(Received for publication, April 13, 1995; and in revised form, October 9, 1995)
From the
Internalization of the ternary vitronectin-thrombin-antithrombin (VN-TAT) complex by human umbilical vein endothelial cells was investigated. Radiolabeled VN-TAT was bound to the cell surface at 4 °C, and internalization was initiated by increasing the temperature to 37 °C. After 30 min about half of the VN-TAT complex disappeared from the cell surface and accumulated in the subendothelial matrix.
Translocation of VN-TAT complex from the luminal to the basolateral side was confirmed by electron microscopic evaluation of cross-sections of endothelial cells incubated with gold-conjugated VN-TAT complex. Furthermore, cells cultured in VN-TAT deficient serum, incubated with purified VN-TAT, and subsequently assayed for fluorescent staining using a monoclonal antibody directed against thrombin-modified antithrombin and a polyclonal antibody against vitronectin showed co-localization of both antibodies in punctates. Punctates were randomly distributed in both the xy and xz plane of endothelial cells as evidenced by confocal laser scanning microscopy. Trichloroacetic acid precipitation and SDS-polyacrylamide gel electrophoresis showed that VN-TAT was not degraded during translocation and inhibition of the microfilament system reduced release of VN-TAT to the matrix, indicating that transcytosis was responsible for translocation. These findings emphasize that VN-TAT complex is taken up by endothelial cells, not only leading to the removal of inactivated thrombin from the circulation but also to deposition of VN into the subendothelial matrix.
The key enzyme of coagulation, thrombin (T), ()is
rapidly inhibited by its main plasma inhibitor antithrombin (AT), with
which it forms an equimolar complex. In human serum (Ill and Ruoslahti,
1985; Podack et al., 1986; Preissner et al., 1987)
and in human plasma (de Boer et al., 1993), this binary
complex associates with a third plasma protein, vitronectin (VN),
resulting in the formation of a ternary
vitronectin-thrombin-antithrombin (VN-TAT) complex. Upon complex
formation the normally folded plasma form of VN is conformationally
altered, leading to exposure of multiple domains (Tomasini and Mosher,
1988) such as a heparin-binding site and a collagen binding domain
(Gebb et al., 1986). The exposure of the heparin binding
domain has been found to be a prerequisite for some of the
physiological properties ascribed to VN, such as binding and
stabilization of plasminogen activator inhibitor (PAI-1) (Declerck et al., 1988; Wiman et al., 1988; Salonen et
al., 1989) and scavenging and inactivation of the nascent C5b-9
complex of the complement cascade (Podack and
Müller-Eberhard, 1979; Tschopp et al.,
1988). Therefore the extended form of VN is considered to be an
``activated'' form of VN and TAT complex a physiological
inducer of VN extension.
In a previous report we have shown that the heparin binding domain also mediates binding of VN-TAT to EC (de Boer et al., 1992). However, these experiments did not directly address the metabolic fate of VN-TAT complex bound to the endothelial cell surface. The present study provides evidence that following binding, VN-TAT is translocated through the EC and becomes deposited into the extracellular matrix. The mechanism described represents a route by which VN reaches the subendothelial matrix where it may be involved in a number of important physiological functions.
EC of the second passage were subcultured in eight-well strips (Costar) coated with fibronectin (10 µg/ml). At confluence the cells were incubated overnight with serum-free medium (RPMI 1640/penicillin/streptomycin/fungizone) with the addition of 0.5% BSA and media supplement (Sigma) containing insulin (25 µg/ml), transferrin (25 µg/ml), and sodium selenite (25 ng/ml). Each well contained about 20,000 EC as calculated by counting a trypsin-obtained cell suspension in a Bürker-Türk chamber. For fluorescence experiments, endothelial cells were cultured on glass coverslips. Coverslips were pretreated with glutaraldehyde (1%, v/v) and ethanol (70%, v/v, 24 h), transferred to six-well culture plates, coated with 1% gelatin (w/v, 20 min) (Merck, Darmstadt, Germany), and treated with glutaraldehyde (0.5%, v/v).
Time-dependent binding and internalization was studied over
a time period of 30 min to 4 h. EC were cultured in strips containing
eight disconnectable wells and were incubated with radiolabeled VN-TAT
(10 ng/well, 1.25 nM) at 4 and 37 °C. Following washes
with cold Hepes buffer, cells were separated from the matrix by ammonia
extraction (0.1 M) for 15 min at 4 °C. Cell lysates were
collected, and radioactivity was determined in a -scintillation
counter. The wells, containing the remaining extracellular matrix, were
washed three times with cold Hepes buffer, disconnected, and counted.
Distribution of radiolabel is shown as specific binding, expressed in
nanograms of VN-TAT/10
cells.
To study the effect of
heparin on internalization, 13 ng/well (1.65 nM) of
radiolabeled ternary complex was incubated for 3 h at 4 °C in the
absence or presence of 100-fold molar excess of unlabeled VN-TAT or
unfractionated heparin (0.01-100 units/ml) and label distribution
was examined after ammonia extraction. Bound radiolabel was expressed
as specific signal (nanograms of VN-TAT/10 cells).
BSA-coated wells were used as control for binding of radioligand to
plastic in the absence of EC.
To compare different extraction
buffers, confluent EC cultured in eight-well strips were incubated at 4
or 37 °C with 21.4 ng/well (2.7 nM) of I-labeled VN-TAT in a total volume of 50 µl of Hepes
buffer. After 1 h the cells were washed three times with cold Hepes
buffer to eliminate non-bound label. Removal of the cells was performed
by incubation with ammonia (0.1 M in water, pH 11), EDTA (10
mM), or urea (2 M) in Hepes buffer (50 µl/well,
pH 7.4) for 15 min at 4 °C. Cell lysates (in the case of ammonia)
or cell suspensions (in the case of urea or EDTA) were collected, and
radioactivity was determined in a
-scintillation counter. The
wells, containing the isolated EC matrices, were washed three times and
disconnected, and their radioactivity was measured in a
-scintillation counter. Results are expressed as percentage of
total cell-associated signal.
Of the saved media, 40 µl was mixed with 10 µl of pasteurized protein solution and 100 µl of trichloroacetic acid (final concentration 20%, w/v). Trichloroacetic acid-insoluble label was separated from trichloroacetic acid-soluble label by centrifugation and the pellet and supernatant were counted. Degradation was defined as the amount of radioactivity in the medium that was soluble in 20% trichloroacetic acid. Matrix-deposited label was defined as radioactivity remaining associated with the wells after removal of the cells either by ammonia or trypsin.
For SDS-PAGE analysis, cell lysates of four wells were obtained by ammonia extraction. Lysates were pooled to obtain a sufficient signal, the pH was neutralized and SDS-containing sample buffer (Laemmli, 1970) was added. The matrices were collected by scraping in sample buffer. The scraped wells were checked for radiolabel left behind. Proteins were separated on 4-15% gradient acrylamide gels followed by autoradiography.
Figure 1:
Binding and internalization of VN-TAT
complex by endothelial cells. Distribution of VN-TAT complex in the
cell-compartment and extracellular matrix at 4 °C (a and c) and 37 °C (b and d) and in the
absence (a and b) or presence (c and d) of increasing amounts of unfractionated heparin is shown.
Monolayers of cultured EC were incubated with radiolabeled VN-TAT
complex (1.25 or 1.65 nM) for the time-points indicated (panels a and b) or for 3 h (panels c and d) and association of label with the cell compartment (
or
) or with the extracellular matrix (
or
) was
determined as described under ``Distribution Assay.''
Specific binding is shown, expressed in nanograms/10
cells
(± standard error of the mean), which was defined as the
difference in binding in the absence and presence of 100-fold molar
excess of unlabeled VN-TAT complex. Data presented are representative
results of three independent experiments performed in triplicate wells.
Aspecific binding of radiolabeled VN-TAT on BSA-coated wells (
)
is shown in panels c and d. When symbols lack
standard error bars, they are too small to extend the
symbols.
To investigate the involvement of heparin-like structures on the endothelial cell surface, VN-TAT (13 ng/well) was incubated for 3 h in the absence or presence of increasing amounts of heparin (0.05-500 µg/ml) and binding at 4 °C or uptake at 37 °C was measured. At 4 °C, binding of VN-TAT complex to the cell surface was inhibited in the presence of heparin (Fig. 1c). Half-maximal inhibition was obtained in the presence of 200 µg/ml heparin. At 37 °C, heparin inhibited VN-TAT association with the cell compartment and decreased deposition of VN-TAT into the matrix proportionally (Fig. 1d). Half-maximal inhibition was achieved in the presence of 3 and 20 µg/ml of heparin, respectively. No binding occurred to BSA-coated wells.
To compare different extraction methods, detachment of the cells was performed with 0.1 M ammonia, 2 M urea, or 10 mM EDTA. Removal of the cells was checked microscopically; with ammonia the cells were lysed, whereas incubation with urea or EDTA removed the cells predominantly intact (not shown). At 4 °C, 90-93% of the cell-associated label was found associated with the cell compartment, whereas 7-10% was found in the matrix. At 37 °C, 50-59% was found in the cell compartment and 41-50% was associated with the ECM (Table 1).
Figure 2:
Translocation of VN-TAT through the
endothelial cell monolayer. a, a monolayer of EC was preloaded
with radiolabeled VN-TAT (4.4 nM) for 1 h at 4 °C and
washed (designated as time point 0). Cells were incubated with
unlabeled VN-TAT (4.4 nM) to chase the signal, and the
temperature was raised to 37 °C. At the time-points indicated,
radioactivity associated with the well prior to cell removal and in the
media was measured. Incubation at 37 °C in the presence of
unlabeled VN-TAT resulted in release of radioligand from the cell
monolayer () into the medium (
). b, free iodine
in the media was measured by trichloroacetic acid precipitation
(expressed as percentage of total counts/min). During incubation at 37
°C (15-120 min) the amount of free iodine (
) did not
exceed the amount present in the medium after 1 h at 4 °C (time
point 0). c, at the time points indicated cells were washed
and incubated with ammonia (0.1 M for 15 min at 4 °C).
Radioactivity detectable in ammonia lysates represented ligand
associated with the cell compartment (surface + intracellar,
). Matrix-deposited label was defined as radioactivity remaining
associated with the wells after removal of the cells (
). d, cells were treated with trypsin/EDTA/proteinase K (30 min
at 4 °C). Surface-bound ligand was defined as ligand released from
the cells by trypsin (
), whereas ligand not sensitive to trypsin
treatment represented internalized ligand (
). Matrix-deposited
label was defined as radioactivity remaining associated with the wells
after removal of the cells (
). Results in panels c and d are expressed as percentage of cell + ECM-associated
ligand (± standard error of the mean). Representative results of
three independent experiments are shown. When symbols lack standard
error bars, they are too small to extend the
symbols.
Division into cell-associated or ECM-associated label by ammonia treatment showed that after 1 h of incubation at 4 °C (time point 0) 88% was associated with the cell compartment and 12% with ECM. Raising the temperature showed that in time the cell compartment lost label, whereas ECM was enriched (Fig. 2c). To further subdivide cell-associated ligand into surface-bound and intracellular ligand, limited trypsin digestion according to Chappell et al.(1992) was performed. Surface-bound ligand was defined as ligand released from the EC-monolayer by trypsin/EDTA/proteinase K treatment. Ligand not sensitive to enzymatic digestion represented internalized ligand. Partitions, expressed as percentage of the sum of cell- and matrix-associated label, are shown in Fig. 2d. After 1 h at 4 °C (time point 0), 90% was associated with the cell surface, 8% with the matrix, and 2% could be detected intracellularly. During incubation at 37 °C, the amount of surface-bound label decreased in time, whereas the amount of intracellular ligand increased to 19% and matrix-deposited ligand increased up to 50%.
Figure 3: Light microscopic visualization of gold-conjugated VN-TAT bound to the cell surface. A monolayer of EC was incubated for 1 h at 4 °C with gold-conjugated VN-TAT in the absence (panel a) or presence of 10 µg/ml heparin (panel b). Cells were fixed and silverenhanced. Representative micrographs are shown. Bar, 100 µm.
Figure 4: Binding of VN-TAT to preisolated matrix. Preisolated matrix (PI-ma) was prepared by ammonia extraction. EC monolayer and PI-ma was incubated for 1 h at 4 °C with 4.4 nM radiolabeled VN-TAT in the absence or presence of 100-fold molar excess of unlabeled VN-TAT, washed and treated with ammonia. Radioligand associated with the cell monolayer (ec) prior to ammonia treatment, with ammonia lysate (lys), its isolated matrix (ma) and with PI-ma was measured. Specific signals are shown (expressed in cpm/well ± standard error of the mean), defined as the difference in radioactivity in the absence or presence of unlabeled ligand. Experiments were performed on triplicate wells on three different cell batches and a representative experiment is shown. PI-ma bound 23 times more radioligand compared to matrix isolated after incubation with radiolabel (ma).
Figure 5: Electrophoretic analysis of translocated VN-TAT. Label associated with the cell compartment or deposited into the extracellular matrix was subjected to 4-15% SDS-PAGE followed by autoradiography. Radiolabeled VN-TAT is shown (lane 1) associated with the cell compartment (lanes 2-5) or associated with the matrix (lanes 6-9) after 1 h of preincubation at 4 °C (time point 0 min) or after warming the cells to 37 °C for 15, 60, and 180 min, respectively. Molecular markers in thousands are shown on the left margin.
Figure 6: Electron microscopic evaluation of VN-TAT translocation. Time-dependent internalization and translocation of VN-TAT gold-conjugates was visualized by electron microscopy in ultrathin sections of EC. Incubation of EC for 1 h at 4 °C reveals clusters of VN-TAT-gold conjugates close to the cell surface (a) and the same situation at higher magnification (b). After preincubation at 4 °C, the cells were warmed to 37 °C for 1 h. The gold marker is seen in endosomes (c and d) and deposited into the subendothelial matrix (c). Representative micrographs are shown. Bars represent 1.0 µm.
Figure 7:
Confocal microscopy of intracellular and
subcellular distribution of VN-TAT. EC were cultured on glass
coverslips in VN-TAT deficient medium. At confluence, cells were
incubated for 3 h at 37 °C with medium to which purified VN-TAT was
added. After washing, cells were fixed and permeabilized with methanol
enabling the antibodies to enter the cell compartment. Internalized
VN-TAT was detected with monoclonal antibody AT
directed against thrombin-modified antithrombin and a
monospecific polyclonal antibody against VN, followed by anti-mouse IgG
conjugated with FITC and anti-rabbit IgG conjugated with TRITC
fluorescent label. Fluorescent signal was visualized using convocal
laser scanning microscopy and en phase (a-f) or traverse (g-l) micrographs were generated. Panel a-f shows en phase micrographs scanned from the top of the cell (a and d) into the subendothelial matrix (c and f) and in an intermediate section (b and e)
for the presence of thrombin-modified antithrombin (FITC channel, a-c) or vitronectin (TRITC channel, d-f).
In panels g-i triplicate micrographs are shown of the
part of the cell marked by the broken line in micrograph d,
scanned for the presence thrombin-modified antithrombin. Panels
j-l represent a detail of some punctates scanned for
thrombin-modified antithrombin (j) or vitronectin (k)
and the merged signals of micrographs j and k (l), showing complete co-localization of
thrombin-modified antithrombin and vitronectin. Representative
micrographs are shown (amplification 63
1.4).
The clearance of the equimolar thrombin-antithrombin (TAT) complex from the circulation and its distribution into extravascular tissue is coupled to ternary complex formation with a third glycoprotein in plasma, vitronectin (de Boer et al., 1993). In the present study we provide evidence for the presence of these components in the vascular wall and define requirements that lead to translocation of the luminally bound ternary complex to the basolateral side of endothelial cells (EC). Recently, we have shown that VN-TAT complex binds rapidly to EC and that the binding domain of the complex is located in the heparin binding region of the VN moiety (de Boer et al., 1992). Exposure of the heparin binding region occurs upon a conformational transition; native VN has no affinity for heparin. In vitro, this transition can be achieved by denaturation using chaotropes, detergent, low pH, or binding to plastic. In vivo, TAT complex may serve as a ``physiological activator'' of VN, since the interaction between TAT and VN leads to a similar conformational change in VN.
In the present report, we studied the destination of radiolabeled
VN-TAT complex incubated on metabolically inactive EC (4 °C) or
metabolically active cells (37 °C). An assay was designed that
discriminates between VN-TAT associated with the cell compartment or
with extracellular matrix. For this purpose endothelial cells were
cultured on disconnectable wells, which could be placed in a
-scintillation counter and measured separately. Ammonia
extraction, a standard technique in our laboratory (Sixma et
al., 1987), was used to lyse the cells. This method of cell
removal keeps the matrix intact and firmly attached to the entire area
of the culture wells (Vlodavsky et al. 1987).
VN-TAT-associated radioactivity detectable in the cell lysate
represented cell-associated label, whereas matrix-associated label was
defined as the radioactivity left behind on the culture wells after
ammonia extraction. Other extraction buffers containing urea or EDTA,
which removed the cells from the matrix intact, gave similar results
indicating that measurement of matrix-deposited label was not dependent
on the extraction method.
VN-TAT binding to EC incubated at 4 °C occurred in a cell-specific, ligand-specific, time-dependent, and heparin-dependent manner, which is in accordance with previous observations (de Boer et al., 1992). EC incubated at 37 °C bound VN-TAT with similar binding characteristics, but additionally radiolabel was delivered to the extracellular matrix. The appearance of radiolabeled VN-TAT in the subendothelial matrix was not due to direct binding of VN-TAT to exposed extracellular matrix, since the integrity of the monolayer remained intact during the binding assay.
This was deduced from two control experiments. (a) When matrices were preisolated by ammonia extraction and subsequently incubated with radiolabel for 1 h at 4 °C, this matrix contained 27 times more radiolabel compared to the matrix isolated from an EC monolayer which had been incubated with radioligand prior to ammonia extraction. (b) Light-microscopic evaluation of EC incubated with gold-conjugated VN-TAT for 1 h at 4 °C showed a fully intact EC monolayer on which radiolabel was evenly distributed.
To study internalization in more detail, a pulse-chase set-up was used in which the surface of EC was preloaded with VN-TAT at 4 °C. The radioactive signal was then chased by adding unlabeled VN-TAT, and the cells were metabolically activated by raising the temperature to 37 °C. In time, the cell compartment lost radiolabel, whereas the matrix was enriched with ligand. During the pulse-chase experiment, radiolabel initially bound to the cell surface was released to the medium due to the establishment of a new equilibrium in the presence of an excess amount of unlabeled VN-TAT in the medium. Radioactivity remained constant throughout the incubation period, indicating that matrix-deposited radioligand was not derived from the medium, but originated from intracellular pools. To obtain additional data on kinetics of internalization, we performed a cell removal technique using limited proteolytic trypsin cleavage (Chappell et al., 1992) which discriminates between surface-bound and intracellular radioligand. We could trace an intracellular pool of radioligand; internalization reached a steady state level after 90 min of incubation at 37 °C. The trypsin method detected about 10-20% less VN-TAT in the matrix compared to ammonia treatment. Apparently some VN-TAT was liberated from the matrix during the enzymatic treatment with trypsin.
The presence of an intracellular pool was also evidenced by immunofluorescent staining of endothelial cells cultured under VN-TAT deficient conditions and subsequently incubated with medium containing purified VN-TAT. Since no antibodies are available that directly recognize the ternary VN-TAT complex, cells were incubated with a monoclonal antibody directed against thrombin-modified antithrombin and a polyclonal antibody against vitronectin and double-fluorescent staining was performed. Fluorescent label co-localized in a punctated pattern randomly spread intracellularly and associated with the extracellular matrix.
These findings were complemented by electron microscopic evaluation of ultrathin sections of EC incubated with gold-conjugated VN-TAT. EM micrographs illustrated the translocation phenomenon and revealed the presence of gold conjugates in transcytotic endosomes as well as in association with the subendothelial matrix. We cannot rule out transport of VN-TAT complex via cell junctions or through lateral diffusion, although indications for this possibility could not be found in the EM micrographs.
Transcytosis rather than endocytosis seemed to be involved in the translocation of the complex from the luminal to the basolateral side of the EC as was concluded upon several observations. (a) Endosomal structures were involved in the translocation from the luminal to the basolateral side of the EC. (b) Electrophoretic analysis of matrix-deposited complex showed that the complex was released to the matrix fully intact. (c) Trichloroacetic acid precipitation of media collected during the pulse-chase experiment showed a constant level of free iodine, indicating that VN-TAT was not degraded during internalization. (c) Specific inhibitors of lysosomal function and endocytosis such as ammonium chloride, chloroquine, primaquine, or monensin did not influence matrix deposition, whereas specific inhibitors of transcytosis such as cytochalasin B and colchicine partially decreased matrix deposition. It should be noted that treatment with cytochalasin B and colchicine, which may induce cell death in time, was carried out only for a short period of time in order to minimize loss of integrity of the EC monolayer. This may explain their relatively mild effect on matrix deposition compared to the high concentrations used.
Various plasma molecules cross the endothelium by receptor-mediated transcytosis. For instance, insulin (King and Johnson, 1985) as well as some carrier proteins like albumin (Ghitescu et al., 1986), transferrin (Jefferies et al., 1984), or lipoprotein lipase (Saxena et al., 1990) are transported through the endothelium very efficiently without degradation. Especially the processing of lipoprotein lipase by endothelial cells is of interest, since striking similarities are apparent compared to VN-TAT; binding is heparin-dependent, internalization reaches a steady state level (Saxena et al., 1990), and during internalization no degradation occurs. Translocation of lipoprotein lipase is decreased in the presence of cytochalasin B but not affected by chloroquine, indicating that transcytosis is involved as well. The establishment of a steady state level of internalized ligand may be characteristic for transcytosis and may be due to very rapid translocation of ligand. In the case of VN-TAT, label appeared in the matrix almost immediately and exceeded the amount of intracellular ligand.
When incubated on fibroblasts, lipoprotein lipase is internalized and subsequently degraded through a lysosomal pathway (Chappell et al., 1992). This was shown to occur via the low density lipoprotein receptor-related protein pathway.
Lipoprotein receptor-related
protein cannot be responsible for internalization of VN-TAT by
endothelial cells, since HUVEC do not express this receptor (Godyna et al., 1995). Interestingly, fibroblasts are able to process
and degrade conformationally altered, non-complexed VN (Panetti and
McKeown-Longo, 1993). This was shown to be mediated by the
integrin receptor. This receptor
cannot be involved in the above described pathway either, since RGD
peptides have no effect on binding of VN-TAT to HUVEC (de Boer et
al., 1992). Recently Waltz and Chapman(1994) showed that the
urokinase plasminogen activator receptor binds conformationally altered
VN. The involvement of this receptor and possibly others in binding and
internalization of VN-TAT by endothelial cells remains to be
established.
In conclusion, our results indicate that the direct
association of TAT with VN will cause a conformational transition(s) of
VN, leading to the exposure of the heparin binding site, which enables
the complex to bind to EC through high affinity binding sites with
subsequent internalization. This process not only leads to the
clearance of TAT complex from the circulation, but also promotes
deposition of VN-TAT into the extracellular matrix. This mechanism may
supply the matrix with VN, which must originate from plasma, since EC
neither synthesize VN (Seiffert et al., 1991) nor bind native
VN (Hess et al., 1993). Alternatively, heparin-binding VN
multimers found in platelet releasate (Stockmann et al., 1993)
and at low concentrations in plasma may cross the EC monolayer in an
equivalent manner, as suggested from in vitro experiments
(Hess et al., 1993; Völker et
al., 1993). Two important features of VN in the matrix are its
abilities to promote cell attachment and to stabilize PAI-1, a major
inhibitor of plasminogen activation. Purified VN-TAT complex is still
able to bind PAI-1 and promote cell attachment, ()indicating
that the ternary complex may fulfil crucial functions at its final
destination in the vessel wall.