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Cell based therapy with progenitor populations are hampered in patients with diabetes mellitus (DM) due to greater cellular senescence. Therefore, cell enhancement strategies are currently being sought to augment therapeutic benefit.
We isolated endothelial progenitor cells (EPCs) from healthy and DM donors treating them with small molecule glycogen synthase kinase 3β (GSK3β) inhibitors (GSKi). Cell yield and functional assays were performed. Proteomic profiling of ECPs from DM, healthy controls, and GSKi treated cells was performed. Finally, CD1 nude mouse underwent femoral artery wire injury and xenotransplantation of EPCs to evaluate therapeutic efficacy.
Patients with DM had fewer EPCs (14.9 ± 4.6 vs 38.5 ± 6.7 cells, p<0.01) and higher rates of apoptosis as assessed by flow cytometry (9.2 ± 0.9 vs 7.3 ± 0.9%, p=0.02). This was associated with higher levels of GSK3β activity as demonstrated by higher levels of phosphorylated β–catenin (0.55 ± 0.08 vs 0.42 ± 0.04, p=0.04). Proteomics identified 38 non–redundant differentially regulated targets in DM–EPCs that were normalized by GSKi treatment. Multiple targets were validated by Q–PCR and Western blot, including Cathepsin B (CatB). At baseline, CatB activity was 40% (p<0.05) less in DM–EPCs and GSKi treatments raised levels 400% (p<0.01) and attenuated increased rates of cellular apoptosis. Antagonism of CatB using CA074 blocked reductions in apoptosis. Finally, in vivo efficacy of cell based therapy was assessed in a xenotransplant mouse model. Administration of DM–EPCs alone significantly reduced intima:media (IM) ratio (1.37 ± 0.15 vs. 0.81 ± 0.12, p<0.01) at 14 days. Pretreatment of cells with GSKi resulted in a further reduction in IM ratio compared to untreated EPCs (0.50 ± 0.07, p=0.02) – an effect lost when CatB was antagonized by CA074 co–culture (0.77 ± 0.11, p=0.03 vs. EPC+GSKi).
Increased basal GSK3β activity contributes to accelerated EPC apoptosis and cellular senescence in patients with DM. Upregulation of CatB by small molecule antagonism of GSK3β reduces apoptosis and enhances cell based therapy in an arterial mechanical injury model.
Poster Sessions, Expo North
Sunday, March 10, 2013, 3:45 p.m.–4:30 p.m.
Session Title: Cell Therapy and Angiogenesis
Abstract Category: 42. TCT@ACC–i2: Cell Therapy & Angiogenesis
Presentation Number: 2110–231
- 2013 American College of Cardiology Foundation