The ubiquitin-activating enzyme E1 as a novel therapeutic target for [617128]

The ubiquitin-activating enzyme E1 as a novel therapeutic target for
the treatment of restenosis
Zhexue Qina,1, Bin Cuia,1, Jun Jina, Mingbao Songa, Baoshang Zhoub, Hongfeng Guoc,
Dehui Qiana, Yongming Hea, Lan Huanga,*
aDepartment of Cardiology, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, People's Republic of China
bDepartment of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, People's Republic of China
cDepartment of General Medicine, Training Base of Medical Service, Third Military Medical University, Chongqing 400038, People's Republic of China
article info
Article history:
Received 10 November 2015Received in revised form31 January 2016Accepted 12 February 2016Available online 13 February 2016
Keywords:
Ubiquitin-activating enzyme E1Restenosis
Inflammation
Vascular injuryabstract
Aims: The ubiquitin-activating enzyme E1 (UBA1, E1), the apex of the ubiquitin proteasome pathway,
plays a critical role in protein degradation and in pathological processes. Whether UBA1 participates thedevelopment of vascular restenosis remains unknown. This study aims to determine the role of UBA1 in
the development of balloon injury induced neointimal formation.
Methods and results: Immunostaining and western blots were used to examine the expression of the
ubiquitinated protein in the injured carotid after angioplasty. Higher levels of ubiquitinated protein wereobserved in the neointima. Local delivery of potent chemical UBA1 inhibitor PYR-41 (100
mM) and UBA1
shRNA lentivirus both resulted in a substantial decrease in intimal hyperplasia at 2 weeks and 4 weeks
after balloon injury. UBA1 inhibition also reduced Ki-67 positive cell percentage and in flammatory
response in the carotid artery wall. We further determined that in vitro UBA1 inhibition was able to
ameliorate TNF- a-induced nuclear factor-kappa B (NF- kB) activation by reducing I kB degradation in
vascular smooth muscle cells (VSMCs). UBA1 inhibition also led to the accumulation of short-livedproteins such as p53, p21 and c-jun, which may account for the UBA1 inhibition-induced cell cycle
delay. Thus, VSMCs proliferation was blocked.
Conclusions: UBA1 inhibition effectively suppresses neointimal thickening through its anti-proliferative
and anti-in flammatory effects. Our results provide further evidence that the ubiquitin-proteasome sys-
tem is a potential new target for the prevention of vascular restenosis.
©2016 Elsevier Ireland Ltd. All rights reserved.
Restenosis after angioplasty remains a remarkable challenge,
although drug-eluting stents have reduced the incidence of reste-nosis considerably [1]. Vascular smooth muscle cells (VSMCs) play a
pivotal role in the development of intimal thickening and reste-
nosis. VSMCs proliferate and migrate from the media to the intima
[2,3] . Thus, finding new targets against VSMCs is important.
The ubiquitin proteasome system is the main intracellular pro-
tein degradation route by which cells get rid of excess and mis-
folded proteins. A growing body of evidence has implicated the
ubiquitin proteasome system (UPS) as being involved in theregulation of the complex cell signaling processes that are funda-
mental to atherosclerotic diseases. Recent studies have identi fied
the contribution that the UPS makes to the initiation and compli-
cation of atherosclerosis via the regulation of vascular in flamma-
tion, apoptosis, oxidative stress and cholesterol metabolism [4].A n
increased activity in ubiquitination-proteasome in neointimal areas
and the role of ubiquitin gene expression in these settings have
been mentioned [5]. Furthermore, the effects of proteasome inhi-
bition on neointima formation have been well characterized, and
the proteosome is considered as a therapeutic target [4,6] .H o w –
ever, the consequences of blocking protein degradation by inhib-
iting the apex of protein ubiquitination remain largely unknown.
Here, we used chemical and genetic approaches to investigate the
inhibition of protein ubiquitination in VSMCs both in vitro and
in vivo.
The ubiquitin moiety is generally attached via an E1-E2-E3
multi-enzyme cascade. In the first step, the ubiquitin-activating*Corresponding author. Department of Cardiology, No.183, Xinqiao Street, Xin-
qiao Hospital, Third Military Medical University, Chongqing 400037, People's Re-public of China.
E-mail address: huanglan260@126.com (L. Huang).
1These authors contributed equally to this work.
Contents lists available at ScienceDirect
Atherosclerosis
journal homepage: www.elsevier.com/locat e/atherosclerosis
http://dx.doi.org/10.1016/j.atherosclerosis.2016.02.016
0021-9150/ ©2016 Elsevier Ireland Ltd. All rights reserved.Atherosclerosis 247 (2016) 142 e153

enzyme E1, UBA1 (E1), binds ATP $Mg2țand ubiquitin and ca-
talyses C-terminal ubiquitin acyl-adenylation and the binding of
a molecule [7]. This ubiquitin is then available to be transferred
to one of the E2 ubiquitin conjugating enzymes. E2 enzymes then
interact with one of the hundreds of ubiquitin E3 ligases to
transfer the ubiquitin to the ε-amino group of a lysine residue in
the target protein. After several cycles, four or more ubiquitins
linked via lysine-48 of ubiquitin (K48) are attached to the target
protein. The K48-linked polyubiquitination chain is the canonical
ubiquitin chain that targets the ubiquitinated protein for degra-
dation by the proteasome enzyme complex [8].M o n o –
ubiquitination with a single ubiquitin conjugated to a protein
regulates DNA repair, nuclear export and histone regulation
rather than protein degradation [9,10] . To date, dozens of E2
enzymes and hundreds of E3 enzymes have been identi fied,
whereas only two ubiquitin E1 enzymes have been discovered, of
which E1 is the predominant isoform in the UPS pathway.
Because the inhibition of the proteasome effectively reduces
neointima formation in vivo, we hypothesized that inhibition of
UBA1, the apex of the UPS, would also effectively block the UPS
pathway ( Fig. 1 A), as do proteasome inhibition, and may thus
attenuate neointimal hyperplasia.
In the present study, we determined ubiquitinated protein in the
arterial neointima. We also demonstrated that the genetic and
chemical inhibition of the UBA1 enzyme reduced neointimal hy-
perplasia. Furthermore, the inhibition of the UBA1 enzyme caused
cell cycle arrest and blocked protein degradation in VSMCs. Our
findings support the possibility that UBA1 may be a novel thera-
peutic target for neointimal hyperplasia after arterial injury and for
restenosis after angioplasty.
1. Methods
A detailed method section is available in the Supplementary
material online.
1.1. Mice
Adult male Sprague Dawley rats weighing 250 e300 g
(Chongqing, China) were housed in the Center for Experimental
Animals (an Assessment and Accreditation of Laboratory Animal
Care-accredited experimental animal facility) at Third Military
Medical University, Chongqing, China. The rats were anaesthetized
with an intramuscular injection of 100 mg/kg ketamine and 5 mg/
kg xylazine to further harvest the aortas for VSMCs culture or an-
gioplasty with balloon embolectomy catheter (2F, Cordis, USA). All
procedures involving experimental animals were performed in
accordance with protocols approved by the Committee for Animal
Research of Third Military Medical University, China, and con-
formed to the Guide for the Care and Use of Laboratory Animals
(NIH, 2011).
1.2. Rat carotid balloon injury model
See the Supplementary Materials and Methods section for
details.
1.3. Lentivirus-mediated delivery of small interfering RNA
The lentivirus-mediated siRNA construct was designed as
previously described [11].B r i e fly, annealed oligonucleotides
encoding sense and antisense strands linked by the loop
sequence were subcloned into the pSINsi-mU6 vector. We
designed the siRNA sequences as follows: (siRNA-E1-1) 50-
TTAACTTCGTGACATCCCAGG-30,50-CCTGGGATGTCACGAAGTTAA-30,( s i R N A – E 1 – 2 )50-TAAGGAAGT CTTCAACA AGAG-30,50-CTCTTG-
TTAGACTTCCTTA-30. The pSINsi-mU6 vector was introduced into
a lentivirus vector, pLenti6/V5-D-TOPO (Invitrogen, U.S.), and the
recombinant lentiviruses were produced in 293T cells. Concen-
trated lentiviral solutions encoding sh-UBA1 or control (50 ml)
were infused into the injured segment of the common
carotid artery and incubated for 30 min. All experimental pro-
cedures were approved by the Institutional Animal Care and Use
Committee of Third Military Medical University (Chongqing,
China).
1.4. Real-time polymerase chain reaction (PCR)
RNA isolation, cDNA synthesis and real-time PCR were per-
formed as described [12]. The E1 and GAPDH primers used are
detailed in the Supplementary Materials and Methods section.
1.5. Western blot
Western blot analysis was performed as described previously
[13]. The speci fic band was scanned with an imaging analyzer. b-
actin or GAPDH was used as a loading control. See the details in the
Supplementary Materials and Methods section.
1.6. Electrophoretic mobility shift assay
The nuclear binding of NF- kB was analyzed by electrophoretic
mobility shift assays using VSMCs nuclear extracts used as
described previously [14].
1.7. VSMCs proliferation and cell cycle detection
See the details in the Supplementary Materials and Methods
section.
1.8. Immunohistochemistry and immuno fluorescence staining
The immunohistochemical and immunocytochemical methods
are described in the Supplementary Materials and Methods section.
1.9. Detection of apoptosis
Carotid arteries were excised and fixed with acetone. In vivo
apoptosis was evaluated using TUNEL staining (Roche, US), and the
samples were counterstained with DAPI. The in vitro apoptosis rate
was detected with flow cytometry using the Annexin V-FITC and PI
Apoptosis Detection Kit (Abcam, Cambridge, US).
1.10. Statistics
The results were expressed as the means ±SEM. One-way
ANOVA was used to analyze data among three groups. Student's
t-test was used to compare the data between two groups. A P
value <0.05 was considered signi ficant.
2. Results2.1. Expression of ubiquitinated proteins of smooth muscle cells in
injured carotid arteries
First, balloon injury induced neointima formation in rat was
detected with Ub antibody. As shown in Fig. 1 B in neointima,
ubiquitinated proteins were stained in all arterial layers, especially
in the neointima. Meanwhile consecutive slices and fluorescence
confocal staining showed that
a-SMA coincided with ubiquitinatedZ. Qin et al. / Atherosclerosis 247 (2016) 142 e153 143

proteins in the neointima, which mainly consisted of vascular
smooth muscle cells ( Fig. 1 C&D). Besides, Western blot was per-
formed with cellular protein extracts from carotid arteries. Ubiq-
uitinated proteins were detected in both injured and uninjured
carotid artery extracts. The level of ubiquitinated proteins was
lower in uninjured right carotid arteries at 1 week, 2 weeks after
angioplasty ( Fig. 1 E). These results may suggest that proteinubiquitination might contribute to the neointima progression.
2.2. Local delivery of chemical E1 inhibitors attenuated neointima
formation in injured rat carotid arteries
We next assessed the effects of chemical E1 inhibitors on neo-
intima formation after angioplasty in vivo. PYR-41 and PYZD-4409
Fig. 1. Ubiquitinated protein expression in injured carotid artery. A, Scheme of ubiquitin enzyme system E1-E2-E3 pathway that precedes proteasome degrad ation of ubiquitinated
proteins. B, Immuno fluorescent staining of ubiquitinated protein in neointima or shamed carotid with ubiquitin antibody (Green). C, Immunochemical staining of ubiquit inated
protein in consecutive slices of carotid neointima. Ub (Left panel) and a-SMA antibody (Right panel). D, Fluorescent staining of carotid neointima with a-SMA and Ub with confocal
imaging technique. DAPI (blue), a-SMA (green), Ub (red) and merged image were presented. Ubiquitinated proteins were colocalized with a-SMA in neointimal area in the merged
picture. E, Western blot revealed that ubiquitinated protein levels were higher in the injured carotid compared with uninjured group at 1week and 2 we eks post angioplasty. n ¼5,
*P<0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)Z. Qin et al. / Atherosclerosis 247 (2016) 142 e153 144

are two well-recognized E1 inhibitors [9,15] . PYR-41 was chosen to
test the effects of E1 inhibition on neointima formation. We
confirmed their inhibition of E1 enzymatic activity. PYR-41 reduced
the level of Ub ~ E1 thioesters compared with DMSO without
affecting E1 expression ( Fig. 2 A). In addition, PYR-41 blocked
MG132-induced ubiquitin accumulation in VSMCs ( Fig. 2 B). Thus,
we concluded that PYR-41 is effective E1 enzymatic inhibitors.
Local drug delivery was applied to determine the E1 inhibitors'
effects on the neointima in a rat carotid arterial injury model. Aspositive control, MG132 signi ficantly inhibited the neointima for-
mation compared with DMSO group (Supplement Fig. 2 A), which is
consistent with Stangl K0group results [34]. PYR-41 attenuated
neointimal hyperplasia at 2 weeks and 4 weeks after injury, by
59.1% and 43.9%, respectively, relative to the control group
(*P<0.01, n ¼8;Fig. 2 C). The I/M ratio was also decreased signif-
icantly at both 2 and 4weeks in the PYR-41 group (* P<0.01, n ¼8;
Fig. 2 C). Meanwhile, the medial areas remained unaffected by
either vascular injury or PYR-41 ( Supplement Fig. 1 ). PYR-41
Fig. 2. PYR-41 local delivery reduced neointima formation in rat carotid arteries. A, PYR-41 treated VSMCs were collected and lysated and then heated in eith er non-reducing (NR)
or reducing (R) sample buffer, followed by SDS-PAGE and immunoblotting with anti-UBA1. B, PYR-41 at the concentration of 50 mM blocked MG132 induced ubiquitinated protein
accumulation (Control VS PYR41, n ¼4, *P<0.05; MG132 VS MG132 țPYR41, n ¼4, *P<0.05). C, Neointimal hyperplasia stained with hematoxylin and eosin at 2weeks and 4weeks
after carotid balloon injury were ameliorated after local delivery of 100 mM PYR-41 compared with control group (n ¼8). Areas of neointima and medial of the carotid arteries were
quanti fied. Neointima areas at both 2weeks and 4weeks were signi ficantly reduced in the PYR-41 group compared with control group (n ¼8, *P<0.01). D, Local delivery of PYR-41
reduced Ki-67 positive cells relative to control group. n ¼3, *P<0.01.Z. Qin et al. / Atherosclerosis 247 (2016) 142 e153 145

delivered to the injured artery, induced a remarkable reduction in
the number of Ki67-positive cells by 2 weeks in the neointima
relative to the number in the control group (* P<0.01, n ¼3;
Fig. 2 D).
Similarly, another chemical E1 inhibitor, PYZD-4409, at a con-
centration of 150 mM reduced the neointima area signi ficantly
compared with control group at 4 weeks ( Supplement Fig. 2B ).
These results suggest that the chemical inhibition of E1 enzymatic
activity is able to attenuate the neointimal overgrowth that occurs
after vascular injury.
2.3. E1 knockdown reduced the neointimal hyperplasia after
balloon injury
Genetic manipulation was performed to test the role of E1 in
vascular injury. Two shRNA lentiviral vectors against E1 were
generated. Rat cultured VSMCs were used to detect the ef ficiency of
knockdown. The transfection ef ficiency in cultured VSMCs was
90.3±0.9% (Supplement Fig. 3 A). The real-time PCR results in
Fig. 3 A show that the mRNA expression was suppressed by both
shRNAs. The protein expression levels were also reduced by E1knockdown, as illustrated in Fig. 3 B. Subsequently, the accumula-
tion of ubiquitinated protein was blocked as a result of E1 knock-
down after transfection at different days ( Fig. 3 C).
We then delivered sh-UBA1 lentivirus to the local injured rat
carotid artery. Immunostaining with an anti-GFP antibody revealed
that GFP was present from 3 days post angioplasty, and the trans-
fection ef ficiency was 73.5 ±7.6% ( Supplement Fig. 3B ). These re-
sults suggest that the lentivirus-mediated delivery system was
effective in rat carotid arteries.
The effects of E1 knockdown in the carotid artery were also
confirmed. E1 expression were markedly lower at 2 and 4 weeks
after vascular injury ( Supplement Fig. 3C ). Interestingly, the neo-
intimal area in the E1 knockdown group was reduced by 43.3% and
27.8% at 2 weeks and 4weeks, respectively, compared with the area
in the control group ( Fig. 3 D, n ¼8, *P<0.01). I/M ratio were
markedly lower in the E1 knockdown group ( Fig. 3 D, n ¼8,
*P<0.05). Concomitantly, the number of Ki67-positive nuclei
decreased substantially from 43.3 ±5.3% in the control group to
19.12 ±0.74% in the E1 knockdown group ( Fig. 3 E, n¼3, *P<0.01).
Therefore, genetic inactivation of E1 also ameliorates neointimal
hyperplasia after vascular injury.
2.4. E1 inhibition reduced monocyte/macrophage in filtration and
NF-
kB activation
Monocyte/macrophage in filtration contributes to neointima
formation and vascular in flammation [16]. Immuno fluorescence
staining of the vessels was performed in the E1 inhibition groups.
Three days following carotid artery injury, there were substantial
increases in the number of CD68-positive monocytes and macro-
phage in the injured arteries relative to the numbers in arteries of
the E1 knockdown and chemical inhibitor groups ( Fig. 4 A). The
CD68 protein levels were substantially decreased in the E1 knock-
down group ( Fig. 4 B, n¼5,*P<0.05) and the PYR-41 group at 1
week and 2 weeks ( Supplement Fig. 4 ). These findings indicate that
E1 inhibition results in less monocyte/macrophage recruitment to
the vessel wall after injury.
Numerous reports have suggested that NF- kB activation plays an
important role in neointimal vascular hyperplasia [17,18] . The un-
injured carotid artery showed weak expression of NF- kB p65. NF- kB
wasfirst activated at day 1 post angioplasty as reported [19].
Wefirst detected the NF- kB target gene expression. VCAM1 and
ICAM1 were determined with tissue protein extracts. In PYR41
group, the VCAM1 and ICAM1 levels were downregulatedsignificantly at 1week ( Fig. 4 C, n¼5,*P<0.05). UBA1 knockdown
with shRNA lentivirus also exerted inhibitory effects on VACM1 and
ICAM1 compared with control group at 1week ( Fig. 4 C, n¼5,*P<
0.05). Next, MCP1 was measured with ELISA and was attenuated by
chemical inhibition of UBA1 or UBA1 knockdown signi ficantly
1week post angioplasty ( Fig. 4 D, n¼3,*P<0.05). Furthermore, NF-
kB transcription activity was detected with EMSA. After 1week, the
EMSA results showed that the DNA binding activity of NF- kB p65
was reduced in the PYR-41 and E1 knockdown groups, by 56.9% and
34.9%, respectively ( Fig. 4 E, n¼3, *P<0.05). We thus concluded
that E1 inhibition reduced NF- kB activation.
2.5. E1 inhibition attenuated NF- kB activation by blocking I kB
degradation
Next, we tested effects of E1 inhibition on NF- kB activation
in vitro. Cultured VSMCs were treated with PYR-41 or transfected
with lentivirus carrying shRNA against UBA1. PYR-41 inhibited the
phosphorylation of NF- kB p65 and decreased the VCAM1 and
ICAM1 expression ( Fig. 5 A). Similarly, UBA1 knockdown also
resulted in lower level of p-p65, VCAM1 and ICAM1 ( Fig. 5 A, n¼3,
*P<0.05). Meanwhile, the adhered bone marrow derive monocyte
to the plated VSMCs were reduced in the sh-UBA1 group compared
with control vector group ( Fig. 5 B, n¼6,*P<0.05).
The effects of E1 inhibition on NF- kB activation were tested
under TNF- astimulation. TNF- acaused remarkable dose-
dependent I kB-aphosphorylation when VSMCs were incubated
for 20 min at a concentration of 10 ng/mL. Intriguingly, PYR-41 nor
PYZD-4409 treatment affected the I kB-aphosphorylation, a result
that is consistent with the effects of the proteasome inhibitor
MG132 ( Supplement Fig. 5 ). However, chemical E1 inhibitors
blocked TNF- a-induced I kB-aand I kB-bdegradation ( Fig. 5 C, n¼3,
*P<0.01). This effect was also in accordance with the MG132 effect
(Fig. 5 C). To further con firm the effects of E1 inhibition on NF- kB
activation, we employed EMSA to analyze the DNA binging ability
of NF- kB. As shown in Fig. 5 D, chemical E1 inhibitors had signi fi-
cantly inhibitory effects on TNF- a-induced NF- kB nuclear trans-
location and activation. In addition, E1 knockdown exerted a
similar inhibitory effect on TNF- a-induced NF- kB activation
(Fig. 5 D).
2.6. E1 inhibition blocked degradation of the short-lived proteins
Most proteins are normally degraded by the ubiquitin protea-
some system. The inhibition of the E1 enzyme may block
ubiquitination-induced protein degradation and thus increase the
abundance of short-lived proteins. We therefore determined the
levels of short-lived proteins in the different groups submitted to
the genetic and chemical inhibition of the E1 enzyme. Fig. 6 A
showed the accumulation of multi-ubiquitinated forms of p21, p53
and c-jun after 24 h of treatment with PYR-41 at the concentrations
of 0, 30 and 50 mmol/L. The abundances of p21, p53 and c-jun
increased in a dose-dependent manner relative to the PYR-41
concentration. Moreover, 20 min of incubation with PYR-41 at the
higher concentration of 100 mmol/L induced the accumulation of
short-lived proteins ( Supplement Fig. 6A ). PYZD-4409 stimulation
of VSMCs also induced the accumulation of short-lived-proteins
(Supplement Fig. 6B ). Similar effects were also observed with the
E1 knockdown ( Fig. 6 B, n¼3,*P<0.01).
2.7. In vitro analysis of the effect of E1 inhibition on VSMCs
proliferation
PYR-41 treatment at 25 mM for 48 h signi ficantly decreased the
uptake of3H-thymidine by VSMCs ( Fig. 6 C, n ¼3,*P<0.05). InZ. Qin et al. / Atherosclerosis 247 (2016) 142 e153 146

Fig. 3. UBA 1 knockdown effectively blocked balloon injured induced neointimal hyperplasia. A and B, Real time and Western blot revealed lentivirus vector c arrying shRNA against
UBA1 ef ficiently inhibited UBA 1 expression at both mRNA and protein levels (n ¼3, *P<0.01). C, As a consequence, at day 4 and 7 after E1 knockdown in VSMCs, ubiquitinated
proteins were reduced (n ¼5, *P<0.01). D, Hematoxylin and eosin staining after E1 shRNA lentivirus transfection in the injured carotid. Local E1 knockdown in the carotid resulted
in a signi ficant decrease in neointimal hyperplasia at 4weeks after balloon injury and gene knockdown. Areas of neointima and I/M were remarkably inhibited in E1 knockdown
group compared with control group, respectively (n ¼8, *P<0.05). E, Immunochemical staining of Ki-67 in E1 knockdown group and control group. The number of Ki-67-positive
cells dropped in the E1 knockdown group signi ficantly (n ¼3, *P<0.01).Z. Qin et al. / Atherosclerosis 247 (2016) 142 e153 147

addition, the transfection of shRNA-E1 decreased the number of
VSMCs and the level of3H-thymidine uptake at 72 h aftertransfection ( Fig. 6 C, n ¼3,*P<0.05). Cell cycle alteration was
examined by flow cytometry. Fluorescence-activated cell sorting
Fig. 4. E1 inhibition reduced monocyte/macrophage in filtration and NF- kB activation. A, Immuno fluorescent staining of CD68 in the balloon injured carotid arteries treated with
PYR-41 or UBA1 knockdown lentivirus transfection (n ¼3, *P<0.05, compared with control group). B, CD68 protein levels in the carotid artery extract with western blot at 1 week
and 2 weeks. (n ¼5, *P<0.05, sh-UBA1 group compared with scrambled shRNA group). C. Western blots results on the VCAM1, ICAM1 were signi ficantly decreased in the PYR-41
group (n ¼5, *P<0.05) and sh-UBA1 group (n ¼5, *P<0.05) compared with control, respectively at 1 week. Equal loading was con firmed by GAPDH staining. D, Effects of PYR-41
and sh-UBA1 on the MCP1 protein levels in rat carotid artery 1 week after balloon injury evaluated by ELISA. MCP1 levels were normalized with protein co ncentration. (PYR-41 vs.
Control, n ¼3, *P<0.05; sh-UBA1 vs. Scrambled group, n ¼3, *P<0.05). E, NF- kB DNA binding activity after PYR-41 or sh-UBA1 treatment in the carotid arteries was detected by
NF-kB probe with EMSA (n ¼3, *P<0.05 compared with control group).Z. Qin et al. / Atherosclerosis 247 (2016) 142 e153 148

was used to examine the cell-cycle distribution ( Fig. 6 D). The per-
centage of VSMCs progressed to the S phase decreased from 27.36%
in the untreated and 28.56% DMSO groups to 20.79% in the PYR-41
group ( Fig. 6 D, n¼3,*P<0.05). The knockdown of UBA1 with the
lentivirus have similar effect to delay the VSMCs progression to the
S phase ( Fig. 6 D, n¼3,*P<0.05).2.8. Effects of E1 inhibition on apoptosis in the injured carotid and
in vitro
To unmask the mechanisms underlying E1 inhibition in vivo, we
evaluated the local response of the injured carotid artery after E1
inhibition. Apoptosis of the carotid cells was detected by TUNEL.
Balloon injury led to the apoptosis of media layer VSMCs in 30 min
Fig. 5. E1 inhibition reduced in flammation in VSMCs in vitro. A, ICAM1, VCAM1 and p-p65 protein levels were decreased by PYR-41 and sh-UBA1 respectively. PYR-41 vs. Control,
n¼3, *P<0.05; sh-UBA1 vs. Scrambled group, n ¼3, *P<0.05. B, Monocyte adhered to cultured VSMCs treated with scrambled lentivirus or sh-UBA1 lentivirus. Sh-UBA1 vs.
scrambled group, n ¼6, *P<0.05. C, PYR-41 attenuated the TNF ainduced I kBa,bdegradation, which is consistent with the effect of MG132. TNF ațPYR-41 vs. TNF a,n¼3, *P<0.01.
D, PYR-41 treatment and E1 knockdown inhibited TNF ainduced NF- kB DNA binding activity. TNF ațPYR-41 group vs. TNF agroup, n ¼3, *P<0.05. TNF ațsh-UBA1 group vs. TNF a
group, n ¼3, *P<0.01.Z. Qin et al. / Atherosclerosis 247 (2016) 142 e153 149

(Fig. 7 A). The apoptosis of media layer VSMCs was also evident in
the PYR-41- and PYZD-4409-treated groups. At 30 min post an-
gioplasty, the apoptosis rate in the chemical E1 inhibition group
appears higher than that in the control group ( Fig. 7 A). Meanwhile,
there were no marked differences between PYR-41 at 100 mM and
PYZD-4409 at 150 mM. At 8 h post angioplasty, the PYR-41 and
PYZD-4409 treatments prolonged the apoptosis and maintained ahigher apoptosis rate in the chemical E1 inhibition group, whereas
nearly no apoptosis occurred in medial VSMCs ( Fig. 7 A). Simulta-
neously, at 8 h post angioplasty, the number of live VSMCs in the
chemical E1 inhibition group was signi ficantly smaller than that in
the control group.
Subsequently, we investigated whether chemical E1 inhibitors
caused the death of cultured VSMCs. Flow cytometry showed that
Fig. 6. E1 inhibition blocked the degradation of short-lived proteins and inhibits cell cycle progression. A, PYR41 increased the protein levels of p21, p53 and c-jun at 30 mM and
50mM( n ¼3, *P<0.01), compared with control. B, sh-UBA1 lentivirus transfection into VSMCs increased the protein levels of p21, p53 and c-jun compared with scrambl ed shRNA.
(n¼3*P<0.01). C, Cells were starved for 24 h and then incubated with 25 mM PYR-41.3H-thymidine was incorporated and counted in a liquid scintillation counter. (n ¼4,
*P<0.05). E1 knockdown reduced VSMCs number (n ¼3, *P<0.05) and3H-thymidine uptake (n ¼3, *P<0.05). D, VSMCs were first synchronized and subjected to different
treatment such as PYR-41, DMSO or transfected with scrambled siRNA or sh-UBA1 lentivirus. The cells were harvest and the cell-cycle distributions we re detected using flow
cytometry. (n ¼3, *P<0.05).Z. Qin et al. / Atherosclerosis 247 (2016) 142 e153 150

cell death rate up to 59.7 ±2.84% in the VSMCs at the concentration
of 50 mM PYR-41, while PYR-41 at 100 mMc a u s e d8 7 . 7 ±1.4% cell
death ( Fig. 7 B). Nevertheless, shRNA-E1 did not signi ficantly increase
the proportion of VSMCs undergoing apoptosis ( Supplement Fig. 7 ).
3. Discussion
The current study demonstrated that the ubiquitin activating
enzyme E1 is a potential target for the treatment of vascular
restenosis, in line with the therapeutic role of the proteasome in the
progression of atherosclerotic diseases [20]. In this study, we
showed that E1 inhibition, whether chemical or genetic, inhibited
vascular neointimal hyperplasia. As shown in the scheme in Fig. 7 C,
both in vivo and in vitro tests veri fied that the prevention of
restenosis induced by E1 inhibition was attributable to the effects
on the in flammation and protein degradation pathways.
Atherosclerosis is the leading cause of cardiovascular disease
around the world. Stenting is bene ficial to restore the blood flow of
the coronary artery. However, the stenting implantation resulted in
acute vessel injury at the time of PCI and triggered the enhanced
healing responses and finally caused neointimal hyperplasia and
in-stent restenosis. Many different therapeutic strategies were
evaluated to prevent restenosis before the drug-eluting stent era
[21e23]. Some of them have been showed different adverse events,such as late thrombosis, delayed restenosis and aneurysm forma-
tion. The potency of the drugs or the toxic effects may shift the
restenosis to late restenosis or damage of the vascular bed. The first
generation drug-eluting stent such as sirolimus-eluting Cypher
stent and paclitaxel-eluting Taxus stent approved by the FDA in
early 2000s reduced the neointimal hyperplasia [24,25] . Later, the
second generation stent such as everolimus-eluting stent showed
more safety and lower stent thrombosis [26]. Therefore, exploring
the new drugs against neointimal hyperplasia with fewer side ef-
fects is of great value.
The deregulation of the ubiquitin proteasome pathway has been
previously reported to be involved in atherosclerosis. Increased
levels of ubiquitin conjugates have been observed in carotid pla-
ques, particularly in unstable plaques [5,27,28] . Furthermore, pro-
teasome inhibitors such as MG132 and lactacystin have been
shown to ameliorate neointimal hyperplasia and are considered
therapeutic agents [29,30] . Because E1 is the apex of the protea-
some ubiquitin pathway, we hypothesized that E1 may play a
pivotal role in the prevention of restenosis.
We demonstrated that injured carotid arteries and smooth
muscle cells in atherosclerotic lesion have increased levels of
ubiquitin conjugates relative to the uninjured arteries. Ubiquitin
was expressed in all intimal areas, and the level was higher therein
than that in medial layer cells.
EE1chemicalInhibitor
Proteindegradation
d
d
IκBα/β,p65p21,p27,c-jun
Neointimalformation
a
a
t
t
i
i
o
o
n
n
p
p
2
2
7
7
,
,
c
c
ICAM1/VCAM1
Inflammation
m
m
m
m
a
a
t
t
i
i
m
m
a
a
l
l
f
f
Cellcycle
Proliferation
y
y
o
o
l
l
i
i
f
f
e
f
e
r
r
a
a
o
o
n
n30min
8h
A
PYR-41 Control PYZD-4409B
C
E1knockdown
Apoptosisp53
p
p
5
5
3
3
Fig. 7. E1 chemical inhibition induced VSMCs apoptosis in vivo and in vitro. A, PYR-41 (100 mM) and PYZD-4409 (150 mM) induced apoptosis in the carotid arterial wall. Apoptosis
cells (green) were detected with TUNEL. B, PYR-41 induced VSMCs apoptosis at concentration of 50 mM and 100 mM stained with Annexin-V-FITC and PI with cytometry. n ¼3,
*P<0.01 compared with control. n ¼3,*P<0.05, PYR-41 50 mM vs. 100 mM. C. Scheme of the potential mechanisms by which E1 inhibition blocked balloon injury induced
neointima formation. [indicates promoting effects. ⊥represents inhibitory effects. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)Z. Qin et al. / Atherosclerosis 247 (2016) 142 e153 151

Previously, intense nuclear ubiquitin immunostaining has been
found in aortic intimal thickening induced in rats after eight weeks
of exposure to a silastic tube [30,31] . We observed this phenome-
non in some neointimal areas but found that intense nuclear
ubiquitin immunostaining was not always present throughout the
neointima. We observed that the intense nuclear staining was
located in the basal and inner parts of the intimal rather than all
throughout the neointima. The intense nuclear staining in the base
of the neointima is partly in accordance with the results of other
studies [31]. However, we also observed some intense nuclear
staining toward the lumen, which may indicate that the nuclear
staining was not restricted to certain areas. The speci ficity of the
distribution of overexpression, as tied to a neointimal phenotype or
area, obviously remains unknown.
The different phenotypes of intimal and medial VSMCs may
reflect the breakdown of myo fibrillar proteins in VSMCs, eventually
generating a proliferating phenotype [6,32] . Some researchers
maintain that ubiquitin gene expression characterizes the con-
tractile phenotype of VSMCs rather than the proliferating pheno-
type [33]. However, subsequent research has demonstrated that
proteasome inhibitors halt proliferation and induce apoptosis [34].
Regardless, the level of ubiquitinated protein differed between the
intimal and medial VSMCs. The functional role of higher ubiquitin
levels in VSMCs requires further investigation.
E1 is the apex of the ubiquitin proteasome signaling pathway.
Recently, several chemical inhibitors of E1 have been developed. To
date, PYR-41 [15], PYZD-4409 [35] and fungicide ziram [36] are the
known structurally unrelated inhibitors of E1 enzymatic activity.
We determined the effects of PYR-41 and PYZD-4409 on local
neointimal hyperplasia. PYR-41 signi ficantly inhibited neointimal
growth and reduced the proliferation of VSMCs, as demonstrated
by detecting Ki-67. We also veri fied the PYZD-4409-induced inhi-
bition of E1 activity and found that it was able to reduce neointimal
growth. The similar effects of these two chemical inhibitors partly
revealed the anti-neointima formation effects of E1 ligase in-
hibitors, although we were unable to exclude the unknown targets
that may contribute to their cytotoxic effects. Furthermore, we
genetically knocked down E1 with a lentivirus carrying shRNA
targeting E1. We were able to ef ficiently deliver the lentivirus to the
local carotid tissue, resulting in the marked downregulation of E1.
The genetic manipulation of E1 further supported the idea that E1
inhibition can delay neointima formation. Therefore, we concluded
that E1 ligase inhibition blocked the neointimal overgrowth.
Meanwhile, proteasome inhibitors such as MG132 [34] and borte-
zomib [37] have been reported to exert apoptotic effects to prevent
vascular restenosis.
E1 inhibition also reduced the in filtration of macrophages
recruited by the injured tissue via the induction of in flammatory
factors. Both chemical and genetic inhibition of E1 quantitatively
reduced macrophage in filtration and ameliorated the CD68 expres-
sion. The anti-in flammatory effects were not limited to monocyte/
macrophage in filtration but also extended to a decrease in the NF- kB
expression in the local tissue. EMSA results showed us that NF- kB
activity was also inhibited by E1 ligase inhibition. As a result, the
ubiquitin proteasome pathway was associated with protein degra-
dation. We explored the change in NF- kB activation-associated I kB
degradation in vitro. Our in vitro study indicated that E1 inhibition is
able to block TNF- a-induced I kB degradation. The accumulation of
IkB ultimately prevented the activation of NF- kB and was responsible
for the muted in flammatory response in the injured carotid.
E1 enzyme inhibition increased the abundance of short-lived-
proteins such as p53, p21 and c-jun. The accumulation of the
apoptotic accelerator p53 may have facilitated the apoptosis of cells
in carotid vessel walls. In addition, the suppression of NF- kB activity
also reversed its anti-apoptosis effects [38]. Moreover, p21 acts asboth a p53 downstream molecule and a cell cycle inhibitor [39]. The
p21 accumulation is another mechanism responsible for the inhi-
bition of neointimal hyperplasia and the low percentage of Ki-67-
positive cells.
Our in vitro research demonstrated that E1 inhibition blocked
VSMCs growth in a dose-dependent manner. This observation
confirmed the in vivo findings that the local delivery of inhibitor or
E1 knockdown decreased the percentage of Ki-67-positive cells in
the neointima. Proteasome inhibitors were reported to have similar
effects on VSMCs proliferation [34]. In addition, E1 knockdown has
been shown to retard cancer growth [9,40] , highlighting the key role
of E1 in cell proliferation. Meanwhile, cell cycle progression was also
suspended by E1 inhibition. The proportion of cells in the S phase
decreased in the E1 inhibition group. We hypothesized that this cell
cycle blockade was a result of the high levels of p53 and p21, which
are cell cycle checkpoint regulators and cyclin-dependent kinase
inhibitors. All of these points may illustrate the pivotal role of the
ubiquitin proteasome system in the proliferation of VSMCs [34,41] .
E1 inhibition, whether chemical or genetic, does not speci fically
target K-48-linked proteins, which are subjected to proteasomal
degradation. K-63-linked protein polyubiquitination and mono-ubiquitination represent different protein functions. In this study,
we could not exclude the possibility that E1 inhibition altered the
function of these proteins.
The local delivery of chemical E1 inhibitors caused more medial
VSMCs apoptosis than observed in the control group. The instant E1
ligase deactivation induced more VSMCs apoptosis. The cell loss
explained the weaker neointima formation. Interestingly, chemical
E1 inhibitors exerted apoptotic effects similar to those observed
with proteasome inhibitors such as MG132 [34] and bortezomib
[37]. Our in vitro study supported the apoptotic effects of chemical
E1 inhibitors in the local carotid tissue. PYR-41 led to VSMC death
in vitro. All of these ubiquitin proteasome pathway chemical in-
hibitors emerged as inducers of apoptosis, implying that radically
shutting down protein degradation might be lethal to living cells.
However, the E1 shRNA lentivirus transfection was unable to
induce a different level of apoptosis in 8 h. The differences might be
due to the delayed action of shRNA against E1 expression. Still the
concise mechanisms accounting for discrepant effects between
chemical and genetical inhibition needs further investigation.
Although E1 inhibition has a similar effect as proteasome inhibi-
tion, the differences in targets suggest that E1 inhibition may over-
come some of the problems that proteasome inhibitors encounter [9].
The assembled application of E1 and proteasome inhibitors
might enhance the pharmaceutical effects of these inhibitors against
many proliferative diseases. For example, bortezomib was firstly
approved for the treatment of relapsed/refractory multiple myeloma
and signi ficantly improved the survival of patients [42,43] . However,
bortezomib has been reported to be associated with peripheral neu-
ropathy in 37 e44% of the multiple myeloma patients, which caused
severe pain and affect the quality of life of the patients [44].
In conclusion, our study extended the discovery of the protea-
some as a therapeutic target in the prevention of vascular reste-
nosis. We demonstrated that E1 ligase, the apex of the ubiquitin
proteasome pathway, is a potential novel therapeutic target for the
prevention of restenosis. E1 may be a modulator of VSMCs prolif-eration and neointima formation. Speci fic chemical inhibitors and
genetically manipulation, targeted to E1 in the injured carotid,
could be effective in limiting VSMCs proliferation and neointima
formation in individuals undergoing catheter-based therapy.
Sources of funding
This work was supported by the National Natural Science
Foundation of China (No. 31301167, 81370211 and 31300803).Z. Qin et al. / Atherosclerosis 247 (2016) 142 e153 152

Disclosures
None.
Conflict of interest
The authors have no con flicts of interest.
Acknowledgements
We are grateful for Professor Gao Xubin for his critical reading.
We also thank Kang Huali, Peng Jin and Chen Qian for excellent
technical assistance.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.atherosclerosis.2016.02.016 .
References
[1]F. Alfonso, R.A. Byrne, F. Rivero, A. Kastrati, Current treatment of in-stent
restenosis, J. Am. Coll. Cardiol. 63 (2014) 2659 e2673 .
[2]A.C. Newby, A.B. Zaltsman, Molecular mechanisms in intimal hyperplasia,
J. Pathol. 190 (2000) 300 e309.
[3]C.G. Santos-Gallego, B. Picatoste, J.J. Badimon, Pathophysiology of acute cor-
onary syndrome, Curr. Atheroscler. Rep. 16 (2014) 401 .
[4]M.S. Willis, W.H. Townley-Tilson, E.Y. Kang, J.W. Homeister, C. Patterson, Sent
to destroy: the ubiquitin proteasome system regulates cell signaling andprotein quality control in cardiovascular development and disease, Circ. Res.
106 (2010) 463 e478.
[5]J. Herrmann, W.D. Edwards, D.R. Holmes Jr., K.L. Shogren, L.O. Lerman,
A. Ciechanover, et al., Increased ubiquitin immunoreactivity in unstable
atherosclerotic plaques associated with acute coronary syndromes, J. Am. Coll.Cardiol. 40 (2002) 1919 e1927 .
[6]J. Herrmann, A. Ciechanover, L.O. Lerman, A. Lerman, The ubiquitin-
proteasome system in cardiovascular diseases-a hypothesis extended, Car-
diovasc. Res. 61 (2004) 11 e21.
[7]B.A. Schulman, J.W. Harper, Ubiquitin-like protein activation by E1 enzymes:
the apex for downstream signalling pathways, Nat. Rev. Mol. Cell Biol. 10
(2009) 319 e331.
[8]A. Hershko, The ubiquitin system for protein degradation and some of its roles
in the control of the cell division cycle, Cell Death Differ. 12 (2005)
1191 e1197 .
[9]G.W. Xu, M. Ali, T.E. Wood, D. Wong, N. Maclean, X. Wang, et al., The
ubiquitin-activating enzyme E1 as a therapeutic target for the treatment ofleukemia and multiple myeloma, Blood 115 (2010) 2251 e2259 .
[10] M. Kulkarni, H.E. Smith, E1 ubiquitin-activating enzyme UBA-1 plays multiple
roles throughout C. elegans development, PLoS Genet. 4 (2008) e1000131 .
[11] H. Matsumae, Y. Yoshida, K. Ono, K. Togi, K. Inoue, Y. Furukawa, et al., CCN1
knockdown suppresses neointimal hyperplasia in a rat artery balloon injurymodel, Arterioscler. Thromb. Vasc. Biol. 28 (2008) 1077 e1083 .
[12] T.F. Chou, S.J. Brown, D. Minond, B.E. Nordin, K. Li, A.C. Jones, et al., Reversible
inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic
protein clearance pathways, Proc. Natl. Acad. Sci. U. S. A. 108 (2011)
4834 e4839 .
[13] T. Shimoyama, S. Hiraoka, M. Takemoto, M. Koshizaka, H. Tokuyama,
T. Tokuyama, et al., CCN3 inhibits neointimal hyperplasia through modulationof smooth muscle cell growth and migration, Arterioscler. Thromb. Vasc. Biol.
30 (2010) 675 e682.
[14] T. Dschietzig, C. Richter, G. Pfannenschmidt, C. Bartsch, M. Laule, G. Baumann,
et al., Dexamethasone inhibits stimulation of pulmonary endothelins by
proin flammatory cytokines: possible involvement of a nuclear factor kappa B
dependent mechanism, Intensive Care Med. 27 (2001) 751 e756.
[15] Y. Yang, J. Kitagaki, R.M. Dai, Y.C. Tsai, K.L. Lorick, R.L. Ludwig, et al., Inhibitors
of ubiquitin-activating enzyme (E1), a new class of potential cancer thera-
peutics, Cancer Res. 67 (2007) 9472 e9481 .
[16] P. Libby, In
flammation in atherosclerosis, Nature 420 (2002) 868 e874.
[17] Y.J. Wang, J.T. Wang, Q.X. Fan, J.G. Geng, Andrographolide inhibits NF-
kappaBeta activation and attenuates neointimal hyperplasia in arterialrestenosis, Cell Res. 17 (2007) 933 e941.
[18] P. Kanellakis, G. Pomilio, C. Walker, A. Husband, J.L. Huang, P. Nestel, et al.,
A novel antioxidant 3,7-dihydroxy-iso flav-3-ene (DHIF) inhibits neointimal
hyperplasia after vessel injury attenuating reactive oxygen species and nu-
clear factor-kappaB signaling, Atherosclerosis 204 (2009) 66 e72.
[19] B. Liu, M. Han, J.K. Wen, Acetylbritannilactone inhibits neointimal hyperplasiaafter balloon injury of rat artery by suppressing nuclear factor-{kappa}B
activation, J. Pharmacol. Exp. Ther. 324 (2008) 292 e298.
[20] C. Di Filippo, R. Marfella, M. D'Amico, Possible dual role of ubiquitin-
proteasome system in the atherosclerotic plaque progression, J. Am. Coll.
Cardiol. 52 (2008) 1350 e1351 author reply 1351 .
[21] S. Redondo, C.G. Santos-Gallego, P. Ganado, M. Garcia, L. Rico, M. Del Rio, et al.,
Acetylsalicylic acid inhibits cell proliferation by involving transforming
growth factor-beta, Circulation 107 (2003) 626 e629.
[22] S. Redondo, E. Ruiz, C.G. Santos-Gallego, E. Padilla, T. Tejerina, Pioglitazone
induces vascular smooth muscle cell apoptosis through a peroxisome
proliferator-activated receptor-gamma, transforming growth factor-beta1,
and a smad2-dependent mechanism, Diabetes 54 (2005) 811 e817.
[23] M. Roque, E.D. Reis, V. Fuster, A. Padurean, J.T. Fallon, M.B. Taubman, et al.,
Inhibition of tissue factor reduces thrombus formation and intimal hyper-
plasia after porcine coronary angioplasty, J. Am. Coll. Cardiol. 36 (2000)
2303 e2310 .
[24] R. Gallo, A. Padurean, T. Jayaraman, S. Marx, M. Roque, S. Adelman, et al.,
Inhibition of intimal thickening after balloon angioplasty in porcine coronaryarteries by targeting regulators of the cell cycle, Circulation 99 (1999)
2164 e2170 .
[25] M. Poon, J.J. Badimon, V. Fuster, Overcoming restenosis with sirolimus: from
alphabet soup to clinical reality, Lancet 359 (2002) 619 e622.
[26] S.H. Hofma, J. Brouwer, M.A. Velders, A.W. van't Hof, P.C. Smits, M. Quere, et
al., Second-generation everolimus-eluting stents versus first-generation
sirolimus-eluting stents in acute myocardial infarction. 1-year results of the
randomized XAMI (XienceV Stent vs. cypher stent in primary PCI for acute
myocardial infarction) trial, J. Am. Coll. Cardiol. 60 (2012) 381 e387.
[27] R. Marfella, M. D'Amico, C. Di Filippo, A. Baldi, M. Siniscalchi, F.C. Sasso, et al.,
Increased activity of the ubiquitin-proteasome system in patients with
symptomatic carotid disease is associated with enhanced in flammation and
may destabilize the atherosclerotic plaque: effects of rosiglitazone treatment,
J. Am. Coll. Cardiol. 47 (2006) 2444 e2455 .
[28] D. Versari, J. Herrmann, M. Gossl, D. Mannheim, K. Sattler, F.B. Meyer, et al.,
Dysregulation of the ubiquitin-proteasome system in human carotid athero-
sclerosis, Arterioscler. Thromb. Vasc. Biol. 26 (2006) 2132 e2139 .
[29]
S. Meiners, A. Ludwig, V. Stangl, K. Stangl, Proteasome inhibitors: poisons and
remedies, Med. Res. Rev. 28 (2008) 309 e327.
[30] J. Herrmann, A. Ciechanover, L.O. Lerman, A. Lerman, The ubiquitin-
proteasome system emicro target for macro intervention? Int. J. Cardiovasc.
Interv. 7 (2005) 5 e13.
[31] M. Igarashi, T. Kato, H. Ohnuma, Y. Morita, T. Kawanami, H. Sasaki, Ubiquitin
expression in atherosclerotic lesions of wistar fatty and wistar lean rats, Ar-tery 21 (1994) 256 e270.
[32] J. Thyberg, K. Blomgren, Effects of proteasome and calpain inhibitors on the
structural reorganization and proliferation of vascular smooth muscle cells in
primary culture, Lab. Investig. 79 (1999) 1077 e1088 .
[33] P.J. Adam, P.L. Weissberg, N.R. Cary, C.M. Shanahan, Polyubiquitin is a new
phenotypic marker of contractile vascular smooth muscle cells, Cardiovasc.
Res. 33 (1997) 416 e421.
[34] S. Meiners, M. Laule, W. Rother, C. Guenther, I. Prauka, P. Muschick, et al.,
Ubiquitin-proteasome pathway as a new target for the prevention of reste-
nosis, Circulation 105 (2002) 483 e489.
[35] E.A. Obeng, L.M. Carlson, D.M. Gutman, W.J. Harrington Jr., K.P. Lee, L.H. Boise,
Proteasome inhibitors induce a terminal unfolded protein response in mul-tiple myeloma cells, Blood 107 (2006) 4907 e4916 .
[36] A.P. Chou, N. Maidment, R. Klintenberg, J.E. Casida, S. Li, A.G. Fitzmaurice, et
al., Ziram causes dopaminergic cell damage by inhibiting E1 ligase of the
proteasome, J. Biol. Chem. 283 (2008) 34696 e34703 .
[37] W.Z. Ying, H.G. Zhang, P.W. Sanders, EGF receptor activity modulates
apoptosis induced by inhibition of the proteasome of vascular smooth muscle
cells, J. Am. Soc. Nephrol. 18 (2007) 131 e142.
[38] Z.G. Liu, H. Hsu, D.V. Goeddel, M. Karin, Dissection of TNF receptor 1 effector
functions: JNK activation is not linked to apoptosis while NF-kappaB activa-
tion prevents cell death, Cell 87 (1996) 565 e576.
[39] W.K. Wu, J.J. Sung, Y.C. Wu, Z.J. Li, L. Yu, C.H. Cho, Bone morphogenetic protein
signalling is required for the anti-mitogenic effect of the proteasome inhibitorMG-132 on colon cancer cells, Br. J. Pharmacol. 154 (2008) 632 e638.
[40] M.R. Schlabach, J. Luo, N.L. Solimini, G. Hu, Q. Xu, M.Z. Li, et al., Cancer pro-
liferation gene discovery through functional genomics, Science 319 (2008)
620e624.
[41] J. Herrmann, L.O. Lerman, A. Lerman, On to the road to degradation: athero-
sclerosis and the proteasome, Cardiovasc. Res. 85 (2010) 291 e302.
[42] R.C. Kane, P.F. Bross, A.T. Farrell, R. Pazdur, Velcade: U.S. FDA approval for the
treatment of multiple myeloma progressing on prior therapy, Oncologist 8
(2003) 508 e513.
[43] R.C. Kane, A.T. Farrell, R. Sridhara, R. Pazdur, United States food and drug
administration approval summary: bortezomib for the treatment of pro-
gressive multiple myeloma after one prior therapy, Clin. Cancer Res. Off. J. Am.
Assoc. Cancer Res. 12 (2006) 2955 e2960
.
[44] G. Cavaletti, A.J. Jakubowiak, Peripheral neuropathy during bortezomib
treatment of multiple myeloma: a review of recent studies, Leuk. Lymphoma
51 (2010) 1178 e1187 .Z. Qin et al. / Atherosclerosis 247 (2016) 142 e153 153