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Clusterin ameliorates endothelial dysfunction in diabetes by suppressing mitochondrial fragmentation
Journal Pre-proof Clusterin ameliorates endothelial dysfunction in diabetes by suppressing mitochondrial fragmentation Lulu Ren, Feifei Han, Lingling Xuan, Yali Lv, Lili Gong, Yan Yan, Zirui Wan, Lifang Guo, He Liu, Benshan Xu, Yuan Sun, Song Yang, Lihong Liu PII:
S0891-5849(19)31273-0
DOI:
https://doi.org/10.1016/j.freeradbiomed.2019.10.008
Reference:
FRB 14446
To appear in:
Free Radical Biology and Medicine
Received Date: 2 August 2019 Revised Date:
10 October 2019
Accepted Date: 10 October 2019
Please cite this article as: L. Ren, F. Han, L. Xuan, Y. Lv, L. Gong, Y. Yan, Z. Wan, L. Guo, H. Liu, B. Xu, Y. Sun, S. Yang, L. Liu, Clusterin ameliorates endothelial dysfunction in diabetes by suppressing mitochondrial fragmentation, Free Radical Biology and Medicine (2019), doi: https://doi.org/10.1016/ j.freeradbiomed.2019.10.008. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
1
Title Page
2
Clusterin ameliorates endothelial dysfunction in diabetes by
3
suppressing mitochondrial fragmentation
4
Lulu Ren1, Feifei Han1, Lingling Xuan1, Yali Lv1, Lili Gong1, Yan Yan1, Zirui Wan1, Lifang Guo1,
5
2
6
1
7
100020, China
8
2
9
* Corresponding author.
, He Liu1, Benshan Xu1, Yuan Sun 1,Song Yang1, Lihong Liu1, * Department of Pharmacy, Beijing Chao-Yang Hospital, Capital Medical University, Beijing,
School of Life Sciences, Tsinghua University, Beijing 100084, China
10
Department of Pharmacy, Beijing Chao-Yang Hospital, Capital Medical University,
11
8 Gongren Tiyuchang Nanlu, Beijing 100020, China.
12
Tel: 86-010-8523 1464.
13
E-mail: [email protected].
14 15 16 17 18 19 20 21 22 23 24 25 26
1
Abstract
2
Clusterin (CLU) is a stress-responding protein associated with cytoprotection in
3
a broad range of pathological processes. However, clusterin’s function in
4
diabetes-induced endothelial dysfunction has not been defined. Herein, using two
5
diabetes models, we investigated the role of clusterin in endothelial dysfunction
6
triggered by diabetes and the molecular mechanisms involved. The results revealed
7
that clusterin overexpression inhibited ICAM-1/VCAM-1 expression in aortas and
8
improved endothelium-dependent vasodilatation in db/db diabetic mice and
9
streptozotocin (STZ)-induced diabetes models. Consistently, in vitro, adenoviral
10
clusterin overexpression reduced the expression of a range of pro-inflammatory
11
cytokines and suppressed monocyte adhesion to endothelial cells subjected to high
12
glucose and high palmitate. Further study indicated that clusterin overexpression
13
mitigated mitochondrial excessive fission and reduced mitochondrial ROS production.
14
Conversely, silencing clusterin aggravated mitochondrial fission and endothelial
15
inflammatory activation in high glucose-exposed endothelial cells. Accumulating
16
evidence indicates that impaired mitochondrial dynamics plays a considerable role in
17
promoting endothelial dysfunction in diabetic subjects. Therefore, treatments
18
targeting mitochondrial undue fission may be promising measures to prevent vascular
19
complications of diabetes. Furthermore, AMP-activated protein kinase (AMPK)
20
activation contributed to the modulation of mitochondrial dynamics executed by
21
clusterin. Mechanistically, clusterin promoted the phosphorylation of AMPKα and its
22
downstream target acetyl-CoA carboxylase (ACC), while the inhibition of AMPKα
23
negated the improvement in mitochondrial dynamics provided by clusterin
24
overexpression. Over all, these findings suggest that clusterin exerts beneficial effects
25
in endothelial cells under diabetic conditions via inhibiting mitochondrial
26
fragmentation mediated by AMPK.
27 28
Key words： ：Clusterin; CLU; Endothelial dysfunction; Diabetes; Oxidative stress;
29
Mitochondrial fission.
30
Abbreviations: CLU, clusterin; ICAM-1, intercellular adhesion molecule 1; 1
1
VCAM-1, vascular cell adhesion molecule 1; STZ, streptozotocin; ROS, reactive
2
oxygen species; ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase;
3
ECs, endothelial cells; AP-1, activator protein-1; Drp-1, dynamin-related protein;
4
AAV, adeno-associated virus; NC, negative control; FBG, fasting blood glucose; HFD,
5
high fat diet; CDS, coding sequence; TCID50, 50 % tissue culture infective dose;
6
IPGTT, intraperitoneal glucose tolerance test; DM, diabetic mellitus; ITT, insulin
7
tolerance test; NEFA, non-esterified fatty acid; H&E, hematoxylin and eosin; DHE,
8
dihydroethidium; HUVECs, human umbilical vein endothelial cells; Ad, adenoviral
9
virus; DCFH-DA, 2',7'-dichlorodihydrofluorescein diacetate; MDA, malondialdehyde;
10
SOD, superoxide dismutase; T-AOC, total cellular antioxidant capacity; Ach,
11
acetylcholine;
12
voltage-dependent
13
MFN1, mitofusin-1; MFN2, mitofusin-2; MCP-1, monocyte chemoattractant protein 1;
14
MOI, multiplicity of infection; CC, compound C; FBS, fetal bovine serum; PBS,
15
phosphate buffer; MMP/∆Ψm: mitochondrial membrane potential; CCK-8, cell
16
counting Kit-8; LDH, lactate dehydrogenase; ANOVA, analysis of variance.
SNP,
sodium
nitroprusside;
anion-selective
channel
NA, 1;
noradrenaline;
OPA1,
optic
VDAC1,
atrophy
1;
17 18
1. Introduction
19
Recently, sedentary lifestyle, diet, obesity, and aging of the population have led
20
to an increased prevalence of diabetes [1, 2]. Cardiovascular disease, one of the major
21
causes of mortality in diabetics, occurs in over half of diabetic patients [3]. However,
22
deficiencies in prevention and treatment measures remain prevalent. Endothelial
23
dysfunction, the first step of cardiovascular diseases, is a key mediator of vascular
24
disease in diabetes [4]. Hyperglycemia, excessive free fatty acid as well as insulin
25
resistance in diabetes cause increased oxidative stress, chronic inflammation and
26
activation in endothelium, resulting in endothelial dysfunction [5, 6]. Mitochondria
27
are vital organelles with multiple roles in modulating the energy supply, the ROS
28
generation and the dynamics of intracellular calcium [7-10]. The mitochondria
29
continuously change in size, shape and intracellular location instead of being static in
30
order to maintain the homeostasis [8, 11]. In diabetes, mitochondrial dysfunction 2
1
leads to abnormal mitochondrial signaling and the disruption of cellular metabolic
2
homeostasis, inducing and aggravating macrovascular complications [12, 13].
3
Mounting evidence indicated that the dynamics of mitochondrial fission/fusion play a
4
vital role in the modulation of endothelial functions [14, 15]. It has been demonstrated
5
that excessive palmitate and hyperglycemia impaired mitochondrial dynamics by
6
promoting mitochondrial fission and inhibiting mitochondrial fusion in endothelial
7
cells, resulting in increased mito-ROS production and electron transport chain
8
impairment [16, 17]. The morphological changes in mitochondria provoked ROS
9
overproduction in diabetic conditions [18]. Mitochondrial ROS mediates the signaling
10
associated with endothelial inflammatory activation in the early stage of vascular
11
disease by activating proinflammatory transcription factors, provoking elevated
12
expression levels of adhesion molecules and inflammatory cytokines [19, 20].
13
Moreover, as a major headstream of ROS production, mitochondria are susceptible to
14
oxidative damage, which causes a vicious circle of events in endothelial cells (ECs)
15
[21-23]. Thus, reducing the pathological levels of ROS and correcting the imbalance
16
of mitochondrial dynamics in diabetes-induced endothelial dysfunction might
17
improve patient outcomes.
18
Clusterin (CLU), a functional homologue to small heat shock proteins, is a highly
19
sensitive biosensor of increased oxidative stress [24, 25]. This heterodimeric
20
glycoprotein is functionally implicated in numerous pathological processes including
21
inflammation, apoptosis, and aging [26]. The stress-induced transcription of the CLU
22
gene can be mediated by the interaction between a conserved 14-bp “CLU-specific
23
element” and a nuclear transcription factor called heat shock factor-1 [27]. Clusterin
24
expression is also mediated by NF-κB and AP-1 (activator protein-1) associated with
25
oxidative stress [28]. Increasing evidence indicates that clusterin increases under
26
pathophysiological stress, exerting a chaperone activity associated with cytoprotection
27
in various diseases including cancers, ischemic heart failure, and neurodegenerative
28
disorders [29]. Van Dijk et al. reported that intravenous myocardial infarct size in rats
29
can be reduced by clusterin [30]. Clusterin protected cardiomyocytes from apoptosis
30
induced by oxidative stress via the Akt/GSK-3β pathway [31]. Intriguingly, the 3
1
overexpression of human clusterin in drosophila reduces the ROS level and increases
2
resistance to starvation, heat shock, and oxidative stress, extending their lifespan [32].
3
These results imply that clusterin may represent a particular component of antioxidant
4
response networks. However, whether clusterin prevents oxidative stress-induced
5
endothelial dysfunction in diabetes remains unclear.
6
Using db/db diabetic mice and STZ-induced diabetes models in the current study,
7
we found that clusterin overexpression suppressed mitochondrial fission and ROS
8
production, ameliorating diabetes-induced endothelial dysfunction. However,
9
AMPKα-siRNA and the inhibitor of AMPK (compound C) abolished the effect of
10
clusterin on endothelial dysfunction under the high glucose and high palmitate
11
conditions.
12 13
2. Material and Methods
14
2.1 Animals
15
The animals were housed at constant temperatures (22°C) under a cycle with 12
16
h of darkness and 12 h of light. Water and food were available ad libitum. All of the
17
animal treatments were performed in accordance with the Health Guidelines of the
18
Capital Medical University.
19
Male db/db mice and non-diabetic control db/m mice (6 weeks old,
20
C57BLKS/JNju ) were obtained from the Model Animal Research Center of Nanjing
21
University and fed with normal chow. Each group consisted of 8 mice (N=8). After
22
one-week adaption, recombinant adeno-associated virus expressing clusterin
23
(AAV-CLU, 10^10 PFU/mouse) or negative control adeno-associated virus (AAV-NC,
24
1010 PFU/mouse) were administered to the mice via tail vein injection once every six
25
weeks as previously described [33]. Their fasting blood glucose (FBG) was monitored
26
using a glucometer (OneTouch Ultra Easy, LifeScan, Malvern, PA, USA), and the
27
body weights were measured once a fortnight. At the end, the mice were sacrificed
28
under anesthesia with an intraperitoneal injection of sodium pentobarbital at 50 mg/kg,
29
and their plasma and aortas were collected for further experiments.
30
The fat-fed streptozotocin rat is another model for type 2 diabetes. The rats are fed a 4
1
high fat diet to induce insulin resistance and injected with a low dose of STZ to impair β-cell
2
function, resulting in hyperglycemia, associated with hyperlipidemia and insulin resistance
3
[34]. For the STZ-induced diabetes model, male Sprague-Dawley (SD) rats (8 weeks
4
old) were purchased from Huafukang Animal Company (Beijing, China) and fed a
5
high fat diet (HFD, 45 % fat, 35 % carbohydrate, 20% protein; H10045, Huafukang,
6
China) or a control chow (10 % fat, 70 % carbohydrate, 20% protein) for 4 weeks.
7
Each group consisted of 8 rats (N=8). The rats in the diabetes mellitus groups were
8
then intraperitoneally injected with STZ buffer at a single dose of 40 mg/kg, and the
9
control group rats were injected with sodium citrate buffer. Diabetes was defined as
10
blood glucose levels > 200 mg/dL (11.1mmol/L) after injection. Then, 100 µL of
11
adeno-associated virus AAV-CLU or AAV-NC (1012 PFU/mL) were administered via
12
tail vein injection. All of the animals were maintained on the preceding diets
13
respectively, and their FBG and body weights were monitored. After 8 weeks, the rats
14
were sacrificed under anesthesia with an intraperitoneal injection of sodium
15
pentobarbital at 40 mg/kg, and their plasma and aortas were harvested for further
16
experiments.
17 18
2.2 Recombinant adenoviral and adeno-associated virus vectors
19
Recombinant adenovirus vector plasmid encoding full-length human CLU CDS
20
(NM_ 001831.4) was constructed with the HB-Infusion cloning system (Hanbio,
21
Shanghai, China) according to the manufacturer’s directions. Then shuttle plasmid
22
and skeleton plasmid were co-transfected into HEK293A cells for 6 h. Until 80%
23
cytopathic effect was observed, the virus suspension was collected. The CsCl density
24
gradient centrifugation-dialysis combination method was used for adenovirus
25
purification. The viral titer (1.26×10^10 PFU/mL) of recombinant adenoviral virus
26
Ad-CLU was tested using the 50% tissue culture infective dose (TCID50) method.
27
Ad-NC (1.18×10^10 PFU/mL) was used as a negative control.
28
For construction of adeno-associated virus vectors, adenovirus vector plasmid
29
encoding full-length mouse CLU CDS (NM_013492.3) and rat CLU CDS
30
(NM_053021.2) were constructed with the HB-Infusion cloning system. Then the 5
1
shuttle plasmid, pAAV-RC and pHelper were co-transfected into AAV-293 cells. The
2
cells were collected and disrupted after 72 h. An Adeno-Associated Virus Purification
3
Mini-kit (Biomiga, San Diego, CA, USA) was used for purification. The viral titers of
4
AAV-r-CLU (1.5×10^12 PFU/mL) or AAV-m-CLU (1.6×10^12 PFU/mL) were tested
5
using qRT-PCR. AAV-NC (1.3×10^12 PFU/mL) was used as a negative control.
6 7 8
2.3 IPGTT and ITT The intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT)
9
were conducted on the second weekend after STZ injection. The rats were fasted and
10
injected with glucose (2 g/kg) or insulin (1 U/kg). Their blood glucose levels were
11
measured using tail bleeds 0, 20, 40, 60, and 120 min after the administration of
12
glucose or insulin.
13 14
2.4 Histological analysis
15
Hematoxylin and eosin (H&E) staining was applied to characterize the
16
pathological morphology of aorta. Aortic tissue was isolated from the animals and
17
fixed in phosphate-buffered 4 % paraformaldehyde. Then the tissue was embedded in
18
paraffin wax and cut into sections. The H&E stained sections were observed under an
19
optical microscope (Olympus, Tokyo, Japan).
20 21
2.5 Immumohistochemistry
22
Immunohistochemical staining was performed on thoracic aortae to determine
23
clusterin (CLU), VCAM-1 and ICAM-1. Primary antibodies against ICAM-1 (sc8439,
24
1:100), VCAM-1 (sc13160, 1:100), and clusterin (sc166831, 1:50) used in the
25
immunohistochemistry experiment were purchased from Santa Cruz Biotechnology
26
(Dallas, TX, USA). After antigen retrieval, the aortic sections were incubated with the
27
primary antibody at 4 ℃ overnight and incubated with labeled polymer-horseradish
28
peroxidase secondary antibodies. The sections were then counterstained with
29
hematoxylin followed by diaminobenzidine chromogen incubation. The positive
30
staining of the tissue sections were quantized using Image J. 6
1
2.6 Assessment of ROS production
2
Intracellular ROS level in aorta was determined by the superoxide indicator
3
dihydroethidium (DHE, Thermo Fisher Scientific, Waltham, MA, USA). DHE shows
4
blue-fluorescence in the cytosol without oxidization, while oxidized DHE intercalates
5
within DNA, exhibiting a bright fluorescent red. First, the freshly isolated aortas were
6
frozen and sectioned. Then the sections were incubated with DHE solution (5 µM) at
7
37 °C for 30 min in the dark. After washing, the sections were observed and imaged
8
using a fluorescence microscope. Image J was used to analyze the data.
9
To assess the mitochondrial ROS produced in the ECs, the mito-ROS specific
10
indicator Mito-SOX red was used in the study. Mito-SOX red is a fluorogenic dye for
11
highly specific detection of mitochondrial superoxide in live cells. Mito-SOX red
12
reagent is oxidized by superoxide in the mitochondria and exhibits red fluorescence.
13
Before use, a working solution of Mito-SOX red (5 µM) was made with Hank's
14
balanced salt solution. The cells were then incubated with the working solution at
15
37 °C for 10 min without light. The cells were then stained with Hoechst 33342. Then,
16
the cells were imaged using the fluorescence microscope (Olympus, Japan).
17 18
2.7 ELISA
19
8-OHdG (8-Hydroxy-2’-deoxyguanosine) level in the plasma collected from the
20
mice or rats was detected using 8-OHdG ELISA kit (mouse/rat, Nanjingjiancheng,
21
Jiangsu, China). Briefly, 50 µL of plasma or standard samples were added into the
22
microplate wells, and then 50 µL of biotinylated antibody working solution was added
23
into each well. The microplate was incubated at 37 °C for 30 min after shaking gently.
24
After washing five times, horseradish peroxidase conjugated reagent was added into
25
the wells. After 30 min, chromogen solution A and B were applied successively and
26
incubated for 10 min at 37 °C. The reaction was then halted using stop solution. The
27
optical density values were measured by the microplate reader. The 8-OHdG level
28
was calculated according to the manufacturer's protocols. Similarly, levels of TNF-α
29
and IL-6 in supernatant of cell culture medium collected from HUVECs subjected to
30
different treatments were quantified according to the protocols of ELISA kits 7
1
(Biolegend, San Diego, CA, USA). Secreted clusterin in the plasma was detected
2
using a Clusterin ELISA Kit (Abcam, Cambridge, MA, USA) according to the
3
manufacturer’s protocols.
4 5
2.8 Determination of malondialdehyde (MDA), total superoxide dismutase (SOD)
6
activity and total cellular antioxidant capacity (T-AOC)
7
A whole aorta homogenate was prepared with the sample preparation solution in
8
ice-water bath and centrifuged at 4°C (12000 g, 3 min). The protein was quantified
9
according to the BCA Assay Kit instructions (P0009, Beyotime, Nanjing, China).
10
Malondialdehyde (MDA) was detected using a Lipid Peroxidation MDA Assay kit
11
(S0131, Beyotime, China) according to the manufacturer's instructions. The MDA
12
content was determined on a microplate reader at 532 nm and expressed as µmol/mg
13
protien. The total superoxide dismutase (SOD) activity was determined via WST-8
14
assay with a Total Superoxide Dismutase Assay Kit (S0101, Beyotime, China)
15
according to the manufacturer’s protocol. The total SOD activity was calculated and
16
expressed as U/mg protein.
17
A variety of antioxidants including antioxidant molecules and antioxidases can
18
scavenge excessive ROS to prevent oxidative stress in tissues. The total antioxidant
19
capacity (T-AOC) of the tissue is defined as the total levels of various antioxidants.
20
T-AOC was measured using a Total Antioxidant Capacity Assay Kit (S0121,
21
Beyotime, China) with an ABTS (2,2’-azino-bis(3-ethylbenz-thiazoline-6-sulfonic
22
acid) method. When ABTS is incubated with a proper chemical, a blue-green ABTS
23
radical (ABTS•+) is formed. The color production of ABTS•+ can be suppressed by
24
antioxidants in the sample proportionally to a degree. Therefore, the concentrations of
25
antioxidants in tissue samples can be quantified indirectly. According to the
26
manufacturer's protocols, the T-AOC was calculated and expressed as the
27
trolox-equivalent antioxidant capacity according to the manufacturer's protocols.
28 29 30
2.9 Assessment of vasomotor responses Endothelium-intact aortas were isolated gently and placed in ice-cold and 8
1
oxygenated PSS buffer (Nacl, 130 mM; KCl, 4.7 mM; KH2PO4, 1.18 mM;
2
MgSO4•7H2O, 1.17 mM; NaHCO3, 14.9 mM; Glucose, 11.1 mM; EDTA, 0.026 mM;
3
CaCl2, 1.16 mM). The aortas were cut into rings of approximately 3 mm in length.
4
The vasomotor responses to acetylcholine (Ach, 10-9 M-10-5 M) and sodium
5
nitroprusside (SNP, 10-9 M-10-5 M) were monitored by the myograph system
6
(DMT620, Danish Myo Technology, Aarhus, Denmark). Percentage of relaxation
7
relative to the noradrenaline (NA, 10-6 M)–induced vasoconstriction was calculated.
8 9
2.10 Quantitative RT-PCR
10
Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to
11
examine the relative gene expression levels. The primers used in the experiment are
12
shown in Table S1. The total RNA in HUVECs was isolated using RNAiso Plus
13
(Takara, Shiga, Japan), and the total RNA in the aortic tissue was extracted using an
14
RNAsample Total RNA kit (Tiangen, Beijing, China) according to the manufacturer's
15
protocols. Nanodrop ND-2000 (Thermo Fisher Scientific, USA) was used to detect
16
the concentration and quality of the RNA. Reverse transcription was performed using
17
a FastKing RT kit (Tiangen, China). PCR was performed using a TB Green Premix Ex
18
Taq II kit (Takara, Japan). The relative gene expression levels normalized to GAPDH
19
were calculated using ∆∆CT method.
20 21
2.11 Western blotting
22
The total protein was isolated using RIPA buffer (2 % protease inhibitor and 1 %
23
phosphatase inhibitor) and quantified using a BCA Assay Kit according to the
24
manufacturer’s instructions (Beyotime, China). The mitochondrial protein was
25
isolated from the aorta tissue according to the tissue mitochondria isolation protocol.
26
First, protein samples were loaded onto 8 % SDS℃PAGE and migrated under an
27
electric field. Then the protein was transferred to a PVDF membrane (Millipore,
28
Burlington, MA, USA). Before primary antibodies incubation, the membranes were
29
blocked with 5% skim milk. Then the membranes were washed and incubated with
30
fluorescently labeled secondary antibodies. Protein band images were developed 9
1
using an LI-COR Odyssey scanner (LI℃COR, USA). Primary antibodies were
2
purchased from Abcam and Cell Signaling Technology including anti-β-actin
3
(ab8227), GAPDH (ab8245), VDAC1(ab15895), Drp-1 (#8570), OPA1 (#80471),
4
MFN1 (ab57602), MFN2 (#9482), AMPKα (#5831), p-AMPKα (#2535), ACC
5
(#3676), p-ACC (#11818), AMPKβ (#4150), p-AMPKβ1 (#4186), ICAM-1 (sc8439),
6
VCAM-1 (sc13160) and clusterin (ab229127 /sc5289/sc166831).
7 8
2.12 Cell culture and treatments
9
Human Umbilical Vein Endothelial Cells (HUVECs, ScienCell, Carlsbad, CA,
10
USA) were cultured in endothelial cell medium (1% growth supplement and 10%
11
FBS) at 37 °C in a 5% CO2 humidified atmosphere. Passages 3 to 8 cells were used in
12
further experiments. HUVECs were infected with adenovirus Ad-CLU expressing
13
selected clusterin and negative control adenovirus Ad-NC with 100 MOI. After 6 h,
14
fresh medium was substituted for the culture medium, and the cells were cultured for
15
another 18 h. The clusterin expression level was determined using qPCR and Western
16
blotting (Fig.S1). For the knockdown of clusterin, the cells were transfected with
17
CLU-siRNA1 and CLU-siRNA2, silencing the clusterin expression for 48 h. The two
18
separate siRNA target different sequence segments of CLU-mRNA. The sequence of
19
CLU-siRNA1 was 5’ CCCGCCAACAGAAUUCAUATT 3’, the sequence of
20
CLU-siRNA2 was 5’ CCAGGAAGAACCCUAAAUUTT 3’, and the sequence of
21
siRNA-NC was 5’ UUCUCCGAACGUGUCACGUTT 3’. The cells were then
22
subjected to normal glucose medium (5.5 mM D-glucose, NG) with 27.5 mM
23
mannitol for osmotic compensation or high glucose and high palmitate medium (33
24
mM D-glucose and 200 µM palmitic acid, HG/HF) for 24 h as previously described
25
[35, 36]. For the inhibition of AMPK activation, HUVECs transfected with Ad-CLU
26
or Ad-NC were treated with 10 µM of compound C (CC) (MCE, USA) or
27
AMPKα-siRNA1/2.
28
GAGGAGAGCUAUUUGAUUATT 3’, the sequence of AMPKα-siRNA2 is 5’
29
GCGUGUACGAAGGAAGAAUTT 3’, and the sequence of siRNA-NC is 5’
30
UUCUCCGAACGUGUCACGUTT 3’. RPMI-1640 medium with 10 % FBS was
The
sequence
of
10
AMPKα-siRNA1
was
5’
1
used for THP-1 (ATCC, Manassas, VA, USA) cells culture.
2 3
2.13 Monocyte adhesion assay
4
A monocyte adhesion assay was performed to investigate the role of clusterin in
5
the suppression of monocyte adhesion to ECs. First, THP-1 cells were cultured in
6
RPMI-1640 medium with 10% FBS (Gibco, Waltham, MA, USA) at 37 °C and
7
incubated with calcein AM (2 µM) for 30 min. The THP-1 cells were then collected
8
and resuspended. After HUVECs were added in black plates containing 96 wells and
9
pretreated with adenovirus, the THP-1 cells (1×105) were seeded into each well and
10
co-incubated with the HUVECs for 1 h. The unattached THP-1 cells were removed by
11
PBS washing. The plates were photographed under a fluorescence microscope and the
12
fluorescence intensity was assessed using a microplate reader (Thermo Fisher
13
Scientific, USA).
14 15
2.14 Tube formation assay
16
Tube formation assay was performed as previously described [37]. In brief, the
17
HUVECs were infected with adenovirus expressing secreted clusterin (Ad-CLU) or
18
negative control adenovirus (Ad-NC) for 24 h. Then, the cells were incubated with
19
calcein AM (2 µM) for 20 min. For clusterin knockdown, the HUVECs were
20
transfected with siRNA silencing clusterin expression for 48 h using Lipofectamine
21
2000 (Thermo Fisher Scientific, USA). Then, the 96-well plates were coated with 60
22
µL of matrigel matrix (Corning, NY, USA) and incubated at 37°C for 30 min. When
23
the matrigel was polymerized, the HUVECs were collected and seeded (1×104/well)
24
into the wells and subjected to NG or HG/HF treatments for 16 h. Capillary-like tubes
25
were imaged using the fluorescence microscope.
26 27
2.15 Mitochondrial staining and assessment of mitochondrial morphology
28
Mito-Tracker green (Beyotime, China) was applied to characterize the
29
mitochondrial morphology in HUVECs. Briefly, Mito-Tracker green stock solution (1
30
mM) was prepared with anhydrous dimethylsulfoxide and diluted with Hank's 11
1
Balanced Salt Solution /Ca2+/Mg2+. The cells were incubated with Mito-Tracker green
2
staining solution (200 nM) at 37°C for 20 min. The staining solution was then
3
removed, and fresh culture solution was added into the wells. The cells were observed
4
under a fluorescence microscope. Image-Pro Plus software was applied to the
5
quantification of the number and individual volume of the mitochondria.
6
Mitochondrial fragmentation was indicated by a decrease in the mitochondrial density
7
per cell.
8 9
2.16 Assessment of MMP
10
The mitochondrial membrane potential (MMP, ∆Ψm) was monitored using JC-1
11
dyes, exhibiting potential-dependent accumulation in mitochondria. The HUVECs
12
were infected with adenovirus and pretreated with NG or HG/HF, and incubated with
13
JC-1 probe (2 mM) at 37°C for 30 min. The cells were then stained using Hoechst
14
33342. A fluorescence microscope was used to observe the cells. The quantified data
15
are shown as the normalized ratio of green fluorescence to red fluorescence.
16
Mitochondrial depolarization is demonstrated by a lower ratio of red/ green
17
fluorescence intensity.
18 19
2.17 Viability and cytotoxicity of HUVECs
20
CCK-8 (Beyotime,China) was used to evaluate the viability of cells. An LDH
21
Cytotoxicity Assay Kit (Sigma-Aldrich, St. Louis, MO,, USA) was used to determine
22
the cytotoxicity of cells subjected to HG/HF following the manufacturer’s protocols.
23 24
2.18 Statistical analysis
25
All of the data are expressed as the mean ± SD and were analyzed using
26
GraphPad Prism 7.00 and SPSS 21.0. If the data passed equal-variance test, an
27
unpaired two-tailed t-test was conducted for comparisons between two groups;
28
ANOVA and post hoc multiple comparisons test were used for comparisons among
29
three or more groups. Nonparametric tests were used for non-normally distributed
30
data. Statistically significant differences were considered at a value of P < 0.05. 12
1
3. Results
2 3
3.1 Endothelial inflammatory activation, increased ROS generation and mitochondrial fragmentation occurred in diabetes
4
To investigate the role of clusterin in diabetes-induced endothelial dysfunction,
5
the db/db mice were used as diabetes models and the db/m mice were non-diabetic
6
controls (Fig.1A). The male db/db mice were hyperglycemic with increased level of
7
non-esterified fatty acid (NEFA) in the plasma, and their body weights were
8
significantly higher than the db/m mice (Fig.1B-D). First, the endothelial
9
inflammatory activation in the diabetic aortas was determined. Endothelial activation,
10
the onset of endothelial dysfunction, can be induced by the hyperglycemia and
11
hyperlipidaemia in diabetic mice. As expected, the expression of VCAM-1and
12
ICAM-1 adhesion molecules was up-regulated in the db/db mice aortas (Fig.1E-G).
13
Moreover, H&E staining indicated that marked pathomorphological changes occurred
14
in the diabetic aortas (Fig.1E).
13
1 2
Fig.1. Hyperglycemia and hyperlipidemia in diabetic mice leads to endothelial
3
inflammatory activation. (A) Male db/db diabetic mice (left) and non-diabetic
4
control db/m mice (right). (B) Body weights of db/db and db/m mice were measured
5
once a fortnight. (C) Fasting blood glucose (FBG) was measured once every four
6
weeks. Diabetic mice had a higher FBG level than the littermate db/m mice. (D)
7
Diabetic mice had a higher level of non-esterified fatty acid (NEFA) in plasma than
8
the littermate db/m mice. (E) Representative hematoxylin and eosin (H&E) staining 14
1
images (×200, left panel) and immunohistochemical images (×400) for the thoracic
2
aortic sections. (F) Statistical analysis of immunohistochemical staining. Diabetic
3
mice scored higher in the staining of CLU, VCAM-1 and ICAM-1 than the db/m mice.
4
(G) The mRNA levels of ICAM1, VCAM1 and CLU were determined using real-time
5
PCR. (H) Representative Western blotting images of CLU expression. CLU
6
expression in the db/db mice aortas increased compared to the db/m mice. (I) The
7
diabetic mice had a higher level of clusterin in the plasma than the littermate db/m
8
mice. (J) DHE (dihydroethidium) staining for ROS determination in frozen aortic
9
sections. (K) Quantified analysis of fluorescence intensity for the ROS levels. The
10
diabetic mice had a higher ROS level. (L) The level of 8-OHdG was measured by
11
ELISA. Concentrations of MDA (M) and total SOD activity (N) in aorta were
12
measured.
13
malondialdehyde; SOD, superoxide dismutase.
n=5.
*
P<0.05,
**
P<0.01,
***
P<0.001 vs. db/m group. MDA,
14 15
It has been determined that clusterin plays a cytoprotective role as a
16
stress-inducible protein for various diseases [26, 38, 39]. However, the function of
17
clusterin in diabetes-induced endothelial dysfunction has not been defined.
18
Before we dive in, the expression abundance of clusterin in the diabetic aortas was
19
investigated. The qRT-PCR results showed that the mRNA expression level of CLU in
20
the db/db mice aorta was higher than in the db/m mice (Fig. 1G). As expected, the
21
Western blotting and immunohistochemical staining results indicated that the CLU
22
expression in the db/db mice aortas increased under hyperglycaemic and
23
hyperlipidemic stress compared with the db/m mice (Fig. 1E, F, and H). Also, diabetic
24
mice had a higher level of clusterin in the plasma than the littermate db/m mice (Fig.
25
1I).
26
15
1 2
Fig.2. Mitochondrial ROS production and mitochondrial fission were increased
3
in the HUVECs subjected to HG/HF treatment. (A) Mitochondrial ROS
4
(mito-ROS) and nucleus were stained by MitoSOX red and Hoechst 33342,
5
respectively. Scale bar is 100 µm. (B) Quantified analysis of fluorescence intensity for
6
mito-ROS level. HUVECs treated with HG/HF showed an increased level of
7
mito-ROS. (C) Representative images of MitoTracker Green staining characterizing
8
the mitochondrial morphology in the HUVECs subjected to NG or HG/HF treatment
9
and the quantified analysis (D). Under HG/HF challenge, the fibers of the
10
mitochondria in the HUVECs became short and fragmentized, resulting in a decreased
11
mitochondrial density. Scale bar is 50 µm. n=3. *P<0.05,
12
db/m group or NG group. NG, normal glucose; HG/HF, high glucose and high
13
palmitate.
**
P<0.01, ***P<0.001 vs.
14 15
One of the key mediators for diabetic endothelial dysfunction is excessive ROS,
16
and ROS production by mitochondria is the primary intracellular source of ROS [15,
17
40]. In our study, the total ROS and mitochondrial ROS (mito-ROS) were detected by 16
1
DHE staining and MitoSOX red staining, respectively. As shown in Fig.1J-K,
2
compared to the db/m mice, the fluorescence intensity of DHE in the db/db mice was
3
significantly increased, representing a higher level of ROS. Accumulated oxidation
4
damage to the mitochondrial DNA and membrane is the main risk for mitochondrial
5
dysfunction, and mitochondrial injury is a critical factor in endothelial activation and
6
dysfunction in diabetes [8]. 8-OHdG and MDA, the indicators of oxidative DNA and
7
membrane lipids damage, were detected by ELISA. As expected, the levels of
8
8-OHdG and MDA in the db/db mice were higher than in the controls (Fig.1L-M). In
9
contrast, the total activity of the antioxidase superoxide dismutase (SOD) in the db/db
10
mice was lower than in the control db/m mice (Fig.1N). On the other hand, the
11
HUVECs stimulated with high glucose and high fatty acid (HG/HF) exhibited higher
12
mito-ROS levels (Fig.2A-B). Increased production of mitochondrial ROS can be
13
induced by disrupted mitochondrial dynamics and aggravated mitochondrial fission in
14
diabetes. MitoTracker Green staining was used to characterize the mitochondrial
15
morphology. In the HUVECs treated with normal glucose, the mitochondria appeared
16
as long fibers with a dense network, and mitochondria fibers in the HUVECs
17
stimulated with HG/HF were short and fragmentized (Fig.2C-D).
18 19 20
3.2 Clusterin overexpression dysfunction in vitro and in vivo
ameliorates
diabetes-induced
endothelial
21
To further investigate the role of clusterin in endothelial dysfunction induced by
22
diabetes, the spontaneous db/db diabetic mice and STZ-induced diabetic rats were
23
used as models in vivo and HUVECs were used as models in vitro with clusterin
24
overexpression. In vitro, we first profiled the expression of adhesion molecules and
25
other cytokines associated with endothelial inflammatory activation. The mRNA
26
expression of VCAM-1, ICAM-1, MCP-1, IL-6, TNF-α, and ET-1 were increased in
27
the HUVECs stimulated with high glucose and high palmitate (HG/HF) compared to
28
the NG group; in contrast, the HUVECs transfected with the adenovirus expressing
29
clusterin (Ad-CLU) had a lower expression level than the HG/HF control group
30
(Fig.3A-B). We further examined the IL-6 and TNF-α content in the supernatant of 17
1
medium. The results showed that HG/HF promoted the secretion of pro-inflammatory
2
factors IL-6 and TNF-α (Fig.3C). ICAM-1 and VCAM-1 overexpression means that
3
monocyte adhesion and infiltration to ECs will be promoted, therefore, an adhesion
4
assay was performed to examine whether clusterin prevents ECs from recruiting
5
monocytes. As expected, HG/HF increased the adhesion of THP-1cells to HUVECs
6
compared with the NG group. However, clusterin overexpression inhibited the
7
monocyte adhesion to HUVECs (Fig.3D). In addition, the cell viability was decreased
8
and the cytotoxicity was increased following HG/HF stimulation; however, the
9
clusterin overexpressed HUVECs showed a higher viability and a lower cytotoxicity
10
(Fig.S2). Angiogenesis is one of the vascular endothelial functions that can be
11
impaired by oxidative stress. HG/HF impaired the tube formation of the HUVECs on
12
the Matrigel, and the branches shortened in the HG/HF group as shown in Fig.3E,
13
while clusterin markedly reduced the impairment.
18
1 2
Fig.3.
Clusterin
overexpression
suppressed
3
inflammatory activation in vitro. The HUVECs were infected with adenovirus
4
expressing clusterin (Ad-CLU) or negative control adenovirus (Ad-NC) (100 MOI)
5
for 24 h and subjected to NG or HG/HF treatments for another 24 h. (A-B) The
6
mRNA expression levels of the cytokines associated with endothelial inflammatory
7
activation were measured by qRT-PCR, and in (B) expressed as a heat map, with
8
each column representing the mean of three independent experiments. (C)
9
Adenovirus-infected HUVECs were subjected to NG or HG/HF treatments. IL-6 and
10
TNF-α in medium were quantified by ELISA. (D) The adhesion of Calcein
11
AM-labeled THP-1cells to the HUVECs was determined by fluorescence microscopy 19
HG/HF-induced
endothelial
1
and a fluorescence microplate reader. CLU overexpression decreased the monocyte
2
adhesion to the HUVECs. (E) HUVECs infected with Ad-CLU or Ad-NC (100 MOI)
3
were seeded on Matrigel and retained for 12 h. Tube formation of HUVECs on
4
Matrigel was pictured and analyzed by Image J. n=3. #P<0.05,
5
vs. NG control, *P<0.05, **P<0.01, ***P<0.001 vs. HG/HF control. CM, blank culture
6
medium; NG, normal glucose; HG/HF, high glucose and high palmitate; Ad-NC,
7
negative control adenovirus; Ad-CLU, the adenovirus expressing clusterin.
##
P<0.01,
###
P<0.001
8 9
In db/db mice, clusterin overexpression (Fig.4C, Fig.S3A) made no difference on
10
the body weight and FBG (Fig.4A). Compared to the db/db mice infected with
11
AAV-NC (AAV-NC-db/db), the level of NEFA in plasma of the db/db mice infected
12
with AAV-CLU (AAV-CLU-db/db) decreased (Fig.4B). The pathological morphology
13
of the aortas in the clusterin overexpressed db/db mice was improved compared to
14
that in the AAV-NC-db/db mice (Fig.4D). The expression of VCAM-1 and ICAM-1 in
15
the AAV-CLU-db/db mice aortas was markedly down-regulated compared to the
16
AAV-NC-db/db mice (Fig.4F-G). The results indicated that clusterin overexpression
17
alleviated endothelial inflammatory activation in db/db mice aortas.
18
The role of clusterin in other diabetes models was also examined. The
19
STZ-induced diabetic rats fed a high fat diet (HFD) were a drug-induced type 2
20
diabetes model (Fig.S4). Similarly, overexpression of clusterin (Fig.5C) made no
21
difference in the fasting blood glucose (FBG) or body weights of the diabetic rats
22
(Fig.5A). The NEFA level in plasma of the diabetic rats increased compared with the
23
non-diabetic rats (Fig.5B). Endothelial function was further evaluated by measuring
24
the isometric tension of the aortic rings. The endothelium-dependent vasorelaxation
25
responding to acetyl choline (Ach) was impaired in the diabetic rats infected with
26
AAV-NC (AAV-NC/DM), while clusterin mitigated the impairment in the diabetic rats
27
infected with AAV-CLU (AAV-CLU/DM). However, the endothelium-independent
28
vasorelaxation responding to sodium nitroprusside (SNP) showed no significant
29
difference among the groups (Fig.5D). The expression levels of VCAM-1 and
30
ICAM-1 were lower in the aortas of AAV-CLU/DM rats compared with the 20
1
AAV-NC/DM rats (Fig.5E-F). In brief, clusterin overexpression ameliorates
2
endothelial dysfunction in the STZ-induced diabetic rats.
21
1 2
Fig.4. Clusterin overexpression alleviated endothelial inflammatory activation in 22
1
db/db mice aortas. (A) Body weights of mice were measured once a fortnight, and
2
FBG was measured once every four weeks. Clusterin overexpression made no
3
difference on the body weight and FBG. (B) NEFA was detected in plasma of mice
4
collected at the time of sacrifice. (C) Western blotting for clusterin expression in mice
5
aortas (upper panel) and quantitative data of clusterin expression normalized against
6
β-actin
7
immunohistochemical images (×400) for aortic sections. (E) Quantitative data of
8
immunohistochemical staining of CLU, VCAM-1, ICAM-1. The expressions of
9
VCAM-1 and ICAM-1 in the AAV-CLU infected db/db mice aortas were
10
down-regulated, while the clusterin expression increased markedly compared to the
11
AAV-NC-db/db mice. n=6.
12
*
13
adeno-associated virus; AAV-CLU, the adeno-associated virus expressing clusterin.
P<0.05,
(lower
**
panel).
P<0.01,
***
(D)
#
Representative
P<0.05,
##
H&E
P<0.01,
###
staining
images
(×200),
P<0.001 vs. AAV-NC-db/m;
P<0.001 vs. AAV-NC-db/db. AAV-NC, negative control
14
23
1 2
Fig.5. Clusterin overexpression rescued endothelial dysfunction in STZ-induced
3
diabetic rats. (A) Body weights and FBG of rats were measured once every four
4
weeks. Clusterin overexpression made no difference on the body weight and FBG. (B)
5
NEFA was detected in plasma collected at the time of sacrifice. STZ-induced diabetic
6
rats had a higher level of non-esterified fatty acid (NEFA) in plasma than the normal
7
rats. (C) Western blotting for clusterin expression in aortas (upper panel) and
8
quantitative data of clusterin expression normalized against β-actin (lower panel). The
9
clusterin expression increased markedly in the rats injected with AAV-CLU. (D) Ach-
10
and SNP-induced vasorelaxation of aortic rings were recorded by vascular tension
11
measurement system. The endothelium-dependent vasorelaxation responding to Ach
12
was impaired in AAV-NC infected diabetic rats, while clusterin overexpression 24
1
mitigated the impairment in AAV-CLU infected diabetic rats. (E) Representative H&E
2
staining images (×200), immunohistochemical images (×400) for aortic sections. (F)
3
Quantitative data of immunohistochemical staining of VCAM-1, ICAM-1. Expression
4
of VCAM-1 and ICAM-1 in AAV-CLU infected diabetic rat aortas was markedly
5
down-regulated compared to AAV-NC infected diabetic rats. n=5. #P<0.05, ##P<0.01,
6
###
7
fed with normal diet and subjected to no special treatment; HFD, high fat diet; DM,
8
rats treated with HFD/STZ to produce diabetes mellitus models.
P<0.001 vs. Normal; *P<0.05, **P<0.01, ***P<0.001 vs. AAV-NC/DM. Normal, rats
9 10
3.3 Clusterin overexpression inhibits oxidative stress and mitochondrial ROS
11
production induced by diabetes
12
Oxidative stress accumulated in diabetes induces and aggravates endothelial
13
activation
and
dysfunction
[41].
DCFH-DA (2',7'-dichlorodihydrofluorescein
14
diacetate) probe was applied to detect the ROS level in the HUVECs, and the result
15
indicated that the ROS level in the ECs treated with HG/HF increased markedly,
16
while the ECs transfected with Ad-CLU had lower ROS levels (Fig.6A-B). In the
17
diabetic mice and rat aortas, the ROS levels were detected by DHE staining. Similarly,
18
clusterin overexpression significantly reduced the ROS levels (Fig.6C-D and H-I). We
19
further examined 8-OHdG and MDA, indicators of DNA and membrane lipids
20
oxidative damage. As shown in Fig.6E-F, compared with the db/m mice infected with
21
AAV-NC (AAV-NC-db/m), the levels of 8-OHdG and MDA in the db/db mice
22
infecetd with AAV-NC (AAV-NC-db/db) were higher, which were reversed by
23
clusterin overexpression in the AAV-CLU-db/db mice. A similar result was obtained
24
in the diabetic rats (Fig.6J-K). Excessive ROS production beyond the efficiency of
25
available antioxidant defense systems leads to oxidative stress. Meanwhile, increased
26
oxidative stress in diabetes impaired the endogenous antioxidant system composed of
27
antioxidative enzymes and other antioxidants. As expected, the total superoxide
28
dismutase (SOD) activity in the aortas was reduced in the diabetic mice and rats but
29
increased in the AAV-CLU-db/db and AAV-CLU/DM groups (Fig.6G and L). The
30
total cellular antioxidant capacity (T-AOC) and glutathione disulfide (GSSG) /reduced 25
1
glutathione (GSH) in the rat aortas were also measured. In the AAV-NC/DM group,
2
the level of T-AOC declined and GSSG/GSH was elevated, which suggested that the
3
aortas became more susceptible to oxidative damage. However, the level of T-AOC
4
and the GSSG/GSH ratio were partially restored by clusterin overexpression in the
5
AAV-CLU/DM group (Fig.6M-N).
6
Mitochondria-derived ROS was the major intracellular component of ROS,
7
which contributes to oxidative damage. To examine whether clusterin ameliorates
8
diabetes-caused mitochondrial ROS production, the HUVECs were stained by a
9
specific mitochondrial superoxide indicator (MitoSOX red). Compared with the cells
10
exposed to NG, the HG/HF treated cells produced much more mito-ROS, which was
11
partially alleviated by the over-expression of clusterin (Fig.6O-P).
12 13
These data indicated that clusterin suppressed oxidative stress and mitochondrial ROS production induced by diabetes.
14
26
1 2
Fig.6. Clusterin prevented oxidative stress and mitochondrial ROS production.
3
(A) Representative images of ROS stained by DCFH-DA (2',7'-dichlorodihydrofluore
4
-scein diacetate) and the quantified data (B). n=3.
5
control, *P < 0.05, vs. HG/HF control. (C) Dihydroethidium (DHE) staining for ROS
6
determination in frozen mice aortic sections and fluorescence intensity was quantified 27
##
P < 0.01,
###
P < 0.001 vs. NG
1
(D). (E-G) Levels of 8-OHdG, MDA, and total SOD activity were analyzed by ELISA.
2
AAV-NC-db/db mice had a higher level of 8-OHdG, MDA and a decreased total SOD
3
activity, while AAV-CLU-dbdb mice showed a lower level of 8-OHdG, MDA and
4
increased total SOD activity. n=6.
5
AAV-NC-db/m; *P < 0.05,
6
Representative images of the rat aortas ring stained by H&E (×100) and DHE. (I)
7
Quantified data of ROS level indicated by fluorescence intensity of DHE. (J-N)
8
Diabetic rat (AAV-NC/DM) had a higher level of 8-OHdG (J), MDA (K) and
9
GSSG/GSH ratio (N), a decreased total SOD activity (L) and T-AOC (total cellular
10
antioxidant capacity) (M), while AAV-CLU/DM rat showed a lower level of 8-OHdG,
11
MDA and GSSG/GSH ratio, a increased total SOD activity and T-AOC. n=5.
12
0.01, ###P < 0.001 vs. Normal (control); *P < 0.05, ***P < 0.001 vs. AAV-NC/DM. (O)
13
Images of MitoSOX red staining in HUVECs treated with NG or HG/HF and
14
quantified analysis (P). Clusterin overexpression reduced the mitochondrial ROS
15
production in the HUVECs with HG/HF treatment. n=3. ###P < 0.001 vs. Ad-NC /NG,
16
***
17
high glucose and high palmitate; Ad-NC, negative control adenovirus; Ad-CLU,
18
adenovirus expressing clusterin; AAV-NC, negative control adeno-associated virus;
19
AAV-CLU, adeno-associated virus expressing clusterin; HFD, high fat diet; DM, rats
20
treated with HFD/STZ to produce diabetes mellitus models.
#
P < 0.05,
**
P < 0.01,
***
##
P < 0.01,
###
P < 0.001 vs.
P < 0.001 vs. AAV-NC-db/db. (H)
##
P<
P < 0.001 vs. Ad-NC/HG/HF. CM, culture medium; NG, normal glucose; HG/HF,
21 22
28
1 2
Fig.7. Clusterin diminished mitochondrial fragmentation and regulated the
3
expression of proteins responsible for mitochondrial fission and fusion. (A)
4
Mitochondria were stained with MitoTracker Green, and mitochondrial density was
5
quantified (B). Scale bar is 100 µm. n=3.
6
Ad-NC/HG/HF. (C-E) Western blotting analysis of mitochondrial dynamics-related
7
proteins, including mitochondrial fission protein Drp-1 and mitochondrial fusion 29
##
P<0.01 vs. Ad-NC/NG,
**
P<0.01 vs.
1
protein OPA1, MFN1, and MFN2. The expression level of Drp-1 modulating
2
mitochondrial fission in the HUVECs exposed to HG/HF increased notably, while the
3
clusterin expression alleviated the increase. (F) Representative images of the
4
HUVECs stained by JC-1/Hochest 33342 under fluorescence microscopy. (G) The
5
mitochondrial membrane potential (MMP) indicated by the red/green ratio was
6
decreased in the HUVECs treated with HG/HF, while the HUVECs infected with
7
Ad-CLU showed a higher MMP compared to the Ad-NC infected HUVECs. n=3.
8
###
9
blotting analysis of the mitochondrial dynamics related proteins in cell lysate (H-J)
P<0.001 vs. NG control, *P<0.05,
***
P<0.001 vs. HG/HF control. (H-L) Western
10
and mitochondria (K-L) isolated from the mice aortas. n=6. #P<0.05,
11
AAV-NC-db/m; **P<0.01, ***P<0.001 vs. AAV-NC-db/db.
###
P<0.001 vs.
12 13
3.4 Clusterin overexpression prevents mitochondrial fragmentation
14
It has been confirmed that mitochondrial dynamic change and aggravated
15
mitochondrial fragmentation in diabetes contributes to mitochondrial ROS production
16
[16]. Simultaneously, superabundant ROS exacerbates oxidative damage to the
17
mitochondria, which is a fatal factor to endothelial dysfunction in diabetes. First, we
18
examined whether clusterin regulates mitochondrial dynamics under high glucose and
19
high palmitate conditions. MitoTracker Green staining was used to characterize
20
mitochondrial morphology. The mitochondria in HUVECs stimulated with HG/HF
21
became shorter and punctated, which was partially restored by clusterin
22
overexpression (Fig.7A). The quantified data indicated that clusterin elevated the
23
mitochondrial density under HG/HF conditions (Fig.7B). Further, the expression of
24
proteins modulating mitochondrial dynamics including Drp-1, OPA1, MFN1, and
25
MFN2 was determined via Western blotting assay. In vitro, expression level of Drp-1
26
modulating mitochondrial fission in HUVECs exposed to HG/HF increased notably;
27
however, the Ad-CLU infected HUVECs had lower Drp-1 expression levels than the
28
negative adenovirus group (Fig.7C-D). Decreased OPA1 expression in the HG/HF
29
treated ECs was also observed compared with NG treated ECs, while clusterin
30
alleviated the decrease (Fig.7C, E). MFN1 and MFN2 had no notable change after 30
1
HG/HF stimulation. Proteins including OPA1, MFN1, and MFN2 modulated
2
mitochondrial fusion. These results indicated that clusterin overexpression prevents
3
mitochondrial excessive fission triggered by high glucose and high palmitate
4
conditions. ATP synthesis is a crucial function of the mitochondria, and the
5
mitochondrial membrane potential (MMP, ∆Ψm) is a prerequisite for oxidative
6
phosphorylation. The dissipation of MMP implies that mitochondria are disrupted. To
7
determine whether clusterin also affects the MMP, we measured the MMP using a
8
JC-1 fluorescence probe (Fig.7F-G). The MMP in the ECs exposed to HG/HF showed
9
a decreased red/green ratio and was restored partially by clusterin overexpression.
10
The expression abundances of OPA1, Drp-1, MFN1, and MFN2 were also
11
investigated in the diabetic mice. The total and mitochondrial proteins were extracted
12
from the mice aortas. Compared with the db/m mice, Drp-1 expression level in cell
13
lysate (Fig.7H-I) and mitochondria (Fig.7K-L) increased in the AAV-NC-db/db mice,
14
but the AAV-CLU-db/db mice had a lower level. In addition, compared with the
15
non-diabetic mice, decreased expression levels of OPA1 in the cell lysate (Fig.7H, J)
16
and mitochondria (Fig.7K-L) was observed in the AAV-NC-db/db mice, and clusterin
17
overexpression alleviated the decrease. Consistent results were obtained in the
18
STZ-induced diabetic rats (Fig.S5).
19
Together, these results indicated that clusterin plays a vital part in maintaining
20
the balance of mitochondrial fusion/fission, protecting mitochondria from oxidative
21
damage.
22 23
3.5 Clusterin attenuates mitochondrial fragmentation via activation of AMPK
24
Accumulating evidences demonstrated that AMPK is an important metabolism
25
regulator that has attracted extensive attention as a prospective therapeutic target for
26
type 2 diabetes [42]. Recent researches illustrated that AMPK activation also
27
contributes to preventing endothelial dysfunction in diabetes [43, 44]. We investigated
28
whether clusterin plays a role in regulating AMPK signaling. The phosphorylation of
29
AMPKα (Thr 172) was inhibited under hyperglycemia, but this inhibition was
30
partially restored in the AAV-CLU-db/db mice aortas (Fig.8A). The expression and 31
1
phosphorylation (Ser 79) of acetyl-CoA carboxylase (ACC), the down-stream target
2
of AMPK associated with fatty acid metabolism were improved in the
3
AAV-CLU-db/db mice aortas. A similar profile was observed in the STZ-induce
4
diabetic rats (Fig.8B). The phosphorylation of AMPKα on Thr 172 was elevated by
5
clusterin overexpression compared with the diabetic rats treated with negative AAV.
6
Meanwhile, the level of p-ACC (Ser79) was obviously restored in the AAV-CLU/DM
7
group.
8
To identify whether AMPK activation is indispensable for clusterin in preventing
9
mitochondria impairment, we subjected the HUVECs to an AMPK specific inhibitor,
10
compound C. First, mitochondrial ROS production was examined in HUVECs treated
11
with or without compound C (10 µM). The mito-ROS level of clusterin-overexpressed
12
HUVECs exposed to HG/HF was obviously reduced, whereas the reduction was
13
weakened by compound C (Fig.9A and C). On the other hand, mitochondria of the
14
Ad-CLU treated ECs showed a relatively normal network structure despite HG/HF
15
stimulation, which was destroyed by compound C, exhibiting an increase in
16
mitochondrial fragmentation (Fig.9B and D). As predicted, under the HG/HF
17
conditions, the reduction in the Drp-1 expression and the increase in the OPA1
18
expression observed in the Ad-CLU pretreated ECs were wiped out by compound C
19
(Fig.9E), which also indicated that compound C restrained clusterin from attenuating
20
the mitochondrial fragmentation induced by the high glucose and high palmitate
21
conditions. We also investigated the influence of compound C on the suppression of
22
endothelial inflammatory activation in the Ad-CLU treated HUVECs. As expected,
23
although the ECs were clusterin overexpressed, the mRNA expression of ICAM1 and
24
VCAM1 responding to HG/HF was enhanced following compound C treatment
25
(Fig.9F).
26
Furthermore, siRNAs targeting AMPKα mRNA were used to investigate whether
27
knockdown of AMPK in HUVECs under the high glucose and high palmitate
28
conditions
29
AMPKα-siRNA1/2 or siRNA-NC following Ad-CLU infection. As shown in Fig.S6,
block
clusterin’s
effects.
32
HUVECs
were
transfected
with
1
AMPKα-siRNA1 and AMPKα-siRNA2 reversed the clusterin-mediated prevention of
2
mito-ROS production (Fig.S6A and C). Also, AMPKα-siRNA1 and AMPKα-siRNA2
3
reversed the clusterin-mediated inhibition of mitochondrial fragmentation (Fig. S6B
4
and D). In addition, the mRNA expression of ICAM1, VCAM1 and CCL2 responding
5
to HG/HF was enhanced in the ECs treated with AMPKα-siRNA1 or AMPKα-siRNA2
6
despite
7
AMPKα-siRNA blocked clusterin’s effects in HUVECs under the high glucose and
8
high palmitate conditions, which was same as the AMPK inhibitor compound C.
overexpression
(Fig.S6E-G).
These
data
indicated
that
Together, these data demonstrated that clusterin attenuated mitochondrial ROS
9 10
clusterin
production and fragmentation through the activation of AMPK.
11
12 13
Fig.8. Clusterin activated AMPK in db/db and STZ-induced diabetes. (A)
14
Phosphorylation of AMPKα on Thr172 and phosphorylation of ACC on Ser79 in mice
15
aortas were analyzed by western blotting. The ratios of p-AMPKα/AMPKα and
16
p-ACC/ACC were increased in the AAV-CLU infected db/db mice compared to the
17
AAV-NC infected db/db mice. n=4. #P<0.05, ##P<0.01, ###P<0.001 vs. AAV-NC-db/m;
18
*
P<0.05,
**
P<0.01,
***
P<0.001 vs. AAV-NC-db/db. (B) Phosphorylation of AMPKα 33
1
on Thr172 and phosphorylation of ACC on Ser79 in the rat aortas were analyzed by
2
western blotting. The ratios of p-AMPKα/AMPKα and p-ACC/ACC were decreased
3
in STZ-induced diabetes mellitus (DM) models, while clusterin overexpression
4
promoted the phosphorylation of AMPKα and ACC in diabetic rats. n=4. #P<0.05,
5
##
P<0.01, ###P<0.001 vs. Normal (control); *P<0.05, **P<0.01, ***P<0.001 vs. DM.
6
34
1 2
Fig.9. Compound C abolished the attenuated mitochondrial ROS production and 35
1
fragmentation mediated by clusterin under HG/HF conditions and clusterin
2
knockdown aggravated mitochondrial oxidation and endothelial inflammatory
3
activation in the HUVECs. (A) Images of MitoSOX red staining in the HUVECs
4
pretreated with or without compound C or siRNA and quantified analysis(C).
5
(B)Mitochondrial morphology was characterized using MitoTracker Green, and
6
mitochondrial density was quantified (D). Scale bar is 50 µm. (E) Western blotting
7
analysis of mitochondrial dynamics related proteins, including mitochondrial fission
8
protein Drp-1 and mitochondrial fusion protein OPA1, MFN1, and MFN2. (F) The
9
mRNA expression levels of ICAM1, VCAM1 and CCL2 were measured by qRT-PCR.
10
n=3. #P<0.05,
##
11
Ad-CLU-HG/HF. CM, culture medium; NG, normal glucose; CC, compound C;
12
HG/HF, high glucose and high palmitate; Ad-CLU, the adenovirus expressing
13
clusterin; CLU-siRNA1 and CLU-siRNA2, the siRNA targeting CLU-mRNA.
P<0.01,
###
P<0.001 vs. HG/HF, *P<0.05,
**
P<0.01,
***
P<0.001 vs.
14 15
3.6 Clusterin knockdown increased mito-ROS production and aggravated the
16
endothelial inflammatory activation
17
To obtain more evidence to support our conclusion, we knocked down clusterin
18
expression by siRNA (Fig.S1) in the HUVECs. The CLU-siRNA1 and CLU-siRNA2
19
target different sequence segments of CLU-mRNA. As a result, silencing clusterin
20
aggravated mitochondrial ROS production and fragmentation under HG/HF
21
conditions (the last two panels of Fig.9A-D). VCAM-1 and ICAM-1 expression in the
22
clusterin knockdown HUVECs exposed to HG/HF was significantly elevated (Fig.9F).
23
Besides, HG/HF impaired the tube formation of HUVECs on Matrigel, whereas
24
silencing clusterin caused a greater decrease in network formation (Fig.S7). These
25
results suggested that clusterin knockdown aggravated mitochondrial fragmentation
26
and endothelial inflammatory activation in contrast to clusterin overexpression.
27 28 29 30
4. Discussion Accumulating
evidence
revealed
that
preventing
vascular
endothelial
dysfunction caused by diabetes can alleviate cardiovascular complications in diabetic 36
1
patients [3]. In this study, we used two diabetic models of spontaneous and
2
drug-induced diabetes to explore the role of clusterin in endothelial dysfunction
3
caused by hyperglycemia and hyperlipidemia. We determined that clusterin
4
ameliorates diabetes-caused endothelial dysfunction by inhibiting monocyte adhesion
5
and improving endothelium-independent vasorelaxation. Our data also indicated that
6
clusterin overexpression reduced oxidative stress and ROS levels, which is the major
7
cause of endothelial dysfunction under diabetic conditions. The results highlight the
8
previous conclusion that clusterin is a biosensor for oxidative stress and cleaner of
9
oxidized production [45, 46]. Previous reports suggested that clusterin plays a
10
protective role against neointimal hyperplasia by inhibiting the apoptosis of
11
endothelial cells and the proliferation of VSMCs [36]. In another research, clusterin
12
was identified as a mediator of the leptin signaling that plays a key role in the
13
regulation of energy homeostasis [47]. These studies were consistent with the results
14
indicating that clusterin is involved in cytoprotection under a wide variety of stress
15
conditions.
16
On the other hand, clusterin shares functional similarities with small heat shock
17
proteins (sHSPs) as a stress-induced protein [29]. Clusterin and sHSPs increase in
18
response to oxidative stress, regulated by the heat shock factor signaling [24, 48].
19
Thus, it is not surprising that the expression levels of clusterin are up-regulated in
20
metabolic diseases associated with oxidative stress [28]. For example, the clusterin
21
level in plasma is obviously up-regulated in patients with diabetes or heart failure [49].
22
Furthermore, elevated sHSP expression improves cell survival by decreasing the
23
intracellular levels of free radicals and reducing the oxidative damage to DNA,
24
proteins, and lipids. However, sHSPs were inhibited from constant increasing because
25
of a negative feedback between sHSPs and heat shock factor [50]. In our study,
26
similar to sHSPs, clusterin plays a role of cytoprotection but may not be constantly
27
elevated under diabetic conditions and not completely exert its effects, while higher
28
levels of clusterin in the diabetic animal models significantly improved endothelial
29
function with clusterin over-expression.
30
This study demonstrated that clusterin inhibited mitochondrial fragmentation and 37
1
ROS production, which has not previously reported. Mounting evidence has revealed
2
that mitochondrial dysfunction may contribute significantly to endothelial dysfunction
3
[8, 15, 16]. Elevated free fatty acid and hyperglycemia in diabetes contribute to
4
mitochondrial dysfunction, including the perturbation of mitochondrial morphology
5
and dynamics [51-53]. Recent evidence indicates that mitochondrial fission in
6
endothelial cells is promoted under diabetic conditions, resulting in reduced oxidative
7
phosphorylation capacity and increased ROS production [8]. In addition to being a
8
major headstream of ROS, the mitochondria are targets of ROS. Excess ROS is
9
detrimental to mitochondrial proteins, DNA, and membrane lipid, which prompts
10
endothelial dysfunction [54, 55]. Therefore, maintaining mitochondrial homeostasis is
11
necessary to suppress endothelial dysfunction in diabetic states.
12
How does clusterin regulate the balance of mitochondrial dynamics? AMPK
13
signaling attracted our interests. AMPK, an intracellular sensor of energy, has recently
14
been explored as a novel drug target for preventing diabetic complications [42]. ROS
15
overproduction via mitochondria and NADPH oxidase induced by diabetes in
16
endothelial cells can be inhibited by AMPK activation [44]. AMPKα phosphorylation
17
is subdued markedly in diabetes, resulting in disturbed mitochondrial function and
18
energy homeostasis [56]. Previous findings suggested that AMPK deficiency
19
aggravates diabetic vascular injury via promoting excessive fission of mitochondria
20
[12]. Our present study showed that AMPK was activated by clusterin overexpression,
21
and inhibitor of AMPK, compound C, weakened the action of clusterin in the
22
HUVECs subjected to HG/HF. These data revealed that clusterin attenuates
23
mitochondrial impairment through the activation of AMPK. It has been suggested that
24
clusterin inhibits mitochondrial apoptosis by suppressing p53-activating stress signals,
25
inhibiting Bax activation and cytochrome C release [57]. However, the role of AMPK
26
in mitochondrial dynamics modulated by clusterin has not been previously reported,
27
and would be interesting to be investigated in more detail. For example, the mediator
28
between clusterin and AMPK and the specific downstream signaling remain obscured,
29
awaiting further investigation.
30
The role of clusterin in endothelial dysfunction might be beyond inhibiting the 38
1
perturbation of mitochondrial dynamics. We also found that the overexpression of
2
clusterin affects the transcription of proinflammatory genes in endothelial cells
3
stimulated with HG/HF. Previous evidence suggested that clusterin may regulate the
4
function of ECs with TNF-α stimulation via repressing the NF-κB signaling pathway
5
[36]. Another report indicated that up-regulated expression of clusterin triggered by
6
shear stress can restrain atherosclerosis via preventing endothelial activation [58].
7
Thus, the mechanism of the protective role of clusterin in diabetic endothelial
8
dysfunction may be involved in chronic inflammation. It was recently shown that
9
clusterin also facilitates stress induced autophagy to enhance cancer cell survival [59].
10
However, the impact of clusterin on autophagy in ECs remains unkown. Further
11
studies are needed to explore whether the protective role of clusterin observed in
12
diabetes-induced endothelial dysfunction is also mediated in part through autophagy
13
or mitophagy.
14
Lipotoxicity from elevated free fatty acid in diabetes also fosters endothelial
15
dysfunction, and this factor was taken into account in this study. Clusterin
16
overexpression decreased the level of NEFA in db/db diabetic mice (Fig. 4B),
17
implying that clusterin may play a role in lipid metabolism. But more experiments are
18
needed to verify the result and clarify the involved mechanism. A vicious circle of
19
events occurred in the endothelium could be induced by the metabolic milieu in
20
diabetes including hyperglycemia, elevated free fatty acid and insulin resistance, and
21
the endothelial dysfunction induced by the multiple metabolic disturbances is closely
22
related to vascular complications [5]. Hence, understanding and preventing
23
endothelial dysfunction is constructive for the prevention of diabetic complications. In
24
the current study, two diabetes models of spontaneous diabetes with LEPR-deficiency
25
and STZ-induced diabetes were used, but there are still some limitations. First, loss of
26
function using CLU-KO mice has yet to be considered. In addition, clusterin is a
27
complicated protein with multiple physiological functions and subcellular localization,
28
and its crystal structure remains unknown [26]. This study did not determine
29
respectively the roles of clusterin in different subcellular organelles. In light of the
30
published data and our previous work (Fig.S8), clusterin primarily as a secreted form 39
1
exerts cytoprotection under a variety of stress conditions [60]. In our current study,
2
HUVECs were infected with Ad-CLU expressing sCLU precursor (~60 kD) and
3
sCLU, which were detected by Western blotting (Fig.S1B) and ELISA (Fig.S1C). In
4
the db/db mice and STZ-induced rat models, the sCLU precursor (located in the
5
cytosol) of 60 kD in the aorta tissue and the secreted clusterin in the plasma were
6
detected. However, further animal studies are needed to investigate the separate role
7
of clusterin with different subcellular location in diabetes-induced endothelial
8
dysfunction. Moreover, David Bradley et al. suggested that adipocyte-specific sCLU
9
expression impairs hepatic insulin sensitivity, implying that the role of clusterin might
10
be multiple and tissue-specific [61]. However, the gain- or loss- of function
11
experiments supporting the role of clusterin in insulin resistance was absent. To be
12
honest, we have only scratched the surface of the clusterin mystery, further research is
13
necessary.
14
In conclusion, our study provides fresh insight into the role of clusterin and sheds
15
further light on a novel mechanism explaining that clusterin ameliorates endothelial
16
dysfunction in diabetes via suppressing mitochondrial fragmentation. Our study also
17
demonstrated that AMPK activation plays a considerable role in clusterin-mediated
18
mitochondrial homeostasis. Therefore, clusterin may be a promising therapeutic target
19
for endothelial dysfunction induced by diabetes.
20 21
Acknowlegements
22
Thanks a lot for the support and help from Institute of Materia Medica, Chinese
23
Academy of Medical Science and Peking Union Medical College.
24 25
Funding
26
This work was supported by National Science and Technology Major Projects for
27
‘Major New Drugs Innovation and Development’ [2017ZX09101001] and National
28
Natural Science Foundation of China [81641016, 81603197].
29
Conflict of interests
30
None 40
1 2
Author contributions
3
Design of the project: Lihong Liu
4
Experiments: Lulu Ren, Feifei Han, Lingling Xuan, Benshan Xu, Yuan Sun and Song
5
Yang
6
Data analysis: Yali Lv, Lili Gong, Zirui Wan, Lifang Guo and He Liu
7
Figures layout : Lulu Ren and Yan Yan
8
Manuscript writing: Lulu Ren
9
Manuscript revision: Lihong Liu
10 11
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[53] Chong, C. R.; Clarke, K.; Levelt, E. Metabolic Remodeling in Diabetic
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Cardiomyopathy. Cardiovascular research; 2017.
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Breton-Romero, R.; Linder, E. A.; Berk, B. D.; Weisbrod, R. M.; Widlansky, M. E.;
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Gokce, N.; Ballinger, S. W.; Hamburg, N. M. Mitochondrial DNA damage and 46
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disease. Cardiovascular diabetology 15:53; 2016.
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and aging. Free radical biology & medicine 29:222-230; 2000.
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Diabetologia 60:1577-1585; 2017.
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Papassideri, I. S.; Zou, Y.; Margaritis, L. H.; Boothman, D. A.; Gonos, E. S.
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Intracellular clusterin inhibits mitochondrial apoptosis by suppressing p53-activating
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research : an official journal of the American Association for Cancer Research
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15:48-59; 2009.
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[58] Urbich, C.; Fritzenwanger, M.; Zeiher, A. M.; Dimmeler, S. Laminar shear stress
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upregulates the complement-inhibitory protein clusterin : a novel potent defense
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mechanism against complement-induced endothelial cell activation. Circulation
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101:352-355; 2000.
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[59] Zhang, F.; Kumano, M.; Beraldi, E.; Fazli, L.; Du, C.; Moore, S.; Sorensen, P.;
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Zoubeidi, A.; Gleave, M. E. Clusterin facilitates stress-induced lipidation of LC3 and
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autophagosome biogenesis to enhance cancer cell survival. Nature communications
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5:5775; 2014.
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[60] Klock, G.; Baiersdörfer, M.; Koch-Brandt, C. Chapter 7 Cell Protective Functions
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of Secretory Clusterin (sCLU). 104:115-138; 2009.
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Clusterin Impairs Hepatic Insulin Sensitivity and Adipocyte Clusterin Associates With
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Cardiometabolic Risk. Diabetes care 42:466-475; 2019.
47
Clusterin ameliorates endothelial dysfunction in diabetes by suppressing mitochondrial fragmentation Lulu Ren1, Feifei Han1, Lingling Xuan1, Yali Lv1, Lili Gong1, Yan Yan1, Zirui Wan1, Lifang Guo1, 2
1
, He Liu1, Benshan Xu1, Yuan Sun 1,Song Yang1, Lihong Liu1, * Department of Pharmacy, Beijing Chao-Yang Hospital, Capital Medical University, Beijing,
100020, China 2
School of Life Sciences, Tsinghua University, Beijing 100084, China
* Corresponding author. Department of Pharmacy, Beijing Chao-Yang Hospital, Capital Medical University, 8 Gongren Tiyuchang Nanlu, Beijing 100020, China. Tel: 86-010-8523 1464. E-mail: [email protected].
Highlights Clusterin overexpression rescues diabetes-induced endothelial dysfunction. In diabetic models, clusterin inhibits oxidative stress and mitochondrial ROS production. Clusterin suppresses mitochondrial fragmentation via reducing expression level of Drp-1. AMPK activation involved in the molecular mechanisms of clusterin against endothelial dysfunction.
S0891-5849(19)31273-0
DOI:
https://doi.org/10.1016/j.freeradbiomed.2019.10.008
Reference:
FRB 14446
To appear in:
Free Radical Biology and Medicine
Received Date: 2 August 2019 Revised Date:
10 October 2019
Accepted Date: 10 October 2019
Please cite this article as: L. Ren, F. Han, L. Xuan, Y. Lv, L. Gong, Y. Yan, Z. Wan, L. Guo, H. Liu, B. Xu, Y. Sun, S. Yang, L. Liu, Clusterin ameliorates endothelial dysfunction in diabetes by suppressing mitochondrial fragmentation, Free Radical Biology and Medicine (2019), doi: https://doi.org/10.1016/ j.freeradbiomed.2019.10.008. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
1
Title Page
2
Clusterin ameliorates endothelial dysfunction in diabetes by
3
suppressing mitochondrial fragmentation
4
Lulu Ren1, Feifei Han1, Lingling Xuan1, Yali Lv1, Lili Gong1, Yan Yan1, Zirui Wan1, Lifang Guo1,
5
2
6
1
7
100020, China
8
2
9
* Corresponding author.
, He Liu1, Benshan Xu1, Yuan Sun 1,Song Yang1, Lihong Liu1, * Department of Pharmacy, Beijing Chao-Yang Hospital, Capital Medical University, Beijing,
School of Life Sciences, Tsinghua University, Beijing 100084, China
10
Department of Pharmacy, Beijing Chao-Yang Hospital, Capital Medical University,
11
8 Gongren Tiyuchang Nanlu, Beijing 100020, China.
12
Tel: 86-010-8523 1464.
13
E-mail: [email protected].
14 15 16 17 18 19 20 21 22 23 24 25 26
1
Abstract
2
Clusterin (CLU) is a stress-responding protein associated with cytoprotection in
3
a broad range of pathological processes. However, clusterin’s function in
4
diabetes-induced endothelial dysfunction has not been defined. Herein, using two
5
diabetes models, we investigated the role of clusterin in endothelial dysfunction
6
triggered by diabetes and the molecular mechanisms involved. The results revealed
7
that clusterin overexpression inhibited ICAM-1/VCAM-1 expression in aortas and
8
improved endothelium-dependent vasodilatation in db/db diabetic mice and
9
streptozotocin (STZ)-induced diabetes models. Consistently, in vitro, adenoviral
10
clusterin overexpression reduced the expression of a range of pro-inflammatory
11
cytokines and suppressed monocyte adhesion to endothelial cells subjected to high
12
glucose and high palmitate. Further study indicated that clusterin overexpression
13
mitigated mitochondrial excessive fission and reduced mitochondrial ROS production.
14
Conversely, silencing clusterin aggravated mitochondrial fission and endothelial
15
inflammatory activation in high glucose-exposed endothelial cells. Accumulating
16
evidence indicates that impaired mitochondrial dynamics plays a considerable role in
17
promoting endothelial dysfunction in diabetic subjects. Therefore, treatments
18
targeting mitochondrial undue fission may be promising measures to prevent vascular
19
complications of diabetes. Furthermore, AMP-activated protein kinase (AMPK)
20
activation contributed to the modulation of mitochondrial dynamics executed by
21
clusterin. Mechanistically, clusterin promoted the phosphorylation of AMPKα and its
22
downstream target acetyl-CoA carboxylase (ACC), while the inhibition of AMPKα
23
negated the improvement in mitochondrial dynamics provided by clusterin
24
overexpression. Over all, these findings suggest that clusterin exerts beneficial effects
25
in endothelial cells under diabetic conditions via inhibiting mitochondrial
26
fragmentation mediated by AMPK.
27 28
Key words： ：Clusterin; CLU; Endothelial dysfunction; Diabetes; Oxidative stress;
29
Mitochondrial fission.
30
Abbreviations: CLU, clusterin; ICAM-1, intercellular adhesion molecule 1; 1
1
VCAM-1, vascular cell adhesion molecule 1; STZ, streptozotocin; ROS, reactive
2
oxygen species; ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase;
3
ECs, endothelial cells; AP-1, activator protein-1; Drp-1, dynamin-related protein;
4
AAV, adeno-associated virus; NC, negative control; FBG, fasting blood glucose; HFD,
5
high fat diet; CDS, coding sequence; TCID50, 50 % tissue culture infective dose;
6
IPGTT, intraperitoneal glucose tolerance test; DM, diabetic mellitus; ITT, insulin
7
tolerance test; NEFA, non-esterified fatty acid; H&E, hematoxylin and eosin; DHE,
8
dihydroethidium; HUVECs, human umbilical vein endothelial cells; Ad, adenoviral
9
virus; DCFH-DA, 2',7'-dichlorodihydrofluorescein diacetate; MDA, malondialdehyde;
10
SOD, superoxide dismutase; T-AOC, total cellular antioxidant capacity; Ach,
11
acetylcholine;
12
voltage-dependent
13
MFN1, mitofusin-1; MFN2, mitofusin-2; MCP-1, monocyte chemoattractant protein 1;
14
MOI, multiplicity of infection; CC, compound C; FBS, fetal bovine serum; PBS,
15
phosphate buffer; MMP/∆Ψm: mitochondrial membrane potential; CCK-8, cell
16
counting Kit-8; LDH, lactate dehydrogenase; ANOVA, analysis of variance.
SNP,
sodium
nitroprusside;
anion-selective
channel
NA, 1;
noradrenaline;
OPA1,
optic
VDAC1,
atrophy
1;
17 18
1. Introduction
19
Recently, sedentary lifestyle, diet, obesity, and aging of the population have led
20
to an increased prevalence of diabetes [1, 2]. Cardiovascular disease, one of the major
21
causes of mortality in diabetics, occurs in over half of diabetic patients [3]. However,
22
deficiencies in prevention and treatment measures remain prevalent. Endothelial
23
dysfunction, the first step of cardiovascular diseases, is a key mediator of vascular
24
disease in diabetes [4]. Hyperglycemia, excessive free fatty acid as well as insulin
25
resistance in diabetes cause increased oxidative stress, chronic inflammation and
26
activation in endothelium, resulting in endothelial dysfunction [5, 6]. Mitochondria
27
are vital organelles with multiple roles in modulating the energy supply, the ROS
28
generation and the dynamics of intracellular calcium [7-10]. The mitochondria
29
continuously change in size, shape and intracellular location instead of being static in
30
order to maintain the homeostasis [8, 11]. In diabetes, mitochondrial dysfunction 2
1
leads to abnormal mitochondrial signaling and the disruption of cellular metabolic
2
homeostasis, inducing and aggravating macrovascular complications [12, 13].
3
Mounting evidence indicated that the dynamics of mitochondrial fission/fusion play a
4
vital role in the modulation of endothelial functions [14, 15]. It has been demonstrated
5
that excessive palmitate and hyperglycemia impaired mitochondrial dynamics by
6
promoting mitochondrial fission and inhibiting mitochondrial fusion in endothelial
7
cells, resulting in increased mito-ROS production and electron transport chain
8
impairment [16, 17]. The morphological changes in mitochondria provoked ROS
9
overproduction in diabetic conditions [18]. Mitochondrial ROS mediates the signaling
10
associated with endothelial inflammatory activation in the early stage of vascular
11
disease by activating proinflammatory transcription factors, provoking elevated
12
expression levels of adhesion molecules and inflammatory cytokines [19, 20].
13
Moreover, as a major headstream of ROS production, mitochondria are susceptible to
14
oxidative damage, which causes a vicious circle of events in endothelial cells (ECs)
15
[21-23]. Thus, reducing the pathological levels of ROS and correcting the imbalance
16
of mitochondrial dynamics in diabetes-induced endothelial dysfunction might
17
improve patient outcomes.
18
Clusterin (CLU), a functional homologue to small heat shock proteins, is a highly
19
sensitive biosensor of increased oxidative stress [24, 25]. This heterodimeric
20
glycoprotein is functionally implicated in numerous pathological processes including
21
inflammation, apoptosis, and aging [26]. The stress-induced transcription of the CLU
22
gene can be mediated by the interaction between a conserved 14-bp “CLU-specific
23
element” and a nuclear transcription factor called heat shock factor-1 [27]. Clusterin
24
expression is also mediated by NF-κB and AP-1 (activator protein-1) associated with
25
oxidative stress [28]. Increasing evidence indicates that clusterin increases under
26
pathophysiological stress, exerting a chaperone activity associated with cytoprotection
27
in various diseases including cancers, ischemic heart failure, and neurodegenerative
28
disorders [29]. Van Dijk et al. reported that intravenous myocardial infarct size in rats
29
can be reduced by clusterin [30]. Clusterin protected cardiomyocytes from apoptosis
30
induced by oxidative stress via the Akt/GSK-3β pathway [31]. Intriguingly, the 3
1
overexpression of human clusterin in drosophila reduces the ROS level and increases
2
resistance to starvation, heat shock, and oxidative stress, extending their lifespan [32].
3
These results imply that clusterin may represent a particular component of antioxidant
4
response networks. However, whether clusterin prevents oxidative stress-induced
5
endothelial dysfunction in diabetes remains unclear.
6
Using db/db diabetic mice and STZ-induced diabetes models in the current study,
7
we found that clusterin overexpression suppressed mitochondrial fission and ROS
8
production, ameliorating diabetes-induced endothelial dysfunction. However,
9
AMPKα-siRNA and the inhibitor of AMPK (compound C) abolished the effect of
10
clusterin on endothelial dysfunction under the high glucose and high palmitate
11
conditions.
12 13
2. Material and Methods
14
2.1 Animals
15
The animals were housed at constant temperatures (22°C) under a cycle with 12
16
h of darkness and 12 h of light. Water and food were available ad libitum. All of the
17
animal treatments were performed in accordance with the Health Guidelines of the
18
Capital Medical University.
19
Male db/db mice and non-diabetic control db/m mice (6 weeks old,
20
C57BLKS/JNju ) were obtained from the Model Animal Research Center of Nanjing
21
University and fed with normal chow. Each group consisted of 8 mice (N=8). After
22
one-week adaption, recombinant adeno-associated virus expressing clusterin
23
(AAV-CLU, 10^10 PFU/mouse) or negative control adeno-associated virus (AAV-NC,
24
1010 PFU/mouse) were administered to the mice via tail vein injection once every six
25
weeks as previously described [33]. Their fasting blood glucose (FBG) was monitored
26
using a glucometer (OneTouch Ultra Easy, LifeScan, Malvern, PA, USA), and the
27
body weights were measured once a fortnight. At the end, the mice were sacrificed
28
under anesthesia with an intraperitoneal injection of sodium pentobarbital at 50 mg/kg,
29
and their plasma and aortas were collected for further experiments.
30
The fat-fed streptozotocin rat is another model for type 2 diabetes. The rats are fed a 4
1
high fat diet to induce insulin resistance and injected with a low dose of STZ to impair β-cell
2
function, resulting in hyperglycemia, associated with hyperlipidemia and insulin resistance
3
[34]. For the STZ-induced diabetes model, male Sprague-Dawley (SD) rats (8 weeks
4
old) were purchased from Huafukang Animal Company (Beijing, China) and fed a
5
high fat diet (HFD, 45 % fat, 35 % carbohydrate, 20% protein; H10045, Huafukang,
6
China) or a control chow (10 % fat, 70 % carbohydrate, 20% protein) for 4 weeks.
7
Each group consisted of 8 rats (N=8). The rats in the diabetes mellitus groups were
8
then intraperitoneally injected with STZ buffer at a single dose of 40 mg/kg, and the
9
control group rats were injected with sodium citrate buffer. Diabetes was defined as
10
blood glucose levels > 200 mg/dL (11.1mmol/L) after injection. Then, 100 µL of
11
adeno-associated virus AAV-CLU or AAV-NC (1012 PFU/mL) were administered via
12
tail vein injection. All of the animals were maintained on the preceding diets
13
respectively, and their FBG and body weights were monitored. After 8 weeks, the rats
14
were sacrificed under anesthesia with an intraperitoneal injection of sodium
15
pentobarbital at 40 mg/kg, and their plasma and aortas were harvested for further
16
experiments.
17 18
2.2 Recombinant adenoviral and adeno-associated virus vectors
19
Recombinant adenovirus vector plasmid encoding full-length human CLU CDS
20
(NM_ 001831.4) was constructed with the HB-Infusion cloning system (Hanbio,
21
Shanghai, China) according to the manufacturer’s directions. Then shuttle plasmid
22
and skeleton plasmid were co-transfected into HEK293A cells for 6 h. Until 80%
23
cytopathic effect was observed, the virus suspension was collected. The CsCl density
24
gradient centrifugation-dialysis combination method was used for adenovirus
25
purification. The viral titer (1.26×10^10 PFU/mL) of recombinant adenoviral virus
26
Ad-CLU was tested using the 50% tissue culture infective dose (TCID50) method.
27
Ad-NC (1.18×10^10 PFU/mL) was used as a negative control.
28
For construction of adeno-associated virus vectors, adenovirus vector plasmid
29
encoding full-length mouse CLU CDS (NM_013492.3) and rat CLU CDS
30
(NM_053021.2) were constructed with the HB-Infusion cloning system. Then the 5
1
shuttle plasmid, pAAV-RC and pHelper were co-transfected into AAV-293 cells. The
2
cells were collected and disrupted after 72 h. An Adeno-Associated Virus Purification
3
Mini-kit (Biomiga, San Diego, CA, USA) was used for purification. The viral titers of
4
AAV-r-CLU (1.5×10^12 PFU/mL) or AAV-m-CLU (1.6×10^12 PFU/mL) were tested
5
using qRT-PCR. AAV-NC (1.3×10^12 PFU/mL) was used as a negative control.
6 7 8
2.3 IPGTT and ITT The intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT)
9
were conducted on the second weekend after STZ injection. The rats were fasted and
10
injected with glucose (2 g/kg) or insulin (1 U/kg). Their blood glucose levels were
11
measured using tail bleeds 0, 20, 40, 60, and 120 min after the administration of
12
glucose or insulin.
13 14
2.4 Histological analysis
15
Hematoxylin and eosin (H&E) staining was applied to characterize the
16
pathological morphology of aorta. Aortic tissue was isolated from the animals and
17
fixed in phosphate-buffered 4 % paraformaldehyde. Then the tissue was embedded in
18
paraffin wax and cut into sections. The H&E stained sections were observed under an
19
optical microscope (Olympus, Tokyo, Japan).
20 21
2.5 Immumohistochemistry
22
Immunohistochemical staining was performed on thoracic aortae to determine
23
clusterin (CLU), VCAM-1 and ICAM-1. Primary antibodies against ICAM-1 (sc8439,
24
1:100), VCAM-1 (sc13160, 1:100), and clusterin (sc166831, 1:50) used in the
25
immunohistochemistry experiment were purchased from Santa Cruz Biotechnology
26
(Dallas, TX, USA). After antigen retrieval, the aortic sections were incubated with the
27
primary antibody at 4 ℃ overnight and incubated with labeled polymer-horseradish
28
peroxidase secondary antibodies. The sections were then counterstained with
29
hematoxylin followed by diaminobenzidine chromogen incubation. The positive
30
staining of the tissue sections were quantized using Image J. 6
1
2.6 Assessment of ROS production
2
Intracellular ROS level in aorta was determined by the superoxide indicator
3
dihydroethidium (DHE, Thermo Fisher Scientific, Waltham, MA, USA). DHE shows
4
blue-fluorescence in the cytosol without oxidization, while oxidized DHE intercalates
5
within DNA, exhibiting a bright fluorescent red. First, the freshly isolated aortas were
6
frozen and sectioned. Then the sections were incubated with DHE solution (5 µM) at
7
37 °C for 30 min in the dark. After washing, the sections were observed and imaged
8
using a fluorescence microscope. Image J was used to analyze the data.
9
To assess the mitochondrial ROS produced in the ECs, the mito-ROS specific
10
indicator Mito-SOX red was used in the study. Mito-SOX red is a fluorogenic dye for
11
highly specific detection of mitochondrial superoxide in live cells. Mito-SOX red
12
reagent is oxidized by superoxide in the mitochondria and exhibits red fluorescence.
13
Before use, a working solution of Mito-SOX red (5 µM) was made with Hank's
14
balanced salt solution. The cells were then incubated with the working solution at
15
37 °C for 10 min without light. The cells were then stained with Hoechst 33342. Then,
16
the cells were imaged using the fluorescence microscope (Olympus, Japan).
17 18
2.7 ELISA
19
8-OHdG (8-Hydroxy-2’-deoxyguanosine) level in the plasma collected from the
20
mice or rats was detected using 8-OHdG ELISA kit (mouse/rat, Nanjingjiancheng,
21
Jiangsu, China). Briefly, 50 µL of plasma or standard samples were added into the
22
microplate wells, and then 50 µL of biotinylated antibody working solution was added
23
into each well. The microplate was incubated at 37 °C for 30 min after shaking gently.
24
After washing five times, horseradish peroxidase conjugated reagent was added into
25
the wells. After 30 min, chromogen solution A and B were applied successively and
26
incubated for 10 min at 37 °C. The reaction was then halted using stop solution. The
27
optical density values were measured by the microplate reader. The 8-OHdG level
28
was calculated according to the manufacturer's protocols. Similarly, levels of TNF-α
29
and IL-6 in supernatant of cell culture medium collected from HUVECs subjected to
30
different treatments were quantified according to the protocols of ELISA kits 7
1
(Biolegend, San Diego, CA, USA). Secreted clusterin in the plasma was detected
2
using a Clusterin ELISA Kit (Abcam, Cambridge, MA, USA) according to the
3
manufacturer’s protocols.
4 5
2.8 Determination of malondialdehyde (MDA), total superoxide dismutase (SOD)
6
activity and total cellular antioxidant capacity (T-AOC)
7
A whole aorta homogenate was prepared with the sample preparation solution in
8
ice-water bath and centrifuged at 4°C (12000 g, 3 min). The protein was quantified
9
according to the BCA Assay Kit instructions (P0009, Beyotime, Nanjing, China).
10
Malondialdehyde (MDA) was detected using a Lipid Peroxidation MDA Assay kit
11
(S0131, Beyotime, China) according to the manufacturer's instructions. The MDA
12
content was determined on a microplate reader at 532 nm and expressed as µmol/mg
13
protien. The total superoxide dismutase (SOD) activity was determined via WST-8
14
assay with a Total Superoxide Dismutase Assay Kit (S0101, Beyotime, China)
15
according to the manufacturer’s protocol. The total SOD activity was calculated and
16
expressed as U/mg protein.
17
A variety of antioxidants including antioxidant molecules and antioxidases can
18
scavenge excessive ROS to prevent oxidative stress in tissues. The total antioxidant
19
capacity (T-AOC) of the tissue is defined as the total levels of various antioxidants.
20
T-AOC was measured using a Total Antioxidant Capacity Assay Kit (S0121,
21
Beyotime, China) with an ABTS (2,2’-azino-bis(3-ethylbenz-thiazoline-6-sulfonic
22
acid) method. When ABTS is incubated with a proper chemical, a blue-green ABTS
23
radical (ABTS•+) is formed. The color production of ABTS•+ can be suppressed by
24
antioxidants in the sample proportionally to a degree. Therefore, the concentrations of
25
antioxidants in tissue samples can be quantified indirectly. According to the
26
manufacturer's protocols, the T-AOC was calculated and expressed as the
27
trolox-equivalent antioxidant capacity according to the manufacturer's protocols.
28 29 30
2.9 Assessment of vasomotor responses Endothelium-intact aortas were isolated gently and placed in ice-cold and 8
1
oxygenated PSS buffer (Nacl, 130 mM; KCl, 4.7 mM; KH2PO4, 1.18 mM;
2
MgSO4•7H2O, 1.17 mM; NaHCO3, 14.9 mM; Glucose, 11.1 mM; EDTA, 0.026 mM;
3
CaCl2, 1.16 mM). The aortas were cut into rings of approximately 3 mm in length.
4
The vasomotor responses to acetylcholine (Ach, 10-9 M-10-5 M) and sodium
5
nitroprusside (SNP, 10-9 M-10-5 M) were monitored by the myograph system
6
(DMT620, Danish Myo Technology, Aarhus, Denmark). Percentage of relaxation
7
relative to the noradrenaline (NA, 10-6 M)–induced vasoconstriction was calculated.
8 9
2.10 Quantitative RT-PCR
10
Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to
11
examine the relative gene expression levels. The primers used in the experiment are
12
shown in Table S1. The total RNA in HUVECs was isolated using RNAiso Plus
13
(Takara, Shiga, Japan), and the total RNA in the aortic tissue was extracted using an
14
RNAsample Total RNA kit (Tiangen, Beijing, China) according to the manufacturer's
15
protocols. Nanodrop ND-2000 (Thermo Fisher Scientific, USA) was used to detect
16
the concentration and quality of the RNA. Reverse transcription was performed using
17
a FastKing RT kit (Tiangen, China). PCR was performed using a TB Green Premix Ex
18
Taq II kit (Takara, Japan). The relative gene expression levels normalized to GAPDH
19
were calculated using ∆∆CT method.
20 21
2.11 Western blotting
22
The total protein was isolated using RIPA buffer (2 % protease inhibitor and 1 %
23
phosphatase inhibitor) and quantified using a BCA Assay Kit according to the
24
manufacturer’s instructions (Beyotime, China). The mitochondrial protein was
25
isolated from the aorta tissue according to the tissue mitochondria isolation protocol.
26
First, protein samples were loaded onto 8 % SDS℃PAGE and migrated under an
27
electric field. Then the protein was transferred to a PVDF membrane (Millipore,
28
Burlington, MA, USA). Before primary antibodies incubation, the membranes were
29
blocked with 5% skim milk. Then the membranes were washed and incubated with
30
fluorescently labeled secondary antibodies. Protein band images were developed 9
1
using an LI-COR Odyssey scanner (LI℃COR, USA). Primary antibodies were
2
purchased from Abcam and Cell Signaling Technology including anti-β-actin
3
(ab8227), GAPDH (ab8245), VDAC1(ab15895), Drp-1 (#8570), OPA1 (#80471),
4
MFN1 (ab57602), MFN2 (#9482), AMPKα (#5831), p-AMPKα (#2535), ACC
5
(#3676), p-ACC (#11818), AMPKβ (#4150), p-AMPKβ1 (#4186), ICAM-1 (sc8439),
6
VCAM-1 (sc13160) and clusterin (ab229127 /sc5289/sc166831).
7 8
2.12 Cell culture and treatments
9
Human Umbilical Vein Endothelial Cells (HUVECs, ScienCell, Carlsbad, CA,
10
USA) were cultured in endothelial cell medium (1% growth supplement and 10%
11
FBS) at 37 °C in a 5% CO2 humidified atmosphere. Passages 3 to 8 cells were used in
12
further experiments. HUVECs were infected with adenovirus Ad-CLU expressing
13
selected clusterin and negative control adenovirus Ad-NC with 100 MOI. After 6 h,
14
fresh medium was substituted for the culture medium, and the cells were cultured for
15
another 18 h. The clusterin expression level was determined using qPCR and Western
16
blotting (Fig.S1). For the knockdown of clusterin, the cells were transfected with
17
CLU-siRNA1 and CLU-siRNA2, silencing the clusterin expression for 48 h. The two
18
separate siRNA target different sequence segments of CLU-mRNA. The sequence of
19
CLU-siRNA1 was 5’ CCCGCCAACAGAAUUCAUATT 3’, the sequence of
20
CLU-siRNA2 was 5’ CCAGGAAGAACCCUAAAUUTT 3’, and the sequence of
21
siRNA-NC was 5’ UUCUCCGAACGUGUCACGUTT 3’. The cells were then
22
subjected to normal glucose medium (5.5 mM D-glucose, NG) with 27.5 mM
23
mannitol for osmotic compensation or high glucose and high palmitate medium (33
24
mM D-glucose and 200 µM palmitic acid, HG/HF) for 24 h as previously described
25
[35, 36]. For the inhibition of AMPK activation, HUVECs transfected with Ad-CLU
26
or Ad-NC were treated with 10 µM of compound C (CC) (MCE, USA) or
27
AMPKα-siRNA1/2.
28
GAGGAGAGCUAUUUGAUUATT 3’, the sequence of AMPKα-siRNA2 is 5’
29
GCGUGUACGAAGGAAGAAUTT 3’, and the sequence of siRNA-NC is 5’
30
UUCUCCGAACGUGUCACGUTT 3’. RPMI-1640 medium with 10 % FBS was
The
sequence
of
10
AMPKα-siRNA1
was
5’
1
used for THP-1 (ATCC, Manassas, VA, USA) cells culture.
2 3
2.13 Monocyte adhesion assay
4
A monocyte adhesion assay was performed to investigate the role of clusterin in
5
the suppression of monocyte adhesion to ECs. First, THP-1 cells were cultured in
6
RPMI-1640 medium with 10% FBS (Gibco, Waltham, MA, USA) at 37 °C and
7
incubated with calcein AM (2 µM) for 30 min. The THP-1 cells were then collected
8
and resuspended. After HUVECs were added in black plates containing 96 wells and
9
pretreated with adenovirus, the THP-1 cells (1×105) were seeded into each well and
10
co-incubated with the HUVECs for 1 h. The unattached THP-1 cells were removed by
11
PBS washing. The plates were photographed under a fluorescence microscope and the
12
fluorescence intensity was assessed using a microplate reader (Thermo Fisher
13
Scientific, USA).
14 15
2.14 Tube formation assay
16
Tube formation assay was performed as previously described [37]. In brief, the
17
HUVECs were infected with adenovirus expressing secreted clusterin (Ad-CLU) or
18
negative control adenovirus (Ad-NC) for 24 h. Then, the cells were incubated with
19
calcein AM (2 µM) for 20 min. For clusterin knockdown, the HUVECs were
20
transfected with siRNA silencing clusterin expression for 48 h using Lipofectamine
21
2000 (Thermo Fisher Scientific, USA). Then, the 96-well plates were coated with 60
22
µL of matrigel matrix (Corning, NY, USA) and incubated at 37°C for 30 min. When
23
the matrigel was polymerized, the HUVECs were collected and seeded (1×104/well)
24
into the wells and subjected to NG or HG/HF treatments for 16 h. Capillary-like tubes
25
were imaged using the fluorescence microscope.
26 27
2.15 Mitochondrial staining and assessment of mitochondrial morphology
28
Mito-Tracker green (Beyotime, China) was applied to characterize the
29
mitochondrial morphology in HUVECs. Briefly, Mito-Tracker green stock solution (1
30
mM) was prepared with anhydrous dimethylsulfoxide and diluted with Hank's 11
1
Balanced Salt Solution /Ca2+/Mg2+. The cells were incubated with Mito-Tracker green
2
staining solution (200 nM) at 37°C for 20 min. The staining solution was then
3
removed, and fresh culture solution was added into the wells. The cells were observed
4
under a fluorescence microscope. Image-Pro Plus software was applied to the
5
quantification of the number and individual volume of the mitochondria.
6
Mitochondrial fragmentation was indicated by a decrease in the mitochondrial density
7
per cell.
8 9
2.16 Assessment of MMP
10
The mitochondrial membrane potential (MMP, ∆Ψm) was monitored using JC-1
11
dyes, exhibiting potential-dependent accumulation in mitochondria. The HUVECs
12
were infected with adenovirus and pretreated with NG or HG/HF, and incubated with
13
JC-1 probe (2 mM) at 37°C for 30 min. The cells were then stained using Hoechst
14
33342. A fluorescence microscope was used to observe the cells. The quantified data
15
are shown as the normalized ratio of green fluorescence to red fluorescence.
16
Mitochondrial depolarization is demonstrated by a lower ratio of red/ green
17
fluorescence intensity.
18 19
2.17 Viability and cytotoxicity of HUVECs
20
CCK-8 (Beyotime,China) was used to evaluate the viability of cells. An LDH
21
Cytotoxicity Assay Kit (Sigma-Aldrich, St. Louis, MO,, USA) was used to determine
22
the cytotoxicity of cells subjected to HG/HF following the manufacturer’s protocols.
23 24
2.18 Statistical analysis
25
All of the data are expressed as the mean ± SD and were analyzed using
26
GraphPad Prism 7.00 and SPSS 21.0. If the data passed equal-variance test, an
27
unpaired two-tailed t-test was conducted for comparisons between two groups;
28
ANOVA and post hoc multiple comparisons test were used for comparisons among
29
three or more groups. Nonparametric tests were used for non-normally distributed
30
data. Statistically significant differences were considered at a value of P < 0.05. 12
1
3. Results
2 3
3.1 Endothelial inflammatory activation, increased ROS generation and mitochondrial fragmentation occurred in diabetes
4
To investigate the role of clusterin in diabetes-induced endothelial dysfunction,
5
the db/db mice were used as diabetes models and the db/m mice were non-diabetic
6
controls (Fig.1A). The male db/db mice were hyperglycemic with increased level of
7
non-esterified fatty acid (NEFA) in the plasma, and their body weights were
8
significantly higher than the db/m mice (Fig.1B-D). First, the endothelial
9
inflammatory activation in the diabetic aortas was determined. Endothelial activation,
10
the onset of endothelial dysfunction, can be induced by the hyperglycemia and
11
hyperlipidaemia in diabetic mice. As expected, the expression of VCAM-1and
12
ICAM-1 adhesion molecules was up-regulated in the db/db mice aortas (Fig.1E-G).
13
Moreover, H&E staining indicated that marked pathomorphological changes occurred
14
in the diabetic aortas (Fig.1E).
13
1 2
Fig.1. Hyperglycemia and hyperlipidemia in diabetic mice leads to endothelial
3
inflammatory activation. (A) Male db/db diabetic mice (left) and non-diabetic
4
control db/m mice (right). (B) Body weights of db/db and db/m mice were measured
5
once a fortnight. (C) Fasting blood glucose (FBG) was measured once every four
6
weeks. Diabetic mice had a higher FBG level than the littermate db/m mice. (D)
7
Diabetic mice had a higher level of non-esterified fatty acid (NEFA) in plasma than
8
the littermate db/m mice. (E) Representative hematoxylin and eosin (H&E) staining 14
1
images (×200, left panel) and immunohistochemical images (×400) for the thoracic
2
aortic sections. (F) Statistical analysis of immunohistochemical staining. Diabetic
3
mice scored higher in the staining of CLU, VCAM-1 and ICAM-1 than the db/m mice.
4
(G) The mRNA levels of ICAM1, VCAM1 and CLU were determined using real-time
5
PCR. (H) Representative Western blotting images of CLU expression. CLU
6
expression in the db/db mice aortas increased compared to the db/m mice. (I) The
7
diabetic mice had a higher level of clusterin in the plasma than the littermate db/m
8
mice. (J) DHE (dihydroethidium) staining for ROS determination in frozen aortic
9
sections. (K) Quantified analysis of fluorescence intensity for the ROS levels. The
10
diabetic mice had a higher ROS level. (L) The level of 8-OHdG was measured by
11
ELISA. Concentrations of MDA (M) and total SOD activity (N) in aorta were
12
measured.
13
malondialdehyde; SOD, superoxide dismutase.
n=5.
*
P<0.05,
**
P<0.01,
***
P<0.001 vs. db/m group. MDA,
14 15
It has been determined that clusterin plays a cytoprotective role as a
16
stress-inducible protein for various diseases [26, 38, 39]. However, the function of
17
clusterin in diabetes-induced endothelial dysfunction has not been defined.
18
Before we dive in, the expression abundance of clusterin in the diabetic aortas was
19
investigated. The qRT-PCR results showed that the mRNA expression level of CLU in
20
the db/db mice aorta was higher than in the db/m mice (Fig. 1G). As expected, the
21
Western blotting and immunohistochemical staining results indicated that the CLU
22
expression in the db/db mice aortas increased under hyperglycaemic and
23
hyperlipidemic stress compared with the db/m mice (Fig. 1E, F, and H). Also, diabetic
24
mice had a higher level of clusterin in the plasma than the littermate db/m mice (Fig.
25
1I).
26
15
1 2
Fig.2. Mitochondrial ROS production and mitochondrial fission were increased
3
in the HUVECs subjected to HG/HF treatment. (A) Mitochondrial ROS
4
(mito-ROS) and nucleus were stained by MitoSOX red and Hoechst 33342,
5
respectively. Scale bar is 100 µm. (B) Quantified analysis of fluorescence intensity for
6
mito-ROS level. HUVECs treated with HG/HF showed an increased level of
7
mito-ROS. (C) Representative images of MitoTracker Green staining characterizing
8
the mitochondrial morphology in the HUVECs subjected to NG or HG/HF treatment
9
and the quantified analysis (D). Under HG/HF challenge, the fibers of the
10
mitochondria in the HUVECs became short and fragmentized, resulting in a decreased
11
mitochondrial density. Scale bar is 50 µm. n=3. *P<0.05,
12
db/m group or NG group. NG, normal glucose; HG/HF, high glucose and high
13
palmitate.
**
P<0.01, ***P<0.001 vs.
14 15
One of the key mediators for diabetic endothelial dysfunction is excessive ROS,
16
and ROS production by mitochondria is the primary intracellular source of ROS [15,
17
40]. In our study, the total ROS and mitochondrial ROS (mito-ROS) were detected by 16
1
DHE staining and MitoSOX red staining, respectively. As shown in Fig.1J-K,
2
compared to the db/m mice, the fluorescence intensity of DHE in the db/db mice was
3
significantly increased, representing a higher level of ROS. Accumulated oxidation
4
damage to the mitochondrial DNA and membrane is the main risk for mitochondrial
5
dysfunction, and mitochondrial injury is a critical factor in endothelial activation and
6
dysfunction in diabetes [8]. 8-OHdG and MDA, the indicators of oxidative DNA and
7
membrane lipids damage, were detected by ELISA. As expected, the levels of
8
8-OHdG and MDA in the db/db mice were higher than in the controls (Fig.1L-M). In
9
contrast, the total activity of the antioxidase superoxide dismutase (SOD) in the db/db
10
mice was lower than in the control db/m mice (Fig.1N). On the other hand, the
11
HUVECs stimulated with high glucose and high fatty acid (HG/HF) exhibited higher
12
mito-ROS levels (Fig.2A-B). Increased production of mitochondrial ROS can be
13
induced by disrupted mitochondrial dynamics and aggravated mitochondrial fission in
14
diabetes. MitoTracker Green staining was used to characterize the mitochondrial
15
morphology. In the HUVECs treated with normal glucose, the mitochondria appeared
16
as long fibers with a dense network, and mitochondria fibers in the HUVECs
17
stimulated with HG/HF were short and fragmentized (Fig.2C-D).
18 19 20
3.2 Clusterin overexpression dysfunction in vitro and in vivo
ameliorates
diabetes-induced
endothelial
21
To further investigate the role of clusterin in endothelial dysfunction induced by
22
diabetes, the spontaneous db/db diabetic mice and STZ-induced diabetic rats were
23
used as models in vivo and HUVECs were used as models in vitro with clusterin
24
overexpression. In vitro, we first profiled the expression of adhesion molecules and
25
other cytokines associated with endothelial inflammatory activation. The mRNA
26
expression of VCAM-1, ICAM-1, MCP-1, IL-6, TNF-α, and ET-1 were increased in
27
the HUVECs stimulated with high glucose and high palmitate (HG/HF) compared to
28
the NG group; in contrast, the HUVECs transfected with the adenovirus expressing
29
clusterin (Ad-CLU) had a lower expression level than the HG/HF control group
30
(Fig.3A-B). We further examined the IL-6 and TNF-α content in the supernatant of 17
1
medium. The results showed that HG/HF promoted the secretion of pro-inflammatory
2
factors IL-6 and TNF-α (Fig.3C). ICAM-1 and VCAM-1 overexpression means that
3
monocyte adhesion and infiltration to ECs will be promoted, therefore, an adhesion
4
assay was performed to examine whether clusterin prevents ECs from recruiting
5
monocytes. As expected, HG/HF increased the adhesion of THP-1cells to HUVECs
6
compared with the NG group. However, clusterin overexpression inhibited the
7
monocyte adhesion to HUVECs (Fig.3D). In addition, the cell viability was decreased
8
and the cytotoxicity was increased following HG/HF stimulation; however, the
9
clusterin overexpressed HUVECs showed a higher viability and a lower cytotoxicity
10
(Fig.S2). Angiogenesis is one of the vascular endothelial functions that can be
11
impaired by oxidative stress. HG/HF impaired the tube formation of the HUVECs on
12
the Matrigel, and the branches shortened in the HG/HF group as shown in Fig.3E,
13
while clusterin markedly reduced the impairment.
18
1 2
Fig.3.
Clusterin
overexpression
suppressed
3
inflammatory activation in vitro. The HUVECs were infected with adenovirus
4
expressing clusterin (Ad-CLU) or negative control adenovirus (Ad-NC) (100 MOI)
5
for 24 h and subjected to NG or HG/HF treatments for another 24 h. (A-B) The
6
mRNA expression levels of the cytokines associated with endothelial inflammatory
7
activation were measured by qRT-PCR, and in (B) expressed as a heat map, with
8
each column representing the mean of three independent experiments. (C)
9
Adenovirus-infected HUVECs were subjected to NG or HG/HF treatments. IL-6 and
10
TNF-α in medium were quantified by ELISA. (D) The adhesion of Calcein
11
AM-labeled THP-1cells to the HUVECs was determined by fluorescence microscopy 19
HG/HF-induced
endothelial
1
and a fluorescence microplate reader. CLU overexpression decreased the monocyte
2
adhesion to the HUVECs. (E) HUVECs infected with Ad-CLU or Ad-NC (100 MOI)
3
were seeded on Matrigel and retained for 12 h. Tube formation of HUVECs on
4
Matrigel was pictured and analyzed by Image J. n=3. #P<0.05,
5
vs. NG control, *P<0.05, **P<0.01, ***P<0.001 vs. HG/HF control. CM, blank culture
6
medium; NG, normal glucose; HG/HF, high glucose and high palmitate; Ad-NC,
7
negative control adenovirus; Ad-CLU, the adenovirus expressing clusterin.
##
P<0.01,
###
P<0.001
8 9
In db/db mice, clusterin overexpression (Fig.4C, Fig.S3A) made no difference on
10
the body weight and FBG (Fig.4A). Compared to the db/db mice infected with
11
AAV-NC (AAV-NC-db/db), the level of NEFA in plasma of the db/db mice infected
12
with AAV-CLU (AAV-CLU-db/db) decreased (Fig.4B). The pathological morphology
13
of the aortas in the clusterin overexpressed db/db mice was improved compared to
14
that in the AAV-NC-db/db mice (Fig.4D). The expression of VCAM-1 and ICAM-1 in
15
the AAV-CLU-db/db mice aortas was markedly down-regulated compared to the
16
AAV-NC-db/db mice (Fig.4F-G). The results indicated that clusterin overexpression
17
alleviated endothelial inflammatory activation in db/db mice aortas.
18
The role of clusterin in other diabetes models was also examined. The
19
STZ-induced diabetic rats fed a high fat diet (HFD) were a drug-induced type 2
20
diabetes model (Fig.S4). Similarly, overexpression of clusterin (Fig.5C) made no
21
difference in the fasting blood glucose (FBG) or body weights of the diabetic rats
22
(Fig.5A). The NEFA level in plasma of the diabetic rats increased compared with the
23
non-diabetic rats (Fig.5B). Endothelial function was further evaluated by measuring
24
the isometric tension of the aortic rings. The endothelium-dependent vasorelaxation
25
responding to acetyl choline (Ach) was impaired in the diabetic rats infected with
26
AAV-NC (AAV-NC/DM), while clusterin mitigated the impairment in the diabetic rats
27
infected with AAV-CLU (AAV-CLU/DM). However, the endothelium-independent
28
vasorelaxation responding to sodium nitroprusside (SNP) showed no significant
29
difference among the groups (Fig.5D). The expression levels of VCAM-1 and
30
ICAM-1 were lower in the aortas of AAV-CLU/DM rats compared with the 20
1
AAV-NC/DM rats (Fig.5E-F). In brief, clusterin overexpression ameliorates
2
endothelial dysfunction in the STZ-induced diabetic rats.
21
1 2
Fig.4. Clusterin overexpression alleviated endothelial inflammatory activation in 22
1
db/db mice aortas. (A) Body weights of mice were measured once a fortnight, and
2
FBG was measured once every four weeks. Clusterin overexpression made no
3
difference on the body weight and FBG. (B) NEFA was detected in plasma of mice
4
collected at the time of sacrifice. (C) Western blotting for clusterin expression in mice
5
aortas (upper panel) and quantitative data of clusterin expression normalized against
6
β-actin
7
immunohistochemical images (×400) for aortic sections. (E) Quantitative data of
8
immunohistochemical staining of CLU, VCAM-1, ICAM-1. The expressions of
9
VCAM-1 and ICAM-1 in the AAV-CLU infected db/db mice aortas were
10
down-regulated, while the clusterin expression increased markedly compared to the
11
AAV-NC-db/db mice. n=6.
12
*
13
adeno-associated virus; AAV-CLU, the adeno-associated virus expressing clusterin.
P<0.05,
(lower
**
panel).
P<0.01,
***
(D)
#
Representative
P<0.05,
##
H&E
P<0.01,
###
staining
images
(×200),
P<0.001 vs. AAV-NC-db/m;
P<0.001 vs. AAV-NC-db/db. AAV-NC, negative control
14
23
1 2
Fig.5. Clusterin overexpression rescued endothelial dysfunction in STZ-induced
3
diabetic rats. (A) Body weights and FBG of rats were measured once every four
4
weeks. Clusterin overexpression made no difference on the body weight and FBG. (B)
5
NEFA was detected in plasma collected at the time of sacrifice. STZ-induced diabetic
6
rats had a higher level of non-esterified fatty acid (NEFA) in plasma than the normal
7
rats. (C) Western blotting for clusterin expression in aortas (upper panel) and
8
quantitative data of clusterin expression normalized against β-actin (lower panel). The
9
clusterin expression increased markedly in the rats injected with AAV-CLU. (D) Ach-
10
and SNP-induced vasorelaxation of aortic rings were recorded by vascular tension
11
measurement system. The endothelium-dependent vasorelaxation responding to Ach
12
was impaired in AAV-NC infected diabetic rats, while clusterin overexpression 24
1
mitigated the impairment in AAV-CLU infected diabetic rats. (E) Representative H&E
2
staining images (×200), immunohistochemical images (×400) for aortic sections. (F)
3
Quantitative data of immunohistochemical staining of VCAM-1, ICAM-1. Expression
4
of VCAM-1 and ICAM-1 in AAV-CLU infected diabetic rat aortas was markedly
5
down-regulated compared to AAV-NC infected diabetic rats. n=5. #P<0.05, ##P<0.01,
6
###
7
fed with normal diet and subjected to no special treatment; HFD, high fat diet; DM,
8
rats treated with HFD/STZ to produce diabetes mellitus models.
P<0.001 vs. Normal; *P<0.05, **P<0.01, ***P<0.001 vs. AAV-NC/DM. Normal, rats
9 10
3.3 Clusterin overexpression inhibits oxidative stress and mitochondrial ROS
11
production induced by diabetes
12
Oxidative stress accumulated in diabetes induces and aggravates endothelial
13
activation
and
dysfunction
[41].
DCFH-DA (2',7'-dichlorodihydrofluorescein
14
diacetate) probe was applied to detect the ROS level in the HUVECs, and the result
15
indicated that the ROS level in the ECs treated with HG/HF increased markedly,
16
while the ECs transfected with Ad-CLU had lower ROS levels (Fig.6A-B). In the
17
diabetic mice and rat aortas, the ROS levels were detected by DHE staining. Similarly,
18
clusterin overexpression significantly reduced the ROS levels (Fig.6C-D and H-I). We
19
further examined 8-OHdG and MDA, indicators of DNA and membrane lipids
20
oxidative damage. As shown in Fig.6E-F, compared with the db/m mice infected with
21
AAV-NC (AAV-NC-db/m), the levels of 8-OHdG and MDA in the db/db mice
22
infecetd with AAV-NC (AAV-NC-db/db) were higher, which were reversed by
23
clusterin overexpression in the AAV-CLU-db/db mice. A similar result was obtained
24
in the diabetic rats (Fig.6J-K). Excessive ROS production beyond the efficiency of
25
available antioxidant defense systems leads to oxidative stress. Meanwhile, increased
26
oxidative stress in diabetes impaired the endogenous antioxidant system composed of
27
antioxidative enzymes and other antioxidants. As expected, the total superoxide
28
dismutase (SOD) activity in the aortas was reduced in the diabetic mice and rats but
29
increased in the AAV-CLU-db/db and AAV-CLU/DM groups (Fig.6G and L). The
30
total cellular antioxidant capacity (T-AOC) and glutathione disulfide (GSSG) /reduced 25
1
glutathione (GSH) in the rat aortas were also measured. In the AAV-NC/DM group,
2
the level of T-AOC declined and GSSG/GSH was elevated, which suggested that the
3
aortas became more susceptible to oxidative damage. However, the level of T-AOC
4
and the GSSG/GSH ratio were partially restored by clusterin overexpression in the
5
AAV-CLU/DM group (Fig.6M-N).
6
Mitochondria-derived ROS was the major intracellular component of ROS,
7
which contributes to oxidative damage. To examine whether clusterin ameliorates
8
diabetes-caused mitochondrial ROS production, the HUVECs were stained by a
9
specific mitochondrial superoxide indicator (MitoSOX red). Compared with the cells
10
exposed to NG, the HG/HF treated cells produced much more mito-ROS, which was
11
partially alleviated by the over-expression of clusterin (Fig.6O-P).
12 13
These data indicated that clusterin suppressed oxidative stress and mitochondrial ROS production induced by diabetes.
14
26
1 2
Fig.6. Clusterin prevented oxidative stress and mitochondrial ROS production.
3
(A) Representative images of ROS stained by DCFH-DA (2',7'-dichlorodihydrofluore
4
-scein diacetate) and the quantified data (B). n=3.
5
control, *P < 0.05, vs. HG/HF control. (C) Dihydroethidium (DHE) staining for ROS
6
determination in frozen mice aortic sections and fluorescence intensity was quantified 27
##
P < 0.01,
###
P < 0.001 vs. NG
1
(D). (E-G) Levels of 8-OHdG, MDA, and total SOD activity were analyzed by ELISA.
2
AAV-NC-db/db mice had a higher level of 8-OHdG, MDA and a decreased total SOD
3
activity, while AAV-CLU-dbdb mice showed a lower level of 8-OHdG, MDA and
4
increased total SOD activity. n=6.
5
AAV-NC-db/m; *P < 0.05,
6
Representative images of the rat aortas ring stained by H&E (×100) and DHE. (I)
7
Quantified data of ROS level indicated by fluorescence intensity of DHE. (J-N)
8
Diabetic rat (AAV-NC/DM) had a higher level of 8-OHdG (J), MDA (K) and
9
GSSG/GSH ratio (N), a decreased total SOD activity (L) and T-AOC (total cellular
10
antioxidant capacity) (M), while AAV-CLU/DM rat showed a lower level of 8-OHdG,
11
MDA and GSSG/GSH ratio, a increased total SOD activity and T-AOC. n=5.
12
0.01, ###P < 0.001 vs. Normal (control); *P < 0.05, ***P < 0.001 vs. AAV-NC/DM. (O)
13
Images of MitoSOX red staining in HUVECs treated with NG or HG/HF and
14
quantified analysis (P). Clusterin overexpression reduced the mitochondrial ROS
15
production in the HUVECs with HG/HF treatment. n=3. ###P < 0.001 vs. Ad-NC /NG,
16
***
17
high glucose and high palmitate; Ad-NC, negative control adenovirus; Ad-CLU,
18
adenovirus expressing clusterin; AAV-NC, negative control adeno-associated virus;
19
AAV-CLU, adeno-associated virus expressing clusterin; HFD, high fat diet; DM, rats
20
treated with HFD/STZ to produce diabetes mellitus models.
#
P < 0.05,
**
P < 0.01,
***
##
P < 0.01,
###
P < 0.001 vs.
P < 0.001 vs. AAV-NC-db/db. (H)
##
P<
P < 0.001 vs. Ad-NC/HG/HF. CM, culture medium; NG, normal glucose; HG/HF,
21 22
28
1 2
Fig.7. Clusterin diminished mitochondrial fragmentation and regulated the
3
expression of proteins responsible for mitochondrial fission and fusion. (A)
4
Mitochondria were stained with MitoTracker Green, and mitochondrial density was
5
quantified (B). Scale bar is 100 µm. n=3.
6
Ad-NC/HG/HF. (C-E) Western blotting analysis of mitochondrial dynamics-related
7
proteins, including mitochondrial fission protein Drp-1 and mitochondrial fusion 29
##
P<0.01 vs. Ad-NC/NG,
**
P<0.01 vs.
1
protein OPA1, MFN1, and MFN2. The expression level of Drp-1 modulating
2
mitochondrial fission in the HUVECs exposed to HG/HF increased notably, while the
3
clusterin expression alleviated the increase. (F) Representative images of the
4
HUVECs stained by JC-1/Hochest 33342 under fluorescence microscopy. (G) The
5
mitochondrial membrane potential (MMP) indicated by the red/green ratio was
6
decreased in the HUVECs treated with HG/HF, while the HUVECs infected with
7
Ad-CLU showed a higher MMP compared to the Ad-NC infected HUVECs. n=3.
8
###
9
blotting analysis of the mitochondrial dynamics related proteins in cell lysate (H-J)
P<0.001 vs. NG control, *P<0.05,
***
P<0.001 vs. HG/HF control. (H-L) Western
10
and mitochondria (K-L) isolated from the mice aortas. n=6. #P<0.05,
11
AAV-NC-db/m; **P<0.01, ***P<0.001 vs. AAV-NC-db/db.
###
P<0.001 vs.
12 13
3.4 Clusterin overexpression prevents mitochondrial fragmentation
14
It has been confirmed that mitochondrial dynamic change and aggravated
15
mitochondrial fragmentation in diabetes contributes to mitochondrial ROS production
16
[16]. Simultaneously, superabundant ROS exacerbates oxidative damage to the
17
mitochondria, which is a fatal factor to endothelial dysfunction in diabetes. First, we
18
examined whether clusterin regulates mitochondrial dynamics under high glucose and
19
high palmitate conditions. MitoTracker Green staining was used to characterize
20
mitochondrial morphology. The mitochondria in HUVECs stimulated with HG/HF
21
became shorter and punctated, which was partially restored by clusterin
22
overexpression (Fig.7A). The quantified data indicated that clusterin elevated the
23
mitochondrial density under HG/HF conditions (Fig.7B). Further, the expression of
24
proteins modulating mitochondrial dynamics including Drp-1, OPA1, MFN1, and
25
MFN2 was determined via Western blotting assay. In vitro, expression level of Drp-1
26
modulating mitochondrial fission in HUVECs exposed to HG/HF increased notably;
27
however, the Ad-CLU infected HUVECs had lower Drp-1 expression levels than the
28
negative adenovirus group (Fig.7C-D). Decreased OPA1 expression in the HG/HF
29
treated ECs was also observed compared with NG treated ECs, while clusterin
30
alleviated the decrease (Fig.7C, E). MFN1 and MFN2 had no notable change after 30
1
HG/HF stimulation. Proteins including OPA1, MFN1, and MFN2 modulated
2
mitochondrial fusion. These results indicated that clusterin overexpression prevents
3
mitochondrial excessive fission triggered by high glucose and high palmitate
4
conditions. ATP synthesis is a crucial function of the mitochondria, and the
5
mitochondrial membrane potential (MMP, ∆Ψm) is a prerequisite for oxidative
6
phosphorylation. The dissipation of MMP implies that mitochondria are disrupted. To
7
determine whether clusterin also affects the MMP, we measured the MMP using a
8
JC-1 fluorescence probe (Fig.7F-G). The MMP in the ECs exposed to HG/HF showed
9
a decreased red/green ratio and was restored partially by clusterin overexpression.
10
The expression abundances of OPA1, Drp-1, MFN1, and MFN2 were also
11
investigated in the diabetic mice. The total and mitochondrial proteins were extracted
12
from the mice aortas. Compared with the db/m mice, Drp-1 expression level in cell
13
lysate (Fig.7H-I) and mitochondria (Fig.7K-L) increased in the AAV-NC-db/db mice,
14
but the AAV-CLU-db/db mice had a lower level. In addition, compared with the
15
non-diabetic mice, decreased expression levels of OPA1 in the cell lysate (Fig.7H, J)
16
and mitochondria (Fig.7K-L) was observed in the AAV-NC-db/db mice, and clusterin
17
overexpression alleviated the decrease. Consistent results were obtained in the
18
STZ-induced diabetic rats (Fig.S5).
19
Together, these results indicated that clusterin plays a vital part in maintaining
20
the balance of mitochondrial fusion/fission, protecting mitochondria from oxidative
21
damage.
22 23
3.5 Clusterin attenuates mitochondrial fragmentation via activation of AMPK
24
Accumulating evidences demonstrated that AMPK is an important metabolism
25
regulator that has attracted extensive attention as a prospective therapeutic target for
26
type 2 diabetes [42]. Recent researches illustrated that AMPK activation also
27
contributes to preventing endothelial dysfunction in diabetes [43, 44]. We investigated
28
whether clusterin plays a role in regulating AMPK signaling. The phosphorylation of
29
AMPKα (Thr 172) was inhibited under hyperglycemia, but this inhibition was
30
partially restored in the AAV-CLU-db/db mice aortas (Fig.8A). The expression and 31
1
phosphorylation (Ser 79) of acetyl-CoA carboxylase (ACC), the down-stream target
2
of AMPK associated with fatty acid metabolism were improved in the
3
AAV-CLU-db/db mice aortas. A similar profile was observed in the STZ-induce
4
diabetic rats (Fig.8B). The phosphorylation of AMPKα on Thr 172 was elevated by
5
clusterin overexpression compared with the diabetic rats treated with negative AAV.
6
Meanwhile, the level of p-ACC (Ser79) was obviously restored in the AAV-CLU/DM
7
group.
8
To identify whether AMPK activation is indispensable for clusterin in preventing
9
mitochondria impairment, we subjected the HUVECs to an AMPK specific inhibitor,
10
compound C. First, mitochondrial ROS production was examined in HUVECs treated
11
with or without compound C (10 µM). The mito-ROS level of clusterin-overexpressed
12
HUVECs exposed to HG/HF was obviously reduced, whereas the reduction was
13
weakened by compound C (Fig.9A and C). On the other hand, mitochondria of the
14
Ad-CLU treated ECs showed a relatively normal network structure despite HG/HF
15
stimulation, which was destroyed by compound C, exhibiting an increase in
16
mitochondrial fragmentation (Fig.9B and D). As predicted, under the HG/HF
17
conditions, the reduction in the Drp-1 expression and the increase in the OPA1
18
expression observed in the Ad-CLU pretreated ECs were wiped out by compound C
19
(Fig.9E), which also indicated that compound C restrained clusterin from attenuating
20
the mitochondrial fragmentation induced by the high glucose and high palmitate
21
conditions. We also investigated the influence of compound C on the suppression of
22
endothelial inflammatory activation in the Ad-CLU treated HUVECs. As expected,
23
although the ECs were clusterin overexpressed, the mRNA expression of ICAM1 and
24
VCAM1 responding to HG/HF was enhanced following compound C treatment
25
(Fig.9F).
26
Furthermore, siRNAs targeting AMPKα mRNA were used to investigate whether
27
knockdown of AMPK in HUVECs under the high glucose and high palmitate
28
conditions
29
AMPKα-siRNA1/2 or siRNA-NC following Ad-CLU infection. As shown in Fig.S6,
block
clusterin’s
effects.
32
HUVECs
were
transfected
with
1
AMPKα-siRNA1 and AMPKα-siRNA2 reversed the clusterin-mediated prevention of
2
mito-ROS production (Fig.S6A and C). Also, AMPKα-siRNA1 and AMPKα-siRNA2
3
reversed the clusterin-mediated inhibition of mitochondrial fragmentation (Fig. S6B
4
and D). In addition, the mRNA expression of ICAM1, VCAM1 and CCL2 responding
5
to HG/HF was enhanced in the ECs treated with AMPKα-siRNA1 or AMPKα-siRNA2
6
despite
7
AMPKα-siRNA blocked clusterin’s effects in HUVECs under the high glucose and
8
high palmitate conditions, which was same as the AMPK inhibitor compound C.
overexpression
(Fig.S6E-G).
These
data
indicated
that
Together, these data demonstrated that clusterin attenuated mitochondrial ROS
9 10
clusterin
production and fragmentation through the activation of AMPK.
11
12 13
Fig.8. Clusterin activated AMPK in db/db and STZ-induced diabetes. (A)
14
Phosphorylation of AMPKα on Thr172 and phosphorylation of ACC on Ser79 in mice
15
aortas were analyzed by western blotting. The ratios of p-AMPKα/AMPKα and
16
p-ACC/ACC were increased in the AAV-CLU infected db/db mice compared to the
17
AAV-NC infected db/db mice. n=4. #P<0.05, ##P<0.01, ###P<0.001 vs. AAV-NC-db/m;
18
*
P<0.05,
**
P<0.01,
***
P<0.001 vs. AAV-NC-db/db. (B) Phosphorylation of AMPKα 33
1
on Thr172 and phosphorylation of ACC on Ser79 in the rat aortas were analyzed by
2
western blotting. The ratios of p-AMPKα/AMPKα and p-ACC/ACC were decreased
3
in STZ-induced diabetes mellitus (DM) models, while clusterin overexpression
4
promoted the phosphorylation of AMPKα and ACC in diabetic rats. n=4. #P<0.05,
5
##
P<0.01, ###P<0.001 vs. Normal (control); *P<0.05, **P<0.01, ***P<0.001 vs. DM.
6
34
1 2
Fig.9. Compound C abolished the attenuated mitochondrial ROS production and 35
1
fragmentation mediated by clusterin under HG/HF conditions and clusterin
2
knockdown aggravated mitochondrial oxidation and endothelial inflammatory
3
activation in the HUVECs. (A) Images of MitoSOX red staining in the HUVECs
4
pretreated with or without compound C or siRNA and quantified analysis(C).
5
(B)Mitochondrial morphology was characterized using MitoTracker Green, and
6
mitochondrial density was quantified (D). Scale bar is 50 µm. (E) Western blotting
7
analysis of mitochondrial dynamics related proteins, including mitochondrial fission
8
protein Drp-1 and mitochondrial fusion protein OPA1, MFN1, and MFN2. (F) The
9
mRNA expression levels of ICAM1, VCAM1 and CCL2 were measured by qRT-PCR.
10
n=3. #P<0.05,
##
11
Ad-CLU-HG/HF. CM, culture medium; NG, normal glucose; CC, compound C;
12
HG/HF, high glucose and high palmitate; Ad-CLU, the adenovirus expressing
13
clusterin; CLU-siRNA1 and CLU-siRNA2, the siRNA targeting CLU-mRNA.
P<0.01,
###
P<0.001 vs. HG/HF, *P<0.05,
**
P<0.01,
***
P<0.001 vs.
14 15
3.6 Clusterin knockdown increased mito-ROS production and aggravated the
16
endothelial inflammatory activation
17
To obtain more evidence to support our conclusion, we knocked down clusterin
18
expression by siRNA (Fig.S1) in the HUVECs. The CLU-siRNA1 and CLU-siRNA2
19
target different sequence segments of CLU-mRNA. As a result, silencing clusterin
20
aggravated mitochondrial ROS production and fragmentation under HG/HF
21
conditions (the last two panels of Fig.9A-D). VCAM-1 and ICAM-1 expression in the
22
clusterin knockdown HUVECs exposed to HG/HF was significantly elevated (Fig.9F).
23
Besides, HG/HF impaired the tube formation of HUVECs on Matrigel, whereas
24
silencing clusterin caused a greater decrease in network formation (Fig.S7). These
25
results suggested that clusterin knockdown aggravated mitochondrial fragmentation
26
and endothelial inflammatory activation in contrast to clusterin overexpression.
27 28 29 30
4. Discussion Accumulating
evidence
revealed
that
preventing
vascular
endothelial
dysfunction caused by diabetes can alleviate cardiovascular complications in diabetic 36
1
patients [3]. In this study, we used two diabetic models of spontaneous and
2
drug-induced diabetes to explore the role of clusterin in endothelial dysfunction
3
caused by hyperglycemia and hyperlipidemia. We determined that clusterin
4
ameliorates diabetes-caused endothelial dysfunction by inhibiting monocyte adhesion
5
and improving endothelium-independent vasorelaxation. Our data also indicated that
6
clusterin overexpression reduced oxidative stress and ROS levels, which is the major
7
cause of endothelial dysfunction under diabetic conditions. The results highlight the
8
previous conclusion that clusterin is a biosensor for oxidative stress and cleaner of
9
oxidized production [45, 46]. Previous reports suggested that clusterin plays a
10
protective role against neointimal hyperplasia by inhibiting the apoptosis of
11
endothelial cells and the proliferation of VSMCs [36]. In another research, clusterin
12
was identified as a mediator of the leptin signaling that plays a key role in the
13
regulation of energy homeostasis [47]. These studies were consistent with the results
14
indicating that clusterin is involved in cytoprotection under a wide variety of stress
15
conditions.
16
On the other hand, clusterin shares functional similarities with small heat shock
17
proteins (sHSPs) as a stress-induced protein [29]. Clusterin and sHSPs increase in
18
response to oxidative stress, regulated by the heat shock factor signaling [24, 48].
19
Thus, it is not surprising that the expression levels of clusterin are up-regulated in
20
metabolic diseases associated with oxidative stress [28]. For example, the clusterin
21
level in plasma is obviously up-regulated in patients with diabetes or heart failure [49].
22
Furthermore, elevated sHSP expression improves cell survival by decreasing the
23
intracellular levels of free radicals and reducing the oxidative damage to DNA,
24
proteins, and lipids. However, sHSPs were inhibited from constant increasing because
25
of a negative feedback between sHSPs and heat shock factor [50]. In our study,
26
similar to sHSPs, clusterin plays a role of cytoprotection but may not be constantly
27
elevated under diabetic conditions and not completely exert its effects, while higher
28
levels of clusterin in the diabetic animal models significantly improved endothelial
29
function with clusterin over-expression.
30
This study demonstrated that clusterin inhibited mitochondrial fragmentation and 37
1
ROS production, which has not previously reported. Mounting evidence has revealed
2
that mitochondrial dysfunction may contribute significantly to endothelial dysfunction
3
[8, 15, 16]. Elevated free fatty acid and hyperglycemia in diabetes contribute to
4
mitochondrial dysfunction, including the perturbation of mitochondrial morphology
5
and dynamics [51-53]. Recent evidence indicates that mitochondrial fission in
6
endothelial cells is promoted under diabetic conditions, resulting in reduced oxidative
7
phosphorylation capacity and increased ROS production [8]. In addition to being a
8
major headstream of ROS, the mitochondria are targets of ROS. Excess ROS is
9
detrimental to mitochondrial proteins, DNA, and membrane lipid, which prompts
10
endothelial dysfunction [54, 55]. Therefore, maintaining mitochondrial homeostasis is
11
necessary to suppress endothelial dysfunction in diabetic states.
12
How does clusterin regulate the balance of mitochondrial dynamics? AMPK
13
signaling attracted our interests. AMPK, an intracellular sensor of energy, has recently
14
been explored as a novel drug target for preventing diabetic complications [42]. ROS
15
overproduction via mitochondria and NADPH oxidase induced by diabetes in
16
endothelial cells can be inhibited by AMPK activation [44]. AMPKα phosphorylation
17
is subdued markedly in diabetes, resulting in disturbed mitochondrial function and
18
energy homeostasis [56]. Previous findings suggested that AMPK deficiency
19
aggravates diabetic vascular injury via promoting excessive fission of mitochondria
20
[12]. Our present study showed that AMPK was activated by clusterin overexpression,
21
and inhibitor of AMPK, compound C, weakened the action of clusterin in the
22
HUVECs subjected to HG/HF. These data revealed that clusterin attenuates
23
mitochondrial impairment through the activation of AMPK. It has been suggested that
24
clusterin inhibits mitochondrial apoptosis by suppressing p53-activating stress signals,
25
inhibiting Bax activation and cytochrome C release [57]. However, the role of AMPK
26
in mitochondrial dynamics modulated by clusterin has not been previously reported,
27
and would be interesting to be investigated in more detail. For example, the mediator
28
between clusterin and AMPK and the specific downstream signaling remain obscured,
29
awaiting further investigation.
30
The role of clusterin in endothelial dysfunction might be beyond inhibiting the 38
1
perturbation of mitochondrial dynamics. We also found that the overexpression of
2
clusterin affects the transcription of proinflammatory genes in endothelial cells
3
stimulated with HG/HF. Previous evidence suggested that clusterin may regulate the
4
function of ECs with TNF-α stimulation via repressing the NF-κB signaling pathway
5
[36]. Another report indicated that up-regulated expression of clusterin triggered by
6
shear stress can restrain atherosclerosis via preventing endothelial activation [58].
7
Thus, the mechanism of the protective role of clusterin in diabetic endothelial
8
dysfunction may be involved in chronic inflammation. It was recently shown that
9
clusterin also facilitates stress induced autophagy to enhance cancer cell survival [59].
10
However, the impact of clusterin on autophagy in ECs remains unkown. Further
11
studies are needed to explore whether the protective role of clusterin observed in
12
diabetes-induced endothelial dysfunction is also mediated in part through autophagy
13
or mitophagy.
14
Lipotoxicity from elevated free fatty acid in diabetes also fosters endothelial
15
dysfunction, and this factor was taken into account in this study. Clusterin
16
overexpression decreased the level of NEFA in db/db diabetic mice (Fig. 4B),
17
implying that clusterin may play a role in lipid metabolism. But more experiments are
18
needed to verify the result and clarify the involved mechanism. A vicious circle of
19
events occurred in the endothelium could be induced by the metabolic milieu in
20
diabetes including hyperglycemia, elevated free fatty acid and insulin resistance, and
21
the endothelial dysfunction induced by the multiple metabolic disturbances is closely
22
related to vascular complications [5]. Hence, understanding and preventing
23
endothelial dysfunction is constructive for the prevention of diabetic complications. In
24
the current study, two diabetes models of spontaneous diabetes with LEPR-deficiency
25
and STZ-induced diabetes were used, but there are still some limitations. First, loss of
26
function using CLU-KO mice has yet to be considered. In addition, clusterin is a
27
complicated protein with multiple physiological functions and subcellular localization,
28
and its crystal structure remains unknown [26]. This study did not determine
29
respectively the roles of clusterin in different subcellular organelles. In light of the
30
published data and our previous work (Fig.S8), clusterin primarily as a secreted form 39
1
exerts cytoprotection under a variety of stress conditions [60]. In our current study,
2
HUVECs were infected with Ad-CLU expressing sCLU precursor (~60 kD) and
3
sCLU, which were detected by Western blotting (Fig.S1B) and ELISA (Fig.S1C). In
4
the db/db mice and STZ-induced rat models, the sCLU precursor (located in the
5
cytosol) of 60 kD in the aorta tissue and the secreted clusterin in the plasma were
6
detected. However, further animal studies are needed to investigate the separate role
7
of clusterin with different subcellular location in diabetes-induced endothelial
8
dysfunction. Moreover, David Bradley et al. suggested that adipocyte-specific sCLU
9
expression impairs hepatic insulin sensitivity, implying that the role of clusterin might
10
be multiple and tissue-specific [61]. However, the gain- or loss- of function
11
experiments supporting the role of clusterin in insulin resistance was absent. To be
12
honest, we have only scratched the surface of the clusterin mystery, further research is
13
necessary.
14
In conclusion, our study provides fresh insight into the role of clusterin and sheds
15
further light on a novel mechanism explaining that clusterin ameliorates endothelial
16
dysfunction in diabetes via suppressing mitochondrial fragmentation. Our study also
17
demonstrated that AMPK activation plays a considerable role in clusterin-mediated
18
mitochondrial homeostasis. Therefore, clusterin may be a promising therapeutic target
19
for endothelial dysfunction induced by diabetes.
20 21
Acknowlegements
22
Thanks a lot for the support and help from Institute of Materia Medica, Chinese
23
Academy of Medical Science and Peking Union Medical College.
24 25
Funding
26
This work was supported by National Science and Technology Major Projects for
27
‘Major New Drugs Innovation and Development’ [2017ZX09101001] and National
28
Natural Science Foundation of China [81641016, 81603197].
29
Conflict of interests
30
None 40
1 2
Author contributions
3
Design of the project: Lihong Liu
4
Experiments: Lulu Ren, Feifei Han, Lingling Xuan, Benshan Xu, Yuan Sun and Song
5
Yang
6
Data analysis: Yali Lv, Lili Gong, Zirui Wan, Lifang Guo and He Liu
7
Figures layout : Lulu Ren and Yan Yan
8
Manuscript writing: Lulu Ren
9
Manuscript revision: Lihong Liu
10 11
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Clusterin ameliorates endothelial dysfunction in diabetes by suppressing mitochondrial fragmentation Lulu Ren1, Feifei Han1, Lingling Xuan1, Yali Lv1, Lili Gong1, Yan Yan1, Zirui Wan1, Lifang Guo1, 2
1
, He Liu1, Benshan Xu1, Yuan Sun 1,Song Yang1, Lihong Liu1, * Department of Pharmacy, Beijing Chao-Yang Hospital, Capital Medical University, Beijing,
100020, China 2
School of Life Sciences, Tsinghua University, Beijing 100084, China
* Corresponding author. Department of Pharmacy, Beijing Chao-Yang Hospital, Capital Medical University, 8 Gongren Tiyuchang Nanlu, Beijing 100020, China. Tel: 86-010-8523 1464. E-mail: [email protected].
Highlights Clusterin overexpression rescues diabetes-induced endothelial dysfunction. In diabetic models, clusterin inhibits oxidative stress and mitochondrial ROS production. Clusterin suppresses mitochondrial fragmentation via reducing expression level of Drp-1. AMPK activation involved in the molecular mechanisms of clusterin against endothelial dysfunction.