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Downregulation of Inflammation and a Cascade of Proangiogenic Signals Mediate the Beneficial Effects of Gene-Modified Stem Cell Therapy in Hindlimb Ischemia

      Background

      Novel therapies to afford limb salvage in patients with imminent amputation due to critical limb ischemia are needed. We have previously shown E-selectin, a cell adhesion molecule, as an essential participant in neovascularization. Thus, we hypothesized E-selectin supercharged mesenchymal stem cells (E-selectin+/MSC) would augment limb reperfusion, tissue regeneration, and functionality.

      Methods

      C57Bl6 mice underwent femoral artery ligation and received either vehicle (phosphate-buffered saline; n = 9) or syngeneic donor MSCs, transduced ex vivo to express either GFP+ (control; n = 20) or E-selectin-GFP+ (treatment; n = 18). Laser Doppler imaging, confocal laser microscopy, and treadmill exhaustion test were used to determine neovascularization and limb function. Extent of ischemic skeletal muscle atrophy (mean myocyte size in μm2) was assessed via leg muscle histology. RT2 Profiler PCR Array analysis of 84 genes involved in angiogenesis assessed therapeutic mechanism of action. The Student t-test or analysis of variance was used to compare means and significance set at P < .05.

      Results

      Compared with GFP+/MSC and phosphate-buffered saline, treatment with E-selectin-GFP+/MSC increased ischemic leg Laser Doppler imaging reperfusion (55% vs 39% vs 24%; P < .001), ischemic mouse footpad vessel density (23% vs 14% vs 14%; P < .01) and treadmill distance traversed (162 m vs 111 m vs 110 m; P < .01). The ischemic limb in mice treated with E-selectin-GFP+/MSC were less atrophic than controls (793 μm2 vs 556 μm2 vs 546 μm2; P < .001) (Fig). Seven proangiogenic genes were upregulated in E-selectin-GFP+/MSC-treated ischemic leg tissue while tumor necrosis factor was downregulated, when compared with GFP+/MSC-treated tissues.

      Conclusions

      This innovative E-selectin supercharged stem cell therapy confers increased limb reperfusion, function, and decreased atrophy, likely via upregulation of proangiogenic cytokines and downregulated tumor necrosis factor.
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      FigConfocal laser microscopy images of ischemic murine footpads Dill perfusion.