Although several mechanisms have been proposed for the cardioprotective effects of MSCs, including cardiomyocyte regeneration, spontaneous cell fusion and paracrine action [3], there is
Trang 1necrosis factor-a and interleukin-10 by hypoxia- and
serum-deprivation-stimulated mesenchymal stem cells
Implications for their paracrine roles
Zongwei Li, Hua Wei, Linzi Deng, Xiangfeng Cong and Xi Chen
Research Center for Cardiac Regenerative Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
Introduction
Ischaemic heart disease is a life-threatening condition
that may cause sudden cardiac failure and death
Many researchers have investigated cell transplantation
as an alternative treatment for heart disease Bone
marrow-derived mesenchymal stem cells (MSCs) are
easily obtainable and expandable, multipotent
progeni-tor cells [1] that have emerged as attractive candidates for cellular therapies for heart and other organ-system disorders [2] Although several mechanisms have been proposed for the cardioprotective effects of MSCs, including cardiomyocyte regeneration, spontaneous cell fusion and paracrine action [3], there is a growing
Keywords
IL-10; IL-1b; mesenchymal stem cell;
paracrine; TNF-a
Correspondence
X Chen; X Cong, Research Center for
Cardiac Regenerative Medicine, The
Ministry of Health, Cardiovascular Institute
& Fu Wai Hospital, Chinese Academy of
Medical Sciences & Peking Union Medical
College, 167 Beilishilu, Beijing 100037,
China
Fax ⁄ Tel: +86 10 88398584
E-mail: chenxifw@yahoo.com.cn;
xiangfeng_cong@yahoo.com.cn
(Received 26 April 2010, revised 27 June
2010, accepted 10 July 2010)
doi:10.1111/j.1742-4658.2010.07770.x
To understand the potential paracrine roles of interleukin-1b (IL-1b), tumour necrosis factor-a (TNF-a) and interleukin-10 (IL-10), the expres-sion and secretion of these factors by rat bone marrow-derived mesenchy-mal cells stimulated by hypoxia (4% oxygen) and serum deprivation (hypoxia⁄ SD) were investigated We found that hypoxia ⁄ SD induced nuclear factor kappa Bp65-dependent IL-1b and TNF-a transcription Fur-thermore, hypoxia⁄ SD stimulated the translation of pro-IL-1b and its processing to mature IL-1b, although the translation of TNF-a was unchanged Unexpectedly, the release of IL-1b and TNF-a from
hypox-ia⁄ SD-stimulated mesenchymal cells was undetectable unless ATP or lipo-polysaccharide was present This result suggests that IL-1b and TNF-a are not responsible for the paracrine effects of mesenchymal cells under ischae-mic conditions We also found that hypoxia⁄ SD induced the transcription and secretion of IL-10, which were significantly enhanced by lipopolysac-charide and the proteasomal inhibitor MG132 Moreover, both the condi-tioned medium from hypoxia⁄ SD-stimulated mesenchymal cells (MSC-CM) and IL-10 efficiently inhibited cardiac fibroblast proliferation and collagen expression in vitro, suggesting that mesenchymal cell-secreted IL-10 pre-vents cardiac fibrosis in a paracrine manner under ischaemic conditions Taken together, these findings may improve understanding of the cellu-lar and molecucellu-lar basis of the anti-inflammatory and paracrine effects of mesenchymal cells
Abbreviations
BrdU, 5-bromodeoxyuridine; DMEM, Dulbecco’s modified Eagle’s medium; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; hypoxia ⁄ SD, hypoxia and serum deprivation; IL, interleukin; IMDM, Iscove’s modified Dulbecco’s medium;
LPS, lipopolysaccharide; MSCs, mesenchymal stem cells; NF-jBp65, nuclear factor kappa Bp65; p38, p38 mitogen-activated protein kinase; TNF-a, tumour necrosis factor-a.
Trang 2body of evidence supporting the hypothesis that
para-crine mechanisms mediated by MSC-secreted factors
play an essential role in the reparative process [4,5]
It has been reported that MSC-conditioned medium
under normoxic conditions significantly attenuates
car-diac fibroblast proliferation and type I and III collagen
expression, exerting paracrine anti-fibrotic effects
However, researchers did not analyse the active
compo-nents of the conditioned medium [6] Other researchers
have suggested that adrenomedullin and hepatocyte
growth factor are paracrine factors secreted by
trans-planted MSCs, decreasing myocardial fibrosis [7–9]
Whether other paracrine factors released by MSCs
mediate these cells’ anti-fibrotic effects remains largely
unknown
Interleukin-1b (IL-1b) and tumour necrosis factor-a
(TNF-a) are present in the tissues or systemic
circula-tion in many inflammatory condicircula-tions It has also been
reported that the expression of IL-1b and TNF-a in
MSCs can be augmented by exposure to hypoxia [5]
Furthermore, IL-1b can induce cardiomyocyte growth
but inhibits cardiac fibroblast proliferation in culture
[10] By contrast, MSC transplantation in rat models
of myocardial infarction has anti-inflammatory effects,
decreasing protein production and gene expression for
IL-1b and TNF-a [11] To address these paradoxes of
both pro- and anti-inflammatory effects, the secretion
of IL-1b and TNF-a from MSCs under ischaemic
con-ditions must be further characterized
IL-10 is an anti-inflammatory cytokine that has been
reported to be involved in the immunomodulation
mediated by transplanted MSCs [12,13] IL-10 is also a
potential anti-fibrotic factor in the liver and kidney
[14–16] In addition, the protective effect of MSCs
against sepsis is dependent on IL-10, which is not
directly produced by the injected MSCs but rather by
endogenous macrophages [17] However, it is not known whether MSCs can secrete IL-10 under ischae-mic conditions, resulting in a paracrine anti-fibrotic effect in the heart
To assess the paracrine effects of IL-1b, TNF-a and IL-10 released by MSCs on cardiac remodelling under ischaemic conditions, conditioned medium from MSCs (MSCs-CM) was collected during hypoxia and serum deprivation (hypoxia⁄ SD) This medium was used to treat cardiac fibroblasts, enabling observation of the paracrine effects of MSCs The expression and secre-tion of IL-1b, TNF-a and IL-10 by hypoxia⁄ SD-stimu-lated MSCs were also investigated Our data demonstrate that MSCs-CM can inhibit cardiac fibro-blast proliferation and collagen synthesis, with
< 30 kDa molecules as its major active components MSCs did not secrete IL-1b and TNF-a under hypoxia⁄ SD conditions, although MSC-secreted IL-10 hindered cardiac fibroblast proliferation and collagen expression These findings suggest that IL-10 may be
an important paracrine, anti-fibrotic mediator secreted
by MSCs
Results
MSCs-CM inhibits cardiac fibroblast proliferation and collagen synthesis
The effects of MSCs-CM on cardiac fibroblast prolifer-ation and collagen synthesis were detected by [3 H]-thy-midine and [3H]-proline incorporation As shown in Fig 1A, MSC-CM treatment significantly inhibited [3H]-thymidine and [3H]-proline incorporation under normoxic or hypoxic culture conditions To further clarify the molecular mass range of important active factors in the MSCs-CM, the medium was divided into
Fig 1 MSCs-CM inhibits cardiac fibroblast proliferation and collagen synthesis (A) The effects of MSCs-CM on the incorporation of [ 3 H]-thy-midine and [ 3 H]-proline by cardiac fibroblasts under normoxic or hypoxic conditions Each data point represents the mean ± SEM of at least three independent experiments ***P < 0.001 versus normoxic control (Cont) group; ###P < 0.001 and ##P < 0.01 versus hypoxic control (Cont + h) group (B) The effects of the > 30 kDa and < 30 kDa components of MSCs-CM on the incorporation of [ 3 H]-thymidine and [ 3 H]-proline by cardiac fibroblasts under normoxic or hypoxic conditions ***P < 0.001 versus Cont group; ###P < 0.001 versus Cont + h group.
Trang 3> 30 and < 30 kDa components using a 30 kDa
molecular mass cut-off ultrafiltration membrane
Frac-tionation revealed that the < 30 kDa components, but
not the > 30 kDa components, of the MSCs-CM
inhibited cardiac fibroblast proliferation and collagen
synthesis (Fig 1B)
Hypoxia⁄ SD induces NF-jB-dependent IL-1b and
TNF-a transcription
Because transcription of IL-1b and TNF-a can be
aug-mented in MSCs by hypoxia [5], and because the
molecular masss of IL-1b and TNF-a are both 17 kDa
(< 30 kDa), changes in IL-1b and TNF-a gene
tran-scription in hypoxia⁄ SD-stimulated MSCs were
exam-ined As shown in Fig 2A, the increased transcription
of IL-1b and TNF-a occurred after 3 h of hypoxia⁄ SD
with a gradual increase up to 6 h, after which
tran-scription decreased We also found that trantran-scription
of IL-1b and TNF-a was mainly induced by SD,
whereas hypoxia simply augmented this effect (Fig 2B)
It has been reported that the nuclear factor-jB (NF-jB) signalling pathway plays an important role in reg-ulating IL-1b and TNF-a transcription [18,19] To investigate the role of this pathway in hypoxia⁄ SD-induced transcription, MSCs were exposed to BAY 11-7082, an NF-jB pathway inhibitor, followed by hypoxia⁄ SD for 6 h As shown in Fig 2C, the tran-scription of IL-1b and TNF-a was significantly attenu-ated by BAY 11-7082 Interestingly, the proteasomal inhibitor MG132 also abrogated hypoxia⁄ SD-induced IL-1b and TNF-a transcription
Next, to clarify the mechanism by which the NF-jB pathway induces IL-1b and TNF-a transcription, the nuclear translocation of NF-jBp65 was assessed by immunocytochemical staining As shown in Fig 2D, NF-jBp65 was mainly distributed in the cytoplasm
of control cells By contrast, hypoxia⁄ SD treatment significantly stimulated the nuclear translocation of
Fig 2 Hypoxia ⁄ SD induces NF-jB-dependent IL-1b and TNF-a transcription (A) MSCs were incubated under hypoxia ⁄ SD conditions for the indicated number of hours, and the relative mRNA levels of IL-1b and TNF-a were determined by real-time PCR The data are the mean ± SEM of at least three independent experiments *P < 0.05 and **P < 0.01 versus control group (0 h) (B) The relative mRNA levels for IL-1b and TNF-a in MSCs after hypoxia, SD or hypoxia ⁄ SD for 6 h by real-time PCR **P < 0.01 versus Cont group; #P < 0.05 versus SD group (C) MSCs were exposed to BAY 11-7082 or MG132, followed by hypoxia ⁄ SD for 6 h and detection of relative mRNA levels of IL-1b and TNF-a by real-time PCR *P < 0.05 and **P < 0.01 versus hypoxia ⁄ SD treatment group (D) A representative pattern of the nuclear translo-cation of NF-jBp65, as assessed by immunocytochemical staining of MSCs using anti-(NF-jBp65 primary Ig) (red) and nuclear labelling with 4¢,6-diamidino-2-phenylindone (blue).
Trang 4NF-jBp65, indicated by strong immunostaining in the
nucleus Pretreatment with BAY 11-7082 inhibited
hypoxia⁄ SD-induced NF-jBp65 translocation, with
substantial levels of NF-jBp65 staining remaining in
the cytoplasm of most cells These results demonstrate
that hypoxia⁄ SD induces IL-1b and TNF-a
transcrip-tion, which are dependent on activation of the NF-jB
pathway
Hypoxia⁄ SD-induced IL-1b and TNF-a transcription
depend on the extracellular signal-regulated
kinase pathway
The extracellular signal-regulated kinase 1⁄ 2 (ERK1 ⁄ 2)
and p38 mitogen-activated protein kinase (p38)
signal-ling pathways play important roles in hypoxia⁄
SD-induced apoptosis of MSCs [20,21] and may also
affect IL-1b and TNF-a transcriptional regulation [22]
To confirm this, 20 lm U0126 (Fig S1A) was used to
inhibit the ERK1⁄ 2 pathway in MSCs, followed by
measurement of IL-1b and TNF-a mRNA levels by
real-time PCR As shown in Fig 3A, U0126 completely
abolished hypoxia⁄ SD-induced IL-1b and TNF-a
transcriptional upregulation When the MSCs were
exposed to 15 lm SB202190 (Fig S1B), a p38-specific
inhibitor, hypoxia⁄ SD-induced IL-1b transcription
was inhibited by 60%, although TNF-a transcription was not affected (Fig 3B) Like BAY 11-7082, U0126 could also inhibit NF-jBp65 nuclear translocation (Fig 3C), suggesting that hypoxia⁄ SD-induced activa-tion of the NF-jB signalling pathway depends on the ERK1⁄ 2 signalling pathway
Hypoxia⁄ SD increases the translation of pro-IL-1b but not TNF-a
Having demonstrated significant transcriptional upreg-ulation, we next examined protein levels of IL-1b and TNF-a in MSCs-CM Unexpectedly, neither IL-1b nor TNF-a was detectable in MSCs-CM using enzyme-linked immunosorbent assay (ELISA) analysis To determine the reason for this lack of IL-1b and TNF-a secretion by MSCs, changes in these factors’ transla-tion in hypoxia⁄ SD-stimulated MSCs were investi-gated As shown in Fig 4A, hypoxia⁄ SD increased pro-IL-1b translation in a time-dependent manner,
unchanged at each time point Furthermore, MG132, BAY 11-7082 and U0126, all of which abrogated hypoxia⁄ SD-induced IL-1b and TNF-a transcription, also abolished pro-IL-1b translational upregulation (Fig 4B,C) but failed to affect TNF-a translation
A
C
B
Fig 3 IL-1b and TNF-a transcriptional
induction depends on the ERK1 ⁄ 2 pathway.
MSCs were exposed to the ERK1 ⁄ 2
inhibitor U0126 or the p38 inhibitor
SB202190, followed by hypoxia ⁄ SD for 6 h.
(A,B) Relative mRNA levels for IL-1b and
TNF-a, as determined by real-time PCR.
*P < 0.05 versus hypoxia ⁄ SD group.
(C) A representative pattern of the nuclear
translocation of NF-jBp65, as assessed by
immunocytochemical staining of MSCs
using an anti-(NF-jBp65 primary Ig) (red)
and nuclear labelling with
4¢,6-diamidino-2-phenylindone (blue).
Trang 5(Fig 4B) These results demonstrate that in
hypox-ia⁄ SD-stimulated MSCs, IL-1b mRNA can be
effi-ciently translated into pro-IL-1b protein, whereas the
translation of TNF-a mRNA is severely repressed
Hypoxia⁄ SD induces cleavage of pro-IL-1b into
mature IL-1b
Given that hypoxia⁄ SD induced significant
transla-tional upregulation of pro-IL-1b and that processing
of pro-IL-1b into mature IL-1b requires activating
cleavage of pro-caspase 1 [23], the cleavage of both
pro-IL-1b and pro-caspase 1 was examined in
hypox-ia⁄ SD-stimulated MSCs As shown in Fig 5A,
hypox-ia⁄ SD promoted the processing of pro-IL-1b into
mature IL-1b, with a stronger induction effect in the
presence of the endotoxin LPS Consistent with these
data, hypoxia⁄ SD also induced the cleavage of
pro-caspase 1, with stronger activation in the presence of
LPS (Fig 5B)
Hypoxia⁄ SD-stimulated MSCs require a second
signal for IL-1b and TNF-a release
Although significant cleavage of IL-1b and
pro-caspase 1 occurred intracellularly in hypoxia⁄
SD-stim-ulated MSCs, mature IL-1b was undetectable in
MSCs-CM (Fig 6A) However, significant release of
IL-1b by hypoxia⁄ SD-stimulated MSCs in the presence
of ATP was detected Furthermore, when both LPS
and ATP were present, hypoxia⁄ SD-stimulated MSCs
released a larger amount of IL-1b (Fig 6A) We also
examined TNF-a expression in hypoxia⁄ SD-stimulated
MSCs in the presence of LPS As shown in Fig 6B,
LPS relieved the translational inhibition of TNF-a
Moreover, TNF-a release by MSCs was detectable
after hypoxia⁄ SD treatment for 6 h in the presence of LPS (Fig 6C) These findings demonstrate that hypoxia⁄ SD-stimulated MSCs require a second stimulatory signal in order to secrete IL-1b and TNF-a
Hypoxia⁄ SD induces the transcription and secretion of IL-10
Because of the lack of secretion of the inflammatory cytokines IL-1b and TNF-a from hypoxia⁄ SD-stimu-lated MSCs, as well as the significant anti-inflamma-tory effects of MSCs, expression and secretion of the anti-inflammatory cytokine IL-10 by these cells was investigated As shown in Fig 7A, hypoxia⁄ SD
A
C
B
Fig 4 Hypoxia ⁄ SD increases translation of pro-IL-1b but not TNF-a (A) Representative western blots for pro-IL-1b and TNF-a expression in MSCs stimulated by hypoxia ⁄ SD for the indicated number of hours (B) Representative western blots for pro-IL-1b and TNF-a expression in MSCs
in the presence and absence of BAY 11-7082 or MG132 *, nonspecific band (C) Representative western blots for pro-IL-1b expression in MSCs in the pres-ence and abspres-ence of U0126 *, nonspecific band.
A
B
Fig 5 Hypoxia ⁄ SD induces cleavages of pro-IL-1b and pro-cas-pase 1 MSCs were stimulated by hypoxia ⁄ SD in the presence or absence of LPS for the indicated number of hours (A) Representa-tive western blots for pro-IL-1b and mature IL-1b in MSCs (B) Rep-resentative western blots for pro-caspase 1 and cleaved caspase 1
in MSCs.
Trang 6induced significant IL-10 transcription after 3, 6 and
12 h Moreover, the transcriptional induction of IL-10
by hypoxia⁄ SD was abolished by the p38 inhibitor
SB202190 but was unexpectedly augmented by the
pro-teasomal inhibitor MG132 and by LPS (Fig 7B)
Next, the secretion of IL-10 from hypoxia⁄
SD-stimu-lated MSCs was examined by ELISA As shown in
Fig 7C, a small amount of IL-10 release from MSCs
was detected at the 6-h time point, and this release
was elevated at the 12-h time point Furthermore,
IL-10 secretion was augmented by the presence of LPS
at each time point
IL-10 inhibits cardiac fibroblast proliferation and collagen expression
The molecular mass of IL-10 is 19 kDa, which is
< 30 kDa and thus part of the MSCs-CM fraction that inhibited cardiac fibroblast proliferation and colla-gen synthesis (Fig 1B) To investigate the potential contribution of IL-10 to the paracrine effects of MSCs, the influence of IL-10 on cardiac fibroblast prolifera-tion was characterized using a 5-bromodeoxyuridine (BrdU) incorporation assay As shown in Fig 8A,B, different IL-10 concentrations significantly inhibited
A
B
C
Fig 6 Hypoxia ⁄ SD-stimulated MSCs
require a second signal for IL-1b and TNF-a
release (A) The results of ELISA analysis of
supernatants from MSCs after hypoxia ⁄ SD
stimulation for 12 h in the presence and
absence of ATP and LPS (B) Representative
western blots for TNF-a expression in MSCs
stimulated by hypoxia ⁄ SD in the presence
or absence of LPS for the indicated number
of h *, nonspecific band (C) The results of
ELISA analysis of supernatants from MSCs
after hypoxia⁄ SD stimulation for 12 h in the
presence or absence of LPS.
A
C
B
Fig 7 Hypoxia ⁄ SD induces expression and
secretion of IL-10 (A) Relative IL-10 mRNA
levels in MSCs stimulated by hypoxia ⁄ SD
for the indicated number of hours Data are
the mean ± SEM of at least three
indepen-dent experiments *P < 0.05 versus control
group (0 h) (B) Relative IL-10 mRNA levels
in MSCs after hypoxia ⁄ SD treatment for 6 h
in the presence and absence of various
reagents *P < 0.05 versus control group;
##P < 0.01 versus hypoxia ⁄ SD treatment
group (C) The results of ELISA analysis of
supernatants from MSCs after hypoxia ⁄ SD
stimulation for the indicated number of
hours in the presence or absence of LPS.
*P < 0.05 versus 6-h group; **P < 0.01
versus 12 h group.
Trang 7BrdU incorporation into cardiac fibroblast under
nor-mal 10% fetal bovine serum or serum-free culture
con-ditions IL-10 also decreased type I and III collagen
and a-smooth muscle actin mRNA levels in cardiac
fibroblasts (Fig 8C) Moreover, IL-10 effectively limited
angiotensin II-induced type I and III collagen protein
expression (Fig 8D) These results indicate that IL-10
can inhibit cardiac fibroblast proliferation and collagen
expression, suggesting a paracrine, anti-fibrotic role for
this factor
Discussion
In this study, we focused on the paracrine effects of
MSCs on cardiac fibroblast proliferation and collagen
expression, as well as the possible paracrine roles of
IL-1b, TNF-a and IL-10 in cardiac fibrosis First, our
results demonstrate that MSCs-CM have significant
anti-fibrotic effects, as indicated by decreased [3
H]-thy-midine and [3H]-proline incorporation by cardiac
fibroblasts Moreover, we found that < 30 kDa
compo-nents of MSCs-CM play the dominant anti-fibrotic
role, suggesting that these anti-fibrotic factors may be
soluble small molecules Second, our data show that
hypoxia⁄ SD induces NF-jB-dependent IL-1b and
TNF-a trTNF-anscriptionTNF-al upregulTNF-ation However, these two fTNF-ac-
fac-tors are not secreted from hypoxia⁄ SD-stimulated
MSCs unless a second signalling stimulus is present
This finding suggests that the paracrine roles of TNF-a
and IL-1b after MSC transplantation may be negli-gible Third, we determined that hypoxia⁄ SD induces transcription and secretion of IL-10, which signifi-cantly inhibits cardiac fibroblast proliferation and collagen expression MSC-secreted IL-10 may thus play a role in the attenuation of cardiac fibrosis under ischaemic conditions
NF-jB is a ubiquitous protein transcription factor that induces a variety of genes affecting the inflamma-tory processes [24,25] Normally, NF-jB is inactive and coupled to IjB protein [26,27] Based on our study, we hypothesize that hypoxia⁄ SD stimulates the phosphorylation and ubiquitin-related degradation of IjB The active form of NF-jBp65 is then released and translocated into the nucleus to activate the tran-scription of IL-1b and TNF-a In this report,
hypox-ia⁄ SD-induced IL-1b and TNF-a transcription were abolished by the ERK1⁄ 2 inhibitor U0126, suggesting that hypoxia⁄ SD-induced NF-jB activation is depen-dent on ERK1⁄ 2 signalling However, the p38 inhibi-tor SB202190 only partly inhibited hypoxia⁄ SD-induced IL-1b transcription and failed to affect the TNF-a mRNA levels Activation of p38 may thus be involved in the regulation of IL-1b mRNA stability by
a mechanism independent of NF-jB signalling
Pro-IL-1b is synthesized in the cytosol of activated cells without a signal sequence, precluding secretion via the classical endoplasmic reticulum–Golgi route [28] Processing of pro-IL-1b into its active form
Fig 8 MSC-secreted IL-10 is involved in the inhibition of cardiac fibrosis (A) BrdU incorporation in cardiac fibroblasts grown in standard DMEM at 24 h after IL-10 treatment at different concentrations **P < 0.01 and ***P < 0.001 versus Cont group (B) BrdU incorporation in cardiac fibroblasts grown in DMEM with 10% fetal bovine serum at 24 h after IL-10 treatment at different concentrations **P < 0.01 and
***P < 0.001 versus 10% fetal bovine serum treatment group (C) The relative mRNA levels of collagen I, collagen III and a-smooth muscle actin (a-SMA) in cardiac fibroblasts in the presence and absence of IL-10 *P < 0.05 and **P < 0.01 versus Cont group (D) Representative western blots for collagen I and III in the presence and absence of 0.1 l M angiotensin II and IL-10.
Trang 8requires caspase 1 [29], which is itself activated by a
molecular scaffold termed the inflammasome [23] It is
generally accepted that such IL-1b generation and
secretion by monocytes occurs in two steps First, an
inflammatory signal, such as the endotoxin LPS,
pro-motes the synthesis and cytoplasmic accumulation of
pro-IL-1b A second signal, in the form of exogenous
ATP, triggers caspase 1-mediated processing of
pro-IL-1b and secretion of the mature cytokine [30,31] In our
study, hypoxia⁄ SD enhanced the transcription and
translation of IL-1b as well as the cleavage of
pro-IL-1b into mature pro-IL-1b However, pro-IL-1b was not
released from hypoxia⁄ SD-stimulated MSCs unless
ATP or LPS was present
Interestingly, although hypoxia⁄ SD induced
signifi-cant TNF-a transcription, the translation of TNF-a
remained unchanged even when TNF-a transcription
was inhibited by MG132 or BAY 11-7082 The exact
reason for the translational repression of TNF-a is
unclear, but there are at least two possibilities:
micro-RNA-mediated TNF-a mRNA translational silencing
or TNF-a mRNA AU-rich element-mediated
post-transcriptional regulation involving AU-rich
element-binding proteins and processing bodies (P-bodies) [32]
Such AU-rich element-mediated translational
repres-sion of TNF-a may strongly correlate with IL-10
secre-tion by MSCs [33]
LPS preconditioning enhances the efficacy of MSC
transplantation in a rat model of acute myocardial
infarction, resulting in superior therapeutic
neovascu-larization and decreased fibrosis [34] Meanwhile, IL-10
has been reported to inhibit fibrosis in the liver [16],
kidney [15] and airway [35] In this study, we found
that LPS significantly augmented hypoxia⁄ SD-induced
IL-10 transcription and secretion Furthermore, IL-10
effectively inhibited cardiac fibroblast proliferation and
collagen expression in vitro, suggesting that IL-10 has
the potential to prevent cardiac fibrosis Thus, we
hypothesize that the enhanced anti-fibrotic effects of
LPS preconditioning may be because of increased
IL-10 secretion induced by LPS MG132 also
signifi-cantly inhibited hypoxia⁄ SD-induced MSC apoptosis
in vitro (data not shown) and enhanced IL-10
expres-sion Therefore, MG132 preconditioning may provide
another effective strategy of maximizing the viability,
paracrine effects and biological and functional
proper-ties of MSCs
In conclusion, our work demonstrates that
hypox-ia⁄ SD increases the transcription but not the secretion
of IL-1b and TNF-a, suggesting that the roles of these
factors in the paracrine effects of MSCs are negligible
However, hypoxia⁄ SD also enhances the transcription
and secretion of IL-10, which may be an important
mediator of the cells’ paracrine anti-fibrotic effects These findings help to improve our understanding of the cellular and molecular basis of MSCs’ anti-inflam-matory and paracrine effects
Materials and methods
Materials
Iscove’s modified Dulbecco’s medium (IMDM), Dulbecco’s modified Eagle’s medium (DMEM) and Trizol reagent were purchased from Invitrogen (Carlsbad, CA, USA) M-MLV reverse transcriptase was obtained from Promega (Madison,
WI, USA) and Power SYBR Green PCR Master Mix was purchased from Applied Biosystems (Foster City, CA, USA) SB202190, U0126, MG132, BAY 11-7082, LPS and angiotensin II were obtained from Sigma (St Louis, MO, USA) The BrdU cell proliferation assay kit was acquired from Calbiochem (Gibbstown, NJ, USA) ELISA detection kits for IL-1b, TNF-a and IL-10 as well as antibodies against IL-1b and TNF-a were obtained from R&D Sys-tems (Minneapolis, MN, USA), whereas antibodies against ERK, phospho-ERK1⁄ 2, p38 and phospho-p38 were pur-chased from Cell Signalling Technology (Danvers, MA, USA) Antibodies against NF-jBp65, caspase 1, collagen I, collagen III and b-actin and horseradish peroxidise-conju-gated secondary antibodies were manufactured by Santa Cruz Biotechnology (Santa Cruz, CA, USA)
Cell culture, inhibitor treatment and conditioned medium collection
Isolation and expansion of MSCs were conducted as previ-ously reported [20] Briefly, bone marrow was harvested from the tibias and femurs of 80 g rats, plated in IMDM supplemented with 15% heat-inactivated fetal bovine serum and 100 UÆmL)1 penicillin⁄ streptomycin and incubated at
37C in a humidified tissue culture incubator containing 5% CO2 The medium was replaced 4 h after plating and
24 h later to remove nonadherent hematopoietic cells Adherent MSCs were further grown in medium, which was replaced every 48 h The MSCs used in subsequent experi-ments had been passaged one to three times All procedures were approved by the Animal Care Committee of the Cardiovascular Institute and Fu Wai Hospital (Beijing, China) For inhibitor-based studies, 15 lm SB202190 (p38 inhibi-tor) [36,37], 20 lm U0126 (ERK1⁄ 2 inhibitor) [20], 10 lm MG132 (proteasome inhibitor) or 5 lm BAY 11-7082 (NF-jB inhibitor) was preincubated with MSCs in com-plete medium for 1 h The cells were subsequently washed
in serum-free IMDM and exposed to hypoxia⁄ SD in the continued presence of inhibitor Hypoxic conditions were generated by incubating the MSCs at 37C in a sealed hypoxic GENbox jar fitted with a catalyst to scavenge free oxygen, as described previously [20]
Trang 9MSC-CM was generated as follows First, 80% confluent
cells were administered serum-free DMEM and incubated
for 6 h under hypoxic conditions The medium was then
collected, clarified by centrifugation and divided into > 30
and < 30 kDa components using 30 kDa molecular mass
cut-off ultrafiltration membranes (Millipore, Billerica, MA,
USA) if necessary As a control, plates containing medium
alone were also subjected to the same conditions
Neonatal cardiac fibroblasts were isolated from Sprague–
Dawley rats (1–3 days old) and characterized as previously
described [38] All experiments were performed on the
sec-ond or third passage of cardiac fibroblasts after starvation
in serum-free DMEM for 24 h The cells were then treated
with control medium or MSCs-CM
[3H]-Thymidine and [3H]-proline uptake assays
Cardiac fibroblasts were transferred to 24-well plates,
starved of serum for 24 h and then stimulated with
stan-dard medium or MSCs-CM for 24 h [3H]-Thymidine or
[3H]-proline (Institute of High Energy Physics, Chinese
Academy of Sciences, Beijing, China) was added to each
well to a final concentration of 1 lCiÆmL)1 during the last
6 h of incubation Stimulation was terminated by rinsing
the cardiac fibroblasts three times with NaCl⁄ Pi and then
adding ice-cold 10% trichloroacetic acid for 30 min Cell
precipitates were washed three times with ice-cold NaCl⁄ Pi
and then solubilized in 1% SDS with 0.1 m sodium
hydrox-ide overnight at room temperature The radioactivity of
SDS-soluble protein was determined by liquid scintillation
spectrometry (Beckman Model LS6000-SC, Brea, CA, USA)
RNA extraction and real-time PCR analysis
Total RNA was extracted from MSCs using Trizol reagent
according to the manufacturer’s instructions Next, cDNA
was generated from 2 lg of total RNA using M-MLV
reverse transcriptase and oligo(dT)18 primer Real-time
PCR was performed in a total volume of 25 lL containing
0.5 lL RT product, 0.5 lm primers and 12.5 lL Power
SYBR Green PCR Master Mix
Glyceraldehyde-3-phos-phate dehydrogenase mRNA amplified from the same
sam-ples served as an internal control The relative expression
of each targeted gene was normalized by subtracting the
corresponding glyceraldehyde-3-phosphate dehydrogenase
threshold cycle (Ct) values using the DDCt comparative
method The sequences of all primers used in this work are
5¢-GCTGTGGCAGCTACCTATGT-CTTG-3¢ and 5¢-AGGTCGTCATCATCCCACGAG-3¢; TNF-a:
5¢-AACTCGAGTGACAAGCCCGTAG-3¢ and 5¢-GTAC
CACCAGTTGGTTGTCTTTGA-3¢; IL-10: 5¢-CAGACCC
CCCAAGTA-3¢; collagen I: TCCTGGCAATCGTGGTT
CAA and ACCAGCTGGGCCAACATTTC; collagen III:
TGGACAGATGCTGGTGCTGAG and GAAGGCCAG
(a-SMA): AGCCAGTCGCCATCAGGAAC and CCGG AGCCATTGTCACACAC; and glyceraldehyde-3-phosphate dehydrogenase: 5¢-GGCACAGTCAAGGCTGAGAATG-3¢ and 5¢-ATGGTGGTGAAGACGCCAGTA-3¢
Immunocytochemical staining for NF-jBp65
MSCs in IMDM supplemented with 10% fetal bovine serum were plated on six-well glass slides When the cells reached 70–80% confluence, they were preincubated with U0126 or BAY 11-7082 as described above and exposed to hypoxia⁄ SD for 6 h The cells were then fixed in 2% para-formaldehyde in NaCl⁄ Pi for 30 min, washed twice with NaCl⁄ Pi and permeabilized with 0.3% Triton X-100 in NaCl⁄ Pifor 10 min Next, the MSCs were blocked in 2% goat serum for 1 h and incubated with rabbit anti-(NF-jBp65 primary IgG) for 1–2 h The cells were then washed and incubated with rhodamine-labelled goat anti-(rabbit second-ary IgG) After three NaCl⁄ Piwashes and incubation with the nuclear stain 4¢,6-diamidino-2-phenylindone for 20 min, the MSCs were washed in NaCl⁄ Pi for 10 min and mounted in gelvatol for microscopic imaging
Protein extraction and western blotting analysis
Lysates of stimulated cells were prepared and subjected to SDS⁄ PAGE as previously described [20] Briefly, stimulated cells were rinsed twice with ice-cold NaCl⁄ Piand lysed in ice-cold lysis buffer for 30 min Cell lysates were then cen-trifuged at 13 000 g for 10 min at 4C and their protein concentrations were determined by the BCA Protein Assay Lysate amounts allowing equal protein loading between lanes were determined and mixed with 5· SDS sample buf-fer, boiled for 5 min and separated by 10–15% SDS⁄ PAGE before transferring the proteins onto nitrocellulose mem-branes by semi-dry transfer After blocking in 5% skim milk for 1 h, the membranes were rinsed and incubated overnight at 4C with gentle shaking and with the appro-priate diluted primary antibody in 5% BSA, 1· Tris-buf-fered saline (TBS) and 0.1% Tween-20 (TBS⁄ T) Excess antibody was then removed by washing the membranes with TBS⁄ T and subsequent incubation with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature After further washes in TBS⁄ T, the bands were visualized using an enhanced chemiluminescence detection kit and radiographic film exposure
ELISA analysis of IL-1b, TNF-a and IL-10 secretion
by MSCs
The MSCs-CM was concentrated 20· by ultrafiltration using 10 kDa molecular mass cut-off ultrafiltration mem-branes (Millipore) following the manufacturer’s instruc-tions Production of IL-1b, TNF-a and IL-10 by MSCs
Trang 10was then determined by ELISA using the commercially
available kits mentioned earlier according to the
manufac-turer’s instructions Absorbance was measured at 450 nm
using a microplate reader Results were compared with a
standard curve constructed by titrating rat IL-1b, TNF-a
and IL-10
BrdU incorporation assay
Cardiac fibroblasts were transferred to 96-well plates,
starved of serum for 24 h and stimulated with IL-10 for
24 h DNA synthesis at 24 h was measured using a BrdU
ELISA kit Briefly, the cells were incubated for 4 h at
37C with 20 lLÆwell)1of BrdU The supernatant was then
removed and the cells were fixed in 200 lLÆwell)1of
FixDe-nat for 30 min at room temperature Subsequently,
BrdU Ig, horseradish peroxidase-conjugated goat
anti-(mouse IgG) and substrate solution were applied to the
wells The absorbance of the samples was measured at
450 nm using a microplate reader
Statistical analysis
Data are expressed as the mean ± SEM Differences
among groups were tested by one-way analysis of variance
evaluated using Student’s t-test A value of P < 0.05 was
considered statistically significant
Acknowledgement
This study was supported by the National Natural
Science Foundation of China (30871024) and the Major
National Basic Research Program in the People’s
Republic of China (Program 973, 2007CB512108 &
2010CB529508)
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