Katri Pajusola

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.

A model for gene therapy of human hereditary lymphedema

Primary human lymphedema (Milroys 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.

Vascular Endothelial Cell Growth Factor Receptor 3-Mediated Activation of Lymphatic Endothelium Is Crucial for Tumor Cell Entry and Spread via Lymphatic Vessels

Lymphangiogenic growth factors vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to promote lymphatic metastasis by inducing tumor-associated lymphangiogenesis. In this study, we have investigated how tumor cells gain access into lymphatic vessels and at what stage tumor cells initiate metastasis. We show that VEGF-C produced by tumor cells induced extensive lymphatic sprouting towards the tumor cells as well as dilation of the draining lymphatic vessels, suggesting an active role of lymphatic endothelial cells in lymphatic metastasis. A significant increase in lymphatic vessel growth occurred between 2 and 3 weeks after tumor xenotransplantation, and lymph node metastasis occurred at the same stage. These processes were blocked dose-dependently by inhibition of VEGF receptor 3 (VEGFR-3) signaling by systemic delivery of a soluble VEGFR-3-immunoglobulin (Ig) fusion protein via adenoviral or adeno-associated viral vectors. However, VEGFR-3-Ig did not suppress lymph node metastasis when the treatment was started at a later stage after the tumor cells had already spread out, suggesting that tumor cell entry into lymphatic vessels is a key step during tumor dissemination via the lymphatics. Whereas lymphangiogenesis and lymph node metastasis were significantly inhibited by VEGFR-3-Ig, some tumor cells were still detected in the lymph nodes in some of the treated mice. This indicates that complete blockade of lymphatic metastasis may require the targeting of both tumor lymphangiogenesis and tumor cell invasion.

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.

Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are important regulators of blood and lymphatic vessel growth and vascular permeability. The VEGF‐C/VEGFR‐3 signaling pathway is crucial for lymphangiogenesis, and heterozygous inactivating missense mutations of the VEGFR‐3 gene are associated with hereditary lymphedema. However, VEGF‐C can have potent effects on blood vessels because its receptor VEGFR‐3 is expressed in certain blood vessels and because the fully processed form of VEGF‐C also binds to the VEGFR‐2 of blood vessels. To characterize the in vivo effects of VEGF‐C on blood and lymphatic vessels, we have overexpressed VEGF‐C via adenovirus‐and adeno‐associated virus‐mediated transfection in the skin and respiratory tract of athymic nude mice. This resulted in dose‐dependent enlargement and tortuosity of veins, which, along with the collecting lymphatic vessels were found to express VEGFR‐2. Expression of angiopoietin 1 blocked the increased leakiness of the blood vessels induced by VEGF‐C whereas vessel enlargement and lymphangiogenesis were not affected. However, angiogenic sprouting of new blood vessels was not observed in response to AdVEGF‐C or AAV‐VEGF‐C. These results show that virally produced VEGF‐C induces blood vessel changes, including vascular leak, but its angiogenic potency is much reduced compared with VEGF in normal skin.—Saaristo, A., Veikkola, T., Enholm, B. Hytönen, M., Arola, J., Pajusola, K., Turunen, P., Jeltsch, M., Karkkainen, M. J., Kerjaschki, D., Bueler, H., Ylä‐Herttuala, S., Alitalo, K. Adenoviral VEGF‐C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes. FASEB J. 16, 1041–1049 (2002)

Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

Recent work from many laboratories has demonstrated that the vascular endothelial growth factor-C/VEGF-D/VEGFR-3 signaling pathway is crucial for lymphangiogenesis, and that mutations of the Vegfr3 gene are associated with hereditary lymphedema. Furthermore, VEGF-C gene transfer to the skin of mice with lymphedema induced a regeneration of the cutaneous lymphatic vessel network. However, as is the case with VEGF, high levels of VEGF-C cause blood vessel growth and leakiness, resulting in tissue edema. To avoid these blood vascular side effects of VEGF-C, we constructed a viral vector for a VEGFR-3–specific mutant form of VEGF-C (VEGF-C156S) for lymphedema gene therapy. We demonstrate that VEGF-C156S potently induces lymphangiogenesis in transgenic mouse embryos, and when applied via viral gene transfer, in normal and lymphedema mice. Importantly, adenoviral VEGF-C156S lacked the blood vascular side effects of VEGF and VEGF-C adenoviruses. In particular, in the lymphedema mice functional cutaneous lymphatic vessels of normal caliber and morphology were detected after long-term expression of VEGF-C156S via an adeno associated virus. These results have important implications for the development of gene therapy for human lymphedema.

Vascular endothelial growth factors VEGF‐B and VEGF‐C

Vascular endothelial growth factor, which was idenheart cDNA library, respectively, by using a serendipitously found partial mouse cDNA clone as a probe tified almost 10 years ago, has so far been considered as the only growth factor relatively specific for endothelial (20,24). Independently, another group found the same gene when attempting to identify candidate genes for cells. VEGF is an important regulator of endothelial cell proliferation, migration, and permeability during multiple endocrine neoplasia type 1 (MEN1). The product of this alternatively spliced gene was designated as embryonic vasculogenesis and in physiological and pathological angiogenesis [reviewed in (1–3)]. The pivVRF (21). The two currently known isoforms of VEGF-B are otal role of VEGF in embryogenesis is emphasized by the unprecedented result that the inactivation of even a generated by alternative splicing of mRNA from the VEGF-B gene, spanning about 4 kb of DNA. The human single VEGF allele results in embryonic lethality (4,5). VEGF acts through its two known high-affinity recepand murine VEGF-B genes are composed of 7 exons, and their exon–intron organization resembles that of tors Flt1, vascular endothelial growth factor receptor 1 (VEGFR-1) and KDR/Flk1, or VEGFR-2 (6–10). A VEGF and PlGF genes (21,24,25). The mature VEGFB proteins (devoid of signal sequence) have 167 (VEGFthird receptor tyrosine kinase homologous with VEGFR-1 and VEGFR-2, designated Flt4, was cloned B167) and 186 (VEGF-B186) amino acid residues, respectively. VEGF-B186 is generated by using an alternative as an orphan receptor by two research groups and was shown not to bind VEGF (11–14). Additional VEGF splice acceptor site in exon 6, resulting in an insertion of 101 bp between nucleotides 410 and 411 in the coding receptors of unknown nature also exist on endothelial and tumor cells (15,16). sequence of VEGF-B167. This insertion introduces a frame shift and a stop codon at the position correspondThe second member of the VEGF family of growth factors, placenta growth factor (PlGF), is 53% identical ing to nucleotides 521–523 of the coding region of VEGF-B167 cDNA (Fig. 1). Thus, the two VEGF-B isowith VEGF within its platelet-derived growth factorlike region and binds only VEGFR-1 (17–19). Both forms have an identical NH2-terminal domain of 115 aa and different COOH-terminal domains. Although VEGF and PlGF are dimeric glycoproteins related in structure to the platelet-derived growth factors A and the C-terminus of VEGF-B167 is highly basic, that of VEGF-B186 is rich in alanine, proline, serine, and threoB (PDGF-A and PDGF-B). This relation is based on the presence of several conserved amino acid residues nine amino acid residues and has no significant similarity with amino acid sequences of known proteins including 8 equally spaced cysteines. Compared with VEGF, the mitogenic or permeability-enhancing activi(21,24). Unlike other growth factors of the VEGF-family, both isoforms of human and mouse VEGF-B lack ties of PlGF are weak; however, PlGF is able to potentiate the action of VEGF in vivo and in vitro (19). PlGF– the consensus sequence for N-linked glycosylation (NXT/S); instead, VEGF186 is O-glycosylated (24). VEGF heterodimers occur in vivo and have intermediate potency in mitogenic stimulation of endothelial VEGF-B167 remains cell associated with secretion, but it is released into the culture medium with treatcells (35). Two novel endothelial cell-specific growth factors, ment of the producing cells with heparin or high salt. The cell (or matrix) association of VEGF-B167 likely ocstructurally related to VEGF and PlGF, were recently discovered. These factors, designated as vascular endocurs via its basic region, as observed for the highly basic splice variants of VEGF. This notion is supported thelial growth factor B (VEGF-B) or VEGF-related factor (20,21) and vascular endothelial growth factor C by the fact that VEGF-B186 , lacking the highly basic region, is freely secreted from cells and is not bound to (VEGF-C) or VEGF-related protein (22,23) expand the known VEGF family and demonstrate the complexity cell-surface or pericellular heparan sulfate proteoglycans (20,24). of regulation of endothelial functions. This review summarizes the initial studies on VEGF-B and VEGF-C. The apparent molecular masses of the secreted VEGF-B167 and VEGF-B186 polypeptides are 21 kDa VEGF-B/VRF

Stabilized HIF-1α is superior to VEGF for angiogenesis in skeletal muscle via adeno-associated virus gene transfer

Therapeutic angiogenesis provides a potential alternative for the treatment of cardiovascular ischemic diseases. Vascular endothelial growth factor (VEGF) is an important component of the angiogenic response to ischemia. Here we used adeno‐associated virus (AAV) gene delivery to skeletal muscle to examine the effects of VEGF vs. a stabilized form of hypoxia‐inducible factor‐1α (HIF‐1α). The recombinant AAVs were injected into mouse tibialis anterior muscle, and their effects were analyzed by immunohistochemistry and functional assays. These analyses showed that stabilized HIF‐1 α markedly increase capillary sprouting and proliferation, whereas VEGF164 or VEGF120 induced only proliferation of endothelial cells without formation of proper capillary structures. The Evans Blue permeability assay indicated that, unlike VEGF, HIF‐1 α overexpression did not increase vascular leakiness in the transduced muscle. Doppler ultrasound imaging showed that vascular perfusion in the HIF‐1 α treated muscles was significantly enhanced when compared to the controls and not further improved by co‐expression of the arteriogenic growth factors angiopoietin‐1 or platelet‐derived growth factor‐B. Our results show that AAV‐mediated transduction of a stabilized form of HIF‐1 α can circumvent the problems associated with overexpression of individual angiogenic growth factors. HIF‐1 α should thus offer a potent alternative for pro‐angiogenic gene therapy.

Cell-Type-Specific Characteristics Modulate the Transduction Efficiency of Adeno-Associated Virus Type 2 and Restrain Infection of Endothelial Cells

ABSTRACT Adeno-associated viruses (AAVs) are promising vectors for various gene therapy applications due to their long-lasting transgene expression and wide spectrum of target cells. Recently, however, it has become apparent that there are considerable differences in the efficiencies of transduction of different cell types by AAVs. Here, we analyzed the efficiencies of transduction and the transport mechanisms of AAV type 2 (AAV-2) in different cell types, emphasizing endothelial cells. Expression analyses in both cultured cells and the rabbit carotid artery assay showed a remarkably low level of endothelial cell transduction in comparison to the highly permissive cell types. The study of the endosomal pathways of AAV-2 with fluorescently labeled virus showed clear targeting of the Golgi area in permissive cell lines, but this phenomenon was absent in the endothelial cell line EAhy-926. On the other hand, the response to the block of endosomal acidification by bafilomycin A1 also showed differences among the permissive cell types. We also analyzed the effect of proteasome inhibitors on endothelial cells, but their impact on the primary cells and in vivo was not significant. On the contrary, analysis of the expression pattern of heparan sulfate proteoglycans (HSPGs), the primary receptors of AAV-2, revealed massive deposits of HSPG in the extracellular matrix of endothelial cells. The matrix-associated receptors may therefore compete for virus binding and reduce transduction in endothelial cells. Accordingly, in endothelial cells detached from their matrix, AAV-2 transduction was significantly increased. Altogether, these results point to a more complex cell-type-specific mode of transduction of AAV-2 than previously appreciated.

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.