Myeloid Angiogenic Cells Research Articles

The Vasoreparative Function of Myeloid Angiogenic Cells Is Impaired in Diabetes Through the Induction of IL1β.

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 IL1β mRNA and protein expression. Inhibition of IL1β abrogated the antiangiogenic effect induced by high‐d‐glucose. IL1β 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‐1β. Stem Cells 2018;36:834–843

Dysfunction and Therapeutic Potential of Endothelial Progenitor Cells in Diabetes Mellitus.

Diabetes mellitus (DM) is a chronic, multifactorial metabolic disease whereby insulin deficiency or resistance results in hyperglycemia. Endothelial cells (ECs) form the innermost layer of the blood vessel and produce and release a variety of vasoactive substances and growth factors to regulate vascular homeostasis and angiogenesis. Hyperglycemia and insulin resistance can cause endothelial dysfunction, leading to vascular complications such as coronary artery disease, peripheral arterial disease, diabetic nephropathy, neuropathy and retinopathy. The detrimental effect exerted on ECs by hyperglycemia and insulin resistance underlines the importance of reparatory mechanisms in DM. Endothelial progenitor cells (EPCs), derived from bone marrow, have been recognized as endogenous cells involved in endothelial repair and new blood vessel formation. Initially isolated from a subset of circulating CD34+ mononuclear cells, EPCs were found to possess the ability to differentiate into ECs when cultured in vitro and incorporate into newly formed vessels upon transplantation in animal models of ischemia. Due to the low frequency of CD34+ cells in circulation, the vast majority of studies investigating EPC actions have used cells that are generated through the culture of peripheral blood mononuclear cells (PBMNCs) for 4 - 7 days in endothelial selective medium. These cells, mainly of myeloid hematopoietic cell origin, were termed “Early EPCs,” of which, few expressed stem/progenitor-cell markers. Therefore, early EPCs were also termed “myeloid angiogenic cells” (MACs). When PBMNCs are cultured for over 2 weeks, early EPCs gradually diminish while so-called late EPCs appear. Late EPCs share phenotypic features with mature ECs and are therefore also termed blood-derived ECs; they will not be addressed in this review. MAC dysfunction has been observed in a variety of disease conditions including DM. In this article we review the activities and therapeutic potential of MACs in DM. We will interchangeably use “EPCs” and “MACs” to refer to the cells procured by culture of PBMNCs in EC selective medium for approximately 7 days.

Bioavailability of Bergamot (Citrus bergamia) Flavanones and Biological Activity of Their Circulating Metabolites in Human Pro-Angiogenic Cells

Myeloid angiogenic cells (MACs) play a key role in endothelial repairing processes and functionality but their activity may be impaired by the lipotoxic effects of some molecules like stearic acid (SA). Among the dietary components potentially able to modulate endothelial function in vivo, (poly)phenolic compounds represent serious candidates. Here, we apply a comprehensive multidisciplinary approach to shed light on the prospects of Bergamot (Citrus bergamia), a citrus fruit rich in flavanones and other phenolic compounds, in the framework of lipotoxicity-induced MACs impairment. The flavanone profile of bergamot juice was characterized and 16 compounds were identified, with a new 3-hydroxy-3-methylglutaryl (HMG) flavanone, isosakuranetin-7-O-neohesperidoside-6″-O-HMG, described for the first time. Then, a pilot bioavailability study was conducted in healthy volunteers to assess the circulating flavanone metabolites in plasma and urine after consumption of bergamot juice. Up to 12 flavanone phase II conjugates (sulfates and glucuronides of hesperetin, naringenin and eriodyctiol) were detected and quantified. Finally, the effect of some of the metabolites identified in vivo, namely hesperetin-7-O-glucuronide, hesperetin-3′-O-glucuronide, naringenin-7-O-glucuronide and naringenin-4′-O-glucuronide, was tested, at physiological concentrations, on gene expression of inflammatory markers and apoptosis in MACs exposed to SA. Under these conditions, naringenin-4′-O-glucuronide and hesperetin-7-O-glucuronide were able to modulate inflammation, while no flavanone glucuronide was effective in curbing stearate-induced lipoapoptosis. These results demonstrate that some flavanone metabolites, derived from the in vivo transformation of bergamot juice phenolics in humans, may mitigate stearate-induced inflammation in MACs.

Insulinlike Growth Factor-Binding Protein-1 Improves Vascular Endothelial Repair in Male Mice in the Setting of Insulin Resistance.

Insulin resistance is associated with impaired endothelial regeneration in response to mechanical injury. We recently demonstrated that insulinlike growth factor-binding protein-1 (IGFBP1) ameliorated insulin resistance and increased nitric oxide generation in the endothelium. In this study, we hypothesized that IGFBP1 would improve endothelial regeneration and restore endothelial reparative functions in the setting of insulin resistance. In male mice heterozygous for deletion of insulin receptors, endothelial regeneration after femoral artery wire injury was enhanced by transgenic expression of human IGFBP1 (hIGFBP1). This was not explained by altered abundance of circulating myeloid angiogenic cells. Incubation of human endothelial cells with hIGFBP1 increased integrin expression and enhanced their ability to adhere to and repopulate denuded human saphenous vein ex vivo. In vitro, induction of insulin resistance by tumor necrosis factor α (TNFα) significantly inhibited endothelial cell migration and proliferation. Coincubation with hIGFBP1 restored endothelial migratory and proliferative capacity. At the molecular level, hIGFBP1 induced phosphorylation of focal adhesion kinase, activated RhoA and modulated TNFα-induced actin fiber anisotropy. Collectively, the effects of hIGFBP1 on endothelial cell responses and acceleration of endothelial regeneration in mice indicate that manipulating IGFBP1 could be exploited as a putative strategy to improve endothelial repair in the setting of insulin resistance.

Endothelial Progenitors: A Consensus Statement on Nomenclature.

SummaryEndothelial progenitor cell (EPC) nomenclature remains ambiguous and there is a general lack of concordance in the stem cell field with many distinct cell subtypes continually grouped under the term “EPC.” It would be highly advantageous to agree on standards to confirm an endothelial progenitor phenotype and this should include detailed immunophenotyping, potency assays, and clear separation from hematopoietic angiogenic cells which are not endothelial progenitors. In this review, we seek to discourage the indiscriminate use of “EPCs,” and instead propose precise terminology based on defining cellular phenotype and function. Endothelial colony forming cells and myeloid angiogenic cells are examples of two distinct and well‐defined cell types that have been considered EPCs because they both promote vascular repair, albeit by completely different mechanisms of action. It is acknowledged that scientific nomenclature should be a dynamic process driven by technological and conceptual advances; ergo the ongoing “EPC” nomenclature ought not to be permanent and should become more precise in the light of strong scientific evidence. This is especially important as these cells become recognized for their role in vascular repair in health and disease and, in some cases, progress toward use in cell therapy. Stem Cells Translational Medicine 2017;6:1316–1320

Vitamin D improves endothelial dysfunction and restores myeloid angiogenic cell function via reduced CXCL-10 expression in systemic lupus erythematosus.

Patients with systemic lupus erythematosus (SLE) have accelerated cardiovascular disease and dysfunctional endothelial repair mechanisms. Myeloid angiogenic cells (MACs), derived from circulating monocytes, augment vascular repair by paracrine secretion of pro-angiogenic factors. We observed that SLE MACs are dysfunctional and secrete pro-inflammatory cytokines. We also found that the vitamin D receptor was transiently expressed during MAC differentiation and that in vitro, calcitriol increased differentiation of monocytes into MACs in both SLE and in a model using the prototypic SLE cytokine, interferon-alpha. The active form of vitamin D (calcitriol) restored the SLE MAC phenotype towards that of healthy subjects with reduced IL-6 secretion, and normalised surface marker expression. Calcitriol also augmented the angiogenic capacity of MACs via the down-regulation of CXCL-10. In SLE patients treated with cholecalciferol for 12 weeks, the improvement in endothelial function correlated with increase in serum 25(OH)D concentrations independently of disease activity. We also show that MACs were able to positively modulate eNOS expression in human endothelial cells in vitro, an effect further enhanced by calcitriol treatment of SLE MACs. The results demonstrate that vitamin D can positively modify endothelial repair mechanisms and thus endothelial function in a population with significant cardiovascular risk.

Angiogenic Role of MMP-2/9 Expressed on the Cell Surface of Early Endothelial Progenitor Cells/Myeloid Angiogenic Cells.

Since the introduction of angiogenic cell therapy using early endothelial progenitor cells (EPCs), myeloid angiogenic cells (MACs) have been expected to be useful in treating ischemic diseases. In order to elucidate the angiogenic properties of MACs/EPCs, we clarified the characteristics of MACs as compared to M2 macrophages (Mϕs). Comparison of the gene expression profiles of MACs and late EPCs revealed that MACs expressed greater amounts of metalloproteinase (MMP)-9. It should be noted that the profile of MMP-2/9 expression on the cell surface of MACs was similar to that of M2 Mϕs, and that cell surface MMP-2/9 might be an active form based on molecular size. In addition, the invasion of MACs was prohibited not only by MMP-2/9 inhibitor, but also by the hyaluronidase treatment that caused the down-regulation of MMP-9 on the cell surface of MACs and inhibited their invasion activity. These results indicate that cell surface MMP-2/9 plays an important role in the high invasion ability of MACs. The conditioned medium of both MACs and M2 Mϕs stimulated tube formation of endothelial cells in vitro. MACs caused an increase in vessel formation in in vivo models through the production of IL-8. We propose that the role of MACs with cell surfaces expressing MMP-2/9 is rapidly invading ischemic tissue.

Role of vitamin D in endothelial function and endothelial repair in clinically stable systemic lupus erythematosus

BackgroundPatients with systemic lupus erythematosus (SLE) have endothelial dysfunction and increased risk of cardiovascular disease. Endothelium-dependent dilatation (ED) is abnormal in patients with SLE, and endothelial repair mechanisms are also impaired. Myeloid angiogenic cells (MACs) promote angiogenesis to restore damaged vessels. Vitamin D deficiency is associated with cardiovascular disease in the general population and is prevalent in SLE. We aimed to assess the effect of vitamin D on endothelial repair and function. MethodsVitamin D deficient (<20 ng/mL) patients with SLE were treated with cholecalciferol by their physician. Vitamin D replete patients (>30 ng/mL) and healthy controls (>20 ng/mL) were also recruited. Endothelial function was determined by the ratio of ED to independent dilatation (EI). MACs from patients were cultured with and without 10 nM calcitriol, and function determined by migration and angiogenesis assays. Endothelial nitric oxide synthase (eNOS) expression was studied in human aortic endothelial cells treated with tumour necrosis factor α (TNFα) and MAC-conditioned media. FindingsWe studied 22 vitamin D deficient and 18 replete patients. Vitamin D deficient patients had an increased number of MACs compared with controls (p=0·04) but impaired migratory capacity (p=0·001) and reduced angiogenic capacity, although this was not statistically significant (p=0·13). Media from calcitriol-treated MACs significantly increased angiogenesis compared with untreated MACs (p=0·01). Calcitriol reduced IP-10 expression by MACs (p<0·0006), and blockade of IP-10 restored the angiogenic capacity of MACs from patients with SLE. In cholecalciferol-treated patients, change in 25-hydroxyvitamin D was strongly correlated with change in ED:EI (r=0·650, p=0·006) after adjustment for age (odds ratio 1·12, 95% CI 1·02–1·24; p=0·02). Media from calcitriol-treated MACs more strongly attenuated TNFα-mediated downregulation of eNOS in human aortic endothelial cells than did untreated MACs from patients with SLE (p=0·01). InterpretationIn this small experimental study, calcitriol improved endothelial function in patients with stable SLE. This improvement was associated with an increase in MAC number and function. The improved angiogenic capacity in MACs might be mediated via downregulation of IP-10 and changes in ED:EI by MAC regulation of eNOS in endothelial cells. The findings suggest that vitamin D could be a novel therapy to reduce cardiovascular disease in this patient group. FundingNorth West England Medical Research Council Fellowship Scheme in Clinical Pharmacology and Therapeutics (funding from UK Medical Research Council (grant number G1000417/94909), ICON, Astra Zeneca, GlaxoSmithKline, Medicines Evaluation Unit).

Role of vitamin D in endothelial repair mechanisms in systemic lupus erythematosus

BackgroundPatients with systemic lupus erythematosus (SLE) have an increased prevalence of cardiovascular disease (CVD). Vitamin D deficiency is common in SLE and is an independent risk factor for CVD in the general population. We have previously shown that low vitamin D is associated with arterial stiffness in patients with SLE. Myeloid angiogenic cells (MACs) have an important role in endothelial repair in animal models. Preliminary studies have shown impaired function of these cells in SLE. Myeloid cells respond to vitamin D in vitro and express functional vitamin D receptors. We aimed to assess whether vitamin D has beneficial effects on MAC function and thus endothelial repair. MethodsPeripheral blood mononuclear cells (PBMCs) from vitamin D deficient (<20 ng/mL) patients with SLE, or vitamin D replete (>20 ng/mL) healthy controls, were cultured for 7 days on fibronectin in endothelial growth media with or without 1,25(OH)2D3 (0·1–100 nM). For survival studies, the number of cells able to take up LDL was counted in random fields. Surface CD marker expression (CD14, CD68, CD86, CCR7, CD206) in MACs was determined with real-time quantitative PCR. Angiogenic factor secretion was measured with a Bio-Plex human angiogenesis suspension array (Bio-Rad Laboratories, CA, USA). Angiogenic function of MACs was determined with human aortic endothelial cells in a Matrigel angiogenesis model. Data were analysed using linear regression, paired t tests, and one-way ANOVA where appropriate. FindingsMACs expressed high levels of CD206, and low CCR7, compared with PBMCs, consistent with an M2 macrophage phenotype. Phagocytosis was demonstrated by MAC uptake of fluorescein isothiocyanate-labelled beads. MAC secreted interleukin 8 (mean 13·9 ug/mL, SD 20·1), vascular endothelial growth factor (37·2 pg/mL, 26·1), hepatocyte growth factor (1414 pg/mL, 568), leptin (209 pg/mL, 144), and platelet-derived growth factor (564 pg/mL, 365) but not granulocyte-colony stimulating factor or angiopoetin. 1,25(OH)2D3 (0·1–100 nM) increased the number of MACs from patients with SLE after 8 days in those with a low number (<150 per field) at baseline (p=0·037) in a dose-dependent manner. This effect was replicated in healthy MACs treated with 0·1 ng/mL interferon-alpha2b (p≤0·001). Expression of all macrophage markers was reduced by 1,25(OH)2D3 (10 nM) but the M2 marker CD206 was preferentially reduced compared with M1 markers CCR7 (p=0·008) and CD86 (p=0·043). Conditioned media from vitamin D (10 nM) treated MACs increased endothelial tubule network density in the angiogenesis model (mean relative density 1·43 [SD 0·12] vs 1·66 [0·15], p=0·012) whereas vitamin D alone did not (0·96 [0·10], p=0·582). Vitamin D did not, however, increase the concentration of any of the angiogenic factors measured in the Bio-Plex array. InterpretationResults from this study suggest that MACs are a population of M2 macrophages with angiogenic capacity in vitro, and that vitamin D both increases the number of MACs and changes the surface maker profile in SLE. The angiogenic capacity of MACs was increased by vitamin D ex vivo, but this was not due to changes in expression of common angiogenic factors. Further work will identify the mechanism by which vitamin D might augment endothelial repair in patients with SLE. FundingUK Medical Research Council.

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.

Characterisation and therapeutic potential of endothelial progenitor cells

Endothelial progenitor cells (EPCs) may have great potential for use as a cellular therapy for promoting vascular repair of ischaemic tissues, such as complex wounds. The term EPC, however, has been used to describe a diverse variety of cells. This ambiguity has led to conflicting results when comparing results of EPC studies from different research laboratories. Recently, there is consensus that when selective in-vitro cell culture techniques are used, two distinct EPC subpopulations are generated—namely, myeloid angiogenic cells (MACs) and outgrowth endothelial cells (OECs). This study provides a full phenotypic and functional characterisation of MACs and OECs and compares their potential therapeutic role in wound healing.EPC subtypes were isolated from human peripheral blood and umbilical cord blood. MACs appeared early in culture (∼7 days) as elongated cells with multiple fine dendritic processes and low proliferative potential. OEC colonies appeared later in culture (3–5 weeks), and formed a cobblestone-shaped monolayer. Genome-wide transcriptomics demonstrated that MACs and OECs belong to the haemopoietic and endothelial lineages, respectively. MACs cultured in vitro were further analysed, and were found to be phenotypically very similar to M2 alternative activated macrophages.Analysis of functional characteristics showed that MACs have low proliferative potential. However, OECs possess high proliferative potential, enabling them to be single-cell cloned, with an average 60 population doublings in 80 days for cord-derived OECs.Using in-vitro angiogenesis assays, OECs demonstrated de-novo tubulogenesis capacity, and were capable of interacting with mature endothelial cells through adherens and tight junctions. MACs did not form tubes and did not differentiate into endothelial cells, but significantly promoted tubulogenesis of mature endothelial cells by releasing paracrine factors such as interleukin 8 and monocyte chemoattractant protein 1. FundingNorthern Ireland Medical and Dental Training Agency.

35 Paracrine release of interleukin 8 (IL-8) by myeloid angiogenic cells promotes angiogenesis in the ischaemic retina

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,...

Myeloid Angiogenic Cells Act as Alternative M2 Macrophages and Modulate Angiogenesis through Interleukin-8

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.