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Berezin. Vessel Plus 2018;2:22 I http://dx.doi.org/10.20517/2574-1209.2018.23 Page 3 of 10

mune phenotypes of EPCs, a consensus on common strong endothelial markers was initiated, which are
expressed on the surface of these precursors. The cells may express cell markers of endothelial cells and their
precursors, such as CD31, CD144, KDR (CD309, VEGF receptor-2), and CD133, but in absence of CD45 [26,27] .
The CD45(-) cells, which are detected and isolated based on single or combined expression of CD34, CD133
[26]
and CD309, were referred to as EPCs . Three antigens, i.e., CD133, CD34, and CD309, are constitutively
determined on the surfaces of EPCs, while after differentiation EPCs lose CD133 antigen and begin to be
positively present on CD31, vascular endothelial cadherin, endothelial NO synthase (eNOS) and von Wil-
lebrand factor. However, EPCs expressed sufficient distinction in self-renewal ability that was determined
[28]
using colony-forming method . It has been speculated that a specific property may allow a more precise
definition of EPCs, because the majority of antigens used as molecular markers were commonly shared with
[29]
the various cells from hematopoietic lineage . Indeed, some fractions of EPCs with high proliferating ca-
pacity could contain non-hematopoietic population of CD45(-), CD31(+) cells with additional presentation
CD117(+). Taken together, the combination of CD45(-), CD31(+), CD 133(+), CD34(+) and CD309(+) or von
Willebrand factor (+) probably allows for getting reliable purification of progenitor endothelial subsets from
mononuclear cells with presentation of endothelial markers.

Depending on the ability to appear in fibronectin coated dish all EPCs were divided into early outgrowth
(5-7 days after fibronectin plating) or late outgrowth endothelial cells (7-10 days after fibronectin plating).
Interestingly, the late outgrowth precursors originated from peripheral blood mononuclea cells and ex vivo
+
+
+
demonstrated immune phenotypes (CD31 , CD146 , CD105 , and CD309 ) and functional properties suitable
+
as mature endothelial cells. Indeed,two distinguished populations of late outgrowth progenitor cells based
on differential expression of the cell surface marker CD34 have been identified. The population of EPCs with
co-expression of CD34 antigen additionally to CD31(+), CD146(+), CD105(+), and CD309(+) exhibited higher
proliferative capability to CD34(-) EPCs. Therefore, CD34(+) EPCs had reproduced tubes and colony shaping
[28]
in the single-cell colony-formation investigation as well as they responded to angiogenic growth factors .
In contrast, CD34(-) cells had limited capability to reproduce colonies or even had none of these properties
in vitro. There is evidence that the absence of CD34(+) EPCs in the colony leads to cultures collapsing
within one or two passage and confirming a strong hierarchy in self-renewal of EPCs, which may be an
[28]
extremely important functional feature of precursors . Thus, late outgrowth precursors may differentiate
[29]
+
into functionally mature endothelial cells and progenitor-like angiogenesis-promoting cells (CD34 EPCs) . In
fact, the colonies are shaped by heterogeneous populations of EPCs that express different markers suitable
for endothelial and monocyte lineage. Finally, CD34(+) EPCs mediate marginally to angiogenesis and
neovascularization by differentiation, although they are potent triggers and powerful regulators of pro-
inflammatory response and remodeling of vascular wall [29,30] . Other candidates for cell surface molecule
markers and genes’ signatures to a functional determination based surely on self-renewal hierarchy of
EPCs are actively investigated. Thus, there are several populations of endothelial progenitors with different
proliferative activity and angiopoetic capabilities that can represent markers of endothelial reparation/injury
and endothelial dysfunction.

THE MECHANISMS OF ANGIOPOETIC ACTIVITY OF EPCS
Previous preclinical and clinical studies have shown that several factors, such as VEGF, stromal cell-
derived factor-1 (SDF-1) may enhance differentiation, proliferation, adhesion, and incorporation into
endothelial cell monolayers of EPCs through VEGF/eNOS-related and chemokine SDF-1 Akt-related
pathways [31-33] . Moreover, there is evidence that the blockade of VEGF or eNOS prevented all VEGF-induced
and SDF-1alpha-induced effects toward EPCs including ischemia-induced proliferation, vasculogenesis
and angiogenesis [33,34] . In this context, the transfer of genes (cdkn1c and il33) that are coding p57 and an
endothelial cell cycle inhibitor may regulate self-renewal capacity of EPCs. Recent studies confirmed that
increased expression of these target genes of Notch signaling play a pivotal role in regulation of cell cycling
with EPCs and stem cells [35,36] . Thus, EPCs contribute their angiopoetic effect through direct differentiation
[37]
into mature endothelial cells and probably into cells with other phenotypes like smooth muscle cells .