Thrombosis |
From the Diabetes Research Institute at the Heinrich-Heine-University, Düsseldorf, Germany.
Correspondence to H. Hauner, MD, Clinical Department, Diabetes Research Institute at the Heinrich-Heine-University, Aufm Hennekamp 65, 40225 Düsseldorf, Germany. E-mail hauner{at}dfi.uni-duesseldorf.de
| Abstract |
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and interkeukin-1ß (IL-1ß) also
exerted a stimulatory effect on PAI-1 release and increased PAI-1 mRNA
levels. As assessed by a semiquantitative reverse
transcriptionpolymerase chain reaction technique, TGF-ß1 mRNA is
expressed by differentiation of human preadipocytes and is moderately
upregulated by TNF-
and IL-1ß. In conclusion, our results clearly
indicate that TGF-ß1 is a potent inducer of PAI-1 production
in subcutaneous human adipocytes. In addition, data suggest that
TNF-
and IL-1ß also have stimulatory effects on PAI-1 protein
secretion and may contribute to the elevated PAI-1 levels observed
in obesity.
Key Words: obesity tumor necrosis factor growth factors interleukin plasminogen activator inhibitor
| Introduction |
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Elevated plasma levels of PAI-1 were found in obese subjects in many clinical and epidemiological studies, and these appear to be closely correlated with an abdominal pattern of adipose tissue distribution in both men7 and women8 9 10 11 12 but are also positively associated with other components of insulin-resistance syndrome.3 The mechanisms responsible for elevated PAI-1 plasma levels in this syndrome are not yet completely understood. In vitro studies have demonstrated that a variety of cell types, including endothelial and mesothelial cells, are able to produce PAI-1.13 14
In 1991, Sawdey and Loskutoff13 demonstrated for the first time the expression of PAI-1 in mouse adipose tissue. Subsequent studies detected PAI-1 mRNA and protein in adipocytes from other sources as well as in differentiated 3T3-L1 adipocytes.15 16 17 Recently, expression and secretion of PAI-1 was also demonstrated in adipose tissue from humans.18 19 20 Little currently is known about regulation of PAI-1 in adipose tissue. According to animal data, transforming growth factor-ß1 (TGF-ß1) appears to be a major inducer of PAI-1 synthesis in adipose tissue.13 A stimulatory effect of TGF-ß1 on PAI-1 production has recently been described in human adipose tissue explants.18
Studies in murine adipocytes and 3T3-L1 cells demonstrated a
stimulating effect of tumor necrosis factor-
(TNF-
) on PAI-1
expression and secretion,13 21 whereas first experiments
in human adipose tissue gave contradictory results.18 20
TNF-
is expressed and secreted by human
adipocytes,22 23 24 and researchers have hypothesized that
TNF-
is the main mediator of insulin resistance in the obese
state.23 Another interesting cytokine is
interleukin-1ß (IL-1ß),25 which, like TNF-
, is well
known to suppress adipose differentiation and lipoprotein lipase
expression and activity.26 A few studies reported that
IL-1ß is able to stimulate PAI-1 production in human and rat
endothelial cells,27 28 29 but nothing is
currently known about its effects on PAI-1 synthesis in human
adipocytes.
The aim of this study was to further characterize regulation of PAI-1
production in a model of in vitro differentiated human
adipocytes. We were particularly interested to investigate the effects
of TGF-ß1, TNF-
, and IL-1ß on PAI-1 mRNA expression and
secretion.
| Methods |
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from Pepro Tech Inc. The PAI-1 ELISA kit was from
WAK Chemie; the TGF-ß1 ELISA kit was from R&D; and Trizol, reverse
transcriptase (RT) Superscript RT, random hexamers, Taq
polymerase, and oligonucleotides were obtained from
Gibco/BRL. For sequencing of transcripts, we used the ABI Prism Big Dye
Terminator Cycle Sequencing Ready Reaction Kit from Perkin Elmer; the
gel-extraction kit was obtained from Clontech. Immunohistochemistry was
performed with an LSAB-kit from Dako; polyclonal antibody against PAI-1
was obtained from Chemicon; and all other chemicals were from
Boehringer or Merck. Sterile plastic ware for tissue culture
was purchased from Flow Laboratories.
Cell Isolation and Culture
Subcutaneous adipose tissue samples (20 to 80 g) were
obtained from the mammary adipose tissue of young, normal-weight women
(body mass index, <26 kg/m2; age range, 19 to 36
years) undergoing surgical mammary reduction. Stromal cell fraction was
isolated by collagenase digestion as described
previously.30 Samples were dissected from fibrous material
and visible blood vessels minced into small pieces (10 mg) and digested
in 10 mmol/L PBS containing 0.5 mg/mL crude
collagenase and 20 mg/mL BSA, pH 7.4, for
90 minutes.
The stromal cell fraction obtained after a short
centrifugation at 200g was incubated with
erythrocyte-lysing buffer that contained 154 mmol/L
NH4Cl, 5.7 mmol/L
K2HPO4, and 0.1 mmol/L
EDTA to eliminate contaminating erythrocytes. Cells were repeatedly
washed and resuspended in Dulbeccos Eagles/Hams F-12 medium
(vol/vol, 50:50) supplemented with 10% fetal calf serum. After cell
adhesion for 16 hours, cells were cultured under serum-free
hormone-supplemented conditions to allow adipose
differentiation.30
The inoculated cell fraction did not contain adipocytes as assessed by morphological criteria. In vitro differentiated adipocytes were defined as cells, the cytoplasm of which was completely filled with lipid droplets. In addition, GPDH activity was used as a differentiation marker and determined by an established method.31 In undifferentiated cultures, GPDH activity was below detection levels (<20 mU/mg protein) and increased to an average level of 486±73 mU/mg protein on day 16 of culture under standard adipogenic conditions.
Incubation With TNF-
, TGF-ß1, and IL-1ß
During the 16-day culture period, 40% to 70% of the
preadipocytes underwent adipose differentiation. Cultures were then
incubated with TNF-
(0.1 to 5 nmol/L), TGF-ß1 (4 to 400 pmol/L)
and IL-1ß (0.2 to 20 ng/mL) for the time periods indicated. Medium
for the determination of PAI-1 protein was stored at -20°C.
RNA Preparation
RNA was prepared according to the RNA isolation technique
described by Chomczynski and Sacchi.32 Cells were
harvested in Trizol and 200 µL of chloroform was added. After
centrifugation, the aqueous phase was mixed with an
equal volume of isopropyl alcohol. After 12 hours at -20°C, RNA was
pelleted for 15 minutes at 10 000 g, redissolved in LiCl, washed twice
with 70% ethanol, dried, and redissolved in H2O.
The purity of the RNA yield was verified by electrophoresis in a
formaldehyde-containing agarose gel.
cDNA Generation and Polymerase Chain Reaction
Total RNA was diluted to 0.2 µg/µL in
H2O and 1st strand cDNA prepared with 5 µL of
RNA, superscript RT, and random hexamers (both Gibco/BRL) according to
the instructions of the manufacturer. Two and one-half microliters of
cDNA and a polymerase chain reaction (PCR) primer mix that contained 2
U Taq polymerase in PCR buffer, 200 µmol/L each of
dCTP, dGTP, dTTP, and dATP, and 500 pmol/L of each primer in a 50 µL
volume with 50 µL of mineral oil. PCR conditions were a denaturing
step at 95°C for 1 minute, followed by 30 cycles of 94°C, 45
seconds; 57°C, 45 seconds; and 72°C, 45 seconds for the detection
of PAI-1 mRNA and by 26 cycles of 94°C, 45 seconds; 61°C, 45
seconds; and 72°C, 45 seconds for the detection of TGF-ß1 mRNA. PCR
products were transferred onto a 2% Tris-Borate/EDTAagarose gel,
stained with ethidium bromide, and analyzed with the
LumiAnalyst system from Boehringer.
For semiquantitative analysis, 2 primer sets were used simultaneously in the same tube: 1 was specific for PAI-1 and TGF-ß1 cDNA and the other for transcription factor Sp1, which is ubiquitously expressed and was used as an internal standard.33 PAI-1specific primers (301 bp) were 5'- GTG TTT CAG CAG GTG GCG C-3' sense (19 nucleotides) and 5'-CCG GAA CAG CCT GAA GAA GTG-3' antisense (21 nucleotides). TGF-ß1specific primers (288 bp) were 5'-AAC CGG CCT TTC CTG CTT CTC A-3' sense (21 nucleotides) and 5'-CGC CCG GGT TAT GCT GGT TGT A-3' antisense (22 nucleotides). Sp1-specific primers (231 bp) were 5'-GAG AGT GGC TCA CAG CCT GTC-3' sense (21 nucleotides) and 5'-GTT CAG AGC ATC AGA CCC CTG-3' antisense (21 nucleotides).
To determine the presence of mRNA of TGF-ß1 receptor type 2 and 3, we performed a qualitative RT-PCR with specific primers for these products: TGF-ß1 receptor 2 specific primers (324 bp), 5'-TGG CTG TAT GGA GAA AGA ATG ACG-3' sense and 5'-CGG TTA ACG CGG TAG CAG TAG AA-3', and TGF-ß1 receptor 3 specific primers (382 bp), 5'-CCC GCA AGC TGA CAT GGA TAA GA-3' sense and 5'-ACA AGG CCC CCG TCA GGA GTG-3' antisense. Sequences of the transcripts obtained were analyzed by use of ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit according to instructions of the manufacturer. Numbers of PCR cycles were chosen to ensure that amplification of PCR products was within exponential range.
Measurement of PAI-1 and TGF-ß1 Protein
PAI-1 and TGF- ß1 proteins were measured in culture medium by
use of commercially available specific ELISA kits. The interassay and
intra-assay variations of both ELISAs were <10%.
Immunohistochemical Staining of PAI-1 in Newly Differentiated
Adipocytes
Adipocytes were cultured in cell-chamber slides (Becton
Dickinson) for 16 days. After being washed with PBS, newly
differentiated adipocytes were fixed with ethanol that contained 10%
methanol. Cells were subjected to the protocol of Dako (LSAB kit).
Polyclonal antibody directed against PAI-1 was used. Cultures were
counterstained with hematoxylin for 10 seconds, rinsed with tap water,
and mounted in Entellan synthetic resin.
Statistical Analysis
Results are expressed as mean±SEM. Differences between groups
were tested with Students t test for paired data.
Differences with P<0.05 were regarded as significant
| Results |
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Effects of TGF-ß, TNF-
, and IL-1 on PAI-1 Release Into the
Culture Medium From Newly Developed Human Adipocytes
In the primary culture model of human preadipocytes, a high
percentage of cells underwent differentiation and developed biochemical
and morphological characteristics of human adipocytes within 16 days.
These cultures were incubated with cytokines of interest and
for the time periods indicated. As shown in Figure 2
, TGF-ß1 increased accumulation of
PAI-1 in a dose- and time-dependent manner. Four, 40,and 400 pmol/L
TGF-ß1 increased the amount of PAI-1 in the medium after 48 hours by
1.4-fold, 2.7-fold, and 7.4-fold, respectively, to a maximum
concentration of 431±40 ng/mL at 400 pmol/L. A significant stimulatory
effect of 400 pmol/L TGF-ß1 on PAI-1 protein was already seen after
an incubation time of 6 hours (31.9±2.7 versus 9.7±2.7 ng/mL in
control cultures; P<0.05). When TGF-ß1treated
adipocytes were immunostained with specific polyclonal
antibody against human PAI-1, the percentage of immunopositive
adipocytes was not significantly different from control cultures at the
same antibody titer, which indicated that TGF-ß1 increased PAI-1
synthesis in each cell (Figure 1C
).
|
As demonstrated in Figure 3
, both TNF-
and IL-1ß also increased accumulation of PAI-1 in culture medium.
Incubation of newly differentiated human adipocytes with 5 nmol/L
TNF-
for 24 hours stimulated PAI-1 accumulation by 2.7-fold
(87.4±6.9 versus 32.5±6.7 ng/mL in controls; P<0.05). At
a concentration of 20 ng/mL, IL-1ß increased PAI-1 accumulation by
3.1-fold (173.9±17.6 versus 55.4±20.4 ng/mL; P<0.05;
Figure 3
).
|
Effects of TGF-ß, TNF-
, and IL-1 on PAI-1 mRNA in Newly
Differentiated Human Adipocytes
Specific PAI-1 mRNA was assayed after a 24-hour incubation of
newly differentiated human adipocytes with TGF-ß1, TNF-
, and
IL-1ß, respectively, by use of a semiquantitative RT-PCR method. As
demonstrated in Figure 4
, TGF-ß1 led to
an increase of PAI-1 mRNA by
2.3-fold at the highest concentration,
400 pmol/L (P<0.05). TNF-
at a maximal concentration of
5 nmol/L increased the steady-state level of PAI-1 mRNA by 1.7-fold
(P=NS). IL-1ß stimulated PAI-1 mRNA 3.4-fold at 0.2 ng/mL
and 3-fold at the maximum concentration of 20 ng/mL.
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Expression of TGF-ß1 Receptors in Cultured Human
Adipocytes
Because TGF-ß1 was found to be a potent stimulator of PAI-1
production and release, we were interested to study expression
of the 2 TGF-ß1 receptor subtypes that exert the biological effects
of this cytokine.34 By use of specific primers for
TGF-ß1 receptor 2 and 3, we determined that both subtypes are
expressed at the mRNA level in newly differentiated adipocytes (Figure 5
). In contrast, TGF-ß1 receptor
subtype 1 was not detectable (data not shown).
|
Effects of TNF-
and IL-1 on TGF-ß mRNA in Newly Differentiated
Human Adipocytes
Samad et al35 recently postulated that the effect of
TNF-
on PAI-1 secretion may be mediated by an increased
production of TGF-ß1 by adipocytes. To address this aspect,
we studied the effects of TNF-
and IL-1ß on the expression of
TGF-ß1 mRNA. As shown in Figure 6
, both
TNF-
and IL-1ß only moderately increased TGF-ß1 mRNA after 24
hours. TNF-
5 nmol/L increased TGF-ß1 mRNA levels by 37%
(P<0.05); IL-1ß 2 ng/mL, by 44% (P<0.05). We
also determined TGF-ß1 protein in the culture medium by highly
sensitive ELISA. However, TGF-ß1 protein was not detectable either
under basal conditions or in the presence of TNF-
or IL-1ß (data
not shown).
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| Discussion |
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To elucidate regulation of PAI-1 in human adipocytes we focused our
interest on the role of TGF-ß1 and other cytokines. TGF-ß1
is a pleiotropic cytokine that has been implicated in numerous
biological processes, which include wound repair and tissue
remodeling.34 We now can provide extended evidence that
TGF-ß1 is a strong inducer of PAI-1 mRNA synthesis and PAI-1 release
by human adipocytes. At the highest concentration of TGF-ß1 used in
our experiments, a 7.4-fold increase in PAI-1 protein and a 2.3-fold
increase in PAI-1 mRNA levels were observed. These findings point to a
direct and specific effect of TGF-ß1 insofar as our experiments also
revealed that adipose tissue expresses
2 TGF-ß1 receptor subtypes
(type 2 and 3) that are known to mediate most of the biological effects
of this cytokine.34
Apart from TGF-ß1, 2 other ubiquitously detectable cytokines,
TNF-
and IL-1, also seem to play a physiological
role in the regulation of PAI-1. To date, TNF-
is known to be
effective for stimulation of PAI-1 release in many cell types,
especially endothelial cells.13 We now
report that human adipocytes in primary culture also respond to TNF-
with enhancement of PAI-1 release and elevation of PAI-1 mRNA. These
data are in contrast to a recent report by Alessi et al,18
who could not find an effect of TNF-
on PAI-1 production in
cultured human adipose tissue pieces, whereas Cigolini et
al20 recently showed an increase of PAI-1
production after TNF-
incubation. Samad et al17
originally reported that mice treated with TNF-
exhibit elevated
expression of PAI-1 mRNA in adipose tissue. In further experiments,
these authors also were able to demonstrate that 3T3-L1 adipocytes in
culture respond to TNF-
treatment with upregulation of PAI-1 mRNA
expression. However, this latter finding could not be confirmed in a
study in differentiated murine 3T3-L1 cells.16
The effect of TNF-
on PAI-1 synthesis and secretion is of particular
interest, because TNF-
is well established to be expressed in human
adipose tissue.22 23 24 38 Animal studies also suggest that
TNF-
could play an important role in the development of
obesity-linked insulin resistance.23 In addition, the 2
TNF-
receptor subtypes are upregulated in human
obesity.24 Therefore, upregulation of the TNF-
system
could contribute to the elevated levels of PAI-1 protein in obesity
and, furthermore, link insulin resistance with impaired
fibrinolysis. This was recently further substantiated
by a study of Samad et al39 in ob/ob mice; these authors
reported that neutralization of TNF-
results in significantly
reduced levels of plasma PAI-1 and adipose tissue PAI-1.
Detection of TGF-ß1 mRNA by RT-PCR in human adipocyte cultures represents the first preliminary observation that TGF-ß1 is produced at the local level and may contribute to the regulation of PAI-1 production and release in an autocrine and paracrine function. However, we were unable to measure significant amounts of TGF-ß1 protein in the culture medium. Detection of TGF-ß1 mRNA in human adipose tissue is not sufficient to prove production of substantial amounts of functional TGF-ß1 protein. Therefore, further studies are required to finally demonstrate the synthesis of TGF-ß1 protein by adipocytes.
In our experiments, a modest effect of TNF-
exposure on TGF-ß1
mRNA levels occurred, thereby confirming the original observation by
Samad et al35 in obese mice. In the recent study from the
same group in ob/ob mice, neutralization of TNF-
or deletion of both
TNF receptors resulted in reduced adipose tissue TGF-ß mRNA
expression. This effect was exclusively mediated by means of the p55
TNF receptor.39 Further studies are required to
demonstrate clearly the expression of TGF-ß1 by human adipocytes and
to answer the question of whether TNF-
and IL-1ß stimulate PAI-1
production by means of stimulation of local TGF-ß1
expression.
Concerning a possible role of IL-1ß, IL-1ß is known to increase PAI-1 expression in human endothelial cells and rat cardiac microvascular endothelial cells.27 28 29 Our data unequivocally show that IL-1ß is able to increase both PAI-1 protein and PAI-1 mRNA levels in differentiated human adipocytes in primary culture. A specific stimulatory effect was already seen at 0.2 ng/mL IL-1ß, whereas rat endothelial cells showed an increase in PAI-1 production only at a 10-fold higher concentration.29 Again, this effect of IL-1ß in human adipocytes may be mediated by an increase in expression of TGF-ß1.
In conclusion, our present studies indicate that human adipocytes
are an important source of PAI-1 production. TGF-ß1 was found
to be a strong inducer of PAI-1 release and PAI-1 mRNA expression in
cultured human adipocytes. Likewise, TNF-
and IL-1ß stimulate
PAI-1 production in the same fat cell model, which indicates
that PAI-1 production is controlled by a variety of locally
synthesized cytokines or cytokines from other sources.
These findings may lead to a better understanding of why PAI-1
concentrations are upregulated in human obesity.
Received August 23, 1999; accepted February 8, 2000.
| References |
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