAH1 transgenic (DDAH1 Tg) mice and wild-type littermates using a high methionine/low folate (HM/LF) diet. Plasma total homocysteine was elevated approximately 3-fold in both wild-type and DDAH1 Tg mice fed the HM/LF diet compared with the control diet (P<0.001). Plasma ADMA was approximately 40% lower in DDAH1 Tg mice compared with wild-type mice (P<0.001) irrespective of diet. Compared with the control diet, the HM/LF diet diminished endothelium-dependent dilation to 10 µmol/L acetylcholine in cerebral arterioles of both wild-type (12±2 vs. 29±3%; P<0.001) and DDAH1 Tg (14±3 vs. 28±2%; P<0.001) mice. Responses to 10 µmol/L papaverine, a direct smooth muscle dilator, were impaired with the HM/LF diet in wild-type mice (30±3 vs. 45±5%; P<0.05) but not DDAH1 Tg mice (45±7 vs. 48±6%). DDAH1 Tg mice also were protected from hypertrophy of cerebral arterioles (P<0.05) but not from accelerated carotid artery thrombosis induced by the HM/LF diet.
Overexpression of DDAH1 protects from hyperhomocysteinemia-induced alterations in cerebral arteriolar structure and vascular muscle function.
amino acids; nitric oxide synthase; endothelium; vasodilation; thrombosis
Methylated arginines are endogenous analogues of L-arginine, the substrate for nitric oxide (NO) synthase. Asymmetric dimethylarginine (ADMA) interferes with NO formation, causing endothelial dysfunction. ADMA is a predictor of cardiovascular events and mortality in humans. It is eliminated primarily by enzymatic activity of dimethylarginine dimethylaminohydrolase (DDAH).
We investigated whether human DDAH-1 (hDDAH-1) transgenicity protects from ischemic tissue damage in temporary middle cerebral artery occlusion (tMCAO) in mice. Infarct sizes did not significantly differ between hDDAH-1 transgenic (TG) mice and wild-type littermates (WT). As expected, ADMA plasma concentrations were significantly decreased, cerebral hDDAH expression and protein significantly increased in transgenic animals. Interestingly, neither brain tissue DDAH activity nor ADMA concentrations were different between TG and WT mice. In contrast, muscular DDAH activity was generally lower than in brain but significantly increased in TG mice.
Our study demonstrates that hDDAH-1 transgenic mice are not protected from ischemic cerebral tissue damage in tMCAO. This lack of protection is due to high basal cerebral DDAH activity, which is not further increasable by transgenic overexpression of DDAH.
Although embryonal proteins have been used as tumor marker, most are not useful for detection of early malignancy. In the present study, we developed mouse monoclonal antibodies against fetal lung of miniature swine, and screened them to find an embryonal protein that is produced at the early stage of malignancy, focusing on lung adenocarcinoma. We found an antibody clone that specifically stained stroma of lung adenocarcinoma. LC-MS/MS identified the protein recognized by this clone as dimethylarginine dimethylaminohydrolase 2 (DDAH2), an enzyme known for antiatherosclerotic activity. DDAH2 was found to be expressed in fibroblasts of stroma of malignancies, with higher expression in minimally invasive adenocarcinoma (MIA) and invasive adenocarcinoma than in adenocarcinoma in situ (AIS). Moreover, tumors with high stromal expression of DDAH2 had a poorer prognosis than those without. In vitro analysis showed that DDAH2 increases expression of endothelial nitric oxide synthase (eNOS), inducing proliferation and capillary-like tube formation of vascular endothelial cells. In resected human tissues, eNOS also showed higher expression in invasive adenocarcinoma than in AIS and normal lung, similarly to DDAH2. Our data indicate that expression of DDAH2 is associated with invasiveness of lung adenocarcinoma via tumor angiogenesis. DDAH2 expression might be a prognostic factor in lung adenocarcinoma.
Electronic supplementary material
The online version of this article (doi:10.1007/s00428-015-1863-z) contains supplementary material, which is available to authorized users.
DDAH2; Angiogenesis; Adenocarcinoma; Malignant stroma; Prognosis
Supplemental Digital Content is Available in the Text.
Inhibition of dimethylarginine dimethylaminohydrolase 1 attenuates pain-related behavior and hyperexcitability in pain conditions associated with excessive nitric oxide production, representing a novel therapeutic target.
Activation of neuronal nitric oxide synthase, and consequent production of nitric oxide (NO), contributes to spinal hyperexcitability and enhanced pain sensation. All NOS isoforms are inhibited endogenously by asymmetric dimethylarginine, which itself is metabolised by dimethylarginine dimethylaminohydrolase (DDAH). Inhibition of DDAH can indirectly attenuate NO production by elevating asymmetric dimethylarginine concentrations. Here, we show that the DDAH-1 isoform is constitutively active in the nervous system, specifically in the spinal dorsal horn. DDAH-1 was found to be expressed in sensory neurons within both the dorsal root ganglia and spinal dorsal horn; L-291 (NG–[2-Methoxyethyl]-l-arginine methyl ester), a DDAH-1 inhibitor, reduced NO synthesis in cultured dorsal root ganglia neurons. Spinal application of L-291 decreased N-methyl-d-aspartate–dependent postdischarge and windup of dorsal horn sensory neurons—2 measures of spinal hyperexcitability. Finally, spinal application of L-291 reduced both neuronal and behavioral measures of formalin-induced central sensitization. Thus, DDAH-1 may be a potential therapeutic target in neuronal disorders, such as chronic pain, where elevated NO is a contributing factor.
Spinal hyperexcitability; Nitric oxide; Neuronal nitric oxide synthase; Asymmetric dimethylargenine; Dimethylarginine dimethylaminohydrolase inhibition
Background and Aims
Portal hypertension is characterized by reduced hepatic eNOS activity. Asymmetric-dimethylarginine (ADMA), an eNOS inhibitor, is elevated in cirrhosis and correlates with severity of portal hypertension. Dimethylargininedimethylaminohydrolase-1 (DDAH-1) is the key enzyme metabolizing hepatic ADMA. This study characterized DDAH-1 in cirrhosis, and explored hepatic DDAH-1 reconstitution through FXR agonism and DDAH-1 gene therapy.
DDAH-1 Immunohistochemistry was conducted on human cirrhosis and healthy liver tissue. Subsequently, sham-operated or bile-duct-ligated (BDL) cirrhosis rats were treated with FXR agonist Obeticholic acid (OA, 5mg/kg) or vehicle for 5 days. Further animals underwent hydrodynamic injection with DDAH-1-expressing plasmid or saline control. Groups: Sham+saline, BDL+saline, BDL+DDAH-1-plasmid. Portal pressure (PP) measurements were performed. Plasma ALT was measured by Cobas-Integra; DDAH-1 expression by qPCR and Western blot; eNOS activity by radiometric assay.
Immunohistochemistry and Western-blotting confirmed hepatic DDAH-1 was restricted to hepatocytes, and expression decreased significantly in cirrhosis. In BDL rats, reduced DDAH-1 expression was associated with elevated hepatic ADMA, reduced eNOS activity and high PP. OA treatment significantly increased DDAH-1 expression, reduced hepatic tissue ADMA, and increased liver NO generation. PP was significantly reduced in BDL+OA vs. BDL+vehicle (8±1 vs. 13.5±0.6 mmHg; p<0.01) with no change in MAP. Similarly, DDAH-1 hydrodynamic injection significantly increased hepatic DDAH-1 gene and protein expression, and significantly reduced PP in BDL+DDAH-1 vs. BDL+ saline (p<0.01).
This study demonstrates DDAH-1 is a specific molecular target for portal pressure reduction, through actions on ADMA-mediated regulation of eNOS activity. Our data support translational studies targeting DDAH-1 in cirrhosis and portal hypertension.
Portal hypertension; ADMA; DDAH-1; Nitric oxide
Endothelial dysfunction participates in the development and progression of salt-sensitive hypertension. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS). The objectives of this study were to investigate the impact of a high salt diet on the PRMT/ADMA/DDAH (protein arginine methyltransferases; dimethylarginine dimethylaminohydrolase) pathway in Dahl salt-sensitive (DS) rats and SS-13BN consomic (DR) rats, and to explore the mechanisms that regulate ADMA metabolism independent of blood pressure reduction. Plasma levels of nitric oxide (NO) in DS rats given a high salt diet and subjected to intragastric administration of hydralazine (SH + HYD group) were lower than those given a normal salt diet (SN group). There were significant decreases in expression and activity of dimethylarginine dimethylaminohydrolase (DDAH) and endothelial NO synthase (eNOS) in DS rats given a high diet (SH group) in comparison to the SN group. The activity of DDAH and expression of eNOS in the SH + HYD group decreased more significantly than SN group. The mRNA expression of DDAH-1 and DDAH-2 were lowest in the SH group. The results suggest that salt, independent of blood pressure, can affect the PRMT-1/ADMA/DDAH system to a certain degree and lead to endothelial dysfunction in Dahl salt-sensitive rats.
endothelial dysfunction; asymmetric dimethylarginine; dimethylarginine; dimethylaminohydrolase; endothelial nitrite oxide synthase; oxidative stress
Inhibition of nitric oxide (NO) signaling may contribute to pathological activation of the vascular endothelium during severe malaria infection. Dimethylarginine dimethylaminohydrolase (DDAH) regulates endothelial NO synthesis by maintaining homeostasis between asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor, and arginine, the NOS substrate. We carried out a community-based case-control study of Gambian children to determine whether ADMA and arginine homeostasis is disrupted during severe or uncomplicated malaria infections. Circulating plasma levels of ADMA and arginine were determined at initial presentation and 28 days later. Plasma ADMA/arginine ratios were elevated in children with acute severe malaria compared to 28-day follow-up values and compared to children with uncomplicated malaria or healthy children (p<0.0001 for each comparison). To test the hypothesis that DDAH1 is inactivated during Plasmodium infection, we examined DDAH1 in a mouse model of severe malaria. Plasmodium berghei ANKA infection inactivated hepatic DDAH1 via a post-transcriptional mechanism as evidenced by stable mRNA transcript number, decreased DDAH1 protein concentration, decreased enzyme activity, elevated tissue ADMA, elevated ADMA/arginine ratio in plasma, and decreased whole blood nitrite concentration. Loss of hepatic DDAH1 activity and disruption of ADMA/arginine homeostasis may contribute to severe malaria pathogenesis by inhibiting NO synthesis.
During a malaria infection, the vascular endothelium becomes more adhesive, permeable, and prone to trigger blood clotting. These changes help the parasite adhere to blood vessels, but endanger the host by obstructing blood flow through small vessels. Endothelial nitric oxide (NO) would normally counteract these pathological changes, but NO signalling is diminished malaria. NO synthesis is inhibited by asymmetric dimethylarginine (ADMA), a methylated derivative of arginine that is released during normal protein turnover. We found the ratio of ADMA to arginine to be elevated in Gambian children with severe malaria, a metabolic disturbance known to inhibit NO synthesis. ADMA was associated with markers of endothelial activation and impaired tissue perfusion. In parallel experiments using mice, the enzyme responsible for metabolizing ADMA, dimethylarginine dimethylaminohydrolase (DDAH), was inactivated after infection with a rodent malaria. Based on these studies, we propose that decreased metabolism of ADMA by DDAH might contribute to the elevated ADMA/arginine ratio observed during an acute episode of malaria. Strategies to preserve or increase DDAH activity might improve NO synthesis and help to prevent the vascular manifestations of severe malaria.
Dimethylarginine dimethylaminohydrolase (DDAH) metabolizes asymmetric dimethylarginine to generate l-citrulline and is present in large quantities in the kidney. We present a new study that optimizes the Prescott–Jones colorimetric assay to measure DDAH-dependent l-citrulline generation in kidney homogenates. We found that the removal of urea with urease is necessary since urea also produces a positive reaction. Deproteinization with sulfosalicylic acid was found to be optimal and that protease inhibitors were not necessary. All assays were conducted in phosphate buffer, since other common additives can create false positive and false negative reactions. Arginase or nitric oxide synthase isoenzymes were not found to influence l-citrulline production. Our optimized l-citrulline production assay to measure DDAH activity correlated closely with the direct measure of the rate of asymmetric dimethylarginine consumption. Using this assay, we found that both superoxide and nitric oxide inhibit renal cortical DDAH activity in vitro.
asymmetric dimethylarginine; l-citrulline; dimethylarginine dimethylaminohydrolase; nitric oxide; urea
We investigated the effects of hepatic ischemia/reperfusion (I/R) injury on asymmetric dimethylarginine (ADMA, a nitric oxide synthase inhibitor), protein methyltransferase (PRMT) and dimethylarginine dimethylaminohydrolase (DDAH) (involved, resp., in ADMA synthesis and degradation), and the cationic transporter (CAT). Male Wistar rats were subjected to 30 or 60 min hepatic ischemia followed by 60 min reperfusion. ADMA levels in serum and bile were determined. Tissue ADMA, DDAH activity, DDAH-1 and CAT-2 protein, DDAH-1 and PRMT-1 mRNA expression, GSH/GSSG, ROS production, and lipid peroxidation were detected. ADMA was found in bile. I/R increased serum and bile ADMA levels while an intracellular decrease was detected after 60 min ischemia. Decreased DDAH activity, mRNA, and protein expression were observed at the end of reperfusion. No significant difference was observed in GSH/GSSG, ROS, lipid peroxidation, and CAT-2; a decrease in PRMT-1 mRNA expression was found after I/R. Liver is responsible for the biliary excretion of ADMA, as documented here for the first time, and I/R injury is associated with an oxidative stress-independent alteration in DDAH activity. These data are a step forward in the understanding of the pathways that regulate serum, tissue, and biliary levels of ADMA in which DDAH enzyme plays a crucial role.
Nitric oxide (NO) is generated from L-arginine by NO synthases, of which three forms have been identified: endothelial, inducible and neuronal (eNOS, iNOS and nNOS, respectively). The arginine metabolite asymmetric dimethylarginine (ADMA) is a potent, noncompetitive inhibitor of nNOS, while its congener NG-monomethyl-L-arginine (L-NMMA) is a less potent, competitive inhibitor. In rat neurons large amounts of ADMA are found, suggesting its importance in modulatin neuronal activity.
Humans generate approximately 300 μmol (∼60 mg) ADMA per day. It is released from myelin basic proteins that are highly expressed in neuronal tissue. ADMA is mainly degraded by the action of the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which exists in two isoforms. DDAH1 is highly expressed in brain, suggesting specific function in this area. The presence of nNOS and DDAH1 in brain suggests that ADMA may have specific CNS activity and be more than an unregulated metabolite.
Increased NO production - either prior to or concurrently with opioid administration - results in an enhanced rate and extent of development of tolerance to morphine in mice. NO produces an alteration in the μ-opioid receptor that increases constitutive receptor activity. It thereby reduces the ability of a selective μ-opioid agonist to activate the μ-opioid receptor; these in vitro molecular effects occur in a time course consistent with the in vivo development of antinociceptive tolerance in mice. Amongst many other synthetic NOS inhibitors of varying specificity, 7-nitroindazole (7-NI) has been shown to have a high affinity (IC50 0.71 μM) to nNOS. Selective blockade of nNOS by 7-NI attenuated morphine withdrawal in opiate dependent rats, suggesting nNOS as a viable target for development of pharmacotherapies.
We hypothesize that, by inhibiting nNOS and reducing NO levels, ADMA may decrease μ-opiate receptor constitutive activity, resulting in alteration of the analgesic dose-response curve of morphine.
addiction; opiates; heroine; nociception; NOS; Asymmetrical dimethylarginine; NG-monomethyl-L-arginine