Septic shock is a leading cause of death in intensive care units. As part of the septic process, the endothelium becomes activated and propagates the septic condition. It has become evident that reactive oxygen species (ROS) are involved in the signaling of mediators of sepsis, such as tumor necrosis factor-alpha (TNF-alpha) and the lipopolysaccharide coating of gram-negative bacteria (LPS). An important source of these ROS is NADPH oxidase, which is a ubiquitously expressed enzyme complex that also exists in endothelial cells (EC). We showed that O2- from NADPH oxidase was important for LPS, as well as TNF-alpha, induction of two markers of an activated endothelium, interleukin-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVEC). / Expression of a gene can be increased by a rise in transcription as well as post-transcriptional changes, such as mRNA stability modifications. We assessed the role of NADPH oxidase in this process and found a complex interaction. Although LPS increases IL-8 transcription, it also destabilizes IL-8 mRNA in a p38 and extracellular signal-regulated kinase (ERK) MAPK dependent manner, which was only evident after blocking NADPH oxidase. This regulation involved the mRNA de-stabilizing factor tristetraprolin (TTP). In contrast, TNF-alpha enhanced the stability of IL-8, IL-6 and ICAM-1 mRNA in a p38 MAPK dependent, but NADPH oxidase independent manner. Furthermore, LPS did not have an effect on mRNA stability of IL-6 or ICAM-1 in our system. Thus, we conclude from our studies that the NAPDH oxidase is important for the induction of inflammatory molecules in LPS and TNF-alpha treated EC and is also involved in mRNA stability regulation of these molecules in a signal and gene specific fashion.

Skeletal satellite cells are adult stem cells located among muscle fibers. Proliferation, migration and subsequent differentiation of these cells are critical steps in the repair of muscle injury. We document in this study the roles and mechanisms through which the NAPDH oxidase complex regulates skeletal satellite cell proliferation. The NADPH oxidase subunits Nox2, Nox4, p22phox, p47phox and p67 phox were detected in primary human and murine skeletal muscle satellite cells. In human satellite cells, NADPH oxidase-fusion proteins were localized in the cytosolic and membrane compartments of the cell, except for p47 phox, which was detected in the nucleus. In proliferating subconfluent satellite cells, both Nox2 and Nox4 contributed to O2- production. However, Nox4 expression was significantly attenuated in confluent cells and in differentiated myotubes. Proliferation of satellite cells was significantly reduced by antioxidants (N-acetylcysteine and apocynin), inhibition of p22phox expression using siRNA oligonucleotides, and reduction of Nox4 and p47phox activities with dominant-negative vectors resulted in attenuation of activities of the Erk1/2, PI-3 kinase/AKT and NFkappaB pathways and significant reduction in cyclin D1 levels. We conclude that NADPH oxidase is expressed in skeletal satellite cells and that its activity plays an important role in promoting proliferation of these cells.

Blockade of the renin-angiotensin system lowers the rate of cardiovascular events in patients at risk for vascular disease and also improves endothelial function but the mechanism remains unclear. HUVECs were stimulated with Ang II (100 nM). Ang II produced a 2-fold increase in O2- production, which was measured by lucigenin-enhanced chemiluminescence. This increase was blocked by NAD(P)H oxidase inhibitor DPI, but not by eNOS inhibitor L-NAME. Ang II increased monocyte adhesion to ECs by 4.5-fold, and this increase was blocked by candesartan (AT1 receptor antagonist), DPI, L-NAME, wortmannin (PI3K inhibitor), dominant negative-AKT, and p22phox siRNA. Dominant active-AKT increased adhesion by 1.5-fold. Our findings indicate that the simultaneous activation by Ang II of eNOS and NAD(P)H oxidase leads to endothelial activation. This process can partially explain the therapeutic benefits of reducing the action of Ang II.

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