Current concepts on endothelial stem cells definition, location, and markers.

Ischemic vascular disease is a major cause of mortality and morbidity worldwide, and regeneration of blood vessels in perfusion-deficient tissues is a worthwhile therapeutic goal. The idea of delivering endothelial stem/progenitor cells to repair damaged vasculature, reperfuse hypoxic tissue, prevent cell death, and consequently diminish tissue inflammation and fibrosis has a strong scientific basis and clinical value. Various labs have proposed endothelial stem/progenitor cell candidates. This has created confusion, as there are profound differences between these cell definitions based on isolation methodology, characterization, and reparative biology. Here, a stricter definition based on stem cell biology principles is proposed. Although preclinical studies have often been promising, results from clinical trials have been highly contradictory and served to highlight multiple challenges associated with disappointing therapeutic benefit. This article reviews recent accomplishments in the field and discusses current difficulties when developing endothelial stem cell therapies. Emerging evidence that disputes the classic view of the bone marrow as the source for these cells and supports the vascular wall as the niche for these tissue-resident endothelial stem cells is considered. In addition, novel markers to identify endothelial stem cells, including CD157, EPCR, and CD31low VEGFR2low IL33+ Sox9+ , are described.

miR-130a activates the VEGFR2/STAT3/HIF1α axis to potentiate the vasoregenerative capacity of endothelial colony-forming cells in hypoxia.

Hypoxia modulates reparative angiogenesis, which is a tightly regulated pathophysiological process. MicroRNAs (miRNAs) are important regulators of gene expression in hypoxia and angiogenesis. However, we do not yet have a clear understanding of how hypoxia-induced miRNAs fine-tune vasoreparative processes. Here, we identify miR-130a as a mediator of the hypoxic response in human primary endothelial colony-forming cells (ECFCs), a well-characterized subtype of endothelial progenitors. Under hypoxic conditions of 1% O2, miR-130a gain-of-function enhances ECFC pro-angiogenic capacity in vitro and potentiates their vasoreparative properties in vivo. Mechanistically, miR-130a orchestrates upregulation of VEGFR2, activation of STAT3, and accumulation of HIF1α via translational inhibition of Ddx6. These findings unveil a new role for miR-130a in hypoxia, whereby it activates the VEGFR2/STAT3/HIF1α axis to enhance the vasoregenerative capacity of ECFCs.

The Vasoreparative Potential of Endothelial Colony Forming Cells: A Journey Through Pre-clinical Studies.

For over a decade various cell populations have been investigated for their vasoreparative potential. Cells with the capacity to promote blood vessel regeneration are commonly known as endothelial progenitor cells (EPCs); although such a definition is currently considered too simple for the complexity of cell populations involved in the reparative angiogenic process. A subset of EPCs called endothelial colony forming cells (ECFCs) have emerged as a suitable candidate for cytotherapy, primarily due to their clonogenic progenitor characteristics, unequivocal endothelial phenotype, and inherent ability to promote vasculogenesis. ECFCs can be readily isolated from human peripheral and cord blood, expanded ex vivo and used to revascularize ischemic tissues. These cells have demonstrated efficacy in several in vivo preclinical models such as the ischemic heart, retina, brain, limb, lung and kidney. This review will summarize the current pre-clinical evidence for ECFC cytotherapy and discuss their potential for clinical application.

The Vasoreparative Function of Myeloid Angiogenic Cells Is Impaired in Diabetes Through the Induction of IL1ß.

Myeloid angiogenic cells (MACs) promote revascularization through the paracrine release of angiogenic factors and have been harnessed as therapeutic cells for many ischemic diseases. However, their proangiogenic properties have been suggested to be diminished in diabetes. This study investigates how the diabetic milieu affects the immunophenotype and function of MACs. Both MACs isolated from diabetic conditions and healthy cells exposed to a diabetic environment were used to determine the potential of MACs as a cell therapy for diabetic-related ischemia. MACs were isolated from human peripheral blood and characterized alongside proinflammatory macrophages M (LPS + IFNγ) and proangiogenic macrophages M (IL4). Functional changes in MACs in response to high-d-glucose were assessed using an in vitro 3D-tubulogenesis assay. Phenotypic changes were determined by gene and protein expression analysis. Additionally, MACs from type 1 diabetic (T1D) patients and corresponding controls were isolated and characterized. Our evidence demonstrates MACs identity as a distinct macrophage subtype that shares M2 proangiogenic characteristics, but can be distinguished by CD163hi expression. High-d-glucose treatment significantly reduced MACs proangiogenic capacity, which was associated with a significant increase in IL1ß mRNA and protein expression. Inhibition of IL1ß abrogated the antiangiogenic effect induced by high-d-glucose. IL1ß was also significantly upregulated in MACs isolated from T1D patients with microvascular complications compared to T1D patients without microvascular complications or nondiabetic volunteers. This study demonstrates that Type 1 diabetes and diabetic-like conditions impair the proangiogenic and regenerative capacity of MACs, and this response is mediated by IL-1ß. Stem Cells 2018;36:834-843.

Preclinical Evaluation and Optimization of a Cell Therapy Using Human Cord Blood-Derived Endothelial Colony-Forming Cells for Ischemic Retinopathies.

Cell therapy using endothelial progenitors holds promise for vascular repair in ischemic retinopathies. Using a well-defined subpopulation of human cord blood-derived endothelial progenitors known as endothelial colony-forming cells (ECFCs), we have evaluated essential requirements for further development of this cell therapy targeting the ischemic retina, including dose response, delivery route, and toxicity. First, to evaluate therapeutic efficacy relating to cell dose, ECFCs were injected into the vitreous of mice with oxygen-induced retinopathy. Using angiography and histology, we found that intravitreal delivery of low dose (1 × 103 ) ECFCs was as effective as higher cell doses (1 × 104 , 1 × 105 ) in promoting vascular repair. Second, injection into the common carotid artery was tested as an alternative, systemic delivery route. Intracarotid ECFC delivery conferred therapeutic benefit which was comparable to intravitreal delivery using the same ECFC dose (1 × 105 ), although there were fewer human cells observed in the retinal vasculature following systemic delivery. Third, cell immunogenicity was evaluated by injecting ECFCs into the vitreous of healthy adult mice. Assessment of murine ocular tissues identified injected cells in the vitreous, while demonstrating integrity of the host retina. In addition, ECFCs did not invade into the retina, but remained in the vitreous, where they eventually underwent cell death within 3 days of delivery without evoking an inflammatory response. Human specific Alu sequences were not found in healthy mouse retinas after 3 days of ECFC delivery. These findings provide supportive preclinical evidence for the development of ECFCs as an efficacious cell product for ischemic retinopathies. Stem Cells Translational Medicine 2018;7:59-67.

Endothelial cell-derived pentraxin 3 limits the vasoreparative therapeutic potential of circulating angiogenic cells.

AIMS: Circulating angiogenic cells (CACs) promote revascularization of ischaemic tissues although their underlying mechanism of action and the consequences of delivering varying number of these cells for therapy remain unknown. This study investigates molecular mechanisms underpinning CAC modulation of blood vessel formation. METHODS AND RESULTS: CACs at low (2 × 105 cells/mL) and mid (2 × 106 cells/mL) cellular densities significantly enhanced endothelial cell tube formation in vitro, while high density (HD) CACs (2 × 107 cells/mL) significantly inhibited this angiogenic process. In vivo, Matrigel-based angiogenesis assays confirmed mid-density CACs as pro-angiogenic and HD CACs as anti-angiogenic. Secretome characterization of CAC-EC conditioned media identified pentraxin 3 (PTX3) as only present in the HD CAC-EC co-culture. Recombinant PTX3 inhibited endothelial tube formation in vitro and in vivo. Importantly, our data revealed that the anti-angiogenic effect observed in HD CAC-EC co-cultures was significantly abrogated when PTX3 bioactivity was blocked using neutralizing antibodies or PTX3 siRNA in endothelial cells. We show evidence for an endothelial source of PTX3, triggered by exposure to HD CACs. In addition, we confirmed that PTX3 inhibits fibroblast growth factor (FGF) 2-mediated angiogenesis, and that the PTX3 N-terminus, containing the FGF-binding site, is responsible for such anti-angiogenic effects. CONCLUSION: Endothelium, when exposed to HD CACs, releases PTX3 which markedly impairs the vascular regenerative response in an autocrine manner. Therefore, CAC density and accompanying release of angiocrine PTX3 are critical considerations when using these cells as a cell therapy for ischaemic disease.

The role of immune-related myeloid cells in angiogenesis.

Macrophage function is not restricted to the innate and adaptive immune responses, but also includes host defence, wound healing, angiogenesis and homeostatic processes. Within the spectrum of macrophage activation there are two extremes: M1 classically activated macrophages which have a pro-inflammatory phenotype, and M2 alternatively activated macrophages which are pro-angiogenic and anti-inflammatory. An important property of macrophages is their plasticity to switch from one phenotype to the other and they can be defined in their polarisation state at any point between the two extremes. In order to determine what stage of activation macrophages are in, it is essential to profile various phenotypic markers for their identification. This review describes the angiogenic role for myeloid cells: circulating monocytes, Tie-2 expressing monocytes (TEMs), myeloid-derived suppressor cells (MDSCs), tumour associated macrophages (TAMs), and neutrophils. Each cell type is discussed by phenotype, roles within angiogenesis and possible targets as a cell therapy. In addition, we also refer to our own research on myeloid angiogenic cells (MACs), outlining their ability to induce angiogenesis and their similarities to alternatively activated M2 macrophages. MACs significantly contribute to vascular repair through paracrine mechanisms as they lack the capacity to differentiate into endothelial cells. Since MACs also retain plasticity, phenotypic changes can occur according to disease states and the surrounding microenvironment. This pro-angiogenic potential of MACs could be harnessed as a novel cellular therapy for the treatment of ischaemic diseases, such as diabetic retinopathy, hind limb ischaemia and myocardial infarction; however, caution needs to be taken when MACs are delivered into an inflammatory milieu.

Ex vivo expansion of human outgrowth endothelial cells leads to IL-8-mediated replicative senescence and impaired vasoreparative function.

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, ß-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 transcriptome-proteome 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.