Received: 4 May 2016 | Revised: 30 July 2016 | Accepted: 8 August 2016

DOI: 10.1111/1440-1681.12649

INVITED REVIEW

Autophagy in vascular endothelial cells

Fan Jiang

Department of Pathology and

Pathophysiology, School of Basic
Medicine, Shandong University, Jinan,
Shandong Province, China
Correspondence
Fan Jiang, Department of Pathology and
Pathophysiology, School of Basic Medicine,
Shandong University, Jinan, Shandong
Province, China.
Email: fjiang@sdu.edu.cn
Funding information
National Natural Science Foundation of
China, Grant/Award Number: 31471087
and 91539102
Summary
The importance of autophagy in cardiovascular physiology and cardiovascular disease
is increasingly recognized; however, the precise biological effects and underlying
mechanisms of autophagy in the cardiovascular system are still poorly understood. In
the last few years, the effects of autophagy in endothelial cells have attracted great
interests. This article provides a summary of our current knowledge on the regulatory
factors, signalling mechanisms, and functional outcomes of autophagy in endothelial
cells. It is suggested that in most situations, induction of an autophagic response has
cytoprotective effects. The beneficial effects of autophagy in endothelial cells are
likely to be context-­dependent, since autophagy may also contribute to cell death
under certain circumstances. In addition to regulating endothelial cell survival or death,
autophagy is also involved in modulating other important functions, such as nitric
oxide production, angiogenesis and haemostasis/thrombosis. The mounting data will
help us draw a clear picture of the roles of autophagy in endothelial cell biology and
dysfunction. Given the pivotal role of endothelial dysfunction in the pathogenesis of
vascular disease, disruptions of autophagy in endothelial cells are likely to have signifi-
cant contributions. This is supported by some preliminary ex vivo data indicating that
compromised autophagic functions may be important in the development of endothe-
lial dysfunctions associated with diabetes and ageing.

KEYWORDS
autophagy, cell death, cell survival, endothelial cell, stress response

1 | INTRODUCTION energy, thereby maintaining metabolic homeostasis and ­enabling

Autophagy is a highly conserved biological phenomenon, entailing
the formation of membranous intracellular vesicles, and subsequent
engulfment and delivery of various cellular components (proteins,
­organelles, and invading pathogens) to lysosomes for degradation
and material recycling. Readers are referred to some comprehensive
reviews on the basic molecular mechanisms of autophagy and its
­potential implications in various diseases.1–8 It is thought that in most
cells, a basal level of autophagy occurs constantly, which is essential
for maintaining the health of the cell by clearing misfolded proteins
and damaged organelles (for example dysfunctional mitochondria
and ribosomes). An increased autophagic response can be elicited by
nutrient deprivation. In this case, autophagy facilitates digestion of
existing cell components and redistribution of the biomaterials and
adaptation to the stress. However, autophagy is not merely a stress
response to metabolic perturbations; indeed, autophagy can be in-
duced by a variety of cellular stressors, such as hypoxia, reactive
oxygen species (ROS), DNA damage, and aggregation of misfolded
proteins.4,9 Mounting evidence has suggested that there are com-
plex cross-­talks between autophagy and other stress pathways;
hence autophagy may have an integral role in orchestrating the reg-
ulatory network involved in cellular stress response.4 Likewise, nu-
merous studies have indicated that disturbed autophagic responses
may underlie the pathogenesis of various diseases, including neuro-­
degeneration, metabolic disease, kidney disease, inflammation, can-
cer, as well as cardiovascular disease.7,8,10,11 Vascular endothelial cells
have a pivotal role in maintaining normal functions of the cardiovas-
cular system, while endothelial dysfunction has been recognised as a

Clinical and Experimental Pharmacology wileyonlinelibrary.com/journal/cep © 2016 John Wiley & Sons Australia, Ltd | 1021
and Physiology 2016; 43: 1021–1028
1022 | Jiang
common risk factor for virtually all kinds of cardiovascular diseases.
Our understanding on the importance of autophagy in endothelial cell
biology is still incomplete.7 Recent studies have demonstrated that
the autophagic process in vascular endothelial cells can be regulated
by a range of biological factors and chemical compounds, and may
have significant impacts on the fate of endothelial cells. This article
provides a summary of our current knowledge on the regulatory fac-
tors, signalling mechanisms, and functional outcomes of autophagy
in endothelial cells.
2 | OVERVIEW OF THE MECHANISMS OF THE
AUTOPHAGIC RESPONSE
The predominant form of autophagy is macroautophagy (referred to
as autophagy thereafter), in which cytosolic proteins and broken orga-
nelles are enclosed in double-­membrane vesicles (called autophago-
somes) and delivered to lysosomes for degradation. Autophagy is
initiated by the formation of autophagosome precursors (phago-
phores). This process is triggered via two routes: (i) activation of the
ULK1 (unc-­51 like autophagy activating kinase 1 or Atg1) complex; and
(ii) formation of a protein complex containing the class III phosphati-
dylinositol 3-­kinase (PI3K) Vps34 and its positive modulator beclin-
­1 (Atg6). Under normal conditions, the activity of ULK1 is ­repressed
by the mammalian target of rapamycin complex 1 (mTORC1). Under
stress conditions such as nutrient deprivation, mTORC1 is inacti-
vated, thereby ULK1 is activated. The downstream effectors and
mechanisms of ULK1 for autophagy initiation are not totally clear; it is
1–5,12
known that the ULK1 complex can facilitate activation of Vps34.
On the other hand, the autophagy inducing function of the beclin-­1/
Vps34 complex is not strictly dependent on ULK1, since overexpres-
sion of beclin-­1 alone is sufficient to stimulate autophagy.13 The activ-
ity of Vps34 is dependent on beclin-­1, and accumulating evidence has
suggested that the overall function of the beclin-­1/Vps34 complex
can be further regulated through post translational modifications such
as phosphorylation and ubiquitination.14
The expansion of autophagophore is mediated by two ubiquitin-­
like protein conjugation systems. The first pathway starts from Atg12,
through activities of Atg7 and Atg10, and finally results in formation
of the Atg12-­Atg5-­Atg16L complex. The second pathway starts from
the unconjugated form of LC3 (named LC3-­I) (Atg8), through sequen-
tial reactions facilitated by Atg7 and Atg3, leading to formation of the
phosphatidylethanolamine-­conjugated (lipidated) form of LC3 (named
LC3-­II), which is a specific marker of the autophagosome membrane.
The Atg12-­Atg5-­Atg16L1 complex and the lipidated LC3 play essential
1,2,4,12
roles in the elongation and closure of the vesicular membrane.
Mature autophagosomes finally fuse with lysosomes, forming autoly-
sosomes. This process is crucial for clearance of existing autophago-
somes and maintenance of a normal autophagic flux. The mechanisms
of the fusion step in mammalian cells are not clearly understood; there
is evidence suggesting that the small GTPase Rab7 is required for this
process.6 Lysosomal acidification via the lysosomal H+-­ATPase is also
2
required for a proper fusion function.
In addition to macroautophagy, there are other two types of auto-
phagy, namely microautophagy and selective autophagy. In microau-
tophagy, the lysosomes directly engulf cytosolic components through
inward invaginations on the lysosomal membrane.5 Selective autoph-
agy is mediated by specific autophagy receptor proteins, such as p62/
SQSTM1, NBR1 and NDP52, which on one hand can bind to LC3 on the
autophagosome membrane, and on the other hand recognise various
cargoes via ubiquitin-­dependent15 as well as ubiquitin-­independent
signals.5,16 In this article, we will only discuss macroautophagy.
3 | AUTOPHAGY INDUCTION IN
ENDOTHELIAL CELLS
In normally cultured endothelial cells, there is a basal level of
­autophagic flux, since treatment with chloroquine, which blocks lyso-
somal acidification and clearance of the formed autophagosomes,
significantly increases the accumulation of LC3 puncta (a marker of
autophagosomes) in the cytosol.17 Recent studies have shown that
autophagic responses can be induced in endothelial cells by a vari-
ety of naturally occurring compounds with recognized cardiovascular
protective effects. These include epigallocatechin gallate18 and cur-
cumin,19 two polyphenol antioxidants. Curcumin-­induced autophagy
exhibits cytoprotective effects on endothelial cell viability in the pres-
ence of oxidative stress.19 Induction of autophagy by epigallocatechin
gallate attenuates palmitic acid-­induced accumulation of lipid drop-
lets, presumably via increased lipid catabolism by the autophagosome-­
lysosome system.18 Autophagy can also be induced by analogues of
resveratrol, namely pterostilbene and dimethoxystilbene.20,21 While
pterostilbene exerts an anti-­apoptotic effect via autophagy induc-
tion, dimethoxystilbene per se triggers apoptosis in endothelial cells.
However, concomitant inhibition of autophagy augments the pro-­
apoptotic effect of dimethoxystilbene, supporting that the autophagic
response likewise has a cytoprotective action in dimethoxystilbene-­
treated cells. Moreover, it has been demonstrated that the mTOR
inhibitor rapamycin at a concentration range of 100-­1000 nmol/L
counteracts oxygen/glucose deprivation-­induced cell death, and this
effect is abolished by autophagy inhibitors.22 These results collec-
tively suggest that induction of an autophagic response in endothelial
cells exerts cytoprotective actions under stress conditions.
Autophagy in endothelial cells is also regulated by cardiovascular
risk factors. Treatment of different endothelial cell lines with oxidized
low-­density lipoprotein (oxLDL) triggers autophagic responses.23,24
Fluorescence labelling experiments have demonstrated that intracel-
lular oxLDL aggregates co-­localise with the autophagosome marker
LC3 and the lysosome marker LAMP2a.24 Interestingly, several lines
of evidence suggest that increased autophagic flux in endothelial cells
accelerates the catabolism and clearance of lipid substrates,18,20,24
which may prevent lipid overload-­induced endothelial toxicity. Like
oxLDL, advanced glycation end-­products (AGEs) are also implicated in
promoting endothelial cell damage and dysfunction found in diabetes
mellitus and atherosclerosis. Xie et al.25 have shown that incubation of
endothelial cells with AGE increases the formation of autophagosomes
Jiang | 1023

and the expression of LC3-­II, and this response is mediated by produc-

tion of ROS. In addition, inhibition of autophagy with 3-­methyladenine 4 | REGULATION OF ENDOTHELIAL
worsens AGE-­induced cell death, supporting a cytoprotective role of AUTOPHAGY BY MECHANICAL
autophagy in AGE-­challenged endothelial cells.25 Other cardiovascu- SHEAR STRESS
lar risk factors that can modulate endothelial autophagy include lipo-
polysaccharide (LPS),26 cigarette smoke extract27 and ROS.28 Wang Shear stress is the mechanical force caused by the sliding of the blood
28
et al. have shown that increased ROS generation by the glycolysis on the surface of the endothelium. Shear stress has pivotal roles in
inhibitor 2-­deoxy-­d-­glucose, or direct stimulation with exogenous modulating biological functions of endothelial cells including gene
H2O2, can elicit autophagy in endothelial cells, mainly by activating expression, proliferation, migration, morphogenesis, permeability,
AMP-­activated protein kinase (AMPK); this autophagic response is thrombogenicity, and inflammation.29,30 Endothelial cells transduce
associated with enhanced cell survival under stress conditions. In an- the mechanical signals through a network of multiple messenger
other study, it has been demonstrated that cigarette smoke extract molecules and signalling proteins, which is still not completely un-
induces both of unfolded protein response and autophagy in endo- derstood.30 It is thought that unidirectional wall shear stress with a
thelial cells.27 In contrast to the cytoprotective actions of autophagy relatively high magnitude (eg, that produced by laminar blood flow in
as evidenced by the above studies, however, cigarette smoke extract-­ straight parts of the arterial tree), is vascular protective. In contrast,
induced autophagy appears to be detrimental, since treatment with shear stress associated with disturbed blood flow (eg, low shear stress
the autophagy inhibitor 3-­methyladenin or knocking down Atg5 and oscillatory shear stress found in areas with branches or curva-
expression significantly delays the cell death. LPS is an important tures) is pro-­atherogenic.29,30
exogenous proatherogenic factor which induces endothelial inflam- Recently, our group and several other groups have independently
mation and dysfunction. LPS triggers endothelial autophagy, only in reported that the autophagic response in endothelial cells is regulated
cells maintained in a normal proliferating status (cultured in the pres- by shear stress.17,31–33 These studies have shown that laminar shear
ence of 20% serum);26 LPS-­induced autophagy has been shown to be stress increases endothelial autophagy as assessed by LC3 puncta
mediated by the expression of the apoptosis inhibitor protein BIRC2 morphology, LC3-­II formation, and expression of a variety of auto-
(cIAP1).26 However, in LPS-­treated endothelial cells, autophagy seems phagic genes; our data also indicate that this response is reversible
to have an injurious role. Therefore, autophagy appears to have di- since cessation of the flow stimulation in flow-­adapted cells reduces
chotomous effects on endothelial cell viability in response to different the level of autophagy. Moreover, we have confirmed that the increase
cardiovascular risk factors. The different stimuli with autophagy induc- in autophagosome abundance in response to shear stress is not due
ing activities in endothelial cells, and their effects on cellular functions to impaired autophagosome clearance, since flow-­induced autoph-
are summarized in Table 1. agy persists after pre-­treatment with bafilomycin A1, which blocks
lysosomal acidification and subsequent fusion with autophagosomes.
T A B L E 1 Stimuli with autophagy inducing activities in endothelial Functionally, shear stress-­induced autophagy is associated with multi-
cells and their effects on cellular functions ple beneficial effects, including increased cell viability against oxidative
Autophagy inducing Cellular effects of the insults, increased endothelial nitric oxide synthase (eNOS) expression
stimuli autophagic response References and nitric oxide bioavailability, reduced inflammatory reactions, and
Epigallocatechin gallate Increased catabolism and 18 reduced oxidative stress.17,31–33 Our study suggests that shear stress-­
clearance of intracellular induced autophagy is mediated by the Sirt1-­FoxO axis (discussed
lipid substrates below). The data by Yao et al.31 indicate that expression of the small
19
Curcumin Cytoprotection against GTPase Rab4 is also involved in shear stress-­induced autophagy in
oxidative stress
endothelial cells.
20,21
Analogues of resveratrol Anti-­apoptosis; increased The effects of the “bad” shear stress, ie, low shear stress and oscilla-
pterostilbene and clearance of lipid
tory shear stress, on endothelial autophagy appear to be controversial.
dimethoxystilbene) substrates
22 Yang et al. demonstrated that levels of the autophagic markers beclin-­1
Rapamycin Inhibition of oxygen/
glucose deprivation-­ and LC3-­II were lower under a low shear stress condition (5 dyn/cm2)
induced cell death as compared to those under the “physiological” shear stress (15 dyn/
Oxidized low-­density Prevention of lipid 23, 24 cm2),34 and the impaired autophagic function under low shear was as-
lipoprotein overload-­induced cell sociated with reduced expression of eNOS and increased expression of
toxicity endothelin-­1. These data are consistent with our observation that low
25
Advanced glycation Cytoprotection shear or oscillatory shear fails to stimulate significant autophagic re-
end-­products
sponses in endothelial cells.17 On the other hand, Ding et al.35 reported
26
Lipopolysaccharide ? that low shear stress (3 dyn/cm2) could enhance endothelial autophagy,
27
Cigarette smoke extract Promoting cell death as evidenced by the increased expression of LC3-­II, while increasing the
28
Reactive oxygen species Enhanced cell survival magnitude of shear stress resulted in a gradual decline of autophagy.
under stress conditions
The reason(s) for these discrepant observations is not clear, but may
1024 | Jiang
be related to different experimental settings. Interestingly, it has been
noted that in the aortic arch, which is continuously exposed to tur-
bulent or oscillatory shear stress, the level of the autophagy receptor
protein p62 is upregulated, indicating an impaired autophagic flux.36 In
cultured endothelial cells, although oscillatory shear stress significantly
increases the abundance of LC3-­II and the number of autophagosome,
these effects appear to be primarily due to blockade of the end stage
autophagosome clearance, but not an enhanced induction of the auto-
phagic response. In this case, the increased autophagosome accumu-
lation to oscillatory shear stress is associated with increased oxidative
stress, while further induction of autophagy by starvation or rapamycin
normalises the disturbed redox homeostasis.36
5 | SIGNALLING PATHWAYS INVOLVED
IN MEDIATING AUTOPHAGY IN
ENDOTHELIAL CELLS
As mentioned earlier, the mTORC1-­ULK1 pathway is a master regu-
lator responsible for autophagy induction, especially in response to
metabolic stresses such as amino acid starvation and growth factor
9
deprivation. In addition to this pathway, endothelial autophagy is also
under the control of other signalling mechanisms. AMPK is an endog-
enous energy sensor, which is activated when ATP production is re-
duced and AMP concentration is elevated; the AMPK pathway is also
tightly involved in modulating autophagy. AMPK can phosphorylate
and activate TSC2 (tuberous sclerosis 2), leading to the inhibition of
9
mTORC1. AMPK can also directly phosphorylate ULK1 and beclin-­1,
thereby activate the autophagy machinery.37,38 In line with these no-
tions, several studies have demonstrated that AMPK has a crucial role
in mediating autophagy in endothelial cells in response to metabolic
20,21,28
stress or treatments with natural cytoprotective compounds.
Moreover, AMPK in endothelial cells also mediates autophagy induc-
tion induced by decorin, a small extracellular matrix proteoglycan
molecule.39 In a separate study, treatment of endothelial cells with
high concentrations of glucose and fatty acids diminished AMPK
­activity and the phosphorylation of ULK1, which were accompanied
by ­impaired basal autophagy; these changes increased cell apoptosis
and inflammation.40 Intriguingly, reactivation of AMPK with chemi-
cal ­activators AICAR or phenformin failed to restore ULK1 phospho-
rylation or autophagy under these stress conditions. The authors
suggest that AMPK becomes uncoupled from autophagy under over-­
nutrition-­induced stress, which may contribute to the pathogenesis of
endothelial dysfunctions in patients with metabolic syndrome.40
Another signalling pathway that mediates autophagy is the Sirt1-­FoxO
+
axis. The NAD -­dependent deacetylase Sirt1 has been shown by recent
studies to have a pivotal role in modulating stress responses in mamma-
lian cells, including autophagy.41–44 The FoxO family transcription factors,
such as FoxO1 and FoxO3A, are major down-­stream targets of Sirt1,
which have critical roles in cell fate determination and stress adaptation.45
Functions of FoxOs are negatively modulated by Akt-­induced phosphor-
ylation, whereas deacetylation of FoxOs by Sirt1 overrides the inhibitory
effect of Akt, leading to FoxO activation.46 It is well documented that
FoxOs are important regulators of autophagy, and FoxO may induce auto-
phagy by driving the expression of several autophagy-­related genes.42–44
Moreover, it has been shown that FoxOs can directly modulate the tran-
scription of autophagic genes by binding to their promoter regions.43,44
In our study, we have provided evidence supporting that Sirt1-­mediated
FoxO1 activation is involved in mediating shear stress-­induced endothe-
lial autophagy.17 In addition to FoxO activation, Sirt1 may also directly
deacetylate Atg5 and Atg7, and this effect of Sirt1 is presumed to partic-
ipate in promoting autophagy.41,47 Our experiments have confirmed that
shear stress-­induced Sirt1 upregulation is accompanied by reduced Atg5
and Atg7 acetylation; however, it remains to be clarified whether Sirt1-­
dependent deacetylation of Atg proteins has a causal role in shear-­ and
other stimuli-­induced autophagy in endothelial cells.
In endothelial cells, several studies have pointed to a critical role
of calmodulin-­dependent protein kinase kinase-­β (CaMKK-­β) in me-
diating autophagy.18,20 For example, knockdown of CaMKK-­β blunts
the autophagic response induced by epigallocatechin gallate in bo-
vine aortic endothelial cells; CaMKK-­β-­mediated autophagy is likely
to be secondary to the increase in intracellular Ca2+ concentration.18
CaMKK is the upstream kinase of Ca2+/calmodulin-­dependent protein
kinases I and IV, as well as AMPK. It has been shown that in HeLa cells,
amino acid starvation results in an increase in cytosolic [Ca2+] and sub-
sequent activation of CaMKK-­β, which in turn stimulates the AMPK
activation and autophagy induction.48 Alternatively, increased cyto-
solic [Ca2+] may also stimulate the Ca2+-­calmodulin-­regulated kinase
DAPK (death-­associated protein kinase), leading to phosphorylation
and activation of beclin-­1 and Vps34.49
The intracellular redox status may have significant impacts on the pro-
cess of autophagy. In endothelial cells, the autophagic response induced
by various stimuli has been linked to intracellular redox changes and
can be suppressed by antioxidants.17,25,28,50–52 Conversely, ­incubation
of ­endothelial cells with hydrogen peroxide can mimic the autophagy-­
inducing effects of these stimuli.17,28 The precise signalling mechanisms
of the redox-­dependent regulation of autophagy are still poorly defined,
while among the key regulators of autophagy, AMPK has been proposed
to be a potential mechanistic link between ROS and autophagy.53 Indeed,
there is some evidence suggesting that ROS-­dependent activation of
AMPK may explain the induction of endothelial autophagy by metabolic
stressors and by chemerin, an endogenous adipokine.28,51 Apart from
AMPK, the expression level of Sirt1 has been shown to be regulated by
ROS.54,55 We have shown that ROS-­dependent upregulation of Sirt1
may stimulate autophagy in endothelial cells,17 supporting the notion
that Sirt1 has a critical role in regulating autophagy in the presence of
oxidative stress.56 The above signalling mechanisms involved in mediat-
ing autophagy in endothelial cells are illustrated in Figure 1.
6 | FUNCTIONAL OUTCOMES OF
AUTOPHAGY INDUCTION IN ENDOTHELIAL
CELLS
Generally, autophagy is thought to be a pro-­survival stress response
that helps cells to maintain intracellular metabolic homeostasis under
Jiang
F I G U R E 1 Signalling mechanisms involved in mediating
autophagy in endothelial cells. mTORC1, mammalian target of
rapamycin complex 1; ULK1, unc-­51 like autophagy activating kinase
1; AMPK, AMP-­activated protein kinase; TSC2, tuberous sclerosis
2; CaMKK-­β, calmodulin-­dependent protein kinase kinase-­β; DAPK,
death-­associated protein kinase; ROS, reactive oxygen species; Ac,
acetylation; [Ca2+]i, intracellular calcium concentration
stress conditions. Depending on the cellular and environmental context,
however, autophagy may also be involved in promoting cell death.57
Consistent with this notion, most of the current literatures support
that an elevated autophagic response in vascular endothelial cells
­favours enhanced stress tolerance and cell survival. However, in cer-
tain circumstances, autophagy induction may also promote endothelial
cell death. For example, treatment of human endothelial cells with the
­endogenous angiogenic inhibitor endostatin induces autophagy as well
as cell death, which is insensitive to caspase inhibitors, but is suppressed
by the autophagy inhibitor 3-­methyladenine.58 Similarly, inhibition of
autophagy either by pharmacological inhibitors or by genetic silencing
of autophagy-­specific genes effectively prevents endothelial cell death
59
triggered by different stressors, including fatty acid overload, simu-
lated ischemia/reperfusion,52 and the dihydropyridine anti-­hypertensive
drug nicardipine hydrochloride.60 More interestingly, Chen et al.61 have
provided evidence showing that in endothelial cells challenged with
hypoxia, there is a transition from autophagy-­mediated cell survival to
autophagy-­mediated cell death in a time-­dependent manner.
The Nomenclature Committee on Cell Death recommends a defi-
nition of “autophagic cell death” being cell death events that can be
suppressed by inhibition of the autophagic pathway either by phar-
macological inhibitors or by knockout/knockdown of core auto-
phagic genes (such as Atg5/12 and beclin-­1).57 It is recognised that
autophagic cell death is a biological process distinct from apoptosis
and necrosis. However, the mechanism of execution of autophagy-­
induced cell death is not understood. An over-­simplified explanation
is that ­autophagic cell death is caused by the excessive degradation
of essential cellular components that are required for normal cellular
functions;53 however, emerging evidence suggests that orchestrated
signalling events are likely to participate in autophagic cell death. Using
a cell-­penetrating autophagy-­inducing peptide derived from beclin-­1,
| 1025
it has been shown that high levels of autophagy do result in cell death,
which is not blocked by apoptosis or necroptosis inhibitors.62 A term
“autosis” is therefore coined to reflect the unique morphological fea-
tures associated with this type of cell death, including increased auto-
phagosomes/autolysosomes, nuclear convolution, and focal swelling of
the perinuclear space. Interestingly, these authors have discovered that
autosis is sensitive to inhibition of Na+/K+-­ATPase with pharmacologi-
cal antagonists,62 suggesting a crucial role for Na+/K+-­ATPase in medi-
ating autophagic cell death. In addition to inducing cell death directly,
autophagy also cross-­talks with apoptosis. Under most circumstances,
autophagy and apoptosis are mutually exclusive, ie, autophagy blocks
the induction of apoptosis, and vice versa.57,63 However, in some cases,
autophagy may be able to promote cell death by enhancing apopto-
sis.64 Nevertheless, these mechanistic aspects of autophagy-­related
cell death have not been studied in detail in endothelial cells.
7 | EFFECTS OF AUTOPHAGY ON OTHER
FUNCTIONS OF ENDOTHELIAL CELLS
In addition to the regulation of endothelial cell survival or death,
­autophagy is also involved in modulating other important endothelial
functions. As mentioned above, several studies have suggested that
there is a correlation between autophagy and the endothelial NO
function. It has been shown that autophagy induction is associated
with increased eNOS expression,32 whereas decreased autophagy is
accompanied by down-­regulation of the eNOS expression.34 How
­autophagy couples to eNOS expression is unknown. It appears that
a normal (or enhanced) autophagic flux is essential for maintaining
normal eNOS functions, since co-­existence of autophagy induction
and autophagosome clearance blockade is associated with reduced
eNOS activity and NO production.65 This argument is supported by
another study carried out in freshly isolated endothelial cells from
diabetic ­patients. Fetterman et al.66 demonstrated that, as compared
to endothelial cells from healthy controls, diabetic endothelial cells
exhibited an impairment in insulin-­induced eNOS activation, together
with a higher level of p62 (indicating a reduction in autophagosome
clearance); on the other hand, the number of LC3-­bound puncta and
beclin-­1 expression were similar in diabetic and control cells, indicat-
ing that the eNOS dysfunction was related to reduced autophagic
flux, but not autophagy induction. Moreover, inhibition of autophagic
flux with bafilomycin in normal endothelial cells mimicked the eNOS
dysfunction. These data suggest that maintaining an intact autophagic
flux, but not merely autophagy induction, is critical for correcting
hyperglycaemia-­induced eNOS dysfunctions.66
Autophagy may also have significant impacts on the angio-
genic functions of endothelial cells. In bovine aortic endothelial
cells, inhibition of autophagy by 3-­methyladenine or Atg5 silenc-
ing ­reduces the angiogenic functions (tube formation and cell
­migration), whereas induction of autophagy by Atg5 overexpression
­enhances these functions.67 This pro-­angiogenic effect of autoph-
agy ­appears to be related to redox-­dependent activation of Akt,
but not to alterations in the production of pro-­angiogenic factors
1026 | Jiang

such as vascular endothelial growth factor, platelet-­derived growth

8 | CONCLUDING REMARKS
factor, or integrins.67 Similar pro-­angiogenic effects of autophagy
have also been observed in human endothelial cells treated with
The importance of autophagy in cardiovascular physiology and disease is
a resveratrol ­analogue or chemerin (an adipokine associated with
increasingly recognised; however, our understanding on the precise bio-
obesity and metabolic syndrome).21,51 In human microvascular en-
logical effects and underlying mechanisms of autophagy in the cardio-
dothelial cells, autophagy has been shown to have a synergistic
vascular system is far from complete.7,74 Strategies targeting autophagy
effect with high mobility group box 1, a non-­histone chromosome
to treat various cardiovascular diseases remain in a hypothetical stage.
associated protein involved in regulating nucleosome stability, to
In the last few years, the effects of autophagy in endothelial cells have
promote angiogenic activities in response to hypoxia.68 Moreover,
attracted great interests, and the mounting data will help us draw a clear
impaired autophagy may be implicated in ageing-­related angiogenic
picture of the roles of autophagy in endothelial cell biology and dysfunc-
dysfunctions in endothelial cells. Lin et al.69 have shown that senes-
tion. Most of the current experimental data in this area are derived from
cent endothelial cells in culture exhibit an impairment in autophagic
cell culture studies; so far there is little direct evidence showing a causal
flux (as evidenced by the increased number of autophagosomes but
role of aberrant endothelial autophagy in vascular diseases. Given the
not autolysosomes, as well as the increased p62 accumulation) and
pivotal role of endothelial dysfunction in the pathogenesis of vascular
compromised angiogenic functions. Reduced autophagic flux is also
disease, however, disruptions of autophagy in endothelial cells are likely
observed in aortic endothelial cells from aged rats. Overexpression
to have significant contributions. This argument is supported by some
of dynamin-­related protein 1 (a mitochondrial fission factor) in
preliminary ex vivo data indicating that compromised autophagic func-
­senescent cells restores both of the autophagic flux and angiogenic
tions may be important in the development of endothelial dysfunctions
activities. Knock-­down of the expression of dynamin-­related protein
associated with diabetes and ageing.66,69
1 in vivo in the common ­carotid arteries disrupts the autophago-
Obviously, the effects of autophagy in endothelial cells appear to
some clearance; in parallel, this treatment significantly represses the
be variable; conflicting results have been reported by different groups.
angiogenic functions of aortic rings ex vivo.69 These data together
Collectively, it is suggested that in most situations, induction of an au-
highlight a possible role of endothelial cell autophagy in promoting
tophagic response has pro-­survival effects and protects endothelial cells
angiogenesis. On the other hand, it was also found that induction of
from the cytotoxic actions of various stressors. The beneficial effects of
autophagy by ­directly ­inhibiting mTOR with rapamycin reduced the
autophagy in endothelial cells are likely to be context-­dependent, since
regenerative and ­angiogenic capacities of endothelial cells both in
autophagy may also contribute to cell death under certain circumstances.
vitro and in vivo; however, the rate of the overall autophagic flux was
The protective effects of autophagy require a concomitant increase
­ xamined in this study.70
not e
in autophagosome clearance (ie, an increased rate of autophagic flux);
Two of the core functions of vascular endothelial cells are mainte-
­autophagy initiation coupled with an impaired autophagic flux would be
nance of a normal haemostasis function and prevention of the occur-
detrimental for endothelial cells. In addition to the regulation of endo-
rence of thrombosis. Endothelial cells produce von Willebrand factor
thelial cell survival or death, autophagy is also involved in modulating
(vWF), which is stored in Weibel-­Palade bodies (WPBs) intracellularly
other important functions of endothelial cells, such as NO production,
and secreted into the subendothelial space and into the blood flow.
angiogenesis, and haemostasis/thrombosis. Finally, it is important to
Upon vessel wall damage, the GP Ib/IX/V complex expressed on the
note that in different types of cardiovascular cells, autophagy may play
surface of platelets binds to vWF, initiating platelet adhesion and
disparate roles.7,74 Therefore, careful evaluations on the final effects of
the haemostasis cascade.71 In a study by Torisu et al.72, the authors
autophagy in different cardiovascular disease models are needed.
have discovered that in endothelial cells, WPBs and autophagosomes
exist in close proximity, and a significant amount of vWF is located
in mature autophagosomes. Moreover, genetic or pharmacological AC KNOW L ED G EM ENTS
­inhibition of the autophagic process impairs ligand-­stimulated release
This work was supported by research grants from the National Natural
of vWF both in vitro and in vivo, probably due to disruptions in the
Science Foundation of China (31471087 and 91539102). The author
processing and maturation of vWF into WPBs.72 These data highlight
apologises for the fact that many elegant publications in this area can-
a house-­keeping role of autophagy in maintaining normal haemostatic
not be cited because of space limitations.
functions of the endothelium. However, excessive endothelial autoph-
agy under some pathological conditions may contribute to thrombosis.
In vivo studies in a rat model of deep vein thrombosis have shown D I S C LO S U R E
that the thrombus formation is increased after treatment with rapa-
None.
mycin.73 In vitro, the authors have demonstrated that rapamycin en-
hances membrane ruffling of endothelial cells and increases platelet
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