CD34 marks angiogenic tip cells in human vascular endothelial cell cultures - PubMed (original) (raw)

CD34 marks angiogenic tip cells in human vascular endothelial cell cultures

Martin J Siemerink et al. Angiogenesis. 2012 Mar.

Abstract

The functional shift of quiescent endothelial cells into tip cells that migrate and stalk cells that proliferate is a key event during sprouting angiogenesis. We previously showed that the sialomucin CD34 is expressed in a small subset of cultured endothelial cells and that these cells extend filopodia: a hallmark of tip cells in vivo. In the present study, we characterized endothelial cells expressing CD34 in endothelial monolayers in vitro. We found that CD34-positive human umbilical vein endothelial cells show low proliferation activity and increased mRNA expression of all known tip cell markers, as compared to CD34-negative cells. Genome-wide mRNA profiling analysis of CD34-positive endothelial cells demonstrated enrichment for biological functions related to angiogenesis and migration, whereas CD34-negative cells were enriched for functions related to proliferation. In addition, we found an increase or decrease of CD34-positive cells in vitro upon exposure to stimuli that enhance or limit the number of tip cells in vivo, respectively. Our findings suggest cells with virtually all known properties of tip cells are present in vascular endothelial cell cultures and that they can be isolated based on expression of CD34. This novel strategy may open alternative avenues for future studies of molecular processes and functions in tip cells in angiogenesis.

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Figures

Fig. 1

Fig. 1

CD34 is expressed on endothelial filopodial extensions at sites of active angiogenesis in vivo. Immunoperoxidase staining with anti-CD34 monoclonal antibody QBEND-10 of a section of surgically removed human colon carcinoma lesion. Anti-CD34 antibody stains capillaries and numerous endothelial filopodia (arrowheads). ×450

Fig. 2

Fig. 2

Morphology of CD34+ cells in HUVEC cultures shows a strong resemblance with that described for tip cells in vivo. a Flow cytometric dot plots of HUVECs of one representative donor demonstrating the proportion of CD34+ cells in a HUVEC culture at passage 3. b Third passage HUVECs expressed VE-cadherin (red), but only a subset was stained for CD34 (green). Note filopodia extending from elongated CD34+ HUVECs (arrowheads), whereas adjacent CD34− HUVECs show cobblestone morphology and lack of filopodia. DNA was stained with DAPI (blue). (Color figure online)

Fig. 3

Fig. 3

CD34+ cells in HUVEC cultures co-express endothelial markers. CD34+ HUVECs were analyzed by 2-color flow cytometry for the expression of CD31, CD146 and VE-cadherin (endothelial cell specific markers), CD133 and CD117 (stem cell and endothelial progenitor cell specific markers), and CD14 (myelomonocytic specific marker). The results demonstrate that all CD34+ cells are positive for CD31, CD146 and VE-cadherin, but negative for CD133, CD117 and CD14

Fig. 4

Fig. 4

CD34+ cells in HUVEC cultures are actively generated and show a lower proliferation rate. a CD34− cells were sorted from HUVEC cultures using FACS and then cultured again. b After 10 days of culture, cells were analyzed for CD34 expression by FACS showing a subpopulation of 9.8% of CD34+ cells. SSC, side scatter. CD34+ and CD34− FACS-sorted HUVECs were cultured separately for 48 h and images were taken every 15 min at 10 fixed spots in each culture. Time-lapse movies were analyzed by counting the total amount of cells and the number of cells in mitosis, as recognized by the division of one cell into 2 daughter cells. c Numbers of cells at the given time points. d Total number of cells in mitosis. Shown are means and standard error of the means (SEM); * indicates P < 0.05, relative to CD34−, determined by Student t test

Fig. 5

Fig. 5

The number of CD34+ cells on HUVECs in vitro is regulated by mechanisms known to induce or inhibit the tip cell phenotype in vivo_._ The proportion of CD34+ HUVECs was determined by flow cytometry after culturing of cells in the presence or absence of VEGF, bFGF, TNF-α or DLL4 for 48 h. a Dose-dependent increase of CD34 expressing cells when stimulated with VEGF but not bFGF. b Continuous stimulation with TNF-α decreased the number of CD34+ HUVECs. c HUVECs cultured on DLL4-coated plates showed a lower number of CD34+ cells. Shown are mean and SD; * indicates P < 0.05 relative to control, determined by Student t test for paired samples

Fig. 6

Fig. 6

CD34 on HUVECs in vitro is upregulated during angiogenesis and tip cell formation. 3-Dimensional in vitro angiogenesis of collagen-gel embedded HUVEC spheroids cultured in the presence or absence of bFGF after fixation and staining for the expression of CD34 (green). Arrowheads point at filopodial extensions of tip cells. (Color figure online)

Fig. 7

Fig. 7

Gene expression profiles of CD34+ HUVECs show features of a tip cell phenotype. a Pie chart (left) illustrating the number of genes with significantly (Q value <0.15) altered expression (_total circle_) between CD34+ and CD34− fractions, and the 441 genes with significantly (_Q_ value >0.15) increased (yellow part) or decreased (blue part) expression by more than 2-fold. Heat map (right) of the 441 differentially expressed genes in 4 HUVEC donors sorted for CD34 (yellow, induced; blue, repressed; log2 based scale). b GO analysis reveals a significant association of CD34+ cells with terms that are characteristic for tip cell functions and association of CD34− cells with terms of cell proliferation. P values represent a Benjamini–Hochberg–corrected EASE score. (Color figure online)

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