Gebhardt
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Influence of organosulphur compounds from
garlic on the secretion of matrix
metalloproteinases and their Inhibitor TIMP-1 by
cultured HUVEC cells
Katrin Meyer and Rolf Gebhardt
Institute of Biochemistry, Medical Faculty, University of Leipzig,
Liebigstr. 16, 04103 Leipzig, Germany
Keywords: DADS, HUVEC, MMPs, TIMP-1
Abstract
Organosulphur compounds from garlic, especially diallyl disulphide
(DADS), affected the secretion of some matrix metalloproteinases
(MMPs) and of tissue inhibitor of metalloproteinase-1 (TIMP-1), one of
their inhibitors, in human umbilical vein endothelial cells. Addition of
DADS to the culture medium resulted in a concentration-dependent
reduction of MMP-2 and TIMP-1 secretion. In the presence of inducers
(phorbol 12- myristate 13-acetate, forskolin and tumour necrosis factor α)
addition of DADS caused a distinct concentration-dependent decrease of
MMP-9 and TIMP-1 secretion. Other organosulphur compounds like allyl
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mercaptan and S-allylcysteine showed a less clear effect on MMP- or
TIMP-1-secretion. All tested substances seemed to be toxic at high
concentrations only.
These results suggest that DADS, through shifting the MMP-TIMP
balance, might mediate some of the effects ascribed to garlic
preparations.
Abbreviations: AM, allyl mercaptan; AMS, allyl methyl sulphide; DADS,
diallyl disulphide; HUVEC, human umbilical vein endothelial cells; ELISA,
enzyme linked immuno sorbent assay; MMP, matrix metalloproteinase;
MTT, 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide; PMA,
phorbol 12- myristate 13-acetate; SAC, S-allylcysteine; TIMP, tissue
inhibitor of metalloproteinases; TNFα, tumour necrosis factor α
Introduction
Matrix metalloproteinases
(MMPs) are a family of zinc-dependent
proteinases involved in extracellular matrix degradation (Nagase and
Woessner, 1999; Benjamin, 2001). The balance between these
proteinases and their specific inhibitors (TIMPs) is thought to play an
important role in tissue remodelling and pathogenesis of many diseases
(e.g. angiogenesis, arthritis, atherosclerosis, metastasis and fibrosis)
(Brew et al, 2000; Galis and Khatri, 2002; Luttun et al, 2000; Gomez et
al, 1997). Concerning the physiology and pathology of the vascular
system, MMPs and TIMPs produced by endothelial cells are of particular
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interest (Luttun et al, 2000; van Hinsbergh, 1992; Kunz, 2002; Loftus et
al, 2002). Depending on the type of tissue, endothelial cells express a
distinct set of these proteinases and inhibitors that can be modulated by
cytokines and other signals (Hanemaaijer et al, 1993). Because of the
importance of MMPs for pathological events, agents that may affect their
expression or activity are currently discussed and developed as possible
drugs for the treatment of some of these diseases (George, 2000;
Celentano and Frishman, 1997; Demeule et al, 2000).
Accumulating evidence suggests that garlic consumption may improve
the health status in many types of disease including atherosclerosis
(Reuter et al, 1996; Orekhov and Grunwald, 1997). Among the many
components of garlic, the organosulphur compounds are believed to
show an antiatherosclerotic potential (Amagase et al, 2001; Lawson,
1998). In addition, interference with hepatic cholesterol biosynthesis was
demonstrated using rat hepatocytes and human Hep G2 cells (Gebhardt
et al, 1994). However, although several reports have added details about
possible molecular mechanisms of the organosulphur compounds
(Gebhardt 1997b, Gebhardt and Beck, 1996; Gebhardt, 1995), the
complex interactions with various physiological and pathological cellular
processes are far from being understood. Furthermore, aspects of the
bioavailability and biotransformation of these compounds have to be
taken into account. Though allicin is the first product formed by alliinase,
it is doubtful whether allicin occurs in systemic blood, because it is highly
reactive. On the other hand, there is evidence suggesting that diallyl
disulphide (DADS) and allyl mercaptan (AM) represent intermediates
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with detectable concentrations in peripheral blood (Germain et al., 2002).
In the present study, we have investigated whether some of these more
stable organosulphur compounds derived from garlic are able to interfere
with the secretion of MMPs and of TIMP-1 by human umbilical vein
endothelial cells (HUVEC).
Materials and methods
Materials
William’s E medium was purchased from Bio Whittaker (Verriers,
Belgium), foetal calf serum and accutase were from PAA Laboratories
(Linz, Austria), glutamine from Merck (Darmstadt, Germany), HUVEC,
Medium 200 and its supplements were from TEBU GmbH (Frankfurt,
Germany), trypsin and tumour necrosis factor α (TNFα) were obtained
from Boehringer-Mannheim (Mannheim, Germany), dimethyl sulfoxide
(DMSO) was from Carl Roth GmbH+Co (Karlsruhe, Germany), penicillin,
streptomycin, phorbol 12- myristate 13-acetate (PMA), forskolin and 3[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide (MTT) reagent
were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen,
Germany). AM was obtained from Aldrich Chemical Co. Ltd (Gillingham,
England), allyl ethyl ether and allyl methyl sulphide (AMS) were from
Lancaster Synthesis Ltd. (Morecambe, England). Culture flasks, 6-well
plates and other cell culture material were purchased from TPP Techno
Plastic Products (Trasadingen, Switzerland). Enzyme linked immuno
sorbent assay (ELISA) and activity assay test systems were obtained
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from Amersham Pharmacia Biotech (Buckinghamshire, England). DADS
and S-allylcysteine (SAC) were supplied by Jacques Auger (Tours,
France)
Cell Cultures
HUVEC were maintained in 75 cm² culture flasks at 37°C under an
atmosphere of 5% CO₂ and 95% air in a mixture of 2 different media
containing 80% (v/v) William’s E supplemented with 10% (v/v) heatinactivated foetal calf serum, 2 mmol/l glutamine, penicillin (50U/ml) and
streptomycin (50µg/ml) and 20% (v/v) Medium 200 supplemented with
low serum growth supplements consisting of 2% (v/v) foetal bovine
serum, 1.5 µg/ml basic fibroblast growth factor, 5 mg/ml heparin, 100
µg/ml BSA, 1 mg/ml hydrocortisone, 5 µg/ml human epidermal growth
factor, penicillin G (100 U/ml), streptomycin sulphate (100µg/ml) and
amphotericin B (0.25µg/ml). In 75 cm² flasks 15 ml of the medium
mixture were used, in 6-well plates cells were grown in 1 ml of the
medium mixture per well. Culture medium was replaced every second
day. When confluent monolayers were reached, cells were detached by
either trypsin or accutase and seeded into new flasks or 6-well plates at
a density of about 2500 cells/cm².
Incubation of the cell cultures with organosulphur compounds from garlic
For experiments, cultures were used between passages 4 and 15,
reaching about 80% confluency in 6-well plates. DADS, AM and SAC
were dissolved in DMSO or medium and diluted by fresh medium to
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concentrations reaching from 0.2 to 500µM. The concentration of DMSO
in the medium was adjusted to 0.91% in each case. Diluted substances
were added to the cell cultures during a 9 h or 24 h incubation period.
For stimulation of some MMPs and TIMP-1, 10 nM PMA, 25µM forskolin
and 10 ng/ml TNFα were added to the medium during the 9 h or 24 h
incubation period according to Hanemaaijer et al. (1993).
Analytical procedures
Conditioned medium was collected from 6-well plates after an incubation
period of 9 h or 24 h and centrifuged for 5 min at 13000 g to remove cells
and debris. Most samples were used freshly, only some were frozen at –
20°C until use. In some cases samples were diluted 1:4 – 1:10 with
assay buffers from the ELISA or activity assay test kit. Secretion of
individual MMPs and TIMP-1 was determined by ELISA and activity
assay techniques. Respective assays for MMP-1, -2, -3, -8, -9, and -13
ELISA, TIMP-1 ELISA and MMP-2 and –9 activity assay were performed
exactly according to the manufacturer’s recommended procedure. In
case of activity assays, MMPs were further activated during the test
procedure by adding p-aminophenylmercuric acetate. Experiments were
calibrated using internal and external standards. Each concentration of
test substances was measured in two, in some cases in up to eleven
different wells.
Toxicity studies
Cytotoxicity of DADS, AM and SAC was determined by MTT cytotoxicity
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assay. The assay was carried out according to Gebhardt (1997a) with
the following modifications: After the 9 h or 24 h incubation period, the
medium was discarded and 600µl of fresh medium were added to each
well of 6-well plates containing 0.5 mg/ml MTT reagent. After an
incubation period of further 2 h medium was replaced by 800 µl DMSO
for dissolving the formazane produced. The absorbance at 590 nm was
read immediately with a Tecan Spectra Fluor spectrophotometer.
Additionally, lactate dehydrogenase leakage assay was performed as
described (Gebhardt and Fausel, 2000).
Statistical analysis
All measurements were performed at least in duplicate. Data are
expressed as mean ± range. EC50 values were determined by curve
fitting analysis using Origin software on a PC.
Results
Cytotoxicity
In order to evaluate cytotoxic effects of garlic organosulphur compounds
on HUVEC cells, MTT cytotoxicity assays were performed with DADS,
AM and SAC. In HUVEC stimulated with PMA, forskolin and TNFα, no
cytotoxicity could be shown for DADS after a 24 h incubation period,
except for the highest concentration (500µM) where a slight reduction
could be observed (Fig. 1). AM and SAC seemed to be even less toxic.
In non-stimulated cells, results were not as clear as in stimulated due to
Figure 1
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large variations between different cultures. Nevertheless, after 9 h of
incubation they likewise suggest DADS (Fig. 1), AM and SAC (not
shown) to be non-cytotoxic and to affect cells only marginally at high
concentrations. Using lactate dehydrogenase leakage assays, similar
results were obtained (not shown).
Secretion of MMPs and TIMP-1
HUVEC were found to express and secrete mainly MMP-1, MMP-2 and
TIMP-1. After an incubation period of 9 h on average about 58 ng/ml of
MMP-1 (Fig. 2), 16 ng/ml of MMP-2 (not shown) and 44 ng/ml of TIMP-1
(Fig. 2) were secreted by the HUVEC. Treatment with PMA, forskolin and
TNFα for 9 h clearly enhanced the secretion of MMP-1 and TIMP-1 about
2.6- or 3.9-fold respectively (Fig. 2). A possible enhancement of MMP-2
was not tested.
While MMP-3 and MMP-9 secretion were not detectable by ELISA (the
detection limit was 2.5 ng/ml for MMP-3 and 0.5 ng/ml for MMP-9) in the
basal state, about 14 ng/ml MMP-3 and 2 ng/ml MMP-9 were secreted
after stimulating the cells with PMA, forskolin and TNFα for 24 h.
However, especially for MMP-9 the secretion rate considerably differed in
different subcultures. Cultures from lower passage numbers usually
secreted more MMP-9 (not shown). Similar to that, secreted MMP-9
activity measured by activity assay also varied a lot with values of about
5 ng/ml in average after 24 h of incubation including stimulants (not
shown). The differences between the two assays may be due to the fact
that the ELISA mostly detects pro-MMP-9, while the activity assay
Figure 2
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additionally detects activated MMP-9. Because of further activation of the
pro-MMP-9 during the activity assay procedure, all pro and active forms
of MMP-9 could be detected by the activity assay.
In contrast to the others, MMP-8 and MMP-13 secretion was never
detectable after 9 h or 24 h of incubation with or without stimulation (the
detection limit was 0.2 ng/ml for MMP-8 and 0.1 ng/ml for MMP-13) (not
shown). In general, the amount of the MMPs secreted by the HUVEC
increased with time up to at least 24 h (not shown).
Effects of DADS, AM and SAC on MMP- and TIMP-Secretion
Addition of DADS to the culture medium resulted in a slight
Figure 3
concentration-dependent reduction of secreted MMP-2 activity starting
above 10 μM and leading to a loss of approx. 30% at 500 µM after 9 h of
incubation. AM and AMS showed no reducing effect up to 500 µM (Fig.
3). The structural analogues, SAC and allyl ethyl ether, were also without
effect (not shown). Similar results were obtained with a specific ELISA
for MMP-2 after 9 h of cultivation (data not shown).
Using an activity assay for MMP-9 the DADS concentration-dependent
Figure 4
diminution of enzyme activity secreted by HUVEC after stimulation with
PMA, forskolin and TNFα was also evident after 24 h (Fig. 4). Diminution
started at a DADS concentration above 32 µM with an apparent EC-50
value of 65 µM. The MMP-9 ELISA showed similar results with a
reduction of MMP-9 secretion by DADS starting above 8 µM with an
apparent EC-50 value of 24 µM (Fig.5). In the activity assay, AM and
SAC did not show any reduction, whereas in the ELISA a slight reduction
Figure 5
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of MMP-9 secretion seemed to occur at the highest concentration (Fig. 4
and 5).
In contrast to MMP-2 and -9, secretion of MMP-1 was not effected by
DADS, AM or SAC in basal cultivation. Even after stimulation of the
HUVEC, no reducing effect on MMP-1 secretion was detectable below a
DADS concentration of 500 µM after 9 h of incubation (data not shown).
Likewise, neither DADS, nor AM or SAC reduced MMP-3 production of
HUVEC stimulated with PMA, forskolin and TNFα for 24 h (data not
shown).
On the other hand, DADS not only influenced production of certain
MMPs, it also reduced the secretion of TIMP-1 (Fig. 6). The most
prominent effect of DADS could be observed in stimulated cells,
characterized by a reduction of TIMP-1 secretion above 16 µM with an
EC-50 value of about 75 µM. Non-stimulated HUVEC reacted only in a
moderate DADS concentration-dependent TIMP-1 decrease starting
above 50 µM and leading to a maximal reduction by 22-37% (Fig.6).
Again, AM and SAC showed no effect, neither in stimulated HUVEC (Fig.
6), nor in non-stimulated cells (data not shown).
Discussion
The present study describes two main results: (a) cultured HUVEC
secrete a distinct set of MMPs and TIMP-1 which is altered in response
to a cocktail of stimulating signals and (b) DADS is the only of the tested
organosulphur compounds able to downregulate selective members of
Figure 6
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the secreted proteases or the inhibitor in the basal as well as in the
induced state. Concerning induction of MMP and TIMP secretion, several
signalling pathways seem to be important, as all three stimuli, PMA,
forskolin and TNFα, together enhanced the secretion. PMA is a potent
activator of protein kinase C (Liu and Heckman, 1998), forskolin
enhances cyclic AMP (Chabardès et al, 1999) and TNFα is a very
important inflammatory mediator acting through NF-ĸB (Hehlgans and
Männel, 2002). Obviously, these signals induce only a specific set of
proteases that seems to be characteristic of HUVEC. In accordance,
Hanemaaijer et al. (1993) showed that the three single substances
variously influence different MMPs and TIMPs in different cells.
The fact that only DADS interfered with the secretion of MMPs and of
TIMP-1 is remarkable from several points of view. Firstly, secretion by
HUVEC is much more sensitive to DADS than any cytotoxic response.
Secondly, there is a structural aspect. The comparison with some
structural analogues suggests that the disulphide bond and/or two allylgroups are necessary for interference. Of course, additional experiments
are needed to define the exact structural requirements as well as the
mode of action. Nonetheless, it is interesting that AM is inactive,
although it has been shown in several studies to mimic effects of DADS
at least at higher concentrations (Gebhardt and Beck, 1996). Thirdly, the
influence of DADS seems very specific manifesting only on a subset of
MMPs and on TIMP-1. This again raises the question of the molecular
mechanisms behind this effect as interference seems not coupled to
inducibility. On the other hand, it indicates that DADS may change the
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balance between MMPs and inhibitors in endothelial cells independent of
their state of activation. Finally, DADS is derived from allicin, the product
formed from the genuine garlic compound alliin under conditions of an
active alliinase, while SAC is found in ethanol treated garlic preparations
only (Yeh and Liu, 2001). Our results demonstrate that SAC, in contrast
to DADS, does not show any influence on MMP and TIMP secretion
indicating that physiological effects may depend on the type of garlic
material consumed.
Together, these findings add new aspects to the possible role of garlic in
health and disease. The ability of a garlic derived organosulphur
compound to modulate the balance between MMPs and TIMPs renders it
possible that garlic consumption affects the breakdown of extracellular
matrix in or around blood vessels. Since matrix deposition and disruption
are fundamental aspects of arteriosclerosis and plaque rupture (Shah,
2000; Galis and Khatri, 2002; Ye et al, 1998), it seems worth to further
investigate the influence of garlic consumption on these processes in
diseased states.
Acknowledgements
The authors would like to thank F. Struck and Dr. F. Gaunitz for technical
assistance. This work was supported by a grant (QLK1-CT-1999-00498)
from the European Commission.
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Legends to figures
Figure 1. Cytotoxicity of DADS (□) after 9 h of HUVEC cultivation under
basal conditions and of DADS (■), AM (●) and SAC (▲) after 24 h of
cultivation with PMA, forskolin and TNFα determined by MTT cytotoxicity
assay. Results are expressed in percentage of absorbance of control
cultures. Values represent mean ± range from one experiment.
Figure 2. Secretion of MMP-1 and TIMP-1 by HUVEC after 9 h of
incubation without stimulants (
and TNFα (
) or with the stimulants PMA, forskolin
) determined by ELISA. Values represent mean ± range
from one experiment (MMP-1) or two experiments (TIMP-1).
Figure 3. Effect of DADS (□), AM (○) and AMS (⊗) on secreted MMP-2
activity of HUVEC after 9 h of basal cultivation. Activity was measured in
diluted samples and is shown as percentage of corresponding values in
control cultures. Values represent mean ± range from one experiment.
Figure 4. Effect of DADS (■), AM (●) and SAC (▲) on secreted MMP-9
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activity of HUVEC after 24 h of cultivation in presence of PMA, forskolin
and TNFα. Results are expressed in percentage of absorbance of control
cultures. Values represent mean ± range from one experiment.
Figure 5. Effect of DADS (■), AM (●) and SAC (▲) on MMP-9 secretion
of HUVEC after 24 h of cultivation in presence of PMA, forskolin and
TNFα measured by ELISA. Results are expressed in percentage of
absorbance of control cultures. Data represent mean ± range from two
independent experiments.
Figure 6. Effect of DADS (□) after 9 h of basal cultivation and of DADS
(■), AM (●) and SAC (▲) after 24 h of incubation in presence of PMA,
forskolin and TNFα on the secretion of TIMP-1 by HUVEC determined by
ELISA. Stimulated samples were diluted 1:10, non-stimulated were used
undiluted. Results are expressed as percentage of absorbance of control
cultures. Values represent mean ± range from one experiment.
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