Characterization of a distinct population of circulating human non-adherent endothelial forming cells and their recruitment via intercellular adhesion molecule-3

Abstract

Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133(+) population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from 'early' endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis.

Figure 1. Surface expression profiling of freshly isolated CD133⁺ cells, naEFCs and HUVEC.

In (A), freshly isolated CD133⁺ cells were phenotyped for hematopoietic progenitor cell and endothelial cell markers by flow cytometry. In the histograms, the light dotted lines represent unstained cells and the dark lines represent stained cells of one representative experiment from n≥3. In (B), CD133⁺ enriched cells at 4 days of culture (naEFCs) and HUVEC were more extensively assessed for surface antigen phenotype. The histograms show one representative experiment from n≥3 with the light and dark lines as above. In (C), the pan-leukocyte marker CD45 and the myeloid markers CD11b and CD14 were examined with the light dotted lines representing unstained cells and the dark lines representing stained cells of one representative experiment from n≥3.

Figure 2. Vascular properties of naEFCs.

In (A), representative dot plots from one experiment show the incorporation of DiI-Ac-LDL and binding of UEA-1-FITC by naEFCs and HUVEC. The percentage of cells double positive for DiI-Ac-LDL uptake and binding of UEA-1-FITC was quantified. *p<0.05, versus naEFCs, n≥3. In (B), representative image of a Matrigel assay at 6 hours post seeding of CFSE-labeled naEFCs (green) and DiI-Ac-LDL positive HUVEC (red). Results represent one experiment of n = 5 with images captured by transmission and confocal microscopy. In (C), the number of tubes, branches and loops formed in the endothelial tube formation Matrigel assay in vitro with HUVEC alone or HUVEC co-cultured with naEFCs. #p = 0.05 versus HUVEC alone, *p<0.05 versus HUVEC alone, n = 5. In (D), a representative image of the tube formation of HUVEC alone (upper image) or HUVEC co-cultured with naEFCs (lower image) in vitro. Results represent one experiment of n = 5 with images captured by transmission microscopy. In (E), representative histograms showing increased surface expression of VCAM-1 following TNFα administration for 24 hours on naEFCs (left panel) and HUVEC (right panel), n = 4–6.

Figure 3. naEFCs express mature EC markers and form perfused tubes in vivo.

In (A), CFSE-labelled naEFCs mixed with Matrigel prior to injection into the flank of NOD/SCID mice, after 7 days the plugs were removed, processed and sections counterstained for nuclei with DAPI prior to imaging by confocal microscopy. The upper left image shows the cross section of a CFSE-naEFC generated tube-like structure (green) within which the nuclei of cells can be seen (blue) at 60× mag (arrows). The upper right image is the control plug in which no naEFCs were added. Images represent one experiment of n≥3. Similar sections were stained for CD144 and images captured by confocal microscopy with CFSE-naEFCs (green) exhibiting CD144 (red) as a cross section of a tube (lower left image) and CD144 staining in the junctions of the CFSE-naEFCs (lower right panel). Images are a representative of n≥3. In (B), similar experiments were executed and at day 7 post-implant the mice were injected i.v. with TRITC-lectin prior to exsanguinations, plugs removed, processed and sections counterstained for nuclei with DAPI prior to imaging by confocal microscopy. The representative image shows the cross section of a CFSE-naEFC generated tube-like structure (green, upper left image), TRITIC-lectin (red, upper right image), DAPI counterstain (blue, lower left image) and the merged image (lower right). In (C), CFSE-naEFCs were digested from explanted Matrigel plugs using dispase and phenotyped for hematopoietic progenitor cell and endothelial cell markers by flow cytometry (right panel); cells from contra-lateral control Matrigel plugs were similarly examined for antigen expression (left panel). In the histograms, the light dotted lines represent unstained cells and the dark lines represent stained cells of a representative of repeated experiments.

Figure 4. Hematopoietic properties of naEFCs.

naEFCs were seeded in MethoCult and growth factors GM-CSF, IL-3, SCF and EPO for 14 days prior to colony counting and staining with May Grunwald/Giemsa to assess cellular morphology. naEFCs formed blast-forming unit-erythroid (BFU-E), colony-forming units (CFU)-GEMM, -GM, -G and -M colonies in methylcellulose. Colony formation was photographed and quantified after 14 days and compared between naEFCs and freshly isolated CD133⁺ and CD133⁻ cells (mean ± sem, n = 3).

Figure 5. Gene expression analysis of naEFCs versus HUVEC.

In (A), a heat map illustrating the hierarchical clustering of Log2 relative gene expression in 3 separate HUVEC and naEFC samples. In (B), scatter data showing the average gene expression data in naEFCs and HUVEC. The dots represent the gene expression of UCB CD133+ 4 day cultured naEFCs versus HUVEC. The diagonal lines indicate the cut off value of 1.5 fold activation and genes coloured on the basis of expression level (yellow, evenly expressed genes; blue, naEFC upregulated genes; red, naEFC downregulated genes). In (C), ICAM-3 mRNA levels in naEFCs and HUVEC as determined by qPCR with relative gene expression normalised to CycA. Data are expressed as relative fold change (mean ± sem) normalised to HUVEC, n = 3,*p<0.05 versus HUVEC. In (D–F), flow cytometric analysis of ICAM-3 on (D) naEFCs, (E) HUVEC and (F) freshly isolated peripheral blood CD133⁺CD117⁺ gated cells. Light dotted line represents the unstained control and the dark line represents cells stained for ICAM-3. One representative experiment is shown n≥3.

Figure 6. ICAM-3 mediates rolling and adhesion of naEFCs.

In (A), still images of Video S1 illustrate the interaction of naEFCs with untreated (left panel), TNFα treated (5 ng/ml for 5 hours, middle and right panels) where naEFCs were pre-treated with an isotype control antibody (middle panel) or an antibody to ICAM-3 (right panel) prior to perfusion over HUVEC at 2 dynes/cm². In (B and C), data of rolling and adherent naEFCs is represented as the mean ± sem per field of view (fov) for n = 3;*p<0.05 versus untreated; #p<0.05 versus iso ctl. In (D and E), data of rolling and adherent whole blood treated with an isotype control or antibody to ICAM-3 is represented as the mean ± sem per field of view (fov) for n = 4–5;*p<0.05 versus untreated; #p<0.05 versus iso ctl.