Abstract
Ixmyelocel-T, an expanded, autologous, multicellular therapy cultured from bone marrow, has recently demonstrated clinical promise in the treatment of patients with critical limb ischemia. At this time, mechanisms describing its beneficial effects remain unclear. We investigated the potential mechanisms by which ixmyelocel-T may improve recovery from ischemia. In a rat model of hind limb ischemia, ixmyelocel-T treated rats exhibited significantly increased blood flow recovery by comparison to their vehicle control counterparts, as measured by laser Doppler. Ixmyelocel-T treated rats exhibited increased plasma nitrate levels (6.2±2.8 vs 32.8±12.4 μM, p < 0.05) measured with the Greiss reaction, and decreased plasma lipid peroxidation (0.04±0.005 vs.0.03±0.003 μM, p < 0.05) measured with the thiobarbituric acid reactive substances (TBARS) assay, indicating a preservation in the bioavability of nitric oxide. Fluorescent imaging revealed that PKH-labeled ixmyelocel-T co-localizes with eNOS in the blood vessels of the ischemic limb. Co-culture experiments with human umbilical vein endothelial cells (HUVEC) were employed to determine the effect of ixmyelocel-T on the endothelium. Co-culture resulted in increase of eNOS (1516±225 vs.1995±167 pg/ml, p < 0.05), as measured by ELISA, and nitric oxide (NO) production (71±1.4 vs.75±1.3 AU, p < 0.05), as measured with the intracellular NO probe DAF-2DA. Co-culture also resulted in reduced reactive oxygen species in TNFα activated HUVECs measured by an intracellular ROS probe (4.8±1.6 vs.1.4±0.4 nM DCF), as well as increased the release of the anti-inflammatory cytokine IL-10 by ixmyelocel-T. Ixmyelocel-T also promoted HUVEC migration in an in vitro scratch assay. Collectively, our data suggests that ixmyelocel-T stimulates recovery from ischemia through mechanisms involving stimulation of eNOS expression, increasing nitric oxide production, reducing oxidative stress and inflammation, and promoting endothelial migration, thereby promoting the preservation of vascular function. We hypothesize that these functions of ixmyelocel-T are responsible for the recovery of ischemic tissues in patients with CLI.
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