3-nitrotyrosine and Vascular-Diseases

Glycation, oxidation, nitration, and crosslinking of proteins are implicated in the pathogenic mechanisms of type 2 diabetes, cardiovascular disease, and chronic kidney disease. Related modified amino acids formed by proteolysis are excreted in urine. We quantified urinary levels of these metabolites and branched-chain amino acids (BCAAs) in healthy subjects and assessed changes in early-stage decline in metabolic, vascular, and renal health and explored their diagnostic utility for a noninvasive health screen. We recruited 200 human subjects with early-stage health decline and healthy controls. Urinary amino acid metabolites were determined by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry. Machine learning was applied to optimise and validate algorithms to discriminate between study groups for potential diagnostic utility. Urinary analyte changes were as follows: impaired metabolic health-increased N

Topics: Adult; Algorithms; Amino Acids, Branched-Chain; Biomarkers; Body Mass Index; Case-Control Studies; Chromatography, High Pressure Liquid; Female; Glycation End Products, Advanced; Glycosylation; Humans; Kidney; Lysine; Male; Metabolic Diseases; Oxidation-Reduction; Severity of Illness Index; Tandem Mass Spectrometry; Tyrosine; Vascular Diseases

The endothelial dysfunction of Fabry disease results from α-galactosidase A deficiency leading to the accumulation of globotriaosylceramide. Vasculopathy in the α-galactosidase A null mouse is manifested as oxidant-induced thrombosis, accelerated atherogenesis, and impaired arterial reactivity. To better understand the pathogenesis of Fabry disease in humans, we generated a human cell model by using RNA interference. Hybrid endothelial cells were transiently transfected with small interfering RNA (siRNA) specifically directed against α-galactosidase A. Knockdown of α-galactosidase A was confirmed using immunoblotting and globotriaosylceramide accumulation. Endothelial nitric oxide synthase (eNOS) activity was correspondingly decreased by >60%. Levels of 3-nitrotyrosine (3NT), a specific marker for reactive nitrogen species and quantified using mass spectrometry, increased by 40- to 120-fold without corresponding changes in other oxidized amino acids, consistent with eNOS-derived reactive nitrogen species as the source of the reactive oxygen species. eNOS uncoupling was confirmed by the observed increase in free plasma and protein-bound aortic 3NT levels in the α-galactosidase A knockout mice. Finally, 3NT levels, assayed in biobanked plasma samples from patients with classical Fabry disease, were over sixfold elevated compared with age- and gender-matched controls. Thus, 3NT may serve as a biomarker for the vascular involvement in Fabry disease.

Topics: Adolescent; Adult; alpha-Galactosidase; Animals; Biomarkers; Case-Control Studies; Cell Line; Disease Models, Animal; Endothelial Cells; Fabry Disease; Human Umbilical Vein Endothelial Cells; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Nitric Oxide Synthase Type III; RNA, Small Interfering; Tyrosine; Vascular Diseases; Young Adult

The aim of this study was to establish whether the long-term consumption of reused canola oil contributes to the development of dyslipidemia, obesity, and endothelial function.. Canola oil was used for one frying cycle (1 FC) of corn flour dough or reused 10 times (10 FC). Rats received chow diet (control) or supplemented with 7% raw oil (RO), 1 FC or 10 FC oil (n = 10 per group). Food consumption, blood pressure (BP), and body weight plasma glucose, plasma lipids were monitored. Vascular reactivity was analyzed using aorta rings stimulated with phenylephrine and acetylcholine. Nitrotyrosine presence in aorta rings was analyzed by immunohistochemistry.. After 10 wk of follow-up, visceral adipose tissue was significantly more abundant in 1 FC (7.4 ± 0.6 g) and 10 FC (8.8 ± 0.7 g) than the RO (5.0 ± 0.2 g; P = 0.05 versus 10 FC group) or control group (2.6 ± 0.3 g; P = 0.05 versus all groups). Despite similar plasma cholesterol, triglycerides, and BP among groups, a significantly reduced acetylcholine-induced vascular relaxation was observed in the three groups receiving the oil-supplemented diet (47.2% ± 3.6%, 27.2% ± 7.7%, and 25.9% ± 7.6% of relaxation, for the RO, 1 FC, and 10 FC, respectively; P < 0.05 for all versus 62.4% ± 9.7% of the control group). Endothelial dysfunction was concomitant with the presence of nitrotyrosine residues at a higher extent in the groups that received heated oils compared with the RO group.. High canola oil intake over 10 wk was associated with increased adipose tissue and early endothelial dysfunction probably induced by peroxinitrite formation. Such deleterious effects were significantly potentiated when the consumed oil had been used repeatedly for frying.

Topics: Acetylcholine; Adiposity; Animals; Aorta; Brassica rapa; Cooking; Diet; Dietary Fats, Unsaturated; Endothelium, Vascular; Fatty Acids, Monounsaturated; Intra-Abdominal Fat; Male; Obesity, Abdominal; Plant Oils; Rapeseed Oil; Rats, Wistar; Time Factors; Tyrosine; Vascular Diseases; Vasodilation

Enzyme replacement therapy in Fabry disease was initiated in 2001. In a significant proportion of patients, the apparent removal of stored glycosphingolipid from the endothelial cells does not prevent progression of vascular disease. Shu et al. show a link between accumulation of globotriaosylceramide in the endothelial cells and 3-nitrotyrosine formation, indicating endothelial nitric oxide synthase uncoupling. 3-Nitrotyrosine will be useful to better understand Fabry vasculopathy, and to evaluate additional therapeutic interventions targeting oxidative stress.

Topics: Animals; Fabry Disease; Humans; Male; Tyrosine; Vascular Diseases

Gestational diabetes (GDM) is associated with increased oxidative stress and overexpression of inflammatory cytokines, both of which might lead to endothelial dysfunction and vascular disease. As such, GDM could be viewed as a sort of ‘short lived’ metabolic syndrome. As umbilical cord vessels represent a suitable model for the study of vascular alterations brought about by GDM, the aim of the present work was to characterize the phenotype of human umbilical vein endothelial cells (HUVECs) chronically exposed to hyperglycaemia and to a pro-inflammatory environment during pregnancy so as to identify molecular modifications of cellular homoeostasis eventually impacting on endothelial dysfunction.. Tissue specimens and HUVECs were obtained from umbilical cords of GDMand control women. As compared to controls, GD-HUVEC exhibited enhanced monocyte adhesion and vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1(ICAM-1) expression and exposure on plasma membrane after tumour necrosis factor-alpha(TNF-α) stimulation (Western blot, flow cytometer). As compared to control cells, GD-HUVEC in basal conditions exhibited enhanced monocyte adhesion, nitric oxide synthase (NOS) expression and activity (eNOS Real-Time polymerase chain reaction, Western Blot for eNOS total protein and monomers/dimers ratio, conversion of [3H]-L-arginine in [3H]-L-citrulline), increased O(-)(2)egeneration together with increased NT levels (immunofluorescence) and reduced NO bioavailability(guanosine 3',5'-monophosphate (cGMP) production, EIA). Furthermore, immunohistochemistry revealed increased eNOS and NT immunoreactivity in GD umbilical cords.. Endothelial cells exposed in vivo even transiently to hyperglycaemia, oxidative stress and inflammation exhibit durable pro-atherogenic modifications.

Topics: Adult; Atherosclerosis; Blood Glucose; Cell Adhesion; Cyclic AMP; Diabetes, Gestational; Female; Glucose Tolerance Test; Homeostasis; Human Umbilical Vein Endothelial Cells; Humans; Hyperglycemia; Leukocytes; Nitric Oxide; Nitric Oxide Synthase Type III; Oxidative Stress; Pregnancy; Tyrosine; Umbilical Cord; Vascular Diseases

Aging is associated with increased oxidative stress levels and impaired neovascularization following ischemia. Because Nox2-containing NADPH oxidase is a major source of ROS in the vasculature, we investigated its potential role for the modulation of ischemia-induced neovascularization in the context of aging.. Hindlimb ischemia was surgically induced by femoral artery removal in young (2 months) and old (10 months) Nox2-deficient (Nox2(-/-)) and wild type mice. We found that Nox2 expression is increased by aging in ischemic muscles of wild type mice. This is associated with a significant reduction of blood flow recovery after ischemia in old compared to young mice at day 21 after surgery (Doppler flow ratios: 0.51 ± 0.05 vs. 0.72 ± 0.05; p < 0.05). We also demonstrate that capillary and arteriolar densities are significantly reduced in ischemic muscles of old animals, while oxidative stress levels are increased (nitrotyrosine immunostaining). Importantly, Nox2 deficiency reduces oxidative stress levels in ischemic tissues and restores blood flow recuperation and vascular densities in old animals. Endothelial progenitor cells (EPCs) have an important role for postnatal neovascularization. Here we show that the functional activities of EPCs (migration, adhesion to mature endothelial cells) are significantly impaired in old compared to young mice. However, Nox2 deficiency rescues EPC functional activities in old animals. We also demonstrate an age-dependent pathological increase of oxidative stress levels in EPCs (DHE, DCF-DA) that is not present in Nox2-deficient animals.. Nox2-containing NADPH oxidase deficiency protects against age-dependent impairment of neovascularization. Potential mechanisms include reduced ROS generation in ischemic tissues and preserved angiogenic activities of EPCs.

Topics: Age Factors; Aging; Animals; Blood Flow Velocity; Cell Adhesion; Cell Movement; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Hindlimb; Ischemia; Laser-Doppler Flowmetry; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Skeletal; NADPH Oxidase 2; NADPH Oxidases; Neovascularization, Physiologic; Oxidative Stress; Recovery of Function; Regional Blood Flow; Stem Cells; Superoxides; Time Factors; Tyrosine; Vascular Diseases

This study was designed to examine the effects of a high-fat, high-sucrose (HFHS) diet on vascular and metabolic actions of insulin. Male rats were randomized to receive an HFHS or regular chow diet for 4 wk. In a first series of experiments, the rats had pulsed Doppler flow probes and intravascular catheters implanted to measure blood pressure, heart rate, and regional blood flows. Insulin sensitivity and vascular responses to insulin were assessed during a euglycemic hyperinsulinemic clamp performed in conscious rats. In a second series of experiments, new groups of rats were used to examine skeletal muscle glucose transport activity and to determine in vitro vascular reactivity, endothelial nitric oxide synthase (eNOS) protein expression in muscle and vascular tissues and endothelin content, nitrotyrosine formation, and NAD(P)H oxidase protein expression in vascular tissues. The HFHS-fed rats displayed insulin resistance, hyperinsulinemia, hypertriglyceridemia, hyperlipidemia, elevated blood pressure, and impaired insulin-mediated renal and skeletal muscle vasodilator responses. A reduction in endothelium-dependent vasorelaxation, accompanied by a decreased eNOS protein expression in muscles and blood vessel endothelium, and increased vascular endothelin-1 protein content were also noted in HFHS-fed rats compared with control rats. Furthermore, the HFHS diet induced a reduced insulin-stimulated glucose transport activity in muscles and increased levels of NAD(P)H oxidase protein and nitrotyrosine formation in vascular tissues. These findings support the importance of eNOS protein in linking metabolic and vascular disease and indicate the ability of a Westernized diet to induce endothelial dysfunction and to alter metabolic and vascular homeostasis.

Topics: Animals; Blood Pressure; Blotting, Western; Body Weight; Deoxyglucose; Diet; Dietary Fats; Endothelin-1; Endothelium, Vascular; Fatty Acids, Nonesterified; Fluorescent Antibody Technique; Glucose Clamp Technique; Heart Rate; Insulin; Insulin Resistance; Male; Obesity; Organ Size; Rats; Rats, Sprague-Dawley; Sucrose; Triglycerides; Tyrosine; Vascular Diseases; Vascular Resistance

Vascular diseases are important clinical complications of diabetes. Advanced glycation end-products (AGE) are mediators of vascular dysfunction, but their effects on vascular smooth muscle cell (VSMC) ROS production are unclear. We studied the source and downstream targets of AGE-mediated ROS and reactive nitrogen species production in these cells. Significant increases in superoxide production in AGE-treated VSMC were measured using lucigenin (7650+/-433 vs 4485+/-424 LU/10(6) cells, p<0.001) or coelenterazine (277,907+/-71,295 vs 120,456+/-4140 LU/10(6) cells, p<0.05) and confirmed by ESR spectroscopy. These signals were blocked by the flavin-containing oxidase inhibitor diphenylene iodonium (DPI). AGE-stimulated NF-kappaB activity was abolished by DPI and the superoxide scavenger MnTBAP. AGE differentially regulated VSMC NADPH oxidase catalytic subunits, stimulating the transcription of Nox1 (201+/-12.7%, p<0.0001), while having no effect on Nox4. AGE also increased 3-nitrotyrosine formation, which was inhibited by MnTBAP, DPI, or the NOS inhibitor L-NAME. Regarding the source of NO, AGE stimulated inducible nitric oxide synthase mRNA (1 vs 9.7+/-3.0, p=0.046), which was abolished by a NF-kappaB inhibitor, SOD, catalase, or siRNA against Nox1. This study establishes that AGE activate iNOS in VSMC through a ROS-sensitive, NF-kappaB-dependent mechanism involving ROS generation by a Nox1-based oxidase.

Topics: Animals; Cells, Cultured; Glycation End Products, Advanced; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NADH, NADPH Oxidoreductases; NADPH Oxidase 1; NF-kappa B; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Superoxides; Transcription, Genetic; Tyrosine; Vascular Diseases

Insulin resistance may enhance the neointima formation via increased oxidative stress. However, clinical trials investigating the benefit of antioxidant therapy with alpha-tocopherol showed negative results. Recent studies showed that chemical characteristics of gamma-tocopherol are distinct from those of alpha-tocopherol. We hypothesized that gamma-tocopherol is superior to alpha-tocopherol in preventing the neointima growth after arterial injury in insulin resistance. Male rats were fed with standard chow or a high fructose diet for induction of insulin resistance. Thereafter, the left carotid artery was injured with a balloon catheter. After 2 weeks, the carotid arteries were harvested and histomorphometrically analyzed. The neointima-media ratio of the injured artery was significantly greater in insulin resistance group (n=8, 1.33+/-0.12) than in normal group (n=10, 0.76+/-0.11, p<0.01). gamma-Tocopherol (100 mg/kg/day) reduced the ratio (n=5, 0.55+/-0.21, p<0.01 vs. insulin resistance group), while alpha-tocopherol was without effect (n=7, 1.08+/-0.14). The quantification of plasma phosphatidylcholine hydroperoxide, an indicator of systemic oxidative stress, and dihydroethidium fluorescence staining of the carotid artery, an indicator of the local superoxide production, showed that oxidative stress in the systemic circulation and local arterial tissue was increased in insulin resistance. Both tocopherols decreased plasma phosphatidylcholine hydroperoxide, but failed to suppress the superoxide production in the carotid arteries. Increased 3-nitrotyrosine in neointima by insulin resistance was greatly reduced only by gamma-tocopherol. In conclusion, gamma-tocopherol, but not alpha-tocopherol, reduces the neointima proliferation in insulin resistance, independently of its effects on superoxide production. The beneficial effect may be related with its inhibitory effects on nitrosative stress.

Topics: Acridines; alpha-Tocopherol; Animals; Antioxidants; Blood Pressure; gamma-Tocopherol; Insulin; Insulin Resistance; Male; Muscle, Smooth, Vascular; Oxidative Stress; Phosphatidylcholines; Rats; Rats, Sprague-Dawley; Tunica Intima; Tyrosine; Vascular Diseases; Vitamin E

Patients with chronic renal failure (CRF) are at a greatly increased risk of cardiovascular mortality. This fact could be due to the presence of conventional risk factor and specific uremic as increase of oxidative stress, hyperhomocystaenemia, deranged calcium-phosphate metabolism and chronic inflammatory state. In order to analyze the vascular effects of CRF, we studied the histomorphometric characteristics (intima-media thickness and monocyte chemoattractant protein (MCP-1) accumulation (inmunohistochemical) on radial artery from 13 patients with CRF. We determined by Western blot analysis, the vascular nitrotyrosin abundance (footprint of nitric oxide (NO) inactivation by reactive oxygen species (ROS), and the endothelial nitric oxide synthase (eNOS) expression. The NOS activity was, also, determined. The results were compared with those obtained in pudenda artery from a healthy control group (n: 16). The CRF group showed a significant increase in intima and media thickness 108 +/- 16 vs 14 +/- 2.5 microm, p < 0.001 and 291 +/- 19 vs 153 +/- 15 microm, p < 0.001, respectively). The CRF group exhibited a marked elevation of MCP-1 vascular expression (2 +/- 0.15 vs 0.6 +/- 0.12 u, p < 0.001). A significant positive correlation was found between MCP-1 vascular expression and its inmunohistochemical deposits (r: 0.98, p < 0.0001). Nitrotyrosin abundance (western blot) was significantly increased in artery of CRF patients (2.1 +/- 0.1 vs 0.42 +/- 0.1 u, p < 0.0001). No significant differences was found in NOS activity between CRF and control groups. However, eNOS expression was greatly increased in the CRF patients (1.73 +/- 0.1 vs 0.67 +/- 0.1 u, p < 0.001). A significant positive correlation was found between nitrotyrosin and eNOS expression and systolic arterial pressure. However, the differences between CRF and control groups persisted after statistically fitting to arterial pressure. The present study demonstrate that in CRF there are arterial preatherosclerotic changes and an increase of vascular nitrotyrosin accumulation, which is the footprint of NO inactivation by ROS. The secondary NO inactivation can, in turn, contribute to eNOS vascular upregulation.

Topics: Blood Vessels; Cytokines; Endothelium; Female; Humans; Kidney Failure, Chronic; Male; Middle Aged; Nitric Oxide Synthase; Tyrosine; Vascular Diseases

This study investigated the effects of hyperbaric oxygen (HBO) therapy on the cardiovascular alteration (e.g. mean arterial pressure, vascular reactivity of thoracic aorta rings changes) caused by zymosan in rats.. Rats.. University research laboratory.. We investigated the effects of HBO therapy (2 ATA at the fourth and eleventh hours after study onset) on the cardiovascular alteration caused by zymosan (500 mg/kg, administered i.p. as a suspension in saline) in rats. Cardiovascular alterations were assessed 18 h after administration of zymosan and/or HBO therapy.. Treatment of rats with HBO therapy attenuated the vasoplegic response to zymosan. In fact, the analysis of arterial pressure curves revealed no signs of vasoplegic shock. The aorta rings of animals treated with zymosan and HBO had a significantly increased contraction to norepinephrine (NE) and endothelin-1 (ET-1) and dilation to acetylcholine (ACh) compared with the zymosan group. The HBO therapy also attenuated the increase of malondialdehyde (MDA) levels caused by zymosan in the aorta. Immunohistochemical analysis for nitrotyrosine and for iNOS revealed positive staining in the aorta from zymosan-treated rats. The degree of staining for nitrotyrosine and iNOS was markedly reduced in tissue sections obtained from zymosan-rats treated with HBO therapy.. This study provides the first evidence that HBO therapy attenuates the degree of zymosan-induced cardiovascular derangement in the rat.

Topics: Analysis of Variance; Animals; Aorta; Endothelium, Vascular; Hyperbaric Oxygenation; Immunohistochemistry; Male; Malondialdehyde; Multiple Organ Failure; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Random Allocation; Rats; Rats, Sprague-Dawley; Tyrosine; Vascular Diseases; Vasodilation; Zymosan