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Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1304-1317
Studies of Multimerin in Human Endothelial Cells
By
Catherine P. M. Hayward,
Elisabeth M. Cramer,
Zhili Song,
Shilun Zheng,
Roxanna Fung,
Jean-Marc Massé,
Ron H. Stead, and
Thomas J. Podor
From the Departments of Pathology and Medicine, McMaster University,
Hamilton, Ontario, Canada; the Hamilton Health Sciences Corporation,
Hamilton, Ontario, Canada; and INSERM U91, Hôpital Henri Mondor,
Créteil, France.
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ABSTRACT |
Multimerin is a novel, massive, soluble protein that resembles von
Willebrand factor in its repeating, homomultimeric structure. Both
proteins are expressed by megakaryocytes and endothelial cells and are
stored in the region of platelet  -granules resembling Weibel-Palade
bodies. These findings led us to study the distribution of multimerin
within human endothelial cells. Multimerin was identified in vascular
endothelium in situ. In cultured endothelial cells, multimerin was
identified within round to rod-shaped, dense-core granules, some of
which contained intragranular, longitudinally arranged tubules and
resembled Weibel-Palade bodies. However, multimerin was found primarily
in different structures than the Weibel-Palade body proteins von
Willebrand factor and P-selectin. After stimulation with secretagogues,
multimerin was observed to redistribute from intracellular structures
to the external cellular membrane, without detectable accompanied
secretion of multimerin into the culture media. In early passage
endothelial cell cultures, multimerin was associated with extensive,
fibrillary, extracellular matrix structures, in a different
distribution than fibronectin. Although multimerin and von Willebrand
factor are stored together in platelets, they are mainly found within
different structures in endothelial cells, indicating that there are
tissue-specific differences in the sorting of these soluble, multimeric
proteins.
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INTRODUCTION |
REGULATED SECRETORY pathways provide an
important mechanism for responding to external stimuli and for rapidly
altering cellular functions.1-6 Platelets and endothelial
cells are important for hemostasis and both contain secretory granules
that allow sequestered proteins to be released at sites of vessel
injury.7-9 In some cells (including platelets), many
different proteins are stored in a common secretory granule, whereas in
other cells, there are populations of storage granules with different
content proteins.1,3,5,6-8,10-15
Platelet -granules and endothelial cell Weibel-Palade bodies are
secretory granules that share similar features: both contain longitudinally aligned tubular structures and their granule membranes are the storage site for P-selectin.7,8,16-24 The tubules
found in these granules coincide with the presence of ultra-high
molecular-weight von Willebrand factor, and they are absent in severe
von Willebrand disease.7-9,17,18,25-31 Yet, in contrast to
the many different proteins stored in -granules,7,8 only
von Willebrand factor, P-selectin, histamine, and the lysosomal
membrane protein CD63 have been localized to Weibel-Palade
bodies.9,18,22-24,26,32,33
Within -granules, most soluble proteins are found within the central
matrix or electron-dense nucleoid.7,8 Recently, multimerin
and factor V were localized to the eccentric, -granular electron
lucent zone where von Willebrand factor is stored.34,35 Like von Willebrand factor, multimerin is one of the largest proteins found in platelets, and it is composed of variably sized,
disulfide-linked homomultimers, most of which are millions of daltons
in size.36-38 Similar to von Willebrand factor and its
binding of factor VIII,39,40 multimerin also interacts with
a coagulation cofactor, factor V. 35 In resting platelets,
but not in plasma, all of the biologically active factor V is stored
complexed with multimerin.35 However, the complexes of
multimerin and factor V dissociate when platelets are activated by
thrombin, suggesting multimerin may function as an intragranular factor
V-carrier protein.35 Although multimerin resembles von
Willebrand factor in its massive homomultimeric structure36,37,41,42 and these proteins bind homologous
coagulation cofactors,35,39 the primary structures of
multimerin and von Willebrand factor are not related.43-48
The functions of multimerin are largely unknown, but its
Arg-Gly-Asp-Ser site and similarities to the extracellular matrix
protein collagens type VIII and X have suggested multimerin functions
as an adhesive or extracellular matrix protein.43
The colocalization of multimerin and von Willebrand factor in the
Weibel-Palade body-like region of platelet -granules,34 and multimerin's expression in vascular tissue and endothelial cells43 led us to investigate the storage and biosynthesis
of multimerin in human endothelial cells. We report that multimerin is
contained within dense-core granules in human endothelial cells and
within fibrillary structures in the extracellular matrix of cultured
endothelium. However, we found multimerin primarily within different
endothelial cell granules than von Willebrand factor, indicating that
there are differences in the way endothelial cells and megakaryocytes
compartmentalize these multimeric proteins.
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MATERIALS AND METHODS |
Antibodies.
Antibodies used for immunoprecipitation, ELISA, and immunocytochemistry
included monoclonal and polyclonal antimultimerin,38 monoclonal, and polyclonal anti-von Willebrand factor (Dako,
Carpenteria, CA; electron microscopy studies, Dakopatts, Glostrup,
Denmark), polyclonal anti-P-selectin (Cedarlane Laboratories, Hornsby,
Ontario, Canada; electron microscopy studies, a gift from Dr Michael
Berndt, Victoria, Australia49), monoclonal
anti-CD63 (D545 from Dr. S. J. Israels, Winnipeg, Manitoba,
Canada50; and a monoclonal antibody from Caltag
Laboratories, Burlingame, CA), normal mouse IgG (Zymed Laboratories
Inc, San Francisco, CA), polyclonal antifibronectin (Organon Teknika
Inc, Scarborough, Ontario) and sheep antihuman factor V (Affinity
Biologicals, Hamilton, Ontario). For some immunostaining experiments,
polyclonal antimultimerin (0.5 µL) was preadsorbed for 30 minutes
with buffer or with 80 mU of soluble, affinity purified
multimerin.38 One mU of multimerin was defined as the
amount of multimerin antigen in 1 × 106 platelets of
pooled platelet lysate.
Secondary antibodies included Texas Red (TR) and fluorescein
isothiocyanate (FITC) conjugated goat antimouse IgG, goat antirabbit IgG, and donkey antisheep IgG (Jackson Immuno-Research Laboratories, West Grove, PA; antisera with minimal cross-reactivity against the
other species IgG), goat antirabbit IgG coupled to 10 nm and 15 nm
colloidal gold, and goat antimouse IgG coupled to 15 nm colloidal
gold (Amersham, Les Ullys, France). For some studies, the
cell nuclei were counterstained with propidium iodide (2.5 µg/mL;
Sigma, St. Louis, MO).
Cell preparation.
Human umbilical vein endothelial cells were isolated from collagenase
digested umbilical cord segments and grown in Primaria flasks (VWR
Scientific, Toronto, Ontario) containing minimal essential media
supplemented with 20% fetal bovine serum (Gibco BRL, Burlington, Ontario), 20 µg/mL endothelial cell growth factor (Boehringer Mannheim, Laval, Quebec, Canada), 12 U/mL porcine heparin, 2 mmol/L L-glutamine, 0.09% sodium bicarbonate, 10 mmol/L HEPES, and 50 µg/mL
gentamycin. For cell passage studies, cells from 4 to 6 cords were
pooled and passaged every 3 days at 95% to 100% confluence (with
trypsin, EDTA for endothelial cells; Sigma) and samples were harvested
on day 3 at greater than 80% confluence. For immunoblot, immunoprecipitation, and enzyme-linked immunosorbent assays (ELISA), cells were solubilized in lysing buffer containing 1% Triton X-100 and
protease inhibitors (20 mmol/L Tris, 130 mmol/L NaCl, pH 7.4 with 10 mmol/L EDTA, 0.1 µmol/L leupeptin, 0.2 mmol/L phenylmethylsulfonyl fluoride, and 5 mmol/L N-ethyl maleimide; 0.25 mL per T25 flask), scraped from the flask, and Triton-insoluble material was removed by
centrifugation (15,000g × 15 min). For some studies, the
Triton-insoluble pellet was washed twice in lysing buffer with Triton
X-100, then solubilized by boiling in buffer containing 1% sodium
dodecyl sulfate (SDS). In some experiments, passage 1 day 2 endothelial cells were cultured in media containing 0 to 12 µg/mL cycloheximide. For investigations of endocytosis, passage 1 day 2 endothelial cells
were incubated (30 minutes or 18 hours at 37°C) in complete media
with or without 10 µg/mL monoclonal antimultimerin, washed, cultured
for a further 0 to 18 hours, fixed, and labeled (permeabilized and
nonpermeabilized) with TR-antimouse IgG. Some slides were double-immunolabeled with rabbit antimultimerin.
Metabolic labeling studies (18-hour, 3-day, and pulse-chase
studies34) were performed with first passage endothelial
cells (T25 flasks, 80% confluent). Cells were incubated for 0.5 hours in methionine-free media and were labeled in methionine-free media (3 mL) containing 0.1 (3-day and 18-hour labeling) or 0.5 (pulse-chase) mCi/mL 35S-methionine, and 2% dialyzed fetal calf serum
(18-hour and 3-day studies). For studies of regulated protein
secretion, cells were labeled for 3 days in media supplemented with
10%, 20%, or 40% unlabeled methionine, followed by consecutive
18-hour and 3-hour chases in complete medium without label (duplicate
T25 flasks for each condition). The labeled cells were washed twice in
sterile phosphate-buffered saline, incubated (37°C) in serum-free
medium with or without 10 µmol/L ionophore A23187 (Sigma; 0.1%
dimethyl sulfoxide final, all flasks) or 1 to 2 U/mL of human thrombin (Enzyme Research Laboratories, South Bend, IN), and the cell lysates and culture media (collected into the same protease inhibitors as the
lysates) were harvested at 30 minutes. For other investigations of
regulated secretion, the culture media of unlabeled control and
secretagogue-treated cells were analyzed by ELISA, immunoprecipitation, and immunoblotting.
Glycoprotein analyses.
The multimerin content of culture media and cell lysates were
quantitated (neat and 1/2 dilutions) with an ELISA, and pooled platelet
lysate as the standard.51 The multimerin concentrations were expressed as mU/T25 flask. The lower limit of multimerin detection
in the ELISA was approximately 5 mU/mL. To compare the multimerin
antigen content/mg of cellular protein, the total protein content of
platelet and endothelial cell lysates was determined (BioRad DC protein
assay reagent, BioRad, Mississauga, Ontario).
Radioimmunoprecipitations (one mL volumes of culture media and cell
lysates) were prepared as previously described,34,38 by
using JS-1 covalently linked to Sepharose beads (25 µL) or protein A
Sepharose (25 µL), preincubated with polyclonal antimultimerin (25 µL) or anti-von Willebrand factor (15 µL). To reduce nonspecific binding, samples were precleared twice with gelatin agarose (30 µL/mL; Sigma), and once by using protein A Sepharose (25 µL/mL); preliminary experiments indicated that this treatment did not remove
multimerin. For analyses of cell lysates containining radiolabeled multimerin, samples were immunoprecipitated twice, as
described,34 to further reduce nonspecific protein binding.
Immunoprecipitates were analyzed with reduced 4% to 8%, or 7% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
nonreduced multimer gels (1.25% agarose/1.5% acrylamide
gels).37 For some studies, multimerin was deglycosylated
with endoglycosidase H or N-glycosidase F as described,34
and analyzed by autoradiography or by immunoblotting. For immunoblot
analyses of endothelial cell lysates and culture media, samples were
concentrated by immunoprecipitation with 20 µL of JS-1 Sepharose
beads, followed by Western blotting with polyclonal antimultimerin and
chemiluminescent substrate for protein detection.51
Immunohistochemistry.
Immunoperoxidase histochemistry was performed as
described,34 by using monoclonal (JS-1) and polyclonal
antimultimerin, frozen sections of normal tissue (tonsil, lung, liver,
small bowel, aorta, carotid artery, umbilical cord, placenta, and bone
marrow), and cultured endothelial cells. Immunofluorescent
labeling experiments were performed by using endothelial cells cultured
for 1 to 4 days on gelatin-coated, sterile, glass coverslips.
Coverslips were fixed (5 minutes, 3.5% paraformaldehyde), treated with
glycine (0.1 mol/L, 5 minutes), and blocked (30 to 60 minutes in
phosphate-buffered saline containing 50 µg/mL normal goat IgG and 3%
bovine serum albumin) before incubation with antisera in blocking
buffer. Nonpermeabilized cells were labeled with primary antibodies at
4°C before fixation. For double immunolabeling experiments,
antibody incubations were performed sequentially, by using
antimultimerin followed by the relevant fluorescent secondary antibody,
before labeling with the second primary antibody. Controls for the
primary antisera included no primary antibody, normal mouse IgG, and
normal rabbit serum. Controls also included coverslips incubated with a
single primary antibody, and both relevant and irrelevant fluorescent secondary antibodies. Double-labeling experiments were performed in
parallel with single-labeled coverslips, by using dilutions of primary
antisera validated not to give false-positive colocalization by the
irrelevant secondary antibody (monoclonal antibodies: 10 µg/mL;
anti-CD63: pool of two different antibodies, each at 10 µg/mL;
polyclonal antimultimerin: 1/200; monoclonal and polyclonal anti-von
Willebrand factor: 1/1000 and 1/5000; 10 µg/mL polyclonal antihuman
factor V; polyclonal anti-P-selectin: 1/50; polyclonal antifibronectin: 1/200; fluorescent secondary antibodies: 1/50).
Cells were examined with an Axioplan Universal Microscope (Carl Zeiss),
and FITC, TR, and combination filters. Images were acquired with
Northern Exposure Image Analysis Software (version 2.90, Empix Imaging
Inc, Mississauga, Ontario, Canada), were separated into red and green
channel images by using Photoshop 3.0 (Adobe Systems Inc, Mountain
View, CA), and imported into Canvas 5 (Deneba Software, Miami, FL).
Confocal scanning laser microscopy was performed with a Universal
Confocal Laser Scan Research Microscope System (Carl Zeiss), a 100 X
objective, and individual excitation lasers and filters for TR and FITC
fluorochromes. Single-labeled preparations were used to set the levels
for contrast and brightness in the double-labeled preparations, to
ensure that there was no crossover of the fluorochrome into the
opposite channel.
Electron microscopy.
Endothelial cells (T25 flasks) were fixed in situ with 1%
glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4; 1 hour, 4°C). The fixed cells were washed three times (0.1 mol/L phosphate buffer), scraped from the flask, and transported (ambient temperature, pressurized aircraft compartment) in 0.1 mol/L phosphate buffer containing 12.3% sucrose. Cells were embedded in glycol methacrylate upon arrival. Thin sections were immunolabeled as previously
described17,52 and examined under a Philips CM10 electron
microscope. In double-labeled preparations, the sections were labeled
with antimultimerin, followed by 10 nm immunogold antirabbit, before
labeling the opposite side of the section with antibodies against von
Willebrand factor or P-selectin and 15 nm immunogold. Controls included
sections labeled with nonimmune antisera, and for the double-labeling
studies, replacing the second primary antibody with nonimmune or
irrelevant antisera.
Direct-binding studies.
Endothelial cells were grown in 24-well plates for 3 days (first
passage, approximately 90% confluent; seeded at 2.5 × 105 cells/well), were treated with 10 µmol/L A23187 or
control buffer (0.1% dimethyl sulfoxide final in all samples) in
Hank's buffer containing 125I labeled JS-1 (monoclonal
antimultimerin; 0.125 to 11 µg/mL) +/- a 100-fold excess of unlabeled
JS-1. After a 30-minute incubation, the wells were washed gently four
times and the bound counts were solubilized (2% SDS) and counted. Cell
pellets prepared from the wash fluids contained less than 1% of the
total bound counts and the nonspecific binding of JS-1, at the highest
JS-1 concentration, was approximately 16% of the total binding.
| |
RESULTS |
Localization of multimerin in cultured endothelial cells and in
endothelium in situ.
Indirect immunofluorescent labeling studies, with monoclonal and
polyclonal antimultimerin, indicated multimerin was contained within
round to rod-shaped granules within cultured human umbilical vein
endothelial cells, with additional, less intense, perinuclear staining
(Figs 1a, b, d, h, and i
and 2c). The round-shaped granules were
most common (Figs 1b, c, and d and 2c), but more elongated granules
were also found in some cells (Fig 1a, h, and i). These structures were
not evident when nonpermeabilized endothelial cells were labeled (Fig
2a), confirming their intracellular location. No granular labeling was
observed with normal mouse IgG (Fig 1g), preimmune rabbit antisera,
omission of the primary antibodies, or with polyclonal antimultimerin
that was preadsorbed with purified multimerin (Fig 1f).
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| Fig 1.
The intracellular distribution of multimerin in
endothelial cells, evaluated by indirect immunolabeling and
epifluorescent microscopy. Fixed, permeabilized endothelial cells were
labeled with monoclonal and polyclonal antimultimerin, normal mouse
IgG, or polyclonal antimultimerin preadsorbed with purified multimerin or with buffer (N indicates cell nuclei). The granular staining was
similar in cells cultured for 24 hours with (c) or without (b) the
protein synthesis inhibitor cycloheximide. Most of the multimerin-labeled granules were round to slightly elongated (b, c, d),
but rod-shaped granules were also seen in some cells (a, h, i). The
same cytoplasmic structures in endothelial cells were recognized by
monoclonal and polyclonal antimultimerin (h and i show identical fields
of a double-labeled cell). No fluorochrome was detected in the opposite
channel of single labeled cells (d and e are paired images; same
magnification, all panels).
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| Fig 2.
Redistribution of multimerin after treatment of
endothelial cells with secretagogues. Cells were washed and resuspended
in fresh, serum-free culture media before treatment with and without ionophore A23187. Nonpermeabilized (NP) and permeabilized (P) cells
were labeled with antimultimerin (a and b), antimultimerin and
anti-von Willebrand factor (e and f), or with antimultimerin and
propidium iodide to visualize cell nuclei (c and d show the multimerin-labeled structures associated with cells that contained labeled nuclei). Standard immunofluorescent microscopy images of
endothelial cells processed 30 (a-d) and 60 minutes (e and f) after
treatment with buffer or ionophore illustrate the redistribution of
multimerin in response to secretagogues (N indicates cell nuclei) and
the different distributions of multimerin and von Willebrand factor
associated with secretagogue-treated endothelial cells (e and f).
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In quiescent cultures, very little multimerin was detected on the
external membrane of nonpermeabilized endothelial cells (Fig 2a).
However, after treatment with secretagogues (ionophore A23187 or
thrombin) in fresh, serum-free culture media, there was a significant
increase in the multimerin immunolabeling of the external cell membrane
(Fig 2b), and this coincided with a more diffuse staining pattern of
cell-associated multimerin (Fig 2b, d, and f) and the loss of the
discrete intracellular, granule staining for multimerin (Fig 2c and d).
In parallel with the secretion of von Willebrand factor, multimerin
appeared on the external cell membrane 2 to 5 minutes after treatment
with secretagogues (not shown). Multimerin remained in diffuse patches
on the external cell membrane 30 (Fig 2b and d) to 60 (Fig 2f) minutes
after secretion. In preparations counterstained to visualize cell
nuclei, "footprints" resembling the distribution of multimerin on
the membrane of secretagogue-treated endothelial cells were seen in
regions where the endothelial cells had lifted off the coverslip during
processing, suggesting at least part of the multimerin secretion was at
the basal cell surface. Attempts to determine if multimerin was
secreted onto apical and/or basal cell surfaces by using
confocal microscopy were unsuccessful, due to bleaching of the diffuse
multimerin immunolabel during serial optical sectioning. Striking
differences were seen in the distributions of multimerin (Fig 2f) and
von Willebrand factor (Fig 2e) in preparations of secretagogue-treated
endothelial cells (Fig 2e and f compare their distributions 60 minutes
after treatment with secretagogues).
To investigate if the structures containing multimerin could be
constitutive secretory vesicles, endothelial cells were cultured for 24 hours with or without high concentrations (12 µg/mL) of the protein
synthesis inhibitor cycloheximide. Inhibition of protein synthesis
resulted in a loss of perinuclear staining for multimerin (Fig 1c), von
Willebrand factor, and a marked reduction in perinuclear staining for
the constitutively secreted protein, fibronectin (not shown). However,
the granular staining for multimerin (Fig 1c) and von Willebrand
factor, and the secretagogue-induced redistribution of these proteins,
were similar in control and cycloheximide-treated cells.
To determine if the granules containing multimerin might represent
structures containing internalized protein, endothelial cells were
cultured (30 minutes or 18 hours) in media containing antimultimerin
and observed for 0 to 18 hours for evidence of antibody
internalization. Intense labeling of fibrillary structures in the
extracellular matrix of cultured endothelium was observed in these
studies. The distribution of antimultimerin in permeabilized and
nonpermeabilized cells was identical and no antimultimerin was detected
in intracellular granules (not shown).
The elongated shape of some multimerin granules in endothelial cells
suggested multimerin might be stored in Weibel-Palade bodies16 and perhaps in the same location as von Willebrand factor and P-selectin, two proteins known to be stored within these
organelles.9,18,22-24 However, comparison studies indicated that there was more heterogeneity in the number of multimerin granules/cell (0 to >200) and most multimerin granules were not as
elongated as the structures containing von Willebrand factor and
P-selectin (Figs 1, 2, 3,
4, and 8b). To further compare the distributions of
multimerin, von Willebrand factor, and P-selectin, double-labeling
experiments were performed, with stringent conditions to avoid
false-positive colocalization. No fluorescence was detected in the
opposite channel of single primary antibody labeled cells, labeled with
both relevant and irrelevant secondary antibodies (Fig 1d). When
double-labeling was performed with monoclonal and polyclonal
antimultimerin, the same intracellular granules were labeled (Fig 1h
and i). As anticipated, an identical population of intracellular
granules were labeled by antibodies to von Willebrand factor and
P-selectin (Fig 3a). However, the distribution of multimerin in
endothelial cells was different from von Willebrand factor and
P-selectin (Fig 3b and c). To determine if multimerin was stored in
lysosomes, the distribution of multimerin was compared to CD63, a
protein found in lysosomes and in Weibel-Palade bodies.33 CD63 was identified in larger perinuclear structures and in peripheral cytoplasmic structures, but these structures did not contain detectable multimerin (Fig 3d). Although our previous studies indicated multimerin was stored complexed with coagulation factor V in
platelets,35 we were unable to detect factor V within
cultured human umbilical vein endothelial cells.
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| Fig 3.
Comparisons of the intracellular distribution of
multimerin, von Willebrand factor, P-selectin, and CD63. Paired,
epifluorescent microscopic images of structures in the cytoplasm of
double-immunolabeled, quiescent, permeabilized endothelial cells are
shown. Primary antibodies included monoclonal (a, vWf; b and c,
multimerin; d, CD63) and polyclonal antisera (a and c, P-selectin; b,
vWf; d, multimerin). Single-labeled coverslips, processed in parallel, showed the same pattern of labeling. Identical granules were labeled by
antibodies to P-selectin and von Willebrand factor, but multimerin was
found in a different distribution than von Willebrand factor, P-selectin, and CD63 (N indicates cell nuclei). Cells labeled with
polyclonal antimultimerin (d) showed more background labeling of
nongranular structures than the cells labeled with monoclonal antimultimerin (b and c).
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| Fig 4.
Confocal scanning laser microscopy images comparing the
intracellular distributions of multimerin with von Willebrand factor and P-selectin. Cells were prepared as in Fig 3. The images are overlays of matching 250 nm optical sections (taken through the cellular plane showing the maximal intensity granular labeling for both
proteins) and show labeled structures in the cytoplasm of endothelial
cells. Multimerin is shown in red, and P-selectin and von Willebrand
factor are shown in green (N indicates the cell nuclei). Differences
are seen in the distributions of multimerin, compared to von Willebrand
factor and P-selectin. Some of the elongated multimerin-labeled
granules (open arrowheads) did not contain detectable von Willebrand
factor or P-selectin immunolabel. Regions of possible overlap in the
distributions of multimerin and von Willebrand factor or P-selectin are
indicated (solid arrows).
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| Fig 8.
The distribution of multimerin in the extracellular
matrix of endothelial cells. Nonpermeabilized (a and c) and
permeabilized (b and d) endothelial cells were labeled with monoclonal
antibodies to multimerin (a-d), polyclonal antibodies to von Willebrand
factor (b), polyclonal antibodies to fibronectin (c), and propidium
iodide (to visualize cell nuclei; a), and were examined by
epifluorescent microscopy. Panels a to c show paired images of the same
field of primary passage endothelial cells. The cells shown in panel b
contained few multimerin granules but abundant von Willebrand factor
granules (N indicates a cell nucleus). Solid arrows indicate regions
where the von Willebrand factor-labeled Weibel-Palade bodies in the
cell periphery appeared to follow the margins of the
multimerin-containing fibrils. Open arrowheads indicate multimerin in
thicker, intensely labeled structures. Multimerin and fibronectin were
associated with large fibrillary structures in the extracellular matrix
(c), with differences in their distributions. Cell passage in vitro was
associated with reduced extracellular matrix staining for multimerin
(d).
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Confocal microscopy analyses (250 nm optical sections [Fig 4a and b]
and overlays of serial optical sections through the entire cell [not
shown]) confirmed multimerin was contained within discrete, round to
rod-shaped structures. The serial optical sections indicated that most
of the multimerin, von Willebrand factor- and P-selectin-containing granules were at the same cell depth, consistent with the thinness of
the peripheral, endothelial cell cytoplasm. The majority of the
multimerin granules did not contain detectable von Willebrand factor
(Fig 4a) or P-selectin immunolabel (Fig 4b). In cells containing multimerin granules, approximately 1 in 300 of the von Willebrand factor-labeled structures appeared to overlap multimerin-labeled structures (Fig 4a, solid arrows) and similar findings were seen in
cells labeled with antibodies to multimerin and P-selectin (Fig 4b,
solid arrows). Elongated granules containing multimerin but not von
Willebrand factor or P-selectin were identified (Fig 4a and b, open
arrowheads).
In frozen sections of human tissue, multimerin was identified within
the endothelium of different-sized venous and arterial blood vessels,
including capillaries, venules, veins, arterioles, arteries, and vaso
vasorum (Fig 5). The most intense
multimerin immunolabeling was observed within the cell layer adjacent
to the vessel lumen, with less intense staining of the subendothelium (Fig 5). Most vessels examined showed staining for multimerin, but
occasional vessels without multimerin labeling were observed. No
staining of endothelium was observed with the negative controls, processed without the monoclonal antibody or with preimmune rabbit IgG
(Fig 5A). Apart from platelets, megakaryocytes, endothelium, and
adjacent structures, no other cells or structures exhibited staining
for multimerin.
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| Fig 5.
The distribution of multimerin in normal blood vessels.
Frozen sections of normal tissue, including aorta (A and B), small bowel (C), placenta (D and E), carotid artery (F), and umbilical cord
(G; vein is shown) were immunolabeled with monoclonal antimultimerin (B, C, D, F, and G), polyclonal anti-multimerin (E), or the preimmune polyclonal antiserum (A). Bars indicate 30 µm (C and G) and 10 µm
(A, B, D, E, and F).
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The ultrastructure of the multimerin granules in endothelial cells was
investigated by using immunoelectron microscopy and polyclonal
antimultimerin. No gold particles were present in the control sections
labeled with nonimmune antisera. In sections labeled with
antimultimerin, gold particles were identified within small vesicles
and within round, dense-core granules (Fig
6a) that had the appearance of regulated secretory granules. Within the
dense-core granules, multimerin was often in an eccentric position,
resembling its distribution within platelet
-granules.34,35,53 Multimerin was also identified within
elongated structures, limited by a unit membrane and containing
longitudinally aligned tubules similar to Weibel-Palade
bodies16 (Fig 6a and b). Some, but not all, of the
elongated granules within endothelial cells were labeled by the
multimerin antibodies. The round granules containing multimerin were
negative for P-selectin (Fig 6b). However, the P-selectin positive
Weibel-Palade bodies contained either no detectable multimerin (Fig 6b,
inset) or low levels (Fig 6b) of multimerin. In most sections examined
by double immunoelectron microscopy, multimerin and von Willebrand
factor were located within different membrane-bound structures
(Fig 7a and b). Occasionally, multimerin and von Willebrand factor were observed within the same granule, but
within these organelles the two proteins were often located in
different regions, suggesting compartmentalization of proteins within
these structures (Fig 7c). von Willebrand factor was the predominant
labeled protein in the granules that contained multimerin and von
Willebrand factor. In preparations double-labeled with antimultimerin
and 10 nm gold, followed by nonimmune or irrelevant antisera and 15 nm
gold, there was no labeling of granules by the 15 nm gold and only
occasional background labeling that was not associated with specific
structures.
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| Fig 6.
Immunoelectron microscopy studies of human umbilical vein
endothelial cells. (a) Cells labeled with multimerin antibodies. Gold
particles were located in round dense-core granules (arrows) and less
often in elongated structures (arrowheads) that closely resembled
Weibel-Palade bodies. (b and inset) Cells double immunolabeled for
multimerin (10 nm gold) and P-selectin (15 nm gold). Multimerin immunolabel was mainly observed in dense-core granules (arrows) that
were negative for P-selectin. P-selectin- positive Weibel-Palade bodies (WP; arrowheads) displayed either low levels (b) or no detectable (inset) multimerin immunolabel. N indicates the cell nucleus.
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| Fig 7.
Immunoelectron micrographs of human umbilical vein
endothelial cells double-immunolabeled with antibodies to multimerin
(10 nm gold) and von Willebrand factor (15 nm gold). (a) Most of the multimerin-positive granules were round in shape (arrows) and distinct
from the von Willebrand factor-containing elongated Weibel-Palade bodies (WP). (b) Two dense-core granules of similar size with distinct
appearances and contents. One is elongated and contains von Willebrand
factor (arrowheads) whereas the other one is more spherical and
contains multimerin (arrows). (c) Weibel-Palade (WP) bodies containing
von Willebrand factor were generally devoid of multimerin (arrowheads).
Occasionally, both proteins were found in their matrix, but the
labeling for multimerin (arrows) was minimal compared to von Willebrand
factor.
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Studies of multimerin in the extracellular matrix.
In nonpermeabilized and permeabilized preparations of primary and
first-passage cultures there was intense, patchy, fibrillary extracellular matrix staining with both monoclonal
(Fig 8a-d) and polyclonal antimultimerin,
and double-labeling experiments indicated the same extracellular matrix
structures were labeled by these antibodies (not shown). The
extracellular matrix was not labeled by normal rabbit or mouse IgG, or
when the polyclonal antimultimerin was preadsorbed with purified
multimerin (not shown). Extensive, multimerin-labeled fibrillary
structures were seen in some (Fig 8a-d) but not all regions (images in
Figs 1-4) of the culture monolayers, and many contained endothelial
cells enmeshed within their fibrils (Fig 8a). The number, size, and
appearance of the multimerin-containing, fibrillary extracellular
matrix structures were similar in quiescent and secretagogue-treated monolayers, and they were the most intensely labeled structures in
nonpermeabilized (Fig 8a and c) and permeabilized (Fig 8b and d)
quiescent and secretagogue-treated (not shown) endothelial cell
cultures. In addition to fine fibrillary structures (Fig 8b, multimerin
panel, solid arrows), intense labeling of thicker fibrils (Fig 8b,
multimerin panel, open arrowheads) was observed with antimultimerin.
Triton X-100 extraction of the cell monolayers resulted in loss of the
intracellular, multimerin granular staining but it did not alter the
appearance of the multimerin-containing extracellular matrix structures
(not shown), indicating that multimerin was associated with the
Triton-insoluble, extracellular matrix.
In early passage endothelial cell cultures, differences were observed
in the extracellular matrix distributions of multimerin and fibronectin
(Fig 8c), and the multimerin-labeled fibrillary structures contained
weak to no labeling for extracellular von Willebrand factor (Fig 8b).
In many regions, the von Willebrand factor-labeled Weibel-Palade
bodies in the peripheral cytoplasm of endothelial cells appeared to
follow the margins of the multimerin-containing fibrillary structures
(Fig 8b, solid arrows), suggesting a close association between
endothelial cells and the multimerin-containing fibrillary matrix.
Effect of cell passage on multimerin expression.
Variability in the multimerin staining pattern of different endothelial
cell preparations led us to investigate the effect of cell passage on
multimerin. Greater than 99% of the cells in primary to passage 4 cultures contained abundant von Willebrand factor storage granules. In
early passage cultures (primary to passage 2), many (30% to 55%)
cells contained greater than 100 multimerin granules, and most (77% to
87%) contained greater than 20 granules. However, by passage 3 and 4, most (62% to 73%) endothelial cells contained only 0 to 20 multimerin
granules.
Cell passage in vitro was also associated with marked changes in the
extracellular matrix multimerin. The multimerin-labeled extracellular
matrix structures were larger and more numerous in primary cultures
(structures/10 mm2 in primary, passage 1 and passage 4 cultures: 150, 52, and 14; Fig 8d compares typical low power fields of
primary and passage 4 cultures). Although the extracellular matrix
multimerin was most evident in primary cultures, the multimerin antigen
levels in the culture media of primary passage cells were consistently lower than passage 1 and later cultures (multimerin antigen levels in
primary and passage 1 to 4 culture media were 32, 80, 250, 330, and 270 mU/T25 flask; results of a representative experiment with different
passages from the same cell harvest for all analyses). The
corresponding primary, passage 1 and 4 endothelial cell lysates contained only 4.6, 4.6, and 1.5 mU of multimerin per T25 flask (equivalent to 1.5%, 1.5%, and 0.6% of the platelet multimerin content/mg of cell lysate protein), indicating that most of the synthesized multimerin was constitutively secreted.
Investigations of multimerin biosynthesis and secretion by
endothelial cells.
Direct-binding experiments with radiolabeled monoclonal antimultimerin
were used to study the effect of granule secretion on the amount of
extracellular matrix-associated multimerin. Although secretagogue
treatment increased the multimerin immunolabeling of the endothelial
cell external membrane (Fig 2), Scatchard analyses indicated there were
1.6 to 1.8 × 1010 specific-binding sites for
monoclonal antimultimerin per well in both control and
ionophore-treated monolayers (data from two separate experiments;
area/well: 1.9 cm2 containing approximately 2.5 × 105 endothelial cells). Comparisons of untreated and Triton
X-100 or SDS-extracted monolayers indicated greater than 98% of the extracellular multimerin was associated with the Triton-insoluble matrix. These data, together with the immunostaining findings, suggested most of the extracellular multimerin was associated with
fibrillary matrix structures that were not appreciably altered by
treatment with secretagogues. Previous studies of secretagogue-treated platelets indicated that most of the released multimerin remained platelet-associated and only small multimers were found in platelet releasate.37 When washed endothelial cells were treated
with secretagogues in fresh media, there was no detectable increase in
the amount of multimerin in the culture media compared with control
flasks, treated without secretagogues (data from 14 separate experiments, evaluated with ELISA, immunoprecipitation/immunoblot and
radioimmunoprecipitation assays). However, the amount of multimerin contained within the untreated endothelial cell lysates (<5 mU/T25 flask) was close to the limit of detection of the ELISA assay.
Immunoblot and metabolic-labeling experiments were used to evaluate the
biosynthesis and processing of multimerin by endothelial cells.
Previous studies using Dami cells (a megakaryocytic cell line)
indicated multimerin is first synthesized as a promultimerin (proM)
subunit (170 kD), which undergoes extensive glycosylation, subunit
proteolytic processing, and multimerization during biosynthesis to form
the mature, disulfide-linked homomultimeric protein.34 Pulse-chase experiments indicated a similar pattern of multimerin biosynthesis by cultured endothelial cells, and the secretion of
labeled multimerin into the culture media was complete between 3 to 6 hours after synthesis (not shown). Immunoblot analyses (Fig 9A) and 18-hour metabolic labeling
studies (Fig 9A, radioimmunoprecipitation panel) indicated that most of
the multimerin synthesized by endothelial cells in culture was
constitutively secreted into the culture media. Large amounts of
multimerin were also detected in the Triton-insoluble matrix (Fig 9A).
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| Fig 9.
Subunit and multimer composition of endothelial cell and
platelet (Plt) multimerin. The multimerin in the culture media (CM), and cell lysates (Lys) of passage-1 endothelial cells was concentrated by immunoprecipitation and analyzed by immunoblotting with polyclonal antimultimerin or by radioimmunoprecipitation. (A) Reduced (R) 4% to
8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (immunoblot) and nonreduced (NR) multimer gels (origin at
top) comparing the multimerin in 12%, 120%, and 24% of the culture
media, cell lysate, and Triton-insoluble pellet (Mtx) from a T25 flask
(material pooled from 4 flasks, harvested on day 3 at 75% confluence)
with platelet multimerin. The radioimmunoprecipitation panel (18-hour
labeling; 7% SDS-PAGE) compares equivalent volumes of endothelial cell
culture media and cell lysates with 125I-labeled multimerin
purified from platelets. (B) The mobility of reduced multimerin
subunits from platelets and endothelial cell culture media, before
( ) and after (+) deglycosylation with N-glycosidase F. Fully
glycosylated proM, the predominant 155 kD (p155) subunit of mature
platelet multimerin, and cell lysate proM containing
endoglycosidase-H-sensitive forms of N-linked carbohydrate (*, panel
A), are indicated in the panels showing reduced multimerin.
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Similar to Dami cells, 34 the predominant multimerin
subunit in reduced endothelial cell lysates was recently synthesized
proM containing endoglycosidase H sensitive forms of N-linked
carbohydrate (Fig 9A, upper panels; the bands in lanes Lys indicated by
an *). Fully glycosylated forms of proM (Mr 186 kD,
reduced, band indicated as proM in Fig 9) and smaller multimerin
subunits were detected in the culture media (Figs 9A and B and
10A), and in lesser amounts in the cell
lysates (Figs 9A and 10A).
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| Fig 10.
Long-term metabolic labeling studies of endothelial cell
multimerin. Multimerin inmmunoprecipitates of equivalent volumes of
culture media (CM; from 18 hours [18h] and 3 days [3d] of labeling in media supplemented with 40% unlabeled media; 18-hour [C1] and 3-hour [C2] chases in unlabeled media; and ionophore A23187 [+] or buffer [] treated cells) and endothelial cell lysates (Lys; ionophore A23187 and buffer-treated cells) were compared with 125 I labeled platelet multimerin (Plt), and
immunoprecipitates prepared by using normal mouse IgG (mIgG). Reduced
SDS-PAGE (A) and nonreduced multimer gels (B) analyses of single or
double-immunoprecipitates (*) from two experiments are shown. The
images represent 3-day (A, lanes 1-3), 20-day (A, lanes 4-10), and
32-day (A, lanes 11-16; B) exposures. The arrows indicate the location
of the p155 and proM subunits. Small quantities of more fully
proteolyzed multimerin were detected in the cell lysates after
constitutive multimerin secretion was complete. Secretagogue
stimulation did not release detectable amounts of multimerin into the
culture media.
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Consistently, the multimerin subunits from endothelial cells migrated
with an apparent molecular mass that was 8 to 10 kD smaller than the
multimerin subunits from Dami cells. Deglycosylation studies (with
N-glycosidase F) indicated these differences were due to the quantity
of N-linked carbohydrate (not shown). The smaller multimerin subunits
in the culture media of endothelial cells did not comigrate with the
platelet multimerin subunits after removal of N-linked carbohydrate
with N-glycosidase F (Fig 9B), suggesting that there are additional
differences in the post-translational processing of multimerin by these
cells.
Nonreduced multimer gel analyses indicated that the multimerin in the
endothelial cell fractions had a different mobility compared to
platelet multimerin (Figs 9A and 10B), possibily due to differences in
their subunit sizes. The endothelial cell lysates and culture media
(Figs 9A and 10B) contained mainly small multimerin multimers. In
immunoblots of the Triton-insoluble fractions of cultured endothelial
cells, the multimerin in the high molecular weight portion of the gel
migrated as a diffuse band rather than discrete multimers. However,
nonreduced/reduced analyses confirmed the high molecular weight
material in the Triton-insoluble fraction contained disulfide-linked
multimerin subunits (not shown). Immunostaining experiments indicated
that the multimerin within endothelial cell granules was extracted by
Triton-X-100, suggesting that the high molecular weight multimerin in
the Triton-insoluble fraction was associated with the extracellular
matrix.
The storage and regulated secretion of multimerin was investigated with
metabolic labeling (3-day labeling, consecutive 18- and 3-hour chases
in cold media, followed by treatment with or without secretagogues).
Because immunostaining experiments indicated that there was an almost
complete loss of multimerin granules when the cells were grown for 3 days in medium containing less than 20% methionine, some studies were
performed by using labeling media supplemented with 20% to 40%
unlabeled methionine. In all studies, much larger quantities of
radiolabeled von Willebrand factor were detected in the culture media
and cell lysates compared with multimerin. Regulated secretion of
labeled von Willebrand factor into the culture media was readily
evident (2- to 5-day exposures), as previously reported.25
Consistently, radiolabeled multimerin was found in the control cell
lysates long after constitutive multimerin secretion was complete, and
the stored multimerin exhibited more complete proteolytic processing
than constitutively secreted multimerin (Fig 10A). However, regulated
secretion of multimerin from endothelial cells into the culture media
could not be confirmed (Fig 10A), possibly due to the very low
quantities of labeled multimerin contained within the cells and the
association of released multimerin with the endothelial cells and their
matrix.
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DISCUSSION |
A number of parallels exist between multimerin and von Willebrand
factor, including their similar massive multimeric structures, expression by megakaryocytes and endothelial cells, and storage in the
Weibel-Palade body-like region of platelet
-granules.9,17,34-37,41-43 The focus of our current
study was to investigate multimerin in human endothelium. We observed
specific multimerin immunolabeling of the endothelium in large and
small arterial and venous blood vessels in situ, confirming its
distribution in endothelium in vivo. Within cultured endothelial cells,
we identified multimerin in round to rod-shaped, dense core granules.
However, by using light and electron microscopy, we found multimerin
primarily within different structures than the Weibel-Palade body
proteins von Willebrand factor and P-selectin, although the
immunoelectron microscopy studies indicated some Weibel-Palade bodies
contained small amounts of multimerin. These studies provide evidence
for differences in the way multimerin and von Willebrand factor are compartmentalized within endothelial cells and megakaryocytes. Although
multimerin is stored complexed with coagulation factor V in
platelets,35 we were unable to detect factor V within
cultured human umbilical vein endothelial cells, suggesting that
multimerin has functions in endothelium that are independent of its
putative function as an intragranular, factor V carrier protein.
Regulated secretory granules can be defined by three characteristics:
(1) secretion that is dependent on external stimuli, (2) the formation
of specialized, dense-core, membrane-bound structures, and (3)
prolonged intracellular storage of their content
proteins.1,5 Several features of the endothelial cell
granules containing multimerin suggested these structures could be
regulated secretory organelles. First, immunostaining experiments
indicated multimerin was released from these structures onto the
external membrane in response to secretagogues. Second, the dense-core
appearance of the multimerin granules was consistent with a regulated,
rather than a constitutive, secretory vesicle. Third, pulse-chase
experiments indicated radiolabeled multimerin remained in the cell
lysate long after constitutive secretion was complete. Fourth, granules
containing multimerin persisted when endothelial cells were treated for
24 hours with high concentrations of a protein synthesis inhibitor.
Fifth, we could not show any internalization of extracellular
multimerin, excluding endocytic or phagocytic vesicles as the
explanation for the multimerin granules. However, with secretagogue
stimulation, we were unable to confirm multimerin secretion into the
culture media or detect an increase in the extracellular
matrix-associated multimerin, possibly due to the very low levels of
multimerin stored in these cells, the binding of the released
multimerin to the cell membrane, and the very large quantities of
multimerin associated with the extracellular matrix under basal culture
conditions. The detection of protein redistribution by using
immunostaining may be more sensitive for evaluating regulated secretory
proteins, because some proteins localized to endothelial cell secretory granules are secreted in amounts that cannot be measured by
direct-binding assays.33
Multimerin is not detectable in normal plasma,38 even when
tested neat in the multimerin ELISA (unpublished observations). Although we found small amounts of multimerin in intracellular granules
in cultured endothelial cells, the multimerin synthesized by these
cells was mainly constitutively secreted. We observed similar
predominant constitutive secretion of multimerin in studies of a
cultured megakaryocyte cell line.34 The presence of
multimerin in vascular endothelium in situ and the lack of detectable
multimerin in normal plasma suggests that it is rapidly cleared
following its secretion in vivo, or perhaps it remains bound to
platelets, endothelial cells, and/or the extracellular matrix.
It is also possible that the predominant constitutive secretion of
multimerin by cultured cells may not reflect its normal processing by
vascular endothelial cells in situ.
The multimerin transcripts expressed in endothelial cells, platelets,
and Dami cells (a megakaryocyte cell line) are identical in
size36,43 yet differences are seen in the mobility of their multimerin subunit and multimers. The pattern of multimerin
biosynthesis by endothelial cells in culture resembled Dami
cells,34 with minor differences in the extent of multimerin
N-glycosylation by these cells. Similar to Dami cells, the majority of
the multimerin synthesized by endothelial cells in culture was
constitutively secreted, with less complete proteolytic processing of
the multimerin constitutively secreted by these cells, compared with
platelet multimerin. The different mobilities of the N-deglycosylated
multimerin subunits from platelets and cultured endothelial cells
suggest that there may be additional differences in their
post-translational processing of multimerin, perhaps due to different
proteolytic processing. Many regulated secretory proteins undergo
proteolytic processing during storage, although storage of a regulated,
secretory protein may occur without proteolytic processing and some
granular proteins are proteolytically modified only in specific cell
types.1,15,54 After a long-term metabolic labeling in media
supplemented with 20% to 40% methionine and a prolonged chase to
allow complete secretion of constitutively secreted multimerin, we
found small quantities of multimerin persisting in the cell lysates
that were consistently more completely proteolytically processed than
the multimerin constitutively secreted from endothelial cells. A number of ubiquitous and tissue-specific proprotein conversion endoproteases have been identified5; but the endoproteases responsible
for processing regulated and constitutive secretory proteins, including
multimerin, in megakaryocytes and endothelial cells are largely
unknown.
Pathways for regulated and constitutive protein secretion exist in many
cells.1-6,55 The factors that direct soluble proteins to
common or distinct storage granules are largely unknown, but the
ability of proteins to form homoaggregates or coaggregates may be one
of the important determinants.1,5,56,57 The lack of
interactions between multimerin and von Willebrand factor could account
for their sorting to different endothelial cell structures and their
apparent compartmentalization in some organelles that contained both
proteins. In cells that contain different populations of regulated
secretory granules, there is some variability in the completeness of
protein sorting. For example, when von Willebrand |
Abstract
Introduction
Abstract