Introduction. Human cardiac progenitor cells (CPCs) have been identified as an endogenous myocardial cell population, capable of aiding myocardial tissue maintenance, including the potential to differentiate into endothelial and vascular smooth muscle cells. Vascular endothelial growth factor (VEGF) ligands have been identified playing critical roles in angiogenesis. VEGF receptors have three major subtypes (VEGFRs 1, 2 and 3); previous data identified CPC VEGFR expression and pro-angiogenic growth factor secretion.

Aims. This study examined whether human CPCs utilise VEGFR signalling to potentiate angiogenesis, both directly by CPC differentiation and indirectly through pro-angiogenic paracrine signalling.

Methods. Human adult myocardial tissue was collected during cardiac surgery and c-Kit-positive (c-Kit⁺), CD45-negative (CD45^–) CPCs were isolated by immunomagnetic bead sorting; five unique CPC lines were generated from individual donor samples. c-Kit⁺/ CD45^– CPCs were then characterised in vitro by clonogenicity assays, immunocytochemistry and RT-qPCR. Human CPC lines were sorted by FACS into three lineages: endothelial (CD31⁺), smooth muscle (CD91⁺/CD140b⁺/CD31^–) or uncommitted (CD91^–/CD140b^–/CD31^–) groups. VEGFR and marker (SDF1; TGF-β) expression in CPC sub-populations were quantified (qPCR; Western blot; immunocytochemistry). VEGF-A stimulation and effects on signal transduction were examined (Western blot; immunocytochemistry). Statistics: ANOVA plus Tukey’s test, significance: p<0.05; data are mean±SEM. This work was approved by the Faculty Research Ethics Committee at the University of Leeds and by the Wales Research Ethics Committee for NHS clinical tissue samples (NREC 17/WA/0314).

Results. Human CPC lines were isolated (n=5) and the stem cell phenotype confirmed by analyses of differentiation (immunocytochemistry) and self-renewal (clonogenicity: 50-90% of single-cell clones generated clonal colonies; RT-qPCR: ‘stemness’ genes confirmed in all 5 lines, n=3 technical replicates). CPCs from a representative line were then FACS-sorted into populations, separated by markers of: endothelial linage CD31⁺ (1.99% of total cells), smooth muscle lineage CD91⁺/CD140b⁺/CD31^– (13.77%) and CD91^–/CD140b^–/CD31^– (31.28%) cells. Gene expression analyses identified mRNA for VEGFRs 1, 2 and 3 in all sub-populations (n=3). However only VEGFR1 protein expression was confirmed in all three sub-populations, not VEGFR2 or VEGFR3 (n=3). For growth factors previously identified as being secreted by CPCs (SDF, TGF-β, VEGFs, FGF-2), we identified high gene expression levels in human CPCs, with expression seen in all sub-populations (n=3). Application of CPC secretome, from each sub-population, to human endothelial cells on Matrigel in vitro did not show a clear increase in tube junction formation or tube segment length (n=6).

Conclusions. We isolated and analysed human CPCs, in bulk lines and sub-populations, identifying VEGFR1 gene and protein expression, but not VEGFR2 nor VEGFR3. We are building on this work to identify signalling pathways in human CPCs linked to VEGF-A stimulation, and further assessing impacts of VEFG-A stimulation on CPC secretome and associated potential to drive angiogenesis.