eNOS Uncoupling: A Therapeutic Target For Ischemic Foot of Diabetic Rat

Abstract

Aim To determine the relationship between eNOS uncoupling and diabetic ischemic foot and whether reversing eNOS uncoupling by Dihydrofolate reductase (DHFR) transfection or folic acid (FA) supplementation can be beneficiary in diabetic ischemic foot.

Methods The bilateral common iliac artery of diabetic rats were ligated to establish the diabetic ischemic foot animal model. DHFR transfection was implemented via femoral artery and muscle injection of in vivo transfection reagent mixture (GenEscortIII) every 4 days during the 2 weeks intervention. The color doppler flow imaging (CDFI) of femoral artery for RI measurement, triceps and quadriceps structure and histology, eNOS coupling status, DHFR expression level, superoxide, peroxynitrite (ONOO– ) and nitric oxide (NO) production in the presence or absence of L-NAME (eNOS inhibitor) were examined among wild type rats (WT), diabetic sham rats (DM), rats of diabetic ischemic foot (DF) or DF with DHFR transfection (DFT) or DF with FA supplementation (DFF).

Results Dihydroethidium (DHE) fluorescence, as an index of superoxide production was enhanced in the femoral arteries of diabetic rats and even more in those of ischemic foot from diabetic rats. However, the DHE fluorescence was diminished in the presence of L-NAME suggesting eNOS uncoupling is the source of superoxide overproduction which further led to increased peroxynitrite production and decreased NO^. bioavailability. Subsequently, the hind limb muscle became atrophic and the local collateral circulation was defective due to endothelial dysfunction related to eNOS uncoupling. However, all of the above and hemodynamic index (RI) of femoral artery were resumed via restoration of DHFR protein expression by folic acid treatment or DHFR transfection.

Conclusions eNOS uncoupling is involved in diabetic ischemic foot due to DHFR suppression. DHFR restoration can reverse eNOS uncoupling and resume the endothelial dysfunction and pathological changes (increased vasculature resistance, hind limb muscle atrophy and defective collateral circulation) associated with eNOS uncoupling in diabetic ischemic foot. All of which enlightens a novel therapeutic strategy for future diabetic ischemic foot treatments.

Key words

eNOS uncoupling - diabetic foot - ischemia - DHFR

• References

• 1 Liu ZJ, Velazquez OC. Hyperoxia, endothelial progenitor cell mobilization, and diabetic wound healing. Antioxid Redox Signal 2008; 10: 1869-1882 doi: 10.1089/ars.2008.2121
• 2 Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 2000; 87: 840-844
• 3 Gao L, Chalupsky K, Stefani E. et al. Mechanistic insights into folic acid-dependent vascular protection: dihydrofolate reductase (DHFR)-mediated reduction in oxidant stress in endothelial cells and angiotensin II-infused mice: a novel HPLC-based fluorescent assay for DHFR activity. Journal of molecular and cellular cardiology 2009; 47: 752-760 doi: 10.1016/j.yjmcc.2009.07.025
• 4 Gao L, Siu KL, Chalupsky K, Weintraub NL. et al. Role of uncoupled endothelial nitric oxide synthase in abdominal aortic aneurysm formation: treatment with folic acid. Hypertension 2012; 59: 158-166 doi: 10.1161/HYPERTENSIONAHA.111.181644
• 5 Chuaiphichai S, McNeill E, Douglas G. et al. Cell-autonomous role of endothelial GTP cyclohydrolase 1 and tetrahydrobiopterin in blood pressure regulation. Hypertension 2014; 64: 530-540 doi: 10.1161/HYPERTENSIONAHA.114.03089
• 6 Ashor AW, Siervo M, Lara J. et al. Effect of vitamin C and vitamin E supplementation on endothelial function: a systematic review and meta-analysis of randomised controlled trials. The British journal of nutrition 2015; 113: 1182-1194 doi: 10.1017/S0007114515000227
• 7 Wang S, Moustaid-Moussa N, Chen L. et al. Novel insights of dietary polyphenols and obesity. The Journal of nutritional biochemistry 2014; 25: 1-18 doi: 10.1016/j.jnutbio.2013.09.001
• 8 Gao L, Mann GE. Vascular NAD(P)H oxidase activation in diabetes: a double-edged sword in redox signalling. Cardiovascular research 2009; 82: 9-20 doi: 10.1093/cvr/cvp031
• 9 Oak JH, Cai H. Attenuation of angiotensin II signaling recouples eNOS and inhibits nonendothelial NOX activity in diabetic mice. Diabetes 2007; 56: 118-126 doi: 10.2337/db06-0288
• 10 Youn JY, Gao L, Cai H. The p47phox- and NADPH oxidase organiser 1 (NOXO1)-dependent activation of NADPH oxidase 1 (NOX1) mediates endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction in a streptozotocin-induced murine model of diabetes. Diabetologia 2012; 55: 2069-2079 doi: 10.1007/s00125-012-2557-6
• 11 Sugiyama T, Levy BD, Michel T. Tetrahydrobiopterin recycling, a key determinant of endothelial nitric-oxide synthase-dependent signaling pathways in cultured vascular endothelial cells. J Biol Chem 2009; 284: 12691-12700 doi: 10.1074/jbc.M809295200
• 12 Ishak NA, Ismail M, Hamid M. et al. Antidiabetic and Hypolipidemic Activities of Curculigo latifolia Fruit: Root Extract in High Fat Fed Diet and Low Dose STZ Induced Diabetic Rats. Evidence-based complementary and alternative medicine: eCAM 2013; 2013: 601838 doi: 10.1155/2013/601838
• 13 Westvik TS, Fitzgerald TN, Muto A. et al. Fancher TT et al. Limb ischemia after iliac ligation in aged mice stimulates angiogenesis without arteriogenesis. Journal of vascular surgery 2009; 49: 464-473 doi: 10.1016/j.jvs.2008.08.077
• 14 Setsukinai K, Urano Y, Kakinuma K. et al. Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J Biol Chem 2003; 278: 3170-3175 doi: 10.1074/jbc.M209264200
• 15 Corcoran HA, Smith BE, Mathers P. et al. Laser Doppler imaging of reactive hyperemia exposes blood flow deficits in a rat model of experimental limb ischemia. Journal of cardiovascular pharmacology 2009; 53: 446-451 doi: 10.1097/FJC.0b013e3181a6aa62
• 16 Rochette L, Lorin J, Zeller M. et al. Nitric oxide synthase inhibition and oxidative stress in cardiovascular diseases: possible therapeutic targets?. Pharmacology & therapeutics 2013; 140: 239-257 doi: 10.1016/j.pharmthera.2013.07.004
• 17 Zhang T, Xia LH, Bian YY. et al. Blood flow of the acral finger arterioles in patients with type 2 diabetes by quality Doppler profiles. Cell Biochem Biophys 2013; 67 717-725 doi: 10.1007/s12013-013-9561-4
• 18 Kolluru GK, Bir SC, Kevil CG. Endothelial dysfunction and diabetes: effects on angiogenesis, vascular remodeling, and wound healing. International journal of vascular medicine 2012; 2012: 918267 doi: 10.1155/2012/918267
• 19 Spinetti G, Kraenkel N, Emanueli C. et al. Diabetes and vessel wall remodelling: from mechanistic insights to regenerative therapies. Cardiovascular research 2008; 78: 265-273 doi: 10.1093/cvr/cvn039
• 20 Schiekofer S, Balletshofer B, Andrassy M. et al. Endothelial dysfunction in diabetes mellitus. Seminars in thrombosis and hemostasis 2000; 26: 503-511 doi: 10.1055/s-2000-13206