The International Journal of Biochemistry & Cell Biology 55 (2014) 11–23

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The International Journal of Biochemistry

& Cell Biology
journal homepage: www.elsevier.com/locate/biocel

Endotoxin-induced vascular endothelial cell migration is dependent

on TLR4/NF-!B pathway, NAD(P)H oxidase activation, and transient
receptor potential melastatin 7 calcium channel activity
Daniela Sarmiento a , Ignacio Montorfano a , Mónica Cáceres d , César Echeverría a,f ,
Ricardo Fernández c , Claudio Cabello-Verrugio b , Oscar Cerda d , Pablo Tapia e ,
Felipe Simon a,g,∗
a
Laboratorio de Fisiopatología Integrativa, Departamento de Ciencias Biologicas, Facultad de Ciencias Biologicas and Facultad de Medicina,
Universidad Andres Bello, Avenida Republica 239, Santiago, Chile
b
Laboratorio de Biología y Fisiopatología Molecular, Departamento de Ciencias Biologicas, Facultad de Ciencias Biologicas and Facultad de Medicina,
Universidad Andres Bello, Avenida Republica 239, Santiago, Chile
c
Laboratorio de Fisiología, Departamento de Ciencias Biologicas, Facultad de Ciencias Biologicas and Facultad de Medicina, Universidad Andres Bello,
Avenida Republica 239, Santiago, Chile
d
Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago,
Chile
e
Departamento de Medicina Intensiva, Facultad de Medicina, Pontificia Universidad Católica de Chile, Marcoleta 367, Santiago, Chile
f
Laboratorio de Bionanotecnología, Universidad Bernardo O’Higgins, General Gana 1780, Santiago, Chile
g
Millennium Institute on Immunology and Immunotherapy, Santiago, Chile

a r t i c l e i n f o a b s t r a c t

Article history: Endothelial dysfunction is decisive and leads to the development of several inflammatory diseases.
Received 28 February 2014 Endotoxemia-derived sepsis syndrome exhibits a broad inflammation-induced endothelial dysfunc-
Received in revised form 16 July 2014 tion. We reported previously that the endotoxin, lipopolysaccharide (LPS), induces the conversion of
Accepted 4 August 2014
endothelial cells (ECs) into activated fibroblasts, showing a myofibroblast-like protein expression pro-
Available online 12 August 2014
file. Enhanced migration is a hallmark of myofibroblast function. However, the mechanism involved in
LPS-induced EC migration is no totally understood. Some studies have shown that the transient receptor
Keywords:
potential melastatin 7 (TRPM7) ion channel is involved in fibroblast and tumor cell migration through the
Endothelial dysfunction
Inflammation
regulation of calcium influx. Furthermore, LPS modulates TRPM7 expression. However, whether TRPM7
Endothelial cell migration is involved in LPS-induced EC migration remains unknown.
Lipopolysaccharide Here, we study the participation of LPS as an inducer of EC migration and study the mechanism
TRPM7. underlying evaluating the participation of the TRPM7 ion channel.
Our results demonstrate that LPS induced EC migration in a dose-dependent manner. Furthermore,
this migratory process was mediated by the TLR-4/NF-!B pathway and the generation of ROS through
the PKC-activated NAD(P)H oxidase. In addition, LPS increased the intracellular calcium level and the
number of focal adhesion kinase (FAK)-positive focal adhesions in EC. Finally, we demonstrate that using
TRPM7 blockers or suppressing TRPM7 expression through siRNA successfully inhibits the calcium influx
and the LPS-induced EC migration.
These results point out TRPM7 as a new target in the drug design for several inflammatory diseases
that impair vascular endothelium function.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Endothelial dysfunction is a key factor in several inflamma-

∗ Corresponding author. Laboratorio de Fisiopatología Integrativa, Departamento
de Ciencias Biologicas, Facultad de Ciencias Biologicas and Facultad de Medicina,
Universidad Andres Bello, Avenida Republica 239, Santiago, Chile.
Tel.: +562 2661 5653; fax: +562 2698 0414.
E-mail address: fsimon@unab.cl (F. Simon).
tory diseases, including endotoxaemia-derived sepsis syndrome
pathogenesis (Riedemann et al., 2003). Endotoxaemia is a severe
inflammatory disease, resulting from the presence of large amounts
of the gram-negative bacterial endotoxin, lipopolysaccharide (LPS),

http://dx.doi.org/10.1016/j.biocel.2014.08.001
1357-2725/© 2014 Elsevier Ltd. All rights reserved.
12 D. Sarmiento et al. / The International Journal of Biochemistry & Cell Biology 55 (2014) 11–23
in the bloodstream (Grandel and Grimminger, 2003;Peters et al.,
2003).
Despite the detrimental effects of endotoxaemia as a conse-
quence of an overactivation of the immune system, it is well
accepted that LPS-induced tissue damage contributes to the patho-
genesis of sepsis through an immune system-independent pathway
(Dimmeler et al., 1995;Hotchkiss and Karl, 2003).
Circulating LPS interacts with endothelial cells (ECs) through
its receptor toll-like receptor-4 (TLR-4), triggering a intracellular
signaling by means of nuclear factor kappa-B (NF-!B) activation.
This pathway includes reactive oxygen species (ROS) genera-
tion, principally through the PKC-dependent NAD(P)H oxidase
activation promoting EC death, cytokine secretion and increased
hyperpermeability (Becerra et al., 2011;Dauphinee and Karsan,
2006;Huet et al., 2011;Simon and Fernandez, 2009), which may,
at least in part, contribute to the organ dysfunction observed in
sepsis syndrome.
We reported that LPS induces the conversion of ECs into
activated fibroblasts through an endothelial-to-mesenchymal tran-
sition (EndMT)-based mechanism (Echeverria et al., 2013). After
EndMT, endothelial markers are downregulated, and fibroblast-
specific genes are severely increased (Echeverria et al., 2013;Kizu
et al., 2009;Zeisberg et al., 2007). Because EndMT is a cellular mech-
anism for the conversion of polarized endothelial cells into motile
mesenchymal cells, this process has also been characterized by the
acquisition of migratory features (Goumans et al., 2008;Potenta
et al., 2008;Thiery et al., 2009). Therefore, through EndMT, ECs dis-
aggregate from the vascular monolayer to migrate into the adjacent
tissue (Goumans et al., 2008;Potenta et al., 2008;Thiery et al., 2009).
During the development of inflammatory diseases, ECs are persis-
tently exposed to endotoxins and cytokine-like molecules in the
inflammatory environment, thereby maintaining the conditions
for EndMT and promoting the conversion of ECs into fibrotic-like
migrating cells. Recently, it has been reported results showing that
endotoxin is able to induce endothelial migration (Dauphinee et al.,
2013;Lappas, 2012). However, the underlying mechanism the EC
migration induced by LPS is not totally understood.
Cell migration is completely dependent on intracellular cal-
cium changes to generate intracellular [Ca2+ ] gradients (Brundage
et al., 1991;Tran et al., 1999). To promote cell migration, Ca2+
influx from the outside of the cell is mediated through a num-
ber of Ca2+ channels, including voltage-gated L-type Ca2+ channels
(Yang and Huang, 2005) and transient receptor potential (TRP) cal-
cium channels, such as TRPV1, TRPC3, and TRPM7 (Nilius et al.,
2007;Waning et al., 2007;Wei et al., 2009). Numerous studies have
shown that changes in [Ca2+ ]i are important in EC migration. It has
been reported that the intracellular variation of calcium levels is a
decisive step in EC migration (Kimura et al., 2001;Tran et al., 1999).
The inhibition of Ca2+ influx using calcium channel blockers,
markedly abolishes EC migration (Wu et al., 1997;Yoshikawa et al.,
1999). Consistently, the T-type calcium channel has been impli-
cated in angiotensin II-induced EC migration (Martini et al., 2010).
Nonetheless, the precise molecular identity of the calcium channel
that mediates Ca2+ influx during EC migration remains unknown.
The transient receptor potential melastatin 7 (TRPM7) ion
channel is involved in the migration of human nasopharyn-
geal carcinoma cells (Chen et al., 2010), pancreatic cancer cells
(Rybarczyk et al., 2012), and fibroblasts (Wei et al., 2009;Wei
et al., 2010). TRPM7 is a Ca2+ and Mg2+ channel, which is non-
permeable to monovalent cations and is ubiquitously expressed
in most cells, including ECs (Monteilh-Zoller et al., 2003;Nadler
et al., 2001;Schmitz et al., 2003;Simon et al., 2013). TRPM7 is
activated through Mg2+ -ATP (Hermosura et al., 2002;Kozak and
Cahalan, 2003;Nadler et al., 2001;Nunez-Villena et al., 2011), reg-
ulated through H2 O2 and PIP2 (Aarts et al., 2003;Nadler et al.,
2001;Runnels et al., 2002), and blocked through Zn2+ and Gd3+
(Kozak and Cahalan, 2003;Nadler et al., 2001;Nunez-Villena et al.,
2011). Recently, we reported that TRPM7 is involved in the LPS-
induced endothelial fibrosis (Echeverria et al., 2014). However,
the participation of the TRPM7 ion channel in the LPS-induced EC
migration has not been studied.
Therefore, the aim of this study was to study whether LPS pro-
motes EC migration and investigate the mechanism underlying
evaluating the participation of the TRPM7 ion channel.
Our data demonstrated that LPS induced EC migration in a dose-
dependent manner. This process is mediated by the participation
of the TLR-4/NF-!B pathway and the generation of ROS through the
PKC-activated enzyme NAD(P)H oxidase. Furthermore, in associa-
tion with an increase in the migratory phenotype, the LPS increased
the intracellular calcium level and the number of focal adhesion
kinase (FAK)-positive focal adhesions in EC. Moreover, we demon-
strated that the TRPM7 ion channel is critically involved in the
LPS-induced calcium influx and the LPS-induced EC migration.
These results provide further insight into the molecular mech-
anisms involved in LPS-induced endothelial fibrosis, and these
findings could be useful to understand the role of endothelial dys-
function during inflammation.
2. Materials and Methods
Details of all procedures are provided in the Supplementary
Material.
2.1. Primary cell culture
Human umbilical vein endothelial cells (HUVEC) were iso-
lated by collagenase (0.25 mg/mL) digestion from freshly obtained
umbilical cord veins from normal pregnancies, after patient’s
informed consent. The investigation conforms with the principles
outlined in the Declaration of Helsinki. The Commission of Bioethics
and Biosafety of Universidad Andrés Bello approved all experimen-
tal protocols. Cells were grown in gelatin-coated dishes at 37 ◦ C in a
5%:95% CO2 :air atmosphere in medium 199 (Sigma, MO), contain-
ing 100 "g/mL endothelial cell growth supplement (ECGS) (Sigma),
100 "g/mL heparin, 5 mmol/L D-glucose, 3.2 mmol/L L-glutamine,
10% fetal bovine serum (FBS) (GIBCO, NY), and 50 U/mL penicillin-
streptomycin (Sigma-Aldrich, St Louis, USA).
2.2. Cell adhesion assay
For cell adhesion experiments cells were incubated in absence or
presence of different LPS concentrations for 15 min in the presence
of 1% FBS. Then, 10,000 cells per well were seeded over gelatin-
coated 96-well plates (Falcon, Becton Dickinson, CA, USA) at 37 ◦ C
for 20 min in M199 plus 1% FBS. Cell adhesion was stopped by
pouring off the medium. After washing, cells were fixed with 10%
ethanol for 5 min and stained with 0.2% crystal violet for 5 min. After
removing excess of dye, cells were solubilized in 0.1 M NaH2 PO4 in
50% ethanol for 15 min at room temperature (RT). Absorbance at
570 mn was analyzed on a microplate reader. All assays were per-
formed at least in triplicates in three separates sets of experiments.
2.3. Cell-spreading assay
For cell-spreading experiments cells were incubated in absence
or presence of different LPS concentrations for 15 min in the pres-
ence of 1% FBS. Then, 10,000 cells per well were seeded over
gelatin-coated 24-well plates (Falcon) at 37 ◦ C for 40 min in M199
plus 1% FBS. After washing, cells were fixed with 10% ethanol for
5 min and stained with 0.2% crystal violet for 5 min. After remov-
ing excess of dye, spread and non-spread cells were identified
 D. Sarmiento et al. / The International Journal of Biochemistry & Cell Biology 55 (2014) 11–23
through microscopic visualization and counted. Images were cap-
tured through a digital microscope system. The ratio spread/total
cells was calculated for each set of experimental conditions after
counting four fields per set of conditions. All assays were performed
at least in triplicates in three separates sets of experiments.
2.4. Cell migration measurement by Transwell assay
The capacity of ECs to migrate was assayed using Transwell
chambers (Costar, Cambrige, MA, USA) with 8.0-"m-pore poly-
carbonate filters. Cells were seeded in absence or presence of
different LPS concentrations for 24 h in 1% FBS on the upper com-
partment of the chamber. To stimulate cell migration, 10% FBS was
added to the lower compartment of the chamber. Thus, migration
was allowed to occur for 24 h. After washing, non-invading cells
were removed from the upper surface of the membrane with a
cotton swab. The invading cells were fixed with 10% ethanol for
5 min and stained with 0.2% crystal violet for 5 min. Images were
captured through a digital microscope system. Cell migration was
evaluated by counting four fields per chamber in every condition.
All assays were performed at least in triplicates in three separates
sets of experiments.
2.5. Cell migration measurement by wound-closure assay
ECs were seeded until reached a monolayer. Then, cell cultures
were scratched with a 10 "L sterile tip and washed to remove
detached cells and debris. Cells were then incubated in medium
containing 1% FBS and different LPS concentrations. After 8 h cells
were fixed with methanol for 2 min. Images of each wound were
captured through a digital microscope system (Motic microscope
AE31 with moticam 2300, Motic Instruments Inc., Hong Kong,
China). The ratio of wound closure percentage was determined
using a software analysis system (Motic Images Plus 2.0, Motic
Instruments Inc.). In brief, cell margins were outlined in each image,
and the uncovered area of each wound was compared between
LPS-treated cells and vehicle-treated cells. For the quantification of
the mean migrated distance in the wound-closure assay, the lin-
ear migrated distance of several cells was measured at 8 h. Then,
the average of migrated distance was plotted. All assays were per-
formed at least in triplicates in three separates sets of experiments.
2.6. Fluorescent immunocytochemistry
Cells were incubated on M199 plus 1% FBS with or without
LPS for 8 h at 37◦C. After the treatments, the cells were fixed for
15 min at 4 ◦ C in 4% formaldehyde and 4% sucrose in TBS and
washed in TBS. Cells were permeabilized and blocked with 0.1%
Triton X-100, 3% BSA in TBS for 45 min at RT and stained with anti-
phospho-tyrosine 397 FAK (p397FAK) rabbit polyclonal antibody
(Calbiochem, USA). Primary antibody was detected with Alexa 488
goat anti-rabbit (Invitrogen, USA) and F-Actin was detected with
Alexa 555-conjugated Phalloidin (Cytoskeleton, USA). Images were
acquired with a CCD camera installed on a confocal Olympus DSU
system (Olympus, Tokyo, Japan).
2.7. Small interfering RNA against TRPM7 and transfections
SiGENOME SMARTpool siRNA against TRPM7 (four separated
siRNAs per TRPM7 transcript) were purchased from Dharmacon
(Dharmacon, Lafayette, CO). Non-targeting siRNA (siRNA-CTRL)
was used as a control. In brief, HUVEC were plated overnight in
24-well plate and then transfected with 5 nM siRNA of siCTRL or
siTRPM7 using DharmaFECT 4 transfection reagent (Dharmacon)
used according to the manufacturer’s protocols in serum-free
medium in OptiMEM (Gibco) for 6 h.
13
2.8. Quantitative PCR and Western blotting
Cells transfected with siCTRL or siTRPM7 were subjected to qPCR
and Western blotting for TRPM7. QPCR: Total RNA was extracted
with Trizol according to the manufacturer’s protocol (Invitrogen,
Carlsbad, CA). DNAse I-treated RNA was used for reverse transcrip-
tion using the Super Script II Kit (Invitrogen, Carlsbad, CA). Equal
amounts of RNA were used as templates in each reaction. QPCR
was performed using SYBR Green PCR Master Mix (AB Applied
Biosystems, Foster City, CA). Data are presented as relative mRNA
levels of the gene of interest normalized to relative levels of 28S
mRNA. Western blotting: Whole cell extracts were subjected to 12%
SDS-PAGE. Resolved proteins were transferred to a nitrocellulose
membrane, blocked, and then incubated ON with the anti-TRPM7
antibody (Neuromab, CA) or anti-pY397-FAK. Tubulin and total
FAK was used as a loading control. TRPM7 and pY397-FAK protein
content was determined by densitometric scanning of immunore-
active bands and intensity values were obtained by densitometry
of individual bands compared with tubulin or total pY397-FAK,
respectively (normalized against siRNA-CTRL or vehicle condition,
respectively).
2.9. Measurement of Ca2+ concentration by flow cytometry
ECs were treated with LPS for 24 h and incubated with the Ca2+ -
sensitive cell permeant dye 5 "M Fluo-4 (Invitrogen) for 15-30 min
at room temperature in the dark. Then, cells were analyzed immedi-
ately by flow cytometry (FACSCanto, BD Biosciences, San José, CA)
to evaluate the intracellular calcium level. In some experiments,
cells were transfected with siCTRL or siTRPM7 and intracellular cal-
cium level was measured. Experiments were performed and then
intracellular calcium levels were measure using Fluo-4 dye, which
is detected in the FL-1 channel (∼483 nm). A minimum of 10,000
cells/sample were analyzed. Cellular dye intensity analysis was per-
formed using FACSDiva software v4.1.1 (BD Biosciences).
2.10. Reagents
Lipopolysaccharide from E. coli was purchased from Sigma
(0127:B8). Buffers and salts were purchased from Merck Bio-
sciences (Darmstadt). CLI-095 was purchased from InvivoGen.
IKK-16 was purchased from Tocris Bioscience (Minneapolis, MN).
DPI, NAC, catalase, DTT, apocynin, chelerythrine and wedelolactone
were purchased from Sigma-Aldrich (St. Louis, MO).
2.11. Data analysis
All results are presented as means ± SD. Student’s t test (Mann-
Whitney) was used for comparing two groups and considered
significant at p < 0.05. For comparing multiple groups, either one-
way analysis of variance (ANOVA) (Kruskal–Wallis) followed by
Dunn’s post hoc test, or two-way ANOVA followed by Bonferroni
post hoc test were used and considered significant at p < 0.05.
3. Results
3.1. Lipopolysaccharide induced-endothelial cell migration is
dependent on Toll-like receptor 4 activation and NF-!B activity.
To determine whether LPS increases EC migration, we
performed experiments to evaluate adherence and spreading prop-
erties in presence or absence of LPS. As shown in Fig. 1A, LPS induced
a significant increment in cell adhesion. In addition, the cells were
seeded onto collagen-coated plates in the absence or presence of
LPS to determine whether LPS affects the capacity of ECs to spread
over a collagen matrix. The crystal violet-stained cells shown in
14 D. Sarmiento et al. / The International Journal of Biochemistry & Cell Biology 55 (2014) 11–23

Fig. 1. LPS induces EC migration increase. (A) ECs were incubated in the absence or presence of LPS (1, and 10 "g/mL) for 20 min and subjected to cell adhesion assay.
Then, cells were removed and adherent cells were fixed and stained with crystal violet. Cells were solubilized, and the released crystal violet was measured. Determinations
were done at least in triplicates and results are expressed in percentage normalized relative to vehicle-treated condition (0 "g/mL LPS). (B) Representative images obtained
from ECs in the absence or presence of LPS (1, and 10 "g/mL) for 40 min, stained with crystal violet and subjected to spreading assay. Arrows indicate spread cell. Bar scale
represents 25 "m. (C) Bar graph summarizing several experiments as showed in (B). (D) Representative images obtained from ECs incubated in the absence or presence of LPS
(1, and 10 "g/mL) and subjected to Transwell migration assay. ECs were placed in the upper compartment of a Transwell chamber in 1% FBS whereas in the lower chamber
10% FBS was added. Migration was allowed to occur for 24 h and migrated were fixed and stained with crystal violet. Bar scale represents 50 "m. (E) Bar graph summarizing
several experiments as showed in (D). (F) Representative images obtained from ECs incubated in the absence or presence of LPS (1, 10, and 20 "g/mL) and subjected to a
wound-closure assay. Images were taken at 0, 4, 6, and 8 h. To visualize the uncovered area of each wound, cell margins were outlined in each image. Bar scale represents
50 "m. (G) Graph summarizing several experiments as showed in (F), in which ECs were incubated in the absence or presence of LPS (0.1, 1, 5, 10, and 20 "g/mL). (H) Mean
migrated distance at 8 h from several experiments as showed in (F). *: P < 0.05, **: P < 0.01 against vehicle-treated condition (0 "g/mL LPS). Graph bars show the means ± SD
(N = 3-5).

Fig. 1B demonstrate that LPS-treated ECs had higher cell exten-
sions spreading over the collagen matrix than vehicle-treated ECs.
The quantification of this experiment showed a significant incre-
ment in the proportion of spread cells after exposure to LPS (Fig.
1 C),suggesting an increase in cell migration.
Next, we used a transwell cell migration assay in which ECs were
placed on a top chamber and a higher concentration of serum was
added to the lower chamber to create a serum gradient as a chemo-
tactic stimulus. The experiments were performed in the presence
or absence of LPS. Our results showed an increase in the migra-
tion of LPS-treated ECs compared with vehicle-treated condition
(Fig. 1D-E).
In addition, we used a cell monolayer-wounding assay to evalu-
ate whether LPS induced EC migration. Significant increases in cell
migration, measured as wound closure, were observed when ECs
were exposed to 1 to 20 "g/mL of LPS after 6 and 8 h compared with
vehicle-treated cells (Fig. 1F-G). In addition, the mean migrated dis-
tance was severely augmented as a consequence of LPS exposure
(Fig. 1H). These results are in agreement with evidence reported
previously (Dauphinee et al., 2013;Lappas, 2012), demonstrating
that endotoxin is able to induced EC migration.
To demonstrate that our findings are not due to contaminating
cells, we performed an meticulous inspection of the primary EC
culture used. Using VE-cadherin as a specific endothelial marker
 D. Sarmiento et al. / The International Journal of Biochemistry & Cell Biology 55 (2014) 11–23 15

Fig. 2. LPS-induced EC migration is inhibited by TLR-4 blocker and NF-!B inhibitors. (A-C) ECs incubated with (+) or without (-) LPS were subjected to Boyden-chamber
migration assay in the absence or presence of the TLR4 inhibitor CLI-095 (0.1, 1, and 10 "M) (A), and the NF-B inhibitors IKK-16 (0.1, 1, and 10 "M) (B), and wedelolactone (1,
10, and 20 "M) (C). Results are expressed as fold of change relative to vehicle-treated condition (0 "M LPS). (D-I) ECs incubated with (+) or without (-) LPS were subjected to
wound-closure assay in the absence or presence of the TLR4 inhibitor CLI-095 (0.1, 1, and 10 "M) (D), and the NF-B inhibitors IKK-16 (0.1, 1, and 10 "M) (F), and wedelolactone
(1, 10, and 20 "M) (H). Results are expressed in percentage relative to vehicle-treated condition (0 "g/mL LPS). (E, G, and I). Mean migrated distance at 8 h from several
experiments as showed in D, F, and H, respectively. *: P < 0.05, **: P < 0.01 against vehicle-treated condition (0 "g/mL LPS) at 8 h. Graph bars show the means ± SD (N = 3-6).

we found that > 99% of cells in our EC cultures were VE-cadherin
positive, demonstrating that our primary EC cultures are highly
enriched in ECs and virtually devoid of any contaminating cells
(supplementary Fig. S1).
Considering that changes in cell proliferation and viability
interfere with the analysis of cell migration, we established a non-
proliferative culturing system using a low serum culture medium.
We did not observe any significant increase in cell proliferation or
viability between 0 to 10% FBS up to 8 h of culturing (supplemen-
tary Fig. S2A and C), whereas after 24 h, significant differences in
cell proliferation or viability were detected in medium containing
greater than 2% FBS (supplementary Fig. S2B and D). Thus, under all
experimental conditions (1% FBS), changes in cell proliferation or
viability was not detected. In addition, LPS exposure (1 to 20 "g/ml)
did not show any change neither in cell proliferation (supplemen-
tary Fig. S3A) nor viability (supplementary Fig. S3B) up to 8 h of
culturing.
Since the Toll-like receptor 4 (TLR4) is the receptor for LPS, we
assessed whether TLR4 is involved in the LPS-induced EC migration.
To test this, we used the specific TLR4 inhibitor, CLI-095. LPS-
treated ECs in the presence of CLI-095 did not show any increase
in cell migration measured by Boyden chamber assay (Fig. 2A).
Similar results were obtained by means of wound closure strat-
egy. LPS-treated ECs exposed to CLI-095 suppresses cell migration
in a concentration-dependent pattern (Fig. 2D). Furthermore, the
mean migrated distance did not show any increase using CLI-095
(Fig. 2E). Considering that LPS binds endothelial TLR4 promoting
an intracellular signaling through nuclear factor kappa-B (NF-!B)
activation, we further explored the LPS-induced EC migration using
a membrane permeable I!B kinase inhibitors, IKK-16 and wedelo-
lactone, which inhibits LPS-induced I!B degradation by I!B kinase
(IKK), and prevents NF-!B activation. LPS-treated ECs exposed to
IKK-16 and wedelolactone did not show any increase in EC migra-
tion (Fig. 2B and 2 C, respectively). Analogous results were obtained
16 D. Sarmiento et al. / The International Journal of Biochemistry & Cell Biology 55 (2014) 11–23

Fig. 3. LPS-induced EC migration is inhibited by the antioxidant compounds NAC, the enzyme catalase, and the reducing agent DTT. (A-C) ECs incubated with (+) or without
(-) LPS were subjected to Boyden-chamber migration assay in the absence or presence of NAC (1, 5, and 10 "M) (A), catalase (1, 10, and 100 ng/ml) (B), and DTT (0.1, 1,
and 10 "M) (C). Results are expressed as fold of change relative to vehicle-treated condition (0 "M LPS). (D-I) ECs incubated with (+) or without (-) LPS were subjected to
wound-closure assay in the absence or presence of NAC (1, 5, and 10 "M) (D), catalase (1, 10, and 100 ng/ml) (F), and DTT (0.1, 1, and 10 "M) (H). Results are expressed in
percentage relative to vehicle-treated condition (0 "g/mL LPS). (E, G, and I). Mean migrated distance at 8 h from several experiments as showed in D, F, and H, respectively.
*: P < 0.05, **: P < 0.01 against vehicle-treated condition (0 "g/mL LPS) at 8 h. Graph bars show the means ± SD (N = 3-6).

by means of wound closure assay. LPS-treated cells in the presence
of IKK-16 and wedelolactone failed to increase EC migration (Fig. 2F
and 2H, respectively). Further, the LPS-induced mean migrated dis-
tance was unaffected using IKK-16 and wedelolactone (Fig. 2G and
2I, respectively). We did not observe a significant change in cell
viability neither using CLI-095, IKK-16, nor wedelolactone (sup-
plementary Fig. S4). These results are in agreement with evidence
reported previously demonstrating that TLR4/NF-!B pathway is
involved in the LPS-induced EC migration (Dauphinee et al., 2013).
3.2. Lipopolysaccharide induced-endothelial cell migration is
dependent on intracellular ROS.
We previously reported that LPS generates intracellular ROS
in ECs (Simon and Fernandez, 2009). Thus, we were prompted
to explore whether reducing agents and antioxidants inhibit
LPS-induced EC migration as a consequence of decreasing the intra-
cellular oxidative status. First, we studied the effect of the ROS
scavenger N-acetyl cysteine (NAC) in the LPS-induced migration
of endothelial cells. By means of Boyden chamber strategy we
observed that LPS-treated ECs exposed to NAC did not exhibit
any increase in cell migration (Fig. 3A). Concordant results were
obtained by means of wound closure strategy. LPS-treated ECs
exposed to NAC did not show any increase in cell migration reach-
ing similar levels to that observed in ECs in the absence of LPS
(Fig. 3D). Furthermore, the LPS-induced increase in mean migrated
distance was severely abolished with NAC (Fig. 3E). Next, we tested
whether the enzyme catalyzing H2 O2 decomposition, catalase, par-
ticipates in the LPS-induced EC migration. Our results showed
that ECs incubated with catalase in the presence of LPS exhibited
a similar cell migration than that observed in ECs in absence of
LPS, evaluated by Boyden chamber assay (Fig. 3B). In concordance,
 D. Sarmiento et al. / The International Journal of Biochemistry & Cell Biology 55 (2014) 11–23 17

Fig. 4. LPS-induced EC migration is inhibited by the NAD(P)H oxidase inhibitors DPI and Apocynin, and the PCK inhibitor chelerythrine. (A-C) ECs incubated with (+) or
without (-) LPS were subjected to Boyden-chamber migration assay in the absence or presence of DPI (1, 5, and 50 "M) (A), Apo (0.1, 1, and 10 "M) (B), and chelerythrine
(0.1, 1, and 5 "M) (C). Results are expressed as fold of change relative to vehicle-treated condition (0 "M LPS). (D-I) ECs incubated with (+) or without (-) LPS were subjected
to wound-closure assay in the absence or presence of DPI (1, 5, and 50 "M) (D), Apo (0.1, 1, and 10 "M) (F), and chelerythrine (0.1, 1, and 5 "M) (H). Results are expressed in
percentage relative to vehicle-treated condition (0 "g/mL LPS). (E, G, and I). Mean migrated distance at 8 h from several experiments as showed in D, F, and H, respectively.
*: P < 0.05, **: P < 0.01 against vehicle-treated condition (0 "g/mL LPS) at 8 h. Graph bars show the means ± SD (N = 3-6).

experiments performed by wound closure strategy showed that
ECs incubated with catalase failed to increase cell migration when
cells were exposed to LPS (Fig. 3E). Besides, the mean migrated
distance was unchanged (Fig. 3F). In the same line, the reducing
agent dithiothreitol (DTT) was used and Boyden chamber assay was
performed. DTT exposure inhibited the LPS-induced EC migration
(Fig. 3 C). Similarly, LPS-treated cells exposed to DTT did not show
any increase in EC migration and mean migrated distance mea-
sured by the wound closure assay (Fig. 3H-I). We did not observe a
significant change in cell viability neither using NAC, catalase, nor
DTT (supplementary Fig. S5).
3.3. Lipopolysaccharide induced-endothelial cell migration is
dependent on NAD(P)H oxidase activation and PKC activity.
Considering that NAD(P)H oxidase (NOX) is a important source
of LPS-induced intracellular ROS generation (Simon and Fernandez,
2009), we tested whether NOX activity was involved in the LPS-
induced EC migration. To that end, we used the NOX inhibitors
diphenyleneiodonium (DPI) and apocynin (Apo), and the EC migra-
tion in the presence or absence LPS was measured by means of
Boyden chamber strategy. ECs exposed to DPI and Apo did not
show any increase in LPS-induced EC migration (Fig. 4A and 4B,
respectively). Similar results were obtained by means of wound
closure assay. LPS-treated cells in the presence of DPI and Apo
failed to increase EC migration (Fig. 4D and 4F, respectively). Fur-
ther, the mean migrated distance was virtually unaffected using
DPI and Apo (Fig. 4E and 4G, respectively). Besides, inhibition of
xanthine oxidase and mitochondria using allopurinol and rotenone
respectively, did not affect the endothelial migration induced by
LPS (data not shown). Since NOX is activated by phosphorylation
mediated by PKC activity, we used a generic PKC inhibitor, Chel-
erythrine, to evaluated the participation of PKC in the LPS-induced
EC migration. Using the Boyden chamber assay, ECs exposed to
18 D. Sarmiento et al. / The International Journal of Biochemistry & Cell Biology 55 (2014) 11–23

Fig. 5. LPS induces calcium overload and phosphorylation of FAK. (A) ECs were incubated in the absence or presence of LPS (10 "g/mL) and the calcium overloading was
evaluated by means of the Ca2+ -sensitive fluorescent dye fluo-4. (B-I) Representative images of cellular distribution of phosphorylated focal adhesion kinase (pFAK) on Tyr397
from ECs in the absence (vehicle-treated) or presence (LPS-treated) of 10 "g/mL LPS for 8 h. ECs were subjected to immunocytochemistry experiments to identify p397-FAK
(pFAK) in green (B and F), F-Actin in red (C and G), and merged images (D and H). The box depicted in (D and H) indicates the magnification shown in (E and I), respectively.
Arrowheads indicate FAK points. Nuclei were stained using hoestch (blue). Bar scale represents 10 "m. (J) Bar graph summarizing several experiments as showed in (B-I).
Determinations were done at least in triplicates and results are expressed as number of pFAK points per cells normalized relative to vehicle-treated condition (0 "g/mL LPS).
Several independent fields were counted (N = 8-10). **: P < 0.01. Graph bars show the means ± SD. LPS increases pFAK detection. (K) Representative images from western
blot experiments for detection of pFAK in EC in the absence or presence LPS. Total FAK was used as loading control. (L) Densitometic analysis of experiments as shown in K.
Results are expressed as pFAK/total FAK. **: P < 0.01. Graph bars show the mean ± SD (N = 3).

Chelerythrine did not show any increase in LPS-induced EC migra-
tion (Fig. 4 C). Also, Chelerythrine exposure was efficient in to
abolish the LPS-induced EC migration measured by means of
wound closure assay (Fig. 4H). Additionally, the mean migrated dis-
tance was similar compared with vehicle-treated condition (Fig. 4I).
We did not observe a significant change in cell viability neither
using DPI, Apo, nor Chelerythrine (supplementary Fig. S6). These
results suggest that the NAD(P)H oxidase and PKC are involved in
the mechanism of LPS-induced EC migration.
3.4. Lipopolysaccharide increases the intracellular calcium level
and focal adhesion kinase phosphorylation in endothelial cells
migration (Chen et al., 1996;Eide et al., 1995). Because increased
focal adhesion turnover leads to an increase in cell migration,
increases in FAK phosphorylation is a sign of an increment in cell
migration. Thus, we examined whether LPS induces an increase
in pY397-FAK in ECs. As depicted in Fig. 5B-I, LPS-treated ECs
exhibit an increase in pY397-FAK labeling, primarily at the leading
edge of cells (arrowheads), compared with vehicle-treated ECs. The
quantification of pY397-FAK points per cells showed that LPS treat-
ment significantly increased FAK phosphorylation compared with
vehicle-treated ECs (Fig. 5 J). In according, total pY397-FAK detec-
tion measured by western blot experiments showed a pY397-FAK
increases in ECs exposed to LPS (Fig. 5K-L).

Taking into consideration that cell migration is dependent on
intracellular calcium changes, we were prompted to investigate
whether LPS induces intracellular calcium increases in ECs using
the calcium-sensitive fluorescent probes Fluo-4. EC exposed to LPS
showed a significant increase in intracellular calcium as measured
as a higher Fluo-4 fluorescence compared to vehicle-treated cells
(Fig. 5A). This finding is in agreement to the results showing the
participation of PKC in LPS-induced EC migration (Fig. 4 C, H, and I).
Calcium-dependent PKC activates several proteins involved in
cell migration including the focal adhesion kinase (FAK). It is an
ubiquitously expressed tyrosine kinase, which plays a fundamen-
tal role in cell migration (Mitra et al., 2005). FAK downregulation
inhibits the rate of cell migration and decreases cell spreading (Sieg
et al., 2000). Conversely, FAK overexpression enhances cell migra-
tion (Cary et al., 1996). FAK phosphorylation at Tyr397 (pY397-FAK)
regulates the turnover of focal adhesion points to promote cell
3.5. Lipopolysaccharide induced-endothelial cell migration is
dependent on the TRPM7 ion channel
Considering that LPS increases the expression of TRPM7 (Nunez-
Villena et al., 2011) and that TRPM7 is involved in migration of
human cancer cells (Chen et al., 2010;Rybarczyk et al., 2012;Wei
et al., 2009;Wei et al., 2010), we examined whether TRPM7
participates in LPS-induced EC migration. Due to the lack of specific
inhibitors, we first assessed the effect of polyvalent cations, Gd3+
and Zn2+ , which inhibit a range of ion channels, including TRPM7
(Aarts et al., 2003;Inoue et al., 2010). LPS-treated ECs in the pres-
ence of Gd3+ and Zn2+ did not show any increase in cell migration
measured by Boyden chamber assay (Fig. 6A and B, respectively).
Similar results were obtained by means of wound closure strategy.
Gd3+ and Zn2+ inhibited the increase of LPS-induced EC migration
(Figure 6D and F, respectively) and reduced the mean migrated
 D. Sarmiento et al. / The International Journal of Biochemistry & Cell Biology 55 (2014) 11–23 19

Fig. 6. LPS-induced cell migration is inhibited by Gd3+ , Zn2+ , and 2APB. (A-C) ECs incubated with (+) or without (-) LPS (1, and 10 "g/ml) were subjected to Boyden-chamber
migration assay in the absence or presence of 10 "M Gd3+ (A), 10 "M Zn2+ (B), and 200 "M 2-APB (C). Results are expressed as fold of change relative to vehicle-treated
condition (0 "M LPS). (D-I) ECs incubated with (+) or without (-) LPS (1, and 10 "g/ml) were subjected to wound-closure assay in the absence or presence of 10 "M Gd3+
(D), 10 "M Zn2+ (F), and 200 "M 2-APB (H). Results are expressed in percentage relative to vehicle-treated condition (0 "g/mL LPS). (E, G, and I). Mean migrated distance
at 8 h from several experiments as showed in D, F, and H, respectively. *: P < 0.05, **: P < 0.01 against vehicle-treated condition (0 "g/mL LPS) at 8 h. Graph bars show the
means ± SD (N = 3-5).

distance (Figure 6E and G), suggesting the participation of a TRPM7-
like ion channel. Similar results were observed using lower doses
of Gd3+ and Zn2+ (supplemental Figure S7).
Next, we used the calcium channel blocker, 2-
aminoethoxydiphenyl borate (2-APB), which inhibits TRPM7
activity in a different manner than polyvalent cations (Kovacs
et al., 2012;Togashi et al., 2008). Figure 6 C shows that 2-APB
inhibited the increase of LPS-induced EC migration measured by
the Boyden chamber assay. Consistently, using the wound closure
strategy, 2-APB treatment also inhibited the LPS-induced EC migra-
tion (Figure 6H) and the mean migrated distance (Figure 6I). Similar
results were obtained using a lower dose of 2-APB (supplemental
Figure S8).
Considering that TRPM7 blockers are not totally specific, we
used small interference RNA (siRNA) to knockdown TRPM7 expres-
sion and unequivocally demonstrate that TRPM7 participates in
LPS-induced EC migration. ECs were transfected with a specific
siRNA against TRPM7 (siTRPM7). Transfected ECs were exposed
to LPS and subsequently subjected to migration assays. The siRNA
efficiency for the knockdown of mRNA and protein of TRPM7
expression was >95% (Fig. 7A-C). Moreover, as we showed pre-
viously, the siTRPM7 effect is specific as the expression of other
TRPMs channels was not affected (supplemental Figure S9). The
TRPM7 knockdown abolished the LPS-induced calcium influx
suggesting that the intracellular calcium increase is mediated by
TRPM7 (Fig. 7D).
ECs transfected with a non-targeting sequence (siCTRL) showed
an increment in cell adhesion after exposure to LPS (Fig. 8A), as
observed in non-transfected cells. In contrast, ECs transfected with
a siTRPM7 and exposed to LPS showed a significant inhibition in
LPS-induced EC adhesion (Fig. 8A).
Consistent with the cell adhesion experiments, LPS-treated
ECs transfected with siCTRL showed an increase in cell exten-
sion spreading (Fig. 8B-C) similar to non-transfected cells, whereas
20 D. Sarmiento et al. / The International Journal of Biochemistry & Cell Biology 55 (2014) 11–23
Fig. 7. TRPM7 downregulation inhibits calcium overload. (A) Changes in the expres-
sion of mRNA of TRPM7 by qPCR. Endothelial cells were transfected with a specific
siRNA against human isoform of TRPM7 (siTRPM7) and a non-targeting siRNA (siC-
TRL) used as a control. Results are expressed relative to cells transfected with
siRNA-CTRL condition and normalized against 28S. ***: P < 0.001. Graph bars show
the mean ± SD (N = 3). (B-C) Changes in the expression of the protein of TRPM7 by
western blot. Endothelial cells were transfected with siRNA-TRPM7 and siRNA-CTRL
and then protein expression was analyzed. (B) Representative images from west-
ern blot experiments performed for detection of TRPM7. (C) Densitometric analyses
from several experiments, as shown in (B). Protein levels were normalized against
tubulin, and the data are expressed relative to siRNA-CTRL. ***: P < 0.001. Graph
bars show the mean ± SD (N = 3). (D) Endothelial cells were transfected with siRNA-
TRPM7 and siRNA-CTRL and incubated in the absence or presence of LPS (10 "g/mL).
Then, calcium overloading was evaluated by means of the Ca2+ -sensitive fluorescent
dye fluo-4. **: P < 0.01 against vehicle-treated condition (0 "g/mL LPS). Graph bars
show the means ± SD (N = 3).
LPS-treated ECs transfected with siTRPM7 abolished LPS-induced
cell extension spreading (Fig. 8B-C). The quantification of this
experiment showed that TRPM7 downregulation inhibited LPS-
induced cell spreading up to vehicle-treated levels (Fig. 8 C).
Moreover, we assayed migratory behavior using the Boyden-
transwell migration assay. ECs transfected with siCTRL and exposed
to LPS showed migration similar to that observed in non-
transfected cells (Fig. 8D-E), whereas ECs transfected with siTRPM7
and exposed to LPS showed a statistically significant inhibition of
cell migration (Fig. 8D-E).
In addition, ECs transfected with siCTRL and exposed to LPS
showed a wound closure percentage increase (Fig. 8F-G) similar to
that observed in non-transfected cells. In contrast, ECs transfected
with siTRPM7 showed an inhibition in LPS-induced EC wound clo-
sure (Fig. 8F-G), and the mean migrated distance was also decreased
(Fig. 8H).
4. Discussion
Endothelial dysfunction is a key feature in the pathogenesis
of inflammatory diseases. In the context of endotoxemia-derived
inflammatory disease, we recently reported that LPS induces
the conversion of endothelial cells into activated fibroblasts,
showing a protein expression profile characteristic of myofibro-
blasts (Echeverria et al., 2014;Echeverria et al., 2013). However,
whether LPS promotes endothelial migration remains unknown.
Although, the mechanism of LPS-induced EC migration has been
studied, several issues remain to be elucidated.
In this study, we demonstrated that LPS induced a dose-
dependent EC migration. Furthermore, we demonstrated the
participation of the TLR-4/NF-!B pathway and the generation of
ROS through the PKC-activated enzyme NAD(P)H oxidase in the
LPS-induced EC migration. In addition, TRPM7-mediated calcium
influx and phosphorylated FAK was increased in ECs after LPS
exposure. Furthermore, the downregulation of TRPM7 significantly
decreases LPS-induced EC migration, suggesting that TRPM7 is
critically involved in LPS-induced EC migration. Findings showed
here were summarized in a integrative proposed model (Fig. 9).
Cell migration is a major and distinctive feature of fibroblasts.
Fibroblasts migrate to the site of injured tissue to initiate the
healing process. However, during severe inflammation, fibroblasts
promote tissular fibrosis, leading to lost organ function. In con-
trast to previous assumptions, fibroblasts are not the sole source of
pathological fibrosis. Endothelial cells are also sources of activated
fibroblasts during inflammation, revealing a new field of interest
for basic and clinical researchers. In the context of endotoxemia-
derived inflammation, the fact that ECs exposed to LPS acquire a
migratory phenotype suggests a mechanism promoting vascular
dysfunction and subsequent organ failure.
During endotoxemia, ECs exposed to LPS could migrate from
the endothelial monolayer placed in the inner wall of blood ves-
sels to reach more external tissues in the vessel. Furthermore,
migrating ECs could even reach deep tissues in the organs, and
as a consequence of the LPS-induced endothelial fibrosis, the new
myofibroblast could be located not only in the endothelium of ves-
sels but also in external regions and even in several other tissues.
The fibrosed endothelium could promote vascular dysfunction, as
fibrosis avoids the replacement of injured ECs and impairs the
function of the remaining healthy ECs. Once established, vascu-
lar dysfunction could generate severe effects on organ function
through defects in blood perfusion, nutrient supply, and molecule
exchange, among others. However, further experiments are needed
to demonstrate this speculation. On the other hand, several cell
types including endothelial cells show that activation of NF-!B
stimulates cytokine production. Thus, it is plausible to propose that
LPS effects in cell migration are mediated by the cytokine produc-
tion. Additional experiments are needed to elucidate that issue.
Increments in cell adhesion and spreading are indicators of
enhanced cell migration. Consistently, our results showed that LPS
increases cell adhesion and spreading. These findings were con-
firmed using transwell and wound-closure assays to measure cell
migration. Thus, different experimental strategies have revealed
that LPS promotes a significant increase in EC migration.
Because cell migration inhibition experiments involve the use
of calcium channel blockers, it is well accepted that calcium
channels-mediated calcium influx is necessary for cell migration
(Wu et al., 1997;Yoshikawa et al., 1999). Thus, the identification
of these calcium channels is an issue of significant importance.
Here, we demonstrated that TRPM7 is critically involved in LPS-
induced EC migration. These results are consistent with those
showing that TRPM7 regulates the migration of human nasopha-
ryngeal carcinoma cells through Ca2+ influx (Chen et al., 2010).
In addition, our data are consistent with findings showing that
the migration of pancreatic cancer cells and fibroblasts is depend-
ent on TRPM7 (Rybarczyk et al., 2012;Wei et al., 2009;Wei et al.,
2010). Considering that LPS induces ROS generation and TRPM7
activity are regulated through ROS, it is possible to suggest that
LPS-induced ROS generation modulates TRPM7 to promote EC
migration (Nunez-Villena et al., 2011;Sarmiento et al., 2014). How-
ever, our data are in contrast to experiments showing that TRPM7
downregulation increases EC migration (Baldoli et al., 2013). In this
study, the authors performed wound-closure assay as the unique
experimental approach and did not use LPS to induce cell migration.
These differences could explain such discrepancy.
Single cellular processes are frequently performed through dif-
ferent proteins depending on the acting stimulus. Considering that
angiotensin II-induced EC migration is mediated through the T-type
calcium channel (Martini et al., 2010), it is possible that different
calcium channels regulate EC migration induced through differ-
ent stimuli. In the present study, TRPM7 potentially controls EC
migration when an inflammatory stimulus is present. This obser-
vation is consistent with findings showing that TRPM7 regulates
 D. Sarmiento et al. / The International Journal of Biochemistry & Cell Biology 55 (2014) 11–23 21

Fig. 8. TRPM7 downregulation inhibits LPS-induced EC migration. (A) ECs transfected with a non-targeting siRNA (siCTRL) or siRNA against TRPM7 (siTRPM7) were incubated
in the absence or presence of LPS (1, and 10 "g/mL) for 20 min and subjected to cell adhesion assay. Then, cells were removed and adherent cells were fixed and stained with
crystal violet. Cells were solubilized, and the released crystal violet was measured. Determinations were done at least in triplicates and results are expressed in percentage
normalized relative to vehicle-treated condition (0 "g/mL LPS). *: P < 0.05 against vehicle-treated condition. Graph bars show the mean ± SD (N = 3-4). (B) Representative
images obtained from ECs transfected with a siCTRL or siTRPM7 in the absence or presence of LPS (1, and 10 "g/mL) for 40 min, stained with crystal violet and subjected
to spreading assay. Arrows indicate spread cell. Bar scale represents 25 "m. (C) Bar graph summarizing several experiments as showed in (B). (D) Representative images
obtained from ECs transfected with siCTRL or siTRPM7 and incubated in the absence or presence of LPS (10 "g/mL) and subjected to Transwell migration assay. ECs were
placed in the upper compartment of a Transwell chamber in 1% FBS whereas in the lower chamber 10% FBS was added. Migration was allowed to occur for 24 h and migrated
cells were fixed and stained with crystal violet. Bar scale represents 50 "m. (E) Bar graph summarizing several experiments as showed in (D). (F) Representative images
obtained from ECs transfected with siCTRL or siTRPM7 and incubated in the absence or presence of 10 "g/mL LPS and subjected to a wound-closure assay. Images were
taken at 0 and 8 h. To visualize the uncovered area of each wound, cell margins were outlined in each image. Bar scale represents 50 "m. (G) Graph summarizing several
experiments as showed in (F), in which ECs transfected with siCTRL or siTRPM7 were incubated in the absence or presence of LPS (0.1, 1, 5, and 10 "g/mL). (H) Mean migrated
distance at 8 h from several experiments as showed in (F). *: P < 0.05, **: P < 0.01 against vehicle-treated condition (0 "g/mL LPS). Graph bars show the means ± SD (N = 4-6).

the migration of cells in the inflammatory environment of tumors
as pancreatic cancer cells (Rybarczyk et al., 2012) and nasopharyn-
geal carcinoma cells (Chen et al., 2010), and that TRPM7 is involved
in LPS-induced endothelial fibrosis (Echeverria et al., 2014). Fur-
thermore, we observed that inhibition of voltage-gated L-type and
T-type calcium channels, and TRPV1 and TRPC3 calcium channels,
did not show any change in LPS-induced EC migration, suggesting
that these channels are not involved in the endothelial migration
induced by LPS (data not shown). Further experiments are needed
to demonstrate the participation of these channels.
In contrast to studies showing that increases in [Ca2+ ]i pro-
mote cell migration, intracellular Ca2+ concentration is reduced in
22 D. Sarmiento et al. / The International Journal of Biochemistry & Cell Biology 55 (2014) 11–23
Fig. 9. Proposed model of TRPM7-mediated LPS-induced EC migration. i) It has
been extensively reported that LPS performs its actions through its receptor TLR4,
with subsequent NF-!B activation. Thus, it is likely that the downstream effects
of LPS (i.e., its stimulation of EC migration) are initiated by the activation of the
TLR4/NF-!B pathway. ii) The activation of the TLR4/NF-!B pathway involves the
production of ROS derived from NAD(P)H oxidase activation. This ROS production
could be supported by NF-!B through positive feedback. iii) Furthermore, NAD(P)H
oxidase activation is mediated by PKC-dependent phosphorylation. Additionally, it
has been reported that FAK is directly or indirectly activated by PKC. Interestingly,
it has been reported that ROS production could stimulate TRPM7 calcium channel
activity. iv) Our previous studies performed in neurons showed that the LPS-induced
neural calcium increase is dependent on TRPM7 expression. v) In addition, we very
recently reported that LPS-treated HUVECs showed an increased intracellular cal-
cium level that was dependent on TRPM7 during endothelial fibrosis. Thus, it is
plausible that this protein is responsible for the intracellular calcium increase upon
LPS challenge in the context of EC migration. vi) Such calcium increases activate
several process, including PKC activation, NF-!B stabilization, and gene expression
to support the migratory event. LPS, lipopolysaccharide; TRPM7, transient receptor
potential melastatine 7; TLR4, toll-like receptor-4; NF-!B, nuclear factor-kappa B;
PKC, protein kinase C; FAK, focal adhesion kinase; ROS, reactive oxygen species.
migrating ECs, particularly at the leading edge (Kimura et al., 2001).
These findings suggest that a precise time- and/or site-specific reg-
ulation in [Ca2+ ]i is required for EC migration. Therefore, TRPM7
downregulation could modulates EC migration through alterations
in the dynamics of intracellular Ca2+ concentration.
To generate an increased migration phenotype, the LPS dis-
rupted the intracellular signals that control cell migration. Several
proteins regulate cell migration, including the focal adhesion kinase
(FAK), a well-studied cell migration protein. FAK plays a critical role
in the regulation of cell adhesion, spreading, invasion and migra-
tion. Indeed, FAK activation has been associated with increased
migration phenotypes (Mitra et al., 2005;Schlaepfer and Mitra,
2004). The suppression of FAK expression reduced cell migration
and spreading (Sieg et al., 2000). The supplementation with FAK
restores the migrating phenotype in FAK-deficient cells. The re-
introduction of dominant-negative FAK in FAK mutants lacking
kinase activity did not restore cell migration (Schlaepfer et al.,
1999;Sieg et al., 2000), and increased FAK expression enhanced
the migration of CHO cells (Cary et al., 1996). Consistently, FAK
overexpression has been observed in several tumors, such as
endometrial neoplasia and vascular intimal hyperplasia (Livasy
et al., 2004;Owens et al., 1995;Owens et al., 2001). Several phos-
phorylation sites in FAK are crucial to induce cell migration.
Phosphorylation at Tyr397 (pY397-FAK) enhances cell migration
through the recruitment of several focal contact proteins that
regulate the turnover of focal adhesion sites (Chen et al., 1996;Eide
et al., 1995). Thus, increases in pY397-FAK promote increased cell
migration. Our results showed a significant increment in pY397-
FAK after treatment with LPS, suggesting an increment in cell
migration. Alternatively, LPS promotes several genomic and non-
genomic cellular effects. Thus, we cannot exclude the possibility of
complementary pathway activation to generate cell migration. It
has been reported that LPS induces ROS generation in endothe-
lial cells (Simon and Fernandez, 2009). Here, we demonstrated
that NAD(P)H oxidase-mediated ROS generation participates in the
LPS-induced EC migration. Increased intracellular oxidative sta-
tus promotes an increment in protein phosphorylation mediated
through phosphatase activity inhibition. Thus, increases in pY397-
FAK could be promoted by the LPS-induced phosphatase activity
inhibition.
Taken together, the findings presented here provide evidence
that LPS is a sufficient and crucial factor in generating a fibroblast-
like migratory function as part of a major process for the induction
of endothelial fibrosis in which the reprogramming of EC protein
expression converts healthy ECs into pathogenic cells. Further-
more, our findings demonstrate that TRPM7 plays a decisive role
in LPS-induced endothelial cell migration, suggesting a novel drug
target for improving the current treatment against endotoxaemia-
derived sepsis syndrome and several other inflammatory
diseases.
Conflict of interest.
The authors confirm that there are no conflicts of interest.
Acknowledgments
This work was supported by research grants from Fondo
Nacional de Desarrollo Científico y Tecnológico - Fondecyt 1121078
(FS), 11121239 (OC), 1120380 (CCV), 1120976 (RF). Millennium
Institute on Immunology and Immunotherapy P09-016-F (FS),
Association-Francaise Contre Les Myopathies AFM 16670 (CCV),
UNAB-DI-281-13/R (CCV), and UNAB-DI-67-12/I (CE). The funders
had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript. The authors are grateful
to Director Dr. Iván Oyarzún and Dr. Mario Carmona, Dr. Jaime Men-
doza and Mrs. Juana Belmar from Servicio Ginecología y Obstetricia,
Hospital San Jose de Melipilla.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.biocel.
2014.08.001.
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