Fig 2: Advanced glycation end products (AGEs) impaired the viability of bone marrow mesenchymal stromal cells (bMSCs) through increased reactive oxygen species (ROS) production attributable to selective reduction of superoxide dismutase (SOD)‐1 and SOD‐3 expression. A through C, Culture of bMSCs with AGE‐BSA (with concentration from 100 to 400 μg/mL with BSA as control) significantly increased intracellular ROS (A) and early (B) and late apoptosis (C) in a dose‐dependent manner. n=6/group. *P<0.0001 for 0 vs 400 μg/mL AGE‐BSA; # P<0.0001 for 400 μg/mL BSA vs 400 μg/mL AGE‐BSA; + P<0.001 for 0 vs 200 μg/mL AGE‐BSA. D through H, The representative images of flow cytometric analysis (D and E) and quantification of intracellular ROS (F) and early (G) and late apoptosis (H). AGE‐BSA treatment (400 μg/mL with BSA as control) significantly increased intracellular ROS, and apoptosis of bMSCs. Inhibition of ROS production with N‐acetylcysteine (NAC) significantly prevented AGE‐induced increase in intracellular ROS, and apoptosis of bMSCs (n=6/group; *P<0.01 for WT+AGE vs WT+BSA; + P<0.01 for WT+AGE+NAC vs WT+AGE). I through L, The representative images (I and K) and quantification (J and L) of Western blot analysis. AGE‐BSA treatment (400 μg/mL) significantly decreased the protein levels of SOD‐1 and SOD‐3 in bMSCs (n=3; *P<0.05 for WT+AGE vs WT+BSA; J). The expression levels of SOD‐1, SOD‐3, and glutathione peroxidase‐1 (Gpx‐1) were significantly increased in bMSCs from triple‐transgenic (TG) mice compared with bMSCs from wild‐type (WT) mice (n=3; *P<0.05 for TG vs WT; L). To determine if decreased levels of SOD‐1 and SOD‐3 contributed to AGE‐induced increase of ROS production in bMSCs and subsequent increase in apoptosis, experiments were repeated with bMSCs from mice with concomitant overexpression of SOD‐1, SOD‐3, and Gpx‐1 (TG‐bMSCs). Indeed, AGE‐induced ROS and apoptosis were significantly decreased in TG‐bMSCs compared with WT‐bMSCs (n=6/group; # P<0.05 for TG+AGE vs WT+AGE). Statistical differences were determined with 1‐way ANOVA with Bonferroni post hoc test or Kruskal‐Wallis test with Dunn post hoc test. All data were shown as means±SD. FITC indicates fluorescein isothiocyanate; DCF, dichlorofluorescein; PI, propidium iodide; SSC‐A, side scatter area.

Fig 3: Concomitant overexpression of triple antioxidant enzymes effectively preserved the muscle fibres of the ischemic limb. The muscle fibres reduced in size and became largely disconnected in the WT control mice with limb ischaemia. CD31 staining showed that capillary density was low in the limb muscle after ischaemia for 3, 7 and 21 days. On the other hand, the muscle fibres appeared to remain long and large, as well as well-connected in the limb muscles at day 21 after ischaemia in the TG mice with concomitant global overexpression of SOD1, SOD3 and Gpx-1. CD31 staining demonstrated that the capillary density remained largely intact (similar to the non-ischemic limb). Normal: normal right limb; ischaemia; ischemic left limb. Red arrow: capillary vessels. BL: before ligation. Scale bar: 50µm

Fig 4: NAC preserved the expression of antioxidant enzymes in ischemic muscle in type-2 diabetic mice. To exam the effect of NAC on the expression of antioxidant enzymes in diabetic ischemic limbs, ischemic muscle tissue was harvested for Western blot analysis. As demonstrated in panels (A,B), the expression of SOD-1, Gpx-1, and catalase were significantly decreased in diabetic ischemic limbs compared to non-diabetic control (n = 4, * p < 0.05 for TH versus SWR); there was a trend towards a decreased expression of SOD-3, but it was not significant statistically. NAC treatment significantly prevented the reduction in the expression of SOD-1, Gpx-1, and catalase in diabetic ischemic limb muscle tissue (n = 4, # p < 0.05 for TH versus TH & NAC). SOD-1, copper-zine superoxide dismutase in cytoplasm; SOD-3, extracellular SOD; Gpx-1, glutathione peroxidase-1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TH, diabetic TALLYHO/JngJ mice with CLI operation; SWR, non-diabetic SWR/J mice with CLI operation; TH & NAC, TALLYHO/JngJ mice with CLI operation plus NAC treatment. All data are shown as means ± SD. Significant differences were determined with one-way ANOVA with Bonferroni’s post hoc test or Kruskal-Wallis test with Dunn post hoc test.

Fig 5: Combination of triple antioxidant enzyme overexpression in bone marrow mesenchymal stromal cells (bMSCs) and host treatment with N-acetylcysteine (NAC) additively enhanced the survival of bMSCs in diabetic ischemic limb.Dihydroethidium (DHE) staining (from the adductor muscle of ischemic hind limb 21 days after ligation) demonstrated that reactive oxygen species (ROS) production in diabetic ischemic limb was significantly increased compared with the nondiabetic ischemic limb (n=8/group; *P<0.01 for TALLYHO/JngJ mice [TH] vs SWR/J mice [SWR]; A and B). NAC treatment substantially decreased tissue ROS level in the ischemic limb in diabetic mice (n=8/group; # P<0.01 for TH and NAC vs TH). In vivo optical fluorescent imaging analysis showed that concomitant overexpression of superoxide dismutase (SOD)-1, SOD-3, and glutathione peroxidase-1 (Gpx-1) significantly improved the survival of bMSCs (TG-bMSCs) in diabetic ischemic limb by almost 5 times over the control (from 7.63% to 33.52%; # P<0.01 for TG-bMSCs vs wild-type [WT] bMSCs) 1 week after implantation. The survival of bMSCs was substantially increased to >50% in the ischemic limb in diabetic mice with combination of inhibiting intracellular ROS production in bMSCs with concomitant overexpression of SOD-1 and Gpx-1 (TG-bMSCs) and extracellular ROS production with NAC treatment and SOD-3, compared with survival rate of about 30% to 35% for either triple enzyme overexpression or NAC treatment alone at week 1 after cell delivery, and >30% and 10% for the cells with combined treatment at weeks 2 and 3, respectively (n=8/group; + P<0.05 for WT-bMSCs vs WT-bMSCs and NAC; **P<0.05 for TG-bMSCs and NAC vs TG-bMSCs; ## P<0.05 for TG-bMSCs and NAC vs WT-bMSCs and NAC; C and D). Increased cell numbers in TG-bMSCs and TG-bMSCs and NAC group were confirmed with ex vivo histological analysis of DiR+ fluorescence 14 days after transplantation (n=8/group; # P<0.01 for TG-bMSCs vs WT-bMSCs; + P<0.05 for WT-bMSCs vs WT-bMSCs and NAC; **P<0.01 for TG-bMSCs and NAC vs TG-bMSCs; ## P<0.01 for TG-bMSCs and NAC vs WT-bMSCs and NAC; E and F). Bar=50 µm. Statistical differences were determined by 2-way repeated-measures ANOVA followed by Tukey test (D) and 1-way ANOVA with Bonferroni post hoc test (B and F). All data were shown as means±SD. DAPI indicates 4',6-diamidino-2-phenylindole.