Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institute, 17177 Stockholm, Sweden
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ABSTRACT |
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Human umbilical vein smooth muscle cells (HUVSMCs) utilize
extracellular cystine, glutathione (GSH), and
N-acetylcysteine (NAC) to synthesize
cellular GSH. Extracellular cystine was effective from 5 µM, whereas
GSH and NAC were required at 100 µM for comparable effects. The
efficacy of extracellular GSH was dependent on de novo GSH synthesis,
indicating a dependence on cellular -glutamyltransferase (glutamyl
transpeptidase). Coculture of syngenetic HUVSMCs and corresponding human umbilical vein endothelial cells (HUVECs) on porous
supports restricted cystine- or GSH-stimulated synthesis of HUVSMC GSH
when supplied on the "luminal" endothelial side. Thus HUVSMC GSH
rapidly attained a steady-state level below that achieved in the
absence of interposed HUVECs. HUVSMCs also readily utilize
both reduced ascorbate (AA) and oxidized dehydroascorbate (DHAA) over
the range 50-500 µM. Phloretin effectively blocked both AA- and
DHAA-stimulated assimilation of intracellular AA, indicating a role for
a glucose transporter in their transport. Uptake of extracellular AA
was also sensitive to extracellular, but not intracellular, thiol
depletion. When AA was applied to the endothelial side of the coculture
model, assimilation of intracellular AA in HUVSMCs was restricted to a
steady-state level below that achieved by free access.
human vascular smooth muscle cell antioxidant regulation; endothelial cell-smooth muscle cell interactions
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INTRODUCTION |
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OXIDATIVE STRESS ARISES from an imbalance between prooxidant and antioxidant states in the cell. Oxidative stress has been implicated in a growing number of physiological and pathophysiological processes in human cells and tissues (12). For instance, it is now well established that oxidative stress is tightly associated with the injurious events within the cells of the vascular wall occurring during atherosclerosis (25), ischemia-reperfusion (8), and acute or chronic inflammations (16). It is thus of considerable clinical interest to be able to manipulate the antioxidative capacity of cells undergoing oxidative stress during such processes. To this end, many xenobiotic principles have been synthesized that function as antioxidants or antioxidant mimetics. Most notable of this latter class are the superoxide dismutase mimetics (9) and glutathione (GSH) peroxidase mimetics (4). An alternative strategy to the use of xenobiotic principles would be to utilize endogenous antioxidant principles and/or their metabolic precursors. Two antioxidants that have attracted considerable clinical interest as both markers of oxidative stress and potential therapeutic agents are the tripeptide GSH and ascorbate. However, before one can fully realize the potential of manipulating endogenous antioxidant defenses in a "natural" manner, one must understand the basic biochemical processes that control the intercellular supply and disposition of these molecules in human tissues.
The cellular composition of the human vascular wall mainly consists of
the endothelial cells lining the vessel wall and the smooth muscle
cells surrounding these in a variety of layers, separated by various
basal membranes, or intimae. Endothelial cells provide a selective
barrier to the uptake of circulatory substances, such as regulatory
molecules and metabolic precursors, for example, for antioxidant
defense. Furthermore, myoendothelial cooperation in such transport
processes is an essential prerequisite for the further passage of the
substances to the tissues. We have previously studied the
amino acid precursor uptake and specificity for GSH synthesis in human
umbilical vein endothelial cells (HUVECs). These endothelial cells
demonstrated effective uptake of cystine from the circulation (3),
through a combination of the
Xc
transporter and
-glutamyltransferase (glutamyl transpeptidase) (7),
which was optimized to the plasma concentration of the disulfide
(25-50 µM). On the other hand, the uptake of cysteine was
relatively inefficient, and the cells lacked the ability to utilize
extracellular methionine (3). We also demonstrated the ability of the
cells to utilize extracellular GSH itself for the synthesis of the
tripeptide, although the concentrations required for this were
nonphysiologically high, in the millimolar range. The HUVECs are also
able to deacetylate the cysteine precursor thiol drug
N-acetylcysteine (NAC) and utilize the
resultant cysteine for GSH synthesis (6).
In another study, we monitored the characteristics of uptake of reduced ascorbate (AA) and dehydroascorbate (DHAA) into HUVECs. We demonstrated rapid, energy-dependent uptake of AA, with a dependence on a cellular glucose transporter. On the other hand, the uptake of DHAA and its reduction to ascorbate in the cells by GSH- and/or NADPH-dependent processes were shown to be relatively inefficient. The relative efficacies of these uptake processes reflect the respective plasma levels of these essential molecules, with the majority (>95%) of plasma ascorbate being in the reduced form (50-200 µM). Thus it has been suggested that these two redox forms of the antioxidant are transported into cells by distinct transporters (26).
Recently we described the isolation and culture of human umbilical vein smooth muscle cells (HUVSMCs) (1). This provides for several opportunities for further systematic study of the disposition of ascorbate and GSH precursors in the human vessel wall. First, little is known of the amino acid precursor specificity and uptake mechanism for GSH synthesis in human smooth muscle cells (21). Similarly, although the effects of extracellular ascorbate on a number of smooth muscle cell-specific biochemical processes have been studied (11), there have been no studies on the mechanism of uptake of AA and DHAA into human smooth muscle cells. Thus we report on the use of monolayer cultures of HUVSMCs for the study of the mechanism of sulfur amino acid/GSH and AA/DHAA assimilation into human smooth muscle cells. In addition, we have also previously described methods for the coculture of HUVECs and HUVSMCs, derived from the same individual, on either side of fixed, porous supports (1). Thus, using such cocultures, we investigated the potential role of endothelial cells in regulating the flow of metabolic precursors of intracellular AA and GSH in the underlying smooth muscle cells. The results are discussed in terms of the overall mechanisms of intercellular cooperation in the maintenance of ascorbate and GSH levels in the cells of the human vascular wall.
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MATERIALS AND METHODS |
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Chemicals and cell culture reagents.
Reduced GSH, AA, DHAA, all amino acids, monobromobimane, and buthionine
sulfoximine (BSO) were all purchased in >99% purity from Sigma
Chemical (St. Louis, MO). M199 and DMEM cell culture media and
fibronectin (human) were purchased from GIBCO (Inchinnan, UK). Sulfur
amino acid-free M199 (M199) was obtained from Nord Junter and
Cell AB (Bromma, Sweden). Collagenase (fraction 2, trypsin free) was
purchased from Boehringer-Mannheim (Mannheim, Germany). Human serum
(B+ve) was obtained from the blood donor center of
Sabbatsberg Hospital (Stockholm, Sweden). All other materials for the
culture of cells were obtained from Falcon (Becton Dickinson Labware,
Lincoln Oak, NJ). Chemicals and reagents for routine analyses were
obtained from local suppliers in the highest grade available.
Cell isolation and culture. Human umbilical cords were obtained from the delivery center of Karolinska Hospital (Stockholm, Sweden) and were kept on sterile PBS at 4°C until collection. The endothelial cells were obtained by collagenase perfusion of the vein as described by Jaffe et al. (15), with minor modifications (3). Endothelial cells were maintained in M199 supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml), fungizone (1.25 µg/ml), L-glutamine (2 mM), and human serum (10%) and passaged by conventional trypsinization, and the cells were generally frozen, after reaching confluency, for further use in coculture experiments with syngenetic HUVSMCs.
The smooth muscle cells were prepared from the umbilical cord vein, after removal of the endothelial cells, essentially according to Bohlin et al. (1). Briefly, the vein was refilled with collagenase (326 U/mg, 2 mg/ml in PBS, supplemented with 0.5 mM Ca2+ and 0.4 mM Mg2+) and incubated at 37°C for 30 min. The solution was flushed out and the vein was then cut open and trimmed free from the amnion. Sections were placed into explant culture, luminal face down, in DMEM supplemented with antibiotics and human serum as for the endothelial growth medium. The explants were removed after 3 days, and the adherent HUVSMCs were grown to confluency and passaged by trypsinization. Cells at passages 3-7 were routinely used for monolayer experiments and coculture experiments with HUVECs.Cell incubations. Confluent monolayers of HUVSMCs, on either 12- or 24-well plates, were exposed to AA or DHAA at a variety of concentrations in the DMEM culture medium for increasing times up to 24 h. After incubation, cells were washed three times in PBS, and the cell-associated ascorbate level was determined by HPLC as described below. Control experiments were performed in which it was determined that three washes were required to remove "loosely" associated ascorbate from the cells. In some experiments, phloretin (2), a potent inhibitor of the plasma membrane glucose transporter, was introduced during the incubation 5 min before the addition of AA- or DHAA-supplemented medium.
For the utilization of sulfur amino acid precursors of GSH biosynthesis, HUVSMCs were depleted of >90% of their GSH by an 18-h incubation in sulfur amino acid-free M199 supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml), and human serum (10%). The medium was then changed, and the same medium was reapplied to the cells but was supplemented with cysteine, cystine, or methionine at increasing concentrations. The cells were then incubated for increasing times up to 24 h and analyzed for intracellular GSH content as described below. Control measurements of cellular GSH levels before and after the initial depletion of GSH were also taken. In some experiments, HUVECs and HUVSMCs were cocultured on polyethylene teraphthalate permeable supports (Costar; 3-µm pore diameter, 8 × 105 pores/cm2) precoated with human plasma fibronectin (5 µg/ml in PBS) for 30 min at 37°C. The smooth muscle cells were seeded first at a density of 5 × 104 cells/cm2 on the outside of the insert. The cells were allowed to attach for 4 h, and then the insert was inverted, placed in a culture well in a 12-well plate, filled with 1 ml of the M199 endothelial cell culture medium, and seeded with HUVECs at a density of 5 × 104 cells/ml. The cells were then cultured until both cell layers approached confluency. Parallel plates of each cell type were cultured to estimate confluency of each cell type, which was finally assessed by the measurement of transmembrane resistance as described previously (1). A cutoff resistance of 12-14Biochemical assays. AA was assayed in cells and medium according to Honneger et al. (13) after extraction of the samples with TCA (1.25%). The molecule was separated by HPLC (Waters Associates) on a Supelcosil L-18 column (150 × 4.5 mm; 3 µm) and quantitated by electrochemical detection using an Antec Model CU-03 detector, with the electrode set in the oxidative mode at +0.5 V. Samples were analyzed as soon as possible after generation. Intracellular GSH was analyzed following in situ derivatization of the cells with the membrane-permeant thiol reagent monobromobimane, exactly as described previously (5). Cellular protein was determined by the method of Peterson (23).
Statistical methods. As appropriate, groups of data were compared for significant differences using a two-tailed, paired Student's t-test using an Instat software package. Significance was noted at the level of P < 0.05.
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RESULTS |
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To study the sulfur amino acid precursor specificity of GSH synthesis
in HUVSMCs, the cells were initially depleted of GSH either by BSO (25 µM in control medium) treatment or by incubation in M199 for
18 h, from 35.2 ± 2.1 or 32.6 ± 1.7 nmol GSH/mg protein (n = 3 for all observations)
to 2.2 ± 0.2 or 3.6 ± 0.3 nmol GSH/mg protein, respectively,
without morphological changes to the cells. In both cases, the
half-life of turnover of GSH in HUVSMCs appears to lie between 3 and 6 h. Reincubation in control medium of the cells depleted in M199
caused an immediate and linear increase in cellular GSH to 34.3 ± 1.8 nmol/mg protein over the initial 9 h of incubation, rising to 37.7 ± 1.4 nmol/mg protein by 24 h. On the other hand, the levels of GSH
in BSO-pretreated cells remained essentially unaltered after washing
and reincubation with control medium for up to 9 h (5.8 ± 0.4 nmol/mg protein) and rose to 29.4 ± 2.1 nmol/mg protein by 24 h of
incubation.
The incubation of GSH-depleted HUVSMCs with M199 containing
cystine as the sole sulfur source resulted in rapid resynthesis of GSH.
The initial levels of GSH achieved with cystine between 10 and 50 µM
were similar. However, after reaching a plateau at 6 h, cellular GSH
levels began to decline, but they were maintained at higher levels with
increasing concentrations of the disulfide (Fig.
1). It will be noted that
methionine (1 mM) induced only minimal resynthesis of GSH in HUVSMCs
under these conditions (Fig. 1). Interestingly, the thiol drug NAC was
also shown to be a precursor of GSH synthesis; however, the efficacy of
the compound was inferior to that of cystine or cysteine, based on the
number of cysteine equivalents in the medium (Fig.
2). Finally, the presence of extracellular GSH supported the elevation of intracellular GSH in depleted HUVSMCs in
a concentration-dependent manner. It will be noted that 100 µM GSH
was nearly as effective as 50 µM cystine in this respect (Fig.
3 vs. Fig. 1). The presence of BSO (25 µM) in the
extracellular medium greatly reduced the capacity of the cells to
utilize extracellular GSH to elevate intracellular levels of the
tripeptide, indicating the need for the activity of glutamate-cysteine
ligase (
-glutamyl cysteine synthetase) in this effect (Fig. 3).
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The HUVSMCs lack measurable levels of AA when cultured under normal conditions. However, the presence of extracellular AA facilitated the assimilation of AA within the cells. The initial rates of appearance of AA in the cells over the first 1 h were fairly similar over the range 50-500 µM extracellular AA, whereas by 6 h a clear dose-dependent correlation between extracellular and intracellular ascorbate was evident (Fig. 4). It will be noted that control experiments demonstrated that the cells required two washes in PBS before assay to remove AA loosely associated with the cells (data not shown). In contrast, the presence of DHAA in the extracellular medium also stimulated the appearance of AA associated with the cells, in a concentration-dependent manner. However, the kinetics of AA assimilation were somewhat different from those achieved with AA itself, with maximum concentrations of cellular AA being achieved by 60 min of incubation and declining thereafter (Fig. 5). Additionally, on a molar basis, DHAA was less effective than AA in supporting the elevation of intracellular AA in the cells (Fig. 4 vs. Fig. 5). Both DHAA-supported and AA-supported assimilation of AA into the cells were almost totally inhibited by the presence of the glucose transporter inhibitor phloretin (1 mM) in the medium (Fig. 6).
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Interestingly, when cells were incubated with AA or DHAA in M199 lacking all sulfur amino acid precursors, the levels of AA that accumulated in the cells in each case were far lower than for control medium (Fig. 7, A and B, respectively). However, cells depleted of their GSH by an overnight incubation with M199 lacking sulfur precursors before incubation with AA or DHAA in the presence of BSO (25 µM) also displayed normal accumulation of AA in the cells in each case (Fig. 7, A and B, respectively).
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In the intact blood vessel wall, the circulation almost certainly
serves as the major source of continued supply of metabolic precursors
of smooth muscle cell GSH and AA. Thus it follows that the endothelial
cells lining the vessel wall play an important role in regulating the
levels of these antioxidants in smooth muscle cells. To begin probing
the complexities of these interactions in the intact vessel wall, we
have employed syngenetic cocultures of HUVECs and HUVSMCs on fixed,
porous supports, in order to establish the role of an intermittent
endothelial cell layer in controlling the disposition of GSH and AA in
smooth muscle cells supplied with precursors from the "luminal"
side of the endothelial cells. It will be noted that, under the
conditions of coculture, conditions of high electrical resistance of
the bilayer were maintained throughout, indicating tight-junctional
integrity, particularly in the endothelial layer. To study GSH
assimilation in the smooth muscle cells of the bilayer, both cell types
were depleted of their GSH by an overnight incubation in medium lacking
sulfur amino acids (M199) (3). Control experiments showed that
this treatment did not affect the morphology of either cell type on the
filter. In Fig. 8A, it can
be seen that resynthesis of HUVSMC GSH using extracellular cystine or
GSH as a precursor on the luminal side of the endothelial cells was
retarded in both cases, compared with the respective rates in HUVSMCs
cultured alone on the filters. Similarly, Fig. 8B shows that, when AA was supplied to
the luminal side of the HUVECs, the assimilation of AA in smooth muscle
cells was again retarded, reaching a steady-state level by 60 min of
incubation, which was maintained for up to 3 h and declined thereafter.
In the absence of HUVECs, AA levels in HUVSMCs rose linearly over the
5-h test incubation period. In both cases, control experiments indicated that HUVSMCs did not release their complement of AA or GSH
during their removal from the supports by trypsinization (data not
shown).
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DISCUSSION |
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Previous studies in monolayer cultures of HUVECs have shown that the
synthesis of intracellular GSH is largely dependent on the uptake of
cystine and its reduction to cysteine (3). The uptake of the disulfide
was shown to be matched to the plasma concentration of the disulfide
and also to be dependent on the activity of both
-glutamyltransferase and the
Xc
carrier (7). Interestingly, we show here that the smooth muscle cells,
positioned immediately under the endothelial cells, also readily
utilize cystine as a precursor of GSH synthesis. However, the cells
appear to have an ability to utilize the disulfide far more efficiently
than do the endothelial cells (3), with considerable GSH synthesis
supported by levels as low as 5 µM. This indicates that HUVSMCs in
the intact vessel seem to be adapted to levels of cystine in the
extracellular fluid around them that are lower than the levels to which
endothelial cells are normally exposed in the plasma. This
perhaps indicates a reliance on carriers other than the
-glutamyltransferase and
Xc
systems operating in HUVECs (7).
Other differences in precursor uptake behavior also exist between
HUVSMCs and HUVECs. For instance, like HUVECs, HUVSMCs can utilize
extracellular GSH for the synthesis of intracellular GSH. However,
considerable resynthesis of GSH in GSH-depleted cells was achieved at a
reduced tripeptide level of 100 µM (Fig. 4), whereas millimolar
concentrations were required with HUVECs (3). Additionally,
extracellular GSH was only effective in HUVSMCs if cellular
glutamate-cysteine ligase was operative. This is unlike the situation
with HUVECs, in which BSO had no inhibitory effect on the ability of
extracellular GSH to elevate intracellular levels of the tripeptide
(3). These data strongly suggest that -glutamyltransferase is
expressed on the surface of HUVSMCs and is active in the initial uptake
of GSH from the extracellular space. Furthermore, as with HUVECs, it
appears that HUVSMCs lack one or more enzymatic components of the
cystathionase pathway and are unable to utilize extracellular methionine for GSH biosynthesis. Finally, the drug NAC was a less effective precursor of GSH synthesis in HUVSMCs, on a molar thiol basis, than cystine or GSH (Fig. 2 vs. Figs. 1 and 3). However, considerably lower concentrations of the thiol were required to support
substantial GSH in the smooth muscle cells than in HUVECs (6). This
suggests that the activity of the
N-deacetylase enzyme liberating
cysteine from the drug may be higher in HUVSMCs than in HUVECs. The
therapeutic significance of this during systemic NAC therapy is
uncertain. However, if NAC passes the endothelium intact, it may reach
the smooth muscle cells and provide considerable clinical benefit by
protecting smooth muscle cell GSH levels.
The above experiments detailing the mechanisms of uptake of precursors
of intracellular GSH and AA into smooth muscle cells lack one important
dimension. In the intact blood vessel wall, the supply of
sulfur-containing amino acid precursors to the intracellular environment of the smooth muscle cells may be achieved by active uptake
from the pericellular fluid on specific membrane-bound carriers, e.g.,
-glutamyltransferase or the
Xc
carrier, as discussed above. These precursors may be maintained in the
intimal fluid either indirectly, by active uptake into endothelial
cells from the circulation and subsequent secretion over the basal side
of the plasma membrane, or directly, via alterations in
tight-junctional integrity. In the former process, one particular mechanism that is of particular current interest is that of vesicular transport in and secretion from the endothelium, mediated by the vesiculo-vacuolar organelle (VVO) pathway (10, 19). Alternatively, after active uptake into the endothelial cells, metabolic cooperation may occur via myoendothelial gap junctions (14). In any event, the use
of simple monolayer culture models neither gives an accurate insight
into the true levels of GSH in smooth muscle cells in the intact vessel
wall nor allows us to delineate any intercellular interactions with
endothelial cells that may control precursor supply to smooth muscle
cells.
The data detailed in Fig. 8A show the influence of an intermittent HUVEC layer on extracellular cystine- and GSH-dependent assimilation of GSH in HUVSMCs. In both cases, intracellular GSH levels were rapidly restricted to a plateau level that was considerably lower than those levels achieved during direct cellular contact with extracellular precursors. Thus the endothelial cells seem to exert a controlling influence on the actual levels of GSH achieved in the smooth muscle cells. It will be noted, however, that the efficacy of resynthesis of GSH was inferior to that achieved with cells grown on plastic. Indeed, culture-specific alterations of the GSH levels of HUVECs have been previously noted (27). Irrespective of the molecular mechanism by which this control is achieved, these data clearly show that GSH levels achieved in cultured monolayers of HUVSMCs may have little relevance to the actual levels of the tripeptide maintained in these cells in the intact vessel wall.
The mechanisms underlying the effects of the intermittent endothelial
cell layer are, as yet, obscure. It may be suggested that the
endothelial cells simply offer a physical barrier to the passage of
precursors of HUVSMC GSH biosynthesis. On the other hand, a mechanism
involving uptake by the endothelial cells, followed either by secretion
into the intima and subsequent uptake of precursors into the smooth
muscle cells or by direct passage of GSH and/or its precursors
via myoendothelial gap junctions, may be postulated. Preliminary data
(unpublished observations) demonstrate that neither lipopolysaccharide
(300 ng/ml) nor tumor necrosis factor- (20 ng/ml) is able to affect
the kinetics of reassimilation of GSH in the HUVSMCs in the bilayer
coculture model, in response to either GSH or cystine supplied on the
luminal side of the HUVECs. Under these conditions, the agents are
known to cause total closure of myoendothelial gap junctions in a mixed
monolayer coculture (14). It may be argued, however, that such gap
junctional contacts are not present in the bilayer model presently
employed. However, ultra-structural studies of similar smooth muscle
cell-endothelial cell models, often of discordant vessel and genetic
origins, clearly demonstrate the presence of profuse numbers of gap
junctions spanning the gap between the cells within the
pores of membranes similar to those used in the present studies (24).
However, this matter awaits further clarification in the present model,
as does the possible involvement of VVO-dependent transport, although
the latter process is generally regarded as being specific for
macromolecules (10).
Thus it seems likely that HUVSMCs, which are clearly able to utilize lower concentrations of extracellular sulfur-containing precursors than the overlying HUVECs, assimilate their intracellular GSH in the intact vessel wall primarily as a result of active transport from a pool of sulfur amino acid precursors in the intimal fluid, which is maintained by secretion from overlying endothelial cells. In view of the potential role of a pool or pools of thiol precursors in the intimal fluid between endothelial and smooth muscle cells, it should be mentioned that very little is known of the actual levels of cystine and GSH in this extracellular compartment. This hampers prediction of which concentrations of the agents are relevant for the characterization of the respective active uptake processes. Thus experimental concentrations over a wide range were used in each case, the upper levels of which are clearly not relevant with respect to plasma concentrations of the respective agents. This quandary does not apply to endothelial cells, as they are in direct contact with the circulation, where relevant concentrations are easily amenable to assay.
Turning to ascorbate, from the data in Fig. 4 it is clear that AA is readily assimilated into HUVSMCs, in a concentration-dependent manner, when supplied extracellularly. However, unlike the case with HUVECs, levels reached saturation after 4-6 h and were generally much lower, on a nanomole AA per milligram protein per hour basis, than those achieved with corresponding concentrations of AA in the case of the HUVECs (26). Interestingly, on a molar basis, HUVSMCs were also able to utilize extracellular DHAA for the assimilation of intracellular AA nearly as effectively as extracellular AA itself (Fig. 5). The kinetics of assimilation were, however, somewhat different, with peak levels of AA achieved within the first 1 h of incubation and declining levels thereafter. This behavior is similar to that detected in HUVECs (26).
In terms of the relationships between the concentrations of AA and DHAA utilized to probe the biochemical processes detailed above, it should be mentioned that normal plasma levels of AA lie between 20 and 50 µM (22), with the corresponding levels of DHAA usually being lower than this (18). In the present experiments, both AA and DHAA have been utilized over the range 50-500 µM, which may be deemed unphysiologically high. However, these concentrations were chosen partly to maintain compatibility with our previous experiments with HUVECs and partly to accentuate those biochemical processes involved in the uptake of these materials into the smooth muscle cells. Additionally, little is known of the actual concentrations of AA and DHAA in the intimal fluid between these cells in vivo. However, AA concentrations in extracellular fluids, such as the epithelial lining fluid of the lung, clearly fall into this range (17). Furthermore, AA concentrations of up to 200 µM are often used in cell culture experiments studying the formation in and secretion of extracellular matrix materials from cells derived from connective tissues (11, 20).
In terms of the mechanism(s) of assimilation of intracellular AA in HUVSMCs, it appears that the activity of a glucose transporter is vital to the process, as the specific hexose transporter inhibitor phloretin (2) considerably hampered both AA- and DHAA-dependent assimilation of AA in the cells (Fig. 6). Previous studies have indicated a role for a glucose transporter(s) in other diploid cell types (2, 28, 29), and a hexose carrier has been shown to be responsible for the uptake of both AA and DHAA in some cell types (28). Interestingly, in the present experiments, both AA-dependent and DHAA-dependent assimilation of intracellular AA were also shown to be sensitive to the presence of sulfur-containing amino acids (reduced and/or oxidized) in the extracellular milieu. However, the depletion of intracellular GSH had no effect on this process (Fig. 7, A and B, respectively). The reasons underlying this are, however, obscure. It may be that optimal activity of the glucose transporter requires the presence of reduced and/or oxidized thiols at its extracellular surface.
Analogously to the above discussion of GSH assimilation in the HUVSMCs, precursors of AA are, in all probability, presented to the cells from the systemic circulation, via the overlying endothelial cells. From our studies using the HUVEC-HUVSMC bilayer cocultures it was again evident that an intermittent HUVEC layer strongly influenced the assimilation of AA in the HUVSMC when AA was supplied to the luminal side of the HUVECs (Fig. 8B). Thus it is again evident that intercellular cooperation between both cell types serves to maintain, over several hours, a steady-state level of AA in the smooth muscle cells that is well below levels achieved by free access to AA at all time points tested. This cooperative regulation may be of prime importance to the antioxidant function of AA in the smooth muscle cells, as previous work in HUVECs has shown that an optimal intracellular AA concentration is required in order for the molecule to elicit a protective antioxidant activity against hydrogen peroxide toxicity. When intracellular concentrations of AA were allowed to rise above this optimal level, then AA actually potentiated peroxide-dependent cytotoxicity, probably by acting as a prooxidant in supporting iron-dependent generation of reactive oxygen metabolites (26).
In terms of the molecular mechanism(s) involved in controlling the
supply of AA equivalents to HUVSMCs in the intact vessel wall,
preliminary experiments indicate that movement of AA via myoendothelial
gap junctions may not be involved, as the assimilation of AA in HUVSMCs
cocultured with HUVECs on fixed, porous supports (1) was not affected
by preincubation with either lipopolysaccharide or tumor necrosis
factor- (14) (unpublished observations). However, the same arguments
and restrictions in interpretation of these data are indicated as for
the case with GSH above. Thus, in view of the apparent efficacies of
the uptake of both AA and DHAA into HUVSMCs, it seems likely that
intracellular AA is assimilated and maintained in the smooth muscle
cells in the intact vessel wall by endothelial cell-dependent secretion
of AA and/or DHAA into the intimal fluid, perhaps as a result
of a VVO-mediated mechanism (10), coupled to active uptake of
these compounds into the smooth muscle cells.
In summary, we show that HUVSMCs are able to maintain their intracellular antioxidative capacity through the active uptake of extracellular precursors of GSH synthesis and AA. However, a number of aspects of these processes differ strongly from the HUVECs with which they have intimate contact in the intact vessel wall. These differences may represent adaptations to the availability of these endogenous agents in the extracellular fluid at the intima, compared with the plasma, which seems to be the major source of supply of these important antioxidant precursors to this particular cell. At a more integrated level of biological organization, however, the data also suggest that contact with an overlying endothelial cell layer may exert a controlling influence on the assimilation of these important water-soluble antioxidants in the smooth muscle cells, at least in the case of those juxtaposed to the endothelium. This may perhaps occur through endothelial cell-specific regulation of intimal concentrations of the appropriate metabolic precursors by active secretion processes. Further studies with this syngenetic human endothelial-smooth muscle cell coculture system may provide insight into the mechanisms of these cooperative metabolic interactions between the cells of the intact blood vessel wall, as well as revealing the overall regulatory role of vessel wall cells in the supply of systemic precursors of AA and GSH assimilation in underlying tissues. This is vital in order to develop a rational use of endogenous water-soluble antioxidants in the therapy of human disease.
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ACKNOWLEDGEMENTS |
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These studies were supported by grants from the Swedish Medical and Natural Sciences Research Councils, as well as from the Swedish Committee for Alternatives to Laboratory Animals in Research.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: I. A. Cotgreave, Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institute, Box 210, 17177 Stockholm, Sweden.
Received 24 February 1998; accepted in final form 25 June 1998.
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REFERENCES |
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1.
Bohlin, K.,
L. Olsson,
and
I. A. Cotgreave.
The isolation, culture and characterisation of human smooth muscle cells from umbilical vein and their reconstitution into a functional syngenetic co-culture model with endothelial cells.
Methods Cell Sci.
18:
329-341,
1996.
2.
Cornu, M. C.,
G. A. Moore,
Y. Nakagawa,
and
P. Moldéus.
Ascorbic acid uptake by isolated rat hepatocytes.
Biochem. Pharmacol.
46:
1333-1338,
1993[Medline].
3.
Cotgreave, I. A.,
D. Constantin,
and
P. Moldéus.
Nonxenobiotic manipulation and sulfur precursor specificity of human endothelial cell glutathione.
J. Appl. Physiol.
70:
1220-1227,
1991
4.
Cotgreave, I. A.,
and
L. Engman.
The development of diaryl chalcogenides and -(phenylseleneyl) ketones with antioxidant and glutathione peroxidase-mimetic properties.
In: Handbook of Synthetic Antioxidants, edited by L. Packer,
and E. Cadenas. New York: Marcel-Dekker, 1996, p. 305-320.
5.
Cotgreave, I. A.,
and
P. Moldéus.
Methodologies for the application of monobromobimane to the simultaneous analysis of soluble and protein thiol components of biological systems.
J. Biochem. Biophys. Methods
13:
231-249,
1986[Medline].
6.
Cotgreave, I. A.,
P. Moldéus,
and
I. Schuppe.
The metabolism of N-acetylcysteine by human endothelial cells.
Biochem. Pharmacol.
42:
13-16,
1991[Medline].
7.
Cotgreave, I. A.,
and
I. Schuppe-Koistinen.
A role for -glutamyltranspeptidase in the transport of cystine into human endothelial cells: relationship to intracellular glutathione.
Biochim. Biophys. Acta
1222:
375-382,
1994[Medline].
8.
Crompton, M.,
and
L. Andreeva.
On the involvement of a mitochondrial pore in reperfusion injury.
Basic Res. Cardiol.
88:
513-523,
1993[Medline].
9.
Day, B. J.,
S. Shawen,
S. I. Liochev,
and
J. D. Crapo.
A metalloporphyrin superoxide dismutase mimetic protects against paraquat-induced endothelial cell injury, in vitro.
J. Pharmacol. Exp. Ther.
275:
1227-1232,
1995[Abstract].
10.
Feng, D.,
J. A. Nagy,
J. Hipp,
H. F. Dvorak,
and
A. M. Dvorak.
Vesiculo-vacuolar organelles and the regulation of venule permeability to macromolecules by vascular permeability factor, histamine, and serotonin.
J. Exp. Med.
183:
1981-1986,
1996[Abstract].
11.
Graham, M. F.,
A. Willey,
J. Adams,
D. Jager,
and
R. F. Diegelmann.
Role of ascorbic acid in procollagen expression and secretion in human intestinal smooth muscle cells.
J. Cell. Physiol.
162:
225-233,
1995[Medline].
12.
Halliwell, B., and C. E. Cross. Oxygen-derived
species: their relation to human disease and environmental stress.
Environ. Health Perspect. 102, Suppl. 10: 5-12, 1994.
13.
Honneger, C. G.,
H. Langemann,
W. Krenger,
and
A. Kempf.
Liquid chromatographic determination of common water-soluble antioxidants in biological samples.
J. Chromatogr. B Biomed. Appl.
487:
463-468,
1989.
14.
Hu, J.,
and
I. A. Cotgreave.
Differential regulation of gap junctions by pro-inflammatory mediators in vitro.
J. Clin. Invest.
99:
2312-2316,
1997
15.
Jaffe, E. A.,
R. L. Nachman,
C. G. Becker,
and
C. R. Minick.
Culture of human endothelial cells derived from umbilical cord vein.
J. Clin. Invest.
52:
2745-2756,
1973[Medline].
16.
Janssen, Y. M.,
B. Van Houten,
P. J. Borm,
and
B. T. Mossman.
Cell and tissue responses to oxidative damage.
Lab. Invest.
69:
261-274,
1993[Medline].
17.
Kelly, F. J.,
M. Cotgrove,
and
I. S. Mudway.
Respiratory tract lining fluid antioxidants: the first line of defence against gaseous pollutants.
Cent. Eur. J. Public Health
4:
11-14,
1996[Medline].
18.
Koshiishi, I.,
and
T. Imanari.
Measurement of ascorbate and dehydroascorbate contents in biological fluids.
Anal. Chem.
69:
216-220,
1997[Medline].
19.
Marcus, B. C.,
C. W. Wyble,
K. L. Hynes,
and
B. L. Gewertz.
Cytokine-induced increases in endothelial permeability occur after adhesion molecule expression.
Surgery
120:
411-416,
1996[Medline].
20.
McDevitt, C. A.,
J. M. Lipman,
R. J. Ruemer,
and
L. Solokoff.
Stimulation of matrix formation in rabbit chondrocyte cultures by ascorbate. 2. Characterization of proteoglycans.
J. Orthop. Res.
6:
518-534,
1988[Medline].
21.
Newman, W. H.,
L. M. Zhang,
M. H. McDonald,
D. J. Jollow,
and
M. R. Castresana.
Tolerance to nitroglycerin in vascular smooth muscle cells is not affected by the level of intracellular glutathione or L-cysteine.
Anesth. Analg.
81:
1229-1234,
1995[Abstract].
22.
Nyssonen, K.,
M. T. Parvianen,
R. Salonen,
J. Tuomilehto,
and
J. T. Salonen.
Vitamin C deficiency and risk for myocardial infarction: prospective study of men from eastern Finland.
Br. Med. J.
314:
634-638,
1997
23.
Peterson, G. L.
A simplification of the protein assay method of Lowry, which is more generally applicable.
Anal. Biochem.
83:
346-350,
1977[Medline].
24.
Roth, D. R.,
D. I. Axel,
and
E. L. Betz.
In vitro model of the inner parts of a vessel wall with cultured human vascular cells.
Coron. Artery Dis.
4:
283-291,
1993[Medline].
25.
Spiteller, G.
On the chemistry of oxidative stress.
J. Lipid Mediators
7:
199-221,
1993[Medline].
26.
Ström, K.,
L. Olsson,
P. Moldéus,
A. Lövquist,
and
I. A. Cotgreave.
The uptake of ascorbic acid into human endothelial cells and its effect on oxidant insult.
Biochem. Pharmacol.
50:
1339-1346,
1995[Medline].
27.
Tu, B.,
A. Wallin,
P. Moldéus,
and
I. A. Cotgreave.
Individual culture-specific alterations in the human endothelial glutathione system. Relationships to oxidant toxicity.
Pharmacol. Toxicol.
75:
82-90,
1994[Medline].
28.
Vera, J. C.,
C. I. Rivas,
J. Fischberg,
and
D. W. Golde.
Mammalian facilitated hexose transporters mediate the transport of dehydroascorbate.
Nature
364:
79-82,
1993[Medline].
29.
Washko, P.,
and
M. Levine.
Inhibition of ascorbic acid transport in human neutrophils by glucose.
J. Biol. Chem.
269:
23568-23574,
1992.