NOX 100, a nitric oxide scavenger, enhances cardiac allograft survival and promotes long-term graft acceptance.

BACKGROUND: We examined the role of nitrosative stress in allograft destruction. METHODS: Rats undergoing cardiac transplants received NOX-100, a water-soluble nitric oxide (NO) scavenger with antioxidant properties, with or without low-dose cyclosporine (CsA). Graft survival, NO production, and nuclear factor kappa B (NF-kappaB) activity were studied. RESULT: Using NOX-100 daily until rejection prolonged graft survival (11.6+/-0.6 vs. 7.4+/-0.2 days; P<0.05). Daily low-dose CsA (2.5 mg/kg im) for 7 days or until rejection also prolonged survival (12.6+/-0.5 and 21.6+/-1.6 days, respectively; P<0.01 vs. Controls). Low-dose CsA for 7 days and NOX-100 for 30 days prolonged graft survival (45.0+/-4.7 days; P<0.01 vs. all groups.). NOX-100 had no effect on whole blood CsA levels. Combination therapy until Day 100 resulted in 1 graft loss at Day 116 and indefinite survival in 3 animals (>300 days), which accepted a second WF strain heart without further immunosuppressive therapy but promptly rejected a third party (ACI) cardiac allograft. NOX-100 and CsA reduced nitrate and nitrite, and combination therapy completely normalized NO through to Day 30. Electron paramagnetic resonance spectroscopic analysis demonstrated reduction of signals for nitrosylmyoglobin and nitrosyl-heme with NOX-100 and elimination of signals with CsA alone or combination therapy. Activity of myocardial NF-kappaB decreased with monotherapy vs. untreated allografts. Combination therapy resulted in further inhibition of NF-kappaB up to Day 30. The extent of graft survival correlated with the extent of NO scavenging and NF-kappaB inhibition. Short-term combination therapy had no effect on graft lymphocytic infiltrate on Days 15, 20, and 30. CONCLUSION: These data support a role for both oxidative and nitrosative stress in rejection and the immunoregulatory potential of antioxidant therapy after transplantation.

Myocardial nuclear factor-kappaB activity and nitric oxide production in rejecting cardiac allografts.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is a rapid response transcription factor for genes whose products are critical for inflammation and immunity. In a rat model of heterotopic cardiac transplantation, we studied NF-kappaB DNA binding activity and nitric oxide (.NO) production in untreated allografts and whether inhibition of NF-kappaB suppresses .NO production and prolongs graft survival. METHODS: In allograft recipients and isograft controls, NF-kappaB was assayed by electrophoretic mobility shift assay, daily from transplant until rejection. Myocardial .NO was directly detected in explanted allografts by electron spin resonance spectroscopy on day 6 after transplant. The potent inhibitor of NF-kappaB, pyrrolidine dithiocarbamate (PDTC; 250 mg/kg s.c.) was administered daily from transplant until day of rejection. The extent of graft lymphocytic infiltrate was assessed by routine hematoxylin and eosin staining. Immunohistochemical staining of NF-kappaB was per formed to identify the cell type responsible for NF-kappaB activity. RESULTS: A time-dependent increase in myocardial NF-kappaB activity was seen in untreated allografts as compared with isografts as determined by PhosphorImage analysis. Peak NF-kappaB activity occurred in allografts on day 4 with a ninefold increase as compared with isografts (24.0+/-3.7% vs. 2.7+/-0.5; P<0.05). On posttransplant day 6, electron spin resonance spectroscopy analysis of allografts demonstrated .NO identified by a triplet nitrogen signal centered at g=2.012 with hyperfine splitting of 17.5 Gauss, which is consistent with nitrosoheme formation and low-field signals at g=2.08 and g=2.03 consistent with nitrosomyoglobin. These signals were not seen in native hearts of allograft recipients. With PDTC administration, a threefold decrease in NF-kappaB activity within the transplanted heart was observed on posttransplant day 5 as compared with untreated allografts (9.7+/-1.6% vs. 23.5+/-2.5%; P<0.01). PDTC prolonged graft survival as compared with untreated allografts (11.7+/-0.3 vs. 6.6+/-0.2 days; P<0.05) and reduced the intensity of the nitrosoheme and nitrosomyoglobin signals. Allograft mononuclear cell infiltrate correlated with peak NF-kappaB activity with peak infiltrate on posttransplant day 4. PDTC treatment had no effect on the extent of infiltrate. Immunohistochemical staining localized NF-kappaB to the infiltrating mononuclear cells on posttransplant day 5. CONCLUSION: These data support a role for NF-kappaB in allograft rejection.

Long-term treatment in vivo with NOX-101, a scavenger of nitric oxide, prevents diabetes-induced endothelial dysfunction.

Substantial evidence exists that diabetes results in impaired endothelial dysfunction suggesting diminished nitric oxide production from diabetic endothelium. It is not known what factors contribute to the development of this defect. In this study, we tested whether chronic treatment in vivo with NOX-101, a water-soluble nitric oxide scavenger, prevents endothelial dysfunction in diabetes. Sprague-Dawley rats were made diabetic by an intravenous injection of streptozotocin. A subgroup of control or diabetic animals received twice daily subcutaneous injections of 80 mg/kg NOX-101 beginning at 48 h after streptozotocin was injected and throughout 8 weeks of diabetes. Body weights and glucose concentrations were monitored weekly. At the end of 8 weeks, blood glucose and glycosylated haemoglobin was raised in diabetic rats but serum insulin concentrations were reduced. Treatment with NOX-101 did not alter glucose or insulin concentrations in control or diabetic rats; however, total glycosylated haemoglobin was partially reduced compared with untreated rats. In a subgroup of 2-week diabetic and age-matched rats fasted for 24 h, NOX-101 abolished total urinary nitrate plus nitrite (an index of nitric oxide production in vivo). In isolated tissue baths, relaxation to the endothelium-dependent vasodilator, acetylcholine, was impaired in diabetic aortic rings and relaxation to nitroglycerin was unaltered. Treatment of control rats with NOX-101 did not alter maximum relaxation to acetylcholine but shifted the response curve slightly to the right. In contrast in diabetic rats, NOX-101 prevented the impairment in endothelium-dependent relaxation but had no effect on relaxation induced by nitroglycerin. These data suggest the possibility that diabetes-induced endothelial dysfunction in diabetes results, in part, from a paradoxical increase in nitric oxide production during the course of the disease. This suggests a novel pathway of vascular complications.

Neurophysiological investigation of diabetic impotence. Are sacral response studies of value?

The role of electrophysiologically elicited sacral responses in the detection of the neurological component of diabetic impotence has been evaluated in a detailed study using properly defined diagnostic criteria. The results prove that these tests are not reliable indicators of neuropathy and their relevance in the routine investigation of diabetic impotence is of questionable value.