Erythropoietin and a nonerythropoietic peptide analog promote aortic endothelial cell repair under hypoxic conditions: role of nitric oxide

The cytoprotective effects of erythropoietin (EPO) and an EPO-related nonerythropoietic analog, pyroglutamate helix B surface peptide (pHBSP), were investigated in an in vitro model of bovine aortic endothelial cell injury under normoxic (21% O2) and hypoxic (1% O2) conditions. The potential molecular mechanisms of these effects were also explored. Using a model of endothelial injury (the scratch assay), we found that, under hypoxic conditions, EPO and pHBSP enhanced scratch closure by promoting cell migration and proliferation, but did not show any effect under normoxic conditions. Furthermore, EPO protected bovine aortic endothelial cells from staurosporine-induced apoptosis under hypoxic conditions. The priming effect of hypoxia was associated with stabilization of hypoxia inducible factor-1α, EPO receptor upregulation, and decreased Ser-1177 phosphorylation of endothelial nitric oxide synthase (NOS); the effect of hypoxia on the latter was rescued by EPO. Hypoxia was associated with a reduction in nitric oxide (NO) production as assessed by its oxidation products, nitrite and nitrate, consistent with the oxygen requirement for endogenous production of NO by endothelial NOS. However, while EPO did not affect NO formation in normoxia, it markedly increased NO production, in a manner sensitive to NOS inhibition, under hypoxic conditions. These data are consistent with the notion that the tissue-protective actions of EPO-related cytokines in pathophysiological settings associated with poor oxygenation are mediated by NO. These findings may be particularly relevant to atherogenesis and postangioplasty restenosis

Low oxygen tension primes aortic endothelial cells to the reparative effect of tissue-protective cytokines

Erythropoietin (EPO) has both erythropoietic and tissue-protective properties. The EPO analogues carbamylated EPO (CEPO) and pyroglutamate helix B surface peptide (pHBSP) lack the erythropoietic activity of EPO but retain the tissue-protective properties that are mediated by a heterocomplex of EPO receptor (EPOR) and the β common receptor (βCR). We studied the action of EPO and its analogues in a model of wound healing where a bovine aortic endothelial cells (BAECs) monolayer was scratched and the scratch closure was assessed over 24 h under different oxygen concentrations. We related the effects of EPO and its analogues on repair to their effect on BAECs proliferation and migration (evaluated using a micro-Boyden chamber). EPO, CEPO and pHBSP enhanced scratch closure only at lower oxygen (5%), while their effect at atmospheric oxygen (21%) was not significant. The mRNA expression of EPOR was doubled in 5% compared to 21% oxygen, and this was associated with increased EPOR assessed by immunofluorescence and Western blot. By contrast βCR mRNA levels were similar in 5% and 21% oxygen. EPO and its analogues increased both BAECs proliferation and migration, suggesting that both may be involved in the reparative process. The priming effect of low oxygen tension on the action of tissue-protective cytokines may be of relevance to vascular disease, including atherogenesis and restenosis

Redox proteomics of the inflammatory secretome identifies a common set of redoxins and other glutathionylated proteins released in inflammation, influenza virus infection and oxidative stress

Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions

Assessment of HIF-1α expression and release following endothelial injury in-vitro and in-vivo

Background: Endothelial injury is an early and enduring feature of cardiovascular disease. Inflammation and hypoxia may be responsible for this, and are often associated with the up-regulation of several transcriptional factors that include Hypoxia Inducible Factor-1 (HIF-1). Although it has been reported that HIF-1α is detectable in plasma, it is known to be unstable. Our aim was to optimize an assay for HIF-1α to be applied to in vitro and in vivo applications, and to use this assay to assess the release kinetics of HIF-1 following endothelial injury. Methods: An ELISA for the measurement of HIF in cell-culture medium and plasma was optimized, and the assay used to determine the best conditions for sample collection and storage. The results of the ELISA were validated using Western blotting and immunohistochemistry (IHC). In vitro, a standardized injury was produced in a monolayer of rat aortic endothelial cells (RAECs) and intracellular HIF-1α was measured at intervals over 24 hours. In vivo, a rat angioplasty model was used. The right carotid artery was injured using a 2F Fogarty balloon catheter. HIF-1α was measured in the plasma and in the arterial tissue (0, 1, 2, 3 and 5 days post injury). Results: The HIF-1α ELISA had a limit of detection of 2.7 pg/ mL and was linear up to 1000 pg/ mL. Between and within-assay coefficient of variation values were less than 15%. HIF-1α was unstable in cell lysates and plasma, and it was necessary to add a protease inhibitor immediately after collection, and to store samples at -800C prior to analysis. The dynamics of HIF-1α release were different for the in vitro and in vivo models. In vitro, HIF-1α reached maximum concentrations approximately 2h post injury, whereas peak values in plasma and tissues occurred approximately 2 days post injury, in the balloon injury model. Conclusion: HIF-1α can be measured in plasma, but this requires careful sample collection and storage. The carotid artery balloon injury model is associated with the transient release of HIF-1α into the circulation that probably reflects the hypoxia induced in the artery wall

Automatic Optimization of Input Split and Bias Voltage in Digitally Controlled Dual-Input Doherty RF PAs

Digitally controlled Dual-Input Doherty Power Amplifiers (DIDPAs) are becoming increasingly popular due to the flexible input signal splitting between the main and auxiliary stages. Nevertheless, the presence of many degrees of freedom, e.g., input amplitude split and phase displacement as well as biasing for multiple stages, often involves inefficient trial-and-error procedures to reach a suitable PA performance. This article presents automated parameter setting based on coordinate descent or Bayesian optimizations, demonstrating an improvement in the performance in terms of RF output power and power-added efficiency (PAE) in the presence of broadband-modulated signals, yet maintaining suitable linear behavior for, e.g., communications applications

Protective effect of chlorpromazine on endotoxin toxicity and TNF production in glucocorticoid-sensitive and glucocorticoid-resistant models of endotoxic shock

The present study was designed to define the potential ofchlorpromazine (CPZ) as a protective agent against lipopolysaccharide (LPS) toxicity in comparison with glucocorticoids, and to obtain initial correlations with its effects on the levels of tumor necrosis factor (TNF), a pivotal mediator of endotoxic shock. It was found that CPZ protects mice, normal or adrenalectomized, and guinea pigs against lethality of LPS, and inhibited TNF serum levels, like dexamethasone (DEX), a well-known inhibitor ofTNF synthesis. CPZ protected against LPS lethality when administered 30 minutes (min) before, simultaneously, or up to 10 min after LPS and was ineffective when given 30 min after LPS, paralleling the inhibitory effect on TNF production. In another experimental model, where mice were sensitized to LPS toxicity by actinomycin D, CPZ significantly inhibited LPS lethality and hepatotoxicity, whereas under these conditions DEX was inactive. These experiments indicate that CPZ has a protective action in both glucocorticoid-sensitive and -resistant models of endotoxic shock