Abstract
BACKGROUND: This study tested whether combined shock wave (SW)-facilitated melatonin (Mel) delivered into endothelial progenitor cells (EPCs) (EPC(SW-Mel)) plus S-nitroso-N-acetyl-DL-penicillamine (SNAP) was superior to merely one modality alone for improving critical limb ischemia (CLI) in rats. METHODS: SD rats (n = 50) were equally categorized into group 1 (sham-control), group 2 (CLI), group 3 (CLI + SNAP), group 4 (CLI + EPC(SW-Mel)), and group 5 (CLI + EPC(SW-Mel) + SNAP), and ischemia-involved quadriceps were harvested by day 14. RESULTS: An in vitro study showed that at time points of 24/48/72 h, the cell viability/protein expression of endothelial nitric oxide synthase (eNOS)/and cellular expression of nitric oxide (NO) were highest in EPCs, lowest in EPCs + menadione, and much higher in EPC(SW-Mel) + Mena than in EPCs + Mena + Mel. Protein levels of oxidative-stress (NOX-1/NOX-2/oxidized protein)/early (AN-V(+)/PI(-))/late (AN-V(+)/PI(+)) apoptosis and total intracellular/mitochondrial reactive oxygen species ROS exhibited an antithetical trend of cell viability among the groups (all P<0.0001). Matrigel assay of angiogenesis/positively-stained NO cells showed that they were much higher in EPCs + SNAP than in EPCs only (all P<0.0001). Ex vivo angiogenesis/arterial relaxation of carotid-artery rings were highest in left-common-carotid-artery (LCCA) + SNAP, lowest in LCCA + Mena, and notably higher in LCCA than in LCCA + Mena + SNAP (all P<0.0001). Laser Doppler showed ischemic to normal-blood-flow (INBF) ratio was highest in group 1, lowest in group 2, and it progressively increased from groups 3 to 5 (all P<0.0001). The protein levels of oxidative-stress (NOX-1/NOX-4/oxidized protein)/apoptotic [cleaved-caspase-3/cleaved apoptosis/mitochondrial-damage (cytosolic-cytochrome-C/p-DRP-1)]/fibrotic (Smad3/TGF-β)/inflammatory (MMP-9/IL-1β/TNF-α/NF-κB) biomarkers, exhibited an opposite trend, whereas the protein level of endothelial-cell surface markers (CD31/vWF/eNOS) and number of small vessels exhibited an identical pattern of INBF ratio among the groups (all P<0.0001). CONCLUSIONS: Combined EPC(SW-Mel) and SNAP therapy offered a synergic effect toward rescuing from CLI.