Dimethylarginine dimethylaminohydrolase-2 overexpression improves impaired nitric oxide synthesis of endothelial cells induced by glycated protein.

Nitric oxide (NO) synthesis is modulated by dimethylarginine dimethylaminohydrolase (DDAH) via metabolizing asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor. This study investigated whether glycosylated bovine serum albumin (GBSA) could impair NO synthesis by inhibition of DDAH expression and activity, and whether DDAH2 overexpression could reverse the impaired NO synthesis induced by GBSA in endothelial cells. Overexpression of DDAH2 gene was established by liposome-mediated gene transfection in ECV304 endothelial cell line. Cells were incubated with 1.70 mmol/L GBSA for 48h. And the expressions of DDAH1 and DDAH2, gene activities of DDAH and NOS in cells, as well as concentrations of ADMA and NO in media were assayed. The activity of DDAH and expression of DDAH2 gene but not DDAH1 gene were inhibited in endothelial cells after exposure to GBSA, whereas the concentrations of ADMA were increased concomitantly with the decrease of NOS activity in cells and NO production in media. Overexpression of DDAH2 gene could prevent the inhibition of DDAH activity induced by GBSA (0.55+/-0.02 vs 0.42+/-0.02U/g pro; n=3; P<0.05), decrease ADMA concentration (0.59+/-0.04 vs 1.13+/-0.11 micromol/L; n=3; P<0.01), and increase NOS activity and NO production (53.77+/-3.40 vs 34.59+/-2.57 micromol/L; P<0.05) compared with untransfected cells treated with GBSA. These results suggest that decreased DDAH activity and subsequent elevated endogenous ADMA are implicated in the inhibition of NO synthesis induced by GBSA, and overexpression of DDAH2 gene can prevent these changes in DDAH/ADMA/NOS/NO pathway of endothelial cells exposed to GBSA.     

Involvement of the endothelial DDAH/ADMA pathway in nitroglycerin tolerance: the role of ALDH-2

Previous studies have shown that nitroglycerin (GTN) tolerance is closely related to an oxidative stress-induced decrease in activity of mitochondrial isoforms of aldehyde dehydrogenase (ALDH-2), and prolonged GTN treatment causes endothelial dysfunction. Asymmetric dimethylarginine (ADMA), a major endogenous NO synthase (NOS) inhibitor, could inhibit NO production and induce oxidative stress in endothelial cells. ADMA and its major hydrolase dimethylarginine dimethylaminohydrolase (DDAH) have recently been thought of as a novel regulatory system of endothelium function. The aim of the present study was to determine whether the DDAH/ADMA pathway is involved in the development of GTN tolerance in endothelial cells. Tolerance, reflected by the decrease in cyclic GMP (cGMP) production, was induced by exposure of human umbilical vein endothelial cells (HUVECs) to GTN (10 microM) for 16 h. While the treatment increased reactive oxygen species (ROS) production/malondialdehyde (MDA) concentration and decreased ALDH-2 activity as well as cGMP production, it markedly increased the level of ADMA in culture medium and decreased DDAH activity in endothelial cells. Exogenous ADMA significantly enhanced ROS production/MDA concentration and inhibited ALDH-2 activity, and overexpression of DDAH2 could significantly suppress GTN-induced oxidative stress and inhibition of ALDH-2 activity, which is also attenuated by L-arginine. Therefore, our results suggest for the first time that the endothelial DDAH/ADMA pathway plays an important role in the development/maintenance of GTN tolerance.     

Evidence for the pathophysiological role of endogenous methylarginines in regulation of endothelial NO production and vascular function

In endothelium, NO is derived from endothelial NO synthase (eNOS)-mediated L-arginine oxidation. Endogenous guanidinomethylated arginines (MAs), including asymmetric dimethylarginine (ADMA) and NG-methyl-L-arginine (L-NMMA), are released in cells upon protein degradation and are competitive inhibitors of eNOS. However, it is unknown whether intracellular MA concentrations reach levels sufficient to regulate endothelial NO production. Therefore, the dose-dependent effects of ADMA and L-NMMA on eNOS function were determined. Kinetic studies demonstrated that the Km for L-arginine is 3.14 microM with a Vmax of 0.14 micromol mg-1 min-1, whereas Ki values of 0.9 microM and 1.1 microM were determined for ADMA and L-NMMA, respectively. EPR studies of NO production from purified eNOS demonstrated that, with a physiological 100 microM level of L-arginine, MA levels of >10 microM were required for significant eNOS inhibition. Dose-dependent inhibition of NO formation in endothelial cells was observed with extracellular MA concentrations as low 5 microm. Similar effects were observed in isolated vessels where 5 microm ADMA inhibited vascular relaxation to acetylcholine. MA uptake studies demonstrated that ADMA and L-NMMA accumulate in endothelial cells with intracellular levels greatly exceeding extracellular concentrations. L-arginine/MA ratios were correlated with cellular NO production. Although normal physiological levels of MAs do not significantly inhibit NOS, a 3- to 9-fold increase, as reported under disease conditions, would exert prominent inhibition. Using a balloon model of vascular injury, approximately 4-fold increases in cellular MAs were observed, and these caused prominent impairment of vascular relaxation. Thus, MAs are critical mediators of vascular dysfunction following vascular injury.     

Protective effect of neferine on endothelial cell nitric oxide production induced by lysophosphatidylcholine: the role of the DDAH-ADMA pathway

Neferine, extracted from the seed embryo of Nelumbo nucifera Gaertn., has multiple cardiovascular pharmacological effects. The dimethylarginine dimethylaminohydrolase (DDAH) - asymmetric dimethylarginine (ADMA) system is a novel pathway for modulating nitric oxide (NO) production. The aim of this study was to investigate whether the protective effect of neferine on endothelial NO production was related to the DDAH-ADMA pathway. Human umbilical vein endothelial cells (HUVECs) were first exposed to neferine (0.1, 1.0, or 10.0?μmol/L) for 1?h, and then incubated with lysophosphatidylcholine (LPC; 10?μg/mL) in the presence of neferine for 24?h. The medium was collected for measuring the levels of NO, maleic dialdehyde (MDA), as well as ADMA. The endothelial cells were collected for measuring DDAH activity and the level of reactive oxygen species (ROS). LPC significantly decreased NO concentration and DDAH activity and increased the levels of ADMA, ROS, and MDA. Neferine could partially counteract the changes induced by LPC. These findings suggested that neferine could modulate the DDAH-ADMA pathway via its antioxidant properties, which was involved in its beneficial effect on endothelial NO production.     

Dimethylarginine dimethylaminohydrolase (DDAH): expression, regulation, and function in the cardiovascular and renal systems

Asymmetric (N(G),N(G))-dimethylarginine (ADMA) inhibits nitric oxide (NO) synthases (NOS). ADMA is a risk factor for endothelial dysfunction, cardiovascular mortality, and progression of chronic kidney disease. Two isoforms of dimethylarginine dimethylaminohydrolase (DDAH) metabolize ADMA. DDAH-1 is the predominant isoform in the proximal tubules of the kidney and in the liver. These organs extract ADMA from the circulation. DDAH-2 is the predominant isoform in the vasculature, where it is found in endothelial cells adjacent to the cell membrane and in intracellular vesicles and in vascular smooth muscle cells among the myofibrils and the nuclear envelope. In vivo gene silencing of DDAH-1 in the rat and DDAH +/- mice both have increased circulating ADMA, whereas gene silencing of DDAH-2 reduces vascular NO generation and endothelium-derived relaxation factor responses. DDAH-2 also is expressed in the kidney in the macula densa and distal nephron. Angiotensin type 1 receptor activation in kidneys reduces the expression of DDAH-1 but increases the expression of DDAH-2. This rapidly evolving evidence of isoform-specific distribution and regulation of DDAH expression in the kidney and blood vessels provides potential mechanisms for nephron site-specific regulation of NO production. In this review, the recent advances in the regulation and function of DDAH enzymes, their roles in the regulation of NO generation, and their possible contribution to endothelial dysfunction in patients with cardiovascular and kidney diseases are discussed.     

Tissue-specific downregulation of dimethylarginine dimethylaminohydrolase in hyperhomocysteinemia

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, has been proposed to be a mediator of vascular dysfunction during hyperhomocysteinemia. Levels of ADMA are regulated by dimethylarginine dimethylaminohydrolase (DDAH). Using both in vitro and in vivo approaches, we tested the hypothesis that hyperhomocysteinemia causes downregulation of the two genes encoding DDAH (Ddah1 and Ddah2). In the MS-1 murine endothelial cell line, the addition of homocysteine decreased NO production but did not elevate ADMA or alter levels of Ddah1 or Ddah2 mRNA. Mice heterozygous for cystathionine beta-synthase (Cbs) and their wild-type littermates were fed either a control diet or a high-methionine/low-folate (HM/LF) diet to produce varying degrees of hyperhomocysteinemia. Maximal relaxation of the carotid artery to the endothelium-dependent dilator acetylcholine was decreased by approximately 50% in Cbs(+/-) mice fed the HM/LF diet compared with Cbs(+/+) mice fed the control diet (P < 0.001). Compared with control mice, hyperhomocysteinemic mice had lower levels of Ddah1 mRNA in the liver (P < 0.001) and lower levels of Ddah2 mRNA in the liver, lung, and kidney (P < 0.05). Downregulation of DDAH expression in hyperhomocysteinemic mice did not result in an increase in plasma ADMA, possibly due to a large decrease in hepatic methylation capacity (S-adenosylmethionine-to-S-adenosylhomocysteine ratio). Our findings demonstrate that hyperhomocysteinemia causes tissue-specific decreases in DDAH expression without altering plasma ADMA levels in mice with endothelial dysfunction.     

The DDAH/ADMA pathway is a critical regulator of NO signalling in vascular homeostasis

NO is an important regulator of cardiovascular remodelling and function. ADMA, an endogenous L-arginine analogue, reduces NO production by inhibiting the activity of NOS. ADMA levels in turn, are regulated by DDAH, which metabolises ADMA. High levels of ADMA and dysregulated DDAH activity are risk factors for cardiovascular disease and morbidity. To investigate this link, the DDAH I null mouse has been recently generated and has a lethal phenotype. Studies on vascular function in the DDAH I heterozygous knockout mouse, which is viable, demonstrates a causal link between reduced DDAH I activity, increased ADMA levels and reduced NO signalling and vascular dysfunction. In another study, detailed in vitro analyses reveal that the DDAH/ADMA pathway critically regulates endothelial cell motility and angiogenesis and establishes some of the molecular mechanisms involved. These studies highlight the importance of DDAH and ADMA in regulating NO dependent vascular homeostasis.Key words: asymmetric dimethylarginine (ADMA), dimethylarginine dimethylaminohydrolase (DDAH), nitric oxide (NO), angiogenesis, endothelial, motilityNO is generated from L-arginine by NOS; a process which is competitively inhibited by the arginine analogues ADMA and L-NMMA. These endogenous factors are products of proteolytic degradation of methylated proteins. ADMA and L-NMMA are metabolised by DDAH I and II, thereby enhancing NO generation. Of relevance to vascular biology, dysfunctional DDAH activity and ADMA accumulation are risk factors for cardiovascular disorders, including hypertension, artherosclerosis, diabetes, insulin resistance, hypercholesterolemia and homocysteinemia (reviewed in ref. ¹).The DDAH I null mouse was generated recently by Leiper et al.² to facilitate investigation of the role of the DDAH/ADMA pathway in the pathology of cardiovascular disorders. While the absence of DDAH I causes a lethal phenotype, heterozygotes (HT) did not display any obvious abnormalities. However, ADMA levels were raised in tissues and plasma, in association with raised blood pressure and systemic vascular resistance, and reduced cardiac output and heart rate. Synthetic DDAH I inhibitors were designed by the authors and were shown by crystallography to bind to the active site of the enzyme and induce local distortions at this region. Confirming that loss of DDAH I was responsible for ADMA accumulation, these inhibitors enhanced ADMA levels in wildtype mice, and resulted in cardiovascular changes similar to those seen in the HT background. Inhibitor treatment also promoted ADMA release from wildtype blood vessels maintained ex vivo, indicating that the DDAH/ADMA pathway is directly responsible for maintaining cardiovascular function in this model.Evidence was also presented for a causal link between ADMA metabolism and reduced NO levels. In an ex vivo model, aortic rings from HT mice displayed enhanced phenylephrine-induced contraction and reduced acetylcholine-induced relaxation, while DDAH I inhibitors induced similar responses in aortic rings from wildtype mice; indicative of reduced levels of endothelial-derived NO. Further demonstrating an ADMA/NO-dependent mechanism, exogenous L-arginine restored a normal response to these vasomodulators in the HT model (by competing with ADMA for interaction with NOS). Similarly, cultured endothelial cells from HT vessels produced more ADMA and less NO than cells from wildtype vessels, and DDAH I inhibitors induced a similar phenotype in wildtype endothelial cells. The significance of DDAH I/ADMA and NO in vascular disease was tested in a disease model. Endotoxic shock was induced in rats by intravenous infusion of LPS, which induces excess NO production, resulting in systemic hypotension. After blood pressure had fallen by 20%, infusion of a DDAH I inhibitor was able to rapidly stabilise blood pressure, in accordance with inhibition of NO production through reduced ADMA metabolism. Thus, when DDAH I is reduced, ADMA is increased and endogenous NO inhibited, resulting in altered vascular function.Another related study investigated a mechanistic understanding of the role of ADMA/DDAH/NO in angiogenesis.³ The authors demonstrated that ADMA regulates endothelial cell motility and phenotype by inhibiting NO-dependent changes in activity of Rho-GTPases; key mediators of cytoskeletal dynamics and motility. Treatment of pulmonary artery endothelial cells with ADMA enhanced stress fibres and focal adhesion formation in conjunction with increased activity of RhoA in pull-down assays. In accordance with these observations, motility, tracked by time-lapse microscopy, was inhibited by ADMA treatment, and ADMA effects were reversed by a Rho kinase inhibitor (Y-27632) or by adenoviral-mediated gene transfer of a dominant negative RhoA mutant. RhoA activity is mediated by PKG, which mediates RhoA-Ser188 phosphorylation, preventing RhoA localization to the membrane and inhibiting its activity.⁴ In further support of a RhoA-dependent mechanism, ADMA reduced phosphorylation at RhoA-Ser188, while a PKG activator was also able to revert ADMA effects on motility. Further, a non-phosphorylatable mutant of RhoA, Ala188RhoA, or a specific PKG inhibitor, each inhibited cell motility to a similar level as ADMA treatment alone. Inhibition of NO production and endothelial cell motility by ADMA was also reversed by a NO donor, SNAP, or by DDAH I or II overexpression via adenovirus-mediated gene transfer. Thus, reduction of NO/PKG levels by ADMA reduces RhoA phosphorylation at Ser188 resulting in enhancement of RhoA activity and inhibition of cell motility.The significance of these molecular mechanisms to angiogenesis was demonstrated using endothelial cells and aortic ring explants from HT DDAH I and wildtype mice. HT endothelial cells, which secrete more ADMA and produce less NO than their wildtype counterparts, exhibit enhanced RhoA activity and stress fibre formation in conjunction with reduced motility. Reduced sprouting from ex vivo aortic rings was also observed in the HT model, which was mimicked by addition of exogenous ADMA in the wildtype background. These data demonstrate that in vivo, DDAH/ADMA levels are likely to play a key role in control of endothelial cell motility and angiogenesis by regulating NO production.     

Role of Dimethylarginine Dimethylaminohydrolases in the Regulation of Endothelial Nitric Oxide Production

Reduced NO is a hallmark of endothelial dysfunction, and among the mechanisms for impaired NO synthesis is the accumulation of the endogenous nitric-oxide synthase inhibitor asymmetric dimethylarginine (ADMA). Free ADMA is actively metabolized by the intracellular enzyme dimethylarginine dimethylaminohydrolase (DDAH), which catalyzes the conversion of ADMA to citrulline. Decreased DDAH expression/activity is evident in disease states associated with endothelial dysfunction and is believed to be the mechanism responsible for increased methylarginines and subsequent ADMA-mediated endothelial nitric-oxide synthase impairment. Two isoforms of DDAH have been identified; however, it is presently unclear which is responsible for endothelial ADMA metabolism and NO regulation. The current study investigated the effects of both DDAH-1 and DDAH-2 in the regulation of methylarginines and endothelial NO generation. Results demonstrated that overexpression of DDAH-1 and DDAH-2 increased endothelial NO by 24 and 18%, respectively. Moreover, small interfering RNA-mediated down-regulation of DDAH-1 and DDAH-2 reduced NO bioavailability by 27 and 57%, respectively. The reduction in NO production following DDAH-1 gene silencing was associated with a 48% reduction in l-Arg/ADMA and was partially restored with l-Arg supplementation. In contrast, l-Arg/ADMA was unchanged in the DDAH-2-silenced cells, and l-Arg supplementation had no effect on NO. These results clearly demonstrate that DDAH-1 and DDAH-2 manifest their effects through different mechanisms, the former of which is largely ADMA-dependent and the latter ADMA-independent. Overall, the present study demonstrates an important regulatory role for DDAH in the maintenance of endothelial function and identifies this pathway as a potential target for treating diseases associated with decreased NO bioavailability.     

秦皮甲素对AGEs致损的ECV304细胞增殖及氧化应激的影响

为研究秦皮甲素对血管内皮细胞的保护作用,采用CCK-8法观察秦皮甲素对体外AGEs培养的人脐静脉内皮细胞增殖的影响。检测不同浓度AGEs以及秦皮甲素作用后对内皮细胞一氧化氮(NO)、不对称二甲基精氨酸(ADMA)水平的影响以及内皮细胞氧化应激有关指标:活性氧簇(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD);脂肪代谢相关指标:乳酸脱氢酶(lactic dehydrogenase,LDH)、总胆固醇(total cholesterol,CHO)、甘油三酯(triglyceride,TG)和低密度脂蛋白(low density lipoprotein,LDL),同时分别检测粘附相关因子:血管细胞粘附分子-1(VCAM-1)和细胞间粘附分子-1(ICAM-1)的表达水平。结果显示200 mg/L AGEs对人内皮细胞ECV304增殖有显著抑制作用,秦皮甲素可对抗AGEs导致的内皮细胞增殖抑制,并呈浓度依赖性。在25 mg/L时,保护效应达到最高。秦皮甲素可抵抗ROS生成。同时可改善细胞的脂类代谢:胆固醇、LDL以及TG含量在秦皮甲素作用后改善明显。秦皮甲素可显著抑制内皮粘附因子VCAM-1的表达。秦皮甲素还可上调NO水平,下调ADMA水平。总之,秦皮甲素可有效促进人血管内皮细胞增殖并在改善氧化应激,脂代谢,粘附因子和NO释放等方面发挥作用。     

Relationship between protective effect of xanthone on endothelial cells and endogenous nitric oxide synthase inhibitors

1,3,5,6-tetrahydroxyxanthone was synthesized. The relationship between protective effect of xanthone on endothelial cells and endogenous nitric oxide synthase inhibitors was investigated. Endothelial cells were treated with ox-LDL (100 microg/mL) for 48 h. Adhesion of monocytes to endothelial cells and release of lactate dehydrogenase (LDH) was determined. Levels of tumor necrosis factor-alpha (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1), nitric oxide (NO) and asymmetric dimethylarginine (ADMA, an endogenous inhibitor of nitric oxide synthase) in conditioned medium and activity of dimethylarginine dimethylaminohydrolase (DDAH) in endothelial cells were measured. Incubation of endothelial cells with ox-LDL (100 microg/mL) for 48 h markedly enhanced the adhesion of monocytes to endothelial cells, increased the release of LDH, the levels of TNF-alpha, MCP-1 and ADMA, and decreased the content of NO and the activity of DDAH. Xanthone (1,3,5,6-tetrahydroxyxanthone) (1, 3 or 10 micromol/L) significantly inhibited the increased adhesion of monocytes to endothelial cells and attenuated the increased levels of LDH, MCP-1 and ADMA induced by ox-LDL. Xanthone (1,3,5,6-tetrahydroxyxanthone) (3 or 10 micromol/L) significantly attenuated the increased level of TNF-alpha and decreased level of NO and activity of DDAH by ox-LDL. The present results suggest that xanthone (1,3,5,6-tetrahydroxyxanthone) preserves endothelial cells and inhibits the increased adhesion of monocytes to endothelial cells induced by ox-LDL, and that the protective effect of xanthone (1,3,5,6-tetrahydroxyxanthone) on endothelial cells is related to reduction of ADMA concentration via increase of DDAH activity.     

Epigallocatechin gallate preserves endothelial function by reducing the endogenous nitric oxide synthase inhibitor level

Asymmetric dimethylarginine (ADMA), the endogenous nitric oxide synthase inhibitor, is thought to be a key factor contributing to endothelial dysfunction. Tea catechins can cause an endothelium-dependent vasorelaxation. The present study examined the effect of epigallocatechin gallate (EGCG), the major component of tea catechins, on endothelial dysfunction induced by native low density lipoprotein (LDL) in rats and oxidized LDL (ox-LDL) in cultured endothelial cells, and whether the protective effect of EGCG is related to reduction of ADMA level. A single injection of LDL (4 mg x kg(-1), i.v.) markedly reduced endothelium-dependent relaxation and the serum nitrite/nitrate (NO) level, and increased serum concentrations of ADMA, malondialdehyde (MDA), and tumor necrosis factor-alpha (TNF-alpha). EGCG (10 or 50 mg x kg(-1), i.p.) significantly attenuated the inhibition of vasodilator response to acetylcholine and the decreased serum nitrite/nitrate level, and reduced the elevated levels of ADMA, MDA, and TNF-alpha. Exposure of endothelial cells to ox-LDL (100 microg x mL(-1)) for 24 h markedly increased the medium levels of lactate dehydrogenase (LDH), ADMA, TNF-alpha, and MDA, and decreased the level of nitrite/nitrate in the medium and the activity of dimethylarginine dimethylaminohydrolase (DDAH) in the endothelial cells. EGCG (10 and 100 microg x mL(-1)) significantly decreased the levels of LDH, ADMA, TNF-alpha, and MDA, and increased the level of nitrite/nitrate and the activity of DDAH. These results suggest that EGCG protects endothelial dysfunction induced by native LDL in vivo or by ox-LDL in endothelial cells, and the protective effect of EGCG on the endothelium is related to decrease in ADMA level via increasing of DDAH activity.     

Role of endogenous nitric oxide synthase inhibitor in gastric mucosal injury

To explore the role of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) in gastric mucosal injury, 3 models of gastric mucosal injury induced by ethanol, indomethacin, or cold stress were used in rats. The cultured human gastric mucosal epithelial cell line GES-1 infected by Helicobacter pylori (Hp) was selected to mimic human gastric mucosal injury. Gastric mucosal ulcer index (UI), levels of ADMA and NO, and activity of dimethylarginine dimethylaminohydrolase (DDAH) were determined in the mucosal injury models; in Hp-infected or ADMA-treated GES-1 cells, levels of ADMA, NO, and TNF-alpha and activity of DDAH were measured. The results showed that UI and levels of ADMA were markedly increased and accompanied by significantly decreased DDAH activity in the mucosal injury models. Incubation of GES-1 cells with Hp increased levels of TNF-alpha and ADMA and decreased activity of DDAH. Administration of ADMA also increased levels of TNF-alpha. The results suggest that ADMA plays an important role in facilitating gastric mucosal injury, an effect which is associated with inhibiting NO synthesis and inducing inflammatory reaction.     

Protective effect of HDL on endothelial NO production: the role of DDAH/ADMA pathway

Accumulating studies have demonstrated that the dimethylarginine dimethylaminohydrolase/asymmetric dimethylarginine (DDAH/ADMA) system is a novel pathway for modulating nitric oxide (NO) production. The aim of this study was to investigate whether the protective effect of high density lipoprotein (HDL) on endothelial NO production was related to its effect on DDAH/ADMA pathway. Human umbilical vein endothelial cells (HUVECs) were prior exposed to HDL (10, 50, or 100 μg/ml) for 1 h, and then incubated with oxidized low density lipoprotein (ox-LDL) (100 μg/ml) for 24 h. The cultured medium was collected for measuring the concentration of NO and ADMA. The cells were collected for measuring the mRNA and protein expression of DDAH-II as well as DDAH activity. HUVECs treated with ox-LDL (100 μg/ml) for 24 h significantly decreased the concentration of NO, the mRNA and protein expression of DDAH-II as well as DDAH activity and increased the level of ADMA. Pretreatment with HDL (10, 50, or 100 μg/ml) could counteract these changes induced by ox-LDL (100 μg/ml). HDL significantly increased the attenuated endothelial cell NO production induced by ox-LDL, which was attributed to its effect on DDAH/ADMA pathway.     

Dimethylarginine dimethylaminohydrolase activity modulates ADMA levels,VEGF expression,and cell phenotype

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase and is metabolised by dimethylarginine dimethylaminohydrolase (DDAH). Elevated levels of circulating ADMA correlate with various cardiovascular pathologies less is known about the cellular effects of altered DDAH activity. We modified DDAH activity in cells and measured the changes in ADMA levels, morphological phenotypes on Matrigel, and expression of vascular endothelial growth factor (VEGF). DDAH over-expressing ECV304 cells secreted less ADMA and when grown on Matrigel had enhanced tube formation compared to untransfected cells. VEGF mRNA levels were 2.1-fold higher in both ECV304 and murine endothelial cells (sEnd.1) over-expressing DDAH. In addition the DDAH inhibitor, S-2-amino-4(3-methylguanidino)butanoic acid (4124W 1mM), markedly reduced human umbilical vein endothelial cell tube formation in vitro. We have found that upregulating DDAH activity lowers ADMA levels, increases the levels of VEGF mRNA in endothelial cells, and enhances tube formation in an in vitro model, whilst blockade of DDAH reduces tube formation.     

High-throughput liquid chromatographic-tandem mass spectrometric determination of arginine and dimethylated arginine derivatives in human and mouse plasma

The balance between nitric oxide (NO) and vasoconstrictors like endothelin is essential for vascular tone and endothelial function. L-Arginine is converted to NO and L-citrulline by NO synthase (NOS). Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are endogenous inhibitors of NO formation. ADMA is degraded by dimethylamino dimethylhydrolases (DDAHs), while SDMA is exclusively eliminated by the kidney. In the present article we report a LC-tandem MS method for the simultaneous determination of arginine, ADMA, and SDMA in plasma. This method is designed for high sample throughput of only 20-mul aliquots of human or mouse plasma. The analysis time is reduced to 1.6 min by LC-tandem MS electrospray ionisation (ESI) in the positive mode. The mean plasma levels of l-arginine, ADMA, and SDMA were 74+/-19 (SD), 0.46+/-0.09, and 0.37+/-0.07 microM in healthy humans (n=85), respectively, and 44+/-14, 0.72+/-0.23, and 0.19+/-0.06 microM in C57BL/6 mice. Also, the molar ratios of arginine to ADMA were different in man and mice, i.e. 166+/-50 and 85+/-22, respectively.     

同型半胱氨酸对内皮细胞一氧化氮合酶系统的损伤机制及叶酸的拮抗效应

本实验探讨同型半胱氨酸(Hcy)对人脐静脉内皮细胞(HUVEC)一氧化氮合酶(eNOS)的损伤机制及叶酸(FA)的拮抗效应。HUVEC原代培养,传至第3代后,将其与不同浓度Hcv(10μmol/L、30μmol/L、100μmol/L和300μmol/L)、FA(100μmol/L)或两者联合共同培养72h,用RT-PCR和免疫组织化学技术分别估测细胞eNOS mRNA水平及eNOS蛋白质量;高效液相色谱测定细胞内不对称二甲基精氨酸(ADMA)含量;并分别测定二甲基精氨酸二甲胺水解酶(DDAH)、eNOS活性及一氧化氮(NO)含量。HUVEC与不同浓度Hcy培养72h后,eNOS mRNA和蛋白质表达皆受到抑制;eNOS活性降低;NO生成减少。同时,DDAH活性降低;细胞内ADMA含量呈剂量依赖性增加。加入FA后,eNOS蛋白质水平上调;eNOS活性增强;NO生成增多。同时,DDAH活性增强,ADMA蓄积减少;但eNOS mRNA表达没有改变。Hcy对内皮细胞eNOS的损伤机制涉及eNOS酶蛋白和eNOS的基因表达两个层面,其对eNOS酶蛋白的抑制机制可能通过DDAH-ADMA通路,FA可拮抗Hcy对eNOS酶蛋白的抑制作用,显示出对HHcy有一定的保护作用。但FA对HHcy所导致的eNOS基因表达的抑制无保护效应。     

Mechanisms of homocysteine-induced oxidative stress

Hyperhomocysteinemia decreases vascular reactivity and is associated with cardiovascular morbidity and mortality. However, pathogenic mechanisms that increase oxidative stress by homocysteine (Hcy) are unsubstantiated. The aim of this study was to examine the molecular mechanism by which Hcy triggers oxidative stress and reduces bioavailability of nitric oxide (NO) in cardiac microvascular endothelial cells (MVEC). MVEC were cultured for 0-24 h with 0-100 microM Hcy. Differential expression of protease-activated receptors (PARs), thioredoxin, NADPH oxidase, endothelial NO synthase, inducible NO synthase, neuronal NO synthase, and dimethylarginine-dimethylaminohydrolase (DDAH) were measured by real-time quantitative RT-PCR. Reactive oxygen species were measured by using a fluorescent probe, 2',7'-dichlorofluorescein diacetate. Levels of asymmetric dimethylarginine (ADMA) were measured by ELISA and NO levels by the Griess method in the cultured MVEC. There were no alterations in the basal NO levels with 0-100 microM Hcy and 0-24 h of treatment. However, Hcy significantly induced inducible NO synthase and decreased endothelial NO synthase without altering neuronal NO synthase levels. There was significant accumulation of ADMA, in part because of reduced DDAH expression by Hcy in MVEC. Nitrotyrosine expression was increased significantly by Hcy. The results suggest that Hcy activates PAR-4, which induces production of reactive oxygen species by increasing NADPH oxidase and decreasing thioredoxin expression and reduces NO bioavailability in cultured MVEC by 1) increasing NO2-tyrosine formation and 2) accumulating ADMA by decreasing DDAH expression.     

Detection and quantification of α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid: a metabolite of asymmetric dimethylarginine

Nitric oxide is an ubiquitary cell signaling substance. Its enzymatic production rate by nitric oxide synthase is regulated by the concentrations of the substrate L-arginine and the competitive inhibitor asymmetric dimethylarginine (ADMA). A newly recognized elimination pathway for ADMA is the transamination to α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid (DMGV) by the enzyme alanine-glyoxylate aminotransferase 2 (AGXT2). This pathway has been proven to be relevant for nitric oxide regulation, but up to now no method exists for the determination of DMGV in biological fluids. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of DMGV. D(6)-DMGV was used as internal standard. Samples were purified online by column switching, and separation was achieved on a porous graphitic carbon column. The calibration was linear over ranges of 10 to 200 nmol/L for plasma and 0.1 to 20 μmol/L for urine. The intra- and interday accuracies and precisions in plasma and urine were better than 10%. In plasma samples, DMGV was present in concentrations between 19.1 and 77.5 nmol/L. In urine samples, concentrations between 0.0114 and 1.03 μmol/mmol creatinine were found. This method can be used as a tool for the scientific investigation of the ADMA conversion to DMGV via the enzyme AGXT2.     

Asymmetric dimethylarginine inhibits HSP90 activity in pulmonary arterial endothelial cells: role of mitochondrial dysfunction

Increased asymmetric dimethylarginine (ADMA) levels have been implicated in the pathogenesis of a number of conditions affecting the cardiovascular system. However, the mechanism(s) by which ADMA exerts its effect has not been adequately elucidated. Thus the purpose of this study was to determine the effect of increased ADMA on nitric oxide (NO) signaling and to begin to elucidate the mechanism by which ADMA acts. Our initial data demonstrated that ADMA increased NO synthase (NOS) uncoupling in both recombinant human endothelial NO synthase (eNOS) and pulmonary arterial endothelial cells (PAEC). Furthermore, we found that this endothelial NOS (eNOS) uncoupling increased 3-nitrotyrosine levels preferentially in the mitochondria of PAEC due to a redistribution of eNOS from the plasma membrane to the mitochondria. This increase in nitration in the mitochondria was found to induce mitochondrial dysfunction as determined by increased mitochondrial-derived reactive oxygen species and decreased generation of ATP. Finally, we found that the decrease in ATP resulted in a reduction in the chaperone activity of HSP90 resulting in a decrease in its interaction with eNOS. In conclusion increased levels of ADMA causes mitochondrial dysfunction and a loss of heat shock protein-90 chaperone activity secondary to an uncoupling of eNOS. Mitochondrial dysfunction may be an understudied component of the endothelial dysfunction associated with various cardiovascular disease states.