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[ CA N CER R ES EA RC H 5 1. 3 26 1- 32 66 . J un e 1 5, 1 99
1]
Photosensitized Release of von W illebrand Factor from Cultured
Hum an
Endoth elial C ells1
Thom as H . Foster,2 M elissa C . Prim avera, V ictor J. M
arder, R ussell H ilf, and Lee A nn Sporn
D ep ar tm en ts o f R ad io lo gy [ T. H . F .] , M e di cin e,
H em at olo gy U nit [ M. C . P ., V . J . M ., L . A . S .J , a nd
B io ch em is tr y [ R. H .] , U ni ve rs it y o f R oc he ste r S
ch oo l o f M ed ic in e
a nd Den ti st ry , R oc he st er , N ew Y or k 1 46 42
ABSTRACT
C ultu red e nd othe lia l ce lls fro m th e h um an u mb ilic
al v ein w ere in cu
b at ed w i th l ow con cent ra ti on s ( 1 MU/ in l)o f t he p
ho to se ns it iz er l 'h ot fr i
I I. F oll ow in g a s ub le th al li gh t e xp os ur e, a li gh
t d os e- de pe nd en t r el ea se
o f v on W il le br an d f ac to r ( vVV f) in to th e c ul tu
re m ed ium was o bs er ve d.
A na ly sis o f th e mul tim en e c ompo sit io n o f t he r ele
as ed p ro te in i nd ic at ed
that it originated from the intracellular pool of large vW f m
ultim ers
store d in the W eib el-P alad e b od ies . T his re le ase w as
d etec te d a s e arly
a s 1 h p os tir ra di at io n. R el ea se w as in hib ite d a t
l ow temp era tu re a nd w as
dependen t upon the p re sence o f ex trace llular ca lc ium.
Photo sensi ti za tion
r es ul te d i n a n i nf lu x o f c al ci um who se t im e c ou
rs e p ar all el ed vWf r el ea se
from the cells. S ince vW f m ediates platelet adhesion to the
vascular
s ub endo th el ium , i t i s p os si bl e t ha t i ts p ho to
ch em ica ll y s timul at ed r el ea se
i n v iv o c ou ld c on tr ib ut e t o p la te le t t hr ombus f
ormat io n obs er ve d i n t is su e
fol lowing photodynamic therapy .
INTRODUCTION
T he m echanism s of tum or destruction in response to
photo-
d yn am ic th era py a re th e s ub je ct o f c on sid era ble
in ve stig atio n.
S ing le t o xy ge n ('O 2), fo rm ed as a resu lt o f en erg y
tra nsfer fro m
a photoexcited sensitizer to dioxygen, is highly reactive in
b io lo gical sy stem s an d h as been iden tified as th e ag en
t respo n
sible for m any deleterious effects at the cellular level (1,
2).
These effects have been m ost pronounced in cell m em branes
and have involved direct damage to the membrane (3-5) and
d eactiv ation of m em bran e-assoc iated en zy mes an d tran
spo rt
sys tems (6-8) .
A t a m acro sco pic lev el, P DT 1 p ro duces d estru ctio n of
tum or
and norm al tissue vasculature. A variety of effects have
been
d escrib ed , inc lu din g bloo d flo w stasis, h em orrh ag e,
vaso co n
s tr ic tio n, p la te le t a gg re ga tio n and p la te le t th
rombu s f orma ti on ,
an d b lan ch in g of the m icro vessels (9-1 5). R ece ntly , P
in ga r
e t a l. 16 r epo rte d th e appea ranc e o f in cr ea sed c ir
cu la ting le ve ls
o f thro mb ox an e fo llow in g p ho to dy nam ic therap y in a
rat tum or
m odel. Som e investigators have asserted that the observed
damage to the vasculature is the primary cause of tumor re
sponse to PD T (10). W hile this contention rem ains the
subject
of extensive study, the appearance of pronounced changes in
th e m ic ro va sc ula tu re o f th e tre ate d tis su e h as b
ee n c on vin cin gly
established.
In an effort to better understand some of the processes
in vo lv ed in P DT -in du ced effec ts o n the v ascu lature, w
e u nd er
took experim ents in which cultured hum an endothelial cells
w ere exposed to Photofrin II and light. Few investigations
of
Rece iv ed 12/ 4/ 90 ; a c cept ed 4 /9 /9 1.
T he c osts o f p ub lic atio n o f th is a rtic le w er e d ef
ra ye d in p art b y th e p ay me nt
o f p ag e c ha rg es. T his a rtic le m ust th er efo re b e h
ere by m ark ed a dv ertise me nt in
a cc or da nc e w it h 1 8 U .S .C . S ec tio n 1 73 4 s ole ly
t o i nd ic at e th is f ac t.
' The research presented in this report w as supported by USPH S
G rants
C A3 68 56 , H L0 71 52 , H L4 37 11 , a nd H L3 06 16 a wa rd
ed b y th e N atio na l C an ce r
I ns ti tu te a nd th e N at io na l H ea rt . L un g a nd B lo
od I ns tit ute .
2 Supported in part by the D epartm ent of R adiology at the
University of
R oc he ste r. T o w ho m r eq ue sts fo r re prin ts sh ou ld b
e a dd re ss ed , a t U niv ers ity o f
Roc he ste r S ch oo l o f Medi cin e a nd D en ti st ry . D ep
ar tm e nt o f R ad io lo gy , B ox 6 48 ,
6 01 E lm wo od A ve nu e, R oc he ste r, N Y 1 46 42 .
3 T he a bb re via tio ns u se d a re : P DT , p ho to dy na mic
th er ap y; v Wf . v on W ille -
b rand factor .
endothelial cells have been reported to date. Ben-Hur et al.
(17), using cultured bovine endothelial cells sensitized w
ith
chloroalum inum phthalocyanine tetrasulfonate, reported ef
fects that w ere attributed to a light dose-dependent release
of
c lo ttin g fa cto rs in de pe nd en t o f c ell ly sis. A re po
rt b y C orn er
et al. 18 , using Photofrin II as the sensitizer, dem
onstrated
that bovine endothelial cells w ere m ore sensitive to
photody
nam ic action in vitro than were sm ooth m uscle or
fibroblast
cells from the sam e species. O ur experim ents w ere designed
to
determ ine w hether the biochem ical response of hum an endo
thelial cells to photodynam ic insult included the stim
ulated
re le as e o f vW f.
vW f is a large, adhesive glycoprotein com posed of a series
of
d is ulf id e- bonded mu lt ime rs ( 19 ) s yn th es iz ed by
endot he lia l c ells
(20) and megakaryocytes (21, 22). It is found circulating in
p lasm a an d in p latele t -g ran ules (2 3, 2 4) an d is a co
mpo nen t
o f th e v asc ula r b aseme nt m embra ne (2 5). vW f med ia te
s p la te le t
a dh esio n to th e v es se l w all fo llowin g in ju ry (2 6, 2
7), e sp ec ia lly
under conditions of high sheer stress such as exist in the
m icrovasculature (28). vW f is stored w ithin endothelial
cell-
specific secretory vesicles called W eibel-Palade bodies
(29)
w hich provide a rapidly releasable pool of the largest and m
ost
b io lo gic ally p ote nt m ultim ers (3 0). In v itro , th is
re le ase re su lts
in the appearance of large multimeric forms in the culture
m ed iu m, d ep letio n o f th e in tracellu lar large m ultim
er sto res,
and the appearance of irregular patches containing vW f on
the external cell surface (30). Among the agents known to
induce this rapid vW f release from the W eibel-Palade
bodies
are th ro mb in , h istam in e, fib rin , p horb ol m yristate
acetate, the
c alciu m io no pho re A 23 18 7, an d assem bly o f co mp lem
en t co m
ponents C5b-9 (reviewed in Ref. 31). Stimulated release of
W eib el-P alad e bo dy con te nts is asso ciated w ith a rise
in in tra
c ellu lar c alciu m co nc entra tio n (32 -3 4). In th is stu
dy, w e sh ow
th at p ho to se nsitiz ed d am ag e to e nd oth elia l c ells
trig ge rs re le as e
o f v Wf fro m W eib el-P alad e b od ies o f en do th elial
cells an d th at
release is likely caused by an influx of calcium induced by
pho todynam ic a cti on .
M ATERIALS AND M ETHODS
End oth el ia l C ell C ul tu re a nd Me ta bo li c L ab el in
g. E nd oth eli al c ell s
f rom h uman umbil ic al v ei ns w ere h ar ve ste d a s p re
vio us ly d es cr ib ed ( 29 ,
35) and cultured in 25-cm 2 cell culture flasks in M cC oy's M
edium 5a
(F lo w L abo ra to rie s, M cL ea n, V A) w ith 2 0% fe ta l bo
vine s eru m, 5 0 ig /
m l e ndot he li al c el l g row th s uppl ement (B iomed ic ai
Te chno lo gi es , I nc .,
S toughton, M A), and 100 M g/m l heparin (S igm a Chem ical
Co., St.
L ouis, M O). T he cultures w ere m onitored for the presence of
nonen-
d ot he lia l c ell s b y immun ofl uo re sc en ce s ta in in g
f or vWf a nd w ere f ou nd
to b e free o f c on tam in atio n. C ells w ere ty pica lly u
se d at p assa ge 2 an d
required approxim ately 5 days to reach confluence (3 x IO 6
cells) at
ea ch p ass ag e. P rio r to in cub atio n w ith P ho tofrin , m
etab olic la belin g
w it h [ 15 S] cy st ein e (1 15 3.4 C i/mmo l; N ew Eng la nd
Nuc le ar R es ea rc h
Products, Boston, M A) was carried out for 3 days at 25 nCi/ml
in
co mple te cu ltu re m ed iu m. E xp erim ents w ere p erfo rm
ed o n co nflu en t
c el l mono la ye rs . Endo th el ia l c el l v ia bi li ty wa s
a ss es se d by d et erm in in g
th eir a bility to ex clu de tryp an b lu e. A t v ario us tim
es fo llo win g p ho to -
3261
Research.on January 30, 2014. 1991 American Association for
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8/11/2019 Cancer Res 1991 Foster 3261 6
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1\ tITKO P HO TO SE NSITIZAT1O N O F E ND OTH ELIAL C E LLS
radiation, cells w ere try psiniz ed and incubated f or 1 m in w
ith 0.2%
try pan blu e stain (S ig m a C hem ic al C o.). T he p erc en
tag e o f v iable c ells
was d ete rm i ne d b y u si ng a h emo cy tom et er.
Ph ot of ri n I I T re atm e nt an d L ig ht E x po su re . Ph
oto frin I I was o btain ed
f rom Quad ra L o gi c T e ch no lo gie s, I nc . ( V an co uv
er, B rit is h Columbi a,
C an ad a) an d sto red in th e d ark at 0 C .C ells w ere
incubated w ith 1
M g/m l Photof rin II in serum -f ree culture m edium supplem
ented w ith
Nu trid oma-HU (Boe hrin ge r Man nh eim B i oc hem ic al s, I
nd ian ap oli s,
IN ). Follow ing a 2-h incubation at 37 Cin a 5% C O2, high hum
idity
in cu bato r, ce lls w e re w ash ed tw ice w ith seru m -f ree
m e diu m , an d co m
p lete cu ltu re m e diu m w as rep lace d. Ph oto rad iation w
as d on e w ith th e
unf iltered output f rom a pair of f luorescent lam ps. T he
incident pho-
toradiation pow er density at the lev el of the cells w as 0.2 m
W /cm 2,
u nc orre cte d f or t he ab so rb an ce o f t he c ult ure m e
dium .
An t is era. T h e p reparat io n and charac te ri z at io n o f
monospe ci f ic an ti -
sera agains t hu m an v W f u se d f or im m u nop urif icatio n
w e re p re vio usly
d es cri bed (29 , 3 6) .
C ell L y sis an d v \V 'f Pu rif icatio n. A t s elec te d tim
e po in ts f ollow in g
p ho to radiation , cu ltu re m ed iu m w as co lle cted an d ce
lls w ere ly sed as
p re vio usly d esc rib ed (37 ). T he c ell ly sate or c ultu
re m e diu m sam p les
w ere then incubated w ith gelatin S epharose f or 1.5 h at room
tem per
ature to rem ov e f ibronectin and other contam inating proteins
that
ad he re n on sp ec if ic al ly . G el ati n S ep haro se was
remov ed b y c en tri fu ga-
tion. Protein A - Sepharose C L4B (30 m g/sam ple) (S igm a C
hem ical
C o.) w as preincubated w ith 50 n\ anti-v W f antiserum f or 30
m in, then
added to each sam ple and incubated f or 1.5 h at room tem
perature.Pro tein A - S ep haro se b ead s w e re w ash ed ex ten
siv ely (3 8), the n b oiled
in electrophoresis sam ple buf fer (39), and the collected sam
ple w as
an aly z ed b y g el e le ct ro ph ore sis . vW f immun op urif
ie d f rom e qu al n um
b ers o f c el ls was l oad ed f or an al ys is o f th e c el l
l ys at e s amp le s. S im i larl y,
the total v W f im m unopurif ied f rom culture m edia sam ples
w as sub
j ec te d t o g el anal y si s.
E le ct ro ph ore si s G el s an d v \V f Quan ti tati on . A g
aro se h ori zo ntal s lab
g els w ere p rep ared by us in g a s olu tio n o f 2 % ag aro
se an d 0 .2 % s od iu m
d od ecy l su lf ate in 0 .05 M p hos ph ate b uf fer, p H 7 .0
. E le ctro ph ores is
b uf fe r w as 0 .1 M p ho sp hate, p H 7 .0 , w ith O .I % s od
iu m d od ec yl su lf ate .
E x po su re s o f 2 -3 w e ek s w e re u se d t o p ro du ce t
he au to rad io grap hs u se d
f or q uan tit at iv e d en si tom et ric s can nin g. T h e p
erc en tag e o f to tal ( dim e r
plus large m ultim er) v W f appearing in the culture m edium w
as com
p ute d b y u sin g t he f ol low in g f ormu la:
v W f in culture m edium
v W f in cell ly sate + culture m edium
Im m u nof lu ore sc en ce S tain in g. E nd oth elial c ells c
ultu re d o n g lass
cov erslips w ere w ashed, f ix ed for 20 m in in 3.7% form
aldehy de in
p ho sp hat e- bu ff ere d s al in e, an d p erm e ab il iz ed f
or 1 5 m i n i n 0 .5% T ri to n
X - 1 00 . Fl uo re sc en ce s tain in g u si ng an ti- vW f an
ti se rum was c arri ed o ut
as p re v io us ly d es cri bed (29 ).
4 5Ca2+Inf l ux . S t ud ie s o f 4 5Ca2+inf l ux we re condu ct
ed as d es cri bed
p rev io usly b y B areis e t al. (40 ) an d d eG ro ot e t al.
(32 ). C ells g ro w n to
co nf lu en ce in 2 5-c m 2 ce ll c ultu re f las ks (app ro xim
ately 3 x IO 6 c ells)
w ere incubated f or 2 h w ith Photof rin II as described abov
e, w ashed
tw ice w ith serum -f ree culture m edium , and then ov erlaid w
ith a 2-m l
so lu tio n c ontain ing 4 5C aC l2 (0 .7 15 C i/m m o l, A m e
rsh am , A rlin gto n
H eights, IL ) diluted to 4 /uC i/m l in com plete culture m
edium . C ells
w e re e xp ose d to lig ht, and f ollo w in g in cu bation at 3
7 Cf or v ariou s
tim es, the uptak e of 45C a2+w as stopped by adding 100 n\ of
100 m M
CaCli. T he culture m edium w as im m ediately rem ov ed, cells
w ere
w ashed tw ice w ith cold 150 m M N aC l containing 5 m M C aC
l2 and 10mM 4 -(2- hyd ro x y et hy l )- l- pi pe raz i ne et han
esul f on ic ac id , pH 7 .4 . Ce ll s
w ere ly sed in a 1 m l solution containing 5 m M C aC l2/0.5% T
riton X -
1 00 . R ad io activ ity in th e ce ll ly sate sam p le s w as m
e asu re d b y sc in til
lat ion counting.
RESULTS
S in ce vW f re le ase f rom W e ib el-Palad e b od ie s is an e
xo cy to tic
p ro ce ss re qu irin g main te nan ce o f e nd oth elial c ell
f un ctio n an d
integrity , a study w as conducted to determ ine the v iability
of
p ho to sen sitiz ed cells treated w ith a ran ge o f p hotorad
iatio n
f lu en ces. Fo r at least 3 h f ollow in g ph oto rad iatio n,
lig ht do ses
of 2 m in or less at 0.2 m W /cnr resulted in essentially no
d ec re as e in c ell v iab ili ty as c ompare d w i th unirrad
iate d c on tro ls
(>90% v iable). A t 6 h postphotoradiation, 80% of cells
that
h ad re ce iv ed a 2 -m i n e xp os ure w e re c ap ab le o f e
xc lu din g try pan
blue, w hile v iability of cells treated f or less than 2 m in
did not
dif fer f ro m co ntro ls. S om e dim in ish ed v iab ility w as
o bserv ed in
cells irradiated w ith higher f luences. S ix h af ter 3- and
5-m in
ex po sures, th e p ercen tag e o f to tal cells retain in g th
e ab ility to
ex clu de try pan blu e w as 7 3 an d 5 4% , resp ectiv ely
.
E xposure of cells to Photof rin II and light led to a light
dose-
dependent redistribution of the m ultim eric form s of v W f
be
tw een the cells and culture m edium . A s illustrated by
the
autoradiograph in Fig. 1, an increased light dose caused an
inc re ase in to tal v W f ap pearin g in th e cu ltu re m e diu
m , and th is
secreted v W f co ntain ed a disp ro po rtio nate am o unt o f
the larg e
m ultim eric f orm s. T he am ount of dim eric form s secreted,
how
ev er, d id n ot in crease w ith in creasin g lig ht do se. In
cub atio n o f
c ells w i th Ph oto frin II in th e ab se nc e o f p ho to rad
iatio n re su lte d
in no detectable alteration of the m ultim eric com position
of
cells or culture m edium as com pared w ith control cells
(not
shown).
T he percentage of total labeled v W f appearing as large m
ul-
tim ers or dim ers in the culture m edium at 6 h follow ing
the
large
mu lt ime rs
Fig. 1. S ecretion of large v W f m ultim ers in
response to Ph olof rin II and light ex posure.
C ulture m edium and cell ly sate sam ples w ere
collected 6 h f ollo wing light ex posures of 0, 1.
2. 3. and 5 m in (0.2 m \V /cm 2). v W f w as im m u
n op urif ie d an d an aly z ed as d es crib ed i n M ate
r ia ls and Me thods . Pho to sen s it i/ at ion resu lt ed
i n t he i nc re as ed ap pe aran ce o f l arg e mu lt im e ri
c
f orm s o f v W f in th e cu ltu re m ed iu m , w ith d ep
le
t io n o f t he c el lu lar p oo l. T h ere w as n o d et ec tab
le
ef f ect on the distribution of the dim er pool of
v W f b etw e en c el ls an d c ul tu re m e diu m .
dimer
0
2 3
cell
mm
1 2 3
medium
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