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.

Endothelial Progenitor Cells (EPCs) represent a novel therapeutic approach for the revascularisation of ischaemic tissues. However clinical outcomes have been contradictory due to the lack of a uniform cell definition, resulting in a diverse range of EPC types being utilised for therapeutic applications. This study investigates the phenotype and potential role in retinal angiogenesis of a specific subset known as early EPCs (eEPCs). eEPCs isolated from human peripheral blood were characterised using immunophenotyping and transcriptomics. Tubulogenic potential was determined using a co-culture system with Retinal microvascular endothelial cells (RMECs). Cytokine release was assessed by protein array. IL-8 activity was blocked using the CXCR2 inhibitor SB225002. In vivo angiogenic potential was tested using a murine model of ischaemic retinopathy. Our data indicates that eEPCs are not endothelial progenitors, but represent alternative activated M2 macrophages. These cells were therefore named myeloid angiogenic cells (MACs). MACs significantly induced tube formation in co-cultures (p<0.05) without directly incorporating into a microvascular network. IL-8 was identified in MAC-conditioned media as a key paracrine factor. Interestingly, blockade of IL-8, but not VEGF, prevented MAC-induced angiogenic function. In vivo, MACs significantly reduced avascular areas when compared to controls (p<0.01), despite never incorporating into the resident vasculature. In conclusion, MACs act as alternative M2 macrophages with pro-angiogenic, anti-inflammatory and pro-tissue repair properties in the ischaemic retina. This role in reparative retinal angiogenesis and reversal of ischaemia has been linked to paracrine release of cytokines such as IL-8.

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Purpose Intravitreal neovascularisation (NV) is a serious complication of diabetic retinopathy and occurs in response to tissue ischaemia and VEGF stimulation. eNOS derived NO plays an important role in facilitating VEGF function during NV. Efficient NO production is dependent upon the cofactor tetrahydrobiopterin (BH4) whose deficiency plays a pivotal role in the reduced NO bioavailability observed in diabetic vascular disease. Its role, however, in retinal angiogenesis is still poorly understood. Here, using a murine model partially deficient in BH4 (hph-1), we investigated the role of BH4 in ischaemic retinopathy following oxygen induced retinopathy (OIR). Methods Aortic rings from adult mice or retinas from postnatal day 7 (P7) animals were used to estimate endothelial branch formation and normal vascular coverage. For OIR, animals were exposed to 75% oxygen for 5 days from P7 to P12 and returned to room air. Eyes were collected at various time points between P12 and P17 (maximal neovascular response) and vascular growth quantified by B simplicifolia isolectin staining of retinal flat mounts. Avascular, normal vascular and neovascular areas were quantified using image analysis software. BH4 deficiency was confirmed by HPLC analysis. Results Vascular growth in the absence of BH4 was significantly reduced in aortic explants derived from hph-1 animals which was reversible upon BH 4 supplementation. Retinal vascular development was unaffected in the hph-1 group. However, post OIR, they demonstrated reduced retinal revascularisation and neovascularisation at P17. Conclusions Taken together, our results show that BH4 deficiency attenuates angiogenic drive and neovascularisation in a model of retinal pathology.Heart November 2012 Vol 98 Suppl S5 A9 ABSTRACTS on 9 May 2018 by guest. Protected by copyright.