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The flowing blood generates shear stress at the endothelial cell surface. In endothelial cells, NAD(P)H oxidase complexes have been identified as major sources of superoxide anion (·O 2 − ) formation. In this study, we analysed the effect of laminar shear stress on ·O 2 − formation by cytochrome c reduction assay and on NAD(P)H oxidase subunit expression by standard calibrated competitive reverse transcription-polymerase chain reaction and Western blot in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were exposed to laminar shear stress in a cone-and-plate viscometer for up to 24 h. Short-term application of shear stress transiently induced ·O 2 − formation. This was inhibited by NAD(P)H oxidase inhibitor gp91ds-tat, but NAD(P)H oxidase subunit expression was unchanged. Long-term arterial laminar shear stress (30 dyne cm −2 , 24 h) down-regulated ·O 2 − formation, and mRNA and protein expression of NAD(P)H oxidase subunits Nox2/gp91 phox and p47 phox . In parallel, endothelial NO formation and eNOS, but not Cu/Zn SOD, protein expression was increased. Down-regulation of ·O 2 − formation, gp91 phox and p47 phox expression by long-term laminar shear stress was blocked by L-NAME. NO donor DETA-NO down-regulates ·O 2 − formation, gp91 phox and p47 phox expression in static cultures. In conclusion, our data suggest a transient activation of ·O 2 − formation by short-term shear stress, followed by a down-regulation of endothelial NAD(P)H oxidase in response to long-term laminar shear stress. NO-mediated down-regulation by shear stress preferentially affects the gp91 phox /p47 phox -containing NAD(P)H oxidase complex. This mechanism might contribute to the regulation of endothelial NO/·O 2 − balance and the vasoprotective potential of physiological levels of laminar shear stress.

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The endothelial cell layer plays a major role in the development and progression of atherosclerosis. Endothelial NO synthase (eNOS) produces nitric oxide (NO) from L-arginine. NO can rapidly react with reactive oxygen species to form peroxynitrite. This reduces NO availability, impairs vasodilatation, and mediates proinflammatory and prothrombotic processes such as leukocyte adhesion and platelet aggregation. In the vessel wall, specific NAD(P)H oxidase complexes are major sources of reactive oxygen species. These NAD(P)H oxidases can transfer electrons across membranes to oxygen and generate superoxide anions. The short-lived superoxide anion rapidly dismutates to hydrogen peroxide, which can further increase the production of reactive oxygen species. This can lead to uncoupling of eNOS switching enzymatic activity from NO to superoxide production. This review describes the structure and regulation of different NAD(P)H oxidase complexes. We will also focus on NO/superoxide anion balance as modulated by hemodynamic forces, vasoconstrictors, and oxidized low-density lipoprotein. We will then summarize the recent advances defining the role of nitric oxide and NAD(P)H oxidase-derived reactive oxygen species in the development and progression of atherosclerosis. In conclusion, novel mechanisms affecting the vascular NO/superoxide anion balance will allow the development of therapeutic strategies in the treatment of cardiovascular diseases.

As millions of patients have been infected by SARS-CoV-2 virus a vast number of individuals complain about continuing breathlessness and fatigue even months after the onset of the disease. This overwhelming phenomenon has not been well defined and has been called “post-COVID syndrome” or “long-COVID” [1]. There are striking similarities to myalgic encephalomyelitis also called chronic fatigue syndrome linked to a viral and autoimmune pathogenesis. In both disorders neurotransmitter receptor antibodies against ß-adrenergic and muscarinic receptors may play a key role. We found similar elevation of these autoantibodies in both patient groups. Extracorporeal apheresis using a special filter seems to be effective in reducing these antibodies in a significant way clearly improving the debilitating symptoms of patients with chronic fatigue syndrome. Therefore, such a form of neuropheresis may provide a promising therapeutic option for patients with post-COVID-19 syndrome. This method will also be effective when other hitherto unknown antibodies and inflammatory mediators are involved.

Site-specific functionalization of proteins by bioorthogonal modification offers a convenient pathway to create, modify, and study biologically active biopolymers. In this paper the Staudinger reaction of aryl-phosphonites for the chemoselective functionalization of azido-peptides and proteins was probed. Different water-soluble phosphonites with oligoethylene substituents were synthesized and reacted with unprotected azido-containing peptides in aqueous systems at room temperature in high conversions. Finally, the Staudinger-phosphonite reaction was successfully applied to the site-specific modification of the protein calmodulin.

The main sources of oxidative stress in the vessel wall are nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes. The endothelium mainly expresses the Nox4-containing complex; however, the mechanism by which shear stress in endothelial cells regulates Nox4 is not well understood. This study demonstrates that long-term application of arterial laminar shear stress using a cone-and-plate viscometer reduces endothelial superoxide anion formation and Nox4 expression. In primary human endothelial cells, we identified a 47 bp 5'-untranslated region of Nox4 mRNA by 5'-rapid amplification of cDNA ends (5'-RACE) PCR. Cloning and functional analysis of human Nox4 promoter revealed a range between -1,490 and -1,310 bp responsible for flow-dependent downregulation. Mutation of an overlapping antioxidative response element (ARE)-like and Oct-1 binding site at -1,376 bp eliminated shear stress-dependent Nox4 downregulation. Consistent with these observations, electrophoretic mobility shift assays (EMSA) demonstrated an enhanced shear stress-dependent binding of Nox4 oligonucleotide containing the ARE-like/Oct-1 binding site, which could be inhibited by specific antibodies against the transcription factors nuclear factor erythroid 2-related factor 2 (Nrf2) and octamer transcription factor 1 (Oct-1). Furthermore, shear stress caused the translocation of Nrf2 and Oct-1 from the cytoplasm to the nucleus. Knockdown of Nrf2 by short hairpin RNA (shRNA) increased Nox4 expression twofold, indicating a direct cross-talk between Nrf2 and Nox4. In conclusion, an ARE-like/Oct-1 binding site was noticed to be essential for shear stress-dependent downregulation of Nox4. This novel mechanism may be involved in the flow-dependent downregulation of endothelial superoxide anion formation.