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JACC: BASIC TO TRANSLATIONAL SCIENCE

VOL. 2, NO. 6, 2017

ª 2017 THE AUTHORS. PUBLISHED BY ELSEVIER ON BEHALF OF THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION. THIS IS AN OPEN ACCESS ARTICLE UNDER

ISSN 2452-302X http://doi.org/10.1016/j.jacbts.2017.08.004

THE CC BY-NC-ND LICENSE (http://creativecommons.org/licenses/by-nc-nd/4.0/).

STATE-OF-THE-ART REVIEW

Endothelial- and Immune Cell-Derived Extracellular Vesicles in the Regulation of Cardiovascular Health and Disease Felix Jansen, MD,a Qian Li, MD,a,b Alexander Pfeifer, MD, PHD,c Nikos Werner, MDa

SUMMARY Intercellular signaling by extracellular vesicles (EVs) is a route of cell-cell crosstalk that allows cells to deliver biological messages to speciﬁc recipient cells. EVs convey these messages through their distinct cargoes consisting of cytokines, proteins, nucleic acids, and lipids, which they transport from the donor cell to the recipient cell. In cardiovascular disease (CVD), endothelial- and immune cell-derived EVs are emerging as key players in different stages of disease development. EVs can contribute to atherosclerosis development and progression by promoting endothelial dysfunction, intravascular calciﬁcation, unstable plaque progression, and thrombus formation after rupture. In contrast, an increasing body of evidence highlights the beneﬁcial effects of certain EVs on vascular function and endothelial regeneration. However, the effects of EVs in CVD are extremely complex and depend on the cellular origin, the functional state of the releasing cells, the biological content, and the diverse recipient cells. This paper summarizes recent progress in our understanding of EV signaling in cardiovascular health and disease and its emerging potential as a therapeutic agent. (J Am Coll Cardiol Basic Trans Science 2017;2:790–807) © 2017 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

C

ardiovascular disease (CVD) still represents

vascular diseases associated with systemic endothe-

the leading cause of mortality worldwide.

lial damage, such as atherosclerosis, show signiﬁ-

The underlying disease, atherosclerosis, is

cantly increased levels of circulating EVs (3,4).

initiated and propagated by continuous damage of

However, EVs are not simply inactive debris that

the vascular endothelium, leading to endothelial acti-

reﬂect cellular activation or injury. EVs can transfer

vation and apoptosis, the development of endothelial

proteins, cytokines, mRNA, or noncoding RNA such

dysfunction, and subsequent atherosclerotic lesion

as microRNA (miRNA) or long noncoding RNA to

formation (1). Endothelial cell (EC) injury is a key

target cells and inﬂuence their function and pheno-

element in the complex pathophysiology of athero-

type (5,6). Accordingly, the role of EVs has changed

genesis and triggers the release of EC-derived extra-

from being only a marker of vascular integrity toward

cellular

and

being relevant effectors in intercellular vascular

microvesicles (MVs) (2). Accordingly, patients with

signaling (7,8). In CVD, EVs have been shown to

vesicles

(EVs)

such

as

exosomes

From the aDepartment of Internal Medicine II, Rheinische Friedrich-Wilhelms University, Bonn, Germany; bDepartment of Cardiology, Second Hospital of Jilin University, Nanguan District, Changchun, China; and the cInstitute of Pharmacology and Toxicology, University of Bonn, Bonn, Germany. Drs. Werner, Pfeifer, and Jansen are supported by Deutsche Forschungsgemeinschaft (WE 4139/8-1, JA2352/2-1, DFG GRK1873). Dr. Jansen received support from Medical Faculty of the Rheinische Friedrich-WilhelmsUniversity Bonn, the Familie Schambach foundation and German Society of Cardiology. The authors have reported that they have no relationships relevant to the contents of this paper to disclose. Drs. Jansen and Li contributed equally to this study. All authors attest they are in compliance with human studies committees and animal welfare regulations of the authors’ institutions and Food and Drug Administration guidelines, including patient consent where appropriate. For more information, visit the JACC: Basic to Translational Science author instructions page. Manuscript received July 7, 2017; revised manuscript received August 14, 2017, accepted August 14, 2017.

Jansen et al.

JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 2, NO. 6, 2017 DECEMBER 2017:790–807

contribute to disease development and progression

(21–23). Second, EVs are able to transfer their

ABBREVIATIONS

by promoting initial lesion formation, intravascular

biological content by membrane fusion with

AND ACRONYMS

calciﬁcations, plaque progression, and thrombus for-

the recipient cell. The fusion process is

mation after rupture. In contrast, numerous studies

regulated by the lipid composition of EV

have demonstrated that certain subtypes of EVs can

membrane, and several reports indicate that

mediate vascular protection and endothelial regener-

the presence of phosphatidylserine contrib-

ation (9). In line with these ﬁndings, EVs released by

utes to membrane fusion (24). Third, incor-

progenitor or mesenchymal stem cells have been

poration of EVs into target cells is mediated

shown to improve cardiac function after myocardial

by endocytosis, pinocytosis, or phagocytosis

infarction in experimental studies, highlighting the

(25). Through these interaction routes, EVs

therapeutic potential of EVs in cardiovascular pathol-

transfer their biological contents containing

ogies (10). This review summarizes current knowl-

nucleic acids such as mRNA (26), noncoding

edge of EVs as regulators of cardiovascular health

RNAs (miRNAs [27], long noncoding RNAs

and disease and potential opportunities for therapeu-

[6]), proteins (28), cytokines (29), or bioactive

tic use.

lipids (30) (Figure 1).

BIOGENESIS OF EXTRACELLULAR

EXTRACELLULAR VESICLES AS

CVD = cardiovascular disease EC = endothelial cell EMV = endothelial cell-derived microvesicles

ESCRT = endosomal sorting complex required for transport

IL = interleukin miRNA = microRNA MV = microvesicles NO = nitric oxide PEG = polyethylene glycol TGF = transforming growth factor

VESICLES AND THEIR INTERACTIONS

EFFECTORS OF CARDIOVASCULAR DISEASES

WITH TARGET CELLS In cardiovascular biology, EVs have various physioEVs are membrane vesicles secreted from cells that

logical functions, including activation of platelets and

contain intracellular contents (11). Cells can release a

ECs, as well as regulation of inﬂammation and coag-

broad range of vesicles with diverse features. This

ulation (31–34). Therefore, EVs are emerging as key

review focuses on 2 major types: MVs and exosomes.

players in different stages of CVD development

MVs are large (>150 nm) vesicles that are released by

(31,32,35). The effects of EVs in CVD are extremely

budding from the plasma membrane, whereas exo-

complex and depend on the cellular origin, the

somes are smaller (30 to 100 nm) and originate from

functional state of the releasing cells, the intra-

the endosome (12). However, there is no strict cutoff

vesicular content, and the recipient cells (36,37). The

value that distinguishes MVs from exosomes by

following sections summarize the current knowledge

vesicle size, which can differ in diverse studies (12).

about EVs as effectors of CV disease progression or

Exosomes represent a homogeneous population of

791

Endothelial and Immune Cell EVs in CV Health and Disease

vascular repair.

vesicles that are formed by inward budding of the

DETRIMENTAL EFFECTS OF EXTRACELLULAR VESICLES

multivesicular body (MVB) membrane. Exosome

ON VASCULAR FUNCTION. Endothelial

biogenesis is mediated mainly by the endosomal

occurs as a response to cardiovascular risk factors

sorting complex required for transport (ESCRT) pro-

and represents the initial step in atherosclerosis

tein (13) or lipid ceramide and neutral sphingomyeli-

development, the underlying pathology of CVD

nase, the enzyme that converts sphingomyelin to

(38,39).

ceramide (14). Cargo sorting into exosomes involves

E n d o t h e l i a l d y s f u n c t i o n . Endothelial MVs have

ESCRT and associated proteins such as tumor sus-

been shown to impair vasorelaxation by inhibiting

ceptibility gene 101 protein (TSG101) and ALG-2-

nitric oxide (NO) production in target ECs. This

interacting protein X (ALIX) and small GTPases such

phenomenon is mediated through a decrease in

as Rab7a and Rab27b (15–17). Exosomes are liberated

endothelial NO synthase phosphorylation and activ-

into the extracellular space following fusion of MVBs

ity (40), local oxidative stress (41), or an increased

with the cell membrane, regulated by Rab27A, Rab11,

NADPH oxidase activity with MVs (33) and results in

and Rab31 (18,19). MVs represent a relatively hetero-

impaired vascular relaxation capacities. MVs in the

geneous population of vesicles formed by outward

aforementioned studies are obtained from ECs under

budding of the cell membrane. This process is regu-

physiological (40,41) or pathological (33) conditions,

lated by membrane lipid microdomains and regula-

but they all have detrimental effects on vaso-

tory proteins such as ADP-ribosylation factor 6 (ARF6)

relaxation. Regarding the relation between molecu-

(20). EVs can be regarded as intercellular messengers

lar

for various biological processes. Several routes of

investigation should be conducted to clarify and

interaction between EVs and recipient cells have been

compare the cargoes of EVs derived under different

described. First, EVs can directly activate target cell

conditions (e.g., by RNA sequencing or proteomic

surface receptors by bioactive ligands and proteins

analysis).

contents

and

function

of

dysfunction

EVs,

further

792

Jansen et al.

JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 2, NO. 6, 2017 DECEMBER 2017:790–807

Endothelial and Immune Cell EVs in CV Health and Disease

F I G U R E 1 EV Biogenesis and Interaction With Recipient Cells

Exosome formation starts with endocytosis, a process in which the cell membrane is pinched inward and captures bioactive molecules, resulting in the formation of the endosome. These molecules are sorted into smaller vesicles that bud from the perimeter membrane into the endosome lumen, forming vesicles; this leads to the multivesicular appearance of late endosomes and so they are also known as MVBs. From MSBs, exosome formation occurs by an ESCRT- and ceramide-dependent pathway. Cargo sorting into exosomes involves ESCRT and TSG101, ALIX, and Rab7a, and Rab27b. Exosomes are released into the extracellular space following the fusion of MVBs with the cell membrane, which is regulated by Rab27A, Rab11, and Rab31. Microvesicles are formed by the outward budding of the cell membrane, a process that is regulated by ARF6. Several routes of interaction between EVs and recipient cells have been described. First, EVs can directly activate target cell surface receptors. Second, EVs are able to transfer their biological content by membrane fusion with the recipient cell. Third, incorporation of EVs into target cells is mediated by endocytosis, pinocytosis, or phagocytosis. Using these interaction routes, EVs transfer their biological content containing nucleic acids such as mRNA, noncoding RNAs (microRNAs, long noncoding RNAs), proteins, cytokines, or bioactive lipids. mRNA ¼ messenger RNA; MVB ¼ multivesicular bodies.

In line with the latter ﬁndings, MVs isolated from patients with vascular (acute coronary syndrome

may have diverse biological functions need to be addressed in future studies.

[42]) or predisposing disease (chronic renal failure or

Of interest, storage of human blood under standard

metabolic syndrome [43,44]) were shown to induce

blood-banking conditions results in accumulation of

endothelial dysfunction ex vivo in rat aortic rings.

MV-encapsulated hemoglobin. These erythrocyte-

In contrast, MVs from healthy subjects did not affect

derived MVs react with and degrade NO, inducing

endothelial function (42–44), indicating that the

endothelial

pathophysiological state of the releasing cell de-

derived exosomes derived under septic conditions

termines not only the number of released MVs but

have been shown to mediate septic endothelial

also their content and biological function. Which cell-

dysfunction

speciﬁc MVs mainly inﬂuence endothelial function

involving superoxide, NO, and peroxynitrite produc-

and whether isolated MVs from different cell types

tion (46). Studying EVs derived from patients

dysfunction

by

inducing

(45).

Finally,

endothelial

platelet-

apoptosis

Jansen et al.

JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 2, NO. 6, 2017 DECEMBER 2017:790–807

Endothelial and Immune Cell EVs in CV Health and Disease

represents an important translational approach to

glucose-treated cells, but not from healthy ECs,

exploring the relationship between EVs and certain

facilitated up-regulation of ICAM-1 and VCAM-1 in

diseases. However, comorbidities as well as patients’

endothelial target cells by activating p38 in an reac-

age and sex should be taken into consideration as

tive oxygen species-dependent manner (33). In light

possible

confounders.

and

of these proinﬂammatory effects and the increased

exclusion criteria should be thoughtfully determined,

levels of endothelial MVs in diabetic patients, one

and control groups must be carefully selected.

may speculate that MVs released under pathological

Endothelial

adhesion/

high-glucose conditions might represent a paracrine

i n ﬂ a m m a t i o n . Inﬂammation

mediator transporting proinﬂammatory messages to

plays a pivotal role in CVD (38,47). EVs from various

target cells and thereby foster vascular inﬂammation

cellular sources contribute to vascular inﬂammatory

(61,62). However, while studying the functional ef-

processes including endothelial activation, monocyte

fects of EVs derived under pathological conditions,

adhesion, and transmigration (48–51).

one must consider that the quantity and/or content of

inﬁltration,

Therefore,

activation, and

inclusion

monocyte

In vitro studies have demonstrated that MVs can

EVs may vary with different modes and durations of

induce release of the proinﬂammatory cytokines

stimulation. Therefore, clearly deﬁned pathological

interleukin (IL)-6 and IL-8 from ECs and leukocytes

conditions (including standardized concentrations

(52,53); and promote expression of adhesion proteins

and duration of cell stimulations) as well as a careful

ICAM-1,

facilitating

selection of an adequate control group are mandatory

increased adhesion of monocytes (54,55) and subse-

to elaborate EV functions depending on the parent

quent transmigration, leading to vascular inﬂamma-

cell conditions.

tion

VCAM-1,

and

plaque

and

E-selectin,

development.

Mechanistically,

Compared with the role of MVs, the role of exo-

increased adhesion of monocytes to ECs can be

somes in vascular inﬂammatory processes has been

mediated by the MV-mediated transfer of proin-

less explored (63,64). However, monocyte-derived

ﬂammatory molecules such as oxidized phospho-

exosomes seem to induce vascular inﬂammation and

lipids (56), caspase-3 (57), or RANTES (regulated on

cell death by transferring inﬂammatory miRNAs into

activation, normal T cell expressed and secreted)

ECs resulting in a signiﬁcant up-regulation of ICAM-1,

protein, which is transferred from platelet MVs to

CCL2, and IL-6 levels (65) and provocation of endo-

endothelial target cells (58). Platelet MVs from

thelial apoptosis by tissue factor release (66).

apoptotic platelets also facilitate differentiation be-

Atherosclerotic plaque development, progression, and

tween

professional

rupture. Atherosclerotic plaque rupture with subse-

phagocytes (59). Vascular inﬂammatory processes

quent coronary thrombosis represents the ultimate

involve different cells (e.g., ECs, monocytes, and

step in atherosclerotic lesion progression, leading to

platelets [48–51]). The

aforementioned in vitro

acute myocardial ischemia. Atherosclerotic plaques

studies demonstrated that EVs from diverse parent

can release large amounts of EVs, contributing to

cells can act on various types of target cells. In sum-

plaque progression and instability through various

mary, vascular inﬂammation seems to be regulated by

mechanisms. Plaque EVs originate mainly from leu-

complex

routes

cocytes, reﬂecting the local inﬂammatory environ-

involving a network of cells and EVs. To gain more

ment (67). Plaque EVs express surface antigens

insight into these multifaceted mechanisms, co-

consistent with their leukocyte origin, including ma-

incubation of different EVs with diverse cell types

jor histocompatibility complex classes I and II, and

may be helpful to study this “intercellular commu-

dose-dependently induce T-cell proliferation (68).

nication network.” However, there is a lack of

Antigen-speciﬁc activation of CD4 þ T cells was also

adequate in vitro models, which should be addressed

induced by dendritic exosomes, implicating their

resident

macrophages

intercellular

and

communication

in further studies.

potential involvement in vascular inﬂammation and

Pathological conditions modify EV content and

plaque development (69). Furthermore, plaque MVs

biological functions (60). MVs isolated from athero-

carry catalytically active tumor necrosis factor (TNF)-

sclerotic plaques transfer ICAM-1 to ECs and recruit

a–converting enzyme (TACE/ADAM17) and signiﬁ-

inﬂammatory cells, suggesting that human plaque

cantly enhance the processing of its substrates TNF-a

MVs promote atherogenesis (24).

and TNF receptor, thereby promoting an inﬂamma-

Moreover, oxidatively modiﬁed, but not native,

tory response (70). Importantly, plaque EVs from

EC-derived EVs contain proinﬂammatory oxidized

patients or in vivo models are not single-component

phospholipids that elicit speciﬁc responses in ECs,

molecules, although mainly of leucocyte origin (67),

leading to the adhesion of monocytes (56). In line

and their constitutions may also depend on the stage

with these ﬁndings, EC-derived EVs generated from

of

plaque

progression

(stable

or

unstable?).

793

794

Jansen et al.

JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 2, NO. 6, 2017 DECEMBER 2017:790–807

Endothelial and Immune Cell EVs in CV Health and Disease

Therefore, standard procedures to derive and analyze

inﬂammation, plaque progression, and rupture are

plaque EVs should be established.

illustrated in Figure 1.

Local inﬂammation is enhanced by monocytic MVs

In summary, a broad body of evidence indicates

fostering leucocyte adhesion to postcapillary venules

the active involvement of EVs in plaque develop-

and T-cell inﬁltration in atherosclerotic plaques

ment, progression, and thrombus formation after

in vivo (71,72). Exosomes from T cells also can

rupture. However, there is an urgent need for addi-

contribute to atherosclerotic plaque development by

tional mechanistic studies to explore how their

inducing cholesterol accumulation in human mono-

atheroprone effects can be targeted to decelerate

cytes by the phosphatidylserine-receptor (73).

atherosclerotic lesion formation and rupture.

Vascular smooth muscle cell (VSMC) proliferation plays an important role in atherosclerotic plaque development (39,74). The effect of MVs on VSMC proliferation depends on the cellular origin. In vitrogenerated platelet-derived MVs promoted VSMC proliferation in a platelet-derived growth factor (PDGF)-independent mechanism with minor effects on migratory capacity (75,76). In turn, monocytederived MVs were shown to deliver a lethal message

FAVORABLE EFFECTS OF EXTRACELLULAR VESICLES ON VASCULAR FUNCTION. Despite the various deleterious

effects of EVs in the pathogenesis of CVD, an increasing body of evidence highlights the beneﬁcial effects of certain EVs on vascular function. The following sections summarize the impact of EV on endothelial repair, their inhibitory effect on vascular inﬂammation, and their role in plaque stabilization.

by encapsulated caspase-1–inducing VSMC cell death

Endothelial protection and vascular repair. Given that

(77). More

recently, calciﬁcation-competent EVs

EC injury is not only a key element in the complex

derived from smooth muscle cells, valvular intersti-

pathophysiology of atherogenesis but also in in-stent

tial cells, and macrophages have been described as

restenosis occurring after treatment of coronary ste-

mediators of vascular calciﬁcation that modulate

nosis (90,91), a mechanistic understanding of EC

heart

(78–80).

repair is pivotally important to develop therapeutic

Although EVs show protective effects against heart

strategies to preserve endothelial integrity and

valve calciﬁcation, the potential underlying mecha-

vascular health. Several studies have shown that EVs

nisms are unknown and should be addressed in

particularly of endothelial origin can act as intercel-

future studies.

lular messenger to promote endothelial regeneration

valve

disease

and

atherogenesis

Angiogenesis is a fundamental process in CVD,

and vascular protection in vitro and in vivo.

contributing to plaque instability by promoting neo-

A potential contribution of endothelial MVs in EC

vascularization. Unstable plaques are characterized

survival was shown by Abid Hussein et al. (92), who

by an increased number of vasa vasorum mediating

demonstrated that endothelial MV release is cell

intraplaque hemorrhage (81). MVs isolated from hu-

protective by exporting caspase-3 into MVs and

man atherosclerotic plaques were shown to stimulate

thereby diminishing intracellular levels of proapo-

EC proliferation in vitro after CD40 ligation and to

ptotic caspase-3. Statins seem to facilitate endothelial

enhance in vivo angiogenesis. Interestingly, the pro-

health by promoting endothelial MV release in vitro

liferative effect of MVs isolated from atherosclerotic

(93). Nevertheless, the role of statins in endothelial

plaques was more pronounced using MVs from

MV release is still a matter of debate (94,95). Our

symptomatic patients than from patients without

group has demonstrated that annexin I/phosphati-

symptoms. Therefore, MVs could represent a major

dylserine

determinant of plaque vulnerability (82). Unstable

incorporation

human plaques contain large numbers of procoagu-

endothelial-regenerating cells against apoptosis (96).

lant MVs, originating mostly from leucocytes, eryth-

Inhibition of p38 activity by annexin I-containing

rocytes, and VSMCs localized within the necrotic core

endothelial MV is possibly involved in endothelial

(83). Once plaque rupture occurs, these MVs can

MV-mediated protection. However, whether annexin

initiate the coagulation cascade through different

I also mediates endothelial EV uptake in vivo will be

mechanisms (67,84): ﬁrst, by the expression of tissue

explored in additional animal models. Another study

factor (mainly on monocytic MVs), one major initiator

showed that platelet-derived MVs induced changes in

of blood coagulation (34,85,86); and second, by

the early outgrowth cell, secretome, toward a more

exposure to phosphatidylserine on their outward

proangiogenic

membrane layer (87). Procoagulatory effects of MVs

outgrowth cell-mediated induction of endothelial

have been demonstrated in vitro and in vivo, where

regeneration in vitro and in vivo (97). These studies

they facilitated thrombus formation (88,89). The

indicate that MVs may inﬂuence the endothelial

deleterious

regeneration by the following 2 mechanisms: they

effects

of

EVs

inducing

vascular

receptor-dependent by

ECs

proﬁle

protects

and

endothelial endothelial

ampliﬁed

the

MV and

early

Jansen et al.

JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 2, NO. 6, 2017 DECEMBER 2017:790–807

Endothelial and Immune Cell EVs in CV Health and Disease

F I G U R E 2 Detrimental Effects of Extracellular Vesicles on Vascular Function

The functional effect of EVs in cardiovascular disease is extremely complex and depends on the cellular origin, the functional state of the releasing cells, the biological content, the distinct recipient cell, and the transfer capacity of intravesicular functional bioactive molecules. Here, we illustrate the role of EVs as active promoters of endothelial dysfunction, vascular calciﬁcation, atherogenesis, plaque instability and thrombosis. Endothelial EVs impair vasorelaxation through local oxidative stress or through increased NADPH oxidase activity. EVs released by erythrocytes react with and degrade NO, EVs from platelet and atherosclerotic plaque induce endothelial apoptosis, both mediating endothelial dysfunction. Monocyte-derived EVs transfer inﬂammatory miRNAs into endothelial cells inducing vascular inﬂammation. Vascular smooth muscle cell-derived EVs act as mediators of vascular calciﬁcation modulating atherogenesis. EVs isolated from atherosclerotic plaques transfer ICAM-1 to endothelial cells and recruit inﬂammatory cells, contributing to plaque instability by promoting neovascularization. Once plaque rupture occurs, monocyte EVs and endothelial EVs initiate the coagulation cascade by the expression of tissue factor contributing to thrombosis. EV ¼ extracellular vesicles; miRNA ¼ microRNA; NO ¼ nitric oxide.

could directly interact with ECs and promote vascular

inﬂuencing migration and proliferation capacities in

regeneration, or they may activate endothelial pro-

target cells, in addition to the already described

genitor cells, facilitating endothelial repair (96,98). In

regenerative potential of endothelial MV in interac-

line with these ﬁndings, endothelial MVs carrying

tion

endothelial protein C receptor and activated protein C

miRNA-126 plays an important role in vascular health,

(APC) could also promote cell survival by induction of

miRNA-126–mediated

cytoprotective effects (99).

processing are not clearly identiﬁed and must be

with

progenitor

cells.

However,

downstream

although

signaling

and

Among the biological contents transferred by EVs

addressed in future research. Of note, EVs could

into target cells, miRNAs seem to play a crucial role by

activate EC by mRNA transfer from endothelial pro-

affecting mRNA and protein expression in recipient

genitor cells stimulating angiogenesis (105). In line

cells (100–102). Studies by our group have shown that

with these ﬁndings, MVs from ischemic muscle pro-

endothelial MVs promote vascular endothelial repair

moted progenitor cell differentiation and subsequent

by delivering functional miRNA-126 into recipient

postnatal vasculogenesis (106). Besides MVs, exo-

endothelial

cells

somes also play an important role in cardiovascular

(103,104). Of note, endothelial MV-mediated miRNA-

regeneration. Exosomes derived from mesenchymal

126–induced endothelial repair was altered under

stem cells reduced myocardial ischemia/reperfusion

pathological hyperglycemic conditions (103). These

injury (107). Furthermore, exosomes from cardiac

ﬁndings emphasize the fact that endothelial MVs

progenitor cells increased the migratory capacity of

can stimulate endothelial repair by functionally

ECs

and

vascular

smooth

muscle

in

vitro

and

may

contribute

to

vascular

795

796

Jansen et al.

JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 2, NO. 6, 2017 DECEMBER 2017:790–807

Endothelial and Immune Cell EVs in CV Health and Disease

T A B L E 1 Detrimental and Favorable Effects of Extracellular Vesicles on Vascular Function

Effect

EV Type

Isolation Method

Donor Cell/Origin

In Vitro Experiment

In Vivo Experiment

Effects

Mechanisms

Ref. #

Detrimental effects Injured Centrifugation endothelial MVs 20,000 g

Glucose-treated HCAECs

ApoE/ mice

HCAECs

Induce EC inﬂammation

Up-regulate ICAM-1 and VCAM-1 in EC by activating p38

(33)

Endothelial MVs

Ultracentrifugation RMVECs 100,000 g

Aortic rings from rats

—

Impair vasorelaxation

Local oxidative stress

(41)

Circulating MVs

Centrifugation 13,000 g

Patients with MI

Aortic rings from rats

—

Vasomotor dysfunction

Impair endothelial NO transduction pathway

(42)

Erythrocyte MVs

Differential centrifugation

Human packed red blood cells under standard blood banking conditions

Reduce vasoconstrictor effects

Degrade vasodilator NO

(45)

Platelet exosomes

Ultracentrifugation Platelets from ECs 100,000 g septic patients

—

Induce ECs apoptosis

Superoxide; NO and peroxynitrite production

(46)

PMN MVs

Ultracentrifugation PMNs from 100,000 g healthy volunteers

HUVECs

—

Induce ECs activation

Stimulate EC cytokine release Induction of tissue factor

(53)

Oxidized MVs

Ultracentrifugation Oxidatively 100,000 g modiﬁed HUVECs

Monocytes

—

Stimulate monocytes adhesion to ECs

Contain oxidized phospholipids

(56)

Plaque MVs

Centrifugation 20,500 g

—

Promote inﬂammatory response

Carry catalytically activeTNFalpha converting enzyme (TACE/ADAM17) Enhance the processing of TNF-a and TNF receptor

(70)

Monocyte MVs

Ultracentrifugation Human 100,000 g peripheral blood monocytes

—

Induce VSMCs cell death

Deliver cell death message via encapsulated caspase-1

(77)

CD40 ligand plus plaque MPs

Centrifugation 20,500 g

Stimulate endothelial proliferation and angiogenesis

CD40L signaling

(82)

—

Rat vasoactivity models

HUVECs Human atherosclerotic plaques

VSMCs

Human HUVECs atherosclerotic plaques

Wild-type and BalbC/ Nude mice

Continued on the next page

regeneration in vivo (108). Moreover, CD34þ exo-

secrete

somes promoted angiogenesis and preserved cardiac

inﬂammatory release of transforming growth factor

MVs,

which

in

turn

promote

anti-

function in ischemic myocardium by delivery of sonic

(TGF)-b 1 from macrophages. These ﬁndings suggest

hedgehog (109).

MVs are potent anti-inﬂammatory effectors, which at

In summary, EVs derived from endothelium,

an early stage of inﬂammation could contribute to its

platelets, or endothelium-regenerating cells play a

resolution (112). This effect seems to be mediated by

fundamental role by facilitating regenerative pro-

annexin I expression on the surface of these EVs (113).

cesses after vascular or myocardial injury (10,110,111).

EVs are also taken up by monocytes and B cells

Anti-inﬂammatory

through diverse mechanisms and affect target cells

v e s i c l e s . Several

effects

studies

of

have

extracellular reported

anti-

inﬂammatory effects of EVs. Of interest, neutrophils

toward

an

anti-inﬂammatory

phenotype

(114).

Mesenchymal stem cells contribute to inﬂammatory

797

Jansen et al.

JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 2, NO. 6, 2017 DECEMBER 2017:790–807

Endothelial and Immune Cell EVs in CV Health and Disease

T A B L E 1 Continued

Effect

EV Type

Isolation Method

Donor Cell/Origin

In Vitro Experiment

In Vivo Experiment

Effects

Mechanisms

Ref. #

Favorable effects Endothelial apoptotic bodies

Centrifugation 16,000 g

HUVECs

HUVECs

Endothelial MVs

Centrifugation 20,000 g

HCAECs

HCAECs

Platelet MVs

Centrifugation 20,000 g

Platelets

EOCs

Endothelial MVs

Centrifugation 20,000 g

HCAECs

Endothelial MVs

Centrifugation 20,000 g

Exosomes

Mice models of Promote atherosclerosis atheroprotective effects Increase plaque stability Enhance progenitor cells recruitment —

MiRNA-126dependent inhibition of RGS16 Enhance CXCR4 and CXCL12

(27)

Prevent HCAECs apoptosis

Annexin (96) I/phosphatidylserine receptor– dependent inhibition of p38 activation

Mice models of arterial wire-induced injury

Enhance vasoregenerative potential of EOCs

Enhance EOCs recruitment, migration, differentiation Release proangiogenic factors

HCAECs

Electric injury of murine carotid artery

Promote ECs migration and proliferation Accelerate re-endothelialization

Inhibit SPRED-1 (103) via EMV-mediated transfer of miRNA-126

HCAECs

VSMCs

Wire injury of murine carotid artery

Reduce neointima formation Diminished VSMCs proliferation and migration

Inhibit LRP6 via EMPs-mediated transfer of miRNA-126-3p

(104)

Differential centrifugation

CMPCs

HMECs

—

Stimulate HMECs migration

EMMPRIN-mediated

(108)

Exosomes

HPLC

Mesenchymal stem cells

Mice models of myocardial I/R injury

Reduce local and systemic inﬂammation

Restore bioenergetics Reduce oxidative stress Activate pro-survival signaling

(116)

Endothelial MVs

Centrifugation 20,000 g

HCAECs

ApoE-deﬁcient mice

Promote anti-inﬂammatory effects

Reduce endothelial (118) ICAM-1 expression via the transfer of functional miRNA-222

Endothelial exosomes

Centrifugation 20,500 g

KLF2-transduced HASMCs or shear-stressstimulated HUVECs

Aorta of ApoE knockout mice

Atheroprotection

EV-mediated transfer of miRNA-143/145

Circulating MVs

Ultracentrifugation Blood

ApoE-deﬁcient mice

Penetrate the vascular wall Inhibit VSMCs proliferation and migration

miRNA-223-mediated (121) IGF-1R/PI3K-Akt pathway

—

Monocytes

VSMCs

(97)

(120)

ApoE ¼ apolipoprotein E; CMPC ¼ cardiomyocyte progenitor cell; EC ¼ endothelial cell; EMMPRIN ¼ extracellular matrix metalloproteinase inducer; EMV ¼ endothelial MV; HASMC ¼ human aortic smooth muscle cell; HCAECs ¼ human coronary artery endothelial cells; HMEC ¼ human microvascular endothelial cell; HPLC ¼ high-performance liquid chromatography; HUVEC ¼ human umbilical vein endothelial cell; I/R ¼ ischemia/reperfusion; ICAM ¼ intercellular adhesion molecule; KLF ¼ Krüppel-like factor; MI ¼ myocardial infarction; MV ¼ microvesicles; NO ¼ nitric oxide; PMNs ¼ polymorphonuclear leukocytes; RGS16 ¼ regulator of G-protein signaling; RMVEC ¼ rat renal microvascular endothelial cell; SPRED ¼ sprouty-related EVH1 domain-containing protein; TNF ¼ tumor necrosis factor; VSMC ¼ vascular smooth muscle cell.

repression by releasing exosomes that induce secre-

reperfusion injury model resulted in a signiﬁcant

tion of anti-inﬂammatory cytokines such as IL-10 and

reduction of local and systemic inﬂammation after

TGF- b (115). Administration of mesenchymal stem

24 h (116). In a renal ischemia/reperfusion model in

cell-derived exosomes in a myocardial ischemia/

rats, intravenously administered mesenchymal stem

798

Jansen et al.

JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 2, NO. 6, 2017 DECEMBER 2017:790–807

Endothelial and Immune Cell EVs in CV Health and Disease

cell-derived MVs limited inﬂammation as well as

In conclusion, beneﬁcial and detrimental effects of

renal ﬁbrosis (117). Finally, endothelial MVs promoted

EVs have been described in the regulation of vascular

anti-inﬂammatory effects in vitro and in vivo by

health and disease. However, there are no constant

reducing endothelial ICAM-1 expression by the

rules showing a clear relationship between origin and

transfer of functional miRNA-222 into recipient cells

function of EVs. EVs derived under pathological

(118). In line with these data, ECs suppressed mono-

conditions can induce cardiovascular harm (e.g.,

cyte activation through secretion of exosomes con-

plaque EVs promote inﬂammatory response [70]) but

taining anti-inﬂammatory miRNAs (119). Importantly,

also demonstrate atheroprotective functions (e.g.,

despite a large amount of in vitro cellular studies,

MVs from ischemic muscle induce progenitor cell

more in vivo models exploring local and systemic

differentiation [106]). Even the same original EVs can

inﬂammation should be applied to validate and

show both detrimental and favorable effects (e.g.,

conﬁrm the anti-inﬂammatory effects of EVs.

endothelial EVs impairing vasorelaxation on one

Plaque

stabilization

and

antithrombotic

e f f e c t s . miRNA-containing EVs have been shown to promote vascular protection and plaque stabilization through various mechanisms. Injection of miRNA126-3p–enriched

apoptotic

bodies

of

endothelial

origin promoted atheroprotective effects by limiting plaque

size,

increasing

plaque

stability,

and

enhancing progenitor cell recruitment. miRNA-126– dependent inhibition of regulator of G protein

hand [41] and reducing neointima formation on the other hand [104]). In order to clarify the multifaceted character of EVs and make data more comparable, additional efforts should be put into standardized EV generation techniques. Once they are established, indepth exploration of EV-incorporated and transferred biological molecules and their intracellular processing is necessary to gain more clarity in the understanding of EV function.

signaling (RGS16) and subsequent enhancement of

THERAPEUTIC POTENTIAL OF

CXCR4 and CXCL12 was elaborated as the underlying

EXTRACELLULAR VESICLES IN

mechanism (27). Exosomes from KLF-2–transduced or

CARDIOVASCULAR DISEASES

shear-stress–stimulated ECs are enriched in miRNAs 143 and 145. By transferring functional miRNAs, EVs

EXTRACELLULAR VESICLES AS NOVEL THERAPEUTIC

were shown to control target gene expression in

TOOL? EVs have emerged as vectors for transferring

vascular smooth muscle target cells and reduce

biological information by proteins or genetic mate-

atherosclerotic lesion formation in the aorta of

rial,

apolipoprotein E knockout mice. These ﬁndings sug-

favoring

gest that atheroprotective stimuli induce communi-

atherosclerosis.

thereby

maintaining

endothelial

vascular

repair,

or

homeostasis,

even

limiting

cation between ECs and vascular smooth muscle cells

Due to these beneﬁcial effects, there has been a

through miRNA-transferring EVs (120). Similarly,

rising interest in the potential use of EVs as thera-

circulating miRNA-223–containing exosomes could

peutic vectors in the ﬁeld of cardiovascular medicine

penetrate the vascular wall and inhibit vascular

and regenerative therapy. Multiple studies have

smooth muscle cell proliferation and migration,

shown that the transfer of functional miRNAs into

resulting in decreased plaque size (121). Whereas MVs

target tissue by EVs promotes vascular regeneration

are involved in enhancing blood clotting processes,

and

exosomes seem to suppress platelet aggregation and

lighting

occlusive thrombosis by inhibiting platelet CD36,

transferring EVs. In addition to miRNA-containing

inducing antithrombotic effects. However, further

EVs, many reports describing nanoparticles as a

research is needed to validate these ﬁndings in

new

adequate in vivo models and to understand the

miRNAs to recipient cells have been published

opposing roles of exosomes and MVs in this context.

recently (124–126) (Figure 4).

Finally, platelet-derived exosomes reduced CD36-

atheroprotection the

(27,103,119,120,123),

therapeutic

approach

to

potential

transport

of

miRNAs

highmiRNA-

or

anti-

Chen et al. (124) developed miRNA-34a–containing

dependent oxidized low-density lipoprotein binding

liposome-polycationhyaluronic

and macrophage cholesterol loading, potentially

particle for systemic delivery of miRNA-34a into lung

acid

(LPH)

nano-

contributing to atheroprotection (122). Figure 2 illus-

metastasis of murine melanoma, resulting in signiﬁ-

trates the known beneﬁcial effects of EVs in the

cant down-regulation of surviving expression in the

regulation of vascular integrity. Table 1 summarizes

metastatic

the

studies

Furthermore, biodegradable polymer nanoparticles

exploring the effect of EVs on vascular health and

coated with cell-penetrating peptides for an effective

disease.

delivery

most

important

characteristics

of

of

tumors,

as

chemically

well

as

modiﬁed

reduced

tumor.

oligonucleotide

Jansen et al.

JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 2, NO. 6, 2017 DECEMBER 2017:790–807

Endothelial and Immune Cell EVs in CV Health and Disease

F I G U R E 3 Beneﬁcial Effects of Extracellular Vesicles on Vascular Function

An increasing body of evidence points out the beneﬁcial inﬂuence of certain EVs of diverse cellular sources in cardiovascular biology. This ﬁgure illustrates the favorable effects of EVs on endothelial and vascular function, atherosclerosis, and plaque stabilization. Endothelial EVs reduce endothelial apoptosis by inhibition of p38 activity mediated by an annexin I/phosphatidylserine receptor-dependent mechanism, contributing to endothelial protection. Moreover, endothelial EVs decrease endothelial regenerating cell apoptosis, facilitating endothelial repair. Platelet-derived MVs induce alterations in the endothelial-regenerating cell secretome toward a more proangiogenic proﬁle and amplify vascular protection. Among the biological content transferred by EVs into target cells, miRNAs play a crucial role. Endothelial EVs promote vascular endothelial repair by inhibition of SPRED1 by delivering functional miRNA-126. Endothelial EVs promote anti-inﬂammatory effects by reducing endothelial ICAM-1 expression by the transfer of functional miRNA-222 into recipient cells. Exosomes from KLF-2transduced or shear-stress-stimulated endothelial cells attribute to atheroprotection by transferring miRNA-143/145. Circulating leukocyteand platelet-derived miRNA-223-containing exosomes penetrate the vascular wall, inhibit vascular smooth muscle cell proliferation and migration, resulting in decreased plaque size. Endothelial apoptotic bodies decrease vascular smooth muscle cell proliferation, limit plaque size, and increase plaque stability by miRNA-126-dependent inhibition of G-protein signaling (RGS16) pathway. MV ¼ microvesicle; other abbreviations as in Figures 1 and 2.

analogues have been described. This nanoparticle

therapeutic tool to combat cancer, it is reasonable

system was used to block the activity of the onco-

that nanoparticles can also be used to deliver miRNAs

genic miRNA-155, as well as to attenuate the expres-

to recipient vascular cells for tackling inﬂammation

sion of the proto-oncogene Mcl-1, leading to reduced

and development of atherosclerosis (129). In this

cell viability and pro-apoptotic effects in the recipient

context, magnetic nanoparticle-assisted (circumfer-

cells (127). Another approach targeting miRNA-155

ential) gene transfer into the vascular endothelium

described decelerated tumor growth after applica-

has recently been described as a promising novel

tion of polymer nanoparticles containing antisense

strategy to transfer biological messages into the

peptide nucleic acids with subsequent miRNA-155

diseased vasculature (130–132).

inhibition (125). An integrin a v b3-targeted nanoparticle was used by Anand et al. (128) to deliver anti-

TRANSLATION INTO CLINICAL USE. EVs have mul-

miRNA-132 to the tumor endothelium of human

tiple advantages over currently available drug de-

breast carcinoma in mice, causing restored p120Ras-

livery vehicles, such as their ability to overcome

GAP expression in the tumor endothelium, thereby

natural barriers, their intrinsic cell-targeting proper-

suppressing

ties,

angiogenesis

and

decreasing

tumor

protection

of

their

biological

cargo

from

burden. Although these studies focused on miRNA or

degrading enzymes, and stability in the circulation

anti-miRNA delivery using nanoparticles mainly as a

(133). EV subpopulations could be used as a cargo

799

800

Jansen et al.

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Endothelial and Immune Cell EVs in CV Health and Disease

F I G U R E 4 Potential Therapeutic Use of Extracellular Vesicles

Potential therapeutic application of extracellular vesicles includes the following 4 critical steps: 1) Extracellular vesicles can be modiﬁed by using tissue- or cell-type-speciﬁc ligands present on their surface. Endogenously expressed molecules such as miRNA and noncoding RNAs can be genetically engineered for therapeutic use (e.g., genetic modiﬁcation by overexpression therapeutic nucleic acids). 2) Exogenous loading permits the collection of extracellular vesicles with desired cargo molecules. The collection and puriﬁcation of extracellular vesicles can be carried out by various methods, including differential ultracentrifugation, ultraﬁltration, sucrose gradient centrifugation, or immunoprecipitation. 3) Extracellular vesicles, loaded by any of these strategies, can be delivered into target cells or tissues with different delivery methods (e.g., intravenously injection or intracellular injection). 4) The loaded vesicles can function as favorable effectors in intercellular vascular signaling, contributing to the cardiovascular regeneration in damaged tissue.

system for efﬁcient and selective drug delivery to a

direct EVs to target cells or tissue, cell-speciﬁc ligands

distinct cell type within diseased tissues. This

must be stably expressed on the surface of EVs. Using

approach offers the additional advantages of low

an innovative approach of donor cell engineering

immunogenicity because patient-derived tissue could

with target cell-speciﬁc ligand expression resulted in

be used as the source of individualized and biocom-

targeted delivery of short interfering RNA (siRNA)

patible drug delivery vehicles (134,135) (Figure 3).

and miRNA-loaded EVs to target neurons (138) and

Interestingly, tumor cells incubated with chemo-

breast cancer cells (139).

therapeutic drugs are able to package these drugs into

Despite promising perspectives for the treatment

EVs, which can be collected and used to effectively

of cardiovascular pathologies, EV-based therapies

kill tumor cells in murine tumor models without

still need more investigation to translate experi-

typical side effects (136). Moreover, tumor cell-

mental data into clinical application. One challenge

derived EVs were used as a unique carrier system to

would be to control the fragile equilibrium between

deliver oncolytic adenoviruses to human tumors,

the harmful and beneﬁcial effects reported for EVs in

leading to highly efﬁcient cytolysis of tumor cells.

the context of CVD. Furthermore, the off-side effects

These ﬁndings highlight a novel adenovirus delivery

and clearing mechanisms of EVs need to be better

system with promising clinical applications (137).

explored before they can be seriously considered as a

An important issue regarding EV therapeutics is

novel therapeutic tool for combatting CVD (140).

the biodistribution of EVs. Intravenously injected EVs are of particular interest in the treatment of cardio-

STUDY

vascular alterations, as the entire vascular network

OBSTACLES IN EV RESEARCH. Despite the emerging

LIMITATIONS

AND

METHODOLOGICAL

would be exposed to EVs. However, to selectively

role of EVs as regulators of health and disease, there

Jansen et al.

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Endothelial and Immune Cell EVs in CV Health and Disease

T A B L E 2 Overview of Commonly Used EV Isolation Methods

Method

UC

Principle of Separation

Size and density

Advantages

Disadvantages

Widely used

Ref. #

Relatively long procedure Low throughput Depends on viscosity of biological ﬂuids

(143)

DG

Size and density

High purity of EVs

Time-consuming

(145)

Ultraﬁltration

Size

Time efﬁcient Effective to concentrate EVs

Low purity of EVs

(146)

Precipitation kits

PEG-mediated

High yield Rapid

Low purity of EVs

(142,147)

SEC

Size

Quick procedure Reproducibility

Low purity of EVs

(143,148)

Afﬁnity capture

Binding with EVs surface components

Production of subpopulations of EVs Relatively high purity

High cost (antibody-based) May damage surface components of EVs

(143)

DG ¼ density gradient; PEG ¼ polyethylene glycol; SEC ¼ size-exclusion chromatography; UC ¼ ultracentrifugation; other abbreviations as in Table 1.

are still general limitations in EV research. Within this

mainly on PEG. Although the PEG-mediated tech-

paragraph, we highlight relevant obstacles, which

nique provides a high-yield, rapid, and inexpensive

need to be addressed to better understand EV func-

EV isolation method from both culture media and

tions and move the EV ﬁeld forward.

body ﬂuids, some other contaminants are also cop-

M e t h o d o l o g i c a l o b s t a c l e s i n i s o l a t i n g E V s . Iso-

uriﬁed, leading to the low purity of EVs (142,147). In

lation and puriﬁcation of EVs vary between different

the clinical setting, size-exclusion chromatography

research groups and also depend on the donor cells

and afﬁnity capture are the 2 methods most often

from which they are derived. Therefore, EV classiﬁ-

applied to isolate EVs. Size-exclusion chromatog-

cation, isolation, and puriﬁcation needs to be stan-

raphy can remove most soluble components and is a

dardized to ensure that EV analysis is reproducible

relatively quick procedure with good reproducibility

and internationally comparable among different

(148), but it also faces the possible problem of protein

research groups (141). Furthermore, within EV pop-

or RNA contamination (143). Compared with other

ulations, many distinct subtypes of vesicles exist.

methods,

However, the currently used methods (differential

populations of EVs with relatively high purity, but the

ultracentrifugation,

precipitation,

cost of preparation (antibody based) may limit its

density gradients, microﬁltration, size-exclusion–

applicability and may damage surface proteins and

based approaches, or polyethylene glycol [PEG]-

functionality of EVs (143). Therefore, the develop-

mediated isolation techniques) all have different

ment of new, more selective isolation techniques is

pros and cons in attempting to isolate pure EVs with a

urgently needed to increase the purity of each vesicle

distinct size and surface markers (142). The most

subpopulation (149). The pros and cons of each

commonly used isolation methods are differential

available method are summarized in Table 2.

centrifugation, followed by ultracentrifugation. Two

Size measurement techniques for EV characterization. Size

polymer-based

afﬁnity

capture

can

produce

sub-

major problems with these techniques are the rela-

is an important deﬁning property of EVs, and mea-

tively long procedure times and low throughput,

surement of diameter to determine a size distribution

limiting their application in the clinical setting (143).

is a critical step for EV studies. Size distribution

Moreover, the yield depends on the viscosity of bio-

measurement technologies include electron micro-

logical ﬂuids, so the samples with a relatively high

scopy

viscosity such as plasma would signiﬁcantly reduce

tracking analysis, resistive pulse sensing, and atomic

production (144). Density gradient isolation can pro-

force microscopy (AFM). Although these techniques

mote both the yield and purity of EVs compared with

are commonly used in practice (142), some issues are

ultracentrifugation; however, the procedure is time-

still unsolved. First, the ideal size measurement

consuming and hard to standardize (145). Ultraﬁltra-

should detect EVs with a diameter of 50 nm and larger

tion is time-efﬁcient and can concentrate EVs up to

(150), but most methods, except for EM, cannot detect

240-fold, but the low purity of EVs is an obstacle

the smallest EVs (151). Second, the results of size

(146). Commercial EV precipitation kits are based

distribution, even for the same EV subpopulation,

(EM),

ﬂow

cytometry

(FC),

nanoparticle

801

802

Jansen et al.

JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 2, NO. 6, 2017

Endothelial and Immune Cell EVs in CV Health and Disease

DECEMBER 2017:790–807

C E N T R A L IL L U ST R A T I O N Extracellular Vesicles as Regulators of Vascular Health and Disease

Jansen, F. et al. J Am Coll Cardiol Basic Trans Science. 2017;2(6):790–807.

Many types of cells release EVs, such as exosomes and microvesicles, by different mechanisms. EVs have both favorable and detrimental effects on vascular integrity. The use of genetically modiﬁed EVs might represent a novel therapeutic tool in the ﬁeld of cardiovascular medicine and regenerative therapy. EV ¼ extracellular vesicles; miRNA ¼ microRNA.

Jansen et al.

JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 2, NO. 6, 2017 DECEMBER 2017:790–807

Endothelial and Immune Cell EVs in CV Health and Disease

may show different results depending on the method

such as exosomes can be incorporated into recipient

used (152). Furthermore, there are no standard pro-

cells (160).

tocols (e.g., optimal EM-EV measurement protocols)

Lack

(142).

as

i n v i v o . The secretion of EVs by parent cells and

Importantly,

some

nanoparticle-tracking

methods,

analysis

or

such

resistive

of

EV

secretion

and

uptake

models

pulse

uptake by recipient cells are precisely regulated.

sensing, cannot distinguish membrane vesicles from

However, few data are available studying concrete

nonmembranous particles of similar size, so the re-

mechanisms regulating MV release and clearance.

sults should be compared by using EM, AFM, or other

Moreover, physiological models are not well estab-

microscopy technique (153).

lished (e.g., considering time-dependent release ki-

R e g u l a t i o n o f E V p a c k a g i n g . Although some key

netics of EVs from parent cells). Finally, the absence

players in sorting of cargoes into EVs are revealed

of adequate in vivo models to explore the genera-

(e.g., sumoylated hnRNPA2B1 is reported to control

tion and uptake of cell-speciﬁc EVs limits the

the location of miRNAs into exosomes through bind-

experimental opportunities to study EV functions

ing to speciﬁc motifs [154]), the underlying mecha-

in vivo.

nisms mediating the packaging and loading of

Unclear processing of EVs and their content

selected molecules into EVs remain largely unknown.

a f t e r c e l l u l a r u p t a k e . EVs interact with recipient

Therefore, additional studies exploring EV biogenesis

cells

and mechanisms regulating EV packaging are impor-

through membrane fusion in a ligand-receptor-

tant to understand cardiovascular injury and repair

mediated way or by endocytosis, pinocytosis, or

induced by EVs (155).

phagocytosis (12) (Figure 1). However, processing of

by

transferring

their

biological

contents

experiments

incorporated EVs and their intravesicular content

in vitro. EV uptake experiments are usually performed

after cellular uptake is entirely unknown. To address

Methodological

issues

on

EV

uptake

by direct visualization. Therefore, ﬂuorescent lipid

this point, tracking experiments using ﬂuorescence

membrane dyes, such as PKH26 (156), PKH68 (157),

labeleling represent a possible option to gain further

or rhodamine B (158), are used to stain EV mem-

insight

branes. One potential issue with membrane-binding

mechanisms of biological contents transferred by

dyes is that ﬂuorescent molecules could potentially

EVs (161). Nevertheless, stable labeling of intra-

affect the uptake and biological behavior of EVs.

vesicular contents is highly demanding technically,

However, EV incorporation has been observed with

and further efforts are needed to improve these

many different lipid-binding dyes, suggesting that

techniques (162).

into

time-dependent

cellular

processing

such molecules do not affect internalization of vesi-

Taken together, there are still serious methodo-

cles; nevertheless, additional studies are needed to

logical issues that need to be addressed. Importantly,

verify whether the biological behavior of EVs is

recent position research papers from the Interna-

affected by dyes. Another potential limitation of the

tional Society of Extracellular Vesicles have made a

use of lipophilic dyes is leaching of the ﬂuorescent

ﬁrst step in the right direction to standardize EV

molecules

membranes,

analysis internationally among different laboratories

potentially leading to a pattern of internalization

(141,147,153). In addition, EV-TRACK (163), a novel

that is due to normal membrane recycling rather

crowd-sourcing database, has recently been imple-

than EV uptake. However, direct measurement of the

mented, centralizing knowledge about EV biology

ﬂuorescence transfer rate between EVs and recipient

and methodology with the goal of stimulating

cells support the idea that the increased ﬂuorescence

authors, reviewers, editors, and funders to put

in cells is due to speciﬁc uptake of EVs rather than

experimental guidelines into practice (164).

from

EVs

into

cellular

nonspeciﬁc dye leaching (159). Another issue which must be considered is the fact that most EV uptake

CONCLUSIONS

studies have relied on ﬂuorescence microscopy, which has limited resolution because the wavelength

In the regulation of cardiovascular health and dis-

of visible light is approximately 390 to 700 nm;

ease, EVs act as urgent effectors by transferring

therefore, single EVs or aggregated vesicles, which

bioactive molecules into adjacent and distant re-

are <390 nm in diameter, cannot be distinguished.

cipients. EV-mediated intercellular vascular signaling

This should not affect the assessment of EV uptake

results in detrimental and favorable effects on

in general but may affect the visualization and dy-

vascular integrity. Studies illustrate that EVs can

namic

EVs.

contribute to atherosclerosis development and pro-

Nevertheless, the increasing use of confocal micro-

gression. In contrast, EVs also emerge as crucial

scopy has conﬁrmed that EVs smaller than 390 nm

regulators of vascular homeostasis and mediate

localization

analysis

of

individual

803

804

Jansen et al.

JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 2, NO. 6, 2017 DECEMBER 2017:790–807

Endothelial and Immune Cell EVs in CV Health and Disease

vascular protection. Finally, the use of genetically

53105

modiﬁed EV might represent a novel therapeutic tool

ukbonn.de. OR Dr. Nikos Werner, Medizinische Kli-

in the ﬁeld of cardiovascular medicine and regener-

nik und Poliklinik II, Universitätsklinikum Bonn,

ative therapy (Central Illustration).

Sigmund Freud Strasse 25, 53105 Bonn, Germany.

Bonn,

Germany.

E-mail:

[email protected]

E-mail: [email protected] OR Dr. Qian Li, ADDRESS FOR CORRESPONDENCE: Dr. Felix Jansen,

Department of Cardiology, Second Hospital of Jilin

Medizinische

University, 218 Ziqiang Street, Nanguan District,

Klinik

und

Poliklinik

II,

Uni-

versitätsklinikum Bonn, Sigmund Freud Strasse 25,

Changchun 130000, China.

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KEY WORDS cardiovascular disease, extracellular vesicles, microvesicles

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