Myeloperoxidase attracts neutrophils by physical forces.

Recruitment of polymorphonuclear neutrophils (PMNs) remains a paramount prerequisite in innate immune defense and a critical cofounder in inflammatory vascular disease. Neutrophil recruitment comprises a cascade of concerted events allowing for capture, adhesion and extravasation of the leukocyte. Whereas PMN rolling, binding, and diapedesis are well characterized, receptor-mediated processes, mechanisms attenuating the electrostatic repulsion between the negatively charged glycocalyx of leukocyte and endothelium remain poorly understood. We provide evidence for myeloperoxidase (MPO), an abundant PMN-derived heme protein, facilitating PMN recruitment by its positive surface charge. In vitro, MPO evoked highly directed PMN motility, which was solely dependent on electrostatic interactions with the leukocyte's surface. In vivo, PMN recruitment was shown to be MPO-dependent in a model of hepatic ischemia and reperfusion, upon intraportal delivery of MPO and in the cremaster muscle exposed to local inflammation or to intraarterial MPO application. Given MPO's affinity to both the endothelial and the leukocyte's surface, MPO evolves as a mediator of PMN recruitment because of its positive surface charge. This electrostatic MPO effect not only displays a so far unrecognized, catalysis-independent function of the enzyme, but also highlights a principal mechanism of PMN attraction driven by physical forces.

Myeloperoxidase enhances nitric oxide catabolism during myocardial ischemia and reperfusion.

Impaired microvascular function during myocardial ischemia and reperfusion is associated with recruitment of polymorphonuclear neutrophils (PMN) and has been attributed to decreased bioavailability of nitric oxide (NO). Whereas myeloperoxidase (MPO), a highly abundant, PMN-derived heme protein facilitates oxidative NO consumption and impairs vascular function in animal models of acute inflammation, its capacity to function in this regard during human myocardial ischemia and reperfusion remains unknown. Plasma samples from 30 consecutive patients (61 +/- 14 years, 80% male) presenting with acute myocardial infarction were collected 9 +/- 4 h after vessel recanalization and compared to plasma from healthy control subjects (n = 12). Plasma levels of MPO were higher in patients than in control subjects (1.4 +/- 0.9 vs 0.3 +/- 0.2 ng/mg protein, respectively, p < 0.0001). The addition of hydrogen peroxide to patient plasma resulted in accelerated rates of NO consumption compared to control subjects (0.53 +/- 0.25 vs 0.068 +/- 0.039 nM/s/mg protein, respectively, p < 0.0001). Myocardial tissue from patients with the same pathology revealed intense recruitment of MPO-positive PMN localized along infarct-related vessels as well as diffuse endothelial distribution of non-PMN-associated MPO immunoreactivity. Endothelium-dependent microvascular function, as assessed by an acetylcholine-dependent increase in forearm blood flow in 75 patients with symptomatic coronary artery disease, inversely correlated with MPO plasma levels (r = -0.75, p < 0.005). Plasma from patients undergoing myocardial reperfusion contained increased levels of MPO, which catalytically consumed NO in the presence of H(2)O(2). Given the correlation between intravascular MPO levels and forearm vasomotor function in patients with coronary artery disease, MPO appears to be an important modulator of vasomotor function in inflammatory vascular disease and a potential therapeutic target for treatment.

Non-invasive monitoring of oxidative skin stress by ultraweak photon emission measurement. II: biological validation on ultraviolet A-stressed skin.

BACKGROUND/PURPOSE: Several physical or chemical environmental stressors generate reactive oxygen species, which trigger oxidation reactions of cells or tissues and thereby induce a correlated ultraweak photon emission (UPE) signal. The present study was designed to qualify and validate UPE measurement following ultraviolet (UV) excitation of porcine and human skin as an analytical method to assess the potency of topical antioxidants in vivo. METHODS: UPE of porcine skin in vitro and human skin in vivo following excitation with UVA was recorded using sensitive photomultiplier systems. For validation purposes, the effects of variation of extrinsic and intrinsic parameters encompassing skin thickness, humidity, temperature, pH, and composition of the surrounding atmosphere were assessed. Signals were analyzed with regard to overall signal intensity and spectral distribution. In two clinical trials enrolling 20 volunteers each, the effects of topical antioxidant treatment on UVA-induced UPE were validated. RESULTS: Different stressors encompassing exposition to ozone, UVA irradiation, or even cigarette smoke induced UPE of skin. Critical parameters affecting the quality and quantity of the UPE signal were the spectral composition of the exciting UV light, skin temperature, skin humidity, and the O(2) concentration of the surrounding atmosphere. Generally, UVA-induced UPE decreased with increasing temperature, humidity, and O(2) concentration. Skin pH had no significant effect on UPE with regard to signal quality and quantity over a pH range of 2.8-8.2. In a clinical study UPE measurement following UVA excitation could precisely reflect a dose-dependent antioxidant effect of topically applied vitamin C and alpha-glucosylrutin. CONCLUSION: Our data indicate that UVA irradiation induces UPE especially in deeper (living) skin layers, where antioxidants must be active in order to interfere with accelerated skin ageing. Based on the clinical data, and with knowledge of modulating external variables, UPE measurement following UV excitation can be qualified as a reliable and valid method for the non-invasive measurement of antioxidant efficacy on the skin.

Non-invasive monitoring of oxidative skin stress by ultraweak photon emission (UPE)-measurement. I: mechanisms of UPE of biological materials.

BACKGROUND/PURPOSE: Oxidation of proteins and amino acids is associated with generation of ultraweak photon emission (UPE), which may be used to assess oxidative processes in the skin in a non-invasive way. This first part of a series of reports addresses the physicochemical basis of oxidation-induced UPE in the skin, with a special focus on the contribution of amino acid oxidation. METHODS: UPE of biological samples and protein/amino acid solutions following oxidation with H(2)O(2) in the presence of Fe(2+) was recorded using a sensitive photomultiplier system. Signals were analyzed with regard to overall signal intensity and spectral distribution. RESULTS: Increasing concentrations of H(2)O(2) in aqueous bovine serum albumin solutions induced linearly correlated UPE and protein carbonyl compounds, with a substantially higher sensitivity for the measurement of UPE. In single amino acid solutions, strong UPE signals were generated by oxidation from Phe, Trp, His, and Cys, and weak signals from Lys and Thr. Analysis of reaction products by MS revealed high oxidative material turnover for Cys and His, whereas barely detectable oxidative material turnover seems to be sufficient to generate a UPE signal of similar strength from Trp and Phe. Combination of different amino acids did not result in a simple addition of individual oxidation-induced UPE signals, but in interactions ranging from antagonism to clear synergism. Synergism was evident between Trp- and UPE-generating amino acids such as Thr, Cys, and His, with the strongest synergism by far observed between Trp and His. The strikingly different individual UPE spectra of His and Trp, despite being of comparable overall strength, were congruent with a pure Trp UPE spectrum after combining His with Trp in solution, indicating energy transfer between both amino acids. Combination of Trp and DNA, which also gives UPE signals following oxidation, did not result in a synergistically enhanced or antagonized overall UPE signal, but in a simple addition of individual UPE signals. CONCLUSION: Measurement of UPE could be proven to be a highly sensitive method to assess oxidative processes in biological molecules. The reported data indicate that UPE generated by oxidation stressed skin is mainly due to non-fluorescent photon emission via Trp, whereby Trp acts as an energy receptor from other excited species of oxidation-modified amino acids.

Comparison of activity of immobilised and soluble metalloporphyrins by NO release of oximes by chemiluminescence.

The ongoing interest in very efficient systems for the imitation of cytochrome P-450-dependent monooxygenase reactions, consisting of metalloporphyrin and oxygen donor, prompted us to develop a method to compare the catalytic activity of soluble metalloporphyrins with those which have been immobilised on different silica surfaces. In this article, we describe a method which allows direct comparison between the catalytic activities of soluble and immobilised metalloporphyrins by measuring the NO release of cyclohexanone oxime. We observed that all metalloporphyrins are able to enhance the NO release of cyclohexanone oxime in the presence of iodosobenzene as oxygen donor. In case of cationic metalloporphyrins, no difference in the catalytic activity was determined. In contrast, for perfluorinated metalloporphyrins we ascertained a major loss of activity after immobilisation. These results led us to the assumption that it is not easy to compare quantitative results of NO releases if soluble and immobilised metalloporphyrins were used.

Nitric oxide donors induce neurotrophin-like survival signaling and protect neurons against apoptosis.

Our previous results showed that inhibition of protein tyrosine phosphatases (PTP) by orthovanadate is an appropriate strategy to mimic nerve growth factor (NGF) effects in neurons, including enhanced phosphorylation of TrkA, stimulation of downstream survival signaling pathways, and protection against apoptotic stress. In this study, we wanted to trigger such NGF-like survival signaling in primary hippocampal neurons with the more specific PTP inhibitors ethyl-3,4-dephostatin (DPN), 4-O-methyl-ethyl-3,4-dephostatin (Me-DPN), and methoxime-3,4-dephostatin. It was striking that only the nitric oxide (NO)-releasing dephostatin analogs DPN and Me-DPN, but not the nitrosamine-free methoxime derivative (which did not release NO), enhanced TrkA phosphorylation and protected the neurons against staurosporine (STS)-induced apoptosis. The established NO donor S-nitroso-N-acetylpenicillamine (SNAP) also enhanced TrkA phosphorylation and prevented apoptosis similarly to DPN and Me-DPN. Analysis of the major signaling pathways downstream of TrkA revealed that both SNAP and DPN enhanced phosphorylation of Akt and the mitogen-activated kinases (MAPK) Erk1/2. Blocking of these signaling pathways by the PI3-K inhibitor wortmannin or the MAPK kinase inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene] equally abolished the neuroprotective effect of the NO donors. It was striking that inhibition of the soluble guanylyl cyclase (sGC) by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or protein kinase G (PKG) inhibition by (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo-[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT5823) also blocked the neuroprotective effect of the NO donors, and ODQ clearly attenuated SNAP-induced phosphorylation of TrkA, Akt, and MAPK. In conclusion, NO release by the dephostatin derivatives and subsequent stimulation of sGC and PKG is essential for their neuroprotective effects. In primary neurons, such NO-activated survival signaling involves NGF-like effects, including enhanced phosphorylation of TrkA and activation of PI3-K/Akt and MAPK pathways.