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Enhanced Expression of the tie Receptor Tyrosine Kinase in Endothelial Cells
During Neovascularization
By Jaana Korhonen, Juha Partanen, Elina Armstrong, Anne Vaahtokari, Klaus Elenius, Markku Jalkanen, and Kari Alitalo
We have recently cloned a novel human receptor tyrosine
kinase, tie, from human leukemia cells showing megakaryoblastoid differentiation. We report here that the 4.4-kb tie
messenger RNA (mRNA) is present in all human fetal and
mouse embryonic tissues. By in situ hybridization, the tie
mRNA was localized to the endothelia of blood vessels and
endocardium of 9.5- to 18.5-day mouse embryos. However,
tie was not expressed by endothelial cells of developing
hepatic sinusoids. Increased tie mRNA signal was seen in
proliferating ovarial capillaries during hormone-induced superovulation. Only a weak tie signal was obtained from adult
skin, except during wound healing, when the proliferating
capillaries in the granulation tissue contained abundant tie
RNA. These results suggest that tie may have a role in
neovascularization.
o 1992by The American Society of Hematology.
E
bFGF, colony-stimulating factor-1, platelet-derived growth
factor, and stem cell factor.25
Although the ligand and the biologic function of tie are as
yet unknown, its restricted expression pattern in megakaryoblastoid and endothelial human cell lines indicates
that its functions may involve hematopoietic cell differentiation and/or cell adhesion to the vascular endothelium. We
report here on the cloning of partial cDNAs for mouse tie
and their use in the analysis of its messenger RNA
expression in vivo.
NDOTHELIAL CELLS lining the blood vessels have a
central role in the physiology of the vascular system,
blood clotting, wound healing, reproduction, embryonic
vasculogenesis, and angiogenesis, as well as in several
Growth and differentiation of the endothelial
cells occurs during the formation of primitive blood cells
from the yolk sac mesenchyme. In adult tissues, endothelial
cells proliferate very slowly, except during angiogenesis
associated with vascular regeneration. Among the factors
stimulating angiogenesis, at least four are direct mitogens
for endothelial cells. These are the acidic and basic fibroblast growth factors (aFGF and bFGF),3-7vascular endothelial growth factor/vascular permeability factor,8-I2and transforming growth factor-a (TGFa),13-15which bind to their
specific endothelial cell surface receptor tyrosine kinases
FGF receptor-1 (fIg),16-1*fit-1, l9,*0 and epidermal growth
factor (EGF) receptor,21,22respectively.
The protein product of the novel receptor tyrosine kinase
cDNA, named tie, cloned from K562 and HEL human
leukemia cell lines is N-glycosylated and contains two
Ig-like loops in its extracellular domain. Two or three
so-called EGF homology (EGFH) domains are encoded in
between the first and second Ig loops in different cDNA
variank23This region of the tie receptor thus has structural
similarities with, eg, the EGF, TGFa, and CRIPTO growth
factors, laminin A chain, and blood coagulation factor
IXa.” The cytoplasmic tyrosine kinase domain is about
40% identical at the amino acid level with the corresponding domains of the ret tyrosine kinase and the receptors for
From the Cancer Biology Laboratory, Departments of Pathology
and Krology, and the Department of Pedodontics and Orthodontics,
University of Helsinki, Helsinki; and the Department of Medical
Biochemistry, University of Turku, Turky Finland.
Submitted April 16, 1992; accepted July 15, 1992.
Supported by the Finnish Cancer Organizations, The Finnish
Academy, The Sigrid Juselius Foundation, The Finnish Cultural
Foundation, The Ida Montin Foundation, The Ella and Georg
Ehmrooth Foundation, and The Research and Science Foundation of
Farmos.
Address reprint requests to Kari Alitalo, MD, PhD, Professor of
Cancer Biology, Dept. of Pathology, University of Helsinki, Haartmaninkatu 3, 00290 Helsinki, Finland.
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.
O 1992 by The American Society of Hematology.
0006-4971/92/8010-0019$3.OO/0
2548
MATERIALS AND METHODS
Cloning of mouse tie cDNA probes. Approximately 106 plaques
from two lhgtl0 libraries (a kind gift of Dr Brigitte Galliot,
Zentrum fur Molekiilarbiologie, Heidelberg, Germany) prepared
from 10- and 11-day postcoitum (PC) mouse mRNA were screened
with the human tie receptor C D N A .Two
~ ~ nonoverlapping inserts
encoding the EGFH region and the juxtamembrane region, designated l C l D and D10E5, were subcloned into pGEM3Zf(+)
(Promega, Madison, WI), sequenced using SP6 and T7 primers and
used as probes (Fig 1).
Isolation and analysis of RNA. Total RNA was isolated from the
tissues of 17- to 19-week-old human fetuses (with permission of the
joint ethical committees of University Central Hospital and University of Turku), adult mouse organs, and developing embryos (8- to
18-day PC, newborn, and 2-day-old) according to Chirgwin et a1.26
Total RNA (20 kg) was electrophoresed in 0.8% agarose gels
containing formaldehyde and blotted onto Hybond-N (Amersham,
Arlington Heights, IL) or Gene Screen (Dupont, Wilmington, DE)
filters. The filters were hybridized and washed in stringent conditions.*’
For RNAse protection analysis, RNA probes of 383 and 493 b
were generated from linearized plasmids l C l D and D10E5,
respectively, using [32P]-UTPand T7 and SP6 polymerases:8 and
hybridized at 53°C overnight. Unhybridized RNA was digested
with RNAse A (10 U/pL) and T l ( 1 pg/mL) at 30°C pH 7.5, for 1
hour. The RNAses were inactivated by proteinase K digestion at
37°C for 15 minutes and the samples were analyzed in 8%
sequencing gels.
In situ hybridization and immunohistochemisty. The RNA probes
of 383 and 169 b (antisense and sense) were generated from
linearized plasmid lClD, using T7 and SP6 polymerases and
[35S]-UTP.28In situ hybridization of sections was performed
according to Wilkinson et al?9330with the following modifications:
(1) instead of toluene, xylene was used before embedding in
paraffin wax; (2) 6-km sections were cut and placed on a layer of
diethyl pyrocarbonate-treated water on the surface of glass slides
pretreated with 2% 3-aminopropyltriethoxysilane,(3) alkaline hydrolysis of the probes was omitted; (4) the hybridization mixture
contained 60% deionized formamide; and (5) the high stringency
wash was for 80 minutes at 65°C in a solution containing 50
Blood, Vol80, No 10 (November 15), 1992: pp 2548-2555
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TI€
RECEPTOR TYROSINE KINASE IN VASCULARIZATION
+
EGFH I
Ig-loop
EGF-like
domains
Ig-loop
2549
EGFH I1
PGTAGCRGL'~'C;P~PYGCSCGSGWRGSQCQ~CAPDHFGADCRLQCQCQ
-...-
R
322
--"-.......... *.."".-................"
132
354
x
......+ D10E5
.N**'~'
TM
,..EDPVRESWEEGLDQQUGSVC,TILAALLALVCI
-
I I1 I I I I I I I I I I I I
human tie EGPVQESRAAEEGLDQQ&;;~-
RRSCLHR
RRSCLHR
t
620
DITFFD
100
IIIIIIIIIIIIIIIIIIIIIIIIIIIIII 11l1111111111111111
..-....
*.*"r..*
*.**..*
I
50
IIIIIII
!
- m
RRTFTYQSGSGEETILQFSSGTLTLTRRPKPQPEPLSYPVLE
-5.
TK!2
100
.............
..........
RRTFTYQSGSGEETILQFSSGTLTLTRRPKLQPEPLSYPVLEh DITFED
670
LIGEGNFGQVIRAMIKKDGLKMNAAIKMLKEYASENDHRDFAGELEVLCKL
151
I I I I I I I I I I IIIIIIIIIIIIIIIIIII1111111111111111111II
LIGEGNFGQWRAMIKKDGLKMNAAIKMLKEYASENDHRDFAGELEVLCKL 721
Fig 1. Schematic structure of the human tie receptor tyrosine kinase and comparison of its deduced amino acid sequence with two mouse rie
cDNA clones (1ClD and DlOE5). The rie receptor consists of two Ig-like loops, three EGFH domains followed by three fibronectin Ill-like domains
(FN Ill), a transmembrane region (TM), tyrosine kinase domains (TKl and TK2) interrupted by a kinase insert sequence, and a carboxy terminal tail.
Amino acid sequence homology between mouse and human lie is 96% for both of the segments l C l D and D10E5. Differential splicing is known to
create human rie mRNA forms lacking the EGFH Idomain."
A
B
kb
4.4
-
25
-
kb
4.4
-
-tie
- tie
*
2.2
1.3
c
-
-&actin
- GAPDH
I
P C 8
9
10 11 12 13 14 15 16 17 18 N B 2 d
376 bp
' 376
bp
Fig 2. Expression of rie mRNA in human and mouse tissues. Hybridization of total RNA isolated from 17- t o 19week fetal tissues and
polyadenylated RNAfrom human aduk tissues (MTNB; Clontech) is shown in (A) and (E), respectively. The pactin and GAPDH probes were used
as internel controls for the amount of RNA loaded. Note the strong muscle actin signals in cardiac and skeletal muscles (9.
RNAse protection
assay was used t o detect tie and its possible splicing variants in developing mouse embryo (C) and in aduk tissues (0). Note the presence of some
undigested probe above the protected band.
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KORHONEN ET AL
2550
Fig 3. rie mRNA expression in 12.5-day PC mouse embryo. Shown are photomicrographs of a sagittal section hybridized with lClD antisense
(A and B) and sense (C) probes. Expression of tie mRNA is restricted to the endothelium of blood vessels. Abbreviations: br, brain; mg, meninges;
Ig, lung; mb, mandible; ht, heart; vn, ventricle; at, atrium; sc, spinal cord; pv, prevertebra; cv, posterior cardinal vein.
mmollL dithiothreitol (DTT)and Ix SSC. The sections were
covered with NTB-2 emulsion (Kodak. Rochester. NY) and stored
at 4°C. The slides were exposed for 14 days. developed, and stained
with hematoxylin. Control hyhridizations with sense strand and
RNAse A-trcntcd sections did not give a specific signal a h v e
background.
Factor VI11 was used as a specific marker for endothelial cells.31
Indirect immunoperoxidase staining was performed using rabbit
antibodies to human factor VI11 antigen. followed by peroxidaseconjugated swine antirahhit antibodies (Dako, Glostrup, Denmark).
Prepararion ofmouse ri.wicc.v. Mouse emhryos were derived from
m a t i n p of CBA and NMRl mice. Pregnant micc werc killed hy
cewiciil dislocation and thc embryos werc transferred immediately
via phosphate-huffered saline into 4c6 paraformaldehyde. The
emhryos and isolated mouse organs were fixed for 1X hours at 4°C.
dehydrated. embedded in wax, and cut into 6-bm sections. For
induction of superovulation. S IU of Gestyl (Sigma. St Louis. MO)
was injected intraperitoneally (IP) into 7-week-old NMRI mice on
day I and S IU of Pregnyl (Organon. Oss. Holland) o n day 3. The
ovaries and endometrial samplcs were obtained by killing the mice
on days I. 2.3. and 4. For prcparation of skin wounds. 6-mm long
incisions were made in the dorsal midline in the caudal part of thc
hack skin of adult Ralh/c micc. Unsutured wound tissue samples
were ohtained o n days I. 2.3.4.5.7, and 14 by killing the mice and
excising thc wound with some surrounding tissue.
RESULTS
Ana~sisof tie mRNA in ltumort fetal ti.wm and mouse
emhyo.7. Total RNA isolatcd from various tissucs of 17- to
19-wcck-old human fctuscs was subjcctcd t o Northcrn
blotting and hybridization with thc tie cDNA probc. All
fctal tissuc samplcs tcstcd containcd a 4.4-kb tie mRNA
band that showcd somc variation in intcnsity in diffcrcnt
tissucs (Fig 2A). Adult lung, hcart, and placcnta gavc a
strong tie mRNA signal. lntcrmcdiatc signals wcrc sccn in
kidncy, whcrcas musclc, brain, livcr. and pancrcas containcd considcrably lcss tie mRNA (Fig 2B). Thcsc diffcrcnccs wcrc also apparcnt aftcr dcnsitomctric scanning and
normalization of thc tie signals against thc cxprcssion of
p-actin. Bccausc of inhcrcnt dificultics in studying gcnc
cxprcssion in human tissucs, the dcvclopmcntal pattcrn of
cxprcssion of tie was analyzcd in thc mousc. Thc tie mRNA
was cxprcsscd at rclativcly constant lcvcls during thc wholc
cmbryonic pcriod analyzcd and in thc 2-day postnatal micc
(Fig 2C). Furthcrmorc, no splicing variants of tie mRNA
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2551
riE RECEPTOR TYROSINE KINASE IN VASCULARIZATION
larger veins and arteries as well as the Capillaries of the
dcvcloping and mature alvcoli. In contrast, the liver sinusoids had little if any signal (Fig 4).
Placenta. The placenta of 8-day PC embryo has an
elaborate network of blood vcsscls. The placental circulation is cstablished by day 9 of gestation, when the large
vessels penetrate thc allantois. In a 12.5-day PC embryo,
the labyrinth of thc placcnta is fully devclopcd. In the
placcnta of an 8.5-day PC embryo, the tie signals decorated
the walls of the labyrinthic and allantoic vessels (Fig 5A).
These same structures in adjacent sections stained for the
cndothelial cell marker, factor VI11 antigen (Fig 5B).
Enhanced expression of tie in proliferating capillaries of the
ovary and in granulation ri.rsue. To find out if tic expression
is enhanccd during ncovascularization, we hormonally induccd superovulation by injccting human chorionic gonadotrophin into thc micc. During ovulation, the blood vessels of
thc ovaries and the endometrial mucosa undergo a proliferative and a dcgcncrativc cycIc.32 As can be sccn in Fig 6A
and B, each maturing ovarian follicle is surrounded by
blood vessels which express tie rather weakly. On day 3 after
hormonc trcatmcnt, thc ncwly formcd blood vesscls in the
ovarial stroma show increased amounts of tie mRNA (Fig
6C and D). Figure 6 also shows tie hybridization signals in
thc skin 1 and 7 days aftcr wounding. Normal adult skin
contains few vcsscls that arc all wcakly tie positive (Fig 6E
and F). The cxprcssion of tie mRNA was found to be
maximal on day 7 in ncwly formcd vcsscls of the granulation
tissue and of the wound edge (Fig 6G and H). On day 14,
thc cxprcssion had dccrcascd back to the levcl sccn on day
2 aftcr wounding (data not shown).
DISCUSSION
Fig 3.
(Cont’d).
wcrc dctcctcd in thc RNA protcction analysis, although
such forms havc bccn found in human tie mRNA from
cndothclial cells.1’ Rcsults on adult mousc tissues agrecd
with determinations from human tissucs, cxccpt that thc
hcart had a wcakcr tie signal (Fig 2D).
In situ hybridization of tie mRNA in 12.5-day PC mouse
embryos. To bcttcr assign tie transcripts to cclls and
tissucs, sagittal scctions of 12.5-day PC mousc cmbryos
wcrc hybridized with tie RNAs corrcsponding to thc probcs
l C l D and D10E5 (Fig 1). As sccn in Fig 3A and B, tie
mRNA is ubiquitously cxprcsscd in thc embryonic vcsscls
and in the cndocardium o f thc hcart. Specific patterns of
thcsc signals can bc rccognizcd around thc major largc
vcsscls, thc ccntral ncrvous systcm, corrcsponding to the
mcningcs and blood plcxuscs, along the dcvcloping prcvcrtcbrac, and in thc rcspiratory and digcstivc tracts. Hybridization with thc scnsc probc is shown in Fig 3C. Unspecific
signal is rather cvcnly distributed throughout the sample;
only thc livcr and thc vcsscls containing blood cclls rcflcct
light in thc darkficld image. Thc tie signal was of similar
intensity in both the artcrics and veins. When comparcd in
thc samc in situ hybrization cxpcriment. thc intcnsity of tie
signal was similar in the vcsscls of embryonic and adult
lungs, whcrcas thc tie transcripts wcrc localizcd to thc
The prescnt experiments show that the tie receptor
tyrosine kinasc mRNA is expressed in endothelial cells of
the dcvcloping embryonic vessels. tie mRNA was detected
both in dcvcloping arteries and veins as well as in capillaries. Also, ncwly formed capillaries in hormone-induced,
maturing ovarial folliclcs and in the granulation tissue of
skin wounds showed enhanced tie expression in the adult
mousc.
The cloning of mousc tie shows that its deduced amino
acid scquencc is almost identical with the corresponding
human scqucncc (amino acid identity about 9 6 9 in both
scgmcnts studicd). Furthcr cvidcncc for thc identity of thc
mouse fie cDNA was obtained from Northern hybridization, in which probes from both species yielded the typical
4.4-kb mRNA signal from all human tissucs. The exprcssion
of the 4.4-kb tie mRNA in various human fetal tissues was
surprising, becausc our earlicr mRNA studies suggested
that tie is expressed primarily by human leukemia ccll lines
showing mcgakaryoblastic
Furthcrmore,
tie was exprcsscd in all mousc tissues tested and thcrc was
little variation of transcript abundance during mousc developmcnt from day4 PC to ncwborn and postnatal mice. A
cluc to the specificity of tie expression was shown when we
analyzed culturcd cndothelial cell lines, in which multiple
abundant transcripts wcrc semi'
A good corrclation was obscrvcd bctwccn localization of
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KORHONEN ET AL
2552
u-
--
I
thc in situ tie mRNA signals and immunostaining for factor
VIII, which is a spccific markcr for cndothclial cclls. No
significant diffcrcnccs wcrc ohscrvcd hctwccn tie signals in
thc walls of artcrics and vcins. sugcsting that smooth
musclc cclls do not cxprcss tie. Howcvcr, wc cannot cxcludc
thc possihility that pcricytcs cnvcloping thc capillary hascmcnt mcmhranc and capahlc of diffcrcntiating into smooth
musclc cclls also cxprcss somc tie mRNA. Also. it is
intcrcsting that thc discontinuous cndothclial ccll lining of
livcr sinusoids, which has an incomplctc undcrlying hasal
Fig 5. Expression of ti8 in 8.5day PC mouse placenta. tie transcripts can be seen in endothelial
cells of blood lacunae (bl) (A),
which are also positive for factor
Vlll antigen (B). Scale bar, 0.1
mm.
Fig 4. Localization of tie transcripts in the liver of a 12.5-day
PC mouse embryo. tie is expressed in hepatic vein (hv), but
not in the liver sinusoids (s) (A
through C). Hybridization with
the sense probe shows a low
level of unspecific background
signal (D). Scale bar: 0.1 mm for
(A), (B), and (D); and 0.05 mm for
(C).
lamina, is ncgativc for both tie mRNA and factor VI11
antigcn.
Both Northcrn hyhridization and RNAsc protcction
indicetcd somcwhat dccrcascd tie mRNA amounts in various adult tissucs. This rcsult may partially rcflcct diffcrcnccs in thc dcnsity of vcsscls in cmhryonic vcrsus adult
tissucs. On thc othcr hand, adult human and mousc lung
continucd to cxprcss ahundant tie transcripts. It is intcrcsting to notc that thc prolifcration ratc of vascular cndothclium of thc adult mousc lung is highcr than thc proliferation
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Fig 6. Expressionof fiemRNA
in mouse ovary during superovulation (A through D) and in
wounded skin (E through H).
Small amounts of fie transcripts
appear in the blood vessels of
the ovarial stroma (A and E). On
day 3 after hormone treatment,
fie expression has increased considerably (C and D; arrowheads
indicate the walls of the maturing follicles). Only a few riepositive blood vessels can be
seen near the edge of the wound
(arrow) on day 1 after wounding
(E and F). On day 7, the number
of fie-positive vessels has increased in the granulation tissue
(G and H). Note that the keratinized outer layer of the wound
gives a false-positive signal 11..
Scale bar, 0.1 mm.
..
,_. ..
.. .
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KORHONEN ET AL
2554
rate in the endothelium of the muscle and brain vessels,33in
which lesser tie signals were detected.
Strikingly, tie expression was enhanced during neovascularization associated with the developing ovarian follicles
and granulation tissue in skin wounds. While tie expression
according to our results is typical of endothelial cells, it is
tempting to speculate that the functions of tie are more
important for the growth of new vessels than for the
properties of a resting endothelium. Thus, tie may play a
role in angiogenesis, which is important in solid tumors and
several other angiogenesis-dependent diseases, eg, diabetic
retinopathy, psoriasis, atherosclerosis, and arthritis.34 The
finding of a tie ligand should give further insight to the
function of this interesting receptor.
ACKNOWLEDGMENT
We thank Dr Harri Hiwonen for the Northern blot containing
fetal RNAs, Dr Irma Thesleff and Dr Riitta Alitalo for critical
reading of the manuscript, and Kirsti Tuominen, Minna Ahlstedt,
and Tapio Tainola for expert technical assistance.
NOTE ADDED IN PROOF
Our recent results show that, in in situ hybridization, tie signal
first appears in large, round cells, probably corresponding to
angioblasts of the cephalic mesenchyme of 8.5-day PC mouse
embryo, whereas the mesoderm of 7.5-day embryo is negative. The
differentiating endothelial cells in the heart, dorsal aorta, and sinus
venosus of 9.5-day embryos show abundant tie mRNA signals.
REFERENCES
1. Klagsbrun M: Regulators of angiogenesis. Ann Rev Physiol
53:217, 1991
2. Folkman J, Shing Y:Angiogenesis. J Biol Chem 26710931,
1992
3. Gospodarowicz D: Humoral control of cell proliferation: The
role of fibroblast growth factor in regeneration, angiogenesis,
wound healing and neoplastic growth. Prog Clin Biol Res 9:1, 1976
4. Thomas KA: Fibroblast growth factors. FASEB J 1:434,1987
5. Burgess WH, Maciag T The heparin-binding (fibroblast)
growth factor family of proteins. Ann Rev Biochem 58575,1989
6. Rifkin DB, Moscatelli D: Recent developments in the cell
biology of basic fibroblast growth factor. J Cell Biol 109:1,1989
7. Gospodarowicz D: Fibroblast growth factor and its involvement in developmental processes. Curr Top Dev Biol24:57,1990
8. Connolly DT, Heuvelman DM, Nelson R, Olander JV,
Eppley B L Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest 84:1478,1989
9. Ferrara N, Henzel WJ: Pituitary follicular cells secrete a
novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 161:851, 1989
10. Gospodarowicz D, Abraham JA, Schilling J: Isolation and
characterization of a vascular endothelial cell mitogen produced by
pituitary-derived folliculo stellate cells. Proc Natl Acad Sci USA
1989:7311,1989
11. Levy AP,Tamargo R, Brem H, Nathans D: An endothelial
cell growth factor from the mouse neuroblastoma cell line NB41.
Growth Factors 2:9,1989
12. Tischer E, Gospodarowicz D, Mitchell R, Silva M, Schilling
J, Lau K, Crisp T, Fiddes JC, Abraham J A Vascular endothelial
growth factor: A new member of the platelet-derived growth factor
gene family. Biochem Biophys Res Commun 165:1198,1989
13. Todaro GJ, Fryling C, DeLarco JE: Transforming growth
factors produced by certain tumor cells: Polypeptides that interact
with epidermal growth factor receptors. Proc Natl Acad Sci USA
775258,1980
14. Marquadt H, Hunkapiller MW, Hood LE, Todaro GJ: Rat
transforming growth factor type I: Structure and relationship to
epidermal growth factor. Science 223:1079,1984
15. Schreiber AB, Winkler ME, Dernyck R: Transforming
growth factor-alpha: A more potent angiogenic mediator than
epidermal growth factor. Science 232:1250,1986
16. Ruta M, Burgess W, Givol D, Epstein J, Neiger N, Kaplow J,
Crumley G, Dionne C, Jaye M, Schlessinger J: Receptor for acidic
fibroblast growth factor is related to the tyrosine kinase encoded by
thefms-like gene (FLG). Proc Natl Acad Sci USA 86:8722,1989
17. Dionne CA, Crumley G, Bellot F, Kaplow JM, Searfoss G,
Ruta M, Burgess WH, Jaye M, Schlessinger J: Cloning and
expression of two distinct high-affinity receptors cross-reacting
with acidic and basic fibroblast growth factors. EMBO J 9:2685,
1990
18. Wennstrom S, Sandstrom C, Claesson-Welsh L cDNA
cloning and expression of a human FGF receptor which binds
acidic and basic FGF. Growth Factors 4:197, 1991
19. Shibuya M, Yamaguchi S, Yamane A, Ikeda T, Tojo A,
Matsushime H, Sat0 M: Nucleotide sequence and expression of a
novel human receptor type tyrosine kinase gene ( fit) closely
related to thefms family. Oncogene 5:519, 1990
20. deVries C, Escobedo JA, Ueno H, Houck K, Ferrara N,
Williams LT: Thefms-like tyrosine kinase, a receptor for vascular
endothelial growth factor. Science 255:989, 1992
21. Ushiro H, Cohen S: Identification of phosphotyrosine as a
product of epidermal growth factor-activated protein kinase in
A-431 cell membranes. J Biol Chem 25523363,1980
22. Schlessinger J: The epidermal growth factor receptor as a
multifunctional allosteric protein. Biochemistry 27:3119,1988
23. Partanen J, Armstrong E, Makela TP, Korhonen J, Sandberg M, Renkonen R, Knuutila s, Huebner K, Alitalo K A novel
endothelial cell surface receptor tyrosine kinase with extracellular
epidermal growth factor homology domains. Mol Cell Biol12:1698,
1992
24. Davis CG: The many faces of epidermal growth factor
repeats. New Biol2:410,1990
25. Hanks SK, Quinn AM, Hunter T: The protein kinase family:
Conserved features and deduced phylogeny of the catalytic domains. Science 241:42, 1988
26. Chirgwin JM, Przybyla AE, Mac Donald RJ, Rutter WJ:
Isolation of biologically active RNA from sources enriched in
ribonuclease. Biochemistry 18:5294,1979
27. Sambrook J, Fritsch EF, Maniatis T: Analysis of RNA, in
Ford N, Nolan C (eds): Molecular Cloning: A Laboratory Manual,
vol I. Cold Spring Harbor, NY,Cold Spring Harbor Laboratory,
1989, p 7.37
28. Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K,
Green MR: Efficient in vitro synthesis of biologically active RNA
and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 12:7035, 1984
29. Wilkinson DG, Bailes JA, Champion JE, McMahon AP: A
molecular analysis of mouse development from 8 to 10 days post
coitum detects changes only in embryonic globin expression.
Development 99:493,1987
30. Wilkinson DG, Bailes JA, McMahon AP: Expression of the
proto-oncogene int-1 is restricted to specific neural cells in the
developing mouse embryo. Cell 50:79,1987
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
m RECEPTOR TYROSINE KINASE IN VASCULARIZATION
31. Jaffe EA, Hoyer LW, Nachman R L Synthesis of von
Willebrand factor by cultured human endothelial cells. Proc Natl
Acad Sci USA 71:1906,1974
32. Reynolds LP, Killilea SD, Redmer DA: Angiogenesis in the
female reproductive system. FASEB J 6:886,1992
33. Hobson B, Denekamp J: Endothelial proliferation in tumors
2555
and normal tissues: Continuous labelling studies. Br J Cancer
49405, 1984
34. Klagsbnn M, Folkman J: Angiogenesis, in Sporn MB,
Roberts AB (eds): Peptide Growth Factors and Their Receptors,
vol 11. New York, NY, Springer-Verlag, 1991, p 549
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1992 80: 2548-2555
Enhanced expression of the tie receptor tyrosine kinase in endothelial
cells during neovascularization
J Korhonen, J Partanen, E Armstrong, A Vaahtokari, K Elenius, M Jalkanen and K Alitalo
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