Angiogenic Murine Endothelial Progenitor Cells Are Derived From a Myeloid Bone Marrow Fraction and Can Be Identified by Endothelial NO Synthase Expression

Abstract

Objective-Endothelial progenitor cells (EPCs) contribute to postnatal neovascularization and are therefore of great interest for autologous cell therapies to treat ischemic vascular disease. However, the origin and functional properties of these EPCs are still in debate. Methods and Results-Here, ex vivo expanded murine EPCs were characterized in terms of phenotype, lineage potential, differentiation from bone marrow (BM) precursors, and their functional properties using endothelial NO synthase (eNOS)-green fluorescent protein transgenic mice. Despite high phenotypic overlap with macrophages and dendritic cells, EPCs displayed unique eNOS expression, endothelial lineage potential in colony assays, and angiogenic characteristics, but also immunologic properties such as interleukin-12p70 production and low levels of T-cell stimulation. The majority of EPCs developed from an immature, CD31 ϩ Ly6C ϩ myeloid progenitor fraction in the BM. Addition of myeloid growth factors such as macrophage-colony-stimulating factor (M-CSF) and granulocyte/ macrophage (GM)-CSF stimulated the expansion of spleen-derived EPCs but not BM-derived EPCs. Conclusion-The close relationship between EPCs and other myeloid lineages may add to the complexity of using them in cell therapy. Our mouse model could be a highly useful tool to characterize EPCs functionally and phenotypically, to explore the origin and optimize the isolation of EPC fractions for therapeutic neovascularization. [1][2][3] These cells have the potential to differentiate toward endothelial cells (ECs) and are therefore named endothelial progenitor cells (EPCs). Transplantation of EPCs has been shown to be effective in animal models for re-endothelialization 4,5 and adult neovascularization 6,7 as well as in human patient studies aimed to enhance myocardial regeneration after acute myocardial infarction. 7 Although EPCs are used in clinical trials, the exact phenotypic and lineage/ differentiation parameters of ex vivo-expanded EPCs are poorly defined, and it is not clear which cell populations will be most effective in repair studies. EPCs can be derived from CD34ϩ as well as CD34Ϫ or CD34 low cells and can be isolated and expanded ex vivo using BM aspirates and PB CD14ϩ mononuclear cell fractions. 8 -12 In many studies, EPCs are characterized by their adhesive spindle-like morphology, staining with the EC-binding lectin Ulex europaeus agglutinin (Ulex), and the capacity to endocytose acetylated low-density lipoprotein (acLDL). 2,11 Although this may generally suffice for EPC studies dealing with EPCs obtained from healthy animal models or humans, the different culture conditions and sources used may lead to a large heterogeneity and functionally suboptimal EPC populations. 13,14 It has even been suggested that transplantation of certain cell fractions may contribute to adverse side effects. 15 Clinical studies demonstrated that in patients experiencing diabetes and hypertension, the number of circulating EPCs is severely decreased, and the cells are dysfunctional. 16 -...

Hematopoietic MicroRNA-126 Protects against Renal Ischemia/Reperfusion Injury by Promoting Vascular Integrity

“…In addition, no substantial differences were found in absolute numbers of T cells, B cells, natural killer cells, plasmacytoid dendritic cells, neutrophils, or eosinophils. Because circulating myeloid cells are known to display proangiogenic properties 36 and we previously demonstrated that proangiogenic cells could be cultured from a BMderived immature, CD31 + /Ly6C hi myeloid progenitor cell fraction, 37 the monocytic cells were further subdivided into Ly6C hi , Ly6C med , and Ly6C lo fractions. Again, however, no significant differences could be observed between the experimental groups.…”

Pinocembrin, a major flavonoid in propolis, improves the biological functions of EPCs derived from rat bone marrow through the PI3K-eNOS-NO signaling pathway

“…An in vitro angiogenesis assay was performed as previously described (Loomans et al 2006). EPCs pretreated with LY294002 or L-NAME for 1 h, were treated with PIN (10-20 mg/L) and digested with 0.25 % trypsin.…”

Differentiation of Bone Marrow-Derived Endothelial Progenitor Cells Is Shifted into a Proinflammatory Phenotype by Hyperglycemia

“…We previously provided evidence that early outgrowth EPC, Mph, and DC may share a common myeloid progenitor pool in the BM (25). Therefore, the potential of the BM cells to differentiate into F480 + Mph and CD11c + DC in M-CSF-and GM-CSF-stimulated cultures, respectively, was assessed in parallel to the EPC cultures.…”

“…CD14 + cells have long been known to play a critical, paracrine role in neovascularization (22), and it is highly likely that "early outgrowth" EPC derived from CD14 + cells directly reflect the proangiogenic phenotype of monocytes that have entered ischemic tissue that is rich in vascular endothelial growth factor (23,24). We have previously demonstrated that in mouse BM these angiogenic EPC develop from an immature, CD31 + /Ly-6C + myeloid progenitor fraction (25). Given the proper differentiation factors, however, these BM precursor pools also give rise to important effector cells of the innate immune system such as macrophages (Mph) and dendritic cells (DC) (26,27), suggesting a common myeloid progenitor pool for EPC, Mph, granulocytes, monocytes, and DC.…”