©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of a Naturally Occurring Vascular Endothelial Growth Factor Placenta Growth Factor Heterodimer (*)

(Received for publication, September 12, 1994; and in revised form, January 20, 1995)

Jerry DiSalvo Marvin L. Bayne Greg Conn (§) Perry W. Kwok Prashant G. Trivedi Denis D. Soderman Thomas M. Palisi Kathleen A. Sullivan Kenneth A. Thomas (¶)

From the Department of Biochemistry, Merck Research Laboratories, Rahway, New Jersey 07065

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Vascular endothelial growth factor (VEGF) is a potent and selective mitogen for endothelial cells that is angiogenic in vivo and induced by hypoxia. A homologous protein, placenta growth factor (PlGF), is also reported to be mitogenic for endothelial cells in culture. The rat GS-9L glioma cell line produces not only VEGF homodimers but also PlGF homodimers and a novel heterodimer composed of VEGF and PlGF subunits. All three dimeric forms were purified to apparent homogeneity, and their structures and mitogenic activities were compared. VEGFbulletPlGF heterodimers are vascular endothelial cell mitogens nearly as potent as VEGF homodimers. Therefore, some of the biological activities attributed to VEGF homodimers might be mediated by VEGFbulletPlGF heterodimers. In contrast, pure PlGF homodimers are mitogenic for endothelial cells only at high, possibly non-physiologic concentrations; thus the biological relevance of their mitogenic activity for these cells is not obvious. However, the existence of not only homodimers but also heterodimers clearly extends the similarity between the VEGF/PlGF and the homologous platelet-derived growth factor systems.


INTRODUCTION

Vascular endothelial cells form a luminal non-thrombogenic monolayer throughout the vascular system. Mitogenesis of these cells is required for embryonic vascular development, growth, and repair. Although multiple growth factors have been reported to be mitogenic for vascular endothelial cells(1) , only vascular endothelial growth factor (VEGF) (^1)has been established to be highly selective for these cells(2, 3, 4, 5, 6, 7, 8) . Furthermore, in contrast to the broad spectrum acidic and basic fibroblast growth factors, which do not contain classical secretory leader sequences(9, 10) , the full-length translation products of VEGF and the homologous protein, placenta growth factor (PlGF) include amino-terminal signal sequences consistent with their active secretion.

VEGF (11, 12, 13, 14) and PlGF (15) are glycosylated homodimers that share 46% amino acid sequence identity with each other (16) and are 18-20% identical to the amino acid sequences of platelet-derived growth factor (PDGF) A and B subunits. As in the PDGF system, alternative splicing generates multiple forms of VEGF (17) and PlGF(18) . Human VEGF exists in four mature processed forms containing 206, 189, 165, and 121 amino acids, the most prevalent being the 165-amino acid form(17) . The 206- and 189-amino acid forms each contain a highly basic 24-amino acid insert that promotes tight binding to heparin and, presumably, heparan proteoglycans on cellular surfaces and within extracellular matrices (19) . Differential splicing of human PlGF mRNA leads to either 132- or 153-amino acid residue mature proteins(18) . The 21-amino acid basic insert near the COOH terminus of the longer form also confers heparin binding(16) . Analogous to VEGF, this basic insert could partition the protein onto cell surface and extracellular matrix heparan proteoglycans and, perhaps, might facilitate nuclear binding. It is presently not clear, however, if any biologically functional differences exist among the alternatively spliced forms of VEGF and PlGF.

VEGF appears to be relevant for vascular growth and function. VEGF mRNA expression has been shown to be related temporally and spatially to the development of the embryonic vascular system (20, 21) and to physiological angiogenesis in the female reproductive system(22) . Hypoxia has been shown to increase VEGF mRNA levels in cultured cells (23, 24, 25) , and ischemic cardiac myocytes express elevated VEGF levels both in vitro and in vivo(26) . In addition to its angiogenic properties, VEGF can under certain circumstances induce vascular permeability; hence it is also known as vascular permeability factor(2) .

During purification of VEGF from rat GS-9L glioma cell-conditioned media, we observed that two chromatographic peaks of vascular endothelial cell mitogenic activity eluted from a cation exchange HPLC column(6) . The latter purer peak was the focus of the original purification effort that yielded homodimeric VEGF(14) . We now report the purification to apparent homogeneity of another vascular endothelial cell mitogen from the earlier eluting peak of mitogenic activity. This newly identified protein is a heterodimer composed of both VEGF and PlGF subunits. In addition, we have purified a PlGF homodimer from these same conditioned media. We compare the biological and physical characteristics of this pure heterodimer with VEGF and PlGF homodimers purified from the same source.


EXPERIMENTAL PROCEDURES

Protein Purification

Serum-free conditioned media from rat GS-9L glioma cells were generated, filtered, and sequentially chromatographed on CM-Sephadex C-50 (Pharmacia Biotech Inc.) and concanavalin A-Sepharose lectin affinity (Pharmacia) columns at 4 °C as described for the purification of VEGF homodimers (6) with the exception that media were conditioned by cells maintained in 3% oxygen rather than ambient 20% oxygen. All subsequent chromatographic steps were performed at 20-22 °C. Protein specifically eluted from the lectin affinity column was loaded onto a 25 times 0.46-cm poly(aspartic acid) WCX HPLC column (Nest Group) pre-equilibrated in 50 mM sodium phosphate, pH 6.0, and eluted with a 45-min 0-1 M NaCl linear gradient at a flow rate of 0.75 ml/min monitoring absorbance at 280 nm. Homodimeric VEGF was purified from the second peak of mitogenic activity as described(6) . The first peak of mitogenic activity was pooled, loaded onto a 5 times 0.46-cm Vydac C(4) reversed phase HPLC column (Separations Group) equilibrated in 10 mM trifluoroacetic acid, and eluted with a 60-min linear gradient of 0-67% acetonitrile at a flow rate of 0.5 ml/min monitoring absorbance at 210 nm. Mitogenically active fractions were pooled and rechromatographed (Smart System, LKB) using a 5 times 0.1-cm C(4) microbore column with a linear 60-min gradient from 0 to 50% acetonitrile at a flow rate of 35 µl/min to yield pure VEGFbulletPlGF heterodimers.

Homodimeric PlGF was initially fractionated by sequential chromatography on CM-Sephadex C-50, concanavalin A-Sepharose lectin affinity, and poly(aspartic acid) WCX HPLC columns as described for the VEGF homodimer and VEGFbulletPlGF heterodimer purifications. Further fractionation of homodimeric PlGF relied on the identification of immunocross-reactive protein by Western analysis using antisera made to a synthetic polypeptide corresponding to amino acid residues 31-50 of the longer mature form of rat PlGF conjugated to a tuberculin-purified protein derivative as described(27) . A second antiserum was made to a synthetic polypeptide identical in sequence to residues 1-24 of mature rat VEGF. These two antisera were used to identify chromatographic fractions containing either one or both of the PlGF and VEGF subunits. Fractions from the poly(aspartic acid) column containing PlGF but not VEGF immunocross-reactive bands were pooled and chromatographed by sequential fractionation on a 5 times 0.46-cm Vydac C(4) reversed phase HPLC column followed by a 5 times 0.1-cm C(4) microbore column as described for the heterodimer purification.

Mitogenic Assays

The well surfaces of tissue culture dishes (96-well, Costar) were coated with 100 µl of 10 mg/ml porcine gelatin (Sigma) in phosphate-buffered saline (Life Technologies, Inc.) for 2 h at 25 °C. Human umbilical vein endothelial (HUVE, Clonetics) cells were plated at 4000 cells/well in 100 µl of Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.) and antibiotics (100 units/ml penicillin G, 100 µg/ml streptomycin sulfate; Life Technologies, Inc.) and incubated overnight at 37 °C under 7.5% CO(2). Media were then replaced with fresh supplemented media, and samples to be assayed were added. After 24 h, 0.8 µCi of [methyl-^3H]thymidine (20 Ci/mmol; DuPont NEN; 1 Ci = 37 GBq) was added per well, and the dishes were incubated for an additional 72 h. The cells were then washed with Hanks' balanced salt solution (Life Technologies, Inc.) buffered to pH 7.5 with 25 mM Hepes and supplemented with 0.1% bovine serum albumin. The cells were lysed, and DNA was solubilized by 0.2 M Na(2)CO(3), 0.1 M NaOH, and [^3H]thymidine incorporation was determined by scintillation counting.

Polyacrylamide Gel Electrophoresis and Western Blot Analyses

The homogeneity and apparent mass of the purified proteins and their subunits were determined by SDS-14% polyacrylamide gel electrophoresis (PAGE) (28) with and without prior reduction by dithiothreitol and subsequently visualized by high sensitivity silver staining(29) . The presence of VEGF and PlGF in the chromatographic fractions was determined by Western blot analysis using the antisera described in the preceding purification section. Reduced samples were analyzed as described(27) . Since the peptide-based antisera did not efficiently bind to the nonreduced proteins, samples fractionated on nonreducing SDS-PAGE gels were subsequently reduced by incubation for 15 min in transfer buffer containing 5 mM dithiothreitol and transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore Corp.) in dithiothreitol-containing buffer. After the transfer, membranes were placed in fresh transfer buffer containing 40 mM dithiothreitol and heated for 30 s in a microwave oven at full power. The Western protocol was then completed as described (27) , and the bands were visualized using a PhosphorImager (Molecular Dynamics) after either 1 h or overnight exposure.

Subunit and Polypeptide Amino Acid Composition and Sequencing

Amino acid compositions of pure VEGF and PlGF hetero- and homodimers were determined as described(6) . Pure VEGFbulletPlGF heterodimer was reduced and carboxymethylated with iodo[2-^14C]acetic acid (17.9 mCi/mmol, Amersham Corp.) as described(30) , and the subunits were purified on a 5 times 0.1-cm Vydac C(4) reversed phase microbore column equilibrated in 10 mM trifluoroacetic acid and eluted with a linear 60-min gradient from 0 to 50% acetonitrile at a flow rate of 35 µl/min. Amino acid compositions of the purified subunits were determined. Samples of each pure subunit were applied to Polybrene-coated glass fiber filters, and their amino-terminal sequences were determined by Edman degradation in an Applied Biosystems 470A gas phase microsequencer equipped with an Applied Biosystems 120A phenylthiohydantoin analyzer. Samples (650 ng) of each subunit were also dried by vacuum centrifugation and digested with 50 ng of Lys-C endoprotease (Boehringer Mannheim) in 100 µl of 25 mM Tris, pH 8.5, 0.1% EDTA for 8 h at 37 °C. The digestion products were loaded on a 25 times 0.46-cm Vydac C(18) column equilibrated with 0.1% trifluoroacetic acid and eluted with a 2-h 0-67% linear gradient of acetonitrile at a flow rate of 0.75 ml/min. Amino acid sequences of purified peptides were determined as described for the undigested subunits.

PlGF Cloning

PlGF cDNA was generated by reverse transcriptase from poly(A) RNA isolated from GS-9L cells. Degenerate PCR sense and antisense primers, based on amino acid sequences determined from peptides purified from Lys-C digests of the mature protein, were used to PCR-amplify a region of the cDNA. The 3` and 5` ends of PlGF subunit cDNA were each amplified by the rapid amplification of cDNA ends protocol(31) . Purified PCR products were digested with restriction endonuclease SalI, ligated into pGEM3Zf(+), and used to transform Escherichia coli XL-1 blue. A full-length coding sequence clone was identified in a GS-9L cDNA library by hybridization to a 531-base pair PCR product spanning nucleotides 109-640 of the full-length PCR product. Phage DNA from positive clones was digested with SpeI and ligated into XbaI-digested pGEM3Zf(+). Both plasmid DNA strands were sequenced by the dideoxy chain termination method(32) .


RESULTS

Purification of VEGF Subunit-containing Heterodimer

Conditioned media of rat GS-9L glioma cells were fractionated on CM-Sephadex C-50 and concanavalin A-Sepharose as described for the original purification of homodimeric VEGF(6) . Protein specifically eluted from the lectin column was resolved into two peaks of vascular endothelial cell mitogenic activity, the second of which (Fig. 1A, pool 2) was further fractionated to yield pure homodimeric VEGF. The protein mitogen in the first of the two activity peaks (Fig. 1A, pool 1) has now also been purified to homogeneity by C(4) reversed phase HPLC (Fig. 1, B and C) yielding 150-300 ng of pure protein (denoted VEGFbulletPlGF; see below) per liter of conditioned medium.


Figure 1: Purification of the VEGFbulletPlGF. Serum-free conditioned media were fractionated by CM-Sephadex C-50 and concanavalin A-Sepharose as described(6) . A, active fractions were pooled, loaded onto a poly(aspartic acid) WCX HPLC column in 50 mM sodium phosphate, pH 6.0, and eluted at 0.75 ml/min with a linear gradient of 0-1 M NaCl. Two peaks of HUVE cell mitogenic activity were observed, and each was pooled (horizontalbars). Material in pool 2 was purified to yield homodimeric VEGF(6) . B, fractions from the first active peak were combined (pool 1), loaded onto a C(4) reversed phase HPLC column equilibrated in 10 mM trifluoroacetic acid, and eluted with a linear gradient of 0-67% acetonitrile. C, mitogenically active fractions were again pooled and rechromatographed using a microbore C(4) column equilibrated in 10 mM trifluoroacetic acid and eluted with a linear gradient of 0-50% acetonitrile.



Compared with homodimeric VEGF that has an apparent nonreduced mass of 43.5 kDa, the newly purified nonreduced VEGFbulletPlGF mitogen migrates as major 49.5-kDa and trace 40-kDa bands (Fig. 2A, lanes1 and 2). On reducing SDS-PAGE, pure VEGF homodimer migrates at its subunit mass of 27 kDa, whereas this novel mitogen separates into two principal bands of 27 and 31 kDa (Fig. 2B, lanes1 and 2). Immunocross-reactive VEGF was demonstrated to exist in both nonreduced proteins (Fig. 3A, lanes1 and 2) and to correspond to the reduced 27-kDa but not the 31-kDa band (Fig. 3B, lanes1 and 2). Barely detectable lower mass immunocross-reactive VEGF bands present in the nonreduced (Fig. 3A, lane1, 29 kDa) and reduced (Fig. 3B, lane1, 21 kDa) VEGF lanes are presumable trace degradation products not detectable on the corresponding silver-stained gels (Fig. 2A, lane1; Fig. 2B, lane1). Traces of incompletely reduced immunocross-reactive dimers are also occasionally detected on long exposures (e.g. 50-kDa bands in Fig. 3B, lanes1 and 5). The masses of the dimers, calculated from the more accurate reduced subunit masses, are 54 kDa for the VEGF homodimer and 58 kDa for VEGFbulletPlGF heterodimer.


Figure 2: Purity of VEGF, VEGFbulletPlGF, and PlGF. Purities of VEGF, VEGFbulletPlGF, and PlGF (100 ng each) were determined by electrophoresis through SDS-14% polyacrylamide gels under nonreducing (A) and reducing (B) conditions followed by silver staining. Left side, molecular mass standards; lane 1, VEGF; lane 2, VEGFbulletPlGF; lane 3, PlGF.




Figure 3: Western analysis of VEGF, VEGFbulletPlGF, and PlGF. Pure VEGF, VEGFbulletPlGF, and PlGF (100 ng each) were analyzed using subunit-specific antisera after electrophoresis through SDS-14% polyacrylamide gels under nonreducing (A) and reducing (B) conditions, followed by electrophoretic transfer to Immobilon-P polyvinylidene difluoride membranes. Pure VEGF (lane 1), VEGFbulletPlGF (lane 2), and PlGF (lane 3) were probed with a 1:1000 dilution of VEGF-specific antiserum. Additionally, VEGF (lane 4), VEGFbulletPlGF (lane 5), and PlGF (lane 6) were probed with a 1:1000 dilution of PlGF-specific antiserum. Bound antibody was incubated with I-protein A followed by overnight exposure and visualization using a PhosphorImager.



Despite unequal silver staining intensity (Fig. 2B, lane2), these two reduced and carboxymethylated polypeptides derived from the heterodimeric protein are chromatographically resolved as peaks (Fig. 4, peaks 1 and 2) of virtually identical absorbance (area ratio of 1.00:1.03) at 210 nm eluted from a reversed phase C(4) HPLC column. The second of the two eluted protein peaks is confirmed by NH(2)-terminal (APTTEGEQKAHEVV) and polypeptide sequencing to be the previously identified (14) 164-amino acid subunit present in VEGF homodimers (data not shown). However, the first peak, subsequently identified to be a rat homologue of PlGF, is distinct by both NH(2)-terminal amino acid sequencing (ALSAGNXSTEMEVVPFNEV and an equal amount of this sequence missing the first 3 residues) and sequence analysis of a family of polypeptides purified on a C(18) reversed phase HPLC column from a Lys-C digest. Therefore, this newly purified mitogen appears to be a heterodimer composed of VEGF and PlGF subunits. Amino acid analysis of each reduced and carboxymethylated subunit peak (Fig. 4) combined with their polypeptide masses calculated from the VEGF and subsequently determined PlGF amino acid sequences confirms a very similar molar ratio (1.00:1.05), demonstrating that the heterodimer is composed of one VEGF and one PlGF subunit.


Figure 4: Subunit composition of VEGFbulletPlGF heterodimer. Reduced and carboxymethylated heterodimer subunits were purified on a microbore C(4) reversed phase HPLC column equilibrated in 10 mM trifluoroacetic acid and eluted with a 0-50% linear gradient of acetonitrile. The third major peak, eluting at an effluent volume of 9.3 ml, was present in the reagent blank. Peak 1 was subsequently determined by amino-terminal and peptide sequencing to correspond to the PlGF subunit, and peak 2 was identified as the VEGF subunit.



PlGF cDNA Cloning and Sequencing

Amino acid sequences of purified polypeptides (corresponding to amino acids 33-38 and 58-63 of the mature protein) derived from a Lys-C digest of the 31-kDa PlGF subunit were used to design primers for reverse transcriptase PCR cDNA cloning. The 3` and 5` regions were subsequently amplified(31) , and the PCR-generated sequences were used to screen a rat GS-9L cell cDNA library. The PlGF cDNA was observed to hybridize specifically to a 1.7-kilobase mRNA from GS-9L cells by Northern blot analysis (not shown). The deduced complete 158-amino acid residue sequence from one of the hybridizing clones is shown in Fig. 5. The full-length translation product containing a typical 23- or 26-residue NH(2)-terminal secretory leader sequence corresponding to microheterogeneous mature forms of 135 and 132 residues. Both cleavage sites are compatible with consensus sequences for the proteolytic removal of the secretory leader(33) . Three putative N-glycosylation sites occur at Asn-6, Asn-7, and Asn-74. Both Asn-7 and Asn-74 are not directly observed in the expected microsequencing cycles, implying that they are glycosylated.


Figure 5: Amino acid sequence homology between rat and human PlGF. Complete amino acid sequences of rat and human PlGF were compared. Identical residues are enclosed in boxes. The locations of the mature amino termini of the polypeptides are identified by singlearrowheads, and asparagine residues in potential glycosylation sequences are denoted by asterisks. The location of the polybasic insert is designated by the two-headed arrow.



The fully processed rat subunit amino acid sequence is 60% identical (Fig. 5) to human PlGF (15) increasing to 66% within the putative receptor-binding core structure corresponding to the PDGF homologue v-sis. By both Southern blot analysis and determination of cDNA sequences of several species (^2)this second subunit is identified as the rat counterpart of PlGF. Therefore, we adopted the PlGF nomenclature for consistency despite the glioma cell source of the protein.

The presence of VEGF but not PlGF in the VEGF homodimer and both VEGF and PlGF bands in the VEGFbulletPlGF heterodimer were subsequently confirmed by Western blots of nonreducing (Fig. 3A, lanes1, 2, 4, and 5) and reducing (Fig. 3B, lanes1, 2, 4, and 5) gels using individual peptide-based antisera specific to VEGF and PlGF subunits. Immunostaining in the region of the trace 40-kDa band seen on silver-stained nonreduced SDS-PAGE (Fig. 2, lane2) is also detected on nonreduced Western gels (Fig. 3A, lane2). A corresponding minor band was not detected after reduction, so it might reflect a minor form generated by disulfide shuffling under nonreduced denaturing conditions that is more efficiently recognized by the anti-VEGF than the anti-PlGF antisera. Consistent with the amino acid sequence data, the slower and faster migrating major bands on the blot of the reducing gel of the heterodimer cross-react with the anti-PlGF and anti-VEGF antisera, respectively.

PlGF Homodimer Purification and Characterization

We used peptide-based antisera to search for homodimers of PlGF. The locations of VEGF and PlGF subunits on Western blots of elution fractions from the poly(aspartic acid) WCX cation exchange column confirmed that both VEGF and PlGF were present in the first peak of mitogenic activity, whereas VEGF but not PlGF was found in the second peak of activity. Using a shallower gradient on the cation exchange column, earlier eluting fractions were also identified that contained only PlGF immunocross-reactivity (Fig. 6A) but were not detectably mitogenic in the vascular endothelial cell assay. The fractions from the cation exchange column containing PlGF but not VEGF were pooled and chromatographed on a series of C reversed phase HPLC columns (Fig. 6, B-D), resulting in a single peak containing pure PlGF with a yield of 75-150 ng/liter of conditioned medium. The masses of the nonreduced and reduced PlGF homodimer are 52 kDa (Fig. 2A, lane3) and 31 kDa (Fig. 2B, lane3), respectively. The actual mass of the dimer, determined from the reduced subunit mass, is 62 kDa. As noted for the reduced heterodimer (Fig. 2B, lane2), despite equivalent protein loads the PlGF subunit silver stains noticeably less intensely than the VEGF subunit. These same bands cross-react with anti-PlGF but not anti-VEGF antisera on Western blots of nonreduced (Fig. 3A, lanes3 and 6) and reduced (Fig. 3B, lanes3 and 6) protein.


Figure 6: Purification of the PlGF homodimer. Initial purification of PlGF homodimer by sequential chromatography on CM-Sephadex C-50 and concanavalin A-Sepharose was performed as described for the VEGF homodimer and VEGFbulletPlGF heterodimer purification. A, fractions specifically eluted from the lectin affinity column were chromatographed on a poly(aspartic acid) WCX HPLC column in 50 mM sodium phosphate, pH 6.0, and eluted at 0.75 ml/min with a linear gradient of 0-1 M NaCl over 60 min. Fractions containing PlGF homodimer were identified by Western analysis probing with both VEGF and PlGF antisera (inset). Fractions from the poly(aspartic acid) column that contained PlGF but not VEGF immunocross-reactive bands were pooled (solidbar) and loaded onto a C(4) reversed phase HPLC column (B), and protein was eluted as described for heterodimer purification. Fractions containing PlGF immunocross-reactive bands (B, inset) were pooled and rechromatographed on the same column (C), and the major peak was chromatographed on a microbore C(4) column (D) as described under ``Experimental Procedures.''



Vascular Endothelial Cell Mitogenic Activities

The mitogenic activities of VEGF and PlGF homo- and heterodimers were assayed in parallel as a function of concentration using HUVE cells (Fig. 7). Precise concentrations were measured using extinction coefficients determined by amino acid composition for 54-kDa VEGF homodimer (A = 210), 58-kDa VEGFbulletPlGF heterodimer (A = 220), and 62-kDa PlGF homodimer (A = 250). The VEGF homodimer is the most potent mitogen on these cells, with an ED of 2 ng/ml (37 pM). The VEGFbulletPlGF heterodimer is approximately 3-fold less potent, with an ED of 6 ng/ml (100 pM). On the basis of the increased specific mitogenic activity compared with the crude conditioned media (6) corrected for the amount of VEGF that it contained, the heterodimer was purified at least 2.5 times 10^5-fold to achieve apparent homogeneity. In contrast, the pure rat PlGF homodimer appears to be comparatively inactive, with an estimated ED geq 100 ng/ml (geq1.6 nM) assuming an equivalent peak response.


Figure 7: Mitogenic activity of purified VEGF and PlGF homodimers and heterodimer. Pure VEGF (circle), VEGFbulletPlGF (box), and PlGF (up triangle) were assayed as a function of dose-monitoring [methyl-^3H]thymidine incorporation into the DNA of nearly confluent HUVE cells.




DISCUSSION

We previously identified two resolved peaks of vascular endothelial cell mitogenic activity eluting from a cation exchange column of partially purified conditioned medium from rat GS-9L glioma cells(6) . Active VEGF homodimer in the later eluting peak was purified to homogeneity, sequenced, cloned, and shown to be homologous to PDGF A and B subunits. Similar human VEGFs were concurrently purified and cloned by several other groups(11, 12) . Although we originally purified and partially characterized limited amounts of VEGFbullet PlGF heterodimer from conditioned media of cells grown under ambient oxygen,^2 based on reports of hypoxic induction of VEGF expression (23, 24, 25) we subsequently lowered the O(2) levels used for conditioning media to only 3%. Both previously identified peaks of mitogenic activity eluted from an anion exchange HPLC column (6) increased 5-fold, although the second VEGF homodimer peak typically contained more activity than the preceding VEGFbulletPlGF heterodimer peak.

We have purified the growth factor in the first detectable endothelial cell mitogenic peak eluted from the anion exchange HPLC column. The nonreduced protein migrates with an apparent mass of 49.5 kDa on SDS-PAGE. Upon reduction, the constituent subunits migrate on SDS-PAGE as polypeptides of 27 and 31 kDa, consistent with the possibility that the newly purified mitogen is dimeric. Although the larger mass polypeptide does not stain as intensely with silver as the lower mass band, both reduced and alkylated polypeptides exhibit equal absorbance at 210 nm that, confirmed by mass from amino acid analysis, demonstrates 1:1 stoichiometry of the two chains. Amino-terminal and peptide microsequencing of the purified polypeptides confirms the presence of VEGF and a second distinct subunit. Western blots of nonreduced and reduced protein using specific peptide-based antisera unique for rat VEGF and, after determination of its sequence, PlGF confirm that this molecule contains both subunits. Therefore, based on its mass, subunit content, and stoichiometry, this newly identified endothelial cell mitogen is a heterodimer composed of VEGF and PlGF subunits. Like VEGF homodimers, the VEGFbulletPlGF heterodimer is mitogenic for vascular endothelial cells although with an approximately 3-fold higher ED.

Reverse transcriptase PCR cloning, using primers based on the peptide sequences, establishes rat PlGF as a full-length 158-amino acid residue chain that, based on NH(2)-terminal sequencing, is processed to a 135-residue form that is 60% identical to human PlGF. While lower than the 89% identity between rat and human VEGF, the PlGF homology is still within the range exhibited between rat and human growth factors. We were not able to find a rat gene more closely related to human PlGF by Southern analysis.^2 Furthermore, on the basis of cDNA sequence from several mammalian species, this gene appears to accommodate mutations at a higher frequency than the homologous VEGF gene.^2

Two amino-terminal rat PlGF sequences were observed following removal of either a 23- or a 26-amino acid residue leader sequence. Either these two mature NH(2) termini are generated by alternative signal peptidase cleavage sites or subsequent NH(2)-terminal proteolysis removes 3 amino acid residues after the primary processing event. Despite the shorter polypeptide chain length, the PlGF subunit migrates at a higher mass (31 kDa) than the VEGF subunit (27 kDa). This discrepancy is presumably attributable to more extensive PlGF glycosylation at multiple locations, including at least 2 of 3 consensus N-glycosylation sites, compared with the single N-glycosylation site present in rat VEGF.

The second of the two newly characterized rat proteins, a homodimer of PlGF, was only identified and purified after specific anti-PlGF antisera were generated since it was not detectably active at the concentrations present in partially purified conditioned media. Once purified, detectable but low specific mitogenic activity for HUVE cells was observed. Human PlGF was cloned and expressed, and crude conditioned medium containing recombinant protein was reported to be mitogenic for a bovine aortic endothelial cell-derived line in culture (15, 16) . However, since the protein was not purified, its potency could not be determined and compared with that of VEGF. Furthermore, the presence of some of the recombinant human PlGF in more active heterodimers with host cell VEGF was not excluded.

The low activity of the rat PlGF homodimers is consistent with the very weak mitogenic activity of purified human recombinant homodimeric PlGF^2(34) compared with recombinant human VEGFbulletPlGF heterodimers and VEGF homodimers. The low mitogenic activity of human recombinant PlGF does not appear to be the consequence of inappropriate folding or denaturation since it binds purified soluble FLT VEGF receptors with high affinity(35) . PlGF does not, however, bind tightly to the KDR receptor. In contrast, VEGF binds to extracellular recognition domains of both FLT and KDR. Taken together, these binding and activity data indicate that FLT is not abundant on these endothelial cells, is selectively inactivated by heterodimer formation with a dominant negative soluble FLT expressed by endothelial cells (36) , or, perhaps, cannot efficiently transduce a mitogenic signal. PlGF homodimers might be better mitogens for other types of vascular endothelial cells. If so, then the endothelial cell range of the heterodimers could be broader than that of either of the homodimers. Alternatively, PlGF homodimers might more efficiently stimulate as yet unidentified non-endothelial cell targets.

These data clearly demonstrate that rat glioma cells produce the two related VEGF and PlGF subunits and that all three possible combinations of homodimers and heterodimers are produced. The presence of PlGF and VEGF homodimers and heterodimers is formally analogous to the presence of AA, BB, and AB dimers in the homologous PDGF system. The choice of the type of PDGF dimer predominantly expressed in platelets exhibits species variation. In human platelets AB heterodimers are principally expressed(37) , whereas BB homodimers are most abundant in pigs(38) . The physiological consequences of the type of PDGF dimers that are expressed are not obvious. Similarly, given the nearly equivalent mitogenic activities of VEGF homodimers and VEGFbulletPlGF heterodimers, a substantial advantage of expressing one or the other dimer is not apparent. VEGF has been shown to be very specific for vascular endothelial cells, being inactive on fibroblasts, vascular smooth muscle cells, corneal endothelial cells, adrenal cortical cells, lens epithelial cells, baby hamster kidney cells, granulosa cells, Balb/MK keratinocytes, and Balb/c 3T3 fibroblasts(3, 4, 5, 6) . Although VEGFbulletPlGF is also inactive on Balb/c 3T3 fibroblasts,^2 neither it nor PlGF homodimers have yet been assayed on a wide variety of cells in culture.

The similarity between the PDGF and VEGF/PlGF systems extends to their receptors. Two types of homologous PDGF receptors, alpha and beta, have been described and characterized. Whereas alpha receptors bind all three isoforms with high affinity, beta receptors only recognize PDGF BB with high affinity and PDGF AB with lower affinity but do not bind PDGF AA. In addition, PDGF binding induces receptor dimerization such that PDGF AA dimerizes alpha receptors, AB promotes formation of alphaalpha and alphabeta dimers, and BB induces formation of all three receptor dimers(39) . It is likely that the analogous situation occurs in the VEGFbulletPlGF family that also consists of two known receptors, FLT and KDR, which are as homologous to each other as are the PDGF alpha and beta receptors. We can then speculate that VEGFbulletPlGF heterodimers might be able to induce the formation of not only FLTbulletFLT receptor homodimers but also KDRbulletFLT receptor heterodimers analogous to that observed in the PDGF system. Given that both VEGF and PlGF can exist as alternatively spliced forms and that both homo- and heterodimeric ligands and receptors are possible, the potential exists for extensive modulation of response in the VEGF system. Further functional characterization of the multiple dimeric VEGFs and their interactions with their receptors will be necessary to determine if differential responses to these mitogens occur.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L40030[GenBank].

§
Current address: Regeneron Pharmaceuticals, Tarrytown, NY 10591.

To whom correspondence should be addressed: Merck Research Laboratories, Rm. 80W-243, P.O. Box 2000, Rahway, NJ 07065. Tel.: 908-594-7567; Fax: 908-594-5067; ken_thomas{at}merck.com.

(^1)
The abbreviations used are: VEGF, vascular endothelial growth factor; PlGF, placenta growth factor; PDGF, platelet-derived growth factor; FLT, fms-like tyrosine kinase receptor; KDR, kinase insert domain-containing receptor; HUVE, human umbilical vein endothelial; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; PCR, polymerase chain reaction.

(^2)
J. DiSalvo, M. L. Bayne, G. Conn, B. Shelton-Inloes, P. W. Kwok, P. G. Trivedi, D. D. Soderman, T. M. Palisi, K. A. Sullivan, and K. A. Thomas, unpublished observations.


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