1991;51:3261-3266.

Cancer Res

Thomas H. Foster, Melissa C. Primavera, Victor J. Marder, et al.

Human Endothelial Cells
Photosensitized Release of von Willebrand Factor from Cultured

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[CANCER RESEARCH 51. 3261-3266. June 15, 1991]
Photosensitized Release of von Willebrand Factor from Cultured Human
Endothelial Cells1
Thomas H. Foster,2 Melissa C. Primavera, Victor J. Marder, Russell Hilf, and Lee Ann Sporn
Departments of Radiology [T. H. F.], Medicine, Hematology Unit [M. C. P., V. J. M., L. A. S.J, and Biochemistry [R. H.], University of Rochester School of Medicine
and Dentistry, Rochester, New York 14642
ABSTRACT
Cultured endothelial cells from the human umbilical vein were incu
bated with low concentrations (1 MU/inl)of the photosensitizer l'hot fri n
II. Following a sublethal light exposure, a light dose-dependent release
of von Willebrand factor (vVVf)into the culture medium was observed.
Analysis of the multimene composition of the released protein indicated
that it originated from the intracellular pool of large vWf multimers
stored in the Weibel-Palade bodies. This release was detected as early
as 1 h postirradiation. Release was inhibited at low temperature and was
dependent upon the presence of extracellular calcium. Photosensitization
resulted in an influx of calcium whose time course paralleled vWf release
from the cells. Since vWf mediates platelet adhesion to the vascular
subendothelium, it is possible that its photochemically stimulated release
in vivo could contribute to platelet thrombus formation observed in tissue
following photodynamic therapy.
INTRODUCTION
The mechanisms of tumor destruction in response to photo-
dynamic therapy are the subject of considerable investigation.
Singlet oxygen ('O2), formed as a result of energy transfer from
a photoexcited sensitizer to dioxygen, is highly reactive in
biological systems and has been identified as the agent respon
sible for many deleterious effects at the cellular level (1, 2).
These effects have been most pronounced in cell membranes
and have involved direct damage to the membrane (3-5) and
deactivation of membrane-associated enzymes and transport
systems (6-8).
At a macroscopic level, PDT1 produces destruction of tumor
and normal tissue vasculature. A variety of effects have been
described, including blood flow stasis, hemorrhage, vasocon
striction, platelet aggregation and platelet thrombus formation,
and "blanching" of the microvessels (9-15). Recently, Pingar
et al. (16) reported the appearance of increased circulating levels
of thromboxane following photodynamic therapy in a rat tumor
model. Some investigators have asserted that the observed
damage to the vasculature is the primary cause of tumor re
sponse to PDT (10). While this contention remains the subject
of extensive study, the appearance of pronounced changes in
the microvasculature of the treated tissue has been convincingly
established.
In an effort to better understand some of the processes
involved in PDT-induced effects on the vasculature, we under
took experiments in which cultured human endothelial cells
were exposed to Photofrin II and light. Few investigations of
Received 12/4/90; accepted 4/9/91.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' The research presented in this report was supported by USPHS Grants
CA36856, HL07152, HL43711, and HL30616 awarded by the National Cancer
Institute and the National Heart. Lung and Blood Institute.
2Supported in part by the Department of Radiology at the University of
Rochester. To whom requests for reprints should be addressed, at University of
Rochester School of Medicine and Dentistry. Department of Radiology, Box 648,
601 Elmwood Avenue, Rochester, NY 14642.
3The abbreviations used are: PDT, photodynamic therapy; vWf. von Wille-
brand factor.
endothelial cells have been reported to date. Ben-Hur et al.
(17), using cultured bovine endothelial cells sensitized with
chloroaluminum phthalocyanine tetrasulfonate, reported ef
fects that were attributed to a light dose-dependent release of
"clotting factors" independent of cell lysis. A report by Corner
et al. (18), using Photofrin II as the sensitizer, demonstrated
that bovine endothelial cells were more sensitive to photody
namic action in vitro than were smooth muscle or fibroblast
cells from the same species. Our experiments were designed to
determine whether the biochemical response of human endo
thelial cells to photodynamic insult included the stimulated
release of vWf.
vWf is a large, adhesive glycoprotein composed of a series of
disulfide-bonded multimers ( 19) synthesized by endothelial cells
(20) and megakaryocytes (21, 22). It is found circulating in
plasma and in platelet -granules (23, 24) and is a component
of the vascular basement membrane (25). vWf mediates platelet
adhesion to the vessel wall following injury (26, 27), especially
under conditions of high sheer stress such as exist in the
microvasculature (28). vWf is stored within endothelial cell-
specific secretory vesicles called Weibel-Palade bodies (29)
which provide a rapidly releasable pool of the largest and most
biologically potent multimers (30). In vitro, this release results
in the appearance of large multimeric forms in the culture
medium, depletion of the intracellular large multimer stores,
and the appearance of irregular "patches" containing vWf on
the external cell surface (30). Among the agents known to
induce this rapid vWf release from the Weibel-Palade bodies
are thrombin, histamine, fibrin, phorbol myristate acetate, the
calcium ionophore A23187, and assembly of complement com
ponents C5b-9 (reviewed in Ref. 31). Stimulated release of
Weibel-Palade body contents is associated with a rise in intra
cellular calcium concentration (32-34). In this study, we show
that photosensitized damage to endothelial cells triggers release
of vWf from Weibel-Palade bodies of endothelial cells and that
release is likely caused by an influx of calcium induced by
photodynamic action.
MATERIALS AND METHODS
Endothelial Cell Culture and Metabolic Labeling. Endothelial cells
from human umbilical veins were harvested as previously described (29,
35) and cultured in 25-cm2 cell culture flasks in McCoy's Medium 5a
(Flow Laboratories, McLean, VA) with 20% fetal bovine serum, 50 ig/
ml endothelial cell growth supplement (Biomedicai Technologies, Inc.,
Stoughton, MA), and 100 Mg/ml heparin (Sigma Chemical Co., St.
Louis, MO). The cultures were monitored for the presence of nonen-
dothelial cells by immunofluorescence staining for vWf and were found
to be free of contamination. Cells were typically used at passage 2 and
required approximately 5 days to reach confluence (3 x IO6 cells) at
each passage. Prior to incubation with Photofrin, metabolic labeling
with [15S]cysteine (1153.4 Ci/mmol; New England Nuclear Research
Products, Boston, MA) was carried out for 3 days at 25 nCi/ml in
complete culture medium. Experiments were performed on confluent
cell monolayers. Endothelial cell viability was assessed by determining
their ability to exclude trypan blue. At various times following photo-
3261
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1\ tITKO PHOTOSENSITIZAT1ON OF ENDOTHELIAL CELLS
radiation, cells were trypsinized and incubated for 1 min with 0.2%
trypan blue stain (Sigma Chemical Co.). The percentage of viable cells
was determined by using a hemocytometer.
Photofrin II Treatment and Light Exposure. Photofrin II was obtained
from Quadra Logic Technologies, Inc. (Vancouver, British Columbia,
Canada) and stored in the dark at 20C. Cells were incubated with 1
Mg/ml Photofrin II in serum-free culture medium supplemented with
Nutridoma-HU (Boehringer Mannheim Biochemicals, Indianapolis,
IN). Following a 2-h incubation at 37Cin a 5% CO2, high humidity
incubator, cells were washed twice with serum-free medium, and com
plete culture medium was replaced. Photoradiation was done with the
unfiltered output from a pair of fluorescent lamps. The incident pho-
toradiation power density at the level of the cells was 0.2 mW/cm2,
uncorrected for the absorbance of the culture medium.
Antisera. The preparation and characterization of monospecific anti-
sera against human vWf used for immunopurification were previously
described (29, 36).
Cell Lysis and v\V'f Purification. At selected time points following
photoradiation, culture medium was collected and cells were lysed as
previously described (37). The cell lysate or culture medium samples
were then incubated with gelatin Sepharose for 1.5 h at room temper
ature to remove fibronectin and other contaminating proteins that
adhere nonspecifically. Gelatin Sepharose was removed by centrifuga-
tion. Protein A-Sepharose CL4B (30 mg/sample) (Sigma Chemical
Co.) was preincubated with 50 n\ anti-vWf antiserum for 30 min, then
added to each sample and incubated for 1.5 h at room temperature.
Protein A-Sepharose beads were washed extensively (38), then boiled
in electrophoresis sample buffer (39), and the collected sample was
analyzed by gel electrophoresis. vWf immunopurified from equal num
bers of cells was loaded for analysis of the cell lysate samples. Similarly,
the total vWf immunopurified from culture media samples was sub
jected to gel analysis.
Electrophoresis Gels and v\Vf Quantitation. Agarose horizontal slab
gels were prepared by using a solution of 2% agarose and 0.2% sodium
dodecyl sulfate in 0.05 M phosphate buffer, pH 7.0. Electrophoresis
buffer was 0.1 M phosphate, pH 7.0, with O.I % sodium dodecyl sulfate.
Exposures of 2-3 weeks were used to produce the autoradiographs used
for quantitative densitometric scanning. The percentage of total (dimer
plus large multimer) vWf appearing in the culture medium was com
puted by using the following formula:
vWf in culture medium
vWf in cell lysate + culture medium
Immunofluorescence Staining. Endothelial cells cultured on glass
coverslips were washed, fixed for 20 min in 3.7% formaldehyde in
phosphate-buffered saline, and permeabilized for 15 min in 0.5% Triton
X-100. Fluorescence staining using anti-vWf antiserum was carried out
as previously described (29).
45Ca2+Influx. Studies of 45Ca2+influx were conducted as described
previously by Bareis et al. (40) and deGroot et al. (32). Cells grown to
confluence in 25-cm2 cell culture flasks (approximately 3 x IO6 cells)
were incubated for 2 h with Photofrin II as described above, washed
twice with serum-free culture medium, and then overlaid with a 2-ml
solution containing 45CaCl2 (0.715 Ci/mmol, Amersham, Arlington
Heights, IL) diluted to 4 /uCi/ml in complete culture medium. Cells
were exposed to light, and following incubation at 37Cfor various
times, the uptake of 45Ca2+was stopped by adding 100 n\ of 100 mM
CaCli. The culture medium was immediately removed, cells were
washed twice with cold 150 mM NaCl containing 5 mM CaCl2 and 10
mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.4. Cells
were lysed in a 1 ml solution containing 5 mM CaCl2/0.5% Triton X-
100. Radioactivity in the cell lysate samples was measured by scintil
lation counting.
RESULTS
Since vWf release from Weibel-Palade bodies is an exocytotic
process requiring maintenance of endothelial cell function and
integrity, a study was conducted to determine the viability of
photosensitized cells treated with a range of photoradiation
fluences. For at least 3 h following photoradiation, light doses
of 2 min or less at 0.2 mW/cnr resulted in essentially no
decrease in cell viability as compared with unirradiated controls
(>90% viable). At 6 h postphotoradiation, 80% of cells that
had received a 2-min exposure were capable of excluding trypan
blue, while viability of cells treated for less than 2 min did not
differ from controls. Some diminished viability was observed in
cells irradiated with higher fluences. Six h after 3- and 5-min
exposures, the percentage of total cells retaining the ability to
exclude trypan blue was 73 and 54%, respectively.
Exposure of cells to Photofrin II and light led to a light dose-
dependent redistribution of the multimeric forms of vWf be
tween the cells and culture medium. As illustrated by the
autoradiograph in Fig. 1, an increased light dose caused an
increase in total vWf appearing in the culture medium, and this
secreted vWf contained a disproportionate amount of the large
multimeric forms. The amount of dimeric forms secreted, how
ever, did not increase with increasing light dose. Incubation of
cells with Photofrin II in the absence of photoradiation resulted
in no detectable alteration of the multimeric composition of
cells or culture medium as compared with control cells (not
shown).
The percentage of total labeled vWf appearing as large mul-
timers or dimers in the culture medium at 6 h following the
large
mult i mers
Fig. 1. Secretion of large vWf multimers in
response to Pholofrin II and light exposure.
Culture medium and cell lysate samples were
collected 6 h following light exposures of 0, 1.
2. 3. and 5 min (0.2 m\V/cm2). vWf was immu
nopurified and analyzed as described in "Mate
rials and Methods." Photosensiti/ation resulted
in the increased appearance of large multimeric
forms of vWf in the culture medium, with deple
tion of the cellular pool. There was no detectable
effect on the distribution of the dimer pool of
vWf between cells and culture medium.
dimer
0 2 3
cell
mm
5 1 2 3
medium
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IN VITRO PHOTOSENSITIZATION OF ENDOTHELIAL CELLS
various light doses (see "Materials and Methods" for calcula
tion) is shown graphically in Fig. 2. The data are averages of
densitometric scans of gels from four separate experiments.
Results indicated that a mean of 4% of total large multimers of
vWf were secreted in the absence of photoradiation. Release of
large multimers was significant with respect to controls (P <
0.05) following a 1-min light exposure, and after a 5-min light
exposure, virtually complete release of large multimers oc
curred. The dimer pool distribution was not affected by photo-
radiation. Photosensitization did not induce extensive vWf
degradation since at light doses achieving maximal vWf release,
an average of 80% of metabolically labeled vWf was recoverable
from the culture as compared with unirradiated controls. At
the relatively higher light doses (3- and 5-min exposure, 0.2
mW/cm2), however, there does appear to be some detectable
intracellular degradation of the large multimers of vWf. It is
possible that these fluences damage Weibel-Palade body mem
branes, thereby causing partial depolymerization of the stored
pool of vWf. Such depolymerization could be responsible for
the apparent increase in smaller multimeric forms observed in
the 3- and 5-min cell samples in Fig. 1.
In order to determine the earliest time at which vWf could
be detected in the medium following photoradiation, the time
course of vWf release was determined. Results of this experi
ment are shown in Fig. 3. For each time point, the amount of
total vWf appearing in the culture medium was normalized to
the corresponding untreated control. Following a 5-min light
exposure, there was no detectable increase in vWf release at 15
min postphotoradiation. At 1 h, however, a greater than 2-fold
increase was measured. Medium samples showed increasing
vWf content at the 2- and 3-h time points, beyond which only
a minimal further increase in vWf was detected (data not
shown).
Immunofluorescence staining of cultured endothelial cells
using vWf antiserum revealed the appearance of release
"patches" following photoradiation of Photofrin II-treated
cells. Examples of typical fluorescence micrographs from these
experiments are presented in Fig. 4. These strongly fluorescent
patches are composed of vWf bound to the extracellular cell
surface (30) and characteristically appear on endothelial cells
after release of Weibel-Palade body contents. Release patches
were evident at l h subsequent to light exposure of Photofrin
II-treated cells. Their appearance, therefore, followed a time
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LIGHT DOSE (mi nutes)
Fig. 2. Light dose response of vWf released from Photofrin II-treated cells.
Following densitometric scanning of metabolically labeled vWf analyzed on 2%
agarose gels, the percentage of total large multimers or dimers of vWf appearing
in the culture medium 6 h follow ing exposure to various light doses was computed
(see "Materials and Methods"). Results are presented as means SD from four
independent experiments. A 1-min light exposure resulted in significant release
of large multimers when compared with unirradiated cells (P < 0.05). There was
no change in the distribution of the dimeric pool of v\Vf at any light dose tested.
5
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TIME AFTER TREATMENT (hours)
Fig. 3. Time course of vWf release from photosensitized endothelial cells. The
total amount of vWf released from Photofrin II-treated endothelial cells at various
times following a 5-min photoradiation period was normalized to the amount of
vWf released from the corresponding unirradiated controls. No release was
detected at 15 min after light exposure, but a greater than 2-fold increase in
secreted vWf was detected at l h postphotoradiaton. faints, results of single
experiments. . null result for the normalized data.
course similar to that of the release of large vWf multimers.
Previous studies have shown that the induced release of
Weibel-Palade body contents was almost completely inhibited
when stimulated cells were incubated at temperatures below
20C(41). Our experiments with photodynamic stimulation
were consistent with these findings. When Photofrin II-treated
cells were placed at 4Cimmediately following light exposure,
no vWf release was observed. Release was also inhibited when
extracellular calcium was chelated by incubating cells in the
presence of 2.5 mivi EDTA during and following light exposure.
These findings, summarized in Table 1, provide further evi
dence that an exocytotic process, not general cell lysis, was
induced by Photofrin II and light exposure.
Since photosensitized release of Weibel-Palade body contents
appears to depend, at least in part, on the presence of extracel
lular calcium, studies of calcium influx following light exposure
of Photofrin II-treated cells were conducted as described in
"Materials and Methods." At various times following a 5-min
light exposure, radioactivity in cell lysate samples was deter
mined, and values were normalized to those of unirradiated
controls. A time-dependent influx of 45Ca2+into the cells was
observed (Fig. 5) with no influx detectable at 15 min following
a 5-min light dose. A maximum intracellular concentration of
45Ca2+was observed between 1 and 3 h following photoradia-
tion. The light dose dependence of photosensitized 45Ca2+influx
is presented in Fig. 6. At 2 h postphotoradiation, 45Ca2+influx
was not statistically significant following a 15-s light exposure,
but was significant (P = 0.05) in response to a 1-min exposure.
The influx reached a plateau level of approximately 4 times
that obtained from control cells.
DISCUSSION
Endothelial cells are a fundamental component of the vas
cular system and are important in providing for and maintain
ing a nonthrombogenic vessel wall surface. In order to under
stand details of the mechanisms responsible for the thrombotic
vascular changes observed following PDT, studies of the effects
of photosensitization of this cell are required. Results presented
in this report demonstrate that endothelial cells respond in vitro
to photodynamic action in ways that are consistent with at least
some of the reported macroscopic effects in vivo, namely, for
mation of platelet-rich thrombi in areas of photodynamic insult
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IN VITRO PHOTOSENSmZATION OF ENDOTHELIAL CELLS
Fig. 4. Immunofluoresccnce staining of endothelial cells following photosensitization. Endothelial cells cultured on glass coverslips were treated with Photofrin II
and exposed to light. Cells were then fixed, permeabilized. and stained with fluorescent anti-vVV'fantiscrum. Shown are unirradiated cells (a) and cells fixed at 15 min
(h) and l h (c) following a 2-min light exposure. Arrows point to Weibel-Palade bodies; *. release patches: n. cell nuclei. At 15 min. no release patches were observed,
and cells appeared identical to controls. At I h. numerous release patches were observed. Bar. 10 im.
Table I Inhibitory effects of low temperature anil binding of extracellular Ca~*
on photosensitized release ofrH'f
All cell samples ereexposed to 1 ng/ml Photofrin II for a period of 2 h.
Light-treated samples received a 3-min dose of broad band pholoradiation (0.2
mW/cm:). For samples denoted +EDTA. 2.5 m\t EDTA was introduced into the
cell culture medium immediately prior to photoradiation. In all cases, the large
v\Vf multimers in the medium were assayed 3 h following photoradiation. Data
are results from single experiments.
Protocol
% of total
mullinier in medium
No light. 37'C
Light. 37'C
No light, 4'C
Light, 4'C
No light. +EDTA
Light. +EDTA
3
100
3
0
7
3
<
3
ill
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< + 4
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TIME AFTER TREATMENT (hours)
Fig. 5. Time course of 4!Ca2* influx into photosensitized endothelial cells.
Endothelial cells were incubated with 4 nCi/ml 4!CaCI2 immediately following a
2-h exposure to Photofrin II. Radioactivity in the cell lysate samples was deter
mined at 15 min. l h. and 3 h following a 5-min light exposure. Values were
normalized to those obtained from unirradiatcd controls (cpm irradiated/cpm
control). No detectable influx of 45Ca2*occurred at 15 min postphotoradiation.
Influx of 4*Ca2*was detected at 1 h following light exposure, and reached a peak
between 1 and 3 h posttreatment. . null result.
(15). In particular, we have demonstrated for the first time the
photosensitized release of Weibel-Palade body contents of hu
man endothelial cells. This process results in secretion of large
multimers of vWf, the protein responsible for platelet adhesion
to the vessel wall in areas of injury.
Following exposure of metabolically prelabeled endothelial
cells to Photofrin II, a light dose-dependent release of Weibel-
Palade body contents occurred. This release was characterized
by the increased appearance of only the large multimeric forms
of vWf in the culture medium with depletion of intracellular
stores. No change in the distribution of dimeric forms, localized
predominantly in the endoplasmic reticulum (37), between cells
and culture medium occurred, arguing against cell lysis as a
mechanism for vWf release. Immunofluorescence microscopy
using vWf antiserum revealed that photosensitized release of
Weibel-Palade body contents resulted in the appearance of
characteristic patches of fluorescence on the external cell sur
face. Release patches of this kind were previously described
following exposure of endothelial cells to thrombin, the calcium
ionophore A23187, and fibrin (30, 42). Thus, the observed
cellular changes associated with vWf release occurring in re
sponse to photochemical stimulation appeared similar to those
induced by other known secretagogues. Photosensitization of
endothelial cells resulted in extensive vWf release, since at 6 h
following a 5-min light exposure, cells were virtually depleted
of the Weibel-Palade body stores of vWf.
The release of Weibel-Palade body contents from endothelial
cells is an active process requiring the fusion of the Weibel-
Palade body membrane with the plasma membrane of the cell.
Incubation temperatures below 20Calmost completely inhibit
release of Weibel-Palade body contents induced by the calcium
ionophore A23187 or thrombin, likely by inhibiting the final
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IRRADIATION TIME (mi nutes)
Fig. 6. Light dose dependence of 45Ca:* influx into photosensitized endothelial
cells. Following a 2-h Photofrin II incubation, cells were overlaid with 4 fiCi/ml
45CaCI:. exposed to various light doses, and incubated for 2 h at 37C. 4!Ca2*was
measured in cell lysale samples by scintillation counting. Values obtained were
normalized to those of unirradiated controls and arc presented as means SD
from three or four separate experiments. No significant influx occurred following
a 15-s light exposure. Statistically significant influx of 4'Ca2* occurred following
I nini light exposure, with the influx reaching a plateau level with longer light
exposure. . null result for the normalized data.
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/V VITRO PHOTOSENSIT1ZATION OF ENDOTHEL1AL CELLS
membrane fusion event (41). In addition, vWf release from
Weibel-Palade bodies has been shown to be dependent, at least
in part, on the presence of extracellular calcium (43). In our
studies, photosensitized release was inhibited by both reduced
temperature and by chelation of extracellular calcium with
EDTA. Therefore, at the photodynamic doses used in these
experiments, photosensitization of endothelial cells induced an
exocytotic process triggered by sublethal injury or insult that
did not produce immediate cell death. Thus, even a low, suble
thal drug-light dose delivered to the endothelium appears ca
pable of contributing to tumor destruction through the photo-
chemically stimulated release of vWf.
The time course of photosensitized vWf release was rapid
with respect to that stimulated by ionizing radiation (44). In
the latter case, endothelial cells exhibited secretion that did not
peak until approximately 24 h following exposure. The present
study shows that photoradiation in the presence of Photofrin
II induced a maximum vWf release within 3 h. This difference
in the time course of secretion is most probably a result of the
known differences in the types of intracellular damage caused
by the two therapies. The effects of ionizing radiation, for
example, are understood to be relatively more pronounced in
the cell nucleus, while PDT-mediated damage induced by sin
glet oxygen production tends to be concentrated in the plasma
and organelle membranes (45). Nevertheless, the photosensi
tized release of vWf in these cells was by no means immediate.
No detectable vWf was observed within the first 15 min follow
ing treatment, and only at l h and later could an increase in
release of vWf be detected. vWf release induced by thrombin,
histamine, assembly of complement components C5b-9 or fi
brin, on the other hand, occurs within minutes of cell stimula
tion (31).
The inhibitory effect of the calcium chelator EDTA impli
cates Ca2+ influx as a probable triggering mechanism for the
photosensitized release of vWf. An increased concentration of
intracellular free Ca2+ is known to stimulate many secretory
functions in cells (46), and has been associated directly with the
thrombin (32)-, histamine (33)-, and complement (34)-stimu-
lated secretion of vWf from endothelial cells. Indeed, an influx
of 45Ca2+into photosensitized endothelial cells was observed at
1 h (but not at 15 min) following light exposure. The 45Ca2+
influx therefore coincided with the observed time course for
vWf release. The magnitude of the 45Ca2+influx was roughly
equivalent to that observed following thrombin or 4-phorbol
12-myristate 13-acetate stimulation under essentially identical
experimental conditions (32). It is possible that membrane
depolarization, a general increase in membrane permeability,
damage to the membrane-bound Ca2+-ATPase. or some com
bination of these effects is responsible for influx of Ca2+ into
photosensitized endothelial cells.
The demonstrated capability of the endothelial cells to re
spond to sublethal drug-light doses by releasing the protein
responsible for mediating platelet adherence to the vessel base
ment membrane offers a possible mechanism for the formation
of platelet thrombi in the vicinity of PDT-treated tissue. Such
release could be especially important around the periphery of
tumors where light delivery may not be adequate to accomplish
direct tumor cell kill.
ACKNOWLEDGMENTS
Helpful discussions with Dr. Charles Francis and Scott Gibson are
gratefully acknowledged. The authors wish to thank Carol Weed for
help in the preparation of the manuscript.
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