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From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 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. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 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. .. ,_. .. .. . From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 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. 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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 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 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 Updated information and services can be found at: http://www.bloodjournal.org/content/80/10/2548.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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