Abstract 455: The Roles of VEGF Receptors in Human Cardiac Progenitor Cell Contribution to New Vascular Formation

Abstract

Background: Human cardiac progenitor cells (CPCs) have been shown to play a valuable role in myocardial tissue maintenance, including their ability to develop into endothelial and vascular smooth muscle cells. The vascular endothelial growth factor (VEGF) ligand family has been identified as essential for angiogenesis. VEGF receptors (VEGFRs) comprise three main subtypes, VEGFRs1,2,3; our prior data identified CPC expression of both VEGFRs and of pro-angiogenic secreted growth factors. Hypothesis: Human CPCs utilise VEGFR signalling to potentiate CPC-driven angiogenesis, directly via CPC differentiation and indirectly via secrotome. Methods: Human adult myocardial tissue samples were collected during cardiac surgery and c-Kit-positive (c-Kit + ), CD45-negative (CD45 - ) CPCs isolated by immunomagnetic bead sorting, with five CPC lines generated from individual-donor samples. The c-Kit + / CD45 - CPC population was then characterised by clonogenicity assay, immunocytochemistry (ICC), and real-time RT-qPCR. Human CPC lines were FACS sorted into 3 lineage groups: endothelial (CD31 + ), smooth muscle (CD91 + /CD140b + /CD31 - ) and uncommitted (CD91 - /CD140b - /CD31 - ). Expression of VEGFRs and markers (SDF1; TGF-β) in CPC sub-populations was assessed by: qPCR; Western blot; ICC. Impact on signal transduction by VEGF-A stimulation was identified by Western blot and ICC. Results: Human CPCs were sorted into populations of: endothelial linage CD31 + (1.26% of total cells), smooth muscle lineage CD91 + /CD140b + /CD31 - (13.77%) and CD91 - /CD140b - /CD31 - (31.28%) cells. Analysis of gene expression identified VEGFRs 1, 2 and 3 in all three sub-populations, but only VEGFR1 expression was confirmed at protein level, seen in all three sub-populations. High expression levels of growth factors secreted by CPCs (SDF, TGF-β, VEGFs, FGF-2) were identified in human CPCs, also in all three sub-populations. Conclusion: Human CPC lines were isolated and analysed in bulk and sub-populations, identifying VEGFR1 expression at both gene and protein levels, but not VEGFR2 and VEGFR3. Our further work will identify signalling pathways in human CPCs linked to VEGF-A stimulation, along with the impact of VEFG-A stimulation on CPC secretome and linked angiogenic potential.