Volume 18
Number 11
displayed a relative paucity of pericytes. Retinal
neovascularization no doubt reflects the interplay
of multiple local and systemic processes; initial attempts to understand these phenomena must
begin with a basic understanding of the biochemistry and growth characteristics of retinal vascular
cells. Our tissue culture model and that of Frank
et al. may be quite useful toward achieving this
goal.
Using our present culture conditions, we regularly observed endothelial cell growth from fetal
calf eyes but rarely from adult eyes; pericyte and
smooth muscle cells grew easily from young and
mature tissues. Accordingly, consideration must
be given to conditions and agents capable of
stimulating endothelial growth from adult retinal
vessels in vitro and the implications such means
may have in understanding retinal vascular pathophysiology.
We gratefully acknowledge the helpful advice and encouragement of Drs. Liane Reif-Lehrer and Alice Adler.
Editorial assistance was provided by S. Flavia Blackwell.
From the Department of Retina Research, Eye Research Institute of Retina Foundation, Boston, Mass.
Supported by a grant from The Charles E. Merrill Trust
and NIH grant 5-S07RR0527. Submitted for publication
May 14, 1979. Reprint requests: Sheldon M. Buzney,
Eye Research Institute, 20 Staniford St., Boston, Mass.
02114.
Key words: retinal vessels, vascular endothelium, mural
cells, intramural pericytes, smooth muscle, tissue culture, diabetic retinopathy
REFERENCES
1. Kuwabara T and Cogan DG: Retinal vascular patterns. VI. Mural cells of the retinal capillaries. Arch
Ophthalmol 69:492, 1963.
2. Hogan MJ and Feeney LJ: The ultrastructure of the
retinal blood vessels. I. The larger vessels. J Ultrastruct Res 9:10, 1963.
3. Wise GN, Dollery CT, and Henkind P: The Retinal
Circulation. New York, 1971, Harper & Row Publishers, pp. 39-54, 265-288.
4. Hogan MJ and Zimmerman LE: Ophthalmic Pathology. Philadelphia, 1962, W.B. Saunders Co. pp.
506-510.
5. Buzney SM, Frank RN, and Robison WG: Retinal
capillaries: proliferation of mural cells in vitro. Science 190:985, 1975.
6. Buzney SM, Frank RN, Varma SD, Tanishima T,
and Gabbay KH: Aldose reductase in retinal mural
cells. INVEST OPHTHALMOL VIS SCI 16:392, 1977.
7. Meezan E, Brendel K, and Carlson EC: Isolation of
a purified preparation of metabolically active retinal
blood vessels. Nature 251:65, 1974.
8. Wagner RC and Matthews MA: The isolation and
Reports 1195
culture of capillary endothelium from epididymal
fat. Microvasc Res 10:286, 1975.
9. Durie BGM and Salmon SE: High speed scintillation autoradiography. Science 190:1093, 1975.
10. Gimbrone MA and Cotran RS: Human vascular
smooth muscle in culture. Growth and ultrastructure. Lab Invest 33:16, 1975.
11. Gimbrone MA, Cotran RS, and Folkman J: Human
vascular endothelial cells in culture. Growth and
DNA synthesis. J Cell Biol 60:673, 1974.
12. Macarak EJ, Howard BV, and Kefalides NA: Properties of calf endothelial cells in culture. Lab Invest
36:62, 1977.
13. Weibel ER and Palade GE: New cytoplasmic components in arterial endothelia. J Cell' Biol 23:101,
1964.
14. Folkman J, Haudenschild CC, and Zetter BR: Long
term culture of human and bovine capillary endothelial cells. Proc Natl Acad Sci USA (in press).
15. Gospodarowicz D, Brown KD, Birdwell CR, and
Zetter BR: Control of proliferation of vascular endothelial cells of human origin. J Cell Biol 77:774,
1978.
16. Birdwell CR and Gospodarowicz D: Factors from
3T3 cells stimulate proliferation of cultured vascular
endothelial cells. Nature 268:528, 1977.
17. Suddith RL, Kelly PJ, Hutchison HT, Murray EA
and Haber B: In vitro demonstration of an endothelial proliferative factor produced by neural cell lines.
Science 190:682, 1975.
18. Fenselau A and Mello RJ: Growth stimulation of
cultured endothelial cells by tumor cell homogenates. Cancer Res 36:3269, 1976.
19. Shakib M and de Oliveria L: Studies on developing
retinal vessels. X. Formation of the basement membrane and differentiation of intramural pericytes. Br
J Ophthalmol 50:124, 1966.
20. Jaffe EA: Endothelial cells and the biology of factor
VIII. N Engl J Med 296:377, 1977.
21. Ashton M: The mode of development of the retinal
vessels in man. In The William Mackenzie Centenary Symposium on the Ocular Circulation in
Health and Disease, Cant JS, editor. St. Louis,
1969, The C. V. Mosby Co., pp 7-17.
22. Frank RN, Kinsey VE, Harding CV, and Mikus KP:
In vitro proliferation of endothelial cells from kitten
retinal capillaries.
INVEST.
OPHTHALMOL.
VISUAL
SCI. 18:1195, 1979.
In vitro proliferation of endothelial cells
from kitten retinal capillaries. ROBERT N.
FRANK, V. EVERETT KINSEY,! KARNI W.
FRANK, KEVIN P. MIKUS AND ANN RANDOLPH.
When microvessel (predominantly capillary) fragments
freshly isolated from the retinas of young kittens are int Deceased July 23, 1978.
0146-0404/79/111195+06$00.60/0 © 1979 Assoc. for Res. in Vis. and Ophthal., Inc
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•ii n / -
D
1196
Reports
J.
/noest. Ophthalmol. Visual Sci.
November 1979
cubated in tissue culture medium, we observe the slow
outgrowth of cells that appear to be derived from the
capillary endotlwlimn. The cells grow as a monolayer
with a tightly packed, mosaic-tike array. Electron microscopy of these cells reveals "tight junctions" (zonulae
key retinal capillaries. We now report the growth
in culture of colonies of endothelial cells derived
from the retinal capillaries of young kittens. These
cultures should prove useful for studying biochem. ^ ^ f u n c t i o n a l p r O p e r t i e s t h a t m a y b e unique
occludentes) resembling those found in the endotheluim
. ., ,. , ,, f
.„ . r : ,
.
' ,
„.
. ..
.
to endothelial cells from
capillaries of the
retina as
r
of intact capillaries.
1 lie availability
r .• )
ii
i .i ,. i
,'.
oi m vitro
n i
lir
prepara•;•/ ,
turns of retinal capillary endothehal cells should facilitate
investigations of the causes of abnormal endothelial cell
proliferation in various diseases and offactors that modify cell junctions and capillary permeability in the retina.
Since we have previously demonstrated proliferation in
culture of intramural periajtes ("mural cells") from retino/ capillaries, it should now be possible to carry out
comparative studies of biochemical andfu national properties of retinal capillary pericytes and endothelial cells,
Vascular endothelial cells are known to perform
a wide range of functions. These cells limit the
permeability of the blood vessel wall, synthesize a
number of substances including basement membrane, 1 factor VIII,2 and prostaglandins,3 play an
active role in hemostasis, and provide a smooth
surface for the vascular lumen that permits blood
to flow without turbulence. The endothelial cells
of large and small vessels in many organs including
the retina4 divide infrequently under normal circumstances, but their ability to replicate may increase substantially in certain physiologic conditions and as a result of injury or disease. Vascular
endothelial cells from various locations have important anatomic and functional differences. For
example, by contrast with capillaries from many
organs, the capillaries of the brain and retina are
normally impermeable to most ionic and molecular species, a feature that has been called the
"blood-brain barrier" or "blood-retinal barrier".•'• s
One probable anatomic correlate of this behavior
is the extensive system of zonulae occludentes, or
tight junctions, that join endothelial cells in retinal
and cerebral vessels but are less extensive in other
vascular endothelia.8' 7
Because many features of endothelial cell function are still poorly understood, and because abnormalities of these cells are prominent in several
human diseases, there is considerable value in
having preparations of isolated endothelial cells for
studies of their biochemistry, growth potential,
and physiologic function. Recently, populations of
well-differentiated endothelial cells from large
vessels such as human umbilical vein" and bovine
aorta9 have been grown in culture. Culture of
capillary endothelial cells from vessels of the central nervous system has not yet been accomplisheH, although Buzney et al.'° were able to
obtain in vitro proliferation of "mural cells" (intramural pericytes) from bovine and rhesus mon-
o p p o s e d to e n d o t h e l i a l cells from
"
a r e e r vessels,
6
>
sources outside the central nervous
system.
Methods. Kittens 3 to 4 weeks of age were employed for these experiments. Animals were sacrificed
by decapitation, the eyes were rapidly removed, and retinal microvessel fragments (predominantly capillaries) were isolated under sterile
conditions'with slight modifications of methods
described previously.'" Because eyes from small
numbers of kittens (one to six) were used for each
isolation and because the retinas are small, we
used only a single homogenization step, consisting
of 10 strokes of a Teflon pestile in a hand-held glass
homogenizer (Arthur H. Thomas Co., Philadelphia, Pa.), to fragment the tissue. This minimizes
loss of material, although it increases the proportion of noncapillary material in the final vessel isolate. Homogenization was followed by a single filtration step in which the suspension was decanted
onto a 210 /xm nylon mesh filter mounted on a
metal ring supported on a ring stand. The filtrate
passed onto an 85/U.m mesh filter mounted with a
rubber band on a beaker. The material collected
on the 85 /u,m filter was washed into a fresh receptacle, centrifuged, and cultured as described previously. More recently, we have substituted 53
/am mesh filters for the 85 /xm mesh with improved yields, presumably because the finer filters
retain smaller vessel fragments that are produced
during homogenization of these immature vessels,
Cells were grown in Ham's F-12 medium containing 2 mM glutamine, with the addition of 10%
fetal calf serum and antibiotics (penicillin 150
U/ml and streptomycin 150 fig/ml). Medium,
serum, and antibiotics were purchased from
Grand Island Biological Co., Grand Island, N. Y.
Culture vessels were 35 mm diameter plastic Petri
dishes (Falcon Plastics, Oxnard, Calif). The culhire medium was changed twice weekly. We have
found no apparent difference in retinal capillary
pericyte or endothelial cell proliferation and survival when other enriched media (e.g., TC-199,
Waymouth's) are substituted for F-12, although
detailed quantitative studies of this point have not
been conducted.
For electron microscopy the cells were fixed in
situ in the culture dishes using 2.5% glutaraldehyde buffered with 0.1M sodium cacodylate, pH
a n d
from
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Volume 18
Number 11
Reports
1197
Fig. 1. A, Kitten retinal capillary endothelial cells following 1 month in culture. (Phase contrast, X200.) B, Calf retinal capillary pericytes following 2 weeks in culture. (Phase contrast,
X200.)
Fig. 2. Electron micrograph of portions of two kitten retinal capillary endothelial cells overlying one another and joined by an extensive zonula occludens (tight junction). The cytoplasm
shows ribosomal particles, rough endoplasmic reticulum, and a single endocytic vacuole (arrow). (x86,000.) The inset shows a high magnification view of a tight junction from these
cultured endothelial cells and demonstrates fusion of the outer leaflets of the adjoining plasma
membranes. (x261,000.)
7.3, for 1 hr at 20° C. They were then post fixed
with 1% OsO4 for 1 hr at 20° C, "block" stained
with 0.1% aqueous uranyl acetate for 30 min, dehydrated in graded ethanols, and embedded in
Epon. Thin sections were stained on the grids
with uranyl acetate and lead citrate and viewed
with a Philips 301 electron microscope.
Results. Cells with morphologic characteristics
different from those of the intramural pericytes
described earlier10 were observed to proliferate
extremely slowly from capillary fragments that had
become adherent to the floor of the culture vessel.
One month after isolation of the microvessels,
these cells had only filled an area approximately 3
to 4 medium-power (200x) microscope fields in
diameter in a 35 mm Petri plate. Several features
of these cells strongly suggested that they derived
from capillary endothelium. The proliferating cells
were polygonal, although not regularly hexagonal,
and they formed a mosaic-like array with all of
their sides closely apposed to neighboring cells
(Fig. 1 A). In contrast, pericytes cultured from calf
retinas were more irregular in shape, sometimes
with protruding cytoplasmic processes, and they
formed a monolayer in which neighboring cells did
not form a closely packed array, but may have had
spaces between them or they may have partially
overlapped one another (Fig. 1, B). Although cells
with this "pericyte" growth pattern were the predominant elements that we observed in our cul-
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:st. Ophthalmol. Visual Sri.
November 1979
1198 Reports
O.ljum
Fig. 3. A portion of another kitten endothelial cell culture showing a tonguelike protrusion
from one cell extending above and joined to the cell underneath by a tight junction,
(x 118,000.)
tures of calf retinal microvessels under our usual
conditions of incubation, they were found infrequently in cultures of retinal microvessels from
young kittens.
Electron microscopic examination of cultured
kitten retinal microvessel cells revealed less abundant and varied cytoplasmic organelles than we
had observed in pericytes growing in vitro. There
were moderate numbers of free ribosomes and
a well-developed rough endoplasmic reticulum,
but only occasional micropinocytotic vesicles, and
rare microfilaments, microtubules, andlysosomes.
These cells were distinguished by the presence of
lengthy intercellular junctions, which, with high
magnification (Figs. 2 and 3) were seen to be pentalaminar with fusion of the outer leaflets of the
adjoining plasma membranes. We observed no
other types of junctions in these cultured cells.
Although intercellular junctions have been described in human umbilical vein endothelial cells
in culture,x they appeared to be desmosome-like
contacts, quite unlike the "tight" junctions that we
report here.
The mosaic-like growth pattern and the pres-
ence of extensive tight junctions was strong evidence that the cultured cells derived from capillary endothelium. We have not observed "specific
endothelial organelles" (Weibel-Palade bodies),8' " which are claimed to be characteristic of
vascular endothelium. These organelles have been
observed infrequently in capillary endothelium"
and, with the exception of one report,12 they have
not been described in the endothelial cells of ocular blood vessels, including those in the retina.
In work that is now in progress, we are beginning to investigate other endothelial cell "markers" in these cultures. Although the data are still
preliminary, presumed retinal capillary endothelial cells in culture appear to possess angiotensin-converting enzyme activity. The specific activity is less than that in cultures of aortic
endothelium, but definitely more than that present in pericyte cultures, which demonstrated no
detectable angiotensin-converting enzyme activity. Localization of factor VIII by immunofluorescence is unlikely to be satisfactory in these cultures since an antibody to feline factor VIII has not
been prepared and, in an experiment performed
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Volume 18
Number 11
with Dr. Barry S. Coller of the Division of
Hematology, State University of New York at
Stony Brook, factor VIII in adult cat plasma did
not cross-react with rabbit anti-human factor VIII
antibody.
Discussion. Both our own results and those of
Buzney and Massicotte13 in the accompanying report demonstrate that endothelial cells proliferate
from retinal capillaries whose component cells are
relatively immature. In its state of maturation, the
vascular system of the kitten retina at birth resembles that of the human fetus early in the third
trimester of pregnancy.14"16 The retinal vessels
have not yet extended all the way to the ora serrata, and, although some of the capillary cells in
the posterior pole of the eye have differentiated
into endothelial cells and intramural pericytes,
many of the smallest vessels are composed of cords
of undifferentiated cells."' Growth of the retinal
vessels toward the periphery of the retina and differentiation of the primitive capillary cells into mature forms occurs over the first few months of life.
Our failure to obtain proliferation of well-differentiated endothelial cells from microvessel fragments from the retinas of older animals of several
species (calves, lambs, and adult dogs and rhesus
monkeys) suggests that the endothelial cells of
these more mature retinal vessels have a greatly
reduced ability to replicate, except perhaps in the
presence of unusual stimuli such as trauma or disease. Pericytes appear to behave in the opposite
fashion. Few cells with pericyte morphology survive in our cultures from kitten microvessel fragments, but pericytes compose nearly the entire
population of cells growing from the microvessels
that we obtain from calves as well as from adult
animals of several species.
Our results open a number of possibilities for
investigations that have been difficult to carry out
with in vivo preparations. These include studies of
factors that promote either the selective proliferation or the degeneration of pericytes or endothelial cells in the retinal capillaries, of factors that
affect retinal capillary permeability either by
modifying the structure of intercellular junctions
and/or by altering pinocytic function, and of differences in the biochemical and physiolgoic function of retinal capillary pericytes and endothelial
cells under normal conditions as well as those that
simulate disease states, such as diabetes mellitus.
Reports 1199
stitute of Biological Sciences, Oakland University, Rochester, Mich. This work was supported in part by grant
RO l-EY-01857 (Dr. Frank) from the National Eye Institute and by a grant to Dr. Frank from the Juvenile Diabetes Foundation. Submitted for publication April 27,
1979. Reprint requests: Dr. Robert N. Frank, Kresge
Eye Institute, Wayne State University School of
Medicine, 3994 John R. St., Detroit, Mich. 48201.
Key words: retinal capillaries, endothelial cells, intramural pericytes, tissue culture, zonulae occludentes
(tight junctions)
REFERENCES
V
1. Jaffe EA, Minick R, Adelman B, Becker CG, and
Nachman R: Synthesis of basement membrane collagen by cultured human endothelial cells. J Exp
Med 144:209, 1976.
2. Jaffe EA, Hoyer LW, and Nachman RL: Synthesis of
antihemophilic factor antigen by cultured human
endothelial cells. J Clin Invest 52:2757, 1973.
3. Gimbrone MA Jr and Alexander RW: Angiotensin II
stimulation of prostaglandin production in cultured
human vascular endothelium. Science 189:219,
1975.
4. Engerman RL, Pfaffenbach D, and Davis MD: Cell
turnover in capillaries. Lab Invest 17:738, 1967.
5. Cunha-Vaz J, Faria de Abreu JR, Campos AJ, and
Figo GM: Early breakdown of the blood-retinal
barrier in diabetes. Br J Ophthalmol 59:649, 1975.
6. Brightman MW and Reese TS: Junctions between
intimately apposed cell membranes in the vertebrate brain. J Cell Biol 40:648, 1969.
7. Shakib M and Cunha-Vaz JG: Studies on the permeability of the blood-retinal barrier. IV. Junctional
complexes of the retinal vessels and their role in the
permeability of the blood-retinal barrier. Exp Eye
Res 5:229, 1966.
8. Gimbrone MA Jr, Cotran RS, and Folkman JL:
Human vascular endothelial cells in culture. J Cell
Biol 60:673, 1974.
9. Gospodarowicz D, Moran JS, Braun D, and Birdwell CR: Clonal growth of bovine vascular endothelial cells in tissue culture: Fibroblast growth factor as
a survival agent. Proc Nat Acad Sci USA 73:4120,
1976.
10. Buzney SM, Frank RN, and Robison WG Jr: Retinal
capillaries: Proliferation of mural cells in vitro. Science 190:985, 1975.
11. Weibel ER and Palade GE: New cytoplasmic components in arterial endothelia. J Cell Biol 23:101,
1964.
12. Matsuda H and Sugiura S: Ultrastructure of'tubular
body' in the endothelial cells of the ocular blood
vessels. INVEST OPHTHALMOL 9:919, 1970.
We thank Drs. Clifford V. Harding and Edward Essner for helpful discussions and Stanley Susan for technical advice.
From the Kresge Eye Institute of Wayne State University, School of Medicine, Detroit, Mich., and the In-
13. Buzney SM and Massicotte S: Retinal vessels: proliferation of endothelium in vitro. INVEST OPHTHALMOL VISUAL SCI 18:1191, 1979.
14. Ashton N, Ward B, and Serpell G: Role of oxygen in
the genesis of retrolental fibroplasia. Br J Ophthalmol 37:513, 1953.
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Incest. Ophthalmol. Visual Sci.
November 1979
1200 Reports
15. Patz A, Eastham A, Higgenbotham DH, and Kleh T:
Oxygen studies in retrolental fibroplasia. Am J
Ophthalmol 36:1511, 1953.
16. Wise GN, Dollery CT, and Henkind P: Development of retinal vessels. /;;.- The Retinal Circulation.
New York, 1971, Harper and Row Publishers, pp.
1-18.
Tapetum disorganization in taurine-depleted cats. GUANG Y. W E N , JOHN A. STURMAN,
HENRYK
M.
WISNIEWSKI,
ARNOLD
A.
LIDSKY, ANNE C. CORNWELL, AND KENNETH
C. HAYES.
A severe disorganization of the lattice arrangement of
tapetal rods occurs in cats nutritionally deprived of
taurine. This is the first reported example of a nutritionally related degeneration of tapetal cells. The tapetal cell
degeneration, along with the previously noted photoreceptor degeneration in taurine-depleted cats, suggests
that taurine plays a vital role in maintaining the structural integrity of some biological structures. The visual
evoked potentials were severely reduced in taurine-depleted cats as were the electroretinogratn responses, as
previously noted, indicating that they are deficient in
light processing. These results also emphasize the importance of dietary taurine for the maintenance of normal
structure and function of the eye, at least in the cat.
Taurine is distributed ubiquitously in animals
and is present in especially large concentrations in
retina and in developing brain.' A series of studies
demonstrated that cats fed a synthetic diet containing partially purified casein as the only source
of protein developed abnormalities in retinal
structure and function.2"6 It was subsequently
shown that a deficiency of taurine was responsible
for these changes. These studies confined themselves to structural alterations in retina, concurrent changes in electroretinogram, and concentration of taurine in retina and plasma. More
recent studies have demonstrated that taurine
depletion is far more exaggerated in other tissues,
including brain tissues, but no obvious neurologic
consequences were noted.7' 8 In addition, all previous cats, with a single exception, were maintained on a taurine-free diet for a maximum of 10
months. The present investigation reports the
changes in taurine concentrations, electrophysiological parameters, and eye structures in cats
maintained on a taurine-free diet for 18 months.
Materials and methods. Four cats (two pairs of
littermates, one control and one experimental)
were maintained for 18 months on a synthetic diet
containing casein as the protein source alone or
supplemented with 0.4% taurine.2 All four cats
were examined under neinbutal anesthesia for visual evoked potentials (VEP) as follows: three
small scalp electrodes were fixed along the midline
in occipital, central, and frontal sites. Occipital and
central electrodes were referred to the frontal electrode, and an ear clip served as ground. Both eyelids were retracted with masking tape. A photoflood lamp connected to a Grass photostimulator
was positioned 25 cm in front of the nose. Light
stimulation was presented with the photostimulator set at medium intensity (I = 4), at an average
rate of one flash per second, for a total of 100 trials
per condition. The data were digitized at 1000
points per second with a Hewlett Packard 2115
computer, and 0.5 sec samples were displayed on a
Versatec plotter.
Electroretinogram (ERG) measurements were
performed on both eyes of each cat under nembutal anesthesia after dark-adapting for an hour and
after maximally dilating the pupils with cyclogyl.
One-millisecond light flashes were generated by a
Grass photostimulator behind a ground glass
screen placed 12 cm from the eyes. Each testing
session included 0.5, 1, 5, 10, 20, and 40 Hz at two
intensities (log I = —0.3 and log I = 0).
All cats were anesthetized with nembutal. From
one pair of littermates (one control and one experimental), one eye was removed for perfusion
and fixation, the cats were killed, and tissues were
dissected and frozen in liquid nitrogen. From the
other pair of littermates, samples of visceral tissues (liver, kidney, and heart) were taken, and one
eye was removed and dissected into retina, lens,
and vitreous fluid and frozen in liquid nitrogen
prior to killing by intracardiac perfusion (1%
paraformaldehyde and 2% glutaraldehyde in 0.1M
Sorenson's phosphate buffer, pH 7.4, for 2 min
followed by 4% glutaraldehyde in the same buffer
for 15 min). Retina and tapetum were prepared for
examination by light and electron microscopy as
described previously.9 Each retina was divided
into 18 areas for exact mapping. Results reported
in this communication are taken from the center.
Concentration of taurine in tissue samples was
measured as described previously.10
Results. The tapetum of the taurine-depleted
cats showed a remarkable reduction in size. The
surface area was half that in the taurine-supplemented cats and that found in chow reared cats
(approximately 45 mm2), the thickness was onefifth that found in the taurine-supplemented cats
and in chow reared cats (approximately 65 /Am), and
0146-0404/79/111200+07$00.70/0 © 1979 Assoc. for Res. in Vis. and Ophthal., Inc.
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