Endothelium-specific sepiapterin reductase deficiency in DOCA-salt hypertension.

The endothelial nitric oxide synthase (eNOS) requires tetrahydrobiopterin (H(4)B) as a cofactor and, in its absence, produces superoxide (O(2)(·-)) rather than nitric oxide (NO(·)), a condition referred to as eNOS uncoupling. DOCA-salt-induced hypertension is associated with H(4)B oxidation and uncoupling of eNOS. The present study investigated whether administration of sepiapterin or H(4)B recouples eNOS in DOCA-salt hypertension. Bioavailable NO(·) detected by electron spin resonance was markedly reduced in aortas of DOCA-salt hypertensive mice. Preincubation with sepiapterin (10 µmol/l for 30 min) failed to improve NO(·) bioavailability in hypertensive aortas while it augmented NO(·) production from control vessels, implicating a hypertension-associated deficiency in sepiapterin reductase (SPR), the rate-limiting enzyme for sepiapterin conversion to H(4)B. Indeed, a decreased SPR expression was observed in aortic endothelial cells, but not in endothelium-denuded aortic remains, implicating an endothelium-specific SPR deficiency. Administration of hypertensive aortas with H(4)B (10 µmol/l, 30 min) partially restored vascular NO(·) production. Combined administration of H(4)B and the NADPH oxidase inhibitor apocynin (100 µmol/l, 30 min) fully restored NO(·) bioavailability while reducing O(2)(·-) production. In angiotensin II-induced hypertension, however, aortic endothelial SPR expression was not affected. In summary, administration of sepiapterin is not effective in recoupling eNOS in DOCA-salt hypertension, due to an endothelium-specific loss in SPR, whereas coadministration of H(4)B and apocynin is highly efficient in recoupling eNOS. This is consistent with our previous observations that in angiotensin II hypertension, endothelial deficiency in dihydrofolate reductase is alternatively responsible for uncoupling of eNOS. Taken together, these data indicate that strategies specifically targeting at different H(4)B metabolic enzymes might be necessary in restoring eNOS function in different types of hypertension.

Discovery of Brain-Penetrant Glucosylceramide Synthase Inhibitors with a Novel Pharmacophore.

Inhibition of glucosylceramide synthase (GCS) is a major therapeutic strategy for Gaucher's disease and has been suggested as a potential target for treating Parkinson's disease. Herein, we report the discovery of novel brain-penetrant GCS inhibitors. Assessment of the structure-activity relationship revealed a unique pharmacophore in this series. The lipophilic ortho-substituent of aromatic ring A and the appropriate directionality of aromatic ring B were key for potency. Optimization of the absorption, distribution, metabolism, elimination, toxicity (ADMETox) profile resulted in the discovery of T-036, a potent GCS inhibitor in vivo. Pharmacophore-based scaffold hopping was performed to mitigate safety concerns associated with T-036. The ring opening of T-036 resulted in another potent GCS inhibitor with a lower toxicological risk, T-690, which reduced glucosylceramide in a dose-dependent manner in the plasma and cortex of mice. Finally, we discuss the structural aspects of the compounds that impart a unique inhibition mode and lower the cardiovascular risk.

Aminoguanidine inhibits aortic hydrogen peroxide production, VSMC NOX activity and hypercontractility in diabetic mice.

BACKGROUND: Dysfunctionally uncoupled endothelial nitric oxide synthase (eNOS) is involved in producing reactive oxygen species (ROS) in the diabetic endothelium. The present study investigated whether anti-diabetes drug Aminoguanidine (AG) has any effect on eNOS function and vascular oxidant stress. METHODS AND RESULTS: Blood glucose levels were increased to 452.0 +/- 15.1 mg/dl in STZ-treated male C57BL/6J mice (148.4 +/- 3.2 mg/dl in untreated controls). Aortic productions of NO* and O(2)*- were measured specifically and sensitively using electron spin resonance. Diabetic mice had a marked increase in aortic O(2)*- production. Aortic hydrogen peroxide (H(2)O(2)) production was also increased in diabetic aortas and significantly attenuated by AG. AG however had only a marginal effect in reducing aortic O(2)*- production, which corresponded to a minimal effect in improving aortic nitric oxide (NO*) bioavailability. The endothelium-dependent vasodilatation however was modestly but significantly improved by AG, likely consequent to AG-induced reduction in hyper-contractility. NAD(P)H oxidase (NOX)-dependent O(2)*- production was completely attenuated by AG in endothelium-denuded diabetic aortas. CONCLUSION: In summary, despite that AG is not an effective eNOS recoupling agent presumably consequent to its ineffectiveness in preventing endothelial NOX activation, it is inhibitory of aortic H(2)O(2) production, VSMC NOX activity, and hypercontractility in diabetes.

Attenuation of angiotensin II signaling recouples eNOS and inhibits nonendothelial NOX activity in diabetic mice.

Angiotensin II (Ang II) levels are increased in patients with diabetes, but mechanisms underlying its contribution to diabetic vascular diseases are incompletely understood. We recently reported that in aortic endothelial cells, Ang II induces endothelial nitric oxide synthase (eNOS) uncoupling to produce superoxide (O(2)*(-)) rather than nitric oxide (NO*), upon loss of the tetrahydrobiopterin (H(4)B) salvage enzyme dihydrofolate reductase (DHFR). Here, we found that streptozotocin-induced diabetic mice had a marked increase in aortic O(2)*(-) production, which was inhibited by N-nitro-l-arginine methyl ester hydrochloride, indicating uncoupling of eNOS. Ang II receptor type 1 blocker candesartan or ACE inhibitor captopril markedly attenuated eNOS-derived O(2)*(-) and hydrogen peroxide production while augmenting NO* bioavailability in diabetic aortas, implicating recoupling of eNOS. O(2)*(-) and NO* production were characteristically and quantitatively measured by electron spin resonance. DHFR expression was decreased in diabetic aortas but significantly restored by candesartan or captopril. Either also improved vascular H(4)B content and endothelium-dependent vasorelaxation in diabetes. Rac1-dependent NAD(P)H oxidase (NOX) activity was more than doubled in the endothelium-denuded diabetic aortas but was attenuated by candesartan or captopril, indicating that NOX remains active in nonendothelial vascular tissues, although uncoupled eNOS is responsible for endothelial production of O(2)*(-). These data demonstrate a novel role of Ang II in diabetic uncoupling of eNOS and that Ang II-targeted therapy improves endothelial function via the novel mechanism of recoupling eNOS. Dual effectiveness on uncoupled eNOS and NOX may explain the high efficacy of Ang II antagonists in restoring endothelial function.

The occurrence of a novel hydrophilic hydroperoxide, 3-hydroxy-5-hydroperoxy-2-methyl-5,6-dihydropyran-4-one, as a reactive glycation product in human plasma.

This study describes the occurrence of 3-hydroxy-5-hydroperoxy-2-methyl-5,6-dihydropyran-4-one (HMDP) in plasma obtained from normal subjects and patients with type 2 diabetes. We have shown previously that HMDP is a novel hydrophilic hydroperoxide formed in the in vitro Maillard reaction that could be analyzed using ultrasensitive chemiluminescence detection-high-performance liquid chromatography (CL-HPLC). The HMDP concentration was 273+/-227 nmol/l in normal subjects and 656+/-535 nmol/l in patients with diabetes. The HMDP concentration was proportional to the plasma glucose concentration level (r=0.640; P<0.01) but not with the glycated hemoglobin level. To investigate the in vivo effects of HMDP, a range of concentrations of the compound was incubated for different time periods with human serum albumin and lipoproteins. HMDP was found to induce denaturation of these macromolecules by modifying lysine residues and causing cross-linking and polymerization of proteins. In the presence of metal ions such as iron and copper, HMDP induced peroxidative degradation of lipoprotein lipids as measured by an elevation in thiobarbituric acid reactive substances (TBARS) concentration. These results suggested that HMDP is produced as a consequence of both hyperglycemia and increased oxidative stress, and may have the potential to contribute to the pathogenesis of arterial complications in diabetes mellitus.

A convenient method for preparation of high-purity, Amadori-glycated phosphatidylethanolamine and its prooxidant effect.

Phospholipid-linked glycation has been implicated to play an important role in lipid peroxidation associated with several conditions, such as normal aging and diabetes. In this study, we established a convenient method for the preparation of a high-purity, Amadori-glycated phosphatidylethanolamine (Amadori-PE) standard in a methanol system. We evaluated the prooxidant effect of the Amadori-PE standard. When Amadori-PE was incubated in a micellar and liposome system in the presence of metal ion, a remarkable increase of lipid peroxidation was observed in both systems. In addition, the Amadori-PE-induced lipid peroxidation was effectively inhibited by superoxide dismutase, mannitol, catalase, and EDTA. These results indicate that Amadori-PE is a considerable risk factor that may cause membrane lipid peroxidation in the pathogenesis of diabetes and aging.

Tandem mass spectrometry analysis of Amadori-glycated phosphatidylethanolamine in human plasma.

Amadori-glycated phosphatidylethanolamine (Amadori-PE), a nonenzymatically glycated lipid formed under hyperglycemic conditions, is known as a reliable indicator of lipid glycation in vivo. We have quantified the Amadori-PE concentration in human plasma samples using a reverse-phase liquid chromatography-tandem mass spectrometry with neutral loss scan or multiple reaction monitoring. Amounts of Amadori-PE in plasma of diabetic patients (0.15 mol% of PE), diabetic patients with chronic hemodialysis (0.29 mol% of PE), and nondiabetic patients with chronic hemodialysis (0.13 mol% of PE) are higher than that of the control group (0.08 mol% of PE). In addition, the concentration of Amadori-PE was proportional to that of phosphatidylcholine hydroperoxide, a reliable indicator of membrane lipid peroxidation, in human plasma (P < 0.05). These results indicate that plasma Amadori-PE-glycated lipid product formed under hyperglycemic conditions is an inducer of membrane lipid peroxidation, and therefore lipid glycation plays an active part in the development of human disease.

Angiogenic potency of Amadori-glycated phosphatidylethanolamine.

Glycation has been thought to participate in diabetic vascular diseases. However, there are no reports about the effects of lipid glycation on endothelial dysfunction. In this study, we have evaluated whether Amadori-glycated phosphatidylethanolamine (Amadori-PE), a lipid-linked glycation compound, affected proliferation, migration, and tube formation of cultured human umbilical vein endothelial cells. These three factors involved in angiogenesis were significantly stimulated by Amadori-PE at a low concentration of less than 5 microM. Furthermore, Amadori-PE also stimulated the secretion of matrix metalloproteinase-2 (MMP-2), a pivotal enzyme in the initial step of angiogenesis. Our results indicated for the first time that Amadori-PE would elicit vascular disease through angiogenic potency on endothelial cells, thereby playing an active part in the development and progression of diabetic microangiopathy.

Amadori-glycated phosphatidylethanolamine induces angiogenic differentiations in cultured human umbilical vein endothelial cells.

Glycation has been implicated in the endothelial dysfunction that contributes to both diabetes- and aging-associated vascular complications. The aim of the present study was to determine whether Amadori-glycated phosphatidylethanolamine (Amadori-PE), a lipid-linked glycation compound that is formed at an increased rate in hyperglycemic states, affected proliferation, migration and tube formation of cultured human umbilical vein endothelial cells (HUVEC). Amadori-PE at a low concentration of less than 5 microM significantly enhanced these three factors involved in angiogenesis. Furthermore, stimulation of HUVEC with Amadori-PE resulted in secretion of matrix metalloproteinase 2 (MMP-2), a pivotal enzyme in the initial step of angiogenesis. Our results demonstrated for the first time that Amadori-PE may be an important compound that promotes vascular disease as a result of its angiogenic activity on endothelial cells. We also demonstrated that MMP-2 is a primary mediator of Amadori-PE-driven angiogenesis.

Ion-trap tandem mass spectrometric analysis of Amadori-glycated phosphatidylethanolamine in human plasma with or without diabetes.

Peroxidized phospholipid-mediated cytotoxicity is involved in the pathophysiology of diseases [i.e., an abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) in plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (Amadori-PE; deoxy-D-fructosyl phosphatidylethanolamine), because Amadori-PE causes oxidative stress. However, the occurrence of lipid glycation products, including Amadori-PE, in vivo is still unclear. Consequently, we developed an analysis method of Amadori-PE using a quadrupole/linear ion-trap mass spectrometer, the Applied Biosystems QTRAP. In positive ion mode, collision-induced dissociation of Amadori-PE produced a well-characterized diglyceride ion ([M+H-303]+) permitting neutral loss scanning and multiple reaction monitoring (MRM). When lipid extract from diabetic plasma was infused directly into the QTRAP, Amadori-PE molecular species could be screened out by neutral loss scanning. Interfacing liquid chromatography with QTRAP mass spectrometry enabled the separation and determination of predominant plasma Amadori-PE species with sensitivity of approximately 0.1 pmol/injection in MRM. The plasma Amadori-PE level was 0.08 mol% of total PE in healthy subjects and 0.15-0.29 mol% in diabetic patients. Furthermore, plasma Amadori-PE levels were positively correlated with PCOOH (a maker for oxidative stress). These results show the involvement between lipid glycation and lipid peroxidation in diabetes pathogenesis.

UV analysis of Amadori-glycated phosphatidylethanolamine in foods and biological samples.

Maillard reactions are among the most important of the chemical and oxidative changes occurring in food and biological samples that contribute to food deterioration and to the pathophysiology of human disease. Although the association of lipid glycation with this process has recently been shown, the number of lipid glycation products in food and biological materials has not been clear. In this study, we synthesized the Amadori products derived from the glycation of phosphatidylethanolamine (PE), i.e., Amadori-PEs. Dioleoyl PE was incubated with glucose and lactose for 15 days, and the resultant Amadori-PEs were purified and isolated using solid phase extraction followed by HPLC. With this procedure, essentially pure (>98% purity) Amadori-PEs glycated with glucose (Glc-PE) and with lactose (Lac-PE) were obtained and used as standards in the subsequent studies. To determine the presence of Amadori-PEs in food and biological samples, the carbonyl group of Amadori-PEs was ultraviolet (UV)-labeled with 3-methyl-2-benzothiazolinone hydrazone, and the labeled Amadori-PEs were analyzed with normal phase HPLC-UV (318 nm). The detection limit was 4.5 ng (5 pmol) for Glc-PE and 5.3 ng (5 pmol) for Lac-PE. Among the several food samples examined, infant formula and chocolate contained a high amount of both Glc-PE and Lac-PE over wide concentration ranges, such as 1.5-112 microg/g. Testing biological materials showed Amadori-PE (Glc-PE) was detectable in rat plasma.