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.