The role of alanine glyoxylate transaminase-2 (agxt2) in ß-alanine and carnosine metabolism of healthy mice and humans.

PURPOSE: Chronic ß-alanine supplementation leads to increased levels of muscle histidine-containing dipeptides. However, the majority of ingested ß-alanine is, most likely, degraded by two transaminases: GABA-T and AGXT2. In contrast to GABA-T, the in vivo role of AGXT2 with respect to ß-alanine metabolism is unknown. The purpose of the present work is to investigate if AGXT2 is functionally involved in ß-alanine homeostasis. METHODS: Muscle histidine-containing dipeptides levels were determined in AGXT2 overexpressing or knock-out mice and in human subjects with different rs37369 genotypes which is known to affect AGXT2 activity. Further, plasma ß-alanine kinetic was measured and urine was obtained from subjects with different rs37369 genotypes following ingestion of 1400 mg ß-alanine. RESULT: Overexpression of AGXT2 decreased circulating and muscle histidine-containing dipeptides (> 70% decrease; p < 0.05), while AGXT2 KO did not result in altered histidine-containing dipeptides levels. In both models, ß-alanine remained unaffected in the circulation and in muscle (p > 0.05). In humans, the results support the evidence that decreased AGXT2 activity is not associated with altered histidine-containing dipeptides levels (p > 0.05). Additionally, following an acute dose of ß-alanine, no differences in pharmacokinetic response were measured between subjects with different rs37369 genotypes (p > 0.05). Interestingly, urinary ß-alanine excretion was 103% higher in subjects associated with lower AGXT2 activity, compared to subjects associated with normal AGXT2 activity (p < 0.05). CONCLUSION: The data suggest that in vivo, ß-alanine is a substrate of AGXT2; however, its importance in the metabolism of ß-alanine and histidine-containing dipeptides seems small.

Homoarginine Supplementation Prevents Left Ventricular Dilatation and Preserves Systolic Function in a Model of Coronary Artery Disease.

Background Homoarginine ( hA rg) has been shown to be cardioprotective in a model of ischemic heart failure; however, the mechanism remains unknown. hA rg can inhibit tissue-nonspecific alkaline phosphatase ( TNAP ), an enzyme that promotes vascular calcification. We hypothesized that hA rg will exert beneficial effects by reducing calcification in a mouse model of coronary artery disease associated with TNAP overexpression and hypercholesterolemia. Methods and Results TNAP was overexpressed in the endothelium in mice homozygous for a low-density lipoprotein receptor mutation (wicked high cholesterol [ WHC ] allele). WHC and WHC -endothelial TNAP mice received placebo or hA rg supplementation (14 mg/L in drinking water) starting at 6 weeks of age simultaneously with an atherogenic diet. Outcomes were compared between the groups after 4 to 5 weeks on treatment. Experiments were performed in males, which presented a study limitation. As expected, WHC -endothelial TNAP mice on the placebo had increased mortality (median survival 27 days, P<0.0001), increased coronary calcium and lipids ( P<0.01), increased left ventricular end-diastolic diameter ( P<0.0001), reduced ejection fraction ( P<0.05), and increased myocardial fibrosis ( P<0.0001) compared with WHC mice. Contrary to our hypothesis, hA rg neither inhibited TNAP activity in vivo nor reduced coronary artery calcification and atherosclerosis in WHC -endothelial TNAP mice; however, compared with the placebo, hA rg prevented left ventricular dilatation ( P<0.01), preserved ejection fraction ( P<0.05), and reduced myocardial fibrosis ( P<0.001). Conclusions The beneficial effect of hA rg supplementation in the setting of calcified coronary artery disease is likely due to its direct protective actions on the myocardial response to the ischemic injury and not to the inhibition of TNAP activity and calcification.

CACNA1H missense mutations associated with amyotrophic lateral sclerosis alter Cav3.2 T-type calcium channel activity and reticular thalamic neuron firing.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. In a recent study by Steinberg and colleagues, 2 recessive missense mutations were identified in the Cav3.2 T-type calcium channel gene (CACNA1H), in a family with an affected proband (early onset, long duration ALS) and 2 unaffected parents. We have introduced and functionally characterized these mutations using transiently expressed human Cav3.2 channels in tsA-201 cells. Both of these mutations produced mild but significant changes on T-type channel activity that are consistent with a loss of channel function. Computer modeling in thalamic reticular neurons suggested that these mutations result in decreased neuronal excitability of thalamic structures. Taken together, these findings implicate CACNA1H as a susceptibility gene in amyotrophic lateral sclerosis.

Drug persistence with rivaroxaban therapy in atrial fibrillation patients-results from the Dresden non-interventional oral anticoagulation registry.

AIMS: Worldwide, rivaroxaban is increasingly used for stroke prevention in atrial fibrillation (SPAF) but little is known about the rates of or reasons for rivaroxaban discontinuations in daily care. Using data from a prospective, non-interventional oral anticoagulation (NOAC) registry, we analysed rivaroxaban treatment persistence. METHODS AND RESULTS: Persistence with rivaroxaban in SPAF was assessed in an ongoing, prospective, non-interventional registry of >2600 NOAC patients from daily care using the Kaplan-Meier time-to-first-event analysis. Reasons for and management of rivaroxaban discontinuation were assessed. Potential baseline risk factors for treatment discontinuation were evaluated using Cox regression analysis. Between October 2011 and April 2014, 1204 rivaroxaban SPAF patients were enrolled [39.3% switched from vitamin K antagonists (VKAs) and 60.7% newly treated patients]. Of these, 223 patients (18.5%) stopped rivaroxaban during follow-up (median 544 days), which translates into a discontinuation rate of 13.6 (95% CI 11.8-15.4) per 100 patient-years. Most common reasons for treatment discontinuations were bleeding complications (30% of all discontinuations), followed by other side-effects (24.2%) and diagnosis of stable sinus rhythm (9.9%). A history of chronic heart failure (HR 1.43; 95% CI 1.09-1.87; P = 0.009) or diabetes (HR 1.39; 95% CI 1.06-1.82; P = 0.018) were the only statistically significant baseline risk factors for rivaroxaban discontinuation. After discontinuation of rivaroxaban, patients received antiplatelet therapy (31.8%), VKA (24.2%), another NOAC (18.4%), heparin (9.9%), or nothing (15.7%). CONCLUSION: Our data indicate that overall persistence with rivaroxaban therapy is high, with a discontinuation rate of ∼15% in the first year of treatment and few additional discontinuations thereafter.

The deubiquitinating enzyme USP5 modulates neuropathic and inflammatory pain by enhancing Cav3.2 channel activity.

T-type calcium channels are essential contributors to the transmission of nociceptive signals in the primary afferent pain pathway. Here, we show that T-type calcium channels are ubiquitinated by WWP1, a plasma-membrane-associated ubiquitin ligase that binds to the intracellular domain III-IV linker region of the Cav3.2 T-type channel and modifies specific lysine residues in this region. A proteomic screen identified the deubiquitinating enzyme USP5 as a Cav3.2 III-IV linker interacting partner. Knockdown of USP5 via shRNA increases Cav3.2 ubiquitination, decreases Cav3.2 protein levels, and reduces Cav3.2 whole-cell currents. In vivo knockdown of USP5 or uncoupling USP5 from native Cav3.2 channels via intrathecal delivery of Tat peptides mediates analgesia in both inflammatory and neuropathic mouse models of mechanical hypersensitivity. Altogether, our experiments reveal a cell signaling pathway that regulates T-type channel activity and their role in nociceptive signaling.

S100A1 deficiency impairs postischemic angiogenesis via compromised proangiogenic endothelial cell function and nitric oxide synthase regulation.

RATIONALE: Mice lacking the EF-hand Ca2+ sensor S100A1 display endothelial dysfunction because of distorted Ca2+ -activated nitric oxide (NO) generation. OBJECTIVE: To determine the pathophysiological role of S100A1 in endothelial cell (EC) function in experimental ischemic revascularization. METHODS AND RESULTS: Patients with chronic critical limb ischemia showed almost complete loss of S100A1 expression in hypoxic tissue. Ensuing studies in S100A1 knockout (SKO) mice subjected to femoral artery resection unveiled insufficient perfusion recovery and high rates of autoamputation. Defective in vivo angiogenesis prompted cellular studies in SKO ECs and human ECs, with small interfering RNA-mediated S100A1 knockdown demonstrating impaired in vitro and in vivo proangiogenic properties (proliferation, migration, tube formation) and attenuated vascular endothelial growth factor (VEGF)-stimulated and hypoxia-stimulated endothelial NO synthase (eNOS) activity. Mechanistically, S100A1 deficiency compromised eNOS activity in ECs by interrupted stimulatory S100A1/eNOS interaction and protein kinase C hyperactivation that resulted in inhibitory eNOS phosphorylation and enhanced VEGF receptor-2 degradation with attenuated VEGF signaling. Ischemic SKO tissue recapitulated the same molecular abnormalities with insufficient in vivo NO generation. Unresolved ischemia entailed excessive VEGF accumulation in SKO mice with aggravated VEGF receptor-2 degradation and blunted in vivo signaling through the proangiogenic phosphoinositide-3-kinase/Akt/eNOS cascade. The NO supplementation strategies rescued defective angiogenesis and salvaged limbs in SKO mice after femoral artery resection. CONCLUSIONS: Our study shows for the first time downregulation of S100A1 expression in patients with critical limb ischemia and identifies S100A1 as critical for EC function in postnatal ischemic angiogenesis. These findings link its pathological plasticity in critical limb ischemia to impaired neovascularization, prompting further studies to probe the microvascular therapeutic potential of S100A1.

Aged garlic extract restores nitric oxide bioavailability in cultured human endothelial cells even under conditions of homocysteine elevation.

ETHNOPHARMACOLOGICAL RELEVANCE: Supplementation with aged garlic extract (AGE) has been shown to restore impaired endothelium-dependent vasodilator response in subjects with acutely elevated plasma homocysteine (Hcy) levels after an oral methionine load and in patients with chronic coronary artery disease. Moreover, AGE has been shown to inhibit the progression of coronary calcifications in patients with coronary artery disease. The molecular mechanisms, by which AGE preserves endothelial function is unknown. Our objective was to explore whether AGE preserves endothelial nitric oxide (NO) output even under conditions of elevated Hcy levels by preventing oxidative inactivation of the NO synthase cofactor tetrahydrobiopterin. MATERIAL AND METHODS: Endothelial (EA.hy 926) cells were incubated with hypoxanthine, aminopterin, thymidine and methionine (HAT/MET) to increase cellular Hcy levels, and with and without AGE. Agonist stimulated NO output was measured using the fluorescent probe DAF-2, and cellular thiol levels (Hcy, cysteine, reduced and oxidized glutathione) and cellular tetrahydrobiopterin levels were measured by high performance liquid chromatography. RESULTS: HAT/MET incubation resulted in significantly increased cellular Hcy levels, unaffected by coincubation with AGE. Elevated Hcy went along with significantly decreased NO output (to 34.4 ± 4.4% of control) and levels of tetrahydrobiopterin (from 4.67 ± 2.17 to 2.17 ± 0.97 pmol/mg). Incubation with AGE (5mg/mL) in HAT/MET-treated cells prevented the declines in NO output and tetrahydrobiopterin levels. AGE increased cellular levels of cysteine and total glutathione, and prevented glutathione and tetrahydrobiopterin oxidation induced by elevated Hcy. CONCLUSION: Incubation with AGE preserved normal NO output from endothelial cells even under conditions of elevated Hcy levels by increasing cellular thiol antioxidant and prevention of tetrahydrobiopterin oxidation. This suggests that AGE might be useful in the prevention of endothelial dysfunction.

Aged garlic extract inhibits homocysteine-induced scavenger receptor CD36 expression and oxidized low-density lipoprotein cholesterol uptake in human macrophages in vitro.

AIM OF THE STUDY: Expression of CD36 scavenger receptors on macrophages is involved in oxidized low-density lipoprotein (OxLDL) uptake and foam cell formation during atherosclerotic lesion development. It has been shown previously in vitro and in vivo that the atherosclerotic risk factor homocysteine (Hcy) stimulates macrophage CD36 expression and OxLDL uptake. We now examined the effects of aged garlic extract (AGE), a garlic preparation enriched in water-soluble organic sulfur-containing compounds, on Hcy-induced CD36 expression and foam cell formation in human monocytes/macrophages. RESULTS: Incubation with Hcy (200 µM for 72h) in THP-1-derived macrophages and primary human macrophages caused a 37.8±5.2% and 60.7±4.2% increase in CD36 expression compared to control, respectively. Coincubation with AGE (5mg/ml) significantly suppressed CD36 expression in THP-1 derived macrophages by 48.6±9.0% compared to Hcy-incubated cells only. AGE (1-5mg/ml) dose dependently inhibited Hcy-induced CD36 expression in primary human macrophages, and decreased binding of nuclear proteins to a PPARγ response element. Preincubation with AGE significantly inhibited DiI-labeled OxLDL uptake. CONCLUSIONS: These data indicate that AGE inhibits CD36 expression and OxLDL uptake in human macrophages by modulating the PPARγ pathway, and suggest that the extract could be useful for the prevention of atherosclerotic lesions.

Antipyretic and antinociceptive effects of Nauclea latifolia root decoction and possible mechanisms of action.

CONTEXT: Nauclea latifolia Smith (Rubiaceae) is a small tree found in tropical areas in Africa. It is used in traditional medicine to treat malaria, epilepsy, anxiety, pain, fever, etc. OBJECTIVE: The aim of this study was to investigate the effects of Nauclea latifolia roots decoction on the peripheral and central nervous systems and its possible mechanisms of action. MATERIALS AND METHODS: The analgesic investigation was carried out against acetic acid-induced writhing, formalin-induced pain, hot-plate and tail immersion tests. The antipyretic activity was studied in Brewer's yeast-induced pyrexia in mice. Rota-rod test and bicuculline-induced hyperactivity were used for the assessment of locomotor activity. RESULTS: Nauclea latifolia induced hypothermia and had antipyretic effects in mice. The plant decoction produced significant antinociceptive activity in all analgesia animal models used. The antinociceptive effect exhibited by the decoction in the formalin test was reversed by the systemic administration of naloxone, N(ω)-L-nitro-arginine methyl ester or glibenclamide. In contrast, theophylline did not reverse this effect. Nauclea latifolia (antinociceptive doses) did not exhibit a significant effect on motor coordination of the mice in Rota-rod performance. Nauclea latifolia protected mice against bicuculline-induced behavioral excitation. DISCUSSION AND CONCLUSION: Overall, these results demonstrate that the central and peripheral effects of Nauclea latifolia root decoction might partially or wholly be due to the stimulation of peripheric opioid receptors through the action of the nitric oxide/cyclic monophosphate guanosin/triphosphate adenosine (NO/cGMP/ATP)-sensitive- K(+) channel pathway and/or facilitation of the GABAergic transmission.

Aged garlic extract inhibits CD36 expression in human macrophages via modulation of the PPARgamma pathway.

Expression of CD36 scavenger receptors on macrophages is involved in oxidized low-density lipoprotein uptake and foam cell formation during atherosclerotic lesion development. We examined the effects of aged garlic extract (AGE), a garlic preparation enriched in water-soluble cysteinyl moieties that increases cellular total thiols and glutathione concentrations, on CD36 expression in human monocytes/macrophages (THP-1 cells and primary human monocytes). Compared to control, AGE (1-5 mg/mL) dose-dependently and significantly suppressed CD36 expression up to by 61.8 +/- 7.4% in THP-1-derived macrophages and up to 50.5 +/- 7.1% in primary human macrophages, respectively. Furthermore, AGE prevented induction of CD36 expression by the peroxisome proliferator activator receptor (PPAR) gamma agonist troglitazone, and decreased binding of nuclear proteins to a PPARgamma response element. AGE showed a stronger inhibitory effect on CD36 expression in THP-1 cells during simultaneous incubation with phorbol 12-myristate 13-acetate (PMA) compared to cells that had been pre-incubated with PMA. Furthermore, AGE decreased CD11b expression in a dose-dependent manner. These data indicate that AGE inhibits CD36 expression by modulating the PPARgamma pathway in human macrophages and monocytes differentiation into macrophages, and suggests that the extract could be useful for the prevention of atherosclerotic lesions.

Aged garlic extract improves homocysteine-induced endothelial dysfunction in macro- and microcirculation.

Endothelial dysfunction caused by increases in vascular oxidant stress that decrease bioavailable nitric oxide (NO) plays a critical role in the vascular pathobiology of hyperhomocysteinemia. Boosting cellular glutathione levels or increasing the activity of cellular glutathione peroxidase can compensate for homocysteine's effects on endothelial function. Aged garlic extract (AGE) contains water- and oil-soluble sulfur compounds that modify the intracellular thiol and redox state, minimize intracellular oxidant stress, and stimulate NO generation in endothelial cells and animals. We performed a placebo-controlled, blinded, crossover trial to examine whether AGE reduces macro- and microvascular endothelial dysfunction during acute hyperhomocysteinemia induced by an oral methionine challenge in healthy subjects. Acute hyperhomocysteinemia leads to a significant decrease in flow-mediated vasodilation of the brachial artery as determined by vascular ultrasound, indicative of macrovascular endothelial dysfunction. In addition, acute hyperhomocysteinemia leads to a decrease in acetylcholine-stimulated skin perfusion as measured by laser-Doppler flowmetry. This indicates microvascular endothelial dysfunction, which is presumably a result of impairment of the endothelium-derived hyperpolarizing factor pathway. Pretreatment with AGE for 6 wk significantly diminished the adverse effects of acute hyperhomocysteinemia in both vascular territories. We conclude that AGE may at least partly prevent a decrease in bioavailable NO and endothelium-derived hyperpolarizing factor during acute hyperhomocysteinemia. This pilot study warrants further investigations on the effects of AGE on endothelial dysfunction in patients with other cardiovascular risk factors or established vascular disease and on the clinical outcome of patients with cardiovascular disease.

Aged garlic extract inhibits homocysteine-induced CD36 expression and foam cell formation in human macrophages.

Elevated plasma homocysteine (Hcy) levels have been recognized as an independent risk factor for atherosclerotic vascular disease. During formation of early atherosclerotic lesions, expression of CD36, a class B scavenger receptor on macrophages, is crucially involved in the uptake of oxidized low-density lipoprotein (OxLDL) and foam-cell formation. We therefore determined the effects of Hcy on CD36 expression and foam cell formation in human monocytes/macrophages (THP-1) using flow cytometry, and the effects of aged garlic extract (AGE) on this process. Incubation of THP-1 cells with Hcy (200 micromol/L) for 72 h in the presence of phorbol 12-myristate 13-acetate (PMA) (10 nmol/L) caused a 37.8+/-5.2% increase in CD36 expression compared with PMA-stimulated cells without Hcy (P<0.01). Coincubation with AGE (5 g/L) significantly suppressed CD36 expression by 61.8+/-13.9%, compared with control conditions, and by 48.6+/-9.0% compared with Hcy-incubated cells (P<0.01). THP-1 cells in the presence of PMA (10 nmol/L) were incubated with Hcy or AGE for 72 h followed by incubation with 1,1'-dioctadecyl-3,3,3'3'-tetra-methylindocyanide percholorate (DiI)-labeled OxLDL for 3 h, and fluorescence intensity was measured by flow cytometry. AGE also inhibited DiI-labeled OxLDL uptake into PMA-stimulated THP-1 cells by 85.6+/-2.8% (P<0.01), but Hcy had no effects on it. Our data indicate that AGE inhibits CD36 expression and OxLDL uptake in macrophages and suggest that the extract could modulate the formation of early atherosclerotic lesions.

Endothelial dysfunction and atherothrombosis in mild hyperhomocysteinemia.

Mildly elevated plasma homocysteine levels are an independent risk factor for atherothrombotic vascular disease in the coronary, cerebrovascular, and peripheral arterial circulation. Endothelial dysfunction as manifested by impaired endothelium-dependent regulation of vascular tone and blood flow, by increased recruitment and adhesion of circulating inflammatory cells to the endothelium, and by a loss of endothelial cell antithrombotic function contributes to the vascular disorders linked to hyperhomocysteinemia. Increased vascular oxidant stress through imbalanced thiol redox status and inhibition of important antioxidant enzymes by homocysteine results in decreased bioavailability of the endothelium-derived signaling molecule nitric oxide via oxidative inactivation. This plays a central role in the molecular mechanisms underlying the effects of homocysteine on endothelial function. Supplementation of folic acid and vitamin B12 has been demonstrated to be efficient in lowering mildly elevated plasma homocysteine levels and in reversing homocysteine-induced impairment of endothelium-dependent vasoreactivity. Results from ongoing intervention trials will determine whether homocysteine-lowering therapies contribute to the prevention and reduction of atherothrombotic vascular disease and may thereby provide support for the causal relationship between hyperhomocysteinemia and atherothrombosis.