Abstract 503: Ixmyelocel-T, a Unique Mixed Cellular Therapy for the Treatment of Vascular Diseases Associated with Endothelial Dysfunction

Abstract

Ixmyelocel-T, an expanded autologous multicellular therapy cultured from bone marrow mononuclear cells (BMMNCs), is under investigation for the treatment of vascular diseases associated with endothelial dysfunction. This study examines the mechanisms by which ixmyelocel-T treatment may exert beneficial effects on the endothelium in comparison to BMMNCs.

To determine the effects on endothelial cells, ixmyelocel-T and BMMNCs were co-cultured with human umbilical vein endothelial cells (HUVEC) in non-contacting Transwell inserts. Co-culture with ixmyelocel-T resulted in increased eNOS (1730±141, vs. 1371±135 pg/mL, p < 0.05) measured by ELISA, and nitric oxide (NO) production (1.97±0.2, vs. 1±0.1 relative fluorescence, p < 0.001) measured by DAF-2DA. Co-culture with BMMNCs did not have an effect on eNOS or NO in HUVECs. In TNFα stimulated HUVECs, ixmyelocel-T decreased the generation of reactive oxygen species (46±4 vs. 100±3 % of HUVEC, p < 0.01) measured with DCFH-DA and increased SOD activity (1.3±0.1, vs. 1±0.1 % of HUVEC, p < 0.05), whereas co-culture with BMMNCs didn’t have an effect on ROS or SOD in HUVECs. Apoptosis analyzed by a caspase 3/7 assay demonstrated that ixmyelocel-T decreased apoptosis in TNFα treated HUVECs (0.78±0.02, vs. 1±0.05 relative to HUVEC, p < 0.001). Co-culture with BMMNCs had no effect on HUVEC apoptosis. Real time PCR analysis revealed that ixmyelocel-T decreased the expression of inflammatory markers (ICAM1, VCAM1, and MCP-1) in TNFα treated HUVECs, whereas treatment with BMMNCs had no effect. ELISA analysis indicated that ixmyelocel-T decreased MCP-1 (11983±5357 vs. 23312±11044 pg/mL, p < 0.05) and increased IL-10 secretion (61.3±11.2 vs. 1.2±0.5 pg/mL, p < 0.001), whereas treatment with BMMNCs had no effect.

Ixmyelocel-T stimulated NO production, reduced oxidative stress and inflammation, and prevented apoptosis in endothelial cells. BMMNCs did not exhibit similar results. This is most likely due to the anti-inflammatory cell phenotypes associated with ixmyelocel-T’s expansion process. This study indicates that ixmyelocel-T may be superior to BMMNCs in the treatment of diseases associated with endothelial dysfunction and vascular inflammation.