Association of Genes of the NO Pathway with Altitude Disease and Hypoxic Pulmonary Hypertension

Chronic intermittent hypoxia leads to high-altitude pulmonary hypertension, which is associated with high asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthesis. Therefore, we aimed to understand the relation of single nucleotide polymorphisms in this pathway to high-altitude pulmonary hypertension (HAPH). We genotyped 69 healthy male Chileans subjected to chronic intermittent hypoxia. Acclimatization to altitude was determined using the Lake Louise Score and the presence of acute mountain sickness. Echocardiography was performed after six months in 24 individuals to estimate pulmonary arterial pressure. The minor allele of dimethylarginine dimethylaminohydrolase (DDAH)1 rs233112 was associated with high-baseline plasma ADMA concentration, while individuals homozygous for the major allele of DDAH2 rs805304 had a significantly greater increase in ADMA during chronic intermittent hypoxia. The major allele of alanine glyoxylate aminotransferase-2 (AGXT2) rs37369 was associated with a greater reduction of plasma symmetric dimethylarginine (SDMA). Several genes were associated with high-altitude pulmonary hypertension, and the nitric oxide synthase (NOS)3 and DDAH2 genes were related to acute mountain sickness. In conclusion, DDAH1 determines baseline plasma ADMA, while DDAH2 modulates ADMA increase in hypoxia. AGXT2 may be up-regulated in hypoxia. Genomic variation in the dimethylarginine pathway affects the development of HAPH and altitude acclimatization.

Effect of a Low Dose of Carvedilol on Cyclophosphamide-Induced Urinary Toxicity in Rats—A Comparison with Mesna

One of the major side effects of cyclophosphamide (CPX)—an alkylating anticancer drug that is still clinically used—is urotoxicity with hemorrhagic cystitis. The present study was designed to evaluate the ability of carvedilol to protect rats from cyclophosphamide-induced urotoxicity. Rats were injected intraperitoneally (i.p.) with CPX (200 mg/kg) and administered carvedilol (2 mg/kg) intragastrically a day before, at the day and a day after a single i.p. injection of CPX, with or without mesna (40, 80, and 80 mg/kg i.p. 20 min before, 4 h and 8 h after CPX administration, respectively). Pretreatment with carvedilol partly prevented the CPX-induced increase in urinary bladder and kidney index, and completely protects from CPX-evoked alterations in serum potassium and creatinine level, but did not prevent histological alterations in the urinary bladder and hematuria. However, carvedilol administration resulted in significant restoration of kidney glutathione (GSH) level and a decrease in kidney interleukin 1β (IL-1β) and plasma asymmetric dimethylarginine (ADMA) concentrations. Not only did mesna improve kidney function, but it also completely reversed histological abnormalities in bladders and prevented hematuria. In most cases, no significant interaction of carvedilol with mesna was observed, although the effect of both drugs together was better than mesna given alone regarding plasma ADMA level and kidney IL-1β concentration. In conclusion, carvedilol did not counteract the injury caused in the urinary bladders but restored kidney function, presumably via its antioxidant and anti-inflammatory properties.

Cardiovascular Risk and Endothelial Dysfunction in Primary Sjogren Syndrome Is Related to the Disease Activity

The aim of our study was to evaluate if endothelial-dysfunction (ED) occurs in patients with primary Sjogren syndrome (pSS) and whether it is associated with the disease characteristics and activity. A total of 46 patients with pSS and 30 controls, without known cardiovascular disease, were enrolled in this study. A flow-mediated-dilation (FMD) of the brachial artery, plasma concentrations of the nitric oxide (NO) metabolic pathway (ADMA, L-arginine, SDMA, cGMP), and markers of endothelial inflammatory function (PAI-1, sE-selectin) and angiogenesis (angiostatin, VEGF) were analyzed. The FMD was significantly lower in pSS patients (7.56 ± 3.08 vs. 10.91 ± 1.02%, p = 0.043) and positively correlated with the Ro/SS-A-antibodies (r = 0.34, p = 0.03), pulmonary involvement (r = 0.52, p = 0.001) and inversely with ADMA (r = −0.35, p = 0.04). Plasma ADMA, L-arginine and angiostatin levels were significantly higher in pSS patients (0.39 ± 0.08 vs. 0.36 ± 0.06 µmol/L, p = 0.05; 29.07 ± 6.7 vs. 25.4 ± 5.23 µmol/L, p = 0.01; 152.25 ± 60.99 vs. 120.07 ± 38.7 pg/mL, p = 0.0, respectively). ADMA was associated with ESSDAI (r = 0.33, p = 0.02), SCORE (r = 0.57, p = 0.00003) and focus score (r = 0.38, p = 0.04). In the multiple regression analysis, the ESSDAI was significantly and independently associated with plasma ADMA levels (β = 0.24, p = 0.04). Moreover, plasma cGMP concentrations were negatively correlated with the disease duration (r = −0.31, p = 0.03). Endothelial function is impaired in patients with pSS and associated with the measures of disease activity, which supports the key-role of inflammation in developing and maintaining accelerated atherosclerosis.

Effects of AGXT2 variants on blood pressure and blood sugar among 750 older Japanese subjects recruited by the complete enumeration survey method

Background Alanine:glyoxylate aminotransferase 2 (AGXT2; EC 2.6.1.44) is the only enzyme that degrades the R-form of 3-aminoisobutyrate, an intermediate metabolite of thymine. AGXT2, as well as diaminoarginine dimethylaminohydrolase 1 (DDAH1; EC 3.5.3.18), works as an enzyme that degrades asymmetric dimethylarginine (ADMA), which competitively inhibits the nitric oxide synthase family. Thus, these two enzyme activities may change vascular vulnerability for a lifetime via the nitric oxide (NO) system. We investigated the association between vascular conditions and diseases such as hypertension and diabetes mellitus and polymorphisms of these two genes in 750 older Japanese subjects (mean age ± standard deviation, 77.0 ± 7.6 years) recruited using the complete enumeration survey method in the Nakayama study. Demographic and biochemical data, such as blood pressure (BP) and casual blood sugar (CBS), were obtained. Four functional single nucleotide polymorphisms (SNPs; rs37370, rs37369, rs180749, and rs16899974) of AGXT2 and one functional insertion/deletion polymorphism in the promotor region with four SNPs (rs307894, rs669173, rs997251, and rs13373844) of DDAH1 were investigated. Plasma ADMA was also analyzed in 163 subjects. Results The results of multiple regression analysis showed that a loss of the functional haplotype of AGXT2, CAAA, was significantly positively correlated with BP (systolic BP, p = 0.034; diastolic BP, p = 0.025) and CBS (p = 0.021). No correlation was observed between DDAH1 and either BP or CBS. ADMA concentrations were significantly elevated in subjects with two CAAA haplotypes compared with subjects without the CAAA haplotype (p = 0.033). Conclusions Missense variants of AGXT2, but not DDAH1, may be related to vulnerability to vascular diseases such as hypertension and DM via the NO system.

Overexpression of Alanine-glyoxylate aminotransferase 2 Protects from Asymmetric Dimethylarginine-induced Endothelial Dysfunction and Aortic Remodeling

Objective: Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) are associated with an increased risk of mortality and adverse cardiovascular outcomes. ADMA can be metabolized by dimethylarginine dimethylaminohydrolases (DDAHs) and by alanine-glyoxylate aminotransferase 2 (AGXT2). Deletion of DDAH1 in mice leads to elevation of ADMA in plasma and blood pressure, while overexpression of human DDAH1 is associated with a lower plasma ADMA concentration and protective cardiovascular effects. The possible role of alternative metabolism of ADMA by AGXT2 remains to be elucidated. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of plasma levels of ADMA and protection from vascular damage in the setting of DDAH1 deficiency.Approach and Results: We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of asymmetric dimethylguanidino valeric acid (ADGV), the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling.Conclusions: Upregulation of AGXT2 led to lowering of ADMA levels and protection from ADMA-induced vascular damage in the setting of DDAH1 deficiency. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful.

PHYSICAL THERAPY AFFECTS ENDOTHELIAL FUNCTION IN LYMPHEDEMA PATIENTS

Lymphedema arises due to a malfunction of the lymphatic system and can lead to massive tissue swelling. Complete decongestive therapy (CDT), consisting of manual lymphatic drainage (MLD) and compression bandaging, is aimed at mobilizing fluid and reducing volume in affected extremities. Lymphatic dysfunction has previously been associated with chronic inflammation processes. We investigated plasma ADMA as an indicator of endothelial function/inflammation before-, during- and after-CDT. Also assessed were vascular function parameters such as carotid-femoral pulse wave velocity (PWVcf), flow-mediated dilatation (FMD) and retinal microvasculature analysis. 13 patients (3 males and 10 females, 57 ± 8 years old (mean ± SD), 167.2 ± 8.3 cm height, 91.0 ± 23.5 kg weight), with lower limb lymphedema were included. Vascular function parameters were assessed on day 1, 2, 7, 14 and 21 of CDT, pre- and post-MLD. ADMA was significantly lower post-MLD (p=0.0064) and tended to reduce over three weeks of therapy (p=0.0506). PWVcf weakly correlated with FMD (r=0.361, p=0.010). PWVcf, FMD and retinal microvasculature analysis did not show changes due to physical therapy. The novel results from this study indicate that lymphedema does not affect endothelial function and lymphedema patients may therefore not have a higher risk of cardiovascular diseases. Our results further suggest that manual lymphatic drainage with or without full CDT could have potentially beneficial effects on endothelial function in lymphedema patients (by reducing ADMA levels), which has not been reported previously.

Sequence Variation in the DDAH1 Gene Predisposes for Delayed Cerebral Ischemia in Subarachnoidal Hemorrhage

Delayed cerebral ischemia (DCI) often causes poor long-term neurological outcome after subarachnoidal hemorrhage (SAH). Asymmetric dimethylarginine (ADMA) inhibits nitric oxide synthase (NOS) and is associated with DCI after SAH. We studied single nucleotide polymorphisms (SNPs) in the NOS3, DDAH1, DDAH2, PRMT1, and AGXT2 genes that are part of the L-arginine–ADMA–NO pathway, and their association with DCI. We measured L-arginine, ADMA and symmetric dimethylarginine (SDMA) in plasma and cerebrospinal fluid (CSF) of 51 SAH patients at admission; follow-up was until 30 days post-discharge. The primary outcome was the incidence of DCI, defined as new infarctions on cranial computed tomography, which occurred in 18 of 51 patients. Clinical scores did not significantly differ in patients with or without DCI. However, DCI patients had higher plasma ADMA and SDMA levels and higher CSF SDMA levels at admission. DDAH1 SNPs were associated with plasma ADMA, whilst AGXT2 SNPs were associated with plasma SDMA. Carriers of the minor allele of DDAH1 rs233112 had a significantly increased relative risk of DCI (Relative Risk = 2.61 (1.25–5.43), p = 0.002). We conclude that the DDAH1 gene is associated with ADMA concentration and the incidence of DCI in SAH patients, suggesting a pathophysiological link between gene, biomarker, and clinical outcome in patients with SAH.

Arginine and Arginine/ADMA Ratio Predict 90-Day Mortality in Patients with Out-of-Hospital Cardiac Arrest—Results from the Prospective, Observational COMMUNICATE Trial

(1) Background: In patients with shock, the L-arginine nitric oxide pathway is activated, causing an elevation of nitric oxide, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) levels. Whether these metabolites provide prognostic information in patients after out-of-hospital cardiac arrest (OHCA) remains unclear. (2) Methods: We prospectively included OHCA patients, recorded clinical parameters and measured plasma ADMA, SDMA and Arginine levels by liquid chromatography tandem mass spectrometry (LC-MS). The primary endpoint was 90-day mortality. (3) Results: Of 263 patients, 130 (49.4%) died within 90 days after OHCA. Compared to survivors, non-survivors had significantly higher levels of ADMA and lower Arginine and Arginine/ADMA ratios in univariable regression analyses. Arginine levels and Arginine/ADMA ratio were significantly associated with 90-day mortality (OR 0.51 (95%CI 0.34 to 0.76), p < 0.01 and OR 0.40 (95%CI 0.26 to 0.61), p < 0.001, respectively). These associations remained significant in several multivariable models. Arginine/ADMA ratio had the highest predictive value with an area under the curve (AUC) of 0.67 for 90-day mortality. Results for secondary outcomes were similar with significant associations with in-hospital mortality and neurological outcome. (4) Conclusion: Arginine and Arginine/ADMA ratio were independently associated with 90-day mortality and other adverse outcomes in patients after OHCA. Whether therapeutic modification of the L-arginine-nitric oxide pathway has the potential to improve outcome should be evaluated.

Abstract 13682: Single Nucleotide Polymorphisms in the Ddah1 Gene Are Associated With Delayed Cerebral Ischemia in Patients With Subarachnoid Hemorrhage

Introduction: Delayed cerebral ischemia (DCI) is a major cause of lethality and poor long-term neurological outcome in patients with subarachnoid hemorrhage (SAH). Asymmetric (ADMA) and symmetric dimethylarginine (SDMA) are regulators of vascular NO synthesis from L-arginine. We previously reported that elevated concentrations of both, ADMA and SDMA at ICU admission are associated with the incidence of DCI during follow-up. Dimethylarginines (DMAs) are synthesized by PRMT enzymes; ADMA is metabolized by DDAH1 and DDAH2, whilst SDMA is metabolized by AGXT2. Hypothesis: We hypothesized that common genetic variants in genes encoding the core enzymes of DMA metabolism may predispose individuals for the development of DCI after SAH. Methods: We measured L-arginine, ADMA and SDMA in plasma and cerebrospinal fluid (CSF) of 51 SAH patients at ICU admission and followed them for clinical status and neurological outcome until 30 days post-discharge. Single nucleotide polymorphisms (SNPs) in the NOS3, DDAH1, DDAH2, PRMT1, and AGXT2 genes were analyzed by PCR, and genotypes were related to biomarker levels and outcome. The primary outcome was the incidence of DCI, which was defined as the appearance of new infarctions on cranial computed tomography or magnetic resonance imaging. Results: 18 out of 51 SAH patients developed DCI. DCI patients did not significantly differ from those without DCI in clinical scores. However, patients who developed DCI had higher plasma ADMA and SDMA levels and higher SDMA levels in CSF at admission. DDAH1 gene variation was associated with plasma ADMA (p=0.02), whilst AGXT2 gene variation was associated with plasma SDMA (p=0.02). There was a strong association of all three DDAH1 SNPs that we analyzed with DCI, with carriers of the minor allele of DDAH1 rs233112 having a significantly increased relative risk of developing DCI (RR=2.61 (1.25-5.43), p=0.002). Conclusions: Sequence variation in the DDAH1 gene is associated with the incidence of DCI in SAH patients, suggesting that SNPs in the DDAH1 gene, which regulates plasma ADMA concentration, significantly influences the risk of cerebral ischemia after subarachnoid hemorrhage.

Evaluation of the Correlation between Serum Concentrations of Asymmetric Dimethylarginine and Corrected TIMI Frame Count in Patients with Slow Coronary Flow

Coronary slow flow (CSF) is an important angiographic entity that is characterized by delayed opacification of coronary arteries in the absence of epicardial occlusive disease. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase. Elevated levels of ADMA cause the induction of endothelial dysfunction and thus promote atherosclerosis. This study was aimed at determining the role of ADMA in the development of CSF. One hundred twenty-nine subjects who fulfilled the inclusion criteria were enrolled in this study. According to coronary angiography results, these subjects were divided into five groups. The serum concentration of ADMA was measured in these subjects. In this study, there was no significant correlation between serum concentrations of ADMA and mean corrected TIMI frame count (CTFC) (P>0.05). However, the ADMA level was significantly correlated with CTFC in the left anterior descending (LAD) coronary artery in patients with CSF (r=−0.381, P=0.045). Also, plasma ADMA levels were significantly higher in patients with CSF and without CAD compared to patients without CSF and with CAD (50-90%) (P=0.034). Besides, serum concentrations of ADMA were significantly higher in subjects with BMI<25 kg/m2 compared with those having BMI>30 kg/m2 (P=0.003). It was also shown that the levels of ADMA were significantly higher in subjects with age as a cardiovascular risk factor compared with those without this risk factor (P=0.024). Further studies with larger population sizes are needed to confirm the present findings on the association between the serum concentrations of ADMA and CSF.