Blood outgrowth endothelial cells in patients with severe ischemic cardiomyopathy maintain pro-angiogenic phenotype without senescence

Abstract

Purpose: Clinical translation of cardiovascular cell therapy with proven benefits in experimental models may be hampered by a dysfunctional progenitor cell phenotype in heart failure patients, as reported for bone marrow mononuclear cells. We compared blood outgrowth endothelial cells (BOECs) from patients with severe but stable ischemic heart disease (IHD) and healthy controls (CON). Methods: BOECs were derived from 37 IHD (LVEF≤45%) and 35 CON. Cell surface markers were analysed by flow cytometry to confirm BOEC-phenotype. In vitro functional analysis, including tube formation potential (on matrigel), senescence (SA-β-gal) and Dil-AcLDL uptake (flow cytometry) was performed between passage 3-5 and expression of angiogenic genes was studied using quantitative RT-PCR. Results: An average of 3.4±0.5 BOEC-colony forming units (CFUs)/100×106 mononuclear cells (MNCs) was obtained in IHD (age: 66±2y, LVEF: 31±2%, LVEDD: 56±1mm) vs 2.5±0.4 CFUs/100×106 MNCs in CON (age: 36±2y) (P=0.12). In patients, BOEC-outgrowth did not correlate with the extent of LV systolic dysfunction (R2=0.03) or LV remodeling (R2=0.05), and was not affected by cardiovascular risk factors. A mild correlation was observed between age and CFU number (R2=0.13, P=0.04). Endothelial lineage markers (CD309/CD31/CD146) were comparable in IHD and CON and panleukocyte marker CD45 was absent in all colonies. Progenitor marker CD133 was absent, while CD34 displayed important inter-sample variability and tended to be more expressed in CON (77±10%, n=9) vs IHD (54±8%, n=13) (P=0.06). Dil-AcLDL uptake was more pronounced in BOECs from IHD (98±1%, n=13) vs CON (94±2%, n=9) (P=0.008). In vitro tube formation, total tube length, and number of intersections were comparable in IHD and CON (n=13 vs 9, resp). The percentage senescent BOECs was similar in IHD and CON (7±2%, n=14 vs 8±3%, n=9). Finally, robust expression levels of PLGF, Angiopoietin-2, PDGF-β, FGF-2 and VEGF were comparable in IHD (n=15) and CON (n=9). Conclusions: BOECs can be successfully and consistently derived from patients with IHD and have a preserved angiogenic profile. Therefore, BOECs hold promise for clinical translation of autologous cell therapy in severe ischemic cardiomyopathy.