IAI Accepts, published online ahead of print on 16 June 2014 Infect. Immun. doi:10.1128/IAI.02158-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Endotoxin-induced endothelial fibrosis is dependent on the expression of TGF-β1 and
2
TGF-β2
3 4 5
César Echeverríaa,d, Ignacio Montorfanoa, Pablo Tapiae, Claudia Riedelc, Claudio Cabello-
6
Verrugiob, and Felipe Simona,f,#
7 8
a
Laboratorio de Fisiopatología Integrativa, bLaboratorio de Biología y Fisiopatología
9
Molecular, cLaboratorio de Biología Celular y Farmacología, Departamento de Ciencias
10
Biologicas, Facultad de Ciencias Biologicas and Facultad de Medicina, Universidad Andres
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Bello, Avenida Republica 239, Santiago, Chile.
12 13 14 15 16
d
Laboratorio de Bionanotecnología, Universidad Bernardo O'Higgins, General Gana 1780,
Santiago, Chile. e
Departamento de Medicina Intensiva, Facultad de Medicina, Pontificia Universidad
Católica de Chile, Santiago, Chile. f
Millennium Institute on Immunology and Immunotherapy, Santiago, Chile.
17 18
Running title: Endotoxin-induced endothelial fibrosis via TGF-β
19 20
# Address correspondence to Felipe Simon, [email protected]
1
21
Abstract
22 23
During endotoxemia-induced inflammatory disease, bacterial endotoxins circulate
24
in the bloodstream and interact with endothelial cells (ECs), inducing dysfunction of the
25
ECs. We have previously reported that endotoxins induce the conversion of ECs into
26
activated fibroblasts. Through endotoxin-induced endothelial fibrosis, ECs change their
27
morphology and their protein pattern expression, thereby suppressing endothelial markers
28
and upregulating fibrotic proteins. The most commonly used fibrotic inducers are TGF-β1
29
and TGF-β2. However, whether TGF-β1 and TGF-β2 participate in endotoxin-induced
30
endothelial fibrosis remains unknown. We have shown that the endotoxin-induced
31
endothelial fibrosis process is dependent on the TGF-β receptor, ALK5, and the activation
32
of Smad3, a protein that is activated by ALK5 activation, thus suggesting that endotoxin
33
elicits TGF-β production to mediate endotoxin-induced endothelial fibrosis. Therefore, we
34
investigated the dependence of endotoxin-induced endothelial fibrosis on the expression of
35
TGF-β1 and TGF-β2.
36
Endotoxin-treated ECs induced the expression and secretion of TGF-β1 and TGF-
37
β2. TGF-β1 and TGF-β2 downregulation inhibited the endotoxin-induced changes in the
38
endothelial marker, VE-cadherin, and in the fibrotic proteins, α-SMA and fibronectin.
39
Thus, endotoxin induces the production of TGF-β1 and TGF-β2 as a mechanism to promote
40
endotoxin-induced endothelial fibrosis.
41
To the best of our knowledge, this is the first report showing that endotoxin induces
42
endothelial fibrosis via TGF-β secretion, which represents an emerging source of vascular
43
dysfunction. These findings contribute to understanding the molecular mechanism of
2
44
endotoxin-induced endothelial fibrosis, which could be useful in the treatment of
45
inflammatory diseases.
46 47
Keywords: Endothelial dysfunction; endotoxin; inflammation; fibrosis; TGF-β.
3
48
Introduction
49 50
The initiation and progression of systemic inflammation, including sepsis syndrome
51
and septic shock, result in high morbimortality rates (1,2). During systemic infection, the
52
bloodstream is full of pro-inflammatory cytokines, reactive oxygen species (ROS), and
53
active immune cells. In the case of endotoxemia-induced sepsis syndrome, large amounts of
54
the Gram-negative bacterial endotoxin, lipopolysaccharide (LPS) (3,4), are deposited and
55
circulate in the bloodstream, interacting with the endothelial cells (ECs) located in the
56
internal endothelial monolayer of blood vessels, which induces detrimental effects on
57
endothelium function (5-7). Dysfunction of ECs is a crucial step in the pathogenesis of
58
endotoxemia-derived sepsis syndrome and other inflammatory diseases (5,8).
59
We have previously reported that bacterial endotoxins induce the conversion of ECs
60
into activated fibroblasts, also known as myofibroblasts (9). Endotoxin-induced endothelial
61
fibrosis is mediated through a process known as endothelial-to-mesenchymal transition
62
(EndMT), which has been studied using tumor growth factor-beta 1 and 2 (TGF-β1 and
63
TGF-β2) as EndMT inducers (10,11). However, the role of TGF-β1 and TGF-β2 in
64
endotoxin-induced endothelial fibrosis is currently unknown.
65
Endotoxin-induced endothelial fibrosis is morphologically identified by the change
66
of the round shaped EC monolayer with a short-spindle shape and a cobblestone
67
appearance into a spindle-shaped fibroblast-like phenotype (9,10). At the level of protein
68
expression, endotoxin-induced endothelial fibrosis is characterized by downregulation of
69
the endothelial markers, CD31 and VE۔cadherin, as well as by upregulation of the
70
fibroblast-specific genes, α۔smooth muscle actin (α-SMA) and fibroblast-specific protein-1
4
71
(FSP-1). Additionally, the levels of proteins that form the extracellular matrix (ECM), such
72
as fibronectin (FN) and collagen type III (Col III), are severely enhanced (9,10).
73
The endotoxin-induced endothelial fibrosis process is dependent on the expression
74
of the TGF-β receptor, activin receptor-like kinase 5 (ALK5), because its downregulation
75
and pharmacological inhibition are effective in inhibiting the endothelial fibrosis induced
76
by endotoxins (9). The ALK5 intracellular pathway subsequently activates Smad3 protein
77
by phosphorylation (12,13). Inhibition of Smad3 activation abolishes the endotoxin-
78
induced endothelial fibrosis process (9). This evidence indicates that the ALK5 receptor is
79
involved in endotoxin-induced endothelial fibrosis. Taken together, these findings strongly
80
suggest that endotoxin elicits TGF-β synthesis and secretion, resulting in autocrine and
81
paracrine effects on ECs to mediate endotoxin-induced endothelial fibrosis.
82 83
Therefore, the aim of this study was to investigate if endotoxin-induced endothelial fibrosis is dependent on the expression of TGF-β1 and TGF-β2.
84
Our data demonstrated that when ECs are exposed to endotoxin, the expression and
85
secretion of TGF-β1 and TGF-β2 are induced. In addition, ECs exposed to endotoxin
86
exhibited increased p38 MAPK phosphorylation. Furthermore, suppression of TGF-β1 and
87
TGF-β2 expression was effective in inhibiting the endotoxin-induced decrease of the
88
endothelial marker, VE-cadherin. Additionally, the downregulation of TGF-β1 and TGF-β2
89
was sufficient to abolish the endotoxin-induced increase in the fibrotic marker, α-SMA, and
90
the ECM protein, fibronectin.
91 92
We concluded that endotoxin is capable of inducing the expression and secretion of TGF-β1 and TGF-β2 as a mechanism to promote endotoxin-induced endothelial fibrosis.
93
These results contribute to a better understanding of the molecular basis of
94
endotoxin-induced endothelial fibrosis, which could be useful in improving current 5
95
therapeutic strategies in treating endothelial dysfunction during endotoxemia and other
96
inflammatory diseases.
6
97
Materials and methods
98
Details of all procedures are provided in Additional Supporting Information
99 100
Primary cell culture
101
Human umbilical vein endothelial cells (HUVECs) were isolated by collagenase
102
(0.25 mg/ml) digestion from freshly obtained umbilical cord veins from normal
103
pregnancies, after patients' informed consent. The investigation conforms with the
104
principles outlined in the Declaration of Helsinki. The Commission of Bioethics and
105
Biosafety of Universidad Andres Bello also approved all experimental protocols. Cells
106
were grown in gelatin-coated dishes at 37°C in a 5%:95% CO2:air atmosphere in medium
107
199 (Sigma, MO), containing 100 µg/ml endothelial cell growth supplement (ECGS;
108
Sigma), 100 µg/ml heparin, 5 mM D-glucose, 3.2 mM L-gutamine, 10% fetal bovine serum
109
(FBS; GIBCO, NY), and 50 U/ml penicillin-streptomycin (Sigma).
110 111
Small-interfering RNA and transfections
112
SiGENOME SMARTpool siRNA (four separated siRNAs per each human TGF-β1
113
or TGF-β2 transcript) were purchased from Dharmacon (Dharmacon, Lafayette, CO). The
114
following siRNAs were used: siRNA against human TGF-β1 (siTGFβ1), siRNA against
115
human TGF-β2 (siTGFβ2), and non-targeting siRNA (siCTRL) used as a control. In brief,
116
HUVECs were plated overnight in a 6-well plate and then transfected with 5 nM siRNA
117
using DharmaFECT 4 transfection reagent (Dharmacon) used according to the
118
manufacturer's protocol in serum-free medium for 24 hours. Experiments were performed
119
48 to 72 hours after transfection.
120 7
121
RNA isolation and quantitative real-time PCR
122
QPCR experiments were performed to measure TGF-β1 and TGF-β2 mRNA levels
123
in HUVECs. Total RNA was extracted with Trizol according to the manufacturer's protocol
124
(Invitrogen, Carlsbad, CA). DNAse I-treated RNA was used for reverse transcription using
125
the Super Script II Kit (Invitrogen, Carlsbad, CA). Equal amounts of RNA were used as
126
templates in each reaction. QPCR was performed using the SYBR Green PCR Master Mix
127
(AB Applied Biosystems, Foster City, CA). All reactions were performed in triplicate on an
128
Eco Real-Time PCR System (Illumina, USA). Data are presented as relative mRNA levels
129
of the gene of interest normalized to relative levels of 28S mRNA.
130 131
Western blot procedures
132
Cells were lysed in cold lysis buffer, and proteins were then extracted. Supernatants
133
were collected and stored in the same lysis buffer. Protein extract and supernatant were
134
subjected to SDS-PAGE, and resolved proteins were transferred to a nitrocellulose or
135
PVDF membrane. The blocked membrane was incubated with the appropriate primary
136
antibody, washed twice, and incubated with a secondary antibody. Bands were detected
137
using
138
chemiluminescence (Thermo Scientific, USA). Tubulin was used as a loading control.
139
Images were acquired using the Fotodyne FOTO/Analyst Luminary Workstation
140
(Fotodyne, Inc., Hartland, WI). Protein content was determined by densitometric scanning
141
of immunoreactive bands, and intensity values were obtained by densitometry of individual
142
bands normalized against control. For a detailed list of antibodies used, see Supplementary
143
Table S1.
a
peroxidase-conjugated
IgG
antibody
and
visualized
by
enhanced
144 8
145
Fluorescent Immunocytochemistry
146
ECs were washed twice with PBS and fixed. The cells were subsequently washed
147
again and incubated with primary antibodies. Cells were then washed twice and incubated
148
with secondary antibodies. Samples were mounted with ProLong Gold antifade mounting
149
medium with DAPI (Invitrogen). For a detailed list of antibodies used, see Supplementary
150
Table S2.
151 152
Collagen gel contraction assay
153
Twenty-four well plates were precoated with 1% agarose. Type I collagen was
154
diluted in medium and adjusted to pH 7.4. (collagen I in 0.02 M acetic acid, 0.1 M NaOH,
155
serum-free culture medium containing sodium bicarbonate). ECs were trypsinized when
156
80% confluent and resuspended at 8x105 cells/ml. Equal volumes of collagen and cells
157
were then combined to give 1 mg collagen and 4x105 cells/ml and 500 μL pipetted into
158
each well. Gels were polymerized at 4°C ON. Then, gels were fixed in formaldehyde and
159
washed twice with Tris-buffered saline with 0.2% Tween-20. Detergent was present to
160
reduce surface tension. The area of gels were measured and also gels were weighed on an
161
analytical digital scale with a precision of 0.1 mg, at 24 h after stimulus. Contraction was
162
measured relative to the initial gel weigh and normalized against control condition. All
163
assays were repeated 3 times with triplicate wells per experimental condition.
164 165
Cell migration measurement by transwell assay
166
The capacity of ECs to migrate was assayed using Transwell Boyden chambers
167
(Costar, Cambrige, MA, USA) with 8.0-μm-pore polycarbonate filters. Cells were seeded
9
168
in absence or presence LPS (10 μg/ml) for 24 h in 1% FBS on the upper compartment of
169
the chamber. To stimulate cell migration, 10% FBS was added to the lower compartment of
170
the chamber. Thus, migration was allowed to occur for 24 h. After washing, non-invading
171
cells were removed from the upper surface of the membrane with a cotton swab. The
172
invading cells were fixed with 10 % ethanol for 5 min and stained with 0.2% crystal violet
173
for 5 min. Images were captured through a digital microscope system. Cell migration was
174
evaluated by counting four fields per chamber in every condition. All assays were
175
performed at least in triplicates in three separates sets of experiments.
176 177
Reagents
178
The endotoxin, lipopolysaccharide from E. coli, was purchased from Sigma
179
(0127:B8). Apocynin and NAC were purchased from Sigma-Aldrich. The TLR4 inhibitor,
180
CLI-095 was purchased from InvivoGen. Buffers and salts were purchased from Merck
181
Biosciences (Darmstadt).
182 183
Data analysis
184
All results are presented as the means ± SD. Student's t-test and ANOVA followed
185
by Dunn's post hoc tests were used and considered significant at p 99% of cells in the EC culture were positive for VE-
221
cadherin, whereas those expressing FSP-1 were not detected. Thus, the primary human EC
222
cultures used here were highly enriched in ECs (Supp. Fig. S2).
223 224 225
Expression of TGF-β1 and TGF-β2 is crucial for the modification of endothelial and fibrotic marker expression induced by endotoxin in endothelial cells.
226 227
Considering that the expression of TGF-β1 and TGF-β2 is induced by endotoxin, we
228
investigated if the expression of these cytokines was necessary for the progression of
229
endotoxin-induced endothelial fibrosis. Thus, we used two specific siRNAs against the
230
human TGF-β isoforms, TGF-β1 and TGF-β2. Additionally, a non-targeting siRNA
231
(siCTRL) was used as a control. We studied the specificity and effectiveness of the siRNAs
232
on the inhibition of TGF-β1 and TGF-β2. ECs transfected with the siRNAs against TGF-β1
233
(siTGFβ1) and TGF-β2 (siTGFβ2) showed a significant inhibition of the expression of 12
234
TGF-β1 and TGF-β2, respectively (Fig. 2A-B and Fig. 2C-D, respectively). Noteworthy,
235
siTGFβ1 also inhibited the expression of TGF-β2 (Fig. 2C-D), but siTGFβ2 was specific
236
because the expression of TGF-β1 was not affected (Fig. 2A-B). This finding was in
237
accordance with previously reported data (17). We then tested whether downregulation of
238
TGF-β1 and TGF-β2 changed the protein levels of VE-cadherin, α-SMA, and fibronectin.
239
ECs transfected with siTGFβ1 and siTGFβ2 did not show any change in the protein level of
240
VE-cadherin (Fig. 2E-F), and they showed a slight but significant decrease in the fibrotic
241
proteins, α-SMA (Fig. 2G-H) and FN (Fig. 2I-J), thereby suggesting that the suppression of
242
siTGFβ1 and siTGFβ2 expression is not involved in the fibrotic processes.
243
We then tested whether endotoxin-induced endothelial fibrosis was dependent on
244
the expression of TGF-β1 and TGF-β2. ECs were transfected with siTGFβ1 or siTGF-β2,
245
and the changes in endothelial and fibrotic markers were measured. ECs transfected with
246
siCTRL and exposed to endotoxin exhibited a decrease in the protein level of the
247
endothelial marker, VE-cadherin (Fig. 3A-B and E-F), and an increase in the fibrotic
248
marker, α-SMA (Fig. 3C-D and G-H), which was similar to what has been observed in non-
249
transfected wild type ECs exposed to endotoxin (9,10,18). Of note, ECs transfected with
250
siTGFβ1 or siTGFβ2 and exposed to endotoxin were resistant to fibrosis development
251
because those cells did not show any significant change in the protein levels of VE-
252
cadherin (Fig. 3A-B and E-F) or α-SMA (Fig. 3C-D and G-H). These data suggested that
253
TGF-β1 and TGF-β2 expression is crucial in endotoxin-induced endothelial fibrosis.
254
Next, we evaluated the action of TGF-β1 and TGF-β2 expression on the cellular
255
localization and distribution of endothelial and fibrotic proteins. ECs transfected with
256
siCTRL in the absence of endotoxin showed VE-cadherin (Fig. 4A) and CD31 (Fig. 4B)
257
labeling localized at the plasma membrane. In contrast, the expression of the fibrotic 13
258
markers, FSP-1 (Fig. 4A) and α-SMA (Fig. 4B), was weak. Analogous results were
259
observed for ECs transfected with siTGFβ1 (Fig. 4C-D) and siTGFβ2 (Fig. 4E-F) in the
260
absence of endotoxin. These results were also similar to those observed in non-transfected
261
wild type ECs without LPS showing a round-shaped monolayer with short-spindle
262
morphology and a cobblestone appearance (9,10,18). However, ECs transfected with
263
siCTRL and exposed to endotoxin showed a decrease in the endothelial proteins, VE-
264
cadherin (Fig. 4G) and CD31 (Fig. 4H). Furthermore, endotoxin exposure induced an
265
increase in the fibrotic markers, FSP-1 (Fig. 4G) and α-SMA (Fig. 4H). These effects
266
showed a spindle-shaped, fibroblast-like phenotype with a loss of cell-to-cell connections.
267
Noteworthy, ECs transfected with siTGFβ1 (Fig. 4I-J) or siTGFβ2 (Fig. 4K-L) and exposed
268
to endotoxin were resistant to fibrosis progression because transfected ECs did not exhibit
269
changes in endothelial or fibrotic markers. These results confirmed that TGF-β1 and TGF-
270
β2 expression is crucial in the alteration of the cellular localization and distribution of the
271
proteins involved in endotoxin-induced endothelial fibrosis.
272 273 274
Expression of TGF-β1 and TGF-β2 is crucial for the increase of extracellular matrix proteins induced by endotoxin in endothelial cells.
275 276
It is well-known that oversecretion of ECM proteins is an important factor for the
277
progression of fibrogenesis (16,19). Hence, we tested if the increase in the ECM protein,
278
fibronectin, was dependent on the expression of TGF-β1 and TGF-β2. ECs were transfected
279
with siTGFβ1 or siTGFβ2, and the change in fibronectin levels was measured. ECs
280
transfected with siCTRL and exposed to endotoxin exhibited an increase in the ECM
281
marker, fibronectin (Fig. 5), compared with cells cultured in the absence of endotoxin. 14
282
These findings were similar to non-transfected wild type ECs exposed to endotoxin
283
(9,10,18). Importantly, ECs transfected with siTGFβ1 or siTGFβ2 and exposed to
284
endotoxin did not show any increase in fibronectin levels (Fig. 5A-B or C-D, respectively),
285
thereby showing resistance to fibrosis development. These data suggested that TGF-β1 and
286
TGF-β2 expression is crucial for the increase of endotoxin-induced fibronectin.
287
The cellular localization and distribution of fibronectin were also dependent on
288
TGF-β1 and TGF-β2 expression. ECs transfected with siCTRL in the absence of endotoxin
289
showed VE-cadherin (Fig. 6A) and CD31 (Fig. 6B) labeling localized at the plasma
290
membrane. However, the expression of the ECM protein, fibronectin, was almost absent
291
(Fig. 6A-B). These results were similar in ECs transfected with siTGFβ1 (Fig. 6C-D) and
292
siTGFβ2 (Fig. 6E-F) in the absence of endotoxin as well as in non-transfected wild type
293
ECs without exposure to LPS (9,10,18). However, ECs transfected with siCTRL and
294
exposed to endotoxin showed a decrease in the endothelial proteins, VE-cadherin (Fig. 6G)
295
and CD31 (Fig. 6H). Furthermore, endotoxin exposure induced an increase in the
296
expression of the ECM protein, fibronectin (Fig. 6G-H). Of note, ECs transfected with
297
siTGFβ1 (Fig. 6I-J) or siTGFβ2 (Fig. 6K-L) and exposed to endotoxin were resistant to the
298
decrease in endothelial markers and the increase in fibronectin levels. These results
299
indicated that TGF-β1 and TGF-β2 expression is crucial for the increase in endotoxin-
300
induced ECM expression.
301 302 303
Endotoxin increases collagen gel contraction and migration through a mechanism mediated by the TGF-β receptor, ALK-5.
304
15
305
To evaluate the fibroblast-like motile features we performed collagen gel
306
contraction assays which resemble fibroblast-like collagen fibers reorganization. Cells are
307
able to reorganize the collagen fibers and contract the collagen gel, a process preceding the
308
changes in phenotype. ECs exposed to endotoxin exhibited significant changes in collagen
309
gel contraction compared to non-treated cells, suggesting fibrotic-like collagen fiber
310
reorganization. Since TGF-β induces its actions through its receptor, ALK5, we used the
311
specific ALK-5 inhibitor, SB431542, to evaluate the participation of TGF-β in endotoxin-
312
induced EC migration. LPS-treated ECs in the presence of SB431542, changes in collagen
313
gel contraction were significantly decreased. As expected, ECs treated with SB431542 in
314
the absence of LPS did not show any change in collagen gel contraction compared to
315
untreated cells (Fig. 7A-B). These data suggest that TGF-β participates in the endotoxin-
316
induced ECs collagen gel contraction.
317
Furthermore, we were prompted to investigate whether TGF-β participates in
318
endotoxin-induced ECs migration by means of Boyden chamber transwell cell migration
319
assay, wherein ECs were placed on a top chamber with a higher concentration of serum
320
added to the lower chamber to create a serum gradient for chemotactic stimulation. ECs
321
exposed to endotoxin showed a increased cell migration, whereas endotoxin-treated ECs in
322
the presence of SB431542, showed a similar cell migration than that observed in untreated
323
condition. (Fig. 7C). These data suggest that TGF-β participates in the endotoxin-induced
324
ECs migration.
325
16
326
Discussion
327 328
Dysfunction of endothelial cells is a main feature in systemic inflammation
329
progression during sepsis syndrome, septic shock, and several inflammatory diseases. We
330
have recently reported that endotoxin induces endothelial fibrosis, thereby opening a novel
331
field for biomedical research (9). In this paper, we further investigated the molecular
332
mechanism underlying endotoxin-induced endothelial fibrosis.
333
Here, we demonstrated that exposure to endotoxin induced the expression and
334
secretion of TGF-β1 and TGF-β2 in ECs. Noteworthy, downregulation of TGF-β1 and
335
TGF-β2 expression was effective in inhibiting the endotoxin-induced decrease in the
336
endothelial marker, VE-cadherin. In addition, suppression of TGF-β1 and TGF-β2 was
337
sufficient in abolishing the endotoxin-induced increase in the fibrotic marker, α-SMA, and
338
the ECM protein, fibronectin. Thus, we demonstrated that endotoxin-induced endothelial
339
fibrosis is dependent on the expression of TGF-β1 and TGF-β2.
340
Exposure of ECs to endotoxin was efficient in triggering TGF-β1 and TGF-β2
341
expression at the levels of mRNA and protein. To our knowledge, no previous studies have
342
reported this finding in ECs. The effect of endotoxin in increasing the expression of TGF-
343
β1 and TGF-β2 was mediated through its receptor, TLR-4. These findings were in
344
agreement with those observed in a cell line derived from human prostate epithelial cells in
345
which endotoxin exposure induces TGF-β1 expression (20). Furthermore, the activation of
346
NAD(P)H oxidase and the subsequent generation of ROS were crucial for the increase of
347
endotoxin-induced TGF-β1 and TGF-β2 expression. These results were concordant with the
348
intracellular signaling observed in the activation of the TLR-4 pathway (6,7). We have
349
previously reported that endotoxin-mediated endothelial fibrosis is mediated by the TLR-4 17
350
receptor, the activation of NAD(P)H oxidase, and the generation of ROS (9). Thus, the
351
present results agreed with those previous findings.
352
In fibroblasts, it is well-known that p38MAPK participates in the conversion to
353
myofibroblast (22-26). However, to our knowledge, the participation of p38MAPK in
354
LPS-induced endothelial fibrosis has not been described. It has been shown that LPS
355
induces the NF-κB-dependent production of IL-8 via the activation of p38MAPK (27).
356
Given that cytokines induce endothelial fibrosis, LPS could potentially induce endothelial
357
fibrosis through p38MAPK activation-dependent cytokine production. Additionally, it has
358
been shown that LPS induces leukocyte adhesion proteins in ECs via p38MAPK activation
359
(28), suggesting the potential local secretion of cytokines to mediate endothelial fibrosis.
360
Furthermore, in the absence of LPS, the participation of p38MAPK in endothelial fibrosis,
361
evaluated as EndMT, has been studied. The development of EndMT is abolished using a
362
p38MAPK inhibitor. EndMT induction via the overexpression of HD3-α or by direct
363
stimulation with cytokines is decreased in the presence of a p38MAPK inhibitor,
364
suggesting that p38MAPK is involved in EndMT (14,29). Additionally, it has been reported
365
that p38MAPK activation plays an important role in TGF-β intracellular signaling,
366
promoting fibrotic actions (21,22). Furthermore, TGF-β1 induces the mobilization of
367
p38MAPK to the nucleus (30,31). This evidence suggests that TGF-β1-induced p38MAPK
368
is activated and modulates gene expression to support fibrosis. However, further
369
experiments must be performed to investigate these possibilities in the endothelium.
370
Our results clearly showed that suppression of TGF-β1 and TGF-β2 expression
371
caused ECs to become resistant against the endotoxin challenge. These findings contribute
372
to understanding the molecular mechanism underlying endotoxin-induced endothelial
373
fibrosis. There were no differences in the action of TGF-β1 and TGF-β2 in inducing 18
374
endothelial fibrosis suggesting that both TGF-β isoforms induce the fibrotic changes in the
375
endothelium. These results agreed with data previously reported showing that TGF-β1 and
376
TGF-β2 can induce EndMT (10,14,15).
377
Although the TGF-β isoforms have high similarity between their active domains,
378
TGF-β2 differs in that it binds the TβRII receptor through different residues and is
379
dependent on β-glycan as a co-receptor (32). These results suggest that TGF-β1 and TGF-
380
β2 could perform their activities through different affinities to the receptor. Thus, TGF-β1
381
and TGF-β2 could accomplish different actions based on the spatial and temporal
382
concentration as well as on the density of the receptor at the plasma membrane. We did not
383
observe differences in the expression of TGF-β1 and TGF-β2 at the mRNA or protein
384
levels. However, more detailed studies should be performed to measure and compare the
385
levels of TGF-β1 and TGF-β2 in ECs after several conditions of endotoxin exposure.
386
An interesting finding was that suppression of TGF-β1 decreased the expression of
387
TGF-β2, whereas suppression of TGF-β2 had no effect on TGF-β1 expression. These
388
results were in agreement with those recently reported in cells from human skin melanoma
389
(17). However, the underlying mechanism of TGF-β1 controlling the endogenous levels of
390
TGF-β2 is currently not understood. It is possible that TGF-β1 interacts directly or
391
indirectly with the transcription factor that regulates TGF-β2 expression. Further studies are
392
needed to elucidate this mechanism.
393
In human prostate adenocarcinoma cells, it has been shown that inhibition of TGF-
394
β1 expression also decreases the expression of TGF-β2 and that the expression of TGF-β1
395
is suppressed by siTGFβ2 (17). These different results suggest that the control exerted by a
396
single TGF-β isoform on another isoform could be dependent on the specific cell type
397
where the process occurs. 19
398
Because inhibition of TGF-β1 expression also inhibits TGF-β2 expression, it is
399
difficult to study the effects of TGF-β1 and TGF-β2 individually. Specifically, it is hard to
400
assign functions to TGF-β1 alone. TGF-βs signaling is mediated by smad-family proteins.
401
TGF-β1 and TGF-β2 signaling pathways incorporate different smad components. TGF-β1
402
signaling is mediated by the activation of smad2/3, whereas TGF-β2 signaling uses
403
smad1/5/8 activation (12,13). Therefore, it is possible to dissect the TGF-β1 and TGF-β2
404
pathways by inhibiting select smad proteins. We previously reported that endotoxin-
405
induced EC fibrosis is mediated by smad3 activation, because smad3 inhibition abolished
406
the endothelial fibrosis induced by LPS (9). These findings strongly suggest that TGF-β1,
407
in addition to TGF-β2, is also involved in LPS-induced EC fibrosis.
408
Connective tissue growth factor (CTGF) is a cysteine-rich protein that is induced by
409
transforming growth factor-beta (TGF-β) and is implicated in a variety of fibrotic disorders.
410
Once TGF-β binds its receptor, smad-family proteins are activated and promote the
411
expression of CTGF. Thus, the participation of CTGF in LPS-induced EC fibrosis suggests
412
that fibrotic actions could be mediated by CTGF expression. We previously showed that
413
LPS-induced EC fibrosis is mediated by the activation of smad3 (9). Additionally, we
414
showed that LPS has the capacity to phosphorylate smad2 (9). Furthermore, it has been
415
reported that LPS is able to induce CTGF expression via TGF-β signaling in fibroblasts and
416
neurons (33,34). These data suggest that LPS may be able to induced CTGF expression
417
through the activation of TGF-β-dependent smad proteins. However, similar finding in ECs
418
has not been reported. Further studies are needed to study CTGF signaling in endotoxin-
419
induced endothelial fibrosis.
420
Taken together, these results demonstrated that the expression of TGF-β1 and TGF-
421
β2 is crucial in the development of the endotoxin-induced endothelial fibrosis mechanism. 20
422
Thus, modulation of TGF-β1 and TGF-β2 expression emerges as a novel strategy to
423
improve current treatments against endotoxemia-derived inflammatory diseases.
21
424
Acknowledgments
425 426
This work was supported by research grants from Fondo Nacional de Desarrollo
427
Científico y Tecnológico - Fondecyt 1121078 (FS), 1120380 (CCV), 1130996 (CR),
428
3140448 (CE). Millennium Institute on Immunology and Immunotherapy P09-016-F (FS,
429
CR), Association-Francaise Contre Les Myopathies AFM 16670 (CCV), UNAB-DI-281-
430
13/R (CCV), and UNAB-DI-67-12/I (CE). The authors are grateful to Director Dr. Iván
431
Oyarzún and Dr. Mario Carmona, Dr. Jaime Mendoza and Mrs. Juana Belmar from
432
Servicio Ginecología y Obstetricia, Hospital San Jose de Melipilla.
433 434 435
Conflict of interest.
436 437
The authors confirm that there are no conflicts of interest.
438
22
439
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Figure Legends
554 555
FIG 1 Endotoxin induces the expression and secretion of TGF-β1 and TGF-β2 dependent
556
on TLR-4, NAD(P)H oxidase, and ROS. (A–B) ECs were incubated in the absence (-) or
557
presence (+, 20 μg/ml LPS) of endotoxin for 72 h, and mRNA expression of TGF-β1 (A)
558
and TGF-β2 (B) was then measured by means of qPCR. Determinations were performed in
559
at least triplicates, and the results are expressed normalized relative to 28S mRNA
560
expression. Significant differences were assessed by Student's t-test (Mann-Whitney). **, P
561
< 0.01 against untreated condition. Graph bars show the mean ± SD (N = 3-4). (C–F) ECs
562
were incubated in the absence (-) or presence (+, 20 μg/ml LPS) of endotoxin for 72 h, and
563
the protein secretion of TGF-β1 (C–D) and TGF-β2 (E–F) was then measured in the
564
supernatant. (C and E) Representative images of western blot experiments performed for
565
detection of TGF-β1 (C) and TGF-β2 (E) secretion. (D and F) Densitometric analyses of
566
the experiments shown in C and E, respectively. Protein levels were normalized against
567
tubulin, and data are expressed relative to the untreated condition. (G–J) ECs were
568
incubated with a specific TLR-4 inhibitor (CLI-095, 1 μM; CLI) (G–H), a specific
569
NAD(P)H oxidase inhibitor (Apocynin, 1mM; Apo) (I–J), and an antioxidant (NAC, 1
570
mM) (I–J) as well as exposed to endotoxin (+; 20 μg/ml LPS) for 72 h. Protein secretion of
571
TGF-β1 (G–I) and TGF-β2 (H–J) was then measured in the supernatant. Protein levels were
572
normalized against tubulin, and data are expressed relative to the untreated condition.
573
Significant differences were assessed by Student's t-test (Mann-Whitney). *, P < 0.05 and
574
**, P < 0.01 against untreated condition. Graph bars show the mean ± SD (N = 3-4).
575
28
576
FIG 2 Changes in protein expression during endotoxin-induced endothelial fibrosis are
577
inhibited by transfection of a siRNA against TGF-β1 and TGF-β2. (A–D) ECs were
578
transfected with siRNAs against TGF-β1 and TGF-β2 (siTGFβ1 and siTGFβ1,
579
respectively) or a non-targeting siRNA (siCTRL). Protein expression was then analyzed. (A
580
and C) Representative images of western blot experiments performed for detection of TGF-
581
β1 (A) and TGF-β2 (C). B and D show densitometric analyses of several experiments
582
shown in A and C, respectively. Protein levels were normalized against tubulin, and the
583
data are expressed relative to siCTRL-transfected cells. (E–J) ECs were transfected with
584
siRNAs against TGF-β1 and TGF-β2 (siTGFβ1 and siTGFβ1, respectively) or a non-
585
targeting siRNA (siCTRL). Protein expression was then analyzed. (E, G, and I)
586
Representative images of western blot experiments performed for detection of the
587
endothelial marker, VE-cadherin (VE-cad) (E), the fibrotic marker, α-SMA (G), and the
588
ECM protein, fibronectin (FN) (I). F, H, and J show densitometric analyses of several
589
experiments as shown in E, G, and I, respectively. Protein levels were normalized against
590
tubulin, and the data are expressed relative to siCTRL-transfected cells. Significant
591
differences were assessed by a one-way analysis of variance (ANOVA; Kruskal–Wallis)
592
followed by Dunn's post hoc test. *, P < 0.05 and ***, P < 0.001 against the untreated
593
siCTRL-transfected cells. Graph bars show the mean ± SD (N = 3-5).
594 595
FIG 3 Changes in endothelial and fibrotic marker expression during endotoxin-induced
596
endothelial fibrosis are inhibited by transfection of siRNAs targeting TGF-β1 and TGF-β2.
597
(A–H) ECs transfected with siRNAs against TGF-β1 (A–D) and TGF-β2 (E–H) (siTGFβ1
598
and siTGFβ1, respectively) or a non-targeting siRNA (siCTRL) were incubated in the
599
absence (-) or presence (+; 20 μg/ml LPS) of endotoxin for 72 h, and protein expression 29
600
was then analyzed. (A, C, E, and G) Representative images of western blot experiments
601
performed for detection of the endothelial marker, VE-cadherin (VE-cad) (A and E), and
602
the fibrotic marker, α-SMA (C and G). B, D, F and H show densitometric analyses of
603
several experiments as shown in A, C, E, and G, respectively. Protein levels were
604
normalized against tubulin, and the data are expressed relative to non-treated siCTRL-
605
transfected cells. Significant differences were assessed by a one-way analysis of variance
606
(ANOVA; Kruskal–Wallis) followed by Dunn's post hoc test. **, P < 0.01 against the
607
untreated siCTRL-transfected cells. Graph bars show the mean ± SD (N = 3-5).
608 609
FIG 4 Changes in the distribution of endothelial and fibrotic markers involved in
610
endotoxin-induced endothelial fibrosis are inhibited by transfection of siRNAs targeting
611
TGF-β1 and TGF-β2. (A–L) Representative images from ECs transfected with siRNAs
612
against TGF-β1 (siTGFβ1) (C–D and I–J) and TGF-β2 (siTGFβ2) (E–F and K–L) or a non-
613
targeting siRNA (siCTRL) (A–B and G–H) were incubated in the absence (A–F) or
614
presence of endotoxin (G–L) (20 μg/ml LPS) for 72 h. VE-cadherin/CD31 (red) and FSP-
615
1/α-SMA (green) were detected. Nuclei were stained using DAPI. Bar scale represents 10
616
μm. (N = 4).
617 618
FIG 5 Changes in ECM protein expression during endotoxin-induced endothelial fibrosis
619
are inhibited by transfection of siRNAs targeting TGF-β1 and TGF-β2. (A–D) ECs
620
transfected with siRNAs against TGF-β1 (A–B) and TGF-β2 (C–D) (siTGFβ1 and
621
siTGFβ1, respectively) or a non-targeting siRNA (siCTRL) were incubated in the absence
622
(-) or presence (+; 20 μg/ml LPS) of endotoxin for 72 h, and protein expression was then
623
analyzed. (A and C) Representative images of western blot experiments performed for 30
624
detection of the ECM protein, fibronectin (FN). B and D show densitometric analyses of
625
several experiments as shown in A and C, respectively. Protein levels were normalized
626
against tubulin, and the data are expressed relative to untreated siCTRL-transfected cells.
627
Significant differences were assessed by a one-way analysis of variance (ANOVA;
628
Kruskal–Wallis) followed by Dunn's post hoc test. **, P < 0.01 against the untreated
629
siCTRL-transfected cells. Graph bars show the mean ± SD (N = 3-5).
630 631
FIG 6 Changes in the distribution of ECM proteins involved in endotoxin-induced
632
endothelial fibrosis are inhibited by transfection of siRNAs targeting TGF-β1 and TGF-β2.
633
(A–L) Representative images of ECs transfected with siRNAs against TGF-β1 (siTGFβ1)
634
(C–D and I–J) and TGF-β2 (siTGFβ2) (E–F and K–L) or a non-targeting siRNA (siCTRL)
635
(A–B and G–H) were incubated in the absence (A–F) or presence of endotoxin (G–L) (20
636
μg/ml LPS) for 72 h. VE-cadherin/CD31 (red) and FN (green) were detected. Nuclei were
637
stained using DAPI. Bar scale represents 10 μm. (N = 4).
638 639
FIG 7 Fibroblast-like motility of endotoxin-induced ECs is mediated by ALK-5 activity.
640
ECs were treated with the ALK5 inhibitor, SB431542 (0.5 μM), and incubated in the
641
absence or presence of endotoxin (10 μg/mL LPS) and subjected to collagen gel contraction
642
assay measured as gel area (A), gel weight (B), or by means of transwell migration assay in
643
Boyden chambers (C). Cell movement was allowed to occur for 24 h. Significant
644
differences were assessed by a one-way analysis of variance (ANOVA; Kruskal–Wallis)
645
followed by Dunn's post hoc test. *: P < 0.05, **: P < 0.01 against vehicle-treated condition
646
(0 μg/mL LPS). Graph bars show the means ± SD (N = 3-5).
31
1
Endotoxin-induced endothelial fibrosis is dependent on the expression of TGF-β1 and
2
TGF-β2
3 4 5
César Echeverríaa,d, Ignacio Montorfanoa, Pablo Tapiae, Claudia Riedelc, Claudio Cabello-
6
Verrugiob, and Felipe Simona,f,#
7 8
a
Laboratorio de Fisiopatología Integrativa, bLaboratorio de Biología y Fisiopatología
9
Molecular, cLaboratorio de Biología Celular y Farmacología, Departamento de Ciencias
10
Biologicas, Facultad de Ciencias Biologicas and Facultad de Medicina, Universidad Andres
11
Bello, Avenida Republica 239, Santiago, Chile.
12 13 14 15 16
d
Laboratorio de Bionanotecnología, Universidad Bernardo O'Higgins, General Gana 1780,
Santiago, Chile. e
Departamento de Medicina Intensiva, Facultad de Medicina, Pontificia Universidad
Católica de Chile, Santiago, Chile. f
Millennium Institute on Immunology and Immunotherapy, Santiago, Chile.
17 18
Running title: Endotoxin-induced endothelial fibrosis via TGF-β
19 20
# Address correspondence to Felipe Simon, [email protected]
1
21
Abstract
22 23
During endotoxemia-induced inflammatory disease, bacterial endotoxins circulate
24
in the bloodstream and interact with endothelial cells (ECs), inducing dysfunction of the
25
ECs. We have previously reported that endotoxins induce the conversion of ECs into
26
activated fibroblasts. Through endotoxin-induced endothelial fibrosis, ECs change their
27
morphology and their protein pattern expression, thereby suppressing endothelial markers
28
and upregulating fibrotic proteins. The most commonly used fibrotic inducers are TGF-β1
29
and TGF-β2. However, whether TGF-β1 and TGF-β2 participate in endotoxin-induced
30
endothelial fibrosis remains unknown. We have shown that the endotoxin-induced
31
endothelial fibrosis process is dependent on the TGF-β receptor, ALK5, and the activation
32
of Smad3, a protein that is activated by ALK5 activation, thus suggesting that endotoxin
33
elicits TGF-β production to mediate endotoxin-induced endothelial fibrosis. Therefore, we
34
investigated the dependence of endotoxin-induced endothelial fibrosis on the expression of
35
TGF-β1 and TGF-β2.
36
Endotoxin-treated ECs induced the expression and secretion of TGF-β1 and TGF-
37
β2. TGF-β1 and TGF-β2 downregulation inhibited the endotoxin-induced changes in the
38
endothelial marker, VE-cadherin, and in the fibrotic proteins, α-SMA and fibronectin.
39
Thus, endotoxin induces the production of TGF-β1 and TGF-β2 as a mechanism to promote
40
endotoxin-induced endothelial fibrosis.
41
To the best of our knowledge, this is the first report showing that endotoxin induces
42
endothelial fibrosis via TGF-β secretion, which represents an emerging source of vascular
43
dysfunction. These findings contribute to understanding the molecular mechanism of
2
44
endotoxin-induced endothelial fibrosis, which could be useful in the treatment of
45
inflammatory diseases.
46 47
Keywords: Endothelial dysfunction; endotoxin; inflammation; fibrosis; TGF-β.
3
48
Introduction
49 50
The initiation and progression of systemic inflammation, including sepsis syndrome
51
and septic shock, result in high morbimortality rates (1,2). During systemic infection, the
52
bloodstream is full of pro-inflammatory cytokines, reactive oxygen species (ROS), and
53
active immune cells. In the case of endotoxemia-induced sepsis syndrome, large amounts of
54
the Gram-negative bacterial endotoxin, lipopolysaccharide (LPS) (3,4), are deposited and
55
circulate in the bloodstream, interacting with the endothelial cells (ECs) located in the
56
internal endothelial monolayer of blood vessels, which induces detrimental effects on
57
endothelium function (5-7). Dysfunction of ECs is a crucial step in the pathogenesis of
58
endotoxemia-derived sepsis syndrome and other inflammatory diseases (5,8).
59
We have previously reported that bacterial endotoxins induce the conversion of ECs
60
into activated fibroblasts, also known as myofibroblasts (9). Endotoxin-induced endothelial
61
fibrosis is mediated through a process known as endothelial-to-mesenchymal transition
62
(EndMT), which has been studied using tumor growth factor-beta 1 and 2 (TGF-β1 and
63
TGF-β2) as EndMT inducers (10,11). However, the role of TGF-β1 and TGF-β2 in
64
endotoxin-induced endothelial fibrosis is currently unknown.
65
Endotoxin-induced endothelial fibrosis is morphologically identified by the change
66
of the round shaped EC monolayer with a short-spindle shape and a cobblestone
67
appearance into a spindle-shaped fibroblast-like phenotype (9,10). At the level of protein
68
expression, endotoxin-induced endothelial fibrosis is characterized by downregulation of
69
the endothelial markers, CD31 and VE۔cadherin, as well as by upregulation of the
70
fibroblast-specific genes, α۔smooth muscle actin (α-SMA) and fibroblast-specific protein-1
4
71
(FSP-1). Additionally, the levels of proteins that form the extracellular matrix (ECM), such
72
as fibronectin (FN) and collagen type III (Col III), are severely enhanced (9,10).
73
The endotoxin-induced endothelial fibrosis process is dependent on the expression
74
of the TGF-β receptor, activin receptor-like kinase 5 (ALK5), because its downregulation
75
and pharmacological inhibition are effective in inhibiting the endothelial fibrosis induced
76
by endotoxins (9). The ALK5 intracellular pathway subsequently activates Smad3 protein
77
by phosphorylation (12,13). Inhibition of Smad3 activation abolishes the endotoxin-
78
induced endothelial fibrosis process (9). This evidence indicates that the ALK5 receptor is
79
involved in endotoxin-induced endothelial fibrosis. Taken together, these findings strongly
80
suggest that endotoxin elicits TGF-β synthesis and secretion, resulting in autocrine and
81
paracrine effects on ECs to mediate endotoxin-induced endothelial fibrosis.
82 83
Therefore, the aim of this study was to investigate if endotoxin-induced endothelial fibrosis is dependent on the expression of TGF-β1 and TGF-β2.
84
Our data demonstrated that when ECs are exposed to endotoxin, the expression and
85
secretion of TGF-β1 and TGF-β2 are induced. In addition, ECs exposed to endotoxin
86
exhibited increased p38 MAPK phosphorylation. Furthermore, suppression of TGF-β1 and
87
TGF-β2 expression was effective in inhibiting the endotoxin-induced decrease of the
88
endothelial marker, VE-cadherin. Additionally, the downregulation of TGF-β1 and TGF-β2
89
was sufficient to abolish the endotoxin-induced increase in the fibrotic marker, α-SMA, and
90
the ECM protein, fibronectin.
91 92
We concluded that endotoxin is capable of inducing the expression and secretion of TGF-β1 and TGF-β2 as a mechanism to promote endotoxin-induced endothelial fibrosis.
93
These results contribute to a better understanding of the molecular basis of
94
endotoxin-induced endothelial fibrosis, which could be useful in improving current 5
95
therapeutic strategies in treating endothelial dysfunction during endotoxemia and other
96
inflammatory diseases.
6
97
Materials and methods
98
Details of all procedures are provided in Additional Supporting Information
99 100
Primary cell culture
101
Human umbilical vein endothelial cells (HUVECs) were isolated by collagenase
102
(0.25 mg/ml) digestion from freshly obtained umbilical cord veins from normal
103
pregnancies, after patients' informed consent. The investigation conforms with the
104
principles outlined in the Declaration of Helsinki. The Commission of Bioethics and
105
Biosafety of Universidad Andres Bello also approved all experimental protocols. Cells
106
were grown in gelatin-coated dishes at 37°C in a 5%:95% CO2:air atmosphere in medium
107
199 (Sigma, MO), containing 100 µg/ml endothelial cell growth supplement (ECGS;
108
Sigma), 100 µg/ml heparin, 5 mM D-glucose, 3.2 mM L-gutamine, 10% fetal bovine serum
109
(FBS; GIBCO, NY), and 50 U/ml penicillin-streptomycin (Sigma).
110 111
Small-interfering RNA and transfections
112
SiGENOME SMARTpool siRNA (four separated siRNAs per each human TGF-β1
113
or TGF-β2 transcript) were purchased from Dharmacon (Dharmacon, Lafayette, CO). The
114
following siRNAs were used: siRNA against human TGF-β1 (siTGFβ1), siRNA against
115
human TGF-β2 (siTGFβ2), and non-targeting siRNA (siCTRL) used as a control. In brief,
116
HUVECs were plated overnight in a 6-well plate and then transfected with 5 nM siRNA
117
using DharmaFECT 4 transfection reagent (Dharmacon) used according to the
118
manufacturer's protocol in serum-free medium for 24 hours. Experiments were performed
119
48 to 72 hours after transfection.
120 7
121
RNA isolation and quantitative real-time PCR
122
QPCR experiments were performed to measure TGF-β1 and TGF-β2 mRNA levels
123
in HUVECs. Total RNA was extracted with Trizol according to the manufacturer's protocol
124
(Invitrogen, Carlsbad, CA). DNAse I-treated RNA was used for reverse transcription using
125
the Super Script II Kit (Invitrogen, Carlsbad, CA). Equal amounts of RNA were used as
126
templates in each reaction. QPCR was performed using the SYBR Green PCR Master Mix
127
(AB Applied Biosystems, Foster City, CA). All reactions were performed in triplicate on an
128
Eco Real-Time PCR System (Illumina, USA). Data are presented as relative mRNA levels
129
of the gene of interest normalized to relative levels of 28S mRNA.
130 131
Western blot procedures
132
Cells were lysed in cold lysis buffer, and proteins were then extracted. Supernatants
133
were collected and stored in the same lysis buffer. Protein extract and supernatant were
134
subjected to SDS-PAGE, and resolved proteins were transferred to a nitrocellulose or
135
PVDF membrane. The blocked membrane was incubated with the appropriate primary
136
antibody, washed twice, and incubated with a secondary antibody. Bands were detected
137
using
138
chemiluminescence (Thermo Scientific, USA). Tubulin was used as a loading control.
139
Images were acquired using the Fotodyne FOTO/Analyst Luminary Workstation
140
(Fotodyne, Inc., Hartland, WI). Protein content was determined by densitometric scanning
141
of immunoreactive bands, and intensity values were obtained by densitometry of individual
142
bands normalized against control. For a detailed list of antibodies used, see Supplementary
143
Table S1.
a
peroxidase-conjugated
IgG
antibody
and
visualized
by
enhanced
144 8
145
Fluorescent Immunocytochemistry
146
ECs were washed twice with PBS and fixed. The cells were subsequently washed
147
again and incubated with primary antibodies. Cells were then washed twice and incubated
148
with secondary antibodies. Samples were mounted with ProLong Gold antifade mounting
149
medium with DAPI (Invitrogen). For a detailed list of antibodies used, see Supplementary
150
Table S2.
151 152
Collagen gel contraction assay
153
Twenty-four well plates were precoated with 1% agarose. Type I collagen was
154
diluted in medium and adjusted to pH 7.4. (collagen I in 0.02 M acetic acid, 0.1 M NaOH,
155
serum-free culture medium containing sodium bicarbonate). ECs were trypsinized when
156
80% confluent and resuspended at 8x105 cells/ml. Equal volumes of collagen and cells
157
were then combined to give 1 mg collagen and 4x105 cells/ml and 500 μL pipetted into
158
each well. Gels were polymerized at 4°C ON. Then, gels were fixed in formaldehyde and
159
washed twice with Tris-buffered saline with 0.2% Tween-20. Detergent was present to
160
reduce surface tension. The area of gels were measured and also gels were weighed on an
161
analytical digital scale with a precision of 0.1 mg, at 24 h after stimulus. Contraction was
162
measured relative to the initial gel weigh and normalized against control condition. All
163
assays were repeated 3 times with triplicate wells per experimental condition.
164 165
Cell migration measurement by transwell assay
166
The capacity of ECs to migrate was assayed using Transwell Boyden chambers
167
(Costar, Cambrige, MA, USA) with 8.0-μm-pore polycarbonate filters. Cells were seeded
9
168
in absence or presence LPS (10 μg/ml) for 24 h in 1% FBS on the upper compartment of
169
the chamber. To stimulate cell migration, 10% FBS was added to the lower compartment of
170
the chamber. Thus, migration was allowed to occur for 24 h. After washing, non-invading
171
cells were removed from the upper surface of the membrane with a cotton swab. The
172
invading cells were fixed with 10 % ethanol for 5 min and stained with 0.2% crystal violet
173
for 5 min. Images were captured through a digital microscope system. Cell migration was
174
evaluated by counting four fields per chamber in every condition. All assays were
175
performed at least in triplicates in three separates sets of experiments.
176 177
Reagents
178
The endotoxin, lipopolysaccharide from E. coli, was purchased from Sigma
179
(0127:B8). Apocynin and NAC were purchased from Sigma-Aldrich. The TLR4 inhibitor,
180
CLI-095 was purchased from InvivoGen. Buffers and salts were purchased from Merck
181
Biosciences (Darmstadt).
182 183
Data analysis
184
All results are presented as the means ± SD. Student's t-test and ANOVA followed
185
by Dunn's post hoc tests were used and considered significant at p 99% of cells in the EC culture were positive for VE-
221
cadherin, whereas those expressing FSP-1 were not detected. Thus, the primary human EC
222
cultures used here were highly enriched in ECs (Supp. Fig. S2).
223 224 225
Expression of TGF-β1 and TGF-β2 is crucial for the modification of endothelial and fibrotic marker expression induced by endotoxin in endothelial cells.
226 227
Considering that the expression of TGF-β1 and TGF-β2 is induced by endotoxin, we
228
investigated if the expression of these cytokines was necessary for the progression of
229
endotoxin-induced endothelial fibrosis. Thus, we used two specific siRNAs against the
230
human TGF-β isoforms, TGF-β1 and TGF-β2. Additionally, a non-targeting siRNA
231
(siCTRL) was used as a control. We studied the specificity and effectiveness of the siRNAs
232
on the inhibition of TGF-β1 and TGF-β2. ECs transfected with the siRNAs against TGF-β1
233
(siTGFβ1) and TGF-β2 (siTGFβ2) showed a significant inhibition of the expression of 12
234
TGF-β1 and TGF-β2, respectively (Fig. 2A-B and Fig. 2C-D, respectively). Noteworthy,
235
siTGFβ1 also inhibited the expression of TGF-β2 (Fig. 2C-D), but siTGFβ2 was specific
236
because the expression of TGF-β1 was not affected (Fig. 2A-B). This finding was in
237
accordance with previously reported data (17). We then tested whether downregulation of
238
TGF-β1 and TGF-β2 changed the protein levels of VE-cadherin, α-SMA, and fibronectin.
239
ECs transfected with siTGFβ1 and siTGFβ2 did not show any change in the protein level of
240
VE-cadherin (Fig. 2E-F), and they showed a slight but significant decrease in the fibrotic
241
proteins, α-SMA (Fig. 2G-H) and FN (Fig. 2I-J), thereby suggesting that the suppression of
242
siTGFβ1 and siTGFβ2 expression is not involved in the fibrotic processes.
243
We then tested whether endotoxin-induced endothelial fibrosis was dependent on
244
the expression of TGF-β1 and TGF-β2. ECs were transfected with siTGFβ1 or siTGF-β2,
245
and the changes in endothelial and fibrotic markers were measured. ECs transfected with
246
siCTRL and exposed to endotoxin exhibited a decrease in the protein level of the
247
endothelial marker, VE-cadherin (Fig. 3A-B and E-F), and an increase in the fibrotic
248
marker, α-SMA (Fig. 3C-D and G-H), which was similar to what has been observed in non-
249
transfected wild type ECs exposed to endotoxin (9,10,18). Of note, ECs transfected with
250
siTGFβ1 or siTGFβ2 and exposed to endotoxin were resistant to fibrosis development
251
because those cells did not show any significant change in the protein levels of VE-
252
cadherin (Fig. 3A-B and E-F) or α-SMA (Fig. 3C-D and G-H). These data suggested that
253
TGF-β1 and TGF-β2 expression is crucial in endotoxin-induced endothelial fibrosis.
254
Next, we evaluated the action of TGF-β1 and TGF-β2 expression on the cellular
255
localization and distribution of endothelial and fibrotic proteins. ECs transfected with
256
siCTRL in the absence of endotoxin showed VE-cadherin (Fig. 4A) and CD31 (Fig. 4B)
257
labeling localized at the plasma membrane. In contrast, the expression of the fibrotic 13
258
markers, FSP-1 (Fig. 4A) and α-SMA (Fig. 4B), was weak. Analogous results were
259
observed for ECs transfected with siTGFβ1 (Fig. 4C-D) and siTGFβ2 (Fig. 4E-F) in the
260
absence of endotoxin. These results were also similar to those observed in non-transfected
261
wild type ECs without LPS showing a round-shaped monolayer with short-spindle
262
morphology and a cobblestone appearance (9,10,18). However, ECs transfected with
263
siCTRL and exposed to endotoxin showed a decrease in the endothelial proteins, VE-
264
cadherin (Fig. 4G) and CD31 (Fig. 4H). Furthermore, endotoxin exposure induced an
265
increase in the fibrotic markers, FSP-1 (Fig. 4G) and α-SMA (Fig. 4H). These effects
266
showed a spindle-shaped, fibroblast-like phenotype with a loss of cell-to-cell connections.
267
Noteworthy, ECs transfected with siTGFβ1 (Fig. 4I-J) or siTGFβ2 (Fig. 4K-L) and exposed
268
to endotoxin were resistant to fibrosis progression because transfected ECs did not exhibit
269
changes in endothelial or fibrotic markers. These results confirmed that TGF-β1 and TGF-
270
β2 expression is crucial in the alteration of the cellular localization and distribution of the
271
proteins involved in endotoxin-induced endothelial fibrosis.
272 273 274
Expression of TGF-β1 and TGF-β2 is crucial for the increase of extracellular matrix proteins induced by endotoxin in endothelial cells.
275 276
It is well-known that oversecretion of ECM proteins is an important factor for the
277
progression of fibrogenesis (16,19). Hence, we tested if the increase in the ECM protein,
278
fibronectin, was dependent on the expression of TGF-β1 and TGF-β2. ECs were transfected
279
with siTGFβ1 or siTGFβ2, and the change in fibronectin levels was measured. ECs
280
transfected with siCTRL and exposed to endotoxin exhibited an increase in the ECM
281
marker, fibronectin (Fig. 5), compared with cells cultured in the absence of endotoxin. 14
282
These findings were similar to non-transfected wild type ECs exposed to endotoxin
283
(9,10,18). Importantly, ECs transfected with siTGFβ1 or siTGFβ2 and exposed to
284
endotoxin did not show any increase in fibronectin levels (Fig. 5A-B or C-D, respectively),
285
thereby showing resistance to fibrosis development. These data suggested that TGF-β1 and
286
TGF-β2 expression is crucial for the increase of endotoxin-induced fibronectin.
287
The cellular localization and distribution of fibronectin were also dependent on
288
TGF-β1 and TGF-β2 expression. ECs transfected with siCTRL in the absence of endotoxin
289
showed VE-cadherin (Fig. 6A) and CD31 (Fig. 6B) labeling localized at the plasma
290
membrane. However, the expression of the ECM protein, fibronectin, was almost absent
291
(Fig. 6A-B). These results were similar in ECs transfected with siTGFβ1 (Fig. 6C-D) and
292
siTGFβ2 (Fig. 6E-F) in the absence of endotoxin as well as in non-transfected wild type
293
ECs without exposure to LPS (9,10,18). However, ECs transfected with siCTRL and
294
exposed to endotoxin showed a decrease in the endothelial proteins, VE-cadherin (Fig. 6G)
295
and CD31 (Fig. 6H). Furthermore, endotoxin exposure induced an increase in the
296
expression of the ECM protein, fibronectin (Fig. 6G-H). Of note, ECs transfected with
297
siTGFβ1 (Fig. 6I-J) or siTGFβ2 (Fig. 6K-L) and exposed to endotoxin were resistant to the
298
decrease in endothelial markers and the increase in fibronectin levels. These results
299
indicated that TGF-β1 and TGF-β2 expression is crucial for the increase in endotoxin-
300
induced ECM expression.
301 302 303
Endotoxin increases collagen gel contraction and migration through a mechanism mediated by the TGF-β receptor, ALK-5.
304
15
305
To evaluate the fibroblast-like motile features we performed collagen gel
306
contraction assays which resemble fibroblast-like collagen fibers reorganization. Cells are
307
able to reorganize the collagen fibers and contract the collagen gel, a process preceding the
308
changes in phenotype. ECs exposed to endotoxin exhibited significant changes in collagen
309
gel contraction compared to non-treated cells, suggesting fibrotic-like collagen fiber
310
reorganization. Since TGF-β induces its actions through its receptor, ALK5, we used the
311
specific ALK-5 inhibitor, SB431542, to evaluate the participation of TGF-β in endotoxin-
312
induced EC migration. LPS-treated ECs in the presence of SB431542, changes in collagen
313
gel contraction were significantly decreased. As expected, ECs treated with SB431542 in
314
the absence of LPS did not show any change in collagen gel contraction compared to
315
untreated cells (Fig. 7A-B). These data suggest that TGF-β participates in the endotoxin-
316
induced ECs collagen gel contraction.
317
Furthermore, we were prompted to investigate whether TGF-β participates in
318
endotoxin-induced ECs migration by means of Boyden chamber transwell cell migration
319
assay, wherein ECs were placed on a top chamber with a higher concentration of serum
320
added to the lower chamber to create a serum gradient for chemotactic stimulation. ECs
321
exposed to endotoxin showed a increased cell migration, whereas endotoxin-treated ECs in
322
the presence of SB431542, showed a similar cell migration than that observed in untreated
323
condition. (Fig. 7C). These data suggest that TGF-β participates in the endotoxin-induced
324
ECs migration.
325
16
326
Discussion
327 328
Dysfunction of endothelial cells is a main feature in systemic inflammation
329
progression during sepsis syndrome, septic shock, and several inflammatory diseases. We
330
have recently reported that endotoxin induces endothelial fibrosis, thereby opening a novel
331
field for biomedical research (9). In this paper, we further investigated the molecular
332
mechanism underlying endotoxin-induced endothelial fibrosis.
333
Here, we demonstrated that exposure to endotoxin induced the expression and
334
secretion of TGF-β1 and TGF-β2 in ECs. Noteworthy, downregulation of TGF-β1 and
335
TGF-β2 expression was effective in inhibiting the endotoxin-induced decrease in the
336
endothelial marker, VE-cadherin. In addition, suppression of TGF-β1 and TGF-β2 was
337
sufficient in abolishing the endotoxin-induced increase in the fibrotic marker, α-SMA, and
338
the ECM protein, fibronectin. Thus, we demonstrated that endotoxin-induced endothelial
339
fibrosis is dependent on the expression of TGF-β1 and TGF-β2.
340
Exposure of ECs to endotoxin was efficient in triggering TGF-β1 and TGF-β2
341
expression at the levels of mRNA and protein. To our knowledge, no previous studies have
342
reported this finding in ECs. The effect of endotoxin in increasing the expression of TGF-
343
β1 and TGF-β2 was mediated through its receptor, TLR-4. These findings were in
344
agreement with those observed in a cell line derived from human prostate epithelial cells in
345
which endotoxin exposure induces TGF-β1 expression (20). Furthermore, the activation of
346
NAD(P)H oxidase and the subsequent generation of ROS were crucial for the increase of
347
endotoxin-induced TGF-β1 and TGF-β2 expression. These results were concordant with the
348
intracellular signaling observed in the activation of the TLR-4 pathway (6,7). We have
349
previously reported that endotoxin-mediated endothelial fibrosis is mediated by the TLR-4 17
350
receptor, the activation of NAD(P)H oxidase, and the generation of ROS (9). Thus, the
351
present results agreed with those previous findings.
352
In fibroblasts, it is well-known that p38MAPK participates in the conversion to
353
myofibroblast (22-26). However, to our knowledge, the participation of p38MAPK in
354
LPS-induced endothelial fibrosis has not been described. It has been shown that LPS
355
induces the NF-κB-dependent production of IL-8 via the activation of p38MAPK (27).
356
Given that cytokines induce endothelial fibrosis, LPS could potentially induce endothelial
357
fibrosis through p38MAPK activation-dependent cytokine production. Additionally, it has
358
been shown that LPS induces leukocyte adhesion proteins in ECs via p38MAPK activation
359
(28), suggesting the potential local secretion of cytokines to mediate endothelial fibrosis.
360
Furthermore, in the absence of LPS, the participation of p38MAPK in endothelial fibrosis,
361
evaluated as EndMT, has been studied. The development of EndMT is abolished using a
362
p38MAPK inhibitor. EndMT induction via the overexpression of HD3-α or by direct
363
stimulation with cytokines is decreased in the presence of a p38MAPK inhibitor,
364
suggesting that p38MAPK is involved in EndMT (14,29). Additionally, it has been reported
365
that p38MAPK activation plays an important role in TGF-β intracellular signaling,
366
promoting fibrotic actions (21,22). Furthermore, TGF-β1 induces the mobilization of
367
p38MAPK to the nucleus (30,31). This evidence suggests that TGF-β1-induced p38MAPK
368
is activated and modulates gene expression to support fibrosis. However, further
369
experiments must be performed to investigate these possibilities in the endothelium.
370
Our results clearly showed that suppression of TGF-β1 and TGF-β2 expression
371
caused ECs to become resistant against the endotoxin challenge. These findings contribute
372
to understanding the molecular mechanism underlying endotoxin-induced endothelial
373
fibrosis. There were no differences in the action of TGF-β1 and TGF-β2 in inducing 18
374
endothelial fibrosis suggesting that both TGF-β isoforms induce the fibrotic changes in the
375
endothelium. These results agreed with data previously reported showing that TGF-β1 and
376
TGF-β2 can induce EndMT (10,14,15).
377
Although the TGF-β isoforms have high similarity between their active domains,
378
TGF-β2 differs in that it binds the TβRII receptor through different residues and is
379
dependent on β-glycan as a co-receptor (32). These results suggest that TGF-β1 and TGF-
380
β2 could perform their activities through different affinities to the receptor. Thus, TGF-β1
381
and TGF-β2 could accomplish different actions based on the spatial and temporal
382
concentration as well as on the density of the receptor at the plasma membrane. We did not
383
observe differences in the expression of TGF-β1 and TGF-β2 at the mRNA or protein
384
levels. However, more detailed studies should be performed to measure and compare the
385
levels of TGF-β1 and TGF-β2 in ECs after several conditions of endotoxin exposure.
386
An interesting finding was that suppression of TGF-β1 decreased the expression of
387
TGF-β2, whereas suppression of TGF-β2 had no effect on TGF-β1 expression. These
388
results were in agreement with those recently reported in cells from human skin melanoma
389
(17). However, the underlying mechanism of TGF-β1 controlling the endogenous levels of
390
TGF-β2 is currently not understood. It is possible that TGF-β1 interacts directly or
391
indirectly with the transcription factor that regulates TGF-β2 expression. Further studies are
392
needed to elucidate this mechanism.
393
In human prostate adenocarcinoma cells, it has been shown that inhibition of TGF-
394
β1 expression also decreases the expression of TGF-β2 and that the expression of TGF-β1
395
is suppressed by siTGFβ2 (17). These different results suggest that the control exerted by a
396
single TGF-β isoform on another isoform could be dependent on the specific cell type
397
where the process occurs. 19
398
Because inhibition of TGF-β1 expression also inhibits TGF-β2 expression, it is
399
difficult to study the effects of TGF-β1 and TGF-β2 individually. Specifically, it is hard to
400
assign functions to TGF-β1 alone. TGF-βs signaling is mediated by smad-family proteins.
401
TGF-β1 and TGF-β2 signaling pathways incorporate different smad components. TGF-β1
402
signaling is mediated by the activation of smad2/3, whereas TGF-β2 signaling uses
403
smad1/5/8 activation (12,13). Therefore, it is possible to dissect the TGF-β1 and TGF-β2
404
pathways by inhibiting select smad proteins. We previously reported that endotoxin-
405
induced EC fibrosis is mediated by smad3 activation, because smad3 inhibition abolished
406
the endothelial fibrosis induced by LPS (9). These findings strongly suggest that TGF-β1,
407
in addition to TGF-β2, is also involved in LPS-induced EC fibrosis.
408
Connective tissue growth factor (CTGF) is a cysteine-rich protein that is induced by
409
transforming growth factor-beta (TGF-β) and is implicated in a variety of fibrotic disorders.
410
Once TGF-β binds its receptor, smad-family proteins are activated and promote the
411
expression of CTGF. Thus, the participation of CTGF in LPS-induced EC fibrosis suggests
412
that fibrotic actions could be mediated by CTGF expression. We previously showed that
413
LPS-induced EC fibrosis is mediated by the activation of smad3 (9). Additionally, we
414
showed that LPS has the capacity to phosphorylate smad2 (9). Furthermore, it has been
415
reported that LPS is able to induce CTGF expression via TGF-β signaling in fibroblasts and
416
neurons (33,34). These data suggest that LPS may be able to induced CTGF expression
417
through the activation of TGF-β-dependent smad proteins. However, similar finding in ECs
418
has not been reported. Further studies are needed to study CTGF signaling in endotoxin-
419
induced endothelial fibrosis.
420
Taken together, these results demonstrated that the expression of TGF-β1 and TGF-
421
β2 is crucial in the development of the endotoxin-induced endothelial fibrosis mechanism. 20
422
Thus, modulation of TGF-β1 and TGF-β2 expression emerges as a novel strategy to
423
improve current treatments against endotoxemia-derived inflammatory diseases.
21
424
Acknowledgments
425 426
This work was supported by research grants from Fondo Nacional de Desarrollo
427
Científico y Tecnológico - Fondecyt 1121078 (FS), 1120380 (CCV), 1130996 (CR),
428
3140448 (CE). Millennium Institute on Immunology and Immunotherapy P09-016-F (FS,
429
CR), Association-Francaise Contre Les Myopathies AFM 16670 (CCV), UNAB-DI-281-
430
13/R (CCV), and UNAB-DI-67-12/I (CE). The authors are grateful to Director Dr. Iván
431
Oyarzún and Dr. Mario Carmona, Dr. Jaime Mendoza and Mrs. Juana Belmar from
432
Servicio Ginecología y Obstetricia, Hospital San Jose de Melipilla.
433 434 435
Conflict of interest.
436 437
The authors confirm that there are no conflicts of interest.
438
22
439
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Figure Legends
554 555
FIG 1 Endotoxin induces the expression and secretion of TGF-β1 and TGF-β2 dependent
556
on TLR-4, NAD(P)H oxidase, and ROS. (A–B) ECs were incubated in the absence (-) or
557
presence (+, 20 μg/ml LPS) of endotoxin for 72 h, and mRNA expression of TGF-β1 (A)
558
and TGF-β2 (B) was then measured by means of qPCR. Determinations were performed in
559
at least triplicates, and the results are expressed normalized relative to 28S mRNA
560
expression. Significant differences were assessed by Student's t-test (Mann-Whitney). **, P
561
< 0.01 against untreated condition. Graph bars show the mean ± SD (N = 3-4). (C–F) ECs
562
were incubated in the absence (-) or presence (+, 20 μg/ml LPS) of endotoxin for 72 h, and
563
the protein secretion of TGF-β1 (C–D) and TGF-β2 (E–F) was then measured in the
564
supernatant. (C and E) Representative images of western blot experiments performed for
565
detection of TGF-β1 (C) and TGF-β2 (E) secretion. (D and F) Densitometric analyses of
566
the experiments shown in C and E, respectively. Protein levels were normalized against
567
tubulin, and data are expressed relative to the untreated condition. (G–J) ECs were
568
incubated with a specific TLR-4 inhibitor (CLI-095, 1 μM; CLI) (G–H), a specific
569
NAD(P)H oxidase inhibitor (Apocynin, 1mM; Apo) (I–J), and an antioxidant (NAC, 1
570
mM) (I–J) as well as exposed to endotoxin (+; 20 μg/ml LPS) for 72 h. Protein secretion of
571
TGF-β1 (G–I) and TGF-β2 (H–J) was then measured in the supernatant. Protein levels were
572
normalized against tubulin, and data are expressed relative to the untreated condition.
573
Significant differences were assessed by Student's t-test (Mann-Whitney). *, P < 0.05 and
574
**, P < 0.01 against untreated condition. Graph bars show the mean ± SD (N = 3-4).
575
28
576
FIG 2 Changes in protein expression during endotoxin-induced endothelial fibrosis are
577
inhibited by transfection of a siRNA against TGF-β1 and TGF-β2. (A–D) ECs were
578
transfected with siRNAs against TGF-β1 and TGF-β2 (siTGFβ1 and siTGFβ1,
579
respectively) or a non-targeting siRNA (siCTRL). Protein expression was then analyzed. (A
580
and C) Representative images of western blot experiments performed for detection of TGF-
581
β1 (A) and TGF-β2 (C). B and D show densitometric analyses of several experiments
582
shown in A and C, respectively. Protein levels were normalized against tubulin, and the
583
data are expressed relative to siCTRL-transfected cells. (E–J) ECs were transfected with
584
siRNAs against TGF-β1 and TGF-β2 (siTGFβ1 and siTGFβ1, respectively) or a non-
585
targeting siRNA (siCTRL). Protein expression was then analyzed. (E, G, and I)
586
Representative images of western blot experiments performed for detection of the
587
endothelial marker, VE-cadherin (VE-cad) (E), the fibrotic marker, α-SMA (G), and the
588
ECM protein, fibronectin (FN) (I). F, H, and J show densitometric analyses of several
589
experiments as shown in E, G, and I, respectively. Protein levels were normalized against
590
tubulin, and the data are expressed relative to siCTRL-transfected cells. Significant
591
differences were assessed by a one-way analysis of variance (ANOVA; Kruskal–Wallis)
592
followed by Dunn's post hoc test. *, P < 0.05 and ***, P < 0.001 against the untreated
593
siCTRL-transfected cells. Graph bars show the mean ± SD (N = 3-5).
594 595
FIG 3 Changes in endothelial and fibrotic marker expression during endotoxin-induced
596
endothelial fibrosis are inhibited by transfection of siRNAs targeting TGF-β1 and TGF-β2.
597
(A–H) ECs transfected with siRNAs against TGF-β1 (A–D) and TGF-β2 (E–H) (siTGFβ1
598
and siTGFβ1, respectively) or a non-targeting siRNA (siCTRL) were incubated in the
599
absence (-) or presence (+; 20 μg/ml LPS) of endotoxin for 72 h, and protein expression 29
600
was then analyzed. (A, C, E, and G) Representative images of western blot experiments
601
performed for detection of the endothelial marker, VE-cadherin (VE-cad) (A and E), and
602
the fibrotic marker, α-SMA (C and G). B, D, F and H show densitometric analyses of
603
several experiments as shown in A, C, E, and G, respectively. Protein levels were
604
normalized against tubulin, and the data are expressed relative to non-treated siCTRL-
605
transfected cells. Significant differences were assessed by a one-way analysis of variance
606
(ANOVA; Kruskal–Wallis) followed by Dunn's post hoc test. **, P < 0.01 against the
607
untreated siCTRL-transfected cells. Graph bars show the mean ± SD (N = 3-5).
608 609
FIG 4 Changes in the distribution of endothelial and fibrotic markers involved in
610
endotoxin-induced endothelial fibrosis are inhibited by transfection of siRNAs targeting
611
TGF-β1 and TGF-β2. (A–L) Representative images from ECs transfected with siRNAs
612
against TGF-β1 (siTGFβ1) (C–D and I–J) and TGF-β2 (siTGFβ2) (E–F and K–L) or a non-
613
targeting siRNA (siCTRL) (A–B and G–H) were incubated in the absence (A–F) or
614
presence of endotoxin (G–L) (20 μg/ml LPS) for 72 h. VE-cadherin/CD31 (red) and FSP-
615
1/α-SMA (green) were detected. Nuclei were stained using DAPI. Bar scale represents 10
616
μm. (N = 4).
617 618
FIG 5 Changes in ECM protein expression during endotoxin-induced endothelial fibrosis
619
are inhibited by transfection of siRNAs targeting TGF-β1 and TGF-β2. (A–D) ECs
620
transfected with siRNAs against TGF-β1 (A–B) and TGF-β2 (C–D) (siTGFβ1 and
621
siTGFβ1, respectively) or a non-targeting siRNA (siCTRL) were incubated in the absence
622
(-) or presence (+; 20 μg/ml LPS) of endotoxin for 72 h, and protein expression was then
623
analyzed. (A and C) Representative images of western blot experiments performed for 30
624
detection of the ECM protein, fibronectin (FN). B and D show densitometric analyses of
625
several experiments as shown in A and C, respectively. Protein levels were normalized
626
against tubulin, and the data are expressed relative to untreated siCTRL-transfected cells.
627
Significant differences were assessed by a one-way analysis of variance (ANOVA;
628
Kruskal–Wallis) followed by Dunn's post hoc test. **, P < 0.01 against the untreated
629
siCTRL-transfected cells. Graph bars show the mean ± SD (N = 3-5).
630 631
FIG 6 Changes in the distribution of ECM proteins involved in endotoxin-induced
632
endothelial fibrosis are inhibited by transfection of siRNAs targeting TGF-β1 and TGF-β2.
633
(A–L) Representative images of ECs transfected with siRNAs against TGF-β1 (siTGFβ1)
634
(C–D and I–J) and TGF-β2 (siTGFβ2) (E–F and K–L) or a non-targeting siRNA (siCTRL)
635
(A–B and G–H) were incubated in the absence (A–F) or presence of endotoxin (G–L) (20
636
μg/ml LPS) for 72 h. VE-cadherin/CD31 (red) and FN (green) were detected. Nuclei were
637
stained using DAPI. Bar scale represents 10 μm. (N = 4).
638 639
FIG 7 Fibroblast-like motility of endotoxin-induced ECs is mediated by ALK-5 activity.
640
ECs were treated with the ALK5 inhibitor, SB431542 (0.5 μM), and incubated in the
641
absence or presence of endotoxin (10 μg/mL LPS) and subjected to collagen gel contraction
642
assay measured as gel area (A), gel weight (B), or by means of transwell migration assay in
643
Boyden chambers (C). Cell movement was allowed to occur for 24 h. Significant
644
differences were assessed by a one-way analysis of variance (ANOVA; Kruskal–Wallis)
645
followed by Dunn's post hoc test. *: P < 0.05, **: P < 0.01 against vehicle-treated condition
646
(0 μg/mL LPS). Graph bars show the means ± SD (N = 3-5).
31
