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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2696-2703
Targeting of a Heterologous Protein to a Regulated Secretion
Pathway in Cultured Endothelial Cells
By
Yvonne H. Datta,
Hagop Youssoufian,
Peter W. Marks, and
Bruce M. Ewenstein
From the Division of Hematology, Brigham and Women's Hospital,
Boston, MA.
 |
ABSTRACT |
The stimulation of regulated exocytosis in vascular endothelial
cells (EC) by a variety of naturally occurring agonists contributes to
the interrelated processes of inflammation, thrombosis, and fibrinolysis. The Weibel-Palade body (WPB) is a well-described secretory granule in EC that contains both von Willebrand factor (vWF)
and P-selectin, but the mechanisms responsible for the targeting of
these proteins into this organelle remain poorly understood. Through
adenoviral transduction, we have expressed human growth hormone (GH) as
a model of regulated secretory protein sorting in EC.
Immunofluorescence microscopy of EC infected with GH-containing recombinant adenovirus (GHrAd) demonstrated a granular distribution of
GH that colocalized with vWF. In contrast, EC infected with an rAd
expressing the IgG1 heavy chain (IG), a constitutively secreted protein, did not demonstrate colocalization of IG and vWF. In
response to phorbol ester, GH as well as endogenously synthesized vWF
were rapidly released from GHrAd-infected EC. By
immunofluorescence microscopy, granular
colocalization of GH with endogenous tissue-type plasminogen activator
(tPA) was also demonstrated, and most of the tPA colocalized with vWF.
These data indicate that EC are capable of selectively targeting
heterologous proteins, such as GH, to the regulated secretory pathway,
which suggests that EC and neuroendocrine cells share common protein targeting recognition signals or receptors.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
REGULATED EXOCYTOSIS provides a means by
which endothelial cells (EC) can very rapidly and selectively alter the
microenvironment of individual vascular beds and modulate the
interrelated processes of coagulation, fibrinolysis, and inflammation.
Weibel-Palade bodies (WPB) are secretory granules in EC in which von
Willebrand factor (vWF) and P-selectin are stored.1-4 vWF
is synthesized exclusively by EC and megakaryocytes, and the vWF stored
in WPB consists primarily of very high molecular weight
multimers3,5 that, when released, bind avidly to the
extracellular matrix and to platelet receptors.6 P-selectin
is a type 1 membrane protein that promotes the binding and rolling of
monocytes and neutrophils before leukocyte migration into sites of
inflammation.7 Plasma vWF levels increase rapidly in
response to physiologic stimuli such as exercise and adrenaline in vivo
and in response to the clinical administration of DDAVP, a vasopressin
analog.8 In cultured EC, exocytosis of WPB results in the
release of vWF and surface expression of P-selectin within minutes of
exposure to a number of naturally occurring agonists, including
thrombin,9 peptido-leukotrienes,10 and
histamine.11 EC in vivo also contain large stores of active
and releasable tissue-type plasminogen activator (tPA),12
the primary initiator of plasma fibrinolysis.13 Plasma
levels of tPA increase rapidly after the administration of DDAVP in
vivo, and regulated secretion of tPA from EC has also been demonstrated
in response to thrombin in vitro.14,15
The mechanisms responsible for the sorting of EC proteins into
secretory granules are not well understood. Cultured human EC are
difficult to transfect at high efficiency by standard
means16,17; therefore, previous transfection studies of
regulated EC secretory proteins have been performed in neuroendocrine
cell lines.18-21 vWF expressed in AtT-20 (pituitary) cells
is found in granular structures that are similar in appearance to WPB,
but the vWF cannot be mobilized by agonists that induce secretion of
endogenous peptides in this cell type.18 In contrast,
P-selectin is targeted to functional secretory granules when expressed
in AtT-20 cells,19 and there is evidence that the
cytoplasmic domain is responsible for sorting to the regulated pathway
in this cell type20 and in EC, but not in
platelets.22 It is not known whether the results of these
studies can be extended to the targeting of endogenous proteins in EC.
Human growth hormone (GH) is a 191 amino acid protein normally stored
in dense granules of the somatotroph cells of the anterior pituitary23 and is secreted in response to GH-releasing
hormone. GH has been used as a model of sorting and secretion in AtT-20 cells and PC12 cells.24,25 Through the use of a recombinant adenoviral vector (rAd), we have expressed GH in human umbilical vein
EC (HUVEC) as a model for the sorting of proteins into endothelial regulated secretory granules. In this report, we demonstrate that GH
expressed in HUVEC colocalized with vWF in granules and that the GH was
secreted in a regulated fashion in response to agonists that mobilize
WPB. We also provide evidence that tPA resides at least in part in the
WPB. These data indicate that EC are capable of targeting a
heterologous secretory protein to functional exocytic granules,
suggesting that EC and neuroendocrine cells share protein targeting
recognition signals or receptors.
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MATERIALS AND METHODS |
Materials.
The human growth hormone radioisotopic assay (RIA) kit was purchased
from Nichols Institute (San Juan Capistrano, CA). Rabbit-anti-GH and
anti-vWF polyclonal antibodies were purchased from Dako (Carpinteria, CA), sheep anti-tPA antibody from Enzyme Research Laboratories, Inc
(South Bend, IN), and biotinylated sheep anti-tPA and fluorescein isothiocyanate (FITC)-conjugated goat anti-vWF antibody from The Binding Site (Birmingham, UK), anti-lysosomal-associated membrane protein-1 (LAMP-1) monoclonal antibody (H4A3) from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA).
Fluorophore-conjugated avidin and secondary antibodies were purchased
from Vector Laboratories (Burlingame, CA), ICN/Cappel (Costa Mesa, CA),
or Sigma Chemical Co (St Louis, MO). Sheep antirabbit Ig
antibody-coated magnetic beads were obtained from Dynal (Lake Success,
NY). Human  -thrombin was purchased from Enzyme Research
Laboratories, fibronectin from Collaborative Biomedical Products
(Bedford, MA), and endothelial mitogen from Biomedical Technologies,
Inc (Stoughton, MA). Other tissue culture reagents were purchased from
GIBCO-BRL (Frederick, MD) and Bio-Whittaker (Walkersville, MD).
Restriction enzymes were purchased from Pharmacia (Piscataway, NJ), and
all other reagents were purchased from Sigma Chemical Co. The
polymerase chain reaction (PCR) was performed with reagents from the
GeneAmp PCR Reagent kit (Perkin Elmer, Roche Molecular Systems, Inc,
Branchburg, NJ).
Construction of the GH and IgG adenoviral vectors.
A human GH-expressing rAd was constructed using methods modified from
Becker et al.26 A 2.1-kb BamHI/EcoRI GH
genomic DNA fragment was excised from p0GH27 and ligated
into Bluescript KS( ) (Stratagene, La Jolla, CA). The insert was
excised from Bluescript KS() with Xba I and
HindIII, gel-purified, and ligated into the Xba I and
HindIII sites between the cytomegalovirus (CMV) promoter and SV40 polyadenylation sequences of the rAd transfer vector
pACCMV.pLpASR(+).26 This transfer plasmid containing GH was
cotransfected with the pJM1728 plasmid into 293 cells29 by use of Lipofectamine (GIBCO-BRL). Single clones
were obtained by limiting dilution of viral lysates obtained from the
cotransfected 293 cells. One clone, proven to express GH in 293 cells
as measured by RIA, was amplified by infection of 293 cells in T175
flasks, and the rAd was purified from crude cell lysates by
centrifugation twice through CsCl followed by dialysis in
phosphate-buffered saline (PBS)/10% glycerol.26
A control rAd expressing the signal peptide from P-selectin fused to
the human IgG1 heavy chain constant region (IGrAd) was constructed in a manner similar to the GHrAd. The signal peptide sequence of P-selectin was amplified from human cDNA by PCR with primers incorporating EcoRI and Xba I sites. The
IgG1 heavy chain segment (IG) including the stop codon was
amplified with primers containing Xba I and HindIII
sites from a plasmid containing the cDNA.30 Digests of the
PCR fragments were sequentially cloned into the pACCMV.pLpASR(+) vector
so that the IgG sequence was directly 3' to the P-selectin signal
peptide sequence. IgGrAd was then generated in 293 cells as described above.
Cell culture.
EC were isolated from 2 to 4 human umbilical vein segments by
collagenase digestion and serially subcultured (2 or 3 passages) in
M199 containing 20% heat-inactivated fetal calf serum, 100 µg/mL of
porcine heparin, 50 µg/mL of endothelial cell mitogen, and
penicillin/streptomycin. Final plating was onto gelatin-coated tissue
culture-treated plastic 60-mm or 100-mm dishes or C-24 wells or onto
ultrasonically cleaned, fibronectin-coated glass coverslips in C-24
wells. 293 cells were cultured in gelatin-coated T75 or T175 flasks,
C-6 wells, or microtiter plates (96 wells) in Dulbecco's modified
Eagle's medium (DMEM) containing 5% heat-inactivated fetal calf serum
and penicillin/streptomycin.
Immunofluorescence staining and microscopy.
HUVEC cultured on glass coverslips in 24-well plates were either
mock-infected or infected with approximately 5 to 10 plaque-forming units (pfu) of GHrAd or IGrAd per cell. After approximately 72 hours of
incubation at 37°C, the cells were washed in Dulbecco's phosphate-buffered saline (DPBS), fixed in 3.7% formaldehyde/DPBS for
15 minutes, permeabilized with acetone for 20 minutes, and blocked with
3% bovine serum albumin/1% normal goat serum in DPBS. The cells were
then incubated with unconjugated primary antibodies, washed 4 times
with DPBS, and then incubated with secondary antibodies or avidin
conjugated to fluorophore or with FITC-conjugated anti-vWF antibody, as
indicated in the figure legends. The coverslips were then washed,
dried, and mounted on glass slides for visualization on an Olympus BH2
fluorescence microscope (Olympus Corp, Lake Success, NY).
Some specimens were visualized using an MRC-1024 laser scanning
confocal microscope (Bio-Rad, Hercules, CA); images were
obtained at 0.36-µm intervals in the Z-axis using a 100× objective.
Analysis of GH secretion by RIA.
Confluent HUVEC grown in 24-well plates were infected with
approximately 0.5 or 5 pfu/cell GHrAd or were mock-infected. To measure
regulated secretion, the cells were washed 3 times with Hanks'
balanced salt solution (HBSS)/HEPES/0.1% gelatin 68 to 72 hours after
infection and then treated with agonist for 15 minutes. The
supernatants were collected and stored at 20°C for subsequent analysis. RIA was performed in duplicate on the supernatants following the manufacturer's instructions. To assess constitutive GH
secretion, 50-µL aliquots of conditioned growth medium were removed
from wells twice a day for 3 days after infection, GH was measured by
RIA, and total GH secreted into the media was calculated.
Pulse-chase labeling, immunoprecipitation, and gel electrophoresis.
For radiolabeling experiments, HUVEC were infected with approximately 3 pfu/cell GHrAd or IGrAd or were mock-infected and were then maintained
for 3 days in cysteine- and methionine-deficient medium, supplemented
with 250 µCi of [35S]-cysteine/methionine (NEN, Boston,
MA) per 60-mm plate. Cell supernatants were subjected to
immunoprecipitation onto antirabbit Ig-coated magnetic beads
(PerSeptive Biosystems, Framingham, MA) with rabbit antihuman GH
antibody (Dako) or rabbit antihuman IgG (ICN/Cappel) and
electrophoresed through a 5% to 15% gradient sodium dodecyl sulfate
(SDS) polyacrylamide gel to confirm the presence of GH or IG at the
approximate sizes of 22 and 34 kD, respectively. Regulated secretion
experiments were then conducted on GHrAd- or IGrAd-infected,
radiolabeled HUVEC that were chased for 16 hours with unlabeled OptiMEM
(GIBCO-BRL). The cells were then incubated for 30 minutes at 37°C
with control OptimMEM or OptimMEM containing 100 nmol/L phorbol
myristate acetate (PMA). Chase and treatment supernatants
were collected and analyzed by electrophoresis in 5% to 20% gradient
gels. Gels to be imaged by radiographic film were treated with ENHANCE
(NEN) and dried. For quantitative analysis, bands were either evaluated
by densitometry or by scanning on a phosphorimaging device (Storm 840 PhosphoImager; Molecular Dynamics, Sunnyvale, CA). The relative
targeting efficiency of GH compared to vWF within individual
experiments was calculated by the following formula: relative
efficiency
([GHpma/GHchase]/[vWFpma/vWFchase]). GHpma is the densitometric value assigned to the amount of
GH released into the treatment supernatant in response to PMA;
vWFpma is the value of vWF present in that same lane.
GHchase is the value of GH constitutively released into the
chase medium; vWFchase is the value of vWF present in the
same lane (see Fig 3C). Because GH and vWF were present in the same
samples and comparisons were made from the same gel lanes, no
correction is needed for sample volumes.
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RESULTS |
Colocalization of GH and vWF in GHrAd-infected EC.
Mock- and GHrAd-infected HUVEC were examined for vWF and GH
distribution by conventional and confocal fluorescence microscopy. Staining with FITC-conjugated anti-vWF antibody demonstrated punctate granules corresponding to WPB, similar to those described
previously.3,18 In GHrAd-infected cells stained with
anti-GH antibody and rhodamine-conjugated secondary antibody, we
observed punctate granules similar to those containing vWF. Most of the
granular immunoreactive GH colocalized with vWF, consistent with
targeting of GH to WPB
(Fig 1). There was also occasional staining for GH in smaller granules that did not
colocalize with vWF, suggesting that a small proportion of GH was
targeted to EC granules that were distinct from WPB. As anticipated,
there were numerous granules staining for vWF, but not GH. GH was not
detected in uninfected cells, and GHrAd-infected cells did not stain
with the rhodamine-conjugated antibody in the absence of primary
anti-GH antibody (data not shown). Unpermeabilized GHrAd-infected HUVEC
did not stain to any significant degree for either GH or vWF (data not
shown).
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| Fig 1.
Colocalization of GH with vWF. GHrAd-infected
HUVEC were fixed, permeabilized, and stained with rabbit anti-GH
antibody followed by rhodamine-conjugated sheep antirabbit antibody and
then FITC-conjugated sheep anti-vWF antibody. Images were obtained by
scanning confocal microscopy at 0.36-µm intervals in the Z-axis using
a 100× objective. Shown is a representative scan from the approximate
midsection of the Z-axis. The red and green images were merged to
demonstrate colocalization, which appears yellow.
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The patterns of lysosomal and GH staining were also compared in
GHrAd-infected HUVEC. Although some perinuclear staining for GH
corresponding with LAMP-1 was observed, staining with this lysosomal
marker did not colocalize with the distinct peripheral granules of GH
(Fig 2A). HUVEC infected with the control IGrAd demonstrated a diffuse, fine granular pattern of cytoplasmic staining that did not colocalize with vWF (Fig 2B). HUVEC cotransduced with
GHrAd and IGrAd did not demonstrate any significant colocalization of
these 2 proteins by dual-label immunofluorescence microscopy (Fig 2C).
These results argue against the possibility of heterologous proteins
expressed in EC being nonspecifically targeted to the WPB.
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| Fig 2.
Specificity of GH targeting to WPB. (A) GH (red) versus
LAMP-1 (green). GHrAd-infected HUVEC were fixed, permeabilized, and
stained with rabbit anti-GH and mouse anti-LAMP-1 (H4A3) antibodies,
followed by rhodamine-conjugated antirabbit and FITC-conjugated
antimouse antibodies. Several images were obtained by scanning confocal
microscopy at 0.36-µm intervals in the Z-axis and then combined into
a single image. (B) IG (red) versus vWF (green). IGrAd-infected HUVEC
were fixed, permeabilized, and stained with rhodamine-conjugated
antihuman IgG and FITC-conjugated anti-vWF antibodies. Shown is a
representative confocal scan from the approximate midportion of the
Z-axis. (C) GH (red) versus IG (green). HUVEC transduced with both
GHrAd and IGrAd were fixed, permeabilized, and stained with rabbit
anti-GH antibody and biotinylated goat antihuman IgG antibody, followed
sequentially with rhodamine-conjugated goat antirabbit antibody and
FITC-conjugated avidin. Shown is a representative confocal scan from
the approximate midsection of the Z-axis. In each of these images,
there are few yellow granules, indicating minimal colocalization of
proteins in each case.
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Constitutive and regulated secretion of GH from GHrAd-infected HUVEC.
Confluent HUVEC mock-infected or infected with 0.5 to 5 pfu/cell of
GHrAd demonstrated constitutive secretion of GH into the medium, the
rate of which increased gradually over 3 days, but varied somewhat from
experiment to experiment (data not shown). When GHrAd-infected cells
were treated for 30 minutes with PMA, secretion was modestly increased
compared with controls (fold increase 1.52 ± 0.16 [mean ± SEM]
for 0.5 pfu/cell and 1.37 ± 0.11 for 5 pfu/cell [n = 4, each
experiment performed in triplicate]), suggesting release of GH from an
agonist-sensitive pool.
To further demonstrate that a stored pool of GH was being mobilized,
pulse-chase experiments were performed. GH and IG were immunoprecipitated from both [35S]-cysteine/methionine
labeling medium and chase supernatants of infected HUVEC.
Immunoprecipitates were then analyzed by SDS polyacrylamide gel
electrophoresis (PAGE) to confirm their approximate molecular weights
(Fig 3A). Pulse-chase experiments of
mock-infected or GHrAd-infected HUVEC showed that GH secretion
increased by 3- to 4-fold in response to PMA, in conjunction with a 4- to 5-fold increase in vWF secretion (Fig 3B). These data support the
view that a portion of the GH heterologously expressed in HUVEC was sorted into functional secretory granules. In contrast, similar experiments with HUVEC infected with IGrAd demonstrated only
constitutive secretion of radiolabeled IG, which was not increased
further in response to PMA (Fig 3C).
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| Fig 3.
Regulated secretion of GH and vWF from HUVEC. (A) HUVEC
were infected with IGrAd (1 and 2) or GHrAd (3 and 4) and labeled with
[35S]-methionine/cysteine for 3 days. Labeling medium (1 and 3) and chase supernatants (2 and 4) were then subjected to
immunoprecipitation and SDS-PAGE as described in Materials and Methods,
indicating sizes of approximately 22 and 34 kD, respectively. (B and C)
HUVEC were infected and labeled with
[35S]-methionine/cysteine, and after 16 hours of chase
with unlabeled medium, the cells were treated for 30 minutes with
control buffer or 100 nmol/L PMA. Supernatants were not subjected to
immunoprecipitation before loading onto gels. (B) Treatment
supernatants were analyzed by SDS-PAGE and phosphorimaging. Shown is a
gel representative of 3 similar experiments, with mock-infected HUVEC
treated with control buffer (1) or PMA (2) and GHrAd-infected HUVEC
treated with control buffer (3) or PMA (4) (m-vWF, mature vWF;
propeptide, the cleaved vWF propeptide). (C) Shown are chase and
treatment supernatants analyzed by SDS-PAGE and densitometry,
representative of 3 different experiments; chase supernatants from
IGrAd (1 and 2) and GHrAd (3 and 4), control treatment supernatants
from IGrAd (5) and GHrAd (7), and PMA treatment supernatants from IGrAd
(6) and GHrAd (8). The relative sorting efficiency or GH compared with
vWF was calculated from densitometric analysis of pro-vWF and GH in
lane 4 and m-vWF and GH in lane 8.
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To assess the relative efficiency of targeting, further pulse-chase and
secretion experiments of GHrAd or IgGrAd-infected HUVEC were performed
(Fig 3C). Densitometric analysis of SDS-PAGE gels indicated that the
relative efficiency of GH compared with vWF targeting was 0.2 ± 0.14 (mean ± SD, n = 3). Essentially none of the IG was targeted to
the WPB as measured by this method. These data indicate that the
targeting efficiency of GH is low compared with vWF, although greater
than the targeting efficiency of IG.
Colocalization of tPA with vWF and GH.
Uninfected and GHrAd-infected HUVEC were examined for the intracellular
location of tPA, which, similar to vWF, is stored in EC and secreted in
a regulated fashion.15,31 In contrast to the findings of
Emeis et al,31 we found numerous granules in which tPA and
vWF colocalized, as assessed by conventional and confocal
immunofluorescence microscopy
(Fig 4A). There were also
occasional smaller tPA-containing granules in which vWF was not
detected. We confirmed the colocalization of tPA and vWF by repeating
the immunostaining successively with sheep anti-tPA, followed by Texas
Red-conjugated rabbit antisheep, followed by FITC-conjugated goat
anti-vWF or with biotinylated sheep anti-tPA plus mouse anti-vWF,
followed by rhodamine-conjugated avidin and then FITC-conjugated goat
antimouse antibody (data not shown). There was considerable
heterogeneity among EC both within individual cultures and among
different EC platings with respect to the number of tPA-containing
granules present. In GHrAd-infected cells, many of the granules
staining for GH also stained for tPA (Fig 4B). The total number of GH
and tPA granules and the degree of colocalization varied among cells.
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| Fig 4.
Colocalization of tPA with vWF and GH. (A) tPA
versus vWF. Uninfected HUVEC were fixed, permeabilized, and stained
with biotinylated sheep anti-tPA antibody followed by
rhodamine-conjugated avidin and then FITC-conjugated goat anti-vWF. (B)
tPA versus GH. GHrAd-infected HUVEC were fixed, permeabilized,
and stained with biotinylated sheep anti-tPA and rabbit anti-GH
antibodies, followed by rhodamine-conjugated avidin and then
FITC-conjugated antirabbit antibody. Scanning confocal microscopy
images were obtained at 0.36-µm intervals in the Z-axis using a
100× objective. Shown are representative scans from the approximate
midsection of the Z-axis. The red and green images were merged to
demonstrate colocalization, which appears yellow.
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DISCUSSION |
The targeting of secretory proteins into regulated exocytotic granules
has not been extensively studied in EC. Results obtained primarily in
cell lines of neuroendocrine origin have supported several different
theories regarding the sorting of proteins between the constitutive and
regulated secretory pathways. These include selective condensation and
concentration of the secretory product within the Golgi cisternae,
selective protein targeting through binding to specific granule
receptors, carrier vesicle-mediated targeting to the secretory granule,
and temporal regulation of granule protein
biosynthesis.24,32-34 These mechanisms may not be mutually
exclusive within a given cell type. Selective protein aggregation in
the trans-Golgi network is thought to play an important role in the
development of immature secretory granules and may depend in part on an
acidic milieu and high calcium concentration.35,36 In EC,
both vWF multimer and WPB formation is abolished by the presence of a
weak base in the culture medium.37 However, selective condensation is unlikely to be an exclusive sorting mechanism, because
constitutively secreted proteins have also been found in immature
secretory granules in some cell types.38
The primary amino acid sequences of numerous regulated secretory
proteins have been analyzed in search of a transport sorting signal
similar to those described for sorting between other intracellular compartments,39 but to date no such consensus sequence has
been found. It is likely that secondary or higher protein structures determine sorting into secretory granules. Analysis of the predicted secondary structure of a variety of secretory proteins has shown the
presence of an N-terminal hydrophobic domain that may be both necessary
and sufficient for their sorting to the regulated secretory pathway.40 Also, chromogranin B36 and
pro-opiomelanocortin (POMC)41 contain N-terminal
amphipathic loop conformational motifs that are necessary for sorting.
The structure responsible for the sorting of GH is unknown.
In this study, we expressed GH to determine whether EC possess the
cellular apparatus necessary to target heterologous secretory proteins
to functional exocytotic granules. GH has served as a model of sorting
and secretion in the PC12 (pheochromocytoma) and AtT-20 (pituitary)
cell lines. In these cell types, heterologously expressed GH was found
to reside in secretory granules that contain the endogenous secretory
proteins and was released in a regulated fashion together with the
endogenous proteins.24,25 In this report, we demonstrate
that GH expressed in HUVEC was targeted to functional secretory
granules that contain endogenous vWF and that GH was secreted rapidly
in response to PMA, along with vWF. Pulse-chase experiments clearly
demonstrated that a portion of the GH was secreted in a regulated
manner, along with secretion of mature vWF and the cleaved vWF
propeptide (Fig 3). HUVEC infected with the control IGrAd did not
demonstrate colocalization of IG with vWF, and HUVEC infected with both
GHrAd and IGrAd did not demonstrate colocalization of GH with IG. These
data support the notion that the heterologously expressed GH was
specifically targeted to the WPB; however, the efficiency of this
targeting was low.
The mechanism by which GH is sorted to regulated exocytotic granules
has not been fully elucidated, although carboxypeptidase E appears to
play a role in the anterior pituitary. Membrane-associated carboxypeptidase E has been shown to act as a sorting receptor for POMC
in mouse neuroendocrine cells.42 Mice deficient in carboxypeptidase E secrete GH constitutively, and secretion is not
increased in response to specific agonists.43
Carboxypeptidase E is present in HUVEC44 and may therefore
be responsible for the sorting of GH to the WPB when expressed in
HUVEC. Alternatively, GH expressed in EC may be targeted to the WPB
through coaggregation or specific interaction with vWF or other
endogenous protein constituents of this granule. There is in vitro
evidence that GH forms aggregates with other pituitary secretory
proteins under mildly acidic conditions, but undergoes only minimal
self-aggregation under similar conditions.45 Procoagulant
factor VIII (FVIII) is sorted to vWF-containing granules when expressed
in bovine aortic endothelial cells or in AtT-20 cells stably
transfected with vWF.46 This sorting appears to be due to
specific coaggregation, because mutation of the FVIII binding site in
vWF abolishes the sorting of FVIII to granules.
The results of the immunofluorescence microscopy experiments suggest
that GH was located in more than 1 granule compartment within
transduced EC. Dual-label immunofluorescence staining of GH and LAMP-1
indicated that some of the non-WPB staining may be due to the
processing of a small amount of GH into lysosomes, and staining of GH
in transit through the endoplasmic reticulum and Golgi would also be
expected. We also found that some GH-containing granules stained
positively for tPA. In contrast to the reports by Emeis et
al31,47 suggesting that tPA in EC resides in granules that
are wholly distinct from WPB, our data indicate that tPA is located in
the WPB. Different culture conditions may possibly account for this
discrepancy. Our data are most consistent with the notion that GH
expressed in HUVEC is targeted primarily to the WPB pool, a portion of
which may contain both tPA and vWF.
Knowledge of the basic mechanisms of protein sorting and regulated
secretion in EC may aid in understanding the pathophysiology of certain
thrombotic and inflammatory conditions and in developing potential
therapies. To date, our knowledge of protein sorting to the regulated
pathway in EC has been largely inferred from studies conducted in
neuroendocrine cell lines. We have now shown the feasibility of using
adenoviral vectors to express heterologous secretory proteins in human
EC with the intention of selective targeting to functional secretory
granules. Significantly, there was no discernible interference of the
targeting of endogenous proteins in rAd-infected EC. Our studies
demonstrate that EC have the cellular apparatus necessary to sort a
model secretory protein of neuroendocrine origin to the regulated
pathway of secretion. However, the use of GH to direct heterologous
proteins of potential therapeutic benefit to the WPB may not be
desirable, due to the low sorting efficiency observed. Sequences from
endogenous EC proteins are likey to prove to be of more value in this strategy.
| |
ACKNOWLEDGMENT |
The authors thank Andrew Ritchie for technical assistance and Andrew
McShea and Christopher Wrighton for advice regarding rAd propagation.
| |
FOOTNOTES |
Submitted December 2, 1998; accepted June 1, 1999.
Supported by National Institutes of Health Grants No. K08 HL03499-01A1
and HL-15157.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Bruce M. Ewenstein, PhD, Hematology
Division, Brigham and Women's Hospital, Longwood Medical Research
Center, Room 617, 221 Longwood Ave, Boston, MA 02115; e-mail:
bmewenstei{at}bics.bwh.harvard.edu.
| |
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ABSTRACT
INTRODUCTION