The related FLT4, FLT1, and KDR receptor tyrosine kinases show distinct expression patterns in human fetal endothelial cells.

The growth factor receptors expressed on endothelial cells are of special interest because of their potential to program endothelial cell growth and differentiation during development and neovascularization in various pathological states, such as wound healing and angiogenesis associated with tumorigenesis. Vascular endothelial growth factor ([VEGF] also known as vascular permeability factor) is a potent mitogen and permeability factor, which has been suggested to play a role in embryonic and tumor angiogenesis. The newly cloned FLT4 receptor tyrosine kinase gene encodes a protein related to the VEGF receptors FLT1 and KDR/FLK-1. We have here studied the expression of FLT4 and the other two members of this receptor family in human fetal tissues by Northern and in situ hybridization. These results were also compared with the sites of expression of VEGF and the related placenta growth factor (PlGF). Our results reveal FLT4 mRNA expression in vascular endothelial cells in developing vessels of several organs. A comparison of FLT4, FLT1 and KDR/FLK-1 receptor mRNA signals shows overlapping, but distinct expression patterns in the tissues studied. Certain endothelia lack one or two of the three receptor mRNAs. These data suggest that the receptor tyrosine kinases encoded by the FLT gene family may have distinct functions in the regulation of the growth/differentiation of blood vessels.

Cardiovascular failure in mouse embryos deficient in VEGF receptor-3.

Vascular endothelial growth factor (VEGF) is a key regulator of blood vessel development in embryos and angiogenesis in adult tissues. Unlike VEGF, the related VEGF-C stimulates the growth of lymphatic vessels through its specific lymphatic endothelial receptor VEGFR-3. Here it is shown that targeted inactivation of the gene encoding VEGFR-3 resulted in defective blood vessel development in early mouse embryos. Vasculogenesis and angiogenesis occurred, but large vessels became abnormally organized with defective lumens, leading to fluid accumulation in the pericardial cavity and cardiovascular failure at embryonic day 9.5. Thus, VEGFR-3 has an essential role in the development of the embryonic cardiovascular system before the emergence of the lymphatic vessels.

FLT4 receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissues and cell lines.

The fms-like tyrosine kinase 4 (FLT4) complementary DNA was cloned from a human HEL erythroleukemia cell library by polymerase chain reaction-amplification. We previously reported a partial sequence of FLT4 and showed that the FLT4 gene maps to chromosomal region 5q33-qter (O. Aprelikova, K. Pajusola, J. Partanen, E. Armstrong, R. Alitalo, S. Bailey, J. McMahon, J. Wasmuth, K. Huebner, and K. Alitalo, Cancer Res., 52: 746-748, 1992). Here we present the full-length sequence of the predicted FLT4 protein. The extracellular domain of FLT4 consists of 7 immunoglobulin-like loops, including 12 potential glycosylation sites. On the basis of structural similarities FLT4 and the previously known FLT1 and kinase insert domain-containing receptor tyrosine kinase/fetal liver kinase 1 (KDR/FLK1) receptors constitute a subfamily of class III tyrosine kinases. FLT4 was expressed as 5.8- and 4.5-kilobase mRNAs which were found to differ in their 3' sequences and to be differentially expressed in the HEL and DAMI leukemia cells. Interestingly, a Wilms' tumor cell line, a retinoblastoma cell line, and a nondifferentiated teratocarcinoma cell line expressed FLT4, whereas differentiated teratocarcinoma cells were negative. Most fetal tissues also expressed the FLT4 mRNA, with spleen, brain intermediate zone, and lung showing the highest levels. In in situ hybridization the FLT4 autoradiographic grains decorated bronchial epithelial cells of fetal lung. No evidence was obtained for the expression of FLT4 in the endothelial cells of blood vessels.

FLT4, a novel class III receptor tyrosine kinase in chromosome 5q33-qter.

The receptors for at least two hematopoietic growth factors, namely the stem cell factor and colony-stimulating factor 1, belong to class III receptor tyrosine kinases. Here we describe cloning of a partial complementary DNA for FLT4, an additional member of this gene family from human leukemia cells. The FLT4 tyrosine kinase domain is 79% homologous with the previously cloned FLT1 (M. Shibuya et al., Oncogene, 5: 519-524, 1990) tyrosine kinase and maps to the chromosomal region 5q33-qter. We have found FLT4 expression in human placenta, lung, heart, and kidney, whereas the pancreas and brain appeared to contain very little if any FLT4 RNA. The results suggest that FLT4 functions in multiple adult tissues.

Two human FLT4 receptor tyrosine kinase isoforms with distinct carboxy terminal tails are produced by alternative processing of primary transcripts.

FLT4 is a recently cloned gene encoding a transmembrane tyrosine kinase related to the FLT1 and KDR/FLK1 vascular endothelial growth factor receptors. We have previously shown that FLT4 is expressed as transcripts of 4.5 and 5.8 kb in several human fetal and adult tissues. Here we show that these transcripts encode two polypeptides, FLT4s (short) and FLT41 (long), which are proteolytically processed in transfected cells and leukemia cells and which have different carboxy terminal tails. The 3' coding region of the 5.8 kb mRNA was found to be 65 codons longer than that of the the 4.5 kb mRNA. Analysis of the genomic structure of the region encoding the two carboxy termini revealed that the two transcripts are generated by alternative polyadenylation and subsequent alternative splicing during RNA processing. Our findings thus show regulation of FLT4 structure in the carboxy terminal tail considered important for receptor function. The significance of the two forms may relate to the role of additional potential autophosphorylation sites in the FLT4 long form.

Signalling properties of FLT4, a proteolytically processed receptor tyrosine kinase related to two VEGF receptors.

The FLT4, FLT1 and KDR/FLK1 genes encode structurally similar endothelial cell receptor tyrosine kinases. Recently it has been shown that the FLT1 and KDR/FLK-1 proteins function as high-affinity receptors for vascular endothelial growth factor (VEGF). Here we show that FLT4 does not act as a receptor for VEGF, as VEGF did not show specific binding to the FLT4 tyrosine kinase or induce its autophosphorylation. Also, FLT4 did not interact with KDR in response to VEGF. However, when fused with the ligand binding domain of the colony stimulating factor-1 receptor (CSF-1R), the FLT4 tyrosine kinase was specifically activated by CSF-1. The activated FLT4 tyrosine kinase domain was found to interact with the Src homology 2 domains of the SHC and GRB2 adaptor proteins in vitro and with SHC in cells. CSF-1 stimulation of the CSF-1R/FLT4 receptor chimera induced thymidine incorporation in serum-starved NIH3T3 fibroblasts, but not in porcine aortic or murine lung capillary endothelial cells, although tyrosyl phosphorylation of the receptor and SHC occurred in these cells as well. These results suggest that the endothelial cell FLT4 receptor tyrosine kinase transmits signals for an as yet unidentified growth factor.

Genomic organization of the mouse and human genes for vascular endothelial growth factor B (VEGF-B) and characterization of a second splice isoform.

A second isoform and the genomic structures of mouse and human vascular endothelial growth factor B are described. Both genes consist of seven coding exons and span about 4 kilobases of DNA. The two identified isoforms of vascular endothelial growth factor B are generated by alternative splicing where different splice acceptor sites in exon 6 introduce a frameshift and a partial use of different but overlapping reading frames. Consequently, the COOH-terminal domains in the two isoforms show no resemblance. Mouse and human cDNA clones for the novel isoform of vascular endothelial growth factor B encoded a secreted protein of 186 amino acid residues. Expression in transfected cells generated a protein of 25 kDa which upon secretion was modified by O-linked glycosylation and displayed a molecular mass of 32 kDa under reducing conditions. The protein was expressed as a disulfide-linked homodimer, and heterodimers were generated when coexpressed with vascular endothelial growth factor. The entirely different COOH-terminal domains in the two isoforms of vascular endothelial growth factor B imply that some functional properties of the two proteins are distinct.

A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases.

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, and the permeability of blood vessels are regulated by vascular endothelial growth factor (VEGF) via its two known receptors Flt1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2). The Flt4 receptor tyrosine kinase is related to the VEGF receptors, but does not bind VEGF and its expression becomes restricted mainly to lymphatic endothelia during development. In this study, we have purified the Flt4 ligand, VEGF-C, and cloned its cDNA from human prostatic carcinoma cells. While VEGF-C is homologous to other members of the VEGF/platelet derived growth factor (PDGF) family, its C-terminal half contains extra cysteine-rich motifs characteristic of a protein component of silk produced by the larval salivary glands of the midge, Chironomus tentans. VEGF-C is proteolytically processed, binds Flt4, which we rename as VEGFR-3 and induces tyrosine autophosphorylation of VEGFR-3 and VEGFR-2. In addition, VEGF-C stimulated the migration of bovine capillary endothelial cells in collagen gel. VEGF-C is thus a novel regulator of endothelia, and its effects may extend beyond the lymphatic system, where Flt4 is expressed.

Vascular endothelial growth factor B, a novel growth factor for endothelial cells.

We have isolated and characterized a novel growth factor for endothelial cells, vascular endothelial growth factor B (VEGF-B), with structural similarities to vascular endothelial growth factor (VEGF) and placenta growth factor. VEGF-B was particularly abundant in heart and skeletal muscle and was coexpressed with VEGF in these and other tissues. VEGF-B formed cell-surface-associated disulfide-linked homodimers and heterodimerized with VEGF when coexpressed. Conditioned medium from transfected 293EBNA cells expressing VEGF-B stimulated DNA synthesis in endothelial cells. Our results suggest that VEGF-B has a role in angiogenesis and endothelial cell growth, particularly in muscle.

Novel human vascular endothelial growth factor genes VEGF-B and VEGF-C localize to chromosomes 11q13 and 4q34, respectively.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an important regulator of endothelial cell proliferation, migration, and permeability during embryonic vasculogenesis as well as in physiological and pathological angiogenesis. The recently isolated VEGF-B and VEGF-C cDNAs encode novel growth factor genes of the VEGF family. METHODS AND RESULTS: Southern blotting and polymerase chain reaction analysis of somatic cell hybrids and fluorescence in situ hybridization (FISH) of metaphase chromosomes were used to assess the chromosomal localization of VEGF-B and VEGF-C genes. The VEGF-B gene was found on chromosome 11q13, proximal to the cyclin D1 gene, which is amplified in a number of human carcinomas. However, VEGF-B was not amplified in several mammary carcinoma cell lines containing amplified cyclin D1. The VEGF-C gene was located on chromosome 4q34, close to the human aspartylglucosaminidase gene previously mapped to 4q34-35. CONCLUSIONS: The VEGF-B locus in 11q13 and the VEGF-C locus in 4q34 are candidate targets for mutations that lead to vascular malformations or cardiovascular diseases.

A model for gene therapy of human hereditary lymphedema.

Primary human lymphedema (Milroy's disease), characterized by a chronic and disfiguring swelling of the extremities, is associated with heterozygous inactivating missense mutations of the gene encoding vascular endothelial growth factor C/D receptor (VEGFR-3). Here, we describe a mouse model and a possible treatment for primary lymphedema. Like the human patients, the lymphedema (Chy) mice have an inactivating Vegfr3 mutation in their germ line, and swelling of the limbs because of hypoplastic cutaneous, but not visceral, lymphatic vessels. Neuropilin (NRP)-2 bound VEGF-C and was expressed in the visceral, but not in the cutaneous, lymphatic endothelia, suggesting that it may participate in the pathogenesis of lymphedema. By using virus-mediated VEGF-C gene therapy, we were able to generate functional lymphatic vessels in the lymphedema mice. Our results suggest that growth factor gene therapy is applicable to human lymphedema and provide a paradigm for other diseases associated with mutant receptors.

VEGFR3 gene structure, regulatory region, and sequence polymorphisms.

Vascular endothelial growth factor receptor 3 (VEGFR-3) is required for cardiovascular development during embryogenesis. In adults, this receptor is expressed in lymphatic endothelial cells, and mutant VEGFR3 alleles have been implicated in human hereditary lymphedema. To better understand the basis of its specific endothelial lineage-restricted expression, we have characterized the VEGFR3 gene and its regulatory 5' flanking region. The human gene contains 31 exons, of which exons 30a and 30b are alternatively spliced. The VEGFR3 proximal promoter is TATA-less and contains stretches of sequences homologous with the mouse Vegfr3 promoter region. In transfection experiments of cultured cells, the Vegfr3 promoter was shown to control endothelial cell-specific transcription of downstream reporter genes. This result was further confirmed in vivo; in a subset of transgenic mouse embryos, a 1.6 kb Vegfr3 promoter fragment directed weak lymphatic endothelial expression of the LacZ marker gene. This suggests that endothelial cell-specific elements occur in the proximal promoter, although further enhancer elements are probably located elsewhere. The sequence, organization, and variation in the VEGFR3 gene and its regulatory region provide important tools for the molecular genetic analysis of the lymphatic system and its disorders.