Endotoxin Induces Fibrosis in Vascular Endothelial Cells
through a Mechanism Dependent on Transient Receptor
Protein Melastatin 7 Activity
Cesar Echeverrı́a1, Ignacio Montorfano1, Tamara Hermosilla3,4, Ricardo Armisén4,5, Luis A. Velásquez6,7,
Claudio Cabello-Verrugio1, Diego Varela3,4, Felipe Simon1,2*
1 Departamento de Ciencias Biologicas, Facultad de Ciencias Biologicas and Facultad de Medicina, Universidad Andres Bello, Santiago, Chile, 2 Millennium Institute on
Immunology and Immunotherapy, Santiago, Chile, 3 Centro de Estudios Moleculares de la Celula, Facultad de Medicina, Universidad de Chile, Santiago, Chile, 4 Instituto
de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile, 5 Centro de Investigacion y Tratamiento del Cancer, Facultad de Medicina,
Universidad de Chile, Santiago, Chile, 6 Center for Integrative Medicine and Innovative Science (CIMIS), Facultad de Medicina, Universidad Andres Bello, Santiago, Chile,
7 Centro para el Desarrollo de la Nanociencia y Nanotecnologı́a, Universidad de Santiago de Chile, Santiago, Chile
Abstract
The pathogenesis of systemic inflammatory diseases, including endotoxemia-derived sepsis syndrome, is characterized by
endothelial dysfunction. It has been demonstrated that the endotoxin lipopolysaccharide (LPS) induces the conversion of
endothelial cells (ECs) into activated fibroblasts through endothelial-to-mesenchymal transition mechanism. Fibrogenesis is
highly dependent on intracellular Ca2+ concentration increases through the participation of calcium channels. However, the
specific molecular identity of the calcium channel that mediates the Ca2+ influx during endotoxin-induced endothelial
fibrosis is still unknown. Transient receptor potential melastatin 7 (TRPM7) is a calcium channel that is expressed in many
cell types, including ECs. TRPM7 is involved in a number of crucial processes such as the conversion of fibroblasts into
activated fibroblasts, or myofibroblasts, being responsible for the development of several characteristics of them. However,
the role of the TRPM7 ion channel in endotoxin-induced endothelial fibrosis is unknown. Thus, our aim was to study
whether the TRPM7 calcium channel participates in endotoxin-induced endothelial fibrosis. Using primary cultures of ECs,
we demonstrated that TRPM7 is a crucial protein involved in endotoxin-induced endothelial fibrosis. Suppression of TRPM7
expression protected ECs from the fibrogenic process stimulated by endotoxin. Downregulation of TRPM7 prevented the
endotoxin-induced endothelial markers decrease and fibrotic genes increase in ECs. In addition, TRPM7 downregulation
abolished the endotoxin-induced increase in ECM proteins in ECs. Furthermore, we showed that intracellular Ca2+ levels
were greatly increased upon LPS challenge in a mechanism dependent on TRPM7 expression. These results demonstrate
that TRPM7 is a key protein involved in the mechanism underlying endotoxin-induced endothelial fibrosis.
Citation: Echeverrı́a C, Montorfano I, Hermosilla T, Armisén R, Velásquez LA, et al. (2014) Endotoxin Induces Fibrosis in Vascular Endothelial Cells through a
Mechanism Dependent on Transient Receptor Protein Melastatin 7 Activity. PLoS ONE 9(4): e94146. doi:10.1371/journal.pone.0094146
Editor: Liwu Li, Virginia Polytechnic Institute and State University, United States of America
Received November 12, 2013; Accepted March 14, 2014; Published April 7, 2014
Copyright: ß 2014 Echeverrı́a et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by research grants from Fondo Nacional de Desarrollo Cientı́fico y Tecnológico - Fondecyt 1121078 (FS), 1120240 (DV),
1120286 (RA), 1120712 (LAV), and 1120380 (CCV). Millennium Institute on Immunology and Immunotherapy P09-016-F. Centro para el Desarrollo de la
Nanociencia y Nanotecnologia (CEDENNA) FB0807 (LAV). Association-Francaise Contre Les Myopathies AFM 16670 (CCV). UNAB-DI-281-13/R (CCV). UNAB-DI-6712/I (CE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: fsimon@unab.cl
(ECs) located in the internal endothelial monolayer of blood
vessels, inducing detrimental effects on endothelium function [9–
11].
It is well accepted that the endothelial dysfunction is an
important factor in the pathogenesis of endotoxemia-derived sepsis
syndrome as well as other inflammatory diseases [9,12]. We
reported that LPS induces at least two main effects in vascular
ECs. First, endotoxin promotes endothelial cell death [13].
Second, LPS is able to induce the conversion of ECs into activated
fibroblasts, also known as myofibroblasts [14]. Endotoxin-induced
endothelial fibrosis is mediated through a process known as
endothelial-to-mesenchymal transition (EndMT) in a similar way
that observed using the best-studied EndMT inducers, tumor
growth factor- beta 1 and 2 (TGF-b1 and TGF-b2) [15,16].
Endotoxin-induced endothelial fibrosis is characterized by downregulation of the endothelial markers CD31/PECAM and VE-
Introduction
Sepsis syndrome is the most prevalent cause of mortality in
critically ill patients admitted to intensive care units [1]. The
pathogenesis of sepsis syndrome develops through an overactivation of the immune system, which involves activation of immune
cells, secretion of pro-inflammatory cytokines and generation of
reactive oxygen species (ROS) [1,2]. Despite numerous basic and
clinical studies addressing sepsis syndrome, current therapies for
treating it and its sequelae are unsatisfactory, exhibiting high
morbimortality rates [3,4].
Endotoxemia-derived sepsis syndrome is a important cause of
sepsis syndrome. It is frequently characterized by deposition of
large amounts of the Gram-negative bacterial endotoxin, lipopolysaccharide (LPS) [5–8]. During endotoxemia, the endotoxin
circulating in the bloodstream interacts with the endothelial cells
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TRPM7-Mediated Endotoxin-Induced EC Fibrosis
cadherin, whereas the fibroblast-specific genes a-smooth muscle
actin (a-SMA) and fibroblast-specific protein 1 (FSP-1) are
upregulated. Furthermore, the levels of proteins that form the
extracellular matrix (ECM), such as fibronectin (FN) and type III
collagen (Col III), are greatly increased [14].
It has been reported that Ca2+ influx is absolutely required for
fibrosis development. The generation of myofibroblasts from
cultured rat cardiac fibroblasts is inhibited by chelating external
Ca2+ [17,18], while decreasing the intracellular Ca2+ concentration improves liver and muscle fibrosis [19,20]. Furthermore,
increases in intracellular oxidative stress and pro-inflammatory
cytokine synthesis and secretion, both of which are major features
of fibrosis, are attenuated by inhibition of the Ca2+ influx
[17,18,21]. In addition, increased cell migration, a distinctive
attribute of activated fibroblasts, is also dependent on Ca2+ entry
[22,23]. Therefore, Ca2+ entry is an essential step in the
development of the characteristics of fibrosis.
Determining the molecular entity that mediates the Ca2+ influx
during fibrogenesis is an issue of great importance due to its
therapeutic implications. It has been reported that L-type calcium
channels modulate perivascular fibrosis in the kidney [24].
Similarly, it has been reported that blocking of T-type and Ltype calcium channels is effective in decreasing tubulointerstitial
fibrosis [25]. Furthermore, cardiac fibrosis was found to be
decreased when calcium channel blockers were used in addition to
complementary treatments [26,27]. Accordingly, inhibition of
calcium channels is effective in attenuating liver fibrogenesis [28].
These data suggest that calcium channels are required for Ca2+
influx to promote fibrosis. However, the molecular identity of the
calcium channel that mediates the Ca2+ influx during endotoxininduced endothelial fibrosis remains unknown.
It has been shown that transient receptor potential melastatin 7
(TRPM7) is the main Ca2+-permeable channel involved in the
TGF-b1-induced activation of human atrial fibroblasts into
myofibroblasts, promoting atrial fibrillation [29]. In addition,
TRPM7 is involved in the migration of fibroblasts, which is a
typical feature of myofibroblasts [30,31]. The TRPM7 ion channel
is permeable to the divalent cations Ca2+ and Mg2+ but is
impermeable to monovalent cations. This channel is ubiquitously
expressed in a broad range of cell types, including ECs [32–36].
These findings suggest that TRPM7 could be involved in the
conversion of endothelial cells into myofibroblasts upon endotoxin
challenge. However, the role of the TRPM7 ion channel in
endotoxin-induced endothelial fibrosis is currently not known.
Therefore, the aim of this study was to investigate whether the
TRPM7 protein is involved in endotoxin-induced endothelial
fibrosis.
Our data demonstrated that the TRPM7 ion channel plays a
crucial role in endotoxin-induced endothelial fibrosis. Suppression
of TRPM7 expression protected ECs from the endotoxin-induced
fibrogenic process. Additionally, we demonstrated that intracellular Ca2+ levels were increased in ECs exposed to endotoxin. This
endotoxin-induced Ca2+ increase was abolished by inhibition of
TRPM7 expression, suggesting that the TRPM7-mediated Ca2+
increase is involved in the mechanism underlying endotoxininduced endothelial fibrosis.
The results provided here contribute to our understanding of
the molecular basis of endotoxin-induced endothelial fibrosis,
revealing a novel target that could be useful in drug development
for treating endothelial dysfunction during endotoxemia and other
inflammatory diseases.
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Materials and Methods
Details of all procedures are provided in Methods S1.
Ethics Statement
The investigation conforms with the principles outlined in the
Declaration of Helsinki. The Commission of Bioethics and
Biosafety of Universidad Andres Bello also approved all experimental protocols. Human umbilical cord were obtained from
patients after written patient’s informed consent. The individual in
this manuscript has given written informed consent (as outlined in
PLOS consent form) to publish these case details.
Primary cell culture
Human umbilical vein endothelial cells (HUVEC) were isolated
by collagenase (0.25 mg/mL) digestion from freshly obtained
umbilical cord veins from normal pregnancies, after patient’s
informed consent. Cells were grown in gelatin-coated dishes at
37uC in a 5%:95% CO2:air atmosphere in medium 199 (Sigma,
MO), containing 100 mg/mL endothelial cell growth supplement
(ECGS) (Sigma), 100 mg/mL heparin, 5 mmol/L D-glucose,
3.2 mmol/L L-gutamine, 10% fetal bovine serum (FBS) (GIBCO,
NY), and 50 U/mL penicillin-streptomycin (Sigma).
Small interfering RNA and transfection
SiGENOME SMARTpool siRNA (four separated siRNAs per
human TRPM7 transcript) were purchased from Dharmacon
(Dharmacon, Lafayette, CO). The following siRNA were used:
human TRPM7 (siRNA-TRPM7) and non-targeting siRNA
(siRNA-CTRL) used as a control. In brief, HUVEC were plated
overnight in 24-well plate and then transfected with 5 nM siRNA
using DharmaFECT 4 transfection reagent (Dharmacon) used
according to the manufacturer’s protocols in serum-free medium
for 6 hours. After 48 to 72 h after transfection, experiments were
performed.
Western blot procedures
Vehicle-treated or endotoxin-treated ECs in non-transfected or
transfected conditions were lysed in cold lysis buffer, and then
proteins were extracted. Supernatants were collected and stored in
the same lysis buffer. Protein extract and supernatant were
subjected to SDS-PAGE and resolved proteins were transferred to
a nitrocellulose or PVDF membrane. The blocked membrane was
incubated with the appropriate primary antibody, washed twice,
and incubated with a secondary antibody. Bands were revealed
using a peroxidase-conjugated IgG antibody. Tubulin was used as
a loading control. For a detailed list of antibodies used, see Table
S1.
Fluorescent Immunocytochemistry
ECs were washed twice with PBS and fixed. The cells were
subsequently washed again and incubated with the primary
antibodies. Then, cells were washed twice and incubated with the
secondary antibodies. Samples were mounted with ProLong Gold
antifade mounting medium with DAPI (Invitrogen). For a detailed
list of antibodies used see Table S2.
Calcium Imaging
Plated ECs were mounted in a perfusion chamber on the stage
of an inverted microscope (Olympus IX-81, UPLFLN 40XO 40
x/1.3 oil-immersion objective). Cells were incubated with 1 mM
Fura-2 AM (Molecular Probes) for 30 min and then washed with
Hank’s solution. ECs were transfected (with siRNA-TRPM7 and
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TRPM7-Mediated Endotoxin-Induced EC Fibrosis
siRNA-CTRL) or non-transfected. Fura-2 was alternately excited
at 340 and 400 nm, and the fluorescence filtered at 510 nm was
collected and recorded at 5 Hz using a CCD-based imaging
system (Olympus DSU) running CellR software (Olympus). At the
end of each experiment, maximal fluorescence was obtained by
treating the cell with 1 mM ionomicin. For every experiment,
signals were recorded and the background intensity was subtracted, using a same-size region of interest outside the cells [37].
Results are expressed as the ratio between the 340 nm and
400 nm (R340/400) signals.
Measurement of [Ca2+] by flow cytometry
ECs were harvested with trypsin/EDTA, washed twice in icecold PBS, resuspended and loaded with the Ca2+-sensitive cell
permeant dye Fluo-4 (5 mM) for 15–30 min at room temperature
in the dark. Then, cells were exposed to LPS for 90 s and analyzed
immediately by flow cytometry (FACSCanto, BD Biosciences, San
José, CA). ECs were transfected (with siRNA-TRPM7 and siRNACTRL) or preincubated with L-NAME, MCI-186, NAC, and
GSH. Experiments were performed and then intracellular calcium
levels were measured using Fluo-4 dye. Using the FACSDiva
software, a population of red-positive cells (transfected cells) was
defined, and calcium levels for this population were analyzed. A
minimum of 10,000 cells/sample were analyzed. Cellular dye
intensity analysis was performed using FACSDiva software v4.1.1
(BD Biosciences).
Reagents
Lipopolysaccharide from E. coli was purchased from Sigma
(0127:B8). Fura-2 and Fluo-4 were purchased from Invitrogen. LNAME, cobinamide, NAC and GSH were purchased from Sigma.
PTIO and L-NMMA were purchased from Tocris Bioscience
(Bristol, UK). MCI-186 was purchased from Calbiochem (San
Diego, CA). Human TGF-b1 and TGF-b2 were purchased from
R&D Systems. Buffers and salts were purchased from Merck
Biosciences (Darmstadt).
Data analysis
All results are presented as the means 6 SD. One-way analysis
of variance (ANOVA) (Kruskal–Wallis) followed by Dunn’s post hoc
test were used and considered significant at p,0.05.
Results
Endothelial cells convert into activated fibroblasts upon
endotoxin challenge
Figure 1. Endotoxin-induced fibroblast-like morphology in
endothelial cells. (A–F) Morphological changes in human ECs
resembling fibroblast. Figures show representative phase-contrast
images from at least three separates experiments of non-transfected
ECs exposed to vehicle (A), 20 mg/mL LPS for 24 h (B), 20 mg/mL LPS for
48 h (C), 20 mg/mL LPS for 72 h (D), 2.5 ng/mL TGF-b1 (E), 2.5 ng/mL
TGF-b2, (F). Bar scale represents 50 mm. (G) Endothelial cell length
distribution of cells exposed to vehicle (filled bars), 20 mg/mL LPS for
72 h (empty bars), 2.5 ng/mL TGF-b1 (dashed bars), and 2.5 ng/mL TGFb2, (dashed bars). N = 3-6. (H) Endothelial cell length in which length/
width .2, for cells exposed to vehicle, 20 mg/mL LPS for 72 h, 2.5 ng/
mL TGF-b1, and 2.5 ng/mL TGF-b2. Statistical differences were assessed
by a one-way analysis of variance (ANOVA) (Kruskal–Wallis) followed by
Dunn’s post hoc test. **: p,0.01 against the vehicle-treated condition.
Graph bars show the mean 6 SD (N = 3-6).
doi:10.1371/journal.pone.0094146.g001
Endothelial cells normally exhibit a round, short-spindle
morphology, with a cobblestone appearance (Figure 1A). In
contrast, ECs exposed to the endotoxin LPS showed a spindleshaped, fibroblast-like phenotype (Figure 1B–D), similar to what
has been reported previously [14]. This phenotype is similar to
that obtained using the best-studied EndMT inducers, TGF-b1
(Figure 1E) and TGF-b2 (Figure 1F), suggesting that LPS induces
endothelial fibrosis through EndMT.
To study these phenotypic changes in detail, we measured the
length of cells in the absence or presence of LPS. The results
revealed a differential distribution of cell lengths in ECs in the
presence of vehicle compared to cells exposed to LPS. The mean
cell length of ECs exposed to LPS was ,4-fold higher than
observed in the absence of LPS. Interestingly, ECs exposed to the
typical transforming inducers TGF-b1 and TGF-b2 exhibited a
similar cell length distribution as LPS-treated cells (Figure 1G).
Next, we counted only cells whose length was twice their width
(length/width .2) because in the cell length analysis showed in
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Figure 1G, the length measurements were merged for a significant
portion of the cells. The results showed that the length of
endotoxin-treated cells was ,12-fold higher than that of ECs in
the absence of LPS. Similar results were obtained in ECs exposed
to TGF-b1 and TGF-b2 (Figure 1H).
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TRPM7-Mediated Endotoxin-Induced EC Fibrosis
Figure 2. Endotoxin-induced endothelial fibrosis through changes in endothelial and fibrotic markers is dependent on TRPM7
expression. (A–B) TRPM7 expression downregulation by siRNA. ECs were transfected with a specific siRNA against the human TRPM7 isoform
(siRNA-TRPM7) or a non-targeting siRNA (siRNA-CTRL). (A) Representative images from western blot experiments performed for detection of TRPM7 in
cells transfected with siRNA-TRPM7 or siRNA-CTRL. (B) Densitometric analyses from several experiments, as shown in (A). Protein levels were
normalized against tubulin, and the data are expressed relative to cells transfected with siRNA-CTRL condition. Statistical differences were assessed by
student’s t-test (Mann-Whitney). ***: p,0.001. Graph bars show the mean 6 SD (N = 3). (C–J) ECs were exposed to LPS for 72 h and protein
expression was analyzed. (C–F) Representative images from western blot experiments performed for detection of endothelial markers CD31 (C) and
VE-cadherin (VE-Cad) (D), and fibrotic markers a-SMA (E) and FSP-1 (F). (G–J) Densitometric analyses of the experiments shown in (C–F) respectively.
Protein levels were normalized against tubulin and data are expressed relative to siRNA-CTRL transfected cells without endotoxin condition. Statistical
differences were assessed by a one-way analysis of variance (ANOVA) (Kruskal–Wallis) followed by Dunn’s post hoc test. *: p,0.05 and **: p,0.01
against to siRNA-CTRL transfected cells without endotoxin condition. NS: non-significant. Graph bars show the mean 6 SD (N = 3–6).
doi:10.1371/journal.pone.0094146.g002
To demonstrate that our results were obtained from cultures of
ECs without contamination from fibroblasts or mesenchymal-like
cells, we performed a detailed examination of our EC cultures.
Using VE-cadherin as a specific endothelial marker, we found that
.99% of the cells in our EC cultures were positive for VEcadherin, demonstrating that our primary EC cultures were highly
enriched in ECs (Figure S1).
experimental strategy to achieve downregulation of TRPM7
expression. Thus, we used a specific small interfering RNA
(siRNA) targeting the human isoform of TRPM7 (siRNATRPM7). The efficiency of the siRNA in achieving downregulation of TRPM7 channel expression was .90% compared to ECs
transfected with a non-targeting siRNA sequence used as a control
(siRNA-CTRL) (Figure 2A-B).
As expected, LPS-treated ECs transfected with siRNA-CTRL
showed similar characteristics to those previously observed in nontransfected wild-type endothelial cells exposed to LPS [14]. ECs
transfected with siRNA-CTRL and exposed to endotoxin exhibited a decrease in the expression of the endothelial proteins CD31
(Figure 2C and G) and VE-cadherin (Figure 2E and H).
TRPM7 expression is crucial for the alteration of
endothelial and fibrotic marker expression induced by
endotoxin in endothelial cells
To test whether the TRPM7 channel is involved in endotoxininduced endothelial fibrosis, we applied a molecular biological
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TRPM7-Mediated Endotoxin-Induced EC Fibrosis
Figure 3. Cellular distribution of endothelial and fibrotic markers involved in endotoxin-induced endothelial fibrosis. Representative
images from experiments of vehicle-treated (A–H) or endotoxin (20 mg/mL LPS)-treated (I–P) ECs for 72 h. Endothelial markers CD31 or VE-cadherin
(red), and the fibrotic markers a-SMA, or FSP-1 (green) were detected. In vehicle-treated cells: the box depicted in (A, C, E, and G) indicates the
magnification shown in (B, D, F, and H), respectively. Arrows indicate CD31 (B, F) or VE-cadherin (D, H) labeling at the plasma membrane, whereas
arrowheads indicate a-SMA (B, F) or FSP-1 (D, H) staining, indicating basal expression of fibrotic markers (B, D). In endotoxin-treated cells: the box
depicted in (I, K, M, and O), indicates the magnification shown in (J, L, N, and P) respectively. Arrows indicate a-SMA (J, N) or FSP-1 (L. P), whereas
arrowheads indicate CD31 (J, N) or VE-cadherin (L, P) staining from residual endothelial marker expression indicating EndMT. Nuclei were stained
using DAPI. Bar scale represents 10 mm.
doi:10.1371/journal.pone.0094146.g003
Furthermore, the protein expression of the fibrotic markers aSMA (Figure 2E and I) and FSP-1 (Figure 2F and J) was
significantly increased upon endotoxin challenge. In contrast,
LPS-treated ECs transfected with siRNA-TRPM7 were resistant
to endotoxin challenge. ECs transfected with siRNA-TRPM7 did
not show any decrease in the expression of the endothelial proteins
CD31 (Figure 2C and G) and VE-cadherin (Figure 2E and H).
Accordingly, the levels of the fibrotic markers a-SMA (Figure 2E
and I) and FSP-1 (Figure 2F and J) were not increased in LPStreated ECs transfected with siRNA-TRPM7. ECs transfected
with siRNA-TRPM7 in the absence of LPS did not show any
difference in the expression of endothelial and fibrotic markers
compared to that observed in vehicle-treated cells transfected with
siRNA-CTRL (Figure 2C–J). Similar results were founded using
the non-specific TRPM7 blockers Zn2+ and Gd3+ (Figure S2)
[33,38–40].
To study the participation of TRPM7 in the cellular localization
and distribution of endothelial and fibrotic proteins, we carried out
immunocytochemistry experiments. ECs transfected with siRNACTRL or siRNA-TRPM7 in the absence of endotoxin showed
typical CD31 labeling, localized predominantly to the plasma
membrane, whereas a-SMA was weakly expressed (Figure 3A and
E). VE-cadherin labeling was also detected at the plasma
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membrane, while FSP-1 expression was undetectable (Figure 3C
and G). ECs transfected with siRNA-CTRL and exposed to LPS
showed increased a-SMA labeling in fibrotic-like stress fibers and
decreased CD31 expression (Figure 3I). Additionally, FSP-1
labeling was greatly increased, whereas VE-cadherin was virtually
absent (Figure 3K). This distribution was analogous to that
previously reported in LPS-treated, non-transfected wild-type
endothelial cells [14]. Conversely, endotoxin-treated ECs transfected with siRNA-TRPM7 did not show changes in the
localization and distribution of endothelial and fibrotic markers,
showing similar results to those observed in the absence of LPS.
Endotoxin-treated ECs transfected with siRNA-TRPM7 exhibited
CD31 localized mainly at the plasma membrane, while a-SMA
was weakly detected (Figure 3M). Moreover, VE-cadherin was also
detected at the plasma membrane, but FSP-1 expression was
undetectable (Figure 3O).
TRPM7 expression is crucial for the increase of
extracellular matrix proteins induced by
lipopolysaccharide in endothelial cells
Oversecretion of ECM proteins is a key feature of fibrogenesis
[41,42]. Thus, we measured fibronectin and collagen protein levels
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TRPM7-Mediated Endotoxin-Induced EC Fibrosis
in the supernatants of EC cultures. LPS-treated ECs transfected
with siRNA-CTRL showed an increase in fibronectin (Figure 4A
and C) and type III collagen (Figure 4B and D) levels. These
results were similar to those previously detected in LPS-treated,
non-transfected wild-type endothelial cells [14]. However, the
oversecretion of these ECM proteins was abolished by treatment
with siRNA-TRPM7. ECs transfected with siRNA-TRPM7 and
exposed to LPS did not exhibit any increase in either fibronectin
(Figure 4A and C) or type III collagen (Figure 4B and D). These
findings are in accord with those obtained using the non-specific
TRPM7 blockers Zn2+ and Gd3+ (Figure S3) [33,38–40]. ECs
transfected with siRNA-TRPM7 in the absence of LPS did not
show any differences in the expression of ECM markers compared
to those observed in vehicle-treated cells transfected with siRNACTRL (Figure 4A-D).
Next, we evaluated the role of TRPM7 expression in the cellular
localization and distribution of ECM proteins. ECs transfected
with siRNA-CTRL or siRNA-TRPM7 in the absence of
endotoxin showed typical CD31 (Figure 5A and E) and VEcadherin (Figure 5C and G) labeling, localized predominantly at
the plasma membrane, whereas FN (Figure 5A, E, C and G) was
expressed at low levels. ECs transfected with siRNA-CTRL and
exposed to LPS showed increased FN labeling (Figure 5I and K)
and decreased expression of CD31 (Figure 5I) and VE-cadherin
(Figure 5K), similar to what has previously been reported in
endotoxin-treated non-transfected wild-type endothelial cells [14].
In contrast, LPS-treated ECs transfected with siRNA-TRPM7
were resistant to this endotoxin-induced cellular conversion,
showing CD31 (Figure 5M) and VE-cadherin (Figure 5O) labeling
that was restricted to the plasma membrane, while FN (Figure 5M
and O) was weakly expressed.
Figure 4. Endotoxin-induced endothelial fibrosis through ECM
proteins increase are dependent on TRPM7 expression. ECs
were exposed to LPS for 72 h and protein expression was analyzed. (A–
B) Representative images from western blot experiments performed for
detection of ECM proteins fibronectin (FN) (A) and type III collagen (Col
III) (B). (C–D) Densitometric analyses of the experiments shown in (A–B)
respectively. Protein levels were normalized against tubulin and data
are expressed relative to siRNA-CTRL transfected cells without
endotoxin condition. Statistical differences were assessed by a oneway analysis of variance (ANOVA) (Kruskal–Wallis) followed by Dunn’s
post hoc test. *: p,0.05 and **: p,0.01 against to siRNA-CTRL
transfected cells without endotoxin condition. NS: non-significant.
Graph bars show the mean 6 SD (N = 3–6).
doi:10.1371/journal.pone.0094146.g004
TRPM7 mediates the endotoxin-induced increase in
intracellular Ca2+ in endothelial cells
As an increase in the intracellular Ca2+ concentration ([Ca2+]i)
has been shown to be necessary for the initiation and development
of the fibrotic process [17–19], we prompted to investigate
whether LPS is able to induce an increase in [Ca2+]i. To this end,
ratiometric Ca2+-imaging analyses were performed. Our results
showed that ECs exposed to LPS exhibited a transient rise in Ca2+
levels (Figure 6A), reaching a maximal level within 90 seconds and
slowly returning to basal levels with a half-time constant of 8267
seconds (Figure 6C). No changes in [Ca2+]i were observed in cells
treated with vehicle alone (Figure 6A).
To determine whether the TRPM7 channel is involved in the
endotoxin-induced intracellular Ca2+ increase, we examined
whether downregulation of TRPM7 expression via specific siRNA
treatment would modify the observed pattern of the increase in
intracellular calcium levels. In LPS-treated ECs transfected with
siRNA-TRPM7 (Figure 6B), a transient [Ca2+]i increment was
observed, reaching maximal levels within 90 seconds, and the
main differences observed compared to either LPS-treated ECs
transfected with siRNA-CTRL (Figure 6B) or LPS-treated nontransfected wild-type ECs (Figure 6A) concerned the [Ca2+]i
reached (Figure 6C) and the half-time constant for returning to
basal levels (Figure 6D). Again, no changes in [Ca2+]i were
observed in cells treated with vehicle alone (not shown).
Next, we were prompted to study the participation of reactive
molecules in the TRPM7-mediated LPS-induced Ca2+ level
increase. To that end, we monitored changes in the intracellular
calcium level by means of the Ca2+-sensitive dye Fluo-4
fluorescence. First, we probe that Fluo-4 dye was able to detect
changes in the Ca2+ level only when endotoxin was added
(Figure 6E). Furthermore, the Fluo-4-based assay was able to
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demonstrate that endotoxin-induced Ca2+-increase was dependent
on TRPM7 ion channel expression since transfection of siRNATRPM7 abolished the calcium increase (Figure 6E). Then, ECs
were incubated in the presence of MCI-186, a free radical
scavenger which acts over hydroxyl radical, super oxide and
peroxynitrite among others. Our results demonstrated that MCI186 abolished the calcium level increase induced by LPS
(Figure 6F). Furthermore, ECs were exposed to the antioxidant
N-acetyl cysteine (NAC) or with the reducing agent glutathione in
its reduced form (GSH). Results showed that NAC and GSH
treatment significantly reduced the LPS-induced calcium increase
(Figure 6F). To address definitely the participation of ROS, we
exposed ECs to the oxidant agent hydrogen peroxide (H2O2). Our
results showed that ECs exposed to H2O2 increase the intracellular
calcium signal similarly to observed using the endotoxin
(Figure 6G). Of note, the H2O2-induced Ca2+-increase was also
dependent on TRPM7 ion channel expression because the
transfection of siRNA-TRPM7 significantly decreased the H2O2induced calcium increase (Figure 6G). Besides, ECs were
incubated with the generic inhibitors of all three nitric oxide
synthase (NOS) isoforms, L-NG-Nitroarginine Methyl Ester (LNAME) or with N-monomethyl-L-arginine monoacetate (LNMMA). Our results demonstrated that the treatment with L-
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Figure 5. Cellular distribution of ECM proteins involved in endotoxin-induced endothelial fibrosis. Representative images from
experiments of vehicle-treated (A–H) or endotoxin (20 mg/mL LPS)-treated (I–P) ECs for 72 h. Endothelial markers CD31 or VE-cadherin (red), and the
ECM protein FN (green) were detected. In vehicle-treated cells: the box depicted in (A, C, E, and G) indicates the magnification shown in (B, D, F, and
H), respectively. Arrows indicate CD31 (B, F) or VE-cadherin (D, H) labeling at the plasma membrane, whereas arrowheads indicate FN (B, D, F, and H)
staining, indicating basal expression of fibrotic markers (B, D). In endotoxin-treated cells: the box depicted in (I, K, M, and O), indicates the
magnification shown in (J, L, N, and P) respectively. Arrows indicate FN (J, L, N, and P), whereas arrowheads indicate CD31 (J, N) or VE-cadherin (L, P)
staining from residual endothelial marker expression indicating EndMT. Nuclei were stained using DAPI. Bar scale represents 10 mm.
doi:10.1371/journal.pone.0094146.g005
suppression of TRPM7 expression efficiently inhibits the endotoxin-induced conversion of ECs into activated fibroblasts.
TRPM7 downregulation prevented the endotoxin-induced decrease of endothelial markers and the increase of fibrotic genes
induced by LPS in ECs. Furthermore, TRPM7 downregulation
inhibited the endotoxin-induced increase of ECM proteins.
Finally, we demonstrated that endotoxin is able to induce a
significant increase in the intracellular Ca2+ concentration in ECs.
This endotoxin-induced Ca2+ increase was prevented by inhibition
of TRPM7 expression, suggesting that the endotoxin-induced
Ca2+ increase is mediated by the TRPM7 calcium channel.
In endotoxemia-derived sepsis syndrome, large amounts of LPS
are found in the bloodstream, directly interacting with ECs.
Because ECs express the toll-like receptor 4 (TLR4) LPS receptor,
LPS is able to exert its action in the endothelium of blood vessels.
Hence, endotoxin-induced endothelial fibrosis emerges as an
important potential mechanism for generating detrimental effects
in the organs of patients experiencing endotoxemia-derived sepsis
syndrome.
TRPM7 has been found to be involved in a number of human
diseases and pathological conditions, including the activation of
immune system cells [43,44], brain ischemia [45], Guamanian
lateral sclerosis and Parkinson’s [46], atrial fibrillation [29], and
NAME or L-NMMA did not induce any change in the endotoxininduced Ca2+-increase (Figure 6H). In addition, cells were
incubated with the nitric oxide (NO) scavengers cobinamide
(Cobin) or with 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3oxide (PTIO), which not affect NO synthesis. Data showed that
neither cobinamide nor PTIO modify the endotoxin-induced
Ca2+-increase (Figure 6H). These results suggest that ROS, but not
NO generation, is involved in the TRPM7-mediated intracellular
calcium changes induced by endotoxin.
Discussion
Endothelial dysfunction is a hallmark of the progression of sepsis
syndrome and several inflammatory diseases. Because the current
available therapies are often not satisfactory, the identification of
key proteins involved in these pathologies is essential for improving
their treatment. We previously reported that the endotoxin LPS
induces endothelial fibrosis [14]. Here, we delved deeper into the
molecular mechanism underlying in the endotoxin-induced
endothelial fibrosis.
In this study, we demonstrated that the Ca2+-permeable
channel TRPM7 is a crucial protein in the development of
endotoxin-induced endothelial fibrosis. Our results showed that
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TRPM7-Mediated Endotoxin-Induced EC Fibrosis
Figure 6. Endotoxin-induced intracellular Ca2+ increase is mediated by TRPM7 in endothelial cells. (A) Representative time courses for
normalized florescence in ECs loaded with Fura-2 and treated with vehicle (empty circles) or 20 mg/ml LPS (filled circles). The arrow represent the time
for external solution change containing vehicle (empty circles) or LPS (filled circles). (B) Representative time courses for normalized florescence in ECs
transfected with non-targeting siRNA (siRNA-CTRL, filled circles) or with siRNA for TRPM7 (siRNA-TRPM7, empty circles) and loaded with Fura-2. The
arrow indicates the time for LPS application. (C) Summary bar graph for maximal amplitude of normalized florescence in wild type ECs (WT) or
transfected with siRNAs. (D) Summary of the data for the time constant (t) of calcium return to basal levels, obtained after fitting the data to a single
exponential for wild type ECs (WT) or transfected with siRNAs. (E–H) Endothelial cells were incubated in the absence (2) or presence (+) of 20 mg/mL
endotoxin and the calcium overloading was evaluated by means of the Ca2+-sensitive fluorescent dye fluo-4. (E) ECs were transfected with siRNATRPM7 and siRNA-CTRL and incubated in the absence (2) or presence (+) of endotoxin. (F) ECs were preincubated with 0.5 mM MCI-186, 1 mM NAC
or 2 mM GSH for 1 hr and then incubated in the absence (2) or presence (+) of endotoxin. (G) ECs were transfected with siRNA-TRPM7 and siRNACTRL and incubated in the absence (2) or presence (+) of 10 mM H2O2. (H) ECs were preincubated with 5 mM L-NAME, 1 mM L-NMMA, 50 mM
cobinamide, or 500 mM PTIO for 1 hr and then incubated in the absence (2) or presence (+) of endotoxin. Statistical differences were assessed by a
one-way analysis of variance (ANOVA) (Kruskal–Wallis) followed by Dunn’s post hoc test. *: p,0.05; **: p,0.01 against vehicle-treated condition.
Graph bars show the mean 6 SD (N = 3–4).
doi:10.1371/journal.pone.0094146.g006
calcium homeostasis to promote physiological and pathological
processes.
ECM proteins are produced and secreted in balance with their
degradation in healthy cells, whereas during fibrosis, activated
fibroblasts oversecrete ECM proteins, thereby overwhelming the
capability for ECM degradation [59,60]. Thus, increases in ECM
proteins are a sign that a fibrogenic process has taken place. On
the other hand, ECM proteins may play a role in bacterial
adherence and invasion to promote endotoxin-mediated endothelium damage [61–63], as overexpression of ECM proteins
facilitates the pathogenic mechanism. The fact that inhibition of
the TRPM7 channel abolished the endotoxin-induced increase in
ECM expression in ECs indicates that this channel is a key protein
involved in the progression of endothelial fibrosis under endotoxemia-like conditions. Further studies must be performed to
evaluate whether TRPM7 may be useful as a therapeutic tool.
Taken together, these findings demonstrated that the TRPM7
channel plays a critical role in the mechanism underlying
endotoxin-induced endothelial fibrosis. Thus, TRPM7 emerges
as a novel target for drug design to improve current treatments
against endotoxemia-derived sepsis syndrome and further inflammatory diseases.
cancer [47,48]. The majority of these pathologies are characterized by an increase in the intracellular level of ROS. Similarly,
increases in oxidative stress are a common consequence of
inflammatory processes, and it has been reported that LPS
induces an increase in intracellular ROS levels in ECs [10,49]. In
addition, TRPM7 activity has been found to be regulated by
oxidative stress [33,38], and the expression of TRPM7 is increased
in cells exposed to oxidant agents [39,50]. Interestingly, endotoxin-induced endothelial fibrosis is prevented by treatment with the
reducing agent N-acetylcysteine [14]. Considering these findings, it
can be suggested that the participation of TRPM7 in the
endotoxin-induced endothelial fibrosis could be mediated by the
ROS generated by LPS challenge. Further experiments are
needed to shed light on this issue.
An interesting feature of TRPM7 is that it contains a C-terminal
Ser/Thr kinase domain [36,51]. As we demonstrated that
suppression of TRPM7 expression is necessary for endotoxininduced endothelial fibrosis in the present study, it is possible that
the kinase activity of the channel could also be involved in the
induction of endothelial fibrosis. However, further experiments
will certainly be needed to verify this hypothesis.
Changes in intracellular Ca2+ concentrations are essential for
diverse cellular processes to occur normally. However, several lines
of evidence suggest that alterations of Ca2+ levels are also essential
for the development of pathological conditions [20,52–55].
Considering that an increase in intracellular Ca2+ is fundamental
for fibrogenesis to take place, we sought to study this issue. The
endotoxin-induced intracellular Ca2+ increase was characterized
by a transient elevation of Ca2+ and a rapid return to basal
calcium levels, suggesting the existence of a negative feedback
mechanism regulating the Ca2+ increase. The experiments
involving siRNA-TRPM7 demonstrated that the endotoxininduced Ca2+ increase was mediated through TRPM7. Experiments performed using MCI-186 and L-NAME suggest that
hydroxyl radical, super oxide and peroxynitrite, but not nitric
oxide, could be involved in the endotoxin-induced Ca2+ increase.
Furthermore, since the inhibition of the endotoxin-induced Ca2+
increase produced by NAC and GSH, it is possible to hypothesize
that the oxidative modifications produced in TRPM7 could be
performed in thiol groups from cysteine residues. These findings
suggest either that TRPM7 directly participates in the mechanism
regulating the endotoxin-induced Ca2+ increase or that other
TRPM7-related proteins are involved in the regulation of the
TRPM7-mediated calcium influx. In this context, a non-selective
cation channel, TRPM4, has been shown to be involved in the
regulation of intracellular calcium overloading and oscillations by
controlling the plasma membrane potential [56–58]. Thus,
TRPM4 activity indirectly controls the calcium influx, which is
mediated by additional calcium channels regulating intracellular
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Supporting Information
Figure S1 Primary HUVEC cultures were subjected to
immunocytochemistry experiments to identify ECs as
VE-Cad positive cells (VE-Cad+) and non-endothelial as
VE-Cad negative cells (VE-Cad2). Data are expressed as
percentage of total cells counted. Several independent experiments
were counted (N = 10). Statistical differences were assessed by
student’s t-test (Mann-Whitney). ***: p,0.0001.
(PDF)
Figure S2 Endotoxin-induced endothelial fibrosis
through changes in endothelial and fibrotic markers
are inhibited by using the non-specific TRPM7 blocker
Zn2+ and Gd3+. (A–D) ECs were exposed to LPS for 72 h in the
presence of Zn2+ (A–B) or Gd3+ (C–D), and protein expression of
endothelial marker CD31 (A and C) and fibrotic markers a-SMA
(B and D). Statistical differences were assessed by a one-way
analysis of variance (ANOVA) (Kruskal–Wallis) followed by
Dunn’s post hoc test. **: p,0.01 against to siRNA-CTRL
transfected cells without endotoxin condition. NS: non-significant.
Graph bars show the mean 6 SD (N = 3).
(PDF)
Figure S3 Endotoxin-induced endothelial fibrosis
through ECM proteins increase are inhibited by using
the non-specific TRPM7 blocker Zn2+ and Gd3+. (A–D)
ECs were exposed to LPS for 72 h in the presence of Zn2+ (A–B)
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TRPM7-Mediated Endotoxin-Induced EC Fibrosis
or Gd3+ (C–D), and ECM proteins fibronectin (FN) (A and C) and
type III collagen (Col III) (B and D). Statistical differences were
assessed by a one-way analysis of variance (ANOVA) (Kruskal–
Wallis) followed by Dunn’s post hoc test. **: p,0.01 against to
siRNA-CTRL transfected cells without endotoxin condition. NS:
non-significant. Graph bars show the mean 6 SD (N = 3).
(PDF)
Methods S1 Supporting expanded methods.
(PDF)
Acknowledgments
The authors are grateful to Director Dr. Iván Oyarzún and Dr. Mario
Carmona, Dr. Jaime Mendoza and Mrs. Juana Belmar from Servicio
Ginecologı́a y Obstetricia, Hospital San Jose de Melipilla.
Table S1 Primary and secondary antibodies used in
western blot experiments.
(PDF)
Author Contributions
Conceived and designed the experiments: CE DV FS. Performed the
experiments: CE IM TH DV. Analyzed the data: CE IM DV FS.
Contributed reagents/materials/analysis tools: CCV RA LAV DV FS.
Wrote the paper: CCV RA DV FS.
Table S2 Primary and secondary antibodies used in
immunocytochemistry experiments.
(PDF)
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