PEP-1-SOD1 fusion proteins block cardiac myofibroblast activation and angiotensin II-induced collagen production
Tan et al. BMC Cardiovascular Disorders
PEP-1-SOD1 fusion proteins block cardiac myofibroblast activation and angiotensin II-induced collagen production
Li-Guo Tan 0 1
Jun-Hui Xiao 0
Dan-Li Yu 2
Lei Zhang 1
Fei Zheng 1
Ling-Yun Guo 1
Jian-Ye Yang 1
Jun-ming Tang 0 1
Shi-You Chen 3
Jia-Ning Wang 0 1
0 Department of Cardiology, Renmin Hospital, Hubei University of Medicine , Shiyan, Hubei 442000 , P. R. China
1 Institute of Clinical Medicine, Renmin Hospital, Hubei University of Medicine , Shiyan, Hubei 442000 , P. R. China
2 Department of Emergency, Renmin Hospital, Hubei University of Medicine , Shiyan, Hubei 442000 , P. R. China
3 Departments of Physiology and Pharmacology, University of Georgia , Athens, GA 30622 , USA
Background: Oxidative stress is closely associated with cardiac fibrosis. However, the effect of copper, zincsuperoxide dismutase (SOD1) as a therapeutic agent is limited due to the insufficient transduction. This study was aimed to investigate the effect of PEP-1-SOD1 fusion protein on angiotensin II (ANG II)-induced collagen metabolism in rat cardiac myofibroblasts (MCFs). Methods: MCFs were pretreated with SOD1 or PEP-1-SOD1 fusion protein for 2 h followed by incubation with ANG II for 24 h. Cell proliferation was measured by Cell Counting Kit-8. Superoxide anion productions were detected by both fluorescent microscopy and Flow Cytometry. MMP-1 and TIMP-1 were determined by ELISA. Intracellular MDA content and SOD activity were examined by commercial assay kits. Protein expression was analyzed by western blotting. Results: PEP-1-SOD1 fusion protein efficiently transduced into MCF, scavenged intracellular O2−, decreased intracellular MDA content, increased SOD activity, suppressed ANG II-induced proliferation, reduced expression of TGF-β1, α-SMA, collagen type I and III, restored MMP-1 secretion, and attenuated TIMP-1 secretion. Conclusion: PEP-1-SOD1 suppressed MCF proliferation and differentiation and reduced production of collagen type I and III. Therefore, PEP-1-SOD1 fusion protein may be a potential novel therapeutic agent for cardiac fibrosis.
Cell-penetrating peptide; Copper; Zinc-superoxide dismutase; Cardiac myofibroblasts; Collagen
Cardiac fibrosis, characterized by abnormal proliferation
of cardiac myofibroblasts (MCF) and excessive deposition
of extracellular matrix (ECM), is a leading cause of
progressive deterioration of cardiac function and structure in
a number of chronic heart diseases including
hypertension, coronary heart diseases, and cardiomyopathies.
However, no effective therapeutics is currently available. MCFs
are the most prevalent cell type in the heart that plays
pivotal roles in cardiac fibrosis. Under a number of stimuli
such as cytokines (e.g., TGF-β1, IL-1, or endothelin-1),
hormones (e.g., angiotensin II (Ang II) or noradrenaline),
and mechanical stretch, cardiac fibroblasts can be
converted into MCFs exhibiting increased migratory,
proliferative, and secretion properties. MCFs are the major source
of ECM in the heart with cardiac fibrosis .
A growing number of studies have suggested that
oxidative stress induced by reactive oxygen species (ROS).
(ROS) is associated with cardiac fibrosis. ROS is
significantly elevated in hearts of patients and animal models
with cardiac fibrosis [2, 3]. Increased ROS triggers a
series of cellular events such as differentiation,
proliferation, secretion and gene expression. The elevated ROS
also increases myofibroblast proliferation and ECM
synthesis [4, 5]. Moreover, ROS reduces ECM degradation by
regulating the level of matrix metalloproteinases (MMPs)
and tissue inhibitor of metalloproteinases (TIMPs) .
Superoxide dismutases (SOD) are important antioxidant
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enzymes that accept an electron from superoxide anion
(O2−) and H2O to generate hydrogen peroxide (H2O2).
Cytoplasm-located SOD1 is the major isoform of SODs in
mammalian, and angiotensin II (ANG II)-induced ROS
has been found to down-regulate SOD1 activity in rat
MCF . These studies suggest that SOD1 is a potential
target for preventing cardiac fibrosis. However, exogenous
SOD1 cannot be delivered into cells to block oxidative
stress because of the lack of specific membrane channel
or receptor for SOD1.
PEP-1 ((KETWWETWWTEWSQPKKKRKV), a novel
peptide carrier designed by Morris group , consists of
three domains: a hydrophobic tryptophan rich motif (KET
WWETWWTEW), a spacer (SQP), and a hydrophilic
lysine-rich domain (KKKRKV). A number of proteins fused
with PEP-1 have been efficiently delivered into cells or
tissues. Our previous studies have shown that PEP-1-SOD1
fusion protein can be transduced into myocardial and
cerebral tissues to protect against oxidative stress induced by
ischemia-reperfusion injury [9–11]. In the present study,
we found that PEP-1-SOD1 fusion protein, when
transduced into rat cardiac myofibroblast, mediates collagen
metabolism by blocking ANG II-induced ROS production.
Since collagen deposition is a major factor causing fibrosis,
PEP-1-SOD1 fusion protein may be a potential therapeutic
agent for treating cardiac fibrosis.
Expression and purification of SOD1 and PEP-1-SOD1
SOD1 and PEP-1-SOD1 infusion protein were expressed
and purified as previously described [9, 10]. Briefly, two
prokaryotic expression plasmids, pET15b-SOD1 and
pET15b-PEP-1-SOD1, were constructed with TA-cloning
method. The two recombinant plasmids were respectively
transformed into E. Coli BL21 (DE3) bacteria (Novagen,
USA). Bacteria that have been successfully transformed
were grown in 100 ml LB medium containing 100 ug/ml
ampicillin at 37 °C to an OD600 value of 0.5-1.0 and
induced by 0.83 mM isopropyl-β-D-thiogalactoside (IPTG)
(Promega, USA) at 25 °C for 12 h. The bacteria were then
harvested and lysed by sonication at 4 °C in a binding
buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris–HCl,
pH 7.9) for 30 min. The supernatant after centrifugation
was loaded onto a Ni2+-nitrilotriacetic acid sepharose
affinity column (Qiagen, USA) under native conditions.
The column was washed with 10 volumes of the binding
buffer and 6 volumes of wash buffer (60 mM imidazole,
500 mM NaCl, 20 mM Tris–HCl, pH 7.9), and eluted by
an eluting buffer (1 M imidazole, 500 mM NaCl, 20 mM
Tris–HCl, pH 7.9). The fusion proteins were collected at
the peak of OD280. The salts of fusion proteins were
removed using a PD-10 column, and the fusion proteins
were identified by SDS-PAGE and western blot analysis.
The protein concentration was assayed with BCA assay
kit (Beyotime Institute of Biotechnology, China).
Isolation and culture of primary rat cardiac MCFs
All animal procedures were carried out in accordance with
Regulations of Good Laboratory Practice for non-clinical
laboratory studies of drugs issued by the State Food and
Drug Administration of China, and the experimental
protocol was approved by the Institutional Animal Care and Use
Committee of Hubei University of Medicine. Cardiac
ventricular myofibroblasts were obtained from male adult
Sprague–Dawley rats. Briefly, adult rats were sacrificed by
cervical dislocation under ether anesthesia, and the hearts
were excised, rinsed with growth media (Dulbecco’s
modified essential media (DMEM) supplemented with 10 % fetal
bovine serum, 100 U/ml penicillin G and 100 μg/ml
streptomycin) and trimmed of connective tissue and fat.
The atria were removed, and the ventricle was cut into
small pieces with about 1 mm3 size. The ventricle pieces
were transplant into 25 cm2 culture flasks, allowed to dry
briefly and then flooded with 5 ml growth media. Culture
flasks were then incubated at 37 °C with 5 % CO2 balanced
air for 1 week. Cell outgrowths from explanted tissues were
digested and passaged when grew to 70 % confluent. The
growth media with suspended cells were discarded after
90 min, the fresh growth media was added, and the adhered
cells were cardiac myofibroblasts. Cells from passages 1–6
were used in this study. Cells from passage 2 were detected
by immunofluorescent staining with von Willebrand factor
(VWF), desmin, and vimentin antibodies for identification
of endothelial cells, muscle cells and fibroblasts,
respectively, as previously described .
Transduction of PEP-1-SOD1 protein into MCF
MCF were grown to confluence on 6-well cell culture
plates, and then pretreated with PEP-1-SOD1 fusion
proteins or SOD1 proteins at different dosages (0–4 μmol/L)
for 0–24 h. Cells were then washed with
phosphatebuffered saline (PBS), harvested and lysed for western blot
or enzyme activity assay. The SOD activity was measured
with a SOD Activity Assay Kit by following the
manufacture’s protocols (JianCheng Bioengineering Institute,
China). To further confirm transduction of PEP-1-SOD1
fusion proteins, MCF were cultured to confluence in
24well plates, pretreated with 2 μmol/L PEP-1-SOD1 fusion
proteins or SOD1 proteins for 2 h, washed twice with 1 x
PBS, and immunostained with anti-His-tag antibody.
Measurement of malondialdehyde (MDA) content,
superoxide dismutase (SOD) activity, and superoxide
MDA reflects the peroxide production of cell membrane
lipids caused by oxidative stress. MDA content and SOD
activity were used as indicators of oxidative damage. The
MDA content and SOD activity in MCF were
determined with a MDA Assay Kit and SOD Activity
Assay Kit (JianCheng Bioengineering Institute, China),
respectively. The levels of O2− in cells were detected
with oxidation-sensitive fluorescent probe
dihydroethidium (DHE) (Beyotime Institute of Biotechnology,
China) and measured with fluorescent microscopy
and flow cytometry as described previously .
MCF proliferation assay
To investigate the effect of PEP-1-SOD1 on MCF
proliferation induced by Ang II, MCF were cultured in
96well cell culture plates, and divided into six groups:
vehicle-treated (CTL), SOD1 with vehicle, PEP-1-SOD1
with vehicle, Ang II-treated group, SOD1 with Ang II,
and PEP-1-SOD1 with Ang II. The cells were starved in
serum-free DMEM for 24 h to mimic the ischemic
condition (depletion of nutrients) followed by pretreatment with
or without 2 μmol/L of PEP-1-SOD1 or SOD1 for 2 h. The
media were replaced with DMEM containing 10 % fetal
bovine serum to mimic the reperfusion condition. The cells
were then incubated with vehicle or 100 nmol/L Ang II for
24 h. MCF proliferation was assayed with Cell Counting
Kit-8 (CCK-8) by following the manufacture’s protocols
(Beyotime Institute of Biotechnology, China).
Fluorescent immunocytochemistry was performed on
24-well cell culture plates as previously described .
Briefly, after treatment as described above, cells were
washed twice with 1 x PBS, fixed with 1 %
paraformaldehyde for 15 min at room temperature, and incubated with
rabbit anti-His-tag (diluted 1:100), goat anti-VWF (diluted
1:100), mouse anti-desmin (diluted 1:100), or mouse
antivimentin antibody (diluted 1:100) (Santa Cruz
Biotechnology, USA) at 4 °C overnight. Cells were then incubated
with FITC-conjugated second antibodies (diluted 1:250)
(Zhongshan Biotechnology, China) at 25 °C for 2 h. Nuclei
were stained with DAPI (Sigma, USA). The results were
observed under a fluorescent microscope (Nikon, Japan).
MCF were harvested and lysed in a lysis buffer. Western
blots were performed as previously described . Equal
amount of proteins from each sample were subjected to
SDS-PAGE, and then transferred to nitrocellulose
membranes. The membranes were blocked with 5 % BSA
and incubated at 4 °C overnight with specific primary
antibodies: rabbit anti-PCNA (diluted 1:100), rabbit
His-tag (diluted 1:100), goat anti-collagen I (diluted
1:200), mouse anti-α-SMA (diluted 1:100), rabbit
antiTGF-β1 (diluted 1:200), goat anti-gp91phox (diluted
1:200) (Santa Cruz Biotechnology, USA), or mouse
anti-collagen III (diluted 1:200) (Sigma, USA). The
horseradish peroxidase-conjugated second antibodies
(diluted 1:5000) (Zhongshan Biotechnology, China)
were incubated for 2 h at room temperature. Proteins
were detected by ECL detection system.
MMP-1 and TIMP-1 expression assay
The content of matrix metalloproteinase-1 (MMP-1) or
tissue inhibitor of metalloproteinase-1 (TIMP-1) in
MCFconditioned media was examined using rat MMP-1 and
TIMP-1 ELISA kits (BioSwamp, China).
Transduction of PEP-1-SOD1 fusion protein into cardiac
SOD1 and PEP-1-SOD1 fusion proteins were successfully
expressed and purified as shown in Additional file 1: Figure
S1. MCF were isolated and cultured from rat heart. MCF
were identified by positive-staining of vimentin and
negative staining of endothelial cell and muscle markers vWF
and desmin, respectively (Additional file 1: Figure S2).
To test the transduction efficiency of PEP-1-SOD1
fusion proteins into cardiac MCF, we used anti-His tag
antibody to detect its protein level. As shown in Fig. 1a
and c, the fusion protein was observed in MCF 15 min
after incubation with 2 μM of PEP-1-SOD1, and the
protein level gradually increased when the incubation time
increased. However, incubation with SOD1 protein did
not result in accumulation of SOD1 in the cells (data
not shown). Moreover, the transduction of PEP-1-SOD1
fusion protein, but not SOD1, occurred in a
dosedependent manner (Fig. 1b and d). To further confirm
the transduction, PEP-1-SOD1 or SOD1 was conjugated
to FITC fluorescein, and the transduction was observed
by immunofluorescent microscopy after incubation with
the cells. As shown in Fig. 1e, strong green fluorescent
signals were observed in cells pretreated with
PEP-1SOD1, but not with SOD1 protein, demonstrating that
PEP-1-SOD1, but not SOD1, can enter cardiac
myofibroblasts. Importantly, PEP-1-SOD1 is functionally
active. As shown in Fig. 1f, SOD1 activity of PEP-1-SOD1
was 2.5-fold higher compared to the untransduced cells,
and this high enzyme activity can last for 12–24 h.
PEP-1-SOD1 fusion protein decreased O2− levels and MDA
content while enhanced SOD activity
To determine if PEP-1-SOD1 transduction blocks ROS
in MCF, we used Ang II to induce ROS production. As
shown in Fig. 2a-d, Ang II increased the production of
Fig. 1 Transduction and enzyme activities of PEP-1-SOD1 fusion protein in MCF. a-d: Time and dose-dependent transduction of PEP-1-SOD1.
a, b: 2 μM of PEP-1-SOD1 was incubated with cardiac myofibroblast for 0–2 h as indicated, and western blot was performed using anti-His tag
antibody (a). b: Semi-quantitative assay was done in figure A. #P < 0.01 vs. untreated cells (CTL) group; @P > 0.05 vs. 0.25 h group; &P < 0.01 vs.
0.5 h group; *P < 0.01 vs. 1.0 h group. (n = 5). c: 0-2 μM of PEP-1-SOD1 was incubated with cells for 2 h. Western blots were performed the same
as in A. d: Semi-quantitative assay was done in figure C. #P < 0.01 vs. untreated cells (CTL) group; $P < 0.05 vs. 0.25 h group; &P < 0.01 vs. 0.5 h
group; *P < 0.01 vs. 1.0 h group. (n = 5). e: FITC-conjugated PEP-1-SOD1 transduction was detected by immunofluorescent microscopy. f:
PEP-1SOD1 exhibited SOD enzymatic activity in MCF. #P < 0.01 vs. untreated cells (CTL) group; @P > 0.05 vs. 2 h group; $P < 0.05 vs. 4 h group; &P < 0.05
vs. 6 h group; *P < 0.01 vs. 12 h group. (n = 5)
O2− and MDA content while decreasing SOD activity in
MCF. PEP-1-SOD1 fusion protein pretreatment,
however, reduced Ang II-induced O2− production and MDA
content. PEP-1-SOD1 also enhanced SOD activity in
cells. SOD1 pretreatment appeared to have no significant
effects on the levels of O2−, MDA content, or SOD
activity mediated by Ang II. These results suggest that
PEP1-SOD1 fusion protein transduction protects MCF from
Ang II-induced ROS through the enhanced SOD activity.
SOD1 transduction is clearly unable to increase the SOD1
activity in MCF. PEP-1-SOD1 specifically increased SOD1
activity, but did not affect NADPH oxidases because
PEP1-SOD1 did not alter the expression of gp91phox, a key
subunit of NAPDH oxidases although Ang II also increased
gp91phox expression (Fig. 2e-f ). As expected, SOD1
transduction had no effect on Ang II-induced gp91phox
expression (Fig. 2e-f ).
PEP-1-SOD1 fusion protein attenuated Ang II-induced
Ang II is a potent factor inducing MCF proliferation.
We confirmed that Ang II promotes MCF proliferation
(Fig. 3a). Pretreatment of SOD1 slightly, but
transduction of PEP-1-SOD1 fusion proteins dramatically
Fig. 2 Effect of PEP-1-SOD1 on ANG II-induced ROS in MCF. a-b: O2− levels were detected by fluorescent microscopy (a) and quantified by Flow
Cytometry (b), respectively. Red color was indicated as the changes of O2− levels in cells detected with oxidation-sensitive fluorescent
probe dihydroethidium (DHE), Blue color was indicated as cell nucleus by DAPI staining. c: PEP-1-SOD1, but not SOD1, blocked Ang
II-induced increase of MDA content. d: PEP-1-SOD1, but not SOD1, restored Ang II-blocked SOD activity. *P < 0.01 vs. Vehicle-treated cells
(−), #P < 0.01 vs. Ang II-treated cells, &P < 0.01 vs. SOD1-treated cells (n = 5). e-f: gp91phox expression was analyzed by western blot (e) and
normalized to α-Tubulin (f). *P < 0.01 vs. vehicle-treated cells (−) (n = 5)
attenuated the Ang II-induced cell proliferation.
Proliferating cell nuclear antigen (PCNA) is a biomarker of cell
proliferation. To further determine the effect of
PEP-1SOD1 fusion protein on MCF proliferation, we detected
the PCNA expression. Ang II up-regulated PCNA
expression (Fig. 3b-c). SOD1 transduction, slightly inhibited, but
Fig. 3 Effect of PEP-1-SOD1 on Ang II-induced MCF proliferation. a, MCF proliferation was assayed with CCK-8. b-c, PCNA expression was measured by
western blot (b) and quantified by normalizing to α-Tubulin (c). *P < 0.01 vs. vehicle-treated cells (−), #P < 0.01 vs. Ang II-treated cells, &P < 0.05 vs.
SOD1-treated cells (n = 5)
PEP-1-SOD1 fusion proteins dramatically blocked
PCNA expression (Fig. 3b-c). These results indicate
that Ang II induces MCF proliferation through
increasing ROS production, which can be diminished
by the transduction of PEP-1-SOD1.
Ang-II-induced synthesis of both Col I and Col III
(Fig. 5a-c). The effect of PEP-1-SOD1 was much greater
compared to that of SOD1. These results demonstrated
that delivery of PEP-1-SOD1 can effectively block the
excessive production of collagen in activated MCF.
PEP-1-SOD1 fusion protein attenuated Ang-II-induced
Excessive MCF activation accompanied by excessive
collagen production is the main mechanism underlying the
onset of cardiac fibrosis. Ang II markedly stimulated
expression of smooth muscle α-actin (α-SMA) and
TGFβ1 in MCF (Fig. 4a-c), indicating a MCF activation. It is
known that Ang II induces fibrosis through TGF-β
signaling pathway. SOD1 slightly reduced Ang II-induced
α-SMA and TGF-β expression. PEP-1-SOD1 infusion
protein, however, dramatically blocked Ang II induction
of these two genes (Fig. 4). Importantly, PEP-1-SOD1
had a much greater effect as compared to SOD1 (Fig. 4).
PEP-1-SOD1 blocked Ang-II-induced production of type I
and III collagen
Activation of MCF leads to production of collagen. To
determine if PEP-1-SOD1 fusion plays a role in collagen
synthesis, we used Ang II to treat MCF and detected type
I (Col I) and III collagen (Col III) protein expression,
respectively. As shown in Fig. 5, both the Col I and Col III
production were significantly increased over 2.5 fold by
Ang II induction. SOD1 pretreatment of MCF marginally,
while PEP-1-SOD1 pretreatment dramatically, suppressed
PEP-1-SOD1 increased Ang-II-mediated blockade of MMP-1
secretion and increase of TIMP-1 production
MMP-1 is a crucial enzyme degrading ECM, and
TIMP1 is a key inhibitor of MMPs. The production of MMP-1
and TIMP-1 is associated with cardiac fibrosis. We
found that Ang II reduced the MMP-1 secretion and
stimulated production of TIMP-1 in MCF (Fig. 6a-b),
consistent with the increased production of Col I and
Col III. Pretreatment of SOD1 only marginally reversed
Ang II-mediated increase of MMP-1 and reduction of
TIMP-1 (Fig. 6a-b). Pretreatment of PEP-1-SOD1,
however, almost completely restored Ang II-blocked MMP-1
secretion, and dramatically inhibited Ang II-mediated
increase of TIMP-1 secretion (Fig. 6a-b).
Antioxidants have been shown to suppress cardiac
fibrosis and improve cardiac function in animal models .
Therefore, antioxidant enzymes have been considered as
promising therapeutic agents to prevent cardiac fibrosis.
SOD1 is a key superoxide dismutase localized in the
cytoplasm in mammalian tissues , and thus a potential
agent for cardiac fibrosis therapy . However, exogenous
SOD1 is unable to penetrate into cells or organs to block
Fig. 4 Effect of PEP-1-SOD1 on α-SMA and TGF-β1 protein expression. a α-SMA and TGF-β1 protein expression was detected by western blot.
b Quantification of α-SMA expression by normalizing to α-Tubulin. c Quantification of TGF-β1 expression by normalizing to α-Tubulin. *P < 0.01 vs.
vehicle-treated cells, #P < 0.01 vs. Ang II-treated cells. &P < 0.01 vs. SOD1-transduced cells n = 5
Fig. 5 Effect of PEP-1-SOD1 on type I (Col I) and III (Col III) collagen metabolism. a Col I and Col III production was analyzed with
western blot. b Quantification of Col I by normalizing to α-Tubulin. c Quantification of Col I by normalizing to α-Tubulin. *P < 0.01 vs.
vehicle-treated cells, #P < 0.01 vs. Ang II-treated cells, &P < 0.01 vs. SOD1 transduced cells n = 5
Fig. 6 Effect of PEP-1-SOD1 on matrix metalloproteinase-1 (MMP-1) and tissue inhibitors of metalloproteinase 1 (TIMP-1) secretion. The levels of
MMP-1 (a) and TIMP-1 (b) in culture media were examined by ELISA. *P < 0.01 vs. vehicle-treated cells, #P < 0.01 vs. Ang II-treated cells, &P < 0.01
vs. SOD1- transduced cells n = 5
oxidative stress due to the lack of permeability. Although a
number of approaches have been studied for delivering
proteins into cells including lipid-, polycationic-, nanoparticle-,
and peptide-based methods, these technologies is less
effective in pre-clinical or clinical applications because of
the poor stability of the complexes formed, the rapid
degradation of cargos, or insufficient ability to reach its
target. Cell-penetrating peptides are powerful tools used to
improve cellular uptake of therapeutic molecules and show
bright promise in the clinic application . Our previous
studies have demonstrated that PEP-1-SOD1 can be
efficiently transduced into cardiomyocytes or neuron cells to
prevent these cells from oxidative injury [9, 10, 13]. Our
current study demonstrates that PEP-1-SOD1 can also be
delivered into MCF and protect MCF from Ang II-induced
transdifferentiation. Therefore, PEP-1-SOD1 may be used
as a potential therapeutics to treat cardiac fibrosis.
MCF proliferation and activation are the main factors
contributing to cardiac fibrosis. Ang II appears to
promote both the proliferation and activation because Ang
II stimulates MCF growth, PCNA expression, and
myofibroblast marker α-SMA expression [19–21]. Although
SOD1 transduction only marginally attenuates Ang II
function in MCF proliferation and activation,
PEP-1SOD1 exhibits a dramatic effect in blocking Ang II
activity. In addition, PEP-1-SOD1 blocks collagen production
that is induced by Ang II, suggesting that PEP-1-mediated
SOD1 delivery are multi-functional in MCF, i.e., regulating
MCF proliferation, differentiation and ECM production.
Mechanistically, PEP-1-SOD1 appears to regulate collagen
production by modulating MMP1 and TIMP-1 expression
because PEP-1-SOD1 reverses Ang II-induced
downregulation of MMP1 and upregulation of TIMP-1. Therefore,
PEP-1-SOD1 is likely to block TIMP-1 expression, causing
increased MMP1 expression. The increased MMP1 then
degrades excessive collagen production, leading to the
resolution of fibrosis.
Interestingly, although we are unable to detect the
SOD1 transduction or activity in MCF, pretreatment
with SOD1 exhibits a slight but significant effect on Ang
II-induced expression of α-SMA, TGF-β, Col I, Col II,
MMP1, and TIMP-1. This is likely due to SOD1 activity
in improving ROS status in extracellular environment
[22, 23]. It is also possible that SOD1 may slightly affect
Ang II interaction with its receptor although the
interference does not produce a major impact to Ang II activity.
This potential mechanism is obviously a interesting
subject for future study.
Our study show that PEP-1-SOD1 fusion protein can be
successfully delivered into MCF to scavenge ROS
production and block collagen production, suggesting that
PEP-1-SOD1 may be a promising therapeutic agent for
treating ROS-mediated cardiac fibrosis.
Additional file 1: Figure S1. Expression and purification of SOD1 and
PEP-1-SOD1 fusion proteins. Protein extracts and the purified fusion proteins
were resolved in 12% SDS-PAGE (A) and subjected to Western blot analysis
with rabbit anti-His-tag antibody (B). Lane 1: pre-stained protein marker; lane
2: total protein extracts for SOD1, lane 3: purified SOD1 proteins; lane 4: total
protein extracts for PEP-1-SOD1; lane 5: purified PEP-1-SOD1 fusion proteins.
Figure S2. Identification of cardiac myofibroblasts. Rat cardiac myofibroblasts
were identified by positive staining with anti-vimentin and negative staining
with anti-desmin and anti-vWF antibodies. (DOCX 176 kb)
PEP-1: Cell-penetrating peptide 1; SOD1: Copper, zinc-superoxide dismutase;
ANG II: Angiotensin II; MCF: Cardiac myofibrolast ; MMP-1: Matrix
metalloproteinases 1; TIMP-1: Tissue inhibitor of metalloproteinases 1;
TGFβ1: Transforming growth factor-β1; α-SMA: Smooth muscle α-actin;
ECM: Extracellular matrix; ROS: Reactive oxygen species; IPTG:
isopropyl-β-Dthiogalactoside; DMEM: Dulbecco’s modified essential media; VWF: Von
Willebrand factor; MDA: Malondialdehyde; O2−: Superoxide anion;
PCNA: Proliferating cell nuclear antigen; gp91phox: Cytochrome b-245, beta
polypeptide; Col I: Collagen type I; Col III: Collagen type III.
LGT has made substantial contributions to conception and design of the
study, and carried out the cell experimental studies participated in MMP-1
and TIMP-1 ELISA assay and drafted the manuscript. JHX carried out MDA
and SOD1 measurement, and has made substantial contributions to
conception and design of the study. DLY carried out MMT assay. LZ participated
in cell culture and PEP1-SOD1 protein purification. FZ carried out Western
Blotting assay. YJY participated in immunofluorescence assay. LYG carried
out PEP1-SOD1 protein purification. JMT participated in drafting and
interpretation. JNW participated in drafting and interpretation and has
made substantial contributions to conception and design of the study of
data. SYC has made substantial contributions to conception and design of
the study of data and critically revised the manuscript. All authors read
and approved the final manuscript.
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