Vascular protective actions of a nitric oxide aspirin analog in both in vitro and in vivo models of diabetes mellitus.
BACKGROUND
Defective endothelium-dependent relaxation is observed in experimental and human diabetes mellitus. The nature of this defect is not fully understood but may involve decreased nitric oxide (NO) bioactivity due to enhanced production of reactive oxygen species (ROS). In this paper, we examine the benefits and actions of a novel NO-donating, antioxidant called 2-acetoxybenzoic acid 2-(2-nitrooxymethyl) phenyl ester, and denoted as NCX4016, on NO-mediated endothelium-dependent relaxation in normal arteries exposed to acute elevations in glucose or in arteries derived from chronic diabetic animals.


METHODS
Intrinsic free radical scavenging by NO-NSAIDs in solution were evaluated using electron paramagnetic resonance (EPR) spectroscopy and spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In acute studies, normal rat aortas were exposed in tissue culture for 18 h to 5.5 mM or 40 mM in the presence or absence of NCX4016, a NO-donating NSAID unrelated to aspirin (NCX2216) or aspirin. Vascular reactivity of thoracic aortic rings to endothelium-dependent relaxation to acetylcholine in vitro was determined. For chronic hyperglycemia, diabetes was induced in rats by intravenous injection with streptozotocin. Vascular reactivity of thoracic aortic rings to endothelium-dependent relaxation to acetylcholine in vitro was determined after 8 wks in untreated animals or animals chronically-treated with NCX4016. Antioxidant efficacy in vivo was determined by measurement of plasma isoprostanes and by nuclear binding activity of NF-kappaB in nuclear fractions of aortae.


RESULTS
Incubation with NCX4016 and NCX2216 produced a concentration-dependent inhibition of DMPO-OH formation indicating scavenging of hydroxyl radicals (HO(*)). In contrast, little efficacy to scavenge superoxide anion radicals was noted. Acute incubation of normal arteries with elevated glucose concentration caused inhibition of normal relaxation to acetylcholine. This impairment was prevented by co-incubation with NCX4106 but not by mannitol, the parent compound (aspirin) or by NCX2216. In addition, chronic treatment with NCX4016 prevented the development of defective endothelium-dependent relaxation to acetylcholine. This protection did not occur as a result to any changes in blood glucose concentration or hemoglobin glycation. Treatment with NCX4016 did decrease the elevation in plasma isoprostanes and normalized the diabetes-induced increase in NF-kappaB binding activity in nuclear fractions derived from aortic tissue.


CONCLUSIONS
Collectively, these studies suggest that antioxidant interventions using NO-donating NSAIDs may provide an important novel therapeutic strategy to protect the diabetic endothelium.
Pieper
Galen M
GM
Division of Transplant Surgery, Medical College of Wisconsin, Milwaukee, WI 53226, USA. gmpieper@mcw.edu
Siebeneich
Wolfgang
W
Olds
Cara L
CL
Felix
Christopher C
CC
Del Soldato
Piero
P
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology / Q000627:therapeutic use*; D001013:Aorta, Thoracic / Q000187:drug effects; D001241:Aspirin / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use*; D003921:Diabetes Mellitus, Experimental / Q000188:drug therapy* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D024202:Electrophoretic Mobility Shift Assay; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D016166:Free Radical Scavengers / Q000494:pharmacology / Q000627:therapeutic use*; D015870:Gene Expression / Q000187:drug effects; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D017665:Hydroxyl Radical / Q000378:metabolism; D006943:Hyperglycemia / Q000188:drug therapy / Q000378:metabolism; D028421:Isoprostanes / Q000378:metabolism; D009126:Muscle Relaxation / Q000187:drug effects*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D009638:Norepinephrine / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013481:Superoxides / Q000378:metabolism; D014664:Vasodilation / Q000187:drug effects
D000894:Anti-Inflammatory Agents, Non-Steroidal; D016166:Free Radical Scavengers; D028421:Isoprostanes; D016328:NF-kappa B; D013481:Superoxides; D009569:Nitric Oxide; D017665:Hydroxyl Radical; C102148:nitroaspirin; D005947:Glucose; D000109:Acetylcholine; D001241:Aspirin; D009638:Norepinephrine