Oxidative stress, AGE, and atherosclerosis.

Numerous reports on the molecular mechanism of atherogenesis indicate an increase in oxidative stress, formation of advanced glycoxidation end products (AGEs), chronic inflammation, and activated cellular response particularly in diabetic patients. To elucidate the initiating and early accelerating events this review will focus on the molecular causes of the induction of these stress factors, their interactions, and their contribution to atherogenesis. Metabolic factors such as elevated free fatty acids, high glucose levels or AGEs induce reactive oxygen species (ROS) in vascular cells leading to ongoing AGE formation and to gene induction of proinflammatory cytokines. Vice versa, numerous cytokines found elevated in obesity and diabetes may also induce oxidative stress thus a circulus vitious may be initiated and accelerated. Increased production of ROS, mainly from mitochondria and NAD(P)H oxidase, stimulates signaling cascades including protein kinase C and mitogen-activated protein kinase pathway leading to nuclear translocation of transcription factors such as nuclear factor-kappaB (NF-kappaB), activator protein 1, and specificity protein 1. Subsequently, the expression of numerous genes including cytokines is rapidly induced, which, in turn, may act on vascular cells promoting the deleterious effects. From animal models of accelerated atherosclerosis a causal role of NAD(P)H oxidase and the AGE/RAGE/NF-kappaB axis to atherogenesis is suggested. Because all factors involved form a highly interwoven network of interactions, the blockade of ROS or AGE formation at different sites may interrupt the vicious cycle. Promising candidate agents are, currently on trial. Most important to clinical practice, a number of drugs commonly used in the treatment of diabetes, hypertension, or cardiovascular disease, such as angiotensin-converting enzyme inhibitors, AT(1) receptor blockers, 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins), and thiazolidindiones have shown promising 'preventive' intracellular antioxidant activity in addition to their primary pharmacological actions.

Multi axis differential optical absorption spectroscopy (MAX-DOAS) of gas and aerosol distributions.

We present and demonstrate a relatively simple algorithm, which converts a set of slant column density measurements of oxygen dimers (O4) and NO2 at several different elevation angles to determine the atmospheric aerosol extinction and the absolute concentration and mixing ratio of NO2 within the atmospheric boundary layer. In addition the height of the atmospheric boundary layer can usually be derived, also the technique can be readily extended to determine the concentration of several other trace gases including SO2, CH2O, or glyoxal. The algorithm is based on precise radiation transport modelling determination, taking into account the actual aerosol scenario as determined from the O4 measurements. The required hardware is simple encompassing essentially a miniature spectrometer, a small telescope, a pointing mechanism, and a Personal Computer (PC). Effectively the technique combines the simplicity of a passive MAX-DOAS observation with the capability of a much more complex active DOAS instrument to determine path-averaged, absolutely calibrated mixing ratios of atmospheric trace gases at relatively high accuracy.

The RAGE pathway in inflammatory myopathies and limb girdle muscular dystrophy.

Oxidative stress and nuclear factor-kappaB (NF-kappaB) activation are linked to the pathogenesis of many metabolic, degenerative, and chronic inflammatory diseases. Activation of the receptor for advanced glycation end products (RAGE) by its specific ligand N(epsilon)-carboxymethyllysine (CML) results in the activation of NF-kappaB and the production of proinflammatory cytokines. To determine whether engagement of RAGE contributes to the pathogenesis of inflammatory myopathies, we performed immunohistochemical studies on the presence of CML-modified proteins, RAGE and activated NF-kappaB in muscle biopsies of patients with polymyositis (PM, n=10), dermatomyositis (DM, n=10), limb girdle muscular dystrophy (LGMD, n=10) and in 10 controls with normal muscle biopsy results. In inflammatory myopathies CML, RAGE and NF-kappaB were detected in mononuclear cells and in regenerating muscle fibers. CML, NF-kappaB and, to a lesser extent, RAGE were also found in degenerating muscle fibers, but colocalization of CML, RAGE and NF-kappaB was only seen in infiltrating mononuclear cells and regenerating muscle fibers. Immunofluorescence double labeling demonstrated an expression of CML, RAGE and NF-kappaB in CD4-, CD8-, CD22- and CD68-positive mononuclear cells. Western blot analysis showed an increased immunoreactivity for CML-modified proteins in PM and DM. In LGMD, CML, RAGE and NF-kappaB were found in regenerating muscle fibers and less frequently in degenerating muscle fibers, and with lower staining intensities than in inflammatory myopathies. Our data suggests that the CML-RAGE-NF-kappaB pathway is an evident proinflammatory pathomechanism in mononuclear effector cells in PM and DM. RAGE-mediated NF-kappaB activation may be involved in muscle fiber regeneration in inflammatory myopathies and LGMD.

Failure of arginine-vasopressin and other pressor hormones to increase in severe recurrent dialysis hypotension.

BACKGROUND: Recurrent dialysis hypotension is common in long-term dialysis patients. Arginine-vasopressin (AVP) is a potent vasoconstrictor hormone, release of which is stimulated in hypotension. STUDY DESIGN: We measured AVP as well as adrenaline and noradrenaline in 23 patients with recurrent dialysis hypotension during severe symptomatic episodes of dialysis hypotension (BP syst. < 70 mmHg). We also tested autonomic function (amyl nitrate inhalation, cold pressor test) during the interdialytic interval. RESULTS: We observed that systolic blood pressure decreased from 127 +/- 8 (at the end of the first hour of dialysis; taken as control state) to 64 +/- 1 mmHg (symptomatic hypotension) in 23 patients. In six of the 23 patients hypotension was accompanied by nausea, which is a known direct stimulus of AVP. In these six patients, plasma AVP showed a large increase: control state, 6.2 +/- 0.9; hypotension, 130.4 +/- 51.1 pg/ml; P < 0.5). Of the remaining 17 patients without nausea, AVP fell moderately in nine and increased in eight. Taken together, this group of 17 hypotensive patients failed to show significant AVP stimulation: control state, 9.0 +/- 1.4; hypotension, 13.8 +/- 3.8 pg/ml, NS). Adrenaline and noradrenaline did not change during hypotension. During autonomic testing the patients with recurrent dialysis hypotension (compared to healthy controls) showed blunted baroreflex response (assessed by amyl nitrate inhalation) but intact sympathetic outflow (assessed by cold pressor test). CONCLUSION: The observations of AVP are taken as further evidence of defects in the afferent rather than the efferent limb of autonomic reflexes in dialysis patients with recurrent dialysis hypotension.

N(epsilon)-Carboxymethyllysine in diabetic and non-diabetic polyneuropathies.

Increased oxidative stress and advanced glycosylation are important factors in the development of diabetic neuropathy. In non-diabetic neuropathies their influence has not been investigated in detail so far. We studied the localisation of N(epsilon)-carboxymethyllysine (CML) - a biomarker for oxidative stress - by immunohistochemistry in sural nerve biopsies of 31 patients with different polyneuropathies [diabetic polyneuropathy (n=5), alcohol-associated polyneuropathy (n=4), vitamin B12-deficient polyneuropathy (n=6), chronic inflammatory demyelinating polyneuropathy (CIDP) (n=6), vasculitic neuropathy (n=6), Charcot-Marie-Tooth disease type I (CMT I) (n=4)] and 4 normal controls. CML was detected in the perineurium of patients with diabetic, alcohol-associated, vitamin B12-deficient and vasculitic polyneuropathies. Epineurial, perineurial and endoneurial vessels were CML positive in diabetic, vitamin B12-deficient and vasculitic polyneuropathies. CML was also found in mononuclear inflammatory cells in vasculitic neuropathy. In CIDP and normal controls there was only marginal perineurial CML deposition in 2/6 and 1/4 cases. In CMT I no CML was detected. Immunohistochemical results were confirmed by immunoblot. Our data suggest a role of oxidative stress in the pathogenesis not only of diabetic but also of alcohol-associated, vitamin B12-deficient and vasculitic polyneuropathies. It may be a minor pathogenetic factor in CIDP and may not be involved in CMT I. Underlying causes for increased oxidative stress may be an elevated production of reactive oxygen species and an impairment of antioxidative defences. Therefore, an antioxidative treatment should be considered in alcohol-associated, vitamin B12-deficient and vasculitic polyneuropathy.

Glutamine:fructose-6-phosphate aminotransferase enzyme activity is necessary for the induction of TGF-beta1 and fibronectin expression in mesangial cells.

AIMS/HYPOTHESIS: Increased flux through the hexosamine biosynthetic pathway with glutamine:fructose-6-phosphate aminotransferase (GFAT) as a rate-limiting enzyme has been linked to the enhanced bioactivity of the prosclerotic cytokine TGF-beta1, a key mediator in the development of diabetic nephropathy and possibly other diabetic angiopathies. In this study we investigated the effect of enhanced expression of wild-type GFAT and two enzymatically inactive GFAT mutants on TGF-beta1 synthesis in mesangial cells. METHODS: Mutated human GFAT expression vectors were prepared by PCR-site directed mutagenesis. Wild-type and mutated vectors were transfected into human embryonic kidney 293 cells and mesangial cells and GFAT enzyme activity was assessed by formation of glucosamine-6-phosphate. Production of TGF-beta1 and fibronectin protein was examined by ELISA. RESULTS: Mutation of histidine 577 or lysine 676 to alanine led to a complete loss of GFAT enzyme activity. An increased concentration of wild-type GFAT in mesangial cells enhanced both TGF-beta1 and fibronectin production 1.5-fold, while mesangial cells transfected with the mutated GFAT constructs showed no effect. CONCLUSION/INTERPRETATION: The data indicate that the hexosamine pathway-mediated induction of TGF-beta1 synthesis in mesangial cells is dependent on GFAT enzyme activity. Our results add to previous observations showing that the hexosamine pathway could increase the transcriptional activity of nuclear proteins leading to enhanced cytokine synthesis.