The Effect of C-Reactive Protein Isoforms on Nitric Oxide Production by U937 Monocytes/Macrophages

Inflammation is regulated by many endogenous factors including estrogen, a steroid hormone that declines with increasing age, leading to excessive inflammation in the elderly. C-reactive protein (CRP) is an acute phase inflammatory protein that exists in two forms, native CRP (nCRP) and monomeric CRP (mCRP), which mediate distinct biological activities. It is unclear how each CRP isoform mediates nitric oxide (NO), a signalling molecule generated by NO synthase (NOS). This study investigated whether CRP isoforms have distinct effects on NO production by unstimulated and lipopolysaccharide (LPS)-activated monocytes/macrophages and whether estrogen mediates CRP-induced NO production in an in vitro model of ageing. NO and inducible NOS (iNOS) were measured (n=12) by the Griess assay and an enzyme-linked immunosorbent assay (ELISA) respectively following incubation (24 hours) of human-derived U937 monocytes/macrophages with CRP isoforms ([nCRP] = 500 and 1000µg/ml; [mCRP] = 100 and 250µg/ml) in the absence or presence of 17 beta-estradiol (1x10-7, 1x10-8 and 1x10-9M). The response to each CRP isoform and estrogen was dependent on the differentiation and activation status of cells. Monocytes with or without prior LPS-activation significantly increased (P0.05) on NO/iNOS production by unactivated or LPS-activated macrophages whereas nCRP significantly (P<0.05) reduced NO/iNOS production by macrophages, with or without prior LPS-activation. The nCRP isoform had opposing actions on monocytes, significantly (p<0.01) increasing and reducing NO/iNOS by unactivated and LPS-activated monocytes respectively. Estrogen significantly (P<0.01) reversed nCRP-mediated NO inhibition by unactivated macrophages but decreased CRP-induced NO by unactivated monocytes treated with nCRP or mCRP and LPS-activated monocytes treated with mCRP. NO was differentially mediated by CRP isoforms in a cell-type/state specific manner, with production corresponding to concomitant changes in iNOS levels. Collectively, the findings indicate nCRP and estrogen predominantly reduce NO production whereas mCRP increases NO production. This supports growing evidence that mCRP exacerbates inflammation whilst nCRP and estrogen dampen the overall inflammatory response. Therapeutic strategies that restore estrogen levels to those found in youth and promote the stability of nCRP or/and prevent the formation of mCRP may reduce NO production in age-related inflammatory conditions

Glucose-6-Phosphate Dehydrogenase Deficiency Activates Endothelial Cell and Leukocyte Adhesion Mediated via the TGFβ/NADPH Oxidases/ROS Signaling Pathway

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common genetic inherited trait among humans, affects ~7% of the global population, and is associated with excess risk of cardiovascular disease (CVD). Transforming growth factor-&beta; (TGF-&beta;) regulates immune function, proliferation, epithelial-mesenchymal transition, fibrosis, cancer, and vascular dysfunction. This study examined whether G6PD deficiencies can alter TGF-&beta;-mediated NADPH oxidases (NOX) and cell adhesion molecules (CAM) in human aortic endothelial cells (HAEC). Results show that treatment with high glucose and the saturated free fatty acid palmitate significantly downregulated G6PD; in contrast, mRNA levels of TGF-&beta; components, NOX and its activity, and reactive oxygen species (ROS) were significantly upregulated in HAEC. The expression levels of TGF-&beta; and its receptors, NOX and its activity, and ROS were significantly higher in HG-exposed G6PD-deficient cells (G6PD siRNA) compared to G6PD-normal cells. The protein levels of adhesion molecules (ICAM-1 and VCAM-1) and inflammatory cytokines (MCP-1 and TNF) were significantly increased in HG-exposed G6PD-deficient cells compared to G6PD-normal cells. The adherence of monocytes (SC cells) to HAEC was significantly elevated in HG-treated G6PD-deficient cells compared to control cells. Pharmacological inhibition of G6PD enhances ROS, NOX and its activity, and endothelial monocyte adhesion; these effects were impeded by NOX inhibitors. The inhibition of TGF-&beta; prevents NOX2 and NOX4 mRNA expression and activity, ROS, and adhesion of monocytes to HAEC. L-Cysteine ethyl ester (cell-permeable) suppresses the mRNA levels of TGF-&beta; and its receptors, along with NOX2 and NOX4, and decreases NOX activity, ROS, and adhesion of monocytes to HAEC. This suggests that G6PD deficiency promotes TGF-&beta;/NADPH oxidases/ROS signaling, the expression of ICAM-1 and VCAM-1, and the adhesion of leukocytes to the endothelial monolayer, which can contribute to a higher risk for CVD

Hydrogen Sulfide Regulates Irisin and Glucose Metabolism in Myotubes and Muscle of HFD-Fed Diabetic Mice

Irisin, a novel myokine, is secreted by the muscle following proteolytic cleavage of fibronectin type III domain containing 5 (FNDC5) and is considered a novel regulator of glucose homeostasis. Cystathionine &gamma;-lyase (CSE) produces hydrogen sulfide (H2S) and is involved in glucose homeostasis. We examined the hypothesis that H2S deficiency leads to decreased FNDC5 and irisin secretion, and thereby alters glucose metabolism. High-fat diet-fed mice exhibited elevated blood glucose and significantly reduced levels of CSE, H2S, and PGC-1&alpha;, with decreased FNDC5/irisin levels and increased oxidative stress in the muscle compared with those of normal diet-fed mice (control). High glucose or palmitate decreases CSE/PGC-1&alpha;/FNDC5 levels and glucose uptake in myotubes. Inhibitors (propargylglycine and aminooxyacetate) of H2S producing enzymes or CSE siRNA significantly decreased levels of H2S and FNDC5 along with PGC-1&alpha;; similar H2S-deficient conditions also resulted in decreased GLUT4 and glucose uptake. The levels of H2S, PGC-1&alpha;, and FNDC5 and glucose uptake were significantly upregulated after treatment with l-cysteine or an H2S donor. Myoblast differentiation showed upregulation of PGC-1&alpha; and FNDC5, which was consistent with the increased expression of CSE/H2S. These findings suggest that the upregulation of H2S levels can have beneficial effects on glucose homeostasis via activation of the PGC-1&alpha;/FNDC5/irisin signaling pathway