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

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.Sarah L. Appleby, Michaelia P. Cockshell, Jyotsna B. Pippal, Emma J. Thompson, Jeffrey M. Barrett, Katie Tooley, Shaundeep Sen, Wai Yan Sun, Randall Grose, Ian Nicholson, Vitalina Levina, Ira Cooke, Gert Talbo, Angel F. Lopez and Claudine S. Bonde

Endothelial progenitor cells, uraemic toxins, and the development of endothelial dysfunction in chronic kidney disease.

Morbidity and mortality rates for cardiovascular disease (CVD) are increased among end stage kidney disease (ESKD) patients receiving dialysis treatment, and not corrected with kidney transplantation (KTx). Classic CVD risk factors do not fully predict the increased risk, with novel factors causing endothelial dysfunction (ED), leading to arteriosclerosis, congestive heart failure (CHF) and sudden death, key to disease pathogenesis. These novel factors include bone marrow (BM) derived endothelial progenitor cells (EPCs), which have key roles in maintenance, repair and growth of the endothelium. There is limited data about the role of EPCs and CVD in the ESKD population. This uraemic milieu includes p-cresol (sulfate, PC/S) and indoxyl sulfate (IS), toxins associated with CVD in ESKD. In this thesis, the relationship between CVD and ESKD, and the potential role of EPCs and uraemic toxins was examined from epidemiological, clinical and laboratory perspectives. Data was obtained for the period between 2002-2007 for all hospital separations in Australia. Analysis was performed based on ICD-9/10 coding. This showed (for the first time in an Australian population): (i) an increase in risk for CVD hospital separations among dialysis and KTx, with higher rates for CHF than acute cardiac events (ACE); (ii) an advantage for KTx recipients in regards to ACE, but not CHF hospital separations, over dialysis recipients, and (iii) for CHF, no increase in in-hospital mortality, or length of stay per separation for any ESKD group compared to controls. At a clinical level, in groups of haemodialysis (HDx), KTx patients and controls, low peripheral blood (PB) EPC numbers were correlated with surrogate markers of CVD and ED. No clear relationship of IS and PC/S with ED was seen (although study power was limited). For in vitro studies, techniques were developed for isolation (Flow sort and AutoMACS), enumeration (FACS) and culture expansion of EPCs from BM and umbilical cord blood samples. The effects of uraemic serum and toxins PC and IS on cultured endothelial cells (ECs) and EPCs in vitro was examined, as a model of vascular pathology in ESKD. Greater HUVEC VCAM-1 expression and reduced tube formation in Matrigel were observed in response to increasing PC concentration than IS. The effect of IS (but not PC) at higher concentration in Matrigel was reduced by the addition of EPCs. Akt/ERK expression by western blot, cell migration to VEGF, and supernatant investigation by FlowCytoMix for soluble cell surface markers, were also performed. Testing of HUVEC function post-exposure to sera from control, transplant and HDx recipients did not replicate the above results on the basis of sera PC and IS levels. In summary, this thesis has explored the increased burden of CVD in ESKD patients in Australia, the relationship of EPCs, both in vivo and in vitro, to vascular disease in this setting, and the role of uraemic toxins as agents for CVD. These results underline why certain therapies may not be effective in the ESKD population for CVD prevention, and suggest novel approaches are needed.Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 201

MALDI-tof/tof-MS of N-glysocylated naEFC membrane proteins using hydrazide-bead capture.

<p>The consensus motif for N-linked glycosylation is highlighted in bold.</p

Fold change values of well-established markers in the naEFCs when compared with HUVEC.

↑<p>denotes significantly higher mRNA expression;</p>↓<p>denotes significantly lower mRNA expression.</p

Hematopoietic properties of naEFCs.

<p>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).</p

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

<p>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.</p

Gene expression analysis of naEFCs versus HUVEC.

<p>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,*<i>p</i><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.</p

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

<p>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.</p