Proc. Natl. Acad. Sci. USAVol. 93, pp. 2576-2581, March 1996Cell Biology

Vascular endothelial growth factor B, a novel growth factor forendothelial cells

(angiogenesis/endothelium/mitosis)

BIRGITrrA OLOFSSON*t, KATRI PAJUSOLAtt, ARJA KAIPAINENt, GABRIEL VON EULER*, VLADIMIR JOUKOVt,OLLI SAKSELA§, ARTO ORPANAT, RALF F. PETTERSSON*, KARi ALITALOtt, AND ULF ERIKSSON*tll*Ludwig Institute for Cancer Research, Stockholm Branch, Box 240, S-171 77 Stockholm, Sweden; tMolecular/Cancer Biology Laboratory and §Department ofVirology, Haartman Institute, POB 21 (Haartmaninkatu 3), 00014 Helsinki, Finland; and 11Department of Gynecology and Obstetrics, University of Helsinki,Haartmaninkatu 2, 00290 Helsinki, Finland

Communicated by Sune Bergstrom, Karolinska Institutet, Stockholm, Sweden, December 6, 1995 (received for review October 26, 1995)

ABSTRACT We have isolated and characterized a novelgrowth factor for endothelial cells, vascular endothelialgrowth factor B (VEGF-B), with structural similarities tovascular endothelial growth factor (VEGF) and placentagrowth factor. VEGF-B was particularly abundant in heartand skeletal muscle and was coexpressed with VEGF in theseand other tissues. VEGF-B formed cell-surface-associateddisulfide-linked homodimers and heterodimerized with VEGFwhen coexpressed. Conditioned medium from transfected293EBNA cells expressing VEGF-B stimulated DNA synthesisin endothelial cells. Our results suggest that VEGF-B has arole in angiogenesis and endothelial cell growth, particularlyin muscle.

Angiogenesis, the sprouting ofnew capillaries from preexistingblood vessels, is of central importance in many biologicalprocesses. During embryonic development, vascularization ofmany organs and structures is achieved by angiogenesis fromthe larger vascular network formed by the growth and differ-entiation of both early embryonic and extraembryonicsplanchnic mesoderm in a process called vasculogenesis (1).Furthermore, tissue regeneration and reorganization, as wellas several pathological conditions including growth and me-tastasis formation of solid tumors, have been shown to bedependent on the formation of new blood vessels (2). Theendothelial cell is the major cell type contributing to neovas-cularization. During angiogenesis, endothelial cells, whichform the inner lining of blood vessels, undergo migration withconcomitant proliferation and tube formation and participatein proteolytic degradation of the basement membrane andextracellular matrix. The specific mechanisms regulating an-giogenesis are not fully understood, but several potentialregulators of this process have been described (3). Vascularendothelial growth factor (VEGF) is an endothelial-cell-specific mitogen and has been shown to be a potent angiogenicfactor (4). It exerts its effect through two endothelial receptortyrosine kinases (RTKs) Flk-1/KDR (5-7) and Flt-1 (8, 9),which appear to play a pivotal role in regulation of endothelialcell growth and differentiation and in maintenance of thefunctions of the mature endothelium (10, 11). In addition,three other RTKs have been discovered that are specificallyexpressed in endothelial cells, Flt-4 (12-14), TIE, and TEK(15-17), of which TIE and TEK have been shown to beindispensable for the development of a proper vasculature (18,19). Thus, endothelial cell growth factors have several attrac-tive features as regulators of normal and pathological angio-genesis (4, 20-22). Placenta growth factor (PlGF) (23), whichis a ligand for the Flt-1 RTK (24), is structurally related toVEGF, but its biological function remains obscure at present.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

Both VEGF and PIGF are dimeric glycoproteins that arerelated in structure to platelet-derived growth factor A and Bpolypeptides (PDGF-A and -B), potent mitogens for smoothmuscle cells, glia cells, and several other cell types (25). In thisstudy, we have characterized a novel growth factor for endo-thelial cells VEGF-B,** with structural homology to VEGF,PIGF, and the two PDGF polypeptides. Our results suggestthat VEGF-B plays a role in vascularization of adult andembryonic tissues, in particular of muscle.

MATERIALS AND METHODSCloning and cDNAs Encoding Mouse and Human VEGF-B.

A cDNA clone termed pcif 2, encoding part of mouseVEGF-B, was isolated from a mouse embryonic day (E) 14.5cDNA library cloned in the yeast expression vector pPC67(26). A 0.9-kb Sal I-Not I insert in this cDNA clone was usedto screen an adult mouse heart A ZAP II cDNA library(Stratagene). Several positive clones were subcloned by in vivoexcision into pBluescript SK(+) and the nucleotide sequencesof the inserts were determined (Sequenase 2.0, United StatesBiochemical). To obtain cDNA clones encoding humanVEGF-B, 106 clones of a human fibrosarcoma cDNA libraryHT-1080 in Agtll (Clontech) were screened with the sameinsert. Among several positive clones, one, termed H.1, wasanalyzed and its nucleotide sequence was determined. Basedon this sequence, two oligonucleotides were designed and usedto amplify by reverse transcription-coupled PCR the wholecoding region of human VEGF-B from oligo-(dT)-primedhuman erythroleukemia cell (HEL) RNA. The amplifiedproduct was cloned into the pCRII-vector of the TA cloningkit (Invitrogen) and the nucleotide sequence was determined.Standard molecular biology techniques were used throughoutthis work (27). The multiple amino acid sequence alignmentsand the phylogenetic analysis were done according to Hein(28) by using the PAM 250 distance table.Northern Blot Analysis and in Situ Hybridization. Mouse

and human multiple-tissue Northern blots (Clontech) werehybridized with a 32P-labeled mouse VEGF-B probe (0.9-kbSal I-Not I insert of clone pcif2). VEGF expression wasanalyzed with 32P-labeled VEGF165 cDNA as the probe (a giftfrom Daniel Connolly) (29). The hybridizations were carriedout at 42°C in 50% (vol/vol) deionized formamide/5X SSC,

Abbreviations: VEGF, vascular endothelial growth factor; PDGF,platelet-derived growth factor; PlGF, placenta growth factor; RTK,receptor tyrosine kinase; HUVEC, human umbilical vein endothelialcell; bFGF, basic fibroblast growth factor; E, embryonic day; BCE,bovine capillary cell.tB.O. and K.P. contributed equally to this work, as did K.A. and U.E.ITo whom reprint requests should be addressed.**The sequences reported in this paper have been deposited in theGenBank data base (accession nos. U48800 and U48801 for mouseand human VEGF-B167, respectively).

2576

Proc. Natl. Acad. Sci. USA 93 (1996) 2577

pH 7.0/1% SDS/5X Denhardt's solution/denatured salmonsperm DNA (100 jig/ml). The filters were washed for two30-min periods at 52°C in 2x SSC/0.5% SDS and exposed toKodak XAR film with intensifying screens for 2-5 days at-70°C. In situ hybridization analysis of adult mouse tissuesfrom CBA mice and of embryos derived from matings of CBAand NMRI mice were carried out essentially as described (30).The RNA probes (a 383-bp antisense probe and a 169-bp senseprobe) were generated from a linearized plasmid containing a440-bp Sal I-Sac I fragment derived from the pcif2 cDNAclone. Radiolabeled RNA was synthesized by using T7 and SP6RNA polymerases and 32S-labeled UTP (Amersham). Alka-line hydrolysis of the probes was omitted. Hematoxylin wasused for counterstaining. Control hybridizations with sense-strand, and RNase A-treated sections did not give signalsabove background.

Expression ofVEGF-B and VEGF in 293EBNA Cells. cDNAinserts encoding human VEGF-B and human VEGF165 werecloned into the pREP7 expression vector (Invitrogen). Humanembryo kidney 293EBNA cells (expressing Epstein-Barr virusnuclear antigen 1) were transfected with expression plasmidsby using calcium phosphate precipitation, and the cells wereincubated for 48 h. Monolayers of cells were incubated inmethionine- and cysteine-free medium for 30 min followed bylabeling with [35S]methionine and [35S]cysteine (100 ,tCi/ml)(Promix, Amersham; 1 Ci = 37 GBq) in the same medium for2 h. The labeling medium was replaced with normal mediumwithout serum and labeled proteins were chased for 6 h.Heparin was included during the chase when indicated (100,tg/ml). Medium was collected after the chase period and cellswere solubilized in 10 mM Tris HCl, pH 7.5/50 mM NaCl/0.5% sodium deoxycholate/0.5% Nonidet P-40/0.1% SDS/aprotinin (0.1 unit/ml). Aliquots of the culture supernatantsand the cell lysates were subjected to immunoprecipitationwith specific antisera and analysis by SDS/PAGE. The anti-peptide antiserum to human VEGF-B was generated by im-munizing rabbits with a branched 23-mer oligopeptide com-posed of the N-terminal region (amino acid residues SQP-DAPGHQRKVVSWIDVYTRAT). The antiserum to humanVEGF was from R & D Systems.

Mitogenic Assay for VEGF-B. Conditioned medium con-taining human VEGF-B and human VEGF165 was collectedfrom 293EBNA cells transfected with the appropriate expres-sion vectors or with empty vector (mock) in the presence ofheparin (1 ,Lg/ml) 48 h after transfection. Second-passagehuman umbilical vein endothelial cells (HUVECs) were plated

into 96-well plates (4 x 103 cells per well) in M-199 mediumsupplemented with 10% (vol/vol) fetal bovine serum andincubated for 24 h. Conditioned medium was diluted with thegrowth medium and cells were stimulated for 48 h. Freshconditioned medium containing [3H]thymidine (Amersham;10 ,tCi/ml) was added to the cells and stimulation wascontinued for another 48 h. Cells were washed with PBS andtrypsinized and the incorporated radioactivity was determinedby liquid scintillation counting. Bovine capillary endothelial(BCE) cells were seeded into 24-well plates and grown untilconfluence in minimal essential medium (MEM) supple-mented with 10% fetal calf serum. Cells were starved in MEMsupplemented with 3% fetal calf serum for 72 h, after whichconditioned medium diluted into serum-free medium wasadded to the cells and the cells were stimulated for 24 h.[3H]Thymidine was included during the last 4 h of the stimu-lation (1 ,uCi/ml). Stimulations with basic fibroblast growthfactor (bFGF) were carried out as above by using recombinantbFGF at 6 ng/ml (a gift from Andreas Sommer, Synergen,Boulder, CO). Cells were washed with PBS and lysed withNaOH, and incorporated radioactivity was determined byliquid scintillation counting.

RESULTSPrimary Structures of Mouse and Human VEGF-B. By

using the yeast two-hybrid system for other purposes, weisolated a partial cDNA clone from a mouse E14.5 cDNAlibrary encoding amino acid sequences similar to the VEGFfamily of growth factors. With this cDNA as a probe, severalcDNA clones were isolated from an adult mouse heart cDNAlibrary and from human fibrosarcoma and erythroleukemiatumor cell cDNAs. The mouse and human full-length cDNAclones encoded polypeptides of 188 amino acids containing anN-terminal hydrophobic putative signal sequence (Fig. 1). Inanalogy with VEGF (4), we propose that the signal peptidasecleavage site is located between Ala21 and Pro22. Hence, theprocessed VEGF-B polypeptides contained 167 amino acids.Mouse and human VEGF-B displayed 88% amino acid se-quence identity and were highly basic, especially in theirC-terminal regions. The amino acid replacements were pre-dominantly found in the N- and C-terminal regions, while thecentral portions of the molecules were almost identical. Bothhuman and mouse VEGF-B lacked the consensus sequence forN-linked glycosylation (NXT/S). Pairwise comparisons of theamino acid sequences showed that mouse VEGF-B is -43%

mVB3F-B 167 M S P L L R R L L L V A L L Q L A R T Q A P V S Q F D G P S 30hVEF-B 167 M S P L L R R L L LAALLQLIsPIG 30

mVBIF-B 167 H Q K K V V P W I D V Y A R A T C Q P R E V V V P L S M E L 60hVBF-B 167 H RATCQPREVVVPLTVEL60

mVBGF-B 167 M G N V V K Q L V P S C V T V Q R C G G C C P D D G L E C V 90hVBrF-B 167 g KQLVPSCVT V Q R C G G C C P D D G L E C V 90

mVBXF-B 167 P T G Q H Q V R M Q I L M I Q Y P S S Q L G E M S L E E H S 120hVB3F-B 167 P T G Q H Q V R M Q I L M i|aR Y P S S Q L G E M S L E E H S 120

mVBF-B 167 Q C E C R P K K K E S A V K P D S P R I L C P P C T QRRQ 150hVBF-B 167 Q C E C R P K K KD KSDAV K P D S P R 150

MVEF-B 167 R P D P R T C R C R C R R R R F L H C Q G R G L E L N P D T 180hVBXF-B 167 R P D P R T C R C R C R R RS FLRcQGRGLELNPDT 180

mVB3F-B 167 C R C R K P R KhVB3F-B 167 C R C R R

188188

FIG. 1. Deduced amino acid sequences of mouse and human VEGF-B. Residues identical to mouse VEGF-B are boxed. The arrow indicatesthe putative cleavage site of the signal peptidase.

Cell Biology: Olofsson et al.

2578 Cell Biology: Olofsson et al.

identical to mouse VEGF164 (21), '30% identical to humanPIGF (23), and -20% identical to mouse PDGF-A and -B (31,32) (Fig. 2A). The amino acid sequence motif PXCVXXX-RCXGCC, a hallmark of this family of growth factors, was alsopresent in VEGF-B. In addition, the eight cysteine residues,involved in intra- and intermolecular disulfide bonding, areinvariant among these growth factors. The C-terminal domains ofmouse VEGF-B and VEGF64 displayed a significant similaritywith an additional eight conserved cysteine residues and stretchesof basic amino acids. Phylogenetic analysis verified that VEGF-Bwas most related to VEGF, while PIGF and especially the twoPDGF polypeptides are more distantly related (Fig. 2B).

Tissue Expression of VEGF-B. The expression of VEGF-Btranscripts was examined in mouse and human tissue byNorthern blot analysis and compared with the expression ofVEGF transcripts (Fig. 3A). In mouse tissues, the most abun-dant expression of the 1.4-kb VEGF-B transcript was detectedin heart, brain, skeletal muscle, and kidney. The major 3.7-kbVEGF transcript was expressed in heart, brain, lung, skeletalmuscle, and kidney. In human tissues, the most abundant

AnV EG-B 167 - - - - - - - - - - - - - - - - - - - - - -

nVrJF 164 - - - - - - - - - - - - - - - - - - - -

hP1-GF A - M R T W A C L L L L G C G Y L A H A L A E E A E I P R E L

mDGF B M N R C W A LF L P L C C Y L R L V S A E G D PI P E E L Y

nFf1E-B 167 - - - - - - - - - - - - - - - - - - - - - M S PL L R R L LrrMB 164 - - - - - - - - - - - - - - - - M N F L L S W V H W T L AILhP1GF - - - - - - - - - - - - - - - - - M P V M R L FP C F L Q|LnuA I E R L A R SQ I H S I R D L Q R L L E I DMV G A E D A|LmnDF B E M L S D H SI R S F D D L Q R L L H R D S V D E D G A EL

nTVBF-B 167nfBG 164hPl1Fn Anu,F B

nVB1F-B 167uP3 164hPlcPnU AnE B

nBF-B 167ntJP 164hP1§nm AniPD B

nfJP-B 167nW" 164hP1WnX AiUr B

nNdW-B 167nVl 164hP1WnXGF Airn B

n1BF-B 167n 164

hP1§nG Aun B

nQ"-B 167nUVBK 164hPl1GnPGF AnPIF B

B

LVALL QLARTQAPVS QFDGPSH QKKVVPWIL L Y LH H A K. W SLQA -P - T T EIG E Q K S Hf -E- VFI K F ML AG LA L PMV P PQ Q WA L S - AG HG S S EVE V V PE T S L R A H G S H A I N H V P E K R[B V P I R R K R S I ED L N M T R A H S G V E L ES S S R[l R R S L G S L A A A E

IDVARA Tl-- C P RE V VVPLSMEL M G N V VKQD VETC P E T VVVIF LQ GENY VP D E I E Y I[FlYEVWGRSYC-RALETLUVDVVSEYPSEIEHMF Q E V W G R S Y C R A L E R L V D V V S E Y P S EEl E H ME A I P A V - - -- C K T R I Y E I P R S Q V D P T S A NPAVIAE- - -CKTRTEE FQIrSlRNNIDRTNAN

L- -VPSCVTV RCGGCCPDDGLECVPTG HF - -K P S C V PLRCAGCCDE AL E C VPTSE SF--SPSCVSLLR CTGCCGDENLHCVPV ETAF L I W P P C V E V K R C T G C C N T S S V K C Q S R VHFLVWPEPLC VE RCSGCCNNRNVQ CRASQVQ

IQV§M QE MI QY P S S Q----G E M S L E E H S QN I T|NQ I S P # - - - H L Q HHN!TXQ K I R S G DR P - - - S Y F S Q H VR

H RS VKXV AK VE3V R KK PK LK E VQ VR LEEHL ENMRP VE9 VR K EI VR KK PI FK K AT VT L L A

SC E C R P K K K E S A V K P D S P R I L C P P C T QR R Q RSC E C R PKKDR P|E N H - - IlC R RK HC E C R Pl L R E K M _|K Pl E - - - - RLG G DAVPRRCACATS N L N P D H R E E E T D V R¢KCETI V T P R PrVlT R S P G T S R E Q R A K T P Q AMFR

- - - IPD P R T C R C R C R R R R F L H C QGRG L E L N PL F V Q

V T I R T V[E I R[R P PK G KH[E]KF KH T HD K A ArL K E

LD T C R C R K P R Kl

T L G A

mPa A

-mn~~-B167167F164hPIPGF

I I I

70 60 50 4) 30 2) 10 0

Number of substitution events

FIG. 2. (A) Multiple amino acid sequence alignment of mouse (m)VEGF-B, mouse VEGF164 (21), human (h) PIGF (23), and mousePDGF-A and -B (31, 32). Amino acid residues identical to mouse

VEGF-B are boxed. The invariant cysteine residues in the N-terminaldomains of the five growth factors are on a shaded background. (B)Dendrogram showing the phylogenetic relationship between the fivegrowth factors.

expression of the 1.4-kb VEGF-B transcript and the major3.7-kb and 4.5-kb VEGF transcripts were detected in heart,skeletal muscle, pancreas, and prostate. Although quantitativedifferences exist, VEGF-B and VEGF were coexpressed inmany tissues.The distribution of VEGF-B transcripts was further exam-

ined by in situ hybridization in sections from adult mouse heartand skeletal muscle and from the early (E10) mouse embryo.In the adult heart, VEGF-B transcripts were prominentlyexpressed in the myocardium, while no specific signal wasdetected in arterial smooth muscle (Fig. 3 B and C). In adultstriated muscle, VEGF-B transcripts were expressed by someof the myofibers whereas others seemed to lack the transcript(Fig. 3 D and E). In the E10 mouse embryo, VEGF-Btranscripts were detected mainly in the developing heart (Fig.3 F and G). Thus, we conclude that VEGF-B is predominantlyexpressed in muscular tissues and that expression can bedetected at an early stage during embryonic development.

Expression of VEGF-B in Transfected Cells. The biochem-ical properties of human VEGF-B were examined in trans-fected human embryonic 293EBNA cells. In reducing SDS/PAGE, human VEGF-B migrated at 21 kDa (Fig. 4A).VEGF-B remained cell-associated and was not released intothe culture medium unless the cells were treated with heparin(1-100 jig/ml) or 1.2 M NaCl (data not shown). Undernonreducing conditions, VEGF-B migrated as a dimer of 42kDa in SDS/PAGE analysis. These results suggest thatVEGF-B formed disulfide-linked dimers associated with thecell, probably through ionic interactions with extracellular orcell-surface-bound heparan sulfate proteoglycans.The coexpression of VEGF-B and VEGF in many tissues

and the ability of VEGF to form heterodimers with PIGF (33)suggested to us that VEGF-B could also form heterodimerswith VEGF. To test this possibility, 293EBNA cells werecotransfected with expression vectors encoding humanVEGF165 and human VEGF-B. Metabolically labeled proteinswere chased in the presence of heparin and immunoprecipi-tations were carried out with antisera to either VEGF-B orVEGF (Fig. 4B). The results show that VEGF-B can formdisulfide-linked heterodimers with VEGF, which, in the ab-sence of heparin, remain cell-associated (Fig. 5C). Interest-ingly, since homodimers of VEGF165 are efficiently secretedinto the medium (4), VEGF-B appeared to determine therelease of the heterodimer.VEGF-B Is an Endothelial Cell Mitogen. The ability of

VEGF-B to stimulate endothelial cell proliferation was estab-lished through analysis of [3H]thymidine incorporation inHUVECs and in BCE cells. The results show that conditionedmedium from transfected 293EBNA cells expressing VEGF-Bincreased thymidine incorporation into DNA of bothHUVECs and BCE cells (Fig. 5). As positive controls we usedconditioned medium from transfected 293EBNA cells express-ing VEGF and recombinant bFGF. These results demonstratethat VEGF-B is an endothelial cell growth factor.

DISCUSSIONAngiogenesis is a process involving several endothelial RTKsand their ligands including VEGF and P1GF (34). In this workwe describe a novel growth factor for endothelial cells,VEGF-B, a nonglycosylated highly basic heparin-bindinggrowth factor with close structural similarities to VEGF andP1GF. VEGF-B has a wide tissue distribution but is mostabundant in heart, skeletal muscle, and pancreas. The expres-sion of VEGF-B is different from that of VEGF althoughcoexpression of VEGF-B and VEGF can be seen in manytissues. In situ hybridization analysis of heart and skeletalmuscle identified myocytes as the principle cells expressingVEGF-B transcripts. Thus, VEGF-B, like several other growth

Proc. Natl. Acad. Sci. USA 93 (1996)

Cell Biology: Olofsson et al.

A Mouse

Proc. Natl. Acad. Sci. USA 93 (1996) 2579

Human

*gp *flh*~* Om4bei'sbcMS00 _w0___a

1..

- VEGF-B

- VEGF

FIc;. 3. Exprcssion of VEGF-B iII mouse

and human tissues and a comparison withVEGF expression. (A) Northcrn blot analysisof expression of VEGF-B and VEGF inmouse and l human tissues. (B-G) lit situf hv-bridization analysis of VEGF-B expression in

sections fronm adult mouse hicart (B and C)it s- ;---G iand striated musclC (D and E). and in a

i midsacittal scction from an EIO mouse enm-

bryo (F and G). Dark-field (B, D), and F) andbrigJht-field (C E. and G) microphotogyraphsof the in sitii autoradiograms are shown. Theinsert in C shlows a scction from adult mousehcart hybridized with the VEGF-B scnseprobe. Thc myocardium of the adult mnouse

hcart has a prominient signyal. In striated mus-

cle. VEGF-B cxpression is seen in subpopu-lations of mvofibers (arrow in D). Strongsignals were also obtaincd in the developinghcart of the El) mnouse embryo (arrowheadsin F). Other embrvonic structurcs expressedIower or undctcctable levels of transcripts forVEGF-B.

factors, may act in a paracrine fashion to regulate endothelialcell function.VEGF-B forms disulfide-linked dimers that are secreted

but remain bound to cells or to the extracellular matrix andcan be released by heparin or high salt treatments. Thisassociation is likely to be mediated by the C-terminal basicdomain, as observed for the longer and highly basic splicevariants of VEGF (4, 35, 36). The association of VEGF-B tocells or to the extracellular matrix may have several impor-

tant implications for regulation of its bioavailability andaction in regulation of endothelial cell growth during em-

bryonic development and in maintenance of the vasculaturein adult tissues. Cell-associated VEGF-B may act as a localtrophic factor and as a growth stimulus for endothelial cellsby direct cell-cell interactions. This can be of particularsignificance in developing embryos and in contractile tissues,such as muscle, by providing spatial cues to outgrowingendothelial cells during establishment and maintenance of

VEGF-B- *

.A 0, . , .rv_

X / 6

/ , / -

{, rf8 w* W s@ ,'; e ; *

* s * @

@ #

¼ A

,.'- 4. ,.A.

2580 Cell Biology: Olofsson et al.

A C

4 4

t\

Ay Aw.wt~~~~AX-42 kDaI I I-I - - I

_r~~~_ _~~~~~~~

Ai _"44_

:. - 42 kDa

Heparin 100 Ig/ml

21 kDa -

21 kDa-

B

'4'4,1 '4

'4

- 23 kDa

:I.

I'.f

Antibody toVEGFVB l l + + -| +| -| +| l +

VEGF + - + I- 1+ I 1+ I + I- T+ -

46 kDa42 kDa

23 kDa -21 kDa -

--._. WW_ _ W

FIG. 4. Biochemical properties of VEGF-B and heterodimerization with VEGF. Human VEGF-B and human VEGF165 were expressed, eitherseparately or in combination, in human embryonic kidney 293EBNA cells by transient transfection and the cells were metabolically labeled. Culturesupernatants and detergent-solubilized cell lysates were subjected to immunoprecipitation and SDS/PAGE analysis under reducing conditionsunless otherwise indicated. (A) VEGF-B was expressed as 21-kDa protein in the transfected cells. As a control, cells were transfected with anexpression vector containing the VEGF-B cDNA in reverse orientation (Rev.). By heparin treatment, VEGF-B was released from cells and foundin the supernatant. Analysis of culture supernatants under nonreducing conditions (NR) showed that VEGF-B migrated as a 42-kDa species,indicating a dimeric structure. (B) VEGF-B and VEGF165 were expressed in transfected 293EBNA cells, either separately or in combination.Anti-peptide antisera to VEGF-B or VEGF were used in immunoprecipitation analysis of culture supernatant from heparin-treated cells expressingVEGF-B, from cells coexpressing VEGF-B and VEGF165, from cells expressing VEGF165 alone, and from control cells (Rev.). Under nonreducingconditions (NR), the VEGF-B-VEGF heterodimers migrated as species of 42-46 kDa. (C) VEGF-B was expressed alone or in combination withVEGF. Culture supernatants from cells treated or untreated with heparin were subjected to immunoprecipitation with an anti-peptide antiserumto VEGF-B and analyzed by SDS/PAGE. The data show that VEGF-B homodimers and VEGF-B-VEGF heterodimers are released from cellsby heparin.

the vascular tree. Alternatively, cell association may func-tionally inactivate VEGF-B by making it unaccessible forendothelial cells. Upon trauma or other injuries, it could berapidly released and thus functionally activated. This latterpossibility is attractive in light of the general slow turnoverof endothelial cells in most tissues.The ability of VEGF-B to heterodimerize with VEGF is

consistent with the conservation of the eight cysteine residuesinvolved in inter- and intramolecular disulfide bonding. Fur-thermore, the coexpression of VEGF-B and VEGF in manytissues suggests that VEGF-B-VEGF heterodimers occurnaturally. The formation and cell association of such het-erodimers may affect the formation ofVEGF homodimers and

thus indirectly control release and bioavailability of VEGF.The formation of VEGF-B-VEGF heterodimers imply thatcellular signals via these two growth factors are at least partlyoverlapping. VEGF was recently shown also to form het-erodimers with PIGF (33). The formation of different het-erodimeric complexes of these growth factors, their differen-tial expression patterns during embryonic development and inadults, and different biochemical properties could provide a basisfor a diverse array of regulatory signals for endothelial cells.At least two RTKs, Flk-1/KDR and Flt-1, are involved in

signaling mediated via VEGF and P1GF. Whether VEGF-Bbinds to the same RTKs or whether a novel and yet uniden-tified RTK is the receptor for VEGF-B remains to be eluci-

I I I

Proc. Natl. Acad. Sci. USA 93 (1996)

I I

. +[--I+

Proc. Natl. Acad. Sci. USA 93 (1996) 2581

r.0

10la

L-4

2.0

1.5

1.0

fl nock* VEGF-B 167E VEGF 165O] bFGF

HUVEC BCE

FIG. 5. VEGF-B induces [3H]thymidine incorporation into endo-thelial cells. Conditioned medium from 293EBNA cells transfectedwith expression vectors for VEGF-B, VEGF165, or empty vector(mock) were diluted in respective media, applied to HUVECs and toBCE cells, and incorporation of [3H]thymidine was measured. As a

positive control recombinant bFGF was added to BCE cells. Thecolumns show fold induction of [3H]thymidine incorporation com-pared to basal activity induced by conditioned medium from mock-transfected cells. The bars show the mean ± SD of parallel samples,and similar results were obtained in several independent experiments.

dated. However, with the molecular tools now available, adetailed analysis of the role of VEGF-B in establishing andmaintaining normal and pathological vascularization and en-dothelial cell growth of tissues can be undertaken.

We thank Per 0. Ljungdahl for introducing us to the yeast two-hybrid system and Barbara Akerblom, Tapio Tainola, and MariHelantera for expert technical assistance. This study was partlysupported by grants to K.A. (The University of Helsinki, The FinnishCancer Organizations, The Academy of Finland, and The SigridJuselius Foundation).

1. Poole, T. & Coffin, J. (1989) J. Exp. Zool. 251, 224-231.2. Folkman, J. (1995) Nat. Med. 1, 27-31.3. Klagsburn, M. & D'Amore, P. (1991) Annu. Res. Physiol. 53,

217-239.4. Ferrara, N., Houck, K., Jakeman, L. & Leung, D. (1992) Endocr.

Rev. 13, 18-32.5. Matthews, W., Jordan, G., Gavin, M., Jenkins, N., Copeland, N.

& Lemischka, I. (1991) Proc. Natl. Acad. Sci. USA 88,9026-9030.6. Terman, B., Dougher-Vermazen, M., Carrison, M., Dimitrov, D.,

Armellino, D., Gospodarowicz, D. & Bolhen, P. (1992) Biochem.Biophys. Res. Commun. 187, 1579-1586.

7. Millauer, B., Wizigmann-Voos, S., Schnurch, H., Martinez, R.,Moller, N., Risau, W. & Ullrich, A. (1993) Cell 72, 835-846.

8. Shibuya, M., Yamaguchi, S., Yamane, A., Ikeda, T., Tojo, A.,Hitoshi, M. & Sato, M. (1990) Oncogene 5, 519-524.

9. de Vries, C., Escoedo, J., Ueno, H., Houck, K., Ferrara, N. &Williams, L. (1992) Science 255, 989-991.

10. Shalaby, F., Rossant, J., Yamaguchi, T., Gertenstein, M., Wu,X.-F., Breitman, M. & Schuh, A. (1995) Nature (London) 376,62-66.

11. Fong, G.-H., Rossant, J., Gertsenstein, M. & Breitman, M. (1995)Nature (London) 376, 66-70.

12. Galland, F., Karamysheva, A., Pebusque, M.-J., Borg, M.-P.,Rottapel, R., Dubreuil, P., Rosnet, 0. & Birnbaum, D. (1992)Oncogene 8, 1233-1240.

13. Pajusola, K., Aprelikova, O., Korhonen, J., Kaipainen, A., Per-tovaara, L., Alitalo, R. & Alitalo, K. (1992) Cancer Res. 52,5738-5743.

14. Kaipainen, A., Korhonen, J., Mustinen, T., van Hinsberg, V.,Fang, G.-H., Dumont, D., Breitman, M. & Alitalo, K. (1995)Proc. Natl. Acad. Sci. USA 92, 3566-3570.

15. Partanen, J., Armstrong, E., Makala, T., Korhonen, J., Sandberg,M., Renkonen, R., Knuutila, S., Huebner, K. & Alitalo, K. (1992)Mol. Cell. Biol. 12, 1698-1707.

16. Sato, T., Qin, Y., Kozak, C. & Audus, K. (1993) Proc. Natl. Acad.Sci. USA 90, 9355-9358.

17. Dumont, D., Yamaguchi, T., Conlon, R. & Rossant, J. (1992)Oncogene 7, 1471-1480.

18. Sato, T., Tozawa, Y., Deutsch, U., Wolburg-Buchholz, K., Fuji-wara, Y., Gendron-Maguire, M., Gridley, T., Wolburg, H., Risau,W. & Qin, Y. (1995) Nature (London) 376, 70-74.

19. Dumont, D. J., Gradwohl, G., Fong, G.-H., Puri, M. C., Gert-senstein, M., Auerbach, A. & Breitman, M. L. (1994) Genes Dev.8, 1897-1909.

20. Berse, B., Brown, L., Van der Water, L., Dvorak, H. & Senger,D. (1992) Mol. Biol. Cell 3, 211-220.

21. Breier, G., Albrecht, U., Sterrer, S. & Risau, W. (1992) Devel-opment (Cambridge, U.K) 114, 521-532.

22. Shweiki, D., Itin, A., Soffer, D. & Keshet, E. (1992) Nature(London) 359, 843-845.

23. Maglione, D., Guerriero, V., Vigleitto, G., Delli-Bovi, P. &Persico, M. (1991) Proc. Natl. Acad. Sci. USA 88, 9267-9271.

24. Park, J., Chen, H., Winer, J., Houck, K. & Ferrara, N. (1994) J.Biol. Chem. 269, 25646-25654.

25. Heldin, C.-H. & Westermark, B. (1990) Cell Regul. 1, 555-566.26. Chevray, P. & Nathans, D. (1992) Proc. Natl. Acad. Sci. USA 89,

5789-5793.27. Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J.,

Smith, J. & Struhl, K. (1992) Current Protocols in MolecularBiology (Wiley, New York).

28. Hein, J. (1990) Methods Enzymol. 183, 626-645.29. Keck, P., Hauser, S., Krivi, G., Sanzo, K., Warren, T., Feder, J.

& Connolly, D. (1989) Science 246, 1309-1312.30. Korhonen, J., Partanen, J., Armstrong, E., Vaattokari, A., Ele-

nius, K., Jalkanen, M. & Alitalo, K. (1992) Blood 80, 2548-2555.31. Bonthron, D., Sultan, P. & Collins, T. (1991) Genomics 10,

287-292.32. Mercola, M., Wang, C., Kelly, J., Brownlee, C., Jackson-Grusby,

L., Stiles, C. & Bowen-Pope, D. (1990) Dev. Biol. 138, 114-122.33. DiSalvo, J., Bayne, M., Conn, G., Kwok, P., Trivedi, P., Soder-

man, D. D., Palisi, T., Sullivan, K. & Thomas, K. (1995) J. Biol.Chem. 270, 7717-7723.

34. Mustonen, T. & Alitalo, K. (1995) J. Cell Biol. 129, 895-898.35. Houck, K., Ferrara, N., Winer, J., Cachianes, G., Li, B. & Leung,

D. (1991) Mol. Endocrinol. 91, 1806-1814.36. Tischer, E., Mitshell, R., Hartman, T., Silva, M., Gospodarowicz,

D., Fiddes, J. & Abraham, J. (1991) J. Biol. Chem. 266, 11947-11954.

Cell Biology: Olofsson et al.