PlGF/VEGF dimerization: Two genes code for the two polypeptide chains PDGF-A and PDGF-B. In different cells the three different forms AA, BB and AB are differentially expressed. The three PDGF forms bind with different affinity to the three receptors alpha/alpha, beta/beta and alpha/beta. A similar model is hypothesized for PlGF and VEGF homo and heterodimers

The process in which new blood vessels are formed from pre-existing vessels by vascular sprouting is known as angiogenesis. During angiogenesis, there is an organized proliferation of endothelial cells, which is most likely regulated by polypeptide growth factors. A number of polypeptides are known to stimulate the growth of endothelial cells in vitro: acidic and basic fibroblast growth factors (aFGF and bFGF) (vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). Among these growth factors, only VEGF and PlGF have been reported to be specific for endothelial cells.

The analysis of the function of these growth factors and the signal transduction mechanism stimulating the endothelial cell proliferation, invasion and migration offers the possibility to device therapies for the control of tumor progression and metastasis formation.

Both PlGF and VEGF proteins are present at a high level in many normal tissues. The overlapping expression patterns of PlGF and VEGF suggest the existence of heterodimeric forms of PlGF and VEGF with different role and receptor binding specificity compared to the homodimeric forms.

Previous work

Human PlGF, isolated in our laboratory is a dimeric glycoprotein that shares a number of biochemical and functional features with VEGF. The amino-acid similarity between the two factors in the platelet-derived growth factor-like (PDGF) domain is high (approximately 50%), with a perfect conservation of the characteristically spaced eight cystein residues. Furthermore, the intron-exon structure of VEGF and PlGF genes are similar, consistent with a common evolutionary origin. Analysis of cDNA clones coding for VEGF indicates that alternative splicing of the primary transcript gives rise to at least four forms of the mature protein. We have also found two forms of PlGF generated by an alternative splicing. The two forms, named PlGF-1 and PlGF-2, differ by the insertion of a highly basic 21 amino-acid stretch at the carboxyl-end of the protein. The additional basic region apparently confers to PlGF-2 the property of binding to heparin. Finally, we have demonstrated that human PlGF-1 induces a dose-dependent angiogenic response in the rabbit cornea assay. Overall, it would thus appear that PlGF is an angiogenic factor in many ways similar to VEGF.

Specific aims

The project intends to analyze the mechanism of action and the role of the homodimer PlGF/PlGF and heterodimer PlGF/VEGF in the angiogenesis of the normal tissue and during tumor progression. In particular we intend to:

1) purify the heterodimeric form PlGF/VEGF;

2) identify and characterize the receptor of the homodimeric and heterodimeric forms;

3) study the role of the different forms of PlGF (PlGF-1 and -2);

4) study the function of PlGF and VEGF using transgenic mice and recombinant cells;

5) device systems for the control of tumor angiogenesis and tumor progression.

Phasing of the project

PlGF/VEGF heterodimer isolation

Both VEGF and PlGF contain the 8 cystein motif of PDGF. Two genes code for the two polypeptide chains PDGF-A and PDGF-B. In different cells the three different forms AA, BB and AB are differentially expressed. The three PDGF forms bind with different affinity to the three receptors alpha/alpha, beta/beta and alpha/beta.

Experimental evidences indicate that heterodimeric forms of PlGF and VEGF exist in several cell types such as choriocarcinoma cells JEG-3 and hepatoma cells HepG2.

We intend to construct stable transformants containing both cDNAs coding for the two angiogenic factors in vectors such as pREP4 and pREP9 (Invitrogen). From the conditioned media we will purify the recombinant proteins. The purified proteins will be used to produce monoclonal antibodies able to specifically recognize the heterodimeric form. With these antibodies we will analyze the presence of the heterodimer in speciments of normal and tumor tissues.

The purified heterodimeric protein will tested for the ability to stimulate angiogenesis and vascular permeability in in vitro and in vivo assays.

Identification and characterization of PlGF and VEGF/PlGF receptors

Three receptors, expressed in endothelial cells have been identified FLT1, KDR/FLK1 e FLT4. The angiogenic factor VEGF is the ligand of the receptors FLT1 e KDR/FLK1.

To verify if PlGF is the ligand of these receptors we have started a collaboration with Dr. K. Alitalo (Univ. di Helsinki, Finland), H. Weich (GBF, Dept. of Gene Expression, Braunschweig, Germany) and B. Terman (Cyanamid, Lederle Lab., Pearl River, USA). PlGF-1 is able to compete with VEGF only for the binding to FLT1.

The heterodimer PlGF/VEGF purified by FPLC will be used in binding assays to the known receptors. For these types of experiments we will use the cell lines SK-MEL-37, WM-35 e WM9 that contain the receptors FLT1 and KDR respectively alone and in combination.

PlGF-1 and PlGF-2 function

Alternative splicing mechanisms are responsible for the production of different forms of PlGF and VEGF. Two PlGFs are present in severall tissues and cells with different relative abundance. PlGF-2 has a 21-aminoacid insertion not present in PlGF-1 coding for a highly basic region near the C-terminus. This region confer the ability to PlGF-2 to bind heparin like molecules of the extracellular matrix confering probably a different role to the two forms.

Eukariotic expression vectors containing the cDNAs coding for PlGF-1 and PlGF-2 will be used to generate stable transformant cell lines producing in the media the two growth factor forms. The conditioned media will be tested on endothelial cells derived from different sources to test if these factors are tissue specific. Proliferation of endothelial cells will be monitored in vitro by BrdU incorporation and subsequently monoclonal antibody and peroxidase immunological detection (Amersham). Permeability tests will be performed by culturing the endothelial cells on the Falcon Cyclopore membrane inserts (Becton Dickinson) and monitoring the flow of different molecules through the cell layer.

Use of transgenic mice to study PlGF and VEGF function in angiogenesis

An effective way to test the function of a cloned gene during development is to produce transgenic animals in which the gene is overexpressed or ectopically expressed in vivo. Alternatively, to generate an animal model to investigate the role of PlGF in mammalian development and in tumor formation, we will use homologeous recombination in mouse embryonic stem cells to derive a null allele of the gene. This work willbe done in collaboration with Dr. Carmeliet (Center for Mol. & Vascular Biol., Leuven, Belgium). Mutagenized PlGF and VEGF genes will be also introduced in mice to compete with the wild type factors (see below).

Control of tumor angiogenesis and cell proliferation

An attempt to control tumor angiogenesis will be pursued by dominant negative mutant proteins. After the analysis of the region of PlGF and VEGF important for dimer formation and receptor binding, we will construct recombinant plasmids coding modified proteins able to compete with wild type proteins for receptor binding and dimer formation.