Nitric oxide (NO), the smallest signalling molecule known, is produced by three isoforms of NO synthase (NOS; EC 1.14.13.39). They all utilize l-arginine and molecular oxygen as substrates and require the cofactors reduced nicotinamide-adenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and (6R-)5,6,7,8-tetrahydrobiopterin (BH(4)). All NOS bind calmodulin and contain haem. Neuronal NOS (nNOS, NOS I) is constitutively expressed in central and peripheral neurons and some other cell types. Its functions include synaptic plasticity in the central nervous system (CNS), central regulation of blood pressure, smooth muscle relaxation, and vasodilatation via peripheral nitrergic nerves. Nitrergic nerves are of particular importance in the relaxation of corpus cavernosum and penile erection. Phosphodiesterase 5 inhibitors (sildenafil, vardenafil, and tadalafil) require at least a residual nNOS activity for their action. Inducible NOS (NOS II) can be expressed in many cell types in response to lipopolysaccharide, cytokines, or other agents. Inducible NOS generates large amounts of NO that have cytostatic effects on parasitic target cells. Inducible NOS contributes to the pathophysiology of inflammatory diseases and septic shock. Endothelial NOS (eNOS, NOS III) is mostly expressed in endothelial cells. It keeps blood vessels dilated, controls blood pressure, and has numerous other vasoprotective and anti-atherosclerotic effects. Many cardiovascular risk factors lead to oxidative stress, eNOS uncoupling, and endothelial dysfunction in the vasculature. Pharmacologically, vascular oxidative stress can be reduced and eNOS functionality restored with renin- and angiotensin-converting enzyme-inhibitors, with angiotensin receptor blockers, and with statins.

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Nitric oxide synthases: regulation and function.

Reactive oxygen species (ROS) are generated as by-products of cellular metabolism, primarily in the mitochondria. When cellular production of ROS overwhelms its antioxidant capacity, damage to cellular macromolecules such as lipids, protein, and DNA may ensue. Such a state of “oxidative stress” is thought to contribute to the pathogenesis of a number of human diseases including those of the lung. Recent studies have also implicated ROS that are generated by specialized plasma membrane oxidases in normal physiological signaling by growth factors and cytokines. In this review, we examine the evidence for ligand-induced generation of ROS, its cellular sources, and the signaling pathways that are activated. Emerging concepts on the mechanisms of signal transduction by ROS that involve alterations in cellular redox state and oxidative modifications of proteins are also discussed.

Reactive oxygen species in cell signaling

There is still an unresolved paradox with respect to the immunomodulating role of estrogens. On one side, we recognize inhibition of bone resorption and suppression of inflammation in several animal models of chronic inflammatory diseases. On the other hand, we realize the immunosupportive role of estrogens in trauma/sepsis and the proinflammatory effects in some chronic autoimmune diseases in humans. This review examines possible causes for this paradox. This review delineates how the effects of estrogens are dependent on criteria such as: 1) the immune stimulus (foreign antigens or autoantigens) and subsequent antigen-specific immune responses (e.g., T cell inhibited by estrogens vs. activation of B cell); 2) the cell types involved during different phases of the disease; 3) the target organ with its specific microenvironment; 4) timing of 17beta-estradiol administration in relation to the disease course (and the reproductive status of a woman); 5) the concentration of estrogens; 6) the variability in expression of estrogen receptor alpha and beta depending on the microenvironment and the cell type; and 7) intracellular metabolism of estrogens leading to important biologically active metabolites with quite different anti- and proinflammatory function. Also mentioned are systemic supersystems such as the hypothalamic-pituitary-adrenal axis, the sensory nervous system, and the sympathetic nervous system and how they are influenced by estrogens. This review reinforces the concept that estrogens have antiinflammatory but also proinflammatory roles depending on above-mentioned criteria. It also explains that a uniform concept as to the action of estrogens cannot be found for all inflammatory diseases due to the enormous variable responses of immune and repair systems.

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The complex role of estrogens in inflammation

iNOS-mediated nitric oxide production and its regulation.

Incubation of endothelial cells in vitro with high concentrations of glucose activates protein kinase C (PKC) and increases nitric oxide synthase (NOS III) gene expression as well as superoxide production. The underlying mechanisms remain unknown. To address this issue in an in vivo model, diabetes was induced with streptozotocin in rats. Streptozotocin treatment led to endothelial dysfunction and increased vascular superoxide production, as assessed by lucigenin- and coelenterazine-derived chemiluminescence. The bioavailability of vascular nitric oxide (as measured by electron spin resonance) was reduced in diabetic aortas, although expression of endothelial NOS III (mRNA and protein) was markedly increased. NOS inhibition with N:(G)-nitro-L-arginine increased superoxide levels in control vessels but reduced them in diabetic vessels, identifying NOS as a superoxide source. Similarly, we found an activation of the NADPH oxidase and a 7-fold increase in gp91(phox) mRNA in diabetic vessels. In vitro PKC inhibition with chelerythrine reduced vascular superoxide in diabetic vessels, whereas it had no effect on superoxide levels in normal vessels. In vivo PKC inhibition with N:-benzoyl-staurosporine did not affect glucose levels in diabetic rats but prevented NOS III gene upregulation and NOS-mediated superoxide production, thereby restoring vascular nitric oxide bioavailability and endothelial function. The reduction of superoxide in vitro by chelerythrine and the normalization of NOS III gene expression and reduction of superoxide in vivo by N:-benzoyl-staurosporine point to a decisive role of PKC in mediating these phenomena and suggest a therapeutic potential of PKC inhibitors in the prevention or treatment of vascular complications of diabetes mellitus. The full text of this article is available at http://www.circresaha.org.

Mechanisms Underlying Endothelial Dysfunction in Diabetes Mellitus

Three 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (HCRIs), atorvastatin, pravastatin, and cerivastatin, inhibited phorbol ester-stimulated superoxide anion (O(2)(-)) formation in endothelium-intact segments of the rat aorta in a time- and concentration-dependent manner (maximum inhibition of 70% after 18 hours at 1 to 10 micromol/L). The HMG-CoA reductase product mevalonic acid (400 micromol/L) reversed the inhibitory effect of the HCRIs, which, conversely, was mimicked by inactivation of p21 Rac with Clostridium sordellii lethal toxin but not by inactivation of p21 Rho with Clostridium botulinum exoenzyme (C3). A mevalonate-sensitive inhibition of phorbol ester-stimulated O(2)(-) formation by atorvastatin was also observed in porcine cultured endothelial cells and in a murine macrophage cell line. In the rat aorta, no effect of the HCRIs on protein kinase C, NADPH oxidase, or superoxide dismutase (SOD) activity and expression was detected, whereas that of endothelial nitric oxide (NO) synthase was enhanced approximately 2-fold. Moreover, exposure of the segments to atorvastatin resulted in a significant improvement of endothelium-dependent NO-mediated relaxation, and this effect was abolished in the presence of SOD. Taken together, these findings suggest that in addition to augmenting endothelial NO synthesis, HCRIs inhibit endothelial O(2)(-) formation by preventing the isoprenylation of p21 Rac, which is critical for the assembly of NADPH oxidase after activation of protein kinase C. The resulting shift in the balance between NO and O(2)(-) in the endothelium improves endothelial function even in healthy blood vessels and therefore may provide a reasonable explanation for the beneficial effects of HCRIs in patients with coronary heart disease in addition to or as an alternative to the reduction in serum LDL cholesterol.

Improvement of Nitric Oxide–Dependent Vasodilatation by HMG-CoA Reductase Inhibitors Through Attenuation of Endothelial Superoxide Anion Formation

We investigated the hypothesis that the antiatherosclerotic effect of 17β-estradiol (E2) is due to a shift in the nitric oxide (NO)/superoxide (O2−) balance in the vessel wall, thereby increasing t...

17β-Estradiol inhibition of NADPH oxidase expression in human endothelial cells

1. Formation of nitric oxide (NO) by the constitutive calcium-dependent NO synthase expressed in endothelial cells plays an important role in the control of local blood flow and vascular homeostasis. Expression of the inducible calcium-independent NO synthase (iNOS) in vascular smooth muscle cells (VSMC), on the other hand, is thought to play a potentially detrimental role in the pathogenesis of chronic inflammation or septic shock. In vascular injury, however, iNOS expression in VSMC may be beneficial as a compensatory mechanism for the lack of endothelial NO synthesis, e.g., by preventing restenosis following angioplasty or heart transplant vasculopathy. 2. Because iNOS activity does not seem to be controlled once the enzyme is expressed, regulation of NO release from iNOS-expressing cells predominantly occurs at the transcriptional and/or posttranscriptional level. 3. This review summarizes what is currently known about the regulation of expression of this enzyme in VSMC, details some of the transcription factors involved therein as well as their mode of activation, and highlights some pharmacological strategies based on these findings that may be employed for the control of iNOS expression in VSMC in the clinical arena.

Regulation of inducible nitric oxide synthase gene expression in vascular smooth muscle cells

Objective— 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exert potent anti-inflammatory effects that are independent of their cholesterol-lowering action. We have investigated the effects of these drugs on cytokine-stimulated CD40 expression in human cultured endothelial cells and monocytes. Methods and Results— Reverse transcription-polymerase chain reaction and Western blot analysis revealed that treatment of either cell type with atorvastatin, cerivastatin, or pravastatin (1 to 10 μmol/L) inhibited interferon-γ plus tumor necrosis factor-α-stimulated CD40 expression by ≈50%, an effect that was not reversed by the HMG-CoA reductase product mevalonic acid (400 μmol/L). In contrast, mevalonic acid prevented the inhibitory effect of atorvastatin on cytokine-stimulated vascular cell adhesion molecule-1 expression and subsequent adhesion of THP-1 monocytes to the cultured endothelial cells. Transcription factor analysis revealed an inhibition by atorvastatin of nuclear factor-κB plus signal transducer and activator of transcription-1-dependent de novo synthesis of interferon regulatory factor-1, governing cytokine-stimulated CD40 expression in these cells. One consequence of this statin-dependent downregulation of CD40 expression was a decrease in CD40 ligand-induced endothelial interleukin-12 expression. Conclusions— By interfering with cytokine-stimulated CD40 expression in vascular cells, statins thus seem capable of attenuating CD40 ligand-induced proinflammatory responses, including atherosclerosis. In addition, they point to the coexistence of HMG-CoA reductase-dependent and -independent effects of statins in the same cell type.

3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase-Independent Inhibition of CD40 Expression by Atorvastatin in Human Endothelial Cells

Animal experimental studies have demonstrated that inducible nitric oxide synthase (iNOS) expression correlates with neointima formation and is prevented by HMG-CoA reductase inhibitors (statins). In the present study we have investigated the underlying mechanism of action of these drugs in isolated segments of the rat aorta.




Western blot analysis and immunohistochemistry revealed that tumour necrosis factor α (TNFα) plus interferon-γ (IFNγ) synergistically induce iNOS gene expression in the endothelium but not in the smooth muscle of these segments while constitutive endothelial NO synthase (eNOS) abundance was markedly reduced.




Pre-treatment with 1 – 10 μM atorvastatin, cerivastatin or pravastatin decreased TNFα plus IFNγ stimulated iNOS expression in the endothelium irrespective of the presence of the HMG-CoA reductase product mevalonate (400 μM).




Electrophoretic mobility shift assay experiments confirmed that the combination of TNFα plus IFNγ causes activation of the transcription factors STAT-1 and NF-κB in native endothelial cells. Neutralization of these transcription factors by employing the corresponding decoy oligonucleotides confirmed their involvement in TNFα plus IFNγ stimulated iNOS expression. Translocation of both transcription factors was attenuated by atorvastatin, and this effect was insensitive to exogenous mevalonate.




The present findings thus demonstrate a specific HMG-CoA reductase-independent inhibitory effect of statins, namely atorvastatin, on cytokine-stimulated transcription factor activation in native endothelial cells in situ and the subsequent expression of a gene product implicated in vascular inflammation. This effect may be therapeutically relevant and in addition provide an explanation for the reported rapid onset of action of these drugs in humans.






British Journal of Pharmacology (2002) 136, 143–149; doi:10.1038/sj.bjp.0704678

https://bpspubs.onlinelibrary.wiley.com/doi/pdf/10.1038/sj.bjp.0704678

Andreas H. Wagner

Papers

Improvement of Nitric Oxide–Dependent Vasodilatation by HMG-CoA Reductase Inhibitors Through Attenuation of Endothelial Superoxide Anion Formation

17β-Estradiol inhibition of NADPH oxidase expression in human endothelial cells

Regulation of inducible nitric oxide synthase gene expression in vascular smooth muscle cells

3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase-Independent Inhibition of CD40 Expression by Atorvastatin in Human Endothelial Cells

Atorvastatin inhibition of cytokine-inducible nitric oxide synthase expression in native endothelial cells in situ.