PLoS One. 2016 Aug 25;11(8):e0160153. doi: 10.1371/journal.pone.0160153.

Innovative Flow Cytometry Allows Accurate Identification of Rare Circulating Cells Involved in Endothelial Dysfunction. 

Boraldi F1, Bartolomeo A1, De Biasi S2, Orlando S1, Costa S1, Cossarizza A2, Quaglino D1.

1Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 287, Modena, Italy.
2Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia, Via Campi 287, Modena, Italy.




Although rare, circulating endothelial and progenitor cells could be considered as markers of endothelial damage and repair potential, possibly predicting the severity of cardiovascular manifestations. A number of studies highlighted the role of these cells in age-related diseases, including those characterized by ectopic calcification. Nevertheless, their use in clinical practice is still controversial, mainly due to difficulties in finding reproducible and accurate methods for their determination.


Circulating mature cells (CMC, CD45-, CD34+, CD133-) and circulating progenitor cells (CPC, CD45dim, CD34bright, CD133+) were investigated by polychromatic high-speed flow cytometry to detect the expression of endothelial (CD309+) or osteogenic (BAP+) differentiation markers in healthy subjects and in patients affected by peripheral vascular manifestations associated with ectopic calcification.


This study shows that: 1) polychromatic flow cytometry represents a valuable tool to accurately identify rare cells; 2) the balance of CD309+ on CMC/CD309+ on CPC is altered in patients affected by peripheral vascular manifestations, suggesting the occurrence of vascular damage and low repair potential; 3) the increase of circulating cells exhibiting a shift towards an osteoblast-like phenotype (BAP+) is observed in the presence of ectopic calcification.


Differences between healthy subjects and patients with ectopic calcification indicate that this approach may be useful to better evaluate endothelial dysfunction in a clinical context.

PMID: 27560136




Pathologic mineralization is known to be an active cell-mediated process, nevertheless, the origin of the mineralizing cell population, as well as the type and the source of the initiating stimuli, remain still unclear. Recent evidence indicate that vascular calcification is due to in situ activity of osteoblast-like cells associated with loss of mineralization inhibitors and that, within the vessel wall, endothelial and vascular smooth muscle cells can differentiate into osteoblast-type cells [1]. An additional requirement for ectopic calcification to occur seems the neo-angiogenic process, as newly formed blood vessels are abundantly present closed to areas of calcification [2]. The mechanisms accounting for the co-localization of angiogenesis and calcification are likely multifactorial, involving the coordinated interplay of different cell types (i.e. circulating and resident osteoprogenitors, endothelial and smooth muscle cells) as well as of cytokines and growth factors (i.e. vascular endothelial growth factor, VEGF).

Interestingly, it has been suggested that circulating cells with a chondrocyte/osteoblast-like phenotype, likely contributing to ectopic calcification, can be isolated from peripheral blood. However, investigating circulating progenitor and circulating mature cells is technically challenging due to their low number and the heterogeneity of surface markers assessed so far [3]. Therefore flow cytometry represents the gold standard for the accurate identification of these rare cells. This technique, allowing to simultaneously evaluate the expression of different surface markers, distinguishes between circulating progenitor cells (CPC), capable to sustain either vascular repair or calcification, and circulating mature cells (CMC) that, detached from the intima monolayer, represent a marker of vascular injury and may reveal a pro-osteogenic phenotype (Fig. 1). Therefore, by polychromatic high-speed flow cytometry it was possible to reveal an increase of CPC with a shift towards an osteoblast-like phenotype and an altered balance of CPC/CMC, both CD309+, in patients affected by peripheral vascular manifestation associated with ectopic calcification compared with healthy subjects (Fig. 2).

A number of evidence suggested that angiogenesis can contribute to aberrant mineralization since new vessels can act as a conduit for osteo-progenitor cells including both circulating progenitor cells and pericytes present within vessels. In particular, VEGF can promote angiogenesis and can directly influence several cell types inducing their migration and differentiation [4]. Moreover, growth factors typically exert their function affecting the expression of many other proteins/genes within a complex network. A better understanding of these interactions represents a fundamental requirement to disclose potential therapeutic targets.

Cytoscape plug-in called GeneMANIA [5] was applied to develop a network formed by a ‘seed list’ of genes or by single seed gene provided by a user along with various other genes that are predicted to possess overlapping functions via the ‘guilt-by-association’ function prediction approach. The network for VEGF was obtained by setting the following parameters: physical, genetic and pathway interaction, co-expression, co-localization and source organism Homo sapiens. The network was visualized by Cytoscape v 3.4.0 software, where each gene is represented as a node and the interactions between nodes are defined as edges (Fig. 3). In Figure 3 functions based on GO annotations are also reported.

In summary:

  • The use of a new generation of polychromatic high speed flow cytometry is crucial to accurately identify, by means of multiple markers, poorly represented cells, as circulating progenitor cells (CPC) and circulating mature cells (CMC).
  • Altered ratio of CD309+ on CMC / CD309+ on CPC, suggestive of vascular damage and low repair potential, can be revealed in patients with disease-affected peripheral vessels, also in the absence of a clinically relevant inflammatory condition.
  • An increase of circulating cells with a shift towards an osteoblast-like phenotype (BAP+) might be related to the presence of ectopic calcification.
  • VEGF is increased in patients suffering from ectopic calcification.

These results may pave the way to future studies on a larger cohort of individuals for the potential use of this approach to evaluate endothelial dysfunction in a clinical context.



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  1. Warde-Farley D, Donaldson SL, Comes O, et al. The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res 2010; 38:W214-220.



Figure 1. Cartoon showing the surface markers that can be detected by flow cytometry allowing to identify CPC and CMC. Mobilization of stem and progenitors cells from bone marrow to the periphery is regulated by growth factors (i.e. VEGF). Thereafter, CPC can differentiate towards an endothelial (CD309+) or an osteoblast (BAP+) phenotype. CD309 is a typical endothelial marker, whereas BAP is a glycoprotein normally found on the surface of osteoblasts. Moreover, in response to endothelial damages, detached CMC can be measured in peripheral blood and their pro-osteogenic signature further characterized. 



Figure 2. A) CPC in patients (PT) and in controls (CTR) are characterized by a different BAP+/ CD309+ ratio consistent with the pro-osteogenic phenotype of diseased cells. B) The endothelial marker CD309 indicates that the altered ratio of CPC/CMC can be associated with increased vascular injury in patients (PT) compared to healthy subjects (CTR). *p<0.05



Figure 3. A gene network from GeneMANIA. VEGF is connected to top 20 genes with edges according to the functional association networks from databases: the physical (pink), genetic (green) and pathway (pale blue) interactions, co-expression (purple), co-localization (lilac). Gene name and Uniprot code of top 20 genes linked to VEGF are shown in the upper table. Gene Ontology (GO) annotation of VEGF-connected genes listed in the network reported above. Q-values is a parameter of significance, coverage indicates the number of genes present in the network among all genes associated to a specific biological function as those reported in the GO annotation column.