The Effect of Neutrophil-Derived Products on the Function of Leukocytes Obtained after Titanium Implantation in the Ovine Model

Titanium (Ti) is currently the most common biomaterial used for orthopedic implants; however, these implants may cause deleterious immune response. To investigate the possible mechanisms involved in excessive inflammation, we assessed the activity of neutrophils and monocyte-derived macrophages (MDMs) during the insertion of the Ti implant in a sheep model. The study was conducted on 12 sheep, 4 of which were control animals and 8 were in the experimental group with inserted Ti implant. Neutrophil secretory response was estimated at two time points T0 before surgery and T1 1 h after implantation and was based on the release of enzymes from neutrophil granules and reactive oxygen and nitrogen species (RONS) generation. MDM function was evaluated 5 months after implantation, on the basis of RONS generation arginase activity and morphological changes. Moreover, the influence of some autologous neutrophil derived products, namely, antimicrobial neutrophil extract (ANE) and neutrophil degranulation products (DGP) on leukocytes was estimated. Our study revealed that Ti implant insertion did not cause any adverse effects up to 5 months after surgical procedure. Stimulation of neutrophil cultures with ANE decreased the enzyme release as well as superoxide generation. Treatment of MDM with ANE diminished superoxide and NO generation and increased arginase activity. On the other hand, MDM stimulated with DGP showed elevated superoxide and NO generation as well as decreased arginase activity. To summarize, ANE exerted an anti-inflammatory and pro-resolving effect on studied leukocytes, whereas DGP acted as pro-inflammatory.

Celastrol attenuates fMLP-induced superoxide anion generation, myeloperoxidase production, and elastase release by human neutrophils

Purpose: To investigate the anti-inflammatory effect of celastrol via attenuation of formyl-methionylleucyl-phenylalanine (fMLP)-induced superoxide generation, myeloperoxidase production, and elastase release by peripheral blood neutrophils. Methods: Cytotoxicity of celastrol on human peripheral blood neutrophils was investigated using a 2Htetrazolium hydroxide (XTT) assay. Human neutrophils were stimulated with 100-nM fMLP; the effect of celastrol on superoxide generation was determined via ferricytochrome C reduction, the effect on myeloperoxidase production by tetramethylbenzidine oxidation, and the effect on elastase activity by Boc-Ala-ONp hydrolysis. Results: Treatment of human neutrophils with celastrol showed dose-dependent inhibition of fMLPinduced superoxide generation, myeloperoxidase production, and elastase release with half-maximal inhibitory concentration (IC50) values of 5.9 ± 0.1, 1.9 ± 0.2, and 1.5 ± 0.1 µM, respectively. Conclusion: These results indicate that celastrol possesses anti-inflammatory properties via attenuation of fMLP-induced superoxide generation, myeloperoxidase production, and elastase release by peripheral blood neutrophils.

FXYD1 Is Protective Against Vascular Dysfunction

Nitric oxide (NO) production by eNOS (endothelial NO synthase) is critical for vascular health. Oxidative stress-induced uncoupling of eNOS leads to decreased NO bioavailability, compounded by increased superoxide generation. FXYD1 (FXYD domain containing ion transport regulator 1), a caveolar protein, protects against oxidative inhibition of the Na + -K + -ATPase. We hypothesized that FXYD1 may afford a similar inhibition of oxidative dysregulation of eNOS, providing a broader protection within caveolae. FXYD1-eNOS colocalization was demonstrated by co-immunoprecipitation in heart protein and by proximity ligation assay in human umbilical vein endothelial cells. The functional nature of this partnership was shown by silencing FXYD1 in human umbilical vein endothelial cells, where 50% decreased NO and 2-fold augmented superoxide was shown. Three-dimensional cocultured cardiac spheroids generated from FXYD1 knockout mice were incapable of acetylcholine-induced NO production. Overexpression of FXYD1 in HEK293 cells revealed a possible mechanism, where FXYD1 protected against redox modification of eNOS cysteines. In vivo, vasodilation in response to increasing doses of bradykinin was impaired in knockout mice, and this was rescued in mice by delivery of FXYD1 protein packaged in exosomes. Bloods vessels extracted from knockout mice exhibited increased oxidative and nitrosative stress with evidence of reduce eNOS phosphorylation. Impaired vascular function and augmented superoxide generation were also evident in diabetic knockout mice. Despite this, blood pressure was similar in wildtype and knockout mice, but after chronic angiotensin II infusion, knockout of FXYD1 was associated with a heightened blood pressure response. FXYD1 protects eNOS from dysregulated redox signaling and is protective against both hypertension and diabetic vascular oxidative stress.

Glucose-dependent insulinotropic polypeptide inhibits cardiac hypertrophy and fibrosis in diabetic mice via suppression of TGF-β2

Diabetic cardiomyopathy is associated with an increased risk for heart failure and death in patients with diabetes. We investigated here whether and how GIP attenuated cardiac hypertrophy and fibrosis in diabetic mice with obesity. Diabetic db/db mice at 7 weeks old were infused with vehicle or GIP (50 nmol/kg/day) for 6 weeks, and hearts were collected for histological and RT-PCR analyzes. Cardiomyocytes isolated from neonatal mice were incubated with or without 300 nM [D-Ala2]-GIP, 30 mM glucose, or 100 μg/mL advanced glycation end products (AGEs) for RT-PCR and lucigenin assays. Compared with non-diabetic mice, diabetic mice exhibited larger left ventricle wall thickness and cardiomyocyte sizes and more fibrotic areas in association with up-regulation of myosin heavy chain β (β-Mhc) and transforming growth factor-beta2 (Tgf-β2) mRNA levels, all of which were inhibited by GIP infusion. High glucose increased NADPH oxidase-driven superoxide generation and up-regulated β-Mhc, Tgf-β2, and receptor for AGEs mRNA levels in cardiomyocytes, and augmented the AGE-induced β-Mhc gene expression. [D-Ala2]-GIP attenuated all of the deleterious effects of high glucose and/or AGEs on cardiomyocytes. Our present findings suggest that GIP could inhibit cardiac hypertrophy and fibrosis in diabetic mice via suppression of TGF-β2.