Endothelial progenitor cells (EPCs) promote angiogenesis, and clinical trials have shown such cell therapy to be feasible for treating ischemic disease. However, clinical outcomes have been contradictory owing to the diverse range of EPC types used. We recently characterized two EPC subtypes, and identified outgrowth endothelial cells as the only EPC type with true progenitor and endothelial characteristics. By contrast, myeloid angiogenic cells (MACs) were shown to be monocytic cells without endothelial characteristics despite being widely described as "EPCs." In the current study we demonstrated that although MACs do not become endothelial cells or directly incorporate into a microvascular network, they can significantly induce endothelial tube formation in vitro and vascular repair in vivo. MAC-derived interleukin-8 (IL-8) was identified as a key paracrine factor, and blockade of IL-8 but not vascular endothelial growth factor (VEGF) prevented MAC-induced angiogenesis. Extracellular IL-8 transactivates VEGFR2 and induces phosphorylation of extracellular signal-regulated kinases. Further transcriptomic and immunophenotypic analysis indicates that MACs represent alternative activated M2 macrophages. Our findings demonstrate an unequivocal role for MACs in angiogenesis, which is linked to paracrine release of cytokines such as IL-8. We also show, for the first time, the true identity of these cells as alternative M2 macrophages with proangiogenic, antiinflammatory and pro-tissue-repair properties.

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Harnessing outgrowth endothelial cells (OECs) for vasoreparative therapy and tissue engineering requires efficient ex vivo expansion. How such expansion impacts on OEC function is largely unknown. In this study, we show that OECs become permanently cell-cycle arrested after ex vivo expansion, which is associated with enlarged cell size, b-galactosidase activity, DNA damage, tumor suppressor pathway activation, and significant transcriptome changes. These senescence hallmarks were coupled with low telomerase activity and telomere shortening, indicating replicative senescence. OEC senescence limited their regenerative potential by impairing vasoreparative properties in vitro and in vivo. Integrated transcriptomeproteome analysis identified inflammatory signaling pathways as major mechanistic components of the OEC senescence program. In particular, IL8 was an important facilitator of this senescence; depletion of IL8 in OECs significantly extended ex vivo lifespan, delayed replicative senescence, and enhanced function. While the ability to expand OEC numbers prior to autologous or allogeneic therapy remains a useful property, their replicative senescence and associated impairment of vasorepair needs to be considered. This study also suggests that modulation of the senescence-associated secretory phenotype could be used to optimize OEC therapy.

Endothelial progenitor cells (EPCs) have great clinical value because they can be used as diagnostic biomarkers and as a cellular therapy for promoting vascular repair of ischaemic tissues. However, EPCs also have an additional research value in vascular disease modelling to interrogate human disease mechanisms. The term EPC is used to describe a diverse variety of cells, and we have identified a specific EPC subtype called outgrowth endothelial cell (OEC) as the best candidate for vascular disease modelling because of its high-proliferative potential and unambiguous endothelial commitment. OECs are isolated from human blood and can be exposed to pathologic conditions (forward approach) or be isolated from patients (reverse approach) in order to study vascular human disease. The use of OECs for modelling vascular disease will contribute greatly to improving our understanding of endothelial pathogenesis, which will potentially lead to the discovery of novel therapeutic strategies for vascular diseases.

Numerous studies suggest that endothelial progenitor cells (EPCs) re-vascularise ischaemic tissues and recent clinical trials have highlighted the feasibility, safety and potential therapeutic benefit of an EPC-based cytotherapy for myocardial infarction. However there is still discrepancy about the extent to which these cells incorporate into the vasculature and the level to which they restore functionality to damaged tissue. These controversies are caused by an imprecise EPC definition as many different cell populations are collectively referred to as EPCs. Our aim was to isolate a distinct EPC population named Outgrowth Endothelial Cells (OECs) and test them as a novel cell therapy for ischaemic retinopathy. OECs were isolated from adult human peripheral blood and grown on collagen substrate. They were characterised using immunophenotyping, and genome-wide transcriptomics. Angiogenic activity was assessed using multiple functional assays in vitro, and a mouse model of ischaemic retinopathy in vivo. Our data indicate that OECs possess an unequivocal endothelial phenotype and express progenitor cell markers. They have high proliferative capacity and clonogenic potential. Furthermore, OECs are able to closely interact with mature endothelial cells through adherens and tight junctions. OEC demonstrate de novo tubulogenic potential and fully incorporate into a mature vascular network. This is also demonstrated in vivo, where OECs directly incorporate into resident ischaemic vasculature, and significantly decreased avascular areas (p<0.001) when compared to vehicle-injected retinas. In conclusion, OECs are a distinct EPC sub-population that directly contributes to vascular repair of the ischaemic retina, and have great potential as an alternative treatment for ischaemic retinopathies.

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