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Berezin et al. Vessel Plus 2020;4:15 I http://dx.doi.org/10.20517/2574-1209.2020.03 Page 7 of 13

Mediating angiogenesis and neovascularization
Endothelial cell-derived EVs are involved in the regulation of vascular function, integrity and angiogenesis
through transportation of a wide spectrum of micro-RNA (microRNA-126-3p, microRNA-222-3p,
microRNA-let-7d-5p, microRNA-21-5p, microRNA-26a-5p, microRNA-92a-3p, microRNA-139-5p,
microRNA-30b-5p, microRNA-150 and microRNA-199a-5p) that have been implicated in MAPK/ERK1/2,
c-Jun N-terminal kinases/stress-activated kinases signaling cascade, and nuclear factor-κB signaling
pathway to target cells [progenitor and mature endothelial cells, macrophages, smooth muscle cells (SMC),
fibroblasts] in arterial endothelium [51,52,59,60] . There is evidence that the expression of thrombospondin 1
(THBS1), an inhibitor of angiogenesis and a target for microRNA-92a-3p, was significantly up-regulated
in the endothelial cells in atherosclerosis and that endothelial cell-derived EV transferred microRNA-
[52]
92a-3p to reduce THBS1 expression . Interestingly, the number of activated endothelial cell-derived
EVs decreased, whereas the number of apoptotic endothelial cell-derived EVs increased in patients
with atherosclerosis when compared with healthy individuals. This discrepancy was found to be closely
[61]
associated with impaired repair in atherosclerotic injury of the vasculature .

Plaque formation and vascular calcification
Endothelial cell-derived EVs contain a wide spectrum of intercellular signaling molecules, enzymes,
regulatory proteins, and growth factors, all of which are involved in the regulation of plaque formation and
vascular calcification [61,62] . Transforming growth factor-beta (TGF-β), which induces the expression of von
Willebrand Factor, proliferation of SMCs and progenitor endothelial cells, and recruitment of monocytes/
macrophages, is transferred by activated endothelial cell-derived EVs [63,64] . There is a wide range of
evidence that pro-inflammatory cytokines including tumor necrosis factor-alpha and some adipocytokines
(adiponectin) induce VCAM-1 production in mature endothelial cells, which is then accompanied by
enhanced circulating leukocyte attachment and a weakened ability to release EVs after activation [65,66] .
Therefore, the cellularity of the plaque can be modified by signaling molecules transferred by EVs. Indeed,
deficiencies in endothelial microRNA-126 and microRNA-92a transmitted as cargo with endothelial cell-
derived EVs has demonstrated acceleration of neointimal lesion formation of carotid arteries, increased
smooth muscle cell turnover, and its release was reduced by atheroprotective laminar shear stress [67-69] . It
+
has been established that autophagy of endothelial cells, fibroblasts, and CD45 hematopoietic cells that
accumulates into an atheroma plays a certain role in shaping vulnerable plaques and act as a trigger for
[70]
plaque rupture . There were intriguing findings that clarify a role of down-regulated microRNA-92a-
3p in endothelial cell autophagy through de-repressed autophagy-related gene 4a and increasing activity
of luciferase in autophagy-related gene 4a containing 3’UTR [71,72] . Moreover, EV-derived microRNA-
92a-3p upregulates the expression of cell cycle and mitosis-related genes including claudin-11, and
[73]
downregulates the adhesion-related gene expression in endothelial and foam cells . Thus, the packaging
and transfer of microRNA-92a-3p by endothelial cell-derived EVs ensures both a protective effect and
[74]
induces repair of the endothelium, thereby preventing plaque rupture and intravascular thrombosis .

The mediation of osteogenic trans-differentiation of vascular SMCs by inflammation, endothelial
dysfunction and reactive oxygen species by EVs may be considered as the key regulators of vascular
and plaque calcification. EVs also participate in the formation of microvascular calcifications that are
implicated in atherosclerotic plaque formation and rupture [75,76] . Interestingly, the release of exosomes by
[75]
EVs may promote microvascular calcification in response to environmental calcium stress . Sortilin,
[76]
which is a key regulator of SMC calcification through its recruitment to EVs , has been found to regulate
loading of the calcification protein - tissue nonspecific alkaline phosphatase - into EVs and thereby confer
calcification potency. SMC calcification also requires Rab11-dependent trafficking and FAM20C/casein
kinase 2-dependent C-terminal phosphorylation of sortilin; the deficiency of sortilin was thus found to
suppress ectopic vascular calcification [75,76] . Although it is not clear how EVs influence vascular calcification,
previous clinical studies have shown that cell plaque composition, volume of the plaque, a lipid core and