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| 070P London, UK Pharmacology 2016 |
Modulation of aortic perivascular adipose tissue function by peroxynitrite
Introduction: Peroxynitrite (ONOO-) is formed by the rapid reaction of nitric oxide and superoxide and is an endothelium-independent vasodilator (1). Both superoxide and nitric oxide are produced by the perivascular adipose tissue (PVAT) surrounding blood vessels and the resulting ONOO- formed could modulate the function of the PVAT. In a previous study we demonstrated that treatment of isolated PVAT with ONOO- enhanced the ability of the PVAT to augment vessel relaxation (2). In this study we aimed to further investigate the effect of exogenous ONOO- on PVAT function and release profile.
Methods: Thoracic aortae from male C57BL/6 mice (20-25g) were removed and placed in ice-cold Krebs’ solution. All experiments were in accordance with the Animals (Scientific Procedures) Act 1986 / ASPA Amendment Regulations 2012. The PVAT was removed carefully and treated with either vehicle or ONOO- (10-4M for 1 hour at 37oC in Krebs’ solution). Following this, PVAT was incubated with dihydroethidium (DHE; 10-5M for 30 minutes at 37oC in the dark) to measure the effect of ONOO-on superoxide formation. In some experiments PVAT was homogenized in liquid nitrogen, protein concentration determined and adiponectin content measured using an ELISA kit (Quantikine® ELISA mouse adiponectin kit; R&D Systems). Respirometry (OROBROS Oxygraph-2k) was used to measure the effect of ONOO- on mitochondrial function in the PVAT.
Results: Analysis of the confocal projections of PVAT stained with DHE showed that ONOO-treatment increased superoxide formation (p<0.01, n=6). Incubation with superoxide dismutase (SOD) significantly reduced superoxide formation in PVAT (p<0.01, n=6) (Fig.1). In addition, ONOO--treated PVAT had higher adiponectin content compared to control (72.4 ± 5.5 ng/mg protein vs. 39.7 ± 7.6 ng/mg protein, p<0.01, n=11).
Fig.1 Effect of ONOO- on superoxide formation in PVAT
(# p<0.05 vs Control DHE, ## p<0.01 vs ONOO- DHE,** p<0.01 vs Control DHE, n=6)
Spirometry measurements revealed that ONOO- increased mitochondrial respiration which may be linked to the increased superoxide formation and adiponectin content.
Conclusion: The ability of PVAT to augment relaxation is increased following treatment with ONOO-and may be related to increased adiponectin content caused by increased mitochondrial activity.
References:
1. Ewart MA et al. (2014). Atherosclerosis 234(1): 154-161.
2. Ugusman A et al. (2016). In: EPHAR 2016 Congress Book: Turkey, p295.